Biomedical Chromatography

Ohwaki, Yuichi; Yamane, Tomoko; Ishimatsu, Takashi; Wada, Mitsuhiro; Nakashima, Kenichiro

doi: 10.1002/bmc.742pmid: 17221906

A simultaneous determination of aspirin (ASA) and its metabolite, salicylic acid (SA), in human serum by a semi‐micro column HPLC‐UV was developed. A relatively small size of serum sample (100 µL) containing ASA and SA was cleaned up by a simple solid phase extraction. A good separation of ASA and SA could be achieved within 25 min using a semi‐micro ODS column with an eluent of MeOH/0.7 mm phosphoric acid solution (pH 2.5) = 50:50 (v/v). The calibration curves for ASA and SA showed good linearity (r = 0.999) with the detection limits 114 and 38 ng/mL at a signal‐to‐noise ratio of 3, respectively. ASA and SA in patients' sera administered with low‐dose enteric‐coated aspirin were determined, and the concentration ranges obtained for ASA and SA were 1.2–2.2 and 0.5–57.3 µg/mL, respectively. Copyright © 2007 John Wiley & Sons, Ltd.

Greiner‐Sosanko, Elizabeth; Lower, Darla R.; Virji, Mohamed A.; Krasowski, Matthew D.

doi: 10.1002/bmc.753pmid: 17230449

A high‐performance liquid chromatography assay with ultraviolet detection was developed for the simultaneous determination of the anti‐epileptic drugs lamotrigine, carbamazepine and zonisamide in human plasma and serum. Lamotrigine, carbamazepine, zonisamide and the internal standard chloramphenicol were extracted from serum or plasma using liquid–liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 1–30 µg/mL for lamotrigine, 2–20 µg/mL for carbamazepine, and 1–40 µg/mL for zonisamide. Within‐ and between‐run precision studies demonstrated coefficient of variation <10% at all tested concentrations. Other anti‐epileptic medications tested did not interfere with the assay. The method is appropriate for determining lamotrigine, carbamazepine and zonisamide serum or plasma concentrations for therapeutic monitoring. Copyright © 2007 John Wiley & Sons, Ltd.

Yang, Zhaoyong; He, Xiaobo; Zhang, Yueqin

doi: 10.1002/bmc.725pmid: 17236238

We report a rapid and reliable HPLC‐UV method for determination of raloxifene, a kind of selective estrogen receptor modulator (SERM), in rat tissue. Proteins were precipitated by adding 200 µL of acetonitrile and 50 µL of methanol to 100 µL of the tissue homogenates, following vortex mixing and centrifugation. Separation was carried out on a reversed‐phase C18 column (150 × 4.6 mm, 5 µm) with a mobile phase of acetonitrile:0.05 m ammonium acetate (pH 4.0 ± 0.1; 33:67, v/v) at a flow rate of 1.0 mL/min. The UV detection wavelength was set at 289 nm and the temperature of column was kept at 23°C, without interference from endogenous tissue compounds. The calibration curve was linear from 0.0125 to 10.0 µg/mL with correlation coefficient of over 0.994, while the limit of quantification was 0.008 µg/mL. The intra‐ and inter‐day coefficients of variation were less than 10% (RSD). The recovery of assay was between 95.8 and 104.5%. Furthermore, the method was used to measure the concentration of raloxifene in rat tissue after a simple oral dose. The highest level was observed in liver, lung, spleen, then heart and kidney. The lowest level was found in brain. These results suggest that raloxifene distributes rapidly and moderately into tissues such as liver, lung and spleen. Copyright © 2007 John Wiley & Sons, Ltd.

Mojtahedi, Mohammad Majid; Chalavi, Sohila; Ghassempour, Alireza; Tabar‐Heydar, Kourosh; Sharif, Seyed Javad Ghotb; Malekzadeh, Maryam; Aboul‐Enein, Hassan Y.

doi: 10.1002/bmc.732pmid: 17230451

This work aims to evaluate for the enantiomeric separations of three agrochemical toxins: haloxyfop‐methyl, fenoxaprop‐p‐ethyl and indoxacarb on crystalline degradation products–chiral stationary phase (CDP‐CSP) of high‐performance liquid chromatography (HPLC) under normal and polar organic phases. In the normal phase, the mobile phase was n‐hexane with alcohols including methanol and isopropanol as polar modifiers. In the polar organic phase mode, the mobile phase was methanol with different percentages of triethylammunium acetate. The influence of flow rate (0.3–0.9 mL/min), analyte concentration and silica gel particle sizes (10, 15 and 30 µm) was investigated. This new chiral stationary phase showed excellent stereoselectivity for the two enantiomers of haloxyfop‐methyl and fenoxaprop‐p‐ethyl and chiral recognition for indoxacarb under normal‐phase mode. However, under polar organic phase, only indoxacarb was separated (α < 1.5). The chromatographic results were compared with commercial chiral columns. Copyright © 2007 John Wiley & Sons, Ltd.

