Sensitive and simple determination of mannitol in human brain tissues by gas chromatography–mass spectrometry

A simple, reliable and sensitive gas chromatographic–mass spectrometric method was devised to determine the level of mannitol in various human brain tissues obtained at autopsy. Mannitol was extracted with 10% trichloroacetic acid solution which effectively precipitated brain tissues. The supernatant was washed with tert.-butyl methyl ether to remove other organic compounds and to neutralize the aqueous solution. Mannitol was then derivatized with 1-butaneboronic acid and subjected to GC–MS. Erythritol was used as an internal standard. For quantitation, selected ion monitoring with m/z 127 and 253 for mannitol and m/z 127 for internal standard were used. Calibration curves were linear in concentration range from 0.2 to 20 μg/0.1 g and correlation coefficients exceeded 0.99. The lower detection limit of mannitol in distilled water was 1 ng/0.1 g. Mannitol was detected in control brain tissues, as a biological compound, at a level of 50 ng/0.1 g. The precision of this method was examined with use of two different concentrations, 2 and 20 μg/0.1 g, and the relative standard deviation ranged from 0.8 to 8.3%. We used this method to determine mannitol in brain tissues from an autopsied individual who had been clinically diagnosed as being brain dead. Cardiac arrest occurred 4 days later.     

Liquid chromatography–fast atom bombardment mass spectrometry for detection and determination of pentazocine in human tissues

A reliable and sensitive method was developed for the detection and determination of pentazocine in human solid tissues using liquid chromatography–dynamic fast atom bombardment (FAB) mass spectrometry, combined with a three-step liquid–liquid extraction procedure. Levallorphan tartrate served as an internal standard. The extract was evaporated to dryness and dissolved in the mobile phase, acetonitrile–10 mM ammonium acetate solution (20:80, pH 4.0) containing 0.5% glycerol as FAB matrix. The eluent was pumped at a flow rate of 25 μl/min and split before introduction to FAB mass spectrometer. Quantitative analysis was carried out by means of monitoring quasi-molecular ions with m/z 286 for pentazocine and m/z 284 for levallorphan. The lower limit of detection of pentazocine in each tissue tested was 1 ng/g with scan mode and 0.1 ng/g with SIM mode. Using this method, the concentrations of pentazocine were determined in the tissues of an autopsied individual to perform toxicological evaluation.     

Determination of doxazosin and verapamil in human serum by fast LC-MS/MS: application to document non-compliance of patients

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of doxazosin and verapamil in human serum has been developed. Trimipramine-d₃ as an isotopic labelled internal standard was used for quantification. Serum samples were prepared by simple liquid–liquid extraction with mixture of tert butyl methyl ether and ethyl acetate (1:1, v:v). The analytes and internal standard were separated on C18 column using an isocratic elution with 5 mM ammonium formate with 0.02% formic acid and 0.02% formic acid in acetonitrile (55:45, v:v) at a flow rate of 1.1 mL/min. Positive TurboIonSpray mass spectrometry was used with multiple reaction monitoring of the transitions at: m/z 455.3 → 165.2 and 150.2 for verapamil, m/z 452.2 → 344.4 and 247.4 for doxazosin, m/z 298.2 → 103.1 for trimipramine-d₃. Linearity was achieved between 1 and 500 ng/mL (R² ≥ 0.997) for both analytes. An extensive pre-study method validation was carried out in accordance with FDA guidelines. This assay was successfully applied to determine the serum concentrations of doxazosin and verapamil in suspect non-compliance patients.     

A simple and sensitive HPLC–ESI-MS/MS method for the determination of andrographolide in human plasma

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.     

