Quantitation of acrylamide in food products by liquid chromatography/mass spectrometry

A simple and inexpensive liquid chromatography/mass spectrometry (LC/MS) method was developed for the quantitation of acrylamide in various food products. The method involved spiking the isotope-substituted internal standard (1-C13 acrylamide) onto 6.00 g of the food product, adding 40 mL distilled/deionized water, and heating at 65 degrees C for 30 min. Afterwards, 10 mL ethylene dichloride was added and the mixture was homogenized for 30 s and centrifuged at 2700 x g for 30 min, and then 8 g supernatant was extracted with 10, 5, and 5 mL portions of ethyl acetate. The extracts were combined, dried with sodium sulfate, and concentrated to 100-200 microL. Acrylamide was determined by analysis of the final extract on a single quadrupole, bench-top mass spectrometer with electrospray ionization, using a 2 mm id C18 column and monitoring m/z = 72 (acrylamide) and m/z = 73 (internal standard). For difficult food matrixes, such as coffee and cocoa, a solid-phase extraction cleanup step was incorporated to improve both chromatography and column lifetime. The method had a limit of quantitation of 10 ppb, and coefficients of determination (r2) for calibration curves were typically better than 0.998. Acceptable spike recovery results were achieved in 11 different food matrixes. Precision in potato chip analyses was 5-8% (relative standard deviation). This method provides an LC/MS alternative to the current LC/MS/MS methods and derivatization gas chromatography/mass spectrometry methods, and is applicable to difficult food products such as coffee, cocoa, and high-salt foods.     

Analytical method for the accurate determination of tricothecenes in grains using LC-MS/MS: a comparison between MRM transition and MS3 quantitation

The current food crisis demands unambiguous determination of mycotoxin contamination in staple foods to achieve safer food for consumption. This paper describes the first accurate LC-MS/MS method developed to analyze tricothecenes in grains by applying multiple reaction monitoring (MRM) transition and MS(3) quantitation strategies in tandem. The tricothecenes are nivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, fusarenon X, 3-acetyl-deoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, and HT-2 and T-2 toxins. Acetic acid and ammonium acetate were used to convert the analytes into their respective acetate adducts and ammonium adducts under negative and positive MS polarity conditions, respectively. The mycotoxins were separated by reversed-phase LC in a 13.5-min run, ionized using electrospray ionization, and detected by tandem mass spectrometry. Analyte-specific mass-to-charge (m/z) ratios were used to perform quantitation under MRM transition and MS(3) (linear ion trap) modes. Three experiments were made for each quantitation mode and matrix in batches over 6 days for recovery studies. The matrix effect was investigated at concentration levels of 20, 40, 80, 120, 160, and 200 μg kg(-1) (n = 3) in 5 g corn flour and rice flour. Extraction with acetonitrile provided a good overall recovery range of 90-108% (n = 3) at three levels of spiking concentration of 40, 80, and 120 μg kg(-1). A quantitation limit of 2-6 μg kg(-1) was achieved by applying an MRM transition quantitation strategy. Under MS(3) mode, a quantitation limit of 4-10 μg kg(-1) was achieved. Relative standard deviations of 2-10% and 2-11% were reported for MRM transition and MS(3) quantitation, respectively. The successful utilization of MS(3) enabled accurate analyte fragmentation pattern matching and its quantitation, leading to the development of analytical methods in fields that demand both analyte specificity and fragmentation fingerprint-matching capabilities that are unavailable under MRM transition.     

