Rapid analysis of MMI270B, an inhibitor of matrix metalloproteases in human plasma by liquid chromatography-tandem mass spectrometry: matrix interference in patient samples

A high-throughput method was developed and validated for the quantitative determination of MMI270B, an inhibitor of matrix metalloprotease (MMP) enzymes, in human plasma. The method was based on reverse-phase chromatographic separation of the analyte from plasma extract followed by atmospheric pressure chemical ionization (APCI) and tandem mass spectrometry in the selected reaction monitoring mode (SRM). Extraction was performed using simple protein fi ltration in the 96-well plate format to increase the throughput of the method. Optimised chromatographic separation in a short column (30 x 4.6 mm i.d.) coupled with positive APCI mode of ionization followed by selective SRM mode of detection yielded clean chromatograms with minimal signal suppression. The chromatographic conditions resolved isobaric interference peaks observed in samples from patients dosed with MMI270B. The standard curve was linear (r = 0.997) within the concentration range of 1.04 (lower limit of quanti fi cation) to 1040 ng/mL using 0.1 mL of human plasma. The accuracy of the method varied from 93.6 to 103% with a precision of 2.17-6.71% over the concentration range. The method was simple, rapid, and robust with an analyte recovery of >98%.     

Measurement of 19-nortestosterone and its esters in equine plasma by high-performance liquid chromatography with tandem mass spectrometry

A high-performance liquid chromatographic-tandem mass spectrometric (HPLC/MS/MS) method for the determination of 19-nortestosterone and its esters (cyclopentanepropionate, phenylpropionate, and decanoate) in equine plasma is achieved using an atmospheric pressure chemical ionization (APCI) interface in selected reaction monitoring (SRM) mode. The two internal standards used were 16,16, 17-(2)H(3)-19-nortestosterone for 19-nortestosterone and methenolone acetate for its esters. The steroids studied were extracted from plasma samples with a mixture of diethyl ether/n-hexane (9:1, v/v). The quantification limits for 19-nortestosterone, 19-nortestosterone cyclopentanepropionate, 19-nortestosterone phenylpropionate, and 19-nortestosterone decanoate were 0.16, 5.0, 0.1, and 2.0 ng/mL, respectively, when 2 mL of plasma were used. The recoveries of most of the steroids were 71.6-101.0% except for the decanoate, which could be recovered to about 39.8%. The responses were linear, with correlation coefficients varying from 0.9897 to 0.9999 in the concentration range of 0.1 to 50.0 ng/mL for the steroids studied. When applied to equine (mare) plasma samples, the present method allowed detection of 19-nortestosterone up to 23 days after an intra-muscular injection of 400 mg as the decanoate.     

Method development for the concentration determination of a protein in human plasma utilizing 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometric detection

Although liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology has been widely used for quantitative analysis of small organic molecules, it has been a challenging task to quantitatively analyze protein samples utilizing this technology in biological matrices for pre-clinical and clinical studies. Here we present our initial results in method development for the quantitative determination of rK5 protein concentrations in human plasma samples utilizing LC/MS/MS technology. A protein similar in structure to rK5, but with a slightly reduced molecular weight, was used as internal standard. A 96-well solid-phase extraction procedure was developed to effectively extract protein analytes from plasma samples. Quantitative analysis was obtained by a novel approach of protein monitoring that employed selective reaction monitoring (SRM). Even though mass spectrometry of the internal standard protein gave no fragment ions, SRM monitoring greatly reduced background interference. Using samples prepared in human plasma with sodium EDTA as anticoagulant, a correlation coefficient (r(2)) of 0.9940 was obtained by producing a single standard curve with the injection of six rows of standards with a concentration range from 100 ng/mL to 10 microg/mL. The mean analytical recovery for these standards ranged from 91.5 to 103.6%. The CVs for individual standard levels ranged from 3.7 to 20.9%. The experiment was also repeated using samples prepared in human plasma with sodium heparin as anticoagulant, which produced a correlation coefficient (r(2)) of 0.9952 obtained from a single standard curve with the injection of four rows of standards with a concentration range from 50 ng/mL to 10 microg/mL. The mean analytical recovery for the standards ranged from 96.2 to 104.6%. The CVs for individual standard levels ranged from 2.6 to 15.6%.     

