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protein precipitation method for the determination of oxcarbazepine and its major active metabolite 10-monohydroxy derivative in human plasma by liquid chromatography tandem mass spectrometry was established. analytes were extracted from human plasma samples by protein precipitation with acetonitrile. analyte separation was done using phenomenex Kinetex™ Biphenyl (2.6 µm, 100 x 2.1 mm) column using isocratic elution with a mobile phase of 5 mM ammonium bicarbonate (42.5%) and methanol (57.5%) at a flow rate of 0.3 mL/min and an injection volume of 5 µL. The detection was performed on a triple quadrupole mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 253.1 → 180.0, 257.2 → 184.1 for oxcarbazepine and oxcarbazepine-D4, and 255.1 → 194.2, 259.2 → 198.2 for 10-monohydroxy derivative and 10,11-Dihydro-10-hydroxy carbamazepine-D4 in positive electrospray ionization mode, correspondingly. The method was validated over a concentration range of 20-8000 ng/mL for oxcarbazepine and 37.5-15000 ng/mL for 10-monohydroxy derivative on SCIeX Triple Quad 4500 MS System. Total run time was 4 min. Sample preparation was conducted in ice water bath because of instability of oxcarbazepine in plasma at room temperature. The method was validated in accordance with U.S Food and Drug administration and european Medicines agency guidelines. Method validation has been proved by a variety of tests for matrix effects, extraction efficiency, selectivity, linearity, sensitivity, precision, recovery and stability and can be successfully applied for the bioequivalence/pharmacokinetic studies of oxcarbazepine and its 10-monohydroxy derivative. This method has been designed for bioequivalence study for formulations containing 600 mg of oxcarbazepine. [ABSTRACT FROM AUTHOR]

Highlights • Three analytical methods for mirabegron and metabolites in human plasma validated. • Assays miniaturized for paediatric population by using 96-well plate technology. • Methodology utilized a more sensitive mass spectrometer than previously. • Reduction in both assay volume and/or assay sensitivity was achieved. • The assays enable mirabegron pharmacokinetic assessment in paediatric populations. Abstract Mirabegron is the first registered β3-adrenoceptor agonist for treatment of overactive bladder as an alternative to antimuscarinics. Previously four liquid chromatography-tandem mass spectrometry assays were published to determine mirabegron and eight metabolites (M5, M8, M11–M16) in human plasma. In order to support paediatric development, the assays were further optimized to reduce the required blood volume and increase the sensitivity. The assays were miniaturized by using 96-well supported liquid extraction plates (mirabegron, M5, M16) or 96-well mixed-mode cation exchange solid phase extraction plates (M8, M11–M15) and a more sensitive MS-system was used. For the analytes, up to fivefold increase in assay sensitivity was achieved. The required blood sample volume was reduced from 10 to 2 mL for each timepoint. Validation demonstrated that the assays were accurate, precise and selective in the determination of mirabegron and metabolites. The assays were successfully applied to evaluate the pharmacokinetics of mirabegron in a paediatric population. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • The first bio-analytical assay for a combination of ALK inhibitors is presented. • The first assay for ALK inhibitors in tissue homogenates is reported. • The LC–MS/MS method has successfully been validated in six matrices. • The assay was applied for a pilot study of brigatinib in wild type mice. Abstract Several second and third generation ALK inhibitors have been introduced in recent years. A bioanalytical assay for simultaneous quantification of alectinib, brigatinib, and lorlatinib was developed and validated for human plasma. The method was also partially validated for diluted mouse plasma and tissue homogenates of brain, liver, kidney, and spleen. Samples (40 μl) were pretreated in a 96-well plate by protein precipitation with acetonitrile containing the internal standard [2H 8 ]-alectinib. After chromatographic separation on an ethylene bridged octadecyl silica column by gradient elution at 600 μl/min using 1% (v/v) formic acid (in water) and acetonitrile, compounds were ionized by a turbo electrospray and monitored by selected reaction monitoring on a triple quadrupole mass spectrometer. Validation was performed in a 2–2000 ng/ml concentration range for alectinib and lorlatinib and a 4–4000 ng/ml range for brigatinib. Precisions (within–day and between–day) were in the range 2.2–15.0% and accuracies were in between 87.2 and 110.2% for all matrices and levels. Compounds were sufficiently stable under most investigated conditions. Results of a pilot pharmacokinetic and tissue distribution study for brigatinib in mice are reported. Finally, successful incurred samples reanalysis of tissue homogenate samples containing brigatinib and lorlatinib is presented. Lorlatinib homogenate samples were also successfully reanalyzed using a second independent assay (cross-validation). [ABSTRACT FROM AUTHOR]

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitative analysis of 1,2:5,6-dianhydrogalactitol (DAG) in mouse plasma and tissues. Sodium diethyldithiocarbamate (DDTC) was used as the derivatization reagent to improve its LC-MS/MS behavior. Analytes were separated on a Welch Ultimate XB-CN column with a mobile phase consisting of acetonitrile and 0.1% formic acid solution (65:35). The MS analysis was conducted by positive electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity (r 2  > 0.9958) was observed over the concentration range of 1–1000 ng/mL in plasma and tissue homogenates (brain, liver, heart, spleen, lung and kidney). The intra- and inter-batch precision and accuracy of DAG in plasma and brain samples were all within the acceptable limits. The extraction recovery was stable and no significant matrix effects were observed. The method was successfully applied to study the pharmacokinetic and tissue distribution of DAG in mice after intravenous administration. DAG could cross the blood-brain barrier and had limited liver distribution. Rat primary hepatocytes in vitro experiments demonstrated that DAG had a safe profile in liver. [ABSTRACT FROM AUTHOR]

