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Твое Le ol al fanal Sa we re a Hilde hl ya i EET Elia RY
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DISTR. : LIMITED
7 \ WORLD HEALTH ORGANIZATION DISTR, : LIMITEE
№ y ORGANISATION MONDIALE DE LA SANTE WHO/PHARM/97,595
ORIGINAL : ANGLAIS
CENTRE COLLABORATEUR OMS
FOUR LES SUBSTANCES CHIMIQUES DE REFERENCE
Rapport d'activité pour 1997
par M, Westermark
le rapport annuel du Centre. Ce rapport est communiqué,
de la Pharmatopée internationale et des Préparation
Soigneusement les propositions ainsi que la documentation
au Centre de toute réserve où critique avant la fin septe
consultations ou analyses complémentaires nécessaires p
Les substances chimiques internationales de référence nouvellement établies, proposées par le Centre collaborateur
OMS pour les substances chimiques de référence sur la base d'éssais et d'une caractérisation appropriés, figurent dans
entre autres, aux membres du Tableau consultatif d'experts
5 pharmaceutiques, auxquels I! est demandé d'examiner
jointe concernant les analyses effectuées, et de faire part
more 1998, En de tels cas, le Centre procédera 4 toutes
our la validation de la substance.
>| aucun commentaire négatif n'est reçu dans les trois mols, les nouvelles substances chimiques intemationales de
référence proposées peuvent être considérées comme provisoirement adoptées, Leur adaption définitive fera Poblet d'un
examen au cours de la réunion suivante du Comité d'experts.
Prière d'adresser vos remarques à Mme M. Westermark, Centre collaborateur OMS pour les substances chimiques de
reference, Apoteket AB, Produktion & Laboratorier, Centrallaboratoriet (ACL), Prismavägen 2, 5-14175 Kungens Kurva,
suede.
TABLE DES MATIERES
Pages
Distribution de substances de référence en 1997 ...... aaa e 2
Distribution de spectres de référence en 1997... e A TIT 2
Etablissement de substances de référence en 1997 ...... LL eee aan 2
Etablissement de spectres de référence en 1997 .................. IT 2
Travaux effectués en 1997 sur de nouvelles substances de référence _.................. CE À
Bssaisdestabilitd............... aan Ur TITO 2
Travaux en cours et travaux futurs ..,...... 2.2 ta aa La ea ana III TIT 3
Questions administratives et financiéres ........................ are ТЕ 3
Remerciements ...... iii ee TUTTO 3
Appendice 1. Distribution de substances chimiques de référence en 1997... 1 4
Appendice 2. Distribution de substances chimiques internationales de référence dans les différentes
Regions de POMS en 1997 Lo... ooo eaa ree 9
Appendice 3. Substances chimiques internationales de référence établies en 1997 .......... 10
Appendice 4, Liste des substances chimiques internationales de référence disponibles ............... 11
Appendice 5, Liste des spectres infrarouges internationaux de référence disponibles ................ 17
Appendice 6. Bssalsdestabilité ........ aa arorararea e rra. 20
Appendice 7, Substances chimiques internationales de référence - Liste prévisionnelle pour 1998 ..... 38
Appendice 8, Spectres infrarouges internationaux de référence - Liste prévisionnelle pour 1998 ...... 39
Appendice 9. Acetanilide, N° de contrôle 297171 ............ .......... aradalo. 40
Appendice 10. Captopril, N° de contréle 197214 ............... coco 48
Appendice 11. Chlorhydrate de ciprofloxacine, N* de contréle 197210 ............... ET 57
Appendice 12. Cisplating, N° de contrôle 197207 ......... can Lee TT 66
Appendice 13. Monosulfate de kanamycine, N° de contrôle 197211 ........... =" 73
Appendice 14. Phénacétine, N" de comtróle 297172... e IT 82
Appendice 13, Adipate de pipérazine, N° de contrôle 197212 o.oo... TT 90
Appendice 16. Citrate de pipérazine, N° de contrôle 197213 122 10 7 06
Appendice 17. Sulfate de streptomyeine, N° de contrále 197215... EN 102
Note : Pour des raisons techniques, les appendices 9 a 17 n'ont été établis qu’en anglais.
This document is not issued to the general public, and all rights
are reserved by the World Health Organization (WHO), The
document may not be reviewed, abstracted, quoted, reproduced or
transiated, in part of in whale, without the prior written permission
of WHO. No part of this document may be stored in a retrieval
system or transmitted in any form or by any means - electronic,
mechanical ar other - without the prior written permission of
WHO.
The views expressed in documents by named authors are solely the
responsibility af those authors,
Ge document n'est pas destiné à être distribué au grand public et tous
las droits y afférents sont réservés par l'Organisation mondisie de la
Santé (OMS). Il ne peut être commenté, résumé, cité, reproduit ou
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fécherche documentaire au diffusée sous quelque forme qu per quelque
moyen fue CE soit - électronique, mécañique, ou autre - sans une
autorisation préalable écrite de l'OMS,
Les opinions exprimées dans les documents par des auteurs cités
nómmement n'engagent que lesdits auteurs.
WHO/PHARM/97.585
Page 2
Distribution de substances de référence en 1997
En 1997, le Centre a distribué 1559 substances chimiques internationales de référence. Ce chiffre
représente une augmentation d’environ 34 % par rapport à celui de 1996. Les substances les plus fréquemment
demandées ont été, dans l’ordre, l’acétate de vitamine À (acétate de rétinol), la phénacétine P.F., la vanilline
P.F., la caféine PF. et l'acide folique. On trouvera à l’appendice | le détail de la distribution des diverses
substances.
En 1997, les substances ont été distribuées dans 38 pays. On trouvera à l’appendicée 2 le détail de la
distribution par pays. En ce qui concerné la distribution par Région de l'OMS, on constate qu'environ 14,3 %
des substances ont été fournies 4 la Région africaine, 3,7 % à la Région des Arnériques, 14,4 % à la Région de
la Méditerranée orientale, 60,4 % à la Région européenne, 2,9 % a la Région de 1’ Asie du Sud-Est, et 4,4 % à
la Région du Pacifique occidental,
Distribution de spectres de référence en 1997
Aucun spectre de référence n’a été distribué en 1997.
Etablissement de substances de référente en 1997
Conformément à la procédure recommandée par le Comité OMS d'experts des Spécifications relatives
aux Préparations pharmaceutiques dans son trente-deuxième rapport (Série de Rapports techniques, N° 823),
huit substances chimiques internationales de référence ont été établies en 1997. On trouvera la liste de ces
substances à l’appendice 3.
On trouvera à l’appendice 4 une liste complète de toutes les substances chimiques internationales de
référence détenues par le Centre en janvier 1998, avec indication de la quantité de substance contenue dans
chaque unité de conditionnement et du numéro dé contrôle des lots actuels. Cette liste comprend également
neuf substances mentionnées ci-dessous, dont on peut prévoir qu’elles séront officiellement adoptées par ie
Comité d'experts à sa prochaine réunion.
Etablissement de spectres de référence en 1997
On trouvera À l’appendice 5 une liste complète de tous les spectres infrarouges internationaux de référence
avec indication des nouveaux spectres établis depuis 1993. Aucun nouveau spectre n’a été établi en 1997.
Travaux effectués en 1997 sur de nouvelles substances de reference
Le Centre a poursuivi ses travaux en vue de fournir de nouvelles substances de référence qui seront
nécessaires pour accompagner les spécifications de la troisième édition de la Pharmacopée internationale. En
1997, sept nouvelles substances de référence destinées à accompagner les volumes 3, 4 et 5 ont été examinées :
captopril, chlorhydrate de ciprofloxacine, cisplatine, monosulfate de kanamycine, adipate de pipérazine, citrate
de piperazine et sulfate de streptomycine. Les rapports d’analyse pour ces substances figurent aux
appendices 10-13 et 14-17, Ces substances sont jugées satisfaisantes en vue de leur adoption comme substances
chimiques internationales de référence. Les stocks N° 192171 d’acétanilide et N° 192172 de phénacétine étaient
en cours d'épuisement et ont été remplacées par les stocks N° 297171 d'acétanilide et N° 297172 de phénacétine.
On trouvera aux appendices 9 et 14 les rapports d'analyse dé ces substances.
Essais de stabilité
Le Centre a poursuivi ses examens périodiques de la stabilité des substances chimiques internationales
de référence existantes. En 1997, 33 substances ont été réexaminées, On trouvera les résultats de ce réexamen
à l’appendice 6. On peut obtenir auprès du Centre des détails concernant les méthodes utilisées.
WHO/PHARM/97.595
Page 3
Travaux en cours et travaux futurs
Les travaux sur les substances nécessaires pour accompagner les monographies des volumes 3, 4 et 5 de
la Pharmacopée internationale se poursuivent. Actuellement, le Centre procede a l'étude de 14 des
29 substances énumérées à l’appendice 7.
En 1997, le Centre a poursuivi ses activités de contrôle de la qualité. Un “contrôle des modifications” a,
par exemple, été mis en place. Des modifications ont été apportées à la documentation qui aécompagne les
envois de substances de référence. Par exemple, dépuis juillet 1997 tous les nouveaux certificats sont signés.
1rois boursiers sont venus au Centre en janvier et octobre : le Professeur Kamel Bouzouita et Hassen
Trabelsi, du Laboratoire national de Contrôle des Médicaments, Tunisie, et Vasenta Perera, du National Drug
Quality Assurance Laboratory, Sri Lanka. Ces boursiers ont étudié les aspects administratifs de la manutention
des substances de référence ainsi que différentes techniques d'analyse utilisées dans l'essai des substances
chimiques internationales de référence.
Questions administratives et financières
Le coût de fonctionnement total du Centre en 1997 a été estimé à US $574 447. Le revenu de la vente de
substances de référence a été d'environ US $95 990. Le Siège de l'OMS a apporté une contribution de
US $16 000. Cela laisse un déficit de US $462 457, couvert grâce au soutien d'Apoteket AB.
En 1997, le prix des substances a été maintenu à US $60 par paquet, et les frais d'expédition et de
manutention s'ajoutant à chaque commande ont été maintenus à US $10.
Remerciements
Le Centre remercie les laboratoires qui ont contribué à ses travaux en 1997, notamment le Laboratoire de
la Pharmacopée européenne, Strasbourg, France, I'Institute of Science and Forensic Medicine, Singapour, et le
National Institute for Biological Standards and Control, Londres.
Le Centre désire également remercier les laboratoires pharmaceutiques qui lui ont fourni des substances
pour examen et ont participé aux travaux d'analyse. Cette année, ses remerciements vont en particulier a Bayer,
Leverkusen, Allemagne; Bristol-Myers Squibb, New Brunnswick, Etats-Unis d’ Amérique; et Heraeus GmbH,
Hanau, Allemagne.
AT GIT wk La an rarer as cL lle ITT TT TL TE vr EE EEL IT ey PROTEIN ula ИТ LHL om Lr rermrroarrar nr rama Treat er ham rr rag
WHO/PHARM/S7.595
Page 4
AFPFENDICE 1
DISTRIBUTION DE SUBSTANCES CHIMIQUES DE REFERENCE EN 1997
SCIR Echantillons vendus
Acéclidine, salicylate ]
p-Acétamidobenzalazine 3
Acétazolamide 5
Allopurinol 2
Armidotrizoïque, acide 0
2-Amino-5-nitromhiazole 1
3-Aminopyrazole-4-carboxamide, hémisulfate 3
3-Amino-2,4,6-tritodobenzoíque, acide 0
Amitriptyline, chlorhydrate 6
Amodiaqume, chlorhydrate 2
Amphotéricine B 4
Ampicilline (anhydre) 10
Ampicilline sodique 13
Ampicilline, trihydrate 24
Anhydrotétracyeline, chlorhydrate 16
Atropine, sulfate 4
Azathioprine
Bacitracine zinc
Beclométasone, dipropionate
Bendazol, chlorhydrate
Benzobarbital
Benzylamine, sulfate
Benzylpénicilline potassique
Benzvipénicilline sodique
Béphénium, hydroxynaphtoate
Bétaméthasone
Bétaméthasone, phosphate sodique
Betaméthasone, valérate
Betanidine, sulfate
NN -bis(2,3-xylyDanthranilamide
Bupivacaine, chlorhydrate
Caféine
Calcium, folinate (Leucovorine calcique)
Carbamazépine
Carbénicilline monosodique
Chloramphénicol
Chloramphenicol, palmitate
Chloramphénicol, palmitate (forme A)
3-Chloro-2-méthylaminobenzophénone
Chloroquine, sulfate
2-(4-Chloro-3-sulfamoyIbenzoyl)benzoïque, acide
Chlorphénamine, hydrogénomaléate
Chlorpromazine, chlorhydrate
Chlortalidone
Chlortétracycline, chlorhydrate
Fa LA
A EJ LA a LA A E RIA A EA ES OLA EST LIO = Wr BS EJ Rr СЛ
jm
ba
RE E baso da a tl armo gr tool ae
SCIR
Cimétidine
Clomiféne, citrate
Clomifène, citrate (isomere Z) (voir Zuclomiféne)
Cloxacilliné sodique
Colécalciférol
Cortisone, acétate
Dapsone
Désoxycortone, acétate
Dexaméthasone
Dexaméthasone, acétate
Dexaméthasone - acide phosphorique
Dexaméthasone, phosphate sodique
Diazépam
Diazoxide
Dicloxacilline sodique
Dicolimium, iodure
Dicoumarol
Diéthylcarbamazine, dihydrogénocitrate
Digitoxine
Digoxine
Dopamine, chlorhydrate
Doxorubicine, chlorhydrate
Emétine, chlorhydrate
4-Epianhydrotétracycline, chlorhydrate
4-Epitétracycline, chlorhydrate
Ergocalciférol
Ergométrine, hydrogénomaléate
Ergotamine, tartrate
Erythromycine
Erythromycine B
Erythromycine C
Estradiol, benzoate
Estrone
Etacrynique, acide
Ethambutol, chlorhydrate
Ethinylestradiol
Ethistérone
Ethosuximide
Etocarlide
Flucloxacilline sodique
Flucytosine
Fludrocortisone, acétate
Fluoroutaci!
Fluphénazine, décanoate (dichlorhydrate)
Fluphénazine, énantate (dichiorhydrate)
Fluphénazine, chlorhydrate
Folique, acide
3-Formylrifamycine
WHO/PHARM/97.595
Раде 5
Appendice 1
Echantillons vendus
HA co
pl
BOB OM RRR RR BE) LA NW A e = AS
Bd ot pen
Ls Ch QO =]
da
A DIRE JS EJE IES IA bd BBD BRD o AA
WHO/PHARM/97.595
Page 6
Appendice 1
SCIR
Framycétine, sulfate (Néomycine B, sulfate)
Furosémide
Gentamicine, sulfate
Griséofulviné
Halopénidol
Hydrochlorothiazide
Hydrocortisone
Hydrocortisone, acétate
Hydrocortisone, succinate sodique
(-)-3-(4-Hydroxy-3-méthoxyphényl)-2-hydrazino-2-méthylalanine
(=-)-3-(4-Hydroxy-3-méthoxyphény1)-2-méthylalanine
Ibuprofene
Imipramine, chlorhydrate
Indométacine
o-lodohippurique, acide
Isoniazide
Lanatoside C
Lévodopa
Lévonorgestrel
Lévothyroxine sodique (Thyroxine sodique)
Lidocaïne
Lidocaïne, chlorhydrate
Liothyronine sodique
Lopéramide, chlorhydrate
Mébendazole
Méfénamique, acide
Métazide
Méthaqualone
Methotrexate
Méthyldopa
Méthyltestostérone
Méticilline sodique
Metronidazole
Nafcilline sodique
Néamine, chlorhydrate
Néomycine B, sulfate (voir Framycétine, sulfate)
Néostigmine, métilsulfate
Nicotinamide
Nicotinique, acide
Nifurtimox
Niridazole
Nirndazole-chloréthylcarboxamide
Noréthistérone
Norethistérone, acétate
Nystatine
Ouabaïne
Oxacilline sodique
Oxytétracycline, chlorhydrate
Echantillons vendus
Jal
Ba fa ARCO ES IEA SA LY UA rh = JS EJ OJOS LA LO EMO RJ EF END Lal $ Ll Y TT 6
Ja
MS LA RY LD EA 2 Во © 1 BE Re
Eo LL ad bi Hi] ele [e eb
SCIR
Oxytétracyeline, dihydrate
Papavérine, chlorhydrate
Paracétamo!
Paromornycine, sulfate
Phenéticillme potassique
Phénoxyméthylpénicilline
Phénoxyméthylpénicilline calcique
Phénoxyméthylpénicilline potassique
Phénytoine
Praziquantel
Prednisolone
Predmisolone, acétate
Prednisolone, hémisuceinate
Predmsolone, phosphate sodique
Prednisone
Predrisone, acétate
Probénécide
Procaïne, chlorhydrate
Procarbazine, chlorhydrate
Progestérone
Propicilline potassique
Propranolol, chlorhydrate
Propylthiouracile
Pyrantel, embonate
Pyridostizmine, bromure
Réserpine
Rétinol, acétate (solution a 25 000 UT)
Riboflavine
Rifampicine
Rifampicine quinone
Sodium, cromoglicate
spectinomycine, chlorhydrate
Substances de référence pour le point de fusion
Azobenzène
Vanilline
Benzile
Acétanilide
Phénacétine
Benzanilide
Sulfanilamide
Sulfapyridine
Dicyandiamide
Saccharine
Caféine
Phénolphtaléine
Sulfacétarnide
Sulfaméthoxazole
Sulfaméthoxypyridazine
The LL a Bb TE) в TEE
WHO/PHARM/97,595
Page 7
Appendice 1
Echantillons vendus
11
2
15
0
2
2
bo — e
Fa ba Fs e LH — |
>
ba LA OLA ВО 1 JO RJ RIO EF Ls La
WHO/PHARM/97.595
Page 8
Appendice 1
SCIR
Sulfamilamide
Sulfasalazine
Tamoxiféne, citrate
Tamoxifene, citrate (isomère E)
Testostérone, énantate
Testostérone, propionate
Tétracyclme, chlorhydrate
Thioacétazone
4. 4° -Thiodianiline
Thyroxine sodique (voir Levothyroxine sodique)
Tolbutamide
Tolnaftate
Toluene-2-sulfonamide
Triméthadione
Trimethoprime
Triméthylguanidine, sulfate
Tubocurarine, chlorure
Vincristine, sulfate
Vitamine A, acétate (solution) (voir Rétinol, acétate)
Warfarine
Zuclomiféne
Echantillons vendus
—
т) о) = © © = ю
= в в) ее з © В в
Li La
WHO/PHARM/97.595
Page 9
APFENDICE ?
DISTRIBUTION DE SUBSTANCES CHIMIQUES INTERNATIONALES
DE REFERENCE DANS LES DIFFERENTES REGIONS DE L’OMS EN 1997
Régions OMS
Région africaine (AFRO)
Ethiopie 170
Ouganda 28
Sierra Leone 22
Région des Amériques (AMRO)
Argentine 10
Brésil 7
Cuba 8
Etats-Unis d'Amérique 29
Mexique 3
Région de la Méditerranée orientale (EMRO)
Chypre 4
Libye 14
République arabe syrienne 20
Région européenne (EURO)
Allemagne 266
Autriche 17
Belgique 16
Danemark 18
Espagne 12
Finlande 13
France 69
Hongrie 3
Irtancie 6
Israel 6
Italie 3
Kirghizistan 181
Liechtenstein 3
Norvège 17
Pays-Bas 7
Pologne 15
Royaume-Uni 92
Slovaguie 2
Suede 145
Suisse 37
Region de I'Asie du Sud-Est (SEARO)
Corée 3
Indonésie 3
Sri Lanka 39
Region du Pacifique occidental (WPRO)
Laos 4
Philippines 26
Singapour 35
Taiwan 2
WHO/PHARM/97.595
Page 10
APPENDICE 3
SUBSTANCES CHIMIQUES INTERNATIONALES DE REFERENCE
ETABLIES EN 1997
Substance de référence
Amidotrizoique, acide
3-Amino-2 4 E-triibdobenzolque,
acide
Bétaméthasone, phosphate
sodique
Doxorubicine, chloraydrate
Sulfacétamide
Tarmoxifene, citrate
Tamoxiféne, citrate (isomére E)
Toluéne-2-sulfonamide
N° de contrôle
196205
196206
196203
186202
186200
196208
196209
196204
Rapport d'analyse
WHO/PHARM/S6.587
Appendice 9
WHO/PHARM/96.587
Appendice 10
WHO/PHARM/S6.587
Appendice 11
WHO/PHARM/96.587
Appendice 12
WHO/PRARM/96.587
Appendice 13
WHO/PHARM/S6.587
Appendice 14
WHO/PHARM/96.587
Appendice 15
WHO/PHARM/96.587
Appendice 16
Remarques
Latina 08 Va LL LT I = а нана неее Нал а ad Али, . y EL TDT TE La РАДАР lee poe pot ЛЬ НР АРА a ma at le REE PTE TETE TL Se mr A Tm PUN LUPA NP Lt, ar mm —— TERA о
WHO/PHARM/97.595
Page 11
APPENDICE 4
LISTE DES SUBSTANCES CHIMIQUES
INTERNATIONALES DE REFERENCE DISPONIBLES
1998
Informations générales
Les substances chimiques internationales de référence sont établies conformément à l’avis du Comité
OMS d'experts des Spécifications relatives aux Préparations pharmaceutiques. Elles sont fournies
principalement pour être utilisées dans des épreuves physiques et chimiques ainsi que dans des dosages décrits
dans les spécifications pour le contrôle de la qualité des produits pharmaceutiques publiées dans la Pharmacopée
internationale où proposées sous forme de projets de monographies. Les substances chimiques internationales
de référence sont principalement destinées à servir d’étalons primaires pour l'étalonnage d’étalons secondaires.
