LC–MS/MS method for simultaneous determination of flavonoids and physalins in rat plasma: Application to pharmacokinetic study after oral administration of Physalis alkekengi var. franchetii (Chinese lantern) extract

A rapid and sensitive liquid chromatography with tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous determination of luteolin, luteolin‐7‐O ‐β ‐D‐glucopyranoside, physalin A, physalin D and physalin L in rat plasma. Scutellarein and dexamethasone were used as the internal standards (IS). Plasma samples were prepared by liquid‐liquid extraction with ethyl acetate. The five constituents were separated on an Acquity UPLC BEH C₁₈ column (100 mm × 2.1 mm, 1.7 μm). A gradient elution procedure was used with acetonitrile (A)‐0.1% aqueous formic acid (B). Mass spectrometric detection was performed in negative ion multiple reaction monitoring mode with an electrospray ionization (ESI) source. This method showed good linearity (r ² > 0.997) over a concentration range of 2.0–500 ng/mL with a lower limit of quantification of 2.0 ng/mL for all five compounds. The inter‐ and intra‐day accuracy ranged from 91.7 to 104%, and precisions (RSD) were <6.46% for all analytes. The extraction recoveries of all analytes were >85%. This validated method was successfully applied for the first time to the pharmacokinetic study of five ingredients after oral administration of 70% ethanol extract of Chinese lantern in rats.     

Anesthetic constituents of Zanthoxylum bungeanum Maxim.: A pharmacokinetic study

A sensitive and selective ultra high performance liquid chromatography with tandem mass spectrometry method was established and validated for the simultaneous determination of hydroxy‐α‐sanshool, hydroxy‐β‐sanshool, and hydroxy‐γ‐sanshool in rat plasma after the subcutaneous and intravenous administration of an extract of the pericarp of Zanthoxylum bungeanum Maxim. Piperine was used as the internal standard. The analytes were extracted from rat plasma by liquid‐liquid extraction with ethyl acetate and separated on a Thermo Hypersil GOLD C₁₈ column (2.1 mm × 50 mm, 1.9 μm) with a gradient elution system at a flow rate of 0.4 mL/min. The mobile phase consisted of acetonitrile/0.05% formic acid in water and the total analysis time was 4 min. Positive electrospray ionization was performed using multiple reaction monitoring mode for the analytes. The calibration curves of the three analytes were linear over the tested concentration range. The intra‐ and interday precision was no more than 13.6%. Extraction recovery, matrix effect, and stability were satisfactory in rat plasma. The developed and validated method was suitable for the quantification of hydroxy‐α‐sanshool, hydroxy‐β‐sanshool, and hydroxy‐γ‐sanshool and successfully applied to a pharmacokinetic study of these analytes after subcutaneous and intravenous administration to rats.     

Simultaneous determination of four secoiridoid and iridoid glycosides in rat plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application to a comparative pharmacokinetic study

A simple, reliable and rapid ultra‐performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of four secoiridoid (gentiopicroside, swertiamarin, sweroside) and iridoid glycosides (loganic acid), the bio‐active ingredients in rat plasma. After liquid–liquid extraction, chromatographic separation was accomplished on a Shim‐pack XR‐ODS column with a mobile phase consisting of methanol and 0.1% formic acid in water. A triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source was used as detector operating both in positive and negative ionization mode and operated by multiple‐reaction monitoring scanning. The lower limits of quantitation were 0.25–30 ng/mL for all the analytes. Both intra‐day and inter‐day precision and accuracy of analytes were well within acceptance criteria (±15%). The mean extraction recoveries of analytes and internal standard (amygdalin) from rat plasma were all >71.4%. The validated method was successfully applied to a comparative pharmacokinetic study of four analytes in rat plasma between normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan and Gentiana macrophylla extract, respectively. Results showed significant differences in pharmacokinetic properties of the analytes among the different groups. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of ginsenosides and bufadienolides in rat plasma after the oral administration of Shexiang Baoxin Pill for pharmacokinetic study by liquid chromatography tandem mass spectrometry following solid‐phase extraction