The human health benefits attributed to turmeric/curcumin spice has resulted in its wide utilization as a dietary supplement for companion pets and other animals including horses. While the quantification of free curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin) and their phase-2 metabolites (curcumin- O -sulfate, curcumin- O -glucuronide) have been extensively investigated in human and rodent biological samples (primarily plasma and serum), there is lack of similar data for horses. Herein, we report a validated LC-ESI–MS/MS method for the simultaneous quantification of the aforementioned free curcuminoids and their metabolites in equine plasma. The linearity of the aforementioned curcuminoids and curcumin- O -sulfate was in the range of 0.5–1000 ng/mL and 1–1000 ng/mL for curcumin- O -glucuronide with 85–115% accuracy and <15% precision in equine plasma. The method was validated based on US FDA criteria and applied to characterize the pharmacokinetics of curcumin- O -sulfate in equine plasma. [ABSTRACT FROM AUTHOR]

Levonorgestrel (LNG) is a synthetic progestin that is available in oral contraceptive tablets, a subdermal implant, and an intrauterine system for contraception. LNG pharmacokinetics are a pivotal determinant of contraceptive efficacy and essential in assessing drug-drug interactions influencing LNG exposure following different routes of LNG administration. A highly sensitive LC-MS/MS method was developed and validated to quantify levonorgestrel in human plasma. Liquid-liquid extraction was utilized with a sample volume of 500 μL to extract levonorgestrel from plasma. Chromatographic separation of LNG was achieved with a Fortis™ C18 (3 μm: 100 mm × 2.1 mm) reverse phase analytical column. The mobile phases consisted of de-ionized water plus 0.1% NH 4 OH (100:0.1%, v/v) (A), and methanol plus 0.1% NH 4 OH (100:0.1%, v/v) (B) delivered as a gradient at a flow rate of 400 μL/min. Detection of LNG and internal standard (D-(−)-norgestrel-d7) was achieved using positive polarity mode monitoring at 313.2–245.2 amu and 320.1–251.2 amu, respectively. The assay was linear over the calibration range of 49.6 to 1500 pg/mL. This method was used to quantify plasma LNG released by subdermal implant in support of a drug interaction study among women with HIV receiving efavirenz- or nevirapine-based antiretroviral therapy. [ABSTRACT FROM AUTHOR]

Rotigotine behenate (RGTB), a long chain alkyl ester of the prodrug of rotigotine (RGT), has been synthesized for use in a sustained delivery system. The aim of the present report was to develop and validate a simple, sensitive and reliable LC–MS/MS method for the simultaneous determination of RGT and its prodrug RGTB in rat plasma samples. Detection was performed on a 1290 Infinity UPLC coupled Triple Quad 4500 mass spectrometer operated in positive MRM mode using an Eclipse XDB-CN chromatography column (2.1 mm × 100 mm, 3.5 μm) by isocratic elution using a 0.2% formic acid aqueous solution and acetonitrile, with stable isotope labeled RGT as an internal standard. The sample preparation method employed 50 μL of a plasma sample and liquid-liquid extraction with a mixture of diethyl ether–dichloromethane (3:2, v/v) as the extraction solvent. The proposed method was fully validated by assessing its specificity, linearity, precision and accuracy, recovery, matrix effects and stability. Good linearity was found within the range of 0.1–10.0 ng/mL for both analytes (r > 0.996). This method was successfully applied to a pharmacokinetic study of a slow release RGTB formulation in rats following a single intramuscular injection and biological conversion in vitro. [ABSTRACT FROM AUTHOR]

[Display omitted] • The first LC‐MS/MS method of Isoxazole analogue of curcumin in rat plasma. • The Developed LC‐MS/MS method is a highly sensitive and selective method for quantification of routine pharmacokinetics application. • A simple protein precipitation technique was used with high recovery. • Cost effective in routine analysis as compared to liquid‐liquid extraction (LLE) or solid‐phase extraction (SPE) methods. • The developed method has only five minutes run time, which helps to save time. A novel Isoxazole (ISO) analog of curcumin is synthesized from curcumin and described as having a better pharmacological activity than curcumin, such as anti-cancer, anti-malarial, anti-mycobacterial, and many more. The present research aims to develop a bio-analytical method with a simple, rapid, selective, sensitive, accurate, and precise quantification of ISO by liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS) in rat plasma matrix. The simple plasma protein precipitation method was used for ISO extraction. The ISO was eluted in isocratic mode on a Water symmetry C 18 column (75 × 4.6 mm2, 3.5 μm) at a 600 µL/min flow rate with a 0.1 % formic acid in water and methanol (20:80) as mobile phase. The MS/MS was used as a monitoring tool for the fragmentation of ISO as m / z = 366.1 → 145.1 and m / z = 237.1 → 194.07 for carbamazepine (CBZ; internal standard). The ISO showed good co-relation as (r2 = 0.999) linear and covered a wide range with a lower limit of quantification of 1.0 ng/mL. Finally, the developed method was successfully utilized for oral and intravenous pharmacokinetics of ISO in rats plasma. The absolute bioavailability of ISO was found at about 17.6 % after oral administration. [ABSTRACT FROM AUTHOR]

Efonidipine hydrochloride is a new generation dihydropyridine Ca 2+ channel blocker designed to inhibit both T-type and L-type Ca 2+ channels. Efonidipine possesses a chiral carbon and is clinically administered as a racemate. In the present study, an enantioselective and sensitive LC–MS/MS method of determining efonidipine enantiomers in human plasma was developed and validated to characterize the stereoselective pharmacokinetics. Plasma samples were processed by liquid-liquid extraction (LLE). Chiral separation was optimized on a CHIRALPAK ® ID column using an isocratic mobile phase of acetonitrile/water (60:40, v/v). Detection was using MS in multiple reaction monitoring (MRM) mode, using the transitions of m/z 632.3 → 91.1 for efonidipine enantiomers, and m/z 493.3 → 117.2 for cilnidipine (internal standard). The calibration curves were linear over 0.100–20.0 ng/mL for each enantiomer. The lower limit of quantification (LLOQ) for each enantiomer was established at 0.100 ng/mL. Intra- and inter-day precisions were less than 12.1% for each enantiomer in terms of relative standard deviation (RSD), and accuracies were between −5.0% and 5.0% in terms of relative error (RE) for each enantiomer. No chiral inversion was observed during sample storage, preparation procedure and analysis. The validated method was successfully applied to a stereoselective pharmacokinetic study of efonidipine in healthy subjects after oral administration of 40 mg (20 mg × 2) efonidipine hydrochloride tablets. [ABSTRACT FROM AUTHOR]

