Chromatography-electrospray Mass Spectrometric Method Research Articles

Validation and application of a liquid chromatography–electrospray ionization mass spectrometric method for determination of mazindol in human plasma and urine

IntroductionEven after removal of some stimulants, like fenproporex, amfepramone and mazindol, from Brazilian market, the use of these substances is still high, especially by drivers. Mazindol is the second most used anorectic agent in the world acting as an indirect sympathomimetic agonist, having stimulatory action on central nervous system. Plasma is a good matrix to monitor since it reflects the psychomotor effects of these drugs, but unlike urine has an invasive collection; drug levels and detection time are quite low. MethodThe method involved a liquid–liquid extraction of the samples and a LC–MS analysis was fully validated. Method was used to analyze samples of urine and plasma collected from health volunteers in a period of 24h. Metabolite of mazindol was synthesized using alkaline conditions. ResultsAfter validation the method proved to be adequate to analyze samples collected from health volunteers. Method was linear in the concentration range of 0.1–10ng/mL (r=0.9982) for plasma and 5–50ng/mL (r=0.9973) for urine. DiscussionAnalysis of the samples showed that mazindol can be detected after 1h of administration and that concentration levels in urine were always higher than in plasma. Mazindol metabolite was detected only in urine.

Reversed-phase liquid chromatography with electrospray mass detection and 1H and 13C NMR characterization of new process-related impurities, including forced degradants of efavirenz: related substances correlated to the synthetic pathway.

In this study, a stability-indicating reversed-phase liquid chromatographic electrospray mass spectrometric method was developed and validated for the determination of process-related impurities and forced degradants of Efavirenz in bulk drugs. Efavirenz was subjected to acid, alkaline hydrolysis, H2O2 oxidation, photolysis, and thermal stress. Significant degradation was observed during alkaline hydrolysis, and the degradants were isolated on a mass-based purification system and characterized by high-resolution mass spectrometry, positive electrospray ionization tandem mass spectrometry, and (1)H and (13)C NMR spectroscopy. Accurate mass measurement and NMR spectroscopy revealed the possible structure of process-related impurities and degradant under stress conditions. The acceptable separation was accomplished on Waters bondapak C18 column (250 mm × 4.6 mm; 5 μm), using 5 mM ammonium acetate and acetonitrile as a mobile phase in a gradient elution mode at a flow rate of 1.0 mL/min. The eluents were monitored by diode array detector at 247 nm and quantitation limits were obtained in the range of 0.1-2.5 μg/mL for Efavirenz, degradants, and process-related impurities. The liquid chromatography method was validated with respect to accuracy, precision, linearity, robustness, and limits of detection and quantification as per International Conference on Harmonization guidelines.

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of zofenopril and its active metabolite zofenoprilat in human plasma

A novel, sensitive and rapid liquid chromatographic-electrospray ionization mass spectrometric method was developed and validated for the determination of zofenopril and its active metabolite zofenoprilat in human plasma. The method was based on a single extraction step using methyl tert-butyl ether and did not require chemical derivatization. The chromatographic conditions were optimized; separation was performed on a phenyl–hexyl column (5 μm, 250 mm × 4.6 mm i.d.) with a mobile phase consisting of a solution of methanol and water (95:5, v/v) that also contained 0.1% of formic acid. A flow rate of 1.0 mL/min was used. Zofenopril, zofenoprilat and the internal standard (IS) fosinopril sodium were measured using an electrospray ion source in a positive reaction monitoring mode. Linear calibration curves were generated for zofenopril concentrations between 0.1052 and 1052 ng/mL and for zofenoprilat concentrations between 0.2508 and 2508 ng/mL. In both cases, the coefficients of determination were greater than 0.995. The extraction recovery for zofenopril was 93.5% on average. It was 92.5% for zofenoprilat. The inter- and intra-batch precision and accuracy for both zofenopril and zofenoprilat were higher than 14%. The method was applied to measure the concentrations of zofenopril and zofenoprilat in plasma samples.

