An efficient high-speed counter-current chromatography method for the preparative separation of potential antioxidants from Paeonia lactiflora Pall. combination of in vitro evaluation and molecular docking

Paeonia lactiflora Pall., one of the most famous classical herbal medicine, has been used to treat diseases for over 1200 years. In this research, the functional ingredients were purified by online-switch two-dimensional high-speed counter-current chromatography combined with inner-recycling and continuous injection mode. The antioxidant activity was evaluated by investigating the 2,2′-azobis (2-amidinopropane) dihydrochloride-induced oxidant damage in vitro and confirmed through molecular docking. n-Butanol/ethyl acetate/water (2:3:5, v/v) solvent system was used for the first-dimensional separation and optimized the sample loading. Two pure compounds and a polyphenol-enriched fraction were separated. The polyphenol-enriched fraction was separated with a solvent system n-hexane/ethyl acetate/methanol/water (2:8:4:6, v/v) with continuous injection mode. Five compounds were successfully separated, including gallic acid ( 1 ), methyl gallate ( 2 ), albiflorin ( 3 ), paeoniflorin ( 4 ), and ethyl gallate ( 5 ). Their structures were identified by mass spectrometry and NMR spectroscopy. The results from the antioxidant effect showed that albiflorin had stronger antioxidant activity. Molecular docking results indicated that the affinity energy of the identified compounds ranged from -3.79 to -8.22 kcal/mol and albiflorin showed the lowest affinity energy. Overall, all those findings suggested that the strong antioxidant capacity of albiflorin can be potentially used for the treatment of diseases caused by oxidation.     

Separation and purification of alkaloids and phenolic acids from Phellodendron chinense by pH-zone refining and online-storage inner-recycling counter-current chromatography

In this work, eight compounds from Phellodendron chinense were separated and purified by pH-zone refining counter-current chromatography and traditional counter-current chromatography coupled with online-storage inner-recycling counter-current chromatography (IRCCC). The pH-zone-refining mode was adopted for separating 2.0 g of crude extract with the solvent system of chloroform–methanol–water (4:3:3, v/v), in which 10 mM hydrochloric acid and 10 mM triethylamine were added in the stationary and mobile phases, respectively. Meanwhile, traditional counter-current chromatography coupled with online-storage IRCCC separation was performed by the solvent system of n-hexane-ethyl acetate-methanol-water (5:5:2:8, v/v). Finally, eight compounds, including six alkaloids as 6-methylpiperidin-2-one( 1 ), isoplatydesmine( 4 ), berlambine( 5 ), epiberberine( 6 ), palmatine( 7 ), berberine( 8 ) and two phenolic acids as ferulic acid( 2 ), isoferulic acid( 3 ), were successfully obtained using these three different CCC modes with the purities over 95.0%.     

Complete assignments of 1H and 13C NMR data for two new sesquiterpenes from Cyperus rotundus L.

Two new sesquiterpenes, epi‐guaidiol A (1) and sugebiol (3), together with four known sesquiterpenes, guaidiol A(2), sugetriol triacetate (4), cyperenoic acid (5), and cyperotundone (6) were isolated from the rhizomes of Cyperus rotundus L. Their structures were identified by MS and NMR experiments, and the complete assignments of ¹H and ¹³C NMR data for two new sesquiterpenes were obtained by the aid of two‐dimensional (2D) NMR techniques, including HSQC, HMBC, ¹H‐¹HCOSY and nuclear overhauser enhancement spectroscopy(NOESY). Copyright © 2009 John Wiley & Sons, Ltd.     

Application of one-step inner-recycling counter-current chromatography for the preparative separation and purification of chemical constituents from the rhizome of Bergenia ciliate (haw.) Sternb

Bergenia ciliata (haw.) Sternb, the renowned pharmaceutical plant in Jammu and Kashmir of Pakistan, is widely applied in treating different illnesses including diabetes, diarrhea, and vomiting. This work employed an efficient one-step inner-recycling counter-current chromatography for preparative separating and purifying compounds with similar partition coefficients from the rhizome of Bergenia ciliate (haw.). Five compounds, including quercetin rhamnodiglucoside ( 1 ), quercetin-3-O-rutinoside ( 2 ), bergenine ( 3 ), kaempferol ( 4 ), and palmatic acid ( 5 ), were successfully separated using the optimized biphasic solvent system that contained ter-butylmetylether/n-butanol/acetonitrile/water (2:2:1:5, v/v) with the purities over 98%. Mass spectrometry and nuclear magnetic resonance were conducted for structural identification. As a result, our proposed strategy might be applied in separating compounds with similar partition coefficients, which was advantageous with regard to the less solvent and time consumption, and the increased number of theoretical plates.     