Simultaneous determination of docetaxel and ketoconazole in rat plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for simultaneous quantification of docetaxel and ketoconazole in rat plasma with paclitaxel as internal standard (IS). The analytes were extracted from rat plasma by using a liquid-liquid extraction technique with ethyl acetate and the LC separation was performed on a Cosmosil-C(18) analytical column (150 mm x 2.0 mm i.d., Nacalai Tesque Inc., Japan). The extracted samples were analyzed with LC/MS/MS, operating in selected reaction monitoring (SRM) mode. The SRM transitions of precursor ions to product ions were 830.3-->549.1 (m/z) for docetaxel, 531.2-->489.3 (m/z) for ketoconazole, and 876.7-->307.9 (m/z) for the IS. The calibration curves were linear over the range of 2-500 ng/mL for docetaxel and 50-20 000 ng/mL for ketoconazole, with coefficients of correlation above 0.999. The limits of quantification for docetaxel and ketoconazole were both 2 ng/mL. The limit of detection for each analyte was 1 ng/mL. The intra- and inter-day precision (CV) of analysis were within 7%, and the accuracy ranged from 95 to 110%. The overall recoveries for docetaxel and ketoconazole were about 89.0% and 91.1%, respectively. The total analysis time was only 9.0 min. This quantitation method was successfully applied to the simultaneous determination of docetaxel and ketoconazole in rat plasma and some potential interaction was found in the current coadministration pharmacokinetic study. This established method was also utilized in the in vitro and in vivo drug-drug interaction study of docetaxel and ketoconazole (to be published).     

A rapid and sensitive method for the quantification of ganciclovir in plasma using liquid chromatography/selected reaction monitoring/mass spectrometry

A method using reversed-phase liquid chromatography coupled with electrospray ionization and selected reaction monitoring mass spectrometry has been developed for the quantitative analysis of ganciclovir in rat plasma. Acyclovir, a structurally related analog of ganciclovir, was used as the internal standard. A small volume of plasma (50 microL) was spiked with the internal standard and plasma proteins were precipitated by methanol. The supernatant was dried under nitrogen, and then reconstituted in water. The use of liquid chromatography/selected reaction monitoring/mass spectrometry effectively eliminated potential interference from endogenous constituents in the plasma. This highly selective and sensitive method made it possible to analyze plasma ganciclovir with a lower limit of quantitation of 10 ng/mL. The assay was reproducible and linear in the range 10-10,000 ng/mL. The precision and accuracy values were in the range 2.0-6.9% and 89.0-109.6%, respectively. The analyte recovery was greater than 88%. This method was successfully used to monitor the pharmacokinetic profile of ganciclovir in normal rats following intraperitoneal administration of the drug.     

Direct determination of dialkyl phosphates by strong anion-exchange liquid chromatography/atmospheric pressure chemical ionization mass spectrometry using a quadrupole ion trap instrument

The direct determination of dialkyl phosphates (DAPs) in water by strong anion-exchange (SAX) liquid chromatography/atmospheric pressure chemical ionization (APCI) mass spectrometry was investigated. The SAX high-performance liquid chromatography (HPLC) column was eluted with methanol/water gradients containing ammonium formate (AF) separating the DAPs which included six dimethyl- and diethyl-substituted phosphates, thiophosphates, and dithiophosphates. The high buffer concentrations required for separation were compatible with -ve APCI, but in +ve APCI the DAPs were unstable giving anomalous ions such as [M+15]+ and [M+29]+. These ions are believed to result from ion molecule reactions with CH3OH2+ in the plasma. DAPs are very stable in -ve APCI being detected as abundant [M-H](-) ions, even with 200 mM AF. At higher AF concentration formate clusters ([M+45](-) and [M+91](-)) were seen. Fragmentation by collision-activated dissociation (CAD) was more efficient for deprotonated ethyl-substituted DAPs which lost ethylene followed by ethanol. APCI instrument detection limits were in the low ng/mL range and the response was highly linear. Isotope dilution quantitation using d10-diethyl dithiophosphate (DEDTP) as an internal standard produced an instrument detection limit of 2 ng DEDTP/mL and method detection limit (MDL) of 9.3 ng/mL with accuracy of 99% (spike concentration, 25 ng/mL). DAP mixtures required storage in cold, dry conditions and alcohol solvents should be avoided because of solvolysis reactions.     

