Journal of Chromatography B (v.877, #5-6)
Editorial Board (i).
Application of a liquid chromatography/tandem mass spectrometry method for the pharmacokinetic study of dihydroartemisinin injectable powder in healthy Chinese subjects by Yiming Liu; Xing Zeng; Yuanhui Deng; Lu Wang; Yi Feng; Liu Yang; Dan Zhou (465-470).
A simple, rapid and accurate liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed and validated for quantification of dihydroartemisinin (DHA) in human plasma. Following a simple single-step liquid–liquid extraction with ethyl acetate, the analyte was separated on a C18 column by isocratic elution with methanol–water–10 mM ammonium acetate (80:10:10, v/v/v), and analyzed by mass spectrometry in the positive ion MRM mode. Good linearity was achieved over a wide range of 1.01–2020 ng/mL. Intra- and inter-day precisions were less than 9.0%, and accuracy ranged from 93.0 to 98.2%. The pharmacokinetics of DHA injectable powder was studied for the first time in healthy subjects by this method. After single intravenous infusion of DHA injectable powder 40, 80 and 160 mg, the elimination half-life (t 1/2λZ ) was 1.69, 1.88 and 1.92 h, respectively; mean C max and AUC increased in proportion to the doses. The pharmacokinetics of DHA fit the linear dynamic feature over the DHA dose range studied.
Keywords: Dihydroartemisinin; Injectable powder; LC/MS/MS; Pharmacokinetics; Human plasma;
Remediation of undesirable secondary interactions encountered in hydrophilic interaction chromatography during development of a quantitative LC–MS/MS assay for a dipeptidyl peptidase IV (DPP-IV) inhibitor in monkey serum by Eugene P. Kadar; Chad E. Wujcik (471-476).
PF-00734200 (3,3-Difluoropyrrolidin-1-yl)-((2S,4S)-4-(4-(pyrimidin-2-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone) is an inhibitor of dipeptidyl peptidase IV (DPP-IV) for the treatment of diabetic complications and other disorders. A sensitive and selective LC–MS/MS assay capable of quantifying PF-00734200 in monkey serum was required to support regulated safety studies. Due to the polar nature of this compound and for ease of sample processing, hydrophilic interaction chromatography (HILIC) was identified as an ideal assay technique. During the initial phase of method development significant peak tailing was observed. The effects of polar organic modifier percentage, buffer concentration, column particle size, and flow rate were assessed to determine the final optimal conditions. PF-00734200 demonstrated a strong dependence on buffer concentration with respect to height equivalent to a theoretical plate (HETP), capacity factor (k′), and tailing factor (T). Improvements in chromatography were observed with increasing buffer concentration due to reduction of electrostatic secondary interactions with ionized silanols. A plot of log k′ versus percentage organic modifier at an elevated buffer concentration, produced a linear fit with a correlation coefficient of 0.996, indicating that the primary chromatographic retention mechanism was partitioning. A LC–MS/MS assay was successfully developed and validated for GLP bioanalysis of PF-00734200 in monkey serum utilizing the optimized HILIC conditions. Additionally, carryover was effectively minimized through fortification of ethylene glycol to the sample extract.
Keywords: Dipeptidyl peptidase IV (DPP-IV) inhibitor; Bioanalysis; Hydrophilic interaction chromatography; HILIC; LC–MS/MS;
Determination of asiatic acid in beagle dog plasma after oral administration of Centella asiatica extract by precolumn derivatization RP-HPLC by Xiao-Chun Zheng; Sheng-Hao Wang (477-481).
A novel precolumn derivatization reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV–vis detection for the quantitative determination of total concentration of asiatic acid (AA) in beagle dog plasma is described. AA was extracted with n-hexane-dichloromethane-2-propanol (20:10:1, v/v/v) from plasma, which had been hydrolyzed by acid and derivatized with p-Toluidine. Chromatographic separation was achieved on a C18 column using gradient elution in a water–methanol system. Detection was set at UV wavelength of 248 nm. A calibration curve ranging from 0.01 to 1.5 μg/mL was shown to be linear, and the lower limit of quantification (LLOQ) was 0.01 μg/mL. The intra- and inter-day precisions which were determined by three different concentrations (0.05, 0.2 and 0.8 μg/mL) ranged from 4.4% to 13.1% and 4.6% to 14.2%, respectively. Mean extraction recoveries were no less than 65% for AA and ursolic acid (IS). Plasma samples containing asiatic acid were stable for 30 days at −20 °C. The method was successfully applied to a pharmacokinetic study in beagle dogs after oral administration of Centella asiatica extract, and the main pharmacokinetic parameters obtained were: T 1/2, 4.29 h; T max, 2.70 h; C max, 0.74 μg/mL; AUC0–t and AUC0–∞, 3.74 and 3.82 μg h/mL, respectively.