A sensitive and reliable bioanalytical method was established for quantitati\ve and pharmacokinetic investigation of nine ginsenosides and seven bufadienolides in rat plasma after the oral administration of Shexiang Baoxin Pill by liquid chromatography–electrospray ionization tandem mass spectrometry, using tinidazole and digoxin as internal standards (ISTDs). All of the analytes and ISTDs obtained satisfactory recoveries by solid‐phase extraction using an Oasis HLB μElution Plate, which was eluted with methanol and ethyl acetate successively, and chromatographic separation was achieved on a Shim‐pack XR‐ODSIIcolumn (75 × 2.0 mm, 2.2 μm) with gradient elution using a mixture of acetonitrile–0.1% formic acid solution (v /v) as the mobile phase at a flow rate of 0.3 mL/min. Detection was carried out by a triple‐quadrupole tandem mass spectrometry with positive/negative ion switching multiple reaction monitoring mode. All analytes showed good linearity over a wide concentration range (r ² > 0.99). The lower limit of quantification was in the range 0.625–12.5 ng/mL for bufadienolides and 2–5.5 ng/mL for ginsenosides, and the mean recoveries of all analytes were in the range 78.29–99.35%. The intra‐ and inter‐day precisions (RSD) were in the range 0.08–12.38% with the accuracies between 86.09 and 99.40%. The validated method was then successfully applied to pharmacokinetic study of the above 16 compounds in rat plasma. Pharmacokinetic results indicated that the developed extraction and analytical method could be employed as a rapid, effective technique for pharmacokinetic study of multiple components, especially various polarity that are difficult to extract simultaneously.     

Pharmacokinetic comparison of five tanshinones in normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan or its single herb extract by UPLC‐MS/MS

A fast, sensitive and reliable ultra performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantitation and pharmacokinetic study of five tanshinones (tanshinone I, tanshinone IIA, tanshinone IIB, dihydrotanshinone I, cryptotanshinone), the bio‐active ingredients of Huo Luo Xiao Ling Dan (HLXLD) in rat plasma. After liquid–liquid extraction, chromatographic separation was accomplished on a Shim‐pack XR‐ODS column (75 × 3.0 mm, 2.2 µm particles) and eluted with a mobile phase consisting of acetonitrile–0.05% formic acid aqueous solution (80:20, v/v) at a flow rate of 0.4 mL/min, and the total run time was 7.0 min. The detection was performed on a triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source in positive ionization and multiple reaction monitoring mode. The lower limits of quantification were 0.050–0.400 ng/mL for all the analytes. Linearity, precision and accuracy, the mean extraction recoveries and matrix effects all satisfied criteria for acceptance. This validated method was successfully applied to a comparative pharmacokinetic study of five bio‐active components in rat plasma after oral administration of HLXLD or Salvia miltiorrhiza extract in normal and arthritic rats. The results showed that there were different pharmacokinetic characteristics among different groups. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a UHPLC–MS/MS method for the simultaneous determination of five bioactive flavonoids in rat plasma and comparative pharmacokinetic study after oral administration of Xiaochaihu Tang and three compatibilities

An accurate, rapid, and reliable ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of baicalin, wogonoside, baicalein, wogonin, and oroxylin A in rat plasma. Then, the stability of baicalin and baicalein in the preparation of plasma sample was systematically investigated. The Waters BEH C₁₈ column was used with a gradient mobile phase system of acetonitrile and water containing 0.1% formic acid. The analytes were detected in the multiple reaction monitoring mode with positive electrospray ionization. 100 μL fresh plasma was added with 50 μL antioxidant reagent (1 mol/L HCl containing 0.5% Vitamin C), and liquid–liquid extraction with ethyl acetate was used to extract the analytes from plasma. Lower limits of quantification of baicalin, wogonoside, baicalein, wogonin, and oroxylin A were 21.9, 4.80, 1.20, 0.848, and 0.800 ng/mL, respectively. The mean extract recoveries of five flavonoids were 69.1∼89.2%, and the precision and accuracy were within the acceptable limits. This method was further successfully applied to the comparative pharmacokinetic study of these five flavonoids in rats after oral administration of Xiaochaihutang and three compatibilities. The obtained results may be helpful to reveal the mechanism of Xiaochaihutang formula compatibility.     

