Lansoprazole quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of lansoprazole in human plasma using omeprazole as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid-liquid extraction using diethyl-ether-dichloromethane (70:30; v/v) and chromatographed on a C(18) analytical column. The mobile phase consisted of acetonitrile-water (90:10; v/v)+10 mM formic acid. The method has a chromatographic total run time of 5 min and was linear within the range 2.5-2000 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by Multiple Reaction Monitoring (MRM). The intra- and inter-run precision, calculated from quality control (QC) samples, was less than 3.4%. The accuracy as determined from QC samples was less than 9%. The method herein described was employed in a bioequivalence study of two capsule formulations of lansoprazole.     

Lisinopril quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of Lisinopril in human plasma using Enalaprilat as internal standard. The analyte and internal standard were extracted from the plasma samples by solid-phase extraction using Waters HLB Oasis SPE cartridges and chromatographed on a C8 analytical column. The mobile phase consisted of acetonitrile/water (60:40, v/v) + 20 mM acetic acid + 4.3 mM of triethylamine. The method had a chromatographic total run-time of 6.5 min and was linear within the range 2.00-200 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM). The precision (CV%) and accuracy, calculated from limit of quantification (LOQ) samples (n = 8), were 8.9 and 98.9%, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of Lisinopril 20mg.     

Sensitive method for the determination of ibutilide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for determination of ibutilide in human plasma. The analyte and internal standard sotalol were extracted from plasma samples by liquid-liquid extraction, and separated on a C(18) column, using acetonitrile-water-10% butylamine-10% acetic acid (80:20:0.07:0.06, v/v/v/v) as the mobile phase. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via TurboIonSpray ionization (ESI). Linear calibration curves were obtained in the concentration range of 20-10,000 pg/ml, with a lower limit of quantitation of 10 pg/ml. The intra- and inter-day precision values were below 8% and accuracy was within +/-3% at all three QC levels. The method was utilized to support clinical pharmacokinetic studies of ibutilide in healthy volunteers following intravenous administration.     

Simultaneous determination of flupirtine and its major active metabolite in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective and sensitive HPLC–tandem mass spectrometry method was developed and validated for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The analytes and internal standard diphenhydramine were extracted from plasma samples by liquid–liquid extraction, and chromatographed on a C₁₈ column. The mobile phase consisted of acetonitrile–water–formic acid (60:40:1, v/v/v), at a flow rate of 0.5 ml/min. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method has a limit of quantitation of 10 ng/ml for flupirtine and 2 ng/ml for D-13223, using 0.5-ml plasma sample. The linear calibration curves were obtained in the concentration range of 10.0–1500.0 ng/ml for flupirtine and 2.0–300.0 ng/ml for D-13223. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples was less than 7.2% for flupirtine and D-13223. The accuracy as determined from QC samples was less than 5% for the analytes. The overall extraction recoveries of flupirtine and D-13223 were determined to be about 66% and 78% on average, respectively. The method was applied for the evaluation of the pharmacokinetics of flupirtine and active metabolite D-13223 in volunteers following peroral administration.     

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

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.     

Development and validation of a selective and robust LC-MS/MS method for high-throughput quantifying rizatriptan in small plasma samples: application to a clinical pharmacokinetic study

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of a potent 5-HT(1B/1D) receptor agonist, rizatriptan in human plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 100 microL plasma samples by liquid-liquid extraction (LLE) and chromatographed on a Lichrospher C18 column (4.6mm x 50mm, 5 microm) with a mobile phase consisting of acetonitrile-10mM aqueous ammonium acetate-acetic acid (50:50:0.5, v/v/v) pumped at 1.0 mL/min. The method had a chromatographic total run time of 2 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 270-->201 (rizatriptan) and 313.4-->138 (granisetron) used for quantitation. The assay was validated over the concentration range of 0.05-50 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 98%. The intra-day accuracy of the assay was within 12% of nominal and intra-day precision was better than 13% C.V. Following a 10mg dose of the compound administered to human subjects, mean concentrations of rizatriptan ranged from 0.2 to 70.6 ng/mL in plasma samples collected up to 24h after dosing. Inter-day accuracy and precision results for quality control samples run over a 5-day period alongside clinical samples showed mean accuracies of within 12% of nominal and precision better than 9.5% C.V.     

