Determination of Flavonoids in Costus speciosus and Etlingera elatior by Liquid Chromatography-Mass Spectrometry

In this article, a new and relatively simple liquid chromatography-mass spectrometry (LC-MS) method was developed for the simultaneous determination of five flavonoid compounds. Under optimized conditions, the LC-MS analysis can be achieved on a reverse-phase C₁₈ column (50 × 2.1 mm, 1.8 µm), with a binary mobile phase consisting of 0.1% formic acid in water and methanol under gradient elution conditions. The relative standard deviations (RSDs) of the retention times and peak areas for all analytes were in the range of 0.1–0.5% and 3.2–5.2%, respectively. The LODs and LOQs were in the range of 2.1–7.0 ng mL^?1 and 6.9–23 ng mL^?1, respectively. Furthermore, the developed LC-MS method was successfully applied to analyze flavonoids in different parts of two medicinal Zingiberaceae plants (i.e., Costus speciosus and Etlingera elatior) from different sources. The proposed LC-MS method was simple, effective, and reliable, and thus it has potential to be used for the quality control of other medicinal herbs.     

Qualitative and quantitative analysis of major constituents from Dazhu Hongjingtian capsule by UPLC/Q‐TOF‐MS/MS combined with UPLC/QQQ‐MS/MS

In this work, a sensitive and efficient method was established and validated for qualitative and quantitative analysis of major bioactive constituents in Dazhu Hongjingtian capsule by liquid chromatography tandem mass spectrometry. A total of 32 compounds were tentatively identified using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Furthermore, 12 constituents, namely gallic acid, 3,4‐dihydroxybenzoic acid, salidroside, p‐ coumaric acid‐4‐O ‐β ‐d ‐glucopyranoside, bergeninum, 4‐hydroxybenzoic acid, 4‐hydroxyphenylacetic acid, syringate, 6′′‐O ‐galloylsalidroside, rhodiosin, rhodionin and kaempferol‐7‐O ‐α ‐l ‐rhamnoside, were simultaneously quantified by the developed ultra‐performance liquid chromatography coupled with a triple quadrupole mass spectrometry method in 9 min. All of them were analyzed on an Agilent ZorBax SB‐C₁₈ column (3.0 × 100 mm, 1.8 μm) with linear gradient elution of methanol–0.1% formic acid water. The proposed method was applied to analyze three batches of samples with acceptable linearity (R , 0.9979–0.9997), precision (RSD, 1.3–4.7%), repeatability (RSD, 1.7–4.9%), stability (RSD, 2.2–4.9%) and recovery (RSD, 0.6–4.4%) of the 12 compounds. As a result, the analytical method possessing high throughput and sensitivity is suitable for the quality control of Dazhu Hongjingtian capsule.     

Analysis of phenolic acids and flavonoids in leaves of Lycium barbarum from different habitats by ultra‐high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry

The leaves of Lycium barbarum (LLB) have been utilized as crude drugs and functional tea for human health in China and Southeast Asia for thousands of years. To control its quality, a rapid and sensitive ultra‐high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry method was established and validated for the first time for simultaneous determination of 10 phenolic acids and flavonoids (including neochlorogenic acid , protocatechuic aldehyde, p‐hydroxybenzoic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, p‐coumaric acid, ferulic acid, rutin and kaempferol‐3‐O‐rutinoside) in LLB. The separation was performed on an Acquity UPLC C₁₈ chromatographic column (100 × 2.1 mm internal diameter, 1.7 μm particle size) with 0.1% formic acid in water (A)–acetonitrile (B) as the mobile phase under gradient elution. Multiple reaction monitoring mode was adopted to simultaneously monitor the target components. The developed method was fully validated in terms of linearity (r² ≥ 0.9860), precision (RSD ≤ 6.58%), repeatability (RSD ≤ 6.60%), stability (RSD ≤ 6.17%), recovery (95.56–108.06%, RSD ≤ 4.64%) and limit of detection (0.021–0.664 ng/mL) and limit of quantitation (0.069–2.210 ng/mL), and then successfully applied to evaluate the quality of 64 batches of LLB collected from 41 producing areas in four different provinces of China. The results showed that the LLB, especially collected from Inner Mongolia regions, were rich in the phenolic acids and flavonoids. Rutin, kaempferol‐3‐O‐rutinoside and chlorogenic acid are the predominant compounds contained in LLB. The above findings will provide helpful information for the effective utilization of LLB.     

