Simultaneous determination of cyadox and its metabolites in plasma by high‐performance liquid chromatography tandem mass spectrometry

Cyadox is a novel antimicrobial growth‐promoter of the quinoxalines. For food safety and pharmacokinetic studies, a convenient, sensitive and reproducible LC‐ESI‐MS/MS method was developed for the simultaneous determination of cyadox and its major metabolites, quinoxaline‐2‐carboxylic acid, 1,4‐bisdesoxycyadox, cyadox‐1‐monoxide and cyadox‐4‐monoxide in chicken plasma. Plasma sample was subjected to a simple deproteinisation with acetonitrile. Analysis was performed on a C₁₈ column by detection with mass spectrometry in multiple reaction monitoring mode. A gradient elution program with 0.2% formic acid, methanol and acetonitrile was performed at a flow rate of 0.2 mL/min. The decision limits (CCαs) of five analytes in plasma ranged from 1.0 to 4.0 μg/L, and the detection capabilities (CCβs) were <10 μg/L. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The extraction recoveries of five analytes were between 87.4 and 93.9% in plasma at the spiked levels of 5 (10)–200 μg/L with the relative standard deviations <10% for each analyte. The developed method demonstrated a satisfactory applicability in real plasma samples.     

Simultaneous quantification of l‐tetrahydropalmatine and its urine metabolites by ultra high performance liquid chromatography with tandem mass spectrometry

l ‐tetrahydropalmatine (l ‐THP) is a tetrahydroprotoberberine isoquinoline alkaloid that has been used as an analgesic agent in China for more than 40 years. Recent studies indicated its potential application in the treatment of drug addiction. In this study, a sensitive and rapid method using ultra high performance liquid chromatography with MS/MS was developed and validated for simultaneous quantitation of l ‐THP and its desmethyl metabolites. Enzymatic hydrolysis was integrated into sample preparation to enable the quantitative determination of both free and conjugated metabolites. Chromatographic separation was achieved on an Agilent Poroshell 120 EC‐C₁₈ column. Detection was performed by MS in the positive ion ESI mode. The calibration curves of the analytes were linear (r² > 0.9936) over the concentration range of 1–1000 ng/mL with the lower limit of quantification at 1 ng/mL. The precision for both intra‐ and interday determinations was <8.97%, and the accuracy ranged from ?8.74 to 8.65%. The recovery for all the analytes was >70% without significant matrix effect. The method has been successfully applied to the urinary excretion study of l ‐THP in rats. The conjugates were found to be the major urine metabolites of the drug.     

Simultaneous analysis of ten phytohormones in Sargassum horneri by high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry

Phytohormones have attracted wide attention due to their important biological functions. However, their detection is still a challenge because of their complex composition, low abundance and diverse sources. In this study, a novel method of high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry was developed and validated for the simultaneous determination of ten phytohormones including indole‐3‐acetic acid, isopentenyladenine, isopentenyl adenosine, trans‐zeatin riboside, zeatin, strigolactones, abscisic acid, salicylic acid, gibberellin A3, and jasmonic acid in Sargassum horneri (S. horneri). The phytohormones were extracted from freeze‐dried S. horneri with methanol/water/methanoic acid (15:4:1, v/v/v) analyzed on a Hypersil Gold C₁₈ column and detected by electrospray ionization tandem triple quadrupole mass spectrometry in the multiple reaction monitoring mode. The experimental conditions for the extraction and analysis of phytohormones were optimized and validated in terms of reproducibility, linearity, sensitivity, recovery, accuracy, and stability. Distributions of the phytohormones in the stems, blades, and gas bladder of the S. horneri in drift, fixed, and semi‐fixed growing states were investigated for the first time. The observed contents of the phytohormones in S. horneri range from not detected to 5066.67 ng/g (fresh weight). Most phytohormones are distributed mainly in the stems of S. horneri in drift and semi‐fixed states.     

