Biomedical Chromatography

Paik, Man‐Jeong; Moon, Seung‐Min; Kim, Kyoung‐Rae; Choi, Sangdun; Ahn, Young‐Hwan; Lee, Gwang

doi: 10.1002/bmc.939pmid: 18059066

Metabolic profiling analysis of free amino acids (AAs) in plasma (20 µL) was performed by gas chromatography–mass spectrometry in selected ion monitoring mode after ethoxycarbonyl/tert‐butyldimethylsilyl derivatives. Characteristic fragment ions, including (M − 57) + ions, permitted sensitive and selective detection of most of the AAs in the presence of co‐extracted carboxylic acids, including free fatty acids, at much higher levels. The overall method was linear (r ≥ 0.9991), reproducible (relative standard deviation = 2.3–8.8%) and accurate (relative error = −7.3–7.7%) with detection limits of 0.01–1.9 ng/mL. A total of 18 AAs, 15 protein AAs and three nonprotein AAs were quantitatively screened in a normal human plasma sample. This selective and simple method using a minimal sample volume was effective for the quantitation of plasma free AAs. Copyright © 2007 John Wiley & Sons, Ltd.

Santa, Tomofumi; Fukushima, Takeshi; Ichibangase, Tomoko; Imai, Kazuhiro

doi: 10.1002/bmc.945pmid: 18059058

Chemical derivatization is often used to enhance the detectability of the target compounds and to improve the separation efficiency in high‐performance liquid chromatography (HPLC). In this review, we describe the recent progress in the development of derivatization reagents having a benzofurazan structure, namely, the fluorogenic reagents, water‐soluble reagents, reagents for the analysis of peptides and proteins, and reagents for mass spectrometric detection. The application of these reagents to bio‐samples is also briefly described. Copyright © 2007 John Wiley & Sons, Ltd.

Li, Wei; Li, Jianhua; Hu, Qin

doi: 10.1002/bmc.938pmid: 17939152

A simple, sensitive and selective LC‐MS‐MS method has been developed for the quantification of huperzine A in human plasma. Huperzine A and pseudoephedrine hydrochloride (internal standard) were isolated from human plasma by extraction with ethyl acetate, chromatographed on a C18 column with a mobile phase consisting of 0.2% formic acid–methanol (15:85, v/v) and detected using a tandem mass spectrometer with an electrospray ionization interface. The lower limit of quantification was 0.0508 ng/mL, and the assay exhibited a linear range of 0.0508–5.08 ng/mL (r = 0.9998). The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test vs reference product) in 18 healthy male Chinese volunteers. After a single 0.2 mg dose for the test and reference product, the resulting means of major pharmacokinetic parameters such as AUC0−24, AUC0−∞, Cmax, Tmax and t1/2 of huperzine A were 16.35 ± 3.42 vs 16.38 ± 3.61 ng h/mL, 17.53 ± 3.80 vs 17.70 ± 3.97 ng h/mL, 2.47 ± 0.49 vs 2.51 ± 0.51 ng/mL, 1.3 ± 0.4 vs 1.2 ± 0.3 h and 5.92 ± 0.75 vs 6.18 ± 0.66 h, respectively, indicating that these two kinds of tablets were bioequivalent. Copyright © 2007 John Wiley & Sons, Ltd.

Li, Yun‐Xia; Jiang, Xue‐Hua; Liang, Hu‐Yi; Li, Xue

doi: 10.1002/bmc.940pmid: 18059053

A simple high‐performance liquid chromatographic method was developed to study the pharmacokinetics of forsythiaside in rat plasma after intravenous administration. Hesperidin was used as the internal standard. The drugs were separated on a reversed‐phase C18 column and detected at 332 nm. Good linearity was achieved in the range of 0.067–26.667 µg/mL. The intra‐ and inter‐assay variation coefficients for this analysis were no more than 10.94 and 14.56%, respectively. The average recovery for forsythiaside was 87.42% from plasma. The analytical sensitivity and accuracy of this assay were adequate for characterization of the pharmacokinetics of intravenous administration of forsythiaside to rats and the assay has been successfully applied to provide pharmacokinetic data. The mean t1/2Z was 20.36, 19.40 and 23.62 min for 2, 5 and 20 mg/kg for forsythiaside after i.v. administration, respectively. The AUC0−t increased linearly from 40.64 to 624.14 µg min/mL after administration of the three doses. Copyright © 2007 John Wiley & Sons, Ltd.