Determination of zaltoprofen in human plasma by liquid chromatography with electrospray tandem mass spectrometry: Application to a pharmacokinetic study

In the present study, we developed a method coupling liquid-liquid extraction (LLE) to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) to determine zaltoprofen levels in human plasma, using enalapril as internal standard (IS). The high sensitivity and specificity of MS/MS detection enabled the use of small plasma volumes (250 microL) and a simple LLE procedure. Furthermore, the short run-times (2 min) involved are compatible with the requirements of large-scale clinical studies. Ion acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions m/z 299.3 > 225.0 for zaltoprofen and m/z 377.4 > 234.2 for the IS enalapril. The limit of detection (LOD) was 0.01 microg/mL and the lower limit of quantitation (LLOQ) was 0.05 microg/mL. The devised method was linear over the studied range (0.05-20 microg/mL), with r2 > 0.99 and a run-time of 2 min. Intra-day precisions fell in the range 2.0-13.8%, inter-day precisions in the range 2.1-3.9%, and intra- and inter-day accuracies in the range 102.8-114.1%. The described method provides a fast and sensitive analytical tool for zaltoprofen and was successfully applied to a 24-subject pharmacokinetic study.     

Rapid determination of cyclamate in foods by solid-phase extraction and capillary electrophoresis

A method for the determination of cyclamate in food was developed using solid-phase extraction (SPE) and capillary electrophoresis (CE) with indirect ultraviolet (UV) detection. A 5-10 g sample in 0.1 mol/L hydrochloric acid was homogenized and made up to a volume of 50 mL with 0.1 mol/L hydrochloric acid. After the sample was centrifuged, 25 mL of supernatant was loaded into an Oasis HLB SPE cartridge. The cartridge was washed with 2 mL of demineralized water followed by 2 mL of 50% aqueous methanol, and cyclamate was eluted with 4.5 mL of 50% aqueous methanol. The eluate was added to a solution of sodium propionate (internal standard) for CE analysis. The cyclamate in the eluate was electrophoresed on a fused-silica capillary using 1 mmol/L hexadecyltrimethylammonium bromide and 10 mmol/L potassium sorbate as a running buffer. Detection and reference wavelengths of cyclamate determined with a UV detector were 300 and 254 nm, respectively. The calibration curves for cyclamate showed good linearity in the range of 2-1000 microg/mL and the limits of detection in beverage, fruit in syrup, jam, pickles and confectionary are sample dependent and ranged from 5-10 microg/g. The recovery of cyclamate added at a level of 200 microg/g to various kinds of foods was 93.3-108.3% and the relative standard deviation was less than 4.9% (n=3). A number of commercial samples were analyzed using the proposed method. Cyclamate was detected in one waume, two pickles, and two sunflower seeds. The quantitative values determined with CE correlated to those from high-performance liquid chromatography (HPLC) (the detected values of cyclamate in a sunflower seed measured by CE and HPLC were 3.40 g/kg and 3.51 g/kg, respectively). This analytical method for cyclamate using CE is especially suitable for use in the field.     

反相离子对高效液相色谱法快速分离和定量测定食品中的甜蜜素

采用反相离子对高效液相色谱法快速分离和测定食品中的甜蜜素。在ODS柱上,以V（甲醇）：V（水,含离子对试剂）=30：70的溶液为流动相进行分离,分别考察了流动相中离子对试剂和甲醇浓度对甜蜜素保留行为的影响。检测波长为205nm;采用外标法定量,测得甜蜜素在0．5～2．5g／L范围内具有良好的线性关系;回收率在96．9％～101．7％之间;检测限为0．05g／L。     

Use of a multifunctional column for the determination of deoxynivalenol in grains, grain products, and processed foods

