LC‐MS/MS determination and pharmacokinetic study of lacidipine in human plasma

A robust, specific and fully validated LC‐MS/MS method as per general practices of industry has been developed for estimation of lacidipine (LAC) with 100 μL of human plasma using lacidipine‐¹³C8 as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode. A simple liquid–liquid extraction process was used to extract LAC and IS from human plasma. The total run time was 3.0 min and the elution of LAC and IS occurred at 1.96 and 1.97 min; this was achieved with a mobile phase consisting of 5 mm ammonium acetate buffer–acetontrile (15:85 v/v) at a flow rate of 0.60 mL/min on a Zorbax SB C₁₈ (50 × 4.6 mm, 5 µm) column. A linear response function was established for the range of concentrations 50–15,000 pg/mL (r > 0.998) for LAC. The current developed method has negligible matrix effect and is free from unwanted adducts and clusters which are formed owing to system such as solvent or mobile phase. The developed assay method was applied to an oral pharmacokinetic study in humans and successfully characterized the pharmacokinetic data up to 72 h. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of HPLC‐MS/MS method for the determination of lixivaptan in mouse plasma and its application in a pharmacokinetic study

The aim of the present study was to develop a simple, sensitive and accurate liquid chromatography–electrospray ionization tandem mass spectrometry (ESI‐MS/MS) method for the determination of lixivaptan (LIX) in mouse plasma using vildagliptin as the internal standard (IS). A precipitation procedure was used for the extraction of LIX and vildagliptin from mouse plasma. Chromatographic separation of LIX was achieved using a C₁₈ analytical column (50 × 2.1 mm, 1.8 μm) at 25°C. The mobile phase comprised acetonitrile and ammonium formate (10 mm , pH 3.1; 40:60, v /v) pumped at a flow rate of 0.3 mL min⁻¹. A tandem mass spectrometer with an electrospray ionization source was used to perform the assay. Quantification of LIX at m/z 290 → 137 and IS at 154 → 97 was attained through multiple reaction monitoring. The investigated method was authenticated following the bio‐analytical method of validation guidelines of the US Food and Drug Administration. The developed method showed a good linearity over the concentration range from 5 to 500 ng mL⁻¹, and the calibration curve was linear (r = 0.9998). The mean recovery of LIX from mouse plasma was 99.2 ± 0.68%. All validation parameters for LIX were within the levels required for acceptance. The proposed method was effectively used for a pharmacokinetic study of LIX in mouse plasma.     

Quantification of heteroclitin D in rat plasma: validation of an LC/MS/MS method and its application in a preclinical pharmacokinetic study

A rapid, sensitive and specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of heteroclitin D in rat plasma after using gambogic acid as internal standard (IS). Chromatographic separation was done on a Thermo Hypersil GOLD column (30 × 2.1 mm, 3 µm) using a mobile phase consisting of methanol–water–formic acid (80:20:0.1, v/v/v). The mass spectrometer worked with positive electrospray ionization in multiple reaction monitoring mode, using target ions at [M + H]⁺ m/z 483.3 for heteroclitin D and [M + H]⁺ m/z 629.3 for the IS. The standard curve was linear (R² ≥0.995) over the concentration range 9.98–2080 ng/mL and had good back‐calculated accuracy and precision. The intra‐ and interday precision and accuracy determined on three quality control samples (29.94, 166.4 and 1872 ng/mL) were ≤12.8 and –8.9–3.6%, respectively. The extraction recovery was ≥88.2% and the lower limit of quantification was 9.98 ng/mL. The method was successfully applied to evaluate pharmacokinetics of heteroclitin D in Sprague–Dawley rats following a single intravenous bolus injection of 2.0 mg/kg heteroclitin. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a targeted method for quantification of gypenoside XLIX in rat plasma,using SPE and LC–MS/MS