Application of liquid chromatography–tandem mass spectrometry method for the simultaneous quantitative analysis of propentofylline and its chiral metabolite M1 in rats

A sensitive and selective liquid chromatographic-electrospray ionization mass spectrometric method for the simultaneous determination of propentofylline and enantiomers of its active metabolite M1 in rat serum, cortex and hippocampus was developed and validated according to GLP procedures. Sample preparations were carried out by liquid-liquid extraction using diethyl ether after the addition of the internal standard (pentoxifylline). The dried residue was reconstituted in mobile phase and injected onto a Chiralpak AD column (10 µm, 250 × 4.6 mm i.d.). The limit of quantification for propentofylline in serum, cortex and hippocampus was set at 0.25 ng/mL and for enantiomers of its metabolite M1 at 1.25 ng/mL. The established LC/ESI-MS/MS method has been successfully applied to an initial pharmacokinetic study of propentofylline and also to assessment of distribution of parent drug and enantiomers of its pharmacologically active metabolite M1 to cortex and hippocampus after intravenous administration of propentofylline to rats at a dose of 5 mg/kg.

Usefulness of Sweat Testing for the Detection of Methylphenidate After Fast- and Extended-Release Drug Administration: A Pilot Study

The pharmacokinetics of methylphenidate (MPH), a prescription amphetamine derivative used in the treatment of attention-deficit hyperactivity disorder, has been amply described in conventional biological matrices. Recently, the excretion of MPH and its principal metabolite, ritalinic acid (RA) in oral fluid and plasma after a single drug administration has been described. The aim of this study was to describe the excretion of MPH and RA in sweat after the administration of a single dose of either fast-release or extended-release MPH. Three male subjects received 2 simultaneous oral doses of 10 mg fast-release MPH, and 1 male subject received one dose of 20 mg extended-release MPH. Sweat patches were applied to the back of each participant and removed at timed intervals. MPH and RA were determined in patches using a previously validated liquid chromatography-electrospray ionization mass spectrometric method. MPH was detected in sweat after the administration of fast- and extended-release formulations. For the fast-release formulation, MPH appeared in the sweat patches 2 hours after administration with a maximum of 15.9 nanogram per patch, reached after 24 hours. Mean total MPH excreted was 0.02 mg (about 0.08% of the administered dose). For the extended-release formulation, MPH appeared in the sweat 5 hours after administration and reached a maximum of 34.3 nanogram per patch after 24 hours. Mean total MPH excreted was 0.04 mg (about 0.18% of the administered dose). RA was not detected in either of the sweat patches probably because of its acidic properties. Measuring MPH in sweat patches can be a viable alternative to urine testing for noninvasive monitoring of use and misuse of the drug.

Determination of Nateglinide in Human Plasma by LC-ESI-MS and Its Application to Bioequivalence Study

To evaluate the bioequivalence of nateglinide, a rapid and specific liquid chromatographic-electrospray ionization mass spectrometric method was developed and validated to determine nateglinide for human plasma samples. The analyte was detected using electrospray positive ionization mass spectrometry in the selected ion monitoring mode. Tinidazole was used as the internal standard. A good linear relationship obtained in the concentration ranged from 0.05 to 16 μg mL−1 (r2 = 0.9993). Lower limit of quantification was 0.05 μg mL−1 using 100 μL of plasma sample. Intra- and inter-day relative standard deviations were 2.1–7.5 and 4.7–8.9%, respectively. Among the pharmacokinetic data obtained, Tmax was 2.09 ± 1.06 h for reference formulation and 2.40 ± 0.97 h for test formulation. Cmax was 4.17 ± 1.31 μg mL−1 for reference formulation and 4.37 ± 1.53 μg mL−1 for test formulation. The half-life (t½) was 1.93 ± 0.44 h for reference formulation and 1.92 ± 0.29 h for test formulation. AUC0–10h was 13.67 ± 4.36 μg h mL−1 for reference formulation and 13.21 ± 4.09 μg h mL−1 for test formulation. This method was successfully applied to the pharmacokinetic study in human plasma samples.

Determination of Fluoxetine in Human Plasma by Liquid Chromatography–Mass Spectrometry and Its Application

A rapid, simple, and specific liquid chromatography–electrospray ionization–mass spectrometric method has been developed and validated for the determination of fluoxetine in human plasma. The method was validated with a linear range of 0.5–100 ng mL−1, and the lowest limits of quantification were 0.5 ng mL−1 for fluoxetine. The extraction efficiencies were about 65% and recoveries of method were in the range of 94.0–97.5%. The intraday relative standard deviation (RSD) was less than 11% and interday RSD was within 12%. The method has been successfully applied to the evaluation of pharmacokinetics and bioequivalence of fluoxetine.