® Le mode d’emploi et les données analytiques pour 'usage auquel elles sont destinées dans la spécification
correspondante de la Pharmacopée internationale sont fournis dans les certificats Joints aux substances
distribuées,
Les substances chimiques internationales de référence peuvent être utilisées également dans des épreuves
ét des dosages qui ne sont pas décrits dans la Pharmacopée internationale. Cependant, dans ce cas, il incombe
à l’utilisateur où à la Commission de la Pharmacopée, ou à toute autre autorité qui a prescrit l’utilisation de ces
substances, de vérifier qu’elles conviennent à l’usage qui en est fait.
Il est en général recommandé de conserver les substances à l’abri de la lumière et de l'humidité et de
préférence à une température voisine de +5°C, Lorsque des conditions spéciales de stockage sont nécessaires,
l'indication en est portée sur l’étiquette ou figure dans la notice jointe aux substances. Il est recommandé à
l'utilisateur de n’acheéter que la quantité suffisante pour l'usage immédiat.
La stabilité des substances chimiques internationales de référence conservées au Centre est surveillée par
des examens réguliers et, lorsque cela est nécessaire, les substances détériorées sont remplacées par de nouveaux
lots. Des listes indiquant les numéros de contrôle des lots en cours sont publiées dans les rapports annuels du
Centre et peuvent être obtenues sur demande.
Commandes de substances
Les commandes de substances chimiques internationales de référence doivent être envoyées à :
Centre collaborateur OMS pour les substances chimiques de référence
Apoteket AB
Produktion £ Laboratorier
Centrallaboratoriet, ACL
Prismavägen 2
5-14173 Kungens Kurva
Suede
(Télécopie : + 46 8 740 6040)
Les substances chimiques internationales de référence sont exclusivement fournies par paquets standard
contenant la quantité indiquée sur la liste ci-après.
WHO/PHARM/97.595
Page 12
Appendice 4
Substances de reference
Acéclidine, salicylate
p-Acétamidobenzalazine
Acétazolamide
Aliopurino!
Amdotrizoique, acide
2-Amino-5-nitrothiazole
3-Aminopyrazole-4-carboxamide, hémisulfate
3-Amino-2,4,6-triiodobenzoïque, acide
Amitriptyline, chlorhydrate
Amodiaquine, chiorhydrate
Amphotéricine B
Ampicilline (anhydre)
Ampicilline sodigue
Ampicilline, trihydrate
Anhydrotétracycline, chlorhydrate
Atropine, sulfate
Azathioprine
Bacitracine zinc
Béclométasone, dipropionate
Bendazol, chlorhydrate
Benzobarbital
Benzylamine, sulfate
Benzylpemicilline potassique
Benzylpéniciliine sodique
Béphénium, hydroxynaphtoate
Bétaméthasone
Bétaméthasone, phosphate sodique
Bétaméthasone, valérate
Betanidine, sulfate
NN -bis(2,3-xylyDanthranilamide
Bupivacaine, chlorhydrate
Caféine
Calcium, folinate (Leucovorine calcique)
Captopril
Carbamazepine
Carbénicilline monosodique
Chioramphénico!
Chloramphénicol, palmitate
Chloramphénicol, palmitate (forme A)
5-Chloro-2-méthylaminobenzophénone
Chloroquine, sulfate
2-(4-Chloro-3-sulfamoylbenzoy!)benzoïque, acide
Chlorphénamine, hydrogénomaléate
Chlorpromazine, chlorhydrate
Chlortalidone
Chlortétracycline, chlorhydrate
Cimétidine
Ciprofloxacine, chlorhydrate
Cisplatine
Clomiféne, citrate
Clomiféne, citrate (isomére Z) (voir Zuclomifène)
Cloxacilline sodique
Conditionnement
100 mg
100 mg
100 mg
100 mg
100 mg
23 mg
100 mg
100 mg
100 mg
200 mg
400 mg
200 mg
200 mg
200 mg
25 mg
100 mg
100 mg
200 mg
200 mg
100 mg
100 mg
100 mg
200 mg
200 mg
100 mg
100 mg
100 mg
100 mg
100 mg
50 mg
100 mg
100 mg
100 mg
100 mg
100 mg
200 mg
200 mg
lg
200 mg
100 mg
200 mg
50 mg
100 mg
100 mg
100 mg
200 mg
100 mg
400 mg
100 mg
100 mg
200 mg
N° de contrôle
172048
290042
186128
287049
196205
186131
172050
196206
181101
192160
191153
390001
388002
274003
180096
183111
172060
192174
192175
175066
172051
172052
180099
280047
183112
183115
196203
190145
172053
173067
289054
181102
194188
197214
189143
383043
486004
286072
175073
172061
195201
181106
182109
178080
183114
187138
190150
197210
197207
187136
274005
1
BAG ae Mad at AL 8 nada Lt A ap rr Wr am En
cfa TEA Fr CH bn EY Fit) A ee a Ta
WHO/PHARM/97.595
Page 13
Appendice 4
Substances de référence Conditionnement N° de contrôle
Colécaleiférol (Vitamine D,) 500 mg 190146
Cortisone, acetate 100 mg 167006
Dapsone 100 mg 183115
Desoxycortone, acétate 100 me 167007
Dexaméthasone 100 mg 388008
Dexaméthasone, acétate 100 me 288009
Dexaméthasone < acide phosphorique 100 mg 192161
Dexaméthasone, phosphate sodique 100 mg 192158
Diazépam 100 mg 172062
Diazoxide 100 mg 181105
Dicloxacilline sodique 200 mg 174071
Dicolimum, iodure 100 mg 172055
Dicournarol 100 mg 178077
Disthyicarbamazine, dihydrogénocitrate 100 mg 181100
Digitoxine 100 mg 277010
Digoxine 100 mg 587011
Dopamine, chlorhydrate 100 mg 192159
Doxorubicine, chiorhydrate 100 mg 196202
Emétine, chlorhydrate 100 mg 187134
4-Eptanhydrotétracycline, chlorhydrate 25 mg 288097
4-Epitétracycline, chlorhydrate 25 mg 293098
Ergocalciférol (Vitamine Ds) 500 mg 190147
Ergométrine, hydrogénomaléate 30 mg 277012
Ergotamine, tartrate 50 mg 385013
Erythromycine 250 mg 191154
Erythromycine B 130 mg 194186
Erythromycine C 25 mg 194187
Estradiol, benzoate 100 mg 167014
Estrone 100 mg 279015
Etacrynique, acide 100 mg 281056
Ethambutol, chlorhydrate 100 mg 179081
Ethinylestradiol 100 mg 291016
Ethistérone 100 mg 167017
Ethosuximide 100 mg 17908%
Etocarlide 100 mg 172057
Flucloxacilline sodique 200 mg 195194
Flucytosine 100 mg 18412]
Fludrocortisone, acétate 200 mg 195199
Fluorouracil 100 mg 184122
Fluphénazine, chlorhydrate 100 mg 176076
Fiuphénazine, décanoate (dichlorhydrate) 100 mg 182107
Fluphénazine, énantate (dichlorhydrate) 100 mg 182108
Folique, acide 100 mg 388019
3-Formylrifamycine 200 mg 190149
Framycétine, sulfate (Néomycine B, sulfate) 200 mg 193178
Furosémide 100 mg 171044
CGentamicine, sulfate 100 mg 194183
Griséofulvine 200 mg 280040
Halopérido! 100 mg 172063
Hydrochlorothiazide 100 mg 179087
Hydrocortisone 100 mg 283020
Hydrocortisone, acétate 100 mg 280021
WHO/PHARM/97.595
Page 14
Appendice 4
Substances de référence
Hydrocortisone, succinate sodiqué
(-)-3-(4-Hydroxy-3-méthoxyphényl)-2-hydrazino-
2-méthylalanine (3-0-Méthylcarbidopa)
(-)-3-(4-Hydroxy-3-méthoxyphényl)-2-méthylalanine
(3-0-Méthylméthyldopa)
Ibuprofene
Imipramine, chlorhydrate
Indométacine
o-lodohippurique, acide
Isonjazide
Kanamycine, monosulfate
Lanatoside C
Lévodopa
Lévonorgestrel
Lévothyroxine sodique
Lidocaine
Lidocaine, chlorhydrate
Liothyronine sodique
Lopéramide, chlorbydrate
Mébendazole
Méfénamique, acide
Métazide
Méthaqualone
Méthotrexate
Méthyidopa
Méthyltestostérone
Méticilline sodique
Métronidazole
Nafcilline sodique
Néamine, chlorhydrate (Néomycine A, chlorhydrate)
Néomycine B, sulfate (voir Framycétine, sulfate)
Néostigmine, métilsulfate |
Nicotinamide
Nicotinique, acide
Nifurtimox
Niridazole
Niridazole-chloréthyicarboxamide
Noréthistérone
Noréthistérone, acétate
Nvstatine
Quabaine
Oxacilline sodique
Oxytétracycline, chlorhydrate
Oxytétracycline, dihydrate
Papavérine, chlorhydrate
Paracétamo]
Paromomycine, sulfate
Phénéticilline potassique
Phénoxyméthylpénicilline
Phénoxyméthylpémicilline calcique
Phénoxyméthylpénicilline potassique
Phenytoine
Conditionnement
200 mg
25 mg
25 mg
100 mg
100 mg
100 mg
100 mg
100 mg
12 mg
100 mg
100 mg
200 mg
100 mg
100 mg
100 mg
50 mg
100 mg
200 mg
100 mg
100 mg
100 mg
100 mg
100 mg
100 mg
200 mg
100 mg
200 mg
0,7 mg
100 me
100 me
100 mg
100 mg
200 mg
25 mg
100 mg
100 mg
200 mg
100 mg
200 mg
200 mg
200 mg
100 mg
100 mg
75 mg
200 mg
200 mg
200 mg
200 mg
100 mg
N° de contrôle
194184
193180
179085
183117
172064
178078
171045
185124
197211
281022
295065
194182
189144
181104
181105
193179
194185
195195
173068
172058
173069
194193
179084
167023
274024
183118
272025
193177
187135
179090
179091
194189
186129
186130
186132
185123
191152
283026
382027
189141
189142
185127
195198
195197
167028
179082
179083
176075
179089
de ml a Teen drs momo hal mb ms a Pm] mm Pala LA as um dla]
WHO/PHARM/97.595
CTE (TC FA CA 1 RET fl ta a A A A a A EDT N ,
"ml Bm mb Mo Pu pL ha LA ua al? AL IRE ааа Eee rete Lad Mé dla FO NEAL ee] LAL iE ye EL ET FPR EERO a OP pape Spe er TH PH irra ln cmd [Fn Fd R'm dA dV Rea [rer era TETE
Page 15
Appendice 4
Substances de référence Conditionnement N° de contrôle
Pipérazine, adipate 100 mg 197212
Pipérazine, citrate 100 mg 197213
Praziquantel 100 mg 19419]
Predrusolone 100 me 389029
Prednisolone, acétate 100 mg 289030
Prednisolone, hémisuccinate 200 mg 195196
Prednisoloné, phosphate sodique 200 me 194190
Prednisone 100 me 167031
Prednisone, acétate 100 mg 169032
Probénécide 100 mg 192156
Frocaïne, chlorhydrate 100 mg 183119
Procarbazine, chlorhydrate 100 mg 184120
Progestérone 100 mg 167033
Propicilline potassique 200 mg 274034
© Propranolol, chlorhydrate 100 mg 187139
Propylthiouracile 100 mg 185126
Pyrantel, embonate 500 mg 192157
Pyridostigmine, bromure 100 mg 182110
Réserpine 100 mg 186133
Retinol, acétate (solution) 5 capsules* 791038
Riboflavine 250 mg 382035
Rifampicine 200 mg 191151
Rifampicine quinone 200 mg 190148
Sodium, cromoglicate 100 mg 188140
Spectinomycine, chlorhydrate 200 mg 193176
Streptomycine, sulfate 100 mg 197215
Substances de référence pour le point de fusion
Azobenzene (69°C) 4g 192168
Vanilline (83°C) 48 192169
Benzile (96°C) 4g 294170
Acétanilide (116°C) 4g 297171
Phénacétine (136°C) 4g 297172
@ Benzanilide (165°C) 4g 192173
Sulfanilamide (166°C) 4g 192162
Sulfapyridine (193°C) 42 192163
Dicyandiamide (210°C) 42 192164
Saccharine (229°C) 4e 192165
Caféine (237°C) 4g 192166
Phénolphtaléine (263°C) 4g 192167
Sulfacétamide 100 mg 196200
Sulfaméthoxazole 100 mg 179092
Sulfaméthoxypyridazine 100 mg 178079
Sulfanilamide 100 mg 179094
Sulfasalazine 100 mg 191155
Tamoxifene, citrate 100 mg 196208
Tamoxiféne, citrate (isomére E) 10 mg 196209
Testosterone, énantate 200 mg 194192
Testostérone, propionate 100 mg 167036
Tetracycline, chlorhydrate 200 mg 180093
Thicacétazone 100 mg 171046
4,4'-Thiodianiline 50 mg 183116
Thyroxine sodique (voir Lévothyroxine sodique)
WHO/PHARM/97.595
Page 16
Appendice 4
Substances dé référence
Tolbutamide
Tolnaftate
Toluéne-2-sulfonamide
Triméthadione
Triméthoprime
Triméthylguanidine, sulfate
Tubocurarine, chlorure
Vitamine À, acétate (solution) (voir Rétimol, acétate)
Vinéristine, sulfate
Warfarine
Zuclomiféne
* Par capsule, environ 9 mg dans 250 mg d'huile.
Conditionnement
100 mg
100 mg
100 mg
200 me
100 mg
100 mg
100 mg
9,7 mg/flacon
100 mg
50 me
N° de contrôlé
179086
176074
196204
185125
179093
172059
170037
193181
168041
187137
WHO/PHARM/97.595
Page 17
APPENDICE 5
(3) WORLD HEALTH ORGANIZATION
E ORGANISATION MONDIALE DE LA SANTE
LISTE DES SPECTRES INFRAROUGES INTERNATIONAUX
DE REFERENCE DISPONIBLES
Le Centre collaborateur OMS pour les substances chimiques de référence fournit également 69 spéctres
a infrarouges internationaux de référence,
| Les prix sont actuellement de US $5 par spectre et US $200 par série de 50 spectres au choix (voir liste
ci-dessous) sous classeur cartonné, Le classeur peut étre commandé séparément pour US $10.
Des frais administratifs de US $10 couvrant la manutention et l’expédition par avion (lettre ou paquet-
poste) sont ajoutés à chaque commande.
Les commandes doivent être envoyées à :
Centre collaborateur OMS pour les substances chimiques de référence
Apoteket AB
Produktion & Laboratorier
Centrallaboratonet, ACL
Prismavägen 2
5-14175 Kungens Kurva
Suede
Télex : 11553 APOBOL $
a Télécopic : + 46 8 740 6040,
ВЕ Le paiement doit être effectué dans le délai indiqué sur la facture. Veuillez effectuer le paiement, en
indiquant le numéro de facture, auprès de :
Postgirot Utland
5-10506 Stockholm
Suede
a/c N° : 29840-6, Apoteksbolaget AB/ACL,
SWIFT : PGSI SE 65
FRAT Arbiter mg tad meer A E AMLO A EP ET НдАЕ 3 "АР НЫ лвл читы ama mdd aL bm Ной
WHO/PHARM/97.595
Page 18
Appandice 5
Les spectres infrarouges internationaux de référence suivants sont actuellement disponibles auprés du
Centre :
acéclidine, salicylate
acétazolamide
allopurinol
amiloride, chlorhydrate
amitriptyline, chlorhydrate
ampicilline, trihydrate
béclométasone, dipropionate
benzylpénicilline potassique
bipéridene
bipéridéne, chlorhydrate
bupivacaine, chlorhydrate
caféine (anhvdre)
calcium, folinate
carbidopa
chlorphénamine, hydrogénomaléate
clofazimine
cloxaciliine sodique
colchicine
cytarabine
dexaméthasone
dexaméthasone, acétate, monohydrate
dextrométhorphane, bromhydrate
diazépam
dicolinium, rodure
dicoumarol
diéthylcarbamazine, dihydrogénocitrate
diphénoxylate, chlorhydrate
erythromycine, éthylsuccinate
érythromycine, stéarate
étacrynique, acide
éthionamide
éthosuxumide
furosemide
gallamine, triéthiodure
glibenclamide
halopérido!
hydrochiorothiazide
ibuproféne
imipramine, chlorhydrate
indométacine
isoniazide
hdocaine
lidocaine, chlorhydrate
lindane
metronidazole
miconazole, nitrate
niclosamide
nicotinamide
noscapine
oxamniquine
papavérine, chlorhydrate
phénobarbital
phénoxyméthylpénicilline calcique
phénytoine
primaquine, phosphate
propylthiouracile
protionamide
pyriméthamine
salbutamol
salbutamol, sulfate
sulfadimidine
sulfadoxine
sulfaméthoxazole
sulfaméthoxypyridazine
tiabendazo)
trihexyphénidyle, chlorhydrate
triméthoprime
valproïque, acide
verapamil, chlorhydrate
LE +
WHO/PHARM/97.595
Page 19
Appendice 5
WHO/PHARM/97.595
Page 20
APPENDICE 6
ESSAIS DE STABILITE
La stabilité des substances chimiques internationales de référence pendant leur stockage est surveillée par
un réexamen périodique des substances détenues par le Centre. Les résultats obtenus pour les substances
réexaminées en 1997 sont résumés ci-après. À titre comparatif, on a aussi indiqué les résultats obtenus lors des
réexamens précédents. Les substances ont été conservées dans des recipients étanches a +5°C et sous une
humidité relative inférieure a 30 %. Dans les tableaux, on à adopté les abréviations suivantes :
CE Electrophorése en tube capillaire
DSC Calorimétrie différentielle
DTA Analyse thermique différentielle
GC Chromatographie en phase gazeuse
HPLC Chromatographie liquide à haute performance
IR Spectrophotométrie infrarouge
KF Méthode de Karl Fischer pour la détermination de la teneur en eau
LC-MS Chromatographie en phase liquide/spectrométrie de masse
LOD Perte à la dessiccation
RMN Résonance magnétique nucléaire
PSA Analyse de solubilité par phases
TLC Chromatographie en couche mince
TGA Analyse thermogravimétrique
La valeur estimée des impuretés solides totales, obtenue par HPLC, CE et TLC, est exprimée en aire %,
sauf indication contraire; lorsqu’elle est obtenue par DSC et par DTA, elle est exprimée en mole 9, et par PSA
en poids %. Les pertes de poids, mesurées par LOD et TGA, sont exprimées en poids %. Les valeurs obtenues
par titrage sont calculées par rapport à la substance desséchée ou anhydre, sauf indication contraire.
Pour plus de détails sur les méthodes d'analyse utilisées, on peut s'adresser au Centre.
