specific quality problems with antibiotics and antituberculotics pr a. nicolas, phd head of the...

SPECIFIC QUALITY PROBLEMS

WITH ANTIBIOTICS AND ANTITUBERCULOTICS

Pr A. NICOLAS, PhD

Head of the French Laboratories and Controls Directorate

AFSSAPS

Interregional Seminar for Quality Control Laboratories involved in WHO Prequalification Programme and/or participating in respective sampling and testing projects, Nairobi, Kenya, 23-25 September 2009

1-3

Sampling and testing for Quality Control Laboratories, Nairobi, September 20092 |

ANTIBIOTICS AND ANTITUBERCULOTICS

ANTIBIOTICS AND ANTITUBERCULOTICS

Classification

Origin

Drug quality reports

Fermentation products

Impact on the composition of a monograph

Antituberculosis dosage forms

Conclusion


ANTIBIOTICS - CLASSIFICATION ANTIBIOTICS - CLASSIFICATION

Very diverse class of compounds

Generally grouped according chemical structures– Aminoglysides – Cephalosporins and related beta lactams– Glycopeptides– Macrolides– Penicillins– Quinolones– Sulphonamides and diaminopyrimidines– Tetracyclines


ANTITUBERCULOSIS - CLASSIFICATION ANTITUBERCULOSIS - CLASSIFICATION

Miscellaneous group of antibacterials

Include– Cycloserine – Ethambutol– Isoniazid– Pyrazinamide– Rifampicin


WHO List of Prequalified Medicinal Products

WHO List of Prequalified Medicinal Products

Prequalified antituberculosis products (june 2009)

– Cycloserine

– Ethambutol

– Isoniazid

– Ethionamide

– Pyrazinamide

– Ethambutol + Isoniazid

– Ethambutol + Isoniazid + Rifampicin

– Ethambutol + Isoniazid + Pyrazinamide + Rifampicin

– Isoniazid + Rifampicin

– Isoniazid + Pyrazinamide + Rifampicin


Drug Quality Reports Affecting USAID-assisted Countries (1)


USP Drug Quality and Information Program

– Updated june 2009

– Repository of publicly-available information to raise awereness

of the problem of counterfeit and substandard medicines

– More often cited drugs : antimalarials, antiretrovirals

– But also : antibiotics, antituberculosis products




Ghana

– 13/08/2004 Counterfaiting of Ampicillin (root crop cassava)

Nigeria

– 29/05/2008• Augmentin (amoxicillin/clavulanic acid)• Ampicillin

– 15/05/2009• Amoxicillin, ciprofloxacin, ampicillin, cloxacillin,

erythromycin




Sierra Leone– 17/06 2008 : chloraphecol, tetracycline– 9/06/2008 : amoxicillin, ampicillin

South Africa– 27/05/2009

• Antib-4 (pyrazinamide, ethambutol, isoniazid, rifampicin)

• Ebsar (isoniazid + rifampicin)

Tanzania– 5/10/2008 : antibiotics

Uganda– 1/10/2005 : cloxacillin


ORIGINORIGIN

These active substances could be obtained by :

– synthetic route

– semi-synthetic route derived from fermentation product by

process involving at least cleavage and/or formation of covalent

bonds followed by extraction/purification steps

– fermentation

The origin impacts the purity profile, choice of the analytical methods and their performance


REFERENCES : ICH and Ph. Eur.REFERENCES : ICH and Ph. Eur.

Guideline ICH Q3A(R2) « Impurity in New Drug Substances »

Ph. Eur. Chapter 5.10 «Control of Impurities in substances for pharmaceutical use»

Ph. Eur. General monograph (2034) «Substances for pharmaceutical

use» do not cover « fermentation products and semi-synthetic

products derived therefrom » for application of reporting,

identification and qualification thresholds


THRESHOLDS FOR ORGANIC IMPURITIES IN ACTIVE SUBSTANCES (General case)

THRESHOLDS FOR ORGANIC IMPURITIES IN ACTIVE SUBSTANCES (General case)

UseMaximum

daily dose

Reporting

threshold

Identification

threshold

Qualification

threshold

Human use or human and veterinary use

≤ 2 g/day> 0.05 per cent> 0.10 per cent > 0.15 per cent

Human use or human and veterinary use

> 2 g/day> 0.03 per cent> 0.05 per cent > 0.05 per cent

Veterinary use only

Not applicable> 0.10 per cent> 0.20 per cent > 0.50 per cent


PRODUCTS OF FERMENTATION (1)PRODUCTS OF FERMENTATION (1)

Relevant monograph from Ph. Eur. « Products of fermentation (1468) »

This monograph applies to :

• products obtained by semi-synthesis from a product of fermentation and those obtained by biocatalytic transformation

• whole broth concentrates or raw fermentation products

A complex class of compounds with many analytical challenges



Fermentation processes are biosynthetic pathways consisting of

parallel reactions

– Some antibiotics are mixtures of structurally related substances

– Not always clear which of these substances contribute to activity

(active or impurity ?)

