Accurate Liquid Chromatography-tandem Mass Research Articles

A simple and sensitive liquid chromatography–tandem mass spectrometry method for quantification of multi‐residual pesticides in blood

AbstractPesticides are synthetic chemical compounds that are toxic and may cause toxicity when exposed in large quantities. The leading site of action of these compounds is the inhibition of acetylcholinesterase. Quantifying the pesticide levels in the human body can help understand and study pesticide exposure in humans. The study aimed to develop and validate a rapid and accurate liquid chromatography–tandem mass spectrometry method for the quantitative determination of pesticide concentration in human blood samples. The analytes were separated using an Agilent Stable Bond C18 column and a mobile phase containing 10 mM ammonium formate in water and acetonitrile. The limits of detection and quantitation from the developed method were 0.023–0.161 and 0.072–0.487 ng/mL, respectively, while the recoveries from the spiked blood samples were 78.01–104.36%. The intraday and interday accuracies and precision were 40–105% and 0.5–13 RSD% and 40–113% and 3–14 RSD%, respectively. Linear regression was obtained at a value of > 0.9921, indicating the method to be accurate and precise. Validation was further performed on pesticide exposure and control blood samples effectively. The developed method can be employed to estimate the levels of exposure in blood samples since the method has been found to be accurate and reliable under the European standard guidelines.

LC–MS/MS Determination of Prednisone, A Drug in Phase 4 of Multiple Sclerosis Therapy along with Teriflunomide and Pioglitazone in Rat Plasma

Combination therapy or polytherapy is that uses more than one medication to treat a single disease and associated diseases. Pharmaceutical combination therapy may be achieved by prescribing separate drugs, or, where available, dosage forms that contain more than one active ingredient. A randomized efficacy phase 4 study is going on for prednisone in the treatment of multiple sclerosis, which is a neurodegenerative disease associated with many other disorders. A novel and accurate liquid chromatography tandem mass spectrometry method using electrospray ionization mode has been developed and validated for the simultaneous determination of prednisone (PDN), pioglitazone (PGZ) and teriflunomide (TFM) using imipramine (IMP) and ibuprofen (IBP) as internal standards (IS). The separation was carried on XTerra MS C18 (100 mm x 3.9 mm, 5 µm) reversed phase column using acetonitrile and 0.01M ammonium formate as the mobile phase in gradient mode at 1.0 mL/min. The method was validated in terms of specificity, linearity, accuracy and precision over the concentration range of 1–500 ng/mL. The intra and inter-day precision and accuracy, stability and extraction recoveries of all the analytes were found in the range of 97.2-102.2%. The lower limit of quantitation was 1.0 ng/mL for all the 3 analytes and the extraction recovery values were more than 65%. The method proved highly reproducible and sensitive and was successfully applied to pharmacokinetic study after single dose oral administration of PDN, PGZ and TFM to the rats. Keywords Polytherapy, Teriflunomide, Prednisone, Pioglitazone, Phase 4, Multiple Sclerosis, Pharmacokinetics

Analytical method for sequential determination of persistent herbicides and their metabolites in fish tissues by UPLC–MS/MS

A novel and accurate liquid chromatography–tandem mass spectrometry method was developed to sequentially determine three persistent herbicides (atrazine (ATZ), acetochlor (ACE), and metolachlor (MET)) and seven characteristic metabolites (desethylatrazine (DEA), deisopropylatrazine (DIA), diaminochlorotriazine (DACT), MET–oxanilic acid (MET-OA), MET-ethanesulfonic acid (MET-ESA), ACE-ESA, and ACE-OA) in fresh fish tissues from six fish species. A modified QuEChERS method was conducted to extract the target compounds from fish tissues. Matrix-matched calibrations of the target analytes were carried out at spiking levels of 1, 10, 100, and 1000 ng g−1. The method was validated in accordance with Codex guidelines (CAC/GL 71–2009). Recoveries for the target analytes were 67–120% with relative standard deviations below 20%, and the matrix effects ranged from −58.7% to 59.3%. The limits of detection and quantitation were 0.01–1.90 and 0.02–6.35 ng g−1, respectively. Moreover, the method was successfully applied to analyze the concentrations of the target chemicals in fresh tissue samples of six fish species (n = 67) collected from four markets in Nanning City, Guangxi Province, China. The concentrations in all samples were 1.1–140.5 ng g−1. Interestingly, this study was the first to measure DEA and DIA in fish liver, and their highest concentrations were 10.7 and 14.2 ng g−1, respectively. This method provides a basis for studying the pathways of biotransformation, bioaccumulation, detoxification, and exposure patterns of ACE, ATZ, MET, and their metabolites in aquatic environments.