A sensitive, selective and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantification of gypenoside XLIX, a naturally occurring gypenoside of Gynostemma pentaphyllum in rat plasma and then validated according to the US Food and Drug Administration's Guidance for Industry: Bioanalytical Method Validation . Plasma samples were prepared by a simple solid‐phase extraction. Separation was performed on a Waters XBridgeTM BEH C₁₈ chromatography column (4.6 × 50 mm, 2.5 μm) using a mobile phase of acetonitrile and water (62.5:37.5, v /v). Gypenoside XLIX and the internal standard gypenoside A were detected in the negative ion mode using selection reaction monitoring of the transitions at m/z 1045.6 → 913.5 and 897.5 → 765.4, respectively. The calibration curve was linear (R ² > 0.990) over a concentration range of 10–7500 ng/mL with the lower quantification limit of 10 ng/mL. Intra‐ and inter‐day precision was within 8.6% and accuracy was ≤10.2%. Stability results proved that gypenoside XLIX and the IS remained stable throughout the analytical procedure. The validated LC–MS/MS method was then applied to analyze the pharmacokinetics of gypenoside XLIX after intravenous administration to rats (1.0, 2.0 and 4.0 mg/kg).     

Development and validation of LC‐MS/MS method for simultaneous determination of sofosbuvir and daclatasvir in human Plasma: Application to pharmacokinetic study

A simple and highly sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) bioanalytical method was developed and fully validated for the first time for the simultaneous determination of newly discovered antiviral drugs, namely sofosbuvir (SOF) and daclatasvir (DAC) in human plasma. Tadalafil (TAD) was used as internal standard (IS). SOF, DAC and TAD (IS) were extracted from plasma using liquid–liquid extraction technique with methyl tert‐butyl ether. The chromatographic separation was carried out using ZorbaxSB‐C₁₈ column (4.6 × 50 mm,5 μm) and 5 mm ammonium formate buffer (pH 3.5)–acetonitrile (50:50, v/v) as mobile phase in an isocratic elution mode pumped at a flow rate 0.7 mL min⁻¹. The quantitation was performed on API4500 triple quadrupole tandem mass spectrometer with positive electrospray ionization interface in multiple reaction monitoring mode. Validation was applied according to US Food and Drug Administration guidelines for bio‐analytical methodswith respect to linearity, precision, accuracy, selectivity, carry‐over, stability and dilution integrity. Linearity was obtained over concentration ranges of 0.3–3000 and 3–3000 ng mL⁻¹ for SOF and DAC, respectively, by applying a weighted least‐squares linear regression method (1/x²). The proposed method could be applied successfully in bioequivalence and/or clinical studies for therapeutic drug monitoring of patients undergoing dual combination therapy as the latter combination proved more efficacious and powerful tool for the complete treatment of hepatitis C genotype 3 within 16 weeks. The suggested method has been applied successfully to pharmacokinetic studies with excellent assay ruggedness and reproducibility.     

A sensitive LC‐MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application

An improved, simple and highly sensitive LC‐MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat‐d₇ as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid–liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C₁₈ column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1–6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra‐ and inter‐day precisions (%RSD) were within 1.29–9.19 and 2.85–7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

A quantitative determination of fluorochloridone in rat plasma by UPLC‐MS/MS method: application to a pharmacokinetic study

A precise, high‐throughput and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed for the determination of fluorochloridone (FLC) in rat plasma. The extraction of analytes from plasma samples was carried out by protein precipitation procedure using acetonitrile prior to UPLC‐MS/MS analysis. Verapamil was proved as a proper internal standard (IS) among many candidates. The chromatographic separation based on UPLC was well optimized. Multiple reaction monitoring in positive electrospray ionization was used with the optimized MS transitions at: m/z 312.0 → 292.0 for FLC and m/z 456.4 → 165.2 for IS. This method was well validated with good linear response (r² > 0.998) observed over the investigated range of 3–3000 ng/mL and with satisfactory stability. This method was also characterized with adequate intra‐ and inter‐day precision and accuracy (within 12%) in the quality control samples, and with high selectivity and less matrix effect observed. Total running time was only 1.5 min. This method has been successfully applied to a pilot FLC pharmacokinetic study after oral administration. Copyright © 2016 John Wiley & Sons, Ltd.     

