Journal of Chromatography B (v.919-920, #C)

► Fast and simultaneous quantitation of different classes of drugs in human plasma. ► Simple sample preparation step by protein precipitation. ► LC–MS/MS analysis with online sample clean up. ► Efficient tool for optimization of laboratory resource utilization.High-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) is a standard analytical technique for therapeutic drug monitoring (TDM). A rapid LC–MS/MS method was developed for simultaneous quantitation of 3 antifungals and one active metabolite (posaconazole, voriconazole, itraconazole, and hydroxy-itraconazole), 5 antibiotics (daptomycin, ciprofloxacin, oxacillin, levofloxacin, and rifampicin), an antineoplastic agent (imatinib), and an antiretroviral (raltegravir) in human plasma. Protein precipitation of 10 μL of plasma with acetonitrile was used as a single-extraction procedure. After 2-dimensional LC, all drugs were quantified by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated per FDA recommendations including the study of extraction recovery (from 79.3% to 105.9%) and matrix effect via ion suppression/enhancement phenomenon. This method is precise (intra- and inter-assay coefficients of variation of 1.95–12.77%, 2.56–8.16% and 2.12–11.38% for low, medium and high levels of internal quality controls respectively) and accurate (intra- and inter-assay biases of 0.19–12.67%, 0.04 to −12.17% and 0.22–12.98% respectively). This method is an efficient tool for routine TDM and optimization of laboratory resource utilization.
Keywords: LC–MS/MS; Antifungals; Antibiotics; Raltegravir; Imatinib;

► Gemcitabine is effective in the treatment of malignant gliomas. ► Gemcitabine's metabolite dFdU may contribute to gemcitabine's toxicity. ► A microdialysis technique with UPLC–MS/MS method was developed. ► All the validation data are within the required limits. ► The method was applied to determine gemcitabine and dFdU in rat tumor.The cytotoxic agent Gemcitabine (2′,2′-difluoro-2′-deoxycytidine) has been proved to be effective in the treatment of malignant gliomas. A rapid, sensitive and specific ultra performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) assay using microdialysis sampling was developed and validated to quantify gemcitabine and its major metabolite 2′,2′-difluoro-2′-deoxyuridine (dFdU) in Sprague–Dawley rat bearing 9 L glioma. Microdialysis probes were surgically implanted into the area of rat brain tumor in the striatal hemisphere, and artificial cerebrospinal fluid was used as a perfusion medium. The samples were analyzed directly by UPLC–MS/MS after the addition of 5-bromouracil as an internal standard (IS). Separation was achieved on Agilent SB-C18 (50 mm × 2.1 mm I.D., 1.8 μm) column at 40 °C using an isocratic elution method with acetonitrile and 0.1% formic acid (4:96, v/v) at a flow rate of 0.2 mL/min. Detection was performed using electrospray ionization in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 264.0 → 112.0 (gemcitabine), m/z 265.1 → 113.0 (dFdU) and m/z 190.9 → 173.8 (IS). The calibration curves of gemcitabine and dFdU were linear in the concentration range of 0.66–677.08 ng/mL and 0.31–312.00 ng/mL, respectively. The lower limit of quantification of gemcitabine and dFdU were 0.66 ng/mL and 0.31 ng/mL, respectively. The lower limit of detection of gemcitabine and dFdU were calculated to be 0.2 ng/mL and 0.1 ng/mL, respectively. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. The validated method was simple, precise and accurate, which was successfully employed to determinate the concentrations of gemcitabine and dFdU in the extracellular fluid of rat brain tumor.
Keywords: Microdialysis; UPLC–MS/MS; Extracellular fluid; Gemcitabine; 2′,2′-Difluoro-2′-deoxyuridine;