Simultaneous enantioselective determination of isocarbophos and its main metabolite isocarbophos oxon in rice,soil, and water by chiral liquid chromatography and tandem mass spectrometry

An efficient enantioselective method for the simultaneous determination of isocarbophos and its main metabolite isocarbophos oxon in rice, soil, and water was developed using liquid chromatography with tandem mass spectrometry. The enantioseparation was performed on a Chiralpak AD‐3R column at 30°C using gradient elution. Target compounds were extracted from soil and rice using acetonitrile with omission of a clean‐up procedure, while a C₁₈ solid‐phase extraction column was used for water samples. Quantification was achieved using matrix‐matched calibration. The overall mean recoveries for isocarbophos and isocarbophos oxon enantiomers from the five matrices were 89.7–103 and 90.1–98.7%, with relative standard deviations of 2.1–5.4 and 2.5–4.7%, respectively. Moreover, the absolute configurations of isocarbophos oxon enantiomers were determined by liquid chromatography with tandem mass spectrometry through incubation of each isocarbophos enantiomer in soil, the first eluting enantiomer being confirmed as (R)‐(?)‐isocarbophos oxon. The proposed method was applied to real soil samples and satisfactory results were obtained.     

Simultaneous determination of praziquantel,pyrantel embonate,febantel and its active metabolites,oxfendazole and fenbendazole,in dog plasma by liquid chromatography/mass spectrometry

A liquid chromatography–electrospray–mass spectrometry method (LC/MS) has been developed and validated for determination of praziquantel (PZQ), pyrantel (PYR), febantel (FBT), and the active metabolites fenbendazole (FEN) and oxfendazole (OXF), in dog plasma, using mebendazole as internal standard (IS). The method consists of solid‐phase extractions on Strata‐X polymeric cartridges. Chromatographic separation was carried out on a Phenomenex Gemini C₆‐Phenyl column using binary gradient elution containing methanol and 50 mm ammonium–formate (pH 3). The method was linear (r² ≥ 0.990) over concentration ranges of 3–250 ng/mL for PYR andFEB, 5–250 ng/mL for OXF and FEN, and 24–1000 ng/mL for PZQ. The mean precisions were 1.3–10.6% (within‐run) and 2.5–9.1% (between‐run), and mean accuracies were 90.7–109.4% (within‐run) and 91.6–108.2% (between‐run). The relative standard deviations (RSD) were <9.1%. The mean recoveries of five targeted compounds from dog plasma ranged from 77 to 94%.The new LC/MS method described herein was fully validated and successfully applied to the bioequivalence studies of different anthelmintic formulations such as tablets containing PZQ, PYR embonate and FBT in dogs after oral administration. Copyright © 2015 John Wiley & Sons, Ltd.     

UPLC-PDA Analysis for Simultaneous Quantification of Four Active Compounds in Crude and Processed Rhizome of Polygonum multiflorum Thunb

A novel, rapid and specific ultra performance liquid chromatography-photo diode array detection method was developed for the simultaneous determination of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), emodin-8-O-β-d-glucoside (EMG), emodin (EM) and physcion (PS). The chromatographic separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm i.d., 1.7 μm). The mobile phase was a mixture of 0.3% acetic acid–water and 0.3% acetic acid–acetonitrile employing gradient elution at the flow rate of 0.4 mL min^?1. The four compounds behaved linearly in the concentration range between 60.80–3040.00 μg mL^?1 (TSG), 0.50–25.00 μg mL^?1 (EMG), 2.16–108.00 μg mL^?1 (EM) and 1.56–78.00 μg mL^?1 (PS), respectively with correlation coefficients >0.999. The precision of the method were below 5% RSD. Recoveries of the four compounds ranged from 95.71 to 102.97%, with RSD values less than 2%.     

