Biomedical Chromatography

doi: 10.1002/bmc.918pmid: N/A

Asamoto, Hiromichi; Ichibangase, Tomoko; Saimaru, Hiroshi; Uchikura, Kazuo; Imai, Kazuhiro

doi: 10.1002/bmc.814pmid: 17516464

A highly sensitive and simple method using HPLC‐fluorescence detection with 7‐chloro‐N‐(2‐(dimethylamino)ethyl)‐2,1,3‐benzoxadiazole‐4‐sulfonamide (DAABD‐Cl) as a fluorogenic reagent demonstrated the existence of the low‐molecular‐weight thiols in the extract of Caenorhabditis elegans (C. elegans). The method includes derivatization of the thiols with DAABD‐Cl at 40°C for 10 min in borate buffer (pH 9.0) containing TCEP, CHAPS and EDTA, separation of the derivatives on an ODS column and fluorometric determination of the derivatives at 510 ± 15 nm with excitation at 400 ± 15 nm. The identification of the thiols was made by HPLC‐electrospray ionization mass spectrometry (LC‐MS) following isolation of the derivatives using HPLC‐fluorescence detection. Low‐molecular‐weight thiols were found to exist in the extract of C. elegans, such as cysteine, cysteinylglycine, γ‐glutamylcysteine, reduced glutathione and two other unidentified thiol compounds, confirming the existence of the ‘glutathione cycle’ in C. elegans similar to the mammalian body. Copyright © 2007 John Wiley & Sons, Ltd.

Goda, Ryoya; Sudo, Kenichi

doi: 10.1002/bmc.825pmid: 17549678

During a study of 100 µL aliquots of urocortin containing various acetonitrile contents, we hypothesized that a change in the acetonitrile content in the solution across a specific content of acetonitrile (critical threshold) causes an abrupt change in adsorption capacity to the column packing. Circular dichroism measurements suggest that the conformational change induced by acetonitrile in the solution causes the abrupt change in adsorption capacity, and this solvent‐induced conformational change is reversible across the critical threshold. This hypothesis can apply to various polypeptides with molecular weights range from 1007 to 6789 and to other organic solvents. A new gradient system utilizing the instant recovery of the adsorption capacity across the critical threshold was designed, and applied to the analysis of a 100 µL aliquot of various polypeptide solutions. The results suggest that use of a solution containing organic solvents more than the critical threshold allows successful dilution of polypeptides up to picomolar concentration range without any loss due to its adsorption during handling procedures and loading onto the LC system, and that a new gradient system enables quantitative analysis of polypeptides at picomolar concentrations in such solutions. Copyright © 2007 John Wiley & Sons, Ltd.

Tomita, Mamoru; Nakashima, Mihoko N.; Wada, Mitsuhiro; Nakashima, Kenichiro

doi: 10.1002/bmc.839pmid: 17474141

Simultaneous determination of 3,4‐methylenedioxymethamphetamine (MDMA) and 3,4‐methylenedioxyamphetamine (MDA) in rat blood and brain microdialysates by high‐performance liquid chromatography with fluorescence detection (HPLC‐FL) was developed. Microdialysates were directly subjected to derivatization with 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)benzoyl chloride (DIB‐Cl). The DIB‐derivatives of MDMA, MDA and the internal standard, 1‐methyl‐3‐phenylpropylamine (MPPA), were isocratically separated on an ODS column using a mixture of 50 mm phosphate buffer (pH 7.0)–acetonitrile–methanol–2‐propanol (50:45:5:2, v/v/v/v %) as an eluent at a flow rate of 1.5 mL/min. The calibration curves of MDA and MDMA spiked to blood and brain microdialysates were linear over the ranges 2.5–500 and 5.0–1000 ng/mL, respectively. The detection limits of MDA and MDMA were 1.2 and 4.2 for blood and 1.3 and 4.8 ng/mL for brain, respectively. Additionally, the intra‐ and the inter‐assay precisions were lower than 5.6% for the blood and brain microdialysates (n = 4). The proposed method was successfully applied for the monitoring of MDMA and its metabolite MDA in rat blood and brain microdialysates, and the pharmacokinetic parameters of MDMA and MDA in the microdialysates after administration of MDMA (5 mg/kg, i.p.) with or without caffeine (20 mg/kg, i.p.) were evaluated. Copyright © 2007 John Wiley & Sons, Ltd.

