Biomedical Chromatography

Ye, Guan; Tang, Yi‐Hong; Xia, Guang‐Xin; Sun, Zhao‐Lin; Li, Zhi‐Xiong; Huang, Cheng‐Gang

doi: 10.1002/bmc.1400pmid: 20120039

To profile the anti‐Coxsackie virus B3 constituents of Radix Astragali, an HPLC‐DAD‐MSn analytical method, combined with an in vivo test, has been developed to identify the constituents of the active part, which has been demonstrated to have potency to inhibit the proliferation of virus in cardiac muscle, alleviate infraction in heart and elevate the survival rate of the animal. By comparing their retention time and MS data with those obtained from the authentic compounds and the published data, a total of 19 compounds, including 11 isoflavonoids and eight saponins, were identified, among which one pterocarpane glucoside was reported for the first time. The present study provides an approach to rapidly screening bioactive constituents in traditional Chinese medicines. Copyright © 2010 John Wiley & Sons, Ltd.

Bhushan, Ravi; Agarwal, Charu

doi: 10.1002/bmc.1421pmid: 20954205

Chiral assay of enantiomers of fluoxetine was achieved in pharmaceutical formulations using direct and indirect methods. l‐Tartaric acid was used as a mobile phase additive in thin‐layer chromatography; the enantiomers were separated and isolated and were used to determine the elution order in HPLC. (R,S)‐flouxetine was derivatized with (S)‐N‐(4‐nitrophenoxycarbonyl)phenylalanine methoxyethyl ester ((S)‐NIFE), Marfey's reagent and 1‐fluoro‐2,4‐dinitrophenyl‐l‐methionine amide (FDNP‐l‐Met‐NH2). The diastereomers were separated using RP‐HPLC. The effect of flow rate and TFA concentration on resolution was studied. The diastereomers obtained by derivatization with FDNP‐l‐Met‐NH2 were also separated by RP‐TLC. Copyright © 2010 John Wiley & Sons, Ltd.

Nirogi, Ramakrishna; Kandikere, Vishwottam; Mudigonda, Koteshwara; Ajjala, Devender; Suraneni, Ramakrishna; Thoddi, Parthasarathi

doi: 10.1002/bmc.1422pmid: 20954206

A simple LC‐MS/MS method was developed and validated for the estimation of sarpogrelate in 50 µL of rat plasma. The analyte and internal standard (IS) were extracted from rat plasma by acetonitrile precipitation and they were separated on a reversed‐phase C8 column with gradient program. The MS acquisition was performed with multiple reaction monitoring mode using m/z 430.2 to m/z 135.0 for analyte and m/z 448.2 to m/z 285.3 for IS. The calibration curves were linear over the range of 1–1000 ng/mL with the correlation coefficient greater than 0.999. With dilution integrity up to 20‐fold, the upper limit of quantification was extendable up to 15,000 ng/mL. The method was successfully applied to the analysis of rat plasma samples after single dose oral administration of sarpogrelate at 5 mg/kg to rats for the determination of its pharmacokinetics. Following oral administration the maximum mean concentration in plasma (Cmax, 11514 ng/mL) was achieved at 0.25 h (Tmax) and the area under curve (AUC0–24) was 11051 ± 3315 ng h/mL. The half‐life (t1/2) and clearance (Cl) were 2.9 ± 1.1 h and 490 ± 171 mL/h/kg, respectively. We believe that development of a method in rodent plasma would facilitate the ease of adaptability of sarpogrelate in human plasma. Copyright © 2010 John Wiley & Sons, Ltd.

Gurule, Sanjay; Khuroo, Arshad; Monif, Tausif; Goswami, Dipanjan; Saha, Arabinda

doi: 10.1002/bmc.1423pmid: 20954207

Terbinafine, a widely used antifungal drug, is a challenging molecule for quantitative bioanalysis due to certain factors contributing assay variability. Despite previous attempts at human plasma determination of terbinafine, exhaustive stability of the drug or an internal standard was lacking. Internal standard stability with negligible variation throughout the analysis is an indicator of a reliable bioanalytical method as the majority of LC–MS/MS assays are based on analyte/IS response ratios for quantitation. A newly developed high‐throughput simple LC‐MS/MS method is described for human plasma determination of terbinafine using naftifine internal standard and eluting all compounds within 2 min. A solid‐phase extraction of terbinafine achieving mean recovery of 84.3% (CV < 4%) without compromising sensitivity (limit of quantitation 5.11 ng/mL) or linearity (5.11–3014.19 ng/mL) is delineated in this paper. A heated nebulizer in positive multiple reaction monitoring mode was employed with transitions m/z 292.2 →141.1 and 288.2 →117.0 for terbinafine and naftifine, respectively, resulting in excellent chromatographic separation on a Hypurity Advance (50 × 4.6 mm, 5 µm) column. The developed method was successfully applied to clinical samples and for the first time demonstrated marked improved extraction efficiency and reliable long‐term plasma stability results without any internal standard response variation during the entire course of study. Copyright © 2010 John Wiley & Sons, Ltd.

Huang, Chiung‐Hua; Lee, Shih‐Chang; Chen, Yueh‐Sheng; Yao, Chun‐Hsu

doi: 10.1002/bmc.1424pmid: 20954208

The aim of this study was to evaluate the effect of radiosterilization on 30 Chinese medicines using γ‐rays from the isotope 60Co. Two groups of Chinese medicines, non‐treated and dry samples, were treated using a 60Co irradiation source at the doses 0, 3, 6 and 9 kGy. After storage for 3 months, characterizations of chemical compounds and functional groups were performed by high‐performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy. The results of radiosterlization showed that nearly all of the medicines were decontaminated under the dose of 9 kGy. In most samples, chemical compounds and functional groups were not altered by the irradiation treatment. However, minor changes were found in the molecular structures of 14 medicines under the reported ‘safety dose’ (10 kGy). The drying process before irradiation could decrease the chemical changes caused by γ‐rays to 50%. The HPLC analysis of nine medicines revealed minor changes at a dose of 3 kGy. The findings in this study provide important information that may suggest the need for a re‐evaluation of the reported safety dose. Therefore, further investigation may be warranted to insure the safety of γ‐radiosterlization of Chinese medicines. Copyright © 2010 John Wiley & Sons, Ltd.

