Disclaimer: The views expressed in this article are that of the author and do not reflect the positions of FDA, USP or any other institution.
Introduction
Ionic methods of separation have been used in a variety of applications over several hundreds of years. However, the modern form of ion chromatography (IC) was introduced in 1975 by Small, et al. [1]. The technique, a form of high-performance liquid chromatography (HPLC), has since gained popularity in laboratories for the analysis of inorganic anions and cations, organic acids, carbohydrates, sugar alcohols, aminoglycosides, amino acids, proteins, peptides, glycoproteins and other types of molecules in environmental, agricultural, pharmaceutical, biotechnology, metal plating, power generation, semiconductor fabrication, and other industrial samples. IC-based methods, sometimes in conjunction with mass spectrometry or other hyphenated techniques, have been used in the analysis of active and inactive ingredients, excipients, degradation products, and impurities relevant to pharmaceutical and biotechnological drug quality. A detailed discussion of such applications is beyond the scope of this article. Here, we shall discuss application of ion chromatographic methods in the analysis of therapeutic products and their ingredients included in USP-NF monographs and general chapters, and their importance in setting standards for identity, strength (content), quality and purity of products and ingredients. For more in depth understanding of the principles, instrumentation and applications of IC, readers are encouraged to read the books and other publications on IC that provide in-depth review of this chromatographic technique [2-4].
USP-NF
USP-NF (United States Pharmacopeia-National Formulary) is published by United States Pharmacopeial Convention, Inc., Rockville, MD, in continuing pursuit of their mission to set public standards for medicinal products, dietary supplements, and their ingredients in the United States. USP-NF is recognized in the Federal Food Drug and Cosmetic Act (FD&C) of 1938 as an official compendium of the United States and FDA may enforce compliance with official standards in USP-NF under the adulteration and misbranding provision of the FD&C Act.
USP32-NF27 (official until April 30, 2010) contains more than 4300 monographs and about 220 general chapters (see Front Matters of USP32-NF27). Monographs of products and their ingredients (active and inactive components) include definitions or description of the articles, packaging, storage and other requirements, and specifications. The specification consists of a series of tests, one or more analytical procedure for each test, and the acceptance criteria for each procedure. General chapters provide procedures, sometimes with acceptance criteria and other requirements, in order to compile repetitive information in one location, which would otherwise appear in many monographs.
In most of the other countries, pharmacopeia is part of the government. However, in the United States, the pharmacopeia (USP) is a non-government not-for-profit organization that supports itself from the sale of books (USP-NF and other publications) and reference standards. It does not receive any financial support from federal, state or local governments or non-government organizations.
USP-NF standards are recognized not only in the Unites States but also in many other countries because they are authoritative, science-based, and are established through a transparent and credible process with established integrity. The transparency and credibility of the monographs come through the open review and comment process that takes place when proposed monographs are published in the Pharmacopeial Forum (PF). Although the Council of Experts of USP is the ultimate decision making body for the USP-NF standards, these standards are developed through public involvement and substantial interaction between USP and the stakeholders through consensus building. A stakeholder may be an institution or individual, domestic or international. Interested and knowledgeable stakeholders can provide scientific and regulatory comments to monographs published in PF. The public participation in the monograph development and revision process results in consensus among many individuals and groups, including scientific and trade organizations. The members of the Expert Committees are unpaid volunteers who are experts in their respective fields and who participate in the USP process as individual scientists and not as representatives of their employers or any trade association, thereby eliminating the conflict of interest and providing unbiased authoritative and science-based standards.
Figure 1 - USP Monograph Development and Revision Process
The USP-NF monograph development and revision process is summarized in Figure 1. A manufacturer (sponsor) submits a proposal for a new monograph or a revision to an existing monograph to USP (Request for Revision). The information and data that need to be provided with a Request for Revision is available at the USP website (www.usp.org) under the title Guideline for Submitting Requests for Revisions to USP-NF. USP staff review the proposals for the appropriateness and completeness, and, when satisfied, publish the proposal to PF as In-process Revision for public comment. After the public comment period, members of an appropriate Expert Committee (together called Council of Experts) reviews the proposal and the comments and approves (or disapproves) the request for official adoption. However, it should be noted that the manufacturers are not required at any stage to submit proposals for a new monograph or a revision to an existing monograph. Such submission is discretionary to the manufacturers and they do so voluntarily to support the mission of the development of consensus standards for therapeutic products.
