Throughput Liquid Chromatography-tandem Mass Spectrometry Research Articles

LC-MS/MS-MS3 for the Determination and Quantification of Δ9-Tetrahydrocannabinol and Metabolites in Blood Samples.

Due to the high prevalence of cannabinoids in forensic toxicology analysis, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation, for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry methodology (LC-MS/MS-MS3) was developed for the determination and quantification of ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9- tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH), in blood samples. Chromatographic analysis of THC, THC-OH, and THC-COOH and their deuterated internal standards was preceded by protein precipitation (PPT) of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved by use of an Acquity UPLC® HHS T3 (100 mm x 2.1mm i.d., 1.8 μm) reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate, 0.1% formic acid, and methanol at a flow rate of 0.4 mL/min, with a run time of 10 minutes. For the MS/MS-MS3 analysis, a SCIEX QTRAP® 6500+ triple quadrupole linear ion trap mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction monitoring (MRM) and linear ion trap mode (MS3). The method was validated in accordance with internationally accepted criteria and guidelines, and proved to be selective and linear between 0.5-100 ng/mL (r2>0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were <15%. The mean recoveries were between 88.0-117.2%, for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantification of cannabinoids in blood, using LC-MS/MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC-MS3 technique to determine drugs in biological samples.

Can dried blood spots be used to accurately measure vitamin D metabolites?

BackgroundWhere conventional blood sampling is challenging, dried blood spots (DBS) provide a practical sample alternative for measuring vitamin D levels. Our study aimed to develop and evaluate a clinical pathology service-based assay suitable for measuring vitamin D in batches of DBS samples collected remote to the testing site. MethodsA high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with derivatisation was developed to measure 25-hydroxyvitamin D metabolites (25OHD3, 25OHD2 and 3-epi-25OHD3) in DBS samples. The assay was validated using paired DBS and plasma samples from 37 healthy adults. ResultsThe assay reproducibly (<11.5% coefficient of variation) quantified 25OHD3 (range 1–300 nmol/L), 25OHD2 (range 2–300 nmol/L) and 3-epi-25OHD3 (range 1–200 nmol/L) in DBS samples. The 25OHD3 metabolite was detected in all DBS samples, 3-epi-25OHD3 in six plasma (range 2.1–6.3 nmol/L) and paired DBS samples, and 25OHD2 was not detected. Concentrations of 25OHD3 were highly correlated between paired samples: capillary DBS and venous plasma (r = 0.92), venous DBS and venous plasma (r = 0.93), and capillary DBS and venous DBS (r = 0.97). Ordinary least squares regression was used to characterise (β = 0.81) and correct the systematic bias in DBS data (compared to paired plasma). Thereafter, Bland-Altman analysis demonstrated robust agreement between sample-methods. ConclusionThis simple and rapid DBS-based LC-MS/MS assay accurately quantified serum vitamin D metabolites using a paired-sample ‘bridging strategy’ to correct for the inherent sample-method bias.

Development and validation of a bio-analytical method for simultaneous quantification of nebivolol and labetalol in aqueous humor and plasma using LC-MS/MS and its application to ocular pharmacokinetic studies

