Biomedical Chromatography

Srinivas, NR

doi: 10.1002/bmc.2839pmid: 23172738

ABSTRACT In this study the use of micro‐liquid chromatography coupled to tandem mass spectrometry (μLC‐MS/MS) was investigated in routine bioanalysis application for separation and quantification of pro‐drug AZD6319 (developed for aldezheimer treatment). Microextraction by packed sorbent (MEPS) was used as sample clean‐up method. The focus of this study was put on the evaluation of the usability of smaller column diameters such as 1.0 and 0.3 mm instead of 2.1 mm in bioanalysis application to reduce solvent consumption and sample volumes. Solvent consumption was reduced by 80% when a 1.0 mm column was used compared with 2.1 mm column. Robustness of the micro‐columns in terms of accuracy and precision was investigated. The application of μLC‐MS/MS for the quantitative analysis of AZD6319 in plasma samples showed good selectivity, accuracy and precision. The coefficients of determination (R2) were >0.998 for all runs using plasma samples on the studied micro‐columns. The inter‐day accuracy values for quality control samples ranged from 99 to 103% and from 96 to 105% for 0.3 × 50 mm and 1.0 × 50 mm columns, respectively. The inter‐day precision values ranged from 4.0 to 9.0% and from 4.0 to 8.0% for 0.3 × 50 and 1.0 × 50 mm columns, respectively. In addition the sensitivity was increased by three times using a 1.0 mm column compared with 2.1 mm. Furthermore, robustness of the micro‐columns from different manufacturers was investigated. Copyright © 2012 John Wiley & Sons, Ltd.

Srinivas, NR

doi: 10.1002/bmc.2840pmid: 23165328

ABSTRACT Cholesterol biosynthesis precursors and plant sterols are noncholesterol sterols currently used as relative surrogate markers of cholesterol synthesis and absorption, respectively. Its determination in serum samples is a way of diagnosing inherited disorders and also a tool for health evaluation during lipid‐lowering lifestyle/drug therapy monitoring. This approach is the only one that can be used for large‐scale clinical trials or population based studies, but, nevertheless, there is no reference method for the quantification of noncholesterol sterols in human serum samples and only analysis by GC‐FID and GC‐MS has been reported to be completely validated. Although there has been a wider use of noncholesterol sterols for the measurement and characterization of cholesterol metabolism, there is a lack of harmonization of measurements and of standardization of the methodology, which is essential for routine measurements of diagnostic utility. New recent advances in analytical methods for the determination of serum noncholesterol sterols are highlighted, focusing on the sample preparation, separation and detection techniques, which will enhance the range of applications in clinical practice. Copyright © 2012 John Wiley & Sons, Ltd.

Srinivas, NR

doi: 10.1002/bmc.2898pmid: 23553351

ABSTRACT Azithromycin is one of the best selling antibiotics in the world. It belongs to the new macrolide family of azalides. It is derived from erythromycin and it differs from erythromycin in having a 15‐membered ring and a methyl substituted nitrogen inserted at the 9a position in the aglycone ring. This structural modification confers favourable microbiological and pharmacokinetic characteristics on azithromycin and greater acid stability compared with erythromycin. It is mainly used to treat respiratory infections, sexually transmitted diseases, cutaneous and soft‐tissue infections, etc. This review provides a comprehensive overview of various HPLC, LC‐MS and LC‐MS/MS methods for quantitation of azithromycin in different biological matrices. In addition, it provides general information on extraction steps, internal standard selection, conditions for chromatographic separation, brief validation data and applicable conclusions for reported methods in a defined pattern. Copyright © 2013 John Wiley & Sons, Ltd.

Srinivas, NR

doi: 10.1002/bmc.2907pmid: 23649426

ABSTRACT Clinical investigations of choleteryl ester transfer protein (CETP) inhibitors are still underway owing to its promise of reducing risk factors in patients with cardiovascular disease. Although several CETP inhibitors have reached late phase clinical testing, there is a paucity of publications that describe the determination of various CETP inhibitors in human and/or animal matrices. An attempt is made in this review to collate bioanalytical information on three CETP inhibitors (anacetrapib, dalcetrapib and torcetrapib) and its metabolites, where data were available and reported. As elaborated in the review, owing to numerous structural issues coupled with chromatography/detection challenges indigenous to the class, a wide array of analytical tools, detection systems, interesting process manipulations and separation nuances have been utilized for the quantitative analysis of CETP inhibitors and applicable metabolites. Copyright © 2013 John Wiley & Sons, Ltd.

Srinivas, NR

doi: 10.1002/bmc.2911pmid: 23605782

ABSTRACT Research into biomarkers of autism is a new means of medical intervention in this disease. Chromatographic techniques, especially coupled with mass spectrometry, are widely used in determination of biomarkers and assessment of effectiveness of autism therapy owing to their sensitivity and selectivity. Among the chromatographic techniques gas chromatography and liquid chromatography, especially high‐performance liquid chromatography, have found application in clinical trials. The high‐performance liquid chromatography technique allows an analysis of liquid samples with a wide range of molecules, small and large, providing an opportunity to perform advanced assays within a short time frame. Gas chromatography with the appropriate preparation of samples (gaseous and liquid) and a selection of analysis conditions enables the separation of thermally stable, volatile and non‐volatile organic substances in short runtimes. The chromatographic techniques that are currently used in metabolic studies in autism are designed to identify abnormalities in three areas: the metabolism of neurotransmitters, nutritional and metabolic status and manifestations of oxidative stress. This review presents a necessary theoretical introduction and examples of applications of chromatographic studies of disorder markers in autism. Copyright © 2013 John Wiley & Sons, Ltd.

