Simultaneous determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using ultra-performance liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive, and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method for the determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using sildenafil as an internal standard (IS) was developed and validated. Udenafil, DA-8164 and IS from a 100 microL aliquot of biological samples were extracted by protein precipitation using acetonitrile. Chromatographic separation was carried on an Acquity UPLC BEH C(18) column (50 x 2.1 mm, i.d., 1.7 microm) with an isocratic mobile phase consisting of acetonitrile and containing 0.1% formic acid (75:25, v/v) at flow rate of 0.4 mL/min, and total run time was within 1 min. Detection and quantification was performed by the mass spectrometer using multiple reaction-monitoring mode at m/z 517 --> 283 for udenafil, m/z 406 --> 364 for DA-8164 and m/z 475 --> 100 for IS. The assay was linear over a concentration range of 1-600 ng/mL with a lower limit of quantification of 1 ng/mL in both human plasma and urine. The coefficient of variation of this assay precision was less than 13.7%, and the accuracy exceeded 92.0%. This method was successfully applied for pharmacokinetic study after oral administration of udenafil 100 mg to healthy Korean male volunteers.     

Simple, sensitive and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric method for the quantification of lacidipine in human plasma

A simple, sensitive and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric method was developed and validated for the quantification of lacidipine in human plasma using its structural analogue, amlodipine, as internal standard (IS). The method involves a simple single-step liquid-liquid extraction with tert-butyl methyl ether. The analyte was chromatographed on an Xterra MS C(18) reversed-phase chromatographic column by isocratic elution with 20 mM ammonium acetate buffer-acetonitrile (10:90, v/v; pH 6) and analyzed by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 456.4 --> 354.4 and m/z 409.3 --> 238.3 were used to measure the analyte and the I.S., respectively. The chromatographic run time was 1.5 min and the weighted (1/x(2)) calibration curves were linear over the range 0.1-25 ng ml(-1). Lacidipine was sensitive to temperature in addition to light. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in human plasma were 50 and 100 pg ml(-1), respectively. The within- and between-batch accuracy and precision were found to be well within acceptable limits (<15%). The analyte was stable after three freeze-thaw cycles (deviation <15%). The average absolute recoveries of lacidipine and amlodipine (IS) from spiked plasma samples were 51.1 +/- 1.3 and 50.3 +/- 4.9%, respectively. The assay method described here could be applied to study the pharmacokinetics of lacidipine.     

Determination of malotilate and its metabolites in plasma and urine

A method for the determination of malotilate (I), the corresponding monocarboxylic acid (II) and its decarboxylated product (III) in plasma is described. Plasma was extracted with chloroform spiked with internal standard. The residue, dissolved in methanol, was chromatographed on a reversed-phase column with a mobile phase of 60% acetonitrile and 1% acetic acid in water. The sensitivity limit for I, II and III was 50, 25 and 100 ng/ml of plasma, respectively. Compound I in the same plasma extract was also analysed by gas chromatography--electron-impact mass spectrometry. The base peaks m/z 160 for I and m/z 162 for internal standard (IV) were monitored; the sensitivity limit for I was 2.5 ng/ml of plasma. The determination of the metabolites of I, II and its conjugate (V), and isopropyl-hydrogen malonate (VI) in urine by high-performance liquid chromatography is also described. The limit of quantification for VI was 2.0 micrograms/ml, and the overall coefficient of variation of VI was 4.7%. The limit of quantification for II in urine was 0.5 micrograms/ml and that for V was 1.0 micrograms/ml as total II (II + V). The overall precision of the method was satisfactory. The method was used to determine plasma and urine concentrations in four dogs orally dosed with 100, 200 or 400 mg of malotilate.     

Rapid and sensitive liquid chromatography tandem mass spectrometry method for the quantification of ambroxol in human plasma

A sensitive, specific and rapid high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was described and validated for the quantification of ambroxol in human plasma using enalaprilat as the internal standard (IS). Chromatographic separation was performed on a Lichrospher CN column with a mobile phase of methanol and water (containing 0.1% formic acid) (70:30, v/v). The total run time was 5.0 min for each sample. The analytes was detected by mass spectrometry with electrospray ionization source in positive selected reaction monitoring mode. The precursor-fragment ion reaction for ambroxol was m/z 378.9 --> 263.8, and for IS was m/z 349.0 --> 205.9. The linearity was established over the concentration range of 1.56-400.00 ng/mL. The inter-day and the intra-day precisions were all within 10%. A simple protein precipitation with methanol was adopted for sample preparation. The extraction recoveries of ambroxol and IS were higher than 90.80%. The validated method was successfully applied in pharmacokinetic study after oral administration of 90 mg ambroxol to 24 healthy volunteers.     