Chemical constituents and quality control of two Dracocephalum species based on high‐performance liquid chromatographic fingerprints coupled with tandem mass spectrometry and chemometrics

Two similar Dracocephalum species, namely, Dracocephalum tanguticum Maxim and Dracocephalum moldavica L, are commonly used as ethnic medicines in China. Here we describe a strategy of combining high‐performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry, as well as fingerprints and chemometrics for characterization and discrimination of chemical constituents on the two herbs. A total of 49 compounds including 33 flavonoids, 5 phenylethanol glycosides, 1 coumarin glycoside, 8 organic acids, and 2 other types of compounds were unambiguously or tentatively identified from the two Dracocephalum species. Among the compounds identified, 26 were characterized for the first time and 4 compounds, rosmarinic acid ( 7 ), salvianolic acid B ( 10 ), luteoloside ( 22 ), diosmetin‐7‐O‐glucoside ( 28 ), were inferred as common constituents for the two herbs. Flavonoids featured in these two Dracocephalum species while their types presented significant differences. Acacetin ( 45 ) and acacetin glycosides (acatetin‐7‐O‐glucuronide ( 30 ), acacetin‐7‐O‐(6”‐O‐malonyl) glucoside ( 33 ), buddleoside ( 34 ), tilianin ( 35 ), and agastachoside ( 42 )) were detected only in D. moldavica, which can be used to discriminate two herbal medicines. In addition, six characteristic constitutes in D. tanguticum were simultaneously quantified. Moreover, the induced chemometrics methods including similarity analysis and hierarchical clustering analysis were successfully applied for origin discrimination and quality evaluation of D. tanguticum and D. moldavica.     

Integrating qualitative and quantitative characterization of traditional Chinese medicine injection by high‐performance liquid chromatography with diode array detection and tandem mass spectrometry

The present study aims to describe and exemplify an integrated strategy of the combination of qualitative and quantitative characterization of a multicomponent mixture for the quality control of traditional Chinese medicine injections with the example of Danhong injection (DHI). The standardized chemical profile of DHI has been established based on liquid chromatography with diode array detection. High‐performance liquid chromatography coupled with time‐of‐flight mass spectrometry and high‐performance liquid chromatography with electrospray multistage tandem ion‐trap mass spectrometry have been developed to identify the major constituents in DHI. The structures of 26 compounds including nucleotides, phenolic acids, and flavonoid glycosides were identified or tentatively characterized. Meanwhile, the simultaneous determination of seven marker constituents, including uridine, adenosine, danshensu, protocatechuic aldehyde, p‐coumaric acid, rosmarinic acid, and salvianolic acid B, in DHI was performed by multiwavelength detection based on high‐performance liquid chromatography with diode array detection. The integrated qualitative and quantitative characterization strategy provided an effective and reliable pattern for the comprehensive and systematic characterization of the complex traditional Chinese medicine system.     

Robust method for the analysis of phytochelatins in rice by high‐performance liquid chromatography coupled with electrospray tandem mass spectrometry based on polymeric column materials

A sensitive and robust high‐performance liquid chromatography coupled with electrospray tandem mass spectrometry method for the identification and quantification of glutathione and phytochelatins from rice was developed. Homogenized samples were extracted with water containing 100 mM dithiothreitol, and solid‐phase extraction using polymer anion exchange resin was employed for sample purification. Chromatography was performed on a polymeric column with acetonitrile and water containing 0.1% formic acid as the mobile phase at the flow rate of 300 μL/min. The limit of quantitation was 6–100 nM. This assay showed excellent linearity for both glutathione and phytochelatins over physiological normal ranges, with correlation coefficients (r) > 0.9976. Recoveries for four biothiols were within the range of 76–118%, within relative standard deviations less than 15%. The intraday precision (n = 7) was 2.1–13.3%, and the interday precision over 15 days was 4.3–15.2%. The optimized method was applied to analyze tissue samples from rice grown using nutrient solutions with three different cadmium concentrations (0, 50, and 100 μM). With increasing cadmium concentrations, the content of phytochelatin 2 and phytochelatin 3 in rice roots increased, in contrast to most phytochelatins, and the content of glutathione in rice stems and roots decreased significantly.     

