Nikolaos Grekas

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.

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. 			

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.

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.

 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.

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

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

				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.

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. 

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. 

 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.

 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%.

 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 

) 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 

, while a revision of the monograph entitled 

 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. 

 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. 

 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, 

 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.  

 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.

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 

-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. 

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. 

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

6. J.C. Lindon, J.K. Nicholson and I.D. Wilson, J. Chromatogr. B 

Articles

Chemical purity is the most important quality characteristic of a pharmaceutical substance. This article describes the latest scientific and technological advances to meet recent pharmacopoeial and regulatory requirements regarding the control of organic impurities in synthetically produced active substances. Future developments and suggestions for those working in quality control and raw material selection are discussed.

Organic Impurities in Chemical Drug Substances

A substance is any matter that may be of human, animal, vegetable (plant) or chemical origin. Substances intended for pharmaceutical purposes are used as active ingredients (drug substances) or excipients (auxiliary substances) for the production of medicinal products. They may be obtained from natural sources, by chemical synthesis or by a fermentation procedure. Recombinant proteins and substances derived by biotechnological processes based on genetic modifications are characterized biotechnological substances.  

Human blood, blood products and other substances of animal origin are characterized biological ones. Chemical substances are naturally occurring chemical materials or products obtained by chemical synthesis. As dictated by legislative rules,

 manufacturing and control of chemical materials intended for pharmaceutical purposes are performed using procedures that are designed to ensure a consistent quality and compliance with the requirements of approved specifications. These should be established in accordance with accepted standards and consistent with the manufacturing process of the material.  

A specification is a quality standard. It establishes the criteria to which a substance should conform to be considered acceptable for the manufacture of medicinal products. Thus, specification includes a list of tests, references to analytical procedures and appropriate acceptance criteria for the tests described. If the substance meets the acceptance criteria when tested according to the listed analytical procedures, then it conforms to specification. Approved specifications are provided by Pharmacopoeias in each region, such as the United States Pharmacopoeia (USP), Japanese Pharmacopoeia (JP) and the European Pharmacopoeia (EP).

 The relevant information is given in individual monographs, but also in general monographs and general chapters.

 Excellent technical guidelines have also been published.

Whereas differences in pharmacopoeial specifications among regions usually exist, a harmonized global specification is possible providing that the procedures and acceptance criteria defined are acceptable to regulatory authorities in all regions. Harmonization has already been accomplished in a series of excipient monographs and general chapters. In case there is no available pharmacopoeial monograph, raw material manufacturers must create approved specification and all other scientific data according to the technical guidelines, general monographs and good manufacturing practice (GMP) requirements. 

Although the use of biotech derived products has considerably increased in the last two decades, most of the substances still used for pharmaceutical purposes are chemicals. These chemicals, which are either pharmacologically active or excipients, represent discrete moieties, but are sometimes mixtures. The processes followed for the production of these substances are similar. Therefore, their specifications are similar too. Excipients are included in the medicinal product to facilitate manufacture, enhance stability and to contribute to product attributes such as biopharmaceutical profile, appearance and patient acceptability. Depending on the formulation, certain substances may be added either as active ingredients or excipients. Organic solvents and water are often used and they must meet strict  quality specifications.  			

Water for pharmaceutical purposes is the most common chemical component in pharmaceutical production operations. Specific grades such as purified water, water for injections and highly purified water are used at different stages in the production of raw materials, medicinal products and cleaning operations as well. Medicines other than those requiring sterile or apyrogenic water are prepared with purified water. Approved specifications of purified water in bulk are shown in Table I.

 Table I Pharmacopoeial specifications of purified water in bulk produced on manufacturing site.			

Approximately 5-20 separate tests  are selected to be included in the specification of a chemical substance presented in a pharmacopoeial monograph. These tests should address and confirm appearance, identity; physical and functionality-related characteristics; and chemical and microbiological purity. They are carried out routinely during manufacture (in process testing) or at the time of release. Substances having different grades or forms must be tested according to the same individual monograph. However, additional tests should be applied for the  qualification of the different grades.  