Simultaneous determination of spironolactone and its active metabolite canrenone in human plasma by HPLC-APCI-MS

A sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) method for the simultaneous determination of spironolactone and its active metabolite canrenone in human plasma has been developed and validated. After the addition of estazolam as the internal standard (IS), plasma samples were extracted with methylene chloride : ethyl acetate mixture (20 : 80, v/v) and separated by high-performance liquid chromatography (HPLC) on a reversed-phase C18 column with a mobile phase of methanol-water (57 : 43, v/v). Analytes were determined in a single quadrupole mass spectrometer using an atmospheric pressure chemical ionization (APCI) source. LC-APCI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.25 for spironolactone and canrenone, m/z 295.05 for estazolam. The method was proved to be sensitive and specific by testing six different plasma batches. Calibration curves of spironolactone and canrenone were linear over the range 2-300 ng/ml. The within- and between-batch precisions (relative standard deviation (RSD)%) were lower than 10% and the accuracy ranged from 85 to 115%. The lower limit of quantification (LLOQ) was identifiable and reproducible at 2 ng/ml. The proposed method was successfully applied to study the pharmacokinetics of spironolactone and its major metabolite in healthy male Chinese volunteers.     

Rapid and sensitive determination of udenafil in plasma by LC-MS/MS for intranasal pharmacokinetic study in rats

A rapid and sensitive analytical method for udenafil in rat plasma was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This chromatographic procedure was then applied to the in vivo pharmacokinetic studies in rats for determining the advantages of intranasal administration of the drug over oral administration. Using liquid-liquid extraction (LLE), udenafil and the internal standard (IS) sildenafil were extracted with dichloromethane from 100 μl of plasma samples. Chromatographic separation was performed using Pursuit XRS C?? column (50 mm × 2.1 mm, i.d., 3 μm, Varian Inc., CA, U.S.A.) with an isocratic mobile phase consisting of acetonitrile and 10 mM ammonium acetate (90 : 10, v/v) at a flow rate of 0.2 ml/min over a total run time of 2.5 min. Detection and quantification was performed by mass spectrometry using the multiple reaction-monitoring mode at m/z 517.4→283.1 for udenafil and m/z 475.3→100.0 for IS. Results showed that the developed method was sensitive and specific for udenafil. Linearity was obtained in the range of 0.5-1000 ng/ml. The coefficient of variation of both intra- and inter-day validation were below 11.6% and the intra- and inter-day accuracy ranged from 91.5 to 109.9%. Udenafil concentration was successfully measured from plasma after intranasal as well as after intravenous or oral administration at clinical dose (1.67 mg/kg) in rats. Moreover, the T(max) values obtained from pharmacokinetic studies suggested that administration of udenafil intranasally could be more effective than by the oral route.     

Simultaneous quantitation of nicorandil and its denitrated metabolite in plasma by LC-MS/MS: application for a pharmacokinetic study

A liquid chromatography-electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous quantitation of nicorandil and its denitrated metabolite, N-(2-hydroxyethyl)-nicotinamide, in rat plasma. After a liquid-liquid extraction step, chromatographic separation was performed on a ShinPack C(18) column with an isocratic mobile phase composed of methanol and 2 mM aqueous ammonium acetate containing 0.03% (v/v) formic acid (33:67 v/v). Procainamide was used as an internal standard (IS). Selected reaction monitoring was performed using the transitions m/z 212 → m/z 135, m/z 166 → m/z 106 and m/z 236 → m/z 163 to quantify nicorandil, its denitrated metabolite and IS, respectively. Calibration curves were constructed over the range of 5-15,000 ng.ml(-1) for both nicorandil and its metabolite. The mean relative standard deviation (RSD%) values for the intra-run precision were 5.4% and 7.3% and for the inter-run precision were 8.5% and 7.3% for nicorandil and its metabolite, respectively. The mean accuracy values were 100% and 95% for nicorandil and its metabolite, respectively. No matrix effect was detected in the samples. The validated method was successfully applied to a pharmacokinetic study after per os administration of nicorandil in rats.     

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.     

Simultaneous quantitation of HIV-protease inhibitors ritonavir, lopinavir and indinavir in human plasma by UPLC-ESI-MS-MS

A selective, sensitive and high-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been developed and validated for the quantification of HIV-protease inhibitors ritonavir (RTV), lopinavir (LPV) and indinavir (IDV) in human plasma. Sample clean-up involved protein precipitation of both drugs and fluconazole used as internal standard from 100 μL human plasma. All the analytes were chromatographically separated on a Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm particle size) analytical column using 0.1% formic acid and methanol (40:60, v/v) as the mobile phase. The parent → product ion transitions for ritonavir (m/z 721.40→ 296.10), lopinavir (m/z 629.40→ 447.40) and indinavir (m/z 614.4→ 421.0) IS (m/z 307.10 → 220.10) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was validated over the concentration range of 30-15,000 ng/mL for LPV and IDV and 3-1500 ng/mL for RTV. The method was successfully applied to a pilot bioequivalence study in 36 healthy human subjects after oral administration of lopinavir 200 mg and ritonavir 50 mg tablet formulation under fasting conditions.     

