Development and validation of a highly sensitive LC‐ESI‐MS/MS method for the determination of hyperoside in beagle dog plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the analysis of hyperoside in beagle dog plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves extraction of hyperoside and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB‐C₁₈ (100 × 2.1 mm, 1.8 µm) column by isocratic elution with acetonitrile and water (50:50, v/v) at a flow rate of 0.25 mL/min with a total run time of 2.0 min. The MS/MS ion transitions monitored were 464.4 → 463.4 for hyperoside and 947.12 → 969.60 for IS. Linear responses were obtained for hyperoside ranging from 10 to 5000 ng/mL. The intra‐and inter‐day precisions (RSDs) were <5.38 and 3.39% and the extraction recovery ranged from 94.39 to 100.78% with an RSD <3.82%. Stability studies showed that hyperoside was stable in preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration–time profiles of hyperoside. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of an UPLC‐Q/TOF‐MS assay for the quantitation of neopanaxadiol in beagle dog plasma: Application to a pharmacokinetic study

Neopanaxadiol (NPD), the main panaxadiol constituent of Panax ginseng C. A. Meyer (Araliaceae), has been regarded as the active component for the treatment of Alzheimer's disease. However, few references are available about pharmacokinetic evaluation for NPD. Accordingly, a rapid and sensitive method for quantitative analysis of NPD in beagle dog plasma based on ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry was developed and validated. Analytes were extracted from plasma by liquid–liquid extraction and chromatographic separation was achieved on an Agilent Zorbax Stable Bond C₁₈ column. Detection was performed in the positive ion mode using multiple reaction monitoring of the transitions both at m/z 461.4 → 425.4 for NPD and internal standard of panaxadiol. All validation parameters, such as lower limit of quantitation, linearity, specificity, precision, accuracy, extraction recovery, matrix effect and stability, were within acceptable ranges and the method was appropriate for multitude sample determination. After oral intake, NPD was slowly absorbed and eliminated from circulatory blood system and corresponding plasma exposure was low. Application of this quantitative method will yield the first pharmacokinetic profile after oral administration of NPD to beagle dog. The information obtained here will be useful to understand the pharmacological effects of NPD.     

Development and validation of an LC‐MS/MS method for the determination of mesalazine in beagle dog plasma and its application to a pharmacokinetic study

A simple, specific and sensitive LC‐MS/MS method was developed and validated for the determination of mesalazine in beagle dog plasma. The plasma samples were prepared by protein precipitation, then the separation of the analyte was achieved on a Waters Spherisorb C₆ column (150 × 4.6 mm, 5 µm) with a mobile phase consisting of 0.2% formic acid in water–methanol (20:80, v/v). The flow rate was set at 1.0 mL/min with a split ratio of 3:2. Mass spectrometric detection was achieved by a triple‐quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. Quantitation was performed using selected reaction monitoring of precursor–product ion transitions at m/z 154 → m/z 108 for mesalazine and m/z 285 → m/z 193 for diazepam (internal standard). The linear calibration curve of mesalazine was obtained over the concentration range 50–30,000 ng/mL. The matrix effect of mesalazine was within ±9.8%. The intra‐ and inter‐day precisions were <7.9% and the accuracy (relative error) was within ±3.5%. The validated method was successfully applied to investigate the pharmacokinetics of mesalazine in healthy beagle dogs after rectal administration of mesalazine suppository. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a sensitive and rapid UPLC‐MS/MS method for the determination of koumine in rat plasma: application to a pharmacokinetic study

A rapid, selective and sensitive method using UPLC‐MS/MS was first developed and validated for quantitative analysis of koumine in rat plasma. A one‐step protein precipitation with methanol was employed as a sample preparation technique. Plasma samples were separated on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, i.d. 1.7 µm) with a gradient mobile phase consisting of methanol with 0.1% (v/v) formic acid and water containing 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min. Detection and quantification were performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization. Good linearity (r > 0.9997) was achieved using weighted (1/x²) least squares linear regression over a concentration range of 0.025–15 µg/mL with a lower limit of quantification of 0.025 µg/mL for koumine. The intra‐ and inter‐ precisions (relative standard deviation) of the assay at all three quality control samples were 5.6–14.1% with an accuracy (relative error) of 5.0–14.0%, which meets the requirements of the US Food and Drug Administration guidance. This developed method was successfully applied to an in vivo pharmacokinetic study in rats after a single intravenous dose of 20 mg/kg koumine. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of UPLC‐MS/MS method for determination of domperidone in human plasma and its pharmacokinetic application

