Characterization of eight terpenoids from tissue cultures of the Chinese herbal plant,Tripterygium wilfordii,by high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

In this study, a reliable method for analysis and identification of eight terpenoids in tissue cultures of Tripterygium wilfordii has been established using high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MS). Our study indicated that sterile seedlings, callus cultures and cell‐suspension cultures can rapidly increase the amount of biological materials. HPLC‐ESI‐MS was used to identify terpenoids from the extracts of these tissue cultures. Triptolide, triptophenolide, celastrol and wilforlide A were unambiguously determined by comparing the retention times, UV spectral data, and mass fragmentation behaviors with those of the reference compounds. Another four compounds were tentatively identified as triptonoterpenol, triptonoterpene, 22β‐hydroxy‐3‐oxoolean‐12‐en‐29‐oic acid and wilforlide B, based on their UV and mass spectrometry spectra. The quantitative analysis showed that all three materials contain triptolide, triptophenolide, celastrol, wilforlide A, and the contents of the four compounds in the cell‐suspension cultures were 53.1, 240, 129 and 964 µg/g, respectively, which were at least 2.0‐fold higher than these in the sterile seedlings and callus cultures. Considering the known pharmacological activity of triptolide and celastrol, we recommend the cell‐suspension cultures as biological materials for future studies, such as clinical and toxicological studies. The developed method was validated by the evaluation of its precision, linearity, detection limits and recovery, and it was successfully used to identify and quantify the terpenoids in the tissue cultures. Copyright © 2014 John Wiley & Sons, Ltd.     

Separation and simultaneous determination of seven bioactive components in Tripterygium wilfordii Hook. F. and Tripterygium preparations by micellar electrokinetic capillary chromatography

A simple, comprehensive, and highly selective MEKC method has been developed for simultaneous analysis of seven bioactive components (triptolide, wilfortrine, wilfordine, wilforgine, wilforine, triptophenolide, and triptonide) in the root extracts of Tripterygium wilfordii Hook. F. (TWHF) and Tripterygium preparations (TPs). Optimal BGE consisted of 10 mM sodium tetraborate, 30 mM SDS, and 30% v/v methanol. The separation voltage was 20 kV and the temperature was 25°C. A DAD was used and the detection wavelength was at 218 nm. Under the optimum conditions, the baseline separation of seven components was achieved in less than 26 min. Excellent precision, good stability, and accuracy were obtained. For all analytes, linear calibrations were established within 10–100 μg/mL. The LOD and LOQ were within 1.2–4.2 μg/mL and 4.0–14 μg/mL, respectively. The developed method was suitable for the determination of key components in TWHF and TPs.     

Simultaneous determination of seven major diterpenoids in Siegesbeckia pubescens Makino by high‐performance liquid chromatography coupled with evaporative light scattering detection

A novel HPLC method with evaporative light scattering detection was developed for the simultaneous quantification of seven major diterpenoids of two types, including ent‐pimarane type: Kirenol, Hythiemoside B, Darutigenol, and ent‐kaurane type: ent‐16β,17,18‐trihydroxy‐kauran‐19‐oic acid, ent‐17,18‐dihydroxy‐kauran‐19‐oic acid, ent‐16β,17‐dihydroxy‐kauran‐19‐oic acid, 16α‐hydro‐ent‐kauran‐17,19‐dioic acid in the aerial parts of Siegesbeckia pubescens Makino, an important traditional Chinese medicinal herb. Chromatographic separation was achieved on a Waters Symmetry Shield^TM RP18 column (250 mm× 4.6 mm id, 5 μm) with a gradient mobile phase (A: 0.3% v/v aqueous formic acid and B: acetonitrile) at a flow rate of 1.0 mL/min. The drift tube temperature of evaporative light scattering detection was set at 103°C, and nitrogen flow rate was 3.0 L/min. The method was validated for accuracy, precision, LOD, and LOQ. All calibration curves showed a good linear relationship (r > 0.999) in test range. Precision was evaluated by intra‐ and interday tests that showed RSDs were less than 3.5%. Accuracy validation showed that the recovery was between 96.5 and 102.0% with RSDs below 2.8%. The validated method was successfully applied to determine the contents of seven diterpenoids in the different parts of Siegesbeckia pubescens Makino from two sources and to determine the contents of ent‐pimarane, ent‐kaurane, and total diterpenoids.     

