Pharmaceuticals and Related Drugs - Analytical Chemistry (ACS

REVIEW pubs.acs.org/ac

Pharmaceuticals and Related Drugs R. K. Gilpin Department of Chemistry, Wright State University, Dayton, Ohio 45435, United States

C. S. Gilpin Select-O-Sep, LLC, 111 West Main Street, Freeport, Ohio 43973, United States

’ CONTENTS General Separation-Based Methodology Spectrometric-Based and Other Methodology Biographies References

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4489 4494 4500 4502 4503

he current article is a survey of pharmaceutical and related methodology that has appeared in the literature between January 1, 2009 and December 31, 2010. It is directed exclusively toward the analysis of compounds in either their bulk or formulated forms and focuses on analytical procedures for evaluating quality, purity, strength, and stability. The Review does not deal with biochemical, clinical, metabolic, pharmacokinetic, or related aspects of the topic nor does it include the analysis of pharmaceuticals in the environment or food products. Book and book chapters also are excluded. Further, because of space limitations and the ever expanding base of scientific research and development work in the pharmaceutical industry, within the United States and throughout the World, the cited articles represent only a small fraction of the total number published during the reviewed period. Likewise, even though a cited paper may deal with more than one technique, it is discussed typically only once in the paper. In the selection of appropriate citations, an attempt was made to place greater emphasis on procedures related to newer compounds, emerging and/or highly used techniques, unusual approaches and methodology, and more comprehensive studies with only a few examples cited from the very large number of publications that discuss either more routine procedures, less often used techniques and approaches, or older compounds. The manuscript is organized similar to the last review published in this series that appeared two years ago in Analytical Chemistry,1 which contained an expanded general section and the techniques section combined into two broader areas. This was done to place more emphasis on general trends and emerging techniques and less on more routine methodology. Thus, the article is organized into three major sections: General, Separation-Based Methodology, and Spectrometric-Based and Other Methodology. These sections are arranged in terms of specific approaches and applications. As was noted in the last review, the discovery, development, manufacturing, and sales of pharmaceutical products has become an international industry that must meet the regulatory issues of a global marketplace and its influence on the changing nature of safety and efficacy. Further, r 2011 American Chemical Society

individual market preferences and how these factors are influencing analytical requirements and associated methodologies are evident by the large amount of research papers originating from laboratories throughout the World. This, coupled with the increasing number of journals for reporting the work and their ease of access, has made it increasingly more difficult to pick representative examples from the worldwide body of literature being published.

’ GENERAL During the last two years, numerous reviews were published that either deal exclusively with or contain large sections related to the analysis of pharmaceutical compounds. Many of them focus on general aspects of the technique or instrumentation, especially as they relate to separation-based methodology, mass spectrometry, or vibrational spectroscopy. Common uses of separation-based techniques are to measure content, evaluate standard and chiral purity, and study forced degradation of the pure drug substance and formulations containing it. Similarly, the most common applications of mass spectrometry are in combination with high performance liquid chromatography to profile complex mixtures of medical compounds found in herbal and fermentation products, to characterize biopharmaceuticals, and to elucidate forced degradants and decomposition mechanisms. In the case of vibrational techniques, they have been used most often to monitor and/or study process related questions and to assess crystallinity/polymorphic behavior of the pure compound preformulation and how it is influenced by the manufacturing process. There are a number of reviews published during the last two years that deal with separation-based approaches. One of them considers eluent composition and how it influences the accurate characterization of protein pharmaceuticals by size exclusion chromatography (SEC).2 The mechanisms of protein binding to different separation materials are discussed. Likewise, approaches for minimizing problems associated with protein adsorption are presented, including the use of different additives and cosolvents. The paper contains a number of figures and tables to illustrate how important variables influence the chromatographic process. A second paper has been published that Special Issue: Fundamental and Applied Reviews in Analytical Chemistry Published: April 11, 2011 4489

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Analytical Chemistry emphasizes the importance of employing orthogonal methods to minimize potential inaccuracies when sizing protein aggregates via the use of SEC.3 Some of the techniques that can be employed to validate SEC results are sedimentation velocity analytical ultracentrifugation, flow field flow fractionation, and light scattering. In addition to discussing these orthogonal methods, the paper also considers problems that may be encountered in making SEC protein sizing measurements including: protein adsorption to the column media, dissociation and formation of protein aggregates during analysis, resolution of large protein aggregates, and detection of low levels of aggregates using ultraviolet (UV) monitors. Besides the SEC reviews, two others have addressed (1) the basic principles of hydrophilic interaction liquid chromatography (HILIC) and how it can be employed to assay pharmaceuticals4 and (2) the general aspects of multiple linear regression (MLR) and artificial neural network (ANN) approaches and how they can be used to describe the retention behavior of basic adrenoreceptor agonists and antagonists under HILIC conditions.5 The first of these two articles presents an overview of how bare silica, chemically modified silica, and monolithic columns are used in many different pharmaceutical and biological applications. Of these three column types, the authors note that most of the common HILIC pharmaceutical applications employ unmodified silica as the media of choice in combination with binary mixtures of either acetonitrile and water or acetonitrile and an aqueous buffer as the typical eluents, with methanol as the cosolvent being used only rarely.4 These observations are consistent with those of the authors of the current Review in terms of the most often used eluents for carrying out standard reversedphase liquid chromatography, which are discussed further in the Separation-Based Methodology section. The second manuscript reviews the use of a set of 16 analytes, acebutolol, alprenolol, atenolol, betaxolol, carvedilol, isoxsuprine, methoxamine, midodrine, nadolol, oxymetazoline, phenylephrine, pindolol, ritodrine, salbutamol, sotalol, and timolol, as training solutes for MLR and ANN approaches to model retention behavior as a function of changes in eluent pH and organic cosolvent composition for chromatographic separations carried out on polar HILIC phases. Subsequently, the retention models are used to predict the retention of the four analyte test set solutes, bambuterol, metaproterenol, metoprolol, and propranolol. The manuscript contains a number of log
log correlation plots for the variables examined. Other general papers appearing in the literature related to separation-based techniques include those that deal with the importance of routine stability testing of liquid chromatography (LC) columns,6 operational aspects of two-dimensional LC,7 and general aspects of the chiral analysis of pharmaceuticals by capillary electrophoresis,8,9 simulated moving bed (SMB) chromatography as a process tool,10 and high performance liquid chromatography (HPLC).11
13 The latter citation is a tutorial guide that covers popular chiral eluent additives and stationary phases, mechanisms governing molecular recognition including their thermodynamics. It also discusses the use of spectroscopic and computational methods to elucidate the binding/conformational mechanisms. The SMB paper considers differences between simulated and true moving bed models, design aspects of moving bed separations including graphical representations of the design criteria, performance indicators in terms of purity, recovery, and productivity, and nonlinear adsorption effects of the separations. In one of the above papers,6 a case is made that

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quality-by-design (QbD) not only is important in terms of the development of good pharmaceutical manufacturing practices but also extends into the area of analytical methods development. Thus, procedures and practices that help to verify and facilitate assay reliability are important to the overall methods validation process. For high performance liquid chromatography (HPLC), column performance and consistency in performance are key factors to assuring the ruggedness and reliability of chromatographic methods over time. As such, “HPLC column stability is one of the critical factors that governs the success of a method while it is used to support the life cycle of a pharmaceutical product.” Selected overlays of the chromatograms, obtained using a recommended test mixture of solutes, are provided through a total of 500 injections for several commonly used reversed-phase HPLC columns. Likewise, a color coded twodimensional grid of chromatographic conditions vs column type, constructed on the basis of the 500 injection performance data, is included. The grid evaluates the end performance in terms of no constraints on use, some constraints on use, and not recommended for use. In addition to the above, several other reviews were published that are concerned with detection including two that cover the applications of (1) chemiluminesence to LC14 and CE,15 (2) capacitively coupled contactless conductivity detection to CE,16 evaporative light scattering,17 and charged aerosol18,19 detection to LC. Some of the important topics covered in these papers include (1) the use of different luminescence reagents/reactions including peroxyoxalate, tris(2,20 -bipyridine) ruthenium(II), luminol, and direct oxidations and their application to detecting different classes of compounds; (2) the influence of both liquid chromatographic and evaporative light scattering parameters on the appearance of spike peaks; and (3) advantages and disadvantages of charged aerosol detection compared to other methods. Charged aerosol detectors (CAD) are gaining in popularity, especially in cases where an alternative to standard UV detection is needed. Advantages of CADs are a universal response that is independent of chemical properties of the analyte, dynamic range from the low ng to high μg amounts, good precision for many types of analytes, and simple, reliable operation. Limitations are its limited linearity and it is only useful for nonvolatiles. Specific examples of assays that employ CAD are given in the SeparationBased Methodology section. Besides the more common LC detectors, the use of an electron capture detector (ECD) for the reversed-phase analysis of pharmaceuticals has been evaluated.20 The amount of eluent entering the ECD was reduced via a Scott-type nebulizer spray chamber in combination with lower eluent volumes obtained using 2 mm i.d. columns. The response vs volumetric flow curve included covers a range from 0.15 to 1.0 mL/min and is exponential with rapid increases in sensitivity below 0.30 mL/min. For some pharmaceuticals, impurities in them can be detected at the 0.05% level compared to the active ingredient. As part of the performance evaluation process, comparative measurements were made between the ECD, standard UV detection, and evaporative light scattering detection using chlorpropamide, diclofenac sodium salt, flurbiprofen, nifedipine, bromazepam, sulfadiazine, sulfamerazine, sulfamethazine, sulfamethizole, and sulfaquinazole as test analytes. A review has been published that considers operational issues related to the analysis of natural products via high performance liquid chromatography (HPLC) using UV, photodiode array, electrochemical, fluorescence, refractive index, flame ionization, chemiluminescence, evaporative light scattering, charged aerosol, 4490