A rapid and sensitive UPLC–MS/MS method for the determination of zanubrutinib in beagle plasma and its application in pharmacokinetics

A reliable and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed for the determination of zanubrutinib in the plasma of beagle dogs. The column used was an Acquity BEH C₁₈ column (2.1 mm × 50 mm, 1.7 μm), maintained at 40°C with an injection volume of 2 μl. The gradient elution program was as follows: 0–1 min, 10–10% A; 1–1.1 min, 10–90% A; 1.1–2.1 min, 90–90% A; 2.1–2.2 min, 90–10% A; 2.2–3.0 min, 10–10% A. Mobile phase A was 0.1% formic acid, B was acetonitrile, and the total analysis time was 3 min. The mass spectrometry was performed in positive ion mode, and the scanning mode was multi-reaction monitoring mode with electrospray ionization as the ion source; m/z 472.2 → 455.01 for zanubrutinib and m/z 441.03 → 137.99 for ibrutinib (internal standard). The plasma samples were processed by protein precipitation. The standard curve showed good linearity (r² = 0.999 8) in the range of 1.0–1,000 ng/ml (zanubrutinib) with a low limit of quantification of 1 ng/ml. Also, the intra-day and inter-day precision (RSD) was <5.88% and the accuracy (RE) ranged from −1.56 to 1.08%; the recoveries of zanubrutinib in beagle plasma ranged from 90.12 to 93.53% (RSD 1.67–6.42%) and the ME values of zanubrutinib were 98.70–101.06% (RSD 5.37–8.49%, n = 6). All values meet US Food and Drug Administration requirements. A rapid, highly selective and sensitive method for the determination of zanubrutinib concentration in plasma by UPLC–MS/MS was successfully developed. This method is suitable for pharmacokinetic studies in beagle dogs by following oral administration of zanubrutinib.     

Rapid and sensitive determination of major polyphenolic components in Euphoria longana Lam. seeds using matrix solid‐phase dispersion extraction and UHPLC with hybrid linear ion trap triple quadrupole mass spectrometry

A rapid and sensitive method for the extraction and determination of four major polyphenolic components in Euphoria longana Lam. seeds is presented for the first time based on matrix solid‐phase dispersion extraction followed by ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry. Matrix solid‐phase dispersion method was designed for the extraction of Euphoria longana seed constituents and compared with microwave‐assisted extraction and ultrasonic‐assisted extraction methods. An Ultra high performance liquid chromatography with hybrid triple quadrupole linear ion‐trap mass spectrometry method was developed for quantitative analysis in multiple‐reaction monitoring mode in negative electrospray ionization. The chromatographic separation was accomplished using an ACQUITY UPLC BEH C₁₈ (2.1 mm × 50 mm, 1.7 μm) column with gradient elution of 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile. The developed method was validated with acceptable linearity (r² > 0.999), precision (RSD ≤ 2.22%) and recovery (RSD ≤ 2.35%). The results indicated that matrix solid‐phase dispersion produced comparable extraction efficiency compared with other methods nevertheless was more convenient and time‐saving with reduced requirements on sample and solvent volumes. The proposed method is rapid and sensitive in providing a promising alternative for extraction and comprehensive determination of active components for quality control of Euphoria longana products.     