Simultaneous separation of nine surfactants of various types by HPLC with evaporative light scattering detection

A simultaneous separation of cationic, amphoteric and nonioinc nine surfactants (DMDS, DMDP, DMDM, DMDL, BZC, CDE, A/O, SUNC, IMD) has been performed by a reverse phase-HPLC method utilizing a single J'sphere ODS (250 mm × 4.6 mm, 4 μm) column and a methanol-water containing 0.2% TFA eluent system within 60 min. The observed precision in determination of concentration was less than 5% R.S.D., which revealed that ELSD was an effective tool to detect the various studied surfactants of low volatility without chromophore. In addition, the detection limits were in the concentration range of 3.5-10 μg/mL, and the calibration curves, i.e. the log-log plots, were linear in the working range of 5-4600 μg/mL with the slopes of 1.321-1.668. The application of the analytical procedure to three household products without pretreatment supported that the presented chromatographic method was simple to be practical for a routine analysis of commercial products.     

Fingerprint chromatogram analysis of extracts from the leaves of Tripterygium wilfordii Hook. F. by high performance liquid chromatography

A simple and reliable high performance liquid chromatographic (HPLC) method has been developed and validated for the study of fingerprint chromatograms of extracts from the leaves of Tripterygium wilfordii Hook. F. (TWHF) and for controlling the quality of the herb. HPLC separation of the extracts was performed on a Lichrospher RP-18 column and detected by ultraviolet absorbance at 210 nm. The column temperature was maintained at 35 degrees C. A mobile phase composed of acetonitrile:H2O in the ratio of 39:61 (v/v) was found to be most suitable for this separation at a flow rate of 0.8 mL/min with isocratic elution. Under the chromatographic conditions described, the peak profile of the 10 components collected within 35 min made up the fingerprint of the extracts from leaves of TWHF with universal features. The fingerprint chromatograms had a good stability, precision, and reproducibility. The similarity of the extracts from leaves of TWHF collected in summer and winter was studied with triptolide as a reference peak. The method is suitable for differentiation of extracts from the leaves of TWHF, and can be used as a quality control method for this herb.     

Application of a sensitive and specific LC‐MS/MS method for determination of wilforine from Tripterygium wilfordii Hook. F. in rat plasma for a bioavailability study

A highly selective and specific LC‐MS/MS method was developed and validated for the determination of wilforine in rat plasma. The analyte was separated from plasma matrix by using methyl tertiary butyl ether liquid–liquid extraction with bulleyacinitine A as internal standard (IS). The analysis was carried out on a Sepax GP‐Phenyl column using a mixture of methanol and 10 mmol/L ammonium formate buffer solution containing 0.1% formic acid (75:25, v/v) as the mobile phase pumped at a flow rate of 1.0 mL/min. The detection was operated using a triple‐quadrupole mass spectrometer in multiple selected reaction monitoring with the parent‐to‐product quantifier transitions [M + H]⁺ m/z 867.6 →206.0 for wilforine and 664.1 →584.1 for IS. The main advantage of this method was the high sensitivity (a lower limit of quantification of 0.02 ng/mL) and the small amount of sample (0.1 mL plasma per sample). The method was fully validated to be accurate and precise with a linear range of 0.02–100 ng/mL, and successfully applied to a bioavailability study of wilforine in rats after intravenous and oral administration. The oral absolute bioavailability of wilforine in rats was estimated to be 84%. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous Determination of Triptolide and Tripdiolide in Extract of Tripterygium wilfordii Hook. f. by LC–APCI-MS