 Identification, using specific tests, is the confirmation of a chemical entity. Organic molecules are identified by infrared absorption spectrophotometry (IR). Different procedures, such as liquid chromatography (LC), gas chromatography (GC) and thin layer chromatography (TLC), are also executed. However, the use of at least two different procedures based on a different principle is generally acceptable. If the organic molecule is a salt, specific ion identification testing is necessary.  							

Identification tests based on the measurement of the melting point temperature, freezing point temperature, kinematic viscosity, angle of optical rotation, refractive index and relative density of liquids are frequently applied. Inorganic molecules are identified through the characteristic chemical reactions of their ions. Some EP monographs under the title  "Identification" contain the subdivisions "First Identification" and "Second Identification." The test or tests that constitute the "Second Identification" may only be used if the substance is fully traceable to a batch certified to comply with all the requirements of the monograph, whereas those that constitute the "First Identification" can be used in all cases. 

 Polymorphism is the property of a chemical substance to exist in different crystalline forms. Common methods to determine the existence of multiple forms are X-ray diffraction, IR, nuclear magnetic resonance, scanning electron microscopy and thermal analysis. Polymorphism is an important factor mainly for drug substances. 

 Chemical purity is generally ensured by testing for related substances, residual organic solvents, heavy metals and by the quantitative determination of the content (assay). More specific chemical tests, which ensure a better quality characterization of the sample, are mandatory in certain cases.  

Control on related substances (related compounds) is very significant mainly for organic drug substances. Impurity profiles are better decided by a gas or liquid chromatographic procedure. High performance liquid chromatography (HPLC) is the best analytical method for this purpose. Baseline separation of all the potential organic impurities (impurities arising during the chemical synthesis and degradation products) should be performed. EP monographs report the acceptance criteria for organic impurities in terms of peak area with an indication of the corresponding nominal concentration. Special information and recommendations for interpreting the test for the related substances is also provided in the general chapter concerning this topic.

Organic solvents are used in the production of pharmaceutical substances and medicinal products. These solvents, although volatile, are not completely removed by practical manufacturing and subsequent purification procedures. Residues should be limited to the levels approved by the international Conference on Harmonization (ICH).

 There are three classifications of solvents, which are graded according to their toxicity. Solvents that should be avoided are Class I. Class II solvents should be of limited use, whilst Class III solvents are those with the least toxicity and can be routinely used. Control of the residual solvents is regularly performed by GC with headspace injection. The non-specific test for loss of drying may be applied when only Class III solvents are used in the production process. Periodic or skip testing at release can be applied, but batches that have not been tested for residual solvents must still meet acceptance criteria.  

 Heavy metal testing demonstrates that the level of metallic impurities coloured by the sulfide anion does not exceed the limit specified in the individual monograph regarding parts per million of lead in the substance under examination. Concomitant visual comparison with a control prepared from a standard lead solution is performed in this limit test. Metallic cations, except lead, that typically respond are mercury, cadmium, silver, antimony, bismuth, tin and copper. Quantitative determination of metallic compounds in pharmaceuticals, when necessary, is performed by atomic absorption spectrometry (AAS). An estimation of total inorganic impurities can also be done by determining the residue of ignition or the sulfated ash of the sample.  

Suitable methods must be applied for the quantitative determination of the pharmaceutical substances. HPLC or GC are proposed as the most specific methods for determining the content of a pharmaceutical substance. In many cases, the same stability-indicating procedure for both assay and the quantitation of impurities is employed. In cases where a non-specific analytical procedure (for example, a volumetric titration) is adopted for the assay, other supporting analytical procedures are simultaneously proposed (for example, HPLC determination of the related substances) to achieve overall specifity. Assay results are expressed with reference to the anhydrous or to the dried substance, thus water content or loss on drying tests must be conducted. 

Water content determination is very important when the substance is known to be hygroscopic or a stoichiometric hydrate. Karl Fischer titration is the most widely known specific procedure for water content estimation, but sometimes the non-specific loss on drying test is considered adequate.  

 The microbiological quality of pharmaceutical substances is adequately controlled by performing tests for total viable aerobic count (TVAC) for specified micro-organisms and for sterility — the latter test is performed if the substance is intended for the preparation of sterile dosage forms or if offered as sterile grade. Pyrogen testing or a bacterial endotoxins test should be applied in endotoxin-free grade substances. The type of microbial testing should be based on the nature of the substance, method of manufacture and the intended use. A decision tree that gives further guidance is published in the ICH guideline.