► Development of HPLC–ESI-MS/MS method for the determination of potential biomarkers. ► Optimization of the MS/MS and chromatographic conditions. ► Solid-phase extraction procedure for human urine sample cleanup. ► This method could be useful in the detection of heart failure before symptoms of the disease.A simple, accurate and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantitation of α-ketoglutaric acid (α-KG), l-carnitine (l-CAR) and acetyl-l-carnitine (acetyl-l-CAR) in human urine as potential biomarkers of cardiovascular disease. The separation was performed using an isocratic elution of 0.1% formic acid in water and acetonitrile (97:3, v/v) on an Acclaim 120 C8 column (150 mm × 4.6 mm, 3.0 μm). The flow rate of the mobile phase was 1.2 mL/min and the total assay run time was 3 min. Detection was performed on a triple-quadrupole mass spectrometer in selected reaction monitoring (SRM) mode via an electrospray ionization (ESI) source in positive and negative ion modes. This method covered a linearity range of 0.1–500 ng/mL for l-CAR and acetyl-l-CAR and 1–1000 ng/mL for α-KG with lower limits of quantification (LLOQ) of 0.08 ng/mL for l-CAR, 0.04 ng/mL for acetyl-l-CAR and 0.8 ng/mL for α-KG. The intra-day and inter-day precision and accuracy of the quality control samples exhibited relative standard deviations of less than 5.54% and relative error values from −5.95% to 3.11%. Analyte stability was evaluated under various sample preparation, analysis and storage conditions and varied from −9.89% to −0.47%. A two-step solid-phase extraction (SPE) procedure using silica gel and quaternary amine cartridges was used for urine sample cleanup. The average recoveries for all analyzed compounds were better than 86.64% at three concentrations. The method was successfully applied for the quantitation of α-KG, l-CAR and acetyl-l-CAR in human urine samples.
Keywords: LC–MS/MS; Biomarkers; α-Ketoglutaric acid; l-Carnitine; Acetyl-l-carnitine;

► Quinocetone and its four metabolites. ► Chicken muscle, liver, kidney and fat tissues. ► A rapid and sensitive LC–MS/MS method is firstly developed. ► The elimination curves of quinocetone and its metabolites are obtained.A convenient, rapid and sensitive liquid chromatography-tandem mass spectrometry method was firstly established for the simultaneous determination of quinocetone and its 4 major metabolites: 1-desoxyquinocetone, di-deoxyquinocetone, carbonyl reduced metabolite from di-deoxyquinocetone and 3-methyl-quinoxaline-2-carboxylic acid in chicken muscle, liver, kidney and fat. Sample was extracted with acetonitrile and chloroform, and further purified by Oasis MAX SPE cartridge. Analysis was performed on a C18 column by detection with mass spectrometry in multiple reaction monitoring mode and using a gradient elution program with 0.1% formic acid solution and acetonitrile. The correlation coefficients (r) for each calibration curves are higher than 0.99 within the experimental concentration range. The recoveries of the five target analytes at three spiking levels were between 77.1% and 95.2%, with relative standard deviations less than 15%. The decision limits of the five analytes in chicken edible tissues ranged from 0.24 to 0.76 μg kg−1, and the detection capabilities were below 2.34 μg kg−1. The developed method demonstrated a satisfactory applicability in incurred chicken tissue samples.
Keywords: Quinocetone; Metabolites; Liquid chromatography–tandem mass spectrometry; Chicken; Residues;

