Liquid chromatography tandem mass spectrometry method for determination of N-acetylcysteine in human plasma using an isotope-labeled internal standard

A liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed to determine total N‐acetylcysteine in human plasma. Mass spectrometric detection was achieved in positive electrospray ionization and multiple reaction monitoring mode. The mass transition pairs of N‐acetylcysteine and the isotope‐labeled internal standard d3‐N‐acetylcysteine were 164 → 122 and 167 → 123, respectively. The method was linear over the range of 10–5000 ng/mL in human plasma. The adoption of trichloroacetic acid significantly enhanced the extraction recovery. The blank matrix was screened to minimize the influence of endogenous N‐acetylcysteine. After being fully validated, the method was successfully applied to the pharmacokinetic and bioequivalent study of N‐acetylcysteine after oral administration of 600 mg tablets to 24 healthy Chinese volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

人体血清中3种脂溶性维生素的液相色谱-串联质谱分析方法研究

以人体血清中3种脂溶性维生素检测为例,探讨了一种针对人体内源性代谢物的分析方法。采用液相色谱-串联质谱(LC-MS/MS)对人体血清中维生素A、维生素D_3和维生素E进行检测,分别通过混合人血清基质加入标准品及内标再扣除内源性物质本底的方法,以及与牛血清白蛋白(BSA)模拟基质加标的方法建立标准曲线进行定量分析。结果表明,采用混合人血清基质所建方法对维生素A、维生素D_3和维生素E的检出限分别为4.2、0.8、67.2 ng/mL,定量下限分别为13.7、2.6、221.9 ng/mL。两种方法的线性相关系数均大于0.996;对于实际样品在低、高两个加标浓度下的回收率为90.7%～112%,相对标准偏差(RSD)为1.0%～4.5%,具有良好的准确性和重现性。对40组未知样本的检测结果表明,两种方法无统计学差异。因此,对于人体内源性代谢物采用混合人血清基质扣除本底的方法,可以实现标准品与待测样品基质匹配的准确分析,有利于临床相关疾病的便捷诊断。     

The validation of a simple LC/MS/MS method for determining the level of mevalonic acid in human plasma

A simple plasma extraction method coupled with liquid chromatography–tandem mass spectrometry (LC/MS/MS) detection was developed and validated for the analysis of endogenous mevalonic acid (MVA), a biomarker indicative of the rate of cholesterol biosynthesis, in human plasma samples. The analyte was extracted from the plasma matrix using a straightforward liquid–liquid sample preparation procedure. The extract supernatants were evaporated, reconstituted in aqueous solvent and injected into the LC/MS/MS system without further processing. The chromatographic separation was achieved on a reverse‐phase high‐performance liquid chromatography column. The accuracy and precision of the method was determined over the concentration range 0.25–25 ng/mL MVA from human plasma extracts in three validation batch runs. Inter‐assay precision (%CV) and accuracy (%RE) of the quality control samples were ≤7.00% (at lower limit quality control) and ≤6.10%, respectively. The sensitivity and throughput of this assay was significantly improved relative to previously published methods, resulting in smaller sample requirements and shorter analysis time. Assay results from a clinical study following the oral administration of an exploratory statin demonstrate that this procedure could potentially be used in the investigation of therapies associated with hypercholesterolemia. Copyright © 2010 John Wiley & Sons, Ltd.     

Validation and application of a liquid chromatography‐tandem mass spectrometric method for the determination of GDC‐0152 in human plasma using solid‐phase extraction

A liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of GDC‐0152 in human plasma to support clinical development. The method consisted of a solid‐phase extraction for sample preparation and LC‐MS/MS analysis in the positive ion mode using TurboIonSpray^TM for analysis. d₇‐GDC‐0152 was used as the internal standard. A linear regression (weighted 1/concentration²) was used to fit calibration curves over the concentration range of 0.02–10.0 ng/mL for GDC‐0152. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 99.3% with a precision (%CV) of 13.9%. For quality control samples at 0.0600, 2.00 and 8.00 ng/mL, the between‐run %CV was ≤8.64. Between‐run percentage accuracy ranged from 98.2 to 99.6%. GDC‐0152 was stable in human plasma for 363 days at ?20°C and for 659 days at ?70°C storage. GDC‐0152 was stable in human plasma at room temperature for up to 25 h and through three freeze–thaw cycles. In whole blood, GDC‐0152 was stable for 12 h at 4°C and at ambient temperature. This validated LC‐MS/MS method for determination of GDC‐0152 was used to support clinical studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Validation of a liquid chromatography‐triple quadrupole mass spectrometric method for the determination of 5‐nitro‐5′‐hydroxy‐indirubin‐3′‐oxime (AGM‐130) in human plasma and its application to microdose clinical trial

