MT-177-Dushyant Thesis-2009 - Ganpat University Institutional

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION
FOR ESTIMATION OF GABAPENTIN IN HUMAN PLASMA
AND PHARMACEUTICAL DOSAGE FORMS
A
Thesis Submitted to
GANPAT UNIVERSITY, KHERVA
In Partial Fulfillment of the requirement for the degree of
MASTER OF PHARMACY
IN
THE FACULTY OF PHARMACY
(QUALITY ASSURANCE)
APRIL-2009
Guided By:
Submitted By:
Dr. S. A. Patel
DUSHYANT PATEL
M. Pharm., Ph. D.
B. Pharm.
S. K. PATEL COLLEGE OF PHARMACEUTICAL EDUCATION AND RESEARCH
GANPAT UNIVERSITY, KHERVA - 382711
DIST. – MEHSANA, GUJARAT, INDIA.
CERTIFICATE
This is to certify that the research work embodied in this thesis entitled
“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR
ESTIMATION OF GABAPENTIN IN HUMAN PLASMA AND PHARMACEUTICAL
DOSAGE FORMS” was carried out by Mr. Dushyant B. Patel under my guidance.
The work is original, bonafide and it is up to my satisfaction.
Guided by:
Head of the Department:
Dr. S. A. Patel
Dr. S.S.Pancholi
M.Pharm., Ph. D. (Asst. Professor)
Dept. of Pharm. Quality Assurance,
Shree S. K. Patel College of
Pharmaceutical Edu. & Res.
Kherva, Mehsana
M.Pharm., Ph. D. (Professor)
Dept. of Pharm. Quality Assurance,
Shree S. K. Patel College of
Pharmaceutical Edu. & Res.
Kherva, Mehsana
Principal:
Dr. N.J.Patel
M.Pharm, Ph. D.
Shree S. K. Patel College of
Pharmaceutical Edu. & Res.
Kherva, Mehsana
Date:
Place:
DECLARATION
E
I hereby declare that this thesis entitled “ANALYTICAL METHOD
DEVELOPMENT AND VALIDATION FOR ESTIMATION OF GABAPENTIN IN
HUMAN PLASMA AND PHARMACEUTICAL DOSAGE FORMS” is based on
original work carried out by me in the laboratories of Torrent Research Centre,
Gandhinagar and Shree S.K.Patel College of Pharmaceutical Education And
Research, Kherva. This work has not been submitted earlier to any other
University or College.
Date:
(Dushyant B. Patel)
`
ACKNOWLEDGMENT
A single flower cannot make a garland or a single star can not make the beautiful shiny sky at
the night, same way a research work can never be outcome of a single individual’s talent or
efforts. It is just like climbing a high peak, step by step, accompanied with bitterness, hardships,
frustration, encouragement and trust and with so many people’s kind help. When I found
myself at the top enjoying beautiful scenery, I realized that it was, in fact, teamwork that got
me there. Though it will not be enough to express my gratitude in words to all those people who
help me, I would still like to give my many, many thanks to all these people.
First of all, I would like to give my sincere thanks to my honorific guide, Dr. S. A. Patel, M.
Pharm., Ph. D., (Asst. Professor) Dept. of Pharmaceutical Quality Assurance, S. K. Patel
College Of Pharmaceutical Education & Research, Kherva and Mr. Hiten Shah, Scientist-I,
Torrent Research Centre, Gandhinagar for offering me his valuable advice, patiently supervising
me, and always guiding me in the right direction. Without their help I could not have finished
my dissertation successfully.
I would like to give my sincere and heartfelt thanks to Dr. S.S.Pancholi, M. Pharm., Ph.D., and
Head of the Dept. of Pharmaceutical Quality Assurance and Dr.P.U.Patel, M.Pharm., Ph.D.,
Dept. of Pharmaceutical Quality Assurance, S.K.Patel College of Pharmaceutical Education &
Research, Kherva, and also thanks to faculty member of Department of pharmaceutical
chemistry of this college Dr. L.J.Patel, Dr. B.G.Chaudhry, Mr. A.M.Prajapati, Mrs. D.B.Patel,
Mrs. S.K.Patel, and Mrs. H.J.Panchal who offered me a lot of friendly help; they transferred to
me their research links and suggested to me the necessary readings for pursuing Dissertation
research. With reverence and gratitude, I would like to thank Dr. N.J.Patel, M.Pharm., Ph.D.,
Principal, S.K.Patel College of Pharmaceutical Education & Research, Kherva, for providing
facilities through out this course in this college.
I owe a special word of thanks to Mr. Jignesh Bhatt, Dr. Deepak Jain, Dr. Tushar Mehta and
Mr. Mohan Kundlik for their profound guidance and sharing of knowledge.
In addition to my advisors, a number of other Torrentians at Torrent Research Center have also
had a large impact on my work. Amongst the endless list I would like to thanks Mr. Aniraban
Chowdhary, Mr. Hitesh Parmar, Mr. Ketal Prajapati, Mr. Mehul Patel, Mr. Nitesh Patel, Mr.
Shailesh Ghataliya and Mr. Shuja Khan. I thank them for their helpful comments and
encouragement.
I wish to express my deep sense of gratitude and indebtness to Mr. Jaimin Patel, Mr. Bipin
Patel, Mr. Raj Patel, Mr. Tarun Agrawal, Mr. Naresh Kataria and Mr. Kush Shah for their
selfless guidance.
I am extremely thankful to all my colleagues at S.K.Patel College of Pharmaceutical Education
& Research, Kherva, for providing a good working atmosphere. I am very grateful to Riddhi,
Dipen, Jignesh, Brijesh, Kaushik, Snehal, Lav, Birva, Ankur, Shweta, Hetal, Samixa, Arati,
Jigna, Vikas, Gaurang and Hemant for their generous help and cooperation.
For construction of any strong building, we need a strong foundation, that is why I would like
to have opportunity to owe my thanks to management of TORRENT RESEARCH CENTRE for
giving me chance to perform my dissertation work in such a delightful and friendly environment.
I sincerely thank to Mr. P. I. Patel, Librarian, Mahadevbhai and Mukeshbhai of S. K. Patel
College of Pharmaceutical Education and Research, Kherva, for providing me library facilities
and constant encouragement during my work. For help rendered by Non-teaching staff
especially Mr. Jayeshbhai and Mr. Sushilbhai (Lab. Assistant), Ms. Chaula Patel (Computer
lab), Mr. Dineshbhai Patel (Store in charge), Pravin Bhai and Pankaj Bhai are sincerely
acknowledged.
“May the candle be lightened forever, the joy is not of light alone, but of presence of those, who
played the role behind the curtain.”
Last, but not least, I would like to thanks my parents and sister. I am very grateful to my
Parents and brother HASMUKH, their understanding and love encouraged me to work hard.
Their firm and kind hearted personality has affected me to be steadfast and never bend to
difficulty. They always let me know that they are proud of me, which motivates me to work
harder and do my best.
April 2009
(DUSHYANT B. PATEL)
Dushyant Patel
Index
INDEX
SR NO.
1
PAGE
TITLE
NO.
INTRODUCTION
1
1.1
INTRODUCTION TO EPILEPSY
1
1.2
DRUG PROFILE
4
1.3
REFERENCES
7
REVIEW OF LITERATURE
8
2.1
REVIEW OF LITERATURE
8
2.2
REFERENCES
14
AIM OF THE PRESENT WORK
17
AIM OF THE PRESENT WORK
17
2
3
3.1
4
DEVELOPMENT AND VALIDATION OF LC-MS/MS
METHOD FOR ESTIMATION OF GABAPENTIN IN HUMAN
18
PLASMA
4.1
INTRODUCTION TO LC-MS/MS
18
4.2
EXPERIMENTAL WORK
31
4.3
RESULTS AND DISCUSSION
51
4.4
REFERENCES
66
5
DEVELOPMENT AND VALIDATION OF EXTRACTIVE
SPECTROPHOTOMETRIC METHODS FOR ESTIMATION OF
68
GABAPENTIN IN PHARMACEUTICAL DOSAGE FORMS
5.1
INTRODUCTION TO EXTRACTIVE SPECTROSCOPY
68
5.2
EXPERIMENTAL WORK
68
5.3
RESULTS AND DISCUSSION
74
5.4
REFERENCES
78
SUMMARY AND CONCLUSION
79
6.1
SUMMARY
79
6.2
CONCLUSION
79
PRESENTATIONS AND PUBLICATIONS
81
PAPER COMMUNICATED FOR PUBLICATION
81
6
7
7.1
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List of Tables
List of Tables
Page
Table
Title
No.
No.
2.1
Official analytical method
8
2.2
Reported analytical methods
8
4.1
Different scan mode (MS & MS/MS)
26
4.2
Reference/working standards
31
4.3
Apparatus
31
4.4
Instrumentation
31
4.5
Reagents and chemicals
31
4.6
Preparation of calibration standard and quality control spiking solutions
34
4.7
Preparation of calibration standards and quality control samples in
biological matrix with respect to 5 % spiking.
35
4.8
Tuning parameters for GBP & LEV (IS)
36
4.9
Method development trials
37
4.10
Selection of calibration standard
43
4.11
Specificity and selectivity of blank human plasma for GBP & LEV
53
4.12
Sensitivity (LLOQ of GBP)
53
4.13
Summary of calibration curve parameters of GBP
54
4.14
Back calculated concentration of calibration standards from calibration
curve of GBP
54
4.15
Within-batch or intra-batch accuracy and precision of GBP
55
4.16
Between-batch or inter- batch accuracy and precision of GBP
56
4.17
Recovery of GBP
58
4.18
Variability across QC levels of GBP
58
4.19
Recovery of internal standard (LEV)
59
4.20
Dilution integrity of GBP
59
4.21
Matrix effect for GBP
60
4.22
Main stock solution stability of GBP (93 hrs at room temp.)
61
4.23
Main stock solution stability of LEV (93 hrs at room temp.)
62
4.24
Main stock solution stability of GBP (93 hrs at 2-8 °C)
62
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List of Tables
4.25
Main stock solution stability of LEV (93 hrs at 2-8 °C)
62
4.26
Bench top stability of GBP (20 hrs at room temp.)
63
4.27
Process stability of GBP (at 5°C in auto sampler for 76 hrs)
64
4.28
Freeze and thaw stability of GBP (after 4th cycle at -20°C)
64
4.29
Freeze and thaw stability of GBP (after 4th cycle at -70°C)
65
4.30
Carryover check for GBP
66
5.1
Regression analysis data and summary of validation parameters for the
proposed methods
75
5.2
Drug recovery study in capsule dosage form
76
5.3
Drug recovery study in tablet dosage form
76
5.4
Precision data for GBP
76
5.5
5.6
Analysis of marketed formulation (capsule dosage form) of GBP by
proposed methods (n = 6)
Analysis of marketed formulation (tablet dosage form) of GBP by
proposed methods (n = 6)
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List of Figures
List of Figures
Page
Figure
Title
No.
No.
4.1
Schematic diagram of mass spectroscopy
20
4.2
ESI process in the positive ion polarity mode
23
4.3
APCI process in the positive ion polarity mode
25
4.4
Schematic diagram of tandem mass spectrometer using triple quadrupole
instrument
31
4.5
Tuning spectrum of GBP & LEV
36
4.6
Representative chromatogram of blank plasma
51
4.7
Representative chromatogram of zero standard
51
4.8
Representative chromatogram of LLOQ
51
4.9
Representative chromatogram of ULOQ
52
4.10
Representative chromatogram of LQC
52
4.11
Representative chromatogram of MQC
52
4.12
Representative chromatogram of HQC
52
4.13
Representative calibration curve of GBP
54
5.1
Optimization of pH of buffer for (A) GBP-BCG (B) GBP-BTB complex
71
5.2
Optimization of volume of buffer for (A) GBP-BCG (B) GBP-BTB
complex
71
5.3
Representative spectra of GBP-BCG showing λmax at 416 nm
72
5.4
Representative spectra of GBP-BTB showing λmax at 421 nm
73
5.5
Calibration curve for GBP-BCG
74
5.6
Calibration curve for GBP-BTB
75
S.K.P.C.P.E.R., Kherva
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List of Abbreviations
List of Abbreviations
Abbreviations
Description
arb
Arbitrary Unit
ACN
Acetonitrile
β
Beta
BA
Bioanalytical
BCG
Bromocresol Green
BLQ
Below Limit of Quantification
BTB
Bromothymol blue
Cmax
Concentration Maximum
cc
Cubic centimeter
CDC
Centers for Disease Control and Prevention
Conc.
Concentration
CS
Calibration Standard
CV
Coefficient of Variance
FDA
Food and Drug Administration
GBP
Gabapentin
HPLC
High Performance Liquid chromatography
hrs
Hours
HQC
High Quality Control
ICH
International Conference on Harmonization
i.d.
Internal Diameter
IS
Internal Standard
LC-MS/MS
Liquid Chromatography-Mass Spectrometry- Mass
Spectrometry
LEV
Levetiracetam
LLOQ
Lower Limit of Quantification
LQC
Low Quality Control
min
Minutes
ml
Milliliter
mm
Millimeter
MP
Mobile phase
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List of Abbreviations
mTorr
Milli Torr
MS
Mass Spectrometry
MQC
Medium Quality Control
NA
Not Available
ng
Nanogram
NIST
National Institute of Standards and Technology
No.
Number
OPA
O-phosphoric acid
QC
Quality Control
r
Correlation Coefficient
RPM
Revolution per minute
RT
Retention Time
SD
Standard Deviation
sec
Seconds
SOP
Standard Operating Procedure
SOTP
Standard Operating Test Procedure
temp.
Temperature
ULOQ
Upper Limit of Quantification
V
Volts
VGB
Vigabatrin
v/v
Volume/Volume
v/v/v
Volume/Volume/Volume
Vol
Volunteer
µg
Microgram
µl
Microliter
%
Percentage
mM
Milli molar
STD
Standard
SYS SUIT
System Suitability
S.K.P.C.P.E.R., Kherva
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Introduction
CHAPTER 1: INTRODUCTION
1.1 INTRODUCTION TO EPILEPSY:
¾ Epilepsy is a common chronic neurological disorder that is characterized by recurrent
unprovoked seizures1, 2. These seizures are transient signs and/or symptoms due to
abnormal, excessive or synchronous neuronal activity in the brain3. About 50 million
people worldwide have epilepsy at any one time4. Epilepsy is usually controlled, but not
cured, with medication, although surgery may be considered in difficult cases. However,
over 30% of people with epilepsy do not have seizure control even with the best
available medications 5. Not all epilepsy syndromes are lifelong – some forms are
confined to particular stages of childhood. Epilepsy should not be understood as a single
disorder, but rather as a group of syndromes with vastly divergent symptoms but all
involving episodic abnormal electrical activity in the brain.
1.1.1 CLASSIFICATION:
Epilepsies are classified in six ways:
1. By their first cause (or etiology).
2. By the observable manifestations of the seizures, known as semiology.
3. By the location in the brain where the seizures originate.
4. As a part of discrete, identifiable medical syndromes.
5. By the event that triggers the seizures, as in primary reading epilepsy
6. Musicogenic epilepsy.
1.1.2 DIAGNOSIS:
¾ The diagnosis of epilepsy requires the presence of recurrent, unprovoked seizures;
accordingly, it is usually made based on the medical history. Imaging and measurement
technologies such as electroencephalography (EEG), magnetic resonance imaging
(MRI), single photon emission computed tomography (SPECT), positron emission
tomography (PET), and magneto encephalography (MEG) may be useful to discover an
etiology for the epilepsy, discover the affected brain region, or classify the epileptic
syndrome, but these studies are not useful in making the initial diagnosis.
¾ Long-term video-EEG monitoring for epilepsy is the gold standard for diagnosis, but it is
not routinely employed owing to its high cost, low availability and inconvenience.
Convulsive or other seizure-like activity, non-epileptic in origin, can be observed many
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Introduction
other medical conditions. These non-epileptic seizures can be hard to differentiate in and
may lead to misdiagnosis.
¾ Epilepsy covers conditions with different etiologies, natural histories and prognoses,
each requiring different management strategies. A full medical diagnosis requires a
definite categorization of seizure and syndrome types6.
¾ Many people are misdiagnosed, because doctors unfamiliar with the symptoms believe
that their patients have another illness, because they are not adequately trained to
recognize the early symptoms including odd tastes or smells. Approximately 80% have
petit mal seizures which are harder to spot.
¾ According to the National Institute of Neurological Disorders and Stroke, if a person has
had two or more seizures they are considered to have epilepsy. There are however
exceptions: seizures caused by fever (febrile seizures), those not due to abnormal
electrical activity in the brain (no epileptic events), and seizures that occur during
pregnancy (eclampsia) are not counted.
1.1.3 PRECIPITANTS:
¾ The diagnosis of epilepsy usually requires that the seizures occur spontaneously.
Nevertheless, certain epilepsy syndromes require particular precipitants or triggers for
seizures to occur. These are termed reflex epilepsy. For example, patients with primary
reading epilepsy have seizures triggered by reading. Photosensitive epilepsy can be
limited to seizures triggered by flashing lights. Other precipitants can trigger an epileptic
seizure in patients who otherwise would be susceptible to spontaneous seizures. For
example, children with childhood absence epilepsy may be susceptible to
hyperventilation. In fact, flashing lights and hyperventilation are activating procedures
used in clinical EEG to help trigger seizures to aid diagnosis.
¾ Finally, other precipitants can facilitate, rather than obligatory trigger, seizures in
susceptible individuals. Emotional stress, sleep deprivation, sleep itself, and febrile
illness are examples of precipitants cited by patients with epilepsy. Notably, the
influence of various precipitants varies with the epilepsy syndrome 7.
1.1.4 SEIZURE TYPES:
¾ Seizure types are organized firstly according to whether the source of the seizure within
the brain is localized (partial or focal onset seizures) or distributed (generalized seizures).
Partial seizures are further divided on the extent to which consciousness is affected. If it
is unaffected, then it is a simple partial seizure; otherwise it is a complex partial
(psychomotor) seizure. A partial seizure may spread within the brain - a process known
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Introduction
as secondary generalization. Generalized seizures are divided according to the effect on
the body but all involve loss of consciousness. These include absence (petit mal),
myoclonic, clonic, tonic, tonic-clonic (grand mal) and atonic seizures.
1.1.5 TREATMENT 8-14:
¾ Epilepsy is usually treated with medication prescribed by a physician; primary
caregivers, neurologists, and neurosurgeons all frequently care for people with epilepsy.
In some cases the implantation of a stimulator of the vagus nerve, or a special diet can be
helpful. Neurosurgical operations for epilepsy can be palliative, reducing the frequency
or severity of seizures; or, in some patients, an operation can be curative.
1.1.5.1 Medication for Epilepsy:
1. Drugs acting through Na+ channels
Examples:
¾ Phenytoin
¾ Diphenylhydantoin (DPH)
¾ Phenobarbitone
¾ Trimethadione
¾ Carbamazepine
¾ Lamotrigine
2. Drugs acting through GABA
Examples:
¾ Sodium valproate
¾ Diazepan and Clonazepam
¾ Midazolam
¾ Gabapentin
¾ Vigabatrin
¾ Clobazam
3. Drugs acting through NMDA receptor inhibition
Examples:
¾ Flunarizine
¾ Zonisamide
¾ Topiramate
4. Non pharmacological therapy
Examples:
¾ Vagus nerve stimulation
¾ Ketogenic diet
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Introduction
¾ Surgery
1.2 DRUG PROFILE15-17:
¾ Drug name: Gabapentin
¾ Proprietary name:
o Neurontin , Aclonium
¾ Category: Antiepileptic drug
¾ Iupac name:
o 2-[1-(aminomethyl)cyclohexyl]acetic acid
¾ Structure:
1.2.1 PHYSICOCHEMICAL PROPERTIES:
¾ Molecular Formula: C 9H17NO2
¾ Molecular Weight: 171.24 gm/mol
¾ Melting Point: 162-166° C
¾ pKa: 3.68, 10.70
¾ Isoelectric Point: 7.14
¾ Solubility:
o Ethanol : 1 in 90
o Water : 1 in 10
¾ Partition Coefficient:
o Octanol/Water: 0.075
¾ Log P: -1.10
¾ It is white to off-white crystalline solid
1.2.2 PHARMACOKINETICS PROPERTIES:
¾ Oral Absorption
: ~60%(Dose Dependent)
¾ Presystemic Metabolism : NIL
¾ Plasma Half Life
: 5-7 hrs
¾ Volume Of Distribution
: 0.6 to 0.8 l/kg
¾ Plasma Protein Binding
: < 3%
¾ Dose(mg) : 300 mg
600 mg
900 mg
¾ Cmax(mg/l) : 2.7 mg/l
3.29 mg/l
4.02 mg/l
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Introduction
1.2.3 MECHANISM OF ACTION:
¾ The mechanism by which GBP exerts its analgesic action is unknown, but in animal
models of analgesia, GBP prevents allodynia (pain-related behavior in response to a
normally innocuous stimulus) and hyperalgesia (exaggerated response to painful
stimuli). In particular, GBP prevents pain-related responses in several models of
neuropathic pain in rats or mice (e.g. spinal nerve ligation models, streptozocin-induced
diabetes model, spinal cord injury model, acute herpes zoster infection model). GBP also
decreases pain-related responses after peripheral inflammation (carrageenan footpad test,
late phase of formalin test). GBP did not alter immediate pain-related behaviors (rat tail
flick test, formalin footpad acute phase, acetic acid abdominal constriction test, footpad
heat irradiation test). The relevance of these models to human pain is not known.
