technical_guide_for_the_elaboration_of_monographs_7th_editio

ELABORATION OF MONOGRAPHSTechnical guide for the European PharmacopoeiaEDQM7th Edition2015 ELABORATION OF MONOGRAPHS

Technical guide for the

European Pharmacopoeia7th Edition

2015

European Directorate for the Quality of Medicines & HealthCare

English version

2015

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European Directorate for the Quality of Medicines & HealthCare (EDQM)

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FRANCE Cover image: ? EDQM - Council of Europe Director of the Publication: Dr S. Keitel

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? Council of Europe, 2015

TECHNICAL GUIDE FOR THE ELABORATION OF MONOGRAPHS

7th Edition – 2015

TABLE OF CONTENTS

I. I.1. I.2. I.3. I.4. I.5. I.6. I.7. II.

INTRODUCTION ............................................................................................................................................... 1 PURPOSE OF THE GUIDE .............................................................................................................................. 1 TEST PROCEDURES ...................................................................................................................................... 1 EQUIPMENT .................................................................................................................................................... 2 QUANTITIES ................................................................................................................................................... 2 REAGENTS ...................................................................................................................................................... 4 COMMERCIAL NAMES ................................................................................................................................. 4 REFERENCE STANDARDS ........................................................................................................................... 4 MONOGRAPH ON A SUBSTANCE FOR PHARMACEUTICAL USE ........................................................ 5

II.1. TITLE ................................................................................................................................................................ 5 II.2. DEFINITION .................................................................................................................................................... 6 II.2.1. Combinations ............................................................................................................................................ 7 II.2.2. Content ..................................................................................................................................................... 7 II.3. CHARACTERS ................................................................................................................................................ 8 II.3.1. Appearance ............................................................................................................................................... 8 II.3.2. Taste ......................................................................................................................................................... 9 II.3.3. Odour ....................................................................................................................................................... 9 II.3.4. Solubility ................................................................................................................................................... 9 II.3.5. Stability factors ....................................................................................................................................... 10 II.3.6. Hygroscopicity ........................................................................................................................................ 10 II.3.7. Solid-state properties .............................................................................................................................. 10 II.3.8. Other characteristics .............................................................................................................................. 10 II.3.9. Behaviour in solution .............................................................................................................................. 11 II.4. IDENTIFICATION ......................................................................................................................................... 11 II.4.1. General ................................................................................................................................................... 11 II.4.2. Second Identification series .................................................................................................................... 12 II.4.3. Infrared absorption spectrophotometry .................................................................................................. 13 II.4.4. Ultraviolet and visible absorption spectrophotometry ............................................................................ 13 II.4.5. Melting point, freezing point and boiling point ....................................................................................... 14 II.4.6. Specific optical rotation .......................................................................................................................... 15 II.4.7. Thin-layer chromatography .................................................................................................................... 15 II.4.8. Gas chromatography and liquid chromatography .................................................................................. 16 II.4.9. Chemical reactions ................................................................................................................................. 16 II.5. TESTS ............................................................................................................................................................. 16 II.5.1. General ................................................................................................................................................... 16 II.5.2. Title of tests ............................................................................................................................................ 17 II.5.3. Solution S ................................................................................................................................................ 18 II.5.4. Appearance of solution ........................................................................................................................... 19

II.5.4.1. Clarity and degree of opalescence (2.2.1.) ................................................................................................................. 19 II.5.4.2. Degree of coloration (2.2.2.) ...................................................................................................................................... 19

II.5.5. II.5.6. pH and Acidity or alkalinity .................................................................................................................... 20 Optical rotation (2.2.7.) .......................................................................................................................... 21

II.5.7. II.5.8.

II.5.8.1. Thin-layer chromatography (TLC) (2.2.27.) .............................................................................................................. 27 II.5.8.2. Liquid chromatography (LC) (2.2.29.) ...................................................................................................................... 28 II.5.8.3. Gas-liquid chromatography (GC) (2.2.28.) ................................................................................................................ 33 II.5.8.4. Capillary electrophoresis (CE) (2.2.31.) .................................................................................................................... 33

Absorption spectrophotometry (ultraviolet and visible) (2.2.25.) ........................................................... 22 Related substances .................................................................................................................................. 23

II.5.9. Readily carbonisable substances ............................................................................................................ 34 II.5.10. Foreign anions and/or cations ................................................................................................................ 35 II.5.11. Heavy metals – Elemental Impurities ..................................................................................................... 35 II.5.12. Loss on drying (2.2.32.) .......................................................................................................................... 36 II.5.13. Thermogravimetry (2.2.34.) .................................................................................................................... 36 II.5.14. Semi-micro determination of water (2.5.12.) – (volumetric Karl Fischer) .............................................. 37 II.5.15. Micro determination of water (2.5.32.) – (coulometric Karl Fischer) .................................................... 37 II.5.16. Gas chromatographic determination of water ........................................................................................ 37 II.5.17. Determination of water by distillation (2.2.13.) ...................................................................................... 38 II.5.18. Sulfated ash (2.4.14.) .............................................................................................................................. 38 II.5.19. Residue on evaporation .......................................................................................................................... 38 II.5.20. Residual solvents (2.4.24.) ...................................................................................................................... 38 II.5.21. Bacterial endotoxins ............................................................................................................................... 38 II.6. ASSAY ............................................................................................................................................................ 39 II.6.1. Ultraviolet and visible spectrophotometry (2.2.25.) ............................................................................... 40

II.6.1.1. Direct measurement ................................................................................................................................................... 40 II.6.1.2. Measurement after a colour reaction.......................................................................................................................... 40

II.6.2. Volumetric analysis................................................................................................................................. 40 II.6.3. Chromatography ..................................................................................................................................... 41 II.6.4. Determination of nitrogen by sulfuric acid digestion (2.5.9.) ................................................................. 41 II.7. STORAGE ...................................................................................................................................................... 41 II.8. LABELLING .................................................................................................................................................. 42 II.9. IMPURITIES .................................................................................................................................................. 42 II.10. FUNCTIONALITY-RELATED CHARACTERISTICS ................................................................................ 43 III.

ANALYTICAL VALIDATION ........................................................................................................................ 44

III.1. DEFINITIONS AND TERMINOLOGY ......................................................................................................... 44 III.1.1. Introduction ............................................................................................................................................ 44 III.1.2. Types of analytical procedures to be validated ....................................................................................... 44 III.1.3. Validation characteristics and requirements .......................................................................................... 45 III.1.4. Glossary .................................................................................................................................................. 46 III.2. METHODOLOGY .......................................................................................................................................... 47 III.2.1. Introduction ............................................................................................................................................ 47 III.2.2. Specificity ............................................................................................................................................... 48

III.2.2.1. Identification ............................................................................................................................................................ 48 III.2.2.2. Assays and impurity tests ......................................................................................................................................... 49

III.2.3. III.2.4. III.2.5.

III.2.5.1. Assay ........................................................................................................................................................................ 51 III.2.5.2. Impurities (quantification) ........................................................................................................................................ 51 III.2.5.3. Recommended data .................................................................................................................................................. 51 III.2.6.1. Repeatability ............................................................................................................................................................ 52 III.2.6.2. Intermediate precision .............................................................................................................................................. 52 III.2.6.3. Reproducibility ......................................................................................................................................................... 52 III.2.6.4. Recommended data .................................................................................................................................................. 52 III.2.7.1. Based on visual evaluation ....................................................................................................................................... 52 III.2.7.2. Based on signal-to-noise ratio .................................................................................................................................. 52 III.2.7.3. Based on the standard deviation of the response and the slope ................................................................................ 53 III.2.7.4. Recommended data .................................................................................................................................................. 53 III.2.8.1. Based on visual evaluation ....................................................................................................................................... 53 III.2.8.2. Based on signal-to-noise ratio .................................................................................................................................. 53 III.2.8.3. Based on the standard deviation of the response and the slope ................................................................................ 54

Linearity ................................................................................................................................................. 49 Range ...................................................................................................................................................... 50 Accuracy ................................................................................................................................................. 51

III.2.6. Precision ................................................................................................................................................. 51

III.2.7. Detection limit ........................................................................................................................................ 52

III.2.8. Quantitation limit.................................................................................................................................... 53

III.2.8.4. Recommended data .................................................................................................................................................. 54

III.2.9. Robustness .............................................................................................................................................. 54 III.2.10. System suitability testing ......................................................................................................................... 55 III.3. SPECIFIC APPLICATION TO METHODS USED IN THE PH. EUR. ......................................................... 55 III.3.1. Optical rotation (2.2.7.) .......................................................................................................................... 55

III.3.1.1. Introduction .............................................................................................................................................................. 55 III.3.1.2. Identification ............................................................................................................................................................ 55 III.3.1.3. Tests ......................................................................................................................................................................... 55 III.3.2.1. Identification ............................................................................................................................................................ 56 III.3.2.2. Limit test .................................................................................................................................................................. 56 III.3.2.3. Assay ........................................................................................................................................................................ 56 III.3.3.1. Appearance of solution (2.2.1. and 2.2.2.) ............................................................................................................... 57 III.3.3.2. Acidity or alkalinity ................................................................................................................................................. 57 III.3.3.3. Limit tests for anions/cations (2.4.) .......................................................................................................................... 57 III.3.4.1. Specificity ................................................................................................................................................................ 58 III.3.4.2. Calibration ................................................................................................................................................................ 58 III.3.4.3. Matrix effects ........................................................................................................................................................... 59 III.3.4.4. Detection and quantification limit (based on the standard deviation of the blank) ................................................... 59 III.3.5.1. Thin-layer chromatography (2.2.27.) ....................................................................................................................... 59 III.3.5.2. Liquid chromatography (2.2.29.).............................................................................................................................. 60 III.3.5.3. Gas chromatography (2.2.28.) .................................................................................................................................. 62

III.3.2. Ultraviolet spectrophotometry (2.2.25.).................................................................................................. 56

III.3.3. Non-instrumental limit tests .................................................................................................................... 57

III.3.4. Atomic absorption spectrometry (2.2.23.)............................................................................................... 58

III.3.5. Separation techniques ............................................................................................................................. 59

III.3.6.