Keywords: Precolumn derivatization HPLC; Asiatic acid; Centella asiatica extract; Beagle dog; Pharmacokinetics;
Quantitative analysis of short-chain acyl-coenzymeAs in plant tissues by LC–MS–MS electrospray ionization method by M. Ann D.N. Perera; Suh-Yeon Choi; Eve Syrkin Wurtele; Basil J. Nikolau (482-488).
Because acyl-CoAs play major roles in numerous anabolic and catabolic pathways, the quantitative determination of these metabolites in biological tissues is paramount to understanding the regulation of these metabolic processes. Here, we report a method for the analysis of a collection of short-chain acyl-CoAs (<6 carbon chain length) from plant extracts. Identification of each individual acyl-CoA was conducted by monitoring specific mass-fragmentation ions that are derived from common chemical moieties of all Coenzyme A (CoA) derivatives, namely the adenosine triphosphate nucleotide, pantothenate and acylated cysteamine. This method is robust and quick, enabling the quantitative analysis of up to 12 different acyl-CoAs in plant metabolite extracts with minimal post-extraction processing, using a 30 min chromatographic run-time.
Keywords: Coenzyme A; Acyl-CoA; LC–MS–MS; Plants; Metabolites;
Determination of the residues of 50 anabolic hormones in muscle, milk and liver by very-high-pressure liquid chromatography–electrospray ionization tandem mass spectrometry by Yi Yang; Bing Shao; Jing Zhang; Yongning Wu; Hejun Duan (489-496).
A sensitive and specific method for determination of the residues of 50 anabolic hormones in muscle (pork, beef, shrimp), milk and pig liver was developed. Analytes were separated and acquired by liquid chromatography coupled with an electrospray ionization tandem mass spectrometer (LC–ESI–MS/MS). Target compounds were simultaneously extracted with methanol after enzyme hydrolysis, and purified using a graphitized carbon-black solid-phase extraction (SPE) and followed by NH2 SPE cartridge. Limits of quantification were 0.04–2.0 μg kg−1; average recoveries were 76.9–121.3%; and the relative standard deviation was 2.4–21.2%. This method has been successfully applied in real samples.
Keywords: Anabolic hormones; LC–ESI–MS/MS; Foods of animal origin; Solid-phase extraction;
Quantitative analysis of RU38486 (mifepristone) by HPLC triple quadrupole mass spectrometry by Natalie Z.M. Homer; Rebecca M. Reynolds; Cecilia Mattsson; Matthew A. Bailey; Brian R. Walker; Ruth Andrew (497-501).
A sensitive liquid chromatography–mass spectrometric method was validated for the quantification of RU38486 (mifepristone) in human and murine plasma. The analyte and internal standard (alfaxolone) were extracted by liquid–liquid extraction with diethyl ether, resolved on a C18 column using gradient elution with methanol and ammonium acetate and detected after positive electrospray ionization (m/z 430 → 372; m/z 333 → 297, respectively). Quantification was linear over the range 0.5–500 ng (r 2 > 0.997), precise and accurate (intra-assay RSD ≤ 7.2%, RME ≤ 8.2%; inter-assay RSD ≤ 15.7% RME ≤ 10.2%). The limit of quantification (LOQ) was 50 pg injected on column, permitting reproducible analysis of RU38486 in small volumes of plasma.
Keywords: RU38486; Mifepristone; Steroid; Mass spectrometry; Liquid chromatography;
A simple and sensitive HPLC–ESI-MS/MS method for the determination of andrographolide in human plasma by Li Xu; Da-wei Xiao; Sheng Lou; Jian-jun Zou; Yu-bing Zhu; Hong-wei Fan; Guang-ji Wang (502-506).
