Biomedical Chromatography

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.

A simple and extractionless HPLC method using fluorescence detection was developed for the determination of rosiglitazone in human plasma. After deproteinization using perchloric acid the plasma samples were directly injected onto the HPLC system. The mobile phase was composed of acetonitrile (52%) and 20 mm ammonium acetate (48%, pH 7.5), and analysis was run at a flow rate of 0.2 mL/min with the detector operating at 247 nm for excitation wavelength and at 367 nm for emission wavelength, respectively. The method has a mean recovery of 97%, while the intra‐day and inter‐day precisions were all less than 7%. This method is simple, specific, sensitive and requires only a small plasma volume with short analytical time, and is suitable for the determination of plasma rosiglitazone in routine measurements for pharmacokinetic studies. Copyright © 2004 John Wiley & Sons, Ltd.

To the best of our knowledge, bioanalytical methods to determine rosiglitazone in human plasma reported in literature use internal standards that are not commercially available. Our purpose was to develop a simple method for the determination of rosiglitazone in plasma employing a commercially available internal standard (IS). After the addition of celecoxib (IS), plasma (0.25 mL) samples were extracted into ethyl acetate. The residue after evaporation of the organic layer was dissolved in 750 µL of mobile phase and 50 µL was injected on to HPLC. The separation was achieved using a Hichrom KR 100, 250 × 4.6 mm C₁₈ with a mobile phase composition potassium dihydrogen phosphate buffer (0.01 m, pH 6.5):acetonitrile:methanol (40:50:10, v/v/v). The flow‐rate of the mobile phase was set at 1 mL/min. The column eluate was monitored by fluorescence detector set at an excitation wavelength of 247 nm and emission wavelength of 367 nm. Linear relationships (r² > 0.99) were observed between the peak area ratio rosiglitazone to IS vs rosiglitazone concentrations across the concentration range 5–1000 ng/mL. The intra‐run precision (%RSD) and accuracy (%Dev) in the measurement of rosiglitazone were <±10.69 and <−12.35%, respectively across the QC levels (50–1000 ng/mL). The extraction efficiency was >80% for both rosiglitazone and IS from human plasma. The lower limit of quantitation of the assay was 5 ng/mL. In summary, the methodology for rosiglitazone measurement in plasma was simple, sensitive and employed a commercially available IS. Copyright © 2003 John Wiley & Sons, Ltd.

A liquid chromatography/tandem mass spectrometric method for the quantification of 6‐(3‐benzoyl‐ureido)‐hexanoic acid hydroxyamide (EX‐2), a novel histone deacetylase (HDAC) inhibitor, in mouse plasma was developed to support in‐house pharmacokinetic (PK) studies in the lead optimization stage. In order to determine the PK parameters for EX‐2 in comparison to other HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA), PXD‐101 and LBH‐589, which are currently in different stages of clinical trials, research‐grade bio‐analytical method validations were carried out for EX‐2 and these reference HDAC inhibitors, which were synthesized by in‐house medicinal chemists. The components of validation consisted of specificity, extraction efficiency, signal–response of calibration standards, lower limit of quantification, autosampler stability and accuracy and precision of quality control samples. The validated LC/MS/MS methods were accurate and precise. The calibration curve ranged from 1 to 1600 ng/mL for all the analytes. The methods developed were used to quantify EX‐2 and other HDAC inhibitors in mouse plasma obtained from pharmacokinetic studies. The results suggest that EX‐2 has better PK parameters compared with the reference drugs and is a promising drug development candidate. Copyright © 2007 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.

A method suitable for the determination of unmetabolized polycyclic aromatic hydrocarbons (PAHs) excreted at trace levels (ng/L) in human urine for the monitoring of exposure of the general population to PAH contamination was developed. PAHs were determined, after enrichment by solid‐phase extraction on polyurethane foam (PUF) chips, by HPLC with fluorescence detection. Different parameters affecting analyte extraction to the PUF, including urine salting‐out and organic additives, and optimization of conditions for clean‐up and desorption have been investigated. Optimized conditions were 40 mL acidified urine sample, added with magnesium sulfate, tetrahydrofuran and a 2 cm³ PUF chip, and extracted by shaking at 30 rpm for 1 h at ambient temperature. Desorption was performed, after a clean‐up step with diluted sodium hydroxide, using a small amount of diethyl ether. The recovery of PAH congeners from spiked urines was >90% in the 2–100 ng/L range; the detection limit was 0.1–0.5 ng/L, depending on the considered PAH congener; day‐to‐day precision, at 50 ng/L native PAH content, was CV = 10–20%. The proposed technique provides a simple, economical and effective procedure for the determination of trace amounts of unmetabolized PAHs excreted in human urine spot samples. Copyright © 2006 John Wiley & Sons, Ltd.

Tyrosine kinase inhibitor treatments for chronic myeloid leukaemia based on nilotinib (NIL), dasatinib (DAS) and imatinib (IMA) have improved patient quality of life and have turned chronic myeloid leukemia from a fatal disease into a chronic disease. Dandelion is a rich source of phenolic compounds with strong biological properties, and the effects of using this plant in the treatment of different illnesses can be linked to the presence of various polyphenols found in the different parts of the plant. Thus, dandelion can potentially be used as a nutraceutical (dietary antioxidant) to prevent different disorders associated with oxidative stress, i.e. cardiovascular disorders, cancer and inflammatory processes. Mutual interference between a drug and a food constituent may result in altered pharmacokinetics of the drug and undesired or even dangerous clinical situations. In the present study, a bioanalytical ultra performance liquid chromatography–tandem mass spectrometer (UPLC–MS/MS) method was developed and validated for the quantification of DAS, IMA and NIL in rat plasma. Sample preparation was carried out using solid‐phase extraction with C₁₈ cartridges with a good extraction recovery of ≥94.37% for the three drugs. The method was fully validated as per the US Food and Drug Administration guidelines.