A novel method using liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry in the negative selected ion monitoring mode has been developed and validated for rapid simultaneous determination of triptolide and tripdiolide in the extract of Tripterygium wilfordii Hook. f. The molecular ions m/z [M–H]⁻ 359 and 375 were selected for the quantification in selected ion monitoring mode for triptolide and tripdiolide. Standard calibration curve was linear over the concentration range of 0.12–24 and 0.15–30 μg mL⁻¹ for triptolide and tripdiolide. The relative standard deviations of intra- and inter-day were in the range of 4.7–9.9 and 8.9–12.6%. The average recoveries were between 96.4 and 104.6%. The limits of quantitation were 2.0 × 10⁻³ and 2.5 × 10⁻³ μg mL⁻¹ for triptolide and tripdiolide.     

Development of a rapid and convenient method to separate eight ginsenosides from Panax ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detection

Ginsenosides exhibit diverse biological activities and are major well-known components isolated from the radix of Panax ginseng C.A. Meyer. In the present work, a rapid and facile method for the separation and purification of eight ginsenosides from P. ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detector (HSCCC-ELSD) was successfully developed. The crude samples for HSCCC separation were first purified from ginseng extract using a macroporous resin; the extract was loaded onto a Diaion-HP20 column and fractionated by methanol and water gradient elution. The ginsenosides-protopanaxadiol (PPD) and protopanaxatriol (PPT) fractions were subsequently eluted with 65 and 80% methanol and water gradient elution, respectively. Furthermore, these two fractions were separated by HSCCC-ELSD. The two-phase solvent system used for separation was composed of chloroform/methanol/water/isopropanol at a volume ratio of 4:3:2:1. Each fraction obtained was collected and dried, yielding the following eight ginsenosides: Rg(1), Re, Rf, Rh(1), Rb(1), Rc Rb(2) and Rd. The purity of these ginsenosides was greater than 97% as assessed by HPLC-ELSD, and their structures were characterized by electrospray-ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy. This is the first report regarding the separation of the ginsenosides Rh(1), Rb(2) and Rc from P. ginseng by HSCCC.     

Simultaneous determination of seven triterpenoids and triterpenoid saponins in Folium llicis Purpureae by high performance liquid chromatography coupled with evaporative light scattering detection

A new HPLC coupled with evaporative light scattering detection method was established for the simultaneous determination of seven triterpenoids and triterpenoid saponins in Folium Ilicis Purpureae traditional Chinese medicinal herb derived from the leaf of Ilex purpurea, namely, 3-omicron-beta-D-glucopyranosyl (1-->2)-beta-D-[6-omicron-methyl-glucuronopyranosyl]-28-omicron-beta-D-glucopyranosyl oleanolic acid (SQ-1), pedunculoside (SQ-2), 23-hydroxytormentic acid 28-omicron-beta-D-glucopyranoside (SQ-3), 23-hydroxytormentic acid (SQ-4), rutundic acid (SQ-5), ilexoside B (SQ-6), and ursolic acid (SQ-7). The optimal chromatographic conditions were achieved on a C18 column with a linear gradient elution of mobile phase A: deionized water-isopropyl alcohol-THF-acetic acid (90:10:6:1, v/v) and B: methanol-isopropyl alcohol-THF-acetic acid (90:10:6:1, v/v) at the flow rate of 1.0 mL/min; temperature for drift tube was set at 98degreesC and nitrogen flow rate was 2.8 L/min. All calibration curves of the seven compounds showed good linear regression (r2 > 0.995) within test ranges. The developed method has good repeatability for quantitation of all analytes interested with overall intraday and interday variation of less than 4.1%. The LOD (S/N = 3) and LOQ (S/N = 10) were less than 0.13 and 0.26 microg/mL, respectively, for all analytes. The established method was successfully used to evaluate the quality of Folium Ilicis Purpureae samples of different collections.     