For the first time, a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of fraxin and its metabolite, fraxetin, in rat plasma, using esculin as the internal standard (IS). The plasma samples were precipitated with methanol before separation on an Nova-Pak C 18 column (150 mm × 3.9 mm, 3 μm) using a mobile phase consisting of 0.1% formic acid and methanol (55:45) at a flow rate of 0.8 mL/min. The analytes were detected by multiple reaction monitoring in the negative ion mode with the mass transitions at m / z 368.9→ m / z 191.9 (fraxin), m / z 206.9→ m / z 191.8 (fraxetin) and m / z 339.0→ m / z 176.9 (esculin, IS). The results demonstrated that the calibration curves for both analytes have good linearity (r ≥ 0.995) over a concentration range of 5.00–3000 ng/mL. The assay was validated according to the regulatory bioanalytical guidelines and proved acceptable. The intra- and inter-day precisions (R.S.D.%) were within 10.9% for both analytes, whereas the deviation of assay accuracies (R.E.%) ranged from −5.3 to 1.0%. The method was successfully applied to a pharmacokinetic study after a single oral dose of fraxin at 50 mg/kg to rats. [ABSTRACT FROM AUTHOR]

Etodolac is a non-steroidal anti-inflammatory drug with preferential inhibition of cyclooxigenase-2 and is widely used in the management of pain in patients with inflammatory arthritis. Etodolac is available as a racemic mixture of (−)-( R )-Etodolac and (+)-( S )-Etodolac; cyclooxigenases inhibition is attributed to (+)-( S )-Etodolac. According to our knowledge, this is the first method for determination of etodolac enantiomers in plasma using LC–MS/MS. Plasma extraction were performed with 25 μL of plasma and 1 mL of n-hexane:ethyl acetate (95:5); racemic ibuprofen was used as internal standard. Resolution of enantiomers were performed in a Chiralcel ® OD-H column; deprotonated [M-H] − and their respective ion products were monitored at transitions of 286 > 242 for etodolac enantiomers and 205 > 161 for ibuprofen. The quantitation limit was 3.2 ng/mL for both enantiomers in plasma. The method was applied to study the pharmacokinetics of etodolac enantiomers after the administration of a 300 and 400 mg dose of racemic drug to a healthy volunteer. Analysis of plasma samples showed higher plasma concentration of (−)-( R )-Etodolacfor both doses (300 mg dose: AUC 0–∞ 49.80 versus 4.55 ug h/mL;400 mg dose: AUC 0–∞ 63.90 versus 6.00 ug h/mL) with an (R)-(+)/(S)-(−) ratio of approximately 11. [ABSTRACT FROM AUTHOR]

A rapid, selective, and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneously determine resveratrol levels in plasma and brain tissue in mice for supporting pharmacokinetic and brain distribution studies. Analytes were separated using a Sepax BR-C18 analytical column (5 μm, 120 Å, 1.0 × 100 mm) and eluted using an isocratic elution mobile phase acetonitrile and 0.01% formic acid [60:40, v/v] at a flow rate of 0.1 mL/min. Precursor and product ion transitions for analyte resveratrol m / z 226.9 > 184.8 and curcumin m / z 367.1 > 148.9 were monitored using triple quadrupole mass spectrometer with multiple reaction monitoring (MRM) in negative ionization mode. The method was validated with respect to accuracy, within- and between-day precision, linearity, limit of quantification, recovery, and matrix effects of analyte. The inter- and intra-day accuracy and precision were within the range of the US Food and Drug Administration (FDA) acceptance criteria, for both matrices. The method was also successfully applied to pharmacokinetic and brain distribution studies of resveratrol after intravenous administration of free resveratrol and resveratrol-loaded solid lipid nanoparticles to mice. The combined use of serial blood sampling, small sample volume, simple extraction, and capillary depletion method drastically improved resveratrol analysis from biological matrices. [ABSTRACT FROM AUTHOR]

We developed and validated a high performance liquid chromatographic method coupled with triple quadrupole mass spectrometry for analysis of nizatidine in human plasma and urine. The biological samples were precipitated with methanol before separation on an Agilent Eclipse Plus C 18 column (100 mm × 46 mm, 5 μm) with a mixture of methanol and water (95:5, plus 5 mM ammonium formate) as the mobile phase at 0.5 mL/min. Detection was performed using multiple reaction monitoring modes via electrospray ionization (ESI) at m / z 332.1 → 155.1 (for nizatidine) and m / z 335.1 → 155.1 (for [ 2 H 3 ]-nizatidine, the internal standard). The linear response range was 5–2000 ng/mL and 0.5–80 μg/mL for human plasma and urine, with the lower limits of quantification of 5 ng/mL and 0.5 μg/mL, respectively. The method was validated according to the biological method validation guidelines of the Food and Drug Administration and proved acceptable. This newly developed analytical method was successfully applied in a pharmacokinetic study following single oral administration of a 150 mg nizatidine capsule in to 16 healthy Chinese subjects. Maximum and endpoint concentrations in plasma and urine were quantifiable, suggesting our method is appropriate for routine pharmacokinetic analysis. [ABSTRACT FROM AUTHOR]