Development and validation of a liquid chromatographic/tandem mass spectrometric method for the determination of sertraline in human plasma

A sensitive and rapid liquid chromatographic/tandem mass spectrometric method was developed and validated for the determination of sertraline in human plasma. The analyte and internal standard (IS, diphenhydramine) were extracted with 3 mL of diethyl ether/dichloromethane (2:1, v/v) from 0.25 mL plasma, then separated on a Zorbax Eclipse XDB C18 column using methanol/water/formic acid (75:25:0.1, v/v/v) as the mobile phase. The triple quadrupole mass spectrometry was applied via an atmospheric pressure chemical ionization (APCI) source for detection. The fragmentation pattern of the protonated sertraline was elucidated with the aid of product mass spectra of isotopologous peaks. Quantification was performed using selected reaction monitoring of the transitions of m/z 306 --> 159 for sertraline and m/z 256 --> 167 for the IS. The method was linear over the concentration range of 0.10-100 ng/mL. The intra-day and inter-day precisions, expressed by relative standard deviation, were both less than 6.7%. Assay accuracies were within +/-6.9% as terms of relative error. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.10 ng/mL with a precision of 8.3% and an accuracy of 9.6%. The validated method has been successfully applied for the pharmacokinetic study and bioequivalence evaluation of sertraline in 18 healthy volunteers after a single oral administration of 50 mg sertraline hydrochloride tablets.     

Determination of miglitol in human plasma by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method for the determination of miglitol in human plasma using voglibose as internal standard has been developed and validated. Samples of plasma were deproteinated with acetonitrile and washed with dichloromethane before being analyzed by reversed-phase high-performance liquid chromatography (HPLC). Separation was carried out on a short Nucleosil C(18) column (5 microm, 50 x 4.6 mm i.d.) using 10 mmol/L ammonium acetate at 1.0 mL/min as mobile phase. The detector was an Applied Biosystems Sciex API 4000 mass spectrometer using atmospheric pressure chemical ionization (APCI) for ion production. The instrument was operated at unit resolution in the multiple reaction monitoring mode. The assay was linear over the range 5.00-2000 ng/mL with a limit of detection of 1.00 ng/mL. Intra- and inter-day precision were <2.82% and <2.92%, respectively, with accuracy of 93.3-106%. The assay was successfully applied to a clinical pharmacokinetic study of miglitol given as a single oral dose (50 mg) to healthy volunteers.     

Quantitative determination of fluvastatin in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry using [18O2]-fluvastatin as an internal standard

A sensitive and specific method for the quantitative determination of fluvastatin in human plasma is presented. The drug was isolated from plasma by extractive alkylation with pentafluorobenzyl bromide and further derivatized to the bis-trimethylsilyl derivative. [18O2]-Fluvastatin was prepared from unlabelled fluvastatin and used as an internal standard. Gas chromatography/mass spectrometry under negative ion chemical ionization conditions was used for quantitative measurement of the drug, using m/z 554.26 and 558.26 for target and internal standard, respectively. Calibration graphs were linear within a range of 2 and 512 ng mL(-1) plasma. Intra-day precision was 0.94% (2 ng mL(-1)), 2.53% (8.2 ng mL(-1)), 2.16% (81.9 ng mL(-1)) and 3.26% (409.6 ng mL(-1)); inter-day variability was found to be 1.64% (2 ng mL(-1)), 0.97% (8.2 ng mL(-1)), 1.97% (81.9 ng mL(-1)) and 2.01% (409.6 ng mL(-1)). Intra-day accuracy showed deviations of 0.6% (2 ng mL(-1)), 0.37% (8.2 ng mL(-1)), -1.52% (81.9 ng mL(-1)) and -1.67% (409.6 ng mL(-1)); inter-day accuracy was of -1.64% (2 ng mL(-1)), -1.13% (8.2 ng mL(-1)), -2.28% (81.9 ng mL(-1)) and -0.46% (409.6 ng mL(-1)). The stable isotope labelled standard was found to be stable under the analytical conditions.     