Combined application of chromatographic techniques for the separation of phenolic compounds from Stenoloma chusanum Ching

High‐speed countercurrent chromatography combined with preparative high‐performance liquid chromatography was successfully used to separate seven phenolic compounds from Stenoloma chusanum Ching. A biphasic solvent system composed of hexane/ethyl acetate/methanol/water (1:2:1:2, v/v) was used for the first step high‐speed countercurrent chromatography separation in elution–extrusion mode. A mobile phase composed of acetonitrile (18%) and pure water (82%) was used for further preparative high‐performance liquid chromatography purification. In total, the combined separation yielded seven compounds, including 3,4‐dihydroxy benzoic acid, 3,4‐dihydroxy benzaldehyde, esculetin, caffeic acid, syringic acid, luteolin, and apigenin, at a purity of over 90%. Esculetin was separated from Stenoloma chusanum Ching for the first time. The results suggest that the proposed combination method is a useful strategy for separating compounds from complex samples.     

CE‐ESI‐MS coupled with dynamic pH junction online concentration for analysis of peptides in human urine samples

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE‐ESI‐MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol–water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L ‐Ala‐L ‐Ala (pI=5.57), L ‐Leu‐D ‐Leu (pI=5.52), Gly‐D ‐Phe (pI=5.52) and Gly‐Gly‐L ‐Leu (pI=5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2–2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45–3.57 and 4.93–6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.     

A Three-Phase Solvent System in High-Speed Counter-Current Chromatographic for the Separation and Purification of Bioactive Constituents from Acanthus ilicifolius

A new three-phase solvent system, n-hexane–acetonitrile–dichloromethane–water–ethyl acetate (5:5:1:5:1.5, v/v/v/v/v) was developed for the high-speed counter-current chromatographic (HSCCC) separation and purification of five bioactive constituents, syringic acid (1), vomifoliol (2), vanillic acid (3), 6-hydroxy-2-benzoxazolinone (4), and 2-benzoxazolinone (5), from Acanthus ilicifolius.

     

Counter‐current chromatographic method for preparative scale isolation of picrosides from traditional Chinese medicine Picrorhiza scrophulariiflora

Apocynin, androsin, together with picroside I, II and III from crude extracts of Picrorhiza scrophulariiflora were isolated by means of high‐speed counter‐current chromatography (CCC) combining elution‐extrusion (EE) and cycling‐elution (CE) approach. The EECCC took full advantages of the liquid nature of the stationary phase for a complete sample recovery and extended the solute hydrophobicity window, while CECCC showed its unique advantage in achieving effective separation of special compounds through preventing stationary phase loss. In the present work, the biphasic liquid system composed of n‐hexane/ethyl acetate/methanol/water (1:2:1:2, v/v/v/v) was used for separation of apocynin and androsin, ethyl acetate/n‐butanol/water/formic acid (4:1:5:0.005, v/v/v/v) for picroside I, II and III. However, due to the extremely similar K values (K₁/K₂≈1.2), picroside I and III were always eluted together by several biphasic solvent systems. In this case, the CECCC exhibited great superiority and baseline separated in the sixth cycle using ethyl acetate/water (1:1, v/v) as biphasic liquid system. Each fraction was analyzed by UPLC‐UV and ESI‐MS analysis, and identified by comparing with the data of reference substances. Compared with classical elution, the combination of EE and CE approach exhibits strong separation efficiency and great potential to be a high‐throughput separation technique in the case of complex samples.     

Development and validation of a UPLC–MS method for the determination of galantamine in guinea pig plasma and its application to a pre‐clinical bioavailability study of novel galantamine formulations

To evaluate the bioavailability and pharmacokinetic profiles of two novel galantamine formulations as medical countermeasure products, an ultra‐performance liquid chromatography–single quadrupole mass spectrometry (UPLC–MS) method was developed and validated for quantifying galantamine in guinea pig plasma using solid‐phase extraction with a mixed mode strong cation exchange reversed‐phase cartridge. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C₁₈ column maintained at 40°C. The mobile phases were solution A, acetonitrile–water, 5:95 (v/v) and solution B, acetonitrile–water 90:10 (v/v), both containing 2 mM ammonium formate and 0.2% formic acid. The mobile phase was delivered utilizing a 3 min gradient program start with 95%A–5%B at a flow rate of 0.6 mL/min. The analyte and internal standard, galantamine‐d3, were detected by selected ion monitoring mode on a Waters 3100 single quadrupole mass spectrometer with positive electrospray ionization. The method was validated according to the US Food and Drug Administration bioanalytical guidance. The method was selective and was linear over the analytical range of 2–2000 ng/mL. Accuracy and precision were acceptable with intra‐ and inter‐day accuracies between 96.8 and 101% and precisions (RSD) <4.88%. The method was successfully implemented to measure galantamine plasma levels in a series of pre‐clinical bioavailability studies for the evaluation of novel galantamine formulations.     

