The use of acetone as a substitute for acetonitrile in analysis of peptides by liquid chromatography/electrospray ionization mass spectrometry

The recent worldwide shortage of acetonitrile has prompted interest in alternative solvents for liquid chromatography/mass spectrometry (LC/MS). In this work, acetone was substituted for acetonitrile in the separation of a peptide mixture by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and in the positive electrospray ionization mass spectrometry (ESI‐MS) of individual peptides. On both C12 and C18 stationary phases, the substitution of acetone for acetonitrile as the organic component of the mobile phase did not alter the gradient elution order of a five‐peptide retention standard, but did increase peak width, shorten retention times, and increase peak tailing. Positive ESI mass spectra were obtained for angiotensin I, bradykinin, [Leu⁵]‐enkephalin, and somatostatin 14 dissolved in both acetonitrile/water/formic acid (25%/75%/0.1%) and acetone/water/formic acid (25%/75%/0.1%). Under optimized ESI‐MS conditions, the mass spectral response of [Leu⁵]‐enkephalin was increased two‐fold when the solvent contained acetone. The substitution of acetone for acetonitrile resulted in only slight changes in the responses of the remaining peptides. A higher capillary voltage was required for optimum response when acetone was used. Compared with acetonitrile/water/formic acid (50/50/0.1%), more interfering species below m/z = 140 were found in the ESI‐MS spectra of acetone/water/formic acid (50/50/0.1%). Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid and sensitive determination of aucubin in rat plasma by liquid chromatography-tandem mass spectrometry and its pharmacokinetic application

A sensitive, accurate, rapid and robust LC‐MS‐MS method for the quantification of aucubin, a major bioactive constituent of Aucuba japonica, Eucommia ulmoides and Plantago asiatica, was established and validated in rat plasma. Plasma samples were simply precipitated by adding methanol and the supernatant was chromatographed by a Diamonsil® C₁₈(2) column with the mobile phase comprising a mixture of 10 mm ammonium acetate in methanol and that in water with the ratio of 50:50 (v/v). Quantification of aucubin was performed by mass spectrometry in the multiple‐reaction monitoring mode with positive atmospheric ionization at m/z 364 → 149 for aucubin, and m/z 380 → 165 for catalpol (IS), respectively. The retention time was 2.47 and 2.44 min for aucubin and the IS, respectively. The calibration curve (10.0–30,000 ng/mL) was linear (r² > 0.99) and the lower limit of quantification was 10.0 ng/mL in the rat plasma sample. The method showed satisfactory results such as sensitivity, specificity, precision, accuracy, recovery, freeze–thaw and long‐term stability. This simple LC‐MS method was successfully applied in a pharmacokinetic study carried out in Sprague–Dawley rats after oral administration of aucubin at a single dose of 50 mg/kg. Herein the pharmacokinetic study of aucubin is reported for the first time. Copyright © 2011 John Wiley & Sons, Ltd.     

An LC‐MS/MS method for determination of curculigoside with anti‐osteoporotic activity in rat plasma and application to a pharmacokinetic study

A rapid, simple, selective and sensitive LC‐MS/MS method was developed for the determination of curculigoside in rat plasma. The analytical procedure involves extraction of curculigoside and syringin (internal standard, IS) from rat plasma with a one‐step extraction method by protein precipitation. The chromatographic resolution was performed on an Agilent XDB‐C₁₈ column (4.6 × 50 mm, 5 µm) using an isocratic mobile phase of methanol with 0.1% formic acid and H₂O with 0.1% formic acid (45:55, v/v) at a flow rate of 0.35 mL/min with a total run time of 2.0 min. The assay was achieved under the multiple‐reaction monitoring mode using positive electrospray ionization. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over 4.00–4000 ng/mL (R = 0.9984) for curculigoside with a lower limit of quantification of 4.00 ng/mL in rat plasma. The intra‐ and inter‐day precisions and accuracies were 3.5–4.6 and 0.7–9.1%, in rat plasma, respectively. The validated LC‐MS/MS method was successfully applied to a pharmacokinetic study of curculigoside in rats after a single intravenous and oral administration of 3.2 and 32 mg/kg. The absolute bioavailability of curculigoside after oral administration was 1.27%. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of pesticides in vegetables using large-volume injection column liquid chromatography-electrospray tandem mass spectrometry

