Biomedical Chromatography

α‐Glucosidase plays important roles in the digestion and absorption of carbohydrates in the small intestine. The inhibition of α‐glucosidase is regarded as a potential way to treat diabetes. We established an approach to screening α‐glucosidase inhibitors from medicinal plants using enzyme‐coated magnetic bead. Using 1‐(3‐dimethyl‐aminopropyl)‐3‐ethylcarbodiimide and N‐hydroxysuccinimide as reaction reagents, α‐glucosidase was immobilized on the magnetic beads by covalent linkage. The conjugation of α‐glucosidase to the magnetic beads was characterized using scanning electron microscope and X‐ray diffractometer. The proposed approach was applied in fishing potential α‐glucosidase inhibitors from extract of Morus alba, a Chinese medicinal plant. The structures of potential active compounds were identified via liquid chromatography–mass spectrometry and nuclear magnetic resonance. The results demonstrated that two flavonoids (isoquercitrin and astragalin) could bind to α‐glucosidase, which was confirmed via conventional α‐glucosidase inhibitory assay. Our findings suggested that enzyme‐coated magnetic beads may be suitable for discovering active compounds from medicinal plants. Copyright © 2012 John Wiley & Sons, Ltd.

This treatise summarizes the underlying principle and the road map for systematic LC‐MS/MS bioanalytical method development. The three themes that have recently emerged as central to building quality during method development—phospholipids, incurred sample and sound chromatographic considerations—are the main focus of this article. In order to reduce the bioanalytical risk associated with plasma phospholipids, a dual approach involving extraction and chromatography is recommended. The use of incurred sample during method development is essential to avoid interference arising from drug‐related components. This is different from the current practice of incurred sample reanalysis, which tests reproducibility during method application. LC column/mobile phase optimization is needed to achieve appropriate peak shape, sensitivity and the separation of the analyte from interfering metabolites and phospholipids. Related to sound chromatographic considerations, we lay out facts and myths related to UPLC, vis‐à‐vis HPLC. Incorporation of quality during method development avoids the costly experience of finding out by chance about the invalidity of a method after it has been used in support of a number of pivotal clinical and non‐clinical studies. To this end, we put forth an outline of a protocol for a systematic LC‐MS/MS bioanalytical method development. Copyright © 2009 John Wiley & Sons, Ltd.

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI‐MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI‐MS/MS is presented, focusing on the applications to low molecular weight compounds. Copyright © 2010 John Wiley & Sons, Ltd.

Acetonitrile, an organic solvent miscible with aqueous phase, has seen thousands of publications in the literature as an efficient deproteinization reagent. The use of acetonitrile for liquid–liquid extraction (LLE), however, has seen very limited application due to its miscibility with aqueous phase. The interest in LLE with acetonitrile has been pursued and reported in the literature by significantly lowering the temperature of the mixture or increasing the salt concentration in the mixture of acetonitrile and aqueous phase, resulting in the separation of the acetonitrile phase from aqueous phase, as observed in conventional LLE. However, very limited application of these methods has been reported. The throughput was limited. In this report, we report a new sample preparation technique, salting‐out assisted liquid–liquid extraction with acetonitrile, for high‐thoughput good laboratory practice sample analysis using LCMS, Two compounds from an approved drug, Kaletra^®, were used to demonstrate the extractability of drugs from human plasma matrix. Magnesium sulfate was used as the salting‐out reagent. Extracts were diluted and then injected into a reversed phase LC‐MS/MS system directly. One 96‐well plate was extracted with this new approach to evaluate multiple parameters of a good laboratory practice analytical method. Results indicate that the method is rapid, reliable and suitable for regulated bioanalysis. With minimal modification, this approach has been used for high‐throughput good laboratory practice analysis of a number of compounds under development at Abbott. Copyright © 2008 John Wiley & Sons, Ltd.

The chiral resolution of some clinically used drugs namely metoprolol, teratolol, tolamolol, nebivolol (β‐adrenergic blockers), econazole, miconazole (anti‐fungal agents), cromakalim (anti‐hypertensive agent) and etodolac (anti‐inflammatory agent) was achieved on cellulose tris (3,5‐dichlorophenylcarbamate) chiral stationary phase. The mobile phase used was 2‐propanol at 0.5 mL/min with detection at 220 nm. The separation factors (α) of these drugs ranged from 1.24 to 3.90 while the resolution factors were from 1.05 to 5.0. The chiral recognition mechanisms between the racemates and the chiral selector are discussed. Copyright ­© 2003 John Wiley & Sons, Ltd.

Fabric phase sorptive extraction (FPSE), a recently introduced novel sample preparation technology, has been evaluated for the extraction of benzodiazepines from human blood serum. FPSE utilizes a flexible fabric surface as the substrate platform for creating sol‐gel hybrid organic‐inorganic sorbent coatings. FPSE media can be introduced directly into the sample containing the target analyte(s), requiring no need for prior sample pretreatment or clean‐up. Benzodiazepines were selected as model analytes because they represent one of the most widely used therapeutic drugs in psychiatry and are also amongst the most frequently encountered drugs in forensic toxicology. The chromatographic separation of target analytes was performed on a LiChroCART‐LiChrospher®100 RP‐18e (5 µm, 250 × 4 mm) analytical column, operated at room temperature. Ternary gradient elution was applied with a mobile phase that consisted of acetonitrile, methanol and ammonium acetate (0.05 M), which was delivered at a flow rate of 1.0 mL/min. Diode array detection was performed with monitoring at 240 nm. FPSE was performed using cellulose fabric extraction media coated with sol‐gel poly(ethylene glycol) (sol‐gel PEG). Absolute recovery values in the equilibrium state for the examined benzodiazepines were found to be 27% for bromazepam, 63% for lorazepam, 42 % for diazepam and 39% for alprazolam. Copyright © 2015 John Wiley & Sons, Ltd.