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Analytical Chemistry mass spectrometric, and nuclear magnetic resonance methods of detection.21 The review provides a comprehensive overview of each of the listed detection methods and has an extensive set of references. It is a useful starting point for those not well versed in the operation of different types of detectors, advantages and disadvantages of each, and their application to analyze natural products. However, for those more versed in general aspects of detection or the fundamental and applied aspects of a particular type of detector, the article is of less use in providing detailed information. Due to increasing complexities of many problems and availability of reliable and easy to operate instruments, there is greater and greater use of mass spectrometry (MS) in pharmaceutical research, especially in combination with a preintroduction separation method. The most common hardware configuration (i.e., experimental design) employs an initial reversed-phase HPLC separation connected to either a time-of-flight or quadrupole MS using an electrospray ionization (ESI) interface. Likewise, the most common eluent/introduction solvent is a binary mixture of either acetonitrile/water or methanol/water that often contains acetic acid, formic acid, an ammonium acetate buffer, or an ammonium formate buffer. HPLC-ESI-MS approaches are used most often to characterize complex mixtures such as natural products and herbal medicines or investigate the active pharmaceutical in the presence of impurities and degradation products. Many studies have appeared that employ either HPLC-MS or HPLC-MS/MS to study forced/ stressed degradation under differing conditions of pH, light, oxidation, and temperature. The general goals of these types of studies are to (1) identify decomposition products, (2) elucidate the breakdown pathways (i.e., mechanisms involved in their formation), and (3) measure the kinetics of degradation and hence predict the long-term stability of the compound being studied. Likewise, a large portion of these types of studies also have been carried out concurrently using HPLC in combination with diode array detection (DAD). Examples of both the natural product work and stressed degradation studies are found in the Separation-Based Methodology section. Many reviews have been published that cover a range of topics related to chromatography-mass spectrometry, including comprehensive discussions of the fundamental and applied aspects of different coupling/interfacing approaches,22
25 the analysis of traditional Chinese medicines by both gas26 and liquid27 chromatography-MS, and the use of derivatization reagents for improving LC-MS sensitivity of difficult to ionize analytes.28,29 In the first of the latter two citations, a variety of derivatizing reagents are considered in terms of their usefulness in making noncharged analytes suitable for ESI-MS analysis. Both conventional derivatizing reagents and those specifically designed for mass spectrometric analysis of various classes of compounds are discussed. In addition to the general chemical aspects, examples of the expected major ions and loss ions in the mass spectra are included. In the second citation, derivatization strategies that can be employed to identify potential organic impurities produced during the manufacturing process are provided, including those related to the active compound, starting materials, intermediates, and byproducts. The paper specifically targets compounds that are neutral and hard to detect by ESI-MS and ones that are genotoxic. In addition to this work, there have been several papers published that specifically target the analytical aspects of detecting and quantifying genotoxic contaminants. These are discussed in greater detail in remaining parts of the Review.

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In terms of chromatographic-mass spectrometric coupling approaches, two of the reviews cited above23,24 discuss the fundamental aspects of atmospheric pressure photoionization (APPI) including ionization mechanisms, influence of eluent composition on sensitivity, and performance comparisons with other spray ionization approaches. Although electrospray ionization and atmospheric pressure chemical ionization (APCI), to a lesser extent, continue to be the most often employed approaches for coupling HPLC instruments to mass spectrometers, APPI is growing in popularity as a soft ionization alternative.23,24 Photoionization was first used in gas chromatography in the seventies, later adapted to liquid chromatography and ion mobility mass spectrometry equipment and more recently to HPLC-MS instrumentation. The cited reviews cover a variety of topics including reaction mechanisms, light sources, and the influence of solvents and solvent coadditives on the APPI process. Likewise, they provide comparative performance information for APPI vs ESI and APPI vs APCI. The reviews are a good starting point to learn more about APPI. In terms of other coupled approaches, two of the papers cited previously describe (1) procedures for the use of a liquidjunction nanospray to interface a capillary electrochromatography instrument to a mass spectrometer25 and (2) major advances in the field of ultrahigh pressure LC-MS as they relate to increasing sample throughput and enhancing resolution.22 Although the focus of the first paper is on the optimization of the operational parameters of the interface, it also presents useful information related to the separation of the enantiomers of acebutolol, alprenolol, atenolol, clenbuterol, fluoxetine, metoprolol, mianserin, mirtazapine, nadolol, norfluoxetine, oxprenolol, propranolol, and venlafaxine. The separations were carried out using a 100 μm i.d.  260 mm capillary packed with 5 μm vancomycin modified silica and filled with an ammonium acetate hydroorganic running buffer and an applied voltage of 20 kV. Many of the analytes were separated with resolutions greater than 1.5 with typical separation times between about 10 to 17 min for a given compound’s enantiomers. The second article contains a histogram of the number of publications vs year for the time period from 2003 to 2009 and it shows an almost exponential growth in the use of UHPLC. However, most of the UHPLC work currently being published that is related to pharmaceutical research and development involves bioanalysis, drug metabolism studies, and more complex natural product profiling of traditional Chinese medicines, whereas the number of articles that describe UHPLC methods for carrying out more common content and stability assays represent only a small portion of the total body of work appearing in the literature. This aspect of UHPLC is discussed in greater detail in the Separation-Base Methodology section of the current Review. It is important to note that when referring to operating pressures greater than 400 bar, ultrahigh pressure liquid chromatography (UHPLC) will be used throughout the current Review not UPLC. The latter term, UPLC, is a company trademark of the Waters Corporation and refers to ultraperformance liquid chromatography. A number of companies now market UHPLC systems that can deliver upper pressures in the 600 to 1200 bar range as well as 2.1 mm and smaller i.d. columns packed with sub-2 μm materials. The net result (i.e., separation advantage) is highly efficient shorter columns that can be operated at high linear eluent velocities with almost no loss in column efficiency due to mass transfer effects. Perhaps the most problematic issue associated with UHPLC separation is fluid compressibility, 4491

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Analytical Chemistry which can be and generally is ignored in conventional HPLC. The result of the use of ultrahigh pressure is nonlinear compression and decompression heating that respectively occurs in the pump and column. Investigations of these effects, which are often large, and the development of appropriate physicochemical descriptions of retention mechanisms are emerging areas of research. Besides the more common topics covered above, an extensive review has been published that specifically discusses microchip technology in mass spectrometry.30 It contains a comprehensive set of references and many illustrations, photographs, and micrographs that show important features of the technology. The article is a good starting point to obtain a better understanding of the “state-of-the-art” of microchip technology as it relates to on-chip ionization. Similarly, the review discusses the development of fully microfabricated systems where both the ionization source and mass analyzer are integrated with the microfluidic separation. Currently, many of the operational features of microionization technology are similar to larger scale conventional ionization approaches (i.e., ESI, APCI, and APPI) in that nanoelectrospray ionization on a chip is the most often used approach compared to other micro/nanoscale ionization mechanisms. Likewise, the chip technology has been shown to provide performance similar to that obtained by conventional ESI interfaces. Although not yet available, the end goal of work in the microchip technology area is to produce an extremely small, highly portable, all in one separation-mass spectrometer that can be used at the “point-of-sampling” as is now possible with miniaturized optical spectrometers. As was the case two years ago, vibrational-based spectrometric methods continue to increase in number, especially as they relate to the quality control of pharmaceutical formulations pre-, during, and postmanufacturing. Much of the continued growth in use has resulted from the Food and Drug Administration’s 2003 Process Analytical Technology (PAT) Initiative in combination with the desire to decrease and/or eliminate manufacturing problems, ensure drug physical and chemical purity, and reduce costs, as well as, the increased ease of operation, reliability, and portability of modern vibrational instrumentation. Additionally, the technique is noninvasive, can be carried out in seconds, and often requires little to no sample workup. In terms of vibrational methods, near-infrared (NIR) and Raman spectroscopies are the most often used approaches outside the research and development laboratory. Many papers have appeared that deal with both general and specific aspects of these techniques as they are used for (1) characterizing the physical properties of the active pharmaceutical ingredient (API) and coingredients; (2) measuring total and spatial content distribution; (3) monitoring the API chemical degradation during manufacturing; and (4) detecting changes in processing variables. PAT is an increasingly important part of the quality assurance of pharmaceutical products, and many articles have appeared that specifically address various problems and applications related to understanding the total manufacturing process as it relates to maintaining a product’s physical and chemical quality. Two of them have appeared that discuss the general use of near-infrared (NIR) spectroscopy for the quality control of pharmaceuticals from the laboratory to the production line.31,32 Currently, NIR is the most widely used technique to monitor drug manufacturing. The first article presents a general strategy for measuring the concentrations of the API and excipients simultaneously in solid dosage forms. It focuses on the limitations associated with different procedures of obtaining appropriate calibration standards