Simultaneous determination and pharmacokinetics study of four quinones in rat plasma by ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry after the oral administration of Qianzhi capsules

A sensitive, specific, and accurate ultra high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous quantification of purpurin, munjistin, mollugin, and alizarin from Qianzhi capsules in rat plasma. Chromatographic separation was performed on an Agilent Eclipse Plus C₁₈ RRHD column with a mobile phase consisting of methanol and 5 mM ammonium acetate/water with gradient elution. The analytes were quantified on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode and switching the electrospray ion source polarity with positive electrospray ionization in a single run. Samples were pretreated by liquid–liquid extraction with cyclohexane. The intra‐ and interday precision and accuracy of the assay were within acceptable ranges. Matrix effects for all of the analytes were between 90.16 and 100.21%. The average recovery ranged from 75.38 to 88.96%. This method was successfully applied to study the pharmacokinetic parameters of the four compounds in rat plasma after oral administration of Qianzhi capsules. Four quinones could be rapidly absorbed into blood (t_max, 0.80–1.93 h) and eliminated relatively slowly (t_1/2, 8.07–11.97 h). The results might be helpful for guiding the clinical application of Qianzhi capsules in the future.     

Simultaneous determination of cucurbitacin IIa and cucurbitacin IIb of Hemsleya amabilis by HPLC–MS/MS and their pharmacokinetic study in normal and indomethacin‐induced rats

A selective and sensitive HPLC–MS/MS method was developed for the simultaneous determination of cucurbitacin IIa (cuIIa) and cucurbitacin IIb (cuIIb), the major bioactive cucurbitacins of Hemsleya amabilis, in rat plasma using euphadienol as internal standard (IS). After liquid–liquid extraction with dichloromethane, separation was achieved on a Syncronis HPLC C₁₈ column (150 mm × 4.6 mm, 5 μm) using an isocratic mobile phase system consisting of acetonitrile–water (85:15, v/v) at a flow rate of 0.6 mL/min with a split ratio of 1:2. Detection was performed on a TSQ Quantum Ultra mass spectrometer equipped with an positive‐ion electrospray ionization source. The lower limits of quantification (LLOQs) were 0.25 and 0.15 ng/mL for cuIIa and cuIIb, respectively. The intra‐ and inter‐day precision was <11.5% for the LLOQs and each quality control level of the analytes, and accuracy was between ?9.1 and 7.6%. The extraction recoveries of the analytes and IS from rat plasma were all >87.1%. The method was fully validated and applied to compare the pharmacokinetic profiles of the two cucurbitacins in rat plasma after oral administration of H. amabilis extract between normal and indomethacin‐induced rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of sophoraflavanone G and kurarinone in rat plasma by UHPLC–MS/MS and its application to a pharmacokinetic study

This study aimed to develop and validate a simple and sensitive ultra high performance liquid chromatography tandem mass spectrometry method for the simultaneous determination of sophoraflavanone G and kurarinone in rat plasma by using rutin as the internal standard. Then, the developed method was applied to investigate the pharmacokinetics of sophoraflavanone G and kurarinone in rats after dosing the flavonoid extract from Sophora flavescens. Plasma samples were processed using a liquid–liquid extraction procedure with ethyl acetate. The analysis was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring with an electrospray ionization source in negative ionization mode. Quantitative ion transitions of m/z 423.2→161.2, 437.2→161.1, and 609.3→300.3 were monitored for sophoraflavanone G, kurarinone, and rutin, respectively. The calibration curves of the two analytes exhibited good linearity (r²>0.9923) over the range of 0.1–200 ng/mL for sophoraflavanone G and 0.1–1000 ng/mL for kurarinone. Relative standard deviations were less than 13.2% for the intra‐ and inter‐day precisions and no more than 12.6% for the recovery, showing good precision and satisfactory accuracy of the developed method. The validated method was successfully applied to the pharmacokinetic study of sophoraflavanone G and kurarinone after a single intravenous (25 mg/kg) and oral (500 mg/kg) administration of the flavonoid extract from S. flavescens, and the absolute bioavailability for sophoraflavanone G and kurarinone was about 36 and 17%, respectively.     