Beta blockers is the class of choice of drugs in treatment of open angle Glaucoma. However, many of these drugs suffer from systemic side effects due to their absorption into systemic circulation via nasolachrymal duct. To evaluate the safety and efficacy of nebivolol and labetalol for the treatment of open angle glaucoma, it is important to have a bioanalytical method for measuring the drug concentrations both in aqueous humor and plasma. A simple, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with protein precipitation technique was developed for simultaneous quantification of nebivolol and labetalol using nebivolol-d4 and metoprolol, respectively, as internal standards in aqueous humor and plasma. Nebivolol and labetalol were monitored in electrospray positive ionization (ESI) mode at transition 406.2/151.1 and 329.2/162.0, respectively. Mobile phase comprised of mixture of aqueous buffer (solvent A) and organic phase (solvent B) (mixture of A:B in the ratio of 30:70, v/v). The aqueous buffer was 5 mM ammonium acetate buffer adjusted to pH 3.5 ± 0.05 with formic acid while the organic phase was a mixture of methanol and acetonitrile in the ratio of 25:75, v/v. Chromatographic separation was achieved using reverse phase Zorbax SB-C18 column (4.6 × 100 mm, 3.5 µm). Method was linear in both the matrices in the concentration range of 0.43–750 ng/mL for nebivolol and 0.39–668 ng/mL for labetalol with r2 > 0.99. Accuracy values, expressed in terms of bias (%), for nebivolol in aqueous humor and plasma were ≤9.6% and ≤11.4% and for labetalol were ≤8.6% and ≤5.9%, respectively. Inter-day and intra-day precision values, expressed in terms of RSD (%), for both the drugs were within 11.4%. No interference was obtained due to matrix components. Mean recovery (%) values in aqueous humor and plasma were 72.4% and 73.0% for nebivolol and 56.7% and 54.4% for labetalol, respectively. No significant degradation was observed in both the drugs in both the matrices when stored at −20 °C for 1 month. Aqueous humor and plasma samples of nebivolol and labetalol on bench top were stable for 18 h and 8 h, respectively. The developed method was applied for determining pharmacokinetic parameters of both drugs in aqueous humor following single dose ocular administration in rabbits.

Decreased levels of guanosine 3', 5'-monophosphate (cGMP) in cerebrospinal fluid (CSF) are associated with cognitive decline and amyloid pathology in Alzheimer's disease.

Levels of the cyclic nucleotides guanosine 3', 5'-monophosphate (cGMP) or adenosine 3', 5'-monophosphate (cAMP) that play important roles in memory processes are not characterized in Alzheimer's disease (AD). The aim of this study was to analyse the levels of these nucleotides in cerebrospinal fluid (CSF) samples from patients diagnosed with clinical and prodromal stages of AD and study the expression level of the enzymes that hydrolyzed them [phosphodiesterases (PDEs)] in the brain of AD patients vs. For cGMP and cAMP CSF analysis, the cohort (n = 79) included cognitively normal participants (subjective cognitive impairment), individuals with stable mild cognitive impairment or AD converters (sMCI and cMCI), and mild AD patients. A high throughput liquid chromatography-tandem mass spectrometry method was used. Interactions between CSF cGMP or cAMP with mini-mental state examination (MMSE) score, CSF Aβ(1-42) and CSF p-tau were analysed. For PDE4, 5, 9 and 10 expression analysis, brains of AD patients vs. controls (n = 7 and n = 8) were used. cGMP, and not cAMP levels, were significantly lower in the CSF of patients diagnosed with mild AD when compared with nondemented controls. CSF levels of cGMP showed a significant association with MMSE-diagnosed clinical dementia and with CSF biomarker Aβ42 in AD patients. Significant increase in PDE5 expression was detected in temporal cortex of AD patients compared with that of age-matched healthy control subjects. No changes in the expression of others PDEs were detected. These results support the potential involvement of cGMP in the pathological and clinical development of AD. The cGMP reduction in early stages of AD might participate in the aggravation of amyloid pathology and cognitive decline.

Determination of Urinary Heterocyclic Aromatic Amines in Smokers and Non-Smokers by Using Sensitive and High Throughput Liquid Chromatography-Tandem Mass Spectrometry