Urine drug testing for opioids,cocaine, and metabolites by direct injection liquid chromatography/tandem mass spectrometry

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantification of opioids, cocaine, and metabolites in urine was developed and validated. A 10-microL aliquot of urine was injected directly onto the LC/MS/MS system. The lack of sample preparation substantially reduced total analysis time. Separation was performed by reversed-phase chromatography with gradient elution for all analytes in 26 min. Atmospheric pressure chemical ionization (APCI) was a rugged and efficient ionization technique for basic drugs. Identification and quantification was based on selected reaction monitoring (SRM). Calibration, with deuterated internal standards, was performed by linear regression analysis (weighting factor 1/x). Limits of quantitation (LOQ) were established between 10-100 ng/mL and linearity was obtained up to a maximum of 10 000 ng/mL with an average correlation coefficient (R(2)) > 0.99. Analytical validation criteria for specificity, precision, accuracy, dilution integrity, matrix effect, and stability were fulfilled. The method proved to be simple and time efficient, and was applicable for illicit drug use monitoring and methadone treatment compliance in clinical research projects at the National Institute on Drug Abuse (NIDA).     

Use of 18O3-clodronate as an internal standard for the quantitative analysis of clodronate in human plasma by gas chromatography/electron ionisation mass spectrometry

A sensitive and specific method for the quantitative determination of clodronate in human plasma is presented. The drug was extracted from plasma by anion-exchange chromatography and derivatised to the tetra-tert-butyldimethylsilyl derivative. 18O3-Clodronate was prepared from unlabeled clodronate and used as an internal standard. Gas chromatography/mass spectrometry (GC/MS) under electron ionisation conditions was used for quantitative measurement of the drug, using m/z 643.16 and 651.17 for target and internal standard, respectively. Calibration graphs were linear within the range of 10-1280 ng/mL plasma. Intra-day precision was 1.8% (10 ng/mL), 0.5% (40 ng/mL), 1.0% (120 ng/mL), 0.5% (200 ng/mL), 0.5% (400 ng/mL) and 2.7% (800 ng/mL) and inter-day variability was found to be 0.7% (10 ng/mL), 1.6% (40 ng/mL), 1.3% (120 ng/mL), 2.3% (200 ng/mL), 2.5% (400 ng/mL) and 1.2% (800 ng/mL). Intra-day accuracy showed deviations of 0.8% (10 ng/mL), 0.8% (40 ng/mL), 0.9% (120 ng/mL), 0.9% (200 ng/mL), 1.9% (400 ng/mL) and 0.3% (800 ng/mL) and intra-day accuracy was of -1.4% (10 ng/mL), 0.0% (40 ng/mL), -0.7% (120 ng/mL), -0.4% (200 ng/mL), -1.2% (400 ng/mL) and -3.3% (800 ng/mL). The stable isotope labeled standard was found to be stable under analysis conditions.     

Quantification of zolpidem in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple and robust method for quantification of zolpidem in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Es-citalopram was used as an internal standard. Zolpidem and internal standard in plasma sample were extracted using solid-phase extraction cartridges (Oasis HLB, 1 cm3/30 mg). The samples were injected into a C8 reversed-phase column and the mobile phase used was acetonitrile-ammonium acetate (pH 4.6; 10 mm) (80:20, v/v) at a flow rate of 0.7 mL/min. Using MS/MS in the selected reaction-monitoring (SRM) mode, zolpidem and Es-citalopram were detected without any interference from human plasma matrix. Zolpidem produced a protonated precursor ion ([M+H]+) at m/z 308.1 and a corresponding product ion at m/z 235.1. The internal standard produced a protonated precursor ion ([M+H]+) at m/z 325.1 and a corresponding product ion at m/z 262.1. Detection of zolpidem in human plasma by the LC-ESI MS/MS method was accurate and precise with a quantification limit of 2.5 ng/mL. The proposed method was validated in the linear range 2.5-300 ng/mL. Reproducibility, recovery and stability of the method were evaluated. The method has been successfully applied to bioequivalence studies of zolpidem.     

Determination of M+4 stable isotope labeled cortisone and cortisol in human plasma by microElution solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive microElution solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of M+4 stable isotope labeled cortisone and cortisol in human plasma. In this method, M+4 cortisone and M+4 cortisol were extracted from 0.3 mL of human plasma samples using a Waters Oasis HLB 96-well microElution SPE plate using 70 microL methanol as the elution solvent, and chromatographed on a Waters Symmetry C18 column (4.6 x 50 mm, 3.5 microm). M+9 cortisone and M+9 cortisol were used as the internal standards. A PE Sciex API 4000 tandem mass spectrometer interfaced with the liquid chromatograph via a turboionspray source was used for mass analysis and detection. The selected reaction monitoring (SRM) of precursor --> product ion transitions were monitored at m/z 365.2 [M+H](+) --> 167.0 and at m/z 367.3 [M+H](+) --> 125.1 for M+4 cortisone and M+4 cortisol, respectively. The lower limit of quantitation was 0.1 ng mL(-1) and the linear calibration range was from 0.1 to 100 ng mL(-1) for both analytes. This method demonstrated to be very reproducible and reliable.     