III.3.7. III.3.8. Semi-micro determination of water (2.5.12.) .......................................................................................... 63 Volumetric titrations (2.5.11. - 2.2.19. - 2.2.20.) .................................................................................... 64 Peptide identification by nuclear magnetic resonance spectrometry (2.2.64.) ....................................... 66

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I. INTRODUCTION

I.1. PURPOSE OF THE GUIDE This document is a guide for the authors of monographs and also a means of communicating the principles for the elaboration of monographs to the users of the European Pharmacopoeia (Ph. Eur.), especially industry, licensing authorities and official medicines control laboratories. Since the principles applied and guidance given for the elaboration of monographs should be the same as those applied by licensing authorities, the Technical Guide may also serve as a guideline in the elaboration of specifications intended for inclusion in licensing applications.

It is necessary to bear in mind that a monograph will be a mandatory standard and must be applicable in licensing procedures in all Member States of the Convention on the Elaboration of a European Pharmacopoeia.

I.2. TEST PROCEDURES The methods chosen for the identification tests, purity tests and assay(s) constituting the bulk of a pharmacopoeial monograph are preferably those already described and utilised in the Ph. Eur.. In this context, the author of a monograph is referred not only to the General Methods of the Ph. Eur.. but also to published monographs on similar materials. The above considerations aim at ensuring a reasonable degree of harmonisation within the Ph. Eur. and they only apply in cases where the methods are found to be adequate for the specific purposes. However, due attention is also to be paid to the development of new methods that offer significant improvements in terms of sensitivity, precision, accuracy or discriminating power (selectivity).

Methods included in monographs must be validated as described in the chapter on analytical validation and other relevant specific chapters of this guide. Validation reports are provided to the EDQM but are not published or otherwise provided to users.

The test procedures included in a monograph should be verified in 2 or more laboratories and the laboratory reports on this verification should be provided to the EDQM to ensure future traceability.

The instructions describing any method of analysis cover all factors that can influence the results and that are deemed essential to enable an experienced analyst working according to acknowledged laboratory practices, yet without necessarily having any prior knowledge of the investigation in question, to perform the analysis. Variations in the description of similar methods are to be avoided.

If an analytical procedure is expected to be used generally or if it requires a lengthy description and is used more than once, it may be proposed for inclusion in the general chapters of the Ph. Eur., to be referred to in the individual monographs. The methods are prescribed on the scale conventionally applied in the Ph. Eur. except in cases where for reasons of availability of the material to be analysed, or because of its toxicity or its cost, work on a small scale would be advantageous.

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I.3. EQUIPMENT If the equipment utilised for a method of analysis is not generally available in the States party to the European Pharmacopoeia Convention, it must be possible to have it constructed according to its description in the Ph. Eur.

I.4. QUANTITIES In prescribing the quantities, i.e. masses and volumes, of substances, reagents, and solvents to be taken for identifications, tests and assays, it is the practice of the Ph. Eur. to indicate the accuracy with which they are to be measured (see General Notices). It is therefore necessary to take this aspect into consideration when drafting pharmacopoeial texts.

As guidance to minimise errors in the preparation of analytical solutions, Table 1, giving estimations of the relative uncertainty, is to be consulted.

In order to avoid either the use of extremely low amounts or an unnecessarily large expenditure of solvents, a dilution series will often have to be prescribed for the preparation of dilute solutions used particularly for spectrophotometric measurement. In this context not all combinations of (usually 2 or 3) dilution steps will contribute equally to the random error of the dilution procedure. If critical for the purpose, the optimal dilution is prescribed in consideration of the relative errors (capacity tolerance divided by nominal volume) associated with the various sizes of volumetric pipettes and volumetric flasks commonly used for these operations (taking the usual formula: square root of the sum of the squares of individual relative errors, to estimate the relative dilution error).

Tables giving the optimal number and nature of dilution steps needed to achieve a given dilution ratio, based upon given specifications for the capacity tolerances of volumetric glassware, are available in the literature. For guidance see Table 2 (it is to be noted that these factors do not include reading errors).

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Table 1 – Relative uncertainties in the preparation of analytical solutions

Concentration to be prepared

Preparation of solution

10 g/1000 mL 1 g/100 mL 0.5 g/50 mL 0.25 g/25 mL 0.1 g/10 mL 1 g/1000 mL 0.5 g/500 mL 0.25 g/25 mL 100 mg/100 mL 50 mg/50 mL 10 mg/10 mL 100 mg/1000 mL 50 mg/500 mL 25 mg/250 mL 10 mg/100 mL 5 mg/50 mL 1 mg/10 mL 10 mg/1000 mL 5 mg/500 mL 1 mg/100 mL

Percentage relative uncertainty Mass Volume Total < 0.01 0.02 0.04 0.08 0.02 0.02 0.04 0.08 0.2 0.4 2.0 0.2 0.4 0.8 2.0 4.0 20.0 2.0 4.0 20.0

0.05 0.12 0.17 0.23 0.50 0.05 0.07 0.23 0.12 0.17 0.50 0.05 0.07 0.08 0.12 0.17 0.50 0.05 0.07 0.12

0.05 0.12 0.17 0.24 0.54 0.05 0.08 0.24 0.23 0.43 2.06 0.21 0.41 0.80 2.0 4.0 20.0 2.0 4.0 20.0

10 g/1000 mL

1g/1000 mL

0.1 g/1000 mL

0.01 g/1000 mL

An uncertainty of 0.2 mg for the weighing procedure has been assumed for the calculations of the percentage relative uncertainties.

Table 2 – Relative errors for dilution with analytical glassware (pipettes P/flasks F)

Concentration ratio No. of steps

1/2 1/2.5 1/5 1/10 1/12.5 1/30 1/50 1/100 1/125 1/160 1/200 1/250 1/400 1/500 1/1000

1 1 1 1 1 1 1 1 2 2 2 2 2 2 2

Step 1 P 25 20

F 50 50

Step 2 P

F

Relative error

0.16 0.18 0.17 0.13 0.16 0.20 0.15 0.18 0.20 0.19 0.18 0.20 0.18 0.20 0.20

20 100 25 250 20 250 15 500 20 1000

25 250 25 250 20 250 25 250 25 1000 25 100 25 500 25 100 20 250 25 500 25 250 25 1000 20 500 25 500 20 1000 25 500

Adapted from R.B. Lam and T.L. Isenhour, Minimizing relative error in preparation of standard solutions by judicious choice of volumetric glassware, Analytical Chemistry, 1980, 53, 1158-1161.

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I.5. REAGENTS When the quality of a reagent substance in one or more respects is critical for its intended use, it must be carefully defined, when necessary by prescribing appropriate tests to demonstrate its suitability. Normally, analytical grade reagents are employed in which case it is sufficient to give the name of the reagent, the CAS number and its formula.

Whenever possible, the reagent substances, reagent solutions, volumetric solutions and standard solutions for limit tests already described in the chapter 4. Reagents of the Ph. Eur. are to be employed. Simple solutions of reagent substances or solutions that are prepared for use on a single occasion are to be described in the monograph itself.

The use of reagents that are acknowledged to be extremely toxic or otherwise hazardous (e.g. carcinogenic), is to be avoided, especially in circumstances where their dangerous properties are difficult to control, e.g. when handled as fine powders or in spray reagents. The use of those substances that are prohibited or restricted in one or more of the States party to the European Pharmacopoeia Convention is also to be avoided (e.g. mercury containing reagents, REACH regulation annex XIV).

I.6. COMMERCIAL NAMES Commercial names are given systematically as footnotes in draft monographs for chromatography columns/plates and based on usefulness for the analysts in other cases (e.g. test kits, reagents that are available from a single supplier, etc.). Commercial names are not included in the text published in the Ph. Eur. but are transferred to the EDQM Knowledge Database after adoption of the monograph.

I.7. REFERENCE STANDARDS The policy and procedures regarding reference standards are described for information in general chapter 5.12. Reference standards. Procurement, establishment, storage and monitoring of reference standards are the responsibility of the EDQM. Many reference standards, notably impurity standards, are available only in limited quantities, and the amount prescribed for preparation of solutions must be kept to a minimum. Before publication of a monograph in Pharmeuropa, the required quantities of reference standards should be supplied to the EDQM, who will advise on the best strategy for optimising the use of substances that are available in limited quantities (for example, preparation of a spiked substance rather than supply of the single impurity). The aim of the EDQM is to present the reference standards for adoption at the same time as the monograph or, failing that, by the time of publication at the very latest.

For IR identification, preference is given to chemical reference substances over reference spectra, except in special cases, for example where provision of a reference substance entails practical difficulties.

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II. MONOGRAPH ON A SUBSTANCE FOR PHARMACEUTICAL USE

Monographs are based on the specifications for substances used in medicinal products approved in Member States. When a monograph is added to the work programme, enquiries are made by the EDQM to identify manufacturers of such substances and all data received is taken into account for preparation of the monograph. Interested parties should be invited to participate in the elaboration of the monograph before publication in Pharmeuropa, since the 3-month public period will often be too short for all interested parties to check the draft monograph.