Rapid determination of quinolones in cosmetic products by ultra high performance liquid chromatography with tandem mass spectrometry

This study developed an improved analytical method for the simultaneous quantification of 13 quinolones in cosmetics by ultra high performance liquid chromatography combined with ESI triple quadrupole MS/MS under the multiple reaction monitoring mode. The analytes were extracted and purified by using an SPE cartridge. The limits of quantification ranged from 0.03 to 3.02 μg/kg. The precision for determining the quinolones was <19.39%. The proposed method was successfully developed for the determination of quinolones in real cosmetic samples.     

Rapid screening for anthocyanins and anthocyanin dimers in crude grape extracts by high performance liquid chromatography coupled with diode array detection and tandem mass spectrometry

A rapid and efficient method using high performance liquid chromatography coupled with diode array detection and tandem mass spectrometry (HPLC-DAD–MS/MS) for fast screening large numbers of anthocyanins and anthocyanin dimers in different grape skin extracts, without further sample clean-up procedures, was developed. A good separation of most detected anthocyanins was achieved in a run time of 15 min. Identification of anthocyanin pigments required a combination of several information: UV–vis spectra, MS and MS/MS spectra, and elution pattern. Many compounds have been here detected for the first time and their structures tentatively elucidated.     

Coupling nanoliter high‐performance liquid chromatography to inductively coupled plasma mass spectrometry for arsenic speciation

Nanoliter high‐performance liquid chromatography shows low consumption of solvents and samples, offering one of the best choices for arsenic speciation in precious samples in combination with inuctively coupled plasma mass spectrometry. A systematic investigation on coupling nanoliter high‐performance liquid chromatography to inductively coupled plasma mass spectrometry from instrument design to injected sample volume and mobile phase was performed in this study. Nanoflow mobile phase was delivered by flow splitting using a conventional high‐pressure pump with reuse of mobile phase waste. Dead volume was minimized to 60 nL for the sheathless interface based on the previously developed nanonebulizer. Capillary columns for nanoliter high‐performance liquid chromatography were found to be sensitive to sample loading volume. An apparent difference was also found between the mobile phases for nanoliter and conventional high‐performance liquid chromatography. Baseline separation of arsenite, arsenate, monomethylarsenic, and dimethylarsenic was achieved within 11 min on a 15 cm C₁₈ capillary column and within 12 min on a 25 cm strong anion exchange column. Detection limits of 0.9–1.8 μg/L were obtained with precisions variable in the range of 1.6–4.2%. A good agreement between determined and certified values of a certified reference material of human urine (GBW 09115) validated its accuracy along with good recoveries (87–102%).     

Chemical fingerprint of Ganmaoling granule by double‐wavelength ultra high performance liquid chromatography and ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A method incorporating double‐wavelength ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was developed for the investigation of the chemical fingerprint of Ganmaoling granule. The chromatographic separations were performed on an ACQUITY UPLC HSS C₁₈ column (2.1 × 50 mm, 1.8 μm) at 30°C using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. A total of 11 chemical constituents of Ganmaoling granule were identified from their molecular weight, UV spectra, tandem mass spectrometry data, and retention behavior by comparing the results with those of the reference standards or literature. And 25 peaks were selected as the common peaks for fingerprint analysis to evaluate the similarities among 25 batches of Ganmaoling granule. The results of principal component analysis and orthogonal projection to latent structures discriminant analysis showed that the important chemical markers that could distinguish the different batches were revealed as 4,5‐di‐O‐caffeoylquinic acid, 3,5‐di‐O‐caffeoylquinic acid, and 4‐O‐caffeoylquinic acid. This is the first report of the ultra high performance liquid chromatography chemical fingerprint and component identification of Ganmaoling granule, which could lay a foundation for further studies of Ganmaoling granule.     