¾ The mechanism by which GBP exerts its anticonvulsant action is unknown, but in animal
test systems designed to detect anticonvulsant activity, GBP prevents seizures as do other
marketed anticonvulsants. GBP exhibits antiseizure activity in mice and rats in both the
maximal electroshock and pentylenetetrazole seizure models and other preclinical
models (e.g., strains with genetic epilepsy, etc.). The relevance of these models to human
epilepsy is not known.
¾ GBP is structurally related to the neurotransmitter GABA (gamma-aminobutyric acid)
but it does not modify GABAA or GABAB radioligand binding, it is not converted
metabolically into GABA or a GABA agonist, and it is not an inhibitor of GABA uptake
or degradation. GBP was tested in radioligand binding assays at concentrations up to 100
µM and did not exhibit affinity for a number of other common receptor sites, including
benzodiazepine, glutamate, N-methyl-D-aspartate (NMDA), quisqualate, kainate,
strychnine-insensitive or strychnine-sensitive glycine, alpha 1, alpha 2, or beta
adrenergic, adenosine A1 or A2, cholinergic muscarinic or nicotinic, dopamine D1 or
D2, histamine H1, serotonin S1 or S2, opiate mu, delta or kappa, cannabinoid 1, voltagesensitive calcium channel sites labeled with nitrendipine or diltiazem, or at voltagesensitive sodium channel sites labeled with batrachotoxinin A 20-alpha-benzoate.
Furthermore, GBP did not alter the cellular uptake of dopamine, noradrenaline, or
serotonin.
¾ In vitro studies with radiolabeled GBP have revealed a GBP binding site in areas of rat
brain including neocortex and hippocampus. A high-affinity binding protein in animal
brain tissue has been identified as an auxiliary subunit of voltage-activated calcium
channels. However, functional correlates of GBP binding, if any, remain to be
elucidated.
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Introduction
1.2.4 RECOMMENDED DOSAGE:
¾ People over the age of 12 should be started on 300 mg GBP taken three times a day. The
dose can be increased up to a total of 1,800 mg per day. In some instances, doses of up to
3,600 mg per day have been tolerated.
¾ Children should receive a dosage of 10–15 mg per kg of body weight per day, divided
into three equal doses.
¾ Chronic pain may be treated with 300–3,600 mg per day, divided into three equal doses.
¾ When GBP is used for bipolar disorder, the starting dose is usually 300 mg taken at
bedtime. Depending on the patient's response, the dose can be increased every four to
seven days. Many people receive maximum therapeutic benefit at 600 mg per day,
although some people have required up to 4,800 mg per day.
1.2.5 SIDE EFFECTS:
¾ Patients who experience the following side effects of GBP should check with their doctor
immediately. These include more common side effects, such as unsteadiness, clumsiness,
and uncontrollable back-and-forth eye movements or eye rolling. Less common side
effects include depression, irritability, other mood changes or changes in thinking, and
decreased memory. Rare side effects include pain in the lower back or side, difficulty
urinating, fever and/or chills, cough, or hoarseness.
¾ Children under age 12 who have the following more common side effects should also
check with their doctor immediately: aggressive behavior, irritability, anxiety, difficulty
concentrating and paying attention, crying, depression, mood swings, increased
emotionality, hyperactivity, suspiciousness or distrust.
¾ Multiple side effects often occur when a patient starts taking GBP. While these side
effects usually go away on their own, if they last or are particularly troublesome, the
patient should consult a doctor. More common side effects that occur when first starting
to take GBP include blurred or double vision, muscle weakness or pain, swollen hand,
feet, or legs, trembling or shaking, and increased fatigue or weakness. Less common side
effects that occur when initiating GBP treatment include back pain, constipation,
decreased sexual drive, diarrhea, dry mouth and eyes, frequent urination, headache,
indigestion, low blood pressure, nausea, ringing in the ears, runny nose, slurred speech,
difficulty thinking and sleeping, weight gain, twitching, nausea and/or vomiting,
weakness.
S.K.P.C.P.E.R., Kherva
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Introduction
1.3 REFERENCES:
1. Commission on Epidemiology and Prognosis, International League Against Epilepsy,
Guidelines for epidemiologic studies on epilepsy. Commission on Epidemiology and
Prognosis, International League against Epilepsy. Epilepsia 1993; 34(4):592–6.
2. Blume W, Luders H, Mizrahi E, Tassinari C, van Emde Boas W, Engel J. Glossary of
descriptive terminology for ictal semiology: report of the ILAE task force on
classification and terminology. Epilepsia 2001; 42(9):1212–8.
3. Fisher R, Blume W, Elger C, Genton P, Lee P, Engel J. Epileptic seizures and epilepsy:
definitions proposed by the International League Against Epilepsy (ILAE) and the
International Bureau for Epilepsy (IBE). Epilepsia 2005; 46(4):470–2.
4. Epilepsy:
aetiogy,
epidemiology
and
prognosis.
World
Health
Organization.
http://www.who.int/mediacentre/factsheets/fs165/en/. 2001.
5. Cascino GD. Epilepsy: contemporary perspectives on evaluation and treatment. Mayo
Clinic Proc 1994; 69:1199-1211.
6. Panayiotopoulos CP, Koutroumanidis M. The significance of the syndromic diagnosis of
the epilepsies. National Society for Epilepsy; 2005.
7. Frucht MM, Quigg M, Schwaner C, Fountain NB. Distribution of seizure precipitants
among epilepsy syndromes. Epilepsia 2000; 41:1534–9.
8. Eadie MJ, Bladin PF. A Disease Once Sacred: a History of the Medical Understanding of
Epilepsy; 2001.
9. Theodore WH, Fisher RS. Brain stimulation for epilepsy, Lancet Neurol 2004; 3: 111-8.
10. Regis J, Rey M, Bartolomei F, Vladyka V, Liscak R, Schrottner O, Pendl G. Gamma
knife surgery in mesial temporal lobe epilepsy: a prospective multicenter study.
Epilepsia 2004; 45: 504-15.
11. Cheuk D, Wong V. Acupuncture for epilepsy. Cochrane Database Syst Rev 2006; 5062.
12. Ramaratnam S, Baker GA, Goldstein LH. Psychological treatments for epilepsy.
Cochrane Database Syst Rev 2005; 2029.
13. Ranganathan LN, Ramaratnam S. Vitamins for epilepsy. Cochrane Database Syst Rev
2005; 4304.
14. Ramaratnam S, Sridharan K. Yoga for epilepsy. Cochrane Database Syst Rev 2000;
1524.
15. The Merck’s Index. 14th ed. Merck and co., USA; 2006:p. 742.
16. Dollery C. Therapeutic drugs. 2nd ed. Churchill Livingstone; 1999: p. G1.
17. Physician’s Desk Reference. 62th ed. Thomson Healthcare Inc., NJ; 2008: p. 2462-67.
S.K.P.C.P.E.R., Kherva
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Review of Literature
CHAPTER 2: REVIEW OF LITERATURE
2.1 REVIEW OF LITERATURE:
TABLE NO: 2.1 OFFICIAL ANALYTICAL METHOD
SR
TITLE
METHOD
EXPERIMENTAL CONDITION
NO
1.
USP official Method 1
HPLC
Column: 4.6 mm×25 cm with L1 packing
MP: ACN: phosphate buffer
(24:76, v/v) (pH:1.8)
Detection method: UV detection (215 nm)
Flow rate: 1ml/min
TABLE NO: 2.2 REPORTED ANALYTICAL METHODS
SR
TITLE
METHOD
EXPERIMENTAL
NO
1.
CONDITION
Rapid quantification of gabapentin
LC-MS/MS
2
in human plasma by LC- MS/MS .
Column: Waters symmetry
C18 RP
MP: ACN: 10 mM Amm.
formate (80:20, v/v, pH:3.0)
IS: 1,1-Cyclohexane diacetic
acid monoamide
MS mode: MRM
2.
Determination of gabapentin in
LC-MS/MS
human plasma using hydrophilic
Column: Atlantis HILIC silica
MP: ACN: 100 mM Amm.
3
interaction LC- MS/MS .
formate (85:15, v/v) (pH:3.0)
IS: Metformin
MS mode: MRM
3.
Validated LC-MS/MS method for
LC-MS/MS
Column: Gemini C18
quantification of gabapentin in
MP: ACN: 10 mM Amm.
human plasma: application to
acetate (20:80, v/v, pH:3.2)
pharmacokinetic and BE studies in
IS: (S)-(+)-alpha-amino
4
Korean volunteers .
cyclohexanepropionic acid
hydrate
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4.
Review of Literature
Gabapentin quantification in human
LC-MS/MS
Extraction method: Protein
plasma by high-performance liquid
precipitation
chromatography coupled to
Column: C4 RP
electrospray tandem mass
MS mode: MRM
spectrometry. Application to
bioequivalence study5.
5.
Quantitative determination of
GC-MS
Column: polydimethylsiloxane
vigabatrin and gabapentin in human
IS: Cyclobarbital
serum by gas chromatography -
MS mode: SIM
6
mass spectrometry .
6.
Analysis of gabapentin in serum
GC-MS
Extraction method: SPE
and plasma by solid-phase
Derivatization reagent :
extraction and GC-MS for
MTBSTFA
therapeutic drug monitoring7.
7.
Determination of gabapentin in
HPTLC
Plate: Aluminum-backed silica
pharmaceutical preparations by
gel 60 F254 HPTLC plates
HPTLC8.
MP: n-butanol: water :glacial
acetic acid ( 3:3:2, v/v )
IS: Histamine hydrochloride
8.
Development and application of a
HPLC
Column: Brownlee spheri-5
validated HPLC method for the
cyano
determination of gabapentin and its
MP: ACN:10 mM KH2PO4/10
major degradation impurity in drug
mM K2HPO4 (pH 6.2,8:92 v/v)
9
products .
Detection method: UV
detection at 210 nm
9.
A HPLC micro method for the
HPLC
Column:125 x 3-mm ID
simultaneous determination of
Hypersil BDS C-18 column,
vigabatrin and gabapentin in
3-µ mini-bore
10
serum .
MP : phosphate buffer
(pH 6.5):ACN:methanol:water
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10.
Automated microanalysis of
Review of Literature
HPLC
Extraction method: PP
gabapentin in human serum by
Derivatization reagent: o-
high-performance liquid
phthaldialdehyde reagent
chromatography with fluorometric
Column: Waters 5 µ RP
detection11.
column (10 cmx4.6 mm)
IS: 1-(aminomethyl)
cycloheptane acetic acid
MP: 0.02 M phosphate buffer
(pH 4.5):ACN (50:50, v/v)
Detection : Fluorimetry
(EX: 230 nm, EM: 420 nm)
11.
Determination of gabapentin in
HPLC
Derivatization reagent: o-
plasma by high-performance liquid
phthalaldehyde-3-
chromatography12.
mercaptopropionic acid
IS: [1-(aminomethyl)cycloheptaneacetic acid
Column: Beckman
Ultrasphere 5 microns RP
MP: 0.33 M acetate buffer
(pH 3.7): methanol : ACN
(40:30:30, v/v/v)
Detection method :
Fluorimetry
(EX: 330 nm, EM: 440 nm)
12.
Optimization of an HPLC method
HPLC
IS: Amlodipine
for determination of gabapentin in
Derivatization reagent : 1-
dosage forms through derivatization
fluoro-2,4-dinitrobenzene
with 1-fluoro-2, 4-dinitrobenzene13.
Column : Nova-Pak C(18)
MP: ACN: sodium
dihydrogenphosphate (pH 2.5;
0.05 M) (70:30, v/v)
Detection: UV( 360 nm)
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13.
Determination of gabapentin in
Review of Literature
HPLC
Extraction method: SPE C18
human plasma and urine by high-
cartridge
performance liquid chromatography
Derivatization reagent: 1,2-
with UV-vis detection14.
naphthoquinone-4-sulphonic
acid sodium salt (NQS)
Column: C18
MP: ACN:10mM OPA
(pH 2.5,v/v)
IS: Baclofen
14.
Sensitive high-performance liquid
HPLC
Extraction method: LLE
chromatographic quantitation of
Extracting solvent:
gabapentin in human serum using
dichloromethane : 2 propanol
liquid-liquid extraction and pre-
(1:1, v/v)
column derivatization with 9-
Derivatization reagent: 9-
fluorenylmethyl chloroformate15.
fluorenyl methyl chloroformate
(FMOC-Cl)
Column: Shimpack CLC-C18
(150 mm x 4.6 mm)
MP: methanol:0.05 M sodium
phosphate buffer (73:27, v/v;
pH of 3.9) containing 1 ml/l
triethylamine
15.
Simple and sensitive liquid
HPLC
Derivatization reagent: (2-
chromatographic method with
naphthoxy)acetyl chloride
fluorimetric detection for the
(NAC)
analysis of gabapentin in human
Column: phenyl-hexyl column
plasma16.
MP: sodium acetate buffer
(100 mM; pH 5.0):methanol
(32:68, v/v)
Detection method:
Fluorimetry
(EX: 225 nm, EM: 360 nm)
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16.
Review of Literature
Simultaneous isocratic HPLC
HPLC
Derivatization reagent: Dansyl
determination of vigabatrin and
chloride
gabapentin in human plasma by
Column: Micro bondapak C-
dansyl derivatization17.
18, 10 µ , 300 x 3.9 mm
MP: 50 mmol/l NaH2PO4 in
40% ACN
Detection method: Fluorimetry
(EX: 318 nm, EM: 510 nm)
17.
Determination of gabapentin in
GLC
Extraction method: SPE on
serum using solid-phase extraction
C18 solid-phase column
and gas-liquid chromatography18.
Derivatization reagent:
MTBSTFA plus 1% tBDMCS
Column : HP-1
Detector: FID
IS: [(1-aminomethyl-1cycloheptyl) acetic acid]
18.
Determination of gabapentin in
CE
Derivatization reagent:
serum by capillary
fluorescamine
electrophoresis19.
Detection method: UVfluorescence detection
19.
Determination of gabapentin in
CE
Derivatization reagent:
human plasma by capillary
6-Carboxyfluorescein
electrophoresis with laser-induced
succinimidyl ester (CFSE)
fluorescence detection and
Buffer: 50 mM sodium borate
20
acetonitrile stacking technique .
(pH 9.5)
Detection method:
Fluorimetry (EX: 488 nm, EM:
520 nm)
20.
Spectrofluorimetric determination
Spectrofluor
Derivatization reagent:
of vigabatrin and gabapentin in
imetry
fluorescamine
urine and dosage forms through
Detection method:
21
derivatization with fluorescamine .
Spectrofluorimetry
(EX: 390 nm, EM: 472 nm)
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21.
22.
Review of Literature
Spectrofluorimetric determination
Spectrofluor
Derivatization reagent:
of vigabatrin and gabapentin in
imetry
4-chloro-7-nitrobenzo-2-oxa-
dosage forms and spiked plasma
1,3-diazole
samples through derivatization with
Detection method:
4-chloro-7-nitrobenzo-2-oxa-1,3-
Fluorimetry
diazole22.
(EX: 465 nm, EM: 532 nm)
Colorimetric determination of
Colorimetry
gabapentin in pharmaceutical
Based on following reaction
1) GBP
23
formulation .
with
vanillin
(Duquenois reagent) in the
presence of McIlvain buffer
pH
7.5
and
the
color
developed was measured at
376 nm.
2) The primary amino group of
GBP with ninhydrin reagent
in N, N’-dimethylformamide
(DMF) medium producing
colored
product
which
absorbs maximally at 569
nm.
3) The reaction of gabapentin
with p-benzoquinone (PBQ)
to form a colored product
with λmax at 369 nm.
23.
Determination of the antiepileptics
Colorimetry
Reaction: condensation of the
vigabatrin and gabapentin in dosage
drugs through their amino
forms and biological fluids using
groups with acetylacetone and
Hantzsch reaction24.
formaldehyde according to
Hantzsch reaction yielding the
highly fluorescent dihydropyridine derivatives.
Color developed: yellowish
orange measured at 410 nm &
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Review of Literature
415 nm for VGB and GBP,
respectively.
24.
Rapid chemiluminometric
Chemilumin
Reaction: The luminol-
determination of gabapentin in
ometry
hypochlorite reaction in the
pharmaceutical formulations
presence of GBP.
exploiting pulsed-flow analysis25.
`
2.2 REFERENCES:
1. United States Pharmacopeia, 30th ed., The United States Pharmacopeial convention Inc.,
Rockville. 2007; 2200.
2. Jiang Q, Li S. Rapid high-performance liquid chromatographic determination of serum
gabapentin. J Chromatogr B Biomed Sci Appl 1999; 30:727(1-2), 119-23.
3. Ji HY, Jeong DW, Kim YH, Kim HH, Yoon YS, Lee KC, Lee HS. Determination of
gabapentin in human plasma using hydrophilic interaction liquid chromatography with
tandem mass spectrometry. Rapid Commun Mass Spectrom 2006; 20(14):2127-32.
4. Park JH, Jhee OH, Park SH, Lee JS, Lee MH, Shaw LM, Kim KH, Lee JH, Kim YS,
Kang JS. Validated LC-MS/MS method for quantification of gabapentin in human
plasma: application to pharmacokinetic and bioequivalence studies in Korean volunteers.
Biomed Chromatogr 2007; 21(8):829-35.
5. Ifa DR, Falci M, Moraes ME, Bezerra FA, Moraes MO, de Nucci G. Gabapentin
quantification in human plasma by high-performance liquid chromatography coupled to
electrospray tandem mass spectrometry: Application to bioequivalence study. J Mass
Spectrom 2001; 36(2):188-94.
6. Borrey DC, Godderis KO, Engelrelst VI, Bernard DR, Langlois MR. Quantitative
determination of vigabatrin and gabapentin in human serum by gas chromatographymass spectrometry. Clin Chim Acta 2005; 354(1-2):147-51.
7. Kushnir MM, Crossett J, Brown PI, Urry FM. Analysis of gabapentin in serum and
plasma by solid-phase extraction and gas chromatography-mass spectrometry for
therapeutic drug monitoring. J Anal Toxicol 1999; 23(1):1-6.
8. Saner RT, Pendse U, Moghe A, Khedkar S, Patil P. Determination of gabapentin in
pharmaceutical preparations by HPTLC. Indian drugs 2003; 40(9): 547-8.
9. Ciavarella
AB, Gupta A, Sayeed VA, Khan MA, Faustino PJ. Development and
application of a validated HPLC method for the determination of gabapentin and its
S.K.P.C.P.E.R., Kherva
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Review of Literature
major degradation impurity in drug products.
J Pharm Biomed Anal 2007; 11,
43(5):1647-53.
10. Ratnaraj N, Patsalos PN. A high-performance liquid chromatography micromethod for
the simultaneous determination of vigabatrin and gabapentin in serum. Ther Drug Monit
1998; 20(4):430-4.
11. Tang PH, Miles MV, Glauser TA, DeGrauw T. Automated microanalysis of gabapentin
in human serum by high-performance liquid chromatography with fluorometric
detection. J Chromatogr B Biomed Sci Appl 1999; 30,727(1-2):125-9.
12. Forrest G, Sills GJ, Leach JP, Brodie MJ. Determination of gabapentin in plasma by
high-performance liquid chromatography. J Chromatogr B Biomed Appl 1996;
7,681(2):421-5.