Liquid chromatography mass spectrometry for the determination of salvianolic acid B, a natural compound from the herb Danshen in rat plasma and application to pharmacokinetic study

A sensitive and specific liquid chromatographic-electrospray ionization mass spectrometric method was developed for quantification of salvianolic acid B in rat plasma with resveratrol as the internal standard. The analytes were separated on a reversed-phase column with acetonitrile (40%) and water (60%) containing 0.75% formic acid as mobile phase at a flow rate of 1 mL/min. Liquid-liquid extraction was adopted for the sample preparation, and the analytes were determined using electrospray negative ionization mass spectrometry in the selective monitoring mode. The method was validated over the concentration range 0.1-40 microg/mL using 0.1 mL of plasma with coefficients of correlation >0.999. The intra- and inter-day precisions of analysis were <10%, and accuracy ranged from 94 to 101%. This method was successfully applied to a pharmacokinetics of salvianolic acid B in rats.

High‐performance liquid chromatography and LC‐ESI‐MS method for identification and quantification of two isomeric polyisoprenylated benzophenones isoxanthochymol and camboginol in different extracts of Garcinia species

A rapid, sensitive and simple reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometric method has been developed for the identification and quantification of two isomeric polyisoprenylated benzophenones, isoxanthochymol and camboginol, in the extracts of the stem bark, seeds and seed pericarps of Garcinia indica and in the fruit rinds of Garcinia cambogia. The separation of isoxanthochymol and camboginol was achieved on a Perkin Elmer RP(8) column (10 x 2.1 mm with 5.0 microm particle size) using a solvent system consisting of a mixture of acetonitrile-water (80:20, v/v) and methanol-acetic acid (99.0:1.0, v/v) as a mobile phase in a gradient elution mode. The limits of detection and quantification were 5 and 10 microg/mL for isoxanthochymol and 50 and 100 microg/mL for camboginol, respectively. The intra- and inter-day precisions were 2.34 and 3.41% for isoxanthochymol and 3.35 and 3.66% for camboginol. The identity of the two isomeric compounds in the samples was determined on a triple quadrupole mass spectrometer with ESI interface operating in the negative ion mode. The method was used to identify and quantify isoxanthochymol and camboginol in the different extracts of two Garcinia species, Garcinia indica and Garcinia cambogia.

Liquid Chromatography–Tandem Mass Spectrometry Method for the Determination of Meloxicam and its Metabolite 5-Carboxymeloxicam in Human Plasma

To develop and validate a rapid, sensitive and selective liquid chromatography-electrospray ionization mass spectrometric method for the determination of meloxicam and its metabolite 5-carboxymeloxicam in human plasma. A liquid extraction method was chosen for sample clean-up. Meloxicam, 5-carboxymeloxicam and isoxicam (internal standard) were analyzed on an XBridge™ C18 column with 65% methanol in 10 mM ammonium formate (pH 3.0) and detected in selected reaction monitoring mode. The standard curves were linear over the concentration range 10-2500 ng/ml for meloxicam and 2-100 ng/ml for 5-carboxymeloxicam. Matrix effects were practically absent. This method has been successfully applied to the pharmacokinetic study of meloxicam and 5-carboxymeloxicam after oral administration of meloxicam (15 mg) to 30 volunteers.

Liquid chromatography–tandem mass spectrometry method of loxoprofen in human plasma

A rapid, sensitive and selective liquid chromatography-electrospray ionization mass spectrometric method for the determination of loxoprofen in human plasma was developed. Loxoprofen and ketoprofen (internal standard) were extracted from 20 microL of human plasma sample using ethyl acetate at acidic pH and analyzed on an Atlantis dC(18) column with the mobile phase of methanol:water (75:25, v/v). The analytes were quantified in the selected reaction monitoring mode. The standard curve was linear over the concentration range of 0.1-20 microg/mL with a lower limit of quantification of 0.1 microg/mL. The coefficient of variation and relative error for intra- and inter-assay at four quality control levels were 2.8-5.2 and 4.8-7.0%, respectively. The recoveries of loxoprofen and ketoprofen were 69.7 and 67.6%, respectively. The matrix effects for loxoprofen and ketoprofen were practically absent. This method was successfully applied to the pharmacokinetic study of loxoprofen in humans.