Sensitive liquid chromatography-tandem mass spectrometry method for quantification of hydrochlorothiazide in human plasma

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of hydrochlorothiazide (I), a common diuretic and anti-hypertensive agent. The analyte and internal standard, tamsulosin (II) were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reversed-phase column (Waters symmetry C18) with a mobile phase of 10 mm ammonium acetate-methanol (15:85, v/v). The protonated analyte was quantitated in negative ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 296.1 solidus in circle 205.0 and m/z 407.2 solidus in circle 184.9 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/mL for hydrochlorothiazide in human plasma. The lower limit of quantitation was 500 pg/mL, with a relative standard deviation of less than 9%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Rapid liquid chromatography-tandem mass spectrometry method for quantification of ziprasidone in human plasma

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of ziprasidone (ZIP) in human plasma was developed. ZIP and N-methyl ziprasidone as internal standard (IS) were extracted from alkalinized plasma using tert- butyl methyl ether. Separation was performed isocratically on a C8 column with 90% acetonitrile containing 2 mmol/L ammonium acetate as a mobile phase with a total run time of 2.5 min. MS/MS transitions of m/z 413 --> 194 and m/z 427 --> 177 of the analyte and internal standard were used for quantification. Confirmatory ions of m/z 413 --> 177 and m/z 427 --> 180 were collected as well. The calibration curve based on peak-area ratio was linear up to at least 200 ng/mL with a detection limit of 0.1 ng/mL. The method showed satisfactory reproducibility with a coefficient of variation of less than 5%. The method was successfully applied to the analysis of ZIP in spiked human plasma.     

Validation and application of a high-performance liquid chromatography--tandem mass spectrometry assay for mosapride in human plasma

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of mosapride (I), a novel and potent gastroprokinetic agent that enhances the upper gastrointestinal motility by stimulating 5-HT(4) receptor. The analyte and internal standard, tamsulosin (II), were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reversed-phase Waters symmetry C(18) column with a mobile phase of 0.03% formic acid-acetonitrile (10:90, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 422.3 -->198.3 and m/z 409.1 -->228.1 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-100.0 ng/mL for mosapride in human plasma. The lower limit of quantitation was 500 pg/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.0 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Simultaneous quantification of fexofenadine and pseudoephedrine in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: method development, validation and application to a clinical study

To support the pharmacokinetic and bioavailability study of a once-daily fexofenadine/pseudoephedrine combination, a high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) method for the simultaneous quantification of fexofenadine and pseudoephedrine was developed and validated with 500 microL human plasma using mosapride as an internal standard (IS). Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 502/466 for fexofenadine, m/z 166/148 for pseuoephedrine and m/z 422/198 for the IS. The method exhibited linear dynamic ranges of 1-500 ng/mL and 2-1000 ng/mL for fexofenadine and pseudoephedrine, respectively, in human plasma. The lower limits of quantification were 1 and 2 ng/mL with a relative standard deviation of less than 10% for fexofenadine and pseudoephedrine, respectively. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time was 2 min and more than 400 human plasma samples could be analyzed in one day by running the system overnight. The method is precise and sensitive enough for its intended purpose.     

Rapid, simple and highly sensitive LC-ESI-MS/MS method for the quantification of tamsulosin in human plasma

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of tamsulosin (I), a highly selective alpha1-adrenoceptor antagonist used for the treatment of patients with symptomatic benign prostatic hyperplasia. The analyte and internal standard, mosapride (II) were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reverse phase Waters symmetry C18 column with a mobile phase of 0.03% formic acid-acetonitrile (30:70, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 409.1 solidus in circle 228.1 and m/z 422.3 solidus in circle 198.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.1-50.0 ng/mL for tamsulosin in human plasma. The lower limit of quantitation was 100 pg/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.0 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

A rapid and highly sensitive UPLC-MS-MS method for the quantification of zolpidem tartrate in human EDTA plasma and its application to pharmacokinetic study