Deoxynivalenol (DON), also known as vomitoxin, belongs to a class of naturally occurring mycotoxins produced by Fusarium spp. DON, 12, 13-epoxy-3,7 trihydroxytrichothec-9-en-8-one, is one of the most frequently detected mycotoxins in agricultural commodities worldwide. A method consisting of extraction, filtration, column cleanup, and RP-HPLC-UV separation and quantitation was validated for the determination of DON in grains (rice and barley), grain products (whole wheat flour, white flour, wheat germ, and wheat bran), and processed foods (bread, breakfast cereals, and pretzels). A 25 g test portion was extracted with 100 mL acetonitrile-water (84 + 16, v/v). After blending for 3 min, the supernatant was applied to a multifunctional column (MycoSep 225). The purified filtrate (2 mL) was evaporated to dryness and redissolved in the mobile phase. The toxins were then subjected to RP-HPLC-UV analysis. The accuracy and repeatability characteristics of the method were determined. Recoveries of DON added at levels ranging from 0.5 to 1.5 microg/g for all test matrixes were from 75 to 98%. SD and RSD(r) ranged from 0.7 to 11.6% and 0.9 to 12.7%, respectively. Within-laboratory HorRat values were from 0.1 to 0.7 for all matrixes analyzed. The method was found to meet AOAC method performance criteria for grains, grain products, and processed foods. The identity of DON in naturally contaminated test sample extracts was confirmed by HPLC/MS/MS analysis.     

婴幼儿奶瓶中迁移双酚A的高效液相色谱-电喷雾串联质谱检测方法研究

建立了婴幼儿奶瓶中双酚A(BPA)迁移量的高效液相色谱-电喷雾串联质谱(HPLC-ESI-MS/MS)测定方法。奶瓶食品模拟浸泡液经过弗罗里硅土玻璃层析柱净化,高效液相色谱分离,采用选择反应性监测模式(SRM)检测。以一级质谱得到的准分子离子m/z 227作为母离子,进行二级质谱(MS2)分析。选择MS2的碎片离子m/z 212、133、93定性确证,m/z 212作为定量离子定量。实验优化了质谱条件,并对二级质谱碎裂机理和特征离子进行了研究。测定结果的相对标准偏差不大于8.2%(n=7),回收率在87.7%～105%之间;检出限为2μg/L,能够满足欧盟、美国等对奶瓶中双酚A的限制要求。该法已成功应用于婴幼儿奶瓶中BPA迁移量的测定。     

液相色谱-电喷雾串联质谱法测定人血浆中的艾芬地尔

建立了测定人血浆中艾芬地尔的液相色谱-串联质谱方法。血浆样品用乙酸乙酯液-液提取后,以甲醇-6 mmol/L乙酸铵溶液(pH 7.40)(体积比为90∶10)为流动相进行分离。在Q TRAPTM串联质谱仪上,以选择性反应离子监测(SRM)方式进行定量分析,用于监测的离子为m/z 326.1→308.2 (艾芬地尔)和m/z 531.0→82.1 (酮康唑,内标)。在6 min内完成了艾芬地尔的检测,工作曲线的线性范围为0.25～50 μg/L,日内、日间精密度分别小于2.7%和6.5%,平均回收率为101.3%～105.0%,检测限为0.08 μg/L。本方法灵敏度高,特异性好,可以用于临床试验的血浆样品的检测。     

Detection and identification of zeranol in chicken or rabbit liver by liquid chromatography-electrospray tandem mass spectrometry

A sensitive, specific, and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for detection and identification of zeranol in chicken or rabbit liver. A homogenized liver sample was hydrolyzed with beta-glucuronidase/arylsulfatase, and the hydrolysate was extracted with ethyl ether. The supernatant was evaporated to dryness, and the residue was dissolved in chloroform and re-extracted with sodium hydroxide. After acidification, the extract was cleaned up on a C18 solid-phase extraction cartridge and analyzed by electrospray LC-MS/MS in the negative ion mode. The multiple reaction monitoring transition from both m/z 321 to 277 and m/z 321 to 303 was monitored for confirmation, and the product ion of 277 was used for quantitation. Separation was performed on a Waters XTettra C18 column (50 x 2.1 mm, 3.5 microm) combined with a safeguard column (Symmetry C18, 20 x 3.9 mm, 5 microm), using a gradient elution with acetonitrile and 20 mM ammonium acetate. Calibration curves were prepared and good linearity was achieved over the concentration ranges tested. For all liver samples fortified at 3 different levels of 1, 5, and 50 microg/kg, the overall recoveries and relative standard deviations were in the range of 61-90 and 8-13%, respectively. The limit of quantitation based on the assay validation was 1 microg/kg. The method had been used on a routine basis for detection and identification of zeranol in liver samples.     