Nirogi, Ramakrishna V. S.; Kandikere, Vishwottam N.; Shukla, Manoj; Mudigonda, Koteshwara; Maurya, Santosh; Komarneni, Prashanth

doi: 10.1002/bmc.745pmid: 17230461

A simple, sensitive and rapid high‐performance liquid chromatography/positive ion electrospray tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of pseudoephedrine in human plasma using mosapride as internal standard. Following solid‐phase extraction, the analytes were separated using an isocratic mobile phase on a reverse‐phase column and analyzed by MS/MS in the multiple‐reaction monitoring mode using the respective (M + H)+ ions, m/z 166/148 for pseuoephedrine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 2–1000 ng/mL pseudoephedrine in human plasma. The lower limit of quantification was 2 ng/mL with a relative standard deviation of less than 9% for pseudoephedrine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. Copyright © 2007 John Wiley & Sons, Ltd.

Li, Wenkui; Luo, Suyi; Hayes, Michael; He, Handan; Tse, Francis L. S.

doi: 10.1002/bmc.746pmid: 17221915

A liquid chromatographic method with tandem mass spectrometric detection (LC‐MS/MS) for the determination of N‐methyl‐4‐isoleucine‐cyclosporin (NIM811) was developed and validated over the concentration range 1–2500 ng/mL in human whole blood using a 0.05 mL sample volume. NIM811 and the internal standard, d12‐cyclosporin A (d12‐CsA), were extracted from blood using MTBE via liquid–liquid extraction. After evaporation of the organic solvent and reconstitution, a 10 µL aliquot of the resulting extract was injected onto the LC‐MS/MS system. Chromatographic separation of NIM811 and internal standard was performed using a Waters Symmetry RP‐8 (50 × 4.6 mm, 3 µm particle size) column. The mobile phase consists of 10 mm ammonium acetate in water (A) and acetonitrile (B), with 45% B from 0 to 0.2 min, 45 to 85% B from 0.2 to 0.8 min and 85% B from 0.8 to 2.2 min. The total run time was 3.5 min with a flow rate of 0.8 mL/min. The method was validated for sensitivity, linearity, reproducibility, stability, dilution integrity and recovery. The precision and accuracy of quality control samples at low (2.00 ng/mL), medium (20.0 and 400 ng/mL) and high (2000 ng/mL) concentrations were in the range 1.1–4.3% relative standard deviation (RSD) and −2.5–10.0% (bias), respectively, from three validation runs. The method has been used to measure the exposure of NIM811 in human subjects. Copyright © 2007 John Wiley & Sons, Ltd.

Chen, Xijing; He, Hui; Wang, Guangji; Yang, Bing; Ren, Weichao; Ma, Le; Yu, Qiaoling

doi: 10.1002/bmc.747pmid: 17294509

A simple, accurate, precise, specific and reproducible high‐performance liquid chromatography (HPLC) method was developed for simultaneous determination of resveratrol isomers in rat plasma. Cis‐resveratrol was made by exposure of a trans‐resveratrol solution to sunlight for 5 days followed by separation by HPLC and identification by mass spectrometry (MS). The assay procedure involved simple liquid–liquid extraction of resveratrol isomers and internal standard (IS, caffeine) from a small plasma volume directly into acetonitrile. The supernatant liquid was added an equal volume of water and injected onto a Hypersil ODS2 C18 column (5 µm, 4.6 × 250 mm). Mobile phase consisting of methanol and distilled water was used at a flow rate of 1.0 mL/min for the effective separation of cis‐, trans‐resveratrol and caffeine (IS). The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of cis‐, trans‐resveratrol and IS were 3.2, 4.3 and 6.1 min, respectively. The calibration curve was linear ranging from 0.066 to 6.64 and 0.134 to 13.4 µg/mL with correlation coefficients of 0.9998 and 0.9997 for trans and cis isomers, respectively. The absolute recovery of both isomers was more than 85%. The inter‐ and intra‐day precisions in the measurement of quality control (QC) samples, 0.066, 0.664 and 6.64 µg/mL of trans‐resveratrol, were in the range 2.37–6.95% relative standard deviation (RSD) and 0.77–6.97% RSD, respectively. The inter‐ and intra‐day precisions in the measurement of quality control (QC) samples, 0.134, 1.34 and 13.4 µg/mL of cis‐resveratrol, were in the range 1.93–3.72% relative standard deviation (RSD) and 1.13–6.57% RSD, respectively. Both analytes and IS were stable in the battery of stability studies and freeze–thaw cycles. Resveratrol isomers were found to be stable for a period of 30 days on storage at −20°C. The application of the assay to determine the pharmacokinetic disposition after a single oral dose to rats is described. Copyright © 2007 John Wiley & Sons, Ltd.