LC/MS/MS measurement of penicillin G in bovine plasma, urine, and biopsy samples taken from kidneys of standing animals

Methods for the measurement of penicillin concentration in bovine plasma, kidney and urine were developed and validated. Detection was based on liquid chromatography/tandem mass spectrometry (LC/MS/MS). Phenethecillin was used as an internal standard. Plasma was extracted with acetonitrile using a method with a calculated limit of quantitation (LOQ) of 12 ng/mL. Kidney samples were homogenized in water and acetonitrile, then cleaned up on C18-bonded silica SPE cartridges. The LOQ of this procedure was 10 ng/g. Urine samples were diluted, filtered, and analyzed directly. The LOQ of this procedure was 63 ng/mL. The overall accuracy for plasma was 103% with coefficient of variation (CV) of 3%; for kidney, 96% and 11%, respectively, and for urine, 98% and 4%, respectively. These methods were applied to the analysis of plasma, urine, and kidney biopsy samples taken from standing animals that had been dosed with penicillin.     

Quantitative analysis of Variolin analog (PM01218) in mouse and rat plasma by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

PM01218 is a novel marine-derived alkaloid and has shown potent growth inhibitory activity against several human cancer cell lines. A rapid and sensitive high performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to quantify PM01218 in mouse and rat plasma. The lower limit of quantitation (LLOQ) was 0.05 ng/mL. The calibration curve was linear from 0.05 to 100 ng/mL (R(2)>0.999). The assay was specifically based on the multiple reaction monitoring (MRM) transitions at m/z 278.4-->184.2, no endogenous material interfaced with the analysis of PM01218 and its internal standard from blank mouse and rat plasma. The mean intra- and inter-day assay accuracy remained below 15 and 8%, respectively, for all calibration standards and QC samples. The intra- and inter-day assay precision was less than 12.8 and 8.5% for all QC levels, respectively. The utility of the assay was demonstrated by pharmacokinetics studies of i.v. (bolus) PM01218 on SD rats.     

Determination of tamsulosin in dog plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A rapid, sensitive and accurate liquid chromatographic-tandem mass spectrometric method is described for the determination of tamsulosin in dog plasma. Tamsulosin was extracted from plasma using a mixture of hexane-ethyl acetate (2:1, v/v) and separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase consisting of a mixture of methanol, water and formic acid (80:20:1, v/v/v) was delivered at a flow rate of 0.5 ml/min. Atmospheric pressure chemical ionization (APCI) source was operated in positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 409-->m/z 228 and m/z 256-->m/z 166.9 were used to quantify tamsulosin and the internal standard, respectively. The linearity was obtained over the concentration range of 0.1-50.0 ng/ml for tamsulosin and the lower limit of quantitation was 0.1 ng/ml. For each level of QC samples, inter- and intra-run precision was less than 5.0 and 4.0% (relative standard deviation (R.S.D.)), respectively, and accuracy was within +/-0.3% (relative error (R.E.)). This method was successfully applied to pharmacokinetic study of a tamsulosin formulation product after oral administration to beagle dogs.     

Simultaneous determination of hydrocodone and hydromorphone in human plasma by liquid chromatography with tandem mass spectrometric detection