Validation of an analytical method using UPLC–MS/MS to quantify four bioactive components in rat plasma and its application to pharmacokinetic study of traditional and dispensing granules decoction of Ziziphi Spinosae Semen

A rapid and sensitive UPLC–MS/MS method was established for the simultaneous quantification of 6′′′-feruloylspinosin, spinosin, jujuboside A, and jujuboside B in rat plasma after the oral administration of traditional and dispensing granules (DG) decoction of Ziziphi Spinosae Semen (ZSS). The four components were separated using 0.1% formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.3 mL/min equipped with a C₁₈ column (2.1 × 50 mm, 1.7 μm particle size, Acquity BEH C₁₈). The mass spectrometer was operated under multiple reaction monitoring mode. An aliquot of 100 μL rat plasma was deproteinized by 300 μL methanol. The supernatant was injected into the UPLC–MS/MS system for analysis. The calibration curves displayed good linearity. The intra-day and inter-day precisions (RSD) were less than 7.3%. The accuracies ranged from −1.3 to 6.1%. The extraction recoveries ranged from 95.8 to 101.9%, and the matrix effects were satisfactory. For DG, half-life values (t_1/2) of 6′′′-feruloylspinosin and C_max of jujuboside A were elevated remarkably. MRT_0–t of jujuboside B was significantly increased. No significant variation was observed for the pharmacokinetic parameters of spinosin. The results could provide a scientific basis for the clinical application of traditional and DG decoction of ZSS.     

Determination of curdione in rabbit plasma by liquid chromatography mass spectrometry

A sensitive and selective liquid chromatography mass spectrometry method for determination of curdione in rabbit plasma was developed. After addition of tramadol as internal standard (IS), protein precipitation by acetonitrile was used for sample preparation. Chromatographic separation was achieved on a Zorbax SB‐C₁₈ (2.1 × 50 mm, 3.5 µm) column with acetonitrile–0.1% formic acid as mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive‐ion mode; selective ion monitoring was used for quantification using target fragment ions m/z 237 for curdione and m/z 264 for the IS. Calibration plots were linear over the range of 20–4000 ng/mL for curdione in plasma. The lower limit of quantification for curdione was 20 ng/mL. Mean recovery of curdione from plasma was in the range 94.3–98.4%. The RSD of intra‐day and inter‐day precision were both less than 9%. This method is simple and sensitive enough to be used in pharmacokinetic research for the determination of curdione in rabbit plasma. Copyright © 2011 John Wiley & Sons, Ltd.     

Solid-phase extraction and ultra high-performance liquid chromatography tandem mass spectrometry analysis of the gastrointestinal absorption of emodin in different digestive segments of rats

A rapid, simple and sensitive ultra high‐performance liquid chromatography (UHPLC‐MS/MS) method was established for determining the absorption amount of emodin in the five digestive segments of rat. Plasma samples were pre‐purified by solid‐phase extraction (SPE) with Oasis MAX cartridge. Separation of emodin and 1,8‐dihydroxyanthraquinone (internal standard) was performed on an Acquity BEH UHPLC C₁₈ column (100 mm×2.1 mm, 1.7 μm) with gradient elution of methanol and 0.1% formic acid aqueous solution. The LC/MS system was operated under multiple reaction monitoring mode using electrospray ionization (ESI) in negative ion mode. The results showed that this established method was valid and sensitive for emodin within 0.04–16.4 μg/mL, with low limits of detection and quantification of 0.6 ng/mL and 2.4 ng/mL, respectively and upper limit of quantification of 220.0 ng/mL. The intra‐ and interday variations were below 4.9% of RSD. The extraction recoveries were 98.9–106.1% with RSD of 1.9–3.2%. The plasma concentration‐time relationship showed that the absorption of emodin in stomach was faster than in intestine segments. The sequence of absorption amount was: ileum>jejunum>colon≈duodenum>stomach. Most of emodin was absorbed in ileum, and the absorption amount was increased with prolonged retention of drug form in intestine, especially in ileum and jejunum. The developed UHPLC‐ESI‐MS/MS method was appropriate for determining the in vivo absorption of emodin in other herbal medicines or preparations containing this compound.     