A sensitive and specific method for the quantitative determination of gamithromycin in animal plasma, lung tissue and pulmonary epithelial lining fluid (PELF) using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC–MS/MS) was developed. The sample preparation was rapid, straightforward and consisted of a deproteinization and phospholipid removal step using an Oasis ® Ostro™ 96-well plate (chicken, turkey and calf plasma) or HybridSPE ® -Phospholipid SPE cartridges (pig plasma and turkey lung tissue), while a liquid–liquid extraction with diethyl ether in alkaline medium was used for PELF of turkey poults. Chromatography was performed on a C18 Hypersil GOLD column using 0.01 M ammonium acetate in water with a pH of 9, and acetonitrile as mobile phases. The MS/MS instrument was operated in the positive electrospray ionization mode and the following selected reaction monitoring transitions were monitored for gamithromycin (protonated molecule > product ion): m / z 777.45 > 619.35 and m / z 777.45 > 157.80 for quantification and identification, respectively. The method was validated in-house: matrix-matched calibration graphs were prepared and good linearity ( r ≥ 0.99) was achieved over the concentration ranges tested (2.5–10,000 ng mL −1 for chicken, pig and calf plasma; 5.0–2500 ng mL −1 for turkey plasma; 50–10,000 ng g −1 for turkey lung tissue and 20–1000 ng mL −1 for turkey PELF). Limits of quantification (LOQ) were 2.5 ng mL −1 for chicken, pig and calf plasma and 5.0 ng mL −1 for turkey plasma, while the limits of detection (LOD) ranged between 0.007 and 0.07 ng mL −1 . For lung tissue and PELF, respective LOQ and LOD values of 50 ng g −1 and 0.76 ng g −1 (lung tissue) and 20 ng mL −1 and 0.1 ng mL −1 (PELF) were obtained. The results for the within-day and between-day precision, expressed as relative standard deviation (RSD), fell within the maximal RSD values. The accuracy fell within −30% to +10% (concentrations 1–10 ng mL −1 ) or −20% to +10% (concentrations > 10 ng mL −1 or ng g −1 ) of the theoretical concentration. The method was successfully applied for the quantitative determination of gamithromycin in plasma samples of chickens, turkeys, pigs and calves; and in lung tissues and PELF of turkeys, all derived from pharmacokinetic studies in these animal species. [ABSTRACT FROM AUTHOR]

Efficacy assessments using a combination of ibrutinib and lenalidomide necessitate the development of an analytical method for determination of both drugs in plasma with precision. A high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of lenalidomide, ibrutinib, and its active metabolite PCI45227 in rat plasma. Extraction of lenalidomide, ibrutinib, PCI45227 and tolbutamide (internal standard; IS) from 50 μl rat plasma was carried out by liquid–liquid extraction with ethyl acetate:dichloromethane (90:10) ratio. Chromatographic separation of analytes was performed on YMC pack ODS AM (150 mm × 4.6 mm, 5 μm) column under gradient conditions with acetonitrile:0.1% formic acid buffer as the mobile phases at a flow rate of 1 ml/min. Precursor ion and product ion transition for analytes and IS were monitored on a triple quadrupole mass spectrometer, operated in the selective reaction monitoring with positive ionization mode. Method was validated over a concentration range of 0.72–183.20 ng/ml for ibrutinib, 0.76–194.33 ng/ml for PCI-45227 and 1.87–479.16 ng/ml for lenalidomide. Mean extraction recovery for ibrutinib, PCI-45227, lenalidomide and IS of 75.2%, 84.5%, 97.3% and 92.3% were consistent across low, medium, and high QC levels. Precision and accuracy at low, medium and high quality control levels were less than 15% across analytes. Bench top, wet, freeze–thaw and long term stability was evaluated for all the analytes. The analytical method was applied to support a pharmacokinetic study of simultaneous estimation of lenalidomide, ibrutinib, and its active metabolite PCI-45227 in Wistar rat. Assay reproducibility was demonstrated by re-analysis of 18 incurred samples. [ABSTRACT FROM AUTHOR]

This paper describes a sensitive, reliable method to determine pilsicainide (PLC) levels in microscale sample volumes of human biological fluids using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI). PLC and quinidine as an internal standard were extracted with diethylether from 0.1 mL of alkalinized biological fluids. The extract was injected into an analytical column ( l -column 2 ODS, 75 mm × 2.1 mm i.d.). The mobile phase for separation consisted of 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v) and was delivered at a flow rate of 0.2 mL/min. The drift voltage was 100 V. The sampling aperture was heated at 120 °C and the shield temperature was 260 °C. The ion transitions used to monitor analytes were m / z 273 → m / z 110 for PLC and m / z 325 → m / z 79 for quinidine. The total time for chromatographic separation was less than 8 min. The validated concentration ranges of this method for PLC were 5–2000 ng/mL in plasma, 5–500 ng/mL in ultrafiltered plasma solution, and 25–2000 ng/mL in urine. Mean recoveries of PLC in plasma, ultrafiltered plasma solution, and urine were 93.2–99.7%, 91.4–100.6%, and 93.9–104.7%, respectively. Intra- and interday coefficients of variation for PLC were less than 6.0% and 4.3% in plasma, 6.1% and 3.7% in ultrafiltered plasma solution, and 5.4% and 2.5% in urine at the above concentration ranges, respectively. The lower limit of quantification for PLC in plasma, ultrafiltered plasma solution, and urine were 5 ng/mL, 5 ng/mL, and 25 ng/mL, respectively. This method can be applied to pharmacokinetic study and therapeutic drug monitoring in special populations such as neonates, infants, and the elderly by making effective use of residual samples used for general clinical laboratory testing. [ABSTRACT FROM AUTHOR]