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of xanthinol in human plasma and its application in a bioequivalence study of xanthinol nicotinate tablets

A sensitive, rapid liquid chromatographic–electrospray ionization mass spectrometric method for the determination of xanthinol in human plasma was developed and validated. Xanthinol nicotinate in plasma (0.5 mL) was pretreated with 20% trichloroacetic acid for protein precipitation. The samples were separated using a Lichrospher silica (5 μm, 250 mm × 4.6 mm i.d.). A mobile phase of methanol–water containing 0.1% formic acid (50: 50, v /v) was used isocratically eluting at a flow rate of 1 mL/min. Xanthinol and its internal standard (IS), acyclovir, were measured by electrospray ion source in positive selected reaction monitoring mode. The method demonstrated that good linearity ranged from 10.27 to 1642.8 ng/mL with r = 0.9956. The limit of quantification for xanthinol in plasma was 10.27 ng/mL with good accuracy and precision. The mean plasma extraction recovery of xanthinol was in the range of 90.9–100.2%. The intra- and inter-batch variability values were less than 4.8% and 7.9% (relative standard deviation, R.S.D.), respectively. The established method has been successfully applied to a bioequivalence study of two xanthinol nicotinate tablets for 20 healthy volunteers.

Determination and pharmacokinetics of gastrodin and p-hydroxybenzylalcohol after oral administration of Gastrodia elata Bl. extract in rats by high-performance liquid chromatography–electrospray ionization mass spectrometric method

In this report, a high-performance liquid chromatography-electrospray ionization mass spectrometric (HPLC-MS) method was used to determine gastrodin (GAS) and p-hydroxybenzylalcohol (HBA) in rat plasma after oral administration of Gastrodia elata Bl. (Chinese name: Tianma) extract. Up to 200 microl of plasma containing GAS, HBA and pyromucic acid (as internal standard, IS) were deproteinized with six volumes of methanol. Calibration curves showed linearity within the concentration range tested 2.00-200.00 microg/ml for GAS and 0.832-104.00 microg/ml for HBA in plasma with a correlation coefficient (r) greater than 0.9997. The limit of quantification of 2.00 and 0.83 microg/ml for GAS and HBA had been achieved, respectively. The intra-day and inter-day precisions of the method were determined to be less than 17.82% for GAS and 10.21% for HBA. The recoveries were in the range of 91.12-108.64% with RSD less than 7.80% for GAS and 92.91-106.14% with RSD less than 4.30% for HBA. Evidence showed that a rapid, simple and reproducible LC-MS assay was established to determine GAS and HBA in rat plasma.

Quantitative determination of erythromycylamine in human plasma by liquid chromatography–mass spectrometry and its application in a bioequivalence study of dirithromycin

A sensitive, rapid liquid chromatographic–electrospray ionization mass spectrometric method for determination of erythromycylamine in human plasma was developed and validated. Erythromycylamine in plasma (0.2 mL) was extracted with ethyl acetate, the organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness under gentle nitrogen stream at 45 °C, and the residue was dissolved in 100 μL of mobile phase. The samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm × 2.1 mm I.D., 5 μm). A mobile phase containing 10 mM of ammonium acetate (pH = 6.4)-acetonitrile-methanol (50:10:40, v/v/v) was used isocratically eluting at a flow rate of 0.2 mL/min. Erythromycylamine and its internal standard (IS), midecamycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 4.5 to 720 ng/mL with r = 0.9997. The limit of quantification for erythromycylamine in plasma was 4.5 ng/mL with good accuracy and precision. The mean extraction recovery of the method was higher than 75.1% and 72.7% for erythromycylamine and IS, respectively. The intra-day and inter-day precision ranged from 5.2% to 6.4% and 5.6–9.3% (relative standard deviation, RSD), respectively. The established method has been successfully applied to a bioequivalence study of two dirithromycin formulations for 18 healthy volunteers.

Quantitative determination of azithromycin in human plasma by liquid chromatography–mass spectrometry and its application in a bioequivalence study

A sensitive, rapid liquid chromatographic–electrospray ionization mass spectrometric method for determination of azithromycin in human plasma was developed and validated. Azithromycin in plasma (0.2 mL) was extracted with methyl tert-butyl ether–hexane (50:50, v/v), organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness at 40 °C and dissolved in mobile phase, samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm × 2.1 mm i.d., 5 μm), together with a mobile phase containing of 20 mM ammonium acetate (pH 5.2)–acetonitrile–methanol (50:40:10, v/v/v) and was isocratically eluted at a flow rate of 0.2 mL/min. Azithromycin and its internal standard, clarithromycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 2 to 1000 ng/mL with r = 0.9977. The limit of quantification for azithromycin in plasma was 2 ng/mL with good accuracy and precision. The higher mean extraction recovery, say 81.2% and 75.5% for azithromycin and internal standard (IS), respectively, was obtained in this work. The intra-day and inter-day precision ranged from 4.8% to 8.6% and 6.4% to 10.7% (R.S.D.), respectively. The established method has been successfully applied to bioequivalence study of 2 azithromycin formulations for 24 healthy volunteers.