An LC–MS/MS method for rapid and sensitive high‐throughput simultaneous determination of various protein kinase inhibitors in human plasma

A reliable, high‐throughput and sensitive LC–MS/MS procedure was developed and validated for the determination of five tyrosine kinase inhibitors in human plasma. Following their extraction from human plasma, samples were eluted on a RP Luna®‐PFP 100 Å column using a mobile phase system composed of acetonitrile and 0.01 m ammonium formate in water (pH ~4.1) with a ratio of (50:50, v /v) flowing at 0.3 mL min⁻¹. The mass spectrometer was operating with electrospray ionization in the positive ion multiple reaction monitoring mode. The proposed methodology resulted in linear calibration plots with correlation coefficients values of r ² = 0.9995–0.9999 from concentration ranges of 2.5–100 ng mL⁻¹ for imatinib, 5.0–100 ng mL⁻¹ for sorafenib, tofacitinib and afatinib, and 1.0–100 ng mL⁻¹ for cabozantinib. The procedure was validated in terms of its specificity, limit of detection (0.32–1.71 ng mL⁻¹), lower limit of quantification (0.97–5.07 ng mL⁻¹), intra‐ and inter assay accuracy (−3.83 to +2.40%) and precision (<3.37%), matrix effect and recovery and stability. Our results demonstrated that the proposed method is highly reliable for routine quantification of the investigated tyrosine kinase inhibitors in human plasma and can be efficiently applied in the rapid and sensitive analysis of their clinical samples.     

Development and validation of UHPLC‐MS/MS assay for rapid determination of a carvone Schiff base of isoniazid (CSB‐INH) in rat plasma: application to pharmacokinetic study

In this study, a fast UHPLC‐MS/MS method was developed and validated for the determination of a novel potent carvone Schiff base of isoniazid (CSB‐INH) in rat plasma using carbamazepine as an internal standard (IS). After a single‐step protein precipitation by acetonitrile, CSB‐INH and IS were separated on an Acquity BEH^TM C₁₈ column (50 × 2.1 mm, 1.7 µm) under an isocratic mobile phase, consisting of acetonitrile: 10 mM ammonium acetate (95:5, v/v), at a flow rate of 0.3 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring mode by using positive electrospray ionization source. The precursor to product ion transitions were set at m/z 270.08 → 79.93 for CSB‐INH and m/z 237.00 → 178.97 for IS. The proposed method was validated in compliance with US Food and Drug Administration and European Medicines Agency guidelines for bioanalytical method validation. The method was found to be linear in the range of 0.35–2500 ng/mL (r² ≥ 0.997) with a lower limit of quantification of 0.35 ng/mL. The intra‐ and inter‐day precision values were ≤12.0% whereas accuracy values ranged from 92.3 to 108.7%. In addition, other validation results were within the acceptance criteria and the method was successfully applied in a pharmacokinetic study of CSB‐INH in rats. Copyright © 2014 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.     

Quantitative bioanalysis of bavachalcone in rat plasma by LC–MS/MS and its application in a pharmacokinetics study

This study aims to develop and validate a simple and sensitive liquid chromatography with tandem mass spectrometry (LC–MS/MS) method for investigating the pharmacokinetic characteristics of bavachalcone. Liquid–liquid extraction was used to prepare plasma sample. Chromatographic separation of bavachalcone and IS was achieved using a Venusil ASB C₁₈ (2.1 × 50 mm, 5 μm) column with a mobile phase of methanol (A)–water (B) (70:30, v /v). The detection and quantification of analytes was performed in selected‐reaction monitoring mode using precursor → product ion combinations of m/z 323.1 → 203.2 for bavachalcone, and m/z 373.0 → 179.0 for IS. Linear calibration plots were achieved in the range of 1–1000 ng/mL for bavachalcone (r ² > 0.99) in rat plasma. The recovery of bavachalcone ranged from 84.1 to 87.0%. The method was precise, accurate and reliable. It was fully validated and successfully applied to pharmacokinetic study of bavachalcone.     

