Organic Impurities in Chemical Drug Substances

Many pharmacologically active substances are totally synthetic organic chemicals, which are produced in bulk quantities by the active pharmaceutical ingredients (APIs) manufacturers to comply with good manufacturing practices (GMPs). Some are also highly purified and well-characterized, naturally occurring active substances. Chemical purity of a synthetic API, a characteristic with a significant impact on the drug product quality, is accomplished only if impurities are each present at a nominal concentration less than or equal to a predefined limit.

Impurities are unwanted coexisting components in bulk pharmaceutical chemicals that arise during manufacture and/or subsequent storage. According to the definition given by the International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use, impurity is any component of a substance for pharmaceutical use that is not the chemical entity defined as the substance. Excluding enantiomers, polymorphic forms and extraneous contaminants — the presence of the last is inconsistent with GMP — ICH classifies impurities associated with a chemical API as inorganic, organic (process- and drug-related) and residual solvents.¹

However, some medicinal substances are mixtures of closely related compounds. These compounds have similar activity; they contribute to the assay result and are not regarded as impurities (such an example is cefamandole free acid in the pure form of cefamandole nafate). Budesonide epimer A is not regarded as an impurity although it must be controlled to ensure batch-to-batch consistency and uniformity among different manufacturers. Wherever a substance is supplied in pure form as an organic or inorganic salt, the organic acid or the inorganic counter ion are not considered impurities (examples include maleic acid in enalapril maleate, benzene sulfonate in amlodipine besilate and chlorides in substances supplied as hydrochloride salts). Coexisting water in pure APIs is not an impurity either.

Table 1 EP specifications for the organic-related impurities in selected drug substances.

Organic Impurities in Synthetic APIs

ICH further classifies organic impurities as starting materials, by-products, intermediates, degradation products, reagents, ligands and catalysts. Enantiomers, which are not included in the previous classifications, are stereoisomers with the same molecular formula as the drug substance, differing only in the spatial arrangement of atoms within the molecule and having a nonsuperimposable mirror image relation (chirality). Enantiomers have the same physical and — in an achiral environment — chemical properties, except the optical rotation.

Diastereoisomers (isomers of drugs with more than one chiral centre) and geometric isomers are both chemically distinct, pharmacologically different and readily separated.²

Many of the marketed drugs are chiral and are often supplied as mixtures of enantiomers (racemates) rather than single enantiomers.

However, certain single enantiomeric forms of chiral drugs are regarded as improved chemical entities with a better pharmacological profile. Some of the chiral drug substances offered as pure enantiomers are naturally biosynthetic products. In these cases, the presence of the enantiomeric impurities is excluded because of the high level of enantioselectivity of their biosyntheses.

However, technological advances such as chiral separation or assymetric synthesis permit commercial production of several single enantiomer drugs. In the manufacture and control of these drugs the other enantiomer (antipode) is an undesirable organic impurity.³ For this reason, chiral chromatographic tests are included in the pharmacopoeial monographs of some single enantiomeric drugs. In the future such tests will become more common. A presentation somewhat different from that of ICH, but very detailed, which also gives useful information on the chemical and analytical characterization of the related organic impurities in drug substances and drug products, has recently been published.⁴

Each impurity must be investigated with respect to both chemistry and safety aspects. The former include identification (structural characterization), reporting and quantitation using suitable analytical procedures, while the latter include a process of acquiring and evaluating data concerning the biological safety of an impurity (qualification). Individually listed impurities, limited with specific acceptance criteria, are referred to as specified and they can be either identified or unidentified.

Unspecified impurities are limited by a general acceptance criterion. A decision tree for the identification and qualification along with the corresponding thresholds, which are dependent on the maximum permitted daily dose (MDD), is given by ICH. Summing up, the following list of organic impurities must be presented in the specification of a synthetic drug substance:

• each specified identified or unidentified impurity

• any unspecified impurity

• total impurities.

Specified unidentified impurities are referred to by an appropriate qualitative analytical description (e.g., relative retention time).

Control of Organic Impurities

A description of the identified and unidentified existing impurities in a chemical drug substance is referred to as the impurity profile (IP).

Impurity profiling includes the procedure aimed at the detection, structure elucidation/identification and the quantitative determination of these impurities. Efforts are mainly focussed on the profiling of the organic impurities as the other possible groups, such as inorganic impurities and residual solvents, are easily identified and their toxicity is known. The presence of organic impurities in a drug substance is closely dependent on the process of manufacture. A different route of synthesis will tend to lead to a different IP.