A liquid chromatography–triple quadrupole mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of 5‐nitro‐5′‐hydroxy‐indirubin‐3′‐oxime (AGM‐130) in human plasma to support a microdose clinical trial. The method consisted of a liquid–liquid extraction for sample preparation and LC‐MS/MS analysis in the positive ion mode using TurboIonSpray^TM for analysis. d₃‐AGM‐130 was used as the internal standard. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 10–2000 pg/mL for AGM‐130. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 96.6% with a precision (coefficient of variation, CV) of 4.4%. For quality control samples at 30, 160 and 1600 pg/mL, the between run CV was ≤5.0 %. Between‐run accuracy ranged from 98.1 to 101.0%. AGM‐130 was stable in 50% acetonitrile for 168 h at 4°C and 6 h at room temperature. AGM‐130 was also stable in human plasma at room temperature for 6 h and through three freeze–thaw cycles. The variability of selected samples for the incurred sample reanalysis was ≤12.7% when compared with the original sample concentrations. This validated LC‐MS/MS method for determination of AGM‐130 was used to support a phase 0 microdose clinical trial. Copyright © 2015 John Wiley & Sons, Ltd.     

A quantitative LC–MS/MS method for determination of a small molecule agonist of EphA2 in mouse plasma and brain tissue

Compound 27 {1, 12‐bis[4‐(4‐amino‐6,7‐dimethoxyquinazolin‐2‐yl)piperazin‐1‐yl]dodecane‐1,12‐dione} is a novel small molecule agonist of EphA2 receptor tyrosine kinase. It showed much improved activity for the activation of EphA2 receptor compared with the parental compound doxazosin. To support further pharmacological and toxicological studies of the compound, a method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC–MS/MS) has been developed for the quantification of this compound. Liquid–liquid extraction was used to extract the compound from mouse plasma and brain tissue homogenate. Reverse‐phase chromatography with gradient elution was performed to separate compound 27 from the endogenous molecules in the matrix, followed by MS detection using positive ion multiple reaction monitoring mode. Multiple reaction monitoring transitions m/z 387.3 → 290.1 and m/z 384.1 → 247.1 were selected for monitoring compound 27 and internal standard prazosin, respectively. The linear calibration range was 2–200 ng/mL with the intra‐ and inter‐day precision and accuracy within the acceptable range. This method was successfully applied to the quantitative analysis of compound 27 in mouse plasma and brain tissue with different drug administration routes.     

Sensitive and rapid simultaneous quantitation of leucovorin and its major active metabolite 5-methyl-tetrahydrofolate in human plasma using a liquid chromatography coupled with triple quadruple mass spectrometry

Bioanalysis of an endogenous compound such as leucovorin is never an easy task on a liquid chromatography tandem mass spectrometer (LC–MSMS). Unless it is necessary, regulatory guidance discourages working with surrogate matrices for calibration curve standard preparation. Herein, a selective and sensitive liquid chromatography–tandem mass spectrometry method for simultaneous determination of leucovorin and 5-methyl tetrahydrofolic acid in human plasma was developed and validated. Stable labeled internal standards, i.e. leucovorin D₄ and 5- methyl tetrahydrofolic acid ¹³C₅, were used as internal standards to track and compensate the parent compounds during processing and extraction from plasma. The method involves a rapid solid-phase extraction from plasma followed by reverse-phase gradient chromatography and mass spectrometry detection with a total run time of 5 min. The method was developed and validated from 5 to 2,202 ng/ml for leucovorin and from 5 to 1,300 ng/ml for 5-methyl tetrahydrofolic acid. The mean recoveries for leucovorin and 5-methyl tetrahydrofolic acid were 100.4 and 100.9% respectively. The validated method enabled the simultaneous analysis of leucovorin and 5-methyl tetrahydrofolic acid in samples from clinical pharmacokinetic studies of leucovorin. The peak concentrations of leucovorin and 5-methyl tetrahydrofolic acid were 651–883 and 518–635 ng/ml, respectively, in fasted and fed conditions. The terminal half-life values for leucovorin and 5-methyl tetrahydrofolic acid were 9.3–10.5 and 9.2–17.6 h, respectively.     