The monitoring of the plasmatic concentrations of cardiovascular drugs is crucial for understanding their pharmacokinetics and pharmacodynamics. A simple, sensitive, specific, and high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous estimation and pharmacokinetic study of losartan (LOS), losartan carboxylic acid (LCA), ramipril (RAM), ramiprilate (RPT), and hydrochlorothiazide (HCZ) in rat plasma using irbesartan (IBS) and metolazone (MET) as internal standards (ISs). After solid phase extraction (SPE), analytes and ISs were separated on an Agilent Poroshell 120, EC-C18 (50 mm × 4.6 mm, i.d., 2.7 μm) column with a mobile phase consisting of methanol/water (85:15, v/v) containing 5 mmol/L ammonium formate and 0.1% formic acid at a flow rate of 0.4 mL/min. The precursor → product ion transitions for the analytes and ISs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) mode and switching the electrospray ionization (ESI) mode during chromatography from positive (to detect LOS, LCA, RAM, RPT, and IBS) to negative (to detect HCZ and MET). The method was validated as per the FDA guidelines and it exhibited sufficient specificity, accuracy, and precision. The method was found to be linear in the range of 3-3000 ng/mL for LOS and LCA, 0.1-200 ng/mL for RAM and RPT, and 1-1500 ng/mL for HCZ. The described method was successfully applied to the preclinical pharmacokinetic study of analytes after oral administration of a mixture of LOS (10 mg/kg), RAM (1 mg/kg), and HCZ (2.5 mg/kg) in rats. [ABSTRACT FROM AUTHOR]

A simple, sensitive, and selective LC-MS/MS method was developed and validated for the quantification of carbocisteine in human plasma. Rosiglitazone was used as the internal standard and heparin was used as the anticoagulant. The chromatographic separation was performed by using the Waters Symmetry Shield RP 8, 150 x 3.9 mm, 5 μm column at 40°C with a mobile phase consisting of a mixture of methanol and 0.5% formic acid solution in a 40:60 proportion. The flow rate was 500 μl/min along with a 5 μl injection volume. Protein precipitation was used as the extraction method. Mass spectrometric data were detected in positive ion mode. The MRM mode of the ions for carbocisteine was 180.0 > 89.0 and for rosiglitazone it was 238.1 > 135.1. The method was validated in the concentration curve range of 50.000 ng/mL to 6000.000 ng/mL. The retention times of carbocisteine and the internal standard rosiglitazone were approximately 2.20 and 3.01 min, respectively. The overall run time was 4.50 min. This method was found suitable to analyze human plasma samples for the application in pharmacokinetic and BA/BE studies. [ABSTRACT FROM AUTHOR]

This study describes for the first time a method for the sequential analysis of the enantiomers of cyclophosphamide (CY) and its metabolite carboxyethylphosphoramide mustard (CEPM) in human plasma. The CY and CEPM enantiomers were extracted from plasma using only ethyl acetate and separated on a Chiralpak ® AD-RH column using a mixture of water:acetonitrile:ethanol (45:30:25, v/v/v) plus 0.1% trifluoroacetic acid as the mobile phase at a flow rate of 0.5 mL/min. No matrix effect was observed in the analysis of the enantiomers of both analytes and the analytical method was linear in the range of 0.05–25.0 μg and 250–1000 ng of each enantiomer/mL plasma. The coefficients of variation and relative errors obtained for the assessment of intra- and interassay precision and accuracy were less than 15%. CY and CEPM were found to be stable in human plasma after three successive freeze/thaw cycles, during storage for 4 h at room temperature, and after 24 h inside the autosampler at 4 °C, with deviations less than 15%. The method was applied to the study of the pharmacokinetics of CY and its metabolite CEPM in patients with multiple sclerosis ( n = 10) who received a CY pretransplant conditioning regimen for hematopoietic stem cell transplantation. The pharmacokinetic parameters showed plasma accumulation of the ( S )-(−)-CY enantiomer (S/R ratio = 1.3) and lack of enantioselective exposure to the CEPM metabolite (S/R ratio = 1.0). [ABSTRACT FROM AUTHOR]

Clodronate belongs to the class of bisphosphonates which are used for the treatment of bone disorders. Due to its high polarity it has a low and highly variable oral bioavailability which results in low plasma concentrations and requires sensitive bioanalytical methods to characterize its pharmacokinetics in human. Here, we describe for the first time the development and validation of a LC–MS/MS assay for the quantification of clodronate in human plasma. The bisphosphonate was isolated from the biological matrix by protein precipitation using perchloric acid (10%), and derivatized with trimethylorthoacetate prior sample clean-up with liquid–liquid extraction using methyl tert -butyl ether. The chromatography was performed using an isocratic elution with ammonium acetate 5 mM (85% v/v, pH 3.8) and acetonitrile (15% v/v) as mobile phase with a flow rate of 300 μl/min on a reversed-phase column (Supelco Ascentis ® , C18) temporized at 50 °C. The mass spectrometric detection was done using the API4000 triple quadruple mass spectrometer monitoring the mass/charge transitions 301.0/145 for clodronate and 305.2/137.1 for the internal standard etidronate. The analytical range was set to 5–800 ng/ml, allowing an evaluation of the plasma concentration–time profiles of clodronate for approximately 7–8 half-life (∼24 h). The method was validated according to current FDA/EMA guidelines on bioanalytical method validation with respect to specificity, linearity, intra- and inter-day accuracy and precision, matrix effect, recovery as well as stability. The precision of the assay was 0.6–6.9% and 0.6–8.1% for the intra-day and inter-day variability, respectively. The intra-day and inter-day accuracy (error) was 0.6–8.8% and 2.2–4.5%. The recovery of the analyte was low (2–3%) but reproducible over the entire validation range and sufficient to monitor the target concentrations in human plasma. The drug was shown to be stable in plasma at room temperature for at least 3 h (96.0 ± 6%) and for at least 24 h when stored in the cooled autosampler at 4 °C (102.4 ± 4.5%). Clodronate can also undergo up to three freeze–thaw cycles without impaired stability. Thus, the method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to measure plasma concentrations of clodronate. Finally, the developed method was successfully applied to study the clodronate serum levels in a pharmacokinetic study in healthy volunteers. [ABSTRACT FROM AUTHOR]