In pharmaceutical research and development, IP is often decided by using high performance liquid chromatography (HPLC), mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectrometry. Direct coupling or multiple hyphenation of these techniques along with the use of modern software for spectral/ chromatographic searching is a valuable tool for the detection of impurities at trace levels. In case of volatile, but thermally stable compounds gas chromatography (GC) coupled with various detection systems still plays an important role. Investigation of the impurities in complex natural products by using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS has been proposed. Capillary electrophoresis and solid phase microextraction/GC–MS have also been successfully used. Normally, more than one analytical system is applied for the confirmation of an IP.^5–9

Isocratic and gradient reversed-phase HPLC with ultraviolet-visible (UV-Vis) detection remains the most suitable analytical procedure for routine impurity testing. Baseline separation of all the potential organic impurities and the active substance should be performed. Better specificity is established by using photodiode array detectors, when the method is under development. In certain applications ion pairing offers better peak separation and post-column derivatization lowers detection limits. GC and thin layer chromatography (TLC) are often applied in the industrial quality control (QC) laboratories for impurity testing. TLC determinations have a semi-quantitative nature, but allow the detection of impurities completely retained or those not retained at all by the stationary phase.

Pharmacopoeial Status

The quality of a chemical active substance with respect to organic impurities is controlled by a set of tests within a pharmacopoeial monograph. Individual monographs are periodically updated to keep pace with scientific progress and regulatory developments. Following the revised ICH Q3A impurity testing guideline major pharmacopoeias will continue publishing new or revised relevant monographs and general chapters. Active substances found to contain an organic impurity not detected by the relevant pharmacopoeial tests prescribed below are not of pharmacopoeial quality, unless the amount and the nature of this impurity is compatible with GMP.

Two general chapters of the US Pharmacopeia (USP) deal with organic impurity testing.¹⁰ Concepts and definitions are clearly described although a somewhat different terminology from that of ICH is used. Until now, one of three types of tests in bulk pharmaceutical chemicals is ordered:

• a chromatographic purity test coupled with a non-specific assay

• a chromatographic purity-indicating method that also serves as an assay

• a specific test and limits for known impurities, a procedure that requires reference standards for these impurities.

In the future, new and revised USP individual monographs will include tests that actually control specified and unspecified organic impurities. Where different routes of synthesis yield different IPs, perhaps different analytical procedures will be proposed. All specified impurities will be separately limited, with a further limit of 0.10% for any unspecified (unknown) impurity. Total impurities above the disregard limit should be less than 1.0%.¹¹ USP also proposes that a suitable test for detecting impurities that may have been introduced from extraneous sources, should be employed in addition to tests provided in a specific monograph.

The European Commission decided that the principles and terminology of the revised ICH Q3A should be implemented in the European Pharmacopoeia (EP) monographs of the active substances, both new and already published. A new general chapter concerning the control of impurities in pharmaceutical substances was introduced in the fifth edition of the EP, while a revision of the monograph entitled Substances for Pharmaceutical Use has also been done.¹² According to the policy of EP control of the relevant organic impurities in synthetic drug substances is often accomplished by the test of related substances. Currently, it is a limit test (comparison of the peak areas), but will progressively be changed to utilize a quantitative acceptance criterion.

Some individual monographs already satisfy this demand. More tests are ordered, if the general test does not control a given impurity or there are other special reasons. Potential impurities with a defined structure that are known to be detected by the tests in a monograph, but are not known to be present in medicinal substances above the identification threshold, are referred to as detectable impurities. They are limited by a general acceptance criterion. EP individual monographs published in the new format include a separate section in which all impurities (specified and detected) are listed. Unidentified specified impurities are not listed in this section, but their specific acceptance criteria along with appropriate analytical characteristics (e.g., retention time) are reported in the text, wherever it is applicable.

However, previous EP monographs not having a related substances test in the new explicit style are to be read and interpreted according to the recent amendments. During the coming years, EP individual monographs now published in the old format will be revised to contain related substances tests and lists on specified and other detectable impurities. Monographs containing tests for related substances based on TLC will also be revised.

