Biomedical Chromatography

Elution profiles of kynurenic acid (KYNA) and 7‐chlorokynurenic acid (Cl‐KYNA) were examined by high‐performance liquid chromatography (HPLC) using a triazole‐bonded stationary phase column (Cosmosil® HILIC) under isocratic elution of a mobile phase consisting of CH₃CN–aqueous 10 mm ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl‐KYNA varied with both the CH₃CN content and the pH of the mobile phase. The elution order of KYNA and Cl‐KYNA was reversed between the CH₃CN‐ and H₂O‐rich mobile phases, suggesting that hydrophilic interactions and anion‐exchange interactions caused retention of KYNA and Cl‐KYNA in the CH₃CN‐ and H₂O‐rich mobile phases, respectively. The present HPLC method using a triazole‐bonded column and fluorescence detection (excitation 250 nm, emission 398 nm) was applied to monitor in vitro production of KYNA from d‐kynurenine (d‐KYN) by d‐amino acid oxidase (DAO) using Cl‐KYNA as an internal standard. A single KYNA peak was clearly observed after enzymatic reaction of d‐KYN with DAO. Production of KYNA from d‐KYN was suppressed by the addition of commercial DAO inhibitors. The present HPLC method can be used to evaluate DAO activity and DAO inhibitory effects in candidate drugs for the treatment of schizophrenia. Copyright © 2015 John Wiley & Sons, Ltd.

A method for simultaneous determination of clobazam (CBZ) and its active metabolite N‐desmethylclobazam (DCBZ) in various biological samples by RP‐HPLC with UV detection is described. The determination of both CBZ and DCBZ is performed without derivatization. The internal standard is diazepam. The method is rapid and simple with sensitivity limits of 10 ng/mL for both CBZ and DCBZ and is suitable for routine analysis as well as for animal studies.

Midazolam concentrations in patients' plasma was determined after extraction with high performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). GC was selected for routine plasma assays in terms of selectivity, simplicity, precision, accuracy and sensitivity (0.02 μg/ml); HPLC analysis was less sensitive (0.1 μg/mL) than GC; GC/MS was used for analysis validation. Plasma protein binding of midazolam was determined by GC in patients' plasma after in vitro incubation with midazolam, ultrafiltration and extraction; 5% of the drug was unbound to plasma proteins. Midazolam distribution in lipoprotein fractions separated by ultracentrifugation of plasma obtained from patients on prolonged midazolam treatment was also assayed by GC.

Peroxyoxalate chemiluminescence may be used for sensitive postcolumn detection of phenothiazine analytes separated by high performance liquid chromatography with appropriate optimization of measurement conditions such as solvent, pH and oxalate ester. Detectability of fluorescent analytes by chemical excitation varies greatly, but analytes with low oxidation potentials are generally more readily detected at low levels, as demonstrated for phenothiazines, an important class of fluorescent drugs. Some improvement in detection limits is observed for fluphenazine when chemiluminescence detection is compared to conventional fluorescence detection. Because of the specificity of chemical excitation, fewer interferences from fluorescent impurities in a urine matrix are observed.

The aim of this study was to establish and validate a rapid, selective and reliable ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for simultaneous quantitations of morin and morusin, and to investigate their pharmacokinetics difference between normal and diabetic rats after oral administration. Plasma samples were pretreated via protein precipitation with acetonitrile. Genkwanin was used as internal standard (IS). Analytes and IS were separated on a Thermo Hypersil Gold C₁₈ column (50 × 4.6 mm, 3 μm) using gradient elution. The mobile phase consisted of acetonitrile and 0.1% formic acid in water at a flow rate of 0.5 mL/min. Mass spectrometry detection was carried out by means of negative electrospray ionization source and multipe‐reaction monitoring mode. The transitions of m/z 300.9 → 151.2 for morin, m/z 419.2 → 297.1 for morusin and m/z 283.1 → 268.2 for IS were chosen for quantification. Calibration curves were linear in the range of 1.01–504.2 ng/mL (r² ≥ 0.99) for morin and 1.02–522.3 ng/mL (r² ≥ 0.99) for morusin. The lower limit of quantification was 1.02 ng/mL for morin and 1.05 ng/mL for morusin. The extraction recovery was >85.1% for each analyte. No obvious matrix effect was observed under the present UPLC–MS/MS conditions during all of the bioanalysis. The stability study demonstrated that morin and morusin remained stable during the whole analytical procedure. The method was successfully applied to support the pharmacokinetic comparisons of morin and morusin between normal and diabetic rats.