13. Souri E, Jalalizadeh H, Shafiee A. Optimization of an HPLC method for determination
of gabapentin in dosage forms through derivatization with 1-fluoro-2, 4-dinitrobenzene. J
Pharm Biomed Anal Chem Pharm Bull 2007; 55(10):1427-30.
14. Sagirli O, Cetin SM, Onal A. Determination of gabapentin in human plasma and urine by
high-performance liquid chromatography with UV-vis detection. J Pharm Biomed Anal
2006; 42(5):618-24.
15. Bahrami G, Kiani A. Sensitive high-performance liquid chromatographic quantitation of
gabapentin in human serum using liquid-liquid extraction and pre-column derivatization
with 9-fluorenylmethyl chloroformate. J Chromatogr B Analyt Technol Biomed Life Sci
2006; 1,835(1-2):123-6.
16. Chung TC, Tai CT, Wu HL. Simple and sensitive liquid chromatographic method with
fluorimetric detection for the analysis of gabapentin in human plasma. J Chromatogr A
2006; 30, 1119(1-2):294-8.
17. Krivanek P, Koppatz K, Turnheim K. Simultaneous isocratic HPLC determination of
vigabatrin and gabapentin in human plasma by dansyl derivatization. Ther Drug Monit
2003; 25(3):374-7.
18. Wolf CE, Saady JJ, Poklis A. Determination of gabapentin in serum using solid-phase
extraction and gas-liquid chromatography. J Anal Toxicol 1996; 20(6):498-501.
19. Garcia LL, Shihabi ZK, Oles K. Determination of gabapentin in serum by capillary
electrophoresis. J Chromatogr B Biomed Appl 1995; 7,669(1):157-62.
20. Chang SY, Wang FY. Determination of gabapentin in human plasma by capillary
electrophoresis with laser-induced fluorescence detection and acetonitrile stacking
technique. J Chromatogr B Analyt Technol Biomed Life Sci 2004; 25,799(2):265-70.
S.K.P.C.P.E.R., Kherva
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Review of Literature
21. Belal F, Abdine H, Al-Majed A, Khalil NY. Spectrofluorimetric determination of
vigabatrin and gabapentin in urine and dosage forms through derivatization with
fluorescamine. J Pharm Biomed Anal 2002; 1, 27(1-2):253-60.
22. Hassan EM, Belal F, Al-Deeb OA, Khalil NY. Spectrofluorimetric determination of
vigabatrin and gabapentin in dosage forms and spiked plasma samples through
derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. J AOAC Int 2001;
84(4):1017-24.
23. Abdellatef HE, Khalil HM. Colorimetric determination of gabapentin in pharmaceutical
formulation. J Pharm Biomed Anal 2003; 5, 31(1):209-14.
24. Al-Zehouri J, Al-Madi S, Belal F. Determination of the antiepileptics vigabatrin and
gabapentin in dosage forms and biological fluids using Hantzsch reaction.
Arzneimittelforschung 2001; 51(2):97-103.
25. Manera M, Miro M, Ribeiro MF, Estela JM, Cerda V, Santos JL, Lima JL. Rapid
chemiluminometric determination of gabapentin in pharmaceutical formulations
exploiting pulsed-flow analysis. Luminescence; 2008.
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Aim of The Present Work
CHAPTER 3: AIM OF THE PRESENT WORK
3.1 AIM OF THE PRESENT WORK:
¾ Nowadays it is important to estimate drug concentration in biological fluid to achieve
better therapeutic effect and to check out content of drug in pharmaceutical dosage
forms.
¾ Many methods are available to estimate GBP in human plasma, but our aim behind this
project is to develop accurate, precise and validated method for estimation of GBP which
has higher selectivity and specificity, accuracy and precision which can give reliable
results without interference from biological matrix.
¾ Also no single extractive spectrophotometric method is available to estimate GBP in
pharmaceutical dosage forms.
The specific aim of the research work was
™ To develop and validate LC-MS/MS method for estimation of GBP in human plasma.
™ To develop and validate spectrophotometric method for determination of GBP in
pharmaceutical dosage forms.
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LC-MS/MS Method
CHAPTER: 4 DEVELOPMENT AND VALIDATION OF
LC-MS/MS METHOD FOR ESTIMATION OF GABAPENTIN
IN HUMAN PLASMA
4.1 INTRODUCTION TO LC-MS/MS:
4.1.1 MASS SPECTROMETRY (MS):
¾ Mass spectrometry (MS) has an important place amongst the various spectrometric
techniques for molecular analysis. The phenomenon of deflection of ions in electric or
magnetic field first was proposed by Wien in 1898. A mass spectrometer for general
use however available after 1930 only. At present it is most sensitive method of
molecular analysis. Moreover, it has the potential to yield information on the relative
molecular mass and the structure of the analyte. The principle of MS is production of
ions that are subsequently separated or filtered according to their mass-to-charge (m/z)
ratio and detected. The resulting mass spectrum is a plot of the (relative) abundance of
the generated ions as a function of m/z ratio1. Thus MS is a very sensitive, highly
selective and quantitative analytical technique. Sample size is usually in microgram to
the nanogram range and fragmentation patterns are highly reproducible even for multicomponent mixtures.
¾ The Mass spectrometer nowadays is a highly sophisticated and computerized
instrument1-4. It basically consists of five parts: sample introduction, ionization, mass
analysis, ion detection and data handling. Sample introduction systems comprise
controlled leaks, through which a sample vapor is introduced from a reservoir, various
direct insertion probes for the introduction of solids and low-volatility liquid and
combination with various chromatographic techniques. The ionization of the analytes
can be performed in a number of ways the available ionization techniques can be
classified in four groups, i.e. electron ionization, chemical ionization, desorption
ionization and nebulization ionization1,5. Proton transfer, charge exchange,
electrophilic addition and anion abstraction reaction used to produce positively
charged ions , M+ or MH+. Negative ions are produce by proton transfer or
abstraction and anion attachment. After the production of ions, these are analyzed
according to their m/z ratio in time or space with the mass analyzer. This means that a
singly charged molecule with molecular mass of 400 will give a peak at m/z 400,
while a molecule carrying 40 charges and a molecular mass of 16,000 will also give a
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LC-MS/MS Method
peak at 400 m/z. Five types of mass analyzers are currently available, i.e., magnetic
sector 6, quadrupole mass filter 6, quadrapole ion trap
6,7
, time-of-flight6,8 and fourier-
transform ion-cyclotron resonance instruments6,9. There is not one ideal mass analyzer.
The choice depends on the application.
¾ In practice, most interfaces for coupling of LC with MS have been developed on
quadrupole instrument. The detection of ions after the mass analysis is mostly performed
by means of an electron multiplier. The signal of the electron multiplier is fed to
multichannel analyzer to perform ion counting. In most modern instruments a highvoltage conversion dynode is used in both positive-ion and negative-ion detection. A
mass spectrometer produces an enormous amount of data. The most important type of
output are mass spectrum, mass chromatogram and total ion chromatogram. The mass
spectrum is a plot of relative intensity as a function of the m/z. It contains a base peak,
which is the peak with highest intensity used to normalize the mass spectrum. The mass
spectrum is also contains fragment peaks, isotope peak and background peak.
¾ It is an important spectrometric technique for molecular mass analysis. At present MS is
the most sensitive method for molecular analysis. Mass spectrometry could be
considered as an analytical technique that involves the study in the gas phase of ionized
molecules with the aim of one or more of the following:
•
Molecular weight determination.
•
Structural characterization.
•
Gas phase reactivity study.
•
Qualitative and quantitative analysis of components in a mixture.
¾ In analysis by LC/MS, a sample is injected onto an LC column. The sample is then
separated into its various components. The components elute from the LC column and
pass into the mass spectrometer where they are analyzed. Analysis by direct infusion or
flow injection provides no chromatographic separation of components in the sample
before it passes into the mass spectrometer. The data from the mass spectrometer are
then stored and processed by the data system. In LC-MS sample is introduce by three
ways. They are as •
Using the syringe pump (Direct infusion).
•
Using the divert/inject valve fitted with a sample loop and LC (flow injection
analysis).
•
Using the divert/inject valve and HPLC fitted with column (LC-MS).
Ionization sources:
¾ In LC-MS various types of ionization sources are use. They are as-
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LC-MS/MS Method
(A) For small volatile molecules
•
Electron Ionization (EI).
•
Chemical Ionization (CI).
(B) For small or non-volatile molecules
•
Electrospray ionization (ESI).
•
Matrix-assisted Laser Desorption Ionization (MALDI)
•
Field desorption (FD).
•
Fast Atom Bombardment (FAB).
Instrumentation:
Figure 4.1 Schematic diagram of Mass spectroscopy
¾ The mass spectrometer consists of an ionization source, ion guides, triple-stage mass
analyzer, and ion detection system. The ion guides, mass analyzer, ion detection system,
and part of the ionization source are enclosed in a vacuum manifold. Ionization of the
sample takes place in the ionization source. The specific process used to ionize the
sample is referred to as the ionization mode. The ions produced in the ionization source
are transmitted by the ion guides into the mass analyzer, where they are separated
according to their mass-to-charge ratio. The polarity of the potentials applied to the
lenses in the ionization source and ion guides determines whether positively charged ions
or negatively charged ions are transmitted to the mass analyzer. It can analyze positively
or negatively charged ions (called the positive or negative ion polarity mode).
¾ The ions produced in the ionization source are filtered according to their mass-to charge
ratios (mass analyzed) by the triple-stage mass analyzer, which can perform either one or
two stages of mass analysis. When the system is operated as a conventional mass
spectrometer with one stage of mass analysis, the sample is ionized in the ion source and
the ion products are subjected to mass analysis in the first rod assembly. The resulting
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LC-MS/MS Method
mass-selected ions are then transmitted (through the second and third rod assemblies) to
the ion detection system.
¾ When the system is operated as a tandem mass spectrometer, as before, the sample is
ionized in the ion source and is mass analyzed by the first rod assembly. In this case,
however, mass-selected ions exiting the first rod assembly are allowed to collide with an
inert gas in the second rod assembly and fragment to produce a new set of ion products.
(The second rod assembly is surrounded by a chamber called the collision cell. The
collision cell can be pressurized with an inert gas.) The secondary ion products then
undergo a further mass analysis in the third rod assembly and selected ions are detected.
In this mode, two stages of analysis are performed. In the first stage, ion production
occurs in the ion source, and the first mass analyzer carries out mass analysis. In the
second stage of analysis, formation of new ionic species occurs in the collision cell, and
the second mass analyzer performs mass analysis. Two stages of mass analysis yield far
greater chemical specificity than a single stage can achieve, because two discrete but
directly related sets of masses can be selected and determined.
¾ Systems can be used to elucidate the structures of pure organic compounds and the
structures of the components within mixtures. Each ionic fragment of a molecule formed
in the ion source and separated in a first stage of mass analysis can be further fragmented
and then further separated in a second stage of mass analysis to build up an entire
structure for the molecule, piece by piece. Thus, all pathways for the formation and
fragmentation of each ion in the mass spectrum can be investigated.
¾ The two stages of mass analysis, with resultant reduction of chemical noise in the final
mass spectrum, make analysis very selective and sensitive.
¾ Each sequence of single or triple-stage mass analysis of the ions is called a scan. The
system uses several different scan modes and different scan types to filter, fragment, or
transmit ions in the mass analyzer. The ability to vary the scan mode and scan type, as
well as the ionization and ion polarity modes, affords the user great flexibility in the
instrumentation for solving complex analytical problems.
Ion polarity modes:
¾ Two ion polarity modes: Positive or negative.
¾ Both positively charged and negatively charged ions are formed in the ionization source
of the mass spectrometer. The mass spectrometer can control whether positive ions or
negative ions are transmitted to the mass analyzer for mass analysis by changing the
polarity of the potentials applied to the ionization source and ion guides. The ion guides
are located between the ionization source and the mass analyzer.
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LC-MS/MS Method
¾ The information obtained from a positive-ion mass spectrum is different from and
complementary to that obtained from a negative-ion spectrum.
¾ Thus, the ability to obtain both positive-ion and negative-ion mass spectra aids you in the
qualitative analysis of your sample. You can choose the ion polarity mode and ionization
mode to obtain maximum sensitivity for the particular analysis of interest.
Ionization modes:
¾ Two ionization modes:
•
Electrospray ionization (ESI)
•
Atmospheric pressure chemical ionization (APCI)
Electrospray ionization:
¾ The electrospray ionization (ESI) mode transforms ions in solution into ions in the gas
phase. Many samples that previously were not suitable for mass analysis (for example,
heat-labile compounds or high molecular weight compounds) can be analyzed by the use
of ESI. ESI can be used to analyze any polar compound that makes a preformed ion in
solution.
¾ With ESI, the range of molecular weights that can be analyzed by the mass spectrometer
is greater than 100,000 U, due to multiple charging. ESI is especially useful for the mass
analysis of polar compounds, which include biological polymers (for example, proteins,
peptides, glycoproteins, and nucleotides), pharmaceuticals & their metabolites and
industrial polymers (for example, polyethylene glycols).
In ESI, ions are produced and analyzed as follows:
•
The sample solution enters the ESI needle, to which a high voltage is applied.
•
The ESI needle sprays the sample solution into a fine mist of droplets that are
electrically
•
charged at their surface.
The electrical charge density at the surface of the droplets increases as solvent
evaporates from the droplets.
•
The electrical charge density at the surface of the droplets increases to a critical point
known as the Rayleigh stability limit. At this critical point, the droplets divide into
smaller droplets because the electrostatic repulsion is greater than the surface tension.
The process is repeated many times to form very small droplets.
•
From the very small, highly charged droplets, sample ions are ejected into the gas
phase by electrostatic repulsion.
•
The sample ions enter the mass spectrometer and are analyzed.
¾ The ESI mode use in either positive or negative ion polarity mode. The ion polarity
mode of choice is determined by the polarity of the preformed ions in solution: Acidic
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
molecules form negative ions in solution, and basic molecules form positive ions. The
ejection of sample ions from droplets is facilitated if the ionic charge and surface charge
of the droplet are of the same polarity. Thus, a positively charged needle is used to
analyze positive ions and a negatively charged needle is used to analyze negative ions.
¾ Sample ions can carry a single charge or multiple charges. The number of charges
carried by the sample ion depends on the structure of the analyte of interest and the
carrier solvent. The ESI process is affected by droplet size, surface charge, liquid surface
tension, solvent volatility, and ion solvation strength. Large droplets with high surface
tension, low volatility, strong ion solvation, low surface charge, and high conductivity
prevent good electrospray.
Figure 4.2 ESI process in the positive ion polarity mode
¾ Organic solvents such as methanol, acetonitrile, and isopropyl alcohol are superior to
water for ESI. Volatile acids and bases are good, but salts above 10 mM concentration
and strong acids and bases are extremely detrimental.
Atmospheric pressure chemical ionization:
¾ Atmospheric pressure chemical ionization (APCI) is a soft ionization technique, but not
as soft as ESI. APCI is used to analyze compounds of medium polarity that have some
volatility.
¾ In APCI, ions are produced and analyzed as follows:
•
The APCI nozzle sprays the sample solution into a fine mist of droplets.
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
•
The droplets are vaporized in a high temperature tube (the vaporizer).
•
A high voltage is applied to a needle located near the exit end of the tube. The
high voltage creates a corona discharge that forms reagent ions through a series
of chemical reactions with solvent molecules and nitrogen sheath gas.
•
The reagent ions react with sample molecules to form sample ions.
•
The sample ions enter the mass spectrometer and are analyzed.
¾ APCI is a gas phase ionization technique. Therefore, the gas phase acidities and
basicities of the analyte and solvent vapor play an important role in the APCI process.
¾ In the positive-ion mode, sample ionization occurs in a series of reactions that start with
the electron-initiated cation formation. Typical examples of primary, secondary, and
adduct ion formation are shown below:
Primary ion formation
e−+ N2 →N2+. + 2e−
Secondary ion formation
N2++ H2O →N2 + H2O+.
H2O+. + H2O →H3O+ + HO.
Proton transfer
H3O+ + M → (M+H) + + H2O
¾ In negative-ion mode, (M-H) - is typically formed by the abstraction of a proton by OH−.
¾ APCI is typically used to analyze small molecules with molecular weights up to about
1500 U. APCI is a very robust ionization technique. It is not affected by minor changes
in most variables, such as changes in buffers or changes in buffer strength.
¾ APCI use in positive or negative ion polarity mode. For most molecules, the positive-ion
mode produces a stronger ion current.
¾ This is especially true for molecules with one or more basic nitrogen (or other basic)
atoms. An exception to the general rule is that molecules with acidic sites, such as
carboxylic acids and acid alcohols, produce more negative ions than positive ions.
¾ Although, in general, fewer negative ions are produced than positive ions, negative ion
polarity is sometimes the mode of choice.
¾ This is because the negative ion polarity mode sometimes generates less chemical noise
than does the positive mode. Thus, selectivity might be better in the negative ion mode
than in the positive ion mode.
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
Figure 4.3 APCI process in the positive ion polarity mode
Scan modes:
¾ The mass spectrometer can be operated in a variety of scan modes. The most commonly
used scan modes can be divided into two categories: single mass spectrometer scan
modes and MS/MS scan modes. The scan modes in each category are as follows:
•
Mass spectrometer scan modes: Q1MS and Q3MS
•
MS/MS scan modes: Product, Parent, Neutral Loss
•
Data dependent scan mode
¾ The scan modes that can be employed depend on the number and type of rod assemblies
and the voltages applied to the rod assemblies. The mass analyzer has three rod
assemblies. The first and third rod assemblies, Q1 and Q3, are Hyper Quads (hyperbolicprofile quadrupoles), and the second rod assembly, Q2, is a square-profile quadrupole.
¾ Rod assemblies can be operated in either of two capacities:
•
Ion transmission devices
•
Mass analyzers
¾ If only RF voltage is applied, a rod assembly serves as an ion transmission device that
passes all ions within a large range of mass-to-charge ratios (i.e., virtually all ions
present).
¾ When both RF and DC voltages are applied to a rod assembly, the separation of ions of
different mass-to-charge ratios occurs. This separation allows the rod assembly to serve
as a mass analyzer.
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LC-MS/MS Method
¾ On the mass spectrometer, the hyper quad rod assemblies can be operated with RF and
DC voltages or with only RF voltage. That is, Q1 and Q3 can act either as mass
analyzers or ion transmission devices. The Q2 rod assembly always operates with only
RF voltage. Thus, Q2 is always an ion transmission device. For a summary of how the
rod assemblies function in several of the major scan modes,
Table 4.1 Different Scan Mode (MS & MS/MS)
aScan = full scan or transmission of selected ions
bPass all ions or fragments = pass ions or fragments within a wide range of mass-to-charge
ratios
cFragment ions = collisions with argon gas cause ions to fragment
dSet = set to pass ions of a single mass-to-charge ratio or a set of mass-to-charge ratios
Scan types:
¾ Mass spectrometer can be operated with a three scan types.
•
Full scan
•
Selected ion monitoring (SIM)
•
Selected reaction monitoring (SRM)
Full scan:
¾ The full scan type provides a full mass spectrum of each analyte. With full scan, the
scanning mass analyzer is scanned from the first mass to the last mass, without
interruption, in a given scan time.
¾ Full scan experiments are used to determine or confirm the identity of unknown
compounds or the identity of each component in a mixture of unknown compounds.
¾ The full scan type gives you more information about an analyte than does SIM, but a full
scan does not yield the sensitivity that can be achieved by the other two scan types. With
full scan, you spend less time monitoring the signal for each ion than you do in SIM or
SRM. Thus, full scan provides greater information but lower sensitivity than the other
two scan types.
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LC-MS/MS Method
Selected ion monitoring:
¾ Selected ion monitoring (SIM) is a technique in which a particular ion or set of ions is
monitored. SIM experiments are useful in detecting small quantities of a target
compound in a complex mixture when the mass spectrum of the target compound is
known. Thus, SIM is useful in trace analysis and in the rapid screening of a large number
of samples for a target compound.
¾ Because only a few ions are monitored, SIM can provide lower detection limits and
greater speed than the full scan modes. Lower detection limits are achieved because
more time is spent monitoring significant ions that are known to occur in the mass
spectrum of the target analyte. Greater speed is achieved because only a few ions of
interest are monitored; regions of the spectrum that are empty or have no ions of interest
are not monitored.