Ultra‐high performance liquid chromatography with tandem mass spectrometry method for the simultaneous quantitation of five phthalides in rat plasma: Application to a comparative pharmacokinetic study of Huo Luo Xiao Ling Dan and herb‐pair extract

A fast, sensitive, and reliable ultra‐high performance liquid chromatography with tandem mass spectrometry method has been developed and validated for the simultaneous quantitation and pharmacokinetic study of five phthalides (senkyunolide A, ligustilide, butylidenephthalide, 3‐butylphthalide, and levistilide A) in rat plasma after oral administration of Huo Luo Xiao Ling Dan (HLXLD) or Angelica sinensis‐‐Ligusticum chuanxiong herb pair (DG‐CX) between normal and arthritis rats. After extraction from blood, the analytes and internal standard were subjected to ultra‐high performance liquid chromatography with a Shim‐pack XR‐ODS column (75 × 3.0 mm², 2.2 μm particles) and mobile phase was composed of methanol and water (containing 0.05% formic acid) under gradient elution conditions, with an electrospray ionization source in the positive ionization and multiple reaction monitoring mode. The lower limits of quantification were 0.192–0.800 ng/mL for all the analytes. Satisfactory linearity, precision, accuracy, mean extraction recovery, and acceptable matrix effect have been achieved. The validated method was successfully applied to a comparative pharmacokinetic study of five bioactive components in rat plasma after oral administration of HLXLD or DG‐CX alone, respectively, between normal and arthritic rats. The results showed that there were unlike characters of pharmacokinetics among different groups.     

Simultaneous determination of five isoflavones in rat plasma by LC‐MS/MS: Comparative pharmacokinetic characteristics of Puerariae lobatae radix in normal and type 2 diabetic rats

A rapid and sensitive liquid chromatography with tandem mass spectrometry method was developed and validated for simultaneous determination of puerarin, daidzin, daidzein, 3′‐hydroxy puerarin, and genistein in rat plasma after oral administration of Puerariae lobatae radix extract. The method of protein precipitation with acetonitrile was used for sample preparation. Chromatographic separation was achieved on a C₁₈ column with the mobile phases of acetonitrile/water containing 0.1% formic acid. The analytes were detected by mass spectrometer with an electrospray ionization source operating in the negative ion mode. The linearity, precision, accuracy, dilution reliability, recovery, matrix effects, and stability of the method were within acceptable ranges. The developed method was successfully used to compare the pharmacokinetic characteristics of five analytes in normal and type 2 diabetics rats after oral administration of Puerariae lobatae radix extract. Several pharmacokinetic alterations were observed and this might be caused by the pathological state of type 2 diabetes.     

Liquid chromatography mass spectrometry for the determination of salvianolic acid B,a natural compound from the herb Danshen in rat plasma and application to pharmacokinetic study

A sensitive and specific liquid chromatographic–electrospray ionization mass spectrometric method was developed for quantification of salvianolic acid B in rat plasma with resveratrol as the internal standard. The analytes were separated on a reversed‐phase column with acetonitrile (40%) and water (60%) containing 0.75% formic acid as mobile phase at a flow rate of 1 mL/min. Liquid–liquid extraction was adopted for the sample preparation, and the analytes were determined using electrospray negative ionization mass spectrometry in the selective monitoring mode. The method was validated over the concentration range 0.1–40 µg/mL using 0.1 mL of plasma with coefficients of correlation >0.999. The intra‐ and inter‐day precisions of analysis were <10%, and accuracy ranged from 94 to 101%. This method was successfully applied to a pharmacokinetics of salvianolic acid B in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

The biotransformation of oleuropein in rats

A highly sensitive and specific LC‐MS/MS method was developed to investigate the in vivo bio‐transformation of oleuropein in rat. Rat feces and urine samples collected after oral administration were determined by liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves a simple liquid–liquid extraction of parent oleuropein and the metabolite from rat feces and urine with ethyl acetate. Chromatographic separation was operated with 0.1% formic acid aqueous and methanol in gradient program at a flow rate of 0.50 mL/min on an RP‐C₁₈ column with a total run time of 31 min. This method was successfully applied to simultaneous determination of oleuropein and its metabolites in rat feces and urine. De‐glucosylation, hydrolysis, oxygenation and methylation were found to comprise the major metabolic pathway of oleuropein in rat gastrointestinal tract and three metabolites were absorbed into the blood circulatory system within 24 h after oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Ultra‐performance liquid chromatography–tandem mass spectrometry assay for determination of plasma nomegestrol acetate and estradiol in healthy postmenopausal women

