Liquid-liquid extraction of strongly protein bound BMS-299897 from human plasma and cerebrospinal fluid, followed by high-performance liquid chromatography/tandem mass spectrometry

BMS-299897 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Liquid–liquid extraction (LLE), chromatographic/tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for the quantitation of BMS-299897 in human plasma and cerebrospinal fluid (CSF). Both methods utilized ¹³C₆-BMS-299897, the stable label isotope analog, as the internal standard. For the human plasma extraction method, two incubation steps were required after the addition of 5 mM ammonium acetate and the internal standard in acetonitrile to release the analyte bound to proteins prior to LLE with toluene. For the human CSF extraction method, after the addition of 0.5 N HCl and the internal standard, CSF samples were extracted with toluene and no incubation was required. The organic layers obtained from both extraction methods were removed and evaporated to dryness. The residues were reconstituted and injected into the LC/MS/MS system. Chromatographic separation was achieved isocratically on a MetaChem C18 Hypersil BDS column (2.0 mm × 50 mm, 3 μm). The mobile phase contained 10 mM ammonium acetate pH 5 and acetonitrile. Detection was by negative ion electrospray tandem mass spectrometry. The standard curves ranged from 1 to 1000 ng/ml for human plasma and 0.25–100 ng/ml for human CSF. Both standard curves were fitted to a 1/x weighted quadratic regression model. For both methods, the intra-assay precision was within 8.2% CV, the inter-assay precision was within 5.4% CV, and assay accuracy was within ±7.4% of the nominal values. The validation and sample analysis results demonstrated that both methods had acceptable precision and accuracy across the calibration ranges.     

Development and validation of a simple and sensitive liquid chromatography–tandem mass spectrometry method for quantifying trimetazidine in human plasma

1. A simple and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS‐MS) method for quantifying trimetazidine in human plasma was developed and validated. Sample preparation was based on deproteinating with acetonitrile. 2. Chromatography was performed on a C18 analytical column (5 μm; 150 × 2.1 mm i.d.) and the retention times for trimetazidine and cetirizine (used as the internal standard) were 1.8 and 3.0 min, respectively. The ionization was optimized using an electrospray ionization source and enhanced selectivity was achieved using tandem mass spectrometry. The calibration curve ranged from 0.1 to 200 ng/mL. The inter‐day precision, accuracy and the relative standard deviation (RSD) were all < 15%. The analyte was shown to be stable over the time‐scale of the entire procedure. 3. The robustness of the method was demonstrated by the good reproducibility of the results obtained during the analysis of clinical samples.     

Determination of dabigatran in dog plasma by LC-MS/MS and its application to a pharmacokinetic study of dabigatran etexilate nanosuspension

In this study, a sensitive and rapid LC-MS/MS method was developed and validated to determine dabigatran in plasma of beagle dogs after oral administration of dabigatran etexilate nanosuspension (DABE-NS). The analytes (dabigatran) and sertraline hydrochloride (internal standard, IS) were separated on a Kromasil C₁₈ column using gradient elution consisting of methanol and formate buffer at a flow rate of 0.4 mL/min in 20 min. Detection and quantitation were carried out by multiple reaction monitoring following the transitions: m/z 472.17→289.07 and 305.98→275.00 for dabigatran and IS at positive ion mode, respectively. The calibration curves were linear from 1.0 to 500.0  ng/mL for dabigatran with r  = 0.9995. The accuracy of each analyte ranged from 94.8% to 107.1%, and the precision was within 6%. Besides, this method was successfully applied in the investigation of the pharmacokinetic profile of dabigatran in beagle dogs after oral administration ofDABE-NS. The maximum concentration and the areas under curves of dabigatranfor DABE-NS were significantly higher than those of control formulation, indicating improved oral absorption.     

