Biomedical Chromatography

Beaudry, Francis; Vachon, Pascal

doi: 10.1002/bmc.906pmid: 17849505

Glucosamine is an amino sugar involved in the biosynthesis of glycosylated proteins and lipids. Recently, with increased public interest in natural products medicine, glucosamine has been widely used to treat osteoarthritis, even though demonstrations of its actual efficacy remain relatively unknown. Information related to the pharmcokinetics of glucosamine is sparse. A recent analytical method published used 13C‐glucosamine as an internal standard to analyse study samples. The method lacked accuracy owing to an important natural isotopic contribution of glucosamine to 13C‐glucosamine ion abundance. This manuscript describes a simple method to quantify glucosamine in horse plasma. Glucosamine was extracted by protein precipitation with acetonotrile containing 0.1% formic acid. The chromatography was performed on a Agilent Hypersil‐ODS 100 × 2.1 mm column with a mobile phase composed of acetonitrile and 0.5% formic acid in water (45:55) at a flow rate of 0.3 mL/min. A linear (1/x) relationship was used to perform the calibration over an analytical range of 10–1000 ng/mL. The inter‐batch precision and accuracy ranged from 5.3 to 11.3% and from 87.8 to 107.2% in horse plasma, respectively. The mean endogenous level of glucosamine in horse plasma was 14.4 ng/mL (n = 6). This LC‐ESI/MS/MS method for the determination of glucosamine in horse plasma provided results within generally accepted criteria used for bioanalytical assay. Copyright © 2007 John Wiley & Sons, Ltd.

Gao, Shouhong; Chen, Wansheng; Tao, Xia; Miao, Haijun; Yang, Shaolin; Wu, Rong

doi: 10.1002/bmc.886pmid: 17604362

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry (LC‐MS/MS) quantitative detection method, using cefalexin as internal standard, was developed for the analysis of faropenem in human plasma and urine. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C18 reversed‐phase column with 0.1% formic acid–methanol (45:55, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves with good linearities (r = 0.9991 for plasma sample and r = 0.9993 for urine sample) were obtained in the range 5–4000 ng/mL for faropenem. The limit of detection was 5 ng/mL. Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of faropenem in humans, and to our knowledge, it is the first time the pharmacokinetic of faropenem has been elucidated in vivo using LC‐MS/MS. Copyright © 2007 John Wiley & Sons, Ltd.

Shimada, Yayoi; Goto, Takeshi; Kawamoto, Seiji; Kiso, Takashi; Katayama, Akiko; Yamanaka, Yasushi; Aki, Tsunehiro; Chiang, Kuei‐Chen; Nakano, Toshiaki; Goto, Shigeru; Chen, Chao‐Long; Ohmori, Naoya; Ono, Kazuhisa; Sato, Shuji

Mudigonda, Koteshwara; Jukanti, Raju; Apte, Shashank S.; Kishan, Veerabrahma; Maurya, Santosh; Kandikere, Vishwottam; Nirogi, Ramakrishna

doi: 10.1002/bmc.888pmid: 17604365

A high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of zidovudine in rat plasma. 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 268/127 for zidovudine and m/z 230/112 for the internal standard. The method exhibited a linear dynamic range of 5–500 ng/mL for zidovudine in rat plasma. The lower limit of quantification was 5 ng/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 1.5 min for each sample made it possible to analyze more than 400 plasma samples per day. The validated method was applied for pharmacokinetic studies of the novel drug delivery systems of zidovudine in rats. Copyright © 2007 John Wiley & Sons, Ltd.