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for production samples, as well as, the necessity of obtaining standards that cover wide enough concentration ranges. Different approaches were evaluated using mixtures of acetaminophen, the API/test compound, and three common excipients, microcrystalline cellulose, talc, and magnesium stearate. The second article deals with instrument design, specifically as it relates to equipment that is used to carry out in-line process analysis. In order to achieve greater instrument stability over the more common Michelson interferometers, newer process instruments often have different optical configurations that are based on double-pendulum, transept, or birefringent optical designs. Advantages of NIR vs mid-infrared approaches are (1) the ability to make in situ measurements without sample preparation; (2) optical-fiber probes can be used and manipulated in hazardous environments; (3) large path lengths can be employed to enhance sensitivity; and (4) measurements can be made on aqueous solutions. Many papers have been published that deal with calibration, as well as, the physical and computational factors that affect calibration when making NIR quantative measurements. Examples of these include an article that discusses how various physical factors can influence the accuracy of calibration models, and it specifically concentrates on the in-line monitoring of tablet production.33 As part of the study, particle size, galenic form (i.e., absorption characteristics), compaction pressure, and coating thickness were the factors evaluated and the model system used to carry out the work was 63.5% ibuprofen in combination with four excipients, maize starch (i.e., the other major component), Avicel PH 102, aerosol, and magnesium stearate. Spectral data were collected over various wavelength ranges and processed using different spectral algorithms. The joint use of the whole wavelength range (1100
2500 nm) in combination with a standard normal variate (SNV) treatment provided the best predictive approach. A second article provides procedures for analyzing pharmaceuticals by NIR spectroscopy without the use of a reference method;34 a third discusses how to avoid chemometric pitfalls,35 and a fourth describes dynamic calibration approaches for measuring in-line film thickness of pharmaceutical tablets with regression fits better than 0.99 in some instances.36 In the latter approach, the dynamic calibrations were performed using both averaging and clustering of the NIR spectra acquired during in-line monitoring. Besides the methods discussed above, a net analyte signals (NAS) approach has been used to improve the quality of NIR measurements in samples containing spectral interferences from other components present.37 NAS employs only that part of the spectrum unique to the target compound irrespective of having spectra for the coingredients in the formulation. In carrying out the study, laboratory samples of acetaminophen formulations with known spectra for both it and the excipients were prepared and used to evaluate two methods of NAS processing. Subsequently, the best correlations were obtained without preprocessing using a second derivative Savitzky
Golay numerical filter (15 points filter width), which was used to evaluate four commercial formulations of acetaminophe, acetylsalicylic acid, folic acid, and neomycin. Similarly, scattering orthogonalization, a preprocessing technique, has been found to reduce physical interferences and maintain the information content of the NIR spectra acquired from pharmaceutical tablets produced using different amounts of compression.38 This latter study was carried on 30 different compressed formulations (i.e., six blends and five compression forces) of theophylline, lactose, and microcrystalline cellulose. The resulting NIR spectra were orthogonalized against 4492

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Analytical Chemistry the reduced scattering coefficients, and the content of each sample was determined with good reliability using a Monte Carlo, simulation-based, partial least-squares (PLS) calibration model. Another study has been carried out to evaluate the suitability of different multivariate calibration approaches for analyzing Raman spectra collected from the backside of tablet samples.39 Since the light collected passes through the entire tablet, the resulting signal is more representative of the total composition versus the light collected using normal front-side scattering geometry. The two best methods of calibration were PLS and multivariate curve resolution (MCR). Both were found to give comparable quantative results for measuring acetaminophen in tablet formulations. Another important advantage of the backside vs normal front-side signal collection geometry is an increase in the volume sampled and hence better sensitivity. The authors point out that, for samples where Raman spectroscopy can be applied, it has advantages of speed and simplicity over competing methods like HPLC, especially when multiple single tablet content measurements are needed. Multivariate PLS also has been used to monitor blend uniformity and to establish when the end-point is reached during the manufacturing process via continuous in-line measurements.40 The underlying premise of the approach is that during blending the endpoint is reached/detected when spectral variations between successive steps have reached a minimal predefined limit or a limit determined “on-the-fly.” In the latter instance, a common numerical approach used to evaluate the sequentially generated spectral information is via a moving comparison of standard deviations (s.d.) from blocks of data. However, it is often difficult to determine the exact end-point of blending depending on the data block size used to compute the s.d. and the amount of smoothing used. These and others factors are discussed. During the methods development stage of the reported work, a calibration model was developed using 21 off-line static standards. Also as part of the study, the reliability and accuracy of the in-line NIR content measurements were validated by carrying out HPLC assays on tablets as they were being produced. Subsequently, the procedure was transferred directly to a 15-fold scale-up manufacturing facility. Additional examples of manuscripts that report PAT methodology are ones that employ NIR in-line measurements to characterize powder voiding41 and to measure moisture content during fluidized-bed drying.42 In the first citation, two approaches are presented for analyzing the data. One is based on the analysis of noise in the spectra and the other evaluates the changes in the spectral baseline. The underlying basis of both methods is the observations that large changes in the spectral baseline and increased noise occur when powders flow poorly. Both approaches are reported to be adaptable to other applications that involve powder flow. Besides the above numerical processing methods, a Monte Carlo approach has been used to simulate photon migration in pharmaceutical materials as a means of modeling NIR absorbance and scattering phenomena as a function of sample penetration depth.43 These same authors also have published a paper that reviews the fundamental aspects of NIR absorption and scattering in order to better understand the fundamental interactions of near-infrared radiation with pharmaceuticals and by so doing to develop better assay procedures.44 The paper presents an overview of the relevant theory that controls each process as well as a discussion of techniques that can be used to separate the adsorption and scattering components. Included among these are integrating sphere-based, time-resolve, and

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frequency domain photon migration approaches. Likewise, the salient mathematical relationships are provided in the review. Besides its many daily applications for structural elucidation, compound verification, and to a lesser extent, the measurement of polymorphism, the mid-infrared region, commonly referred to as Fourier transform infrared spectroscopy (FT-IR) has been used to measure changes in protein secondary structure that can occur during the formulation process.45 The approach is based on changes in the amide I bands as the native protein’s structure (i.e., R-helix 1650
1660 cm
1 and intramolecular β-sheet 1683
1695 cm
1) forms aggregated strands (intermolecular β-sheet 1620
1644 cm
1). During the methods development stage, infrared measurements were made on a total of 16 different proteins. While the work described in the manuscript is interesting, very little experimental information is included in terms of the spectrometer settings used to acquire and process the data. Limitations of the approach are the spectral complexity of the infrared band profiles used to characterize protein degradation and potential fitting errors associated with the algorithms used to separate them into their individual spectral components. Many additional mid-infrared papers are included as examples in the Spectrometric and Other Methodology section later in this Review. Many general papers and reviews have appeared for a number of other techniques. Some examples of them are ones that consider various aspects of quantitative nuclear magnetic resonance (NMR) spectroscopy,46
48 application of plasmon resonance biosensors to monitor the processing of biopharmaceuticals,49 recent developments in UV
visible spectrophotometry,50 analytical applications of flow injection analysis using chemiluminescence51 and amperometric52 detection, transmission electron microscopy (TEM) of pharmaceutical materials,53 and advances in microfluidic immunoassays.54 The first of the NMR papers listed above deals with the selection of an appropriate internal quantitative standard and suggests 8 from a list of 25 possible compounds to be the most versatile. These are 2,4,6-triiodophenol, 1,3,5-trichloro-2-nitrobenzene, 3,4,5-trichloropyridine, dimethyl terephthalate, 1,4-dinitrobenzene, 2,3,5-triiodobenzoic acid, maleic acid, and fumaric acid. Selection of the standards was made on the basis of the following criteria: (1) unique chemical shift and stable signals, (2) high purity, (3) solubility in NMR solvents, (4) low volatile and easily weighable, (5) nonreactive and long-term stability, and (6) relatively low molecular weight. The paper also provides spectral information (i.e., chemical shift and integration values) for the eight standards obtained in four different common NMR solvents, D2O, DMSO-d6, CD3OD, and CDCl3. The remaining NMR papers present, respectively: (1) a general overview of recent progress in quantitative applications of the technique and (2) the technique’s application to measuring biologically active substances and excipients. Two of the other papers49,53 are concerned, respectively, with the characterization of the physical properties of formulated products as they are being manufactured and the factors influencing reliability of the TEM measurements. In the latter paper, issues related to sample preparation and electron beam damage are discussed with the goal of providing possible approaches that can be used to minimize these difficulties. Besides the many technique-related papers, a host of others have appeared that present comprehensive overviews of analytical methodology associated with particular compounds or classes of compounds. A few examples of these are ones that are concerned with the (1) acid
base properties of cephalosporins and penicillins,55 (2) stability of indinavir during processing,56 (3) dehydration kinetics of nitrofurantion monohydrate,57 (4) characterization of 4493

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Analytical Chemistry the solid state behavior of propranolol hydrochloride,58 (5) release of nonsteroidal anti-inflammatories from various lipophilic suppository media,59 (6) strategies for identifying counterfeit products containing sildenafil, vardenafil, and tadalafil,60 and (7) interactions of cisplatin with DNA.61 The latter citation discusses the application of immunochemical, 32P-post-labeling, and mass spectrometry methods as tools to correlate molecular dose with the effects of treatment. Most of the other cited work was carried out using a combination of techniques including HPLC, FT-IR, NIR and Raman spectroscopy, differential scanning calorimetry (DSC), and X-ray powder diffraction (XRPD). In the case of indinavir, DSC, XRPD, and NIR measurements were used to study its crystalline and amorphous behavior as a function of temperature, relative humidity, and compression. Similarly, DSC and XRPD in combination with hot-stage microscopy (HSM) and thermogravimetric analysis (TGA) were used to characterize the mechanism of dehydration of nitrofurantoin monohydrate. Both model-fitting, using various nucleation, geometrical contraction, diffusion, and reaction-order relationships, and model-free approaches were used to analyze the kinetic data. Although several of the model-fitting approaches resulted in similar estimates of the activation energy for dehydration, they were ambiguous in terms of elucidating the exact mechanism of dehydration. However, the model-free approach showed that dehydration occurs in two steps, an initial rapid nucleation followed by two/three-dimensional nuclei growth, and coalescence. Still other compound/class related reviews have been published that cover general analytical aspects of oxicams, nimesulide, and nabumetone62 and thyrostatic agents,63 as well as a cross-section of separation-based techniques to measure epoxide and hydroperoxide genotoxic impurities in active pharmaceutical ingredients and products.64 Similarly, other papers and reviews have appeared that deal with comprehensive aspects of separation-based methodology for specific analytes or classes of analytes. Examples of these types of papers include (1) advances in the separation, detection, and characterization of heparin and heparan sulfate including the analysis of contaminants in pharmaceutical preparations,65 (2) the separation and quantitation of tropane alkaloids by gas chromatography, liquid chromatography, capillary electrophoresis (CE), and microemulsion electrokinetic chromatography (MEKC) singularly and when these separation techniques are used in combination with mass spectrometry,66 (3) the CE of sulfonamides using different separation modes as well as in combination with MS and MS-MS,67 (4) advances in the analysis of bioactive fatty acids68 and vitamin B12,69 and (5) separation approaches used to assay triazole antifungal agents over the past decade.70 In the latter citation, the techniques of gas, liquid, thin-layer, and supercritical fluid chromatography are discussed as is capillary electrophoresis. It specifically covers eight drugs, fluconazole, itraconazole, and terconazole, which are first generation compounds, and albaconazole, isavuconazole, posaconazole, ravuconazole, and voriconazole, which are second generation antifungals. In addition to the above compound related publications, many other articles and reviews were published over the last two years that deal with various aspects of sample preparation,71
75 PAT for biopharmaceuticals76
78 and related biopharmaceutical questions,79
83 characterization of solid dosage forms,84 miscellaneous quality assurance and quality-by-design strategy,85
89 stability monitoring of compound collections,90 strategies for evaluating pharmaceutical purity,91
93 computer-aided design of process monitoring and analytical systems,94 estimation of the