A sensitive, rapid and selective ultra‐performance liquid chromatography–tandem mass spectrometric (UPLC‐MS/MS) method was developed for the determination and pharmacokinetic study of domperidone in human plasma. Diphenhydramine was used as the internal standard. Plasma sample pretreatment involved a one‐step liquid–liquid extraction with a mixture of diethyl ether–dichloromethane (3:2, v/v). The analysis was carried out on an Acquity UPLC^TM BEH C₁₈ column. The mobile phase consisted of methanol–water containing 10 mmol/L ammonium acetate and 0.5% (v/v) formic acid (60:40, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionizationsource with positive mode. Each plasma sample was chromatographed within 2.1 min. The standard curves for domperidone were linear (r² ≥ 0.99) over the concentration range of 0.030–31.5 ng/mL with a lower limit of quantification of 0.030 ng/mL. The intra‐ and inter‐day precision (relative standard deviation) values were not higher than 13% and accuracy (relative error) was from ?7.6 to 1.2% at three quality control levels. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of domperidone in healthy Chinese volunteers after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

UPLC‐MS/MS determination of voriconazole in human plasma and its application to a pharmacokinetic study

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method was developed to determine voriconazole in human plasma. Sample preparation was accomplished through a simple one‐step protein precipitation with methanol. Chromatographic separation was carried out on an Acquity UPLC BEH C₁₈ column using an isocratic mobile phase system composed of acetonitrile and water containing 1% formic acid (45:55, v/v) at a flow rate of 0.50 mL/min. Mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electrospray ionization source in the positive ion mode. The multiple reaction monitoring transitions of m/z 351.0 → 281.5 and m/z 237.1 → 194.2 were used to quantify voriconazole and carbamazepine (internal standard), respectively. The linearity of this method was found to be within the concentration range of 2.0–1000 ng/mL with a lower limit of quantification of 2.0 ng/mL. Only 1.0 min was needed for an analytical run. This fully validated method was successfully applied to the pharmacokinetic study after oral administration of 200 mg voriconazole to 20 Chinese healthy male volunteers. Copyright © 2014 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.     

Analysis and pharmacokinetic study of polyphyllin H in beagle dog plasma after oral administration of Rhizoma Paridis extracts by LC‐MS/MS

A highly sensitive, rapid assay method has been developed and validated for the analysis of polyphyllin H in beagle dog plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves extraction of polyphyllin H and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB‐C₁₈ (100 × 2.1 mm, 1.8μm) column by isocratic elution with acetonitrile and water (50:50, v/v) at a flow rate of 0.25 mL/min with a total run time of 2.5 min. The MS/MS ion transitions monitored were 870.46 → 869.6 for polyphyllin H and 947.12 → 969.60 for IS. Linear responses were obtained for polyphyllin H ranging from 1 to 50 ng/mL. The intra‐and inter‐day precisions (RSDs) were <1.77 and 3.39% and the extraction recovery ranged from 91.89 to 93.33% with RSD <2.68%. Stability studies showed that polyphyllin H was stable in the preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration–time profiles of polyphyllin H. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination and validation of chikusetsusaponin IVa in rat plasma by UPLC‐MS/MS and its application to pharmacokinetic study