Tobacco smoking is the single leading cause of preventable death in the United States. A number of heterocyclic aromatic amines (HCAA) are harmful and potentially harmful constituents of tobacco smoke. In the present study, a sensitive, precise and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with high sample throughput was developed to quantitate HCAA in urine from both smokers and nonsmokers. We find that the concentrations of 2-amino-9H-pyrido[2,3-b] indole (AC) and 2-amino-3-methyl-9H-pyrido[2,3-b] indole (MeAC) in smokers were significantly higher than those in non-smokers (P&lt;0.05). No significant differences were found in urinary level of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) between smokers and non-smokers (P&gt;0.05). Two beta-carboline HCAA, harman and norharman, were present at similar concentrations in both smokers and non-smokers (P&gt;0.05). The urinary levels of AC and MeAC were also found to be highly correlated with tobacco-specific carcinogen exposure as assessed by urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, NNAL) (r2 &gt;0.7). Except for AC, MeAC, PhIP, harman, and norharman, other HCAA in the urine samples of both smokers and non-smokers were too low to be detected. In conclusion, a sensitive and high throughput analytical method has been established to monitor urinary HCAA in smokers and non-smokers. AC and MeAC are the most prevalent HCAA resulting from tobacco smoke exposure.

Sensitive, selective and rapid determination of lafutidine in human plasma by solid phase extraction-liquid chromatography-tandem mass spectrometry

A simple, sensitive and high throughput liquid chromatography-tandem mass spectrometry method has been developed for the determination of lafutidine in human plasma. Sample clean-up involved solid phase extraction of lafutidine along with ranitidine as the internal standard from 100 μL of human plasma. The chromatographic separation is achieved within 2.5 min on a Grace Denali C18 (50 × 4.6 mm, 5 μ) column using 2 mM ammonium acetate, pH 3.0 adjusted with acetic acid and acetonitrile (20: 80, v/v) as the mobile phase. The precursor → product ion transitions for lafutidine (m/z 432.2 → 351.4) and IS (m/z 315.3 → 176.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method is validated over a wide dynamic concentration range of 0.25–1000 ng/mL. The mean relative recovery for lafutidine across quality controls is 97.9%. The relative matrix effect between eight different plasma lots, expressed as coefficient of variation of the slopes of the calibration lines is 1.94. The method is applied to a bioequivalence study of 10 mg lafutidine tablet formulation in 26 healthy Indian male subjects under fasting condition. The reproducibility of study data is demonstrated by analysis of 93 incurred samples.

High throughput liquid chromatography-tandem mass spectrometry assay for mercapturic acids of acrolein and crotonaldehyde in cigarette smokers' urine.

3-Hydroxypropylmercapturic acid (3-HPMA) and 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) are urinary metabolites of the toxicants acrolein and crotonaldehyde, respectively. Virtually all human urine samples contain these metabolites, resulting from the action of glutathione-S-transferases on acrolein and crotonaldehyde, which are lipid peroxidation products, environmental and dietary contaminants, and constituents of cigarette smoke. We have developed a high throughput liquid chromatography-tandem mass spectrometry method for quantitative analysis of 3-HPMA and HMPMA in large numbers of small urine samples, as would be required in molecular epidemiology and clinical studies relating levels of these metabolites to cancer risk. Solid-phase extraction on mixed mode reverse phase-anion exchange 96-well plates provided sufficient purification for LC-MS/MS analysis, which was performed by auto-injection using a 96-well format, and resulted in clean, readily interpretable chromatograms, with detection limits of 4.5pmol/mL urine for 3-HPMA and 3.5pmol/mL urine for HMPMA. Accuracy was 92% for 3-HPMA and 97% for HMPMA while inter-day precision was 9.1% (coefficient of variation) for 3-HPMA and 11.0% for HMPMA. The method was applied to more than 2600 urine samples from smokers; mean levels of 3-HPMA and HMPMA were 4800±5358 (S.D.)pmol/mL and 3302±3341pmol/mL, respectively.

Development of a sensitive and rapid method for quantitation of (S)-(−)- and (R)-(+)-metoprolol in human plasma by chiral LC–ESI–MS/MS

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC–ESI–MS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 (250mm×4.6mm, 5μm) column. Solid phase extraction of (S)-(−)- and (R)-(+)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200μL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15mM ammonium acetate in water, pH 5.0 and 0.1% (v/v) diethyl amine in acetonitrile (50:50, v/v) as the mobile phase within 7.0min. The precursor→product ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500–500ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of 200mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison with different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices.