LC–ESI–MS/MS determination of copanlisib,a novel PI3K inhibitor,in mouse plasma and its application to a pharmacokinetic study in mice

A sensitive, selective and rapid LC–ESI–MS/MS method has been developed and validated for the quantification of copanlisib in mouse plasma using enasidenib as an internal standard (IS) as per regulatory guideline. Copanlisib and the IS were extracted from mouse plasma using ethyl acetate as an extraction solvent and chromatographed using an isocratic mobile phase (0.2% formic acid–acetonitrile; 25:75, v/v) on a HyPURITY C₁₈ column. Copanlisib and the IS eluted at ~0.95 and 2.00 min, respectively. The MS/MS ion transitions monitored were m/z 481.1 → 360.1 and m/z 474.0 → 456.0 for copanlisib and the IS, respectively. The calibration range was 3.59–3588 ng/mL. The intra‐ and inter‐batch accuracy and precision (RE and RSD) across quality controls met the acceptance criteria. Stability studies showed that copanlisib was stable in mouse plasma for one month. This novel method has been applied to a pharmacokinetic study in mice.     

Application of a UPLC‐MS/MS method for the analysis of alosetron in human plasma to support a bioequivalence study in healthy males and females

A simple, rapid and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for the determination of alosetron (ALO) in human plasma. The assay method involved solid‐phase extraction of ALO and ALO 13C‐d3 as internal standard (IS) on a LichroSep DVB‐HL (30 mg, 1 cm³) cartridge. The chromatography was performed on an Acquity UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 µm) column using acetonitrile and 2.0 mm ammonium formate, pH 3.0 adjusted with 0.1% formic acid (80:20, v/v) as the mobile phase in an isocratic mode. For quantitative analysis, the multiple reaction monitoring transitions studied were m/z 295.1/201.0 for ALO and m/z 299.1/205.1 for IS in the positive ionization mode. The method was validated over a concentration range of 0.01–10.0 ng/mL for ALO. Post‐column infusion experiment showed no positive or negative peaks in the elution range of the analyte and IS after injection of extracted blank plasma. The extent of ion‐suppression/enhancement, expressed as IS‐normalized matrix factor, varied from 0.96 to 1.04. The assay recovery was within 97–103% for ALO and IS. The method was successfully applied to support a bioequivalence study of 1.0 mg alosetron tablets in 28 healthy Indian male and female subjects. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of an LC‐ESI‐MS/MS method for the quantitation of hemslecin A in rhesus monkey plasma and its application in pharmacokinetics

In this study, a new LC‐ESI‐MS/MS‐based method was validated for the quantitation of hemslecin A in rhesus monkey plasma using otophylloside A as internal standard (IS). Hemslecin A and the IS were extracted from rhesus monkey plasma using liquid–liquid extraction as the sample clean‐up procedure, and were subjected to chromatography on a Phenomenex Luna CN column (150 × 2.0 mm, 3.0 µm) with the mobile phase consisting of methanol and 0.02 mol/mL ammonium acetate (55:45, v/v) at a flow rate of 0.2 mL/min. Detection was performed on an Agilent G6410B tandem mass spectrometer by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 580.5 [M + NH₄]⁺ → 503.4 and m/z 518.2 [M + NH₄]⁺ → 345.0 for hemslecin A and IS, respectively. The assay was linear over the concentration range of 0.5–200 ng/mL and was successfully applied to a pharmacokinetic study in rhesus monkeys. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid and sensitive determination of hypoxic radiosensitizer agent nimorazole in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