Quantification of Tofacitinib in Human Plasma Samples Using Radio-Labeled Internal Standard

A simple, sensitive and accurate liquid chromatography tandem mass spectrometric method has been developed and validated for determination of Tofacitinib in human plasma. The method was developed on Agilent Zorbax SB-C8 150×4.6 mm, 5 µm column using 10mM ammonium formate: acetonitrile (40:60 v/v) mobile phase for Chromatographic separation of Tofacitinib. The Tofacitinib and Tofacitinib- C3 were monitored by electrospray ionization in positive ion multiple reactions monitoring mode to detect the Tofacitinib at mass/charge 313.200/149.200 and Tofacitinib-13C3 (Internal Standard) at 316.500/149.200. Liquid-liquid extraction was employed in the extraction of analytes from human plasma. Both drug and internal standards were stable in plasma samples. The proposed method was validated as per international council of harmonization guidelines over a linear concentration range of 0.2ng/mL to 100.0ng/mL with a correlation coefficient (r) of ≥ 0.9983. Bioavailability and Bioequivalence studies of tofacitinib in biological samples can be achieved by analyzing them using the validated developed method. This study plays a key role in determining routine therapeutic drug monitoring of tofacitinib drug.

Validated Liquid Chromatography-Tandem Mass Spectroscopic Method for Simultaneous Determination of Certain Drugs Used in Cataract Surgery in Rabbit Aqueous Humor.

A sensitive, selective, accurate and precise ultra-high performance liquid chromatography-Tandem mass spectrometry (MS/MS) method was developed and validated for the simultaneous determination of drugs used as eye drops in cataract surgery in aqueous humor. Cataract surgery requires a powerful mydriatic eye drops combination such as cyclopentolate hydrochloride and phenylephrine hydrochloride to dilate the pupil and facilitate eye lens replacement and also requires strong fluoroquinolone antibiotic such as lomefloxacin hydrochloride. The method was performed with positive ion electrospray ionization and the analytes were quantified and monitored on a triple quadrupole mass spectrometer using multiple reaction monitoring scanning mode. Liquid-liquid extraction was used for the purification and preconcentration of analytes from rabbit aqueous humor matrix. Chromatographic elution was performed using an Phenomenex Luna® C18 (150mm×2.1mm, 1.6μm) column and moxifloxacin hydrochloride as internal standard with a mobile phase consisting of methanol:water:formic acid (70:29:1, by volume) at flow rate of 0.2mL/min. Satisfactory results regarding linearity, recovery, stability, accuracy and precision of the analytes were obtained. Full validation of the procedure was performed according to the US Food and Drug Administration guidance for industry: bioanalytical method validation and European Medicines Agency (EMA) guideline on bioanalytical method validation.

Two rapid, accurate liquid chromatography tandem mass spectrometry methods for the quantification of seven uremic toxins: An application for describing their accumulation kinetic profile in a context of acute kidney injury