Rapid determination of sumatriptan in human plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application to clinical pharmacokinetic study by Jeong Ju Seo; Jeonghyeon Park; Min Ho Bae; Mi-sun Lim; Sook Jin Seong; Joomi Lee; Sung Min Park; Hae Won Lee; Young-Ran Yoon (38-42).
► An U-HPLC–MS/MS analysis method for the quantification of sumatriptan was developed and validated using human plasma. ► This analysis method has been successfully validated in the 0.5–50 ng/mL calibration range. ► The assay method precision was within 11.8%, and accuracy was between 97.4 and 102.9%. ► The developed and validated method was successfully applied to a pharmacokinetic study of sumatriptan.A sensitive and simple detection method coupling ultra-performance liquid chromatography with tandem mass spectrometry was developed and validated to analyze sumatriptan levels in human plasma. The plasma sample preparations for the analysis were based on liquid–liquid extraction with ethyl acetate, evaporation, and reconstitution. MS/MS detection was performed on a triple-quadrupole tandem mass spectrometer by monitoring the protonated parent → daughter ion pairs at m/z 296 → 58 and m/z 388 → 71 for sumatriptan and terazosin (internal standard), respectively. The method was validated with respect to its specificity, linearity, sensitivity, accuracy, precision, recovery, and stability. The calibration curve was linear from 0.5 to 50 ng/mL (r  > 0.999). The mean extraction recovery for sumatriptan was higher than 62.3%. The method accuracy was within 97.4%, and the relative standard deviation of the intra- and inter-day precision values was within 11.7% at all quality control levels. Plasma samples that contained sumatriptan were stable under three freeze–thaw cycles, short- and long-term storage, and autosampler conditions. This method was successfully applied to a pharmacokinetic study conducted with 10 healthy volunteers. After oral administration of 50-mg sumatriptan and serial blood sampling over 12 h, the mean area under the plasma concentration–time curve from time 0 to 12 h and the maximum plasma concentration were 116.2 ng h/mL and 33.2 ng/mL, respectively.
Keywords: U-HPLC–MS/MS; Sumatriptan; Human plasma; Pharmacokinetic study; Method validation;

► Quantification of nucleoside and nucleotide analogs in plasma using HPLC–MS/MS. ► Zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin. ► Validation of the assay according to FDA guidelines.This paper describes the development and validation of an assay for the simultaneous quantification of the antiviral and antiretroviral drugs zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma using liquid chromatography coupled to tandem mass spectrometry. Sample pretreatment consisted of protein precipitation with 0.1% (v/v) formic acid in methanol, evaporation and reconstitution. Chromatographic separation was performed on a Synergy Polar reversed phase C18 column (150 mm × 2.0 mm ID, particle size 4 μm) using a stepwise gradient with 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in methanol at a flow rate of 300 μL/min. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for drug detection and quantification. Isotopically labeled zidovudine, lamivudine, tenofovir and ribavirin were used as internal standards. The method was validated over a clinical range of 20–2500 ng/mL for zidovudine, lamivudine and tenofovir, 4–500 ng/mL for abacavir and emtricitabine and 160–20,000 ng/mL for ribavirin. The inter and intra-assay accuracies and precisions were between −8.47% and 14.2% for zidovudine, emtricitabine and ribavirin. For abacavir, lamivudine and tenofovir, the inter and intra-assay accuracies and precisions at the lower limit of quantification were between −11.0% and 18.3%, whereas at all other levels these accuracies and precisions were between −11.7% and 12.0%. The described method is suitable for the determination of zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma in clinical practice to monitor plasma concentrations in selected cases to optimize therapy.
Keywords: HIV; HCV; HBV; Nucleoside reverse transcriptase inhibitor; Nucleotide reverse transcriptase inhibitor; Bioanalysis;