Amodiaquine, chlorhydrate, N° de contrôle 192160
Prémier rapport d'analyse : WHO/PHARM/93.564, appendice 7
Année d'examen : 1992 1997
IR conforme -
TLC, % environ 0,6 -
HPLC, % 0,3 0,4
TGA, % 7,9 7,6
Eau (KF), % 8.0 -
Titrage (spectrophotométrique), % 99.9 -
La a lla ry mmr a Pd o A ph ый WL я
A all To la Sh Le EET Er baal POLA LEO UTE E a TA ET TT Pa her ae UE AN A VE TR
WHO/PHARM/S7.595
Page 21
Appendice 6
Ampicilline, trihydrate, N° de contrôle 274003
Premier rapport d'analyse : WHO/PHARM/75.485, appendice 6
Année d'examen : 1974 1939 1992 1997
IR conforme - - conforme
HPLC, % - 0,3 0,3 0,5*
TGA, % - 13,9 13,8 13,7
Eau (KF), % 13,9 - a -
Titrage (alcalimétrique), % 98,5 - - -
Titrage (mercurimétrique), % - - 95,8 -
* Nouvelle colonne ayant une meilleure efficacité de séparation.
Atropine, sulfate, N? de contróle 183111
Premier rapport d'analyse : WHO/PHARM/84.513, appendice 5
Année d'examen : 1983 1990 1997
IR, conforme - | conforme”
TLC, % 3 taches secondaires <0,1 <0,1*
faibles
HPLC, % 0,6 0,5 0,5
TGA, % - 3,2 3,7
Eau (EF), % 3.1 - 3.8
LOD, % 3,0 - -
Fitrage (potentiométrique), % 100,4 - -
* Le réexamen de cette substance à été en partie réalisé par le Department of Scientific Services, Institute of Science and
Forensic Medicine, Singapour.
WHO/PHARM/97.595
Page 22
Appendice 6
Azathioprine, N° de contrôle 172060
Premier rapport d’ analyse : WHO/PHARM/72.471, appendice 11, WHO/PHARM/73.473, appendice 3
Annce d'examen : 1973 1979 1981 1997
IR. conforme - conforme
TLC, % environ 0,5 ] tache 1 tache -
HPLC, % - - 0,5
TGA, % - - 0,6
LOD, % 0,7 0,7 - -
PSA, 7 environ | - -
Benzobarbital, N° de contrôle 172051
Premier rapport d'analyse : WHO/PHARM/72.471, appendice 13
Année d'examen : 1972 1977 198] 1987 1997
IR ) conforme - - conforme -
TLC, % 0 tache 1 tache 2 taches environ 0,5 -
secondaire secondaire secondaires
HPLC, % - - a 0,3 0,7
TGA, % - - - - 0,1
Eau (KF), % - - - 0,1 -
LOD, % 0,4 0,1 0,1 - .
DTA, % - - - environ 1 -
Titrage (titrimétrique), % 100,0 100,2 - 100,3 -
1
ris a te PP rat le la MSE TPR IR SRE PR ES a | HH DAP i ELLE ОТО? TTT TIAL
WHO/PHARM/97.595
Page 23
Appendice 6
Benzylamine, sulfate, N° de contrôle 172052
Premier rapport d’analyse : WHO/PHARM/72.471, appendice 14
Année d’examen : 1972 1977 1979 1997
IR conforme = - conforme
TEC, % | tache 1 tache -
secondaire secondaire
HPLC, % = - <0,05
TGA, % - - - <0, 1
LOD, % <0, 1 - _ .
O LC-MS - - - confirme
l’identité
Carbamazépine, N° de contrôle 189143
Premier rapport d'analyse : WHO/PHARM/90.547, appendice 7
Année d'examen : 1989 1997
IR conforme I
TLC, % <0,01 _
HPLC, % <0,005 0,06%
@ TGA, % <0,1 0,1
Titrage (spectrophotométrique), % 100,2 -
* Nouveau systéme chromatographique ayant une meilleure efficacité de séparation.
WHO/PHARM/97.595
Page 24
Anpendice 6
Chloramphénicol, N° de contrôle 486004
Premier rapport d'analyse : WHO/PHARM/87.532, appendice 8
Année d'examen : 1986 1992 1997
IR conforme - ) conforme*
TLC, Ya 0,2 - 1 tache trés faible*
HPLC, % 0,2 0,3 0,2 (0,3*)
TGA, % - <0,1 <0,1
ГОР, % 0,05 - -
Titrage (spectrophotométrique), % 99,8 100,0 99.6%
* Le réexamen de cette substance a été en partie réalisé par le Department of Scientific Services, Institute of Science and
Forensic Medicine, Singapour.
Chloramphénicol, palmitate, N° de contrôle 286072
Premier rapport d'analyse : WHO/PHARM/87.532, appendice 9
Année d'examen : 1986 1992 1997
IR a a conforme, pas de conforme, pas de -
forme A forme A
DSC, % 2,7 2,2 -
TLC, % environ 2,7 - -
HPLC, % 3,1 3,1 2,9
TGA, % 0,1 <0,1 <0,1
Eau (EF), % 0,2 <0,1 -
LOD, % <0,1 - -
Titrage (spectrophotométrique), % 100,2 100,0 -
CIN TENE a CC на ные
WHO/PHARM/97,595
Page 25
Appendice 6
Chloramphénicol, palmitate (forme A), N° de contrôle 175073
Premier rapport d'analyse : WHO/PHARM/75.485, appendice 10
Année d'examen : 1974 1992 1997
IR conforme ‘ conforme —— -
DSC, % 0,6 0,7
TLC, % 2 taches - =
HPLC, % - environ 2 1,6
TGA, % - <0,] 0,1
Ss Бан (КР), % - <0,1 -
LOD, % 0,26 - -
Titrage (titrimétrique), % 100,9 - -
Titrage (spectrophotométrique), % - 100,0 -
>-Chloro-2-méthylaminobenzophénone, N° de contrôle 172061
Premier rapport d’analyse : WHO/PHARM/73.475, appendice 4
Année d'examen : 1972 1985 1991 1997
IR conforme - - ‘ conforme
a TLC, % 0 tache 0 tache 0 tache | -
secondaire secondaire secondaire
HPLC, % - - 0,8 1,0
TGA, % - - <0,1 -
LOD, % 0,1 - - -
Eau (KF), % - - - 0,3
DTA, % 0,4 0,5 - -
DSC, % : 0,5
WHO/PHARM/97.595
Page 26
Appendice 6
2-(4-Chloro-3-sulfamoyIbenzoyDbenzoique, acide, N° de contrôle 181106
Premier rapport d'analyse : WHO/PHARM/82.509, appendice 8
Année d'examen : 1981 1997
IR conforme conforme |
TLC, % 2 taches secondaires 2 taches secondaires
HPLC, % 0.4 0,2 (7 limite de dosage)
TGA, % - <0,1
LOD, % 0,2 -
Titrage (potentiométrique), 98,9 -
Chlorpromazine, chlorhydrate, N° de contrôle 178080
Premier rapport d'analyse : WHO/PHARM/79,499, appendice 4
Année d'examen : 1978 1984 1990 1997
IR conforme - - -
TLC, % <0,5 <0,5 environ 0,2 -
HPLC, % - я - 0,06
TGA, % - - <0,1 <0,1
LOD, % 0,1 <0,1 - -
DTA, % 0,37 0,33 - -
PSA, %
environ 0,7
o
Chlortalidone, N° de contrôle 183114
Premier rapport d’analyse : WHO/PHARM/84.513, appendice 9
i A mm —— | Л
"mI NN по но я ив о тн me ns YY Ls se mem
WHO/PHARM/97.595
Page 27
Appendice 6
Annee d'examen : 1983 1997
IR conforme -
TLC, % 0.3 -
HPLC, % 0,5 0,8
HPLC, acide 2-(4-chloro-3-sulfamoy]- 0,1 0,]
benzoyl) benzoique, %
LOD, % 0,1 -
@ TGA, % - <0,1
PSA, % 0,9 -
Titrage (potentiométrique), % 99.5 -
Clomifene, citrate, N° de contrôle 187136
Premier rapport d'analyse : WHO/PHARM/88.537, appendice 8
Année d'examen : 1987 1997
IR conforme -
TLC, % 0 tache secondaire -
en HPLC, % 0,3 0,9*
[somère Z (HPLC), % environ 35 (aire %) 32,6 (p/p %)**
Isomere Z (RMN), % 33
TGA, % 0,3 -
Eau (KF), % = 0,7
Titrage (potentiométrique), %
97,6
* Nouvelle methode plus sélective. Optimisation de la longueur d'onde de 254 nm à 263 nm.
** Avec la SCIR 187137 comme étalon externe,
WHO/PHARM/97.595
Page 25
Appendice 6
Clormifêne, citrate (isomèrée Z), N° de contrôle 187137
Premier rapport d'analyse : WHO/PHARM/88.537, appendice 9
Année d'examen : 1987 1997
IR conforme -
TLC, % <0,1 _
HPLC, % 0,3 0,3*
Isomeére E (HPLC), % environ 2 (aire %) 2,1 (p/p Yo)**
TGA, % 0,2 -
Eau (KF), % - 0,2
Titrage (potentiométrique), % 99,2 -
* Nouvelle méthode plus sélective. Optimisation de la longueur d'onde de 254 nm à 263 nm.
** Par rapport 2 un étalon externe.
Dapsone, N° de controle 183115
Premier rapport d analyse : WHO/PRARM/85.513, appendice 10
Année d’examen : 1983 1988 1997
IR conforme - -
TLC, % 2-4 taches ] -
HPLC, % 1,2 1,9 0,77
LC-MS (identification de Vimpureté identifiée comme
principale) 4-aminodiphény!-
sulfone
PSA, % 1,0 - .
TGA, % - <0,1 0,1
LOD, % 0,1 - .
Titrage (potentiométrique), % 99,9 100,0 -
* Nouvelle méthode HPLC. L'ancienne n’était pas lméaire.
WHO/PHARM/97.595
Page 29
Appendice 6
Désoxycortone, acétate, N° de contrôle 167007
Premier rapport d'analyse : WHO/PHARM/66.431, appendice 1
Année d’examen : 1965 1975 1980 1984 1997
IR conforme - conforme - т
TLC, % aucune aucune - 2 taches a
impureté impureté détectées
détectée détectée
HPLC, % - - - 0,2 0,77
TGA, % - - - - <0,1
LOD, % <0,1 0,1 . <0,1 -
DTA, % - - 0,7 - -
PSA. % <] - - . -
Titrage (spectrophotométrique), % — - - 100 - -
* Nouveau système de chromatographie en phase liquide &yant une meilleure efficacité de séparation.
Dexaméthasone — acide phosphorique, N° de contrôle 192161
Premier rapport d’ analyse : WHO/PHARM/93.564, appendice 10
Année d’ examen : 1992 1997
IR conforme -
TLC, % 0,1 .
HPLC, % 0,1 0,2
TGA, % 1 environ 1
Eau (KF), % 0,7 0,7
LOD, % 1,0 "
Titrage (spectrophotométrique), % 100,2 -
WHO/PHARNM/97.5935
Page 30
Appendice 6
Diazépam, N° de contrôle 172062
Premier rapport d'analyse : WHO/PHARM/73.475, appendice 5
Année d'examen : 1972 1982 1991 1997
IR conforme - conforme -
TLC, % | tache | tache 2 taches -
secondaire secondaire secondaires
très faibles
HPLC, % - - <0,05 <0,1
DTA, % 0,05 0,04 - -
DSC, % - - 0,05 -
TGA, % - - - <0,1
LOD, % <0,1 <0,1 - -
Eau (KF), % - - - 0,1
Titrage (potentiometrique), % 100,0 100,0 - -
Dicloxacilline sodique, N° de contrôle 174071
Premier rapport d'analyse : WHO/PHARM/74.478, appendice 5
Année d'examen : 1974 1982 1984 1989 1995 1997
IR I conforme - - - conforme -
TLC, % - . - - 3 taches -
secondajres
HPLC, % - 0,3 0,4 0,3 0,5 0,4
TGA, % - - - 3,9 3,9 3,9
Eau (KF), % 3,8 3,9 3,8 - - -
Produits de degradation - - 0,6 - - -
(mercurimétrie}, %
Titrage (alcalimétrique), % 99,5 99,4 - - - -
Titrage (mercurimétrique), % - - 99,5 - - -
het ofl epi Nun be eds ЧН ato o A CL retin TA =D я
WHO/PHARM/97.595
Page 31
Appendice 6
Dicoumarol, N° de contrôle 178077
Premier rapport d'analyse : WHO/PHARM/79.499, appendice 5
Année d'examen : 1978 1997
IR conforme -
TLC, % 0 tache secondaire -
HPLC, % <0,05 <0,04 (limite de détection)*
<0,1 (limite de dosage)
TGA, % - <0,1
LOD, % <0,1 -
Titrage (potentiométrique), % 100,0 -
PSA, % 0,3 -
* Nouvelle méthode de chromatographie en phase liquide (passage des phases normales aux phases inversées). Limite de
dosage 0,1 %, limite de detection 0,04 %,
Erythromyecing, N° de contrdle 191154
Premier rapport d'analyse : WHO/PHARM/92.558, appendice 8
Année d'examen : 1991 1997
IR conforme - a
TLC, % environ 1 ,
HPLC, % environ 1,6 1,5
TGA, % 2,2 2,6
Eau (KF), % - 1,4
Titrage (titrimétrique), % 97,6 -
Activité, HPLC 962 ug/mg -
Titrage (microblologigne) 915 Ul/mg -
WHO/PHARM/97.595
Page 32
Appendice 6
Hydrochlorothiazide, N° de controle 179087
Premier rapport d'analyse : WHO/PHARM/80.504, appendice 8
Année d’examen : 1979 1987 1997
IR conforme conforme -
DTA, % 0,4 0,7 -
HPLC, % 0,4 0,4 0,5
TGA, % - - 0,2
LOD, % <0, 1 0,2 -
Titrage (titrimétrique), % 100,0 - -
Méthyldopa, N° de contrôle 179084
Premier rapport d’analyse : WHO/PHARM/80.504, appendice 10
Année d'examen : 1979 1984 1992 1997
IR conforme - - -
TLC, % 0.2 - 0,1 =
HPLC, % <0,2 0,2 0,1 0,1
TGA, % - - 11,7 11,4
Eau (EF), % 11,5 11,5 - 11,3
Titrage (potentiométrique), % 99,7 - -
Méticilline sodique, N° de contrôle 274024
Premier rapport d’analyse : WHO/PHARM/74.478, appendice 9
ditt жен о Де ul or LT a ALT ад, lil
WHO/PHARM/97.595
Page 33
Appendice 6
Année d'examen ; 1974 1978 1984 1997
IR conforme - - -
HPLC, % - <0,1l 0,2 0,2
TGA, % - - - 4,5
Eau (EF), % 4,7 - 4,6 -
Titrage (alcalimétrique), % 99,9 - - -
a Tirage (mercurimétrique), % - y 99,5 -
Nafcilline sodique, N° de contrôle 272025
Premier rapport d'analyse ; WHO/PHARM/72,471, appendice 6
Année d'examen : 1972 1978 1984 1997
IR conforme - - -
HPLC, % - <0,1 0,1 0,6*
TGA, % - - - 4,7
Eau (KF), % 4,0 - 4,2 4,1
Composés organiques volatils, - - - 0,6
® GC, %
Titrage (mercurimétrique), % - - 99,1 -
Titrage (alcalimétrique), % 999 -
* Nouveau système chromatographique avec détection à 225 nm.
WHO/PHARM/97.535
Page 34
Appendice 6
Nystatine, N° de contrile 191152
Premier rapport d'analyse : WHO/PHARM/92.558, appendice 10
1991
Année d'examen : 1992 1993 1994 1995 1996 1997
IR. conforme - - - u -
HPLC, 9%, 5,9 - 7,9" 8,7 9,1 8,8 8,5
impuretés, 304 nm §,3%*¥¥ (mars)
11,2
(octobre)
CE, %, impuretés, - - - - - 7
304 nm (octobre)
HPLC, titrage, % 100,0 - 100,0 100,5 =99 =97 -
TGA, % 5,0 - 5,2 5,3 5,1 4,9 5,1
(mars)
3,0
(octobre)
Eau (BF), % 4,8 - - - 5,3 5,1 -
Titrage 6 382** 5 208%%% 40624" 4 86844 4728*** 4690 4 586%**
microbiologique, (mars)
Ul/mg 4 622%
(octobre)
TLC, % 4.1 я 3 taches - „ - -
(3 taches secondaires
secondaires)
UV, titrage, 100,0 - 100,0 - - =98 -
304 nm,
* Ces valeurs élevées sont dues à un nouveau système chromatographiqué avant une meilleure efficacité de séparation.
+ L'EPCRS, dont la teneur figurant sur l'étiquette est fausse, à été utilisée comme étalon.
“** Le 2° étalon biologique OMS, dont la teneur déclarée est de 4855 Ul/mg, a été utilisé comme étalon.
Hu MM
Nouvelle colonne HPLC.
Remarques concernant le réexamen d'octobre 1997 : une nouvelle méthode HPLC validée, donnant une
méilleure sélectivité, a été adoptée. La tendance observée au niveau des impuretés détectées par HPLC ne
traduit pas une dégradation, mais l’utilisation de méthodes d'analyse de plus en plus sélectives. L'intention
était de changer de lot de nystatine, mais les autres substances examinées contenalent davantage
d’impuretés. Par exemple, le 2° étalon biologique OMS contient 16,8 % d'impuretés. La SCIR actuelle est
donc la plus pure chimiquement, Le Centre continuera a rechercher des lots encore plus purs.
Une méthode de CE a également été développée, mais la méthode HPLC à une sensibilité et une sélectivité
supérieures.
En ce qui conceme le titrage microbiologique, un calcul a montré, que lorsqu’on comparait la valeur obtenue
en 1995 (4728 UV/mg) à celles de 1996 et 1997, la différence n’était pas statistiquement significative au
niveau de confiance 95 % avec un test / par séries non appariées.
tak Ta De
т TA PIP La CET AL HAC ECTS
WHO/PHARM/97.595
Page 35
Appendice 6
Quabaïne, N° de contrôle 283026
Premier rapport d'analyse : WHO/PHARM/84.513, appendice 14
Année d'examen : 1983 1987 1997
IR conforme - -
TLC, % 0,2 0,3 -
HPLC, % 0,4 0,7 0,6
TGA, % - “ 19,9
LOD, % 20,0 19,9 -
— Titrage (colorimétrique), Yo 100,1 100,0 -
Oxaciltine sodigue, N* de contrále 382027
Premier rapport d'analyse : WHO/PHARM/85,510, appendice 8
Année d'examen : 1982 1984 1987 1990 1997
IR conforme - - - -
HPLC, % 1,1 1,0 1,3 0,7 1,4*
TGA, % - - - 4,2
Eau (KF), % 4,3 4,5 4.1 4,2 -
PSA, % 0,9 - - - -
Titrage (mercurimétrique), % - 99.4 99.4 98,9 -
- 1,1
GC,% 1,1 - -
* Nouveau systéme de chromatographie en phase liquide ayant une meilleure efficacité de séparation,
WHO/PHARM/97.595
Page 36
Appendice 6
Fhénéticilline potassique, N° de contrôle 167028
Premier rapport d'analyse : WHO/PHARM/68.448, appendice 7
Année d'examen : 1967 1978 1982
1984
1997
IR conforme - - - conforme
TLC, % Z impuretés - - - a
HPLC, % . 0,4 0,5 0,6 0,7
TGA, % - - - - 0,2
Eau (KF), % 0,5 - 0,3 0,4 -
PSA, % 56,5 - - - -
isomére L
Titrage (mercurimétrique), % 99.1 - - 99.5 -
Phénytoïne, N° de contrôle 179089
Premier rapport d'analyse : WHO/PHARM/80.504, appendice 13
Année d'examen : 1979 1985 1992 1997
IR, conforme - - conforme”
HPLC, % environ 0,02 - environ 0,02 <0,04 (0,02*)
TGA, % - - 0,1 <0,1
Eau (KF), % . - 0,1 -
LOD, % <0,1 0,17 - -
DSC, % - . <0,1 -
Titrage (potentiométrique), % 100,1 - - -
Titrage (chromatographie en phase - - - 100,0*
liquide), %
* Lerecxamen de cette substance a été en partie réalisé par le Department of Scientific Services, Institute of Science and
Forensic Medicine, Singapour.