Quantitative analysis of diclazuril in animal plasma by liquid chromatography/electrospray ionization mass spectrometry

A novel, sensitive and specific method for the quantitative determination of diclazuril in animal plasma using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with negative ion detection is presented. Extraction of the samples was performed with a rapid deproteinization step using acetonitrile. Chromatography of diclazuril and the internal standard was achieved on a Nucleosil ODS 5-microm column, using a gradient elution with 0.01 M ammonium acetate and acetonitrile. To obtain the highest sensitivity and specificity possible, the mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The method was validated according to the requirements defined by the European Community. Calibration curves using plasma fortified between 1-100 ng/mL and 100-2000 ng/mL showed good linear correlation (r >or= 0.9991, goodness-of-fit coefficient 相似文献   

Rapid and sensitive analysis of azadirachtin and related triterpenoids from Neem (Azadirachta indica) by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

Based on reversed-phase high-performance liquid chromatography (RP-HPLC) and atmospheric pressure chemical ionization (APCI) mass spectrometry, a HPLC-MS method was developed to permit the rapid qualitative and quantitative analysis of azadirachtin and related tetranortriterpenoids from seeds and tissue cultures of Neem (Azadirachta indica). APCI+ standard scanning mass spectra of the major Neem triterpenoids were recorded and utilized to select suitable ions for selected ion monitoring (SIM). Transitions for selective reaction monitoring (SRM) were based on MS-MS experiments. Using SIM, major Neem triterpenoids were detected in callus culture material and seed kernels of A. indica. The limit of detection for azadirachtin in extract samples (approximately 1 ng ml(-1) or 10 pg in SIM mode) was determined to be (with respect to injected absolute amounts) approximately 1000-times lower than values quoted in the literature for existing HPLC methods (approximately 200 ng ml(-1) or 10 ng). In addition to high sensitivity, the HPLC-MS method is able to tolerate minimal sample preparation and purification, dramatically reducing total analysis time.     

Quantitative high-throughput determination of endogenous retinoids in human plasma using triple-stage liquid chromatography/tandem mass spectrometry

A high-throughput ultrasensitive analytical method based on liquid chromatography with positive ion atmospheric pressure chemical ionization (APCI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans-retinol (atROH) in human plasma. A stable isotope of atRA was used as internal standard (IS). The analytes and IS were isolated from 100 microL plasma by acetonitrile mono-phase extraction (MPE) performed in black 96-well microtiterplates. A 100 microL injection was focused on-column and chromatographed on an Agilent ZORBAX SB-C18 rapid-resolution high-throughput (RRHT) column with 1.8-microm particles (4.6 mmx50 mm) maintained at 60 degrees C. The initial mobile phase composition was acetonitrile/water/formic acid (10:90:0.1, v/v/v) delivered at 1.8 mL/min. Elution was accomplished by a fast gradient to acetonitrile/methanol/formic acid (90:10:0.1, v/v/v). The method had a chromatographic total run time of 7 min. An Applied Biosystems 4000 Q TRAP linear tandem mass spectrometer equipped with a heated nebulizer (APCI) ionization source was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 315.4-->297 (4-oxo-retinoic acids), 301.2-->205 (retinoic acids), 305.0-->209 (IS) and 269.2-->93 (retinol) used for quantification. The assay was fully validated and found to have acceptable accuracy, precision, linearity, sensitivity and selectivity. The mean extraction recoveries from spiked plasma samples were 80-105% for the various retinoids at three different levels. The intra-day accuracy of the assay was within 8% of nominal and intra-day precision was better than 8% coefficient of variance (CV) for retinoic acids. Inter-day precision results for quality control samples run over a 12-day period alongside clinical samples showed mean precision better than 12.5% CV. The limit of quantification was in the range of 0.1-0.2 ng/mL and the mass limit of detection (mLOD) was in the range 1-4 pg on column for the retinoic acids. The assay has been successfully applied to the analysis of 1700 plasma samples.     

Quantitative determination of medroxyprogesterone acetate in plasma by liquid chromatography/electrospray ion trap mass spectrometry.