A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the determination of andrographolide in human plasma was established. Dehydroandrographolide was used as the internal standard (I.S.). The plasma samples were deproteinized with methanol and separated on a Hanbon C18 column with a mobile phase of methanol–water (70:30, v/v). HPLC–ESI-MS/MS was performed in the selected ion monitoring (SIM) mode using target ions at [M−H2O–H]−, m/z 331.1 for andrographolide and [M−H]−, m/z 331.1 for the I.S. Calibration curve was linear over the range of 1.0–150.0 ng/mL. The chromatographic separation was achieved in less than 6.5 min. The lower limits of quantification (LLOQ) was 1.0 ng/mL. The intra and inter-run precisions were less than 6.95 and 7.22%, respectively. The method was successfully applied to determine the plasma concentrations of andrographolide in Chinese volunteers.
Keywords: Andrographolide; HPLC–ESI-MS/MS; Determination; Pharmacokinetics;
Determination of biogenic amines in beer with pre-column derivatization by high performance liquid chromatography by Tao Tang; Tianyu Shi; Kun Qian; Pingliang Li; Jianqiang Li; Yongsong Cao (507-512).
Eighteen samples of commercially available Chinese beer were analyzed in order to determine the content of biogenic amines. The method involves pre-column derivatization of the amines with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) and subsequent analysis by RP-HPLC (reversed phase-high performance liquid chromatography) with diode array detection. The labeled biogenic amines were separated on a Kromasil C18 column (250 mm × 4.6 mm, 5 μm) at room temperature and UV detection was applied at 254 nm. The separation of seven labeled biogenic amines was achieved within 22 min by elution acetonitrile and HAc–NaAc buffers. The method linearity, calculated for each biogenic amine, has a correlation coefficient higher than 0.9925, in concentrations ranging from 2.9 μmol L−1 to 565 μmol L−1. Detection limits of biogenic amines were 0.056–0.87 μmol L−1, at a signal-to-noise ratio of 3. The proposed method has been applied to the quantitative determination of spermine, phenethylamine, spermidine, histamine, tyramine, tryptamine and putrescine in beer with recoveries of 91.9–103.1% and R.S.D. of 2.86–5.63%. Quantitation is relative to external standards. The results showed that each kind of beer examined contained at least three biogenic amines. Putrescine, histamine and tyramine were detected in all samples. Spermidine was detected in 89% of the beers. Spermine, tryptamine and phenylethylamine occurred in 78%, 61% and 44% of the beers examined, respectively. These levels were below the level that may elicit direct adverse reactions for most consumers.
Keywords: Biogenic amines; Pre-column derivatization; 4-Chloro-3,5-dinitrobenzotrifluoride (CNBF); High performance liquid chromatography; Chinese beer;
Development and validation of an LC–MS/MS method for quantification of cyclic guanosine 3′,5′-monophosphate (cGMP) in clinical applications: A comparison with a EIA method by Yanhua Zhang; Dawn Dufield; Jon Klover; Wenlin Li; Gabriella Szekely-Klepser; Christopher Lepsy; Nalini Sadagopan (513-520).
An LC–MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3′,5′-monophosphate (cGMP) in human plasma. The LC–MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC–MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, 13C10,15N5 -cGMP, which was biosynthesized in-house, was used in the LC–MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6–10.1% CV and −3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6–8.1% CV and −2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9–27.1% CV and −4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1–39.5% CV and −30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R 2 = 0.197, P = 0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5–20 ng/mL. The LC–MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.
Keywords: cGMP; Biomarker; Human plasma; LC–MS/MS; EIA; Comparison;
Expanded bed adsorption of an alkaline lipase from Pseudomona cepacia by Giovana da Silva Padilha; José Carlos Curvelo-Santana; Ranulfo Monte Alegre; Elias Basile Tambourgi (521-526).
An extracellular lipase was isolated from Pseudomona cepacia by expanded bed adsorption on an Amberlite 410 ion-exchange resin. Enzyme characterization and hydrodynamic study of a chromatography column were done. Enzyme purification was done at three condition of expanded bed height (H): at one and half (6 cm), at two (8 cm) and at three (12 cm) times the fixed bed height (H 0 = 4 cm). The results showed that the experimental data was fitted to the Richardson and Zaki equation, and the comparison between the experimental and calculated terminal velocities showed low relative error. In enzyme purification for better condition, a purification factor of about 80 times was found at 6 cm of expanded bed height, or 1.5 times of expansion degree. Purified lipase had an optimal pH and a temperature of 8 and 37 °C, respectively.
Keywords: Expanded bed; Chromatography; Lipase; Pseudomona cepacia; Richardson–Zaki; Characterization;
Determination of eptifibatide concentration in human plasma utilizing the liquid chromatography–tandem mass spectrometry method by Jia Liu; Xiaotao Duan; Xiaoyan Chen; Dafang Zhong (527-532).