РИН Ч en tanh] SES md
[Yer
WHO/PHARM/97.595
Page 37
Appendice 6
Propicilliné potassique, N° de contrôle 274034
Premier rapport d'analyse : WHO/PHARM/75.485, appendice 8
Année d'examen : 1974 1984 1987 1990 1997
IR | conforme - “ - -
HPLC, isomére L, % - - environ 60 environ 60 -
HPLC, % - 0,8 0,5 0,7 0,7
TGA, % - - 0,3 0,3 0,4
Eau (KF), % 0,3 0,4 0,2 0,3 -
PSA, % 57 % я - - -
isomère L
Titrage (mercurimétrique), % - 08.2 98,3 97,9 -
Produits de dégradation - - 0,8 0,8 -
(mercurimétrie), %
Titrage (alcalimétrique), % 99,5 - - - -
Titrage (iodométrique), % 98,3 a - -
Titrage (potentiométrique), % 98,3 - - - -
WHO/PHARNM/97.595
Раде 38
APPENDICE 7
SUBSTANCES CHIMIQUES INTERNATIONALES DE REFERENCE
Liste prévisionnelle pour 1998
Les substances chimiques intemationales de référence ci-après sont nécessaires pour accompagner les
spécifications qui figurent dans la troisième édition de la Pharmacopée internationale :
Volume 3
Noroxymorphone, chlorhydrate
{impureté du chlorhydrate de naloxone)
Volume 4
Dactinomycine
lohexol
Médroxyprogestérone, acétate (*)
Néomycine, sulfate (*)
Volume 5
Albendazole
Aleuronium, chlorure
Amoxicilline, trihydrate
Artééther
Artéméther
Artémisinine
Artésunate
Aténolol
Aténolol pour validation des colonnes
Benznidazole
9,9'-Bisanthracéne-10,10°(9H, 9°H)-dione
Captopril, disulfure (*)
Céfadroxil, monohydrate
Chloramphénicol, succinate sodique
Ciclosporine
Ciclosporine U
Clindamycine, chiorhydrate
Clindamycine, phosphate
1-Cyclopropyl-1,4-dihydro-4-oxo-6-fluord-7-
((2-aminoéthyl)amino)-3-quinolime
carboxylique, acide (dérivé de
l’éthylénédiamine) (*)
!-Cyclopropy!-1,4-dihydro-4-oxo-6-
(1-pipérazinyl)-7-chloro-3-quinoline
carboxylique, acide (*)
]-Cyclopropyl-1,4-dihydro-4-oxo-7-
(1-pipérazinyl)-3-quinoline carboxylique,
acide (*)
Dacarbazine
Dacarbazine, substance apparentée A
Dacarbazine, substance apparentée B
Sodium, amidotrizoate (*)
Thiopental sodique
Vinblastine, sulfate (*)
Dihydroartémisinine
Diméthyl 2,6-diméthvl-4-(2-nitrophényl)
pyridine-3,5-dicarboxylate
Diméthyl 2,6-diméthyi-4-(2-nitrosophényl)
pyridine-3,5-dicarboxylate
|,2-Diphényléthylammonium-3-mercapto-2-
méthylpropanoate (*)
Dithranol
Doxycycline, hyclate (*)
Econazole, nitrate
6-Epidoxycycline, chlorhydrate
Erythromycine, éthylsuccinate
Erythromycine, stéarate
Etoposide
Fluoroquinolinique, acide (*)
1-Hydroxy-9-anthrone
Idoxuridine
Kétoconazole
Lévamisole, chlorhydrate
Lincomycine, chlorhydrate (¥)
Méfloquine, chiorhydrate
Métacycline, chlorhydrate
DL-Methionine
2-Méthyl-3-acétylthiopropionique, acide (*)
Metronidazole, benzoate
Nifédipine
Nonoxino] 9
Octoxinol 9
Rétinol, palmitate
Rétinol, propionate
(*) Indique que des travaux sont en cours au Centre sur cette substance,
ALL AD riada Fad mld kl hi A ET ata ba ВЫ dk Pm lud pS [TE aj EB da al ms dl DP ALF ml mrss mb alas Da VIVA DADA | IE Pe oy vm sets be pm
WHO/PHARM/97.595
Page 39
APPENDICE 8
SPECTRES INFRAROUGES INTERNATIONAUX DE REFERENCE
Liste prévisionnelle pour 1998
Les spectres infrarouges internationaux de référence ci-après sont nécessaires pour accompagner les
spécifications qui figurent dans la troisième édition de la Phermacopée internationale :
Volume 3
Diloxanide, furoate
Volume 4
Disodium, édetate
Metoclopramide, chlorhydrate (*) Ephédrine, sulfate
Naloxone, chlorhydrate (*)
Nitrofurantoine
Pyrazinamide (*)
Spironolactone (*)
Volume 5
Azote, protoxyde
Benzoyle, peroxyde
Ciprofloxacine
Codéine, phosphate
Colchicine
Drethyltoluamide
Diloxanide, furoate
Erythromycine, lactoblonate
Isosorbide, dinitrate
Morphine, sulfate
Péthidine, chiorhydrate
Phénobarbital
Proguanil, chlorhydrate
Sulfadiazine
Tropicamide
(*) Travaux, en cours.
lopanoïque, acide
lotroxique, acide
Kétamine, chlorhydrate
Noréthistérone, énantate
Pentamidine, isétionate
Timolol, maléate
WHO/PHARM/97.595
page 40
APPENDIX 9
Acetanilide
WHO Melting Point Reference Substance
Melting temperature 116 °C
Control No 297171
Analytical Report
Intended use
The stock of the current batch of the WHO Melting Point Reference Substance for acetanilide,
Control No 192171, 1s depleted and has to be replaced.
The WHO Melting Point Reference Substance for acetanilide is supplied primarily for calibration
of different instruments and methods used for determination of melting temperatures against the
method of The International Pharmacopoeia, Third Edition, Volume 1.
Material
About 1000 g of the sample (manufacturers batch no K22532110) were received at the WHO
Centre in May 1997. The material is being stored in tightly closed containers at + 20 °C, protected
from hight.
Analytical data
Description
A white crystalline powder.
Evidence of chemical structure
Infrared spectrum
The spectrum is concordant with the spectrum of the previous lot of the International Chemical
Reference Substance (ICRS) of acetanilide with Control No 192171. An infrared spectrum 15 given
in Figure 1 (No W297171T).
abe) Fb ase] TL La PEN a dalla A o LA o de ads dead Cae] pot | eo pe re al rb, " "aa = = af lem op] ploy py mk md be pe USBI AA
WHO/PHARM/97.595
page 41
Mi CAT SP Te
MIT ILL
4000 0 3500 3000 500 2000.0 1800 1600 1400 1200 1000 800 500 450.0
(+!
Figure 1. IR-spectrum of 1.0 mg of acetanilide Control No 297171 in 300 mg of potassium
bromide recorded against a potassium bromide disc.
Instrument; Perkin-Elmer 1600 FTIR.
Purity
Assigned melting point: 116 °C
Based on results from a collaborative study. See under Collaborative study at the end of this
Appendix.
Thermogravirnetric analysis
The substance is already volatile at 90 °C, therefore thermogravimetric analysis 1s not a suitable
method.
Water
< 0.1 % (n=2) determined by Karl Fischer titration.
High performance liquid chromatography
No impurities above the limit of quantification (0.08 %) were found. A chromatogram 1s given in
Figure 2.
пл св
Detector Reapanse
A
WHO/PHARM/97.595
page 42
13:46:12 57/02/18
—#Z97171 Sug
Er
E"
=
1.
ре;
7
A
3
‚3
A
acetanilide
tb
É
Minutes
97/07/17
Vial: q
Elution Time
VHFO249
Folds?
Taject timer 14:39:18
Inntrument: 103
Figure 2. Chromatogram of acetanilide Control No 297171 monitored at 240 nm.
The following conditions were used:
Eluent: Acetonitrile:0.05 M phosphate buffer pH 3.0 (15:83)
Column: Genesis C18, 4 um, 4.6 x150 mm |
Detector: Gynkotek UVD 3405 operated at 240 nm. ©
Pump: Gynkotek M480 operated at a flow rate of 1.0 ml/min.
Injector: Gynkotek Gina 50 operated at 8 °C,
Integrator: PeakPro (Beckman)
Sample: Acetanilide was dissolved in the eluent at a concentration of
1.0 mg/ml. 5 ul corresponding to 5 ug were injected.
Stability in the eluent:
Limit of detection:
Limit of quantification:
A sample was stored in daylight for one week in the eluent.
No signs of degradation were observed.
1 ng (0.02 %) at 240 nm
4 ng (0.08 %) at 240 nm
The same result, < 0.08 % impurities, was observed for ICRS 192171.
WHO/PHARM/97.595
Diode-array detection
page 43
The chromatographic system described above was used to record UV-spectra for the detected
peaks. The spectrum of the main peak, given in Figure 3, showed a UV-maximum at 240 nm.
The main peak was investigated by the peak purity program and showed no signs of co-eluting
impurities.
| = Acerontiide - 235.7 nm = 1000
100
30
во
20
60
20
#0
30
20
=
180 220 240 2&0 эВ ana 120 vin 220
Figure 3. UV-spectrum of acetanilide recorded in the eluent.
CP
WHO/PHARM/97.595
page 44
Capillary electrophoresis
No impurities above the limit of quantification (0.15 %) were found.
An electropherogram 15 given in Figure 4,
Opera Trace
aent 3009. , a hanlA
ав, Ga A
0.0244
5
0.0224
e
A
о бы
0.018
©
=
TAE
0.014 а 5,3
=
ui
ed
D
©
9,913 ©:
0.010
=
ОНИ f
D.C
1
p.004
aa ,
blank
0.0
A
0.007 я
o = 10 15 20
Adi tam
Figure 4. Electropherogram of acetanilide Control No 297171 monitored at 240 nm.
cP
o
Eluent:
Capillary:
— Detector:
CE Instrument:
Run temperature:
Injection:
Integrator:
Sample:
Stability mn the eluent:
Limit of detection:
Limit of quantification:
md Re rp eg mr PP Da] mean hb me Lem A mm umd muda mal mm em ao ml tam mm le anal MEE Lill eee Fe ee lee mo
WHO/PHARM/97.595
page 45
11.7 mM borate, 8.3 mM phosphate and 50 mM SDS at
pH 9.20
Fused silica (total length 67 cm, length to detector 60 cm, ID:
75 um
Beckman P/ACE Diode Array Detector operated at 240 nm
and 200 nm.
Beckman P/ACE system 5510 operated with an applied
voltage of 30 kV for 20 minutes.
50 °С.
10 seconds pressure injection.
Beckman P/ACE Station Software.
Acetanilide was dissolved in water at a concentration of
1.0 mg/ml. 10 seconds high pressure injection corresponds to
39 ng injected sample.
A sample was stored for 2 days. No signs of degradation were
observed.
0.05 % at 7240 nm
0.15 % at 240 nm
Differential scanning calorimetry
The purity was estimated to about 99.97 mol % (n=10, RSD=0.02 %) determined by differential
scanning calorimetry. The determination was performed on 2 mg using a heating rate of 2 °C per
minute.
Melting temperature (Ty):
Onset:
Instrument:
113.9 °C (n=10, R5D=0.1 %)
113.7 °C (n=10, RSD=0.09 7)
Perkin Elmer DSC7 Differential Scanning Calorimeter
No signs of polymorphic forms were found when the substance was heated from 40 °C to
200 °C.
The previous batch ICRS 192171 showed a purity of 99.95 mol %.
WHO/PHARM/97.595
page 46
Data given by the manufacturer
Identification IR: Conforms.
Assay: 98.5-101.0 %
Anilin: < 0.01 %
Phenol: < 0.002 %
Sulphated ash: < 0.1%
Water: < 0.72 %
Collaborative study
The melting point was assigned after a collaborative study including four laboratories. The melting
point was determined according to The International Pharmacopoeia, Third Edition, Volume 1,
as a capillary melting point determination.
Participating laboratories:
Lab |: Bjôrn Egil Olsen
Norsk Medicinaldepot AS
Sven Oftedalsvej 10
PO Box 100
N 0518 OSLO
NORWAY
Lab 2: Camilla Alvesson
Mettler Toledo AB
Hangpilsgatan 36
42677 Västra Frôlunda
GOTEBORG
SWEDEN
Lab 3: Dr Ulrich Rose
Service de la Qualite "du Medicament
European Pharmacopoeia Laboratory
B.P.907
F-67029 Strasbourg Cedex 1
FRANCE
Lab 4: WHO Collaborating Centre for Chemical Reference Substances
STOCKHOLM
SWEDEN
e
WHO/PHARM/97.595
page 47
RESULTS
Method _ — [labl [12352 |[Lab3 [Labd | Mean
Capillary melting point, °C 115.7 115.5 116.0 115.5 115.7
а 6 6 6 6 4
RSD, % 007 | 0.1 0.3 0.3 0.3
Assigned melting point:116 °C
Stability
No special stability studies were performed as it was considered that this substance, based on the
experience of the stability of the previous lot, was stable and showed no signs of degradation when
stored for 34 years at + 20 °C, Regular re-examinations of this ICRS when stored in the dry state
will be performed.
Conclusion
Acetanilide, Control No 297171, can be considered suitable as WHO Melting Point Reference
Substance for the intended purpose with the melting point set to 116 °C.
WHO/PHARM/97.595
page 48
Captopril
Control No 197214
Analytical Report
Intended vse
The monograph for Captopril in The International Pharmacopoeia, Third Edition, Volume 5,
requires a reference substance of captopril to be used in the infrared spectrophotometric and thin-
layer chromatographic tests for identity as well as in the liquid chromatographic system suitability
test under related substances.
Material
About 100 g of the sample (manufacturers batch no SQ 14225 Capto-1-Re) were received at the
WHO Centre in November 1997. The material is being stored in tightly closed containers at + 5 °C,
protected from light.
Analytical data
Description
A white powder, with characteristic sulfide-odour,
Evidence of chemical structure
Infrared spectrum
An infrared spectrum is given in Figure 1 (No W970028T). The spectrum is concordant with the
spectrum of the United States Pharmacopeia Reference Standard (USPRS) lot H of captopril.
WHO/PHARM/97.595
page 49
100.0
Ad LNT ATR
40
20
0.0
40.0 200 3000 PK 000.0 12800 1600 1400 1200 1000 SO BN 450.0
Get
Figure 1. IR-speCtrum of 1.1 mg of captopril Control No 197214 in 300 mg of potassium bromide
recorded against a potassium bromide disc.
Instrument: Perkin-Elmer 1600 FTIR.
High pe ance Наша с
A spectrum of the main peak (captopril) was recorded by atmospheric pressure chemical ionization
(APcD) in the positive ion mode. The spectrum, given in Figure 2, shows an [M + ET ion of 218.2,
which supports the identity of captopril.
Eluent:
Column:
Columna temperature:
Pump:
Detector:
Operating conditions:
Sample:
atoeranhv wi s-sbectrometric detect
Methano!:0.5 % glacial acetic acid (44:56)
Kromasil, C18, 4.6x150 mm, 5 um particles
40 °C
Hewlett Packard 1050 operated at a flow rate of 1 ml/min.
After splitting the flow rate into the mass spectrometer was
0.1 ml/min.
Fisons Platform II (quadrupole mass spectrometer)
Cone voltage 20 V
Source temperature 180 °C
Captopril was dissolved in the eluent at a concentration of
I mg/ml. After splitting about 2 Lts reached the mass
spectrometer.
Captopril ICRS 197214
WHO/PHARM/97.595
page 50
CAPTOP20 346 (6,410) Cm (318:284-(208:246+467: 03), Scan AP+
190- 6.3 1.1304
|
|
1
|
|
70.5
%-
172.2 [М + H]*
| 218.2
200.2
114.3 | 117.4
103.3 138.3 1702 | 173.2
* вая. в6.4 113.3 - - 498.2 | 2014 219.1
ло oa dat bre Sion ag
Cuy paa Ces pa cest Ip dra dy of 4 dd er A a A um wi dud mead rd it Fr rim aa RET wrayer dg de PEE aa DRE
о Eg de ET do eT TR еда lese
Figure 2. Positive-ion atmospheric pressure chemical ionization mass spectrum of captopril
Control No 197214.
V-snect
A UV-spectrum in 0.1 M NaOH was recorded on a Varian Cary 5 spectrophotometer. The spectrum
is given in Figure 3.
À, shoulder was observed around 235 nm.
A: Вы =259 at 235 nm (n=6, RSD=0.6 7%), calculated with reference to the dried substance,
Ы ve Pa aa
Am mt] ppl dpe HITS ea HR DT La 0a E10 100102 fal lr TR АРАРАТ СР РРР EEE yy ry AUOT LU lili menos mareo abren hn
WHOQ/PHARM/97 595
page 51
ABS
1.0000 +
0.3000 -
235 nm
0.0000 —— — — — are .
250.00 350.00 450.00
NM
—_— Captopril 1
Figure 3. UV-spectrum of captopril Control No 197214, 11 ug/ml in 0.] M NaOH.
Thin-laver chromatography
For the identity of captopril see results under Purity/Thin-layer chromatography.
E
Assay
spectrophotometric assay
99.8 % (n=6, RSD=0.6 %). The determination was performed according to the method described
above under Evidence of chemical structure/UV-spectrum. The USPRS lot H of captopril was used
as standard and regarded as 100 %. The difference between the proposed ICRS and the USPRS lot
H is, statistically, not significant at the 95 % confidence level using unpaired t-test.
hermoeravi 1c analvsi
This technique was not suitable to use as stabilization of weight during the analysis never occured.
Captopril melts at 107 °C. Loss on drying was used instead.
WHO/PHARM/97,595
page 52
Water
It was not possible to use Karl Fischer titration as captopril reacted with the reagent resulting in
values that are too high,
Less on drying
0.1 % determined at 60 °C under reduced pressure over phosphorus pentoxide.
< 0.1 %. The test included methanol, ethanol, acetone, acetonitrile, dichloromethane, pyridine,
chloroform, benzene, trichloroethylene and dioxan. They were all estimated to be < 100 ppm.
The content of organic solvents was tested by gas chromatography with the following conditions:
Instrument: Hewlett Packard 6890
Column: HP-5 (30 mx 0.53 mm, 2.65 um film)
Camier gas: _ Helium (8 ml/min)
Detector: FID
Injection volume: 2 pl
Injector temperature: 200 °C
Detector temperature: 250 °C
Temperature program: 40 °С for 10 minutes, 40 °C/min and holding 240 °C for
5 minutes.
Sample: 20 mg were shaken with 1 ml of benzyl alcohol and another
20 mg were shaken with 1 ml of Milli ) purified water each
for about five minutes. The sample shaken with benzyl alcohol
was filterad.
Purity
3-Mercapto-2-methyipropanoic acid: « 0.01 % determined by gas chromatography according to
The International Pharmacopoeia, Third Edition, Volume 5.
['un-layer chromatography
One secondary spot, with the same Ry as captopril disulfide, was detected. When determined
against an external standard its amount was roughly estimated to 0.7 %. The following thin-layer
chromatographic system according to The International Pharmacopoeia, Third Edition, Volume 5,
was used.
Thin-layer: Silica gel 60 F-254 (Merck) TLC and HPTLC
Eluent: Toluene:glacial acetic acid:methanol (75:25:1)
Sample: 100 pg of captopril dissolved in methanol were applied.
Visualization: Scanning at 200 nm with Camag TILC-scanner 3 was
performed as well as visualization in day-light after spraying
with 2, 2-dinitro-5,5"-dithiodibenzoic acid/methanol.
e
CTD
Petector Besponae
Laptapril LORS 10ug
Y
AE
Le qu a hea
WHO/PHARM/97.595
page 53
Ry (captopril)=0.3 (TLCY/0.4 (HPTLC)
Ry (captopril disulfide)=0.1 (TLC/HPTLC)
The limit of detection of the impurity captopril disulfide was about 0.1 Hg (0.1 %) when scanning
at 200 nm.
The spot of captopril corresponds in position and appearance with that of the USPRS lot of
captopril.
High ormance liguid chroma h
One impurity above the limit of quantification was found. It was identified by LC-MS as
captopril disulfide and estimated to be 0.5 % (n=6, RSD=10.8 %) against an external
standard. A chromatogram is shown in Figure 4,
| | 13:48:58 97/12/18
captopril
|
La hrs Lu al tad bat a Pb Id modo M fella ыы ld bell [e TIA Dro tario] alma ab] SE bd Es ETE Ta ha ni ET il TEE AE. ATL emba I A 001 ee ener or E Y NETO ter ay et РНИИ
Per a "nl " и —]—L— TTD) ul
da
=
be
al > а
с = na
= . | in + |
м y =
= wi En
= Е ©
= 3 =
i pi
| =
Сы TE EE pr rr x —
+ т — т -
y — т T |
10 =0
Elution Time Mimicasg
VRV03132/1 Inject time: 20:26:57 97/12/01
SEracck method Instrument: 102 vial: 8
Figure 4. Chromatogram of captopril Control No 197214 monitored at 200 nm.