Direct injection of a large volume (900 microl) of a sample extract onto a liquid chromatographic (LC) column, LC separation and electrospray tandem mass spectrometric detection were used for the quantitative analysis of a wide polarity range of pesticides in carrots and potatoes. Rapid sample preparation involved extraction of a small amount of sample (2 g) with a small volume of organic solvent (3 ml), clean-up over a filter and dilution of the organic extract with the aqueous LC eluent. The extraction efficiency for the selected pesticides was studied using methanol, acetone and acetonitrile as solvents. Evaluation of the performance of the overall method, using extraction with acetonitrile and detection in the selected-reaction-monitoring mode, showed excellent linearity in the range of 2-100 microg/kg with limits of detection of 0.5-2 microg/kg for both types of vegetable. With relative standard deviations of the MS peak area measurements of less than 6.5% (n=8) the repeatability of the method was fully satisfactory.     

Simultaneous determination of gelsemine and koumine in rat plasma by UPLC‐MS/MS and application to pharmacokinetic study after oral administration of Gelsemium elegans Benth extract

A simple, rapid and sensitive method using UPLC‐MS/MS was established and validated for simultaneous determination of gelsemine and koumine in rat plasma after oral administration of Gelsemium elegans Benth extract. Plasma was performed with methanol precipitation and berberine was chosen as the internal standard. Plasma samples were separated on an Acquity UPLC® BEH C₁₈ column (3.0 × 50 mm, 1.7 μm) with gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase at a flow rate of 0.4 mL/min. Multiple reaction monitoring mode in positive ion mode was utilized for detection. The calibration curves were linear over the range of 0.2–100 ng/mL for gelsemine and 0.1–50 ng/mL for koumine, with the lower limits of quantification 0.2 and 0.1 ng/mL, respectively. The intra‐ and inter‐precision and accuracy were well within the acceptable ranges. The developed method was successfully applied to an in vivo pharmacokinetic study in rat after oral administration of 10 mg/kg Gelsemium elegans Benth extract.     

A validated LC‐MS/MS method for rapid determination of brazilin in rat plasma and its application to a pharmacokinetic study

Brazilin is a major homoisoflavonoid component isolated from the dried heartwood of traditional Chinese medicine Caesalpinia sappan L., which is a natural red pigment used for histological staining. Herein a sensitive, specific and rapid analytical LC‐MS/MS method was established and validated for brazilin in rat plasma. After a simple step of protein precipitation using acetonitrile, plasma samples were analyzed using an LC‐MS/MS system. Brazilin and the IS (protosappanin B) were separated on a Diamonsil C₁₈ analytical column (150 × 4.6 mm, 5 µm) using a mixture of water and 10 mm ammonium acetate in methanol (20:80, v/v) as mobile phase at a flow rate of 0.6 mL/min. The method was sensitive with a lower limit of quantitation of 10.0 ng/mL, with good linearity (r² ≥ 0.99) over the linear range 10.0–5000 ng/mL. All the validation data, such as accuracy and precision, matrix effect, extraction recovery and stability tests were within the required limits. The assay method was successfully applied to evaluate the pharmacokinetics parameters of brazilin after an oral dose of 100 mg/kg brazilin in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of an LC‐MS/MS method for quantification of kadsurenone in rat plasma and its application to a pharmacokinetic study

A sensitive and rapid LC‐MS/MS method was developed and validated for the determination of kadsurenone in rat plasma using lysionotin as the internal standard (IS). The analytes were extracted from rat plasma with acetonitrile and separated on a SB‐C₁₈ column (50 × 2.1 mm, i.d.; 1.8 µm) at 30 °C. Elution was achieved with a mobile phase consisting of methanol–water–formic acid (65:35:0.1, v/v/v) at a flow rate of 0.30 mL/min. Detection and quantification for analytes were performed by mass spectrometry in the multiple reaction monitoring mode with positive electrospray ionization m/z at 357.1 → 178.1 for kadsurenone, and m/z 345.1 → 315.1 for IS. Calibration curves were linear over a concentration range of 4.88–1464 ng/mL with a lower limit of quantification of 4.88 ng/mL. The intra‐ and inter‐day accuracies and precisions were <8.9%. The LC‐MS/MS assay was successfully applied for oral pharmacokinetic evaluation of kadsurenone using the rat as an animal model. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantification and pharmacokinetics of crizotinib in rats by liquid chromatography–tandem mass spectrometry