– Separation not always achievable



Biosynthetic pathways difficult to control (variation from batch to batch)

– Different strains of same species may lead to different impurity profiles

– Large differences in media components in fermentation may lead to different impurity profiles


MONOGRAPH – DEFINITION (1)MONOGRAPH – DEFINITION (1)

Substances obtained by synthetic or semi-synthetic route correspond to a major known compound

Substances obtained by fermentation are often but not always defined as mixtures

The name of the strain is indicated



Synthetic : ciprofloxacin

1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid.

Semisynthetic : rifampicin

2S,12Z,14E,16S,17S,18R,19R,20R,21S,22R,23S,24E)-5,6,9,17,19-Pentahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-8-[[(4-methylpiperazin-1-yl)imino]methyl]-1,11-dioxo-1,2-dihydro-2,7-(epoxypentadeca[1,11,13]trienimino)naphto[2,1-b]furan-21-yl acetate

Semisynthetic antibiotic obtained from rifamycin SV.



Fermentation leading to a known major compound : josamycin

Josamycin is a macrolide antibiotic produced by certain strains of

Streptomyces narbonensis var. josamyceticus var. nova, or

obtained by any other means. The main component is……

Fermentation leading to a mixture : gentamicin

Mixture of the sulphates of antimicrobial substances produced by

Micromonospora purpurea, the main components being

gentamicins C1, C1a, C2, C2a and C2b.


MONOGRAPH - IDENTIFICATIONMONOGRAPH - IDENTIFICATION

Synthetic and semisynthetic – Identification by IR spectroscopy (2.2.24)

Mixtures obtained by fermentation– IR if structures of the individual compounds are very similar :

erythromycin

– Identification by examination of the chromatograms obtained in the test for composition

• Gentamicin : the chromatogram obtained with the test solution shows 5 principal peaks having the same retention times as the 5 principal peaks in the chromatogram obtained with reference solution (gentamicin sulphate CRS).


MONOGRAPH - TESTSMONOGRAPH - TESTS

Composition– Used to specify complex mixture such as some fermented

polypeptides (tyrothricin) or aminoglycosides (gentamicin)

Related substances

– ICH impurity limits/thresholds cannot be applied because of wide variety in purity and complexity

– Not possible to establish one fixed set of thresholds for identification and qualification

– Specifications should be derived on a case by case basis considering batch results of various suppliers


MONOGRAPH - TESTS COMPOSITION

MONOGRAPH - TESTS COMPOSITION

In the case of mixture a « composition test » is described

Composition. Liquid chromatography (2.2.29): use the normalisation procedure taking into account only the peaks due to gentamicins C1, C1a, C2, C2a and C2b; use the chromatogram supplied with gentamicin sulphate CRS to identify the corresponding peaks.

Limits

– gentamicin C1 : 20.0 per cent to 40.0 per cent,

– gentamicin C1a : 10.0 per cent to 30.0 per cent,

– sum of gentamicins C2, C2a, and C2b: 40.0 per cent to 60.0 per cent


COMPOSITION – GENTAMICIN (1)COMPOSITION – GENTAMICIN (1)

Known structures of 5 components


COMPOSITION – GENTAMICIN (2) COMPOSITION – GENTAMICIN (2)

J. Pharm. Biomed. Analysis, 2007, 45, 257-262


MONOGRAPH - TESTS RELATED SUBSTANCES (1)


Same method as for composition but different specifications

Content in impurities can be very high and chemical structures are not always known

Gentamicin

Limits (for related substances eluting before gentamicin C1a):

– any impurity: maximum 3.0 per cent

– total: maximum 10.0 per cent




Threshold for « any unspecified impurity » in current antibiotics monographs from Ph. Eur.