Highlights: [•] An LC–MS/MS method for the determination of zonisamide (ZNM) in plasma was developed. [•] The method was successfully applied for ZNM pharmacokinetics in rabbits. [•] The method is suitable for routine TDM of ZNM. [Copyright &y& Elsevier]

Highlights: [•] Highly selective method for the determination of ruxolitinib and nilotinib in plasma. [•] This is the first LC–MS/MS-based method for simultaneous determination of ruxolitinib and nilotinib in plasma. [•] Plasma sample clean-up involves a simple protein precipitation procedures. [•] The developed LC–MS/MS based method was applied successfully for pharmacokinetic study. [Copyright &y& Elsevier]

Highlights: [•] Simultaneous quantification of fludarabine and clofarabine. [•] Sensitive LC–MS/MS method suitable for pediatric studies. [•] The method was applied to a clinical pharmacokinetic study in pediatric patients. [ABSTRACT FROM AUTHOR]

Triptorelin, a gonadotropin-releasing hormone agonist, has been used in the treatment of hormone-responsive prostate cancer by inducing testosterone suppression. Research on the relationship between the time courses of triptorelin and testosterone is very important, but accurate quantification of triptorelin and testosterone simultaneously in biological specimens is a challenging analytical problem. In the present study, a rapid, sensitive, and selective method for simultaneous determination of triptorelin and testosterone in rat plasma by solid-phase extraction and liquid chromatography-tandem mass spectrometry was developed using a ZORBAX RRHD Eclipse Plus C8 column (2.1 × 50 mm, 1.8 μm) with a 0.05 % propionic acid/methanol gradient. In view of the polarity difference between the two analytes, two internal standards, i.e., leuprolide and testosterone-C, were used for individual quantitation of triptorelin and testosterone. Endogenous testosterone was determined by reference to a calibration curve prepared using testosterone-D as a surrogate analyte. The method exhibits excellent linearity over three orders of magnitude for each analyte. The lower limit of quantification was 0.01 ng/mL for triptorelin and 0.05 ng/mL for testosterone, with consumption of 100 μL of plasma. The method was successfully applied to characterize the pharmacokinetics and pharmacodynamics of slow-release 28-day form triptorelin acetate biodegradable microspheres in rats after intramuscular injections of three consecutive doses of 0.6 mg/kg per 28 days. The results revealed that the pharmacokinetic profile of triptorelin produced an initial flare-up in testosterone levels, rapid castration within 5 days after injection, and long-term castration until the next dose. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of cyclobenzaprine in dog plasma. After extracted with organic solvent, post-treatment samples were separated on an Agela C18 column interfaced with a triple quadrupole tandem mass spectrometer in positive electrospray ionization mode. Multiple reaction monitoring was performed using the transitions of m/z 276.2 → 216.1 and m/z 325.1 → 109.0 to quantify cyclobenzaprine and escitalopram (internal standard), respectively. The mobile phase consisted of acetonitrile: 5 mM ammonium acetate: formic acid (90:10:0.01, v/v/v) at a flow rate of 0.3 ml/min. The total analysis time was 2.4 min. The method was linear over the concentration range of 0.0200-10.0 ng/ml. The intra- and inter-day precision was within 12.8% in terms of relative standard deviation (RSD%) and the accuracy within 5.6% in terms of relative error. This method was successfully applied in a pharmacokinetic study of extended-release cyclobenzaprine in dogs. [ABSTRACT FROM AUTHOR]

Highlights: [•] Reversed phase HPLC coupled to MS is a powerful method to quantify almorexant and its primary metabolites in human plasma. [•] Liquid–liquid extraction is superior to protein precipitation when ng/mL or sub-ng/mL sensitivity needs to be achieved. [•] Sample preparation by SLE and LLE yield comparable results, LLE is easy to automate. [ABSTRACT FROM AUTHOR]

Highlights: [•] An LC–MS/MS was developed and validated for the determination of PGE1 in beagle dog plasma. [•] An LLOQ of 10pg/mL was achieved. [•] Indomethacin was added to plasma to avoid significant endogenous interfere. [•] A simple one-step sample extraction was used followed by a short cycle time of 3min. [•] The method was applied to a pharmacokinetic study in dogs at a clinical relevant dose. [ABSTRACT FROM AUTHOR]

[Display omitted] • First report for quantification of bexarotene in biological matrix by LC–MS/MS. • LLOQ of 10.0 ng/mL was achieved with consumption of only 20 μL of plasma or tissue homogenate. • Simple sample pretreatment with a single-step protein precipitation method was employed. • First investigation of bexarotene pharmacokinetics in mice using a fully validated LC–MS/MS method. Emerging evidence has suggested that bexarotene, a nearly 20-year-old skin cancer drug, may be a potential drug candidate to treat Alzheimer's disease (AD) and other neurodegenerative disorders. As described in this study, a highly sensitive and rapid method, using liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine bexarotene in mouse plasma and brain tissue, was established and validated for the first time. Single-step protein precipitation utilizing methanol solution (containing 0.05 % acetic acid) as precipitation agent was employed to prepare the samples of plasma and brain tissue. Chromatographic separation in gradient elution mode was conducted via an Agilent ZORBAX SB-C18 column (50 mm × 4.6 mm, 5 µm) employing methanol–ammonium acetate buffer (5 mM, pH adjusted to 4.6 with acetic acid) as mobile phase which flowed at 0.45 mL/min. The total run time was 6 min for each sample. Detection through mass spectrometric technique was operated by selected reaction monitoring (SRM) in negative electrospray ionization mode. The method was linear within the range of 10.0–15000 ng/mL for plasma and 10.0–600 ng/mL for brain tissue homogenate with the lower limit of quantification of 10.0 ng/ml. The plasma or tissue homogenate was only required 20 μL. The intra- and inter-day precision were less than 13.8 %, and the RE was between –7.4 % and 3.4 %. The method was applied to investigate the plasma pharmacokinetics and brain distribution of bexarotene in mice after being intragastrically administered with bexarotene at the dosage of 100 mg/kg. The results showed that both brain and plasma concentrations of bexarotene peaked at 1.0 h. Bexarotene was rapidly eliminated with a half-life of 2.0 h. [ABSTRACT FROM AUTHOR]