A novel method for the direct determination of kanamycin B in the presence of kanamycin A in fermentation broth using high performance liquid chromatography with evaporative light scattering detector (HPLC‐ELSD) was developed. An Agilent Technologies C₁₈ column was utilized, evaporation temperature of 40°C and nitrogen pressure of 3.5 bar, the optimized mobile phase was water–acetonitrile (65:35, v/v), containing 11.6 mm heptafluorobutyric acid (isocratic elution with flow rate of 0.5 mL/min) with the gain 11. Kanamycin B was eluted at 5.6 min with an asymmetry factor of 1.827. The method showed good linearity over the concentration range of 0.05 to 0.80 mg/mL for the kanamycin B (r² = 0.9987). The intra‐day and inter‐day coefficients of variation obtained from kanamycin B were less than 4.3%. Mean recovery of kanamycin B from spiked fermentation broth was 95%. The developed method was applied to the determination of kanamycin B without any interference from other constituents in the fermentation broth. This method offers simple, rapid and quantitative detection of kanamycin B. Copyright © 2014 John Wiley & Sons, Ltd.

Một phương pháp HPLC‐PDA đơn giản, isocratic, có độ phân giải cao và nhanh chóng đã được phát triển và xác thực để định lượng đồng thời prilocaine (PCL) và lidocaine (LCL) hydrochlorides trong các nghiên cứu thấm qua niêm mạc miệng được thúc đẩy bởi iontophoresis in vitro. Một cột pha đảo C₁₈ (250 mm x 4.6 mm, 3μm, 110Å) đã được sử dụng để phân tách sắc ký. Pha động gồm có acetonitrile: 0.1M dung dịch đệm phosphate sodium, pH 7.0 (1:1, v/v), cộng với 0.05% (v/v) diethylamine. Tốc độ dòng isocratic được thiết lập ở 1 mL/phút và bước sóng phát hiện là 203 nm. PCL và LCL lần lượt được eluate sau 8.9 phút và 13 phút, và các thông số phù hợp của hệ thống nằm trong khoảng chấp nhận được. Phương pháp này có tính chọn lọc, nhạy cảm, chính xác, hiệu quả và mạnh mẽ, tạo ra một đồ thị tuyến tính trong khoảng nồng độ từ 0.25 đến 10 µg/mL. Ứng dụng của phương pháp này được chứng minh bằng sự tăng cường đáng kể thấm của PCL và LCL với việc thực hiện iontophoresis (1 mA/cm² trong 1 giờ) qua niêm mạc thực quản heo đã được tách biệt. Khối lượng của thuốc được giữ lại trong niêm mạc cũng tăng lên với việc áp dụng dòng điện. Bản quyền © 2015 John Wiley & Sons, Ltd.

Analytical conditions of prepurification extraction and HPLC separation were optimized for determination of urinary serotonin and tryptamine. Under optimal conditions, serotonin, tryptamine and an internal standard were extracted with 15% v/v n‐propanol in diethyl ether from urine samples alkalized with a phosphate buffer (0.75 mol/L, pH 10.0), and then they were re‐extracted into an HCI solution (0.1 mol/L). Purified indoleamines were simultaneously separated by reversed‐phase ion‐pair HPLC with native fluorescence detection. Urinary serotonin and tryptamine were selectively determined within about 45 min per sample for the whole procedure. Analytical recovery, reproducibility and detection sensitivity were satisfactory for pursuing time‐dependent changes in indoleamine levels. Urinary excretion profiles of serotonin and tryptamine in subjects dosed with L‐tryptophan were successfully analyzed by our method.

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.

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.