– Ciprofloxacine : 0.1 %– Cefradine : 0.25 %– Amoxicillin trihydrate : 1 %– Gentamicin sulphate : 3.0 %


MONOGRAPH ASSAY – MICROBIOLOGICAL TITRATION

MONOGRAPH ASSAY – MICROBIOLOGICAL TITRATION

Microbiological assay of antibiotics (2.7.2)

– Two methods• A. Diffusion method (19 antibiotics)• B. Turbidimetric method (14 antibiotics)

– Indication of the reagents, micro-organisms, pH, temperature

– Chapter 5.3. Statistical analysis of results of biological assays and tests

– Use of CombiStats www.edqm.eu


MONOGRAPH - ASSAY HPLC (1)


ASSAY – HPLC (2.2.29)

– For synthetic and semi-synthetic drugs

– For mixtures obtained by fermentation if similar activity of the individual compounds

– Possibilty of sums of active substances

HPLC/UV

– For chromophoric compounds (general case)

– Or after derivatization (amikacine)




Erythromycin

– Content:• sum of the contents of erythromycin A, erythromycin B and

erythromycin C: 93.0 per cent to 102.0 per cent (anhydrous substance),

• erythromycin B: maximum 5.0 per cent,

• erythromycin C: maximum 5.0 per cent.




Other detection modes (aminosides)

– HPLC/PAD (pulsed amperometric detection), Ph. Eur and US, Ph. Int.

• Difficult to handle, poor precision • Noisy, high disregard limit (1.0 % for framycetine and

neomycine sulphate)

– HPLC/ELSD

• Gentamicin sulphate (Chinese Ph.)• Simultaneous determination of content in sulphates (in

place of titration)


MONOGRAPH - STORAGEMONOGRAPH - STORAGE

General conditions : in airtight container

Specific conditions– Protected from light: tiamuline– Multiple conditions

• In an airtight container, at a temperature of 2 °C to 8 °C : potassium clavulanate

• Under nitrogen in an airtight container, protected from light, at a temperature not exceeding 25 °C : rifampicin


MONOGRAPH - TRANSPARENCY LIST (IMPURITIES)

MONOGRAPH - TRANSPARENCY LIST (IMPURITIES)

List of specified and other detectable impurities (for definition see Ph. Eur. General monograph 2034)

All cases– Absence to a very long list

• Nystatin, Josamycin, tyrothricin : no list• Tiamuline : 6 specified impurities, 12 other detectable

impurities• GRAHEK R and ZUPANCIC-KRALJ

J. Pharm. Biomed. Anal. XXX(2009)xxx in press

Identification of gentamicin impurities by LC tandem mass spectrometry : 17 impurities


ANTITUBERCULOSIS DOSAGE FORMSANTITUBERCULOSIS DOSAGE FORMS

Ph. Int., 1st Suppl : 6 new monographs including 2, 3 and 4 component fixed-dose preparations

– Doxycycline capsules– Isoniazid and ethambutol hydrochloride tablets– Rifampicin capsules and tablets– Rifampicin and isoniazid tablets– Rifampicin, isoniazid and pyrizinamide tablets– Rifampicin, isoniazid, pyrizinamide and ethambutol

hydrochloride tablets

Introduction of dissolution test in monographs for tablets and capsules : doxycycline, isoniazid


4 COMPONENTS ANTITUBERCULOSIS TABLETS

4 COMPONENTS ANTITUBERCULOSIS TABLETS

Rifampicin (150 mg), isoniazid (75 mg), pyrizinamide (400 mg) and ethambutol hydrochloride (275 mg) tablets

Rifampicin : semisynthetic antibiotic obtained from rifamycin SV.

Isoniazid, pyrizinamide and ethambutol hydrochloride : synthetic compounds

Monographs in Ph. Eur., USP, Ph. Int., Chinese


ETHAMBUTOL, HClETHAMBUTOL, HCl

Ph. Eur.USP Ph. Int.Chinese

Content99.0 – 101.0 %

Titrimetry

98.0 – 100.5 %

Titrimetry

98.0 – 100.5 %

Titrimetry

Min 98.5 %

Titrimetry

ImpuritiesHPLC 215 nm

Imp B : 1.0 %

Other : 0.10 %

Total : 1.0 %

GC (2.4.24)

Imp D : 5 ppm

(1-2 dichloroethane)