Crizotinib is a small molecule inhibitor of anaplastic lymphoma kinase (ALK) and can be used to treat ALK‐positive nonsmall‐cell lung cancer. A rapid and simple high‐performance liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of crizotinib in rat plasma using a chemical synthetic compound buspirone as the internal standard (IS). The plasma samples were pretreated by a simple protein precipitation with methanol–acetonitrile (1:1, v/v). Chromatographic separation was successfully achieved on an Agilent Zorbax XDB C₁₈ column (2.1 × 50 mm, 3.5 µm). The gradient elution system was composed of 0.1% formic acid aqueous solution and 0.1% formic acid in methanol solution. The flow rate was set at 0.50 mL/min. The multiple reaction monitoring was based on the transitions of m/z = 450.3 → 177.1 for crizotinib and 386.2 → 122.2 for buspirone (IS). The assay was successfully validated to demonstrate the selectivity, matrix effect, linearity, lower limit of quantification, accuracy, precision, recovery and stability according to the international guidelines. The lower limit of quantification was 1.00 ng/mL in 50 μL of rat plasma. This LC‐MS/MS assay was successfully applied to the quantification and pharmacokinetic study of crizotinib in rats after intravenous and oral administration of crizotinib. The oral absolute bioavailability of crizotinib in rats was 68.6 ± 9.63%. Copyright © 2015 John Wiley & Sons, Ltd.     

LC‐MS/MS determination of pogostone in rat plasma and its application in pharmacokinetic studies

Pogostone is an important constituent of Pogostemon cablin (Blanco) Benth., and possesses various known bioactivities. A rapid, simple and sensitive liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for the analysis of pogostone in rat plasma using chrysophanol as internal standard (IS). The analytes were extracted with methanol and separated using a reversed‐phase YMC‐UltraHT Pro C₁₈ column. Elution was achieved with a mobile phase consisting of methanol–water (75:25, v/v) for 5 min at a flow rate of 400 μL/min. The precursor/product transitions (m/z) under MS/MS detection with negative electrospray ionization (ESI) were 223.0 → 139.0 and 253.1 → 224.9 for pogostone and IS, respectively. The calibration curve was linear over the concentration range 0.05–160 µg/mL (r = 0.9996). The intra‐ and inter‐day accuracy and precision were within ±10%. The validated method was successfully applied to the preclinical pharmacokinetic investigation of pogostone in rats after intravenous (5, 10 and 20 mg/kg) and oral administration (5, 10 and 20 mg/kg). Finally, the oral absolute bioavailability of pogostone in rats was calculated to be 70.39, 78.18 and 83.99% for 5, 10 and 20 mg/kg, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative determination of euphol in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

Euphol is a potential pharmacologically active ingredient isolated from Euphorbia kansui. A simple, rapid, and sensitive method to determine euphol in rat plasma was developed based on liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) for the first time. The analyte and internal standard (IS), oleanic acid, were extracted from plasma with methanol and chromatographied on a C₁₈ short column eluted with a mobile phase of methanol–water–formic acid (95:5:0.1, v/v/v). Detection was performed by positive ion atmospheric pressure chemical ionization in selective reaction monitoring mode. This method monitored the transitions m/z 409.0 → 109.2 and m/z 439.4 → 203.2 for euphol and IS, respectively. The assay was linear over the concentration range 27–9000 ng/mL, with a limit of quantitation of 27 ng/mL. The accuracy was between –7.04 and 4.11%, and the precision was <10.83%. This LC‐MS/MS method was successfully applied to investigate the pharmacokinetic study of euphol in rats after intravenous (6 mg/kg) and oral (48 mg/kg) administration. Results showed that the absolute bioavailability of euphol was approximately 46.01%. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of MK-0767 and seven metabolites in rat urine using liquid chromatography/tandem mass spectrometry