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of anatoxins in cyanobacteria and drinking water

Anatoxin-a (AN) and homoanatoxin-a (HMAN) are potent neurotoxins produced by a number of cyanobacterial species. A new, sensitive liquid chromatography/multiple tandem mass spectrometry (LC/MS(n)) method has been developed for the determination of these neurotoxins. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer in positive ion mode. The [M+H](+) ions at m/z 166 (anatoxin-a) and m/z 180 (homoanatoxin-a) were used as the precursor ions for multiple MS experiments. MS(2)bond;MS(4) spectra displayed major fragment ions at m/z 149 (AN), 163 (HMAN), assigned to [Mbond;NH(3)+H](+); m/z 131 (AN), 145 (HMAN), assigned to [Mbond;NH(3)bond;H(2)O+H](+), and m/z 91 [C(7)H(7)](+). Although the chromatographic separation of these neurotoxins is problematic, reversed-phase LC, using a C(18) Luna column, proved successful. Calibration data for anatoxin-a using spiked water samples (10 mL) in LC/MS(n) modes were: LC/MS (25-1000 microg/L), r(2) = 0.998; LC/MS(2) (5-1000(microg/L), r(2) = 0.9993; LC/MS(3) (2.5-1000 microg/L), r(2) = 0.9997. Reproducibility data (% RSD, N = 3) for each LC/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L. The detection limit (S/N = 3) for AN was better than 0.03 ng (on-column) for LC/MS(3) which corresponded to 0.6 microg/L.     

Liquid chromatography/electrospray ionization tandem mass spectrometry analysis of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)

A quantitative liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed for the analysis of the explosive, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). In negative ionization mode, HMX forms an acetate adduct ion [M + CH(3)COO](-), m/z 355, in the presence of a small amount of acetic acid in the mobile phase. The ESI collision-induced dissociation (CID) spectrum of m/z 355 was acquired and the transitions m/z 355 --> 147 and m/z 355 --> 174 were chosen for the determination of HMX in samples. Using this quantification technique, the method detection limit was 1.57 microg/L and good linearity was achieved in the range 5-500 microg/L. This method will help to unambiguously analyze environmentally relevant concentrations of HMX.     

高效液相色谱-线性离子阱串联质谱技术用于白酒中甜蜜素测定的确证分析

建立了白酒中甜蜜素的高效液相色谱-线性离子阱串联质谱测定方法。该方法可同时准确定性和定量。样品无需前处理,过膜后直接进样,由C18色谱柱分离,采用多反应监测(MRM)和触发增强子离子扫描模式检测,采集到的MRM数据用于定量测定,同时得到的高质量子离子谱图用于谱图库检索的方法进行定性确证分析。本文采用外标法定量,方法的线性范围为1.320～132.0 μg/L（r=0.9991）;检出限(信噪比为3)为0.1 μg/L;添加水平分别为2.640、26.40、100.0 μg/L的3个样品的加标回收率为96.38%～107.2%,相对标准偏差均小于9%;阳性样品的谱图匹配度均高于92%。该方法简便、准确、高效,适用于白酒中甜蜜素的测定及阳性样品的确证分析。     

Quantitative LC/MS/MS method and in vivo pharmacokinetic studies of vitexin rhamnoside, a bioactive constituent on cardiovascular system from hawthorn

A simple and accurate liquid chromatography coupled with tandem mass spectrometry method was developed for determination and in vivo pharmacokinetic studies of vitexin rhamnoside in rat plasma. After protein precipitation using methanol, the analytes were separated by a Luna C(18) column with an isocratic elution and analyzed by mass spectrometry in multiple reaction monitoring mode using the respective negative ion at m/z 577.2-293.0 for vitexin rhamnoside and m/z 593.2-413.0 for internal standard (IS) vitexin glucoside. The method was validated systematically within the concentration range 5-5000 microg/L (R > 0.996) and the lower limit of quantitation was 5 microg/L. Acceptable precision and accuracy were acquired for concentrations over the standard curve range. It was further applied to assess pharmacokinetics and bioavailability of vitexin rhamnoside after intravenous and oral administration to rats. The oral bioavailability of vitexin rhamnoside was only 3.57%, which indicated that vitexin rhamnoside had poor absorption or underwent extensive first-pass metabolism. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.     