A highly sensitive, accurate and robust LC‐MS/MS method was developed and validated for determination of nimorazole (NMZ) in rat plasma using metronidazole (MNZ) as internal standard (IS). The analyte and IS were extracted from plasma by precipitating protein with acetonitrile and were chromatographed using an Agilent Poroshell 120, EC‐C₁₈ column. The mobile phase was composed of a mixture of acetonitrile and 0.1 % formic acid (85:15 v/v). The total run time was 1.5 min and injection volume was 5 μL. Multiple reaction monitoring mode using the transitions of m/z 227.1 → m/z 114.0 for MNZ and m/z 172.10 → m/z 128.1 for IS were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The calibration curve was linear in the range of 0.25–200 ng/mL (r² > 0.9996) and the lower limit of quantification was 0.25 ng/mL in the rat plasma samples. Recoveries of NMZ ranged between 88.05 and 95.25%. The precision (intra‐day and inter‐day) and accuracy of the quality control samples were 1.25–8.20% and ?2.50–3.10, respectively. The analyte and IS were found to be stable during all sample storage and analysis procedures. The LC‐MS/MS method described here was validated and successfully applied to pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of LC–MS/MS method for amlexanox in rat plasma and its application in preclinical pharmacokinetics

Amlexanox, an anti-inflammatory and anti-allergic agent, has been widely used clinically for the treatment of canker sores, asthma, and allergic rhinitis. Recently, amlexanox has received considerable attention in curing nonalcoholic fatty liver diseases and hepatitis virus infection. Herein, we first established a sensitive high-performance liquid chromatography-tandem mass spectrum (LC–MS/MS) method for the determination of amlexanox in rat plasma. Propranolol was used as the internal standard (IS). Using a simple protein precipitation method, the amlexanox and IS were separated with Capcell Pak C18 column (2.0 × 50 mm, 5 μm) and eluted with water and acetonitrile each containing 0.1% formic acid using gradient elution condition at a flow rate of 0.4 mL·min⁻¹. Amlexanox and IS were detected by a triple quadrupole mass in multiple reactive monitoring (MRM) under the transitions of m/z 299.2 → 281.2 and m/z 259.9 → 116.1 with positive electrospray ionization, respectively. The calibration curves of amlexanox were established with the range of 50 to 2000 ng·mL⁻¹ (r² > 0.99). The validation method consisted of selectivity, accuracy, precision, carryover effect, matrix effect, recovery, dilution effect, and stability. The fully validated method was successfully applied to the pharmacokinetic study of amlexanox in Wistar rats.     

Validation of an enantioselective LC–MS/MS method to quantify enantiomers of (±)‐OTX015 in mice plasma: Lack of in vivo inversion of (−)‐OTX015 to its antipode

A highly sensitive, specific and enantioselective assay has been validated for the quantitation of OTX015 enantiomers [(+)‐OTX015 and (−)‐OTX015] in mice plasma on LC–MS/MS‐electrospray ionization as per regulatory guidelines. Protein precipitation was used to extract (±)‐OTX015 enantiomers and internal standard (IS) from mice plasma. The active [(−)‐OTX015] and inactive [(+)‐OTX015] enantiomers were resolved on a Chiralpak‐IA column using an isocratic mobile phase (0.2% ammonia/acetonitrile 20 : 80, v /v) at a flow rate of 1.2 mL/min. The total run time was 6.0 min. (+)‐OTX015, (−)‐OTX015 and IS eluted at 3.34, 4.08 and 4.77 min, respectively. The MS/MS ion transitions monitored were m/z 492 → 383 for OTX015 and m/z 457 → 401 for IS. The standard curves for OTX015 enantiomers were linear (r ² > 0.998) in the concentration range 1.03–1030 ng/mL. The inter‐ and intraday precisions were in the range 2.20–13.3 and 8.03–12.1% and 3.80–14.4 and 8.97–13.6% for (+)‐OTX015 and (−)‐OTX015, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method has been applied to the study of stereoselective oral pharmacokinetics of (−)‐OTX015 and unequivocally demonstrated that (−)‐OTX015 does not undergo chiral inversion to its antipode in vivo in mice.     