Enantiomeric determination of tramadol and O-desmethyltramadol in human plasma by fast liquid chromatographic technique coupled with mass spectrometric detection

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for enantiomeric determination of tramadol and its primary phase metabolite O-desmethyltramadol in human plasma has been developed. Tramadol hydrochloride – ¹³C, d_3, was used as an isotopic labeled internal standard for quantification. The method involves a simple solid phase extraction. The analytes and internal standard were separated on Lux Cellulose-2 packed with cellulose tris(3-chloro-4-methylphenylcarbamate) using isocratic elution with hexane/isopropanol/diethylamine (90:10:0.1, v/v/v) at a flow rate of 1.3 mL/min. The APCI positive ionization mass spectrometry was used with multiple reaction monitoring of the transitions at m/z 264.2 → 58.2 for tramadol, m/z 250.1 → 58.2 for O-desmethyltramadol and m/z 268.2 → 58.2 for internal standard. Linearity was achieved between 1–800 ng/mL and 1–400 ng/mL (R² ≥ 0.999) for each enantiomer of tramadol and O-desmethyltramadol, respectively. Intra-day accuracies ranged among 98.2–102.8%, 97.1–109.1% and 97.4–102.9% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged among 95.5–104.1%, 99.2–104.7%, and 94.2–105.6% at the lower, intermediate, and high concentration for all analytes, respectively. This assay was successfully used to determine the concentration of enantiomers of tramadol and O-desmethyltramadol in a pharmacogenetic study.     

Enantioselective assay of nisoldipine in human plasma by chiral high-performance liquid chromatography combined with gas chromatographic−mass spectrometry: applications to pharmacokinetics

Nisoldipine, a second-generation dihydropyridine calcium antagonist, is a racemate compound used in the treatment of hypertension and coronary heart disease. This study presents an enantioselective HPLC-GC–MS method for the analysis of nisoldipine in human plasma and establishes confidence limits for its application to pharmacokinetic studies. Plasma samples were basified and extracted with toluene. The enantiomers were resolved on a Chiralcel^® OD-H column using hexane–ethanol (97.5:2.5, v/v) and the (+)- and (−)-fractions were collected separately with the diode array detector switched off. For the quantification of the nisoldipine enantiomers a GC–MS with an Ultra 1 Hewlett-Packard column was used with the detector operated in the single-ion monitoring mode with electron-impact ionization (m/z 371.35 and 270.20 for nisoldipine and m/z 360.00 for the internal standard, nitrendipine). The method proved to be suitable for pharmacokinetic studies based on the low quantification limit (0.05 ng/ml for each enantiomer) and the broad linear range (0.05–50.0 ng/ml for each enantiomer). Low coefficients of variation (<15%) were demonstrated for both within-day and between-day assays. No interference from drugs associated with nisoldipine treatment was observed. The enantioselective pilot study on the kinetic disposition of nisoldipine administered in the racemic form to a hypertensive patient using a multiple dose regimen revealed the accumulation of the (+)-enantiomer with an AUC^0–24 (+)/(−) ratio of approximately 8. Both enantiomers were quantified in plasma at a time interval of 24 h. This HPLC-GC–MS method is reliable, selective and sensitive enough to be used in clinical pharmacokinetic studies on the enantioselective disposition of nisoldipine in humans.     

Simultaneous determination of triprolidine and pseudoephedrine in human plasma by liquid chromatography–ion trap mass spectrometry

A highly efficient, selective and specific method for simultaneous quantitation of triprolidine and pseudoephedrine in human plasma by liquid chromatography–ion trap-tandem mass spectrometry coupled with electro spray ionization (LC–ESI-ion trap-tandem MS) has been validated and successfully applied to a clinical pharmacokinetic study. Both targeted compounds together with the internal standard (gabapentin) were extracted from the plasma by direct protein precipitation. Chromatographic separation was achieved on a C₁₈ ACE^® column (50.0 mm × 2.1 mm, 5 μm, Advance Chromatography Technologies, Aberdeen, UK), using an isocratic mobile phase, consisting of water, methanol and formic acid (55:45:0.5, v/v/v), at a flow-rate of 0.3 mL/min. The transition monitored (positive mode) was m/z 279.1 → m/z 208.1 for triprolidine, m/z 165.9 → m/z 148.0 for pseudoephedrine and m/z 172.0 → m/z 154.0 for gabapentin (IS). This method had a chromatographic run time of 5.0 min and a linear calibration curves ranged from 0.2 to 20.0 ng/mL for triprolidine and 5.0–500.0 ng/mL for pseudoephedrine. The within- and between-batch accuracy and precision (expressed as coefficient of variation, %C.V.) evaluated at four quality control levels were within 94.3–106.3% and 1.0–9.6% respectively. The mean recoveries of triprolidine, pseudoephedrine and gabapentin were 93.6, 76.3 and 82.0% respectively. Stability of triprolidine and pseudoephedrine was assessed under different storage conditions. The validated method was successfully employed for the bioequivalence study of triprolidine and pseudoephedrine formulation in twenty six volunteers under fasting conditions.     