MK-0767, 5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide (I, Table 1), is a dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist previously studied for the treatment of type 2 diabetes and dyslipidemia. To support further toxicological studies in one of the animal species used in chronic testing of I, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantification of I and seven metabolites in rat urine was developed and validated. In this method, urine samples were diluted with acetonitrile/methanol (50:50, v/v) and injected directly onto the column of an LC system. Detection was achieved by MS/MS using a turbo ion spray probe monitoring precursor --> product ion combinations in selected reaction monitoring (SRM) mode. The linear range for I and three metabolites was 0.8-800 ng/mL, and 8-8000 ng/mL for four other metabolites found to be present in urine at higher concentrations than I. Intra-day and inter-day variation using this method were < or = 13.0%. The method exhibited good linearity, reproducibility, specificity and sufficient sensitivity when used for the analysis of rat urine samples. Concentrations of I and its major metabolites in rat urine were determined in samples collected between 0-24 h after dosing on the last day of administration of nine daily oral doses to three male (1000 mg/kg/day) and three female (300 mg/kg/day) Sprague-Dawley rats. The urinary concentrations of I and its metabolites were similar in male and female rats. The average concentrations of I were 0.51 and 0.33 microg/mL in male and female rats, respectively. Concentrations of four of the seven metabolites quantified were 6- to 45-fold higher than those of I. The most abundant metabolite, with concentrations of 24.2 and 13.3 microg/mL in male and female rat urine, respectively, was a methyl sulfoxide derivative formed by oxidative cleavage of the thiazolidinedione ring, followed by S-methylation and oxidation of the sulfide intermediate.     

Analysis of Chlormequat residues in grain using liquid chromatography-mass spectrometry (LC-MS/MS)

A fast, sensitive and specific method for routine determination of residues from Chlormequat (CAS no. 7003-89-6) is described. The method is based on a simple clean-up using an SPE-C₁₈ cartridge, high-performance liquid chromatography on a standard C₁₈ column (Spherisorb S5 ODS1) and specific detection and quantification by electrospray mass spectrometry (LC-MS/MS). ¹³C-Chlormequat was synthesised for use as internal standard. Samples were extracted with methanol – water – acetic acid. Internal standard and ammonium acetate were added before C₁₈-cartridge clean up and residues eluted with methanol – water – acetic acid, containing 50 mM ammonium acetate. Chromatographic separation was achieved using a solvent composed of acetonitrile – methanol – water – acetic acid (53:21:25:1 by volume), containing 50 mM ammonium acetate. Electrospray ionisation mass spectrometry was employed using m/z 58 (daughter ion of the Chlormequat quaternary ammonium ion, m/z 122) and m/z 61 (daughter ion of the ¹³C-Chlormequat quaternary ammonium ion, m/z 125) for quantification. The LC analysis time was 15 min and the limit of detection of the analytical method was 9 μg/kg. The performance of the method was demonstrated analysing grain material from an inter-comparison study. In Denmark the primary use of Chlormequat chloride (CCC, cycocel, or chlorocholin chloride, CAS no. 999-81-5) is for winter cereals and 11 such winter wheat samples from the Danish National Pesticide Survey were analysed. Residue contents were from below 0.01 up to 0.45 mg/kg, and thus below the EU maximum residue level of 2.0 mg/kg for wheat.     

Determination of 3-hydroxy pterocarpan, a novel osteogenic compound in rat plasma by liquid chromatography-tandem mass spectrometry: application to pharmacokinetics study

A rapid, sensitive and selective LC‐MS/MS method for the quantitative analysis of 3‐hydroxy pterocarpan (S006‐1709) in female rat plasma has been developed and validated. A Discovery RP₁₈ column was used for the chromatographic elution using acetonitrile and 0.1% acetic acid in water as mobile phase (80:20 v/v) at the flow rate of 0.5 mL/min. MS/MS analysis was performed using a triple quadrupole mass spectrometer with electrospray ionization in negative ion mode using biochanin as an internal standard (IS). Extraction of S006‐1709 and IS from rat plasma was done by liquid–liquid extraction method using diethyl ether. The LC‐MS/MS method was sensitive with 1.95 ng/mL as the limit of detection and 3.9 ng/mL as the lower limit of quantification. The method was linear in the concentration range of 3.9–1000 ng/mL. The percentage bias for intraday and interday accuracy was not greater than 4.2 and the %RSD for intraday and interday precision was not greater than 13.2. The recoveries of S006‐1709 and IS were 73.9–79.3 and 85.7%, respectively. S006‐1709 was found to be stable in various stability studies. The validated LC‐MS/MS method was successfully applied for the oral pharmacokinetics study of S006‐1709 at 10 mg/kg in female Sprague–Dawley rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of zidovudine and lamivudine from rat tissues by liquid chromatography/tandem mass spectrometry