¾ SIM can improve the detection limit and decrease analysis time, but it can also reduce
specificity. In SIM, only specific ions are monitored. Therefore, any compound that
fragments to produce those ions will appear to be the target compound. Thus, a false
positive result could be obtained.
Selected reaction monitoring:
¾ In selected reaction monitoring (SRM), a particular reaction or set of reactions, such as
the fragmentation of an ion or the loss of a neutral moiety, is monitored.
¾ In SRM, a limited number of parent / product-ion pairs are monitored. In Product-type
experiments, a parent ion is selected as usual, but generally only one product ion is
monitored. SRM experiments are normally conducted with the product scan mode.
¾ Like SIM, SRM allows for the very rapid analysis of trace components in complex
mixtures. However, because two sets of ions are being selected, the specificity obtained
in SRM can be much greater than that obtained in SIM. Any interfering compound
would not only have to form an ion source product (parent ion) of the same mass-tocharge ratio as the selected parent ion from the target compound, but that parent ion
would also have to fragment to form a product ion of the same mass-to-charge ratio as
the selected product ion from the target compound.
Data types:
¾ You can acquire and display mass spectral data (intensity versus mass-to charge ratio)
with the mass spectrometer in one of two data types: Profile data type & Centroid data
type
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LC-MS/MS Method
Profile data type:
¾ In the profile data type, you can see the shape of the peaks in the mass spectrum. Each
atomic mass unit is divided into many sampling intervals. The intensity of the ion current
is determined at each of the sampling intervals. The intensity at each sampling interval is
displayed with the intensities connected by a continuous line. In general, the profile scan
data type is used when you tune and calibrate the mass spectrometer so that you can
easily see and measure mass resolution.
Centroid data type:
¾ In the centroid data type, the mass spectrum is displayed as a bar graph. In this scan data
type, the intensities of each set of multiple sampling intervals are summed. This sum is
displayed versus the integral center of mass of the sampling intervals. In general, the
centroid scan data type is used for data acquisition because the scan speed is faster and
the disk space requirements are smaller. Data processing is also much faster for centroid
data.
4.1.2 TANDEM MASS SPECTROMETRY (MS/MS):
¾ All the mass spectrum tells us is the molecular weight, but it does not provide structure
information for the component of interest. Efforts to remove this limitation took the
form of the development of tandem mass spectrometric methods (MS/MS). Two
stages of mass analysis are required, one to select the precursor ion from other ions
generated in the ion source and one to analyze the product ions after collisions
therefore this approach is called tandem mass spectrometry (MS/MS)
10, 11
. The
schematic of tandem mass spectrometer is presented in Figure 4.4.
¾ In this technique ions are separated, identify and fragmented in a single instrument. A
single molecular ion (precursor) that is characteristic of a given analyte is pass through a
region where they are activated that causes them to fall apart to produce fragment ions
(product). This is usually done by colliding the ions with a neutral gas in a process called
collisional activation or collision-induced dissociation. It is a fast collision event, where
ion translational energy is converted into ion internal energy to obtain an ion in the
excited state, and slow unimolecular decomposition.
¾ The fragment ions are separated accordingly to their mass to charge ratio (m/z). The
resulting MS/MS spectrum consists only of product ions from selected precursor.
MS/MS can be performed in a number of single-stage instruments and a variety of twostage instruments. The most versatile and most widely used MS/MS configuration is the
triple quadrupole mass analyzer instrument as compared to other type of mass analyzer.
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LC-MS/MS Method
Recently different types of hybrid mass analyzers are developed for specific
application for e.g. quadrupole-linear trap, quadrupole-time-of-flight; ion trap-time-offlight.
¾ In triple quadrupole instruments mass analysis is performed in the first and third
quadrupole while the second quadrupole is used as collision cell in the RF only mode.
Different scan modes are available like product ion scan mode- for structure elucidation,
parent ion scan mode and neutral scan mode -for screening, selected ion monitoring
(SIM) and selected reaction monitoring (SRM) scan mode for quantitative analysis. The
tandem mass spectrometry is a very specific and selective technique for quantitation of
drug in different matrix.
4.1.3 LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY
(LC-MS/MS):
¾ By putting a separating chemistry from an HPLC in front of a specialized detector such
as the mass spectrometer, one has a true multidimensional analytical technique that
raises the level of the confidence of what you are looking at. Combining HPLC and
MS offer the possibility of taking advantage of both HPLC as a powerful separation
technique and MS as a powerful and sensitive detection and identification technique.
HPLC chromatographic peaks may contain unresolved components that non-specific
detectors cannot differentiate. This coupled with the need for more sensitive and
specific detector for HPLC has generated considerable interest in the development of
routine liquid chromatography-mass spectrometry (LC-MS)37. The mass spectrometer
can be considered as a universal, selective and specific detector depending on
selective mode of operation. An HPLC followed by mass spectrometric detection
enables the quantification of therapeutically relevant drug concentration (µg/ml or ng/ml
range) in small volume of biological samples and determinations of the chemical
structures of biotransformation. The principle advantage of HPLC separation followed
by the MS detection is that analyte molecular weight is identified by both
chromatographic retention time and by molecular weight & fragmentation pattern. Twodimensional separation is particularly important for the analysis of complex biological
samples.
¾ The coupling between LC & MS has not been straight forward since the normal operation
condition of both HPLC and MS are different high pressure/ high vacuum, low
temperature/high temperature, liquid phase/ gas phase, high flow/low flow to achieve
and to cope with these problem different LC-MS interface have been developed. The
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LC-MS/MS Method
constant limiting factor is the amount of liquid that can be introduced into the ion source
of a MS prevented the wide spread development and use of LC-MS. But now many of the
difficulties associated with it have been solved and there are now several interface
designs and LC-MS is in routine use in analytical laboratories worldwide.
¾ The diversity of available interfaces: capillary inlet
15
interfaces , moving belt
16-18
, direct liquid interface
flow fast atom bombardment- FAB
23-25
19-21
, particle beam
12-14
, pneumatic nebulizer
, thermospray
26,27
22
, continuous -
and atmospheric pressure
28
ionization- API . Currently API based LC/MS interfaces i.e. electrospray ionization
(ESI) and atmospheric pressure chemical ionization (APCI) are the most widely
approaches, while other has limited extent. A high electrical potential applied to a
solvent emerging from a capillary causes the solvent to break into fine threads which
disintegrate into small droplets, this phenomenon is called electrospray28, 29.
¾ In APCI the column effluent is nebulized into a heated vaporizer tube and after
desolvation the gas-vapour mixture enters the API source. In API source APCI is
initiated by electrons generated at a corona discharge needle in combination with
solvent vapours28,
30
. More than 99% of LC-MS performed today is based on API
interfacing. These interfaces in combination with tandem mass spectrometry of
single stage and double stage fragmentation instrument enables a LC-MS/MS
technique.
¾ ESI is one of the most exciting ionization methods due to direct ionization from
solution and therefore its use as an LC-MS/MS interface, production of multiply
charged ions that extends the effective mass range of the mass analyzer, introduction of
methods to aid in desolvation of the analyte, low background from the ionization process
are the advantages over other ionization. Extensions of the ESI interface led to
miniaturized formats, microelectrospray and nanoelectrospray.
¾ The most important application area of LC-MS is in the pharmaceutical field, where
LC-MS is involved in almost every step of drug development, testing and formulation.
Because of its specificity and sensitivity LC-MS, especially in combination with LCMS/MS has rapidly growing importance in other drug research area.
¾ LC-MS/MS is widely applied for drug development as well as routine analysis. In drug
discovery activities like lead identification, lead optimization, in vitro and in vivo drug
screening, preclinical activities like metabolite identification, impurity screening and
degradant screening, clinical activities like quantitative bioanalysis and metabolite
identification application of LC-MS and LC-MS/MS enhanced extensively.
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LC-MS/MS Method
Figure 4.4 Schematic diagram of tandem mass spectrometer using triple quadrupole
instrument.
4.2 EXPERIMENTAL WORK:
4.2.1 MATERIALS:
TABLE NO: 4.2 REFERENCE/WORKING STANDARDS
Sr. No.
Name of Standard
Batch no.
1.
Gabapentin (Working
standards)
GP0381107
Assay
99.97 %
2.
Levetiracetam
(Reference standards)
LR0520907
99.00 %
Name of manufacturer
Matrix Laboratories Ltd.,
Secunderabad
Hetero Labs Ltd.,
Hyderabad
TABLE NO: 4.3 APPARATUS
Apparatus
Volumetric flask
Glass beakers
Glass bottles
Solvent Filtration
Grade/Capacity
Appropriate volumes (Class A)
Appropriate volumes
Appropriate volumes
Appropriate volumes
Measuring cylinder
Micropipette
Micropipette
Micropipette
Micropipette
Microtube
Appropriate volumes (Class A)
Eppendorf, 2-20µl
Eppendorf, 10-100µl
Eppendorf, 20-200µl
Eppendorf, 100-1000µl
MCT-175, 1.7ml clear
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Manufacturer/ Supplier
Borosil Glassworks Ltd., Mumbai
Borosil Glassworks Ltd., Mumbai
Borosil Glassworks Ltd., Mumbai
Millipore, India/
Borosil Glassworks Ltd., Mumbai
Borosil Glassworks Ltd., Mumbai
Eppendorf India Ltd., Chennai
Eppendorf India Ltd., Chennai
Eppendorf India Ltd., Chennai
Eppendorf India Ltd., Chennai
Axygen, Scientific, USA
M .Pharm. Thesis
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LC-MS/MS Method
TABLE NO: 4.4 INSTRUMENTATION
Instrument
Auto sampler
Solvent delivery module
MS Detector
Vortexer
Analytical balance
Solid Phase Extraction (SPE)
Unit
Freezer (-70°C)
Freezer (-20°C)
Refrigerator(2-8°C)
Ultrasonic bath
Water purification system
Vacuum Pump
Brand/Model
CTC PAL
Accela pump
TSQ Quantum
ultra
Spinix
Mettler Toledo
Waters/
Orochem
U725-86
375 W
RT34M
JAC 4020
Milli-Q
Millipore
Manufacturer/ Supplier
CTC PAL Ltd., UK
Thermo Finnigan Ltd., UK
Thermo Finnigan Ltd., UK
Barnstead International, USA
Mettler Toledo India Pvt. Ltd., Mumbai
Waters, USA/Orochem, India
New Brunswick Scientific co.inc., USA
Vest frost, UK
Samsung India Electronics Ltd., India
Bio-sci instruments, India
Millipore, USA
Millipore, India
TABLE NO: 4.5 REAGENTS AND CHEMICALS
Chemicals/Reagents
Methanol
Water
Ammonium Formate
Grade
LCMS grade
Milli Q
For Molecular biology
Manufacturer/ Supplier
Sigma-Aldrich, Germany
Millipore, USA
Sigma-Aldrich, Germany
4.2.2 PREPARATION OF SOLUTIONS:
a) Preparation of 5 mM Ammonium formate:
¾ Ammonium formate (315.30 mg) was weighed accurately and transferred in 1000 ml
glass bottle. Water (about 1000 ml) was added using graduated cylinder and sonicated to
dissolve.
¾ pH was adjusted to 3.0 with OPA.
b) Elution solution / Blank solution (Methanol 100 %):
c) Auto sampler rinsing solution (Methanol 100%):
d) Mobile phase preparation [Methanol: 5 mM Ammonium formate (80:20,% v/v)]
¾ In 1000 ml glass bottle, Methanol (800 ml) and 5mM Ammonium formate (200 ml) were
added using graduated cylinder. Mixed well. The solution was filtered on a 0.45µm
filter using solvent filtration apparatus by applying vacuum and sonicated it.
e) Preparation of diluent [Water : Methanol, (50: 50, % v/v)]:
¾ In 1000 ml glass bottle, water (500 ml) was taken using graduated cylinder. Methanol
(500 ml) was added using graduated cylinder. Mixed well.
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LC-MS/MS Method
4.2.3 PREPARATION OF ANALYTE SOLUTIONS:
a) Preparation of GBP main stock solution (5.00 mg/ml):
¾ GBP working/reference standard (equivalent to 50 mg) was weighed accurately.
Transferred it in 10 ml volumetric flask. Diluent (50 ml) was added and sonicated to
dissolve. Diluted up to mark with diluent.
b) Preparation of GBP intermediate stock solutions:
1. GBP intermediate stock solution (2.50 mg/ml )
¾ Calculated volume of GBP main stock solution (5 ml) was transferred to 10 ml
volumetric flask and diluted up to mark with diluent.
2. GBP intermediate stock solution (0.50 mg/ml)
¾ Calculated volume of above GBP intermediate stock solution (5 ml) was transferred to
make conc. 0.50 mg/ml of GBP in 25 ml volumetric flask and diluted up to mark with
diluent.
Note: a) Percentage purity/ assay of standard were considered while weighing.
b) Salt equivalency was considered while weighing.
c) Weight and volume of solution might be changed as per the requirement
without altering the concentration for analysis.
4.2.4 PREPARATION OF INTERNAL STANDARD SOLUTION:
a) Preparation of LEV main stock solution (1.00 mg/ml):
¾ LEV working/reference standard (equivalent to 10 mg) was weighed accurately.
Transferred it in 10 ml volumetric flask. Diluent (about 5.0 ml) was added and sonicated
to dissolve. Diluted up to mark with diluent.
b) Preparation of LEV intermediate stock solution (0.20 mg/ml):
¾ The calculated volume of LEV main stock solution (2 ml) was transferred to make conc.
0.20 mg/ml in 10 ml volumetric flask and diluted up to mark with diluent.
c) Preparation of LEV spiking stock solution (1.50 µg/ml):
¾ The calculated volume of LEV intermediate stock solution (0.188 ml) was transferred to
make conc. 1.50 µg/ml of LEV in 25 ml volumetric flask and diluted up to mark with
diluent.
Note: a) Percentage purity/ assay of standard were considered while weighing.
b) Salt equivalency was considered while weighing.
c) Weight and volume of solution might be changed as per the requirement
without altering the concentration for analysis.
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LC-MS/MS Method
4.2.5 PREPARATION OF CALIBRATION STANDARD AND QUALITY CONTROL
SPIKING SOLUTIONS:
TABLE NO: 4.6
Spiking stock solution
Volume of GBP
intermediate stock
solution (0.50 mg/ml)
used (ml)
Diluent
up to (ml)
GBP Conc. (µg/ml)
Sample Name
0.02
10
1.00
CSS-01
0.04
10
2.00
CSS-02
0.12
10
6.00
CSS-03
0.24
10
12.00
CSS-04
0.48
10
24.00
CSS-05
0.96
10
48.00
CSS-06
1.44
10
72.00
CSS-07
1.92
10
96.00
CSS-08
2.40
10
120.00
CSS-09
0.06
10
3.00
LQCS
0.80
10
40.00
MQCS
1.80
2.40
10
5
90.00
240.00
HQCS
2ULOQCS
4.2.6 PREPARATION OF BIOLOGICAL MATRIX CALIBRATION AND QUALITY
CONTROL SAMPLES IN MATRIX:
¾ The blank biological matrix (plasma) taken out from –20°C/-70°C deep freezer and kept
at room temperature for thawing and vortexed adequately before pipetting.
a) Preparation of blank plasma:
¾ To analyte free plasma (95 µl) in micro tube diluent (5 µl) was added and vortexed to
mix. Diluent (25 µl) was added and followed the sample treatment procedure as per
section 4.2.12.
b) Preparation of zero standard:
¾ To analyte free plasma (95 µl) in micro tube diluent (5 µl) was added and vortexed to
mix. Internal standard spiking stock solution (25 µl) was added and followed the sample
treatment procedure as per section 4.2.12.
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LC-MS/MS Method
c) Preparation of calibration standards and quality control samples in biological
matrix with respect to 5 % spiking:
TABLE NO: 4.7
Details of spiking stock solution used
Plasma
volume
(µl)
GBP Conc. (µg/ml)
Sample Name
GBP Conc. (µg/ml)
Volume (µl)
1.00
5
95
0.05
CS-1
2.00
5
95
0.10
CS-2
6.00
5
95
0.30
CS-3
12.00
5
95
0.60
CS-4
24.00
5
95
1.20
CS-5
48.00
5
95
2.40
CS-6
72.00
5
95
3.60
CS-7
96.00
5
95
4.80
CS-8
120.00
5
95
6.00
CS-9
3.00
5
95
0.15
LQC
40.00
5
95
2.00
MQC
90.00
5
95
4.50
HQC
240.00
5
95
12.00
2ULOQ
d) System suitability:
¾ Prepared MQC level sample was used in system suitability and followed the sample
treatment procedure as per section 4.2.12.
4.2.7 TUNING OF GBP AND LEV IN MS:
a) Preparation of tuning GBP solution ( 50 ng/ml):
¾ GBP (5 mg) was taken in 10 ml vol. flask and made up the volume up to mark with
methanol. Above solution (0.01 ml) was taken in 100 ml volumetric flask and diluted
with methanol to 100 ml.
b) Preparation of tuning LEV solution ( 50 ng/ml):
¾ LEV (5 mg) was taken in 10 ml vol. flask and made up the volume up to mark with
methanol. Above solution (0.01 ml) was taken in 100 ml volumetric flask and diluted
with methanol to 100 ml.
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LC-MS/MS Method
TABLE NO: 4.8 TUNING PARAMETERS FOR GBP & LEV (IS)
SR NO.
PARAMETERS
GBP
LEV
1.
Parent ion
172.15
171.13
2.
Product ion
154.15
126.06
3.
Collision energy
12 V
15 V
4.
Tube lens
78.59
66.83
5.
Q1pw
0.70
0.70
6.
Q3pw
0.70
0.70
7.
Spray voltage
4000 V
4000 V
8.
Spray capillary temp
390 °C
390 °C
GBP
LEV
Figure 4.5 Tuning Spectrum of GBP & LEV
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
4.2.8 METHOD DEVELOPMENT TRIALS:
TABLE NO: 4.9 METHOD DEVELOPMENT TRIALS
SR
NO
TRIAL
COLUMN
MOBILE
PHASE
SAMPLE
OBSERVATION
1.
TRIAL-1
Betabasic C8,
50 × 4.6 mm,
5µ
ACN: 10 mM
Amm. formate
( 80:20)
Aqueous
MQC
peak shape was not
good
2.
TRIAL-2
Betabasic C8,
50 × 4.6 mm,
5µ
ACN: 5 mM
Amm. formate
( 80:20)
Aqueous
MQC
IS peak shape was
not good
3.
TRIAL-3
Betabasic C8,
50 × 4.6 mm,
5µ
ACN: 1 mM
Amm. formate
( 80:20)
Aqueous
MQC
peak shape was
good , but higher
tailing observed
4.
TRIAL-4
Betabasic C8,
50 × 4.6 mm,
5µ
ACN: Methanol:
5 mM Amm.
formate
( 60:20:20)
Aqueous
MQC
peak shape was
good , but higher
tailing observed
5.
TRIAL-5
Betabasic C8,
50 × 4.6 mm,
5µ
ACN: Methanol:
1 mM Amm.
formate
( 60:20:20)
Aqueous
MQC
peak shape was
good, but higher
tailing was
observed
6.
TRIAL-6
Betabasic C8,
50 × 4.6 mm,
5µ
ACN: Methanol:
1 mM Amm.
formate
( 40:40:20)
Aqueous
MQC
peak shape was
good, slight higher
tailing was
observed
7.
TRIAL-7
Symmetry C8,
150 × 4.6 mm,
5µ
ACN: Methanol:
1 mM Amm.
formate
( 40:40:20)
Aqueous
MQC
peak shape was
good, slight less
tailing was
observed
8.
TRIAL-8
Symmetry C8,
150 × 4.6 mm,
5µ
Methanol :
1 mM Amm.
formate
( 80:20)
Aqueous
MQC
peak shape was
good, tailing was
not observed
9.
TRIAL-9
Symmetry C8,
150 × 4.6 mm,
5µ
Methanol :
1 mM Amm.
formate
(80:20)
Aqueous
MQC &
External
spiked
MQC
peak shape was
good, ion
suppression was
observed
S.K.P.C.P.E.R., Kherva
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M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
10.
TRIAL10
Symmetry C8,
150 × 4.6 mm,
5µ
Methanol :
5 mM Amm.
formate
(80:20)
Aqueous
MQC &
External
spiked
MQC
peak shape was
good, ion
suppression was not
observed
11.
TRIAL11
Symmetry C8,
150 × 4.6 mm,
5µ
Methanol :
5 mM Amm.
formate
(80:20)
Linearity
& P&A
(Waters
Cartridge)
Within acceptance
criteria
12.