Development and validation of liquid chromatography–mass spectrometry method for the determination of telmisartan in human plasma

A sensitive liquid chromatographic–electrospray ionization mass spectrometric method was developed and validated for fast determination of telmisartan in human plasma. Plasma of 0.1 mL was deprotienated with methanol, centrifugation, evaporation to dryness and dissolving in mobile phase, samples were separated using a Hypersil-Keystone C18 reversed-phase column (150 mm × 2.1 mm i.d., 5 μm), together with a mobile phase containing of acetonitrile–10 mM ammonium acetate (42:58, v/v), 0.2% acetic acid and was isocratically eluted at a flow rate of 0.2 mL/min. Telmisartan and its internal standard, valsartan, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated linearity from 1 to 2000 ng/mL ( r = 0.9988). The limit of quantification for telmisartan in plasma was 1 ng/mL with good accuracy and precision. The mean sample extract recovery of the method were higher than 82 and 78% for telmisartan and internal standard (IS), respectively. The within-run and between-run precision ranged from 3.4 to 8.9% and 5.9 to 11.2% (relative standard deviation, R.S.D.), respectively.

Rapid, simple, specific liquid chromatographic-electrospray mass spectrometry method for the determination of glibenclamide in human plasma

A sensitive liquid chromatographic-electrospray ionization mass spectrometric method was developed and validated for fast determination of glibenclamide in human plasma. After deprotienation with methanol, centrifugation, evaporation to dryness and dissolving in mobile phase, samples were separated using a Hypersil-Keystone C18 reversed-phase column ( 150 mm ×2.1 mm ID, 5 μm), together with a mobile phase containing of acetonitrile–10 mM ammonium acetate (55:45, v/v), 0.2% acetic acid and 0.1% trifluoracetic acid. Glibenclamide and its internal standard, glipizide, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated linearity from 1 to 400 ng/mL ( r =0.998). The limit of quantification for glibenclamide in plasma was 1 ng/mL with good accuracy and precision. The recovery of the method was over 91.5%. Intra- and inter-day precision ranged from 3.42 to 5.57 and 7.49 to 8.64% (RSD), respectively.

Rapid, simple, specific liquid chromatographic-electrospray mass spectrometry method for the determination of glibenclamide in human plasma

A sensitive liquid chromatographic-electrospray ionization mass spectrometric method was developed and validated for fast determination of glibenclamide in human plasma. After deprotienation with methanol, centrifugation, evaporation to dryness and dissolving in mobile phase, samples were separated using a Hypersil-Keystone C18 reversed-phase column ( 150 mm×2.1 mm ID, 5 μm), together with a mobile phase containing of acetonitrile–10 mM ammonium acetate (55:45, v/v), 0.2% acetic acid and 0.1% trifluoracetic acid. Glibenclamide and its internal standard, glipizide, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated linearity from 1 to 400 ng/mL ( r=0.998). The limit of quantification for glibenclamide in plasma was 1 ng/mL with good accuracy and precision. The recovery of the method was over 91.5%. Intra- and inter-day precision ranged from 3.42 to 5.57 and 7.49 to 8.64% (RSD), respectively.

Development and validation of a liquid chromatography–electrosprayionization mass spectrometric method for the determination of dexamethasone in sheep plasma

A quantitative liquid chromatographic–electrospray ionization mass spectrometric method for the determination of dexamethasone in sheep plasma has been developed and validated. The samples were extracted using solid-phase extraction cartridges with mixed reversed-phase materials (oasis-HLB). The chromatographic separation was performed on a reversed-phase XTerrra MS C 18 column ( 3.0 mm×150 mm; 5 μm) using a mobile phase consisting of 65% methanol in water containing 0.1% (v/v) formic acid, pumped at a flow rate of 0.30 ml min −1. The analyte was detected after positive electrospray ionization using selected ion monitoring (SIM) mode. The probe heater temperature was set at 260 °C, the capillary voltage was set at 3.5 kV and the source block voltage (AQA max) was set at 30 V. The method was fully validated. Calibration graphs were linear ( r better than 0.998, n=11), in concentration ranges 6–1000 ng ml −1 for dexamethasone. The intra- and inter-day RSD values were less than 24.1% ( n=6). The limits of detection and quantitation for dexamethasone were found to be 1 and 6 ng ml −1, respectively. The efficiency of the solid phase extraction procedure was found to be 92.4% for dexamethasone. The method was further applied to a pilot kinetic study in order to assess the main pharmacokinetic parameters of dexamethasone in sheep.