Prior to the preparation of any monograph, it is essential to gather as much information as possible on the substance in question. In particular it is necessary to ascertain:

? ? ? ? ?

whether the substance is of natural, synthetic or semi-synthetic origin; whether the substance is a mixture or a single entity; the method(s) of preparation in detail;

whether there are different crystalline forms, since the properties of the substance may vary in accordance with this parameter;

whether both an enantiomer as well as the racemate or other mixtures of enantiomers are available;

whether different hydrates are available;

whether different entities (acid, base, salt, etc) are available.

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The Ph. Eur. and other relevant documents on the state of work must be consulted to see if monographs on similar substances exist or are being elaborated. If this is the case, it is important to ensure that similar monographs follow the same approach unless there are good reasons to deviate, e.g. developments in analytical techniques.

When a substance exists both in a water-free form and in the form of (a) hydrate(s) with different water contents, and if all these forms are used, they are normally treated as individual substances requiring separate monographs. The same rule applies for other solvates.

Substances that are to be described in a monograph may be members of a group of very similar substances (family). This holds true especially for excipients such as macrogols. A master monograph (family monograph) is to be drafted clearly stating the attributes common to all members of the family and that can be used to identify single members of the family.

All active substances and excipients described in the Ph. Eur. are subject to the provisions of the general monograph Substances for pharmaceutical use (2034).

II.1. TITLE The International Nonproprietary Name (INN) established by the World Health Organization should be used wherever it is available; it is supplemented as appropriate by the name of the anion or cation and by the degree of hydration. Anions and cations are indicated as “mono-, di-, tri-, etc.”, as appropriate.

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The following rules apply for the degree of hydration.

? In the case of a well-defined hydrate, “mono-, di-, tri-, etc…hydrate” is added to the title

whereas if the monograph covers more than one degree of hydration, the general term “hydrate” is used. In the latter case, a sentence is added to the DEFINTION section (see chapter II.2). For monographs published prior to the 9th edition of the Ph. Eur., retrospective introduction of the degree of hydration in titles would only be made after careful consideration.

? As of the 9th edition of the Ph. Eur, monographs referring to “anhydrous” substances will

no longer specify this in their title with the exception of a few monographs where the information has a recognised added value and/or is used in common scientific language (e.g. Ethanol, anhydrous).

? For monographs covering substances that can be either water-free or with, a defined or

variable, degree of hydration, no mention will be added to the title. This information will be supplemented in the DEFINITION section of the monographs (see part II.2.). Where a substance is used in approved medicinal products for veterinary use only in Member States, “for veterinary use” is included in the title.

II.2. DEFINITION The chemical structure must be ascertained with the greatest possible care in order to establish the exact:

? ?

graphic formula;

empirical formula and relative molecular mass. The latter is calculated as follows: first, the relative atomic masses, or multiples thereof, are added together using all the figures of the International Table of Relative Atomic Masses; the total is then rounded off to 4 significant figures if the initial digit is 1, 2, 3, 4 or 5, or to 3 significant figures if the initial digit is 6, 7, 8 or 9; the last figure is increased by 1 when the part rejected exceeds one half-unit. When the part rejected is equal to or less than one half-unit, the last figure taken is not modified;

chemical name. This involves investigating in particular:

o the possible existence of isomers so as to be able to specify which isomer is used

or, otherwise, to state that the product is a mixture of isomers;

o in the case of an optical isomer, it is insufficient to take into account only the

direction of the optical rotation. The absolute configuration is given by the R/S system at the asymmetrical centre(s) or any other appropriate system (e.g., for carbohydrates and amino acids);

o ascertaining the state of hydration so as to distinguish clearly between the well-defined hydrates (mono-, di-, tri-, etc… hydrate) and the products that contain variable quantities of water. In the latter case, the term “x-hydrate” is introduced in the chemical name.

?

If the substance contains a variable quantity of water, or refers to both water-free and hydrate form, a sentence is added to the DEFINITION section to explain the exact scope of the monograph.

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Some chemical substances, particularly those obtained from raw materials of natural origin and substances produced by fermentation may not be easily separated from certain related substances (for instance, quinine salts). These may be treated as:

? ? ?

a chemical product when obtained in a very pure state and when they can be assayed by a physico-chemical method;

a substance accompanied by a certain proportion of related substances, giving an exact definition of the main component only (e.g. neomycin);

a mixture of several components, sometimes difficult to define, where an overall description may suffice (e.g. nystatin).

Where applicable, the origin of the substance must be specified (name and strain of the organism from which the substance is derived). Where applicable, the monograph indicates that the substance is semi-synthetic and derived from a fermentation product [to clarify application of the general monograph Substances for pharmaceutical use (2034)].

II.2.1. Combinations

In therapeutics, more or less well-defined chemical combinations (for instance, theophylline- ethylenediamine) or even mixtures are sometimes used. In such cases, it is necessary to specify precisely each component of the combination or mixture, with its chemical structure and the proportion in which it is present.

II.2.2. Content

The substance described by a monograph is never a wholly pure substance but contains a limited proportion of impurities. The content is therefore an important part of the definition. Assay limits are specified between which the content must fall.

In setting these limits for the active substance content, account is taken of:

? ? ? ? ?

the method of preparation, which determines the degree of purity that may be reasonably required;

the reproducibility and accuracy of the analytical method; current batch data of about 10 production batches at release; the evaluation of batch stability data;

a sufficient number of experimental results obtained on several batches (at least 3), if possible, of different origins and ages.

For a non-specific assay by titrimetry the limits are set according to the table provided in part III.3.7 i.e. usually 99.0-101.0 %. Some monographs still include an assay by UV-Vis spectrophotometry usually bearing wider limits.

For a specific assay using a separation technique (for example, liquid or gas chromatography), the upper assay limit is normally 102.0 %; the lower assay limit will take any necessary account of the impurities present based on the available batch data and approved specifications. It may therefore be lower than 98.0 %.

When the substance to be examined contains only impurities that do not interfere with the

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assay, or when it contains only a very low proportion of impurities interfering with the assay, the results of the assay can be used directly. It will then be stated that: “[the substance] contains not less than x per cent and not more than the equivalent of y per cent (at least 100.5 %, but often a little more) of [chemical definition of the pure product]”. The content of the substance is usually expressed with reference to the anhydrous or dried substance. According to the general monograph Substances for pharmaceutical use (2034) the content of residual solvent is taken into account for calculation of the assay content of the substance, therefore no reference in the DEFINITION section of the individual monograph is made.

In cases where the water content is high (e.g. in the case of disodium phosphate dodecahydrate), limits of content may be expressed with reference to the hydrate form of the substance, taking into account the molecular mass of the hydrate form (only for well-defined hydrates) or with reference to the substance on the anhydrous/dried basis in combination with determination of water content/loss on drying.

When the substance to be examined contains a relatively large proportion (a few %) of impurities, which are determined at the same time as the active ingredient, an appropriate wording is to be used (for instance, in the case of quinine salts: “x per cent of total alkaloid salts, expressed as quinine salts”).

Exceptionally reference is made to only a part of the molecule or to an element (for example assay of magnesium oxide in light magnesium carbonate or assay of magnesium in magnesium stearate).

In the case of antibiotics determined by microbiological assays, the active ingredient content is expressed in International Units, where these exist, and only a minimum value is given. See also under part II.6.

II.3. CHARACTERS As defined in the General Notices, statements under the heading CHARACTERS are not to be interpreted in a strict sense and are not regarded as analytical requirements. The principal items that may be referred to under this heading are the following.

II.3.1. Appearance

This description will normally embrace colour and physical form. The term “white” is not used without qualification since, if viewed against a standard white material, very few pharmaceutical materials will appear truly white. It is, of course, not intended that such a comparison be made but experience shows that certain users of the Ph. Eur. may insist on doing so as part of a purchasing contract. The term “white or almost white” is used instead. Where positive colours are to be described, this is done in terms of primary colours or combinations of primary colours.

Colour: the following descriptive terms are used: black, orange, blue, pink, brown, red, colourless, violet, green, white/almost white, grey, yellow

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Compound terms may be used: English

French bleu-vert vert-bleu rouge-violet violet-rouge rouge-brun brun-rouge

greenish-blue bluish-green violet-red

reddish-violet brownish-red reddish-brown

It can be noted that in English, the dominant is placed second, whereas in French, it is placed first. Expressions such as lemon-yellow, buff, salmon-pink are to be avoided; standard dictionaries give equivalents for such terms as spectral colours with suitable qualifiers (for example, buff is described as “dull yellow”). The following adjectives are also used; light, slight, fluorescent, intense, pale, dull, deep, dark.

It is to be noted that the allowed colours and colour combinations also apply to the description of the colour changes of indicators when used in acid/alkalinity tests or in titrimetric assay procedures.

II.3.2. Taste

The taste is not to be taken into consideration.

II.3.3. Odour

In general, no reference is made to odour. In particular no reference to odour is made for those materials that would constitute a hazard if inhaled. Mention of odour in other cases must be justified.

II.3.4. Solubility

A method recommended for the estimation of solubility is given in general chapter 5.11. Characters section in monographs. All solubilities are quoted in the general terms defined in the General Notices. Solvents quoted are normally confined to water, an alcohol and a lipophilic solvent (e.g. water, ethanol, heptane). Solubilities in chloroform and ether are not mentioned and the use of hexane is discouraged. In special cases the solubility of different samples of a material may vary rather considerably even though their composition is still within the limits set by the monograph. The solubilities in the solvents thereby affected are then given to cover more than one solubility class, e.g. “sparingly soluble to soluble in...”. The solubilities or miscibilities in other solvents with which the material is often combined in practice such as fatty oils, etc., may also be mentioned. In some cases it may be useful to specify solubility in alkalis or acids and, particularly in cases of materials that are very insoluble in the above-mentioned solvents, a special solvent may be indicated, e.g. dimethylformamide or dimethyl sulfoxide. It is not necessary to specify the solubility in every solvent that is used in performing the tests of the monograph itself.