Ion Chromatography
The IC involves separation based on ionic interactions between ionic or polar analytes, ions present in the eluent, and ionic functional groups derivatized to the chromatographic support. This can lead to two distinct mechanisms of separation— (a) ion exchange due to competitive ionic binding (attraction) to the chromatographic support (column resin), and (b) ion exclusion due to repulsion between similarly charged analyte ions and the ions derivatized on the chromatographic support. Separation based on ion exchange has been used for a long time and is the predominant form of IC to-date. However, increasing applications of ion exclusion chromatography have been reported more recently. In addition, chromatographic methods in which the separation due to ion exchange or ion exclusion is modified by the hydrophobicity of the analytes and the chromatographic support materials, presence of the organic modifiers in the eluent or due to ion-pair agents, resulting in better resolution of analytes or separation that were not achieved before, have gained popularity recently due to increased applications of mixed mode columns.
Ion-exchange chromatography involves separation of ionic and polar analytes on a chromatographic support, which is derivatized with ionic functional groups that have charges opposite that of the analyte ions. Thus, a column used to separate cations, called a cation-exchange column, contains negative ions. Similarly, an anion-exchange column, which separates anions, is derivatized with positively charged ions. The separation is effected by repeated binding of the analyte ion to the ionic sites on the chromatographic support and desorption by the ions present in the mobile phase. The ion-exchange method of separation is widely used in the analysis of anions and cations, including metal ions, mono- and oligosaccharides, sugar alcohols and other polyhydroxy compounds, aminoglycosides (antibiotics), amino acids and peptides, organic acids, amines, alcohols, phenols, thiols, nucleotides and nucleosides, and other polar molecules.
Ion-exclusion Chromatography uses strong cation- or anion-exchange chromatographic supports to separate ionic analytes from polar, weakly polar and neutral analytes, and has been used typically in the analysis of organic acids, alcohols, glycols, sugars, and other weakly polar compounds. In contrast to the ion-exchange chromatography, the charge on the functional groups on the chromatographic support is same as the charge on the analyte ion. That is, to separate negatively charged or negatively polarized analytes, the chromatographic supports are derivatized with negatively charged functional groups. Similarly, analytes with positive charge or polarity are separated using a chromatographic support that carries positive charges.
Any suitable detector can be used for the detection and quantitation of analytes in IC. The choice depends upon the nature of the analyte molecules. This may include Refractive Index (RI), UV or fluorescence detectors. However, traditionally, IC is associated with electrochemical detectors. Two types of electrochemical detectors are widely used in IC—conductivity (suppressed and nonsuppressed) and pulsed amperometry.
When a constant voltage is applied across two electrodes between which the effluent from a column flows, a current is generated because the effluent contains ions or polar molecules. The strength of the current is proportional to the conductivity of the solution, which, in turn, is proportional to the concentration of ionic/polar species in solution, leading to the detection and quantitation of analytes by a conductivity detector. The problem, however, is that the conductivities of mobile phases are significantly higher than the conductivities of the analytes, simply because the concentrations of ions in the former solutions are 104-105 higher than that of the analytes. The suppressed conductivity detection permits detection and quantitation of analytes at near zero background (baseline) conductivity of the mobile phases.
Used typically in combination with high-performance anion-exchange chromatography (HPAEC, originally introduced as high-pH anion-exchange chromatography), Pulsed Amperometry Detection (PAD) has proved to be a powerful tool in the detection of mono- and oligosaccharides, sugar alcohols, aminoglycosides, amino acids and other molecules that do not have a suitable chromophore, without requiring any sample derivatization. A detailed discussion on the mechanism of action of the suppressed conductivity and the pulsed amperometry detectors is beyond the scope of this article. The interested analysts are encouraged to read books and other publications that provide details of the principles of actions and suitability of selection for use in different types of IC-based chromatographic procedures [2-4].