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of yessotoxins in shellfish

Yessotoxins are a group of large polyether toxins, produced by marine dinoflagellates, which cause widespread contamination of filter-feeding shellfish. A new, sensitive liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of yessotoxin (YTX) and 45-hydroxy-yessotoxin (45-OHYTX), a major metabolite in shellfish. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap MS in negative mode. The molecular related ion species at m/z 1141 [M-2Na+H]- was used as the parent ion for multiple MS experiments. MS-MS and MS3 gave major fragment ions at m/z 1061 [1141-SO3H]- and m/z 945 [1061-C9H12O]-. Predominant ions, that are due to the fragmentation of the backbone structure of YTXs, were observed at the MS4 stage. Reversed-phase LC using a C16 amide column was preferable to C18 phases for the separation of YTX and 45-OHYTX. Optimum calibration and reproducibility data were obtained for YTX using LC-MS-MS; r 2=0.9960, RSD < or = 6.3% at 0.25 microg YTX/g (n=5). The detection limit (S/N=3) was 30 pg YTX on-column which corresponded to 3 ng/g shellfish tissue.     

Development and validation of a high-throughput based on liquid chromatography with ultraviolet absorption and mass spectrometry detection method for quantitation of cichoric acid in Echinacea purpurea aerial-based dietary supplements

A new, rapid, and reproducible reversed-phased liquid chromatography (LC) method with ultraviolet (UV) absorption and/or mass spectrometry (MS) detection has been developed and validated for quantitation of cichoric acid, a major constituent of Echinacea spp. The method involves the use of a short Phenomenex Hydro-RP C18 column (4 microm, 50 mm x 3.0 mm id) and a simple isocratic mobile phase profile. Both UV (diode array detector) and selective-ion monitoring (SIM) at m/z 472.8 were used for quantitation of cichoric acid. The limit of detection was 0.75 ng for UV and 0.15 ng for MS-SIM, and the limit of quantitation was is 2.5 ng for UV and 0.5 ng for MS-SIM. Water-methanol (1 + 1) soluble extracts of 6 commercially available Echinacea purpurea aerial parts-based dietary supplements (EPADS). EPADS were first profiled using a traditional HPLC-UV method. Their UV chromatograms were compared, and cichoric acid was identified to be a key biomarker for EPADS. Then the samples were analyzed by the fast LC-UV/MS method. The turnaround time for a single analysis was 3 min, compared to 15 to 60 min needed for traditional reported LC methods. The high-throughput method was able to separate the cichoric acid peak from peaks of other components in extracts of complex matrixes of EPADS.     

液相色谱串联质谱法测定冷冻饮品中环己基氨基磺酸钠

建立了液相色谱～串联质谱测定冷冻饮品中环己基氨基磺酸钠的方法。冷冻饮品经净化、冷冻离心后用水稀释定容,采用C18色谱柱（50mm×2．1mm,1．7μm）,以乙腈-0．01mol／L乙酸铵缓冲溶液为流动相,电喷雾离子源负离子模式（MRM）定性、定量测定环己基氨基磺酸钠。环己基氨基磺酸钠的质量浓度在2—1000μg／L范围内与峰面积呈线性关系,相关系数,=0．9999,方法的定量限为0．02mg／kg。在10—100μg／L范围内,3水平加标回收率为87．5％～101．8％,测定结果的相对标准偏差为3．0％（H=6）。该方法净化效果好,灵敏度高,能快速完成冷冻饮品中环己基氨基磺酸钠的测定。     