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determination is not accompanied by destruction to a degree that renders them extremely dependent on the actual mode of operation. The possible existence of polymorphism must also be taken into account; differences in the melting point must be indicated even when given under CHARACTERS. In exceptional cases, when the distinction of a specific crystalline form is necessary, determination of the melting point can aid in excluding the unwanted form(s). However, it should be kept in mind that an apparent melting point may be observed: a solid- solid polymorphic transition may take place during testing and the melting point of the resultant form is measured.

For first identification, neither the melting point alone nor the addition of a chemical reaction is sufficient to verify identity of a substance. However, the addition of another identification test such as TLC will often suffice. For second identifications, please refer to part II.4.2.

The melting point determined by the capillary method is defined in the Ph. Eur. (see method 2.2.14. Melting point – capillary method) as the last particle melting point (i.e. clear point or liquefaction point). It must not be confused with the melting interval even though both are given as a range.

II.4.6. Specific optical rotation

When an enantiomer is described in a monograph, a test for optical rotation is given in the IDENTIFICATION section or a cross-reference is made to the test for enantiomeric purity in the TESTS section. When both the racemate (or the racemic mixture) and the enantiomer are available, then, in the monograph of the racemate, an angle of rotation will be given in the TESTS section and will be referred to in the IDENTIFICATION section. When only the racemate is available the angle of rotation will be given in the TESTS section, provided the specific optical rotation of the chiral form is known and is of sufficient magnitude to provide a meaningful test for racemic character.

II.4.7. Thin-layer chromatography

This identification method requires the use of reference substances. Selectivity may be improved by combining TLC with chemical reactions in situ, i.e. by employing appropriate spray reagents. In the latter case, the same or a similar reaction is not to be repeated on a test-tube scale.

It is very important to ensure the separation of a critical pair in a related substances test but such a separation plays a minor role in an identification test. The separation of a critical pair in the individual identification tests is no longer required but the separation of a critical pair in the TESTS section is maintained. However, during development and validation, separation of the substance from similar substances must be demonstrated.

A chromatographic separation test for TLC plates is described in general chapter 4.1.1. Reagents to verify the performance of the plate type concerned. The test is intended to be a quality control procedure, carried out from time to time by the TLC plate user. It is clear that such a general procedure is not appropriate for every thin-layer separation problem and that the description of a separation criterion might still be necessary to ensure the identification of the substance. In these exceptional cases, a separation criterion is described in the IDENTIFICATION section.

16

A TLC system applied to purity testing in a monograph is preferred for identification when suitable. For the latter purpose the concentration of the solution to be examined and the corresponding reference solution is generally reduced so that 5 to 20 μg of each is deposited on the plate or sheet. It may also be necessary to change from a general to a more discriminating detection system.

Further technical requirements on these chromatographic methods are to be found in part II.5.8 on related substances.

II.4.8. Gas chromatography and liquid chromatography

The basic principles mentioned under thin-layer identification apply mutatis mutandis. Gas and liquid chromatography are rarely used for identification and where they are applied, it is by reference to a test or assay that applies the method elsewhere in the monograph. These methods are used only if there is no suitable alternative and are not used as the only identification test.

Further technical requirements on gas and liquid chromatography are to be found in part II.5.8 on related substances.

II.4.9. Chemical reactions

Several commonly applied identification reactions of a chemical nature are included amongst the general methods of the Ph. Eur. and these are to be used, whenever appropriate. Where several reactions for an ion or group are given in general chapter 2.3.1. Identification reactions of ions and functional groups, it is normally necessary to prescribe only one in the monograph. Note the need to specify the amount of material, or solution of it, to be taken for the identification test in question. The same holds true for tests that have to be described in full in the monograph. Identification reactions using toxic reagents are being slowly phased out; special care should be taken when choosing a chemical reaction to be added to a monograph.

Identification criteria that call for the recognition of an odour or a taste are to be avoided.

Each chemical reaction is to be chosen to demonstrate the presence of a different part of the molecule to be identified.

To differentiate substances within a group (family) which differ either by the extent of condensation or by the length of the hydrocarbon chain (e.g. fatty acids), it is necessary to cross-reference to the appropriate purity test(s) where values are determined (e.g. iodine value, saponification value, etc.).

II.5. TESTS II.5.1. General

The TESTS section is principally directed at limiting impurities in chemical substances. General chapter 5.10. Control of impurities in substances for pharmaceutical use gives details of the policy to be applied.

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While the monograph must ensure adequate purity in the interests of public health, it is not the aim of the Ph. Eur. to impose excessive requirements that restrict unnecessarily the ability of manufacturers to produce compliant products.

In the interests of transparency, information is included wherever possible on: the impurities controlled by a test; the approximate equivalent (percentage, ppm, etc.) of the prescribed limit in terms of the defined impurities or class of impurities. The information on the limit imposed may be a nominal content inferred from the conditions of the test or may be based on data from recovery experiments.

Acceptance criteria and limits are set on the basis of analytical data at hand i.e. batch results provided by manufacturers and data produced during monograph elaboration by the testing laboratories. In order to define limits for tests (e.g. loss on drying, residual water, etc), the empirical “3-sigma” rule may be used. In a normal distribution of values the confidence interval defined by the mean of the values ± 3 times the standard deviation accounts for 99.7 % of the data population. A minimum of 10 test results must be available to calculate the mean. However this rule is not systematically applied, especially for the related substances test where impurity limits should reflect more closely their real content in substances used in approved medicinal products.

Example 1: Determination of specification for water content (2.5.12) ? Batch data provided by manufacturer: 10 batches ? Min. value: 3.2 %, max. value: 5.4 % ? Mean + 3 sigma = 6.1 %

Conclusion: The limit for water is set at 6.1% according to “3-sigma” rule.

Example 2: Determination of specification for impurity X limit

? Batch data for level of impurity X provided by manufacturer: 57 batches ? 52 batches around or less 0.05 % , 4 batches about 0.08 %, 1 batch 0.09 %, ? Mean + 3 sigma = 0.11 %

Conclusion: The rule of “mean + 3 sigma” is not applied. The limit for impurity X is set at 0.10 %, based on batch data.

Certain tests may apply to special grades (parenteral, dialysis solutions, etc.) or a test may have a special limit for a particular use: the particular application of a test/limit is indicated within the test.

II.5.2. Title of tests

Wherever possible, the title includes the impurity or class of impurities limited by the test (e.g. Oxalic acid, Potassium, Copper, Chlorides, etc.). Non-specific tests carry a more general title appropriately chosen from the standard terminology of the Ph. Eur. (e.g. Appearance of solution, pH, Acidity or alkalinity, etc.) or a similar designation. Titles that merely refer to the methodology employed in the test (e.g. Absorbance, Specific optical rotation) are to be avoided wherever possible.

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II.5.3. Solution S

A solution of the substance to be examined, designated “Solution S”, is prepared whenever this can be used to perform more than one test (and/or identification).

If necessary, several solutions S, (designated S1, S2...) may be prepared in various ways, each being used for at least 2 tests.

For insoluble substances, solution S may be prepared by an extraction process.

The solvent used depends on the purpose of the tests and the solubility of the substance to be examined and that of its potential impurities. It may be:

?

water (usually):

o carbon dioxide-free water R in cases where the presence of carbon dioxide can

appreciably influence the outcome of a test, e.g. for pH or Acidity or alkalinity (see part II.5.5);

o distilled water R if solution S is used in the tests for barium, calcium and sulfates; o carbon dioxide-free water R prepared from distilled water when both previous cases

apply;

a dilute acid or an alkaline solution;

more rarely, other solvents (alcohols, tetrahydrofuran...) that give solutions with a narrower field of application than aqueous solutions.

? ?

The solvent must make it possible to carry out the specified tests, either directly, or after suitable dilutions explicitly specified in each test. Generally the concentration is around 20 to 50 g/L but may be lower (e.g. 10 g/L) or higher (100 g/L and, exceptionally, more). The quantity of solution S prepared must be sufficient to carry out each of the tests for which it has been prepared. If solution S is to be filtered, account must be taken of the loss on filtering and when the insoluble portion thus separated is to be used for another test, this is clearly indicated. While several tests may be carried out on the same portion of solution S, this is only done for substances where there are good reasons to economise (expensive products or products whose use is subject to restrictions) and this is then clearly indicated in the monograph.

Depending on the particular tests, the concentration of solution S is defined with varying accuracy:

? ? ?

for “Appearance of solution”, “pH” and some identifications, an accuracy of 5 to 10 % is sufficient;

for most limit tests an accuracy of about 2 % is appropriate;

for some cases such as the determination of specific rotation, specific absorbance, various chemical values and, more generally, tests where the result is obtained by calculation, a greater accuracy is needed.

The accuracy with which the concentration of solution S is defined is that required by the most exacting test for which it is intended. The description of the preparation of solution S thus specifies:

?

the quantity of substance to be examined with the required accuracy (see General

19

?

Notices);

the volume, to 1 decimal place (10.0 mL, 25.0 mL...) when the concentration must be known to within less than 1 %, without a decimal (10 mL, 25 mL...) when a lower accuracy is adequate.

II.5.4. Appearance of solution

This test makes it possible to ascertain the general purity of a substance by the detection of impurities insoluble in the solvent selected, or of coloured impurities.

The “Appearance of solution” test is practically always prescribed for substances intended for preparations for parenteral use. Apart from this, it is to be applied only if it yields useful information about specific impurities.

It can comprise one or both of the following tests:

? clarity and degree of opalescence of liquids (2.2.1.); ? degree of coloration of liquids (2.2.2.).

The 2 tests are practically always carried out on identical solutions, usually solution S, but they may be performed on different solutions.