WHO/PHARM/97.595
page 54
The following conditions were used:
Eluent: Methanol:0.1 % concentrated phosphoric acid in
water pH 2.1 (44:56, mixed by hand).
Column: Kromasil, C8, 4.6 x 250 mm, 5 um particles
Column temperature: 40 °C
Detector: Varian Polychrom 9065 operated at 200 nm.
Pump: Varian 9012 operated at a flow rate of 1.0 ml/min.
Injector: Carnegie Med. CMA/200 operated at § °C,
Integrator; PealPro (Beckman)
Sample: Captopril was dissolved in the eluent at a concentration of
1.0 mg/ml. 20 ul corresponding to 20 pe were injected,
Stability in the eluent; When a sample was stored for 24 hours in the dark at 8 °C, a
degradation of 1.3 % was observed. The solutions should be
analyzed within two hours, when stored at 8 °C.
Limit of detection: 5 ng (0.03 %) at 200 nm
Limit of quantification: 17 ng (0.09 %) at 200 nm
Diode-array detection
The chromatographic system described above was used to record UV-spectra for the detected
peaks. The spectra of the impurity peaks, as well as the main peak, were similar with UV-maxima
at about 200 nm, The wavelength of 200 nm was chosen since in the purity method, it gave the best
sensitivity for all peaks. The main peak was investigated by the peak purity program and showed
no signs of co-eluting impurities. One possible impurity, 2-methyl-3-acetyIthiopropionic acid, has
another UV-spectrum with a maximum around 230 nm. This impunty was, however, not found in
this ICRS. |
Impurities identified by LC-M
The method described under Evidence of chemical structure/High-performance liquid
chromatography with mass-spectrometric detection was used to identify the impurity eluting at
15 minutes in Figure 4. The impurity was identified as captopril disulfide, which was also verified
by an external standard.
Differential scanning calorimetry
The purity was estimated to about 99.5 mol % (n=6, RSD=0.04 %). The determination was
performed on 2 mg using a heating rate of 2 °C per minute.
Melting temperature (TM): 106.7 °C (n=6, RSD=0,05 %)
Onset: 105,6 °C (n=6, RSD=0.06 %)
Instrument: Perkin Elmer DSC7 Differential Scanning Calorimeter
No polymorphism was found, when measuring between 40 and 125 °C,
lon €
The ICRS
WHO/PHARM/97,595
page 55
atograph
was screened for the following anions: chlorides, bromides, nitrates, phosphates and
sulfates. < 10 ppm of bromides, < 30 ppm of nitrates and < 100 ppm of sulfates were found.
Chlorides and phosphates were not possible to determine as they co-elute with captopril.
The following conditions were used:
Eluent 1: Sodium carbonate/sodium bicarbonate 1.8 mM/ 1.7 mM
Eluent 2: Sodium bicarbonate (for the determination of chloride)
2.5 mM
Column: Dionex HPIC AS4A
Supressor: ASRS-1
Detector: Dionex 2000i, conductivity
Range: 3 us
Pump: - Dionex 2000;
Injector: Beckman System Gold Autoinjector 507
Integrator: PeakPro (Beckman)
Sample: Captopril was dissolved in the eluent at a concentration of
1.0 mg/ml in water. 50 pl corresponding to 50 pg were
injected.
lon Chlorides Bromides Nitrates Phosphates Sulfates
Limit of detection, ppmin = 10 10 30 100 30
the sample: o
Limit of quantification, 30 50 100 500 100
ppm In the sample:
Data given by the manufacturer
Identification IR: Spectrum is consistent with structure.
Assay HPLC: 99.5 % "as is”
lodometric titration: 99.5 % "dried basis”, 99.4 % "as is”
Chromatographic purity
(HPLC):
Captopril disulfide 0.4 %
Individual impurities Complies
Unknown Complies
Total other impurities Complies
WHO/PHARM/97.595
page 56
Chromatographic impurities
(TLC):
Captopril disulfide < 0.5 %
L-Proline (5024836) ND
50 14224 №
5Q 25487 (2-methyl-3- ND
acetylthiopropionic acid)
3-Mercapto-2-
Gey, panas Acid 0.001 %
Loss on drying: 0.06 %
Melting point: 108 °C
Specific optical rotation: -129°
Heavy metals: < 30 ppm
Restdue on ignition: 0.0 7%
Appearance: Crystalline powder, practically free from
visible impurities
Color: White to off-white
Odor: Characteristic sulfide-odor
Stability
Regular re-examinations of the ICRS when stored in the dry state will be performed.
Conclusion
Captopril, Control No 197214, can be considered suitable as International Chemical Reference
Substance for the intended purpose.
т: plus grey gn ee pm ТТТ У aaa ar ie ar
WHO/PHARM/97 595
page 57
APPENDIX 11
Ciprofloxacin hydrochloride
Control No 197210
Analytical Report
Intended use
Bam onograph or Ciprofloxacin hydrochloride in The International Pharmacopoeia, Third
> YOlume 2, requires à reference substance of ciprofloxacin hydrochlori | |
Infrared spectrophotometric and thin-la yerochionide to be used in the
"ayer chromatographic tests for identir Il as j qui
chrotmatographic assay. The mono . E y as well as in the liquid
| ; graph for Ciprofloxacin in The International Ph ;
Third Edition Volume 5 requires à refe т ‘ona armacopoela,
e > rence substance of ciprofloxacin h i
In the liquid chromatographic test for related substances. P ydrochloride to be used
Material
ow: 0 g of the sample (manufacturers batch no R-124-2) were received at the WHO Centre in
uly . The material is being stored in tightly closed containers at + 5 °C, protected from licht
ght.
Analytical data
Description
A pale yellow powder.
Evidence of chemical structure
Infrared spectru
Ar infrared spectrum js given in Figure 1 (No W.197210T).
€ spectrum is concordant with the spectrum of the European Pharma | |
со
Substance (EPCRS) lot 1 of ciprofloxacin hydrochloride. - POS Chemical Reference
AL и hu PA pala mp IO, [queda qe el ere mr
WHO/PHARM/97.595
page 56
was 77 TT
A |
E Par
APRA ОНО
: M
4000.0 3500 3000 2500 2000.0 1800 1600 1400 1200 1000 800 500 AD
C1 +
100.01
Figure 1. IR-spectrum of 1.0 mg of ciprofloxacin hydrochloride Control No 197210 in 300 mg of
potassium bromide recorded against a potassium bromide disc.
Instrument: Perkin-Elmer 1600 FTIR.
High performance liquid chromatoaraphy with mass- hectrometric detectic
A spectrum of ciprofloxacin was recorded by electrospray (ESI) in the positive ion mode. The
spectrum shows an [M + H]" ion of 332.0, which supports the identity of ciprofloxacin. The
spectrum, given in Figure 2, is concordant with the spectrum of the European Pharmacopoeia
Chemical Reference Substance (EPCRS) lot 1 of ciprofloxacin hydrochloride,
Eluent: Acetonitrile:1 % glacial acetic acid in water (50:50)
Column: Direct inlet
Pump: Hewlett Packard 1050 operated at a flow rate of 0.2 ml/min
Detector: | Fisons Platform II (quadrupole mass spectrometer)
Operating conditions: Cone voltage 65 V
Source temperature 140 °C
Sample: Ciprofloxacin hydrochloride was dissolved in the eluent at a
concentration of 0.01 mg/ml. 20 pl corresponding to 200 ng
were injected.
AA Tea oi e Bs Tm mm [HTH ml ee il ili a ef adr th Lin ama
ta ud ll TNA TITAN TT ATADO AN Cp Ly FE ara a UE CA CLP TL LT E ЦА СЫ НАЗ AT ЧЕРТА] MEN derriere a ved}
WHO/PHARM/97 595
page 59
Ciprofloxacin HCI ICRS 147210 ÉS 140 € CVE5 ACN-4 e en
CIPROFL2 46 (3,935) Gm (40:54-(25;31+13:20)) "1 % НАс (50:50)
100- 314,0 ma
245.0
+
+
788.0 | м A |
332,0
@ e 231.0 `
|
‘ 289.0 315.0
69.9 - |
: 232.0 268.0
В 9 147.8 162.9 4749 190. я, 286.0
160.3 174,
EN 50. pus a 1278. oN ) | N 6 233.0 || 249.0 ma 294.0 330,0 {{334.0
0 Liem here a crue “100 mA a 120 he ча ig и | oll 4 К “а ая f. +" рее т \ ho... We ele . Dale
240 260 280 300 320 340
Figure 2. Positive-ion electrospray mass spectrum of ciprofloxacin hydrochloride
Control No 197210.
ect
A UV-spectrum In water was recorded on a Varian Cary 5 spectrophotometer, The spectrum is
given in Figure 3.
A UV-maximum was observed at 275 nm.
А кт =1079 at 275 nm (n=6, RSD=0.4 %),
Calculations were performed with reference to the dried substance.
WHO/PHARM/97.595
page 60)
ABS
1.0000
275 nim
0.5000
0.0000 -
200.00 300.00 a 400.00 500.00
NM
—— Ciprofloxacin HCl 2
Figure 3. UV-spectrum of ciprofloxacin hydrochloride Control No 197210. 4.6 ug/ml in water.
Thin-lav aph
For the identity of ciprofloxacin hydrochloride see results under Purity/T hin-layer chromatography.
Chloride
For the identity and estimation of chloride see under Purity/Ion Chromatography.
Assay
ICH] toeraphic a
100.5 % (a=6, RSD=0.5 %). The determination was performed at 278 nm according to the method
described below under Purity/High performance liquid chromatography. The EPCRS lot 1 of
ciprofloxacin hydrochloride was used as standard and regarded as 100 %. The result was calculated
on the dried substances. The difference between the proposed ICRS and the EPCRS lot 1 is,
statistically, not significant at the 95 % confidence level using unpaired t-test,
a
100.0 % (n=6, R3D=0.4 %). The determination was performed according to the method described
above under Evidence of chemical structure/UV-spectrum. The EPCRS lot 1 of ciprofloxacin
hydrochloride was used as standard and regarded as 100 %. Calculations were performed with
reference to the dried substances,
WHO/PHARM/97 595
page 61
When the substance was heated to 120 °C, a loss of 6.0 % (w/w) was observed (n=6, RSD=2.2 %).
Instrument: Perkin-Elmer TGA 7
Sample weight: 3 mg
Heating program: 3 °C/min from 20-120 °C and then holding 120 °C for
40 minutes
Melting point: 253-257 °C (decomposition) for ciprofloxacin
The corresponding result for the EPCRS lot 1 was 6.4 % (w/w), (1=6, RSD=0.9 %).
Water
"6.1 % (n=2) determined by Karl Fischer titration,
Organic volatile compounds
< 0.1 %. The test included methanol, ethanol, acetone, acetonitrile, dichloromethane, pyridine,
chloroform, benzene, trichloroethylene and dioxan. Each of them were estimated to be <100 ppm.
The content of organic solvents was tested by gas chromatography with the following conditions:
Instrument: Hewlett Packard 6890
Column: HP-5 (30 m x 0.53 mm, 2.65 um film)
Carrier gas: Helium (8 ml/min)
Detector: FID
Injection volume: 2 ul
Injector temperature: 200 °C
Detector temperature: 250 °C
Temperature programm: 40 °C for 10 minutes, 40 °C/min and holding 240 °C for
5 minutes. |
Sample: 20 mg were shaken with 1 ml of benzyl alcohol and another
20 mg were shaken with 1 ml of Milli Q purified water each for
about five minutes. If undissolved particles were found, filtration
was performed.
Purity
Thin- 0 graph
One secondary spot, probably the ethylenediamine-compound was detected, but estimated to be
< limit of quantification (0.1 %). No detectable amounts of fluoroquinolonic acid were found, not
even when scanning at 254 nm where a higher UV-absorbance was obtained. The following thin-
layer chromatographic system according to The International Pharmacopoeia, Third Edition,
Volume 3 was used.
WHO/PHARM/97.395
page 62
Thin-layer: Silica gel 60 F-254 (Merck) TLC.
Eluent: Acetonitrile:ammonia (260g/1):methanol:dichloromethane
(1:2:4:4)
Sample: 100 and 50 ng of ciprofloxacin hydrochloride dissolved in
acetic acid were applied.
Visualization: Scanning at 278 nm with a CAMAG TLC Scanner 3 was
performed as well as visualization in UV-light of 254 nm,
Re (ciprofloxacin hydrochloride)=0.54
Ry Giuoroquinolonic acid)=0.68
Ry (desfluoro-compound)=0.47
Ry(ethylenediamine-compound)=0.43
Ry (by-compound A)=0.49
The spot of ciprofloxacin hydrochloride corresponds in position and appearance with that of the
EPCRS lot 1 of ciprofloxacin hydrochloride.
No secondary spots were detected in EPCRS lot 1.
E ce liquid ¢ atogra
No impurities above the limit of quantification were found, which means < 0.1 % when determined
by peak area normalization. External reference substances of four possible impurities were injected.
They were all estimated to be below the limit of detection or quantification which means for
fluoroquinolonic acid < 0.04 % (limit of detection), for the desfluoro-compound < 0.02 % (limit of
detection), for the ethylenediamine-compound < 0.06 % (limit of quantification) and for by-
compound A < 0.06 % (limit of detection).
A chromatogram is shown in Figure 4.
#
o
Y PA 30 PSP SE EP sp
[DR E я
Patector HAedpornas
134 Г
рН
| 110
_Piprofli NCi 54g
Hr aba Vds idad fda mr lat be ru fe ee EE
WHO/PHARM/97.595
page 63
15131115 97/04/02
by-compound A
esfiucra-compound
ethyelenediamine-compound
ciprofloxacin hydrochloride
unknown < LOQ
pr
=
Ea
=
Élution Time Minutes
fnjedt timer 23:35:70 37/03/14
THHOISÜ
vial: Ÿ
Scratch method Inarrumedndt: 103
Figure 4. Chromatogram of ciprofloxacin hydrochloride Control No 197210 monitored ar 278 nm.
The following conditions were used:
Eluent: Acetonitrile:Phosphate buffer with triethylamine (10:90)
Buffer: Phosphoric acid (2,45 8/1) mixed with triethylamine to
get a pH of 3.0. |
Column: Genesis C18, 4 pm, 4.6 x 150 mm
Column temperature: 40 °C
Detector: Gynkotek UVD 3408 operated at 278 nm.
Pump: Gynkotek M480 operated at a flow rate of 1.5 ml/min.
Injector: — Gynkotek Gina 50 operated at 8 °C.
Integrator: PeakPro (Beckman)
Sample: Ciprofloxacin hydrochloride was dissolved in the eluent at a
concentration of 0.5 mg/ml. 10 pl corresponding to 5 ug were
injected. |
Stability in the eluent: A sample was stored for about 15 hours. No signs of
degradation were observed.
Limit of detection: 2 ng (0.04 %) at 278 nm.
Limit of quantification: > ng (0.1 %) at 278 nm.
WHO/PHARM/97.595
page 64
In this system fluoroquinolonic acid elutes late. In order to elute it within 30 minutes à gradient was
used. The amount of fluoroquinolonic acid was determined by another liquid chromatographic
method optimized for fluoroquinolonic acid.
The same result, < 0.1 % impurities, was observed for EPCRS lot 1.
Diode- | .
The chromatographic system described above was used to record UV-spectra for the main peak of
ciprofloxacin and for the potential impurities. Ciprofloxacin showed UV-maxima at 205 and
278 nm. Fluoroguinolonic acid showed main maxima at 255 and 262 nm, by-compound À at
255 and 270 nm, desfluoro-compound at 283 nm and the ethylenediamine-compound at 276 nm.
The wavelength of 278 nm was chosen in the purity method. The main peak was investigated by
the peak purity program and showed no signs of co-eluting impurities. The impurities mentioned
will be available as separate impurity reference substances.
Differential scanning calorimetry
The thermogram of ciprofloxacin hydrochloride showed a small exotherm at about 160 *C which
indicates the presence of another polymorphic form. The melting temperature (peak maximum) is
about 325 °C for ciprofloxacin hydrochloride.
Similar results were obtained for the EPCRS lot 1.
Ion chromatography
The ICRS was screened for the following anions: chlorides, bromides, nitrates, phosphates and
sulfates. Less than 10 ppm of bromides, 100 ppm of nitrates and less than 500 ppm of phosphates
were found. Sulfates were found and estimated to 350 ppm. Chlorides were found and roughly
estimated to be 9.7 % (theoretical value 9.2 %).
The following conditions were used:
Eluent 1: Sodium carbonate/sodium bicarbonate 1.8 mM/ 1.7 mM
Eluent 2: Sodium bicarbonate (for the determination of chloride)
2.5 mM
Column: Dionex HPIC AS4A
Supressor: ASRS-1
Detector: Dionex 20001, conductivity
Range: 3 uS (for determination of chloride 300 us)
Pump: Dionex 20001
Injector: Beckman System Gold Autoinjector 507
Integrator: PeakPro (Beckman)
Sample: Ciprofloxacin hydrochloride was dissolved in the eluent at a
concentration of 1,0 mg/ml in water. 50 ul corresponding to
50 ug were injected.
ul ms ms Y mo ml mm mo coo 1
WHO/PHARM/97 595
page 65
Ion Chlorides Bromides Nitrates Phosphates Sulfates
Limit of detection, ppmin 10 10 30 100 30
the sample:
Limit of quantification, 50 50 100 500 100
ppm in the sample:
Data given by the manufacturer
Appearance: Complies
Identity IR: Complies
Identity TLC: Complies
Identity chloride: Complies
® Clarity of solution: Complies
Colour of solution: Complies
pH value: 3.8 (limits 3.0-4.5)
Sulfate: < 200 ppm (limit max 400 ppm)
Heavy metals: < 10 ppm (limit max 20 ppm)
Water: 6.03 % (limits 4.7-6.7 %)
Sulfated ash: < 0.05 % (limit max 0.1 %)
By-products HPLC
Fluoroquinolonic acid: < 0,05 % (limit max 0.2 %)
By-product À < 0.1 %(limit max 0.2 %)
Destluoro-compound < 0.05 % (limit max 0.2 %)
Ethylenediamine-compound < 0.05 % (limit max 0.2 7%)
Other by-products individual: «0.05 % (limit max 0.2°%)
a sum of by-products: 0.1 % (limit max 0.5 %)
Assay, calculated on the dried 99.9 % (limit 98.0-102.0 %)
substance:
Stability
Stability studies were not performed as this substance was not suspected to degrade easily
according to WHO/PHARM/86,529. Regular re-examinations of the ICRS when stored in the dry
state will be performed.
Conclusion
Ciprofloxacin hydrochloride, Control No 197210, can be considered suitable as International
Chemical Reference Substance for the intended purpose. When used in the liquid chromatographic
assay according to The International Pharmacopoeia, Third Edition, Volume 5, the content of
ciprofloxacin hydrochloride is taken to be 100.0 % calculated with référence to the dried substance
which corresponds to 94.0 % on the "as 15” basis.
WEO/PHARM/97.595
page 66
APPENDIX 12
Cisplatin
Control No 197207
Analytical Report
Intended use
The monograph for Cisplatin in The International Pharmacopoeia, Third Edition, Volume 4,
requires a reference substance of cisplatin to be used in the infrared spectrophotometric and thin-
layer chromatographic tests for identity as well as in the spectrophotometric assay.
Material
About 10 g of the sample (manufacturers batch no 89 730 021/33/94 ) were received at the WHO
Centre in June 1995. The material is being stored in tightly closed containers at + 5 °C, protected
from light.
CAUTION: As cisplatin is a cytotoxic drug it should be handled with care, Avoid contact with the
skin and inhalation of airborne particles.
Analytical data
Description
À yellow crystalline powder, o
Evidence of chemical structure
Infrared spectrum
An infrared spectrum is given in Figure 1 (No W197207). The spectrum is concordant with the
spectrum of the European Pharmacopoeia Chemical Reference Substance (EPCRS) lot 1 of
cisplatin.
WHO/PHARM/97.595
page 67
N19/207.5P an a [H
100.0
80
CARAT
7
IT
0.0.
4000.0 3500 3000
2500 000.0 1800 1600 1400 1200 1000 800 600 45 9
1
Figure 1. [R-spectrum of | mg of cisplatin Control No 197207 in 300 mg of potassium
bromide recorded against a potassium bromide disc.