EP specifications for the coexisting organic-related impurities in selected drug substances are summarized in Table 1. The maximum daily dose (MDD) of all the substances presented therein is ≤2.0 g. Acceptance criteria, as shown, are to be tabulated after having interpreted the information given in each separate monograph in conjunction with the general chapters mentioned.¹²

Clarithromycin, a semisynthetic macrolide antibiotic, is also included in this table. Total clarithromycin impurities are limited to a nominal concentration of less than or equal to 3.5%. In certain EP-specific monographs related substances tests cover different IPs. In these cases only impurities for the known profile from a single source need to be reported in the specifications shown in the certificate of analysis (CoA), unless the same master form of CoA is issued for a specific active substance produced by more than one chemical pathway and thus having different IPs.

Pharmacopoeial chromatographic purity tests must be routinely checked for correct performance. Identification of peaks must not be based on absolute terms (retention time), as these are system dependent. Relative retention times for each named impurity are usually quoted. Reference substances for peak identification are offered in certain applications. The chromatograms obtained with the solutions prepared from these reference materials are used for the correct peak identification and for system suitability testing too.

The quantification of an impurity, wherever possible, is performed by using a solution of the reference substance of the impurity. Alternatively, a reference solution of the substance being examined, containing a known amount of the impurity, may be used. Using either of the procedures the result obtained expresses a real percentage of the impurity in the sample. When neither of these procedures is possible and the impurity in question has a response similar to that of the substance under examination, a dilution of the solution of the substance (the test solution or a solution made of the reference substance) is used as a reference solution. The result so obtained expresses a nominal percentage of the impurity in the sample. If a named impurity is known to have a significantly different response a correction factor is used for the calculation of the content of this impurity in the substance being examined.

The correction factor is the reciprocal of the response factor. For the calculation of the nominal percentage of the impurity in the substance being examined, the area of the peak because of the impurity in question in the chromatogram obtained with the test solution must be multiplied by this factor. The peak to which the factor is applied must be identified unambiguously. In all approaches described earlier numerical results should be reported to two significant figures. Conventional rules should be used for rounding.

Conclusion

The implementation of the new pharmacopoeial requirements concerning organic impurities in synthetic drug substances aims at better quality characterization of these raw materials and thus at better medicinal products in the market. Although the relevant technical and regulatory information has been clearly written, difficulties usually arise during the transfer of these requirements to the industrial QC laboratories because special knowledge concerning the detailed attributes of the active substance is necessary.

This knowledge is usually available to key personnel of the API manufacturers, but not to the end-users. End-users must extract all the necessary information on method of manufacture and IP of the active substance from the open part of the drug master file, which is available from the manufacturer. Moreover, people who engaged in QC are urged to apply all the available scientific and technical knowledge during the implementation of a new pharmacopoeial purity test in their laboratories to ensure suitable performance, accurate results and correct interpretation.

Nikolaos Grekas is a senior scientist at Elpen Pharmaceutical Co. Inc., Greece.

References

1. www.ich.org

2. www.fda.gov/cder/guidance/stereo.htm

3. www.aapspharmscitech.org

4. S. Görög, Anal. Bioanal. Chem. 377, 852–862 (2003).

5. A.L. Freed et al., J. Pharm. Biomed. Anal. 35(4), 727–738 (2004).

6. J.C. Lindon, J.K. Nicholson and I.D. Wilson, J. Chromatogr. B 748(1), 233–258 (2000).

7. I.D. Wilson, J. Chromatogr. A 892(1–2), 315–327 (2000).

8. J. Bertram, Pharmeuropa 15.2, (April) 280–289 (2003).

9. R. Nageswara Rao and V. Nagaraju, J. Pharm. Biomed. Anal. 33(3), 335–377 (2003).

10. General chapters <466>, "Ordinary impurities" and <1086>, "Impurities in official articles," in USP 28 – NF 23 (US Pharmacopeia, 12601 Twinbrook Parkway, Rockville, Maryland 20852, USA, 2004).

11. USP Guideline for Submitting a Request for Revision of the USP/NF, Chapter One, Non-Complex Drug Substances and Products, http://www.usp.org

12. General chapter 5.10, Control of impurities in substances for pharmaceutical use and general monograph 01/2005:2034, Substances for pharmaceutical use, in European Pharmacopoeia, 5th Edition (EDQM, 226 avenue de Colmar BP 907, F-67029 Strasbourg, France, 2004), pp 559–561 and 586–587.