The solvent employed is usually water but other solvents may be preferred depending on the solubility of the substance to be examined.

When an organic solvent is used to prepare solution S, it may be necessary to ensure that the solvent also complies with the test, especially where there is a very stringent requirement. The more concentrated the solution the stricter the test. For very pure substances or those used in high doses, the concentration chosen is 50 to 100 g/L, whereas for less pure substances or substances administered in small doses the concentration is 10 to 20 g/L. II.5.4.1. Clarity and degree of opalescence (2.2.1.)

This test is mainly performed on colourless substances or those that give only slightly coloured solutions in order to permit valid comparison with reference suspensions. Newer instruments with ratio selection are capable to measure coloured substances

The quantity of solution required depends on the diameter of the comparison tubes used; it varies from 7 mL to 20 mL for tubes with a diameter of 15 mm to 25 mm prescribed in the general methods. It is therefore necessary to take the latter volume into account.

Most often the solution examined must be “clear” (as defined in the Ph. Eur.). However, in certain cases for substances that are not intended to be used in solution, a more marked opalescence may sometimes be permitted.

II.5.4.2. Degree of coloration (2.2.2.)

The test applies to essentially colourless substances that contain, or may degrade to form, coloured impurities that can be controlled by limiting the colour of solution of the substance. Two methods are described in general method 2.2.2. Degree of coloration of liquids :

? ?

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Method I only requires 2 mL of solution but is seldom prescribed except for substances that give highly coloured solutions;

Method II, which is more discriminating and therefore more frequently used, requires the larger volume of solution employed for the clarity test.

The results given by these 2 methods do not necessarily coincide so the one to be used is specified in the monograph.

The solution is described as colourless when it is less coloured than reference solution B9. When the solution is slightly coloured, the appropriate reference solution is given. When the shade of colour varies according to the samples, 2 or more reference solutions of the same degree of colour may be mentioned, or even only the degree of coloration without specifying the actual colour.

For material intended for parenteral use and for highly coloured solutions, especially when the use of Method I is contemplated, it is preferable to apply a limit of absorbance measured with a spectrophotometer at a suitable wavelength (usually between 400 nm and 450 nm). The concentration of the solution and the limit of absorbance must be stated. The conditions and limit must be based on knowledge of the absorbance curve in the range of 400 nm to 450 nm and on results obtained with appropriate samples, including stored and degraded samples, as necessary.

II.5.5. pH and Acidity or alkalinity

This test allows the limitation of acidic or alkaline impurities stemming from the method of preparation or purification or arising from degradation (e.g. from inappropriate storage) of the substance. The test may also be used to verify the stoichiometric composition of certain salts. Two types of test for protolytic impurities are used in the Ph. Eur.: a semi-quantitative titration experiment using indicators or electrometric methods to define the limits, the Acidity or alkalinity test; or a pH measurement.

pH measurement is included if the material has buffering properties, otherwise a titrimetric procedure is recommended.

The question of whether to prescribe an Acidity or alkalinity test or a pH measurement in a pharmacopoeial monograph can be decided on the basis of an estimation of the buffering properties of the material. To this end, a titration curve can be constructed for an aqueous solution (or, if necessary, an extract) in the intended concentration (10 to 50 g/L) of a specimen, preferably pure, of the substance to be examined, using 0.01 M hydrochloric acid and 0.01 M sodium hydroxide, respectively, and potentiometric pH measurement.

The inflexion point of the titration curve is the true pH of the solution and will, for a pure substance, be at the point of intersection with the pH-axis. The measure of the buffering capacity of the solution to be examined is the total shift in pH, (?pH), read from the titration curve as the result of adding on the one hand 0.25 mL of 0.01 M sodium hydroxide to 10 mL of the solution, and on the other hand 0.25 mL of 0.01 M hydrochloric acid to another 10 mL portion of the same solution. The buffering capacity is inversely proportional to the ?pH. For a sample that is not quite pure, carry out a parallel displacement of the titration curve so that the true pH of the solution is on the pH-axis before the ?pH is read from the curve.

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The magnitude of ?pH of the solution to be examined determines the choice of method for the limitation of protolytic impurities according to the following scheme. The classification is based upon the observation that the colour change for most indicators takes place over a pH range of 2 units.

Class A Class B Class C

?pH > 4 4 > ?pH > 2 2 > ?pH > 0.2

Acidity-alkalinity test utilising 2 appropriate indicators. Acidity-alkalinity test utilising a single appropriate indicator. Direct pH measurement.

The protolytic purity cannot be reasonably controlled. Substances that are salts consisting of ions with more than 1 acidic and/or basic function belong to this class and for these a pH measurement can contribute to ensuring the intended composition if the limits are sufficiently narrow.

Class D ?pH < 0.2

It is evident that by changing the concentration of the solution to be examined, the class of buffering properties as set out above into which the substance will fall can to some extent be altered, since the shape of the titration curve will then also be modified. The concentration range given above is not to be exceeded however, unless poor water solubility makes it unavoidable to use a more dilute solution.

In certain cases a test for acidity-alkalinity cannot be performed with the use of indicators due to coloration of the solution to be examined or other complications, and the limits are then controlled electrometrically. If on the other hand, the addition of standard acid/or base results in decomposition or precipitation of the substance to be examined, it may be necessary to prescribe a pH test regardless of the buffering properties.

If, for special reasons, as mentioned above, a pH measurement has to be prescribed for solutions with little or no buffering capacity, the solution to be examined is prepared with carbon dioxide-free water R. Conversely, the use of carbon dioxide-free water R for preparing solutions that have sufficient buffering capacity to warrant a direct pH measurement is not necessary since the required accuracy, which seldom exceeds 1/10th of a pH unit, will not be affected. When an acidity requirement corresponds to not more than 0.1 mL of 0.01 M sodium hydroxide per 10 mL of solution to be examined, the latter must be prepared using carbon dioxide-free water R. These considerations are to be borne in mind when prescribing the composition of solution S if it is to be used in a test for protolytic impurities.

II.5.6. Optical rotation (2.2.7.)

The optical rotation test, though sometimes useful for identification purposes, is mainly used as a purity test:

? ?

either to assess the general purity of an optically active substance (a liquid or a solid in solution), by calculating the “Specific optical rotation” (title of the test);

or to limit the presence of optically active impurities in any optically inactive substance (racemate or racemic mixtures), provided that the specific optical rotation at 589.3 nm is sufficient to ensure adequate sensitivity. In this case the range normally given should be ? 0.10° to + 0.10° (covering the substances that are not true racemates). In this case the “Angle of rotation” (title of the test) of the liquid or of a solution of the solid, is

measured under defined conditions.

22

It is usually more appropriate to control these impurities by chiral separation methods since the specific optical rotation is often insufficient to limit the presence of the unwanted enantiomer (distomer) in the presence of the active enantiomer (eutomer).

The test is not suitable for highly coloured or opalescent solutions (in the latter case a filtration can sometimes make the determination possible). The following aspects are taken into account in describing the test:

?

the solvent, which depends on the solubility of the substance to be examined and the rotatory power in that solvent. In the case of non-aqueous solvents, their purity and especially their contents of water must often be carefully defined;

the concentration of the solution: it must be high enough to give a reliable reading of the angle of rotation;

the quantity of substance to be used, determined with sufficient accuracy (generally 1 %), as is also the volume to be obtained (given with 1 decimal figure);

the volume required which depends on the apparatus used, but since it rarely exceeds 25.0 mL, that volume is usually prescribed;

the degree of hydration or organic solvation of the substance must be taken into account in calculating the result;

the result is the mean of at least 5 measurements when evaluated visually, with an apparatus giving readings to the nearest 0.01°;

measured angles of rotation (rarely more than 2°) are given to 2 decimal places;

specific optical rotation values are given with 2 or 3 significant figures. Values less than 10 are given to 2 significant figures, while values of 10 and over are given to 3 significant figures;

composition limit for racemates or racemic mixture.

? ? ? ? ? ? ?

?

The value of the optical rotation is calculated with reference to the dried or anhydrous substance

II.5.7. Absorption spectrophotometry (ultraviolet and visible) (2.2.25.)

The absorption of electromagnetic radiation may be used in purity tests as a limit test for certain impurities. The typical case is that of impurities that absorb in a region where the substance to be examined is transparent. It is then the absorbance of a solution of the substance to be examined that is measured. This test may be performed in the following ways:

? ?

by direct measurement on the solution, where the absorbance measured is a maximum absorbance at a given wavelength, or over a wavelength range;

after carrying out a chemical reaction that forms, with the impurity, a substance that absorbs at a wavelength where the substance to be examined is transparent, a maximum value at the given wavelength being prescribed.

For measurements in the ultraviolet, it is advisable not to measure below 230 nm.

It is important to describe precisely the operational conditions to be observed, in particular the preparation of those solutions that are prepared by successive dilutions.

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II.5.8. Related substances

The policy on control of impurities is described in general chapter 5.10. Control of impurities in substances for pharmaceutical use and in the general monograph Substances for pharmaceutical use (2034). Monographs should be elaborated accordingly. Monographs are designed to take account of substances used in approved medicinal products in Member States and should provide adequate control of all impurities occurring in these substances, insofar as the necessary information and samples (substance and impurities) are available from the producers. Where the required information and samples are not provided for a substance synthesised by a given method, the monograph will not necessarily cover the corresponding impurity profile.

The provisions for related substances of the general monograph Substances for pharmaceutical use (2034) apply to all active substances covered by a Ph. Eur. monograph, unless otherwise stated. The following are given as exceptions in the general monograph: biological and biotechnological products, peptides, oligonucleotides, radiopharmaceuticals, products of fermentation and semi-synthetic products derived therefrom, to crude products of animal or plant origin or herbal products.