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bioequivalence of drugs,95 and characterization of thixotropic properties of pharmaceutical formulations.96 Many of the sample preparation articles cover various aspects of microextraction and related techniques including automation and improvements in sample throughput as well as off-line and online operations. The PAT articles deal with topics ranging from production monitoring of the active compound to storage problems including stability and shelf life, losses through adsorption, and contamination from storage container leachables. The articles dealing with purity cover a wide range of topics from (1) strategies for chemical and structural elucidation of the unknown contaminants, (2) potential sources and mechanisms of formation, (3) the general use of various hyphenated analytical techniques, (4) chemometrics and chromatographic purity, and (5) systematic procedures for detecting genotoxic impurities. One of the articles72 contains many useful figures and tables as well as providing specific extraction procedures for many pharmaceutical compounds. As noted at the beginning of this section, the articles included are a small sampling of the total body of analytical work published during 2009 to 2010. Some of the important factors affecting the work are (1) increasing globalization of the pharmaceutical industry in terms of research and development, regulatory affairs, and marketing; (2) PAT and QbD initiatives mandating more attention to assuring/monitoring of the total process from drug discovery to delivery of a final therapeutic product; (3) miniaturization/portability of instrumentation especially as it relates to the vibrational techniques; (4) increasing interest in biopharmaceutical and nontraditional products; and (5) greater automation and higher throughput assays that can provide near-immediate results.

’ SEPARATION-BASED METHODOLOGY The bulk of the articles included in the current Review that deal with separation-based methodology fit into four broad categories: (1) those that describe specific assays for measuring content, stability, and purity including the development and validation of the methods; (2) procedures for measuring chiral purity and factors that can influence it; (3) developments in instrumentation and related applications of the emerging technology; and (4) general aspects of separation techniques including both fundamental and applied considerations. The first three of these are discussed in this section and the fourth has been reviewed in the previous General section. As has been the case for over two decades, separation-based methodologies, excluding process monitoring, continue to be the approaches of choice for analyzing pharmaceutically active compounds in the bulk and in formulated products. Likewise, excluding methods designed to assess either chiral purity, characterize biopharmaceuticals, or measure charged or highly polar analytes, a majority of the separation-based assays continue to be done by reversed-phase high performance liquid chromatography (RP-HPLC). By far, the most common RP-HPLC approach is to carry out the separation using some type of octadecyl modified packing (i.e., porous silica-based) in combination with either a binary aqueous-acetonitrile97
131 or aqueous-methanol132
141 eluent. Examples of these methods are given in Table 1. Even though higher performance columns filled with sub-2 μm packings are increasing in popularity, from the literature examined in preparing the current review, it appears that the bulk of the common assay procedures are still being performed using either 4.6  150 mm or 4.6  250 mm columns packed with 5 μm of completely porous 4494

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Table 1. Examples of HPLC Assays for Measuring/Characterizing Bulk Pharmaceuticals and Formulated Products compound

sample

study

ref

abacavir, didanosine, lamivudine, and zidovudine

bulk drug

content

110

alizapride and degradation products

bulk drug

stability

122

aspirin and folic acid

nanoparticulate systems

release

127

betamethasone dipropionate and related compounds

dermatological products

stability

102

bisoprolol fumarate and hydrochlorothiazide

tablets

content

101

buprenorphine HCl, naloxone HCl and noroxymorphone

tablets

content

100

capreomycin sulfate

bulk drug

purity

104

carvedilol caffeine

dosage forms dosage forms

stability dissolution

108 121

chloroquine

bulk drug

stability

131

defibrase

dosage forms

content

124

diazepam, clonazepam, flunitrazepam, and nitrazepam

bulk drug

content

105

diphenhydramine citrate

tablets

stability

107

doxofylline

bulk drug and dosage forms

content

106

enalapril and lercanidipine

binary mixtures

fundamental

134

emtricitabine and related compounds erythromycin and related compounds

bulk drug bulk drug

content purity

97 115

gatifloxacin

bulk drug

purity

114

lornoxicam

dosage forms

stability

125

losartan potassium, atenolol, and hydrochlorothiazide

dosage forms

stability

99

metronidazole and spiramycin

tablets

content

103

mexiletine

dosage forms

content

117

nicorandil and degradation products

bulk drug and tablets

stability

133

olmesartan oxytocin

bulk drug and dosage forms injectables

stability content

123 112

prednicarbate, mupirocin and ketoconazole

topicals

content

132

pseudoephdrine, pheniramine, guaifenisin, pyrilamine chlorpheniramine and dextromethorphan

cough cold products

content

135

riluzole

bulk drug and tablets

stability

129

salicylic acid, betamethasone dipropionate and related compounds

lotions

stability

130

thioctic acid, benfotiamine and cyanocobalamin

capsules

content

109

tolperisone HCl

tablets

content

113

tramadol chlorhydrate and metoclopramide hydrochloride vardenafil, sildenafil, tadalafil, procaine, lidocaine prilocaine and benzocaine

infusion solutions creams

stability content

98 128

zidovudine

microemulsions

release

118

C18-silica materials. Noticeably absent in the papers that deal with common questions like stability and content of the APIs has been the widespread use of monolith columns, which in more recent past reviews (1 and other papers in the series) appeared to be growing in popularity. This is not to imply they are not being used in specific applications like determining the chiral purity of compounds or general analytical methodology, but they have not been the column of choice in the common pharmaceutical assay methods being developed. Some examples of other less often used reversed-phase approaches include those that employ (1) different stationary phases like amide, octyl, and phenyl modified surfaces,142
145 (2) ternary hydroorganic eluents,146
154 or (3) ion-pairing eluent additives.155
160 Most of the examples cited above are representative of the larger body of RP-HPLC assays published over the past two years, and they are more often isocratic methods that employ phosphate buffers to control the eluent’s pH.98
110,132
136,142,143,146,151,156,158
160 In addition to the use of phosphate salts, other common eluent additives for pH control are acetic acid and/or ammonium acetate,120
124,139,149,150,157 trifluoroacetic acid,125
128,148,153 and formic acid and/or ammonium formate.140,141 These latter volatile

compounds were the most commonly used additives in HPLC assays that employed mass spectrometry (MS) for analyte detection161
177 and are discussed later in this section of the Review. On the basis of the HPLC-MS papers included as examples, most describe gradient elution methods that use water containing either formic acid or an ammonium formate buffer161,163
171 in combination either acetonitrile161,162,164,167
170,172,173,175
177 or methanol163,165,166,171,174 to analyze complex mixtures. The three most common types of samples assayed were natural and herbal products, pharmaceuticals that potentially can contain a number of impurities and/or degradation products, or samples from forced degradation studies. The greater use of acetonitrile vs methanol in both the simpler isocratic and more complex gradient elution methods represents a changing trend in the organic cosolvent most commonly employed to prepare the chromatographic eluents. On the basis of fundamental considerations, it is tempting/reasonable to suggest the cause of this is the need for less viscous eluents and the increasing use of smaller diameter columns packed with 4495

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Analytical Chemistry sub-2 μm materials that are operated at higher linear eluent velocities in order to obtain shorter separation times (i.e., commonly referred to as high throughput assays). The growing interest in high throughput assays is the result of the increasing numbers and complexity of samples being produced by modern combinatorial synthetic procedures. A single synthetic medicinal chemist can produce 20 to 40 times the number of compounds annually using combinatorial techniques compared to the number of compounds possible by traditional organic approaches. A more extensive discussion of the design of high-throughput HPLC assays can be found elsewhere.178 Nevertheless, when a correlation was attempted between the use of acetonitrile vs methanol and column dimension and/or particle diameter, this explained only a small percentage of the increase but not the bulk of the change. Rather, the only explanation that seemed plausible was the increasing globalization of pharmaceutical research and development and the expanding body of scientific experiments carried out internationally. Other than a few exceptions, almost all of the work originating from India, which was a significant portion of the common assay papers appearing in the literature, employed acetonitrile as the organic cosolvent. Whereas, the remaining assays preferentially employ methanol other than those that were carried out with reduced diameter columns or sub-5 μm packings.99,102,116,120,141,155,156,164,168 A few examples of separations performed on columns packed with sub-5 μm materials are ones developed to determine losartan, atenolol, and hydrochlorothiazide in pharmaceutical dosage forms99 and three curcuminoids in Curcuma longa Linn,120 to measure betamethasone dipropionate in a dermatological product,102 to separate the nonpolar ginsenosides in red ginseng,116 to measure desloratadine and related compounds155 and bupropion hydrochloride156 using gradient ion-pairing conditions, and to quantitate the isomers of atracurium, cisatracurium, and mivacurium in pharmaceutical preparations.141 The first two of the above methods employ sub-2 μm packings in combination with ultrahigh pressure liquid chromatography to simultaneously measure the target analyte in less than 3 minutes. In the latter paper,121 comparative chromatograms are included for the separation of curcumin, desmethoxycurcumin, and bismethoxycurcumin using standard HPLC and fast analysis UHPLC conditions with an approximate 15-fold difference in total separation time between the two. In addition to this comparison, four C18 columns and six different operating temperatures ranging from 20 to 45 °C were studied. Plots are given for resolution, symmetry factor, plate count, and retention vs temperature. The optimum separation/resolution was achieved with a BEH column operated at a temperature of 30 °C. Four additional examples of high efficiency LC methods are (1) two carried out using columns pack with sub-2 μm RP materials in combination with UHPLC tandem MS and (2) two with 3.5 μm C18 modified silica in combination with conventional HPLC-MS. These methods were used, respectively, to assay terpene trilactones in Ginkgo biloba extracts and related pharmaceutical formulations,165 to identify fentanyl homologues and analogs,166 to characterize clindamycin phosphate and possible impurities in injection solutions,163 and to determine 13 arylamines and aminopyridines.164 In the latter case, the two assays are carried out sequentially. The first involves a standard HPLC-MS analysis on the sample that is followed by a second HPLC-MS analysis after derivatizing the sample using hexylchloroformate under alkaline conditions. The reaction takes