► Development and validation of fast, reproducible, and accurate LC-UV method. ► Simultaneous determination Nevirapine, Efavirenz and Lumefantrine in human plasma. ► The method is applicable to clinical samples from malaria and HIV patients. ► The method is easy to implement in laboratories with limited resources.A fast and reliable high performance liquid chromatography (HPLC) method with UV diode array detection for simultaneous quantitative analysis of the anti-retroviral drugs, nevirapine (NVP) and efavirenz (EFV) and the anti-malarial, lumefantrine (LUM) in human plasma has been developed and validated. The sample preparation consisted of a plasma protein precipitation with 0.5% acetic acid acetonitrile solution containing the internal standard halofantrine (HALO) prior the LC-analysis. Chromatographic separation was carried out on a Acclaim Polar Advantage C16, column (150 mm × 4.6 mm, particle size, 3 μm) using a gradient of mobile phase made of 0.01% TFA in 0.1 M ammonium acetate (solvent A) and 0.1% TFA in acetonitrile (solvent B). The separation of NVP, EFV, LUM and HALO was achieved within 17 min at a flow rate of 1.0 mL/min and detections were initially performed at three wavelengths, 275 nm (NVP), 255 nm (EFV), and 300 nm (LUM). The method selectivity was demonstrated in six different human plasma batches. In addition, several concomitant drugs were analyzed under our experimental conditions and none of them co-eluted with EFV, NVP and LUM. This demonstrated that our method is highly selective. Calibration graphs plotted with seven concentrations in duplicate for each compound were linear between the selected ranges with a regression coefficient (R 2) greater than 0.998. Absolute extraction recovery for NVP, EFV and LUM were 99%, 98.6 and 102%, respectively. Inter- and intra-day coefficients of variation for LUM, EFV and NVP were ≤10%. The lower limits of quantification were 0.125 μg/mL for LUM and 0.250 μg/mL for both EFV and NVP. Intra- and inter-assay relative standard deviation values were found to be less than 15% at the concentrations examined (0.125–10.0 μg/mL for LUM and 0.250–15.0 μg/mL for both EFV and NVP). The present method was successfully implemented in Tanzania and only one wavelength (255 nm) was used to measure samples of patients receiving either NVP or EFV in combination with LUM. The concentration found in human plasma samples for all three compounds were within the calibration range. This makes our method particularly applicable and useful to resource-limited settings.
Keywords: HPLC-UV; Validation; Sample preparation; Anti retroviral drugs; Anti malarial drug;

Validation of an efficient LC-microdialysis method for gemifloxacin quantitation in lung, kidney and liver of rats by Bibiana Verlindo de Araújo; João Victor Laureano; Lauren Dockhorn Grünspan; Teresa Dalla Costa; Leandro Tasso (62-66).
► An efficient method for quantitation of unbound gemifloxacin concentrations is reported. ► Gemifloxacin was successfully determined in kidney, lung and liver of rats. ► Gemifloxacin unbound tissue concentrations were kidney > liver > lung.A liquid chromatography method has been established for the reliable determination of unbound gemifloxacin concentrations in kidney, lung and liver microdialysates of rats. Microdialysis probes were inserted into tissues of rats, and then dialysates were collected at regular time intervals after intravenous administration of gemifloxacin (40 mg kg−1). A pilot study was performed to assess gemifloxacin penetration in lung, kidney and liver of rats. Gemifloxacin was separated on a C18 column eluted using triethylamine solution (0.5%, v/v), adjusted to pH 3.0 ± 0.1 with 85% phosphoric acid, methanol and acetonitrile (71:15:14, v/v/v) as mobile phase at a flow rate of 1.1 mL min−1. The fluorescence detector was set at excitation and emission wavelengths of 344 nm and 399 nm, respectively. The limit of quantitation was found to be 50 ng mL−1. Linearity was found to be over a concentration range of 50–2000 ng mL−1. The intra-assay and inter-assay precision and accuracy values were determined from the analysis of six quality control samples. The results obtained at three concentration levels showed R.S.D. values lower than 6.06% and 4.10% for repeatability and intermediate precision, respectively. The accuracy (R.E.%) ranged from 90.0 to 106.5%. The chromatographic run time of each sample was performed in 9 min. Drug stability in microdialysates was shown at room temperature for 8 h, after three freeze–thaw cycles, in freezer at −80 °C for 14 days, and in the autosampler after processing for 8 h. The relative recoveries determined by extraction efficiency (EE) and retrodialysis (RD) in vitro employing a flow rate of 1.5 μL min−1 were 29.24 ± 3.67% and 23.67 ± 3.31%, respectively. In vivo recoveries determined by RD in Wistar rats’ kidney, lung and liver were 27.69 ± 2.09%, 23.12 ± 3.79% and 17.38 ± 0.68%, respectively. The method was successfully applied to investigate tissue penetration of unbound gemifloxacin into the kidney, lung and liver of rats.
Keywords: Gemifloxacin; Liquid chromatography; Microdialysis; Probe recovery; Tissue penetration; Validation;

Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate by Nemailla Bonturi; Vanessa Soraia Cortez Oliveira Radke; Sônia Maria Alves Bueno; Sindélia Freitas; Adriano Rodrigues Azzoni; Everson Alves Miranda (67-74).
► Alternative salts can be used in pDNA HIC and TAC as buffer systems. ► pDNA purification with HIC and TAC can be done directly with neutralized lysate. ► Mercaptopyrimidine agarose/sodium citrate system recovered 91.1% of the pDNA. ► Mercaptopyrimidine agarose/potassium phosphate system resulted in 98.8% pDNA purity.The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography. Hydrophobic interaction chromatography (HIC) and thiophilic aromatic chromatography (TAC), both using ammonium sulfate buffers, are commonly employed with success. This work was aimed at studying the feasibility of utilizing alternative salts in the purification of pDNA from neutralized lysate with phenyl-agarose (HIC) and mercaptopyrimidine-agarose (TAC) adsorbents. Their selectivity toward sc pDNA was evaluated through adsorption studies using 1.5 mol/L sodium citrate and 2.0 mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5 mol/L sodium citrate was able to recover 91.1% of the pDNA with over 99.0% removal of gDNA and endotoxin. This represents a potential alternative for the primary recovery of sc pDNA. However, the most promising result was obtained using 2.0 mol/L potassium phosphate buffer and a mercaptopyrimidine-agarose column. In a single chromatographic step, this latter buffer/adsorbent system recovered 68.5% of the pDNA with 98.8% purity in accordance with the recommendations of regulatory agencies with regard to RNA and endotoxin impurity.
Keywords: Aromatic thiophilic chromatography; Hydrophobic interaction chromatography; pDNA; Neutralized lysate;

Determination of ginsenoside Rc in rat plasma by LC–MS/MS and its application to a pharmacokinetic study by Yang Chu; Hong-chao Zhang; Shu-ming Li; Jun-mei Wang; Xiang-yang Wang; Wei Li; Lan-lan Zhang; Xiao-hui Ma; Shui-ping Zhou; Yong-hong Zhu; Chang-xiao Liu (75-78).
► It is the first analytical method (LC–MS/MS) for determining ginsenoside Rc in rat. ► It is the first report of bioavailability and pharmacokinetics of ginsenoside Rc. ► It provides the necessary information for the further investigation of ginseng.Ginsenoside Rc (GRc) is a potential pharmacologically active ingredient isolated from ginseng (Panax ginseng C.A. Meyer, Araliaceae). A simple, rapid and sensitive method for determination of GRc in rat plasma was developed based on liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (IS), ginsenoside Rb1 (GRb1), were extracted from plasma with n-butanol and chromatographied on a C18 column eluted with a mobile phase of methanol and water containing 0.1% formic acid. The detection was performed by positive ion electrospray ionization in selective reaction monitoring mode (SRM), monitoring the transitions m/z 1101.6 → 789.3 and m/z 1131.7 → 364.7 for GRc and IS, respectively. The assay was linear over the concentration range of 5–5000 ng/mL with a limit of quantitation (LOQ) of 5 ng/mL. The accuracy was between 86.7% and 114.9%, and the precision was less than 9.7%. This method was successfully applied to investigate the pharmacokinetic study of GRc in rats after intravenous (2 mg/kg) and oral (20 mg/kg) administration, and the result showed that the ginsenoside was poorly absorbed with an absolute bioavailability being approximately 0.17%.
Keywords: Ginsenoside Rc; LC–MS/MS; Pharmacokinetics; Bioavailability;