If an exception is to be made for some other substance, the following statement is included in the specific monograph: “The thresholds indicated under Related substances (Table 2034.-1) in the general monograph Substances for pharmaceutical use (2034) do not apply.” Monographs should include acceptance criteria for:

? ?

each specified impurity;

unspecified impurities (previously referred to as “any other impurities”), normally set at the identification threshold; the total of impurities.

?

Impurities to be controlled include: intermediates and by-products of synthesis, co-extracted substances in products of natural origin, degradation products. Monographs on organic chemicals usually have a test entitled “Related substances” (or a test with equivalent purpose under a different title), designed to control organic impurities. Inorganic impurities are usually covered, where applicable, by other tests. Residual solvents are covered by specific provisions [see below and in general chapter 5.4. Control of residual solvents and general monograph Substances for pharmaceutical use (2034)].

Genotoxic impurities. Based on the EMA Committee for medicinal products for human use (CHMP) Guideline on the limits of genotoxic impurities (CPMP/SWP/5199/02, EMEA/CHMP/QWP/251344/2006), the following pragmatic approach should be followed when elaborating or revising monographs. New monographs should be based on an evaluation for the presence of potentially genotoxic impurities (PGIs) during marketing authorisation according to the principles of the CHMP guideline or similar evaluation principles for non-EU member states. For active substances included in medicinal products authorised by the competent authorities before application of the CHMP guideline, the specifications as described in the dossier for marketing authorisation should be followed. Action is needed only where there is study data demonstrating genotoxicity of the impurity. The existence of structural alerts alone is considered insufficient to trigger follow-up measures. If a new synthetic route is used that may give rise to different PGIs or to higher levels of previously recognised PGIs then the evaluation by a

24

Competent Authority should be used as the basis for the PGI in question.

Where an issue concerning a PGI is raised by a Competent Authority (notably for revision of a monograph or in comments on a Pharmeuropa draft), this will be dealt with on the basis of data provided to the European Pharmacopoeia Commission by the Competent Authority.

The policy described applies to substances for human use. Where a substance is used in veterinary medicine, the Competent Authority will decide for each particular case the requirements to be applied for PGIs.

The table shown below gives an outline of some common situations faced by groups of experts and suggested action.

Decision table for use during elaboration or revision of monographs

Status Action Substance included in a medicinal product authorised after application of the Monograph should be based on marketing authorisation(s). CHMP guideline*.

Substance included in a medicinal product authorised before application of No action needed, monograph based on marketing authorisation. the CHMP guideline*:

?

no PGI expected from synthetic route.

Substance included in a medicinal product authorised before application of No action needed during elaboration of monograph (based on the CHMP guideline*: marketing authorisation), no revision of existing monographs.

?

? ?

PGI expected from synthetic route of 1st authorised product and subsequently authorised products (if any) have no expected PGI or same PGI as the 1st authorised product at same or lower level and no data showing genotoxicity.

Substance included in a medicinal product authorised before application of Monograph should be elaborated or revised based on evaluation the CHMP guideline*: by the Competent Authority.

?

?

PGI expected from synthetic route of an authorised product and data showing genotoxicity of an expected PGI.

Substance included in a medicinal product authorised before application of Monograph should be elaborated or revised based on evaluation the CHMP guideline*: of new PGI or high level of previously known PGI by the Competent Authority.

? PGI expected from synthetic route of 1st authorised product, and ? subsequently authorised products have a new expected PGI or same PGI as 1st authorised product at a higher level and ? data showing genotoxicity of an expected PGI. Substance included in a medicinal product authorised before issuance of the Monograph should be elaborated or revised based on evaluation CHMP guideline*: of new PGI by the Competent Authority.

? ?

?

PGI not expected from synthetic route of 1st authorised product, and

subsequently authorised product(s) have a new expected PGI and data showing genotoxicity of an expected PGI.

* or similar evaluation principles for non-EU member states

It should be noted that as of 1 January 2016 the new ICH guideline M7 assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk (EMA/CHMP/ICH/83812/2013) will enter into force and replace the current EMA guideline. Control of impurities. The most common and preferred method for control of organic impurities is LC; GC or CE may be the preferred method in some instances. Although there are still some monographs applying thin-layer chromatography, in the future this method should be reserved for

25

control of specific impurities that cannot conveniently be controlled by LC or GC. Existing TLC tests that do not follow this recommendation will be replaced gradually as soon as information on suitable LC or GC tests becomes available.

Where the counter-ion of an active substance is formed from a lower organic acid, a test for related substances of the organic moiety is usually not considered necessary (for example, magnesium lactate used as a source of magnesium).

Monographs frequently have to be designed to cover different impurity profiles because of the use of different synthetic routes and purification procedures by producers. The usual practice is to include a general LC test, supplemented where necessary by other tests (LC, GC, CE, TLC, or other techniques) for specific impurities. It is, however, becoming increasingly impractical in some cases to design a single general test and in such cases more than one general test is included and the scope of the different tests is defined in the tests themselves with a cross-reference in the IMPURITIES section.

Monographs cover a number of specified impurities designated in the IMPURITIES section. Specified impurities are those that occur in current batches of the substances used in approved products and for which an individual acceptance criterion is provided. Wherever feasible, monographs also have an acceptance criterion for other impurities (at the identification threshold for the substance) and a limit for the total of impurities (or a limit for the total of impurities other than a number of identified specified impurities) above the reporting threshold. The acceptance criterion for specified impurities may be set at the identification threshold for the substance. The acceptance criteria for specified impurities take account of both:

?

qualification data, where applicable, the limit being set at a level not greater than that at which the impurity is qualified; the information on qualification is provided by the producer and the compatibility of the limit with the qualification data and approved specifications is checked by the competent authorities during elaboration of the monograph and/or during the Pharmeuropa comment phase; and

batch analysis data, the acceptance criteria being set to take account of normal production; data is provided by the producer for typical batches and checked during elaboration of the monograph on not fewer than 3 batches.

?

All decisions on impurity acceptance criteria should be based on the real impurity content (meaning after application of correction factors (CF)) in representative batches examined. Impurities need to be specified and located appropriately in the chromatogram if the reported batch values for an impurity are:

? ?

above the applicable limit for unspecified impurities before correction and cross the limit downwards when corrected (overestimation, CF<1), or

below the limit for unspecified impurities before correction and cross this limit upwards when corrected (underestimation, CF>1).

Usually, no correction factor will be given if the reported batch values for an impurity are below the applicable limit for unspecified impurities before correction and below the reporting threshold (disregard limit) after correction.

In any case, correction factors between 0.8 and 1.25 (corresponding to response factors of 1.2 to

26

0.8) are not given in monographs. Further information on the indication of correction factors is given in chapter II.5.8.2.b.

Response and correction factors. According to general chapter 2.2.46. Chromatographic separation techniques, the relative detector response factor, commonly referred to as response factor, expresses the sensitivity of a detector for a given substance relative to a standard substance. The correction factor given in the monograph is the reciprocal value of the response factor. The response factor can be calculated from the following formula:

??????=

????????× ????????

RRF = response factor

Ai = area of the peak due to the impurity

As = area of the peak due to the test substance

Cs = concentration of the test substance in milligrams per millilitre Ci = concentration of the impurity in milligrams per millilitre.

For the calculation, the mean of the area ratios over the whole range of linearity or the ratio of the slopes of the respective linearity regression equations may be used.

The response factor can be determined from the formula above, by preparing solutions of defined concentrations of the impurity and the test substance and measuring them by LC/UV at a given wavelength and flow rate. The concentration of the impurity and that of the test substance should be in the same order of magnitude and the measurement should be carried out using a calibration curve determined at several points around the concentration which corresponds to the acceptance criterion of the impurity. The weighings of impurity and test substance should both be corrected for the purity. Ideally, the chromatographic purity and water/solvent content of the impurity and the test substance should be determined beforehand. A provisional value might be assigned on the basis of the formula:

??????????????(%)=[100?(??????????+????????????????)]×

???????????????????????????? ???????????? (%)

100Suitable methods should be chosen when only a small amount of the impurity is available, e. g. TGA, coulometry for water/solvents, and LC to estimate its purity by injecting a concentrated solution of the impurity. If the available amount of impurity is still too small, values given in the certificate of analysis may be used.

It is also important to consider the form (base/acid or salt) of both the impurity and the test substance and to introduce an additional correction factor for the molecular mass ratio when impurity and test substance used for the determination are present in different forms. Preferably the response factors should be determined in two laboratories using the same protocol. If different detector types (diode-array detector (DAD) and variable wavelength detector (VWD)) are available, these may also be considered for the measurement of response factors.

Separation methods. For pharmacopoeial purposes the objective of a purity test using a separation method will usually be the control of impurities derived from one or more known manufacturing processes and decomposition routes. However, the experimental conditions are chosen for the test, especially the detection system, so as not to make it unnecessarily narrow in

27

scope. Chromatographic purity tests may often be the best means of providing a general screening of organic impurities derived from new methods of manufacture or accidental contamination. It may be advantageous to supplement a chromatographic test with other chromatographic or non-chromatographic tests.

As mentioned above (part II.4.8), a chromatographic system applied to purity testing may, when suitable, be applied also for identification.

When a related substances test based on a chromatographic technique is carried out, a representative chromatogram is published with the monograph in Pharmeuropa. Ultimately, the chromatogram will not be published in the Ph. Eur. but will be transferred to the EDQM Knowledge Database.

When no individual impurity is available as a reference substance or when a large number of impurities may be detected in the substance, a representative chromatogram will be supplied with the available reference substance (e.g. substance to be examined spiked with the impurities). Monographs should provide a reliable means of locating all specified impurities on the chromatogram. Identification of unspecified impurities is necessary if a correction factor is to be applied. Peaks may be located using:

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a reference standard for each impurity;

a reference standard of the substance to be examined containing some or all of the specified impurities, provided with a chromatogram.