A novel, sensitive and rapid ultra‐performance liquid chromatography–tandem mass spectrometric method for the quantification of chikusetsusaponin IVa (CHS‐IVa) in rat plasma was established and validated. Plasma samples were pre‐treated by precipitation of protein with acetonitrile and chromatographed on a Waters Symmetry C₁₈ analytical column (4.6 × 50 mm, i.d., 3.5 μm) using a mobile phase consisting of methanol and water containing 0.05% formic acid (55:45, v/v) at a flow rate of 0.4 mL/min. The deprotonated molecular ions [M ? H]^– were employed in electrospray negative ionization mode and selected reaction monitoring transitions were performed for detection. The calibration curves exhibited good linearity (r > 0.99) over the range of 0.5–1000 ng/mL for CHS‐IVa. The recoveries of CHS‐IVa were >92.5% and exhibited no severe matrix effect. This method was successfully applied in the pharmacokinetic study of CHS‐IVa in rats. For oral administration, the plasma concentrations of CHS‐IVa increased to a peak value at 0.35 ± 0.14 h, followed by a gradual decrease to the lower limit of quantitation in 24 h. For intravenous administration, the plasma concentrations of CHS‐IVa decreased quickly (t_1/2, 1.59 ± 0.25 h). The absolute bioavailability of CHS‐IVa in rats was 8.63%. Copyright © 2016 John Wiley & Sons, Ltd.     

A UPLC‐MS/MS method for simultaneous determination of five flavonoids from Stellera chamaejasme L. in rat plasma and its application to a pharmacokinetic study

Stellera chamaejasme L. has been used as a traditional Chinese medicine for the treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, cancer and tuberculosis. A sensitive and selective ultra‐high liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for the simultaneous determination of five flavonoids (stelleranol, chamaechromone, neochamaejasmin A, chamaejasmine and isochamaejasmin) of S. chamaejasme L. in rat plasma. Chromatographic separation was accomplished on an Agilent Poroshell 120 EC‐C₁₈ column (2.1 × 100 mm, 2.7 μm) with gradient elution at a flow rate of 0.4 mL/min and the total analysis time was 7 min. The analytes were detected using multiple reaction monitoring in positive ionization mode. The samples were prepared by liquid–liquid extraction with ethyl acetate. The UPLC‐MS/MS method was validated for specificity, linearity, sensitivity, accuracy and precision, recovery, matrix effect and stability. The validated method exhibited good linearity (r ≥ 0.9956), and the lower limits of quantification ranged from 0.51 to 0.64 ng/mL for five flavonoids. The intra‐ and inter‐day precision were both <10.2%, and the accuracy ranged from −11.79 to 9.21%. This method was successfully applied to a pharmacokinetic study of five flavonoids in rats after oral administration of ethyl acetate extract of S. chamaejasme L.     

Determination of armepavine in mouse blood by UPLC‐MS/MS and its application to pharmacokinetic study

The purpose of this study was to develop an ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC‐MS/MS) method to determine armepavine in mouse blood. Nuciferine was used as internal standard. Chromatographic separation was performed on a UPLC BEH (2.1 × 50 mm, 1.7 μm) column with a gradient elution of acetonitrile and 10 mmol/L ammonium acetate solution (containing 0.1% formic acid). The quantitative analysis was conducted in multiple reaction monitoring mode with m/z 314.1 → 106.9 for armepavine and m/z 296.2 → 265.1 for nuciferine. Calibration curves were linear (r > 0.995) over the concentration range 1–1000 ng/mL in mouse blood with a lowest limit of quantitation of 1 ng/mL. The intra‐ and inter‐day precisions of armepavine in mouse were < 13.5 and 10.8%, respectively. The accuracy ranged between 86.8 and 103.3%. Meanwhile, the average recovery was >70.7% and the matrix effect was within the range 109.5–113.7%. All of the obtained data confirmed the satisfactory sensitivity and selectivity of the developed method which was then successfully applied to evaluate the pharmacokinetic behavior of armepavine in mouse for the first time. The bioavailability of armepavine in mouse was calculated to be 11.3%.     

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.     