Simultaneous determination of thirteen main components and identification of eight major metabolites in Xuebijing Injection by UPLC/Q-TOF

An ultra performance liquid chromatographic method was used for the simultaneous identification and quantification of thirteen main components in Xuebijing Injection, including uridine, gallic acid, guanosine, danshensu, protocatechualdehyde, oxypaeoniflorin, hydroxysafflor yellow A, paeoniflorin, ferulic acid, safflor yellow A, senkyunolide I, senkyunolide H and salvianolic acid B. The chromatographic separation was performed on an Acquity UPLC BEH C₁₈ column (1.7-μm, 2.1 × 100 mm, i.d.) with a gradient elution of acetonitrile and 0.2% acetic acid at a flow rate of 0.4 mL/min. The method was validated for linearity (r ² > 0.9990), intra- and inter-day precision (RSD < 1.94%), accuracy (91.8–99.7%), recovery (96.8–103.8%), limits of detection (0.16–8.0 ng), and limits of quantification (0.54–26.8 ng). At least eight metabolites in prototype were found in rat plasma and urine after intravenous injection of 4 mL/kg doses of Xuebijing Injection. The proposed method could be utilized to qualify and control Xuebijing Injection to ensure its safety and efficacy in application.     

Fast determination of anthocyanins and free pelargonidin in fruits,fruit juices,and fruit wines by high‐performance liquid chromatography using a core–shell column

Anthocyanins are water‐soluble pigments that are liable for colors ranging from red to blue of most fruits, vegetables, and flowers. A novel and fast method was developed for the determination of five anthocyanins and free pelargonidin by high‐performance liquid chromatography coupled to photodiode array detection. A 10% formic acid and acetonitrile mixture was employed as mobile phase in the gradient elution mode. Mobile phase composition, column temperature, flow rate, injection volume, and column conditioning time were optimized by employing a stepwise strategy. Using a C₁₈ core–shell column (100 × 4.6 mm, 2.7 μm), the separation of six analytes was accomplished in less than 9.5 min with a run‐to‐run analysis time of 19 min. The developed method was validated with respect to linearity (r > 0.9999), limit of detection, limit of quantification, intra‐/interday precision (<2%), accuracy (98.6–104.4%), and specificity. Afterwards, the method was applied to the determination of anthocyanins present in 15 different samples including fruits, fruit juices, and fruit wines.     

Simultaneous determination of six components in commercial Roukou Wuwei pills using ultra‐high‐performance liquid chromatography with diode‐array detector

An efficient ultra‐performance liquid chromatography with diode‐array detector method was established for simultaneous determination of six active components in Roukou Wuwei pills, namely gallic acid, piperine, costundide, dehydrocostus lactone, isoalantolactone and alantolactone. Chromatographic separation of six components was successfully achieved on an Waters BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) with a mobile phase composed of acetonitrile and water using a gradient elution. Gallic acid and piperine were detected at 270 nm and 343 nm, respectively; while costundide, dehydrocostus lactone, isoalantolactone and alantolactone were simultaneously measured at 225 nm. All six calibration curves showed good linearity (R² ≥ 0.9994) between the peak area of each component and corresponding concentration. Relative standard deviations for inter‐ and intra‐day precisions were <0.45 and 0.77%, respectively. The mean recovery rates ranged from 96.72 to 102.2% with relative standard deviations <2.07%. The developed method was validated in terms of linearity, precision and accuracy and then successfully applied for the quality control of commercial Roukou Wuwei samples.     

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.     

Development of a validated UPLC–MS/MS method for PK/PD analysis of SYL930 and its two major metabolites in dogs