Acute kidney injury (AKI) is a frequent, serious complication in critically ill patients. Even if renal replacement therapy is rapidly initiated, AKI may lead to the acute accumulation of metabolic waste products called uremic toxins (UTs). Although the accumulation and effects of UTs have been extensively described in the setting of chronic kidney disease (CKD), few data are available for AKI. A rapid, sensitive, specific method with simple sample preparation is required to facilitate routine blood monitoring of UTs in a context of acute accumulation.We have developed and validated two fast liquid chromatography tandem mass spectrometry methods for the quantification of seven UTs in human serum. The first method (in negative ionization mode) enables the quantification of five UTs (hippuric acid (HA), indoxyl sulfate (IxS), para-cresyl sulfate (pCS), para-cresyl glucuronide (pCG), 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF)). The second method (in positive ionization mode) enables the quantification of two UTs (indole-3-acetic acid (IAA), and trimethylamine N-oxide (TMAO)). Sample preparation consisted of the deproteinization of a small volume of serum (50 µL). The run-times required to assay all the UTs in negative and positive ionization modes were only 2.5 and 2 min, respectively. In order to obtain a reliable, toxin-free matrix for the preparation of calibration standards and quality controls, serum was pretreated with activated charcoal. We used these methods to determine the time course of UT accumulation in eight patients who developed an AKI after cardiac surgery.The calibration curves ranged from 0.1 to 100 µg mL−1 for all the UTs (except for IAA: 0.5 to 100 µg mL−1), and the correlation coefficients were above 0.999 for all. The methods were reproducible, repeatable, and accurate, with all coefficients of variation and biases below 15%. The highest concentrations measured in patients with AKI were lower than those reported in CKD stages 4 and 5 but higher than those observed in patients with no impairment of renal function (particularly for IxS and pCS). Our results also highlighted low accumulation of the other toxins (IAA, HA, TMAO, pCG, and CMPF). The UT concentrations did not rise earlier than that of creatinine; although the return to baseline took longer than for creatinine for some compounds. Lastly, assessment of the time course of UT accumulation as a prognostic marker for AKI (particularly for pCS and IxS) appears to be promising and should be continued in a larger number of patients.

LC-MS/MS METHOD FOR L-DOPA QUANTIFICATION IN DIFFERENT TISSUES OF VICIA FABA

A rapid, sensitive, precise and accurate liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for levo 3,4-dihydroxyphenylalanine (L-DOPA) determination in Vicia faba during different growth stages. The method applied a simple sample preparation step followed by a chromatographic separation on a Kinetex XB Core-Shell C18 (100 mm x 4.6 mm, 2.6 µm) column, using a mixture of ultrapure pure water (A) with 0.5 % (v/v) formic acid and methanol (B) as mobile phase. Analysis of L-DOPA was carried out by MS/MS applying a Multiple Reaction Monitoring (MRM) method using the transition m/z 198 → m/z 152. This LC-MS/MS method allowed a well-resolved detection of L-DOPA in ca. 2 min within 6 min run. Method was validated showing a linear range from 0.05 to 10 mg L-1 (R2 = 0.99); repeatability showed RSD value of 1.40%. Recoveries ranged from 94.14 to 116.62% with RSD values ≤ 5.66% and detection and quantification limits were 0.01 and 0.05 mg L-1, respectively. Applying this validated method, L-DOPA was determined in Vicia faba samples to determined its tissue distribution. As expected, a broad range of L-DOPA content finding values from 4.72 (in seeds) up to 133.60 mg g-1 (sprouts).

Determination of Midodrine and its Active Metabolite Desglymidodrine by LC-MS/MS and its Application in Human Pharmacokinetic Studies

Background: Midodrine (MD) is a prodrug which is converted into Desglymidodrine (DMD) after oral administration. </P><P> Objective: The aim of the present study is to develop and validate a precise, accurate liquid chromatography- tandem mass spectroscopy (LC-MS/MS) method for the separation and detection of Midodrine and Desglymidodrine. Methods: The quantification of prodrug Midodrine (MD) and its active metabolite Desglymidodrine (DMD) in human plasma was performed using simple and economical protein precipitation method. Caffeine was used as an internal standard (IS). LC separation was carried out using Jones C18 column (4.6mm x 150mm, 3µm). Isocratic elution was performed using 10mM ammonium formate (pH 4.0 adjusted with formic acid): methanol 30:70, v/v as a mobile phase, at a constant of flow of 0.5 ml/min. Results: Mass spectrometric detection was carried out at positive electrospray ionization with proton adducts at m/z 255.0˃237.1, 198.1˃180.2 and 195.0˃138.1 for Midodrine, Desglymidodrine and caffeine (IS) respectively, in MRM mode. The method was validated over a linear concentration range of 0.3-110 ng/ml (r2 =0.996 and r2 =0.9988) for both Midodrine and Desglymidodrine. The recovery of Midodrine and Desglymidodrine were found to be 99 ± 0.12%. The precision (intra-day and inter-day) and accuracy studies fulfilled the acceptance criteria. Conclusion: The method shows to be stable for the studied stability parameters and was successfully applied for clinical pharmacokinetic studies.