Sora, Daniela Iuliana; Udrescu, Stefan; David, Victor; Medvedovici, Andrei

doi: 10.1002/bmc.845pmid: 17497754

Inter‐ and intra‐individual variability of the loratadine (LOR) metabolism in Caucasian subjects was assessed during a bioequivalence study for two pharmaceutical formulations (solid oral dosage forms) containing 10 mg of the active substance. The analytical data were obtained by applying a reliable, low‐cost and sensitive ion pair liquid chromatography/fluorescence (IPLC/FLD) method for determination of both loratadine and descarboethoxyloratadine (DCL) in human plasma samples. The sample preparation procedure is based on liquid–liquid extraction of the target analytes from alkalinized plasma using diethyl‐ether. The separation of the analytes and 8‐chloroazatadine as internal standard (IS) was achieved through an isocratic ion pair (IP) elution on a Purospher® STAR RP‐18 column. The mobile phase containing sodium dodecyl sulfate (SDS) as ion pairing agent was pumped at a flow rate of 1 mL/min. Fluorescence detection (FLD) was achieved at 280 nm (excitation) and 440 nm (emission) wavelengths. The increased sensitivity of the method is also based on a large sample injected volume (250 µL). Linear response was found over the 0.5–20 ng/mL concentration interval for both target compounds. Low limits of quantification (LLOQ) around 0.3 ng/mL were found for LOR and DCL. Method validation is presented. Copyright © 2007 John Wiley & Sons, Ltd.

Hammad, Sherin Farouk; Mabrouk, Mokhtar Mohamed; Habib, Ahmed; Elfatatry, Hamed; Kishikawa, Naoya; Nakashima, Kenichiro; Kuroda, Naotaka

doi: 10.1002/bmc.848pmid: 17516468

A highly selective and sensitive method was developed for simultaneous determination of the antihistaminic drug hydroxyzine (HZ) and its pharmacologically active metabolite cetirizine (CZ) in human serum using haloperidol as internal standard. The method was based on fluorescence labeling of both drugs with a fluorescent arylboronic acid 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenyl boronic acid followed by separation on silica column using a mobile phase consisting of acetonitrile and water (90:10, v/v%) containing triethylamine and acetic acid. The labeling reaction conditions were optimized and the liquid–liquid extraction method was successfully applied to extract the both drugs from serum. The linearity range was 0.025–2.00 µg/mL for HZ and CZ. The limit of detection (S/N = 3) was 10 and 5 ng/mL for HZ and CZ, respectively. Copyright © 2007 John Wiley & Sons, Ltd.

Zhang, Jun; Rodila, Ramona; Watson, Pamela; Ji, Qin; El‐Shourbagy, Tawakol A.