Determination of Aprepitant in Human Plasma by Using LC-MS/MS with Electrospray Ionization

A precise, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of Aprepitant (APT) in human plasma was developed and validated using Quetiapine (QTP) as internal standard. The analyte and internal standard were extracted from human plasma using liquid-liquid extraction. Chromatographic separation was performed on Discovery C18 10 cm×4.6 mm, 5 μm column with an isocratic mobile phase composed of 5 mM Ammonium Acetate (pH 4.00):Acetonitrile (10:90), at a flow-rate of 0.9 ml/ min. The MS-MS detection was performed on a AB Sciex API 3200 tandem mass spectrometer operated in Multiple reaction monitoring (MRM) at positive mode at m/z 535.10/277.10 and 384.00/253.10 for APT and QTP respectively. A linear dynamic range of 10.004-5001.952 ng/ml for APT was evaluated with mean correlation coefficient (r) of 0.9991. The precision of the assay (expressed as coefficient of variation, CV) was less than 15% at concentrations of LQC, MQC, HQC and was less than 20% for LLOQQC. Percent recoveries for APT at high, middle and low quality control samples was found to be 71.9%, 68.0%, and 63.8% respectively and for internal standard 77.7%. The analyte was found to be stable throughout five freeze-thawing cycles, bench top, wet extract, dry extract, auto sampler and interim stability studies. Therefore, the proposed method was found to be suitable for the routine quality control analysis of Aprepitant in human plasma in bioequivalence studies.

Erratum to “Development and Validation of a LC/MS/MS Method for the Determination of Duloxetine in Human Plasma and Its Application to Pharmacokinetic Study”

A selective, high sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of duloxetine in human EDTA plasma using fluoxetine (IS) as an internal standard. Analyte and IS were extracted from human plasma by liquid-liquid extraction using MTBE-n Hexane (80:20).The eluted samples were chromatographed on X-terra RP8 (50 mmx4.6 mm, 5 μm particle size) column by using mixture of 30 mM ammonium formate (pH-5.0±0.05) and acetonitrile as an isocratic mobile phase at a flow rate of 0.40 mL/min and analyzed by mass spectrometer in the multiple reaction monitoring (MRM) using the respective m/z 298.08→154.0 for duloxetine and 310.02→148.07 for IS. The linearity of the response/ concentration curve was established in human plasma over the concentration range 0.100-100.017 ng/mL. The lower detection limit (LOD,S/N>3) was 0.04 ng/mL and the lower limit of quantization (LOQ,S/N>10) was 0.100 ng/mL. This LC-MS/MS method was validated with Intra-batch and Inter-batch precision of 5.21-7.02. The Intra-batch and Inter-batch accuracy was 97.14-103.50 respectively. Recovery of duloxetine in human plasma is 80.31% and ISTD recovery is 81.09%. The main pharmacokinetic parameters were Tmax (hr) = (7.25±1.581), Cmax (ng/mL) (44.594±18.599), AUC0→t, = (984.702±526.502) and AUC0→∞, (1027.147±572.790) respectively.

Separation of all-trans- and 13-cis-isomers of acitretin on a reversed-phase column, optimization of stability conditions and their simultaneous quantitation in human plasma by liquid chromatography-tandem mass spectrometry