Bioanalytical method development and validation of moxidectin in plasma by LC–MS/MS: Application to in vitro metabolism

Moxidectin (MOX) has recently been approved by the US Food and Drug Administration for the treatment of river blindness in select populations. It is also being evaluated as an alternative for the use of ivermectin, widespread resistance to which is becoming a global health issue. Moreover, MOX is becoming increasingly used as a prophylactic antiparasitic in the cattle industry. In this study, we developed and validated an LC–MS/MS method of MOX in human, monkey and mouse plasma. The separation was achieved on an ACE C₁₈ (50 × 3.0 mm, 3 μm) column with isocratic elution using 0.1% acetic acid and methanol–acetonitrile (1:1, v/v) as mobile phase. MOX was quantitated using MS/MS with an electrospray ionization source operating in negative multiple reaction monitoring mode. The multiple reaction monitoring precursor ion → product ion transitions for MOX and abamectin (IS) were m/z 638.40 → 236.30 and m/z 871.50 → 565.35 respectively. The MS/MS response was linear over the concentration range 0.1–1000 ng/mL in plasma with a correlation coefficient (r²) of 0.997 or better. The within‐ and between‐day precision (relative standard deviation, RSD) and accuracy were within the acceptable limits per US Food and Drug Administration guidelines. The method was successfully applied to an in vitro metabolic stability study of MOX.     

A highly sensitive LC‐MS/MS method for the determination of S‐citalopram in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive, rapid assay method has been developed and validated for the estimation of S‐citalopram (S‐CPM) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of S‐CPM and phenacetin (internal standard, IS) from rat plasma with t‐butyl methyl ether. Chromatographic separation was operated with 0.2% formic acid:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Symmetry Shield RP₁₈ column with a total run time of 3.0 min. The MS/MS ion transitions monitored were 325.26 → 109.10 for S‐CPM and 180.10 → 110.10 for IS. Method validation and pre‐clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.5 ng/mL and the linearity was observed from 0.5 to 5000 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.14–5.56 and 0.25–12.3%, respectively. This novel method has been applied to a pharmacokinetic study and to estimate brain‐to‐plasma ratio of S‐CPM in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

A simple LC–MS/MS method facilitated by salting‐out assisted liquid–liquid extraction to simultaneously determine trans‐resveratrol and its glucuronide and sulfate conjugates in rat plasma and its application to pharmacokinetic assay

A simple LC–MS/MS method facilitated by salting‐out assisted liquid–liquid extraction (SALLE) was applied to simultaneously investigate the pharmacokinetics of trans‐ resveratrol (Res) and its major glucuronide and sulfate conjugates in rat plasma. Acetonitrile–methanol (80:20, v /v) and ammonium acetate (10 mol L⁻¹) were used as extractant and salting‐out reagent to locate the target analytes in the supernatant after the aqueous and organic phase stratification, then the analytes were determined via gradient elution by LC–MS/MS in negative mode in a single run. The analytical method was validated with good selectivity, acceptable accuracy (>85%) and low variation of precision (<15%). SALLE showed better extraction efficiency of target glucuronide and sulfate conjugates (>80%). The method was successfully applied to determine Res and its four conjugated metabolites in rat after Res administration (intragastric, 50 mg kg⁻¹; intravenous, 10 mg kg⁻¹). The systemic exposures to Res conjugates were much higher than those to Res (AUC_0–t , i.v., 7.43 μm h; p.o., 8.31 μm h); Res‐3‐O‐β ‐d ‐glucuronide was the major metabolite (AUC_0–t , i.v., 66.1 μm h; p.o., 333.4 μm h). The bioavailability of Res was estimated to be ~22.4%. The reproducible SALLE method simplified the sample preparation, drastically improved the accuracy of the concomitant assay and gave full consideration of extraction recovery to each target analyte in bio‐samples.