Lamotrigine analysis in plasma by gas chromatography–mass spectrometry after conversion to a tert.-butyldimethylsilyl derivative

Lamotrigine (lamictal) is a new anticonvulsant drug recently approved by the FDA for clinical use. Therapeutic monitoring of lamotrigine is useful for patient management (therapeutic range 1–4 μg/ml). Here we describe a gas chromatography–mass spectrometric identification and quantitation of lamotrigine after extraction from human serum and derivatization. Lamotrigine was extracted from alkaline serum with chloroform and derivatized with N-methyl-N-(tert.- butyldimethysilyl) trifluoroacetamide containing 2% tert.-butyldimethylchlorosilane. Oxazepam-d₅ was used as an internal standard. The tert.-butyldimethylsilyl derivative of lamotrigine showed distinct molecular ions at m/z 483 and 485 as well as other peaks at m/z 426, 370 and 334 for unambiguous identification. The base peak was observed at m/z 199. Similarly, the tert.-butyldimethysilyl derivative of oxazepam-d₅ showed molecular ions at m/z 519 and 521 along with other characteristic peaks at m/z 462, 376 and 318. For the analysis of lamotrigine, the mass spectrometer was operated in the selective ion monitoring mode. The within-run and between-run precisions were 4.3% (mean=3.01, S.D.=0.13 μg/ml) and 5.1% (mean=2.93, S.D.=0.15 μg/ml), respectively at a serum lamotrigine concentration of 3.0 μg/ml. The within-run and between-run precisions were 8.2% (mean=0.49, S.D.=0.04 μg/ml) and 10.6% (mean=0.47, S.D.=0.05 μg/ml), respectively at a serum lamotrigine concentration of 0.5 μg/ml. The assay was linear for serum lamotrigine concentrations of 0.5–20 μg/ml. The detection limit was 0.25 μg/ml. The assay was free from interferences from common tricyclic antidepressants, benzodiazepines, other common anticonvulsants, salicylate and acetaminophen.     

Highly sensitive determination of TS-962 (HL-004), a novel acyl-CoA:cholesterol acyltransferase inhibitor,in rat and rabbit plasma by liquid chromatography and atmospheric pressure chemical ionization-tandem mass spectrometry combined with a column-switching technique

A quantitative bioanalytical method with excellent specificity using liquid chromatography (LC) atmospheric pressure chemical ionization-tandem mass spectrometry (APCI-MS–MS) combined with a column-switching technique has been developed for the highly sensitive and reliable determination of TS-962 (HL-004), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, in rat and rabbit plasma. The method involves protein precipitation of a 25-μl aliquot of plasma sample with eight volumes of methanol containing a deuterium-labeled internal standard, the direct injection of a methanolic supernatant into the analytical instrumentation with no sample evaporation and reconstitution steps, automated on-line clean-up on a C₁₈ short trapping column (10 mm×4.0 mm I.D.) followed by separation on a C₁₈ analytical column (50 mm×4.6 mm I.D.), and detection with APCI-MS–MS using m/z 448 ([M+H]⁺) as a precursor ion and m/z 178 as a product ion in a selected reaction monitoring mode. The lower limit of quantification was 1 ng/ml, and good linearity of the calibration graph was obtained in the range of 1∼490 ng/ml with excellent reliability. The developed method enabled pharmacokinetic profiles to be determined for rats and rabbits with sequential plasma collection from an individual animal.     