A sensitive and specific UPLC‐MS/MS method was developed and validated for the simultaneous determination of 2‐amino‐2‐(2‐(4′‐(2‐propyloxazol‐4‐yl)‐[1,1′‐biphenyl]‐4‐yl)ethyl)propane‐1,3‐diol (SYL930), phosphorylated metabolite (SYL930‐P) and hydroxylated metabolite (SYL930‐M) in dog blood using SYL927 and SYL927‐P, analogues of SYL930, as the internal standards. Analytes were extracted with protein precipitation followed by chromatographic separation on a ZorbaxSB‐C₁₈ column (3.5 μm, 2.1 × 100 mm) with a gradient elution of methanol–water containing 0.1% formic acid (v /v). A triple quadrupole tandem mass spectrometer operating in the positive electrospray ionization mode was used to detect SYL930, SYL930‐P, SYL930‐M and IS transitions of 381.2 → 364.2, 461.2 → 334.2, 397.3 → 380.3, 367.1 → 350.4 and 447.5 → 320.2, respectively. The linear calibration curves for SYL930, SYL930‐P and SYL930‐M were 0.5–500, 0.2–100 and 0.5–100 ng/mL, respectively (r ² > 0.99). The intra‐day and inter‐day precisions (RSD, %) of analytes did not exceed 9.16% except for low QCs (≤16.22%), and the accuracy (RE, %) ranged from −14 to 11.4%. The mean recoveries for SYL930, SYL930‐P and SYL930‐M in dog blood were 85.13–107.94, 73.84–80.08 and 85.64–95.44%, respectively. The validated method was successfully applied to pharmacokinetic and PK/PD studies of SYL930 and its two major metabolites in dogs after an oral administration of SYL930.     

Simultaneous determination of yohimbine,sildenafil, vardenafil and tadalafil in dietary supplements using high‐performance liquid chromatography‐tandem mass spectrometry

A simple and sensitive method was developed for determination of illegal adulterants (yohimbine, sildenafil, vardenafil and tadalafil) in dietary supplements by HPLC‐MS/MS. The separation was achieved on a C₁₈ column with the mobile phase consisting of acetonitrile and 0.1% acetic acid aqueous solution with a gradient elution at a flow rate of 0.5 mL/min. The analytes were quantified and identified by two characteristic transitions using the multiple‐reaction monitoring mode. The recoveries of the analytes ranged from 77.5 to 109.3% with the RSD less than 8.1% (n=6). The method has been successfully applied to screen illegal adulterations of natural dietary supplements.     

Determination of paeoniflorin,calycosin‐7‐O‐β‐d‐glucoside,ononin, calycosin and formononetin in rat plasma after oral administration of Buyang Huanwu decoction for their pharmacokinetic study by liquid chromatography–mass spectrometry

The purpose of this study was to simultaneously investigate the pharmacokinetics of five bioactive compounds in rat plasma after oral administration of Buyang Huanwu decoction (BYHWD) using high‐performance liquid chromatography coupled with mass spectrometry (HPLC‐MS). The separations were performed on a Thermo Hypersil Gold C₁₈ analytical column (50 × 2.1 mm, 3 µm) with the column temperature kept at 30°C. The quantitative analysis was performed using a quadrupole mass spectrometer detector operated under selected ion monitoring mode. A linear gradient elution of A (0.1% formic acid solution) and B (100% acetonitrile) was used at a flow rate of 0.2 mL/min. The method was validated within the concentration ranges 1.8–450, 6.0–1500, 2.0–500, 1.2–300 and 1.2–150 ng/mL for paeoniflorin, calycosin‐7‐O‐β‐d ‐glucoside, ononin, calycosin and formononetin, respectively. The calibration curves were linear with correlation coefficients > 0.99. The lower limits of quantitations were < 6.0 ng/mL. The method was further applied to assess the pharmacokinetics of the five bioactive constituents of BYHWD in rat plasma. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous ultra‐performance liquid chromatography–tandem mass spectrometry determination of six components in rat plasma after oral administration of Smilacis glabrae Roxb. extract

In this study, an accurate and reliable method of ultra‐performance liquid chromatography coupled with a triple‐quadrupole tandem mass spectrometry was firstly developed and fully validated for the simultaneous determination of epicatechin, neoastilbin, astilbin, isoastilbin, engeletin and resveratrol in rat plasma after administration of Smilacis glabrae Roxb. extract. Naringenin was used as an internal standard (IS). The analyte and IS were separated on a C₁₈ column by gradient elution with a mobile phase of acetonitrile–0.3% acetic acid at a flow rate of 0.25 mL/min for a total run time of 8 min. The method was validated in terms of selectivity, linearity, precision, accuracy, extraction recovery, matrix effect and stability. The developed method was successfully applied to determine the main pharmacokinetic parameters of six components in rat plasma.