doi: 10.1002/bmc.849pmid: 17590864

Sirolimus, an effective immunosuppressive agent, is used for drug eluting stents. During stent development, an analytical method for the determination of sirolimus in tissue needs to be established. Normally, tissue samples are homogenized and then analyzed against the calibration standards prepared in a tissue homogenate. This approach provides insufficient control of the homogenization process. In this paper, tissue quality control samples were introduced for the optimization of the homogenization process during method development, but also allowance for the performance evaluation of the entire analytical process. In addition, a new approach using rabbit blood as a homogenization medium was developed to stabilize sirolimus in rabbit tissue homogenates. Calibration standards and quality controls were prepared by spiking different sirolimus working solutions into rabbit blood. Homogenization quality control samples were prepared by injecting other sirolimus working solutions into empty test tubes and pre‐cut arteries within pre‐defined masses. A high‐throughput homogenization procedure was optimized based on the specific chemical properties of sirolimus. The linear dynamic range was between 49.9 pg/mL and 31.9 ng/mL to accommodate the expected artery homogenate concentrations. Additionally, quality controls in rabbit blood were also used in the extraction to support the calibration standards. The accuracy and precision of the quality controls in rabbit blood reflect the extraction performance and the accuracy and precision of the homogenization tissue quality controls reflect the overall performance of the method. The mean bias was between −4.5 and 0.2% for all levels of quality controls in the blood and between 4.8 and 14.9% for all levels of the homogenization tissue quality controls. The CVs of all concentration levels were ≤5.3% for the quality controls in blood and ≤9.2% for the homogenization tissue quality controls. The method was successfully applied to determine the concentration of sirolimus in the rabbit arteries. Copyright © 2007 John Wiley & Sons, Ltd.

Zhou, Zhi‐Ling; Yang, Min; Yu, Xi‐Yong; Peng, Huai‐Yan; Shan, Zhi‐Xin; Chen, Shu‐Zhen; Lin, Qiu‐Xiong; Liu, Xiao‐Ying; Chen, Tie‐Feng; Zhou, Shu‐Feng; Lin, Shu‐Guang

doi: 10.1002/bmc.851

Xu, Man; Fu, Gang; Qiao, Xue; Wu, Wan‐Ying; Guo, Hui; Liu, Ai‐Hua; Sun, Jiang‐Hao; Guo, De‐An

doi: 10.1002/bmc.852pmid: 17549679

A sensitive and selective high‐performance liquid chromatography method was developed and validated to determine the prototype of salvianolic acid B and the metabolites of phenolic acids (protocatechuic acid, vanillic acid and ferulic acid) in rat tissues after oral administration of total phenolic acids and salvianolic acid B extracted from the roots of Salvia miltiorrhiza, respectively. The tissue samples were treated with a simple liquid–liquid extraction prior to HPLC. Analysis of the extract was performed on a reverse‐phase C18 column with a mobile phase consisting of acetonitrile and 0.05% trifluoracetic acid. The calibration curves for the four phenolic acids were linear in the given concentration ranges. The intra‐day and inter‐day relative standard deviations in the measurement of quality control samples were less than 10% and the accuracies were in the range of 88–115%. The average recoveries of all the tissues ranged from 78.0 to 111.8%. This method was successfully applied to evaluate the distribution of the four phenolic acids in rat tissues after oral administration of total phenolic acids of Salvia miltiorrhiza or salvianolic acid B and the possible metabolic pathway was illustrated. Copyright © 2007 John Wiley & Sons, Ltd.

Bhushan, R.; Brückner, H.; Kumar, Virender

doi: 10.1002/bmc.854pmid: 17516470

TLC and HPLC methods were developed for indirect chiral separation of penicillamine (3,3‐dimethylcysteine) enantiomers after derivatization with Marfey's reagent (FDNP‐Ala‐NH2) and two of its structural variants, FDNP‐Phe‐NH2 and FDNP‐Val‐NH2. The binary mobile phase of phenol–water (3:1 v/v) and solvent combinations of acetonitrile and triethylamine phosphate buffer were found to give the best separation in normal and reversed‐phase TLC, respectively. The diastereomers were also resolved on a reversed‐phase C18 HPLC column with gradient elution of acetonitrile and 0.01 m trifluoroacetic acid. The results due to these three reagents were compared. The method was successful for checking the enantiomeric impurity of l‐penicillamine in d‐penicillamine and to check the enantiomeric purity of pharmaceutical formulations of d‐penicillamine. The method was validated for linearity, repeatability, limit of detection and limit of quantification. Copyright © 2007 John Wiley & Sons, Ltd.