Simultaneous determination of bioactive constituents in Danggui Buxue Tang for quality control by HPLC coupled with a diode array detector,an evaporative light scattering detector and mass spectrometry

Danggui Buxue Tang (DBT), a classical traditional Chinese formula comprising Radix Angelicae Sinensis (RAS) and Radix Astragali (RA), has been widely used to treat menopausal irregularity in Chinese women for nearly 800 years. In this study, a comprehensive analytical method of simultaneously determining the main types of bioactive constituents, eighteen in all from the formula, involving flavonoids, saponins, organic acid and some volatile compounds, was developed. This method was based on HPLC coupled to a diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) on a common reverse-phase C(18) column. Liquid chromatography coupled with on-line electrospray ionization mass spectrometry (LC-ESI-MS) was also used to further validate and analyze the constituents. It was found that 0.3% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution. This method, which showed good precision and accuracy, was successfully used to quantify the bioactive constituents in six products. As a result, the validated HPLC method, together with the LC-ESI-MS analysis, provided a new basis for assessing the quality of traditional Chinese medicinal compound preparations (TCMCPs) consisting of many bioactive components.     

Chemical fingerprinting of Shexiang Baoxin Pill and simultaneous determination of its major constituents by HPLC with evaporative light scattering detection and electrospray mass spectrometric detection

High-performance Liquid Chromatography (HPLC) with evaporative light scattered detection (ELSD) and electrospray ionization mass spectrometric detection (ESI-MS) was employed to establish chemical fingerprint of Shexiang Baoxin Pill (SBP) and to simultaneously determinate its seven major constituents, including cholic acid, deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, cinobufagin, recibufogenin, and ginsenoside Rb1. The analysis was performed on a C18 column with water-acetonitrile gradient elution, and the investigated constituents were authenticated by comparing their retention times and mass spectra with those of reference compounds. The proposed method was applied to analyze nine SBP samples and produced data with acceptable linearity, precision, stability and accuracy. Both the chemical fingerprints and quantification data were used to evaluate the quality of various SBP products. The proposed method allows obtaining chemical fingerprint and quantification of multi-components in one run, and therefore can be readily utilized as a comprehensive quality control approach for traditional Chinese medicine.     

Purification and determination of stachyose in Chinese artichoke (Stachys Sieboldii Miq.) by high-performance liquid chromatography with evaporative light scattering detection

A high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was applied for simultaneous determination of stachyose, sucrose and raffinose in Chinese artichoke (Stachys Sieboldii Miq.). Carbohydrates were separated on a Bondapak NH₂ column using a ternary solvent mixture of methanol-acetonitrile-water (55:25:20, v/v/v) as mobile phase. Two different crafts (method 1 and method 2) for extraction and purification of stachyose in Chinese artichoke tubers were detailed and evaluated. Method 1 can meet the purpose of quantification and method 2 is appropriate for the purpose of purification. The content of stachyose in dry tubers was determined to be 236.0 mg/g (from method 1) and the purity of the extracted stachyose flour was calculated to be 87.34% (from method 2), respectively. The analytical method fulfills all the standard requirements of linearity, accuracy and precision. Therefore, it is suitable for purification and routine quantification of stachyose in Chinese artichoke.     

Efficient protocol for purification of diosgenin and two fatty acids from Rhizoma dioscoreae by SFE coupled with high-speed counter-current chromatography and evaporative light scattering detection

Supercritical fluid extraction (SFE) was used to extract diosgenin, linoleic acid, and linolenic acid following acid hydrolysis from Rhizoma dioscoreae, a famous traditional Chinese medicine. The process was performed using a preparative SFE system under 35 MPa of pressure, 65 degrees C of temperature, and modified CO(2) with 95% ethanol for 180 min dynamic extraction. Then, the crude extract was successfully isolated and separated by high-speed counter-current chromatography (HSCCC) with evaporative light scattering detection (ELSD). A two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water was used for HSCCC separation in a stepwise elution mode. The upper phase of the solvent system at the volume ratio of 1:1:1.4:0.6 by volume was used as the stationary phase, and the mobile phase after 200 min was changed into the lower phase of the solvent system at the volume ratio of 1:1.2:1.4:0.6 by volume. The separation produced a total of 20.8 mg diosgenin, 12.1 mg linoleic acid, and 18.4 mg linolenic acid from 300 mg crude extract in one-step purification. The purities of the products were 98.9, 99.0, and 99.4%, respectively, as determined by HPLC. Their chemical structures were identified by MS, UV, and the standards.     