Instrument: Perkin-Elmer 1600 FTIR.
Eluent:
Column:
Pump:
Detector:
Operating conditions:
Sample:
ectrometric detection
A spectrum of the main peak (cisplatin) was recorded by electrospray (ESI) 1n the positive ion
mode. The spectrum shows an [M + H] jon of 300.9, which supports the identity of cisplatin. The
spectrum 1s given in Figure 2. The EPCRS lot 1 of cisplatin shows a similar mass spectrum as the
ICRS 197207,
Water 100 %
Hypercarb, 100 mm (Shandon)
Hewlett Packard 1050 operated at a flow rate of 1 m/min.
After splitting, the flow rate into the mass spectrometer was
about 0.07 ml/min.
Fisons Platform II (quadrupole mass spectrometer)
Cone voltage 35 V
Source temperature 150 °C
Cisplatin was dissolved in the eluent at a concentration of
1 mg/ml. After splitting about | Lig was injected into the mass
spectrometer.
WEO/PHARM/97.595
page 6%
Claplatin ICRS ESl+ Hyperezrb 100% Н20
АТО 170 (3.943) Cm (145:180-(V 31: 15742837152) Sean ES+
100 0.9 dd
;
202,9
| 192.3
| 201.9,
790,01
| 281.9
Я
| 4
|
{
« 173
204.0 316.9
^^ 9,
[M + Aj.
vi A
Г.
re 223.0
A 31.8 278.0 247,0 1.9
wa ¡oa EE : 245.9 5
Dima | 20 | 30а [29
fund | ih {tartan 214.4 i! |
gpd be Lh eel HEHE ”“ ay [I À. Ï | : М | бы
60 EC TOO 120 TA 180 180 20 Th 1 245 hn + “200 т 200 ye! = a
Figure 2. Positive-ion electrospray mass spectrum of cisplatin Control No 197207,
Identification of a degradation product in solution
The chromatographic system described above was used to check the identity of a peak appearing
when cisplatin was stored in an aqueous solution. A peak eluting at about
2 minutes was identified as the monohydrated complex with an [M + EH] ion of 284.
Cisplatin elutes at about 2.9 minutes. One potential impurity, transplatin, was also injected and
found to élute at about 5.6 minutes. It showed an [M + 5] ion of 300.9.
Mass spectrometry of the monohydrated complex has been reported by H. Ehrsson, I. Wallin, A.
Andersson and P-O. Edlund; Cisplatin, transplatin and their hydrated complexes: Separation and
identification using porous graphitic carbon and electrospray ionization mass spectrometry, Anal.
Chem., 67(1995)3608-361 1.
Assay
See results from the manufacturer. Assay performed according to the European Pharmacopoeia and
the United States Pharmacopeia.
avimetric analysi
When the substance was heated to 100 °C, à loss of < 0.1 % (w/w) was observed (n=3).
Instrument: Perkin Elmer TGA 7 Thermogravimetric analyzer.
Sample weight:
Heating program:
Melting point:
Water
2 mg.
5 *C/min from 20-100 °C and then holding 100 °C for 70 minutes.
270 °C with decomposition.
See results from the manufacturer.
SIA aa mul mm nf ld sme sr dd la Y mu mm li mmm Dala ema RO
WHO/PHARM/97.595
page 69
Purity
in-lave o ograph
See results from the manufacturer.
apillarv e esi
Capillary electrophoresis was chosen for the purity determination as cisplatin and the two potential
impurities transplatin and potassium trichloroaminoplatinate were possible to separate within
reasonable time and to detect without derivatization. Only one impurity was found, namely the
monohydrated complex which was estimated to 0.77 % by peak area normalization (n=9,
R3D=0.14 % for the main peak, RSD=10.4 % calculated on the 0.77 % impurity level). It was
earlier identified by LC-MS and found to increase rapidly in water solution. After 24 hours only
about 60 % of intact cisplatin remains, For stability reasons it is better to prepare the sample in
0.5 % sodium chlonde which is chosen in the method. In this solution a degradation of about 0.5 %
was observed, when stored at 15 *C for 24 hours, Electropherograms are given in Fi gure 3 (full
scale) and Figure 4 (details),
Court Traces Adload
a. MVA, E as ча a a
cisplatin
a.
pe
004
>
«>
a.
ac че
A
Ы
Y
Figure 3. Electropherogram of cisplatin Control No 197207 monitored at 210 nm.
WHO/PHARM/97,595
page 70
Crd Trama 2
ОДНО) VA. Chr iL = lagu
espia
Caras Tras A
aie Cr = T —_— mm =
4.55 3 Г 1
cisplatin
an 200 —-
6.2 © 0.00
3
© 0.0 Fan
ET
2 | =
пл g я qa a
=} ; Y
a 3 | | bla : = $ oo
= ВОТ =
5 © | =
= = ê a
= 3 6 do 3 a
Е Teg Ру | = a
2 | 5
a 2
i
I So CE. 5 Му
8
* =
Бы
алан lanos
a ost ah dm
q 3 "o a= le
ernie A A"
= =
Figure 4. Electropherogram (details) of cisplatin Control No 197207 monitored at 210 nm.
Transplatin (limit of detection=1.3 7%) and potassium trichloroaminoplatinate (limit of
detection=0.8 %) were not found in this ICRS.
The following conditions were used:
Eluent:
Capillary:
Detector:
CE Instrument:
Run temperature:
Sample temperature:
100 mM boric acid + 50 mM phosphoric acid in water, pH
adjusted to 7.0 with 2 M sodium hydroxide
Fused silica (total length 60 em, length to detector 50 cm, ID:
50 um)
P/ACE System MDQ operated at 210 nm.
P/ACE System MDQ) operated with an applied voltage of
20 kV for 60 minutes.
23°C
15 °С
We TA A o EC I E CC I PTT di rr rw LARA dE LLL Mm : Le i Pad LS TT
Injection:
Integrator:
Sample:
Stability in 0.5 % NaCl;
Limit of detection:
Limit of quantification:
Silver content
WHO/PHARM/97.595
page 71
20 seconds pressure injection, at a pressure of 0.5 psi, which
corresponds to about 24 nl.
PeakPro (Beckman)
Cisplatin was dissolved in 0.5 % NaCl at a concentration of
1.0 mg/ml.
A sample was stored for 4 h at 15 °C. No signs of degradation
were observed. After 24 h a degradation of about 0.5 % was
observed.
0.4 % at 210 nm (Cisplatin)
1.3 % at 210 nm (Transplatin)
0.8 % at 210 nm (Potassium trichloroaminoplatinate)
1.3 % at 210 nm. (Cisplatin)
4.3 % at 210 nm (Transplatin)
2.3 % at 210 nm (Potassium trichloroaminoplatinate)
See results from the manufacturer.
Platinum content
See results from the manufacturer.
Data given by the manufacturer
Pt content: 65.14 % (limit 64.9-65.2 %)
Characterization according to EP IT.
Identification IR:
Identification TLC:
Appearance of solution 51;
pH of solution S1:
Related substances:
Ag content:
Assay cisplatin:
Meets requirements.
Meets requirements,
Meets requirements.
5.88 (limits 4.5-6.0)
Meets requirements.
18 ppm based on cisplatin (limit < 250 ppm)
100.12 % (limit 97.0-102.0 %)
Characterization according to USP XXII.
Identification HPLC:
Identification IR:
Identification TLC:
Crystallinity:
Meets requirements.
Meets requirements.
Meets requirements.
Crystalline
WHO/PELARM/97.595
page 72
Water content: 0.1 % (limit ] 7%)
UV Purity, E 301/E 246: 5.12 (limit > 4.5)
Potassium 0.1 % (limit < 1%)
trichloroaminoplatinate:
Transplatin: 0.06 % (limit < 27)
Assay cisplatin: 100.77 % (limit >98 %)
stability
Regular re-examinations of the ICRS when stored in the dry state will be performed.
For stability in solution please see under capillary electrophoresis.
Conclusion ©
Cisplatin, Control No 197207, can be considered suitable as International Chemical Reference
Substance for the intended purpose. When used in the spectrophotometric assay according to
The International Pharmacopoeia, Third Edition, Volume 4, the content of cisplatin is taken to be
100 % calculated with reference to the dried substance which corresponds to 100 % on the "as 1s”
basis.
В
WHO/PHARM/97.595
page 73
APPENDIX 13
Kanamycin monosulfate
(anhydrous)
Control No 197211
Analytical Report
Intended use
The monograph for Streptomycin sulfate powder for injections in The International
Pharmacopoeia, Third Edition, Volume 4, requires a reference substance of kanamycin
monosulfate to be used in the thin-layer chromatographic test for identity.
Material
About 3.6 g of the sample, dispensed in ampoules of about 12 mg/ampoule, were received from the
National Institute for Biological Standards and Control in January 1998. The material is being
stored at -20 °C, protected from light.
This ICRS is of the same origin as that of the 1st International Biological Standard for Kanamycin.
Analytical data
Description
A white and very fluffy powder.
Evidence of chemical structure
infrared spectrum
An infrared spectrum of the untreated substance is given in Figure 1 (No W960042T).
WHO/PHARM/97.595
page 74
100.0
JA UM /
/ |
Figure 1. IR-spectrum of 1.5 mg of untreated kanamycin monosulfate Control No 197211 in 300 mg
он!
of potassium bromide recorded against a potassium bromide disc,
Instrument: Perkin-Elmer 1600 FTIR.
The spectrum in Figure | is concordant with the spectra of the United States Pharmacopeia
Reference Standard (USPRS) lot I and of the European Pharmacopoeia Chemical Reference
substance (EPCRS) batch 2 of kanamycin monosulfate when they are re-crystallized in water.
The proposed ICRS was also stored for 24 hours in 98 % relative humidity in order to pick up
moisture to re-crystallize. Before preparing an IR-disc the substance was dried. An infrared
spectrum of the substance after this re-crystallization is given in Figure 2 (960042RH).
Da | SAA, JE
100.01
Aa
ol A
4 | _ ‚ Y
7 A
yoy N
Pep 200 3000 BN gh, 180 1600 140 1200 1000 800 60 gg
Figure 2. IR-spectrum of 1.8 mg moisture treated kanamycin monosulfate Control No [97211 in
300 mg of potassium bromide recorded against a potassium bromide disc.
a
Eo ta UY Lu ap, iid nL
a aa Tr LTT] TTR) MOTE TPT TE e rm ar var eu ry | EL RAI IE
WHO/PHARM/97.595
page 75
The spectrum in Figure 2 is concordant with the spectra of the United States Pharmacopeia
Reference Standard (USPRS)
lot I and of the European Pharmacopoeia Chemical Reference
substance (EPCRS) batch 2 of kanamycin monosulfate,
High performance liquid chromatography with mass-spectrometric detection
A spectrum of kanamycin was recorded by electrospray (ESI) in the positive ion mode. The
spectrum shows an [M + HJ" ion of 485.1, which supports the identi ty of kanamycin. The spectrum,
given in Figure 3, is concordant with the spectrum of the European Pharmacopoeia Chemical
Reference Substance (EPCRS) batch 2.
Eluent:
Column:
Pump:
Detecror:
+
+
Operating conditions
Sample:
Acetonirile:1 % glacial acetic acid in water (50:50)
Direct inlet
Hewlett Packard 1050 operated at a flow rate of 0.2 ml/min
Fisons Platform II (quadrupole mass spectrometer)
Cone voltage 25 V
Source temperature 140 °C
Kanamycin sulfate was dissolved in the eluent at a
concentration of 0.01 mg/ml. 20 ul corresponding to 200 ng
were injected.
Kanamycin sulfate ICRS 197211 ESi+ 140 C CVZ5 ACN:1 % HAc (50:50)
KAMAMYCS 26 (2,235) Gm (21.33-(14:17 +10) ECcam E»
100- 217.1 1.0684
1048
[
|
O
|
161.9
%- |
|
|
|
| 230.8
203.5 |
179.9 |
143.9 263.5
162.9 |
145.8 i
| | +
194.3 | 243.1 284.2 324.4 [M + H}
j | | |
y 285.1
| в |
! i : Ad 292.0 Eo
d'u 4 ei [pose [3380 e
1 e Po Tal {db pb AH encres ETE proteina в Cerner Tagen Dale
160 180 200 770 ad 260 230 300 320 340 380 380 400 420 440 1460 480
Figure 3. Positive-ion electrospray mass spectrum of kanamycin sulfate Control No 197211.
WHO/PHARM/97,595
page 76
UV-spectrum
À UV-spectrum in water was recorded on a Varian Cary 5 spectrophotometer, No maximum above
190 nm was observed, the absorbance was calculated on the slope at 200 nm.
A [en =12 at 200 nm (n=1).
The calculation was performed on the "as 15” basis.
in-la a
For the identity of kanamycin monosulfate see results under Purity/Thin-layer chromatography.
Sulfate
For the identity and estimation of sulfate see under Purity/Ion Chromatography.
Assay
Microbiological ,
See results from collaborating laboratories. e
Thermogravimetric
When the substance was heated to 105 °C, a loss of 4.1 % (w/w) was observed (n=3, RSD=3.1 %).
The substance was analyzed immediately after opening of the ampoule, but as the substance 1s
extremely hygroscopic it readily picks up moisture before the analysis starts. The relative humidity
at the analysis occasion was 25 %.
Instrument: Perkin Elmer TGA 7 Thennogravimetric analyzer.
sample weight: 2 mg.
Heating program: 5 *C/min from 20-105 *C and then holding 105 °C for 180
minutes.
Melting point: About 270 °C with decomposition.
It was not possible to determine the content of water by Karl Fischer titration due to the high
hygroscopicity and the small guantity of sample available.
Organic volatile compounds
< 0,1 %. The test included methanol, ethanol, acetone, acetonitrile, dichloromethane, pyridine,
chloroform, benzene, trichloroethylene and dioxan. Each of them were estimated to be <100 ppm.
The content of organic solvents was tested by gas chromatography with the following conditions:
Instrument: Hewlett Packard 6890
Column: HP-5 (30 m x 0.33 mm, 2.65 um film)
Cartier gas: Helium (8 ml/min)
Detector: FID
Injection volume: 2 ul
Injector temperature: 200 °C
WHO/PHARM/97.595
page 77
Detector temperature: 250 °C
Temperature program: 40 °C for 10 minutes, 40 °C/min and holding 240 °C for
5 minutes.
Sample: 20 mg were shaken with 1 mi of benzylalcohol and another
20 mg were shaken with 1 ml of Milli Q purified water each
for about five minutes. The sample shaken with benzylalcoho!
was filtered.
Purity
Thin-laver chromatography
Two secondary spots were detected, Their total amount was estimated to be about 2.1 %. One of the
spots was identified as kanamycin B sulfate and roughly estimated to be 0.1 %. The following thin-
layer chromatographic system according to The International Pharmacopoeia, Third Edition,
Volume 4 was used.
Thin-layer: Silica gel (Merck) TLC.
Eluent: 7 % potassium dihydrogen phosphate.
Sample: 100 pg of kanamycin monosulfate dissolved in water were
applied.
Visualization: Visualization in day-light after spraying with naphthalene-
1,3-diolethanol and sulfuric acid, as well as scanning at
400 nm with a CAMAG TLC Scanner 3.
Ry (kanamycin sulfate)=0.2
Ку(Капатуст В sulfate)=0.1
The detection limit of the system was about 0.1 ug (0.1 %) both after visual inspection and when
scanning at 400 nm.
The spot of kanamycin monosulfate ICRS Control No 197211 corresponds in position and
appearance with that of the EPCRS batch 2 and USPRS lot [ of kanamycin sulfate.
The EPCRS contained two secondary spots. Their total amount was estimated to about 5.7 %.
One of the spots was identified as kanamycin B sulfate and roughly estimated to be 0.5 %.
The USPRS contained two secondary spots, Their total amount was estimated to about 5.2 %
One of the spots was identified as kanamycin B sulfate and roughly estimated to be 0.8 %.
High performance liqui omatogra
As kanamycin sulfate has practically no UV-absorbance other detection principles had to he
"chosen. Pre-column derivatization using 2,4-dinitrofluorobenzene as denoted in the monograph for
Tobramycin of the USP XXII, was found satisfactory for the detection of kanamycin.
The total amount of impurities estimated by peak area normalization was about 6.7 % (n=24,
R3D=0.13 % for the main peak, RSD=2 % calculated on the 2.4 % impurity level).
Velie
datéctoT Esdponas
WHO/PHARM/97.595
page 78
As can be seen In Figure 4 thirteen impurities above the limit of quantification were found.
One of them was identified as kanamycin B sulfate (eluting at 30 minutes). It was estimated to
0.4 % against an external standard. All peaks eluting before 23 minutes are blank peaks originating
from the derivatization agents.
8:47:08 97/11/19
=
a =
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= E
| 0
| a
7 ind
_ =
* 3 —
de E”
ra = q &
" wl un = и
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= Я "о on = A
3 An =
+ + 25 E Ze
= pu nn "= à
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J . $ | к : @
4 = = pi Е
с T = = =
J | a 1 L = = я
г = | =
ul k = 5
mn пу $ " FE —
ar El = == = Ti
; = e = = её
+ = = 5
7 = A = 2
np
J 5 = = a
ur ‚
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1 S A o
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1 Hol ' Foo | 7 - |
lay
k
TT] " { T T T T T ï 1 Ne ur" T 7 T T -
21 Aa 43
Elution Time
Figure 4. Chromatogram of kanamycin monosulfate Control No 197211 as 2,4-dinitro-
fluorobenzenederivative monitored at 365 nm.
The following conditions were used:
Eluent:
A: Acetonitrile
B: 2.0 g of tnis(hydroxymethyDaminomethane was dissolved
in water then 10.0 mi 1 M sulfuric acid was added and diluted
with water to 1000 ml.
Minutes
e
ты EEE EVE
' tel E D [D
UT alto AT 1 ed ae a LA mili] Pr LP a EA A o A "da LLE u o mmm ml ml a Pat
WHO/PHARM/97 595
page 79
Gradient: % А % В Time, Type
minutes
30 70 0-10 Isocratic
30-80 70—20 10-30 linear
80—30 20—70 30-31 linear
30 70 31-45 | isocratic
equilibration
Column: Brownlee Labs spheri 5 RP-18 at 40 °C, 4.6 x 250 mm, 5 um
particles.
Detector: Waters Lambda-Max mode! 481 operated at 365 nm.
Pump: Waters 600E operated at a flow rate of 1.0 ml/min.
Injector: Carnegie CMA 200 operated at § °C.
Integrator: PeakPro (Beckman)
Sample: Kanamycin sulfate was derivatized (see below), Sample
concentration was about 0.15 mg/ml. 20 ul corresponding to
3 yg were injected.
Derivatization:
2,4-Dinitrofluorobenzene reagent: 10 mg 2 4-dinitrofluorobenzene per ml of ethanol,
Tris(hydroxymethyl)aminomethane reagent: 20 ml of a solution containing 15 mg
tris(hydroxymethyl)aminomethane per ml of water were transferred into a 100-ml volumetric flask
and diluted to volume with dimethylsulfoxide. This reagent was used within 4 hours,
Purity determination: 1.50 ml of a sample solution containing 2.50 mg/ml water, were transferred
into a 25-ml volumetric flask and 7 ml of the 2 4-dinitrofluorobenzene reagent and 7 ml of the
tris(hydroxymethyl)aminomethane reagent were added. The solution was heated for 60 minutes in a
water bath at 60 °C. The flask was left at room temperature for 10 minutes to cool before dilution to
volume with acetonitrile.
N.B. As cooling occurs on mixing with acetonitrile, it is advisable to add the acetonitrile slowly,
permitting the mixture to reach room temperature, before the final dilution.
Stability 1n the eluent: A sample was stored for 24 hours at 8 °C in the dark. No signs
of change in the derivatized sample were observed.
Limit of detection: 0.3 ng (0.02 %) at 365 nm
Limit of guantification: 2 ne (0.06 %) at 365 nm
Ton chromatography
The ICRS was screened for the following anions: chlorides, bromides, nitrates, phosphates and
sulfates. Less than 10 ppm of bromides, less than 100 ppm of nitrates and less than 500 ppm of
phosphates were found, Chlorides were found and estimated at 500 ppm. Sulfates were found and
roughly estimated to be 16.2 % (theoretical value 16.0 %).