Rapid determination of finasteride in human plasma by UPLC–MS/MS and its application to clinical pharmacokinetic study

A rapid, specific, and sensitive method utilizing reversed-phase ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed and validated to determine finasteride levels in human plasma. The plasma samples were prepared by liquid–liquid extraction with ethyl acetate, evaporation, and reconstitution. MS/MS analyses were performed on a triple–quadrupole tandem mass spectrometer by monitoring protonated parent → daughter ion pairs at m/z 373 → 305 for finasteride and m/z 237 → 194 for carbamazepine (internal standard, IS). The method was validated with respect to linearity, recovery, specificity, accuracy, precision, and stability. The method exhibited a linear response from 0.1 to 30 ng/mL (r² > 0.998). The limit of quantitation for finasteride in plasma was 0.1 ng/mL. The relative standard deviation (RSD) of intra- and inter-day measurements was less than 15% and the method was accurate within −6.0% to 2.31% at all quality-control levels. The mean extraction recovery was higher than 83% for finasteride and 84% for the IS. Plasma samples containing finasteride were stable under the three sets of conditions tested and the processed samples were stable up to 29 h in an autosampler at 5 °C. Detection and quantitation of both analytes within 3 min make this method suitable for high-throughput analyses. The method was successfully applied to a pharmacokinetic study of finasteride in healthy volunteers following oral administration.     

Determination of phenethylamine,a phenethyl isothiocyanate marker,in dog plasma using solid-phase extraction and gas chromatography-mass spectrometry with chemical ionization

Phenethyl isothiocyanate is unstable in aqueous media and at low pH, and rapidly degrades to phenethylamine. Concentrations of phenethylamine, a phenethyl isothiocyanate marker, in dog plasma, were determined utilizing solid-phase extraction and gas chromatography–mass spectrometry with chemical ionization using acetone as the reagent gas. Deuterated d₅-amphetamine was used as an internal standard. After extraction, phenethylamine and d₅-amphetamine were derivatized using MBHFBA. Ions monitored for d₅-amphetamine were m/z 337 and 338; and for phenethylamine were m/z 318 and 319. Precision and accuracy were studied using control solutions prepared in naive dog plasma (80 and 300 ng/ml). Intra-day variability was determined using six replicates of each control solution analyzed on a single day. The relative standard deviation for the 80 ng/ml control was 12.9% and for the 300 ng/ml it was 12.1%. Relative accuracy was 10.9% for the low control and −4.1% for the high control. Inter-day variability was determined over a 6-day period. For the 80 and 300 ng/ml control solutions, the relative standard deviations were 15.8 and 9.1%, respectively, and relative accuracy values were 10.1 and −5.2%, respectively. Standard curves were prepared in naive dog plasma and were linear over the range of phenethylamine assayed (10–500 ng/ml). The results of this study indicate that the proposed method is simple, precise, accurate and sensitive enough for analysis of large numbers of plasma samples.     

Determination of free concentrations of ropivacaine and bupivacaine in plasma from neonates using small-scale equilibrium–dialysis followed by liquid chromatography-mass spectrometry

Attempts to determine a safe plasma concentration of ropivacaine and bupivacaine in neonates have not been consistent. This might be due to an underestimation of free drug in small plasma samples by currently used techniques, e.g., ultrafiltration. We describe a simple microscale equilibrium–dialysis technique for the separation of free and bound ropivacaine and bupivacaine. The free drug in the dialysate was determined using solid-phase extraction and liquid chromatography with mass spectrometry. Pentycaine was used as an internal standard and added to the dialysates prior to extraction. The method is very selective and sensitive, as no compounds other than the analyte and internal standard were observed in the resulting chromatograms at low ng/ml levels. The limit of quantitation was 2.5 ng/ml. The calibration curve was linear in the range of 2 to 1000 ng/ml. The precision of the whole procedure was 8.1% (n=10) and 6.5% (n=7) for ropivacaine and bupivacaine, respectively. The method was tested in the analysis of plasma samples taken from neonates who had received epidural injections.     

Determination of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine using high-performance liquid chromatography and electrospray ionization mass spectrometry