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of all-trans-acitretin and 13-cis-acitretin in human plasma. Sample clean-up involved solid phase extraction of both the isomers and their deuterated analogs (all-trans-acitretin-d3 and 13-cis-acitretin-d3) used as internal standards from 300 μL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.3 on a Gemini C18 (50 × 2.0 mm, 3 μ) analytical column using 10 mM ammonium acetate and acetonitrile (60:40, v/v) as the mobile phase. The selectivity factor (α) of the column for the separation was 1.28, based on the capacity factors of 4.56 and 5.84 for (all-trans) and (13-cis)-isomers respectively. The parent → product ion transitions for both the isomers (m/z 325.2 → 266.0) and IS (m/z 328.0 → 266.0) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and negative ion mode. The method was validated over the concentration range of 0.3–500 ng mL−1 for both the analytes. Stability studies were conducted in plasma to check the extent of photoisomerization under different conditions. Quantitative and consistent recovery (>92%) was obtained in the presence of L-ascorbic acid and yellow light (589 nm) for both the analytes. The matrix effect was assessed by post-column analyte infusion and the precision value (% CV) for a relative matrix effect in six different lots of plasma samples was ≤3.9% at the LLOQ levels for both the analytes. The method was successfully applied to a pivotal bioequivalence study in 48 healthy Indian subjects after oral administration of 25 mg acitretin tablet formulation under fasting conditions. To demonstrate the reproducibility in the measurement of study data, an incurred sample reanalysis was done with 270 subject samples and the % change in concentration from the initial analysis was within ±15%.

Development and Validation of a LC/MS/MS Method for the Determination of Duloxetine in Human Plasma and Its Application to Pharmacokinetic Study

A selective, high sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of duloxetine in human EDTA plasma using fluoxetine (IS) as an internal standard. Analyte and IS were extracted from human plasma by liquid-liquid extraction using MTBE-n Hexane (80:20).The eluted samples were chromatographed on X-terra RP8 (50 mmx4.6 mm, 5 μm particle size) column by using mixture of 30 mM ammonium formate (pH-5.0±0.05) and acetonitrile as an isocratic mobile phase at a flow rate of 0.40 mL/min and analyzed by mass spectrometer in the multiple reaction monitoring (MRM) using the respective m/z 298.08→154.0 for duloxetine and 310.02→148.07 for IS. The linearity of the response/ concentration curve was established in human plasma over the concentration range 0.100-100.017 ng/mL. The lower detection limit (LOD,S/N&gt;3) was 0.04 ng/mL and the lower limit of quantization (LOQ,S/N&gt;10) was 0.100 ng/mL. This LC-MS/MS method was validated with Intra-batch and Inter-batch precision of 5.21-7.02. The Intra-batch and Inter-batch accuracy was 97.14-103.50 respectively. Recovery of duloxetine in human plasma is 80.31% and ISTD recovery is 81.09%. The main pharmacokinetic parameters were Tmax(hr) = (7.25±1.581), Cmax(ng/mL) (44.594±18.599), AUC0→t, = (984.702±526.502) and AUC0→∞, (1027.147±572.790) respectively.

Bioanalytical method development and validation for simultaneous estimation of (R)- and (S)-isomers of S002-333: a potent novel anti-thrombotic agent using LC–MS/MS

A selective, sensitive, and high throughput liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the chromatographic separation and quantitation of diastereomers of S002-333, a novel anti-thrombotic agent in rabbit plasma. Sample clean-up involved liquid–liquid extraction (LLE) of both the isomers and internal standard (β-carbolinamide) from 200 μl of rabbit plasma. Both the analytes were chromatographically separated on a Chiralcel OJ-RH column (150 × 4.6 mm, 5 μm particle size) using a gradient flow program comprising 0.1% formic acid, methanol, and acetonitrile as the mobile phase. The parent → product ion transitions (MRM) for both the isomers and IS were 386.4 → 214.2 m/z and 216.1 → 144.2 m/z, respectively, and were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The MS/MS response was linear over the concentration range from 1.56 ng/ml to 400 ng/ml, with a lower limit of detection (LOD) 1.56 ng/ml. The intra- and inter-day precisions (% R.S.D.) between 3.96 and 13.80 for both analytes and the accuracies (% bias) were between −4.05 and 5.93. The validated method can be used in most or all stages of the screening and optimizing process for pharmacokinetic and toxicokinetics studies.