Simultaneous quantitative determination of 13 active components in the traditional Chinese medicinal preparation Suanzaoren oral liquid by HPLC coupled with diode array detection and evaporative light scattering detection

To control the quality of different forms of Suanzaoren decoction, an effective and reliable method for the simultaneous determination of 13 major components (neomangiferin, mangiferin, spinosin, liquiritin apioside, liquiritin, 6′′′‐feruloylspinosin, senkyunolide I, timosaponin BII, isoliquiritoside, timosaponin C, jujuboside A, jujuboside B, and timosaponin AIII) was developed and validated for the first time in this study using high‐performance liquid chromatography with diode array detection and evaporative light scattering detection. The chromatographic separation was performed on a Venusil MP C₁₈ column (250 mm × 4.6 mm, 5 μm) at 30°C with a gradient of acetonitrile/redistilled water as the mobile phase. Diode array detection was carried out at a wavelength of 275 nm. The drift tube temperature and the nitrogen gas flow rate of the evaporative light scattering detection were set at 50°C and 1.6 L/min, respectively. The newly developed method was successfully applied to the determination of 13 components in lab‐prepared Suanzaoren oral liquid, Suanzaoren mixture, and clinical Suanzaoren granules, and this study showed that this was a useful way to comprehensively evaluate the quality of Suanzaoren decoction in different forms of the preparation.     

Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post-column photochemical reaction

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7‐hydroxymethotrexate (7‐OH‐MTX) and 2,4‐diamino‐N^10‐methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7‐OH‐MTX, DAMPA and 2,4‐diaminopteroic acid (DAPA) as an internal standard were separated on a reversed‐phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7‐OH‐MTX and DAMPA from plasma at 100 pmol/mL were 81.5 ± 5.4, 82.5 ± 5.3 and 56.2 ± 7.0%, respectively. The limits of quantification for MTX, 7‐OH‐MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within‐ and between‐day variations for MTX, 7‐OH‐MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of triptolide, tripdiolide and tripterine in human urine by high-performance liquid chromatography coupled with ion trap atmospheric-pressure chemical ionization mass spectrometry

An accurate and selective method for the simultaneous determination of triptolide, tripdiolide and tripterine in human urine using hydrocortisone as an internal standard (IS) by high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization mass spectrometry in negative ion mode has been developed. After triptolide, tripdiolide and tripterine in human urine were extracted with ethyl acetate and cleaned by solid-phase extraction with C(18) cartridges, a satisfactory separation was achieved on an XDB C(18) short column (30 x 2.1 mm i.d., 3 microm) using the mobile phase of acetic acid-ammonium acetate (5 mmol/L, pH = 4.5)-acetonitrile-methanol in gradient elution. Detection was operated by APCI in selected ion monitoring mode. The target ions m/z 359, m/z 375, m/z 449 and m/z 419 were selected for the quantification of triptolide, tripdiolide, tripterine and IS, respectively. The linear range was 1.0-100.0 ng mL(-1), and the limits of quantification in human urine were found to be 0.1-0.5 ng mL(-1) for the three compounds. The precisions (CV%) and accuracies were 6.6-12.9 and 85.1-97.0%, respectively. The developed method could be applied to the determination of triptolide, tripdiolide and tripterine in human urine for diagnosis of the intoxication and for forensic purposes.     