A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of zidovudine (AZT) and lamivudine (3TC) in rat plasma, amniotic fluid, placental, and fetal tissues. Samples were processed by acetonitrile precipitation. Chromatography was performed using a C18 column (5 microm, 150 x 3.9 mm i.d). The mobile phase consisted of 30% methanol and 7.5 mM ammonium acetate (pH 6.5). The method was validated in the range of 0.05-25 microg/mL for both 3TC and AZT in the four biological matrices. Finally, the method was applied to a study involving fetal transport following co-administration of these compounds at a dose of 25 mg/kg each in a pregnant rat.     

Determination of rosamultin in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A specific and reliable LC–MS/MS method for the determination of rosamultin in rat plasma was validated. Plasma samples were prepared with protein precipitation method, and chromatographic separation was performed on a Thermo C₁₈ analytical column (4.6 mm × 50 mm, 3.0 μm). The mass spectrometry (MS) analysis was conducted in positive SRM mode for the transitions of m/z 673.2 → 511.1 for rosamultin and m/z 601.1 → 330.9 for IS. The method validation was conducted over the calibration range of 1.0–500 ng/mL with the precision ≤11.03% and accuracy within ±14.64%. The assay was applied to the pharmacokinetic study after oral administration of rosamultin at a dose of 20 mg/kg in rats.     

LC–MS/MS method for the simultaneous determination of ethyl gallate and its major metabolite in rat plasma

A simple, rapid and sensitive liquid chromatography–tandem mass spectroscopy (LC–MS/MS) method was developed and validated for the determination of ethyl gallate, a pharmacologically active constituent isolated from Lagerstroemia speciosa (Linn.) Pers. This method was used to examine the pharmacokinetics of ethyl gallate and its major metabolite gallic acid in rat plasma using propyl gallate as an internal standard. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a Zorbax SB‐C₁₈ column (3.5 μm, 2.1 × 50 mm) with an isocratic mobile phase consisted of methanol–acetonitrile–10 mM ammonium acetate (10 : 25 : 65, v/v/v) containing 0.1% formic acid at a flow rate of 0.25 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple‐reaction monitoring mode using the electrospray ionization technique in negative mode. The lower limits of quantification of gallic acid and ethyl gallate of the method were 0.5 and 1.0 ng/mL. The intra‐day and inter‐day accuracy and precision of the assay were less than 8.0%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of ethyl gallate to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

The effect and potential of using a temperature controlled separation column with ultra-high pressure microcapillary liquid chromatography/tandem mass spectrometry on proteomic analysis

The microcapillary liquid chromatography (μLC)/tandem mass spectrometry (MS/MS) system has become a prevailing analytical platform in proteomics. Typical proteomic studies aimed at proteome-wide identification of peptides and proteins rely heavily on producing an accurate and reproducible solvent-composition gradient throughout microcapillary separation columns to improve LC separation. With the recent advent of targeted proteomic approaches utilizing the LC retention time as a physicochemical parameter for peptides, high reproducibility of LC separation additionally becomes an important factor. In this study, column temperature elevation is utilized to improve reproducibility and separation efficiency of the μLC-MS/MS system. The simple incorporation of a semi-rigid gas line heater allowed precise control of the temperature of microcapillary columns longer than 70 cm, up to 60 °C. Tryptic enolase peptides were used as a standard sample to evaluate the effect of the controlled temperature elevation on the peptide separation efficiency and reproducibility. In addition to the increased reproducibility in peptide elution time due to the controlled column temperature, the temperature elevation resulted in a decrease in the column operation pressure, which, in turn, allowed a higher solvent flow-rate to be employed using the same LC pumps, leading to further improvements in the performance of μLC systems.     