A fully automated high-throughput liquid chromatography tandem mass spectrometry method for measuring creatinine in urine

Reliable creatinine measurements are important to evaluate kidney function and for creatinine correction to reduce biological variability of other urinary analytes. A high-throughput, accurate liquid chromatography tandem mass spectrometry method for quantitation of human urinary creatinine has been developed and validated.Sample preparation was fully automated including cryovial decapping, sample ID scanning and two serial dilution steps. Quantitation was performed using a stable isotope-labeled internal standard. Multiplexed chromatographic separation of creatinine was achieved within a one-minute analysis and followed by tandem mass spectrometry in positive electrospray ionization mode. The precursor and product ions of creatinine and D3-creatinine were monitored in selected reaction monitoring mode.Method validation results showed reproducibility with within-run precision of 3.59, 3.49 and 2.84% and between-run precision of 4.01, 3.28 and 3.57% for low, medium and high quality control materials prepared from pooled donor urine, respectively. The method showed excellent accuracy with a bias of −1.94%, −0.78% and −1.07% for three levels of certified reference material. The calibration curve was linear throughout a 7.50–300 mg/dL (0.663–26.5 mmol/L) measurement range (R2 = 0.999), with the mean slope of 0.0115 (95%CI, 0.0108–0.0122) and intercept of 0.0027 (95%CI, 0.0003–0.0051). The limit of detection (LOD) of the method was 3.17 mg/dL (0.280 mmol/L). Analytical specificity was achieved by chromatographically separating creatinine from potentially interfering creatine within a one-minute run and monitoring the Quantitation Ion/Confirmation Ion (QI/CI) ratios in samples.A simple, accurate, high-throughput method was successfully developed for measuring creatinine in human urine samples.

Identification of impurities in macrolides by liquid chromatography–mass spectrometric detection and prediction of retention times of impurities by constructing quantitative structure–retention relationship (QSRR)

Macrolides are multicomponent drugs whose impurity control is always a challenge demanding analysis method with good sensitivity and selectivity. Three separate, sensitive, accurate liquid chromatography tandem mass spectrometry methods (LC–MS) were developed for the measurement of three 16-membered ring macrolides (josamycin, josamycin propionate and midecamycin acetate) and related substances in commercial samples. The characteristics of impurities in macrolides were summarized as useful guidance for the impurity analysis of this class of drugs. For each drug, a large number of unknown components have been detected with the high-sensitive MS detector and possible structures of the majority of them were postulated based on the summarized fragmentation rules of 16-membered ring macrolides. A QSRR model was constructed by multilinear regression to predict the retention times of identified impurities which were not detected by the LC–MS methods, without obtaining their reference standards. Satisfactory performance was obtained during leave-one-out cross-validation with a predictive ability (Q2) of 0.95. The generalisation ability of the model was further confirmed by an average error of 2.3% in external prediction. The best QSRR model, based on eight molecular descriptors, exhibited a promising predictive performance and robustness.