A sensitive and rapid liquid chromatography/electrospray ion trap mass spectrometry (LC/MS/MS) method has been developed for the quantitative determination of medroxyprogesterone acetate (MPA) in human plasma. Plasma samples (1.0 mL) were simply extracted with pentane and the extracts were analyzed by HPLC with the detection of the analyte in the selective reaction monitoring (SRM) mode. The determination of MPA was accurate and reproducible, with a limit of quantitation of 0.05 ng/mL in plasma. The standard calibration curve for MPA was linear (r = 0.998) over the concentration range 0.05-6.0 ng/mL in human plasma. Analysis precision over the concentration range of MPA was lower than 18.8% (relative standard deviation, RSD) and accuracy was between 96.2 and 108.7%.     

Quantitative determination of acetylcholine in microdialysis samples using liquid chromatography/atmospheric pressure spray ionization mass spectrometry

A fast, simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of acetylcholine in rat brain microdialysis samples. The chromatographic separation was achieved in 3 min on a reversed-phase column with isocratic conditions using a mobile phase containing 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid (TFA). A stable isotope-labeled internal standard was included in the analysis and detection was carried out with a linear ion trap mass spectrometer using selected reaction monitoring (SRM). Analyte ionization was performed with an atmospheric pressure chemical ionization (APCI) source without applying discharge current (atmospheric pressure spray ionization). This special ionization technique offered significant advantages over electrospray ionization for the analysis of acetylcholine with reversed-phase ion-pairing chromatography. The lower limit of quantification was 0.15 nM (1.5 fmol on-column) and linearity was maintained over the range of 0.15-73 nM, providing a concentration range that is significantly wider than that of the existing LC/MS methods. Good accuracy and precision were obtained for concentrations within the standard curve range. The method was validated and has been used extensively for the determination of acetylcholine in rat brain microdialysis samples.     

Determination of midazolam in human plasma by solid-phase microextraction and gas chromatography/mass spectrometry.

A new method is described for the qualitative and quantitative analysis of midazolam, a short-acting 1,4-imidazole benzodiazepine, in human plasma. It involves a plasma deproteinization step, solid-phase microextraction (SPME) of midazolam using an 85-microm polyacrylate fiber, and its detection by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode, using pinazepam as internal standard. The assay is linear over a midazolam plasma range of 1.5-300 ng/mL, relative intra- and inter-assay standard deviations at 5 ng/mL are below 7%, and the limit of detection is 1 ng/mL. The method is simple, fast and sufficiently sensitive to be applied in clinical and forensic toxicology as well as for purposes of therapeutic drug monitoring.     

Simultaneous analysis of twenty-one glucocorticoids in equine plasma by liquid chromatography/tandem mass spectrometry

A method for the simultaneous separation, identification, quantification and confirmation of the presence of 21 glucocorticoids (GCC) in equine plasma by liquid chromatography coupled with triple stage quadrupole tandem mass spectrometry (LC/TSQ-MS/MS) is described. Plasma sample augmented with the 21 GCC was extracted with methyl tert-butyl ether (MTBE) and analyzed by positive electrospray ionization. Desoxymetasone or dichlorisone acetate was used as the internal standard (IS). Quantification was performed by IS calibration. For each drug, one major product ion was chosen and used for screening for that drug. Analyte confirmation was performed by using the three most intense product ions formed from the precursor ion and the corresponding mass ratios. The recovery of the 21 GCC when spiked into blank plasma at 5 ng/mL was 45-200% with coefficient of variation (CV) from 0.3-18%. The limit of detection (LOD) and that of quantification (LOQ) for most of the analytes were 50-100 pg/mL and 1 ng/mL, respectively, whereas that of confirmation (LOC) was 100-300 pg/mL depending on the analyte. Intra- and inter-day precisions expressed as CV for quantification of 1 and 10 ng/mL was 1.0-17%, and 0.51-19%, respectively, and the accuracy was from 84-110%. The linear concentration range for quantification was 0.1-100 ng/mL (r(2) > 0.997). Estimated measurement uncertainty was from 11-37%. This study was undertaken to develop a method for simultaneous screening, identification, quantification and confirmation of these agents in post-race equine plasma samples. The method has been successfully applied to screening of a large number of plasma samples obtained from racehorses in competition and in pharmacokinetic studies of dexamethasone in the horse and concurrent changes in endogenous GCC, hydrocortisone and cortisone. The method is simple, sensitive, selective and reliably reproducible.