LC-MS/MS测定大鼠血浆中虎杖苷浓度

目的 建立测定大鼠血浆中虎杖苷浓度的LC-MS/MS方法。 方法 采用LC-MS/MS测定大鼠血浆中虎杖苷的浓度,以二苯乙烯苷为内标,血浆样品用乙腈沉淀蛋白,色谱柱为Agilent Zorbax SB-C₁₈ (100 mm×2.1 mm, 3.5 μm),流动相为甲醇-乙腈-0.1%甲酸水溶液(18∶15∶67),流速0.3 ml/min,柱温30 ℃。 结果 虎杖苷的线性范围为1.0~5 000.0 ng/ml (r=0.998 4),最低定量检测浓度为1.0     

Does a stable isotopically labeled internal standard always correct analyte response?: A matrix effect study on a LC/MS/MS method for the determination of carvedilol enantiomers in human plasma

A stable isotopically labeled (SIL) analogue is believed to be the most appropriate internal standard in a quantitative bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay. It is assumed that a SIL internal standard always compensates for variability in chemical derivatization, sample extraction and LC/MS/MS analysis due to its nearly identical chemical and physical properties to the unlabeled analyte. Hence, the analyte to internal standard peak area ratio should be constant despite any variations in sample processing or analysis. However, in our laboratories, a deuterium labeled internal standard of carvedilol demonstrated an unexpected behavior-the analyte to internal standard peak area ratio changed with two specific lots of commercially supplied human plasma. Several experiments, including dilution of the extract with LC mobile phase and post-column infusion of the carvedilol solution followed by the injection of extracted blank plasma, have indicated that a high level of matrix suppression affected the ionization of the carvedilol-S enantiomer and its deuterated internal standard differently in these two lots of plasma. For the first time, it was clearly demonstrated that a slight difference in retention time between the analyte and the SIL internal standard, caused by deuterium isotope effect, has resulted in a different degree of ion suppression between these two analogues. This difference was significant enough to change the analyte to internal standard peak area ratio and affect the accuracy of the method.     

高效液相-质谱串联方法快速测定大鼠血浆中的槲皮素-3’-O-葡萄糖苷含量(英文)

建立大鼠血浆中槲皮素-3’-O-葡萄糖苷的高效液相-串联质谱(HPLC-MS/MS)检测法。生物样品中槲皮素-3’-O-葡萄糖苷和卡马西平(内标)采用液-液萃取的方法提取,乙酸乙酯作为提取溶剂。HPLC-MS/MS方法采用多离子反应模式(MRM)进行检测,被测物质和内标均在3.5min内出峰。槲皮素-3’-O-葡萄糖苷在血浆中的最低定量限是10.625ng/mL,最低检测限是4.25ng/mL,平均回收率大于66.80%,日内、日间精密度和准确度的RSD值均小于10.44%。尾静脉注射10mg/kg槲皮素-3’-O-葡萄糖苷后,其t1/2和AUC分别是(0.02±0.01)h和(1.22±0.28)×104μg/L·h。本法准确、稳定、灵敏度高,适用于槲皮素-3’-O-葡萄糖苷的大鼠药代动力学研究。     

A direct determination of thymidine triphosphate concentrations without dephosphorylation in peripheral blood mononuclear cells by LC/MS/MS

A rapid, sensitive and specific analytical method with minimal sample preparation for the measurement of thymidine triphosphate (TTP) in peripheral blood mononuclear cells (PBMC) by LC/MS/MS has been developed. PBMC were separated from whole blood or buffy coat. The analyte and internal standard were extracted from PBMC with 70% methanol (pH 7.2). These extracts after centrifugation were directly injected onto LC/MS/MS without need for any further sample preparation. The calibration curve was linear over the range 0.8–800 ng/ml. Mean inter- and intra-assay coefficients of variation (CVs) over the range of the standard curve were less than 10%. The overall recovery of TTP was 103.5%.     

A rapid and sensitive LC-MS/MS assay to quantify yonkenafil in rat plasma with application to preclinical pharmacokinetics studies

A novel method for the quantitation of yonkenafil, a new synthetic phosphodiesterase V inhibitor, in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed. The analyte and internal standard (diazepam) were extracted from plasma (100 microl) by liquid-liquid extraction and separated on a C18 column using 10mM ammonium acetate buffer: methanol (15:85, v/v) as mobile phase in a run time of 3.0 min. The detector was a Q-trap mass spectrometer with an ESI interface operating in the multiple reaction monitoring (MRM) mode. The assay was linear over the concentration range 1.0-1000 ng/ml with a limit of detection of 0.20 ng/ml. Intra- and inter-day precision (as relative standard deviation) were both within 8.45% with good accuracy. The method was successfully applied to a preclinical pharmacokinetic study of yonkenafil in rat after sublingual, oral and intravenous administration. The results demonstrate that the sublingual route gives a higher bioavailablity than the oral route and may represent a useful alternative route of yonkenafil administration.     