A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to determine the concentration of eptifibatide in human plasma. Following protein precipitation, the analyte was separated on a reversed-phase C18 column. Acetonitrile:5 mM ammonium acetate:acetic acid (30:70:0.1, v/v/v) was used at a flow-rate of 0.5 mL/min with the isocratic mobile phase. An API 4000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. “Truncated” multiple reaction monitoring using the transition of m/z 832.6 → m/z 832.6 and m/z 931.3 → m/z 931.3 was performed to quantify eptifibatide and the internal standard (EPM-05), respectively. The method had a lower limit of quantification of 4.61 ng/mL for eptifibatide. The calibration curve was demonstrated to be linear over the concentration range of 4.61 − 2770 ng/mL. The intra- and inter-day precisions were less than 10.5% for each QC level, and the inter-day relative errors were 2.0%, 5.6%, and 2.8% for 9.22, 184, and 2490 ng/mL, respectively. The validated method was successfully applied to the quantification of eptifibatide concentration in human plasma after intravenous (i.v.) administration of a 270-μg/kg bolus of eptifibatide and i.v. administration of eptifibatide at a constant rate of infusion of 2 μg/(kg min) for 18 h in order to evaluate the pharmacokinetics.
Keywords: Eptifibatide; LC–MS/MS; Plasma;
Chromatographic separation of (E)- and (Z)-isomers of entacapone and their simultaneous quantitation in human plasma by LC–ESI-MS/MS by Manish Yadav; Pramod Dixit; Vikas Trivedi; Abhishek Gandhi; Arvind Senger; Swati Guttikar; Puran Singhal; Pranav S. Shrivastav (533-540).
A selective, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of (E)-entacapone and (Z)-entacapone in human plasma. Sample clean-up involved liquid–liquid extraction (LLE) of both the isomers and carbamazepine used as internal standard from 500 μL of human plasma. Both the analytes were chromatographically separated with a resolution factor of 3.0 on a Gemini C18 (50 mm × 4.6 mm, 5 μm particle size) analytical column using 1% formic acid and methanol (50:50, v/v) as the mobile phase. The selectivity factor (α) of the column for the separation was 2.0, based on the capacity factors of 2.6 and 1.3 for (E)- and (Z)-isomers respectively. The parent → product ion transitions for both the isomers (m/z 306.1 → 233.0) and IS (m/z 237.3 → 194.2) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 24.3–6076 ng/mL and 23.8–5960 ng/mL for (E)-entacapone and (Z)-entacapone respectively. Matrix effect was assessed by post-column analyte infusion experiment and the process/extraction efficiency found was 94.3% and 89.3% for (E)- and (Z)-isomers respectively. The method was successfully applied to a pivotal bioequivalence study in 36 healthy human subjects after oral administration of 200 mg (E)-entacapone tablet formulation under fasting conditions.
Keywords: (E)-entacapone; (Z)-entacapone; Chromatographic separation; LC–ESI-MS/MS; High throughput; Human plasma;
Confirmatory analysis of firocoxib in bovine milk by rapid resolution liquid chromatography tandem mass spectrometry by Geraldine Dowling; Pasquale Gallo; Liam Regan (541-546).
A rapid method has been developed to analyse for firocoxib (FIRO) residue in bovine milk. Milk samples were extracted with acetonitrile and sample extracts were purified on Evolute™ ABN solid phase extraction cartridges. Aliquots were analysed by rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS). The method was validated in bovine milk, according to the criteria defined in Commission Decision 2002/657/EC. The decision limit (CCα) was 1.18 ng/mL and for the detection capability a (CCβ) value of 2.02 ng/mL was obtained. The measurement uncertainty of the method was 27%. Fortifying bovine milk samples (n = 18) in three separate assays, show the accuracy of the method to be between 96 and 105%. The precision of the method, expressed as RSD values for the within-lab reproducibility at the three levels of fortification (5, 7.5 and 10 ng/mL) was less than 11% respectively.
Keywords: Firocoxib; Bovine milk; Method validation; Decision limit; Detection capability; Mass spectrometry;
Normalization strategies for metabonomic analysis of urine samples by Bethanne M. Warrack; Serhiy Hnatyshyn; Karl-Heinz Ott; Michael D. Reily; Mark Sanders; Haiying Zhang; Dieter M. Drexler (547-552).