Purification and identification of corn peptides that facilitate alcohol metabolism by semi‐preparative high‐performance liquid chromatography and nano liquid chromatography with electrospray ionization tandem mass spectrometry

In this study, peptides that facilitate alcohol metabolism were purified and identified from corn protein hydrolysates. The ultra‐filtered fraction with a molecular weight < 3 kDa (F3) potential activity was separated into six fractions (F3‐H1–F3‐H6) by semi‐preparative high‐performance liquid chromatography. Among the resultant six fractions, F3‐H4 and F3‐H5 exhibited the highest ability to eliminate alcohol in vivo. A total of 16 peptides with strong signal values were identified from F3‐H4 and F3‐H5 fractions by nano liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Several identified peptides were then selected and synthesized to determine their potential to facilitate alcohol metabolism. We found that Leu‐Leu and Pro‐Phe were the key structure units in Gln‐Leu‐Leu‐Pro‐Phe responsible for this peptide's ability to facilitate alcohol metabolism. However, the role of Leu‐Leu and Pro‐Phe may be affected by peptide chain length and hydrophobic properties. Our results have thus provided some insight into the study of the structure–activity relationships of corn peptides.     

Nevirapine quantification in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bioequivalence study

A rapid, sensitive and specific method to quantify nevirapine in human plasma using dibenzepine as the internal standard (IS) was developed and validated. The method employed a liquid-liquid extraction. The analyte and the IS were chromatographed on a C(18) analytical column, (150 x 4.6 mm i.d. 4 microm) and analyzed by tandem mass spectrometry in the multiple reaction monitoring mode. The method had a chromatographic run time of 5.0 min and a linear calibration curve over the range 10-5000 ng ml(-1) (r(2) > 0.9970). The between-run precision, based on the relative standard deviation for replicate quality controls was 1.3% (30 ng ml(-1)), 2.8% (300 ng ml(-1)) and 3.6% (3000 ng ml(-1)). The between-run accuracy was 4.0, 7.0 and 6.2% for the above-mentioned concentrations, respectively. This method was employed in a bioequivalence study of two nevirapine tablet formulations (Nevirapina from Far-Manguinhos, Brazil, as a test formulation, and Viramune from Boehringer Ingelheim do Brasil Química e Farmacêutica, as a reference formulation) in 25 healthy volunteers of both sexes who received a single 200 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 3 week washout interval. The 90% confidence interval (CI) of the individual ratio geometric mean for Nevirapina/Viramune was 96.4-104.5% for AUC((0-last)), 91.4-105.1% for AUC((0-infinity)) and 95.3-111.6% for C(max) (AUC = area under the curve; C(max) = peak plasma concentration). Since both 90% CI for AUC((0-last)) and AUC((0-infinity)) and C(max) were included in the 80-125% interval proposed by the US Food and Drug Administration, Nevirapina was considered bioequivalent to Viramune according to both the rate and extent of absorption.     

Rapid characterization of the chemical constituents and rat metabolites of the Wen‐Jing decoction by ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

The Wen‐Jing decoction, a traditional Chinese medicine formula, has been used as a blood‐activating and stasis‐eliminating drug to treat gynaecological syndromes, such as dysmenorrhea, amenorrhea, and menstrual disorders. However, its pharmacodynamic material basis and mechanism of action have not been thoroughly elucidated to date. The goal of this study was to characterize and identify multiple constituents and metabolites in Wen‐Jing decoction. An ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method was established and validated in the present study for the first time. A total of 101 compounds, including 11 monoterpene glycosides, 19 flavonoids, 49 triterpene saponins, 5 phthalides, 3 phytoecdysones, and 14 others, were unambiguously or tentatively characterized by comparing their retention times and MS data with reference standards or with data reported in the literature. After oral administration of Wen‐Jing decoction, 27 compounds, including nine prototype compounds and 18 metabolites were detected in rat plasma. Thus, the ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method was found to be efficient for in‐depth structural elucidation of chemical compounds in complex matrices of herbal medicines, which will provide useful chemical information for quality control and mechanism‐of‐action research.     