Simultaneous determination of tapentadol and its carbamate prodrug in rat plasma by UPLC‐MS/MS and its application to a pharmacokinetic study

A prodrug of tapentadol, namely tapentadol carbamate (WWJ01), was synthesized to improve the bioavailability of tapentadol owing to its extensive first‐pass metabolism. In this study, a highly rapid and sensitive UPLC‐MS/MS method was developed and validated for the simultaneous determination of tapentadol and WWJ01 in rat plasma with fluconazole as an internal standard. The analytes and internal standard were treated by methanol and then separated on a Phenomenex Kinetex® XB‐C₁₈ (2.1 × 50 mm × 2.6 μm) column at a flow rate of 0.3 mL/min. The mobile phase comprised methanol and water with a gradient elution. The mass transition ion‐pairs were m/z 222.2 → 107.0, m/z 293.2 → 71.9 and m/z 307.1 → 220.0 for tapentadol, WWJ01 and IS, respectively. Excellent linearity was observed over the concentration range of 2–1250 ng/mL (r = 0.995) with a lower limit of quantification of 2 ng/mL for both tapentadol and WWJ01. The intra‐ and inter‐day accuracy and precision for all quality control samples were within ±15%. The validated method was accurate, rapid and reproducible, and was successfully applied to a pharmacokinetic study of tapentadol and WWJ01.     

LC‐ESI‐MS/MS determination of 4‐methylpyrazole in dog plasma and its application to a pharmacokinetic study in dogs

A simple, specific, sensitive and rapid LC‐ESI‐MS/MS method has been developed and validated for the quantification of 4‐methylpyrazole in dog plasma using N‐methylnicotinamide‐d₄ as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a simple protein precipitation. Chromatographic separation of 4‐methylpyrazole and the IS was performed on a monolithic (Chromolith RP_18e) column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (20:80, v/v) at a flow rate of 1.0 mL/min. Elution of 4‐methylpyrazole and the IS occurred at ~1.60 and 1.56 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 4.96–4955 ng/mL. The intra‐ and inter‐day accuracy and precision were in the ranges 1.81–12.9 and 3.80–11.1%, respectively. This novel method has been applied to a pharmacokinetic study in dogs.     

Development and validation of a UPLC‐MS/MS method for the determination of cucurbitacin B in rat plasma and application to a pharmacokinetic study

Cucurbitacin B (CuB), one of the most abundant forms of cucurbitacins, is a promising natural anticancer drug candidate. Although the anticancer activity of CuB has been well demonstrated, information regarding the pharmacokinetics is limited. A rapid, selective and sensitive UPLC‐MS/MS for CuB was developed and validated using hemslecin A (HeA) as internal standard (IS). Plasma samples were pre‐treated by liquid–liquid extraction with dichloromethane. Separation was achieved on a reversed‐phase C₁₈ column (50 × 4.6 mm, 5 µm) at 35°C using isocratic elution with water–methanol (25:75, v/v) at a flow rate of 0.3 mL/min. The analytes were monitored by a triple quadrupole tandem mass spectrometer with positive electrospray ionization mode. The calibration curve was linear (r > 0.995) in a concentration range of 0.3–100 ng/mL with a limit of quantification of 0.3 ng/mL. Intra‐ and inter‐day accuracy and precision were validated by percentage relative error and relative standard deviation, respectively, which were both lower than the limit of 15%. This assay was successfully applied to a pharmacokinetic study of CuB in Wistar rats. Copyright © 2015 John Wiley & Sons, Ltd.     

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 and pharmacokinetic study of polyphyllin I,polyphyllin II,polyphyllin VI and polyphyllin VII in beagle dog plasma after oral administration of Rhizoma Paridis extracts by LC‐MS‐MS

For the first time, a rapid and specific LC‐MS‐MS method has been developed for the analysis of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII in beagle dog plasma. The method was applied to study the pharmacokinetics of Rhizoma Paridis extracts containing polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII. The analysis was carried out on an Agilent Zorbax XDB‐C₁₈ reversed‐phase column (100 × 2.1 mm, 1.8 µm) by isocratic elution with acetonitrile and water (50:50, v/v). The flow rate was 0.25 mL/min. All analytes including internal standards were monitored by selected reaction monitoring with an electrospray ionization source. Linear responses were obtained for polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII ranging from 10 to 5000 ng/mL. The intra‐and inter‐day precisions (RSDs) were less than 6.66 and 9.15%. The extraction recovery ranged from 95.53 to 104.21% with RSD less than 8.69%. Stability studies showed that polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII were stable in preparation and analytical process. The validated method was successfully used to determine the concentration–time profiles of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of a simple,sensitive and accurate LC‐MS/MS method for the determination of guanfacine,a selective α2A‐adrenergicreceptor agonist,in plasma and its application to a pharmacokinetic study