Development of new clean-up method for UPLC–MS/MS analysis of leuprolide

In this study, a sensitive, accurate and cost-effective ultra-performance liquid chromatography-tandem mass spectrometric method was developed using a new clean-up process for the quantification of leuprolide in rat plasma. Chromatography was carried on a HALO peptide ES-C18 column using water and acetonitrile (containing 1% acetic acid) as the mobile phase with a gradient elution. Electrospray ionization was applied and operated in the multiple reaction monitoring positive mode. Leuprolide and an internal standard (leuprolide-d5) were extracted by the liquid–liquid extraction with methylene chloride after protein precipitation by the new clean-up process. In the clean-up process, acetic acid was used for higher reproducibility, and then 10 μL of cleaned aqueous phase was finally injected into the UPLC–MS/MS system. The chromatograms showed good resolution, and no interfering peaks to plasma constituents were observed. The lower limit of quantification in rat plasma was 20 pg/mL for leuprolide. The calibration curves for leuprolide in rat plasma were linear over all the concentration range 20–10,000 pg/mL with a correlation coefficient of 0.991. The intra- and inter-day assay for precision and accuracy fulfilled the international guideline requirements. This validated method was successfully applied to characterize the pharmacokinetics of leuprolide in rats after an intravenous administration of 0.1 mg/kg. Our method allows laboratory researchers to simply determine leuprolide in plasma.

Precise and accurate assay of pregnenolone and five other neurosteroids in monkey brain tissue by LC–MS/MS

A series of steroids present in the brain have been named “neurosteroids” following the possibility of their role in the central nervous system impairments such as anxiety disorders, depression, premenstrual dysphoric disorder (PMDD), addiction, or even neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Study of their potential role requires a sensitive and accurate assay of their concentration in the monkey brain, the closest model to the human. We have thus developed a robust, precise and accurate liquid chromatography–tandem mass spectrometry method for the assay of pregnenolone, pregnanolone, epipregnanolone, allopregnanolone, epiallopregnanolone, and androsterone in the cynomolgus monkey brain. The extraction method includes a thorough sample cleanup using protein precipitation and phospholipid removal, followed by hexane liquid-liquid extraction and a Girard T ketone-specific derivatization. This method opens the possibility of investigating the potential implication of these six steroids in the most suitable animal model for neurosteroid-related research.

Determination of Tetrodotoxin in Carcinoscorpius rotundicauda (Horseshoe Crab) by High-Performance Liquid Chromatography-Tandem Mass Spectrometry

ABSTRACTIn recent years, food poisoning due to the consumption of horseshoe crabs has occurred sporadically in China, and Carcinoscorpius rotundicauda was responsible for most of these events. A poisoning incident due to the ingestion of C. rotundicauda occurred in Fuzhou, China, in August 2014. The remains of the C. rotundicauda were collected from the home of the victims, and six specimens of C. rotundicauda were obtained from the same market from where the victims’ samples were sold. In this study, a rapid and accurate high-performance liquid chromatography-tandem mass spectrometry method was developed to determine tetrodotoxin in C. rotundicauda. The calibration curve of tetrodotoxin was linear from 5.0 to 1000 ng/mL. The limits of detection and quantification were 2.1 and 5.0 ng/mL, respectively. The average recoveries, measured at three concentrations, were from 74.8 to 83.6%, with the corresponding relative standard deviation below 3.8%. The C. rotundicauda samples responsible for poisoning and those purchased from the same market were shown to contain tetrodotoxin.

Sensitive and accurate liquid chromatography–tandem mass spectrometry methods for quantitative determination of a novel hepatitis C NS5B inhibitor BMS-791325 and its active metabolite in human plasma and urine

BMS-791325 is a novel hepatitis C NS5B inhibitor which is currently in clinical development. To support pharmacokinetic (PK) assessments, sensitive, accurate, precise, and reproducible liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods have been developed and validated for the quantitation of BMS-791325 and its active N-demethyl metabolite (BMS-794712) in human plasma and urine. Plasma and urine samples were extracted with methyl-t-butyl ether followed by an LC–MS/MS analysis which was conducted in a multiple reaction monitoring (MRM) mode for the simultaneous detection of the two analytes in human plasma (0.1–50ng/mL) and in human urine (5–2500ng/mL). Intra-run precision (3.0% R.S.D.), inter-run precision (5.3% R.S.D.), and accuracy (±4.7% deviation) from plasma and urine quality control samples provide evidence of the methods accuracy and precision. Selectivity, stability in matrices, extraction recovery, matrix effect on LC–MS detection, and interference of coadministered drugs (famotidine and ritonavir) were all acceptable. Reproducibility of the plasma method was demonstrated by reanalysis of a portion of study samples. The results of cross-validations demonstrated the equivalency of two methods validated in two labs. The plasma method was applied to the analysis of several thousand clinical study samples for PK evaluations of the drug in normal healthy subjects and in patients. The urine method was used in the first in human study to evaluate renal clearance and urinary recovery.