Chromatographic separation of ( E)- and ( Z)-isomers of entacapone and their simultaneous quantitation in human plasma by LC–ESI-MS/MS

A selective, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of ( E)-entacapone and ( Z)-entacapone in human plasma. Sample clean-up involved liquid–liquid extraction (LLE) of both the isomers and carbamazepine used as internal standard from 500 μL of human plasma. Both the analytes were chromatographically separated with a resolution factor of 3.0 on a Gemini C18 (50 mm × 4.6 mm, 5 μm particle size) analytical column using 1% formic acid and methanol (50:50, v/v) as the mobile phase. The selectivity factor ( α) of the column for the separation was 2.0, based on the capacity factors of 2.6 and 1.3 for ( E)- and ( Z)-isomers respectively. The parent → product ion transitions for both the isomers ( m/ z 306.1 → 233.0) and IS ( m/ z 237.3 → 194.2) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 24.3–6076 ng/mL and 23.8–5960 ng/mL for ( E)-entacapone and ( Z)-entacapone respectively. Matrix effect was assessed by post-column analyte infusion experiment and the process/extraction efficiency found was 94.3% and 89.3% for ( E)- and ( Z)-isomers respectively. The method was successfully applied to a pivotal bioequivalence study in 36 healthy human subjects after oral administration of 200 mg ( E)-entacapone tablet formulation under fasting conditions.

Analysis of second-generation antidepressant drug, sertraline and its active metabolite, N-desmethyl sertraline in human plasma by a sensitive and selective liquid chromatography–tandem mass spectrometry method

A precise, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of sertraline (SER) and its primary metabolite, N-desmethyl sertraline (NDS) in human plasma is developed and validated. The analytes and the internal standard-fluoxetine were extracted from 300 μL aliquots of human plasma via liquid–liquid extraction in methyl tert-butyl ether. Chromatographic separation was achieved in a run time of 2.5 min on a Betasil C8 column (100 mm × 2.1 mm, 5 μm) under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for SER, NDS and IS were m/ z 306.2 → 159.0, 292.1 → 159.0 and 310.6 → 148.4, respectively. The method was fully validated for its sensitivity, selectivity, linearity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 0.5–150 ng/mL was established for both the analytes with mean correlation coefficient ( r) of 0.9993 and 0.9980, respectively. The intra-batch and inter-batch precision (%CV) across five quality control levels was ≤10.4% for both the analytes. The method was successfully applied to a bioequivalence study of 100 mg sertraline tablet formulation in 32 healthy Indian male subjects under fasting condition.

High throughput and sensitive determination of trazodone and its primary metabolite, m-chlorophenylpiperazine, in human plasma by liquid chromatography–tandem mass spectrometry

A precise, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of trazodone (TRZ) and its primary metabolite, m-chlorophenylpiperazine ( mCPP), in human plasma was developed and validated. The analytes and the internal standard-nefazodone were extracted from 500 μL aliquots of human plasma via liquid–liquid extraction in n-hexane. Chromatographic separation was achieved in a run time of 2.5 min on a Betabasic cyano column (100 mm × 2.1 mm, 5 μm) under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for TRZ, mCPP and IS were m/ z 372.2 → 176.2, 197.2 → 118.1 and 470.5 → 274.6 respectively. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 10.0–3000.0 ng/mL for TRZ and 0.2–60.0 ng/mL for mCPP was evaluated with mean correlation coefficient ( r) of 0.9986 and 0.9990 respectively. The intra-batch and inter-batch precision (%CV) across five validation runs (LLOQ, lower limit of quantitation; LQC, low quality control; MQC, middle quality control; HQC, high quality control and ULOQ, upper limit of quantitation) was ≤8.4% for both the analytes. The method was successfully applied to a bioequivalence study of 100 mg trazodone tablet formulation in 36 healthy Indian male subjects under fasting and fed conditions.