Unlike plasma and most biological fluids which have solute concentrations that are tightly controlled, urine volume can vary widely based upon water consumption and other physiological factors. As a result, the concentrations of endogenous metabolites in urine vary widely and normalizing for these effects is necessary. Normalization approaches that utilized urine volume, osmolality, creatinine concentration, and components that are common to all samples (“total useful MS signal”) were compared in order to determine which strategies could be successfully used to differentiate between dose groups based upon the complete endogenous metabolite profile. Variability observed in LC/MS results obtained from targeted and non-targeted metabonomic analyses was highly dependent on the strategy used for normalization. We therefore recommend the use of two different normalization techniques in order to facilitate detection of statistically significant changes in the endogenous metabolite profile when working with urine samples.
Keywords: Normalization; Mass spectrometry; Metabonomics; Non-targeted;
A high-performance liquid chromatography method for the quantification of cysmethynil, an inhibitor of isoprenylcysteine carboxylmethyl transferase, in mouse plasma by Mei Wang; Yok Moi Khoo; Jin Zhou; Patrick Casey; How Sung Lee (553-557).
Cysmethynil, a newly identified small molecule inhibitor of isoprenylcysteine carboxylmethyl transferase (Icmt) is involved in the post-translational modification of CaaX proteins. Cysmethynil causes cell death in many human cancer cells in vitro, and inhibits tumor growth in the xenograft mouse model in vivo. A HPLC method for the quantification of cysmethynil in mouse plasma was developed and validated. The lower limit of quantification of this method was 0.01 μg/ml. Inter- and intra-day variability ranged from 0.38–8.5% and accuracy was between 86% and 98%. This sensitive method was used to quantify cysmethynil in plasma of mice after intraperitoneal dosing for preliminary pharmacokinetic studies.
Keywords: Isoprenylcysteine carboxylmethyltransferase (Icmt); Cysmethynil; HPLC; Drug quantification; Preclinical study;
Validation of high performance liquid chromatography–electrochemical detection methods with simultaneous extraction procedure for the determination of artesunate, dihydroartemisinin, amodiaquine and desethylamodiaquine in human plasma for application in clinical pharmacological studies of artesunate–amodiaquine drug combination by Choon-Sheen Lai; N.K. Nair; A. Muniandy; S.M. Mansor; P.L. Olliaro; V. Navaratnam (558-562).
With the expanded use of the combination of artesunate (AS) and amodiaquine (AQ) for the treatment of falciparum malaria and the abundance of products on the market, comes the need for rapid and reliable bioanalytical methods for the determination of the parent compounds and their metabolites. While the existing methods were developed for the determination of either AS or AQ in biological fluids, the current validated method allows simultaneous extraction and determination of AS and AQ in human plasma. Extraction is carried out on Supelclean LC-18 extraction cartridges where AS, its metabolite dihydroartemisinin (DHA) and the internal standard artemisinin (QHS) are separated from AQ, its metabolite desethylamodiaquine (DeAQ) and the internal standard, an isobutyl analogue of desethylamodiaquine (IB-DeAQ). AS, DHA and QHS are then analysed using Hypersil C4 column with acetonitrile–acetic acid (0.05 M adjusted to pH 5.2 with 1.00 M NaOH) (42:58, v/v) as mobile phase at flow rate 1.50 ml/min. The analytes are detected with an electrochemical detector operating in the reductive mode. Chromatography of AQ, DeAQ and IB-DeAQ is carried out on an Inertsil C4 column with acetonitrile–KH2PO4 (pH 4.0, 0.05 M) (11:89, v/v) as mobile phase at flow rate 1.00 ml/min. The analytes are detected by an electrochemical detector operating in the oxidative mode. The recoveries of AS, DHA, AQ and DeAQ vary between 79.1% and 104.0% over the concentration range of 50–1400 ng/ml plasma. The accuracies of the determination of all the analytes are 96.8–103.9%, while the variation for within-day and day-to-day analysis are <15%. The lower limit of quantification for all the analytes is 20 ng/ml and limit of detection is 8 ng/ml. The method is sensitive, selective, accurate, reproducible and suited particularly for pharmacokinetic study of AS–AQ drug combination and can also be used to compare the bioavailability of different formulations, including a fixed-dose AS–AQ co-formulation.