Li, Xiaona; Huo, Changhong; Wang, Qiao; Zhang, Xiaowei; Sheng, Xiaona; Zhang, Lantong

doi: 10.1002/bmc.941pmid: 18059048

Loganin is an important constituent of the traditional Chinese medicine Fructus Corni, with several bioactivities. Microbial metabolism of loganin by intestinal bacteria was investigated. Two metabolites (log‐1 and log‐2) were isolated from anaerobic culture and their structures were identified by means of their ESI‐MS, 1H‐NMR, 13C‐NMR and 2D‐NMR spectral data. Log‐1 was an aglycone of loganin and log‐2 was proved to be a new compound. In vivo metabolites of loganin were detected in rat urine, bile and feces after oral administration of loganin and the structures were proved to be identical with that of the microbial metabolites log‐1 and log‐2 by HPLC–PDA analysis and comparison with the reference standards. Therefore we can prepare metabolites by anaerobic culture with intestinal bacteria. Copyright © 2007 John Wiley & Sons, Ltd.

Li, Juan; Wang, Zhi‐Wei; Zhang, Lei; Liu, Xiao; Chen, Xiao‐Hui; Bi, Kai‐Shun

doi: 10.1002/bmc.942pmid: 18059045

A simple HPLC method was developed for determination of quercitrin and isoquercitrin in rat plasma. Reversed‐phase HPLC was employed for the quantitative analysis using kaempferol‐3‐O‐β‐d‐glucopyranoside‐7‐O‐α‐l‐rhamnoside as an internal standard. Following extraction from the plasma samples with ethyl acetate–isopropanol (95:5, v/v), these two compounds were successfully separated on a Luna C18 column (250 × 4.6 mm, 5 µm) with isocratic elution of acetonitrile–0.5% aqueous acetic acid (17:83, v/v) as the mobile phase. The flow‐rate was set at 1 mL/min and the eluent was detected at 350 nm for both quercitrin and isoquercitrin. The method was linear over the studied ranges of 50–6000 and 50–5000 ng/mL for quercitrin and isoquercitrin, respectively. The intra‐ and inter‐day precisions of the analysis were better than 13.1 and 13.2%, respectively. The lower limits of quantitation for quercitrin and isoquercitrin in plasma were both of 50 ng/mL. The mean extraction recoveries were 73 and 61% for quercitrin and isoquercitrin, respectively. The validated method was successfully applied to pharmacokinetic studies of the two analytes in rat plasma after the oral administration of Hypericum japonicum thunb. ethanol extract. Copyright © 2007 John Wiley & Sons, Ltd.

Baošić, Rada; Radojević, Ana; Radulović, Milanka; Miletić, Srd̄an; Natić, Maja; Tešić, Živoslav