Rapid quantitative analysis of oxiracetam in human plasma by liquid chromatography/electrospray tandem mass spectrometry

A rapid and accurate reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantitative determination of oxiracetam in human plasma. After addition of internal standard (piracetam) plasma was precipitated with two volumes of acetonitrile and the supernatant was evaporated. The residues were dissolved in 0.1% acetic acid and analyzed by reversed-phase HPLC with the detection of the analyte in the multiple reaction monitoring (MRM) mode. This method for the determination of oxiracetam was accurate and reproducible, with a limit of quantitation of 0.2 microg/ml in human plasma. The standard calibration curve for oxiracetam was linear (r2 = 0.999) over the concentration range 0.2-40.0 microg/ml in human plasma. The intra- and inter-day precision over the concentration range of oxiracetam was lower than 8.3% (relative standard deviation, %R.S.D.), and accuracy was between 92.5-106.4%.     

A rapid and sensitive method for determination of veliparib (ABT-888), in human plasma,bone marrow cells and supernatant by using LC/MS/MS

A rapid and sensitive method was developed and validated using a liquid chromatographic method with tandem mass spectrometry detection (LC/MS/MS) for determination of veliparib (ABT-888) in plasma, bone marrow supernatant, and bone marrow cells. Sample preparation involved a single protein precipitation step by the addition of the sample with acetonitrile. Separation of veliparib and the internal standard, A620223.69, was achieved on a Atlantis™ dC₁₈ column (100 mm × 2.1 mm, 3 μm) column using a mobile phase consisting of acetonitrile–ammonium acetate (2 mM) containing formic acid (0.1%, v/v) using isocratic flow at 0.2 mL/min for 3 min. The analyte and internal standard were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 5–1000 nM. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods. This method was subsequently used to measure concentrations of veliparib in cancer patients receiving an oral daily dose of 10 mg with demonstration of drug accumulation in the marrow compartment and in the target leukemia bone marrow cells.     

Validated HPLC-MS/MS method for determination of quetiapine in human plasma

A validated, highly sensitive and selective high-pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitative determination of quetiapine (QUE) in human Na2EDTA plasma with mass spectrometry (MS) detection. Clozapine (CLO) was employed as an internal standard. Samples were extracted using solid phase extraction (SPE). Oasis HLB cartridges and the concentration of quetiapine was determined by isocratic HPLC-MS/MS. The SRM mode was used for MS/MS detection. The method was validated over a concentration range of 1.0-382.2 ng/mL. Inter- and intra-day precision and accuracy of the proposed method were characterized by relative standard deviation (R.S.D.) and the percentage of deviation, respectively; both were lower than 8%. The developed method was employed in the pharmacokinetic study of quetiapine.     

Appropriate calibration curve fitting in ligand binding assays

Calibration curves for ligand binding assays are generally characterized by a nonlinear relationship between the mean response and the analyte concentration. Typically, the response exhibits a sigmoidal relationship with concentration. The currently accepted reference model for these calibration curves is the 4-parameter logistic (4-PL) model, which optimizes accuracy and precision over the maximum usable calibration range. Incorporation of weighting into the model requires additional effort but generally results in improved calibration curve performance. For calibration curves with some asymmetry, introduction of a fifth parameter (5-PL) may further improve the goodness of fit of the experimental data to the algorithm. Alternative models should be used with caution and with knowledge of the accuracy and precision performance of the model across the entire calibration range, but particularly at upper and lower analyte concentration areas, where the 4- and 5-PL algorithms generally outperform alternative models. Several assay design parameters, such as placement of calibrator concentrations across the selected range and assay layout on multiwell plates, should be considered, to enable optimal application of the 4- or 5-PL model. The fit of the experimental data to the model should be evaluated by assessment of agreement of nominal and model-predicted data for calibrators.     