Keywords: Artesunate; Dihydroartemisinin; Amodiaquine; Desethylamodiaquine; Drug combination; Solid phase extraction; HPLC–EC; Pharmacokinetics; Bioavailability;
Capillary zone electrophoresis method development for the analysis of Hippeastrum hybrid agglutinin samples by Alexandre Zatkovskis Carvalho; Tine Dupas; Joachim Brouwers; Jos Hoogmartens; Patrick Augustijns; Ann Van Schepdael (563-567).
A capillary zone electrophoresis (CZE) method was developed aiming the analysis of Hippeastrum hybrid agglutinin (HHA) samples. HHA is presently being tested as a vaginal microbicide to prevent HIV transmission. It acts by direct binding to mannose residues that are abundantly present on the HIV gp120 envelope and so interrupts the virus entry process. The final CZE method employs 50 mM sodium tetraborate (pH 9.9) as background electrolyte. In this condition, a cluster of about 30 isoform peaks is obtained, with very repeatable patterns. RSDs in the order of 0.2% for the migration time and detection sensitivity in the order of 70 μg ml−1 were achieved.
Keywords: Capillary electrophoresis; Hippeastrum hybrid agglutinin; Lectin; Amaryllis; Anti-HIV microbicide;
Liquid chromatography–tandem mass spectrometry method for the simultaneous quantitative determination of the organophosphorus pesticides dimethoate, fenthion, diazinon and chlorpyrifos in human blood by Paul Salm; Paul J. Taylor; Darren Roberts; Janaka de Silva (568-574).
Simultaneous determination of the organophosphorus pesticides dimethoate, fenthion, diazinon and chlorpyrifos in human blood by HPLC–tandem mass spectrometry was developed and validated. The pesticides were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Luna C18 (30 mm × 2.0 mm, 3 μm) column, using a step-gradient at a flow rate of 0.4 ml/min. The assay was linear from 0.5 to 100 ng/ml (r 2 > 0.992, n = 24) for all pesticides. The inter- and intra-day accuracy and precision for the method was 96.6–106.1% and <10%, respectively. The lower limit of quantification was 0.5 ng/ml. In conclusion, the method described displays analytical performance characteristics that are suitable for the quantification of these pesticides in cases of acute poisoning.
Keywords: Organophosphorus pesticides; Dimethoate; Fenthion; Diazinon; Chlorpyrifos; HPLC; Mass spectrometry;
Quantification of HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in peripheral blood mononuclear cell lysate using liquid chromatography coupled with tandem mass spectrometry by R. ter Heine; M. Davids; H. Rosing; E.C.M. van Gorp; J.W. Mulder; Y.T. van der Heide; J.H. Beijnen; A.D.R. Huitema (575-580).
For pharmacokinetic monitoring, measurement of antiretroviral agents in plasma is the gold standard. However, human immunodeficiency virus protease inhibitors (PIs) or non-nucleoside reverse transcriptase inhibitors (NNRTIs) exert their action within the infected cell. Cell-associated concentrations may therefore more adequately reflect therapy outcome. Therefore, for the quantification of nine PIs (amprenavir, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir), 1 active PI metabolite (nelfinavir M8) and 2 NNRTIs (efavirenz and nevirapine) in lysate of peripheral blood mononuclear cells (PBMCs) an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. Analytes were extracted from a PBMC pellet by means of a one-step extraction with 50% methanol containing the internal standards D6-indinavir, D5-saquinavir, 13C6-efavirenz and dibenzepine. Chromatographic separation was performed on a reversed phase C18 column (150 mm × 2.0 mm, particle size 5 μm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25 mL/min. The analytical run time was 10 min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 1–500 ng/mL in PBMC lysate for all analytes. The method was proven to be specific, accurate, precise and robust. The mean precision and accuracy was less than ±12% at all concentration levels. Using the developed assay and a previously developed assay for these analytes in plasma, the relationship between plasma and intracellular pharmacokinetics and their relationship with therapy outcome can now be determined.
Keywords: Antiretroviral; HIV; Pharmacology; LC-MS; HPLC; TDM; Intracellular; LC-MS/MS;
Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma by Dan Zhang; Yingwu Wang; Jiangbin Han; Weisong Yu; Lili Deng; J. Paul Fawcett; Zeyuan Liu; Jingkai Gu (581-585).
This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh2, were extracted from plasma by liquid–liquid extraction and separated on a Zorbax extend C18 analytical column using methanol–acetonitrile-10 mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1–100.0 ng/ml with a limit of detection of 0.05 ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25 mg capsule.
Keywords: 20(S)-Protopanaxadiol; LC–MS/MS; Human plasma; Clinical pharmacokinetics;