Validation and application of a novel LC/MS/MS method for the determination of isoginkgetin in rat plasma

Isoginkgetin is a biflavonoid compound isolated from the leaf extracts of Ginkgo biloba. In this study, an liquid chromatography–tandem mass spectrometry (LC/MS/MS) with liquid–liquid extraction was developed and validated for the analysis of isoginkgetin in rat plasma. In the process of chromatographic separation, selected reaction monitoring transitions for isoginkgetin and IS were m/z 566.8 → 134.7 and m/z 430.8 → 269.3, respectively. The validation parameters including selectivity, linearity, LLOQ, accuracy, precision, matrix effect, stability and recovery were satisfactory. The intra‐ and inter‐batch precision (RSD) were <12.1% in plasma, while the accuracy (RE) was within ±14.3%. This method was employed in a pharmacokinetic study on rats after the intravenous administration of isoginkgetin.     

Development and application of a UHPLC‐MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum

A new, fast and simple analytical method that is able to identify and quantify simultaneously 17 steroid hormones and metabolites (pregnenolone, 17‐OH‐pregnenolone, progesterone, 17‐OH‐progesterone, androsterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisol, corticosterone, aldosterone, 11‐deoxycortisol, 11‐deoxycorticosterone, dihydrotestosterone, estrone and estradiol) has been developed in equine serum using the ultra‐high‐performance liquid chromatography–tandem mass spectrometry technique. A total of 400 µl of sample was deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in ultra‐high‐performance liquid chromatography–tandem mass spectrometry triple quadrupole. The recovery percentage obtained by spiking the matrix at two different concentrations with a standard mixture of steroid hormones was in all cases higher than 85.60% and with the percentage of coefficient of variation lower than 8.37%. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.9922–0.9986, and the limits of detection and limits of quantification were in the range of 0.002–2 and 0.0055–5.5 ng ml⁻¹, respectively. The detected limit of quantification for testosterone (i.e. 50 pg ml⁻¹) is twofold lower with respect to its threshold admitted in geldings plasma (100 pg ml⁻¹ free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of the steroids in equine serum. Once validated, the method was used to quantify 17 steroid hormones in mare, stallion and gelding serum samples. The main steroids detected were corticosterone (range 37.25–51.26 ng ml⁻¹) and cortisol (range 32.57–52.24 ng ml⁻¹), followed by 17‐OH‐pregnenolone, dihydrotestosterone and pregnenolone. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a rapid LC-MS/MS method for the determination of JCC76, a novel antitumor agent for breast cancer, in rat plasma and its application to a pharmacokinetics study

JCC76 is a novel nimesulide analog that selectively inhibits the human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer cell proliferation and tumor progression. To support further pharmacological and toxicological studies of JCC76, a novel and rapid method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) has been developed and validated for the quantification of the compound in rat plasma. A C₁₈ column was used for chromatographic separation, and the mobile phase was aqueous ammonium formate (pH 3.7; 5 mm )–methanol (1:9, v/v) with an isocratic elution. With a simple liquid–liquid extraction procedure using the mixture of methyl tert‐butyl ether–hexane (1:2, v/v), the mean extraction efficiency of JCC76 in rat plasma was determined as 89.5–97.3% and no obvious matrix effect was observed. This method demonstrated a linear calibration range from 0.3 to 100 ng/mL for JCC76 in rat plasma and a small volume of sample consumption. The intra‐ and inter‐assay accuracy and precision were within ±10%. The pharmacokinetics of JCC76 was also profiled using this validated method in rats. In conclusion, this rapid and sensitive method has been proven to effectively quantify JCC76 for pharmacokinetics study. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of penciclovir in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid, specific and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for the determination of penciclovir in human plasma. The method involved simple, one‐step SPE procedure coupled with a C₁₈, 75 × 4.mm, 3µm column with a flow‐rate of 0.5 mL/min, and acyclovir was used as the internal standard. The Quattro Micro mass spectrometry was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. Using 250 µL plasma, the methods were validated over the concentration range 52.555–6626.181 ng/mL, with a lower limit of quantification of 52.55 ng/mL. The intra‐ and inter‐day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a clinical pharmacokinetic study in human volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

A supported liquid extraction LC–MS/MS method for determination of concentrations of GDC‐0425, a small molecule Checkpoint kinase 1 inhibitor,in human plasma