TRIAL12
Symmetry C8,
150 × 4.6 mm,
5µ
Methanol :
5 mM Amm.
formate
(80:20)
Linearity
& P&A
(Orochem
Cartridge)
Within acceptance
criteria
4.2.9 METHOD SUMMARY:
Biological matrix
: Human plasma
Sample volume required
: 0.1 ml
Anticoagulant
: Heparin
Analyte
: Gabapentin
Internal standard
: Levetiracetam
Calibration curve range
: GBP : 0.05 µg/ml to 6.00 µg/ml
Detection mode
: Mass spectrometry
Analytical technique
: Liquid chromatography- Mass
spectrometry
Sample treatment
: Solid phase extraction
Quantitation method
: Peak area ratio
Weighting factor for Calibration curve
: 1/X 2
4.2.10 CHROMATOGRAPHIC CONDITIONS:
Chromatographic
:
Reversed phase
:
Isocratic
Tray temperature
:
5°C
Injection volume
:
5.0 µl
Column
:
Brand
: symmetry
:
Type
: C8
mode
Isocratic/gradient
mode
S.K.P.C.P.E.R., Kherva
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M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
:
Length x i.d. (mm)
: 150 x 4.6
:
Particle size (µ)
: 5.0
Mobile phase
:
Methanol: 5 mM Ammonium formate (80:20, % v/v)
Column Temp.
:
45°C
Flow rate
:
1.0 ml/min with splitter. ( 30% to source & 70% to waste)
Back pressure
:
140 bar (approximately)
Retention times (min)
:
*
GBP
:
1.66 min (± 0.5 min)
LEV
:
1.66 min (± 0.5 min)
Run time
:
3.00 min
•
Retention time of analyte and internal standard may vary between run/batch.
Shimadzu LC method parameters
Pump
:
Shimadzu LC-10ADvp
Pressure range
:
0- 410 Bars
Model
:
SIL-HTc
Rinse volume
:
1000 µl
Needle stroke
:
52 mm
Rinse speed
:
35 µL/sec
Purge time
:
1.0 minutes
Rinse dip time
:
0.0 sec
Rinse mode
:
Before and After aspiration.
Cooler enabled
:
Yes
Cooler temperature
:
5°C
System controller
4.2.11 DETECTOR PARAMETERS:
Ion source parameters :
Ion source
:
ESI mode
Spray voltage
:
4000 v
Sheath gas
:
55 (arb)
Auxiliary gas
:
30 (arb)
Capillary temperature
:
390 °C
Tube lens offset*
:
Tuned value 78.59 V (For GBP)
:
Tuned value 66.83 V (For LEV)
S.K.P.C.P.E.R., Kherva
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M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
Collision energy
:
12 V (For GBP), 15 V (For LEV)
Collision gas pressure
:
1.5 mTorr
MS acquire time
:
3.00 min.
No of segments
:
1
Segment time
:
3.00 min.
Chrom filter
:
10 sec
Peak width
:
0.01 for GBP & LEV.
No of scan events
:
2
Polarity
:
Positive
Data type
:
Centroid
Scan type
:
SRM (Selected Reaction monitoring)
Scan event – 1 (For GBP)
Parent
Product
Width
Time
CE
Q1PW
Q3PW
172.15
154.15
0.010
0.200
12
0.70
0.70
Scan event - 2 (For LEV)
Polarity
:
Positive
Data type
:
Centroid
Scan type
:
SRM (Selected Reaction monitoring)
Parent
Product
Width
Time
CE
Q1PW
Q3PW
171.13
126.06
0.010
0.200
15
0.70
0.70
Where,
Parent
:
Parent ion
Product
:
Daughter ion being monitored
Width
:
Scan width
Time
:
Scan time for event
CE
:
Collision energy
Q1PW
:
Resolution for first quadrupole
Q3PW
:
Resolution for third quadrupole
Divert Valve Settings
:
Not in use.
4.2.12 SAMPLE TREATMENT/SAMPLE PREPARATION:
¾ The study samples along with calibration standards and QC samples were taken out from
the -20°C/-70°C deep freezer and kept at room temperature for thawing. Vortexed each
sample adequately using a vortex mixer before pipetting. Followed the procedure from a
to d as given below.
a) Internal standard spiking solution (25µl) was transferred in micro tube except in blank
S.K.P.C.P.E.R., Kherva
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Dushyant Patel
LC-MS/MS Method
plasma where 25 µl of diluent was added.
b) Plasma sample (100 µl) (blank plasma, zero standard, calibration standard, quality
control samples) and study samples were added in micro tube using a micro pipette.
c) Water (100 µl) was added and vortexed to mix. The above plasma mixture was loaded on
previously conditioned (conditioned with 1 ml of methanol and then with 1 ml of water)
Orochem SPE columns (Orpheus, 30 mg, 1 ml, DVB-LP). The cartridges were washed
with 1 ml of water and eluted with 1ml of elution solution & vortexed it.
d) Samples (5.0 µl) prepared as above was injected.
4.2.13 METHOD VALIDATION OF LC-MS/MS METHOD:
Pre method validation:
1. Pre method validation is a generic that provides concretes, statistical evidence for
acceptable method performance prior to initiating a bioanalytical method validation.
2. A large number of replicate spiked matrix samples were analyzed in a single batch
before method validation.
3. Pre method validation experiment was performed for system suitability, specificity &
selectivity, sensitivity, three linearity, three between-batches or inter-batch
accuracy and precision, recovery and carry over effect check.
4. All pre method validation samples prepared according to describe in sample preparation
section.
5. The utility of this pre method validation approach was illustrated using actual laboratory
data. The process of interpreting the results and drawing conclusions about assay
viability was demonstrated.
6. The resulting conclusions provide sufficient background information to indicate if an
assay procedure can be ready to enter in the validation process.
Method validation and characteristics of the Method:
A. Chromatography:
¾ Representative chromatograms of blank plasma, zero standard, LLOQ, ULOQ, LQC,
MQC, and HQC samples GBP are represented in Figure No 4.6, 4.7, 4.8, 4.9, 4.10, 4.11
and 4.12, respectively.
B. Specificity and selectivity:
¾ Obtain samples of the relevant biological matrix (e.g., plasma, serum, blood, etc.)
collected under controlled conditions.
¾ For plasma matrix, 08 normal plasma lots (04 different plasma lots with the
anticoagulant to be used during method validation and 04 different plasma lots with the
S.K.P.C.P.E.R., Kherva
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Dushyant Patel
LC-MS/MS Method
anticoagulant to be used for study), 0l lipemic plasma and 01 haemolysed plasma lot
were included.
¾ One sample each of the above mentioned 10 plasma lots at blank and LLOQ level was
processed and analyzed as per the procedure described in sample preparation section.
Acceptance criteria:
¾ No interfering peaks from endogenous matrix components, decomposition product etc.,
should be present at the retention time of an analyte and an internal standard in blank
matrix.
¾ If any peak is present at the retention time of analyte in blank matrix, its area response
should be ≤20 % of response of an extracted lowest plasma calibration standard i.e.
LLOQ standard of the same lot.
¾ If any peak is present at the retention time of an internal standard in blank matrix, its area
response should be ≤5 % of the response of an extracted internal standard concentration
of the same lot.
¾ At least 75 % of the buffered plasma and heparinised plasma should meet the acceptance
criteria.
¾ Both lipemic and haemolysed plasma should meet the above three criteria for the
interference at the respective retention time of analyte and internal standard. If the
experiment fails, repeat the experiment and then change the methodology if required.
C. Sensitivity:
Procedure:
¾ Calibration standards, zero standard (matrix spiked only with internal standard) and six
sets of matrix sample spiked at LLOQ concentration were processed and analyzed using
blank matrix lot, as per the procedure described in sample preparation section.
Acceptance criteria:
¾ The analyte area response at the LLOQ should be at least 5 times the response compared
to blank response.
¾ Analyte calculated concentration should be identifiable and reproducible with a precision
of 20% and accuracy of 80- 120%
D. Linearity:
¾ A calibration curve (standard curve) is the relationship between the response of the
instrument and known concentrations of the analyte.
¾ A calibration curve should be prepared for each analyte in the sample.
¾ A sufficient number of standards should be used in order to properly define the
relationship between concentration and response.
S.K.P.C.P.E.R., Kherva
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Dushyant Patel
LC-MS/MS Method
¾ A calibration standard should be prepared in the same biological matrix same as that of
the study samples, by spiking with known concentrations of the analyte.
¾ A calibration curve should be comprised of a “blank matrix” (matrix processed without
analyte and internal standard), a “zero standard” (blank matrix processed only with
internal standard) and six or more calibration standards covering the expected range,
including the LLOQ and ULOQ.
¾ LLOQ should cover at least 4-5 half-life of the reported Cmax.
¾ ULOQ should cover the expected Cmax value. When no reference is available, it should
be decided on basis of development studies results.
¾ Calibration curve must contain minimum six calibration standards. Selection of
calibration standard expressed as multiple of LLOQ and percent of ULOQ are as follows:
TABLE NO: 4.10 SELECTION OF CALIBRATION STANDARD
A 8 Standard
Calibration Curve
CODE
LLOQ
Standard 1 – CS-1
A 9 Standard Calibration
Curve
LLOQ
5% ULOQ or 2 X LLOQ
Standard 2 – CS-2
2 X LLOQ
10% ULOQ
Standard 3 – CS-3
2% to 5% ULOQ
20% ULOQ
Standard 4 – CS-4
10% ULOQ
40% ULOQ
Standard 5 – CS-5
20% ULOQ
60% ULOQ
Standard 6 – CS-6
40% ULOQ
80% ULOQ
Standard 7 – CS-7
60% ULOQ
100% ULOQ
Standard 8 – CS-8
80% ULOQ
Standard 9 – CS-9
100% ULOQ
Procedure:
¾ LLOQ and ULOQ calibration standards in duplicate (add suffix “D” in sample name of
duplicate standard) were prepared, so that if first does not meet acceptance criteria, then
only second standard of the same concentration level should be used in regression
analysis. If both are within acceptance criteria consider the first standard for regression
analysis.
¾ Blank matrix, zero standard and calibration standards were processed and analyzed as per
the procedure described in sample preparation section.
¾ Summary of the curve fitting parameters and of back-calculated concentration at each
standard level from each calibration curve were prepared.
¾ % nominal concentration of back-calculated value at each calibration level to determine
accuracy at each calibration level was calculated.
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
Acceptance criteria:
¾ A correlation coefficient (r) of the calibration curve must be > 0.9900.
¾ The back-calculated concentration of the lowest calibration standard (CS1) must be
within 80-120% of its theoretical concentration.
¾ The back-calculated concentrations of all other calibration standards must be within 85115% of their theoretical concentrations.
¾ The curve must contain at least 75% of the calibration standards for evaluation of curve
fitting.
¾ No two adjacent (or consecutive) calibration standards can be rejected.
¾ Both replicates each of LLOQ and ULOQ standard cannot be rejected, either of the two
replicate must meet the acceptance criteria.
E. Accuracy:
1. Within-batch or intra-batch accuracy:
¾ Calibration standards and six replicates each of LLOQ, LQC, MQC and HQC samples
were processed and analyzed as per the procedure described in sample preparation
section.
¾ % nominal concentration of back-calculated value for LLOQ, LQC, MQC and HQC,
analyzed in single analytical batch was calculated, as per formula to determine withinbatch or intra-batch accuracy.
2. Between -batch or inter-batch accuracy:
¾ Calibration standards and six replicates each of LLOQ, LQC, MQC and HQC samples
were processed and analyzed as per the procedure described in sample preparation
section.
¾ Five different batches on different days were performed.
¾ % nominal concentration of back-calculated value for LLOQ, LQC, MQC and HQC
samples, analyzed on five different batches on different days was calculated.
Acceptance criteria:
¾ The back calculated concentrations of all QC samples (LQC, MQC, and HQC) must be
within 85-115% of their nominal concentration except at LLOQ sample where it should
not deviate by more than 80-120% of its nominal concentration.
¾ At least 67% quality control samples must fall within above-mentioned criteria at each
LLOQ, LQC, MQC, and HQC levels.
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
F. Precision:
1. Within-batch or intra-batch precision:
¾ Calibration standards and six replicates each of LLOQ, LQC, MQC and HQC samples
were processed and analyzed as per the procedure described in sample preparation
section.
¾ Mean, standard deviation and % coefficient of variation for LLOQ, LQC, MQC and HQC
samples, analyzed in single analytical batch were calculated.
2. Between -batch or inter-batch precision:
¾ Calibration standards and six replicates of LLOQ, LQC, MQC and HQC samples were
processed and analyzed as per the procedure described in sample preparation section.
¾ Five different batches on different days were performed.
¾ Mean, standard deviation and % coefficient of variation for LLOQ, LQC, MQC and HQC
samples analyzed on five different batches on different days were calculated.
Acceptance criteria:
¾ The %CV of the back-calculated concentrations of all QC samples (LQC, MQC, and
HQC) must be within 15%, except for LLOQ, which should be within 20%.
¾ At least 67% quality control samples must fall within above-mentioned criteria at each
LLOQ, LQC, MQC and HQC levels.
G. Recovery:
¾ Recovery for analyte and internal standard was performed by comparing the area of
extracted samples at three different concentrations (LQC, MQC and HQC) with unextracted standards area that represents 100% recovery.
Procedure:
¾ Recovery of an analyte was determined at LQC, MQC, HQC levels and recovery of an
internal standard was determined at concentration to be used during method validation.
¾ Six replicates each of LQC, MQC, and HQC samples were processed and analyzed as per
the procedure described in sample preparation section. These were considered as
extracted samples for recovery.
¾ To prepare comparison samples for recovery, extracted solutions of blank matrix were
used, for the preparation of equivalent unextracted samples, to nullify the matrix effect
while calculating the recovery.
¾ For the recovery comparison samples, the spiking stock of standard solution of an analyte
and internal standard were spiked in the extracted blank matrix solution to achieve
concentration equivalent to extracted samples nominal concentration.
S.K.P.C.P.E.R., Kherva
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M .Pharm. Thesis
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LC-MS/MS Method
¾ Unextracted standard solution of an analyte (at LQC, MQC and HQC level) and internal
standard of concentrations equivalent to concentration of extracted samples were
processed and analyzed.
¾ The area for extracted LQC, MQC, HQC samples and internal standard were compared
with areas of unextracted standards and internal standard that represent 100 % recovery.
¾ % recovery of an analyte at LQC, MQC and HQC samples and an internal standard were
calculated.
¾ % recovery for extracted HQC, MQC and LQC samples should not exceed 115%, if it
exceeds then reporting should be justified.
Acceptance criteria:
¾ Recovery of analyte and internal standard should be consistent, precise and reproducible.
¾ Variability within areas at each QC level for analyte should be within CV of 15%.
¾ Variability of the analyte recoveries across all the three QC levels should be within C.V
of 20%.
¾ Variability within areas of IS found with each extracted QC level should be within CV of
15%, while the variability of internal standard area found between all the QC levels
should be within CV of 20%.
H. Dilution integrity:
Procedure:
¾ The stock solutions of analyte and internal standard were prepared as per the procedure
described in sample preparation section.
¾ Analyte spiking stock solution was spiked in blank plasma to get concentration
equivalent to 2 times of ULOQ.
¾ Above spiked samples (2xULOQ) was diluted with blank plasma to get 1/2 and 1/4
concentrations of the spiked sample or as per requirement.
¾ Calibration standards and six aliquots each of diluted samples (1/2 and 1/4 dilutions)
were processed and analyzed as per the procedure as described in sample preparation
section.
¾ %CV for the back calculated concentration was calculated.
¾ % accuracy for the back calculated concentration was calculated.
Acceptance criteria:
¾ Back calculated concentration accuracy should be within 85-115% of theoretical
concentration.
¾ Precision (% CV) for the back calculated concentration should be within 15%.
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
¾ At least 67% of total dilution samples at each dilution level should fall within abovementioned criteria.
I. Matrix effect:
Procedure:
¾ Calibration standards were processed and analyzed in the same matrix which was to be
used during validation experiment and three replicates from all lots described above each
at LQC and HQC levels were processed and analyzed as per the procedure described in
sample preparation section.
Acceptance criteria:
¾ The back calculated concentrations of LQC and HQC must be within 85- 115% of their
theoretical concentration.
¾ At least 67% of LQC and HQC samples must fall within above-mentioned criteria for
each lot of matrix.
¾ At least 75% of the buffered plasma should meet the acceptance criteria.
¾ Both lipemic and haemolysed plasma should meet the above criteria, if the experiment
fails repeat the experiment once or change the methodology.
J. Stability:
1. Stock solution stability:
Procedure:
¾ Fresh stock solution of an analyte and an internal standard were prepared as per the
procedure described in sample preparation section.
¾ Freshly prepared main stock solutions aliquots of analyte and internal standard solution
were stored at 2-8°C in refrigerator or in freezer if required for a relevant period for
short-term and long-term stability.
¾ Freshly prepared main stock solutions aliquots of analyte and internal standard solution
were stored at room temperature for at least 6 hrs or relevant short-term period.
¾ After relevant stability period fresh stock comparison solution of analyte and an internal
standard were prepared as per the procedure.
¾ The main stock aliquots were retrieved from refrigerator and from room temperature.
¾ Appropriately stock solution of analyte was diluted to get concentration equivalent to
ULOQ nominal concentration for both comparison and stability sample solution.
¾ Six injections of freshly prepared diluted analyte and internal standard comparison
solution and stability solutions were performed.
¾ The analytical area results of stability solutions were compared with those of freshly
prepared solutions area results.
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
¾ After relevant long-term stability period at 2-8°C, fresh stock comparison solution of
analyte and an internal standard were prepared as per the procedure.
¾ Above steps were repeated for long-term solution stability.
¾ The area results of long term stability solutions were compared with those of freshly
prepared comparison solutions.
¾ % mean change for analyte and internal standard was calculated.
Acceptance criteria:
¾ % mean change between freshly prepared stock solution (comparison samples) area
results and stability samples area results must be within ± 10%.
¾ If %CV of area of analyte and internal standard for both stability and comparison sample
is greater than 5 % than out of six only one inappropriate sample should be rejected.
¾ If above acceptance criteria are not met, then experiment should be repeated if on
investigation any processing error is found or should be repeated for relevant shorter
storage period or storage temperature.
Main stock solution stability of GBP and LEV (IS) at room temperature for 93 hrs:
¾ Main stock solution of GBP and LEV (IS) were freshly prepared and aliquots of stocks
were kept at room temperature for 93 hrs (stability sample). Aqueous equivalent highest
calibration standards of GBP and LEV (IS) were prepared from the stability samples and
analyzed. Areas of stability samples and freshly prepared samples were compared to
determine % mean change during stability period.
Main stock solution stability of GBP and LEV (IS) at 2-8°C for 93 hrs:
¾ Main stock solution of GBP and LEV (IS) were freshly prepared and aliquots of stocks
were kept at 2-8°C for 93 hrs (stability sample). Aqueous equivalent highest calibration
standards of GBP and LEV (IS) were freshly prepared from the stability samples and
analyzed. Areas of stability samples and freshly prepared samples were compared to
determine % mean change during stability period.
2. Bench top stability of GBP (at room temperature for 20 hrs):
Procedure:
¾ Six aliquots each of LQC and HQC samples were spiked for bench top stability. These
samples were kept at room temperature for 20 hrs.
¾ In case of spiking and keeping the bench top stability samples at room temperature, the
bench top stability period was considered between the time of keeping at room temp.
and the time of start of processing those samples.
¾ Calibration standards and six replicates each of LQC and HQC comparison samples
prepared by spiking with freshly prepared stock solutions or stock solutions with proven
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
stability were processed and analyzed as per the procedure described in sample
preparation section.
¾ The stability samples after relevant period (at room temperature for 20 hrs) were
processed and analyzed.
¾ The mean back-calculated concentration of stability samples were compared with those
of freshly prepared comparison samples.
¾ % mean change as per formula was calculated.
Acceptance criteria:
¾ The back-calculated concentrations of all LQC and HQC samples must be within 85115% of their theoretical concentration.
¾ At least 67% QC samples must fall within above-mentioned criteria at each LQC and
HQC levels.
¾ % Mean change must be within ± 15%.
3. Process stability of GBP at 5°C in auto sampler for 76 hrs:
¾ Process stability of analyte was determined at LQC and HQC levels.