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about 30 min to complete at room temperature, and it produces the corresponding acyl derivatives with an increase in molecular mass of 128 amu for each. The derivatives are more hydrophobic than the parent compounds, which results in longer retention times. The two methods when used together provide complementary information, and it is suggested that they can be utilized as a generic strategy for detecting/identifying arylamines and aminopyridines genotoxic impurities in pharmaceutical products. Although most routine separations continue to be done at ambient temperature, there is growing interest in the use of above ambient conditions as a means of further shorting analysis times and/or to improving chromatographic resolutions via reductions in eluent viscosity. The eluent’s viscosity is about a factor of 2 to 3 less for midrange mixtures of water/acetonitrile and water/ methanol at 80 °C compared to those at ambient temperature. A review has been published that provides both a historical perspective of high temperature liquid chromatography (HTLC) and a comprehensive overview of the topic.179 It covers both fundamental and applied aspects of HTLC separations as they relate to the analysis of pharmaceutically active compounds. Two major chemical limitations of carrying out separations at elevated temperatures are solute and stationary phase instability. For chemically modified silica packing, the latter limitation is increasingly more problematic as the column ages due to phase loss and column voiding as the silica’s structure degrades and collapses. Like many other reemerging/rediscovered approaches, such as (1) the use of ultrahigh pressures, (2) fast analysis using short columns and high linear eluent velocities, (3) RP separations carried out using high to totally aqueous eluents, and (4) pellicular/ porous-shell packings, the use of elevated column temperatures can be traced back to research carried out nearly 40 years ago in the early days of modern HPLC. At that time, one of the principal interests in temperature and temperature control was to ensure reproducibility of solute retention, especially under normal-phase conditions, which is influenced more by thermal fluctuations than RP-HPLC. This is due to two factors, higher solute sorption energies in the NP vs RP mode and adsorption
desorption of the polar modifier under normal-phase conditions. Nevertheless, with the increasing use of higher pressures in liquid chromatography (i.e., UHPLC), the general topic of temperature has become an important area of fundamental investigation. Unlike conventional HPLC where eluent compression and decompression is typically less than a few % and heating due to it can be ignored, the issue of heating is much more problematic in UHPLC. Heating occurs both in the pump as the eluent is compressed and again during decompression as it travels through the column. Of these two effects, decompression is experimentally more difficult to deal with and many times the amount of heating is greater than 30 °C. During the preparation of the current manuscript, a number of reported UHPLC separations were reviewed, some were included as examples and some were not. Several of these papers reported reproducibility problems that appear to be attributable to heating effects. A few examples of the published methods that employ elevated temperatures are ones developed to measure emtricitabine and its related drug substances,97 buprenorphine hydrochloride, naloxone hydrochloride and noroxymorphone in tablets,100 oxytocin and related products,112 erythromycin A, B, C and impurities,115 nonpolar ginsenosides,116 thiol-disulfide exchange reaction products of captopril and thiurams,126 APIs in Diprosalic lotion,130 pKa values for lercanidipine and enalapril in binary mixtures,134 and telithromycin in tablets.136 All but three 4496

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Analytical Chemistry of these methods100,134,136 employ gradient elution conditions and can resolve the listed analytes from a variety of pre- and posteluting impurities and degradation products. Likewise, most of the preceding examples were carried out between about 30 and 40 °C. However, in two instances, operating temperatures of 50 °C136 and 65 °C115 were used to measure antibiotics in the presence of impurities and degradation products. In the first case, forced degradation studies were performed to measure the effects of acid, base, oxidation, UV light, and temperature on the assay’s performance, and in the second, an earlier published isocratic method was modified via the use of gradient elution conditions and subsequently an interlaboratory study carried out to evaluate between-site consistency and thus its suitability as a replacement for accepted standard methodology. The latter study involved five laboratories located in Europe and the United States. Some examples of RP assays that utilize less common phases were ones developed to measure the amount of ketoprofen in creams,142 study the degradation of gemifloxacin mesylate under different stressing conditions,143 assess the stability of sibutramine in both the bulk form and gelatin capsules,144 and separate low levels of dexamethasone and related compounds from betamethasone.145 In the assay published for gemifloxacin, chromatographic profiles are included for acidic, basic, and neutral stress conditions as well as oxidative stress conditions. A total of eight different degradation products were found with the greatest number and amounts of them produced under acidic conditions. However, none of the impurities were identified in the study. For sibutramine, the greatest amounts of degradation were noted when it was either exposed to dilute solutions of hydrogen peroxide or irradiated with 254 nm light. As in the case of the first citation, this paper also does not contain information in terms of the identity of the degradation products. In the last example given above, a large amount of information is included in the manuscript, including structural information and chromatographic profiles obtained using a number of different phenyl modified silica packings. In addition to dexamethasone and betamethasone, a total of 12 additional compounds are observable in the chromatograms with assigned structures for each. A few stability studies have been carried out using ternary eluents. Most of them used a combination of acetonitrile, methanol, and water. Three examples of ternary eluent HPLC assays are ones used to characterize the forced degradation of ropinirole hydrochloride,146 deferasirox,151 and fentanyl152 under conditions of elevated temperature, hydrolysis, oxidation, and photolysis. All of these studies contain useful structural and chromatographic profile information. However, the latter is the most thorough in terms of structural information, mechanisms of degradation, and chromatographic peak assignments. Likewise, it contains useful mass spectrometric and nuclear magnetic resonance (NMR) data. Fentanyl was found to be stable when exposed to light and when treated with base, but under acidic conditions, fentanyl degrades rapidly to N-phenyl-1-(2-phenylethyl)-piperidin-4-amine and when treated with hydrogen peroxide to fentanyl N-oxide. Likewise, the thermal breakdown of the analyte was found to be the most complex process producing five degradants, propionanilide, norfentanyl, 1-phenethylpyridinium salt, 1-phenethyl-1H-pyridin-2-one, and 1-styryl-1H-pyridin-2-one. In addition to these compounds, the method also is capable of separating three known process impurities, acetyl fentanyl, pyruvyl fentanyl, and butyryl fentanyl. Additional examples of ternary eluent methods are ones for quantifying lidocaine and prilocaine,147 gentamicin,148 fleroxacin,149 and boanmycin150 in various pharmaceutical formulations.

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Likewise, a procedure has been developed for measuring polysorbate 80 in solutions containing proteins using a C18 2.1  75 mm column packed with porous shell particles,153 and a stability indicating assay has been reported for determining cholecalciferol in pharmaceutical preparations in the presence of potential degradants and common excipients.154 Two examples of gradient elution ion-pairing (IP) methods published in the last two years are approaches for quantifying desoratadine and related compounds in Clarinex Tablets155 and for simultaneously measuring the intermediates and degradants in duloxetine hydrochloride.158 The IP reagents used were sodium dodecylsulfate and sodium octanesulfonate, respectively. Likewise, isocratic procedures were reported for assaying bupropion hydrochloride in Zyban tablets,156 characterizing low molecular-weight heparins (LMWH) with significant structural diversity,157 measuring chondroitin sulfate sodium, allantion, and pyridoxine hydrochloride in eye drops,159 and determining iron(II) in bisglycinate (Fe-bis-gly) capsule formulations.160 In the latter procedure, the Fe-bis-gly is first treated with ascorbic acid and then 4-(2-pyridylazo) resorcinol (PAR) to produce the Fe(II)-di-PAR complex, which was separated on an C18 column using acetonitrile/phosphate buffer (pH 8.0) containing 1 mM tetrabutylammonium hydrogensulfate. The analyte complex was measured at 706 nm using a diode array detector (DAD). The remaining assays given above all used UV detection ranging from 195 nm for chondroitin to 291 nm for pyridoxine. By far, UV detection, including DAD, continues to be used most often in common assays designed to assess content, stability, and purity. This is followed by mass spectrometry, especially for measuring more complex samples like natural/ herbal products, fermentation derived pharmaceuticals, and biomaterials, as well as to characterize the purity and/or degradation mechanisms of compounds. Other examples of detection methods that have been used to a lesser extent include charged aerosol detection,141,148,153,180 evaporative light scattering detection,157,181
183 electrochemical detection,105,110,111,116 and fluorescence.110,124,140,184 Of these latter approaches, CAD is increasing in popularity for analytes with low volatility. The basic mechanistic steps governing detection are (1) the formation of an aerosol containing the analyte via nebulization; (2) use of a drying gas (i.e., nitrogen) to remove the solvent and form microscopic analyte particles; (3) formation of charged nitrogen molecules using a Corona discharge source; (4) ionization of the aerosol analyte particles via their interaction with the charged nitrogen molecules; and (5) measurement of the charged particles using a collecting electrode electrometer. Some of the analytes measured using CAD were atracurium, cisatracurium, mivacurium, gentamicine sulfate and related substances, polysorbate 80 in solutions containing proteins, and ascorbic acid and dehydroascorbic acid and by ELSD azithromycin, polysorbate 20 and unbound polyethylene
glycol in protein solutions, N,N0 -ethylenebisstearamide in an intravaginal drug delivery device, and LMWHs. In both of the polysorbate studies,153,181 porous shell packings were used to carry out the chromatographic separations, and in the case of azithromycin, it was resolved from artesunate using a porous graphitic carbon packing and binary eluents of acetonitrile and methanol containing different [triethylamine]/[formic acid] ratios.182 The later paper contains useful physicochemical information related to various separation conditions. As noted above, by far, the greatest numbers of HPLC gradient methods using online mass spectrometric detection were 4497

