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 qualitative and quantitative analysis of 28 components in Isodon rubescens by HPLC-ESI-MS/MS.

A novel method, HPLC-MS/MS was developed to qualitatively identify and quantitatively determine the 28 components including 19 diterpenoids, 6 phenolic acids and 3 flavonoids in Isodon rubescens, an important traditional Chinese medicine. The separation was performed on a C(18) column with linear gradient elution with 0.1% aqueous formic acid/methanol containing 0.1% formic acid at a flow rate of 0.7 mL/min. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, LOD and LOQ). The results indicated that the method was simple, rapid, specific and reliable. The proposed method was successfully applied for the qualitative and quantitative analysis of 28 chemical compositions in 21 batches of natural and cultured I. rubescens samples from different sources which had great variation on the contents. The results demonstrated that the method was useful for standardization and differentiation of large numbers of similar samples.

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.

Simultaneous determination of seven components in Qibaomeiran pill by HPLC-DAD.

A high-performance liquid chromatography coupled with photo diode array detection method is developed for simultaneous determination of seven active components in Qibaomeiran pill, including rutin, 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glycoside, ferulic acid, psoralen, isopsoralen, emodin, and physcion. The analysis is performed on a C(18) column using a mobile phase composed of A (0.1% acetic acid) and B (acetonitrile) with linear gradient elution. Four wavelengths at 245, 290, 320, and 350 nm were chosen as the monitoring wavelength to determine seven active components, respectively. All the compounds show good linearity (r > 0.999). The developed method is fully validated in respect to precision, repeatability, and accuracy. The proposed method is successfully applied to quantify the seven active components in different Qibaomeiran pill samples. The results indicate that the developed assay could be considered as a suitable quality control method for the Qibaomeiran pill.

[Studies on fingerprints of Bupleurum chinense from Hebei Province with HPLC-UV].

OBJECTIVE: To establish a HPLC fingerprint analysis method for identification of Bupleurum chinense of Hebei Province and compare the fingerprints of Radix Bupleuri collected from different habitats so as to establish a sensitive and specific method for controlling the quality of Radix Bupleuri. METHODS: The HPLC-UV fingerprints of Radix Bupleuri from different habitats were obtained from Waters 1525 instruments. The HPLC separation was performed on a C18 analytical column gradient eluted with a mixture consisting of acetonitrile and water at a flow rate of 1.0 ml/min with UV detector at 203 nm. The temperature of column was 30 degrees C. RESULTS: The mutual mode of HPLC-UV fingerprints was set up, and the similar degrees to the crude drugs of different habitats were compared. CONCLUSION: It is simple and quick to differ Bupleurum chinense from different habitats with the method that can be used as a quality control item for Radix Bupleuri.

[Study on the metabolite of stilbene glucoside in mice by liquid chromatography tandem-mass spectrometry].

AIM: To study the metabolite of stilbene glucoside in the Chinese traditional medicine Polygonum multiflornm in mice and elucidate its chemical structure by liquid chromatography tandem-mass spectrometry. METHODS: The stilbene glucoside was injected into the tail vein of mice. Blood samples were collected from artery in the eyepit. The methanol-protein-precipitated plasma sample was introduced into the liquid chromatography-tandem mass spectrometer directly. The analytical column was C18 column (250 mm x 4.6 mm ID, 5 microns). The mobile phase consisted of acetonitrile-methanol-water-formic acid (15:18:66:1) for ES+, acetonitrile-methanol-water (15:18:67) for ES-. The UV detection wavelength was set at 320 nm. The mass ion source type was ESI. HV capillary was 3 kV. The dry gas was nitrogen gas and the flow rate was set at 318 L.h-1. The ion source temperature was 150 degrees C. RESULTS: The stilbene glucoside and its metabolite were separated completely under the chromatography condition. The ions at m/z 600 and m/z 605 were detected under positive ion polarity while the ions at m/z 581 and m/z 402 were detected under negative ion polarity. CONCLUSION: It was proposed that the metabolite of stilbene glucoside injected in vein was its glucuronide conjugate.