Wei, Zhang; Bing‐ren, Xiang; Cai‐yun, Wang

doi: 10.1002/bmc.748pmid: 17230450

A sensitive and selective liquid chromatographic–mass spectrometric (LC–MS) method for the determination of venlafaxine in human plasma has been developed. Samples were prepared using liquid–liquid extraction and analyzed on a C18 column interfaced with a triple quadrupole mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase was methanol–water containing 10 mmol/L ammonium acetate, pH 7.9 adjusted with aqueous ammonia (80:20, v/v) at the flow rate of 1.0 mL/min. The analyte and internal standard clozapine were both detected by use of selected ion monitoring mode. The method was linear in the concentration range of 1.0–200.0 ng/mL. The lower limit of quantification (LLOQ) was 1.0 ng/mL. The intra‐ and inter‐day relative standard deviation across three validation runs over the entire concentration range was less than 10.1%. The accuracy determined at three concentrations (5.0, 50.0 and 150.0 ng/mL for venlafaxine) was within ±10.0% in terms of relative error (RE). The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine capsule in 20 healthy volunteers. The results show AUC, Tmax, Cmax and T1/2 between the testing formulation and reference formulation have no significant difference (p > 0.05). Relative bioavailability was 103.4 ± 14.1%. Copyright © 2007 John Wiley & Sons, Ltd.

Zhang, Wei‐Zheng; Lang, Charles; Kaye, David M.

doi: 10.1002/bmc.750pmid: 17236239

Oxidative stress plays an important role in pathogenesis of many diseases. Measurement of 3‐nitrotyrosine (NO2Tyr), as a potential biomarker for nitric oxide‐mediated damage, has recently been the focus of particular attention. We have developed an HPLC method with NBD‐F pre‐column derivatization followed by C18 cartridge cleaning. Using this method we achieved limits of detection of 0.5 and 1.1 nm for NO2Tyr and tyrosine (Tyr), respectively, close to that achieved by LS‐MS/MS. NO2Tyr and tyrosine concentrations were linear over the calibration ranges 0.5–100 nm and 1–320 µm, respectively, with correlation coefficients greater than 0.95. To evaluate the utility of this assay in plasma we analysed samples obtained from smokers and non‐smoking subjects. Consistent with the presence of elevated oxidative stress, the plasma NO2Tyr concentration and NO2Tyr:Tyr ratio of smokers were 17.42 ± 11.6 nm and 0.263 ± 0.192 nm/µm with 3.8 and 3.9 times higher (both p < 0.05), respectively, than that of non‐smoker controls (4.54 ± 2.75 nm and 0.067 ± 0.050 nm/µm, respectively). In conclusion, we have developed a novel HPLC assay for NO2Tyr without MS detection that is applicable to clinical studies addressing the pathophysiology and importance of oxidative stress. Copyright © 2007 John Wiley & Sons, Ltd.

Hao, Kun; Zhao, Xiao‐Ping; Liu, Xiao‐Quan; Wang, Guang‐Ji

doi: 10.1002/bmc.752pmid: 17221914

A rapid, simple and sensitive high‐performance liquid chromatography method for the quantification of gambogic acid in dog plasma was developed and validated. After acidification with hydrochloric acid, dog plasma was extracted with ethyl acetate and determined by HPLC. The analysis was carried out on a reversed‐phase C18 analytical column. The mobile phase consisted of a mixture of methanol–0.05% phosphoric acid (94:6, v/v), and the column temperature was maintained at 35°C. A constant mobile phase flow rate of 1.0 mL/min was employed throughout the analyses. The ultraviolet detector was set at 360 nm. Chromatographic separation was achieved in less than 10 min and the calibration curve was linear over a concentration range of 0.156–20 µg/mL. The intra‐assay and inter‐assay variability values were less than 10.0%. The accuracy ranged from 93.0 to 104.2%. The established method has been successfully applied to a pharmacokinetic study of gambogic acid in dogs. Copyright © 2007 John Wiley & Sons, Ltd.