A simple, practical, accurate and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and fully validated for the quantitation of guanfacine in beagle dog plasma. After protein precipitation by acetonitrile, the analytes were separated on a C₁₈ chromatographic column by methanol and water containing 0.1% (v/v) formic acid with a gradient elution. The subsequent detection utilized a mass spectrometry under positive ion mode with multiple reaction monitoring of guanfacine and enalaprilat (internal standard) at m/z 246.2 → 159.0 and m/z 349.2 → 205.9, respectively. Good linearity was obtained over the concentration range of 0.1–20 ng/mL for guanfacine in dog plasma and the lower limit of quantification of this method was 0.1 ng/mL. The intra‐ and inter‐day precisions were <10.8% relative standard deviation with an accuracy of 92.9–108.4%. The matrix effects ranged from 89.4 to 100.7% and extraction recoveries were >90%. Stability studies showed that both analytes were stable during sample preparation and analysis. The established method was successfully applied to an in vivo pharmacokinetic study in beagle dogs after a single oral dose of 4 mg guanfacine extended‐release tablets. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of etonogestrel and ethinyl estradiol in human plasma by UPLC‐MS/MS and its pharmacokinetic study

A selective, sensitive and rapid ultra‐performance liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of etonogestrel (ENG) and ethinyl estradiol (EE) in human plasma. The analytes and their deuterated internal standards, ENG‐d7 and EE‐d4, were extracted from plasma samples by solid‐phase extraction on HyperSep™ Retain PEP cartridges. The chromatographic analysis was performed on an Acquity UPLC HSS Cyano column, 100 Å (50 × 2.1 mm, 1.8 μm), column using gradient mobile phase, acetonitrile and 2.0 mm ammonium trifluoroacetate at 0–1.7 min (65:35, v/v) and 1.8–2.7 min (95:5, v/v) with 0.250 mL/min flow rate. Analytes and IS protonated precursor → product ion transitions (ENG, m/z 325.2 → 257.2; EE, m/z 530.2 → 171.2; ENG‐d7, m/z 332.2 → 263.2; EE‐d4, m/z 534.2 → 171.2) were monitored on a Triple Quadrupole Mass spectrometer (TQMS), operating in multiple reaction monitoring and positive ionization mode. The calibration curves were established at 10.00–2500 pg/mL for ENG and 1.500–150.0 pg/mL for EE with a correlation coefficient (r²) ≥0.9996 for both. The validated method was successfully applied to support a bioequivalence study of 0.15 mg ENG and EE 0.03 mg tablet formulation, administered in 24 healthy Indian females. Method reliability was assessed by reanalysis of 94 incurred study samples.     

Application of a sensitive and specific LC‐ESI‐MS/MS method for the simultaneous quantification of twelve bioactive components in dog plasma for an intravenous pharmacokinetic study of Yiqifumai Injection in beagle dogs

Yiqifumai Injection is a lyophilized powder preparation widely used to treat coronary heart disease. However, its in vivo bioactive components and pharmacokinetic behavior remain unknown. Therefore a sensitive and specific LC–MS/MS was developed and validated for the simultaneous quantification of eight saponins and four lignans in beagle dog plasma. The plasma samples were pretreated by protein precipitation with methanol–acetonitrile (1:1, v/v). Chromatographic separation of all the 12 analytes and estazolam (internal standard, IS) was successfully accomplished on an Ultimate® XB‐C₈ column (100 × 2.1 mm, 3 μm) with a gradient elution system. The total running time was 8 min with a flow rate of 0.40 mL/min. Acquisition of mass spectrometric data was performed via positive electrospray ionization in multiple reaction monitoring mode. The assay was fully validated in terms of selectivity, linear range, lower limit of quantitation, precision, accuracy, matrix effect, recovery and stability. This validated method was successfully applied to the pharmacokinetics of 12 bioactive components after intravenous administration of Yiqifumai Injection to beagle dogs at a dose of 0.541 g/kg.