Lantong Zhang

Simultaneous characterization and quantitation of 11 coumarins in Radix Angelicae Dahuricae by high performance liquid chromatography with electrospray tandem mass spectrometry

A high performance liquid chromatography with electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method has been developed to characterize and quantify 11 coumarin compounds in Radix Angelicae Dahuricae simultaneously. By using this HPLC-ESI-MS/MS method, all 11 coumarins were separated and determined within 10min. These coumarins were detected by ESI(+) ionization method and quantified by multiple reaction monitor (MRM). The linear regressions were acquired with r(2)>0.995, respectively. The precision was evaluated by intra- and inter-day tests, and relative standard deviation (R.S.D.) values were reported within the range of 1.14-4.42% and 0.37-4.00%. The recovery studies for the quantified compounds were observed over the range of 92.1-105.6% with R.S.D. values less than 4.55%. It demonstrated that the method developed was successfully applied for identification and quantification of 11 coumarins in Radix Angelicae Dahuricae. The results showed that the contents of coumarins in Radix Angelicae Dahuricae were processed differently and varied significantly.

Identification of urinary metabolites of imperatorin with a single run on an LC/Triple TOF system based on multiple mass defect filter data acquisition and multiple data mining techniques

The detection of drug metabolites, especially for minor metabolites, continues to be a challenge because of the complexity of biological samples. Imperatorin (IMP) is an active natural furocoumarin component originating from many traditional Chinese herbal medicines and is expected to be pursued as a new vasorelaxant agent. In the present study, a generic and efficient approach was developed for the in vivo screening and identification of IMP metabolites using liquid chromatography-Triple TOF mass spectrometry. In this approach, a novel on-line data acquisition method mutiple mass defect filter (MMDF) combined with dynamic background subtraction was developed to trace all probable urinary metabolites of IMP. Comparing with the traditionally intensity-dependent data acquisition method, MMDF method could give the information of low-level metabolites masked by background noise and endogenous components. Thus, the minor metabolites in complex biological matrices could be detected. Then, the sensitive and specific multiple data-mining techniques extracted ion chromatography, mass defect filter, product ion filter, and neutral loss filter were used for the discovery of IMP metabolites. Based on the proposed strategy, 44 phase I and 7 phase II metabolites were identified in rat urine after oral administration of IMP. The results indicated that oxidization was the main metabolic pathway and that different oxidized substituent positions had a significant influence on the fragmentation of the metabolites. Two types of characteristic ions at m/z 203 and 219 can be observed in the MS/MS spectra. This is the first study of IMP metabolism in vivo. The interpretation of the MS/MS spectra of these metabolites and the proposed metabolite pathway provide essential data for further pharmacological studies of other linear-type furocoumarins.

LC-MS/MS determination and pharmacokinetic study of five flavone components after solvent extraction/acid hydrolysis in rat plasma after oral administration of Verbena officinalis L. extract.

ETHNOPHARMACOLOGICAL RELEVANCE Traditional Chinese medicine (TCM) has been used in clinical practice for several thousand years. TCM has played an indispensable role in the prevention and treatment of diseases, especially the complicated and chronic ones. Pharmacokinetic study on active constituents in herbal preparations is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of TCM. AIM OF THE STUDY A selective and sensitive HPLC-MS/MS method was first developed and validated for the determination of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin in rat plasma. MATERIALS AND METHODS The LC system consisted of an Agilent Technologies Series 1200 system (Agilent, USA) equipped with an automatic degasser, a quaternary pump, and an autosampler. Chromatographic separations were performed on a Waters SunFire™ C(18) column (150 mm × 4.6mm, 5 μm), and the column temperature was maintained at 25°C and the sample injection volume was 20 μL. The current LC-MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability. RESULTS The validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of five flavone compounds in rat plasma after a single oral administration of Verbena officinalis L. extract with a dosage of 8.0 mL/kg. The time to reach the maximum plasma concentration (T(max1)) was 0.48 ± 2.14 h for luteolin, 0.25 ± 0.13 h for kaempferol, 0.97 ± 1.08 h for apigenin, 1.04 ± 4.25 h for quercetol and 0.25 ± 0.16 h for isorhamnetin, and the maximum plasma concentration (T(max2)) was 3.97 ± 1.48 h, 4.05 ± 0.46 h, 4.33 ± 0.58 h, 2.99 ± 0.48 h and 4.02 ± 0.34 h. The elimination half-time (t(1/2)) of luteolin, kaempferol, apigenin, quercetol and isorhamnetin was 4.02 ± 0.81, 7.65 ± 0.71, 3.30 ± 0.83, 4.55 ± 0.49 and 5.56 ± 1.32 h, respectively. CONCLUSIONS This paper described a simple, sensitive and validated LC-MS/MS method for simultaneous determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma after oral administration of V. officinalis L. extract, and investigated on their pharmacokinetic studies as well, with a short run time of 5 min.