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Table 2. Examples of Separation-Based Assays for Measuring the Chiral Purity of Bulk Pharmaceuticals and Formulated Products compound(s)

technique

additive or column

ref

β-adrenergic antagonists and agonists (18 drugs)

HPLC

AmyCoat phase

185

aminoglutethimide

CE

methylated-β-cyclodextrin

213

anisodamine, benzhexol, fenfluramine, ketoconazole, promethazine, and propranolol

CE

6-O-(2-hydroxybutyl)-β-cyclodextrin

214

2-arylpropionic acid nonsteroidal anti-inflammatories (8 drugs)

HPLC

hydroxypropyl-β-cyclodextrin

186

benzimidazole derivatives

EKC

sulfated cyclodextrins

239

bupropion

CE

sulfated-R-cyclodextrin

215

brompheniramine, pheniramine, cyclopentolate, doxylamine, ketamine, and metoxyphenamine

HPLC

neutral and negatively charged cyclodextrins

188

certirizine, primaquine, sulconazole, trihexyphenidyl chlorpheniramine, citalopram, metoprolol, nefopam, propranolol

CE MEKC

amylose clindamycin phosphate

216 241

cimaterol, formoterol, salbutamol

CE

β-cyclodextrin

217

dorzolamide hydrochloride

HPLC

chiral-R1-acid glycoprotein phase

190

duloxetine, propranolol

CE

erythromycin lactobionate

224

ephedrine and eleven related compounds

CE

sulfated β-cyclodextrin

221

esomeprazole, omeprazole

HPLC

Chiralpak IA phase

193

fenoprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen

CE

eremomycin

218

formoterol cis-β-lactams (19 drugs)

HPLC CE

Chiral-AGP phase several different cyclodextrins

192 219

levodopa

CE

sulfated β-cyclodextrin

222

nateglinide

HPLC

Chiralpak AD-H phase

195

MEKC

methyl-β-cyclodextrin

242

nefopam

MEKC

chondroitin sulfate

240

quinocide, primaquine

CE

β-cyclodextrin and 18-crown-6-ether

220

palonosetron hydrochloride

HPLC

Chiralpak IC phase

196

pheniramine propranolol

CE CE

hydroxypropyl- β-cyclodextrin native and derivatized cyclodextrins

223 225

raltiterxed

MEKC

carboxymethyl-β-cyclodextrin

243

sertraline and related enantiomeric impurities

HPLC

Cyclobond1 2000 DM phase

198

tropic acid

MEKC

sulfated-β-cyclodextrin

244

vigabatrin

HPLC

teicoplanin aglycone phase

199

vincinal diols

CE

aminoethylamino-β-cyclodextrin

226

zolmitriptan

CE

hydroxypropyl-β-cyclodextrin

227

developed to characterize natural products and dietary supplements. Many of these also employed DAD or tandem MS. A few examples include the identification of irioid glycosides and phenylpropanoid glycosides in Radix Scropulariae,161 characterization of the radical scavengers in Shengmai San,162 quantification of terpene trilactones in Ginkgo biloba extracts and pharmaceutical formulations,165 separation of the major constituents in Radix Paeoniae Rubra,167 analysis of the constituent of aqueous formulations of Stachys recta,170 and determination of 14 synthetic antidiabetic drugs in adulterated Chinese drugs.168 Three out of the six examples162,165,168 used MS/MS. In the latter citation, UHPLC gradient conditions were employed to perform the separation in about 4 min with an elution order of metformin, phenformin, rosiglitazone, pioglitazone, chlorpropamide, glipizide, tolbutamide, repaglinide, gliclazide, mitiglinide, glibenclamide, glimepiride, nateglinide, and gliquidone. Similarly, gradient elution UHPLC-MS has been reported for assaying aconitum alkaloids in Yin Chen Si Ni Tang, a traditional Chinese medicine.173 Besides the above examples, LC-DAD and LC-MS methods have been published for assessing the purity of digoxin171 and the structural elucidation of taranabant.176 Additionally, LC-MS/MS assays have been developed to evaluate the purity of injection solutions containing clindamycin and its degradation products and

related impurities,163 identify the photodegradation products of betamethasone dipropionate,175 and measure impurities in low dose tablets containing anastrozole.177 In another study, a combination of both LC-MS and LC-1H NMR were used to investigate the stressed degradation of 4-methyl-2-(3,4-dimethylphenyl)-1-(4-sufamoylphenyl)pyrrole now under development as a selective cyclooxygenase-2 (COX-2) inhibitor.169 Both MS and NMR spectra are included in the paper as well as proposed mechanistic degradation pathways. Likewise, LC-MS and LC-1H NMR have been employed as a means of identifying 400 -isovalerylspiramycin 1 in a genetic engineered strain of S. spiramyceticus F21.172 Another important area for separation-based methods is for evaluating the enantiomeric purity of chiral APIs. During the last two years, several hundred papers have been published that deal with various aspects of chiral separations. A few examples of them include ones that use high performance liquid chromatography,185
204 gas chromatography,205 thin-layer chromatography,191,206 capillary electrochromatography,207
212 capillary electrophoresis213
238 and micellar and related electrokinetic methods239
247 to resolve many different enantiomeric compounds by use of a chiral stationary phase, a chiral additive, or a chiral derivatizing reagent. Summarized in Table 2 are examples of some of the compounds where chiral purity was evaluated in 4498

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Analytical Chemistry either the bulk pharmaceutical or the formulated product using one of these approaches. High performance liquid chromatography and capillary electrophoresis (CE) were used most often and electrokinetic chromatography to a lesser extent. In addition to the more common chiral assay approaches given in Table 2, a detailed study was carried out to evaluate the suitability of both reversed phase and normal phase LC conditions for resolving the enantiomers of 2-amino-N-[2-[1, 2-dihydro-1-(methylsulfonyl)spiro-[3H-indole-3,40 -piperidin]10 -yl]-2-oxo-1-[(phenylmethyloxy) ethyl]-2-methylpropanamide monomethanesulfonate and its neutral precursor using a newly developed polysaccharide stationary phase.187 Although both approaches can be used to separate the enantiomers, the RP conditions give the best resolution and peak shapes. Likewise, a similar, but more comprehensive study was carried out by several of the same investigators to evaluate the separation characteristics of six different polysaccharide chiral stationary phases in both the RP and NP modes of operation.194 A total of 19 neutral pharmaceuticals were used to characterize the retention properties of the phases under a variety of eluent conditions. On the basis of the data obtained, strategies for selecting the initial separation conditions and subsequently approaches for optimizing them were proposed. Two less often used HPLC approaches for evaluating chiral purity are by either circular dichroic detection (CDD) or preanalysis dervatization, and two less often used techniques are thin-layer chromatography and gas chromatography. The first approach has been used to measure the optical purity of carnitine following an achiral LC separation to eliminate interferences from other compounds present in the pharmaceutical formulation.189 Stated advantages of the CDD approach are reduced cost, since special chiral additives and columns are not needed and it can be used to assess simultaneously the chemical purity of the API as its chiral purity is being measured. The second approach, chemical derivatization followed by an HPLC separation, has been used to measure the chiral purity of fluoxetine after reacting it with Marfey’s reagent and (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester.191 In this same paper, a second method is described that uses thin-layer chromatography and L-tartaric acid as a solvent system additive. Likewise, both direct and indirect derivitization approaches, as well as HPLC and TLC chiral assays, have been reported for measuring enantiomeric purity of DL-penicillamine.197 In addition to the above examples, which consider chromatographic methodology for specific chiral compounds, other papers have been published that deal with more general aspects of the topic. Among these are two reviews that discuss monolithic chiral stationary phases201 and the use of microscale chiral HPLC for process analysis.204 Likewise, four other papers have appeared that describe (1) the preparation of a permethyl-β-cyclodextrin bonded phase202 and (2) the online application of circular dichroic and NMR measurements to eliminate the need for authentic standards,203 the properties of eicosa-O-methylβ-cyclodextrin isomers as capillary gas chromatographic chiral stationary phases,205 and the use of thin-layer chromatography for separating optically active pharmaceuticals.206 The first of the reviews presents information about both silica-based and organic monolithic materials and synthetic methods for preparing many different chiral forms of them. Although both types of monoliths are discussed, the paper focuses more on procedures for preparing silica-based chiral phases. The second review is concerned with the miniaturization of the HPLC hardware in order to place