High-performance liquid chromatography with electrospray ionization mass spectrometry was used to determine 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine. After basic hydrolysis of conjugates, the compound was extracted using SPEC-PLUS-3ML-C₁₈ solid-phase extraction columns. A deuterium labelled internal standard (d₃-THC-COOH) was added prior to hydrolysis. Separation was performed on a reversed-phase Zorbax Eclipse XDB-C₈ analytical column (150×3.0 mm I.D.) using a gradient program from 60 to 80% acetonitrile (4 mM formic acid) at a flow-rate of 0.5 ml/min. The compounds were detected by single ion monitoring of m/z 345 and m/z 348 for the protonated molecules [THC-COOH+H]⁺ and [d₃-THC-COOH+H]⁺, respectively. The precision and accuracy were tested on spiked urine samples in the range 2.5–125 ng/ml. The mean recovery was 95% (n=58), coefficients of variations were 2.2–4.3% and the limit of detection 2 ng/ml. Diagnostic qualifying ions of THC-COOH (m/z 327 and m/z 299) and d₃-THC-COOH (m/z 330) were generated using up-front collision-induced dissociation. The relative ion intensities in clinical samples (n=21) were within ±20% deviation compared with standards. Using this tolerance and the presence of the ions m/z 327 and m/z 299 at the correct retention times as the acceptance criteria for identification of THC-COOH positive samples, the limit of detection was 15 ng/ml. The LC–MS method complies with the current recommendations on drugs of abuse testing, in which mass spectrometric detection is emphasized.     

Determination of eptifibatide concentration in human plasma utilizing the liquid chromatography–tandem mass spectrometry method

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.     

Enatiomeric determination of tramadol and O-desmethyltramadol in human urine by gas chromatography–mass spectrometry

A GC–MS assay for stereoselective determination of tramadol and its pharmacologically active phase I metabolite O-desmethyltramadol in human urine was developed. Nefopam was used as internal standard. The method involves a simple solid phase extraction with chiral analysis by gas chromatography–electron ionization mass spectrometry using m/z 263; 58, 249; 58, and 179; 58 for the determination of concentration of tramadol, O-desmethyltramadol and internal standard, respectively. Chromatography was performed on a Rt-βDEXcst column containing alkylated beta-cyclodextrins as a chiral selector. The calibration curves were linear in the concentration range 0.1–20 μg/mL (R² ≥ 0.998). Intra-day accuracies ranged between 97.2–104.9%, 96.1–103.2%, and 97.3–102.8% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged between 95.2–105.7%, 99.1–105.2%, and 96.5–101.2% at the lower, intermediate, and high concentration for all analytes, respectively. This method was successfully used to determine the concentration of enantiomers of T and ODT in a pharmacogenetic study.     

Determination of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry using selected-ion monitoring

A method for the determination of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids by GC-MS with selected-ion monitoring using etorphine as internal standard was established. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after concentration to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed which showed good behavior on GC-MS with electronic-impact ionization. The main fragment, m/z 522, which is the base peak, was selected as the ion for quantitation and the corresponding ion, m/z 520, was selected for monitoring the internal standard, etorphine. The recoveries and coefficients of variation of the whole procedure were determined with five controlled dihydroetorphine-free urine and plasma samples spiked with different concentrations of dihydroetorphine. The concentration of dihydroetorphine for quantitation was in the range 1–20 ng/ml for urine and 2.5–250 ng/ml for plasma. The correlation coefficients of the standard curves are sufficient to determine the dihydroetorphine. The accuracy for quantitation of dihydroetorphine in urine and plasma is less than 10.6%.     

Determination of perfluorodecalin and perfluoro-N-methylcyclohexylpiperidine in rat blood by gas chromatography–mass spectrometry

A gas chromatography–mass spectrometry method (SIM mode) was developed for the determination of perfluorodecalin (cis and trans isomers, 50% each) (FDC), and perfluoromethylcyclohexylpiperidine (3 isomers) (FMCP) in rat blood. The chromatographic separation was performed by injection in the split mode using a CP-select 624 CB capillary column. Analysis was performed by electronic impact ionization. The ions m/z 293 and m/z 181 were selected to quantify FDC and FMCP due to their abundance and to their specificity, respectively. The ion m/z 295 was selected to monitor internal standard. Before extraction, blood samples were stored at −30°C for at least 24 h in order to break the emulsion. The sample preparation procedure involved sample clean-up by liquid–liquid extraction. The bis(F-butyl)ethene was used as the internal standard. For each perfluorochemical compound multiple peaks were observed. The observed retention times were 1.78 and 1.87 min for FDC, and 2.28, 2.34, 2.48 and 2.56 min for FMCP. For each compound, two calibration curves were used; assays showed good linearity in the range 0.0195–0.78 and 0.78–7.8 mg/ml for FDC, and 0.00975–0.39 and 0.39–3.9 mg/ml for FMCP. Recoveries were 90 and 82% for the two compounds, respectively with a coefficient of variation <8%. Precision ranged from 0.07 to 15.6%, and accuracy was between 89.5 and 111.4%. The limits of quantification were 13 and 9 μg/ml for FDC and FMCP, respectively. This method has been used to determine the pharmacokinetic profile of these two perfluorochemical compounds in blood following administration of 1.3 g of FDC and 0.65 g of FMCP per kg body weight, in emulsion form, in rat.     