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of GDC‐0425 concentrations in human plasma has been developed and validated. Supported liquid extraction was used to extract plasma samples (50 μL) and the resulting samples were analyzed using reverse‐phase chromatography and mass spectrometry coupled with a turbo‐ionspray interface. The mass analysis of GDC‐0425 was performed using multiple reaction monitoring transitions in positive ionization mode. The method was validated over the calibration curve range of 1.00–1000 ng/mL using linear regression and 1/x² weighting. Within‐run relative standard deviation ranged from 0.8 to 5.1%, while between‐run RSD varied from 1.9 to 4.7% for QCs. The accuracy ranged from 90.0 to 101.0% of nominal for within‐run and from 94.0 to 100.0% of nominal for between‐run. Overall extraction recovery was 87.4% for GDC‐0425 and 87.9% for GDC‐0425‐d₉. Stability of GDC‐0425 was established in human plasma for 374 days at ?20 and ?70 °C and established in reconstituted sample extracts for 88 h when stored at 2–8 °C. Stable‐labeled internal standard was used to minimize matrix effects. This assay was used to characterize the pharmacokinetics of GDC‐0425 in cancer patients.     

A sensitive and specific liquid chromatography/tandem mass spectrometry method for determination of pinaverium bromide in human plasma: application to a pharmacokinetic study in healthy volunteers

A sensitive and specific method using liquid chromatography–electrospray tandem mass spectrometry (LC‐MS/MS) for the determination of pinaverium bromide in human plasma was developed and validated. Pinaverium bromide and an internal standard (paclitaxel) were isolated from plasma samples by precipitating plasma, and determined by LC‐MS/MS in multiple‐reaction monitoring mode. The main metabolite of pinaverium bromide and endogenous substances in plasma did not show any interference. The calibration curve was linear over the plasma concentration range of 10.0–10000.0 pg/mL with a correlation coefficient of 0.9979. The relative standard derivations intra‐ and inter‐day at 30.0, 300.0 and 8000.0 pg/mL in plasma were less than 15%. The absolute recoveries of pinaverium bromide and the internal standard were 99.7–111.7 and 106.2%, respectively. The lower limit of quantitation was 10 pg/mL. The analytical method was successfully applied to study the pharmacokinetics of pinaverium bromide tablets in healthy Chinese volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

Validation of an ultra‐performance liquid chromatography–tandem mass spectrometry method for the determination of flecainide in human plasma and its clinical application

A simple and reproducible bioanalytical method for the determination of flecainide in human plasma was developed and validated using an ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC‐MS/MS) to obtain higher sensitivity than the current available methods. After simple protein precipitation, flecainide and a stable isotope‐labeled internal standard (IS) were chromatographed on an Acquity UPLC BEH C₁₈ column (2.1 × 100 mm, 1.7 µm) with isocratic elution of mobile phase consisting of 45% methanol containing 0.1% formic acid at a flow rate 0.25 mL/min. Detection was performed in positive electrospray ionization by monitoring the selected ion transitions at m/z 415.4/301.1 for flecainide and m/z 419.4/305.1 for the IS. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve was linear from 2.5 to 1000 ng/mL using 0.1 mL of plasma. No significant interferences were detected in blank human plasma. Accuracy and precision in the intra‐ and inter‐batch reproducibility study were within acceptance criteria. Neither hemolysis effects nor matrix effects were observed. The UPLC‐MS/MS method developed was successfully applied to determine plasma flecainide concentrations to support clinical studies and incurred sample reanalysis also ensured the reproducibility of the method. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Simultaneous determination of morphine‐6‐d‐glucuronide,morphine‐3‐d‐glucuronide and morphine in human plasma and urine by ultra‐performance liquid chromatography–tandem mass spectrometry: Application to M6G injection pharmacokinetic study

A robust ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the determination of morphine‐6‐d ‐glucuronide (M6G), morphine‐3‐d ‐glucuronide (M3G) and morphine (MOR) in human plasma and urine has been developed and validated. The analytes of interest were extracted from plasma by protein precipitation. The urine sample was prepared by dilution. Both plasma and urine samples were chromatographed on an Acquity UPLC HSS T₃ column using gradient elution. Detection was performed on a Xevo TQ‐S tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. Matrix interferences were not observed at the retention time of the analytes and internal standard, naloxone‐D5. The lower limits of quantitation of plasma and urine were 2/0.5/0.5 and 20/4/2 ng/mL for M6G/M3G/MOR, respectively. Calibration curves were linear over the concentration ranges of 2–2000/0.5–500/0.5–500 and 20–20,000/4–4000/2–2000 ng/mL for M6G/M3G/MOR in plasma and urine samples, respectively. The precision was <7.14% and the accuracy was within 85–115%. Furthermore, stability of the analytes at various conditions, dilution integrity, extraction recovery and matrix effect were assessed. Finally, this quantitative method was successfully applied to the pharmacokinetic study of M6G injection in Chinese noncancer pain patients.     