Oxaliplatin (OXP), a third-generation platinum-based chemotherapy drug, was often indirectly analyzed via total platinum by an ICP-MS because it was difficult to directly quantify using an LC-MS/MS method, due to its instability, bad column separability and severe MS signal inhibition. Here, we developed and validated a specific, sensitive and reproducible LC-MS/MS method for the quantification of OXP itself in rat plasma and tongue tissue on a SCIEX 4000 QTRAP® MS/MS system equipped with a Phenomenex Lux 5u Cellulose-1 column (250 × 4.6 mm, 5 μm). This method was validated at the lower limit of detection (LOD) and the lower limit of quantitation (LLOQ) of 5 ng/mL and 10 ng/mL, with linearity of 10–5000 ng/mL (r2 > 0.99) and 10–2500 ng/mL (r2 > 0.99), in rat plasma and tongue homogenates, respectively. The intra- and inter-day precision (CV%) and accuracy (RE%) were within 15% for LLOQ, low-, medium- and high-quality control samples. The mean extraction recoveries were around 50% and 80% for plasma and tongue homogenates, respectively. This assay was successfully applied to pharmacokinetics study following intravenous administration of OXP, as well as tongue tissue distribution after 1 h and 4 h of a novel oral mucosal patch application. [ABSTRACT FROM AUTHOR]

Abstract: This paper reports the development and validation of an assay for ifenprodil based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) and its application to a pharmacokinetic study involving single and multiple intravenous infusions to healthy Chinese volunteers. After sample preparation of plasma by liquid–liquid extraction with ethyl acetate, the analyte and internal standard, urapidil, were separated by reversed phase chromatography in a run time of 4min and detected by positive ion electrospray ionization followed by multiple reaction monitoring of the precursor-to-product ion transitions at m/z 326.2→308.1 for ifenprodil and m/z 388.4→205.3 for IS. The assay was linear in the concentration range 0.2–50.0ng/mL with recovery >76.4%. In the pharmacokinetic study of single intravenous infusions of 5, 10 and 15mg ifenprodil, peak plasma concentrations and areas under the plasma concentration–time curve were both linearly related to dose. In the pharmacokinetic study of multiple once daily intravenous infusions of 10mg ifenprodil for 7 days, pharmacokinetic parameters were similar to those after the single dose showing that ifenprodil does not accumulate on repeated administration. [Copyright &y& Elsevier]

Abstract: A quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay for the mutated BRAF inhibitor dabrafenib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing PLX4720 as internal standard. The extract was directly injected into the reversed-phase chromatographic system after dilution with water. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 2–2000ng/ml calibration range with r 2 =0.993±0.002 for linear regression with quadratic weighting (n =5). Within day precisions (n =6) were 3.3–5.2%, between day (3 days; n =18) precisions 4.7–8.2%. Accuracies were between 95–104% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully used to determine drug pharmacokinetics in mice. [Copyright &y& Elsevier]

The article presents a study that developed and validated a simple and sensitive analytical method for determining metoclopramide (MCP) concentration in plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate pharmacokinetics after an intravenous MCP administration at a clinical dose to cattle. Details related to the reagent and chemicals used in the study are presented. It also discusses the preparation of calibration standards and quality control samples.

Determination of atorvastatin in human plasma using LC-MS/MS has been widely accepted as an effective method owing to its practical applicability in routine drug analysis in pharmacokinetic and pharmacogenetic study. Nevertheless, the method sensitivity can be compromised when limited sample volume is available. This problem is typically encountered in pharmacokinetics studies for which only a small volume of plasma sample can be obtained. Drug extraction from human plasma was performed with the aid of reversed-phase C18 solid phase micro-extraction. The detection was accomplished by LC-MS/MS. The need of plasma volume of less than 250 ul for each sample made it possible to decrease the sample preparation time. The method was successfully validated and proved appropriate for the analysis of atorvastatin in human plasma and can be applied to pharmacokinetics and pharmacogenetics study. [ABSTRACT FROM AUTHOR]

A simple and rapid liquid chromatography-tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantitation of indirubin-5-nitro-3′-monoxime (5-NIO) in rat plasma using 5-chloro-5′-chloro-indirubinoxime as the internal standard. The sample preparation involved protein precipitation using acetonitrile. Separation was achieved on an Xterra column C18 (2.1 mm × 50 mm, i.d. 5 µm) using a mixture of 0.1% formic acid-water/acetonitrile (18:82, v/v) as the mobile phase at a flow rate of 300 µL/min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization and multiple reaction monitoring (MRM) in negative ion mode. The total chromatographic run time was 2.5 min, and the calibration curves of 5-NIO were linear (correlation coefficient range: 0.9989–0.9999) over the concentration range of 0.2–2000 ng/mL in rat plasma. Also, the detection limit of the method was 0.05 ng/mL and it showed good accuracy and precision within the concentration range of 0.45–376 ng/mL. This validated method was successfully applied to estimating the pharmacokinetic parameters of 5-NIO following intravenous administration of 5-NIO to rats. [ABSTRACT FROM AUTHOR]