Chen, Yunyun; Wang, Xueli; Sun, Hong; Xing, Dongming; Hu, Jun; Wai, Zhuohan; Du, Lijun

doi: 10.1002/bmc.889pmid: 17631668

The aim of this study was to investigate the transport behavior and efflux of berberine through the primary culture cortical neurons. High‐performance liquid chromatography coupled with an UV–vis detector at 347 nm was applied. The mobile phase was 0.05 m potassium dihydrogen phosphate solution (containing 0.5% triethylamine, pH 3.0)–acetonitrile (73:27, v/v). Neurons were incubated with Coptidis rhizoma extract 6.5 µg/mL (containing 1.91 µg/mL berberine) and verapamil, KCN or cimetidine for 2 h, and then lysed in methanol to extract intracellular berberine. A 20 µL aliquot of sample was injected into the HPLC system to determine berberine concentration. The results showed that metabolic inhibitor KCN and P‐glycoprotein inhibitor verapamil could increase berberine concentration within the neurons, indicating that efflux of berberine was energy‐dependent and P‐glycoprotein was likely to be involved. Moreover, the organic cation transporter inhibitor cimetidine could decrease berberine concentration within the neurons, suggesting that the organic cation transporter might be involved in the berberine transport process. Copyright © 2007 John Wiley & Sons, Ltd.

Higashi, Tatsuya; Nagahama, Akihiro; Mukai, Yoshiyuki; Shimada, Kazutake

doi: 10.1002/bmc.891pmid: 17624919

A sensitive liquid chromatography–electrospray ionization–tandem mass spectrometric (LC–ESI–MS/MS) method for the simultaneous determination of five 3‐oxo‐4‐ene‐neuroactive steroids, i.e. androstenedione, testosterone (T), progesterone (PROG), 20α‐dihydroprogesterone and 20β‐dihydroprogesterone, in rat brain has been developed and validated. The brain steroids were extracted with methanol‐acetic acid, purified using solid‐phase extraction cartridges and subjected to LC–ESI–MS/MS. The method does not require derivatization. Deuterium‐labeled T and PROG were used as the internal standards, and quantification was based on the selected reaction monitoring mode. This method allowed the reproducible and accurate quantification of the brain neuroactive steroids using 100 mg of tissue; the intra‐ and inter‐assay relative standard deviations were below 4.7 and 4.3%, respectively, and the accuracy values were 97.6–103.2% for all the steroids. The limits of quantitation were 0.1 ng/g tissue for all the steroids. The application of this developed method for the analysis of changes in the brain neuroactive steroid levels by immobilization stress is also presented. Copyright © 2007 John Wiley & Sons, Ltd.

Wei, Chun‐min; Zhang, Rui; Wang, Ben‐jie; Yuan, Gui‐yan; Guo, Rui‐chen

doi: 10.1002/bmc.892pmid: 17849504

A sensitive, simple and selective high‐performance liquid chromatography–tandem mass spectrometry method was developed and applied to the determination of norcantharidin concentration in human serum. Norcantharidin (NCTD) and cyclophosphamide (IS) in serum were extracted with acetone, separated on a C18 reversed‐phase column, gradiently eluted with a mobile phase of acetonitrile–water containing 2 mm ammonium acetate and 0.1% formic acid (pH 3), ionized by positive ion pneumatically assisted electrospray and detected in the multi‐reaction monitoring mode using precursor → product ions of m/z 169.3 → 123.1 for NCTD and 261.2 → 140.2 for IS, respectively. The linear range of the calibration curve for NCTD was 2.5–50 ng/mL, with a lowest limit of quantification of 2.5 ng/mL, and the intra/inter‐day RSD was less than 10%. The method was suitable for determination of low NCTD concentration in human serum after therapeutic oral doses, and has been successfully used for pharmacokinetic studies in healthy Chinese volunteers. Copyright © 2007 John Wiley & Sons, Ltd.