A highly sensitive and selective ultra‐performance liquid chromatography–tandem mass spectrometry method is described for the simultaneous determination of nomegestrol acetate (NOMAC), a highly selective progestogen, and estradiol (E2), a natural estrogen in human plasma. NOMAC was obtained from plasma by solid‐phase extraction, while E2 was first separated by liquid–liquid extraction with methyl tert‐butyl ether followed by derivatization with dansyl chloride. Deuterated internal standards, NOMAC‐d5 and E2‐d4 were used for better control of extraction conditions and ionization efficiency. The assay recovery of the analytes was within 90–99%. The analytes were separated on UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using a mobile phase comprising of acetonitrile and 3.0 mm ammonium trifluoroacetate in water (80:20, v/v) with a resolution factor (R_s) of 3.21. The calibration curves were linear from 0.01 to 10.0 ng/mL for NOMAC and from 1.00 to 1000 pg/mL for E2, respectively. The intra‐ and inter‐batch precision was ≤5.8% and the accuracy of quality control samples ranged from 96.7 to 103.4% for both analytes. The practical applicability of the method is demonstrated by analyzing samples from 18 healthy postmenopausal women after oral administration of 2.5 mg nomegestrol acetate and 1.5 mg estradiol film‐coated tablets under fasting.     

A rapid and sensitive LC‐MS/MS method for the determination of osthole in rat plasma: application to pharmacokinetic study

Osthole, a major component isolated from the fruit of Cnidium monnieri (L.) Cusson, has been widely used in traditional Chinese medicine. We developed and validated a rapid and sensitive LC‐MS/MS method for the quantification of osthole in rat plasma. Sample preparation involved simple liquid–liquid extraction by ethyl acetate after addition of imperatorin as internal standard (IS). The analyte was separated using a C₁₈ column with the mobile phase of methanol–0.1% formic acid (80:20, v/v) at a flow rate of 0.4 mL/min. The elutes were detected under positive electrospray ionization in multiple reaction monitoring mode. The method was sensitive with 0.5 ng/mL as the lower limit of detection. Good linearity was obtained over the range of 1.0–500.0 ng/mL. The intra and inter‐batch accuracy for osthole in rat plasma samples ranged from 99.5 to 108.1% and the variation was <8.9%. The stability, extraction efficiency and matrix effect were also acceptable. This method was successfully applied to the pharmacokinetic study of osthole in rat after intravenous and oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

UPLC–MS/MS assay for simultaneous determination of four compounds in rat plasma: application to pharmacokinetic study after oral administration of Caulis Spatholobi extract

An ultra‐performance liquid chromatography tandem mass spectrometry method was developed for the simultaneous determination of protocatechuic acid, catechin, gallocatechin and formononetin in rat plasma, with genkwanin as the internal standard in this study. Plasma samples were prepared by liquid–liquid extraction with ethyl acetate. The four components were separated on an Agilent Zorbax Eclipse Plus C₁₈ column (2.1 × 50 mm, 1.8 μm) with the mobile phase consisting of water containing 0.05% formic acid and methanol (35:65, v/v), and detected by negative ion electrospray ionization in the selected reaction monitoring mode. The method was linear for all analytes over the investigated ranges, with all correlation coefficients >0.99. The validated lower limit of quantification was 0.5 ng/mL for protocatechuic acid, catechin, and gallocatechin and 0.8 ng/mL for formononetin. The intra‐ and inter‐day precisions (RSD, %) were <13.1%, and accuracy (RE, %) ranged from ?13.8 to 9.9%. The mean absolute extraction recoveries of the analytes and internal standard from rat plasma were all >80.7%. The validated method was successfully applied for the first time to investigate the pharmacokinetics of four chemical ingredients after oral administration of Caulis Spatholobi Extract in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

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

A sensitive and rapid LC‐MS/MS method has been developed and validated for quantifying swertianolin in rat plasma using rutin as an internal standard (IS). Following liquid–liquid extraction with ethyl acetate, chromatographic separation for swertianolin was achieved on a C₁₈ column with a gradient elution using 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B at a flow rate of 0.3 mL/min. The detection was performed on a tandem mass spectrometer using multiple reaction monitoring via an electrospray ionization source and operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 435.1/272.0 for swertianolin and 609.2/300.1 for IS. The lower limit of quantitation was 0.5 ng/mL within a linear range of 0.5–500 ng/mL. Intra‐day and inter‐day precision was less than 6.8%. The accuracy was in the range of ?13.9 to 12.0%. The mean recovery of swertianolin was >66.7%. The proposed method was successfully applied in evaluating the pharmacokinetics of swertianolin after an oral dose of 50 mg/kg Swertia mussotii extract in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of major components of Huangqi–Honghua extract in rat plasma using LC–MS/MS and application to a pharmacokinetic study