TLC Imp A : 1.0%

Aminobutanol

Derivatization by fluorescamine

Fluorimetry

385/485 nm : 1.0 %

Aminobutanol

TLC : 1.0 %

Aminobutanol

TLC : 1.0 %


ISONIAZIDISONIAZID


Content99.0 – 101.0 %

Titrimetry

98.0 – 102.0 %

HPLC

98.0 – 101.0 %

Titrimetry

Min 99.0 %

Titrimetry

ImpuritiesTLC

Hydrazine : 0.05 %

Other : 0.2 %

NONETLC

Hydrazine: 0.02 %

TLC

Hydrazine: 0.02 %


PYRAZINAMIDEPYRAZINAMIDE


Content99.0 – 100.5 %

Titrimetry

99.0 – 101.0 %

Titrimetry

98.5 – 101.0 %

Titrimetry

Min 99.0 %

Titrimetry

ImpuritiesTLC

Any : 0.2 %

NoneNoneTLC

Any : 0.2 %


RIFAMPICINRIFAMPICIN


Content97.0 – 102.0 %

Absorbance 475 nm

95.0 – 103.0 %

HPLC 254 nm

97.0 – 102.0 %

Absorbance 475 nm

Min 93.0 %

HPLC 254 nm

ImpuritiesHPLC 254 nm

Rifampicinquinone : 1.5 %

Other : 1.0 %

Total : 3.5 %

HPLC 254 nm


Other : 1.0 %

Total : 3.5 %

TLC


3-formyl-rifamycin SV : 0.5 %

Other : 1.0 %

HPLC 254 nm


Rifampicin N -Oxide: 0.5 %

3-formyl-rifamycin SV : 0.5 %

Total other:3.0%


4 COMPONENTS ANTITUBERCULOSIS TABLETS – Ph. Int. (1)


Rifampicin (150 mg), isoniazid (75 mg), pyrizinamide (400 mg) and ethambutol hydrochloride (275 mg) tablets

Ph. Int. (4th Edition, 1st supplement 2008)

Identification – Tests A and B (retention time/HPLC for assay)

or– Test C : 2 TLC systems (different stationary phases)




Impurities

– Rifampicin-related substances (RP-HPLC-UV)

• Addition product : 3-formylrifamycin SV (specified impurity of rifampicin limited to 0.5% in the API) and isoniazid : 5.0 %

• Rifampicin quinone : 4.0 %• Any other impurity : 1.5 %• Total : 10.0 %• Disregard limit : 0.1 %

– Impurities in API • 3-formylrifamycin SV : 0.5 %• Rifampicin quinone : 1.5 %• Any other impurity : 1.0 %


4 COMPONENTS ANTITUBERCULOSIS TABLETS - Ph. Int. (3)

4 COMPONENTS ANTITUBERCULOSIS TABLETS - Ph. Int. (3)

No dissolution test

Assay

– First RP-HPLC-UV system for isoniazid, pyrizinamide and ethambutol hydrochloride

– Second RP-HPLC-UV system for rifampicin

– Specifications : 90.0 to 110.0 % of the amounts stated on the label


4 COMPONENTS ANTITUBERCULOSIS TABLETS - USP

4 COMPONENTS ANTITUBERCULOSIS TABLETS - USP

Identification

By retention time (2 HPLC systems)

Dissolution

Not less than 75 % of the labeled amounts of each active substance in 45 min

Impurities

No test

Assay

2 HPLC systems : content 90.0 to 110.0 % in each component


ANTIBIOTICS – COUNTERFAITING (1)ANTIBIOTICS – COUNTERFAITING (1)

Screening of some of the most conterfeited antibiotics in a single chromatographic run

Gaudiano M.C. et coll, J. Pharm. Biomed. Anal., 2008, 48, 303-309

RP-HPLC method, elution by gradient, detection 230 nm

Ampicillin, amoxicillin + clavulanic acid, doxycycline, cloxacillin, chloramphenicol


ANTIBIOTICS – COUNTERFAITING (2)ANTIBIOTICS – COUNTERFAITING (2)


MACROLIDES – COUNTERFAITING (1)MACROLIDES – COUNTERFAITING (1)

Fast chemical identification system for screening of counterfeit drugs of macrolide antibiotics

CHANG-QIN HU et coll.,

J. Pharm. Biomed. Anal., 2006, 540, 68-74– Two color reactions + 2 TLC– Sreening of 10 macrolides


MACROLIDES – COUNTERFAITING (2)MACROLIDES – COUNTERFAITING (2)


CONCLUSIONCONCLUSION

Products from fermentation (antibiotics/antituberculosis) are a challenge for analysts and for the authorities

– Complex mixtures

– All compounds not well known (impurity profile)

– Specific analytical methods (HPLC/amperometry)

– Content determined by activity assay

Products obtained by synthetic routes

– Obey to ICHQ3A (R2)

– Large place for classical HPLC methods coupled to UV detection

– Impurity profile well known

specific quality problems with antibiotics and antituberculotics pr a. nicolas, phd head of the...

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