Simultaneous analysis of bambuterol and its active metabolite terbutaline enantiomers in rat plasma by chiral liquid chromatography–tandem mass spectrometry

A chiral liquid chromatography–tandem mass spectrometry (LC–MS/MS) simultaneous stereoselective analysis of bambuterol and its active metabolite terbutaline enantiomers in Wistar rat plasma has been developed and validated. All analytes and the internal standard were extracted from rat plasma samples by liquid–liquid extraction, separated on macrocyclic glycopeptide teicoplanin column with mobile phase constituted of 20 mM ammonium acetate solution–methanol (10:90, v/v) at a flow-rate of 0.4 mL/min. Detection was performed on an API 3000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The calibration curves in the range 1–800 ng/mL were linear and the accuracy for each analyte was within 8.0%. The intra- and inter-day precision as determined from quality control samples was less than 10.1%. The validated assay was successfully used to determine the enantiomers of bambuterol and terbutaline in rat plasma samples in the pharmacokinetic studies of rac-bambuterol.     

Analytical method for determination of allylic isoprenols in rat tissues by liquid chromatography/tandem mass spectrometry following chemical derivatization with 3-nitrophtalic anhydride

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method following chemical derivatization with 3-nitrophtalic anhydride was developed for the simultaneous determination of farnesol and geranylgeraniol in rat liver and testis. One analogue compound of the analytes, n-pentadecanol, was used as an internal standard (IS) for both analytes in this method. Rat tissues were disintegrated with 8% KOH ethanol solution, and then farnesol, geranylgeraniol and IS were extracted with a mixture of n-hexane-ethanol (98.5:1.5, v/v) in twice. Farnesol, geranylgeraniol and IS were then converted to 3-nitrophtalic derivatives of each analyte, and extracted with n-hexane. A turbo ion spray interface was used as the ionization source of LC-MS/MS and the analysis was performed in the multiple reaction monitoring (MRM) mode. The calibration curve at the spiked concentrations of 0.15-15 microg/g for both analytes showed good linearity. The method was precise; the relative standard deviations of the method for rat liver were not more than 13.4 and 5.4% for farnesol and geranylgeraniol, respectively, and those for rat testis were not more than 8.4 and 8.6% for farnesol and geranylgeraniol, respectively. The accuracies of the method for both rat liver and testis were good, with the deviations between the nominal concentration and calculated concentration of farnesol and geranylgeraniol typically being within 12.3 and 10.2%, respectively. This method provided reliable concentration levels for farnesol and geranylgeraniol in rat liver and testis.     

A sensitive method for the determination of entecavir at picogram per milliliter level in human plasma by solid phase extraction and high-pH LC–MS/MS

Entecavir is a guanine nucleoside analogue used in the treatment of hepatitis B virus (HBV) infection. In this paper, we describe an LC–MS/MS method that was developed and validated for the quantitation of entecavir in human EDTA plasma with both high sensitivity (lower limit of quantitation (LLOQ) of 5 pg/mL) and a wide concentration range (5000-fold) intended for low dose ascending clinical studies. High enrichment was achieved by taking advantage of the excellent loading capacity and reproducibility of Oasis HLB 96-well solid phase extraction plate, which allowed 1 mL of plasma samples to be processed in two equal sequential loading steps. Lobucavir, a structural analogue, was used as the internal standard. A filtration step following the reconstitution proved to be vital for the method robustness. The analyte and internal standard were separated on an Xterra MS C18 column with a gradient elution and high-pH mobile phases. Analytes were detected by positive ion electrospray tandem mass spectrometry. The high-pH mobile phase provided both excellent analyte on-column retention and peak shape, leading to the desired sensitivity. Validation results show good intra-assay (12.3%CV) and inter-assay (3.1%CV) precisions, and good assay accuracy (±7.6%Dev). Recovery was high (∼80%), however, the large volume of plasma used did result in a considerable matrix effect (∼0.45) which was well compensated by the analog internal standard. The method was applied to sample analysis of a Phase I clinical study.     