Srinivas, NR

doi: 10.1002/bmc.2912pmid: 23649485

ABSTRACT Glucuronidation and sulfation represent two major pathways in phase II drug metabolism in humans and other mammalian species. The great majority of drugs, for example, polyphenols, flavonoids and anthraquinones, could be transformed into sulfated and glucuronidated conjugates simultaneously and extensively in vivo. The pharmacological activities of drug conjugations are normally decreased compared with those of their free forms. However, some drug conjugates may either bear biological activities themselves or serve as excellent sources of biologically active compounds. As the bioactivities of drugs are thought to be relevant to the kinetics of their conjugates, it is essential to study the pharmacokinetic behaviors of the conjugates in more detail. Unfortunately, the free forms of drugs cannot be detected directly in most cases if their glucuronides and sulfates are the predominant forms in biological samples. Nevertheless, an initial enzymatic hydrolysis step using β‐glucuronidase and/or sulfatase is usually performed to convert the glucuronidated and/or sulfated conjugates to their free forms prior to the extraction, purification and other subsequent analysis steps in the literature. This review provides fundamental information on drug metabolism pathways, the bio‐analytical strategies for the quantification of various drug conjugates, and the applications of the analytical methods to pharmacokinetic studies. Copyright © 2013 John Wiley & Sons, Ltd.

Srinivas, NR

doi: 10.1002/bmc.2953pmid: 23843248

ABSTRACT Capillary electrophoresis is a fast, inexpensive and low detection limit technique for the analysis of anticancer drugs. It has been used to analyze various anticancer drugs in biological samples, pharmaceutical preparations and environmental matrices. It has also been used to detect various cancer biomarkers in cancer patients. The present article describes the state‐of‐the art of capillary electrophoresis for the analyses of anticancer drugs. Various drugs discussed belong to several groups such as antimitotic agents, nucleoside analogs, antibiotics, topoisomerase inhibitors and DNA intercalating agents. In addition, efforts have also been made to discuss sample preparation, applications of capillary electrophoresis in genomic research, optimization and future perspectives. Copyright © 2013 John Wiley & Sons, Ltd.

Srinivas, NR

doi: 10.1002/bmc.2967pmid: 23813479

ABSTRACT Alkaloids are natural products of metabolism found mainly in plants. Their diversified pharmacological activities as medical agents and extensive occurrence in nutritional products demand the development of reliable and sensitive analytical tools. This review presents the developments in the field of electromigration techniques and specifically capillary and microchip electrophoresis. We include the main aspects of interest to researches over the past 12 years. The scope of this review covers detection, on‐ and off‐line preconcentration techniques, chiral separation and developments in the field of microchip electrophoresis. Their applications in alkaloid determination, capillary electrochromatography and inter‐molecular interactions are also examined. Copyright © 2013 John Wiley & Sons, Ltd.

Srinivas, NR

doi: 10.1002/bmc.2995pmid: 24006302

ABSTRACT Carnitine and its acylesters are a family of compounds that can be used in the early diagnosis of many diseases. Carnitine and acylcarnitines have a crucial role in fatty acid transportation. The increased level of free carnitine, total carnitine, or the acylesters can act as biomarkers for many metabolic disorders, including diabetes, encephalopathy and cardiomyopathy. The determination of these compounds is difficult owing to the simple aliphatic structure, the chiral center and the permanent positive charge. Although MS detection can be enough to differentiate between some carnitine derivatives, closely related structural isomers of the acylcarnitines must be separated before detection because they form the same base peak and second most abundant ion peak. Different separation methods are discussed in this review, including reversed‐phase, hydrophilic interaction, ion exchange, ion pairing, mixed mode liquid chromatography, gas chromatography and electrophoresis. Representative example chromatograms are shown. The sample preparation and the different derivatization reactions are also covered. A table that summarizes the most important analytical methods by detailing the analyte mixture, the sample matrix, the separation mode and the detection method is provided. Copyright © 2013 John Wiley & Sons, Ltd.

Srinivas, NR

doi: 10.1002/bmc.2996pmid: 23939915

ABSTRACT Blood and plasma are the biomatrices traditionally used for drug monitoring and their pharmacokinetic profiling. Blood is the circulating fluid in contact with all organs and tissues of body and thus is the most representative fluid for measuring systemic drug levels. However, venipuncture suffers from the caveat of being an invasive technique which often makes people reluctant to participate in clinical studies. Thus, there is a need for noninvasive bio‐fluids that are ethically appropriate, cost‐efficient and toxicologically relevant. These alternate bio‐fluids may prove clinically useful as alternatives to plasma/serum in therapeutic drug monitoring, pharmacokinetic and toxicokinetic studies, doping control in sports medicine and to monitor local adverse effects. These may be of particular interest in the case of special population groups such as neonates, children, the elderly, terminally ill patients and pregnant or lactating women, and offer the advantage of circumvention of the demand for specialized personnel for sample collection. This review describes such noninvasive bio‐fluids (saliva, sweat, tears and milk) that have been considered for pharmacokinetic drug analysis, emphasizing their sample preparation, its associated difficulties and their correlation with plasma. Copyright © 2013 John Wiley & Sons, Ltd.