Simultaneous quantification of 14 bioactive constituents in Forsythia suspensa by liquid chromatography-electrospray ionisation-mass spectrometry.

INTRODUCTION Forsythia suspensa is a commonly used traditional Chinese medicine including phenylethanoid glycosides, lignans, flavonoids, terpenes and volatile oils. Quantification of multi-components is important for the quality control of Forsythia suspensa. OBJECTIVE To establish a liquid chromatography-electrospray ionisation-mass spectrometry method for simultaneous quantification of 14 bioactive constituents of Forsythia suspensa in different places of China and different parts of this herb. METHODOLOGY The optimal chromatographic conditions were achieved on a Kromasil C(18) column (150 yen 4.6 mm, 5 microm) with gradient elution of methanol, acetonitrile and 0.1% formic acid in 27 min. Detection was performed in negative ionisation mode by monitoring the precursor-product combination in multiple reaction monitoring (MRM) mode. The validation of the method included tests of linearity, sensitivity, precision, repeatability, stability and accuracy. RESULTS All calibration curves showed good linear regression (r > 0.9990) within test ranges. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 0.7-4.3 and 1.1-3.9% respectively, and overall recoveries of 96.65-101.2% for the compounds analysed. CONCLUSION The proposed method was successfully applied for the quantitative analysis of 14 constituents in 12 Forsythia suspensa samples.

A comparative study on the pharmacokinetics of a traditional Chinese herbal preparation with the single herb extracts in rats by LC–MS/MS method

The Er-Mu preparation (EMP) is a well-known traditional Chinese prescription that has been clinically employed for the treatment of asthma and bronchial inflammation for hundreds of years. Neomangiferin, mangiferin, peimine, peiminine, timosaponin BII and timosaponin AIII are the major active ingredients of EMP for their anti-inflammatory or anti-asthmatic effects. The aim of this study was to investigate the pharmacokinetics of the target compounds from the recipe of EMP and the single herb extracts of Anemarrhenae asphodeloides Bge. (ARR) and Fritillariae cirrhosae D.Don (FCB), and the influence of compatibility on the pharmacokinetics of the main active ingredients. The rats were randomly assigned to three groups and orally administered with the recipe of EMP and the single herb extracts of ARR and FCB, respectively. The concentrations of the target compounds in rat plasma were determined by an optimal liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) and multiple reaction monitoring (MRM) with a multi-switching monitoring mode coupled with simple protein precipitation method, and the main pharmacokinetic parameters were estimated. Significant differences (p<0.05) were found in the pharmacokinetic parameters of neomangiferin, mangiferin, peimine and peiminine between the single ARR or FCB extract and the combination treatment (p<0.05). The developed HPLC-ESI-MS method by switching positive and negative ESI sources in a single run was successfully applied to study the pharmacokinetics of six compounds in SD rat, which was powerful in terms of sensitivity, selectivity, time savings and solvent consumption in the quantitative analysis of complex herbal medicines. It was surmised that formula compatibility could significantly influence the pharmacokinetics of EMP and our study has preliminarily elucidated the priority in the compatible administration of EMP based on pharmacokinetic studies.