Quantification of bisphenol A and its selected analogs in serum using pre‐column derivatization with high‐performance liquid chromatography and tandem mass spectrometry

Due to regulation of the use of bisphenol A, several analogs serving as bisphenol A replacements have drawn substantial attention for their adverse health effects. To investigate their occurrence in humans and identify possible pollution sources, it is necessary to develop a sensitive method for total bisphenols detection. Thus, a method based on enzymolysis and liquid‐liquid extraction followed by molecularly imprinted polymer solid‐phase extraction and pre‐column derivatization with high‐performance liquid chromatography and tandem mass spectrometry was proposed. The developed method exhibited superior selectivity and sensitivity. The matrix effect can be eliminated to a great extent. The method detection limits for eight bisphenols were 0.05～0.19 ng/mL. Satisfactory recoveries (71～119%) were obtained by spiking bovine serum at three levels (0.8, 8 and, 20 ng/mL). The method was successfully applied to determine total bisphenols in the serum samples of children. Bisphenol A, bisphenol F, bisphenol S, bisphenol B and bisphenol F were detected with concentrations from below the method detection limit to 1.65, 0.45, 0.79, 2.04 and 0.17 ng/mL, respectively. These results indicate that bisphenol A remains the major pollutant among the studied bisphenols in children, whereas threats from bisphenol A analogs should also be monitored.     

Ultra-high performance liquid chromatography tandem mass spectrometry for comprehensive analysis of urinary acylcarnitines

We report an enabling mass spectrometric method for the analysis of lipid metabolites in order to define better the lipid metabolome in terms of chemical diversity and generate fragment ion spectra of these metabolites as a potential resource for unknown metabolite identification. This work focuses on the analysis of one important class of lipid metabolites, the acylcarnitines. Current analytical methods have only detected and identified a limited number of these metabolites. The method described herein provides the most comprehensive acylcarnitine profile in urine of healthy individuals up to date. It involves an optimized solid phase extraction technique for selective analyte extraction using cartridges containing both lipophilic and cation-exchange properties. The captured analytes are then subjected to ultra-high performance liquid chromatography (UPLC) separation, followed by tandem mass spectrometry (MS/MS) analysis using information-dependent acquisitions and selected reaction monitoring (SRM). The urine of six healthy individuals was analyzed using this method. A total of 355 acylcarnitines were detected; only 43 of them have been previously reported in the urine of healthy individuals. Detection of this large number of acylcarnitines illustrates the great diversity of the lipid metabolome as well as the usefulness of the method for profiling acylcarnitines. Furthermore, the MS/MS spectra of the 355 acylcarnitines will be uploaded to a public human metabolome database as a mass spectrometric resource for unknown metabolite identification.     

Screening anti‐tumor compounds from Ligusticum wallichii using cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry

Tyrosine 367 Cysteine‐fibroblast growth factor receptor 4 cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry was developed. Tyrosine 367 Cysteine‐HEK293 cells were used as the cell membrane stationary phase. The specificity and reproducibility of the cell membrane chromatography was evaluated using 1‐tert‐butyl‐3‐{2‐[4‐(diethylamino)butylamino]‐6‐(3,5‐dimethoxyphenyl)pyrido[2,3‐d]pyrimidin‐7‐yl}urea, nimodipine and dexamethasone acetate. Then, anti‐tumor components acting on Tyrosine 367 Cysteine‐fibroblast growth factor receptor 4 were screened and identified from extracts of Ligusticum wallichii. Components from the extract were retained on the cell membrane chromatographic column. The retained fraction was directly eluted into high‐performance liquid chromatography with mass spectrometry system for separation and identification. Finally, Levistolide A was identified as an active component from Ligusticum wallichii extracts. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide‐formazan colorimetric assay revealed that Levistolide A inhibits proliferation of overexpressing the mutated receptor cells with dose‐dependent manner. Phosphorylation of fibroblast growth factor receptor 4 was also decrease under Levistolide A treatment. Flex dock simulation verified that Levistolide A could bind with the tyrosine kinase domain of fibroblast growth factor receptor 4. Therefore, Levistolide A screened by the cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry can arrest cell growth. In conclusion, the two‐dimensional high‐performance liquid chromatography method can screen and identify potential anti‐tumor ingredients that specifically act on the tyrosine kinase domain of the mutated fibroblast growth factor receptor 4.     