Ion Chromatography in USP-NF
Monographs
USP25-NF20, official between 2000 and 2005, had only 12 monographs that included test methods involving IC and there was no general chapter related to IC or in which IC was cited [5]. However, the number of monographs that include one or more IC-based test procedures increased dramatically in last 10 years. In addition, USP32-NF27 contains two general chapters solely on IC (<345> and <1065>) and at least four general chapters that include IC-based test methods (<1045>, <1052>, <1055>, <1086>), implicating its importance as a chromatographic technique for the analysis of therapeutic products and their ingredients.
USP32-NF27 (official through April 30, 2010) has around 110 monographs that use one or more IC-based test procedures. Such tests include assay (content) of actives in drug substances and products, content of inactive components (excipients) in drug products, purity/impurity tests for drug substances and excipients, and other applications, including dissolution tests. This does not include those published in PF but have not become official yet. An examination of the current USP-NF shows that the application of IC as the procedure for quantitation of ingredients in therapeutic products is significantly higher than its application in other types of tests.
The detection method includes both traditional detectors (UV and RI) as well as the modern conductivity detector, in both suppressed and non-suppressed mode, and the Pulsed Amperometry Detector.
Even though IC-based procedures are used in many monographs, the number of monographs that contain such procedures is very small compared to the total number of monographs in USP-NF. That raises the question of significance of IC in the analysis of pharmaceutical and biotechnological drugs. This will be addressed later in this article.
General Chapters
General Chapter <345> (Assay for citric acid/citrate and phosphate) describe the test procedure that are referenced in several monographs. The procedure uses L61, a hydroxyl-selective weak anion exchange column, and isocratic elution with 20 mM NaOH or KOH for the assay of citrate and phosphate in several drug products. This chapter resulted from a collaboration between USP and Dionex, Inc. and the work was published [6].
The General Chapter <1065> (Ion Chromatography) is an information chapter, which provides a broad overview of IC, including principles of different types of IC, the instrumentation, and stationary and mobile phases. Particular emphasis is given to describe the principles and applications of suppressed and non-suppressed conductivity detector as well as Pulsed Amperometry detector. The chapter discusses commonly used sample preparation techniques and run conditions in different types of IC. The chapter also provides discussion on the selection of column and potential area of applications in the analysis of therapeutic products.
The other four general chapters describe IC-based procedures as part of the chapter or refer to appropriate IC-based procedures. General Chapter <1045> (Biotechnology-derived Articles) describes the application of IC in the analysis of oligosaccharide (glycan) moieties of naturally occurring and recombinant glycoproteins. General Chapter <1052> (Biotechnology-derived Articles—Amino Acid Analysis) describes application of IC in Amino Acid Analysis. General Chapter <1055> (Biotechnology-derived Articles—Peptide Mapping) describes IC-based procedures for the separation of peptides before they are analyzed by more powerful techniques such as mass spectrometry. This provides an example of IC being part of a hyphenated technique. General Chapter <1086> (Impurities in Official Articles) refers to IC as one of the powerful techniques for the detection and limit tests for impurities in active and inactive components of drug products.
Columns in USP-NF
An examination of the description of columns used in chromatographic test procedures in USP-NF shows that out of a total of 70 different column types, 19 are IC columns, indicating the diversity (but not quantity) of applications of IC-based procedures. However, this number appears to be inconsistent with the small number of monographs in USP-NF that uses IC procedure. It may be noted here that L1 column type alone is included in more monographs than all IC-columns combined.
The list of vendors of IC columns in USP-NF shows that, even though Dionex, Inc., who introduced IC system and columns commercially, is still the leading manufacturer, there are 16 other manufacturers, including Metrohm, Agilent Technologies, Waters, TosoHaas, BioRad, and Shodex, all reputed names, who manufacture IC columns. Some of these manufacturers also manufacture IC systems.