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the simultaneous analysis of hydrocodone (HYC) and its metabolite hydromorphone (HYM) in human plasma. A robotic liquid handler and a 96-channel liquid handling workstation were used to aliquot samples, to add internal standard (I.S.), and to extract analytes of interest. A 96-well mixed-mode solid-phase cartridge plate was used to extract the analytes and I.S. The chromatographic separation was on a silica column (50 x 3 mm, 5-microm) with a mobile phase consisting of acetonitrile, water and trifluoroacetic acid (TFA) (92:8:0.01, v/v). The run time for each injection was 2.5 min with the retention times of approximately 2.1 and 2.2 min for HYC and HYM, respectively. The tandem mass spectrometric detection was by monitoring singly charged precursor-->product ion transition 300-->199 (m/z) for HYC, and 28-->185 (m/z) for HYM. The validated calibration curve range was 0.100-100 ng/ml, based on a plasma volume of 0.3 ml. The correlation coefficients were greater than or equal to 0.9996 for both HYC and HYM. The low limit of quantitation (LLOQ) was 0.100 ng/ml for both HYC and HYM with signal-to-noise ratio (S/N) of 50 and 10. respectively. The deuterated analytes, used as internal standards, were monitored at mass transitions 303-->199 (m/z) for HYC-d3 and 289-->185 (m/z) for HYM-d3. The inter-day (n= 17) precision of the quality control (QC) samples were < or = 3.5% RSD (relative standard deviation) for HYC and < or = 4.7% RSD for HYM, respectively. The inter-day accuracy of the QC samples were < or = 2.1% RE (relative error) for HYC and < or = 1.8% RE for HYM. The intra-day (n=6) precision and accuracy of the QC samples were < or = 2.6% RSD and < or = 3.0% RE for HYC, and < or = 4.7% RSD and < or = 2.4% RE for HYM. There was no significant deviation from the nominal values after a 5-fold dilution of high concentration QC samples by blank matrix. The QC samples were stable when kept at room temperature for 24-h or experienced three freeze-thaw cycles. The extraction recoveries were 86% for HYC and 78% for HYM. No detectable carryover was observed when a blank sample was injected immediately after a 2500 ng/ml sample that was 25-fold more concentrated than the upper limit of quantitation (ULOQ).     

Determination of cefaclor in human plasma by a sensitive and specific liquid chromatographic-tandem mass spectrometric method

A sensitive and specific liquid chromatographic-tandem mass spectrometric method is described for the determination of cefaclor in human plasma. The plasma samples were treated by two sample preparation procedures, i.e. protein precipitation (PPT) and solid-phase extraction (SPE). The pretreated samples were analyzed on a C(18) HPLC column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization (ESI) was employed as the ionization source. The analyte and internal standard ampicillin (for PPT) or cefetamet (for SPE) were detected by use of selected reaction monitoring (SRM) mode. The lower limit of quantitation obtained as a result of the PPT procedure was 100 ng/ml. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 12% for cefaclor. The accuracy as determined from QC samples was within +/-3% for the analyte. The SPE procedure could provide the lower limit of quantitation of 2 ng/ml. The precision and accuracy were measured to be below 7.1% and between -3.6% and 1.1%, respectively, for all QC samples. The method was applied for the evaluation of the pharmacokinetic profiles of cefaclor sustained-release formulation.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of levosulpiride in human plasma

A rapid, sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometric (HILIC-MS/MS) method for the determination of levosulpiride in human plasma was developed. Levosulpiride and internal standard, tiapride were extracted from human plasma with ethyl acetate at pH 11 and analyzed on an Atlantis HILIC silica column with the mobile phase of acetonitrile-ammonium formate (190 mM, pH 3.0) (94:6, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r = 0.999) over the concentration range of 1.00-200 ng/ml. The lower limit of quantification for levosulpiride was 1.00 ng/ml using 100 microl plasma sample. The coefficient of variation and relative error for intra- and inter-assay at three quality control (QC) levels were 3.8-9.1 and -2.9 to -0.1%, respectively. The recoveries of levosulpiride ranged from 80.5 to 87.4%, with that of tiapride (internal standard) being 84.6%. This method was successfully applied to the pharmacokinetic study of levosulpiride in humans.     

Sensitive quantification of ranolazine in human plasma by liquid chromatography--tandem mass spectrometry with positive electrospray ionization

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with positive electrospray ionization (ESI) was developed for the quantification of ranolazine in human plasma. After liquid-liquid extraction of ranolazine and internal standard (ISTD) phenoprolamine from a 100 microl specimen of plasma, HPLC separation was achieved on a Nova-Pak C(18) column, using acetonitrile-water-formic acid-10% n-butylamine (70:30:0.5:0.08, v/v/v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 428.5-->m/z 279.1 for ranolazine and m/z 344.3-->m/z 165.1 for the internal standard, respectively. Linear calibration curves were obtained in the concentration range of 5-4000 ng/ml, with a lower limit of quantitation (LLOQ) of 5 ng/ml. The intra- and inter-day precision values were below 3.7% and accuracy was within +/-3.2% at all three quality control (QC) levels. This method was found suitable for the analysis of plasma samples collected during the phase I pharmacokinetic studies of ranolazine performed in 28 healthy volunteers after single oral doses from 200 mg to 800 mg.     