Procedure:
¾ Calibration standards and six replicates each of LQC and HQC comparison samples
prepared by spiking with freshly prepared stock solutions were processed and analyzed as
per the procedure described or use the stored samples.
¾ The above QC samples were kept in the auto sampler for relevant period (at 5°C for 76
hrs) to measure the stability of the processed samples in the auto sampler.
¾ Stability samples after relevant period along with calibration standards and six replicates
each of LQC and HQC comparison samples prepared by spiking with freshly prepared
stock solutions or stock solutions with proven stability were processed and analyzed as
per the procedure.
¾ The process stability period was considered between the time of storing of samples in
auto sampler or refrigerator and the time of analysis of last process stability sample.
¾ The mean back-calculated concentration of stability samples with those of freshly
prepared comparison samples was calculated.
¾ % mean change as per formula was calculated.
Acceptance criteria:
¾ The back-calculated concentrations of all LQC and HQC samples must be within 85115% of their nominal concentration.
¾ At least 67% of quality control samples must fall within above-mentioned criteria at each
LQC and HQC levels.
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
¾ % Mean change must be within ± 15%.
4. Freeze and thaw stability of GBP (after 4th cycle at -20°C and -70°C):
¾ Freeze and thaw stability of analyte was determined after three freeze and thaw cycles at
LQC and HQC levels.
Procedure:
¾ Three sets (set-1, set-2 and set-3) each having six replicates each of LQC and HQC
sample of freeze and thaw stability by spiking with freshly prepared stock solutions or
stock solutions with proven stability were prepared.
¾ All the samples were stored at –20°C and/or –70°C for at least 24 hrs and after this period
all the three sets of samples were thawed unassisted at room temperature.
¾ When completely thawed, set-1, set-2 and set-3 were refrozen for at least 12 hrs under the
same conditions.
¾ Kept set-1 in frozen condition to use for second freeze and thaw cycle when analytical
results of third freeze and thaw cycle were not within the acceptance criteria.
¾ The second freeze - thaw cycle was repeated for set-2 and set-3.
¾ The frozen set-2 and set-3 were thawed for the third freeze and thaw cycle. Set-3 was
thawed.
¾ Calibration standards and six replicates each LQC and HQC comparison samples were
prepared by spiking with freshly prepared stock solutions or stock solution with proven
stability as per the procedure. Those samples were processed and analyzed.
¾ Set-2 stability samples after third freeze and thaw cycle along with freshly prepared low
and high quality control comparison samples were processed and analyzed.
¾ Fourth freeze and thaw cycle for set-3 was repeated.
¾ The mean back-calculated concentration of stability samples with those of freshly
prepared comparison samples was calculated.
¾ % mean change was calculated as per formula.
Acceptance criteria:
¾ The back-calculated concentrations of all LQC and HQC samples must be within 85- 115
% of their nominal concentration.
¾ At least 67 % of QC samples must fall within above-mentioned criteria at each LQC and
HQC levels.
¾ % Mean change must be within ± 15 %.
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
4.3 RESULTS AND DISCUSSION:
4.3.1 Chromatography:
¾ Representative chromatograms of blank plasma, zero standard, LLOQ, ULOQ, LQC,
MQC, and HQC samples GBP are represented in Figure No. 4.6, 4.7, 4.8, 4.9, 4.10, 4.11
and 4.12, respectively.
GBP
LEV
Figure 4.6 Representative chromatogram of blank plasma
GBP
LEV
Figure 4.7 Representative chromatogram of zero standard
GBP
LEV
Figure 4.8 Representative chromatogram of LLOQ
S.K.P.C.P.E.R., Kherva
51
M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
GBP
LEV
Figure 4.9 Representative chromatogram of ULOQ
GBP
LEV
Figure 4.10 Representative chromatogram of LQC
GBP
LEV
Figure 4.11 Representative chromatogram of MQC
GBP
LEV
Figure 4.12 Representative chromatogram of HQC
S.K.P.C.P.E.R., Kherva
52
M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
4.3.2 Specificity and selectivity:
¾ Four different lots of heparinised plasma, one lot of lipemic plasma and one lot of
heamolysed plasma were chromatographed and no area response observed at the RT of
GBP and no area response at the RT of IS (Table No. 4.11).
TABLE NO: 4.11
SPECIFICITY AND SELECTIVITY OF BLANK HUMAN PLASMA FOR GBP AND LEV
Sr.
Sample Name
No.
1
BLANK PLASMA(B225-08)
2
LLOQ(B225-08)
3
BLANK PLASMA(B233-08)
4
LLOQ(B233-08)
5
BLANK PLASMA(B235-08)
6
LLOQ(B235-08)
7
BLANK PLASMA(B257-08)
8
LLOQ(B257-08)
9
BLANK PLASMA(C001-08)
10 LLOQ(C001-08)
11 BLANK PLASMA(D001-08)
12 LLOQ(D001-08)
NA: Not Applicable, NF: Not Found
Area
GBP
NF
161694
NF
156619
NF
168187
NF
155397
NF
161691
NF
155965
% of area
LEV
NF
4789031
NF
4865603
NF
4516163
NF
4764312
NF
4727598
NF
4653627
GBP
0.0
NA
0.0
NA
0.0
NA
0.0
NA
0.0
NA
0.0
NA
LEV
0.0
NA
0.0
NA
0.0
NA
0.0
NA
0.0
NA
0.0
NA
4.3.3 Sensitivity:
¾ The LLOQ was 0.05 µg/ml for GBP. The % CV of GBP at LLOQ was found to be 9.60.
The % nominal concentration for LLOQ samples of GBP was 96.33 (Table No.4.12).
TABLE NO: 4.12
SENSITIVITY (LLOQ OF GBP)
Sr.
No.
1
2
3
4
5
6
n
Mean
SD
%CV
Sample Name
LLOQ
LLOQ
LLOQ
LLOQ
LLOQ
LLOQ
Conc.
(µg/ml)
0.05
0.05
0.05
0.05
0.05
0.05
Area
GBP
196834
152127
195501
187094
200096
218511
LEV
5711167
5403757
5744727
5648930
5738419
5801135
Area Ratio
0.034
0.028
0.034
0.033
0.035
0.038
Calculated
Conc. (µg/ml)
0.049
0.040
0.049
0.047
0.050
0.054
6
0.048
0.005
9.60
% Nominal conc.
98.00
80.00
98.00
94.00
100.0
108.0
96.33
4.3.4 Linearity:
¾ The Linearity of the method was determined by a weighted least square linear regression
analysis of standard plots associated with a nine-point standard calibration curve. Best-fit
calibration curves of peak area ratio versus concentration were drawn. The calibration
curves were linear from 0.05 µg/ml to 6.00 µg/ml with correlation coefficient of r2 ≥
0.9976 for GBP (Table No. 4.13 and 4.14, Figure 4.13).
S.K.P.C.P.E.R., Kherva
53
M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
TABLE NO: 4.13
SUMMARY OF CALIBRATION CURVE PARAMETERS OF GBP
Sr. No.
SLOPE
INTERCEPT
r
r2
1
2
3
4
5
0.7104
0.7685
0.6659
0.6993
0.7235
-0.0004
0.0020
0.0033
0.0029
0.0029
0.9995
0.9997
0.9997
0.9988
0.9988
0.9991
0.9994
0.9994
0.9976
0.9976
TABLE NO: 4.14
BACK CALCULATED CONCENTRATION OF CALIBRATION STANDARDS FROM
CALIBRATION CURVE OF GBP
LINEARITY
Sr.
No.
1
% Nominal
Deviation
2
% Nominal
Deviation
3
% Nominal
Deviation
4
% Nominal
Deviation
5
% Nominal
Deviation
Mean
SD
%CV
% Nominal conc.
CS-1
0.05
0.049
-2.00
0.050
0.00
0.050
0.00
0.051
2.00
0.051
2.00
0.050
0.001
2.00
100.0
CS-2
0.10
0.104
4.00
0.101
1.00
0.100
0.00
0.095
-5.00
0.096
-4.00
0.099
0.003
3.03
99.00
CS-3
0.30
0.293
-2.33
0.294
-2.00
0.293
-2.33
0.281
-6.33
0.283
-5.67
0.289
0.006
2.08
96.33
Conc. (µg/ml)
CS-4
CS-5 CS-6
0.60
1.20
2.40
0.578 1.205 2.331
-3.67
0.42 -2.88
0.609 1.153 2.392
1.50
-3.92 -0.33
0.592 1.189 2.378
-1.33
-0.92 -0.92
0.640 1.177 2.349
6.67
-1.92 -2.12
0.654 1.190 2.373
9.00
-0.83 -1.12
0.615 1.183 2.365
0.029 0.017 0.022
4.72
1.44
0.93
102.5 98.58 98.54
CS-7
3.60
3.715
3.19
3.708
3.00
3.750
4.17
3.762
4.50
3.707
2.97
3.728
0.023
0.62
103.5
CS-8
4.80
4.905
2.19
4.802
0.04
4.747
-1.10
4.775
-0.52
4.777
-0.48
4.801
0.055
1.15
100.0
CS-9
6.00
6.030
0.50
6.075
1.25
6.100
1.67
6.093
1.55
5.864
-2.27
6.032
0.088
1.46
100.5
Figure 4.13 Representative calibration curve of GBP
S.K.P.C.P.E.R., Kherva
54
M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
4.3.5 Accuracy:
Within-batch or intra-batch accuracy:
¾ The % nominal concentration for LLOQ, LQC, MQC and HQC samples of GBP were
found 96.33, 105.0, 104.9 and103.9, respectively (Table No. 4.15).
Between -batch or inter-batch accuracy:
¾ The % nominal concentration for LLOQ, LQC, MQC and HQC samples of GBP were
found 99.00, 101.7, 101.4 and 101.6, respectively (Table No. 4.16).
4.3.6 Precision:
Within-batch or intra-batch precision:
¾ The % CV for LLOQ, LQC, MQC and HQC samples of GBP were 9.60, 4.54, 2.11 and
2.14, respectively (Table No. 4.15).
Between -batch or inter-batch precision:
¾ The % CV for LLOQ, LQC, MQC and HQC samples of GBP were 7.90, 3.38, 4.88 and
2.73, respectively. (Table No. 4.16).
TABLE NO: 4.15
WITHIN-BATCH OR INTRA-BATCH ACCURACY AND PRECISION OF GBP
Sr.
No.
1
2
3
4
5
6
n
Mean
SD
%CV
Sr.
No.
1
2
3
4
5
6
n
Mean
SD
%CV
LLOQ (0.05 µg/ml)
LQC (0.15 µg/ml)
Calculated conc.
% Nominal conc.
0.049
98.00
0.040
80.00
0.049
98.00
0.047
94.00
0.050
100.0
0.054
108.0
6
0.048
96.33
0.005
9.60
MQC (2.00 µg/ml)
Calculated conc.
% Nominal conc.
2.103
105.1
2.172
108.6
2.092
104.6
2.094
104.7
2.088
104.4
2.034
101.7
6
2.097
104.9
0.044
2.11
S.K.P.C.P.E.R., Kherva
Calculated conc.
% Nominal conc.
0.163
108.7
0.166
110.7
0.154
102.7
0.162
108.0
0.148
98.67
0.152
101.3
6
0.158
105.0
0.007
4.54
HQC (4.50 µg/ml)
Calculated conc.
% Nominal conc.
4.726
105.0
4.803
106.7
4.619
102.6
4.734
105.2
4.636
103.0
4.523
100.5
6
4.674
103.9
0.100
2.14
55
M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
TABLE NO: 4.16
BETWEEN-BATCH OR INTER-BATCH ACCURACY AND PRECISION OF GBP
Sr.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Day
Day 1
Day 2
Day 3
Day 4
Day 5
n
Mean
SD
%CV
LLOQ (0.05 µg/ml)
Calculated conc.
0.049
0.040
0.049
0.047
0.050
0.054
0.054
0.049
0.053
0.049
0.046
0.051
0.063
0.052
0.049
0.051
0.044
0.050
0.049
0.049
0.049
0.047
0.048
0.045
0.052
0.050
0.052
0.046
0.048
0.050
30
0.050
0.004
7.90
S.K.P.C.P.E.R., Kherva
LQC (0.15 µg/ml)
% Nominal conc.
98.00
80.00
98.00
94.00
100.0
108.0
108.0
98.00
106.0
98.00
92.00
102.0
126.0
104.0
98.00
102.0
88.00
100.0
98.00
98.00
98.00
94.00
96.00
90.00
104.0
100.0
104.0
92.00
96.00
100.0
99.00
56
Calculated conc.
0.163
0.166
0.154
0.162
0.148
0.152
0.152
0.148
0.158
0.154
0.158
0.156
0.146
0.151
0.155
0.154
0.149
0.156
0.147
0.149
0.149
0.145
0.151
0.146
0.147
0.150
0.153
0.153
0.153
0.152
30
0.153
0.005
3.38
% Nominal conc.
108.7
110.7
102.7
108.0
98.67
101.3
101.3
98.67
105.3
102.7
105.3
104.0
97.33
100.7
103.3
102.7
99.33
104.0
98.00
99.33
99.33
96.67
100.7
97.33
98.00
100.0
102.0
102.0
102.0
101.3
101.7
M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
Table No. 4.16 continue
Sr.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Day
Day 1
Day 2
Day 3
Day 4
Day 5
MQC (2.00 µg/ml)
Calculated conc. % Nominal conc.
2.103
105.1
2.172
108.6
2.092
104.6
2.094
104.7
2.088
104.4
2.034
101.7
2.108
105.4
2.113
105.7
2.075
103.8
1.806
90.30
2.023
101.2
1.819
90.91
2.091
104.6
2.049
102.5
1.806
90.30
1.995
99.75
1.788
89.40
2.062
103.1
2.083
104.2
2.089
104.5
2.095
104.8
2.072
103.6
1.996
99.8
2.038
101.9
2.080
104.0
2.051
102.6
2.015
100.8
2.038
101.9
1.953
97.65
2.018
100.9
n
Mean
SD
%CV
2.028
0.099
4.88
101.4
HQC (4.50 µg/ml)
Calculated conc.
% Nominal conc.
4.726
105.0
4.803
106.7
4.619
102.6
4.734
105.2
4.636
103.0
4.523
100.5
4.597
102.2
4.579
101.7
4.751
105.6
4.632
102.9
4.664
103.6
4.596
102.1
4.536
100.8
4.583
101.8
4.610
102.4
4.567
101.5
4.627
102.8
4.476
99.47
4.477
99.49
4.535
100.8
4.458
99.07
4.587
101.9
4.745
105.4
4.655
103.4
4.407
97.93
4.418
98.18
4.275
95.00
4.346
96.58
4.513
100.3
4.415
98.11
30
4.570
101.6
0.125
2.73
4.3.7 Recovery:
¾ The percentage recovery of GBP was determined by comparing the mean peak area of
GBP in extracted LQC, MQC and HQC samples with freshly prepared Unextracted
LQC, MQC and HQC samples, respectively.
¾ The mean % recovery for LQC, MQC and HQC samples of GBP were 75.16, 74.24 and
74.48, respectively. (Table No. 4.17).
¾ For LEV (IS), mean peak area of eighteen extracted samples was compared to the mean
area peak of eighteen Unextracted LEV (IS) samples. The mean percentage recovery for
LEV (IS) was 104.5 (Table No.4.19).
¾ The % CV Unextracted for LQC, MQC and HQC samples of GBP were 1.37, 0.84 and
1.38, respectively. (Table No. 4.17).
S.K.P.C.P.E.R., Kherva
57
M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
¾ The % CV Extracted for LQC, MQC and HQC samples of GBP were 1.69, 2.20 and
1.95, respectively. (Table No. 4.17).
¾ The % CV of recovery across QC level for GBP was 0.64 (Table No. 4.18).
¾ The % CV within LEV IS concentration of Unextracted and Extracted sample area
across QC level was 1.92 and 2.65, respectively (Table No.4.19).
TABLE NO: 4.17
RECOVERY OF GBP
Sr. No.
1
2
3
4
5
6
n
Mean
SD
%CV
%Recovery
Sr. No.
1
2
3
4
5
6
n
Mean
SD
%CV
%Recovery
Sr. No.
1
2
3
4
5
6
n
Mean
SD
%CV
%Recovery
TABLE NO: 4.18
VARIABILITY ACROSS
QC LEVELS OF GBP
QC LEVELS
%Recovery
LQC
75.16
MQC
74.24
HQC
74.48
Mean
74.63
SD
0.476
%CV
0.64
LQC (0.15 µg/ml)
Area of GBP
Unextracted
Extracted
875947
684127
899832
667856
904803
676503
889850
662878
877122
659813
880627
653359
6
6
888030.17
667422.67
12196.07
11296.22
1.37
1.69
75.16
MQC (2.00 µg/ml)
Area of GBP
Unextracted
Extracted
11855128
8942349
12044387
9307512
12039162
8913235
12160617
8900607
12026886
8741100
12092220
8811499
6
6
12036400.00
8936050.33
101555.12
196631.07
0.84
2.20
74.24
HQC (4.50 µg/ml)
Area of GBP
Unextracted
Extracted
25943141
18948482
26361578
19188069
26357843
19070589
25786432
20011910
25622017
19249242
25534676
19421973
6
6
25934281.17
19315044.17
358083.31
377262.38
1.38
1.95
74.48
S.K.P.C.P.E.R., Kherva
58
M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
TABLE NO: 4.19
RECOVERY OF INTERNAL STANDARD (LEV) (IS AMOUNT 1.50 µg/ml)
Area of LEV
Sr. No.
Unextracted
5878075
5994692
6091646
6089236
5936550
6064166
5728743
5827817
5797186
5811422
5929188
5758359
5770043
5955326
5935133
5850428
5829306
5970797
18
5901006
113183
1.92
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
n
Mean
SD
%CV
%Recovery
Extracted
6484337
6238048
6329058
6365513
6084339
6196985
6125954
6358164
6069732
6130189
6246940
6169999
6046114
6043754
6111543
6231866
5795219
5960257
18
6166001
163172
2.65
104.5
4.3.8 Dilution integrity:
¾
Dilution integrity experiment was carried out at six replicate of two times diluted
2xULOQ (½ dilution) and four times diluted 2xULOQ (¼ dilution) samples. The
samples were prepared and its concentrations were calculated against the previously bulk
spiked calibration standards. The % CV for ½ dilutions and ¼ dilutions samples for GBP
were 3.76 and 2.88, respectively (Table No. 4.20). The mean % nominal concentration
for ½ dilutions and ¼ dilutions samples for GBP were 110.1 and 112.8, respectively
(Table No. 4.20)
TABLE NO: 4.20
DILUTION INTEGRITY OF GBP
Conc.
(µg/ml)
12.000
12.000
12.000
12.000
12.000
12.000
Dilution
Area Ratio
1/4(2ULOQ)
1/4(2ULOQ)
1/4(2ULOQ)
1/4(2ULOQ)
1/4(2ULOQ)
1/4(2ULOQ)
2.314
2.366
2.345
2.471
2.294
2.429
S.K.P.C.P.E.R., Kherva
Calculated
conc. (µg/ml)
13.22
13.52
13.40
14.12
13.10
13.87
59
% Nominal
Conc.
110.1
112.7
111.6
117.6
109.2
115.6
Mean
SD
%CV
112.8
3.25
2.88
M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
Table No. 4.20 continue
12.000
1/2(2ULOQ)
4.788
13.68
114.0
12.000
1/2(2ULOQ)
4.753
13.58
113.2
12.000
1/2(2ULOQ)
4.790
13.69
114.1
110.1
4.14
12.000
1/2(2ULOQ)
4.398
12.57
104.7
12.000
1/2(2ULOQ)
4.478
12.80
106.6
12.000
1/2(2ULOQ)
4.539
12.97
108.1
Underlined Values were outside the acceptance criteria of 85 - 115 % of their Nominal concentration.
3.76
4.3.9 Matrix effect:
¾ In order to ensure the effect of matrix through out the application of the method, plasma
blanks obtained from six different lots (04 normal Heparinised, 01 lipemic and 01
Haemolysed) were spiked with GBP and LEV at LQC and HQC level. Three quality
control samples at each level along with the set of calibration standards were analyzed
and the % nominal concentration of the samples analyzed was represented in Table No.
4.21 for GBP.