Identification of metabolites of oridonin in rats with a single run on UPLC-Triple-TOF-MS/MS system based on multiple mass defect filter data acquisition and multiple data processing techniques.

Oridonin (ORI) is an active natural ent-kaurane diterpenoid ingredient originating from well-known traditional Chinese herb medicine and is expected to be pursued as a new anticancer agent. In the present study, a novel and efficient approach was developed for in vivo screening and identification of ORI metabolites using ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). This analytical strategy was as follows: an effective on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS), was developed to trace all of potential metabolites of ORI. The MMDF and DBS method could trigger an information dependent acquisition scan, which could give the information of low-level metabolites masked by background noise and endogenous components in complex matrix. Moreover, the sensitive and specific multiple data-mining techniques including extracted ion chromatography, mass defect filtering, product ion filtering and neutral loss filtering were employed to identify the metabolites of ORI. Then, structures for the metabolites were successfully assigned based on accurate masses, the mass fragmentation of ORI and metabolic knowledge. Finally, an important parameter Clog P was used to estimate the retention time of isomers. Based on the proposed strategy, 16 phase I and 2 phase II metabolites were detected in rats after oral administration of ORI. The main biotransformation route of ORI was identified as reduction, oxidation, dehydroxylation and glucuronic acid conjugation. This is the first study of ORI metabolism in vivo. This study not only proposed a practical strategy for rapidly screening and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of ORI in vivo. At the same time this methodology can be widely applied for the structural characterization of the metabolites of other ent-kaurane diterpenoid.

Simultaneous determination of five flavonoids from Scutellaria Barbata extract in rat plasma by LC–MS/MS and its application to pharmacokinetic study

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of five flavonoids including scutellarin, naringenin, apigenin, luteoline and wogonin in rat plasma using sulfamethalazole as internal standard (IS). Plasma samples were pretreated with liquid-liquid extraction procedure and acid hydrolysis method was used for converting conjugated flavonoids to their respective free forms. The chromatographic separation was performed on a C(18) column with a linear gradient elution using a mobile phase consisted of 0.01% acetic acid and methanol. The detection was accomplished by multiple-reaction monitoring (MRM) scanning with electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion-pairs (m/z) monitored for scutellarin, naringenin, apigenin, luteoline, wogonin and IS were 461.1/285.1, 271.0/119.0, 269.0/117.0, 285.0/132.9, 283.0/268.0 and 252.0/155.9, respectively. The method was linear for all analytes over investigated ranges with all correlation coefficients greater than 0.9915. The lower limit of quantitation (LLOQ) of scutellarin was 9.15 ng/mL and other compounds were all less than 2.0 ng/mL. The proposed method showed appropriate accuracy and repeatability and was suitable for pharmacokinetic studies of the five flavonoids after oral administration of Scutellaria Barbata extract.

Simultaneous determination and pharmacokinetic study of six flavonoids from Fructus Sophorae extract in rat plasma by LC-MS/MS.

In this study, a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of six flavonoids including sophoricoside, genistin, genistein, rutin, quercetin and kaempferol in rat plasma after oral administration of Fructus Sophorae extract using sulfamethalazole as internal standard (IS). The plasma samples were pretreated and extracted by liquid-liquid extraction. Chromatographic separation was accomplished on a C(18) column with a simple linear gradient elution. The detection was accomplished by multiple-reaction monitoring (MRM) scanning after electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 431.1/267.9 for sophoricoside and genistin, 269.0/133.0 for genistein, 609.2/300.0 for rutin, 301.0/150.9 for quercetin, 284.9/93.0 for kaempferol and 252.0/155.9 for IS. The total run time was 8.0 min. Full validation of the assay was implemented including specificity, linearity, accuracy, precision, recovery and matrix effect. This is the first report on determination of the major flavones in rat plasma after oral administration of Fructus Sophorae extract. The results provided a meaningful basis for the clinical application of this herb.