 Physical and other functionality-related characteristics are different between different grades of the same chemical entity. Such characteristics include  					

Differentiation between different qualities of the same chemical entity is referred to as qualification. Near IR absorption spectrophotometry (NIR) is perhaps the most useful analytical technique for the qualification of chemical substances. NIR can also be used for identification.  

Particle size distribution is conducted by laser diffraction or analytical sieving. Appropriate methods for testing intrinsic dissolution rate, bulk and tapped densities, true density, specific surface area and flowability are described in the pharmacopoeias. Control of these parameters affords an important tool in the characterization of bulk active substances and excipients. 

 Familiarity with the specifications : a key element 

Quality specifications of substances for pharmaceutical use are a very important issue, having a significant impact on medicines. These specifications must be presented in detail on the certificate of analysis (CoA) of the raw material along with reference to the latest pharmacopoeial individual monograph, if it is provided. This is mandatory for both raw material manufacturers and the end-users of pharmaceutical substances. 			

Raw materials manufacturers must routinely test the pharmaceutical substances they produce according to pharmacopoeial instructions. They usually test their products according to all major pharmacopoeias, if harmonized specifications do not exist, to ensure global acceptance. Periodic control (skip testing) of some parameters is allowed only if it is applied by a fully validated manufacturing procedure.  

				End-users initially evaluate their raw material suppliers by carrying out full testing on the first lots of the purchased products; afterwards they only routinely carry out identification testing, accepting the CoA of their suppliers or they perform full testing only on selected batches according to a written standard operating procedure (SOP). 

However, since transport and storage can cause changes that are beyond initial control of the manufacturers, it is better that the materials are further examined by the end-users before use. In either case, QA/QC managers and other personnel involved in testing, the preparation of CoAs and relevant SOP writing must be completely informed and familiar with the afore mentioned topic. The issued CoAs will then be in full compliance with pharmacopoeial and GMP requirements. 

Information on the specifications of the drug substance and the excipients that formulate the medicinal product must also be included in  module 3 of the common technical document (CTD), which is the new format required for all marketing authorization applications. Therefore, personnel involved in the preparation of these documents and regulatory affairs personnel should be similarly informed. Finally, the same applies to personnel who deal with formulation, production, analytical development, and those responsible for the selection of the raw materials. 

Pharmacopoeial monographs, chapters and technical guidelines are regularly revised and updated because of continuous changes in technology and subsequent regulatory demands. Personnel from all sectors of the pharmaceutical industry must be aware of these updates to keep up with the ever changing requirements in the industry. 			

pharmacos.eudra.org/F2/eudralex/index.htm

 (US Pharmacopeia, 12601 Twinbrook Parkway, Rockville, Maryland, 20852, USA, 2003). 

 (EDQM, 226 avenue de Colmar BP 907, F-67029 Strasbourg, France, 2004). 

5. "Substances for Pharmaceutical Use," Monograph Nr 01/2005:2043, 

 (EDQM, 226 avenue de Colmar BP 907, F-67029 Strasbourg, France, 2004) p 586. 

6. "Control of Impurities in Substances for Pharmaceutical use," Monograph Nr 01/2005:51000, 

 (EDQM, 226 avenue de Colmar BP 907, F-67029 Strasbourg, France, 2004) p 559. 

Good Manufacturing Practices for Bulk Pharmaceutical Excipients

, International Pharmaceutical Excipients Council's (IPEC) Revised Guidelines (2001), Pharmeuropa 

8. Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances, ICH Harmonized Tripartite Guideline QA6, 

9. Impurities in New Drug Substances, ICH Tripartite Harmonized Guideline, Q3A(R), 

10. Impurities: Guideline for Residual Solvents, ICH Harmonized Tripartite Guideline Q3C, 

11. Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients, ICH Tripartite Harmonized Guideline, QA7, 

Nikolaos Grekas is a senior scientist at ELPEN A.E. Pharmaceutical Co. Inc., 95 Marathonos Ave., 19009, Pikermi, Attica, Greece. Tel. +302 1 0603 9326, Fax +302 1 0603 9300. 

A harmonized global specification is possible providing that the procedures and acceptance criteria defined are acceptable to regulatory authorities in all regions.