Abstract: A liquid chromatography–electrospray tandem mass spectrometry method was developed and validated to quantitate celecoxib in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography on a C18 column (55mm×2mm, 3μm), the mobile phase consisted of methanol – 10mM ammonium acetate (75:25, v/v). Quantification was performed by mass spectrometry in the multiple reaction monitoring mode with negative electrospray ionization at m/z 380→316 and 384→320 for celecoxib and the internal standard celecoxib-D4, respectively. The lower limit of quantitation was 7.0ng/ml using 0.1ml of plasma and linearity was demonstrated up to 1800ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 4% and inaccuracy did not exceed 6% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study. [Copyright &y& Elsevier]

Abstract: A highly selective, sensitive, and robust liquid chromatography–tandem mass spectrometric method for the determination of 5-thio-d-glucose concentrations in rat and human plasma was developed and validated. The sample preparation procedure involved protein precipitation and solid phase extraction, which efficiently removed sources of interference present in the plasma. Chromatographic separation was obtained using an NH2-column with distilled water and acetonitrile as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in negative ion mode. The selected reaction monitoring (SRM) transitions for 5-thio-d-glucose and an internal standard (5-thio-d-glucose-13C6) were m/z 195→ m/z 105 and m/z 201→ m/z 108, respectively. The correlation coefficients of the calibration curves ranged from 0.9997 to 0.9999 over a concentration range from 10 to 3000ng/mL plasma. The validated method was successfully applied to a pharmacokinetic study in rats. [Copyright &y& Elsevier]

A new simple, sensitive and LC-MS/MS method for quantification of spironolactone and its metabolite, canrenone, in human plasma is proposed. The analytes were analysed on a C18 column at 48 0°C, by using a mobile phase of 58 % methanol, 42 % 10 mmol dm ammonim acetate in water and a flow rate of 1 mL/min. The detection of both analytes in plasma was performed as follows: ESI+, EIC (m/z 169;187;283;305) from m/z 341, after protein precipitation with methanol. Calibration curves were generated over the ranges 2.77-184.50 ng/mL for spironolactone and 2.69-179.20 ng/mL for canrenone by using a weighted (1/y) linear regression. The absolute values of within- and between-run precision and accuracy for both analytes ranged between 3.1 and 13.9 %, and the mean recovery was 99.7 %. The analytes demonstrated good stability in various conditions. The validated method has been applied to a bioequivalence study of 50 mg spironolactone tablets on healthy volunteers. [ABSTRACT FROM AUTHOR]

DA-7010 is a new candidate for an antibacterial agent that targets Gram-negative pathogens by acting as a leucyl-tRNA synthetase inhibitor. In this study, a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to determine DA-7010 levels in the plasma from mice, rats, and dogs. Plasma samples were mixed with methanol for protein precipitation. Chromatographic separation was carried out using a reversed-phase C18 column (Agilent Poroshell 120, 50 × 3.0 mm, 2.7 μm). An isocratic elution of the mobile phase consisting of 5 mM formic acid in water and acetonitrile at a ratio of 84:16 (v/v) was applied at a flow rate of 0.3 mL/min. The total chromatographic run time was 3.5 min. Multiple reaction monitoring (MRM) mode was used for mass spectrometric detection using an Agilent 6460 triple quadrupole coupled with an electrospray ionization (ESI) source operated in positive-ion mode. The MRM transitions analyzed were m/z 220.1→162.1 for DA-7010 and m/z 206.1→170.1 for the internal standard (structural analogue of DA-7010). Calibration curves were constructed in the range of 10–10,000 ng/mL. The intra- and interday precision and accuracy were within 11.3%, excluding those for the lower limit of quantification (LLOQ) samples, which were within 17.1%. The developed LC-MS/MS method was successfully validated and applied in preclinical pharmacokinetic studies of DA-7010 in mice, rats, and dogs following single oral administrations. The oral absorption of DA-7010 was rapid, and the systemic exposure was approximately four times higher in the dogs than in the mice or rats. [ABSTRACT FROM AUTHOR]

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

Rumex acetosa (R. acetosa) has been used in folk remedies for gastrointestinal disorders and cutaneous diseases. Rumex species, in particular, contain abundant anthraquinones. Anthraquinone glycosides and aglycones show different bioactive effects. However, information on the pharmacokinetics of anthraquinone glycosides is limited, and methods to quantify anthraquinone glycosides in plasma are rarely available. A simple and sensitive liquid chromatography-tandem mass spectrometric bioanalytical method for the simultaneous determination of both anthraquinone glycosides and their aglycones, including emodin, emodin-8-O-β-d-glucoside, chrysophanol, chrysophanol-8-O-β-d-glucoside, physcion, and physcion-8-O-β-d-glucoside , in a low volume of rat plasma (20 µL) was established. A simple and rapid sample preparation was employed using methanol as a precipitating agent with appropriate sensitivity. Chromatographic separation was performed on HPLC by using a biphenyl column with a gradient elution using 2 mM ammonium formate (pH 6) in water and 2 mM ammonium formate (pH 6) in methanol within a run time of 13 min. The anthraquinones were detected on triple-quadrupole mass spectrometer in negative ionization mode using multiple-reaction monitoring. The method was validated in terms of selectivity, linearity, accuracy, precision, recovery, and stability. The values of the lower limit of quantitation of anthraquinones were 1–20 ng/mL. The intra-batch and inter-batch accuracies were 96.7–111.9% and the precision was within the acceptable limits. The method was applied to a pharmacokinetic study after oral administration of R. acetosa 70% ethanol extract to rats at a dose of 2 g/kg. [ABSTRACT FROM AUTHOR]