An improved LC‐MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and its active metabolites in human plasma and its application to a pharmacokinetic study in patients

In this study, a sensitive, selective and reproducible liquid chromatography–tandem mass spectrometry method for the simultaneous determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and its active metabolites, 1‐caffeoyl‐5‐feruoylquinic acid and 1,5‐O‐diferuoylquinic acid, in human plasma, using puerarin as internal standard, was developed and validated. Analytes were extracted from plasma samples by liquid–liquid extraction with ethyl acetate, separated on a C₁₈ reversed‐phase column with water containing 5 mM ammonium acetate and acetonitrile as the mobile phase and detected by electrospray ionization mass spectrometry in negative selected reaction monitoring mode. The accuracy and precision of the method were acceptable and linearity was good over the range 1–200 ng/mL for each analyte. In addition, the selectivity, extraction recovery and matrix effect were satisfactory too. The validated LC‐MS/MS method was successfully applied to phase II clinical pharmacokinetic study of 1,5‐DCQA in patients. Copyright © 2010 John Wiley & Sons, Ltd.     

Free mycophenolic acid determination in human plasma ultrafiltrate by a validated liquid chromatography–tandem mass spectrometry method

The aim of this study was to develop and validate fully the liquid chromatography–tandem mass spectrometry method for free mycophenolic acid (MPA) concentration measurements in plasma ultrafiltrate that will be reliable and simple in preparation with deuterated MPA (MPA‐d3) chosen as an internal standard. The chromatographic separation was made with Zorbax Eclipse XDB‐C18 column (4.6 × 150 mm) using a gradient of two solutions as a mobile phase: (A) water and (B) methanol, each containing 0.1% formic acid and 2.5 mm ammonium acetate. Satisfactory repeatability of retention times was achieved with average values of 7.54 ± 0.20 min and 7.50 ± 0.19 min for MPA and MPA‐d3, respectively. The method was selective, with no carry‐over or matrix effect observed. The analytical range was proven for MPA ultrafiltrate concentrations of 1–500 ng/mL. The accuracy and precision fell within the acceptance criteria for intraday (accuracy: 100.63–110.46%, imprecision: 6.23–7.76%), as well as interday assay (accuracy: 98.81–110.63%; imprecision: 5.36–10.22%). The method was used for free MPA determination in plasma samples from patients treated with mycophenolate mofetil. To the best of our knowledge this is the first liquid chromatography–tandem mass spectrometry method for free MPA monitoring using MPA‐d3 that allows to measure plasma ultrafiltrate concentrations as low as 1 ng/mL.     

Application of 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione to analysis of 25-hydroxyvitamin D3 in human plasma by liquid chromatography/electron capture atmospheric pressure chemical ionization-mass spectrometry.

The utility of 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD) as a derivatization reagent in the analysis of 25-hydroxyvitamin D3 [25(OH)D3] using liquid chromatography/electron capture atmospheric pressure chemical ionization-mass spectrometry (LC/ECAPCI-MS) was examined. The derivatives of 25(OH)D3 with NPTAD underwent electron capture in the APCI source in the negative-ion mode and provided 30-fold higher sensitivity compared to an intact compound. This derivatization-LC/ECAPCI-MS method was applied to a plasma 25-hydroxyvitamin D3 assay, and gave satisfactory results in sensitivity, specificity, measurable range and throughput of the analysis.     

Determination of pseudo‐ginsenoside GQ in human plasma by high performance liquid chromatography–tandem mass spectrometry

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC‐MS/MS) was developed for the determination of pseudo‐ginsenoside GQ in human plasma. Liquid–liquid extraction was used to isolate the analyte from biological matrix followed by injection of the extracts onto a C₈ column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer (API‐4000 system) in multiple reaction monitoring mode using negative electrospray ionization. The mobile phase consisted of methanol–10 mm ammonium acetate (90:10, v/v) and the flow rate was 0.3 mL/min. The method was validated over the concentration range of 5.0–5000.0 ng/mL for plasma. Inter‐ and intra‐day precisions (relative standard deviation) were all within 15% and the accuracy (relative error) was ≤9.4%. The lower limit of quantitation was 5.0 ng/mL. The pseudo‐ginsenoside GQ was stable after 8 h at room temperature, 24 h at autosampler and three freeze–thaw cycles (from ?30 to 25 °C). The method was successfully applied to the pharmacokinetic study of pseudo‐ginsenoside GQ in healthy Chinese volunteers. Copyright © 2013 John Wiley & Sons, Ltd.