Screening antiallergic components from Carthamus tinctorius using rat basophilic leukemia 2H3 cell membrane chromatography combined with high‐performance liquid chromatography and tandem mass spectrometry

Carthamus tinctorius, used in traditional Chinese medicine, has many pharmacological effects, such as anticoagulant effects, antioxidant effects, antiaging effects, regulation of gene expression, and antitumor effects. However, there is no report on the antiallergic effects of the components in C. tinctorius. In the present study, we investigated the antiallergic components of C. tinctorius and its mechanism of action. A rat basophilic leukemia 2H3/cell membrane chromatography coupled online with high‐performance liquid chromatography and tandem mass spectrometry method was developed to screen antiallergic components from C. tinctorius. The screening results showed that Hydroxysafflor yellow A, from C. tinctorius, was the targeted component that retained on the rat basophilic leukemia 2H3/cell membrane chromatography column. We measured the amount of β‐hexosaminidase and histamine released in mast cells and the key markers of degranulation. The release assays showed that Hydroxysafflor yellow A could attenuate the immunoglobulin E induced release of allergic cytokines without affecting cell viability from 1.0 to 50.0 μM. In conclusion, the established rat basophilic leukemia 2H3 cell membrane chromatography coupled with online high‐performance liquid chromatography and tandem mass spectrometry method successfully screened and identified Hydroxysafflor yellow A from C. tinctorius as a potential antiallergic component. Pharmacological analysis elucidated that Hydroxysafflor yellow A is an effective natural component for inhibiting immunoglobulin E–antigen‐mediated degranulation.     

Fast quantification of endogenous carbohydrates in plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

Endogenous carbohydrates in biosamples are frequently highlighted as the most differential metabolites in many metabolomics studies. A simple, fast, simultaneous quantitative method for 16 endogenous carbohydrates in plasma has been developed using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. In order to quantify 16 endogenous carbohydrates in plasma, various conditions, including columns, chromatographic conditions, mass spectrometry conditions, and plasma preparation methods, were investigated. Different conditions in this quantified analysis were performed and optimized. The reproducibility, precision, recovery, matrix effect, and stability of the method were verified. The results indicated that a methanol/acetonitrile (50:50, v/v) mixture could effectively and reproducibly precipitate rat plasma proteins. Cold organic solvents coupled with vortex for 1 min and incubated at –20°C for 20 min were the most optimal conditions for protein precipitation and extraction. The results, according to the linearity, recovery, precision, matrix effect, and stability, showed that the method was satisfactory in the quantification of endogenous carbohydrates in rat plasma. The quantified analysis of endogenous carbohydrates in rat plasma performed excellently in terms of sensitivity, high throughput, and simple sample preparation, which met the requirement of quantification in specific expanded metabolomic studies after the global metabolic profiling research.     

Profiling and identification of the absorbed constituents and metabolites of schisandra lignans by ultra‐performance liquid chromatography coupled to mass spectrometry

Schisandra chinensis Baill grows wild in Russia, China, Korea and Japan, and its fruit has been found to be effective in amnesia and insomnia. It is enriched in schisandra lignans (SL) that are major components responsible for therapeutic action. However, there are no reports on the biotransformation analysis of SL. An ultra‐performance liquid chromatography/electrospray‐ionization high‐definition mass spectrometry (UPLC‐Q‐TOF‐HDMS) method was developed to investigate the metabolism of SL in vivo. MS was performed on a Waters Micromass high‐definition system with an electrospray ionization source in positive ion mode and automated MetaboLynx software analysis with excellent MS accuracy and enhanced MS data acquisition. An improved mass defect filter (MDF) method employing both drug and core structure filter templates was applied to the processing of UPLC‐Q‐TOF‐HDMS data for the detection and structural characterization of metabolites. In this study, 30 metabolites were detected and identified in vivo, and demethylation and hydroxylation were confirmed as the primacy metabolic pathway for SL in rat plasma. In conclusion, the presently developed methodology was suitable for biotransformation research of SL and will find wide use in metabolic studies for other herbal medicines. Copyright © 2013 John Wiley & Sons, Ltd.