Significance of IC-Based Methods in USP-NF
As is noted above, the number of monographs that contain IC-based procedures in USP32-NF27 is very small compared to the total number of monographs. This appears to give an impression that there is little interest in IC-based procedures among pharmaceutical and biotech industry. In order to understand this dilemma, readers need to have a more in-depth understanding of the USP-NF monograph development process. The proposals for monographs are submitted by the manufacturers of the drug substances, excipients and products (monograph Sponsors). However, there is no requirement for them to do so. Typically, the approved (NDA, BLA) innovator manufacturers of therapeutic products do not submit proposals during the period they hold the patent for the drug because that would require giving out proprietary information, which might conflict with their business interests. Secondly, there is no public health interest to be served by having monographs in USP-NF during the patent period because the patent holder of a particular therapeutic product is the only manufacturer of that product and the safety and efficacy of the product is assured through the FDA approval process. Generally, the manufacturers submit proposals immediately before the expiration of patents to ensure that the quality of the drugs is maintained or bettered after patent expiration. The manufacturers of generic drugs may also submit proposals after their products are approved (ANDA) by FDA in order to improve the drug quality and to incorporate the introduction of modern technologies in the manufacture and analysis of therapeutic products. Typically, as the former Director of Noncomplex Actives and Excipients Division, it is my experience that monograph proposals are submitted 10-15 years after the NDA/BLA approval, sometimes even later. Therefore, the monographs present a status that reflects the interest and practice of pharmaceutical and biotech communities that are 10-15 years old in terms of application of new technologies. Thus, around 110 monographs, out of a total of 4300, which use IC-based procedures in current USP-NF, really reflect the status that is 10-15 years old. The dramatic increase (about an order of magnitude) of monographs that use IC-based procedure in the last decade is more significant than the actual number of monographs in current USP-NF.
References
- Small, H., Stevens, T.S., Bauman, W.C. (1975) Novel ion-exchange chromatographic method using conductometric detection. Anal. Chem. 47, 1801-1809.
- Weiss, J., Ion Chromatography, 2nd Edn, VCH Verlag, Weinheim, Germany, 1995.
- Haddad, P.R. Jackson, P.E., Ion Chromatography—Principles and Applications, Elsivier, Amsterdam, The Netherlands, 1990.
- Fritz, J. & Gjerde, D.T. Ion Chromatography, 3rd Edn., Wiley-VCH, Weinheim, Germany, 2000.
- Bhattacharyya, L., Ion chromatography in biological and pharmaceutical drug analysis: USP perspectives, presented at the Intl. IC Symp., October 2002, Baltimore.
- DeBorba, B.M., Rohrer, J.S., Bhattacharyya, L. (2004), Development and validation of an assay for citric acid/citrate and phosphate in pharmaceutical dosage forms using ion chromatography with suppressed conductivity detection. J. Pharm. Biomed. Anal. 36, 517-524.
Author Biography
Dr. Bhattacharyya worked for more than 9 years in Merck and Co. as a lead analytical biochemist providing analytical support for vaccines and biotechnology-derived proteins under cGMP and more than 6 years managing the standard setting activities of the US Pharmacopeia in different capacities, including the Director of the Noncomplex Actives (Pharmaceutical Drugs) and Excipients Division in the Dept. of Standards Development. At present, he is an Interdisciplinary Scientist in the Division of Product Quality of the FDA/CBER/QCBQ. He obtained his Ph.D. (1983) from Calcutta University, India. Dr. Bhattacharyya has published several original research papers and review articles in peer-reviewed journals and presented in several conferences.
This article was printed in the March 2011 issue of American Pharmaceutical Review - Volume 14, Issue 2. Copyright rests with the publisher. For more information about American Pharmaceutical Review and to read similar articles, visit www.americanpharmaceuticalreview.com and subscribe for free.