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.