Simultaneous determination of seventeen mycotoxins residues in Puerariae lobatae radix by liquid chromatography–tandem mass spectrometry

This work reported an efficient and accurate liquid chromatography tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of seventeen mycotoxins in Puerariae lobatae radix, a frequently used traditional Chinese medicine (TCM). The effects of four different clean-up methods, including TC-M160, TC-T220, Mycosep 227, and QuEChERS method, on the recoveries of mycotoxins were investigated and compared. Finally, TC-M160 was chosen for better recovery and repeatability for mycotoxins analysis. The analytes were separated on an Agilent ZORBAX SB C18 column (4.6mm×250mm, 5μm particle size), and eluted with a mobile phase consisting of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid at a flow rate of 0.6mL/min. The separated compounds were detected by a triple quadrupole mass spectrometer operating in positive electrospray ionization with multiple reaction monitoring (MRM) mode. The results of method validation accorded with the requirement of analytical method for mycotoxins in COMMISSION REGULATION (EC) No 401/2006. The developed method was successfully applied for determination of mycotoxins in seventeen batches of Puerariae lobatae radix collected from different provinces of China. Three batches of them were found with contamination of mycotoxins AFB1 at (0.751±0.176)μg/kg, T-2 at (1.10±0.01)μg/kg, and T-2 at (0.853±0.044)μg/kg, respectively. The results demonstrated that the proposed method was suitable for monitoring mycotoxins residues in Puerariae lobatae radix.

Simultaneous determination of amlodipine, valsartan and hydrochlorothiazide by LC–ESI-MS/MS and its application to pharmacokinetics in rats

Polypill is a fixed-dose combination that contains three or more active ingredients used as a single daily pill to achieve a large effect in preventing cardiovascular disease with minimal adverse effects. A novel and accurate liquid chromatography tandem mass spectrometry method using electrospray ionization mode has been developed and validated for the simultaneous determination of amlodipine (AMD), valsartan (VAL) using losartan (LOS) as an internal standard (IS), and hydrochlorothiazide (HCT) using furosemide (FSD) as an IS. The separation was carried on Aquasil C18 (50mm×2.1mm, 5µm) reversed phase column using acetonitrile and water containing 0.1% formic acid (50:50, v/v) as the mobile phase. The method was validated in terms of linearity, accuracy and precision over the concentration range of 1–1000ng/mL. The intra and inter-day precision and accuracy, stability and extraction recoveries of all the analytes were in the acceptable range. This method can be successfully applied to the pharmacokinetic study of AMD, VAL and HCT when given as a polypill.

Mitochondrial Coenzyme Q10 Determination by Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry

Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain. Unlike most other respiratory chain disorders, CoQ10 deficiency is potentially treatable. We aimed to develop and validate an accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of mitochondrial CoQ10 in clinical samples. We used mitochondria isolated from muscle biopsies of patients (n = 166) suspected to have oxidative phosphorylation deficiency. We also used fibroblast mitochondria from 1 patient with CoQ10 deficiency and 3 healthy individuals. Samples were spiked with nonphysiologic CoQ10-[(2)H6] internal standard, extracted with 1-propanol and with ethanol and hexane (2 mL/5 mL), and CoQ10 quantified by LC-MS/MS. The method and sample stability were validated. A reference interval was established from the patient data. The method had a limit of quantification of 0.5 nmol/L. The assay range was 0.5-1000 nmol/L and the CVs were 7.5%-8.2%. CoQ10 was stable in concentrated mitochondrial suspensions. In isolated mitochondria, the mean ratio of CoQ10 to citrate synthase (CS) activity (CoQ10/CS) was 1.7 nmol/U (95% CI, 1.6-1.7 nmol/U). We suggest a CoQ10/CS reference interval of 1.1-2.8 nmol/U for both sexes and all ages. The CoQ10/CS ratio was 5-fold decreased in fibroblast mitochondria from a patient with known CoQ10 deficiency due to recessive prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2) mutations. Normalization of mitochondrial CoQ10 concentration against citrate synthase activity is likely to reflect most accurately the CoQ10 content available for the respiratory chain. Our assay and the established reference range should facilitate the diagnosis of respiratory chain disorders and treatment of patients with CoQ10 deficiency.