Separation and purification of steroidal saponins from Paris polyphylla by microwave‐assisted extraction coupled with countercurrent chromatography using evaporative light scattering detection

A method of microwave‐assisted extraction coupled with countercurrent chromatography using evaporative light scattering detection was successfully developed for the separation and purification of steroidal saponins from Paris polyphylla. The main extraction conditions including microwave power, liquid/solid ratio, irradiation time, and extraction temperature were optimized using an orthogonal array design method. A suitable two‐phase solvent system consisting of n‐heptane/n‐butanol/acetonitrile/water (10:19:6:20, v/v/v/v) was employed in the separation and purification of the extracts of P. polyphylla. A total of 7.1 mg polyphyllin VII, 4.3 mg gracillin, 9.2 mg dioscin, and 10.2 mg polyphyllin I were obtained from 1.5 g P. polyphylla in less than 300 min, the purities of which determined by HPLC were 96.7, 97.3, 98.7, and 98.6%, respectively. The identification and characterization of these compounds were performed by LC–ESI‐MS and ¹H NMR spectroscopy. The results demonstrated that the proposed method is feasible, economical and efficient for the extraction, separation and purification of effective compounds from natural products.     

Chromatography of bis-(3-sulfopropyl) disulfide and its breakdown products by HPLC coupled with electrochemical detection

A chromatographic method for the detection of bis-(3-sulfopropyl) disulfide (SPS), a common additive in acidic copper plating baths, and its breakdown products is demonstrated. The detection scheme involves a combination of solid-phase extraction for sample pre-treatment, C(18) reversed-phase high-performance liquid chromatography column for separation, and electrochemical sensor for detection of all non-fully oxidized sulfur-containing compounds. We were able to achieve an effective separation and accurately assign chromatographic peaks to all detectable species. Owing to a high sensitivity of the utilized electrochemical detector, detection in low parts per billion range was possible. This can prove crucial for plating bath control, since minute amounts of certain by-products significantly affect the bath performance.     

Quantification and identification of bioactive metabolites from Kalopanacis Cortex by HPLC with evaporative light scattering detection and ESI quadrupole TOF MS

A method based on HPLC coupled with an evaporative light scattering detection and ESI quadrupole TOF MS was established for the quantification and identification of phenolics and triterpene saponins in Kalopanacis Cortex using a gradient elution of acetonitrile with 0.1% formic acid and water with 0.1% formic acid on an RP C₁₈ column (4.6 × 250 mm, 5 μm). Diverse validation parameters, such as the linearity, LOD and LOQ, accuracy, precision, repeatability, and stability, were successfully obtained. Additionally, the efficiencies of different extraction methods were compared. The developed method was applied for the quantitative analysis of twelve representative metabolites in 61 Kalopanacis Cortex samples. The quantitation results showed that coniferin, kalopanaxsaponin C, septemlosides II, III, C, and D exhibited distinct regional patterns in Kalopanacis Cortex samples. These six compounds including one new triterpene saponin show potential as marker compounds for evaluating the quality of Kalopanacis Cortex and the geographical variation in its chemical composition.     

A simple RP‐HPLC method for quantification of columbianadin in rat plasma and its application to pharmacokinetic study

A rapid and sensitive reversed‐phase high‐performance liquid chromatographic (RP‐HPLC) method was developed to investigate pharmacokinetics of columbianadin, one of the main bioactive constituents in the roots of Angelica pubescens f. biserrata, in rat plasma after intravenous administration to rats at two doses of 10 and 20 mg/kg. The method involves a plasma clean‐up step using liquid–liquid extraction by diethyl ether, followed by RP‐HPLC separation and detection. Separation of columbianadin was performed on an analytical Diamonsil? ODS C₁₈ column, with a mobile phase of MeOH–H₂O (85 : 15, v/v) at a flow‐rate of 1.0 mL/min, and UV detection was set at 325 nm. The retention time of columbianadin and scoparone (internal standard) was 6.7 and 3.5 min, respectively. The calibration curve was linear over the range of 0.2–20.0 μg/mL (r² = 0.9986) in rat plasma. The lower limits of detection and quantification were 0.05 and 0.1 μg/mL, respectively. The extraction recovery from plasma was in the range of 81.61–89.93%. The intra‐ and inter‐day precisions (relative standard deviation) were between 1.01 and 9.33%, with accuracies ranging from 89.76 to 109.22%. The results indicated that the method established was suitable for the determination and pharmacokinetic study of columbianadin in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.