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up to eight parallel systems into a single instrument platform (i.e., design aspects of hardware fabrication) and, subsequently, to use the equipment to carry out simultaneous high throughout chiral assays online. Likewise, during the methods development stage, eight different chromatographic conditions consisting of different column types and/or eluent compositions could be evaluated simultaneously using the same parallel instrument. Column construction, including the use of monolithic layers also has been an important topic, especially as it relates to capillary electrochromatography (CEC). A comprehensive review has been published that discusses “the-state-of-the-art” of monolithic capillary columns for carrying out CEC chiral separations.212 Different types of monolith and the synthetic routes to preparing them are discussed, including the preparation of both organic polymer-based materials and inorganic silica-based materials. The review covers about 150 references on the topic and contains many useful figures, diagrams, and tables. The tables summarize different types of separation strategies/conditions that can be employed to resolve many different types of optically active pharmaceutical compounds. In addition to this review, several other papers have been published that cover particular aspects of chiral separations by CEC. Two papers have appeared that discuss the preparation of S-ketoprofen molecular imprinted open-tubular capillary columns with plate counts ranging from 105 to 106 depending on their length.207,208 In another instance, the use of polysaccharide phases containing chlorinated chiral selectors (i.e., cellulose tris (3-chloro-4-methylphenylcarbamate), amylose tris(5-chloro-2methylphenylcarbamate), cellulose tris(4-chloro-3-methylphenylcarbamate), and cellulose tris(3,5-dichlorophenylcarbamate)) has been evaluated vs more conventional phases that contain nonchlorinated selectors.209 The study was carried out using a total of 48 structurally diverse drugs with the observation that in some cases enantiomeric resolution was possible using the chlorinated selectors when it was not possible with the nonchlorinated selectors. Likewise, the preparation and use of cellulose tris(3,5-dichlorophenylcarbamate) monolithic silica capillary columns using a sol
gel process has been described in a fourth paper.210 Two different immobilization routes were employed, and the subsequent enantiomeric separation properties of both were characterized using 11 chiral compounds. Next to HPLC, capillary electrophoresis (CE) has been an equally important analytical tool for evaluating chiral purity. As noted above, representative CE procedures for assaying various pharmaceutical compounds are given in Table 2. Also included are a few MEKC methods. Besides these compound specific papers, a number of reviews and papers that deal with the general aspects of the technique have been published. They range from recent advances in the field228 to the use of particular classes of chiral selectors like antibiotics,229 cyclodextrins,230 and charged compounds231 or the broad scale application of a particular selector like 6-O-(2-hydroxyl-3-betainylpropyl)-β-cyclodextrin232 and erythromycin lactobionate.233 Two additional articles have been published that cover methods of improving detection when measuring chiral purity by CE. One of them deals with laser-induced phosphorescence,238 and the other is a general review of recent approaches for enhancing sensitivity.234 Among the topics discussed are off-line and online sample treatment methods, preconcentration approaches like stacking and focusing, and alternate methods of detection such as fluorescence spectroscopy and mass spectrometry. Likewise, a general review of the important aspects related to the online coupling of micellar electrokinetic separations with mass spectrometry has been pub4499

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Table 3. Examples of Vibrational and Other Solid-State Assays for Bulk Pharmaceuticals and Formulated Products compound

technique

sample

study

ref

albendazole

XRPD, DSC

bulk drug

polymorph

aripiprazole

FT-IR, Raman, XRPD, DSC

bulk drug

polymorph/stability 249

248

4-(4-chloro-3-fluorophenyl-2-[4-(methoxyloxy)phenyl]-1,3-thiazol-5-yl acetic acid FT-IR, XRPD, ss-NMR

solid formulation

polymorph

250

clopidogrel bisulfate

FT-IR, Raman

bulk drug

polymorph

251

flufenamic acid

XRPD

solid formulation

polymorph/stability 252

ketoprofen

Raman

bulk drug

stability

253

levobupivacaine hydrochloride

FT-IR, Raman, XRPD

bulk drug

polymorph

254

mebendazole mefenamic acid

FT-IR THz, XRPD, DSC

bulk drug bulk drug

polymorph/stability 255 polymorph 256

mitemcinal fumarate

NIR, XRPD

tablets

polymorph

nateglinide

FT-IR, XRPD, DSC

bulk drug

polymorph

Oncohist

FT-IR

protein formulation identity

ranitidine base

FT-IR, ss-NMR, XRPD, DSC bulk drug

polymorph

260

sulfamethoxazole, trimethoprim

FT-IR

powder mixtures

content

261

tiotropium fumarate

FT-IR, Raman, XRPD

bulk drug

polymorph

262

tolbutamide tribenzoatobismuth(III)

XRPD, DSC XRPD

bulk drug bulk drug

polymorph polymorph

263 264

lished,247 as have papers that describe the in situ preparation and use of di-n-butyl L-tartarate-boric acid complexes for carrying out microemulsion separations245 and how the presence of impurities in microemulsions can affect chiral resolution.246 Besides the use of conventional CE methodology, microchip electrophoresis (MCE) is an emerging technique that also has been used to resolve the enantiomers of chiral pharmaceuticals. In one instance, a microchip with a total separation path length of only 2.5 cm in combination with methyl-β-cyclodextrin as the chiral selector, was used to resolve the enantiomers of naproxen.235 The time savings was almost a factor of 10 compared to the same separation being performed with a standard CE instrument equipped with a 50 μm i.d.  48 cm capillary. Two additional examples of MCE papers include one that covers the use of poly(ethylene glycol)-functionalized polymeric chips236 and one that discusses microfluidic picoliter-scale sample introduction.237 The reported sub-100 pL injection volumes are possible using a translational stage that moves the capillary inlet tip through the drop as sample is being applied. The approach is reproducible at the 1.2% to 3.7% RSD level, on the basis of a sampling set of 51 consecutive translational injections.

’ SPECTROMETRIC-BASED AND OTHER METHODOLOGY As noted in the General section of the current Review, the uses of vibrational spectroscopy continue to expand, especially as they relate to monitoring the various stages of manufacturing and to ensure the quality of the product as it is being produced and in its final form. Of the many off-line and in-line process assays that have been developed, a majority of them are based on either NIR or Raman measurements. Unlike many other approaches, both of these techniques are noninvasive, can be carried out in seconds, and often require little to no sample workup. As important PAT tools, they are being used to (1) study both physical and chemical changes in the APIs that can result from different process stressors; (2) characterize the heterogeneity and spatial distribution of the drugs in the final formulation; (3) measure moisture, content, and purity; and (4) evaluate other miscellaneous solid-state

257 258 259

properties. Similarly, during the research and development stages of new pharmaceutical products, knowledge of their solid-state properties is important in arriving at the final formulation, which in most cases are oral solids dispensed as either tablets or capsules. Many vibrational and related solid-state assays have been published over the past two years. Most of them deal with PAT issues; however, a few representative nonprocess related examples are summarized in Table 3.248
264 For each included, there are many similar types of assays for other APIs. FT-IR and XRPD were the two most often used techniques to characterize polymorphism. Typically, these types of studies fit into three broad categories. The focus of some of the studies was to characterize how a particular polymorphic form influences the physical properties of the analyte, like its solubility or dissolution rate. Other studies were concerned with developing procedures for measuring polymorphic purity, and the third type of studies was carried out to investigate how a given polymorphic form affects the thermal and/or chemical stability of the pharmaceutical. Of these three different categories, stability received the greatest attention. In many cases, depending on conformational differences between polymorphic forms of a given compound, the kinetics and thermodynamics of degradation can be altered dramatically. The acid
base reactivity of the different polymorphic forms of flufenamic acid was measured in the presence of magnesium oxide,252 and polymorph I was found to degrade more quickly than polymorph III. These findings are consistent with other previously published degradation/dehydration data for similar fenamate compounds and arise from an internal rotational difference between the two ring system and the proximity of the COOH and NH functional groups within the molecule. The polymorphic purity of mefenamic acid, another fenamate, was measured using tetrahertz spectroscopy.256 However, it is unclear whether the tetrahertz approach provides any advantages over existing FT-IR and DSC methods, since the scatter in data when comparing the known polymorphic composition to the THz predicted were very large. Thus, the method may be useful as a rough scouting approach; it appears to be semiquantative. Several other studies have been concerned with the thermal stability of the different polymorphs of albendazole,248 aripiprazole,249 4500

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Table 4. Examples of Other Spectrometric Assays for Bulk Pharmaceuticals and Formulated Products compound

technique

sample

study

ref

acetaminophen

NIR

syrup formulations

content

265

acetaminophen, aspirin

NMR

tablets

content

266

acetaminophen, aspirin, caffeine

fluorescence

solid formulations

content

267

acidic, basic, neutral drugs

NMR

octanol
water

fundamental

268

5-aminosalicylic acid, citric acid

NMR, LC-SPE-

binary mixtures

fundamental

269

biapenem

NMR, LC-MS

aqueous solutions

purity

270

chlorpheniramine maleate enantiomers

UV

bulk drug

purity

271

chlorpheniramine maleate, phenylephrine hydrochloride chondroitin sulfate

UV NMR, CE, SEC

solid formulation bulk drug

content purity

272 273

dextromethorphan

Raman

liquid mixtures

content

274

NMR

bulk drug

identity

275

didanosine

NMR

bulk drug

polymorph

276

heparins (low molecular weight)

NMR

heparin formulations

content/identity

277 278

NMR

bulk drug

purity/identity

hydrochlorothiazide, medoxomil, olmesartan

UV

tablets

content

279

hydrochlorothiazide, spironolactone hydrochlorothiazide, triamterene

UV UV

solid formulations tablets

content/purity content

280 281

icofungipen

LC-NMR, LC-MS

bulk drug

purity

282

isopropamide iodide, trifluoperazine hydrochloride

UV

binary mixtures

content/stability

283

minoxidil, tretionoin

UV

liquid formulations

content

284

sulfadiazine, sulfamerazine, sulfamethazine

NMR

β-CD complexes

fundamental

285

triamterene, xipamide

UV, TLC

bulk drug, solid formulations

content

286

vestipitant

NMR, LC-NMR

bulk drug

purity

287

levobupivacaine hydrochloride,254 and mebendazole.255 In the first case, recrystallization was investigated as a means of preparing different polymorphic forms of albendazole. When either methanol or N,N-dimethylformamide was used as the recrystallization solvent, polymorph II, a highly stable form of the analyte, was produced. In the next two papers, five different polymorphic forms of both aripirazole and levobupivacaine were prepared as part of the investigation. For aripiprazole, three of the polymorphic forms were found to have high kinetic stability and, thus, are good candidates for the development of solid formulations. However, for levobupivacine hydrochloride, form IV was the most stable polymorph at higher temperatures. In the final paper cited, the stability the commercialized form of mebendazole, polymorph III, was evaluated in tablet products. The influences of both moisture and heat on stability (i.e., the kinetics of the solidstate transformation of polymorph III to I) were studied. Subsequently, the rate constants obtained were used to predict the shelf lives of mebendazole products under various storage conditions. Two of the four studies were carried out using midrange infrared measurements in combination with XRPS and/or DSC, one with only FT-IR, and the fourth with XRPS and DSC. Another study was reported that examines the polymorphic stability of mitemcial fumarate, which is moisture sensitive, under different tableting conditions.257 The goal of the work was to demonstrate that NIR measurements could be used to monitor polymorphic changes in the API as moisture conditions change during the manufacturing process. It is important to point out that the successful application of both NIR and Raman for in-line monitoring is often dependent on the “on-the-fly” calibration, numerical processing, and prediction models used. Other examples of NIR and Raman assays and their general application to PAT are discussed in the General section.