Location by relative retention is not generally considered sufficient for pharmacopoeial purposes, notably for gradient elution. Where a reference standard containing a mixture of impurities is to be used, a sample of each specified impurity should be provided to the EDQM to enable the establishment of the reference standard.

In general, relative retention is given to 1 decimal place. However, it is given to 2 decimal places where necessary to indicate the elution order of closely eluting peaks. General considerations applying to separation techniques:

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high concentrations/loadings are normally used since the symmetry of the principal peak or shape of the spot is not critical in impurity testing so long as there is no interference. When using an external standard in quantitative determinations the response of the principal peak need not be in the linear range of the detector;

in general tests for related substances, the substance to be examined should not to be chemically modified (e.g. derivatisation) before purity testing since the impurity pattern may be modified;

similarly, extraction of the free base or acid prior to impurity testing is to be avoided.

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II.5.8.1. Thin-layer chromatography (TLC) (2.2.27.)

TLC methods should only be used for control of a specified impurity and where LC, GC or CE methods are inappropriate (usually due to a lack of a suitable detection system).

Commercially available pre-coated plates, described in general chapter 4.1.1. Reagents, are to be used; the trade name of the plate found suitable is indicated in a footnote to the draft monograph,

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and posted to the EDQM Knowledge Database after adoption of the monograph. In general chapter 4.1.1. Reagents, besides information on the coating material used (type of coating material, type of binder), a suitability test procedure is described under TLC silica gel plate R. The monograph must describe the type of plate and include a system suitability requirement. Often the substances that would be best suited for a system suitability test will not be readily available individually; a sample of the substance to be examined containing them as contaminants or even a deliberately spiked sample may then be prescribed. Permissible variations to the different parameters are indicated in general chapter 2.2.46. Chromatographic separation techniques.

If any pre-treatment is required or if the chromatography is carried out in unsaturated conditions for the satisfactory conduct of the test, then this information is included in the text of the monograph (especially applicable to the use of reverse-phase plates).

One or more dilutions of the substance to be examined will often prove adequate for reference purposes, provided the impurities to be compared exhibit a similar behaviour under the chosen chromatographic conditions. This implies that the spots to be compared must be sufficiently close in RF value to minimise errors introduced by different diffusion of the substances during their migration. Otherwise, reference solutions containing the specified impurities are to be employed. It may be necessary to instruct the analyst to disregard a spot – often due to the non-migrating counter-ion of a salt – remaining on the starting line.

Summation of the responses exhibited by each individual spot is only acceptable when appropriate equipment is prescribed. It is not recommended to set a limit or limits for the concentration of impurities without a limit on their number, otherwise the total theoretical impurity level would be unacceptably high. This situation may be counteracted by limiting the impurities on 2 or more levels, allowing only a defined number to be at the higher level and the rest below the lower level. As examples, the test may specify that no contaminant may exceed a relative concentration of 1 % and that only 1 may exceed 0.25 % or that no contaminant may exceed a relative concentration of 1 %, only 1 contaminant above 0.5 % and no more than 4 contaminants above 0.25 %.

II.5.8.2. Liquid chromatography (LC) (2.2.29.)

Defining the appropriate chromatographic system will often be one of the major problems to be dealt with in elaborating a pharmacopoeial purity test based on chromatography. In LC the matter is further complicated by the existence of numerous variants of stationary phases, especially amongst the chemically bonded reverse-phase materials for which not only brand-to-brand but occasionally also batch-to-batch variations occur that can influence a given separation. Once the type of stationary phase tested has been found to show a satisfactory separation it must be defined by, for example including the following information: type of particles (irregular or spherical), particle size, specific surface area (m2/g), pore size (nm), and when using reverse-phase columns the extent of carbon loading (%) and whether the stationary phase is end-capped or otherwise treated to inactivate the residual silanol groups (this is particularly important when basic substances are to be examined and there is a risk of peak tailing).The trade name of the stationary phase(s)/column(s) found suitable during elaboration of the monograph is indicated in a footnote to the draft monograph and transferred to the EDQM Knowledge Database after adoption of the monograph.

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In describing the chromatographic system, mention must be made of the column dimensions (length and internal diameter), nature of the stationary phase (as detailed previously) including any steps to prepare or pre-treat it, composition and flow rate of the mobile phase including elution programme (if any), column and autosampler temperature (if differing from room temperature or especially if thermostated), method of injection (if important), injection volume and method of detection.

Depending on the detection wavelength selected, the analyst should choose a suitable grade of solvent when preparing the mobile phase. The following guidance applies for the most frequently used solvents methanol and acetonitrile from the 7th edition of the Technical Guide on. If water is used as a component of the mobile phase, water for chromatography R should be used.

Wavelength intervals λ ≥ 250 nm 220 nm ≥ λ < 250 nm λ < 220 nm Acetonitrile grade Acetonitrile R Acetonitrile for chromatography R Acetonitrile R1 Methanol grade Methanol R Methanol R1 Methanol R2

Permissible variations to the different parameters are indicated in general chapter 2.2.46. Chromatographic separation techniques.

Test and reference solutions are wherever possible prepared using the mobile phase as the solvent in order to minimise peak anomalies.

Since many active pharmaceutical substances are synthesised by a number of synthetic routes, the list of potential impurities to be limited may be large and the analytical challenge to separate them is great. For the sake of robustness and reproducibility, isocratic elution is to be preferred while setting up a compendial method. However, isocratic liquid chromatographic methods may not be sufficiently selective so there is an increasing need to employ gradient methods.

When a gradient system is described, all necessary parameters must be clearly given, e.g. composition of mobile phases, equilibrium conditions, gradient conditions (linear or step), etc. In general the return to the initial conditions is not prescribed in monographs as this is considered to be instrument specific. Should this information be considered important (e.g. ion exchange chromatography), it may be added as a note to the draft monograph and transferred later to the EDQM Knowledge Database.

For gradient elution in LC an important parameter to be considered is the volume between the solvent mixing chamber and the head of the column. This volume is referred to as the dwell volume, D (other terms employed include: effective system delay volume, dead volume and delay volume). The dwell volume is dependent on the configurations of the pumping system including the dimensions of the capillary tubing, the solvent mixing chamber and the injection loop;. Large differences in dwell volume from one pumping system to another will result in differences in elution of peaks. The greatest effect of differing dwell volumes on retention times is for those substances that are not strongly retained. Thus, gradient systems should be conceived with an initial isocratic phase so that analytes are not eluting too close to the injection peak allowing the correction for marked differences in dwell volume from one gradient pumping system to another. The dwell volume of the pumping system employed to develop the method should be equal to or less than 1.0 mL. If the method is developed using a system with a dwell volume greater than

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1.0 mL, then a suitable initial isocratic step is essential. Experts should indicate in their reports the dwell volume of the instrument used for their experimental work. This dwell volume will be stated in a footnote in draft texts and will be transferred to the EDQM Knowledge Database after the monograph is adopted. A method for the determination of the dwell volume is indicated in general chapter 2.2.46. Chromatographic separation techniques.

II.5.8.2.a. System suitability criteria One or more system suitability criteria are to be included in the test. Requirements given in general chapter 2.2.46. Chromatographic separation techniques are also applicable.

Separation capacity. Such a criterion is necessary when separation techniques are employed for assays and tests for related substances. The following approaches, most of which require the separation or partial separation of a critical pair, are acceptable for a system suitability test for selectivity:

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Resolution. As calculated by the formula given in general chapter 2.2.46. Chromatographic separation techniques using 2 closely eluting peaks. In cases where several closely eluting impurities are present, it may be useful to describe more than one resolution requirement. However, when the retention times of the 2 peaks are very different, i.e. when the resolution is large (> 5.0), the use of the resolution as a performance test has little value. It is preferable to use another impurity or another substance chemically related to the substance under study, giving a smaller resolution. Peaks of different heights may be used for calculation of resolution provided the detector is not saturated.

Peak-to-valley ratio. This can be employed when complete separation between 2 adjacent peaks cannot be achieved, i.e. when the resolution factor is less than 1.5. The minimum requirement for peak-to-valley ratio should not be less than 1.5. Often even better separations are necessary to ensure a meaningful integration of impurity peaks.

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In-situ degradation such as oxidation, hydrolysis, Z-E isomerisation or ring closure, offers an alternative approach to define the suitability of the system provided that the solution of the substance can be degraded, in mild “stress” conditions within a reasonably short time, to produce decomposition products, the peaks of which can be used to determine a resolution or a peak-to-valley ratio. This may be a useful approach to avoid the use of impurity reference standards. Chromatogram of a ‘spiked’ or an impure substance can also be employed to define the system. This approach can be employed when it is difficult to isolate an impurity eluting close to the main peak in sufficient quantity to establish a reference substance. In this case a chromatogram can be supplied with the reference substance (for system suitability or for peak identification) or described in the text of the test for related substances. Such chromatograms are not published in the monograph, but provided in the EDQM Knowledge Database.

The use of a spiked or impure substance requires procurement of sufficient material to establish the reference substance used and in the future, replacement of the system suitability test material with material exhibiting the same characteristics.

The methods of choice for defining the performance of the system are the calculation of the resolution and the peak-to-valley ratio and such a requirement is also to be included when using a chemical reference substance (CRS) of a spiked or impure substance. When gradient elution is described, it is preferable to describe a system suitability requirement for each critical gradient

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step.