English

A simple, sensitive and accurate liquid chromatography tandem mass spectrometric (LC/MS/MS) method has been developed and validated for determination of varenicline (VRC) in human plasma and pharmaceutical tablets as a tool for therapeutic drug monitoring. The VRC and internal standard (paracetamol, IS) were extracted by liquid-liquid extraction technique. The separation was achieved on C18 column (150mm × 4.6 mm, 5 μm, maintained at 25°C) by isocratic mode at a flow rate of 0.7 ml/min using a mobile phase consisted of a mixture of 5 mM ammonium formate, pH 7.5 (A) and (acetonitrile: methanol, 50:50, v/v) (B) in a ratio of A:B (15:85, v/v) for 10 min. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Optimization of MRM mode and chromatogrphic conditions were applied to elmeinate the interference peaks and increse of sensitivity. The method was linear (r2 = 0.9998) at concentration range of 20.0 to 500.0 ng/ml with lower limit of detection of 6.0 ng/ml. The method was statistically validated for linearity, accuracy, precision and selectivity following Food and Drug Administration (FDA) guidelines. The mean extraction recovery of VRC from human plasma was 87.06 ± 2.47%. The reproducibility of the method was reliable with the intra- and inter-day precision was < 5% and average accuracy of 103.54%. The validated method was successfully applied to quantify VRC in human plasma as well as bulk and dosage form in quality control laboratory.   Key words: Liquid chromatography tandem mass spectrometric (LC/MS/MS), varenicline, human plasma, dosage form

A rugged and accurate liquid chromatography–tandem mass spectrometry method for the determination of asunaprevir, an NS3 protease inhibitor, in plasma

Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) non-structural protein protease inhibitor currently in Phase III clinical trials for the treatment of HCV infection. A rugged and accurate LC-MS/MS method was developed and validated for the quantitation of asunaprevir in rat, dog, monkey, rabbit and mouse plasma. A systematic method screening and optimization strategy was applied to achieve optimized mass spectrometry, chromatography, and sample extraction conditions. The validated method utilized stable-isotope labeled D9-asunaprevir as the internal standard. The samples were extracted by liquid-liquid extraction using 10% ethyl acetate in hexane. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column. Analyte and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 5.00 to 2000ng/mL for asunaprevir, was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±3.6% CV, inter-assay precision was within ±4.0% CV, and the assay accuracy was within ±8.1% of the nominal values in all the species. The method was successfully applied to support multiple pre-clinical toxicokinetic studies in different species.

Determination of Isotoosendanin in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry: Application to Pharmacokinetics Study

A rapid, sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the quantification of isotoosendanin, an important bioactive component isolated from Meliae cortex. A Capcell PAK C18 column (100 × 4.6 mm) was used for the chromatographic elution using methanol-10 mM ammonium acetate-formic acid (80:20:0.1, v/v/v) as mobile phase at the flow rate of 0.6 mL/min. MS-MS analysis was performed on a triple quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization source in positive ion mode. Extraction of isotoosendanin and genistein (internal standard, IS) from rat plasma was determined by precipitating protein treatment. Quantification was performed by MS in the multiple reaction monitoring mode with positive ionization at m/z 557 → 437 for the analyte and m/z 271 → 215 for IS, respectively. Linear isotoosendanin calibration curves were obtained between 2.0-2,000 ng/mL with a correlation coefficient greater than 0.99. Acceptable precision and accuracy were acquired for concentrations over the standard curve range. Satisfactory results were achieved for sensitivity, specificity, recovery, freeze/thaw and stability. This analytical method was successfully applied to determine the pharmacokinetic parameters of isotoosendanin after an oral administration of 200 mg/kg to rats.