Evaluation of atmospheric pressure ionization interfaces for quantitative measurement of sulfonamides in honey using isotope dilution liquid chromatography coupled with tandem mass spectrometry techniques

A comparison was made between electrospray, atmospheric pressure chemical and atmospheric pressure photospray ionizations to evaluate the MS/MS responses of standard sulfonamides and honey spiked samples. The sample preparation entails an acidic hydrolysis followed by a liquid/liquid extraction. Full method validation was realised by LC-APPI-MS/MS. Decision limit and detection capability were calculated for each analyte (at 50 microg/kg) and ranged between 53.6 and 56.9 and 57.5 and 63.2 microg/kg, respectively. Limits of detection and of quantification ranged, respectively, at 0.4-4.5 and 1.2-15.0 microg/kg. Precursor ion scan experiments of m/z 92 were also carried out as a survey experiment, linked with an enhanced product ion scan experiment to potentially identified additional sulfonamides via a library search.     

Quantitative analysis of betulinic acid in mouse,rat and dog plasma using electrospray liquid chromatography/mass spectrometry

Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.     

An LC–MS/MS assay for the quantitative determination of 2‐pyridyl acetic acid,a major metabolite and key surrogate for betahistine,using low‐volume human K2EDTA plasma

Betahistine is widely used for the treatment of vertigo. Owing to first‐pass metabolism, 2‐pyridyl acetic acid (2PAA, major metabolite of betahistine) was considered as surrogate for quantitation. A specific and sensitive LC–MS/MS method was developed and validated for quantitation of 2PAA using turbo‐ion spray in a positive ion mode. A solid‐phase extraction was employed for the extraction of 2PAA and 2PAA d₆ (IS) from human plasma. Chromatographic separation of analytes was achieved using an ACE CN, 5 μm (50 × 4.6 mm) column with a gradient mobile phase comprising acetonitrile–methanol (90:10% v /v) and 0.7% v/v formic acid in 0.5 mm ammonium trifluoroacetate in purified water (100% v/v). The retention times of 1.15 and 1.17 min for 2PAA and internal standard, respectively, were achieved. Quantitation of 2PAA and internal standard was achieved by monitoring multiple reaction monitoring transition pairs (m /z 138.1 to m /z 92.0 and m /z 142.1 to m /z 96.1, respectively). The developed method was validated for various parameters. The calibration curves of 2PAA showed linearity from 5.0 to 1500 ng/mL, with a lower limit of quantitation of 5.0 ng/mL. The bias and precision for inter‐ and intra‐batch assays were <10%. The developed method was used to support clinical sample analysis.     

Rapid Quantification of Astilbin in Rat Plasma by Liquid Chromatography-tandem Mass Spectrometry and Its Application to Pharmacokinetic Study

Astilbin is a potential immunosuppressive agent with minor cytotoxicity. Its oral bioavailability is supposed to be rather low and therefore a sensitive analytical method is required for its pharmacokinetic study after oral administration. A simple, sensitive and rapid liquid chromatography-tandem mass spectrometry（LC-MS/MS） method was developed and validated for the determination of astilbin in rat plasma. Plasma samples were subjected to liquid-liquid extraction with ethyl acetate and separated by reversed phase high performance liquid chromatography（HPLC） with methanol-0.01%（volume fraction） formic acid（50：50, volume ratio） as mobile phase. Quantitive determination was achieved on negative LC-MS/MS by a multiple reaction moitoring method with transitions m/z 449.1→150.9（quantifier） and m/z 449.1→284.9（qualifier） for astilbin and m/z 128.9→42.0 for internal standard（IS）. A lower limit of quantification（LLOQ） of ng/mL was achieved within a short cycle time of 3.4 min. The method was successfully applied to a pharmacokinetic study involving oral and intravenous administrations of 6 mg/kg astilbin to six rats.