A flexible and high throughput liquid chromatography–tandem mass spectrometric assay for the quantitation of telcagepant in human plasma

Telcagepant (MK-0974) is a novel oral calcitonin gene-related peptide (CGRP) receptor antagonist and is currently under clinical development. Results from phases II and III clinical trials have suggested that telcagepant is effective for migraine treatment. A reliable and high throughput protein precipitation (PPT) method for determination of telcagepant in human plasma using liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry has been developed. Clinical samples, internal standard (IS) and acetonitrile are transferred into 96-well plates using a robotic liquid handling system. An aliquot of 10 μL supernatant is directly injected into the LC–MS/MS system where separation is performed on a FluoPhase RP (150 × 2.1 mm, 5 μm) column with an isocratic mobile phase (60% acetonitrile with 0.1% formic acid and 40% water with 0.1% formic acid) at 0.2 mL/min. The interfering 3S-diastereomer of telcagepant, which is observed in clinical samples, is chromatographically resolved from telcagepant. The PPT procedure significantly reduces the time required for sample processing and the assay is sufficiently sensitive for detection using both API 4000 and API 3000 mass spectrometers. The linear calibration range is 5–5000 nM using 200 μL of plasma. Assay intraday validation was conducted using six calibration curves derived from six lots of human control plasma. Calibration standard accuracy did not deviate by more than 3% and 6% of nominal values, and precision did not exceed 4% coefficient of variation (CV) and 10% CV, respectively on the API 4000 and API 3000. Several clinical phases IIb and III studies have been successfully supported with this assay.     

Using short columns to speed up LC–MS quantification in MS binding assays

The present study describes the use of short columns to speed up LC–MS quantification in MS binding assays. The concept of MS binding assays follows closely the principle of traditional radioligand binding but uses MS for the quantification of bound marker thus eliminating the need for a radiolabelled ligand. The general strategy of increasing the throughput of this type of binding assay by the use of short columns is exemplified for NO 711 binding addressing GAT1, the most prevalent GABA transporter in the CNS. Employing short RP-18 columns with the dimension of 20 mm × 2 mm and 10 mm × 2 mm at flow rates up to 1000 μL/min in an isocratic mode retention times of 8–9 s and chromatographic cycle times of 18 s could be achieved. Based on the internal standard [²H₁₀]NO 711 fast chromatography methods were developed for four different columns that enabled quantification of NO 711 in a range from 50 pM up to 5 nM directly out of reconstituted matrix samples without further sample preparation. A validation of the established methods with respect to linearity, intra- and inter-batch accuracy and precision showed that the requirements according to the FDA guideline for bioanalytical methods are met. Furthermore the established short column methods were applied to the quantification of NO 711 in saturation experiments. The results obtained (i.e., K_d- and B_max-values) were almost identical as compared to those determined employing standard column dimension (55 mm × 2 mm).     

Turbulent-flow chromatography coupled on-line to fast high-performance liquid chromatography and mass spectrometry for simultaneous determination of verticine,verticinone and isoverticine in rat plasma

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC–LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 μm particle size (Agilent Zorbax StableBond-C₁₈, 4.6 mm × 50 mm, 1.8 μm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic–lipophilic balanced copolymer (2.1 mm × 20 mm, 25 μm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC–MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R > 0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.     

Simultaneous determination of cyclophosphamide and carboxyethylphosphoramide mustard in human plasma using online extraction and electrospray tandem mass spectrometry (HTLC–ESI-MS/MS)

A rapid and selective method for simultaneous determination of cyclophosphamide and its metabolite carboxyethylphosphoramide mustard (CEPM) was developed using online sample preparation and separation with tandem mass spectrometric detection. Diluted plasma was injected onto an extraction column (Cyclone MAX 0.5 mm × 50 mm, >30 μm), the sample matrix was washed with an aqueous solution, and retained analytes were transferred to an analytical column (Gemini 3 μm C18 110A, 100 mm × 2.0 mm) using a gradient mobile phase prior to detection by MS/MS. Analytes were detected in an API-3000 LC-MS/MS system using positive multiple-reaction monitoring mode (m/z 261/140 and 293/221 for CTX and CEPM, respectively). Online extraction recoveries were 76% and 72% for cyclophosphamide and CEPM. Within-day and between-day variabilities were <3.0%, and accuracies were between ?6.9% and 5.2%. This method has been used to measure plasma cyclophosphamide and CEPM concentrations in an ongoing Phase II study in children with newly diagnosed medulloblastoma.     

Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography–tandem mass spectrometry

A sensitive and specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 μl was deproteinized by addition of 500 μl methanol which contained 5 μg/ml UCB 17025 as an internal standard. After centrifugation, 50 μl of supernatant was diluted with 1000 μl of 0.1% formic acid–10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 μl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C₁₈ 2.1 mm × 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 μg/ml to 150 μg/ml. Inter-assay inaccuracy was within ±2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC–MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.     

Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: Application to a pharmacokinetic study

A rapid, selective and sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher^® 60 RP Select B column (125 mm × 4 mm i.d., 5 μm particle size) using acetonitrile:5 ± 0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0–15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within ±6.3% of nominal values. No matrix effect was observed in this method. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.     

A sensitive LC–MS/MS method for quantification of a nucleoside analog in plasma: Application to in vivo rat pharmacokinetic studies

A LC–MS/MS method was developed and validated for determination of nucleoside analog (NA) in rat plasma. The method run time was 6 min and the limit of quantification (LOQ) was estimated at 100 pg/mL. The extraction procedure consisted on plasma samples protein precipitation with an acetonitrile solution which contained the stable isotope labeled internal standard (IS). Chromatography was performed on a newly developed C₁₆ column (150 mm × 4.6 mm, 5 μm) in order to avoid the use ion pair salts. The samples were eluted at 0.8 mL/min with a gradient of mobile phase made of water and acetonitrile both acidified with 0.5% acetic acid and 0.025% trifluoroacetic acid (TFA). A tandem mass spectrometer was used as a detector for quantitative analysis. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at four concentration levels in rat plasma, over a concentration ranging between 0.1 and 1000 ng/mL. The data indicate that our LC–MS/MS assay is an effective method for the pharmacokinetics study of NA in rat plasma.     

Validation and application of an LC–ESI-MS method for simultaneous determination of astilbin and its major metabolite 3′-O-methylastilbin in rat plasma

We herein describe the development of an LC–MS method for simultaneous determination of astilbin and 3′-O-methylastilbin in rat plasma. A simple liquid–liquid extraction procedure was followed by injection of the extracts on to a Shim-pack C₁₈ column (150 mm × 2.0 mm I.D., 5 μm) with gradient elution and detection in negative ionization mode. Initially, the method was validated regarding linearity, accuracy and precision. The correlation coefficients of all the calibration curves showed good linearity (r > 0.999) within test ranges, and the relative deviation was less than 10% for intra- and inter-day assays. Besides, this method was also validated for its stability, extraction efficiency, matrix effect and so on. Finally, this proposed method was successfully applied to rat pharmacokinetic study and yielded the most comprehensive data on systemic exposure of them to date.     

Quantitative determination of oxytocin receptor antagonist atosiban in rat plasma by liquid chromatography–tandem mass spectrometry

A kinetic study of atosiban was conducted following repeated intravenous administration in Wistar rats. Sample analysis was performed using liquid chromatography–tandem mass spectrometry (LC–MS/MS) following full validation of an in-house method. Eptifibatide, a cyclic peptide, was used as an internal standard (IS). The analyte and internal standard were extracted using solid phase extraction (SPE) method. Chromatographic separation was carried out using an ACE C18 5 μm 50 mm × 4.6 mm column with gradient elution. Mass spectrometric detection was performed using TSQ Quantum ultra AM. The lower limit of quantification was 0.01 μg/ml when 100 μl rat plasma was used. Plasma concentrations of atosiban were measured at 0 (pre-dose), 2, 15, 30, 45, 60, 120 min at the dosage levels of 0.125 mg/kg (low dose), 0.250 mg/kg (mid dose), and 0.500 mg/kg (high dose), respectively. Atosiban plasma concentration measured at Day 1 showed mean peak atosiban concentration (C_max) 0.40, 0.57, 1.95 μg/ml for low, mid and high dose treated animals and mean peak concentration on Day 28 was 0.41, 0.88, 1.31 μg/ml on Day 28 for low, mid and high dose treated animals.     

Simultaneous determination of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in rat plasma by liquid chromatography–mass spectrometry: Application to pharmacokinetics

A rapid high-performance liquid chromatography–mass spectrometry (HPLC–MS) method was developed and validated for simultaneous quantification of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in rat plasma after oral administration of ginger oleoresin. Plasma samples extracted with a liquid–liquid extraction procedure were separated on an Agilent Zorbax StableBond-C₁₈ column (4.6 mm × 50 mm, 1.8 μm) and detected by MS with electrospray ionization interface in positive selective ion monitoring (SIM) mode. Calibration curves (1/x² weighted) offered satisfactory linearity (r² > 0.995) in a wide linear range (0.0104–13.0 μg/mL for 6-gingerol, 0.00357–4.46 μg/mL for 8-gingerol, 0.00920–11.5 μg/mL for 10-gingerol and 0.00738–9.22 μg/mL for 6-shogaol). The lower limit of quantification (LLOQ) was in a range of 3.57–10.4 ng/mL. The analytes and internal standard can be baseline separated within 6 min. Inter- and intra-day assay variation was less than 15%. This developed method was successfully applied to pharmacokinetic studies of ginger oleoresin after oral administration to rats. Glucuronide of 6-gingerol was determined after β-glucuronidase hydrolysis for more information, and the intestinal glucuronidation was further confirmed by comparison of plasma samples of hepatic portal vein and femoral vein.     