Simultaneous and sensitive determination of xanthotoxin, psoralen, isoimpinellin and bergapten in rat plasma by liquid chromatography-electrospray ionization mass spectrometry

A sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS) method has been developed and validated for the simultaneous determination of xanthotoxin (8-methoxypsoralen), psoralen, isoimpinellin (5,8-dimethoxypsoralen) and bergapten (5-methoxypsoralen) in rat plasma using pimpinellin as an internal standard (IS). The plasma samples were pretreated by protein precipitation with methanol and chromatographic separation was performed on a C(18) column with a mobile phase composed of 1 mmol ammonium acetate and methanol (30:70, v/v). The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization (ESI) source operating in the positive ionization mode. The optimized mass transition ion-pairs (m/z) for quantitation were 217.1/202.1 for xanthotoxin, 187.1/131.1 for psoralen, 247.1/217.0 for isoimpinellin, 217.1/202.1 for bergapten, and 247.1/231.1 for IS. The total run time was 6 min between injections. The calibration curves were linear over the investigated concentration range with all correlation coefficients higher than 0.998. The lower limits of quantitation (LLOQ) of these analytes were less than 1.21 ng/ml. The intra- and inter-day RSD were no more than 9.7% and the relative errors were within the range of -8.1% to 4.5%. The average extraction recoveries for all compounds were between 90.7% and 106.2%. The proposed method was further applied to the determination of actual plasma samples from rats after oral administration of Radix Glehniae extract.

LC-MS/MS determination and pharmacokinetic study of seven flavonoids in rat plasma after oral administration of Cirsium japonicum DC. extract.

ETHNOPHARMACOLOGICAL RELEVANCE Cirsium japonicum DC., a Traditional Chinese Medicine, has the curative effect of antihemorrhagic and antitumor. Pharmacological studies prove that the curative effect may relate to the flavonoids. A simple and rapid resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was first developed and validated for the quantification of seven flavonoids including pectolinarin, linarin, pectolinarigenin, hispidulin, diosmetin, acacetin and apigenin in rat plasma after oral administration of Cirsium japonicum DC. extract. MATERIALS AND METHODS The chromatographic separation was achieved on a C18 column with gradient elution by using a mixture of 0.1% formic acid aqueous solution and methanol as the mobile phase at a flow rate of 0.8mL/min. A tandem mass spectrometric detection was conducted using multiple-reaction monitoring (MRM) via an electrospray ionization (ESI) source in positive and negative ionization mode simultaneously. Samples were pre-treated by a single-step protein precipitation with methanol, and sulfamethoxazole was used as internal standard (IS). RESULTS The optimized mass transition ion-pairs (m/z) for quantization were 623.4/315.2 for pectolinarin, 593.3/285.1 for linarin, 315.3/300.2 for pectolinarigenin, 301.2/286.2 for hispidulin, 301.2/258.2 for diosmetin, 283.0/267.9 for acacetin, 269.0/117.0 for apigenin and 252.2/155.8 for IS. After oral administration of 6mL/kg Cirsium japonicum DC. extract in rats, the maximum plasma concentration (Cmax) of pectolinarin, linarin, pectolinarigenin, hispidulin, diosmetin, acacetin and apigenin were 876.77±97.34ng/mL, 86.79±1.70ng/mL, 6.13±0.12ng/mL, 32.85±2.50ng/mL, 37.2±2.04ng/mL, 19.02±1.29ng/mL and 148.26±20.63ng/mL, respectively. The time to reach the maximum plasma concentration (Tmax) was 5min for pectolinarin, linarin, pectolinarigenin, hispidulin, diosmetin, acacetin and 360min for apigenin. The intra-day and inter-day precisions (RSD%) for seven compounds were less than 13.16% and 7.77% and the accuracy (RE%) range from -7.92% to 14.77%. CONCLUSIONS This is the first research on the pharmacokinetic study of bioactive components in rat plasma after oral administration of Cirsium japonicum DC. extract. The results provided a meaningful basis for better understanding the absorption mechanism of Cirsium japonicum DC. and evaluating the clinical application of this herb medicine.