A rapid and high sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the quantification of zolpidem in human EDTA plasma using ondansetron (IS) as an internal standard. The analyte and IS were extracted from human plasma using ethyl acetate and separated on a C18 column (Inertsil-ODS, 5 μm, 4.6 × 50 mm) interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase, which consisted of a mixture of methanol and 20 mM ammonium formate (pH 5.00 ± 0.05; 75:25 v/v), was injected at a flow rate of 0.40 mL/min. The retention times of zolpidem and IS were approximately 1.76 and 1.22. The LC run time was 3 min. The electrospray ionization source was operated in positive ion mode. Multiple reaction monitoring used the [M + H](+) ions m/z 308.13 → 235.21 for zolpidem and m/z 294.02 → 170.09 for the ondansetron, respectively. Five freeze-thaw cycles was established at -20 and -70°C.The linearity of the response/concentration curve was established in human EDTA plasma over the concentration range 0.10-149.83 ng/mL. The lower detection limit [(signal-to-noise (S/N) > 3] was 0.04 ng/mL and the lower limit of quantification (S/N > 10) was 0.10 ng/mL. This LC-MS-MS method was validated with intra-batch and inter-batch precision of 0.52-8.66.The intra-batch and inter-batch accuracy was 96.66-106.11. Recovery of zolpidem in human plasma was 87.00% and IS recovery was 81.60%. The primary pharmacokinetic parameters were T(max) (h) = (1.25 ± 0.725), C(max) (ng/mL) (127.80 ± 34.081), AUC(0→t), = (665.37 ± 320.982) and AUC(0→∞), 686.03 ± 342.952, respectively.     

Clonazepam quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry in a bioequivalence study

A rapid, sensitive and specific method for quantifying clonazepam in human plasma using diazepam as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a hexane/diethylether (20 : 80, v/v) solution. The extracts were analysed by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed on a Jones Genesis C8 4 microm analytical column (100 x 2.1 mm i.d.). The method had a chromatographic run time of 3.0 min and a linear calibration curve over the range 0.5-50 ng/ml (r2 > 0.9965). The limit of quantification was 0.5 ng/ml. This HPLC/MS/MS procedure was used to assess the bioequivalence of two clonazepam 2 mg tablet formulations (clonazepam test formulation from Ranbaxy Laboratories Ltd and Rivotril from Roche Laboratórios Ltda as standard reference formulation).     

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.     

Simultaneous determination of triptolide, tripdiolide and tripterine in human urine by high-performance liquid chromatography coupled with ion trap atmospheric-pressure chemical ionization mass spectrometry

An accurate and selective method for the simultaneous determination of triptolide, tripdiolide and tripterine in human urine using hydrocortisone as an internal standard (IS) by high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization mass spectrometry in negative ion mode has been developed. After triptolide, tripdiolide and tripterine in human urine were extracted with ethyl acetate and cleaned by solid-phase extraction with C(18) cartridges, a satisfactory separation was achieved on an XDB C(18) short column (30 x 2.1 mm i.d., 3 microm) using the mobile phase of acetic acid-ammonium acetate (5 mmol/L, pH = 4.5)-acetonitrile-methanol in gradient elution. Detection was operated by APCI in selected ion monitoring mode. The target ions m/z 359, m/z 375, m/z 449 and m/z 419 were selected for the quantification of triptolide, tripdiolide, tripterine and IS, respectively. The linear range was 1.0-100.0 ng mL(-1), and the limits of quantification in human urine were found to be 0.1-0.5 ng mL(-1) for the three compounds. The precisions (CV%) and accuracies were 6.6-12.9 and 85.1-97.0%, respectively. The developed method could be applied to the determination of triptolide, tripdiolide and tripterine in human urine for diagnosis of the intoxication and for forensic purposes.     

Liquid chromatography/electrospray ionization mass spectrometry method for the quantification of valproic acid in human plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method was developed and validated for the quantification of valproic acid, an antiepileptic drug, in human plasma using benzoic acid as internal standard (IS). Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the single ion monitoring mode using the respective [M-H]- ions, m/z 143 for valproic acid and m/z 121 for the IS. The assay exhibited a linear dynamic range of 0.5-60 microg/mL for valproic acid in human plasma. The lower limit of quantification was 500 ng/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of valproic acid and the IS from spiked plasma samples were 96.1+/-4.2 and 95.6+/-2.7%, respectively. A run time of 4.5 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability and bioequivalence studies.     

Rapid quantification of lisinopril in human plasma by liquid chromatography/tandem mass spectrometry

An assay based on protein precipitation and liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative analysis of lisinopril in human plasma. After the addition of enalaprilat as internal standard (IS), plasma samples were prepared by one-step protein precipitation using perchloric acid followed by an isocratic elution with 10 mm ammonium acetate buffer (pH adjusted to 5.0 with acetic acid)-methanol (70:30, v/v) on a Phenomenex Luna 5 mu C(18) (2) column. Detection was performed on a triple-quadrupole mass spectrometer utilizing an electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 406 --> 246 for lisinopril and m/z 349 --> 206 for enalaprilat. Calibration curves of lisinopril in human plasma were linear (r = 0.9973-0.9998) over the concentration range 2-200 ng/mL with acceptable accuracy and precision. The limit of detection and lower limit of quantification in human plasma were 1 and 2 ng/mL, respectively. The validated LC-MS/MS method has been successfully applied to a preliminary pharmacokinetic study of lisinopril in Chinese healthy male volunteers.