Development of a sensitive and selective LC-MS/MS method for the determination of α-fluoro-β-alanine, 5-fluorouracil and capecitabine in human plasma

A sensitive and selective quantitative method to determine α-fluoro-β-alanine (FBAL), 5-fluorouracil (5-FU), and capecitabine (Cape) from a single human plasma aliquot (50 μL) has been developed and validated. First, 5-FU and Cape were extracted by liquid–liquid extraction (LLE) using a mixture of acetonitrile and ethyl acetate. This was followed by derivatization with dansyl chloride. The dansyl-derivatives from 5-FU and Cape were further purified using LLE with methyl tertiary-butyl ether (MTBE) and analyzed using a reversed-phase analytical column “Primesep D” (2.1 mm × 50 mm; 5 μm) with embedded basic ion-pairing groups. The remaining aqueous phase containing FBAL was treated with dansyl chloride and the dansyl-FBAL was purified by solid phase extraction. Ultra high pressure liquid chromatography (UPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for analysis of dansyl-FBAL. The method was validated over the concentration ranges of 10–10,000, 5–5000, and 1–1000 ng/mL for FBAL, 5-FU, and Cape, respectively. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day.     

Simultaneous determination of tryptophan and kynurenine in plasma samples of children patients with Kawasaki disease by high-performance liquid chromatography with programmed wavelength ultraviolet detection

A simple, fast, sensitive and specific high-performance liquid chromatography (HPLC) method is developed for simultaneous determination of kynurenine (Kyn) and tryptophan (Trp) with ultraviolet (UV) detection setting programmed wavelength. The separation was carried out on an Agilent Hypersil ODS column (125 mm × 4.0 mm, 5 μm) in less than 6 min and the eluate was monitored by the programmed wavelength detection setting at 360 nm from 0 min to 4 min for Kyn, and at 278 nm from 4 min to 6 min for Trp in a single run with UV detector. The linearities of the method were from 0.20 μmol/L to 21.2 μmol/L for Kyn and 2.25–678.0 μmol/L for Trp, and the detection limits were 0.028 μmol/L for Kyn and 0.053 μmol/L for Trp, respectively. Satisfactory precisions and recoveries were obtained by this method. The assay was employed to analyze plasma samples of children patients with Kawasaki disease (KD). The result showed great difference between Kawasaki disease and control group.     

Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.     

Determination of pyrrole–imidazole polyamide in rat plasma by liquid chromatography–tandem mass spectrometry

Pyrrole (Py)–imidazole (Im) polyamides synthesized by combining N-methylpyrrole and N-methylimidazole amino acids have been identified as novel candidates for gene therapy. In this study, a sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with an electrospray ionization (ESI) source was developed and validated for the determination and quantification of Py–Im polyamide in rat plasma. Py–Im polyamide was extracted from rat plasma by solid-phase extraction (SPE) using a Waters Oasis^® HLB cartridge. Separation was achieved on an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 50 mm) column by gradient elution using acetonitrile:distilled water:acetic acid (5:95:0.1, v/v/v) and acetonitrile:distilled water:acetic acid (95:5:0.1, v/v/v). The method was validated over the range of 10–1000 ng/mL and the lower limit of quantification (LLOQ) was 10 ng/mL. This method was successfully applied to the investigation of the pharmacokinetics of Py–Im polyamide after intravenous administration.     

Performance of tiloronoxim and tilorone determination in human blood by HPLC–MS/MS: Method validation,uncertainty assessment and its application to a pharmacokinetic study

A highly sensitive and selective HPLC–MS/MS method is presented for the quantitative determination of tiloronoxim and its metabolite tilorone in human blood. An aliquot of 200 μl human blood was extracted with a mixture of chloroform/ethyl ether (1/2, v/v), using metoprolol as the internal standard (the IS). Separation was achieved on an Xterra MS C18 column (50 mm × 2.1 mm, 5 μm) with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). Detection was performed using positive MRM mode on a TurboIonSpray source. The mass transitions monitored were m/z 426.3 → 100.0, m/z 411.3 → 100.0 and m/z 268.3 → 116.1 for tiloronoxim, tilorone and the IS, respectively. The method was fully validated using total error theory, which is based on β-expectation tolerance intervals and include trueness and intermediate precision. The method was found to be accurate over a concentration range of 1–100 ng/ml for both compounds. The measurement uncertainty based on β-expectation tolerance intervals was assessed at each concentration level of the validation standards. This method was successively applied to a pharmacokinetic study of tiloronoxim in healthy volunteers.     