Determination of amine-containing phosphonic acids and amino acids as dansyl derivatives

Conditions are selected for the analytical separation of (N-phosphonomethyl glycine), products of its microbiological conversion, glutamic acid, and alanine as dansyl derivatives using reversed-phase HPLC: column (250 × 4.6 mm) ReproSil-PAH EPA; mobile phase, methanol + 20 mM CH₃COONa (pH 5.1) (20: 80); rate of mobile phase, 1 mL/min; working detector wavelength, 330 nm. The duration of separation is 35 min. The lower limits of the analytical range (in ng) for dansyl derivatives are as follows: glyphosate, 8.2; aminomethyl phosphonic acid, 24.2; glutamic acid, 9.4; alanine, 12.6: glycine, 17.7; and sarcosine, 19.3. The TLC study of dansyl derivatives of amino acids was performed on sorbfil plates PTSH-P-V using two-dimensional chromatography in the systems ethyl acetate-isopropanol-24% aqueous ammonia (45: 35: 20) in the first direction and chloroform-methanol-acetic acid (25: 5: 1) in the second one. For determining phosphonic acids (glyphosate and aminomethyl phosphonic acid), a version of one-dimensional chromatography with the sequential use of two systems, chloroform-methanol-acetic acid (25: 5: 0.2) and ethanol-24% aqueous ammonia (7: 3), was proposed.     

Application of off‐line two‐dimensional high‐performance countercurrent chromatography on the chloroform‐soluble extract of Cuscuta auralis seeds

In this study, the chloroform‐soluble extract of Cuscuta auralis was separated successfully using off‐line two‐dimensional high‐performance countercurrent chromatography, yielding a γ‐pyrone, two alkaloids, a flavonoid, and four lignans. The first‐dimensional countercurrent separation using a methylene chloride/methanol/water (11:6:5, v/v/v) system yielded three subfractions (fractions I–III). The second‐dimensional countercurrent separations, conducted on fractions I–III using n‐hexane/ethyl acetate/methanol/water/acetic acid (5:5:5:5:0, 3:7:3:7:0, and 1:9:1:9:0.01, v/v/v/v/v) systems, gave maltol ( 1 ), (−)‐(13S)‐cuscutamine ( 2 ), (+)‐(13R)‐cuscutamine ( 3 ), (+)‐pinoresinol ( 4 ), (+)‐epipinoresinol ( 5 ), kaempferol ( 6 ), piperitol ( 7 ), and (9R)‐hydroxy‐d ‐sesamin ( 8 ). To the best of our knowledge, maltol was identified for the first time in Cuscuta species. Furthermore, this report details the first full assignment of spectroscopic data of two cuscutamine epimers, (−)‐(13S)‐cuscutamine and (+)‐(13R)‐cuscutamine.     

Separation of chlorogenic acid and concentration of trace caffeic acid from natural products by pH‐zone‐refining countercurrent chromatography

Chlorogenic acid and caffeic acid were selected as test samples for separation by the pH‐zone‐refining countercurrent chromatography (CCC). The separation of these test samples was performed with a two‐phase solvent system composed of methyl‐tert‐butyl‐ether/acetonitrile/water at a volume ratio of 4:1:5 v/v/v where trifluoroacetic acid (TFA; 8 mM) was added to the organic stationary phase as a retainer and NH₄OH (10 mM) to the aqueous mobile phase as an eluter. Chlorogenic acid was successfully separated from Flaveria bidentis (L.) Kuntze (F. bidentis) and Lonicerae Flos by pH‐zone‐refining CCC, a slightly polar two‐phase solvent system composed of methyl‐tert‐butyl‐ether/acetonitrile/n‐butanol/water at a volume ratio of 4:1:1:5 v/v/v/v was selected where TFA (3 mM) was added to the organic stationary phase as a retainer and NH₄OH (3 mM) to the aqueous mobile phase as an eluter. A 16.2 mg amount of chlorogenic acid with the purity of 92% from 1.4 g of F. bidentis, and 134 mg of chlorogenic acid at the purity of 99% from 1.3 g of crude extract of Lonicerae Flos have been obtained. These results suggest that pH‐zone‐refining CCC is suitable for the isolation of the chlorogenic acid from the crude extracts of F. bidentis and Lonicerae Flos.     