A sensitive and reliable LC–MS/MS method was developed and validated for simultaneous quantification of the major components of Huangqi–Honghua extact in rat plasma, including hydroxysafflor yellow A (HSYA), astragaloside IV (ASIV), calycosin‐7‐O‐β‐d ‐glucoside (CAG), calycosin, calycosin‐3′‐O‐glucuronide (C‐3′‐G) and calycosin‐3′‐O‐sulfate (C‐3′‐S). After extraction by protein precipitation with acetonitrile and methanol from plasma, the analytes were separated on a Hypersil BDS C₁₈ column by gradient elution with acetonitrile and 5 mM ammonium acetate. The detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source switched between negative and positive modes. HSYA was monitored in negative ionization mode from 0 to 4.9 min, and ASIV, CAG, calycosin, C‐3′‐G and C‐3′‐S were determined in positive ionization mode from 4.9 to 10 min. The lower limits of quantification of the analytes were 6.25 ng/mL for HSYA, 0.781 ng/mL for CAG and 1.56 ng/mL for ASIV and calycosin. The intra‐ and inter‐assay precision (RSD) values were within 13.43%, and accuracy (RE) ranged from ?8.75 to 9.92%. The validated method was then applied to the pharmacokinetic study of HSYA, ASIV, CAG, calycosin, C‐3′‐G and C‐3′‐S in rat after an oral administration of Huangqi–Honghua extract.     

LC‐MS/MS method for the determination of agomelatine in human plasma and its application to a pharmacokinetic study

A sensitive and selective LC‐MS/MS method for the determination of agomelatine in human plasma was developed and validated. After simple liquid–liquid extraction, the analytes were separated on a Zorbax SB‐C₁₈ column (150 × 2.1 mm i.d., 5 µm) with an isocratic mobile phase consisting of 5 mm ammonium acetate solution (containing 0.1% formic acid) and methanol (30:70, v/v) at a flow‐rate of 0.3 mL/min. The MS acquisition was performed in multiple reaction monitoring mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.3 and m/z 285.2 → 193.2 for agomelatine and internal standard, respectively. The methods were validated for selectivity, carry‐over, matrix effects, calibration curves, accuracy and precision, extraction recoveries, dilution integrity and stability. The validated method was successfully applied to a pharmacokinetic study of agomelatine in Chinese volunteers following a single oral dose of 25 mg agomelatine tablet. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of evobrutinib and its metabolite evobrutinib‐diol in dog plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography hyphenated with electrospray ionization tandem mass spectrometric method (LC–ESI–MS/MS) was developed and validated for simultaneous determination of evobrutinib and evobrutinib‐diol in dog plasma. The plasma sample was processed using acetonitrile and chromatographic separation was carried out on a Waters Acquity BEH C₁₈ column (50 × 2.1 mm, 1.7 μm). The mobile phase was composed of 0.1% formic acid and acetonitrile, with an optimized gradient elution at a flow rate of 0.4 mL/min. Detection was accomplished in selective reaction monitoring mode via electrospray ionization interface operated in positive ion mode. The precursor‐to‐product transitions for quantification were m/z 430.2 → 98.1 for evobrutinib, m/z 464.2 → 98.1 for evobrutinib‐diol and m/z 441.2 → 138.1 for ibrutinib (internal standard). The developed assay was linear over the tested concentration ranges with correlation coefficient >0.995. The LLOQ was 0.1 ng/mL for both analytes. The inter‐ and intra‐day precisions were <9.65% and the accuracy ranged from ?3.94 to 6.37%. The extraction recovery was >85.41% and no significant matrix effect was observed. The developed assay was successfully applied to the pharmacokinetic study of evobrutinib and evobrutinib‐diol in dogs after oral administration of evobrutinib at a single dose of 5 mg/kg.