Simultaneous measurement of aldosterone and cortisol by high-performance liquid chromatography–tandem mass spectrometry: Application to dehydration–rehydration studies

Aldosterone and cortisol are useful biomarkers of dehydration and stress, respectively. The aim of this study was to develop an HPLC–tandem mass spectrometric method for the simultaneous measurement of aldosterone and cortisol in human plasma that could be applied to the study of athletes undergoing exercise and rehydration. Samples were prepared and analysed using an on-line sample preparation/HPLC system coupled to a triple quadrupole tandem-mass spectrometer. Samples (200 μL) were pre-treated and extracted on Hysphere C18 HD cartridges (7 μm, Spark Holland). Chromatography was performed on a Sunfire C18 analytical column (50 mm × 3.0 mm, 3 μm, Waters) under isocratic conditions at a flow rate of 0.3 mL/min. The mobile phase consisted of 35% acetonitrile/water. Mass spectrometric detection was by selected reaction monitoring using negative electrospray ionization conditions. The assay had an analytical range of 25–500 pg/mL and 25–500 ng/mL for aldosterone and cortisol, respectively (r² > 0.992, n = 22). Inter-day accuracy and imprecision for quality control samples was 99.4–106% and <16%, respectively (n = 10). In a study of nine human subjects, both aldosterone and cortisol concentrations reflected the expected physiological responses to dehydration, rehydration and exercise when measured by this method. The reported method is suitable to facilitate the study of athletes undergoing dehydration and rehydration protocols.     

A novel rapid method for simultaneous determination of eight active compounds in silymarin using a reversed-phase UPLC-UV detector

A novel rapid chromatographic method based on utilization of UPLC column was developed for the analysis of eight active compounds in silymarin. The analysis was performed on a Waters Acquity UPLC system with an Acquity UPLC^BEH C18 column (5 mm × 2.1 mm I.D., 1.7 μm) and a gradient elution of methanol and water containing 0.01% formic acid with a run time of 9 min, in which the retention time of the last analyte was 5.8 min. And all eight active compounds achieved complete separation. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity. The results indicated that the type of column, the type of mobile phase and the modified addition were significant to the separation of isomeric compounds in herb extracts.     

Effect of disease state on ionization during bioanalysis of MK-7009, a selective HCV NS3/NS4 protease inhibitor,in human plasma and human Tween-treated urine by high-performance liquid chromatography with tandem mass spectrometric detection

HPLC–MS/MS methods for the determination of a Hepatitis C NS3/NS4 protease inhibitor (MK-7009) in human plasma and Tween-treated urine were developed and validated over the concentration range 1–1000 ng/mL and 0.2–100 μg/mL respectively. A stable isotope labeled internal standard (ISTD), D₄-MK-7009, was employed. Analytes were chromatographed by reversed phase HPLC and quantified by an MS/MS system. Electrospray ionization in the positive mode was employed. Multiple reaction monitoring of the precursor to product ion pairs m/z 758.6 → 637.4 MK-7009 and m/z 762.5 → 637.4 ISTD was used for quantitation. Analyte and internal standard were extracted from 250 μL of plasma using an automated 96-well liquid–liquid extraction. Plasma pH adjustment prior to extraction minimized ionization suppression in plasma samples from patients with Hepatitis C. The urine method involved direct dilution in the 96-well format of 0.020 mL Tween-treated urine. These methods have supported several clinical studies. Incurred plasma sample reanalysis demonstrated adequate assay reproducibility and ruggedness.     

Quantitation of polar analytes using column-switching: Application to oxycodone and three metabolites in human plasma

We present herein a sensitive and selective assay for the determination of oxycodone and its main metabolites, oxymorphone, noroxycodone and noroxymorphone in human plasma, using column-switching and liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Sample preparation comprised protein precipitation with perchloric acid. After neutralization, the supernatant was injected without any evaporation step onto a polymeric, pH-resistant cartridge (HySphere Resin GP 10–12 μm) for sample clean-up (Prospekt II). The latter operation was achieved by using alkaline conditions to ensure retention of analytes and methanol for matrix interference removal. More than two hundred plasma samples could be analyzed with a single cartridge. Analytes were desorbed in the backflush mode and were separated on a conventional reversed phase column (XTerra MS 4.6 × 50 mm, 3.5 μm), using an acidic mobile phase (i.e. containing 0.1% of formic acid). Mass spectrometric detection was achieved with a 4000 Q TRAP equipped with an atmospheric pressure chemical ionization (APCI) source, in positive ionization mode, operated in the selected reaction monitoring mode (SRM). Starting from a plasma volume of 250 μl, quantification ranges were 25–10,000 pg/ml for OXM and NOXM and 50–10,000 pg/ml for OXC and NOXC. Accuracy was found to be within 98% and 108% and precision better than 7%. Replicate determination of incurred or study samples ensured the method to be reproducible and usable for clinical studies.