doi: 10.1002/bmc.943pmid: 18059055

The lipophilicity of a series of Schiff base ligands and their complexes with nickel(II) and copper(II) has been determined by reversed‐phase thin‐layer chromatography using binary dioxane–water mobile phase. Chelate ligands were prepared by condensation of diamine and the corresponding β‐diketone. Copper(II) and nickel(II) complexes with chelate ligands containing ethane‐1,2‐diamine or propane‐1,2‐diamine as the amine part and pentane‐2,4‐dione and/or 1‐phenylbutane‐1,3‐dione, pentane‐2,4‐dione and/or 1,1,1‐trifluoropentane‐2,4‐dione, or 1,1,1‐trifluoropentane‐2,4‐dione and/or 1‐phenylbutane‐1,3‐dione as the β‐diketone part were synthesized. Some of investigated compounds were screened for their in vitro antifungal activity against Sacharomyces cerevisiae and antibacterial activity against Escherichia coli. Chromatographically obtained lipophilicity parameters were correlated both with calculated n‐octanol–water partition coefficient C log P and antimicrobial activities. Satisfactory correlations were obtained. Chromatographic data proved to be reliable parameters for describing the lipophilic properties of the investigated compounds. Additionally, the principal components analysis was performed on the data chromatographically obtained. This statistical method was useful for distinguishing compounds and objective comparison of their lipophilicity parameters. Copyright © 2007 John Wiley & Sons, Ltd.

Matsuura, Katsuhiko; Ohmori, Tomofumi; Nakamura, Mitsuhiro; Itoh, Yoshinori; Hirano, Kazuyuki

doi: 10.1002/bmc.944pmid: 18004739

A liquid chromatographic tandem mass spectrometric (LC‐MS/MS) assay was developed and validated to determine valproic acid in human plasma. The method involved a solid‐phase extraction of valproic acid and betamethasone valerate, an internal standard, from plasma and detection using an LC‐MS/MS system with electrospray ionization source in negative ion mode. Separation was achieved within 3 min on a non‐porous silica column with mobile phase containing ammonium acetate and methanol. Multiple reaction monitoring was utilized for detection monitoring at 142.89–142.89 for valproic acid and 457.21–457.21 for the internal standard. The calibration curve for valproic acid was linear over the range of 0.5–150 µg/mL. The limit of detection was 0.17 µg/mL and the lower limit of quantification was 0.5 µg/mL, when 0.2 mL plasma was used for extraction. The percentage coefficient of validation for accuracy and precision (inter‐ and intra‐day) for this method was less than 9.5% with recovery ranging from 82.3 to 86.9% for valproic acid. Copyright © 2007 John Wiley & Sons, Ltd.

Crow, Brian; Bishop, Michael; Kovalcik, Kasey; Norton, Dean; George, Joe; Bralley, J. Alexander

doi: 10.1002/bmc.946pmid: 18004745

A rapid and high‐throughput method for the determination of urinary levels of the oxidative stress biomarker, 8‐hydroxy‐2′‐deoxyguanosine (8‐OH‐dG), has been developed and validated using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC‐MS/MS). The assay features a cheap and readily available non‐isotopic internal standard, a single‐step filtration sample preparation, and a total analysis time of 6 min including column re‐equilibration. The method was validated based on linearity, accuracy (100–106%), precision (CV < 7%), sample preparation stability (≤5%, 72 h). Intra‐laboratory patient ranges were established comparing children and adults (n = 345). Copyright © 2007 John Wiley & Sons, Ltd.

Espada, R.; Josa, J. M.; Valdespina, S.; Dea, M. A.; Ballesteros, M. P.; Alunda, J. M.; Torrado, J. J.

doi: 10.1002/bmc.947pmid: 18059059

A fast and selective HPLC method for assaying amphotericin B in biological samples was developed and validated. The chromatographic separation was achieved in less than 12 min on a reverse‐phase C18 column using an acetonitrile–acetic acid–water (52:4.3:43.7, v/v/v) mixture as mobile phase. The flow rate was 1 mL/min and the effluent was monitored at 406 nm. A linear response over the concentration range 0.1–10.0 µg/mL was obtained. Intra‐day and inter‐day RSDs were below 5% for all the sample types. This new HPLC method was applied to assay amphotericin B in plasma and several tissue samples such as kidney, liver, spleen and bone marrow. Application of this method to pharmacokinetic studies in mice and dog is provided. Copyright © 2007 John Wiley & Sons, Ltd.