Systematic evaluation of acetone and acetonitrile for use in hydrophilic interaction liquid chromatography coupled with electrospray ionization mass spectrometry of basic small molecules

Sub‐2‐µm particle size hydrophilic interaction liquid chromatography [HILIC] combined with mass spectrometry has been increasing in popularity as a complementary technique to reversed‐phase LC for the analysis of polar analytes. The organic‐rich mobile phase associated with HILIC techniques provides increases in compound ionization, due to increased desolvation efficiency during electrospray ionisation mass spectrometric (ESI‐MS) analysis. Although recent publications illustrated selectivity and response comparisons between reversed‐phase LC/MS and HILIC LC/MS, there are limited discussions evaluating the optimisation of the mass spectrometry parameters regarding analytes and alternative mobile phases. The use of acetone as an alternative organic modifier in HILIC has been investigated with respect to signal‐to‐noise in ESI‐MS for a variety of polar analytes. Analyte reponses were measured based on a variety of cone and capillary voltages at low and high pH in both acetone and acetonitrile. In order to visualise compound behaviour in the ESI source, surface plots were constructed to assist in interpreting the observed results. The use of acetone in ESI is complicated at low m/z due to the formation of condensation products. Favourable responses were observed for certain analytes and we envisage offering an insight into the use of acetone as an alternative to acetonitrile under certain analytical conditions for particular compound classifications for small molecule analysis. We also highlight the importance of optimising source voltages in order to obtain the maximum signal stability and sensitivity, which are invariably, highly solvent composition dependent parameters. Copyright © 2011 John Wiley & Sons, Ltd.     

Multiresidue analysis of nine β‐agonists in animal muscles by LC‐MS/MS based on a new polymer cartridge for sample cleanup

A novel, sensitive, and reliable LC‐MS/MS method for multiresidue analysis of nine β‐agonists (salbutamol, terbutaline, cimaterol, fenoterol, clorprenaline, ractopamine, tulobuterol, clenbuterol, and penbuterol) in four farm animal muscles was developed. Muscle matrix was extracted with acetonitrile–10% sodium carbonate solution, and then was subjected to cleanup using a SPE cartridge packed with new polymer synthesized in acetone. Chromatographic separation of the components was performed on a Luna C₁₈ column using 0.1% of formic acid in water and acetonitrile. The mass spectrometer was operated in the positive electrospray mode. Good precision and accuracy were obtained for all analytes (except for fenoterol) at the spiked three levels of 1.0, 10, and 50 μg/kg. The decision limit and detection capability of nine β‐agonists ranged from 0.04 to 0.18 and 0.15 to 0.69 μg/kg, respectively. The method developed was successfully applied to the monitoring of nine β‐agonists in pork, beef, mutton, and chicken from Chinese markets.     

A rapid UHPLC–MS/MS method for the quantification of ARQ531 in rat plasma: Validation and its application to a pharmacokinetic study

A simple and sensitive ultra-high performance liquid chromatography–tandem mass spectrometric (UHPLC–MS/MS) method was developed and validated for the determination of ARQ531, a Bruton’s tyrosine kinase inhibitor in rat plasma. After protein precipitation with acetonitrile, the samples were separated on a UPLC BEH C₁₈ column with 0.1% formic acid in water and acetonitrile as mobile phase at a flow rate of 0.4 ml/min. The mass detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring with precursor-to-product ion transitions of m/z 479.1 > 365.1 and m/z 441.2 > 138.1 for ARQ531 and internal standard, respectively. Good linearity (correlation coefficient > 0.9988) was achieved over the concentration range of 0.5–1,000 ng/ml and the lower limit of quantitation was 0.5 ng/ml. The accuracy ranged from −13.50 to 11.35% and the precision was <8.87%. The extraction recovery was >85.56%. ARQ531 was demonstrated to be stable under the tested conditions. The validated method was further applied to a pharmacokinetic study of ARQ531 in rats after intravenous (1 mg/kg) and oral (1, 3 and 10 mg/kg) administration. The results demonstrated that ARQ531 displayed linear pharmacokinetic profiles over the oral dose range of 1–10 mg/kg and good oral bioavailability (>50%).