Rapid preparative separation of six bioactive compounds from Gentiana crassicaulis Duthie ex Burk. using microwave‐assisted extraction coupled with high‐speed counter‐current chromatography

A rapid method combining microwave‐assisted extraction (MAE) and high‐speed counter‐current chromatography (HSCCC) was applied for preparative separation of six bioactive compounds including loganic acid ( I ), isoorientin‐4′‐O‐glucoside ( II ), 6′‐O‐β‐d ‐glucopyranosyl gentiopicroside ( III ), swertiamarin ( IV ), gentiopicroside ( V ), sweroside ( VI ) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. MAE parameters were predicted by central composite design response surface methodology. That is, 5.0 g dried roots of G. crassicaulis were extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by HSCCC with n‐butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail‐to‐head elution mode. 16.3, 8.8, 12., 25.1, 40.7, and 21.8 mg of compounds I–VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPLC with photo diode array detection and quadrupole TOF‐MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, MAE coupled with HSCCC could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.     

Separation of five flavone glycosides including two groups with similar polarities from Dracocephalum tanguticum by a combination of three high-speed counter-current chromatography modes

The separation of compounds with similar polarities is challenging. In the present study, five flavone glycosides, including two groups with similar polarities, were obtained from Dracocephalum tanguticum by three high-speed counter-current chromatography modes, including flow rate conversion mode, recycling mode, and heart-cut mode. With flow rate conversion mode, compounds 3 and 4 with similar polarities and compound 5 were separated by high-speed counter-current chromatography with ethyl acetate/methanol/water (5.0% acetic acid) (8:2:10, v/v) system. The flow rate was controlled as: 1.8 mL/min for 0–160 min, 2.2 mL/min for 160–200 min, and 2.5 mL/min for 200–400 min. However, compounds 1 and 2 with similar polarities were not separated due to the similar distributive properties. Then, a recycling and heart-cut mode were introduced to improve the separation efficiency. The heart-cut mode was introduced in the second and third cycles, and compounds 1 and 2 were well separated in the fourth cycle. Consequently, five flavone glycosides, including two groups with similar polarities were obtained and identified as cosmosiin (1), pedaliin (2), quercetin-3-O-rutinoside (3), pedaliin-6''-acetate (4), and sorbifolin-6-O-β-glucopyranoside (5). The current strategy provides a reference for separating compounds with similar polarities from a crude sample.     

Quantification of heteroclitin D in rat plasma: validation of an LC/MS/MS method and its application in a preclinical pharmacokinetic study

A rapid, sensitive and specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of heteroclitin D in rat plasma after using gambogic acid as internal standard (IS). Chromatographic separation was done on a Thermo Hypersil GOLD column (30 × 2.1 mm, 3 µm) using a mobile phase consisting of methanol–water–formic acid (80:20:0.1, v/v/v). The mass spectrometer worked with positive electrospray ionization in multiple reaction monitoring mode, using target ions at [M + H]⁺ m/z 483.3 for heteroclitin D and [M + H]⁺ m/z 629.3 for the IS. The standard curve was linear (R² ≥0.995) over the concentration range 9.98–2080 ng/mL and had good back‐calculated accuracy and precision. The intra‐ and interday precision and accuracy determined on three quality control samples (29.94, 166.4 and 1872 ng/mL) were ≤12.8 and –8.9–3.6%, respectively. The extraction recovery was ≥88.2% and the lower limit of quantification was 9.98 ng/mL. The method was successfully applied to evaluate pharmacokinetics of heteroclitin D in Sprague–Dawley rats following a single intravenous bolus injection of 2.0 mg/kg heteroclitin. Copyright © 2014 John Wiley & Sons, Ltd.     

Rapid separation of cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside from crude mulberry extract using high‐performance countercurrent chromatography and establishment of a volumetric scale‐up process

This study describes the rapid separation of mulberry anthocyanins; namely, cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside, using high‐performance countercurrent chromatography, and the establishment of a volumetric scale‐up process from semi‐preparative to preparative‐scale. To optimize the separation parameters, biphasic solvent systems composed of tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, flow rate, sample amount and rotational speed were evaluated for the semi‐preparative‐scale high‐performance countercurrent chromatography. The optimized semi‐preparative‐scale high‐performance countercurrent chromatography parameters (tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 4.0 mL/min; sample amount, 200–1000 mg; rotational speed, 1600 rpm) were transferred directly to a preparative‐scale (tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 28 mL/min; sample amount, 5.0–10.0 g; rotational speed, 1400 rpm) to achieve separation results identical to cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside. The separation of mulberry anthocyanins using semi‐preparative high‐performance countercurrent chromatography and its volumetric scale‐up to preparative‐scale was addressed for the first time in this report.     