TABLE NO: 4.21
MATRIX EFFECT FOR GBP
Sample ID
LQC (B225-08)
LQC (B225-08)
LQC (B225-08)
LQC (B233-08)
LQC (B233-08)
LQC (B233-08)
LQC (B235-08)
LQC (B235-08)
LQC (B235-08)
LQC (B257-08)
LQC (B257-08)
LQC (B257-08)
LQC (C001-08)
LQC (C001-08)
LQC (C001-08)
LQC (D001-08)
LQC (D001-08)
LQC (D001-08)
HQC (B225-08)
HQC (B225-08)
HQC (B225-08)
HQC (B233-08)
HQC (B233-08)
HQC (B233-08)
HQC (B235-08)
HQC (B235-08)
HQC (B235-08)
Area
GBP
650607
545422
640750
610622
676296
597559
640966
626810
649784
635371
605975
619823
613001
615736
626823
600304
599510
620307
17133637
17359078
17579091
17464700
17903117
17661794
10326750
17417496
18019725
S.K.P.C.P.E.R., Kherva
Area Ratio
LEV
5282209
5387477
5168602
5748937
5442613
5371499
5194270
5188691
5296223
5269874
5167406
5085894
5082831
5268006
5246560
5033638
5132926
5208218
5141557
5108035
5219371
5104934
5278421
5241003
5233174
5151214
5319023
0.123
0.101
0.124
0.106
0.124
0.111
0.123
0.121
0.123
0.121
0.117
0.122
0.121
0.117
0.119
0.119
0.117
0.119
3.332
3.398
3.368
3.421
3.392
3.370
1.973
3.381
3.388
60
Calculated
Conc. (µg/ml)
0.158
0.129
0.159
0.136
0.159
0.142
0.158
0.155
0.157
0.154
0.150
0.156
0.154
0.149
0.153
0.153
0.149
0.152
4.333
4.419
4.380
4.449
4.410
4.382
2.565
4.397
4.405
% Nominal
Conc.
105.3
86.00
106.0
90.67
106.0
94.67
105.3
103.3
104.7
102.7
100.0
104.0
102.7
99.32
102.0
102.0
99.33
101.3
96.29
98.20
97.33
98.87
98.00
97.38
57.00
97.71
97.89
M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
Table No. 4.21 continue
HQC (B257-08)
17289568
5028727
3.438
4.471
99.36
HQC (B257-08)
18163954
5325605
3.411
4.435
98.56
HQC (B257-08)
17342054
5405325
3.208
4.172
92.71
HQC (C001-08)
13654431
5373683
2.541
3.303
73.40
HQC (C001-08)
18340751
5109316
3.590
4.668
103.7
HQC (C001-08)
18048440
5248333
3.439
4.472
99.38
HQC (D001-08)
17843374
5101723
3.498
4.548
101.1
HQC (D001-08)
14638922
5142617
2.847
3.701
82.24
HQC (D001-08)
17966219
5142285
3.494
4.543
100.9
Underlined Values were outside the acceptance criteria of 85 – 115 % of their Nominal concentration.
4.3.10
Stability:
4.3.10.1 Stock solution stability.
¾ Stock solution stability was determined by comparing the peak areas of freshly prepared
samples (comparison samples) with stability samples.
Stock solution stability of GBP (93 hrs at room temp. for main stock):
¾ GBP main stock solution was found to be stable at room temperature for 93 hrs with %
mean change of 4.10 (Table No.4.22).
Stock solution stability of LEV (93 hrs at room temp. for main stock):
¾ LEV (IS) main stock solution was found to be stable at room temperature for 93 hrs with
% mean change of -1.29 (Table No. 4.23).
Stock solution stability of GBP (93 hrs at 2-8˚C for main stock):
¾ GBP main stock solution was found to be stable at 2-8˚C for 93 hrs with % mean change
of 5.38 (Table No.4.24).
Stock solution stability of LEV (93 hrs at 2-8˚C for main stock):
¾ LEV Main stock solution was found to be stable at 2-8˚C for 93 hrs with % mean change
of -2.35 (Table No.4.25).
TABLE NO: 4.22
MAIN STOCK SOLUTION STABILITY OF GBP (93 hrs at room temp.)
GBP(12.00 µg/ml)
Sr. No.
1
2
3
4
5
6
n
Mean
SD
% CV
% Mean Change
S.K.P.C.P.E.R., Kherva
Comp. sample area
Stability sample area
37965530
37885805
38242777
38073735
37874277
38241913
6
38047340
167026
0.44
39658803
39813714
39070867
39884983
39330670
39890216
6
39608209
336657
0.85
4.10
61
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LC-MS/MS Method
TABLE NO: 4.23
MAIN STOCK SOLUTION STABILITY OF LEV (93 hrs at room temp.)
LEV(1.50 µg/ml)
Sr. No.
1
2
3
4
5
6
n
Mean
SD
% CV
% Mean Change
Comp. sample area
Stability sample area
6065366
6062324
6155877
5971748
5962427
6044741
6
6043747
70970
1.17
6036516
6008907
5863199
5993892
5891232
6001840
6
5965931
70759
1.19
-1.29
TABLE NO: 4.24
MAIN STOCK SOLUTION STABILITY OF GBP (93 hrs at 2-8˚C)
GBP(12.00 µg/ml)
Sr. No.
1
2
3
4
5
6
n
Mean
SD
% CV
% Mean Change
Comp. sample area
Stability sample area
37965530
37885805
38242777
38073735
37874277
38241913
6
38047340
167026
0.44
39114837
40092928
40095247
40644570
40215347
40394861
6
40092965
522901
1.30
5.38
TABLE NO: 4.25
MAIN STOCK SOLUTION STABILITY OF LEV (93 hrs at 2-8˚C)
LEV(1.50 µg/ml)
Sr. No.
1
2
3
4
5
6
n
Mean
SD
% CV
% Mean Change
Comp. sample area
Stability sample area
6065366
6062324
6155877
5971748
5962427
6044741
6
6043747
70970
1.17
5829432
5914353
5875641
5931620
5958053
5901846
6
5901824
45017
0.76
-2.35
4.3.10.2 Bench top stability of GBP (at room temp. for 20 hrs):
¾ LQC and HQC samples were spiked in human plasma and were kept at room
temperature for 20 hrs and were processed and analyzed along with previously bulk
S.K.P.C.P.E.R., Kherva
62
M .Pharm. Thesis
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LC-MS/MS Method
spiked calibration standards, LQC and HQC samples. Concentrations were calculated to
determine % mean change during stability period.
¾ GBP was found to be stable in LQC and HQC samples for 20 Hrs at room temperature
with % mean change of 4.17 and 3.71, respectively (Table No. 4.26).
TABLE NO: 4.26
BENCH TOP STABILITY OF GBP (20 hrs at room temp.)
LQC (0.15 µg/ml)
Comparison samples
Sr. No.
1
2
3
4
5
6
n
Mean
% Mean Change
Sample conc.
% Nominal Conc.
Sample conc.
% Nominal Conc.
0.147
0.149
0.149
0.145
0.151
0.146
6
0.148
98.00
99.33
99.33
96.67
100.7
97.33
0.160
0.151
0.156
0.156
0.157
0.144
6
0.154
106.7
100.7
104.0
104.0
104.7
96.00
4.17
HQC (4.50 µg/ml)
Comparison samples
Sr. No.
1
2
3
4
5
6
n
Mean
% Mean Change
Stability samples
Stability samples
Sample conc.
% Nominal Conc.
Sample conc.
% Nominal Conc.
4.477
4.535
4.458
4.587
4.745
4.655
6
4.576
99.49
100.8
99.07
101.9
105.4
103.4
4.550
4.639
4.724
4.896
4.935
4.733
6
4.746
101.1
103.1
104.9
108.8
109.7
105.2
3.71
4.3.10.3 Process stability of GBP (at 5°C in auto sampler for 76 hrs):
¾ LQC and HQC samples were prepared and processed. These processed samples were
kept in auto sampler for 76 hrs at 5°C, these samples were analyzed along with
previously bulk spiked calibration standards, LQC and HQC samples concentrations
were calculated to determine % mean change during stability period.
¾ GBP was found to be stable in LQC and HQC samples for 76 hrs at 5°C in auto sampler
with % mean change of 0.77 and 5.44, respectively (Table No. 4.27).
S.K.P.C.P.E.R., Kherva
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LC-MS/MS Method
TABLE NO: 4.27
PROCESS STABILITY OF GBP (at 5°C in auto sampler for 76 hrs)
LQC (0.15 µg/ml)
Comparison samples
Sr. No.
1
2
3
4
5
6
n
Mean
% Mean Change
Stability samples
Sample conc.
% Nominal Conc.
Sample conc.
% Nominal Conc.
0.147
0.150
0.153
0.153
0.153
0.152
6
0.151
98.00
100.0
102.0
102.0
102.0
101.3
0.149
0.153
0.154
0.157
0.152
0.150
6
0.153
99.33
102.0
102.7
104.7
101.3
100.0
0.77
HQC (4.50 µg/ml)
Comparison samples
Sr. No.
1
2
3
4
5
6
n
Mean
% Mean Change
Stability samples
Sample conc.
% Nominal Conc.
Sample conc.
% Nominal Conc.
4.407
4.418
4.275
4.346
4.513
4.415
6
4.396
97.93
98.18
95.00
96.58
100.3
98.11
4.625
4.602
4.720
4.769
4.546
4.548
6
4.635
102.8
102.3
104.9
105.9
101.2
101.1
5.44
4.3.10.4 Freeze and thaw stability of GBP (after 4th cycle at -20°C):
¾ Samples were prepared at LQC and HQC levels, aliquoted and frozen at -20°C. Six
samples from each concentration were subjected to three freeze and thaw cycles
(stability samples). These samples were processed after 4th cycle and analyzed along
with previously bulk spiked calibration standards, LQC and HQC samples (comparison
samples). Concentrations were calculated to determine % mean change after 4th cycle.
¾ GBP was found to be stable in LQC and HQC samples after 4th cycle at -20°C with %
mean change of 0.00 and -14.36, respectively (Table No. 4.28).
TABLE NO: 4.28
FREEZE AND THAW STABILITY OF GBP (after 4th cycle at -20°C)
LQC (0.15 µg/ml)
Sr. No.
1
2
3
4
5
6
n
Mean
% Mean Change
Comparison samples
Stability samples
Sample conc.
% Nominal Conc.
Sample conc.
% Nominal Conc.
0.146
0.151
0.155
0.154
0.149
0.156
6
0.152
97.33
100.7
103.3
102.7
99.33
104.0
0.158
0.150
0.150
0.152
0.148
0.151
6
0.152
105.3
100.0
100.0
101.3
98.67
100.7
S.K.P.C.P.E.R., Kherva
0.00
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LC-MS/MS Method
Table No. 4.28 continue
HQC (4.50 µg/ml)
Comparison samples
Sr. No.
Sample conc.
Stability samples
% Nominal Conc.
Sample conc.
% Nominal Conc.
1
4.536
100.8
2.463
54.73
2
4.583
101.8
4.132
91.82
3
4.610
102.4
4.197
93.27
4
4.567
101.5
4.361
96.91
5
4.627
102.8
4.283
95.18
6
4.476
99.47
4.029
89.53
n
6
6
Mean
4.567
3.911
% Mean Change
-14.36
Underlined Values were outside the acceptance criteria of 85 – 115 % of their Nominal concentration.
4.3.10.5 Freeze and thaw stability of GBP (after 4th cycle at -70°C):
¾ Samples were prepared at LQC and HQC levels, aliquoted and frozen at -70°C. Six
samples from each concentration were subjected to three freeze and thaw cycles
(stability samples). These samples were processed after 4th cycle and analyzed along with
previously bulk spiked calibration standards, LQC and HQC samples (comparison
samples). Concentrations were calculated to determine % mean change after 4th cycle.
GBP was found to be stable in LQC and HQC samples after 4th cycle at -70°C with %
mean change of -5.71 and -6.07, respectively (Table No.4.29).
TABLE NO: 4.29
FREEZE AND THAW STABILITY OF GBP (after 4th cycle at -70°C)
LQC (0.15 µg/ml)
Sr.
No.
1
2
3
4
5
6
n
Mean
% Mean Change
Sr.
No.
1
2
3
4
5
6
n
Mean
% Mean Change
Comparison samples
Stability samples
Sample conc.
% Nominal Conc.
Sample conc.
% Nominal Conc.
0.146
0.151
0.155
0.154
0.149
0.156
6
0.152
103.3
90.00
100.0
88.00
101.3
90.00
0.155
0.135
0.150
0.132
0.152
0.135
6
0.143
103.3
90.00
100.0
88.00
101.3
90.00
-5.71
HQC (4.50 µg/ml)
Comparison samples
Stability samples
Sample conc.
% Nominal Conc.
Sample conc.
% Nominal Conc.
4.536
4.583
4.610
4.567
4.627
4.476
6
4.567
100.8
101.8
102.4
101.5
102.8
99.47
4.259
4.251
4.259
4.396
4.321
4.249
6
4.289
94.64
94.47
94.64
97.69
96.02
94.42
S.K.P.C.P.E.R., Kherva
-6.07
65
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LC-MS/MS Method
4.3.11 Carry over check for GBP:
¾ No area response was observed or area response less than 20% and 5% of mean response
of an extracted LLOQ standard was observed at the RT of analyte and internal standard
(IS), respectively, in respective blank matrix and or blank solution (Table No. 4.30).
TABLE NO: 4.30
CARRY OVER CHECK FOR GBP
Area
Sr.
No.
Sample Name
1
2
3
4
5
6
7
EXTRACTED LLOQ
EXTRACTED LLOQ
BLANK PLASMA
EXTRACTED ULOQ
BLANK PLASMA
EXTRACTED ULOQ
BLANK PLASMA
GBP
LEV
246416
244374
NF
27080421
NF
27496928
NF
6634503
6736462
NF
6731495
NF
6883295
NF
Area Ratio
0.037
0.036
NA
4.023
NA
3.995
NA
Calculated
Conc.
(µg/ml)
0.051
0.049
NA
6.036
NA
5.994
NA
4.4 REFERENCES:
1. Chapman JR. Practical Organic Mass Spectrometry. 2nd ed. Wiley, London; 1993.
2. McLafferty FW, Turecek, F. Interpretation of Mass Spectra. 4th ed. University science
Books, Mill Valley, C.A.; 1993.
3. Lambert JB, Shurvell HF, Lighter D, Cooks RG. Introduction to Organic Spectroscopy.
MacMillan Publishing, New York; 1987: p.313.
4. Hoffmann E, Charette J, Stroobant V. Mass Spectrometry: Principles and Applications.
Wiley, London; 1996.
5. Nibbering NMM. J Chromatogr 1982 ; 251, 93.
6. Niessen WMA, Nibbering M.
Liquid Chromatography-Mass Spectrometry. 2nd ed.
Marcell Decker Inc, New York; 1999: p. 43-51.
7. March RE. J Mass Spectrom 1995; 30:1519.
8. Schlag EW. Time-of –Flight Mass Spectrometry and its application. Elsevier,
Amsterdam; 1994.
9. Brunnee C. Int J Mass Spectrom Ion Proc 1987; 76: 125.
10. Yang L, Amad M, Winnik WM, Schoen AE, Schweingruber H, Mylchreest I, Rudewicz
PJ. Rapid Commun Mass Spectrom 2002; 16: 2060.
11. McLafferty FW. Tandem Mass Spectrometry. Wiley, New York; 1983.
12. Talroze VL, Skurat VE, Gorodetskii IG, Zolotai NB. Russ J Phys Chem 1972; 46: 456.
13. Talroze VL, Skurat VE, Karpov GV. Russ J Phys Chem 1969; 43: 241.
S.K.P.C.P.E.R., Kherva
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M .Pharm. Thesis
Dushyant Patel
LC-MS/MS Method
14. Talroze VL, Gorodetskii IG, Zolotoy NB, Skurat VE, Karpov GV, Maslennikova V.
Adv Mass Spectrom 1978; 7: 858.
15. Apffel JA, Brinkman UA, Frei RW, Evers EIAM. Anal Chem 1983; 55: 2280.
16. McFadden WH, Schwartz HL, Evans S. J Chromatogr 1976; 122: 389.
17. Alcock NJ, Eckers C, Games DE, Games MPL, Lant MS, McDowall MA, Rossiter M,
Smith RW, Westwood SA, Wong H. J Chromatogr 1982; 251:165.
18. Arpino PJ. Mass Spectrom Rev 1989;8:35.
19. Meleara A. Mass Spectrom 1980;8:1597.
20. Niessen WMA. Chromatographia 1986; 21:277.
21. Niessen WMA. Chromatographia 1986; 21:342.
22. Blakley CR, Vestal ML. Anal Chem 1983; 55: 750.
23. Caprioli RM, Fan T, Cottrell JS. Anal Chem 1986; 58: 2949.
24. Caprioli RM. Continuous flow fast- atom bombardment mass spectrometry. Wiley, New
York; 1990.
25. Caprioli RM, Suter MJF. Int J Mass Spectrom Ion processes 1992; 118: 449.
26. Willoughby RC, Browner RF. Anal Chem 1984; 56: 2626.
27. Willoughby RC, Ph.D. Thesis: Studies with an aerosol generating interface for LC-MS.
Georgia Institute of Technology, Atlanta; 1983.
28. Niessen WMA, Nibbering M. Liquid Chromatography-Mass Spectrometry. 2nd ed.
Marcell Decker Inc., New York; 1999: p. 285-345.
29. Zeleny J. Phys Rev 1917; 10:1.
30. Horning EC, Horning MG, Carroll DI, Dzidic I, Stillwell RN. Anal Chem 1973; 45: 936.
S.K.P.C.P.E.R., Kherva
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Extractive Spectrophotometric Method
CHAPTER: 5 DEVELOPMENT AND VALIDATION OF EXTRACTIVE
SPECTROPHOTOMETRIC METHODS FOR ESTIMATION OF
GABAPENTIN IN PHARMACEUTICAL DOSAGE FORMS
5.1 INTRODUCTION TO EXTRACTIVE SPECTROSCOPY1:
¾ The addition of an amine in its ionized form to an ionized acidic dye, e.g. methyl orange
or bromocresol purple, yields a salt (ion-pair) that may be extracted into an organic
solvent such as chloroform or dichloromethane. The indicator dye is added in excess and
the pH of the aqueous solution is adjusted if necessary to a value where both the amine
and dye are in the ionized forms. The ion pair is separated from the excess indicator by
extraction into the organic solvent, and the absorbance is measured at the λmax of the
indicator in the solvent.
¾ Usually, the most intensely absorbing form of the indicator is measured, with the
addition of acidified or basified ethanol. Alternatively, the absorbance of the indicator
may be measured in aqueous solution after back extraction from the organic solvent.
Because of higher molar absorptivity (ion-pairs) obtained by acid-dye method, this
method is very sensitive.
¾ The correct choice of pH may permit the selective assay of the mixture of an amine and a
quaternary ammonium salt.
5.2 EXPERIMENTAL WORK:
5.2.1 APPARATUS:
¾ A double beam UV-Visible Spectrophotometer (Shimadzu model UV-1700, Japan),
attached to a computer software UV probe 2.10, with a spectral width of 2 nm,
wavelength accuracy of 0.5 nm and pair of 1 cm matched quartz cells.
¾ Analytical balance (CP224S, Sartorius, Gottingen, Germany).
¾ Ultrasonic cleaner (Frontline FS 4, Mumbai, India).
¾ Corning volumetric flasks, pipettes of borosilicate glass were used in the study.
5.2.2 REAGENTS AND MATERIALS:
¾ GBP bulk powder was kindly gifted by Torrent research centre, Gujarat (India).
¾ The commercial fixed dose products containing 300 mg GBP were procured from the
local pharmacy.
¾ AR grade chloroform (S.D. Fine Chemical Ltd., Mumbai, India).
S.K.P.C.P.E.R., Kherva
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Extractive Spectrophotometric Method
¾ AR grade Bromocresol Green reagent and Bromothymol Blue reagent ((Finar Chemicals
Ltd., India)
¾ Whatman filter paper no. 41 (Millipore, USA)
5.2.3 PREPARATION OF REAGENTS:
a) Preparation of diluent:
¾ Distilled water was used as diluent.
b) Preparation of buffer solutions:
•
Phosphate buffer solution (pH 2.0):
¾ Potassium dihydrogen phosphate (13.6 mg) was accurately weighed and dissolved in
water (100 ml). pH of solution was adjusted to 2.0 with HCl.
•
Phosphate buffer solution (pH 2.5):
¾ Potassium dihydrogen phosphate (10 g) was accurately weighed and dissolved in water
(100 ml). pH of solution was adjusted to 2.5 with HCl.