Li, Hua; Yan, Zhi‐ying

doi: 10.1002/bmc.1425pmid: 20954209

In this work, focal cerebral ischemia and reperfusion were induced by the model of middle cerebral artery occlusion. The dialysate of extracellular fluid in the hypothalamus of rats were obtained by using brain microdialysis technique. An efficient and sensitive MEKC method for the simultaneous determination of multiple amino acid neurotransmitters in microdialysate was developed by capillary electrophoresis with laser‐induced fluorescence detection and 5‐(4, 6‐dichloro‐s‐triazin‐2‐ylamino) fluorescein derivatization. Different parameters that influenced derivatization reaction and CE separation were studied and optimized. This method was used to investigate the dynamic change of fourteen amino acid neurotransmitters in microdialysates during cerebral ischemia/reperfusion period. Our results reveal that MCAO and reperfusion elicited significant increases in the extracellular levels of Arg, Lys, Trp, Phe, Gln, GABA, Asn, Pro, Ser, Ala, Tau, Gly, Glu and Asp. The excitatory/inhibitory neurotransmitter balance was disturbed during ischemia/reperfusion. The dynamic changes and functional status of releasable neurotransmitters during ischemia/reperfusion were discussed. Copyright © 2010 John Wiley & Sons, Ltd.

Ruan, Hang; Zhang, Zhen; Zhu, Xuan

doi: 10.1002/bmc.1426pmid: 20954210

dl‐Praeruptorin A is a novel drug with valuable apoptosis and inflammation inhibitory effects in cardiac muscle. Previous pharmacokinetic studies of dl‐praeruptorin A have had limited success due to its very low plasma concentrations. In this study, we developed and validated a new rapid, sensitive and specific high‐performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC/ESI–MS/MS) method for quantitative analysis of dl‐praeruptorin A in rat plasma. dl‐Praeruptorin A and diazepam (internal standard) extracted from rat plasma samples with chloroform and analyzed on an XTerra™ RP18 column (150 mm × 4.6 mm i.d., 5 µm) were chromatographically separated within 5.5 min using methanol–water (75:25, v/v; flow rate 1 mL/min) as the mobile phase. dl‐Praeruptorin A was detected in positive ion mode using multiple reaction monitoring. The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ, 2.5 ng/mL), precision (intra‐ and inter‐day <11.0%), accuracy (90.2–96.3%), recovery (>79.2%) and stability were determined. The correlation coefficient (r2) for the linear range of 2.5–2500.0 ng/mL was >0.999. No matrix effects were observed. The validated method was successfully applied to pharmacokinetic studies of dl‐praeruptorin A after intravenous administration to rats. The LLOQ obtained with this method was lower than in previous studies and could be valuable for determination of dl‐praeruptorin A in therapeutic drug monitoring and preclinical studies to establish appropriate dose and frequency. Copyright © 2010 John Wiley & Sons, Ltd.

Noh, Keumhan; Kwon, Kwang‐il; Jeong, Tae Cheon; Kang, Wonku

doi: 10.1002/bmc.1427pmid: 20737450

Sparfloxacin, a fluoroquinolone antibiotic, is used for the treatment of bacterial infection. A quantification method using mass spectrometry was developed for the determination of sparfloxacin in rat plasma. After simple protein precipitation with acetonitrile, the analytes were chromatographed on a reversed‐phase C18 column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma sparfloxacin concentrations after a single oral administration of sparfloxacin in rats. Copyright © 2010 John Wiley & Sons, Ltd.

Li, Hong Y.; Zhao, Yu; Cao, Yang; Wang, Wei N.; Zhao, Da Q.

doi: 10.1002/bmc.1428pmid: 20954211

A superoxide dismutase (SOD) with the molecular weight of 31,079 has been purified as a homodimer from Panax ginseng by employing neutral pH buffer extraction, ammonium sulfate precipitation, isoelectric point precipitation and ion exchange methods. The enzyme's specific activity determined by an improved Marklund method was 9480.43 U/mg. Metal analysis showed that the SOD contained iron with the stoichiometry of 0.9 ± 0.3 Fe/subunit and exhibited high thermal stability (70°C) over the pH range from 4.0 to 9.0. Its maximum absorption wavelength was 278 nm and it was sensitive to hydrogen peroxide, trichloromethane‐ethanol and urea. These results indicate that the enzyme is an iron SOD. Copyright © 2010 John Wiley & Sons, Ltd.

Sørensen, L. K.

doi: 10.1002/bmc.1429pmid: 20954212

A liquid chromatographic/tandem mass spectrometric method using pneumatically assisted electrospray ionization (LC‐ESI‐MS/MS) was developed for determination of N‐acetylneuramic acid and N‐glycolylneuramic acid in infant formula. Reconstituted samples were hydrolysed in dilute sulfuric acid and deproteinized with acetonitrile. The extract was analysed directly without further clean‐up by hydrophilic interaction chromatography. The substances were detected in negative ion mode and matrix matched standards were used for calibration. The relative intra‐laboratory reproducibility standard deviation was better than 6% for both substances. An R2 of 0.985 was obtained by comparison with a classical colorimetric assay based on reaction with resorcinol. The developed method is expected to be applied for accurate routine analysis of infant formulas.