Determination of the stress biomarker corticosterone in serum of tumor‐bearing mice by surrogate‐based liquid chromatography–tandem mass spectrometry

A simple, robust and specific liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed and validated to determine the concentration of corticosterone (Cort) which is usually regarded as a stress biomarker in mouse serum. Since Cort is an endogenous hormone, a ‘surrogate analyte’ strategy was adopted using the stable isotope‐deuterated corticosterone as a surrogate of the authentic analyte to generate the calibration curve. With telmisartan as the internal standard, the analytes were extracted with methanol, ethanol and acetone (1:1:1, v/v/v) and separated on a XTerra C₁₈ (2.1 × 50 mm, 3.5 µm) column using a mobile phase consisting of 0.2% formic acid in water–methanol (30:70, v/v). Detection was performed in multiple reaction monitoring mode with an electrospray ionization source operated in positive ion mode. The standard curves were linear (r² > 0.999) over the dynamic range of 8.60–430 ng/mL, with a lower limit of quantification of 8.60 ng/mL. The intra‐ and inter‐assay precisions were less than 15.0% of the relative standard deviation. This method was further used for analysis of serum samples from C57B/L tumor‐bearing mice before and after the treatment of fluoxetine. Validation of the assay and its application to the analysis demonstrated that the method was applicable to determine meaningful changes in Cort concentrations in serum samples of the tumor‐bearing mice for the stress status evaluation. Copyright © 2013 John Wiley & Sons, Ltd.     

A highly sensitive LC-MS/MS method for the determination of S-raclopride in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive and rapid assay method has been developed and validated for the estimation of S‐(−)‐raclopride (S‐RCP) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive ion mode. The assay procedure involves a simple liquid–liquid extraction technique for extraction of S‐RCP and phenacetin (internal standard, IS) from rat plasma. Chromatographic separation was achieved with 0.2% formic acid : acetonitrile (80:20, v/v) at a flow rate of 0.30 mL/min on a Phenomenex Prodigy C₁₈ column with a total run time of 4.5 min. The MS/MS ion transitions monitored were 347.2 → 112.1 for S‐RCP and 180.1 → 110.1 for IS. Method validation and pre‐clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity range was extended from 0.05 to 152 ng/mL in rat plasma. The intra‐day and inter‐day precisions were 0.23–10.5 and 3.74–7.29%, respectively. This novel method was applied to a pharmacokinetic study of S‐RCP in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High‐speed counter‐current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two‐phase solvent systems composed of CHCl₃–MeOH–(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12‐hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94–99% as determined by HPLC. Their chemical structures were characterized on the basis of ¹H‐NMR, ¹³C‐NMR, and LC‐ESI‐Q‐TOF‐MS/MS analyses.     

Ultra‐high‐performance liquid chromatography tandem mass spectrometry method for the determination of gambogenic acid in dog plasma and its application to a pharmacokinetic study

A highly sensitive and rapid ultra‐high‐performance liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of gambogenic acid in dog plasma. Gambogic acid was used as an internal standard (IS). After a simple liquid–liquid extraction by ethyl acetate, the analyte and internal standard were separated on an Acquity BEH C₁₈ (100 × 2.1 mm, 1.7 µm; Waters ) column at a flow rate of 0.2 mL/min, using 0.1% formic acid–methanol (10:90, v/v) as mobile phase. Electrospray ionization source was applied and operated in the positive ion mode. Multiple reaction monitoring mode with the transitions m/z 631.3 → 507.3 and m/z 629.1 → 573.2 was used to quantify gambogenic acid and the internal standard, respectively. The calibration curves were linear in the range of 5–1000 ng/mL, with a coefficient of determination (r) of 0.999 and good calculated accuracy and precision. The low limit of quantification was 5 ng/mL. The intra‐and inter‐day precisions (relative standard deviations) were <15%. The methodology recoveries were more than 66.63%. This validated method was successfully applied to a pharmacokinetic study after intravenous injection administration of gambogenic acid in dogs at a dose of 1 mg/kg. Copyright © 2014 John Wiley & Sons, Ltd.