Determination of rifalazil, a potent antibacterial agent, in human plasma by liquid-liquid extraction and LC-MS/MS

A sensitive assay for determination of rifalazil (also known as ABI-1648 and KRM-1648) in human plasma is described. The analytical method utilizes liquid-liquid extraction of plasma with methyl tert-butyl ether, followed by reversed-phase liquid chromatography with a C18 column and a mobile phase gradient utilizing 0.1% formic acid in water and acetonitrile, respectively. Electrospray mass spectrometry in the positive ion mode with selected reaction monitoring of rifalazil and an isotope labeled internal standard, 13C4-rifalazil (ABI-9901) was used for selective and sensitive detection. The calibration range was 0.050-50 ng/mL plasma using 200 microL plasma sample volume. The absolute extraction recovery of rifalazil from K2-EDTA plasma, evaluated at three concentration levels, was 88.6-97.3%, and the recovery for the internal standard was 96.8%. A study of plasma matrix effects showed a peak area response at 90-99% compared to neat solutions for both rifalazil and the internal standard. Stability evaluation of rifalazil in plasma, whole blood and methanol showed that the analyte stability was adequate when stored under study conditions. The precision, as evaluated in three validation batches, was consistent for fortified plasma quality control (QC) samples at four concentration levels, with < or =6% R.S.D. except for at the lowest quality control level where it was 10.7% R.S.D. The accuracy for QC samples (difference between found and nominal concentration) ranged from -2.3% to 5.1%. Similar precision and accuracy values were obtained over 6 months of routine application of this method. It was concluded that the performance improved markedly during routine operation by replacing a closely related structural analog internal standard with the stable isotope internal standard.     

Quantitative liquid chromatographic–tandem mass spectrometric determination of reserpine in FVB/N mouse plasma using a “chelating” agent (disodium EDTA) for releasing protein-bound analytes during 96-well liquid–liquid extraction

A sensitive, specific, accurate and reproducible analytical method employing a divalent cation chelating agent (disodium EDTA) for sample treatment was developed to quantitate reserpine in FVB/N mouse plasma. Samples pretreated with 40 μl of 2% disodium EDTA in water were extracted by a semi-automated 96-well liquid–liquid extraction (LLE) procedure to isolate reserpine and a structural analog internal standard (I.S.), rescinnamine, from mouse plasma. The extracts were analyzed by turbo ionspray liquid chromatography–tandem mass spectrometry (LC–MS–MS) in the positive ion mode. Sample preparation time for conventional LLE was dramatically reduced by the semi-automated 96-well LLE approach. The assay demonstrated a lower limit of quantitation of 0.02 ng/ml using 0.1-ml plasma sample aliquots. The calibration curves were linear from 0.02 to 10 ng/ml for reserpine. The intra- and inter-assay precision of quality control (QC) samples ranged from 1.75 to 10.9% for reserpine. The intra- and inter-assay accuracy of QC samples ranged from −8.17 to 8.61%. Reserpine and the I.S. were found to be highly bound to FVB/N mouse plasma protein. This is the first report of disodium EDTA employed as a special protein-bound release agent to recover protein-bound analytes from plasma. These matrix effects and the effects of pH in the HPLC mobile phase on the sensitivities of LC–MS–MS are discussed in this paper.     

Analysis of amphetamines and metabolites in urine with ultra performance liquid chromatography tandem mass spectrometry

A simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and fully validated for the quantitative determination of seven amphetamines and metabolites in urine. The method was validated for selectivity, linearity, LOQ, LOD, imprecision, bias, analyte and processed sample stability, matrix effect, recovery, carryover and dilution integrity. A classic liquid–liquid extraction with ethyl acetate was used as sample preparation procedure. The compounds were separated on an Acquity UPLC HSS C₁₈ column in 6.8 min. The linear dynamic range was established from 25 to 500 ng/mL. The limit of quantification was fixed to the lowest calibrator level and the limit of detection ranged from 0.125 to 2.5 ng/mL. The method presented an excellent intra- and inter-assay imprecision and bias (<10.7%) at each measured concentration of two external quality controls (QC) and three “in house” QC. No matrix effects were observed and good recoveries (>70%) were obtained for all the compounds. No carryover was observed after the analysis of high concentrated samples (8000 ng/mL). The method was subsequently applied to authentic samples.     