WEHO/PELARM/97.595
page 80
The following conditions were used:
Eluent 1:
Eluent 2:
Column:
Supréssor:
Detector:
Range:
Pump:
Injector:
Integrator:
Sample:
lon
Limit of detection, ppm in
the sample:
Limit of quantification,
ppm in the sample:
Sodium carbonate/sodium bicarbonate 1.8 mM/1.7 mM
Sodium bicarbonate (for the determination of chloride)
2.5 mM
Dionex HPIC AS54A
ASRS-1
Dionex 2000i, conductivity
3 uS (for determination of sulfate 300 5)
Dionex 20001
Beckman System Gold Autoinjector 507
PeakPro (Beckman)
Капатуст sulfate was dissolved in the eluent at a
concentration of 1.0 mg/ml in water. 50 pl corresponding to
50 ug were injected.
Chlorides Bromides Nitrates Phosphates Sulfates
10 10 30 100 30
50 30 100 500 100
Data given by collaborating laboratories
NIBSC
Description:
Microbiological assay,
determined in a
collaborative study:
Moisture content:
Stability:
Hygroscopicity:
The first International Standard for
Kanamycin (Established in 1986)
10345 International Units per ampoule.
< 0.2 % in the closed ampoule.
Accelerated degradation studies on the
sealed ampoules at 56 °С for 20 months
showed no loss in potency.
The ampoule contents are extremely
hygroscopic.
WHO/PHARM/97 395
page 31
Stability
Stability studies were not performed as this substance was not suspected to degrade easily when
stored at -20 °C. Regular re-examinations of the ICRS when stored in the dry state will be
performed.
Hygroscopicity
The ampoule contents are extremely hygroscopic. When stored for 7 hours at 98 % relative
humidity the substance gained 30 % in weight. Results are given In Figure 5. After 30 minutes it
gamed 16 % 11 weight. At continued storage for 24 hours to one week at 98 % relative humidity,
the content of water decreases to 3-7 %.
35,0
30,0 +
25,0 т
20,0 +
15,0 +
1607
50 T
0,0
Sala ot welght (%)
0,0 1.0 2,0 3,0 4,0 5,0 6,0 7.0
Time (hours)
Figure 5. Kanamycin monosulfate Control No 197211 stored at 98 % relative humidity,
As the substance is extremely hygroscopic it is advisable to let it equilibrate with the surrounding
atmosphere before weighing it. To
Conclusion
Kanamycin monosulfate, Control No 197211, can be considered suitable as International Chemical
Reference Substance for the intended purpose. As this is a biological substance it 1s difficult to
obtain a better quality. This ICRS is purer than other official reference materials.
WHO/PHARM/97.595
page 82
APPENDIX 14
Phenacetin
WHO Melting Point Reference Substance
Melting temperature 136 °C
Control No 297172
Analytical Report
Intended use
The stock of the current batch of the WHO Melting Point Reference Substance for phenacetin,
Control No 192772, 1s depleted and has to be replaced.
The WHO Melting Point Reference Substance for phenacetin is supplied primarily for calibration
of different instruments and methods used for determination of melting temperatures against the
method of The International Pharmacopoeia, Third Edition, Volume 1.
Material
About 4000 g of the sample (manufacturers batch no 207372) were received at the WHO Centre in
April 1997. The material is being stored in tightly closed containers at + 20 °C, protected from
light.
Analytical data
Descripti
A white powder,
Evidence of chemical structure
Infrared spectrum |
The spectrum is concordant with the spectrum of the previous lot of the International Chemical
Reference Substance (ICRS) of phenacetin with Control No 192172. An infrared spectrum is given
in Figure 1 (No W297172T).
WHO/PHARM/97,595
100 6 Mi — VE - TH =
В TN 0 AA
Lo Val TA
TT ti
| | MR
CM-1
Figure 1. ZR-spectrum of 1.0 mg of phenacetin Control No 297172
in 300 mg of potassium bromide recorded against a potassium bromide disc.
Instrument: Perkin-Elmer 1600 FTIR.
Purity
Assigned melting point: 136 °C
Based on results from a collaborative study. See under Collaborative study at the end of this
Appendix.
Water
< 0.1 % (n=2) determined by Karl Fischer titration.
17h pe
ance land e
oeraph
One impurity was found. It was estimated to be 0.06 % (n=6, RSD=2.6 % when calculated on the
0.06 % Impurity level, KSD< 0.01 % for the mäin peak). À chromatogram is given in Figure 2.
mails
Tetector Ecaponae
WHO/PHARM/97.595
page 84
13:35:22 ÿ#1/06/06
nPhenéé:17172 509
7
on
Le =
=
ть
—
=
=
>,
m
т= Ч
=
— =
E
or
=
pr
=
г
a e
= |
=
“|
—
=" = =
4 = =
= ©
- = Li
= = a
qu = =
J = =
a В че
oo A 206 _ a I _ _ _, —
= _ | я A т т 7 pr. + mr 7 T T
T т т то Нонна Aa L 7 т т da я TT т T 7 7
20
+9 Minczed
Elucion Time arar
: :20 06 3 i
ELIOT _ trject cima: 17 vials ÿ
Eoratch mesked Instruévraes 103 —,
Figure 2. Chromatogram of phenacetin Control No 297172 monitored at 247 nm.
The following conditions were used:
Eluent: Acetonitrile:0.05 M phosphate buffer pH 3.0 (23:77)
Column: Kromasil C18, 5 um, 4.6 x150 mm
Detector: Gynkotek UVD 3405 operated at 247 nm,
Pump: Gynkotek M480 operated at a flow rate of 1.0 ml/min.
Injector: Gynkotek Gina 50 operated at 8 °C.
Integrator: PeakPro (Beckman)
Sample: | Phenacetin was dissolved in the eluent at a concentration of
1.0 mg/ml. 5 pl corresponding to 3 pg were injected.
Stability in the eluent: A sample was stored in the eluent for about ten hours in the
dark at +8 °C. No signs of degradation were observed. When
the sample was stored in daylight for one week at + 20 °C a
degradation of less than 0.06 % was observed.
Limit of detection: I ng (0.02 %) at 247 nm
Limit of quantification: 3 ng (0.06 %) at 247 nm
In the previous batch ICRS 192172 two impurities were found and estimated to be about
0.2 %.
58.1
80.0
50.0
40.0
20.0
-0. 2
WHO/PHARM/97.595
page 85
Diode- et
The chromatographic system described above was used to record UV-spectra for the detected
peaks. The spectra of the main peak and the peak of impurity | were similar with UV-maxima at
247 nm. The main peak was investigated by the peak purity program and showed no signs of co-
eluting impurities. A UV-spectrum of the main peak recorded in the eluent is given in Figure 3.
PF - <unknmsun> - 247.0 Am - 1000
247 nm
1
(IT
193 220 240 260 280 300 320 240 260 280 397
Figure 3. UV-spectrum of phenacetin recorded in the eluent.
WHO/PHARM/97.595
page 86
Capillary electrophoresis
No impurities above the limit of quantification (0.1 %) were found.
An electropherogram is given in Figure 4.
Cheer Tri
Bre 1005 Mu A
pre ET. ET A
РЕ
ao
0,5
0.041
me
son
0 as
=.
—
D
с:
a
=
a
=:
8.024 с, 0,2
a.
aan
oe — + 7 Ar =
| blank 0.0
bo rer rt a —— — ri rar -а,
E, E Lar er”
à = a ma ME
Minutas T= =
Figure 4. Eleciropherogram of phenacetin Control No 297172 monitored at
247 nm,
с»
A de a MTA a Ud lA dal PY Op a "EEE ИГРАТЬ А
Eluent:
Capillary:
Detector:
CE Instrument:
Kun temperature:
Injection:
Integrator:
Sample:
Limit of detection:
Limit of quantification:
WHO/PHARM/97.595
page 87
11.7 mM borate, 8.3 mM phosphate and 50 mM SDS at
pH 9.20
Fused silica (total length 67 cm, length to detector 60 em, ID:
75 um
Beckman P/ACE Diode Array Detector operated at 247 nm
and 200 nm.
Beckman P/ACE system 5510 operated with an applied
voltage of 30 kV for 20 minutes.
50 °C.
10 seconds pressure injection,
Beckman P/ACE Station Software.
Phenacetin was dissolved in water at a concentration of
20.0 mg/ml in methanol and then diluted with water to a
concentration of 2.0 mg/ml (fresh solutions). 10 seconds high
pressure injection corresponds to 118 ng injected sample.
0.03 % at 247 nm
0.10 % at 247 nm
Differential scanning calorimetry
The punty was estimated to about 99.99 mol % (n=10, RSD=0,007 %) determined by differential
scanning calorimetry. The determination was performed on 2 mg using a heating rate of 2 °C per
minute.
Melting temperature (Tm):
Onset;
Instrument:
134.4 °C (n=10, R§D=0.09 %)
134.3 °С (n=10, RSD=0.07 %)
Perkin Elmer DSC7 Differential Scanning Calorimeter
No signs of polymorphic forms were found, when the substance was heated from 40 °C to
200 °C.
The previous batch ICRS 197172 showed the same result.
Data given by the manufacturer
Identification:
Assay:
Chloroacetanilide:
p-Phenetidine:
Heavy metals:
Sulphated ash:
Loss on drymg:
Conforms.
99.3 %
within limits
within limits
within limits
0.0 %
0.0%
WHO/PHARM/97.595
page 8%
Collaborative study
The melting point was assigned after a collaborative study including four laboratories. The melting
point was determined according to The International Pharmacopoeia, Third Edition, Volume 1,
ag a capillary melting point determination.
Participating laboratories:
Lab 1: Björn Egil Olsen
Norsk Medicinaldepot AS
Sven Oftedalsvej 10
PO Box 100
N 0518 OSLO
NORWAY
Lab 2: Camilla Alvesson
Mettler Toledo AB
Hängpilsgatan 56
42677 Vaástra Frólunda
GOTEBORG
SWEDEN
Lab 3: Dr Ulrich Rose
Service de la Qualite du Medicament
European Pharmacopoeia Laboratory
B.P. 907
F-67029 Strasbourg Cedex 1
FRANCE
Lab 4: WHO Collaborating Centre for Chemical Reference Substances
STOCKHOLM
SWEDEN
RESULTS
Method — Lab! |Lab2 |Lab3 |Lab4 | Mean _
Capillary melting point, °C 136.1 135.7 135.9 136.2 136.0
n 6 6 6 6 4
RSD, % 0.06 | 0.04 0.07 0.09 [0.16
“Assigned melting point: 136 °C To
Il MALA ul kat dd
hie | md т EC HT EEE i 0 = FETO Dr jr CEN AAA TUN, y A й DS LT
—]][ As da oid i 3 chaff erate A J] MEME TO TA DTO A aaa a — E at riel hdr ae y rr Ep — 8 TT MTI й —
hide dade "| Add dado Bb eeh aber a UI
WHO/PHARM/97.595
page 89
Stability
No special stability studies were performed as it was considered that this substance, based on
the experience of the stability of the previous lot, was stable and showed no signs of degradation
when stored for 34 years at + 20 °C. Regular re-examinations of this ICRS when stored in the dry
state will be performed.
Conclusion
Phenacetin, Control No 297172, can be considered suitable as WHO Melting Point Reference
Substance for the intended purpose with the melting point set to 136 °C.
WHO/PHARM/97.595
page 90
APPENDIX 15
Piperazine adipate
Control No 197212
Analytical Report
- Intended use
The monograph for Piperazine adipate tablets in The International Pharmacopoeia, Third Edition,
Volume 4, requires a reference substance of piperazine adipate to be used in the thin-layer
chromatographic test for identity.
Material
About 10 g of the sample (manufacturers batch NOENR4786) were received at the WHO Centre in
June 1996. The material is being stored in tightly closed containers at + 5 °C, protected from light.
This ICRS is of the same origin as that of the European Pharmacopoeia Chemical Reference
Substance (EPCRS) lot 1.
Analytical data
Descripti
À white powder.
Evidence of chemical structure
Infrared spectrum
An infrared spectrum is given in Figure 1 (W197212). The spectrum is concordant with the
spectrum of the United States Pharmacopeia Reference Standard (USPRS) lot F of piperazine
adipate.
ST TTT fpr pd A Er ra a A ati o AT pa
Er EE EE Te a im Fla
WHO/PHARM/97 595
page 91
Mores UTTTT | E
Ma | | ara
PAM
LA
100.0
и
=
X
_3
=
—
2 ——————]———————[—]—]lÍ Lu
0.0 LL, _ | |
e 4000.0 300 3000 200 2000.0 4800 1600 1400 1200 1000 ad 500 450.0
C4
Figure |. [R-spectrum of 0.8 mg of piperazine adipate Control No 197212 in 300 mg of potassium
bromide recorded against à potassium bromide disc.
Instrument: Perkin-Elmer 1600 FTIR.
High performance liquid chromatography with mass-spectrometric detection
A spectrum of piperazine adipate was recorded by particle beam electron ionization (PB-EI) in the
positive ion mode. The spectrum, which is given in Figure 2, is concordant with the spectrum of
piperazine adipate published in the National Institute of Standards and Technology (NIST) library.
Eluent: Methanol: Water (50:50)
Column: Direct inlet
Pump: Hewlett Packard 1050 operated at a flow rate of 0.5 ml/min
e Detector: Fisons Platform Y (quadrupole mass spectrometer)
Operating conditions: Electron energy 70 V
Source temperature 250 °C
Sampie: Piperazine adipate was dissolved in water at a concentration
of 0.1 mg/mi. 10 ul corresponding to ! Le were injected.
WHO/PHARM/97.595
page 92
Piperazino adipate JCRS 197212 El+ 250 0
PIPERAAT 54 (4,615) Cim {52:57 -(48:50+51:84))
; Scan El+
100, “E 5.826
56.2
#5 1
= ,
| 82.2
57.2
>
85.1
86.1
45.3
51,7 wh
80.1 84,1
6% 1 77.1
DIT ame me me e mes By
ado na Ara AA eee же , di i pF Ef gE A тесты Per a A Pa pa 0 mp =
40 45 60 5 eo es Ue "o gs д ws 7 740 77445 0 7 5 150
Figure 2. Positive-ion electron ionization mass spectrum of piperazine adipate Control No 197212.
UV-spectrum
Piperazine adipate has practically no UV absorbance except at wavelengths below 210 nm.
A UV-spectrim in water was recorded on a Varian Cary 5 spectrophotometer.
A UV-maximum was observed at 195 nm. A 1, was about 46 (n=1).
l'hin-laver chromatography
For the identity of piperazine adipate see results‘under Purity/Thin-layer chromatography.
Assay
See results from collaborating laboratories.
e VI
When the substance was heated to 105 °C, à loss of < 0.1 % (w/w) was observed (n=6).
Instrument: Perkin Elmer TGA 7 Thermogravimetric analyzer.
Sample weight: 10 mg.
Heating program: 5 *C/min from 20-105 *C and then holding 105 °C for
360 minutes.
Melting point: 250 °C with decomposition.
WHO/PHARM/97.595
page 93
0.1 % (n=2), determined by Karl Fischer titration.
reanic volatile com
< 0.1 %. The test included methanol, ethanol, acetone, acetonitrile, dichloromethane, pyridine,
chloroform, benzene, trichloroethylene and dioxan. Each of them were estimated to be < 100 ppm.
The content of organic solvents was tested by gas chromatography with the following conditions:
Instrument: Hewlett Packard 6890
Column: HP-5 (30 m x 0.53 mm, 2,65 um film)
Carrier gas: Heltum (8 ml/min)
Detector: FID
Injection volume: 2 1
Injector temperature: 200 °C
Detector temperature: 250 °C
Temperature program: 40 °C for 10 minutes, 40 *C/min and holding 240 °C for
5 minutes.
Sample: 20 mg were shaken with 1 ml of benzyl alcohol and another
20 mg were shaken with 1 mi of Milli Q) purified water each
for about five minutes, The sample shaken with benzyl alcohol
was filtered.
Parity
No secondary spots were detected, which means < 0,25 %. The following thin-layer
chromatographic system according to The International Pharmacopoeia, Third Edition, Volume 4,
was used.
Thin-layer: Silica gel G (Merck) TLC
Eluent: Ammonia conc.:acetone (20:80)
Sample: 300 ug of piperazine adipate dissolved in ethanol:ammeonia
conc. (2:3) were applied.
Visualization: Visualization tn day-light after spraying with
triketohydrindenehydrate (3 mg/ml) in glacial acetic acid/1-
butanol (3:100) and then with triketohydrindenehydrate
(1.5 mg/ml) in ethanol and drying at 105 °C, Thereafter
spraying with iodine (0.03 mol).
АМА РАЯ ll mE mo! Paba be am Danko dm milo aime em LEI yp
WHO/PHARM/97.595
page 94
Re(piperazine adipate)=0.1
Rr(ethylenediamine)=0.4
Re(triethylenediamine)=0.2
Re(adipic acid)=0.04
Rr (piperazine)=0.08
The detection limit of the system was about 1.25 pg (0.25 %) when examined visually.
The spot of piperazine adipate corresponds in position and appearance with that of the USPRS lot F
of piperazine adipate.
ton chromatography
The ICRS was screened for the following anions: chlorides, bromides, nitrates, phosphates and
sulfates. Less than 300 ppm of chlorides, less than 10 ppm of bromides, less than 100 ppm of
phosphates and less than 30 ppm of nitrates were found. Sulfates co-eluted with adipate and could
not be determined.
The following conditions were used:
Eluent 1:
Eluent 2:
Column:
Supressor:
Detector:
Range:
Pump:
Injector:
Integrator:
sample:
fon
Limit of detection, ppm in
the sample:
Limit of quantification,
ppm in the sample:
Sodium carbonate/sodium bicarbonate 1.8 mM/ 1.7 mM
Sodium bicarbonate (for the determination of chloride)
2.5 mM
Dionex HPIC AS4A
ASRS-1
Dionex 2000i, conductivity
3 45
Dionex 20001
Beckman System Gold Autoinjector 507
PeakPro (Beckman)
Piperazine adipate was dissolved in the eluent ata
concentration of 1.0 mg/ml in water. 50 pl corresponding to 50
g were injected.
Chlorides Bromides Nitrates Phosphates Sulfates
10 10 30 100 30
50 50 100 500 100
EN
Data siven by collaborating laboratories
European Pharmacopoeia lab.
EPCRS batch 1 1s from the same batch as ICRS 197212.
Appearance:
Water content:
Identity, IR:
Identity, melting point:
Identity, melting point:
Related substances:
Assay:
Stability
Satisfactory
0.1 %
Conforms.
248 °C
151 °C (as piperazine)
0.3 %
100.22 %
WHO/FHARM/97 595
page 95
Stability studies were not performed as this substance was not suspected to degrade easily
according to WHO/PHARM/86.529. Regular re-examinations of the ICRS when stored in the dry
state will be performed.
Conclusion
Piperazine adipate, Control No 197212, can be considered suitable as International Chemical
Reference Substance for the intended purpose,
WHO/PHARM/97.595
page 96
Piperazine citrate
Control No 197213
Analytical Report
Intended use
The monograph for Piperazine citrate tablets in The International Pharmacopoeia, Third Edition,
Volume 4, requires a reference substance of piperazine citrate to be used in the thin-layer
chromatographic test for identity.
Material
About 10 g of the sample (manufacturers batch NOENR4787) were received at the WHO Centre in
June 1996. The material is being stored in tightly closed containers at + 5 °C, protected from light.
This ICRS is of the same origin as that of the European Pharmacopoeia Chemical Reference
Substance (EPCRS) lot 1.
Analytical data
Description
A white granular powder.
Evidence of chemical structure
ed spect
An infrared spectrum is given in Figure 1 (W197213). The spectrum is concordant with the
spectrum of the United States Pharmacopeia Reference Standard (USPRS) lot F of piperazine
citrate.
==] la data a ol tl gi] armada all al д la [AF ST LL LER EE a o Tr a Ae AAA Frid la CU NT ad tt Fr AURA NL al a ii a LITA mr. FE SN A ATI P
WHO/PHARM/97 595
| M97243,8P TT Ts
a
0 — od a | La 1
AAN ТО
Cam
e 4000.0 300 3000 2500 000.0 1800 1500 1400 1200 1000 Boo
Os |
000 450.0
Figure 1. IR-spectrum of 1.1 mg of piperazine citrate Control No 197213 in 300 me of potassium
bromide recorded against a potassium bromide disc.
Instrument: Perkin-Elmer 1600 FTIR.
zh performance liquid ¢ atographv with mass-spectrometric tion
A spectrum of piperazine, given in Figure 2, was recorded by atmospheric pressure chemical
ionization (APcl) in the positive ion mode. The spectrum shows an [M + HT" ion of 87.2, which
supports the identity of piperazine.