In addition to the examples given in Table 3, a NIR method was developed for determining acetaminophen in low-dose pharmaceutical syrups containing 2% w/v of it.265 Several different prediction models were evaluated, and four PLS factors were selected for making the final prediction. Once developed, the NIR approach was used to monitor the real time concentration of acetaminophen, as syrups were prepared under manufacturing conditions. Good agreement was found between the measured and expected values (i.e., blended amounts). In another solution study, the photochemistry of ketoprofen in different solvents was evaluated using timeresolved Raman spectroscopy and density function theory.253 In acetonitrile, 2-propanol, and aqueous-acetonitrile mixtures containing ketoprofen, a ketyl radical species is initially formed that subsequently undergoes a cross-coupling reaction. However, in basic solutions of acetonitrile and water, ketoprofen undergoes a decarboxylation reaction. Besides the expanding number of vibrational methods, many UV and NMR methods also have appeared in the literature for carrying out various pharmaceutical measurements. Examples of some of these are summarized in Table 4.266
287 In one instance, a surface plasmon resonance sensor (PRS) was developed to quantify dextromethorphan in enzyme containing solutions.274 The PRS is based on an optical fiber bundle with its sensing surface coated with a thin layer of molecularly imprinted β-cyclodextrin polymer. The sensing layer is synthesized using complexes of the analyte and β-cyclodextrin with toluene-2, 4-diisocyanate as the cross-linking agent and dimethyl formamide as the reaction solvent. The PRS optical bundle also contained an uncoated fiber used to carry the source radiation that was produced using a Xenon lamp modulated at 40 Hz and a lock-in amplifier for signal acquisition from the sensing bundle. The spectral range studied was from 380 to 820 nm. 4501

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Table 5. Examples of Electrochemical and Other Miscellaneous Assays for Bulk Pharmaceuticals and Formulated Products compound acetaminophen

technique

sample

study

ref

DSC

bulk drug

polymorph

288

FIA-electrode

tablets

content

289

acetaminophen, caffeine

voltammetric

tablets

content

290

azidothymidine

polymer electrode

bulk drug

fundamental

291

benserazide, carbidopa, levodopa

voltammetric

formulations

content

292

carbamazepine

STM

bulk drug

fundamental

293

chlorphenoxamine hydrochloride

voltammetric

bulk drug, tablets

content

294

desipramine hydrochloride dextromethorphan hydrobromide

PVC electrode membrane electrode

tablets formulations

content content

295 296 297

FIA-electrode

formulations

content

diclofenac

voltammetric

bulk drug

content

298

diflunisal

voltammetric

tablets

content

299

fosamprenavir

voltammetric

solid formulations

content

300

imipramine

voltammetric

tablets

content

301

ketoprofen, piroxicam

PVC electrode

solid formulations

content

302

naltrexone naratriptan

voltammetric voltammetric

solid formulations tablets

content content

303 304

paeonol

voltammetric

solid formulations

content

305

rutin

voltammetric

tablets

content

306

spiramycin

voltammetric

bulk drug, solid formulations

content

307

sulfamethoxazole

voltammetric

solid formulations

content

308, 309

terazosin

membrane electrode

bulk drug

fundamental

310

tramadol hydrochloride

voltammetric

solid formulations

content

311

trimethoprim triprolidine hydrochloride

voltammetric ion-exchange electrode

tablets tablets, syrups

content content

312 313

Several NMR papers have been published that cover a range of topics including detecting drug counterfeiting using 13C isotopic measurements,266 a general procedure for evaluating octanol
water partition coefficients,268 degradation269 and purity270,282,287 studies, identity testing of biopharmaceuticals,273,277,278 characterization of crystallinity and polymorphism,275,276 and the complexation of sulfonamides with β-cyclodextrins.285 In the first example, complete isotopic 13C profiles were measured for 20 different aspirin and 16 different acetaminophen samples obtained from manufacturers throughout the World. Unlike approaches that measure only the global 13C value, the 13C isotopic abundance of each of the 9 carbons in aspirin and the 6 carbons in acetaminophen were determined, and subsequently, the data was analyzed by a principal component approach. Each of the 36 samples had unique profiles that allowed them to be traced back to their known “point-of-origin”. Although in carrying out the work, only aspirin and acetaminophen were used as test compounds, the authors’ emphasize that the complete 13C profiling approach (i.e., measurement of the isotopic abundance of each carbon) has the potential of making a significant contribution to other techniques available for detecting counterfeiting and patent infringement in the pharmaceutical industry. Other examples of spectrometric methods are included in Table 4. Likewise, representative example assays based on other techniques are summarized in Table 5.288
313 Although not as popular as they once were, electrochemical methods continue to be developed for many different classes of pharmaceuticals. Most of them are procedures for measuring the content of formulated products. Although some are related to the development of new sensors for the direct potentiometric determination of the API, most involve a voltammetric measurement. A few examples

where new/novel sensors were developed include the use of (1) cobalt oxide nanoparticles to measure acetaminophen; (2) a conducting polymer functionalized with a metal-cyclam complex for assaying azidothymidine; (3) single-wall carbon nanotubes for sensing diclofenac; and (4) a glassy carbon electrode modified with gold nanoparticles/ethylenediamine/carbon nanotubes for determining rutin in the presence of ascorbic acid. During the past two years, a number of other electroanalytical approaches were used to evaluate the properties of many bulk drug substances as well as to measure them in formulated products. Likewise, other techniques, such as colorimetric, atomic adsorption, radiochemical, flow injection methods, etc., also have been and continue to be used daily. Unfortunately, it is not possible to include these in the current Review; however, this is not meant to diminish their importance. Additionally, the compounds cited represent only a sampling of the pharmaceuticals being marketed or under development. The intent of the Review is to present an overview of a variety of topics and techniques that capture current trends in the field, not to exhaustively discuss any one of them. A significant amount of the total body of research and development work is occurring throughout the World and access to it is being increasingly easier to obtain. Clearly, the work being carried out related to process analysis is growing at a rapid pace, and the desire and ability to take the analysis to the “point-of-sampling” has stimulated much research.

’ BIOGRAPHIES Roger K. Gilpin is the Mead Distinguished Professor at Wright State University as well as President and a cofounder of Select-OSep, LLC. Prior to this, he was Dean of the College of Science and 4502

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Analytical Chemistry Mathematics at Wright State University. He also is Director of the Consortium for Environmental and Process Technologies. Dr. Gilpin received his B.S. degree in chemistry from Indiana State University in 1969 and his Ph.D. degree in analytical chemistry from the University of Arizona in 1973. From 1973 to 1978, he was employed as Senior Scientist and then as Group Leader of Analytical Chemistry in the Research Division of McNeil Laboratories. In 1978, Dr. Gilpin joined the faculty of Kent State University and was Professor and Chairman of the Department of Chemistry from 1985 to 1996. His research interests are in fundamental and applied gas and liquid chromatography including UHPLC, chromatographic and spectrometric studies of chemically modified surfaces, characterization of the interfacial properties of materials, fundamental and applied aspects of hyphenated separation-based methods mass spectrometry, and environmental, pharmaceutical, and biomedical analysis. He has published over 200 papers and presented nearly 500 talks at national and international scientific conferences. He is Associate Editor of the Journal of Chromatographic Science and past Associate Editor for the Encyclopedia of Analytical Chemistry. He has served as a member of the Special Emphasis Panel for NIH related to technology transfer for over 25 years. Christina S. Gilpin is Director of Educational Services at Select-O-Sep, LLC as well as CFO and a cofounder of the company in 2004. Prior to this, she was a Senior Research Scientist with Brehm Environmental Research Laboratories. She received her B.S. degree in psychology/sociology and her first Masters in library science/information science from Kent State specializing in the areas of science and technology. While at Kent State, she worked in the Analytical Instrumentation Facility as an Analytical Scientist. Subsequently, she worked as a Research Librarian specializing in science information retrieval and analysis at the Air Force Institute of Technology and then Ohio University. She also holds a second Masters in science teaching from Wright State University and currently is a facilitator/site manager consultant for the online web-based general chemistry courses. Her research interests are in chemical information systems, information mining and processing, and the development of chemical and related science simulation software for enhancing educational delivery and the learning experience. She has published in both the areas of chemical analysis and in science teaching. Likewise, she has given numerous papers and presentations at regional, national, and international scientific conferences based on both her laboratory work and, more recently, research related to the development and management of online courses and student performance/acceptance of new learning tools, as well as coauthored several book chapters. Most recently, she has been the PI on a Fast-Track SBIR contract from the U.S. Department of Education to develop an interactive Electronic Chemistry Laboratory Workbook.

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