Determination of the HIV integrase inhibitor, MK-0518 (raltegravir), in human plasma using 96-well liquid-liquid extraction and HPLC-MS/MS

An HPLC-MS/MS method was developed for the determination of MK-0518 (raltegravir), an HIV integrase inhibitor, in human plasma over the concentration range of 2-1000 ng/mL. Stable isotope labeled (13)C(6)-MK-0518 was used as an internal standard. The sample preparation procedure utilized liquid-liquid extraction with hexane:methylene chloride in the 96-well format with a 200 microL plasma sample size. The compounds were chromatographed on an Ace C(18) (50 x 3.0 mm, 3 microm, titanium frits) column with 42.5/57.5 (v/v %) 0.1mM EDTA in 0.1% formic acid/methanol mobile phase at a flow rate of 0.5 mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for MK-0518 (m/z 445-->109) and (13)C(6)-MK-0518 (m/z 451-->367) on an Applied Biosystem API 4000 HPLC-MS/MS was used for quantitation. Intraday precision of standard curve concentrations in five different lots of control plasma was within 3.2%, while accuracy ranged from 94.8 to 106.8%. The mean extraction recovery of spiked plasma samples was 87%. Quality control (QC) samples were stored at -20 degrees C. Initial within day analysis showed QC accuracy within 7.5% of nominal with precision of 3.1% or less. The plasma QC samples were demonstrated to be stable for up to 23 months at -20 degrees C. The method described has been used to support over 18 clinical studies during Phase I through III of clinical development.     

Determination of dimenhydrinate in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a relative bioavailability study

Here we present a sensitive and specific liquid chromatography-tandem mass spectrometric method for the quantification of dimenhydrinate (I) in human plasma. Sample preparation is conducted using citalopram (II) addition as an internal standard (IS), liquid-liquid extraction with basified plasma using a mixture hexane/acetate (1:1, v/v) as the extracting solvent, and the final extract reconstituted in the mobile phase. I and II (IS) were injected in a C8 column with the mobile phase composed of methanol:isopropanol:water:formic acid (78.00:19.92:2.00:0.08, v/v/v/v) and monitored using a positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 256.0>167.0 and m/z 325.0>109.0 for I and II, respectively. The limit of quantification (LOQ) was 0.4 ng/mL, the dynamic range being 0.4-200 ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of plasma samples taken up to 24 h after oral administration of 100 mg of dimenhydrinate in healthy volunteers demonstrated its applicability to bioavailability studies.     

Determination of talinolol in human plasma using automated on-line solid phase extraction combined with atmospheric pressure chemical ionization tandem mass spectrometry

A specific LC-MS/MS assay was developed for the automated determination of talinolol in human plasma, using on-line solid phase extraction system (prospekt 2) combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. The method involved simple precipitation of plasma proteins with perchloric acid (contained propranolol) as the internal standard (IS) and injection of the supernatant onto a C8 End Capped (10 mmx2 mm) cartridge without any evaporation step. Using the back-flush mode, the analytes were transferred onto an analytical column (XTerra C18, 50 mmx4.6 mm) for chromatographic separation and mass spectrometry detection. One of the particularities of the assay is that the SPE cartridge is used as a column switching device and not as an SPE cartridge. Therefore, the same SPE cartridge could be used more than 28 times, significantly reducing the analysis cost. APCI ionization was selected to overcome any potential matrix suppression effects because the analyte and IS co-eluted. The mean precision and accuracy in the concentration range 2.5-200 ng/mL was found to be 103% and 7.4%, respectively. The data was assessed from QC samples during the validation phase of the assay. The lower limit of quantification was 2.5 ng/mL, using a 250 microL plasma aliquot. The LC-MS/MS method provided the requisite selectivity, sensitivity, robustness accuracy and precision to assess pharmacokinetics of the compound in several hundred human plasma samples.