Eluent: Methanol: Water (50:50)
Column: Direct inlet
e Pump: Hewlett Packard 1050 operated at a flow rate of 0.3 mi/min
Detector: Fisons Platform I (quadrupole mass spectrometer)
Operating conditions: Cone voltage 30 V
Source temperature 180 °C
Sample; Piperazine citrate was dissolved in water at a concentration of
0.01 mg/ml. 20 Ll corresponding to 200 ng were injected.
WHO/PHARM/97.393
page 98 A
| [M + H]
Piperazine citrate ICRS 197213 APci+ 180 C Direct inlat ,
PIPERACS 86 (7.335) Cm (84:07-(106:113+71:78)) Sean AP+
87.2 1.28e4
190
% |
i 44.3
e
70.2
84.2
42,0 58.3 85.2
LE a repre ; J
TA ds TH TR TE TE TE EE E EE E do Te 74 76 7 80 8 = 8 à 8 E:
Figure 2. Positive-ion atmospheric pressure chemical ionization mass spectrum of piperazine
citrate Control No 197213.
[J V-spect HIT] :
Piperazine citrate has practically no UV absorbance except at wavelengths below 210 nm.
A UV-spectrum in water was recorded on a Varian Cary 5 spectrophotometer.
A UV-maximum was observed at 191 nm. A НЫ was about 30 (n=1).
For the identity of piperazine citrate see results under Purity/Thin-layer chromatography.
The identity of citrate was confirmed according to The International Pharmacopoeia, Third
Edition, Volume 1 by the general identification test for citrate.
Assay
See results from collaborating laboratories.
E улей alvsi
When the substance was heated to 105 °C, à loss of 11.1 % (w/w) was observed (n=6,
RSD=0.35%).
EE at ehh La ААС
CCH oC ST” na lng Nr Pe A TT TT DR EE й Eee LL
WHO/PHARM/97.595
page 99
Instrument: Perkin Elmer TGA 7 Thermogravimetric analyzer.
Sample weight: 10 mg.
Heating program: 3 *C/mun from 20-105 °C and then holding 105 °C for
360 minutes.
Melting point; 183-187 °C with decomposition.
Water
11.3 % (n=2), determined by Karl Fischer titration,
Organic volatile compounds
< 0.1 %. The test included methanol, ethanol, acetone, acetonitrile, dichloromethane, pyridine,
chloroform, benzene, trichloroethylene and dioxan. Each of them were estimated to be < 100 ppm.
The content of organic solvents was tested by gas chromatography with the following conditions:
Instrument: Hewlett Packard 6890
Column: HP-5 (30 m x 0.53 mm, 2.65 um film)
Carrier gas: Helium (8 ml/min)
Detector: FID
Injection volume: 2 ul
Injector temperature: 200 °C
Detector temperature: 250 °C
Temperature program: 40 °C for 10 minutes, 40 *C/min and holding 240 °C for
5 minutes.
Sample: 20 mg were shaken with 1 mi of benzyl alcohol and another
20 mg were shaken with 1 mi of Milli Q purified water each
for about five minutes. The sample shaken with benzyl alcohol
was filtered. |
Purity
in-la atograph
No secondary spots were detected, which means < 0.25 %. The following thin-layer
chromatographic system according to The International Pharmacopoei, Third Edition, Volume 4,
Was used.
Thin-layer: Silica gel G (Merck) TLC
Eluent: Ammonia conc.:acetone (20:80)
Sample: 500 pg of piperazine citrate dissolved in ethanol:ammonia
conc. (2:3) were applied.
WHO/PHARM/97,395
page 100
Visualization: Visualization in day-light after spraying with
triketohydrindenehydrate (3 mg/ml) in glacial acetic acid/]-
butano! (3:100) and then with triketohydrindenehydrate
(1.5 mg/ml) in ethanol and drying at 105 °C. Thereafter
spraying with 16dine (0.05 mol/l).
Ry (piperazine citrate)=0.06
Rrcethylenediamine)=0. 18
Re(triethylenediamine)=0.08
Ry(piperazine)=0.06
Ky(citrate)=0.02
The detection limit of the system was about 1.25 не (0.25 %) when examined visually.
The spot of piperazine citrate corresponds in position and appearance with that of the USPRS lot F e
of piperazine citrate.
lon chromatography
The ICRS was screened for the following anions: chlorides, bromides, nitrates, phosphates and
sulfates. < 200 ppm of chlorides, < 10 ppm of bromides, < 30 ppm of nitrates, < 500 ppm of
sulfates and « 600 ppm of one unknown ion were found.
The following conditions were used:
Eluent 1: Sodium carbonate/sodium bicarbonate 1.8 mM/ 1.7 mM
Eluent 2: Sodium bicarbonate (for the determination of chloride)
2.5 mM
Column: Dionex HPIC AS4A
SUpressor: ASRS-1 _ e
Detector: Dionex 20001, conductivity
Range: 3 us
Pump: Dionex 2000:
Injector: Beckman System Gold Autoinjector 307
Intégrator: PeakPro (Beckman)
Sample; Piperazine citrate was dissolved in the éluent at a
concentration of 1.0 mg/ml in water. 50 pl corresponding to
50 ug were injected.
- D [ey I 1
LAGO EI am are, RH EL A CUE mp tT st TT LN E RR Rhy Rm eT TR a p— MF la i LE Td re dada
WHO/PHARM/97.595
page 101
Jon Chlorides Bromides Nitrates Phosphates Sulfates
Limit of detection, ppmin 10 10 30 100 30
‚ the sample:
Limit of quantification, 50 50 100 500 100
ppm in the sample:
Data given by collaborating laboratories
European Pharmacopoeia lab,
EPCRS batch 1 is from the same batch as ICRS 197213.
Appearance: Satisfactory.
e Identity, IR: Conforms.
Water content: 11.3 %
Identity, melting point: 191 °C
TLC: Complies
Stability
Stability studies were not performed as this substance was not suspected to degrade easily
according to WHO/PHARM/86.529. Regular re-examinations of the ICRS when stored in the dry
state will be performed.
Conclusion
Piperazine citrate, Control No 197213, can be considered suitable as International Chemical
© Reference Substance for the intended purpose.
WHO/PHARM/97.595
page 102
Streptomycin sulfate
Control No 197215
Analytical Report
Intended use
The monograph for Streptomycin sulfate powder for injections in The International
Pharmacopoeia, Third Edition, Volume 4, requires a reference substance of streptomycin sulfate to
be used in the thin-layer chromatographic test for identity.
Material
About 21 g of the sample were received at the WHO Centre in January 1998. The material is being
stored in sealed ampoules at -20 °C, protected from light.
This ICRS is of the same origin as that of the 3rd Intemational Biological Standard for
streptomycin sulfate.
Analytical data
À white fluffy freeze-dried sample.
Evidence of chemical structure
Infrared spectrum
An infrared spectrum is given in Figure 1 (No W197215T). The spectrum is concordant with the
spectrum of the European Pharmacopoeia Chemical Reference Substance (EPCRS) lot 1 of
streptomycin sulfate as well as with the spectrum of the United States Pharmacopeia Reference
Standard (USPRS) lot H of streptomycin sulfate.
PALITO Pad md o a o la Parken pad Br ES Ta as old da С Ar rm er TT TT TENTE NS ne ra A УЧ + ETT Es ren pe TO e re а Ll
WHO/PHARM/97.595
page 103
MW ———— _ KiSTaIST SP —— — Ir
"NE TYTN
ОДО
0.04 — 1. . Е, Но
4000.0 3500 3000 200 2000.0 1800 1600 1400 1200 1000 500 600 250.0
(4-1
o
Figure 1. IR-spectrum of 1.1 mg of streptomycin sulfate Control No 197215 in 300 meg of potassium
bromide recorded against a potassium bromide disc.
Instrument: Perkin-Elmer 1600 FTIR.
High performance liquid chromatography with mass-spectrometric detection
A spectrum of streptomycin was recorded by electrospray (ESI) in the positive ion mode. The
spectrum shows an [M+ HI ion of 582.0, which supports the identity of streptomycin. The
spectrum, which is given in Figure 2, is concordant with the spectrum of the European
Pharmacopoeia Chemical Reference Substance (EPCRS) lot 1.
Eluent: Acetonitrile: 1 % glacial acetic acid (50:50)
Column: Direct inlet
а Pump: Hewlett Packard 1050 operated at a flow rate of 0.2 ml/min
Detector: Fisons Platform IT (quadrupole mass spectrometer)
Operating conditions: Cone voltage 65 V
Source temperature 140 °C
Sample: Streptomycin sulfate was dissolved in the eluent at a
concentration of 0,01 mg/ml. 20 Ll corresponding to 200 ng
were injected,
WHO/PHARM/97.595
page 104
Streptomycin sulfate CRS 187215 ESi+ 140 C Direct inlet
STREPTOZ ve as, 240) Cm (173:184-(184:171+161:167)) | Scan ES+
04.3
25763
100-
300.7
"a
100,9
> '
Ч 263,1 [M + aj
705 582.0
: 330.1
' 123.6 176,1 221.0
; 146.1 185,0 200.8 241.9 E 321.1
| И | 271,7 ; oa
| 3738 393.7 4200 4543 485149145755
ol | i bi LL AR | АМ LM. | Pane LA Jul oh yo adm hs YU bide a oe Mb ht iti. Dos
80 80 Tes 120 140 160 220 240 260 280 Lh
Figure 2. Positive-ion electrospray mass spectrum of streptomycin sulfate Control No 197215,
UV-spectrim .
À. UV-spectrum in water was recorded on a Varian Cary 5 spectrophotorneter. Streptomycin sulfate
has no distinct maximum but a slope increasing towards lower wavelengths was observed.
Ar =160 at 200 nm (n=1).
The calculation was performed on the "as is” basis.
in-laver chromatogra
For the identity of streptomycin sulfate see results under Purity/Thin-layer chromatography.
Sulfate
For the identity and estimation of sulfate see results under Purity/lon Chromatography.
Assay
Microbiological assay
See results under collaborating laboratories.
[Ck RR Hare nu apa a a .
y à EE ee tie tee re VCF EFF : = Turn npg pees he TR RTE НЫ agra IST Е A В A Ha NEO Marat Ma a aaa eat В
" ' hms AE eI hein ie Lo Fri! DLO Ll TO erie kn lg RT rar a srl] NE A ARE EA burns tao NORTEL И
AA pl aga E Lil metal ne dlls me Monte ha
WHO/PHARM/97.595
page 105
Thermogravimetric analysis
When the substance was heated to 105 °C, a loss of 1.8 % (w/w) was observed (n=2,
RSD=28.3 %). The substance is hygroscopic and picks up moisture immediately. This
determination was performed on freshly opened ampoules. If the sample was allowed to equilibrate
with the atmosphere for 24 hours at a relative humidity of 22-27 % a loss of 8.1 % (w/w) was
observed (n=2, RSD=18.8 %).
Instrument: Perkin Elmer TGA 7 Thermogravimetric analyzer.
Sample weight: 3-3 me.
Heating program: > “C/min from 20-105 °C and then holding 105 °C for
240 minutes.
Melting реше Indefinite melting range. Streptomycin sulfate becomes slightly
discoloured already when heating to 105 °C.
Water
8.5 % (n=2, RSD=7.7 %) determined by Karl Fischer titration. The determination was performed
on samples that had equilibrated for 24 hours with the atmosphere at a relative humidity of
22-27 %.
Organic volatile compounds
< 0.1 %. The test included methanol, ethanol, acetone, acetonitrile, dichloromethane. pyridine,
chloroform, benzene, trichloroethylene and dioxan. Each of them were estimated to be < 100 ppm.
The content of organic solvents was tested by gas chromatography with the following conditions:
Instrument: Hewlett Packard 6890
Column: HP-5 (30 m x 0.33 mm, 2.65 um film)
Carmier gas: Helium (8 ml/min)
48 Detector: FID
Injection volume: 2 ul
Injector temperature: 200 °C
Detector temperature: 250 °С
Temperature program: 40 °C for 10 minutes, 40 *C/min and holding 240 °C for
5 minutes.
Sample: 20 mg were shaken with 1 ml of benzyl alcohol and another
20 mg were shaken with | ml of Milli Q purified water each
for about five minutes. The sample shaken with benzyl alcohol
was filtered.
WHO/PHARM/97.595
page 106
Purity
Thin-layer chromatography
One secondary unknown spot was detected, it was roughly estimated to 2 %, The following thin-
layer chromatographic system according to The International Pharmacopoeia, Third Edition,
Volume 4 was used.
Thin-layer: Silica gel Merck) HPTLC 60 F254.
Eluent: 7 % potassium dihydrogen phosphate.
Sample: 100 ug of streptomycin sulfate dissolved in water were
applied.
Visualization: Visualization in day-light after spraying with naphthalene-
1,3-diol/ethanol and sulfuric acid.
e
Rr(streptomycin sulfate)=0.60
Re (unknown)=0.14
R;(kanamycin sulfate)=0.28
Rf (neomycin sulfate)=0.09
The detection limit of the system was about 0.5 ug (0.3 %) when examined visually after spraying.
The spot of streptomycin sulfate ICRS Control No 197215 corresponds in position and appearance
with that of the EPCRS lot 1 of streptomycin sulfate,
ig ance liquid e atogra
The total amount of impurities estimated by peak area normalization was about 6.0 % (n=5,
RSD=0.08 % for the main peak, RSD=0.8 % calculated on the 2.6 % impurity level).
A chromatogram is shown in Figure 3. Five impurities above the limit of quantification were
found. One of them was identified as dihydrostreptomycih (eluting at 20.5 minutes) by means ;
of an external standard from National Institute for Biological Standards and Control e
(NIBSC 62/013) and estimated to 1.2 % (n=5, RSD=5.4 %). The main impurity was unknown
4, estimated to 2.6 % by peak area normalization. According to results from the validation of
the method unknown 2, increased when stored in solution. It is here estimated to 1.3 % and
might be stability indicating.
Ea
DeESCLOC Kasponas
aE. Fir AIT a mm um nu ll une ara ay
WHO/PHARM/97.595
page 107
—5trep560W41 32.856
Г To "3 14:06:07 48/02/28
==
= |
Е |
m
|
=)
+
a |
=
= ||
=
Tr
= -
= q
=] = = 8 :
| = 5 = =
= = “I Tn
= = Е = =.
я El , =
7 = el. ©
} = = Wu y
=" = “= — = +=
Fa = = É => =
"E Pr =}
= - 2] 8 a
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= =
E Der as
1 " " — MM — " — — тт
Tiucióon Tine
BARAJA
Terátchl method
Minutes
78/07/05
а]: a
Infect Elmer 8:14:01
Inatrueno: 103
Figure 3. Chromatogram of streptomycin sulfate Control No 197215 monitored at 200 nm.
The following conditions were used:
Eluent:
Column:
Detector:
Pump:
Injector:
Integrator:
Sample:
Stability in the eluent;
Limit of detection:
Lirnit of quantification:
0.1 M phosphate buffer at pH 3.8 was prepared. Then
hexanesulfonic acid sodium salt was added to a concentration
of 0.015 M. 100 ml acetonitrile were mixed manually with
200 ml buffer. (The low wavelength gives noise if the eluent is
not properly mixed. The amount of organic solvent has strong
effect on the retention time and resolution).
Kromasil C18, 4.6 x 250 mm, 5 pm particles.
Gynkotek DAD3405 operated at 200 nm.
Gynkotek 480 operated at a flow rate of 1.0 ml/min.
Gynkotek Gina 50 operated at 8 °C
PeakPro (Beckman)
Streptomycin sulfate was dissolved in water at a concentration
of 2.0 mg/ml. 20 ul corresponding to 40 pg were injected.
A sample was stored for 1 week at room temperature.
No signs of degradation were observed.
50 ng (0.1 %) at 200 nm
100 ng (0.25 %) at 200 nm
WHO/PHARM/97.595
page 108
Diode-array detection
The chromatographic system described above was used to record UV-spectra for the detected
peaks. The spectra of the impurity peaks as well as the main peak were similar. None of them had a
distinct maximum but they all showed a slope increasing towards lower wavelengths. The ,
wavelength of 200 nm was chosen, giving the best sensitivity for all peaks. The main peak was
investigated by the peak purity program and showed no signs of co-eluting impurities.
Capillary Electrophoresis
Three impurities above the limit of quantification were found. Their total amount was estimated to
be 5.2 % by peak area normalization (n=13, RSD=0.5 % for the main peak, RSD=13.6 % at the 2 %
impurity level). Dihydrostreptomycin does not separate from streptomycin in this method, it 1s
determined by liquid chromatography, see Purity/High performance liquid chromatography. An
electropherogram is given in Figure 4.
Clromrian] ria
ZI Crs A | |' a | a 9
"— (= йа. т |
7
1.
streptomycin sulfate 4.20
dl.
|
E
a
Di
OH 5275
2,14
n unknown 2 : Ш sn
unknown 3 ла
de Chal
GT
x 0.10
unknown 1 ' ° |
hi oe
| oa Cl
-
| Qo, Oil
+
Qu
a Fr
ac
5.00
1
q > e 15 E] :
Figure 4. Electropherogram of streptomycin sulfate Control No 1 97215 monitored at 200 nm.
Mr MR erm cel def th A a a or or HL tat et EL nat Rs tes ÖLE ELSE U Le ES VT TOTO da le La PT, ola demi MU : pra TA LAA TAL
ll mp [ie АЛ ТЫ ны a am mm om mur me a af
WHO/PHARM/97.595
page 109
The following conditions were used:
Eluent: 100 mM phosphoric acid and 50 mM triethylamine, pH adjusted to 3.00
with 2 M sodium hydroxide
Rinsing of capillary: 3.0 minutes with 0.1 M sodium hydroxide
3.0 minutes with water
3.0 minutes with running buffer
Capillary: Fused silica (total length 100 cm, length to detector 90 em, ID: 75 HIT)
CE Instrument: Beckman, P/ACE System MDQ) operated with an applied voltage of
20 kV for 45 minutes.
Detector: Beckman P/ACE System MDQ Diode Array Detector operated at
| 200 nm (bandwidth 10 nm).
Run temperature: 50 °С
Sample temperature: 85°C
Injection: ) 20 seconds pressure injection, at a pressure of 0.5 psi.
Integrator: Beckman P/ACE System MDQ) soft ware
Sample: streptomycin sulfate was dissolved in water at a concentration of
3 mg/ml.
Stability in the eluent: A sample was stored for one week at 8 °C in darkness. No signs of
degradation were observed.
Limit of detection: 0.1 % at 200 nm.
Limit of quantification: 0.25 % at 200 nm.
lon chromatography
The ICRS was screened for the following anions: chlorides, bromides, nitrates, phosphates and
sulfates. Less than 10 ppm of bromides, less than 100 ppm of nitrates and less than 500 ppm of
phosphates were found. Chlorides were found and estimated to 300 ppm. Sulfates were found and
roughly estimated to be 21.9 % (theoretical value 19.8 %).
The following conditions were used:
Eluent 1: Sodium carbonate/sodium bicarbonate 1.8 mM/ 1.7 mM
Eluent 2: Sodium bicarbonate (for the determination of chloride)
2.5 mM
Column: Dionex HPIC AS4A
Supressor: ASRS-1
Detector: Dionex 20001, conductivity
Range: 3 iS (for determination of sulfate 300 us)
Pump: Dionex 20001
Injector: Beckman System Gold Autoinjector 507
Integrator: PeakPro (Beckman)
WHO/PHARM/97.595
page 110
Sample: Streptomycin sulfate was dissolved in the eluent at a
concentration of 1.0 mg/ml in water, 50 ul corresponding to
50 pg were injected,
Ion Chlorides Bromides Nitrates Phosphates Sulfates
Limit of detection, ppm in 10 10 30 100 30
the sample:
Limit of quantification, 30 30 100 500 100
ppm in the sample:
Data given by collaborating laboratories
NIBSC |
Description: 7 The third International standard for
Streptomycin established 1979.
Microbiological assay, 78500 International Units per ampoule.
determined in a
collaborative study:
Stability
Stability studies were not performed as this substance was not suspected to degrade easily when
stored at -20 °C. Regular re-examinations of the ICRS when stored in the dry state will be
performed.
Hygroscopicity ‚о
As the substance is very hygroscopic it is advisable to let it equilibrate with the surrounding
atmosphere overnight if accurate weighing is requested.
Conclusion
Streptomycin sulfate, Control No 197215, can be considered suitable as International Chemical
Reference Substance for the intended purpose. As this is a biological substance it is not possible to
obtain a better quality.
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