Simultaneous determination of thiabendazole and its major metabolite, 5-hydroxythiabendazole,in bovine tissues using gradient liquid chromatography with thermospray and atmospheric pressure chemical ionisation mass spectrometry

A novel method is presented for the determination of thiabendazole and 5-hydroxythiabendazole in animal tissues. Samples are homogenised in buffer at pH=7.0, extracted with ethyl acetate and cleaned up using CN solid-phase extraction columns. Thiabendazole and 5-hydroxythiabendazole are separated chromatographically using gradient elution and analysed by liquid chromatography–mass spectrometry. Deuterated thiabendazole is employed as an internal standard for thiabendazole determination; 5-hydroxythiabendazole is quantified via external standards. Samples are screened by monitoring the protonated molecular ions at m/z=202 for thiabendazole, 206 for deuterated thiabendazole and 218 for 5-hydroxythiabendazole using thermospray LC–MS. Positives are confirmed by multiple ion monitoring using APCI LC–MS. Validation of the method was carried out at 50, 100 and 200 μg kg⁻¹. Recoveries for thiabendazole in bovine muscle, liver and kidney ranged from 96–103% with C.V.s between 0.7 and 4.8% and for 5-hydroxythiabendazole recoveries ranged from 70–85% with C.V.s between 3.1 and 11.5%.     

Effect of disease state on ionization during bioanalysis of MK-7009, a selective HCV NS3/NS4 protease inhibitor,in human plasma and human Tween-treated urine by high-performance liquid chromatography with tandem mass spectrometric detection

HPLC–MS/MS methods for the determination of a Hepatitis C NS3/NS4 protease inhibitor (MK-7009) in human plasma and Tween-treated urine were developed and validated over the concentration range 1–1000 ng/mL and 0.2–100 μg/mL respectively. A stable isotope labeled internal standard (ISTD), D₄-MK-7009, was employed. Analytes were chromatographed by reversed phase HPLC and quantified by an MS/MS system. Electrospray ionization in the positive mode was employed. Multiple reaction monitoring of the precursor to product ion pairs m/z 758.6 → 637.4 MK-7009 and m/z 762.5 → 637.4 ISTD was used for quantitation. Analyte and internal standard were extracted from 250 μL of plasma using an automated 96-well liquid–liquid extraction. Plasma pH adjustment prior to extraction minimized ionization suppression in plasma samples from patients with Hepatitis C. The urine method involved direct dilution in the 96-well format of 0.020 mL Tween-treated urine. These methods have supported several clinical studies. Incurred plasma sample reanalysis demonstrated adequate assay reproducibility and ruggedness.     

A fast and sensitive HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of ergone in rats

A fast and sensitive HPLC–APCI-MS/MS method was developed for the determination of ergosta-4,6,8(14),22-tetraen-3-one (ergone) in rat plasma. The plasma sample containing ergone and ergosterol (internal standard) were simply treated with acetone to precipitate and remove proteins and the isolated supernatants were directly injected into the HPLC–APCI-MS/MS system. Chromatographic separation was performed on a 1.8 μm Zorbax SB-C18 column (100 mm × 3.0 mm) with a 97:3 (v/v) mixed solution of methanol and 0.1% aqueous formic acid being used as mobile phase. Quantification was performed by multiple selected reactions monitoring (MRM) of the transitions with (m/z)⁺ 393–268 for ergone and (m/z)⁺ 379–69 for the IS. The method was validated in the concentration range of 5–1600 ng/mL for ergone. The precision of the assay (RSD%) was less than 10.5% at all concentrations levels within the tested range and adequate accuracy, and the limit of detection was 1.5 ng/mL. The absolute recoveries of both ergone and ergosterol from the plasma were more than 95%. The developed method has been successfully applied to the pharmacokinetic study of the drug in SD rats.