The enantio‐separations of eight 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs (2‐APA NSAIDs) were established using reversed‐phase high‐performance liquid chromatography with hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral mobile phase additive for studying the stereoselective skin permeation of suprofen, ketoprofen, naproxen, indoprofen, fenoprofen, furbiprofen, ibuprofen and carprofen. The effects of the mobile phase composition, concentration of HP‐β‐CD and column temperature on retention and enantioselective separation were investigated. With 2‐APA NSAIDs as acidic analytes, the retention times and resolutions of the enantiomers were strongly related to the pH of the mobile phase. In addition, both the concentration of HP‐β‐CD and temperature had a great effect on retention time, but only a slight or almost no effect on resolutions of the analytes. Enantioseparations were achieved on a Shimpack CLC‐ODS (150 × 4.6 mm i.d., 5 μm) column. The mobile phase was a mixture of methanol and phosphate buffer (pH 4.0–5.5, 20 mM) containing 25 mM HP‐β‐CD. This method was flexible, simple and economically advantageous over the use of chiral stationary phase, and was successfully applied to the enantioselective determination of the racemic 2‐APA NSAIDs in an enantioselective skin permeation study. Copyright © 2009 John Wiley & Sons, Ltd.

This report together with the paper by T. Mizuochi, M. W. Spellman, M. Larkin, J. Solomon, L. J. Basa and T. Feizi (1988) Biochem. J. 254, 599–603 describes the structural elucidation of the N‐linked oligosaccharides of the HIV envelope glycoprotein, gp120 (cloned from the HTLV‐III B isolate and expressed as a secreted fusion protein after transfection of Chinese hamster ovary cells), which is known to bind with high affinity to human T4 lymphocytes. Oligosaccharides were released from peptide by hydrazinolysis, fractionated by paper electrophoresis, high performance lectin affinity chromatography and Bio‐Gel P‐4 column chromatography, and their structures determined by sequential exoglycosidase digestions in conjunction with methylation analysis. The glycoprotein was found to be unique in its diversity of oligosaccharide structures. These include high‐mannose type and hybrid type, as well as four categories of complex type chains: mono‐, bi‐, tri‐ and tetra‐antennary, with or without N‐acetyllactosamine repeats, and with or without a core region fucose residue. Among the sialidase‐treated oligosaccharides no less than 29 structures were identified as follows: magnified image where G=galactose; GN=N‐acetylglucosamine; M=mannose; F=fucose; ±=residues present in a proportion of chains. The actual number of oligosaccharide structures is much greater since before desialylation there was evidence that among the hybrid and complex type chains all but 6% contained sialic acid at the C‐3 position of terminal galactose residues, and partially sialylated forms of the bi‐ and multiantennary chains were present.

Phương pháp sắc ký lỏng-khối phổ kép (LC-MS/MS) đơn giản, nhạy cảm, chọn lọc và nhanh chóng đã được phát triển và xác thực để định lượng atorvastatin và các chất chuyển hóa hoạt động ortho-hydroxyatorvastatin và para-hydroxyatorvastatin trong huyết tương người sử dụng rosuvastatin làm chuẩn nội (IS). Sau quá trình chiết xuất lỏng-lỏng đơn giản, các chất phân tích được tách rời sử dụng pha động đồng đẳng trên cột pha đảo C₁₈ và được phân tích bởi hệ MS trong chế độ giám sát nhiều phản ứng sử dụng các ion [M+H]⁺ tương ứng, m/z 559/440 đối với atorvastatin, m/z 575/466 đối với ortho-hydroxyatorvastatin, m/z 575/440 đối với para-hydroxyatorvastatin và m/z 482/258 cho IS. Phương pháp thử nghiệm cho thấy một dải động tuyến tính từ 0.1–20 ng/mL đối với atorvastatin và hai chất chuyển hóa của nó trong huyết tương người. Giới hạn định lượng thấp nhất là 100 pg/mL với độ lệch chuẩn tương đối dưới 8%. Độ chính xác và độ chuẩn chấp nhận được thu được cho các nồng độ trên khoảng đường cong tiêu chuẩn. Tỷ lệ thu hồi tuyệt đối trung bình của atorvastatin, ortho-hydroxyatorvastatin, para-hydroxyatorvastatin và IS từ các mẫu huyết tương thêm chất nhờn lần lượt là 54.2 ± 3.2, 50.1 ± 3.8, 65.2 ± 3.6 và 71.7 ± 2.7%. Thời gian chạy 2.5 phút cho mỗi mẫu cho phép phân tích hơn 300 mẫu huyết tương người mỗi ngày. Phương pháp đã được xác thực này đã được sử dụng thành công để phân tích các mẫu huyết tương người nhằm áp dụng trong các nghiên cứu dược động học, khả dụng sinh học hoặc tương đương sinh học. Bản quyền © 2006 John Wiley & Sons, Ltd.