Wang, Su‐Jun; Ruan, Jin‐Xiu; Zhao, Yan‐Hong; Zhang, Zhen‐Qing

doi: 10.1002/bmc.895pmid: 17853386

A simple and sensitive method was developed for the simultaneous quantification of harpagoside and cinnamic acid in rat plasma using high‐performance liquid chromatography system coupled to a negative ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of two volumes of acetonitrile. The analytes were separated on an Intersil C8‐3 column (2.1 mm i.d. × 250 mm, 5 µm) with acetonitrile–5 mm ammonium formate aqueous solution (60:40, v/v) as mobile phase at a flow‐rate of 0.2 mL/min. Detection was performed on a quadrupole mass spectrometer equipped with electrospray ionization (ESI) source operated under selected ion monitoring (SIM) mode. (M + HCOO)− at m/z 539 for harpagoside, (M − H)− at m/z 147 for cinnamic acid and (M − H)− at m/z 137 for salylic acid (internal standard) were selected as detecting ions, respectively. The method was validated over the concentration range 7–250 ng/mL for harpagoside and 5–500 ng/mL for cinnamic acid. The lower limits of quantitation for harpagoside and cinnamic acid were 7 and 5 ng/mL, respectively. The intra‐ and inter‐day precisions (RSD%) were within 9.5% and the assay accuracies (RE%) ranged from −5.3 to 3.0% for both analytes. Their average recoveries were greater than 86%. Both analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the pharmacokinetic study of harpagoside and cinnamic acid following oral administration of Radix Scrophulariae extract to rats. Copyright © 2007 John Wiley & Sons, Ltd.

Chakradhar, Lagishetty; Kallem, Rajareddy; Karthik, Arumugam; Sundari, Bala Tripura; Ramesh, Srirangam; Mullangi, Ramesh; Srinivas, Nuggehally R.

doi: 10.1002/bmc.896pmid: 17642067

A sensitive and specific liquid chromatography–positive electrospray ionization–tandem mass spectrometry method has been developed and validated for the determination of glimepiride (GPD) in human plasma. GPD and the internal standard (IS, glibenclamide) were extracted from a small aliquot of human plasma (200 µL) by a simple liquid–liquid extraction technique using ethyl acetate as extraction solvent. The compounds were separated on a YMC Propack, C18, 4.6 × 50 mm column using a mixture of ammonium acetate buffer, acetonitrile and methanol (30:60:10, v/v) as mobile phase at 0.5 mL/min on an API 4000 Sciex mass spectrometer connected to an Agilent HPLC system. Method validation and pre‐clinical sample analysis was performed as per FDA guidelines and the results met the acceptance criteria. GPD and IS were detected without any interference from human plasma matrix. The method was proved to be accurate and precise at linearity range of 0.02–100.00 ng/mL with a correlation coefficient of 0.999. The method was robust with a lower limit of quantitation of 0.02 ng/mL. Intra‐ and inter‐day accuracies for GPD were 88.60–113.50 and 96.82–103.93%, respectively. The inter‐day precision was better than 12.21%. This method enabled faster and reliable determination of GPD in a pre‐clinical study. Copyright © 2007 John Wiley & Sons, Ltd.

Zhao, Liang; Lou, Zi‐Yang; Zhu, Zhen‐Yu; Hai‐Zhang, Zhen‐Yu; Zhang, Guo‐Qing; Chai, Yi‐Feng

doi: 10.1002/bmc.897pmid: 17685411

A reliable and rapid method based on rapid‐resolution liquid chromatography–diode array detection (RRLC‐DAD) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF/MS) has been developed for the isolation and characterization of multiple constituents in the root of Stellera chamaejasme L., which was extracted by sonication with methanol in an optimized procedure. Separation of the multiple constituents was achieved on an Agilent Zorbax XDB‐C18 (50 × 3.0 mm i.d.; 1.8 µm) column using a gradient elution at a flow rate of 0.4 mL/min. The detection wavelength was 210 nm. Mass spectra were acquired in both positive and negative modes. A formula database of the known chemical constituents in the root of Stellera chamaejasme L. was established by an Agilent software. Twenty‐two obvious peaks appeared in the total ion chromatogram and nine of them were characterized by TOF/MS. The RRLC‐DAD and ESI‐TOF/MS method with ultrasonic extraction would be useful for rapid and effective characterization of chemical constituents in the root of Stellera chamaejasme L. Copyright © 2007 John Wiley & Sons, Ltd.