Determination of SR 49059 in human plasma and urine by LC-APCI/MS/MS

SR 49 059 ((2S 1-[(2R 3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide) is an orally active non-peptide vasopressin V_1a antagonist. A sensitive, selective, and robust LC-MS/MS method was developed to determine the plasma and urine concentrations of SR 49 059 in support of clinical studies. Plasma samples were prepared based on a rapid extraction procedure using Chem Elut™ cartridges. The extracted samples were analyzed on a C₁₈ HPLC column interfaced with a Finnigan TSQ 700 mass spectrometer. Positive atmospheric chemical ionization (APCI) was employed as the ionization source. The analyte and its internal standard (²H₆-SR 49 059) were detected by use of multiple reaction monitoring (MRM) mode. The plasma matrix had a calibration range 0.2−20 ng ml⁻¹, with within and between run accuracy and precision both less than 10%. The chromatographic run time was approximately 3 min. Urine samples were prepared based on a simple dilution with water, followed by analysis under the same conditions as plasma. The calibration range for urine matrix was 20–5000 ng ml⁻¹, with within and between run accuracy and precision less than 11%. The method has been successfully applied to the clinical sample analysis. The plasma assay was also evaluated on a Finnigan TSQ 7000 mass spectrometer. The performance based on precision and accuracy was virtually identical to that on the TSQ 700, with the exception of linearity in calibration curve (the TSQ 700 was linear, the TSQ 7000 was quadratic).     

Quantification of propiverine by liquid chromatography/electrospray tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy subjects

Here we report on the development and validation of a sensitive and rapid reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of propiverine in human plasma. After adding an internal standard (oxybutynin chloride) to human plasma, samples were extracted using n-hexane/ethylacetate (8:2, v/v). Compounds extracted were analyzed by reversed-phase high-performance liquid chromatography (HPLC) with multiple reaction monitoring (MRM) mode for analyte detection. This method for determination of propiverine proved accurate and reproducible, with a limit of quantitation of 0.5 ng/ml in human plasma. The standard calibration curve for propiverine was linear (r2=0.9988) over the concentration range 0.5-1000.0 ng/ml in human plasma. The intra- and inter-day precision over this concentration range was lower than 8.66% (relative standard deviation, %R.S.D.), and accuracy was between 99.46 and 109.41%, respectively. This method was successfully applied to a bioequivalence study of two propiverine hydrochloride tablet formulations (20 mg) in 24 healthy subjects after a single administration.     

Development and validation of a multi‐analyte LC‐MS/MS approach for quantification of neuroleptics in whole blood,plasma, and serum

Based on a similar approach for quantification of antidepressants, benzodiazepines, and z‐drugs, a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) multi‐analyte approach with simple liquid‐liquid extraction was extended for fast target screening and quantification of neuroleptics in whole blood, plasma, and serum. As this method is part of a multi‐analyte procedure for over 100 analytes from different drug classes and as the extracts were additionally used in the authors' laboratory for gas chromatography‐mass spectrometry (GC‐MS) analysis, one universal stable‐isotope‐labelled internal standard (SIL‐IS) was used to save time and resource. The method was validated with respect to international guidelines. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. Selectivity problems could not be observed, but matrix effects ranged from 68 to 211% in all samples. For the low quality control (QC), recovery ranged from 32 to 112%, process efficiency from 31 to 165% and for the high QC recovery from 42 to 141%, process efficiency from 29 to 154%. In addition statistical data evaluation of the variances of the recovery, matrix effects, and process efficiency data between whole blood vs. plasma, whole blood vs. serum, and plasma vs. serum were done. The presented LC‐MS/MS approach was applicable for selective detection of 33 neuroleptics as well as accurate and precise quantification of 25 neuroleptics in whole blood, 19 in plasma, and 17 in serum. More significant matrix effects (ME) for neuropletic drugs overall in plasma and serum as compared with whole blood were detected. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of LC–MS/MS method for the determination of cyproheptadine in several pharmaceutical syrup formulations

A rapid and sensitive liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the qualitative and quantitative assay of cyproheptadine (CP) in pharmaceutical samples. Diphenylpyraline hydrochloride (DPP) was used as an internal standard (IS). Two multiple reaction-monitoring (MRM) transitions for each analyte were observed: 288.1/96.1 and 288.1/191.2 for CP and 282.1/167.2 and 282.1/116.3 for DPP. The retention time of the drug was 7.29 min. The analytical method was successfully validated for linearity (1–100 ng/ml), intra-day precision, inter-day precision, and accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were 0.86 and 0.98 ng/ml, respectively. The proposed method was applied to analyse the cyproheptadine content from seven different syrup formulations.