It should be noted that the inclusion of retention times or relative retention values are given only for identification of peaks and do not constitute alternative system suitability criteria. Sensitivity. Disregard limit/Reporting threshold serves a 2-fold purpose:

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decision criterion for the user whether a peak area or a corrected peak area of an impurity is to be included in the total of impurities;

general criterion for the user to determine compliance of his actual chromatographic system with the requirement of general chapter 2.2.46. Chromatographic separation techniques (S/N ratio ≥ 10 at the disregard limit/reporting threshold).

Typically, the disregard limit for substances covered by a monograph is set in accordance with the reporting threshold given in Table 2034.-1. (see Substances for pharmaceutical use (2034)) and usually a respective reference solution is prescribed in the monograph. When more than one impurity is limited and a limit for the total of impurities is prescribed, a reporting threshold needs to be included in the test for related substances. This threshold helps to compensate for differences in sensitivity that can be observed when different analytical systems are being employed. However, when only one impurity is limited, no reporting threshold needs to be included (external standardisation only).

For specified impurities with correction factors > 1.25 (i.e. response factors < 0.8), the peak should be quantifiable not only at its limit, but also down to the disregard limit/reporting threshold. The latter is important for the determination of the sum of impurities. Therefore, if the general signal-to-noise requirement of 10 is not applicable, it may be necessary to add a specific sensitivity criterion for this impurity.

Example: impurity X is specified at 0.15 % with a correction factor of 5 and a general disregard limit at 0.05 %. For the impurity X under consideration, the sensitivity of the method is sufficient if:

? (1) a S/N ratio of minimum 10 is obtained with a 0.05 % (relative to the test solution)

solution of impurity X, when impurity X is available as reagent/CRS; or

? (2) a S/N ratio of minimum 50 is obtained with a 0.05 % solution of the active substance

when impurity X is not available. Option (2) is preferred when only limited amounts of the isolated impurity are available and the correction factor of the specified impurity is less or equal to 5. In case of option (2), as the correction factor of the impurity is between 1.25 and 5 (i.e. the response factor is between 0.8 and 0.2) and a dilution of the test solution is used for the quantification, it is recommended that the sensitivity of the method is verified during its validation. The S/N ratio of the impurity peak at the reporting threshold should be at least 10 to be quantifiable. To take account of different sensitivities of equipment used, a minimum S/N ratio should be described in monographs where the observed S/N of the impurity peak is not higher than 50 at the reporting threshold. The introduced S/N ratio requirement should be at least 10 times the correction factor (e. g. correction factor is 4, then S/N requirement should be at least 40).

Example 1: Rosuvastatin Calcium: Impurity C, correction factor 1.4, limit 0.8 %, reporting threshold 0.05 %, quantified using a dilution of the test solution 0.2 % (ref. sol. (b)).

? S/N of impurity C at reporting threshold: 55 (minimum requirement 10 to be quantifiable,

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minimum 50 to take account of the sensitivity of different equipment)

? S/N of principal peak in ref. sol. (b): 361, i.e. 90 at reporting threshold of 0.05 % Conclusion: method is very sensitive so that a minimum S/N is not required in the monograph

Example 2: Correctoprolol (theoretical case): Impurity A, correction factor 2.2, limit 0.2 %, reporting threshold 0.05 %, quantified using a dilution of the test solution 0.1 % (ref. sol. (b)). ? S/N of impurity A is 35 at the reporting threshold (minimum requirement is 10 to be

quantifiable, minimum 50 to take account of different equipment)

? S/N of principal peak in ref. sol. (b): 154, i.e. 77 at the reporting threshold of 0.05 %

(minimum 2.2 x 10 = 22).

Conclusion: based on these results the sensitivity is sufficient but using a less sensitive equipment the minimum requirements might not be fulfilled, recommendation is to include in the monograph a minimum requirement of S/N of 44 for reference solution (b).

Repeatability. In LC with UV detection, it is commonly accepted that the relative standard deviation of the peak response obtained on 3 injections of a reference solution corresponding to 0.1 % of the test solution is not more than 5.0 %.

II.5.8.2.b. Quantification Quantification is required for limits applied to specified impurities, unspecified impurities and total impurities. It is most commonly achieved using an external standard and less commonly by the normalisation procedure.

External standard. A dilution of the test solution/substance to be examined is used, unless there is a large difference in the detector response of a specified (or exceptionally an unspecified) impurity that necessitates the use of a specific external standard, which may be:

? a solution of the impurity, normally in form of a reference standard (preferred option); ? a solution of the substance to be examined containing a known amount of the impurity.

Where a dilution of the substance to be examined is used as external standard, the experts should determine correction factors for the impurities, which are indicated in monographs only if they are outside a range of 0.8 to 1.25 and considered relevant in view of the batch results (see part II.5.8). Correction factors are normally given to only 1 decimal place.

It is recommended not to apply correction factors > 5 for specified impurities, but to use external standards in these cases where possible.

In order to take account of different responses, it is possible to use a wavelength, different from the default wavelength, for the control of particular impurities. It is understood that the test and the reference solutions are recorded at the same wavelength unless otherwise prescribed.

The acceptance criteria for related substances tests may be expressed either in terms of comparison of peak areas (“comparative test style”, which has been used previously) or as numerical values (“quantitative test style”; this is the preferred style to be used for new texts or major revisions).

Based on the requirements of the general monograph Substances for pharmaceutical use (2034):

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in monographs using the comparative style (acceptance criteria expressed in terms of

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peak areas), a disregard limit is usually set with reference to a dilution of the test solution;

in monographs referring to numerical values for acceptance criteria, a reporting threshold is defined.

Normalisation procedure. Quantification by the area normalisation technique requires that all the solutes are known to be eluted and detected, preferably with uniform response factors, and that the detector response is linear up to about 120 % of the concentrations employed. This must be validated.

As indicated in general chapter 2.2.46. Chromatographic separation techniques, peaks due to solvents or reagents or arising from the mobile phase or the sample matrix, and those at or below the reporting threshold, are excluded before calculating the percentage content of a substance by normalisation. An additional reference solution is prescribed to determine the reporting threshold. The corresponding numerical value is stated in the monograph. II.5.8.3. Gas-liquid chromatography (GC) (2.2.28.)

The difficulties met when defining the appropriate chromatographic system are similar in GC purity tests to those mentioned under LC although the emphasis may be on other points. The experimental details to be described in a pharmacopoeial test must, therefore, also here be worded as an example so that the chromatographic parameters may be varied to obtain the required performance. The nature of the stationary phase, i.e. the composition of the coating material (including its concentration) and the inert support (including its particle size and any pre-treatment) must also be given here in general terms but the details are to be recorded for subsequent publication in Pharmeuropa. These pieces of information will be transferred to the EDQM Knowledge Database after adoption of the monograph.

In describing the chromatographic system, mention must be made of essentially the same factors as mentioned under LC with appropriate variations, e.g. temperature programme (if any) instead of elution programme, injection port and detector temperatures, etc. Use of packed columns should be avoided. Permissible variations of the different parameters are indicated in general chapter 2.2.46. Chromatographic separation techniques.

For the sake of robustness and reproducibility isothermal operating conditions are preferred. Quantification is usually based on an internal standard technique or on the area normalisation procedure. The same limitations concerning summation of peak responses as mentioned for LC apply here as well.

For the expression of acceptance criteria, the principles defined in part II.5.8.2 for LC are to be applied. In cases where capillary columns are used, a resolution factor > 5 is accepted. II.5.8.4. Capillary electrophoresis (CE) (2.2.31.)

CE is increasingly employed to separate and control a large number of impurities of vastly different polarities. It is also suitable to control the content of the unwanted enantiomer in chiral therapeutic substances. Where the separation is conducted in a fused-silica capillary, the problem encountered in reverse-phase LC of varying performance from different stationary phases, is avoided.

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Joule heating occurs during a run and to obtain satisfactory reproducibility a defined temperature is maintained using a thermostat; for instruments without a thermostat, a low voltage should be used.

The limit of detection is adversely affected by the small injection volume and the small detection pathway in the capillary, even when stacking techniques are applied. For the control of impurities or assays, the use of an internal standard is recommended to achieve appropriate precision. Otherwise the guidance for the use of this technique is similar to that given previously for LC. For chiral analysis, a chiral reagent is added to the running buffer. The chiral reagent should be carefully described in the monograph or as a reagent, particularly for cyclodextrin derivatives. Since many of the cyclodextrin derivatives are randomly substituted, it is important to give the exact or average degree and location of substitution. During validation of the method more than one batch of the cyclodextrin derivative should be used.

Experimental parameters to be considered for inclusion in the monograph:

? ? ? ? ? ? ? ? ? ?

Instrumental parameters: voltage, polarity, temperature, capillary size (diameter and length ? total and effective – to the detector).

Coating material of the capillary (where applicable). Buffer: pH, molarity, composition. Sample solvent.

Separation: pole outlet, voltage (U), current (I).

Injection: time (t), voltage (U) for electrokinetic injection or pressure difference ?p for hydrodynamic injection.

Detection: wavelength, instrumentation. Temperature.

Shelf-life of solutions.

Rinsing procedures (time, reagents, ?p) needed to stabilise the migration times and the resolution of the peaks:

o pre-conditioning of a new capillary;

o pre-conditioning of the capillary before a series of measurements; o between-run rinsing.

As a footnote for transfer to the EDQM Knowledge Database after publication of the monograph:

? ?

if a coated capillary is used, the trade name of the capillary found suitable during elaboration of the monograph;

for chiral separations, the trade name of the chiral reagent (cyclodextrin or other) found suitable during elaboration of the monograph.

In order to minimise the electro-osmotic flow signal, test and reference solutions are, wherever possible, prepared using water for injections or the running buffer as the solvent.

II.5.9. Readily carbonisable substances

The value of this non-specific test has greatly diminished through the introduction of chromatographic tests providing more information on organic impurities. The major advantage of

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