•
Phosphate buffer solution (pH 3.0):
¾ Potassium dihydrogen phosphate (13.6 mg) was accurately weighed and dissolved in
water (100 ml). pH of solution was adjusted to 3.0 with HCl.
•
Phosphate buffer solution (pH 3.5):
¾ Potassium dihydrogen phosphate (13.6 mg) was accurately weighed and dissolved in
water (100 ml). pH of solution was adjusted to 3.5 with HCl.
•
Phosphate buffer solution (pH 4.0):
¾ Disodium hydrogen phosphate (504 mg) and Potassium dihydrogen phosphate (301 mg)
were accurately weighed and dissolved in water (100 ml). pH of solution was adjusted to
4.0 with glacial acetic acid.
•
Phosphate buffer solution (pH 4.5):
¾ Disodium hydrogen phosphate (504 mg) and Potassium dihydrogen phosphate (301 mg)
were accurately weighed and dissolved in water (100 ml). pH of solution was adjusted to
4.6 with glacial acetic acid.
•
Phosphate buffer solution (pH 5.0):
¾ Potassium dihydrogen phosphate (4 g) was accurately weighed and dissolved in water
(100 ml). pH of solution was adjusted to 5.0 with 10 M KOH.
•
Phosphate buffer solution (pH 5.5):
¾ Potassium dihydrogen phosphate (4 g) was accurately weighed and dissolved in water
(100 ml). pH of solution was adjusted to 5.5 with 10 M KOH.
S.K.P.C.P.E.R., Kherva
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Extractive Spectrophotometric Method
c) Preparation of bromocresol green dye (0.05% w/v):
¾ Bromocresol Green reagent (50 mg) was accurately weighed and 0.1 N NaOH (0.72 ml)
was added. Ethanol (20 ml) was added and shaken well and then volume was made up to
100 ml with water.
d) Preparation of bromothymol blue dye (0.05% w/v):
¾ Bromothymol Blue reagent (50 mg) was accurately weighed and 0.02 M NaOH (4 ml)
was added. Ethanol (95 %) (20 ml) was added and shaken well and then volume was
made up to 100 ml with water.
e) Preparation of standard stock solutions:
¾ GBP (100 mg) was accurately weighed and transferred into 100 ml volumetric flask.
Distilled water (100 ml) was added to get GBP standard stock solution (1 mg/ml).
Working standard solution (200 µg/ml) was prepared by diluting GBP standard stock
solution (20 ml) to 100 ml with diluent.
5.2.4 DEVELOPMENT OF THE METHOD:
¾ In colorimetric method for optimization, different dyes like, bromocresol green,
bromothymol blue, methyl orange, thymol blue and phenolphthalein were prepared and
used. But among them only BCG and BTB gave color with drug, hence BCG (Method I)
and BTB (Method II) were used for development of the methods.
a) Optimization of conditions:
¾ Conditions under which reaction of GBP with dyes fulfill the essential requirements
were investigated. All conditions studied were optimized at room temperature (25±20C).
b) Selection of suitable pH buffer solution:
¾ Buffer solutions of different pH (2.0 to 5.5) were prepared. Working standard solution
(5 ml) was pipette out and added to separating funnels. BCG solution (in excess) and
buffer solutions (2 ml) of different pH were added to each. Shaken well and extracted
with 10 ml of chloroform. Later the extracts were taken into 10 ml volumetric flasks,
treated with anhydrous sodium sulphate and volume was made up with chloroform, and
then absorbances were measured at 416 nm. Same procedure was applied for the BTB
and then absorbances were measured at 421 nm. It was found that drug with BCG and
BTB gave maximum absorbance at pH 4.0 (Figure 5.1 A and B)
c) Optimization of volume of buffer:
¾ Volume of buffer was optimized by changing volume of buffer and other parameters
were kept constant. Buffer solution of pH 4.0 was used for optimization. Working
standard solution (5 ml) was transferred in seven separating funnels, different volume of
S.K.P.C.P.E.R., Kherva
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Extractive Spectrophotometric Method
buffer solution was added in the different separating funnels. BCG solution or BTB
solution was added in excess each. Shaken well and extracted with 10 ml of chloroform.
Later the extracts were taken into 10 ml volumetric flasks, treated with anhydrous sodium
sulphate and volume was made up with chloroform, and then absorbances were
measured at 416 nm and 421 nm for BCG and BTB, respectively. It was found that after
addition of 2 ml of buffer in BCG and 2.5 ml of buffer in BTB, absorbance became
constant. Hence 2 ml and 2.5 ml of buffer solution were optimized for BCG method and
BTB method, respectively (Figure 5.2 A and B).
0.8
0.7
0.7
0.6
0.6
0.5
Absorbance
A bsorbance
0.9
0.5
0.4
0.3
0.2
0.4
0.3
0.2
0.1
0.1
0
0
0
0
2
4
2
4
6
6
pH of buffer
pH of buffer
(A)
(B)
0.8
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.7
0.6
A b so r b a n c e
A b so rb an c e
Figure 5.1 Optimization of pH of Buffer for (A) GBP-BCG (B) GBP-BTB complex
0.5
0.4
0.3
0.2
0.1
0
0
1
2
3
4
0
ml Of Buffer
1
2
3
4
ml Of Buffer
(A)
(B)
Figure 5.2 Optimization of volume of Buffer for (A) GBP-BCG (B) GBP-BTB complex
d) Stability study of drug dye complexes:
¾ The stability of the drug dye complexes was determined individually for both the dyes
(BCG and BTB). Working standard solution (5 ml) was pipette out and added to a
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Extractive Spectrophotometric Method
separating funnels. Buffer solution (2 ml) and BCG solution (in excess) were added to
each. Shaken well and extracted with 10 ml of chloroform. Later the extracts were taken
into 10 ml volumetric flasks, treated with anhydrous sodium sulphate and made up the
volume with chloroform. The absorbances were measured periodically at an interval of
30, 60, 90, 120, 180, 240, 300 and 360 minutes at 416 nm. Same procedure was applied
for the BTB method using buffer solution (2.5 ml) and BTB solution (in excess), and
then absorbances were measured at 421 nm. Finally it was found that BCG-GBP
complex was stable at least for 6 hrs, whereas BTB-GBP complex was stable at least for
4 hrs.
5.2.5 METHOD VALIDATION:
a) Linearity:
Method I (BCG Method)
¾ From the working standard solution, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 ml were
transferred to a series of separating funnels and buffer solution (2 ml) was added to each
separating funnels, then BCG solution was added in excess and shaken well, and then 10
ml of chloroform was added to each and shaken well and kept for few minutes. Later the
extracts were taken into 10 ml volumetric flasks, treated with anhydrous sodium sulphate
and made up the volume with chloroform, and then absorbance of the solution was
measured at 416 nm (Figure 5.3) against reagent blank. Final concentrations of analyzed
solutions were 10 µg/ml to 120 µg/ml. The standard calibration plot was prepared to
calculate the amount of the analyte drug in unknown samples.
Figure 5.3 Representative absorption spectra of GBP-BCG showing λmax at 416 nm
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Method II (BTB Method)
¾ From the working standard solution, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 ml were transferred to
a series of separating funnels and buffer solution (2.5 ml) was added to each separating
funnels, then BTB solution was added in excess and shaken well, and then 10 ml of
chloroform was added to each and shaken well and kept for few minutes. Later the
extracts were taken into 10 ml volumetric flasks, treated with anhydrous sodium sulphate
and made up the volume with chloroform, and then absorbance of the solution was
measured at 421 nm (Figure 5.4) against reagent blank. Final concentrations of analyzed
solutions were 40 µg/ml to 90 µg/ml. The standard calibration plot was prepared to
calculate the amount of the analyte drug in unknown samples.
Figure 5.4 Representative absorption spectra of GBP-BTB showing λmax at 421 nm
b) Accuracy (% Recovery):
¾ The accuracy of the proposed methods was performed by calculating recovery of GBP
by the standard addition method. Known amounts of standard solutions of GBP were
added at 50, 75 and 100% levels to prequantified sample solutions of 40 µg/ml GBP for
BCG and BTB both. At each level of the amount 3 determinations were performed. The
amount of GBP was estimated by applying obtained values to regression equation.
c) Method precision (Repeatability):
¾ The precision of the instrument was checked by repeated scanning and measurement of
the absorbance of solutions of 50 µg/ml GBP (n = 6) and 70 µg/ml GBP (n = 6) for BCG
and BTB, respectively without changing the parameters for the method. The repeatability
was expressed in terms of % relative standard deviation (%RSD).
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Extractive Spectrophotometric Method
d) Intermediate precision (Reproducibility):
¾ The intraday and interday precision of the proposed methods were performed by
analyzing the corresponding responses 3 times on the same day and on 3 different days
over a period of 1 week for 6 different concentrations of standard solutions of GBP (20,
40, 60, 80, 100 and 120 µg/ml) for BCG and GBP (40, 50, 60, 70, 80 and 90 µg/ml) for
BTB. The results were reported in terms of %relative standard deviation (%RSD).
e) Limit of detection and Limit of quantification:
¾ The limit of detection (LOD) and limit of quantification (LOQ) of the drug were derived
by calculating the signal-to-noise (i.e. 3.3 for LOD and 10 for LOQ) ratio using the
following equations designated by International Conference on Harmonization (ICH)
guideline:
LOD = 3.3 Χ σ/S and LOQ = 10 Χ σ/S
Where, σ = the standard deviation of the response, S = slope of the calibration curve
5.3 RESULTS AND DISCUSSION:
METHOD VALIDATION:
5.3.1 Linearity:
¾ Linear correlation was obtained between absorbance versus concentrations of GBP in the
ranges of 10-120 µg/ml for BCG and 40-90 µg/ml for BTB. The linearity of the
calibration curve was validated by the high values of correlation coefficient of regression
line (Table No. 5.1; Figure 5.5 and 5.6).
Absorbance
y = 0.0065x + 0.0895
R² = 0.9966
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
20
40
60
80
100
120
140
Concentra ti on (µg/ ml )
Figure 5.5 Calibration curve for GBP-BCG
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Extractive Spectrophotometric Method
0.9
y = 0.0145x ‐ 0.4744
0.8
R = 0.9954
2
Absorbance
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
30
40
50
60
70
80
90
100
Concentra ti on (µg/ml )
Figure 5.6 Calibration curve for GBP-BTB
TABLE NO: 5.1
REGRESSION ANALYSIS DATA AND SUMMARY OF VALIDATION
PARAMETERS FOR THE PROPOSED METHODS
Parameters
BCG
416
BTB
421
Beer Lambert’s law limits, µg/ml
10-120
40-90
Molar absorptivity*
Sandell’s sensitivity, µg/cm2 /0.001
Absorbance unit*
Regression equation y=mx+c
1627.70
1123.72
0.1138
0.1750
y = 0.0065x + 0.0895
y = 0.0145x - 0.4744
Slope (m)
0.0065
0.0145
Intercept (c)
Correlation coefficient (r2)
Limit of detection (LOD), µg/ml
Limit of quantification (LOQ), µg/ml
Repeatability (%RSD, n=6)
Precision (%RSD)
Interday (n = 3)
Intraday (n = 3)
0.0895
0.9966
2.90
8.77
0.357
-0.4744
0.9954
10.86
32.91
0.330
0.77-1.90
0.14-1.08
0.87-1.77
0.31-1.63
λ max, nm
.
* Mean of six determinations
5.3.2 Accuracy (% Recovery):
¾ The recovery experiments were performed by the standard addition method. The mean
recovery of GBP was 99.23 + 0.77 to 100.4 + 0.77 and 99.23+ 0.77 to 99.91 + 1.07 for
BCG in capsule and tablet dosage form, respectively (Table No. 5.2 and 5.3). The mean
recovery of GBP was 99.62 + 0.29 to 101.6 + 0.18 and 98.78 + 0.11 to 101.4 + 0.05 for
BTB in capsule and tablet dosage form, respectively (Table No. 5.2 and 5.3).The low
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Extractive Spectrophotometric Method
value of standard deviation indicates that the proposed methods are accurate. Results of
recovery studies are shown in Table No. 5.2 and 5.3.
TABLE NO: 5.2
DRUG RECOVERY STUDY IN CAPSULE DOSAGE FORM
Drug-Dye
Complex
GBP-BCG
GBP-BTB
Level
I
II
III
I
II
III
Amount of sample
taken
(µg/ml)
40
40
40
40
40
40
Amount of
standard spiked
(%)
50 %
75 %
100 %
50 %
75 %
100 %
Mean
% Recovery ± SD*
(n=3)
99.23 ± 0.77
100.4 ± 0.77
100.3 ± 1.60
99.62 ± 0.29
99.88 ± 0.25
101.6 ± 0.18
TABLE NO: 5.3
DRUG RECOVERY STUDY IN TABLET DOSAGE FORM
Drug-Dye
Complex
GBP-BCG
GBP-BTB
Level
Amount of
sample taken
(µg/ml)
I
II
III
I
II
III
Amount of
standard
spiked (%)
50 %
75 %
100 %
50 %
75 %
100 %
40
40
40
40
40
40
Mean
% Recovery ± SD*
(n=3)
99.23 ± 0.77
99.36 ± 1.18
99.91 ± 1.07
98.78 ± 0.11
99.12 ± 0.25
101.4 ± 0.05
5.3.3 Method precision (Repeatability):
¾ The RSD value of method precision for GBP was found to be 0.357 % for BCG and
0.330 % for BTB. (Table No. 5.4). Low relative standard deviation was indicates that the
proposed methods are repeatable.
TABLE NO: 5.4
PRECISION DATA FOR GBP
Sr. No.
1
2
3
4
5
6
Mean
SD
% RSD
S.K.P.C.P.E.R., Kherva
Absorbance of GBP
BCG (GBP: 50 µg/ml)
BTB (GBP: 70 µg/ml)
0.412
0.521
0.412
0.414
0.413
0.411
0.415
0.413
0.001
0.357
0.52
0.522
0.524
0.521
0.519
0.521
0.002
0.330
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Extractive Spectrophotometric Method
5.3.4 Intermediate precision (Reproducibility):
¾ The low % RSD values of interday (0.77% -1.90 % for BCG and 0.87% -1.77% for
BTB) and intraday (0.14%-1.08 % for BCG and 0.31%-1.63% for BTB) variations
reveal that the proposed methods provide acceptable intraday and interday variation for
GBP at different concentration levels (calibration range concentration for BCG and
BTB) (Table No. 5.1).
5.3.5 Limit of detection (LOD) and Limit of quantification (LOQ):
¾ LOD and LOQ values for GBP were found to be 2.90 µg/ml and 8.77 µg/ml, respectively
for BCG and 10.86 µg/ml and 32.91 µg/ml for BTB, respectively. These data show that
this method is sensitive for the determination of GBP (Table No. 5.1).
5.3.6 Assay of the pharmaceutical formulations:
¾ The proposed validated methods were successfully applied to determine GBP in its
dosage forms. The results obtained were comparable with the corresponding labeled
amount (Table No. 5.5 and 5.6). No interference of the excipients with the absorbance of
interest appeared; hence the proposed methods are applicable for the routine estimation
of GBP in pharmaceutical dosage forms.
TABLE NO: 5.5
ANALYSIS OF MARKETED FORMULATION (CAPSULE DOSAGE FORM) OF
GBP BY PROPOSED METHODS (n = 6)
Label Claim
Sample No.
GBP
(mg/cap)
300
300
300
300
300
300
1
2
3
4
5
6
Amount Found
% Label Claim
GBP
(mg/cap)
GBP
(mg/cap)
BCG
BTB
BCG
BTB
301.1
304.8
302.0
301.1
297.3
304.8
298.6
297.7
297.0
297.9
296.7
298.8
100.4
101.6
100.7
100.4
99.11
101.6
100.6
0.93
99.52
99.22
99.01
99.32
98.91
99.62
99.27
0.28
Mean
SD
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Extractive Spectrophotometric Method
TABLE 5.6
ANALYSIS OF MARKETED FORMULATION (TABLET DOSAGE FORM)
OF GBP BY PROPOSED METHODS (n = 6)
Label Claim
Sample No.
GBP
(mg/tab)
1
2
3
4
5
6
300
300
300
300
300
300
Amount Found
GBP
(mg/tab)
BCG
BTB
300.5
295.1
294.9
294.2
303.2
293.6
296.8
295.4
294.0
295.4
299.5
294.8
Mean
SD
% Label Claim
GBP
(mg/tab)
BCG
BTB
100.2
98.36
98.31
98.07
101.1
97.87
98.92
98.46
98.00
98.46
99.85
98.27
99.38
98.25
1.18
0.24
5.4 REFERENCES:
1. Beckett AH, Stenlake JB. Practical pharmaceutical chemistry. 4th ed.; 2004: 2, p.304.
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Summary and Conclusion
CHAPTER 6: SUMMARY AND CONCLUSION
6.1 SUMMARY:
¾ Methods including LC-MS/MS, GC-MS, HPLC, HPTLC, Capillary Electrophoresis,
colorimetric methods were reviewed for the estimation of GBP in biological fluids and
pharmaceutical formulations.
¾ LC-MS/MS method for estimation of GBP in human plasma was developed.
¾ The developed LC-MS/MS method was validated for linearity, accuracy, interday and
intraday precision, matrix effect, dilution integrity, freeze and thaw stability, stock
solution stability, recovery, bench top stability, process stability.
¾ Extractive spectrophotometric methods based on formation of ion pair between GBP and
dyes were developed using BCG and BTB dye.
¾ Dye, pH of buffer, volume of buffer, volume of dye was optimized to get maximum
absorbance of resulting solution.
¾ The developed extractive spectrophotometric methods were validated for linearity, limit
of detection, limit of quantification, recovery (accuracy), reproducibility, interday and
intraday precision.
¾ Finally the developed validated extractive spectrophotometric methods were applied in
pharmaceutical dosage forms including capsule and tablets.
6.2 CONCLUSION:
6.2.1 LC-MS/MS METHOD:
¾ The results of the method validation for GBP indicated that the analytical method was
valid for the analysis of GBP with a calibration range of 0.05 µg/ml to 6.00 µg/ml in
human plasma using LEV as internal standard.
¾ From results of matrix effect, it was concluded that the method can be used for
estimation of GBP in human plasma without any interferences of biological matrix.
6.2.2 EXTRACTIVE SPECTROPHOTOMETRIC METHODS:
¾ During the course of study, it was observed that acidic solution of the drug formed
colored ion-association complexes with BCG and BTB which were soluble in
chloroform. This property of the drug was followed for the development of extractive
spectrophotometric methods for analysis of drug. The complex of GBP with BCG and
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Summary and Conclusion
BTB showed λmax at 416 nm and 421 nm, respectively. These developed methods were
used for the estimation
of GBP from two formulations. Both methods involve formation of ion-associated
complex with BCG and BTB at pH 4.0 exhibiting λmax at 416 nm and 421 nm,
respectively.
¾ The proposed methods were based on addition of an amine in its ionized form to an
ionized dye, yield a salt (ion-pair) that was extracted into an organic solvent such as
chloroform or dichloromethane. The indicator dye was added and the pH of the aqueous
solution was adjusted to a value where both the amine and dye were in the ionized forms.
The ion pair was extracted by extraction into the organic solvent. In these methods
Beer’s law was obeyed with BCG and BTB in the concentration range of 10-120 µg/ml
and 40-90 µg/ml, respectively.
¾ The optimum conditions for color development had been established by varying the
different parameters involved. For testing the accuracy and reproducibility of the
proposed methods, recovery studies were performed. The data obtained by recovery
studies indicate non-interference from the excipients used in the formulations. The
percentage recoveries were close to 100%. This study revealed that the common
excipients and other additives such as lactose, starch, gelatin, talc and magnesium
stearate that are usually present in the tablet dosage forms do not interfere in the analysis.
Thus, it can be concluded that the proposed methods are found to be simple, precise and
accurate that can be used for the determination of GBP in their pharmaceutical dosage
forms in a routine manner.
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Presentations and publications
CHAPTER 7: PRESENTATIONS AND PUBLICATIONS
7.1 PAPER COMMUNICATED FOR PUBLICATION:
1. D.B.PATEL*, S.A.PATEL, R.V.SHAH, N.J.PATEL. Extractive Spectrophotometric
Methods for the Determination of Gabapentin in Pharmaceutical Dosage Forms. Indian J
Pharm Sci.
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