LABEL-FREE BIO-AFFINITY MASS SPECTROMETRY FOR SCREENING AND LOCATING BIOACTIVE MOLECULES.

Despite the recent increase in the development of bioactive molecules in the drug industry, the enormous chemical space and lack of productivity are still important issues. Additional alternative approaches to screen and locate bioactive molecules are urgently needed. Label-free bio-affinity mass spectrometry (BA-MS) provides opportunities for the discovery and development of innovative drugs. This review provides a comprehensive portrayal of BA-MS techniques and of their applications in screening and locating bioactive molecules. After introducing the basic principles, alongside some application notes, the current state-of-the-art of BA-MS-assisted drug discovery is discussed, including native MS, size-exclusion chromatography-MS, ultrafiltration-MS, solid-phase micro-extraction-MS, and cell membrane chromatography-MS. Finally, several challenges and limitations of the current methods are summarized, with a view to potential future directions for BA-MS-assisted drug discovery. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

Development of an ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry method for comparative pharmacokinetics of six triterpenoids in rat plasma and application to different forms of Phytolacca acinosa.

Phytolacca acinosa is an herb for treatment of ascites and tumor. Two forms of P. acinosa, i.e. raw and vinegar-processed herb, have been used in clinic. However, pharmacokinetic difference between the two forms of P. acinosa has not been fully understood. Herein, a comparative pharmacokinetic method based on liquid chromatography with tandem mass spectrometry was developed for quantification of six bioactive triterpenoids, including esculentoside H, esculentoside T, esculentoside A, esculentoside B, phytolaccagenic acid, and phytolaccagenin in rat plasma after oral administration of different forms of P. acinosa. Separation was performed on an Acquity BEH C18 column (1.7 µm, 2.1 mm × 50 mm). The method was validated over a linear range of 2.0-5000 ng/mL. Intraday and interday bias were within ±5%. Besides, all triterpenoids were stable in plasma during different storage conditions. The described method was successfully applied to a comparative pharmacokinetic study of raw and vinegar-processed P. acinosa in rats. Notably, double peak phenomenon for six triterpenoids of P. acinosa was observed for the first time. AUC0→t and Cmax values of esculentoside H, esculentoside T, phytolaccagenic acid, and phytolaccagenin were significantly lower in vinegar-processed group than that of raw group, indicating the oral bioavailability of the four triterpenoids was decreased after vinegar processing.

A biochemometrics strategy combining quantitative determination, bioactivity evaluation and relationship analysis for identification of analgesic alkaloids of raw and vinegar-processed Corydalis turtschaninovii.

A biochemometrics strategy combining quantitative determination, bioactivity evaluation, and relationship analysis was proposed for identification of analgesic components of herbs. First, a robust liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of nine major alkaloids in crude and vinegar-processed Corydalis turtschaninovii. Nine alkaloids were separated on a BEH C18 column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid and then detected by multiple reactions monitoring in the positive ion mode. Nitidine chloride was employed as the internal standard. The method displayed good linearity and the precisions of intra-day and inter-day were all within 3.0%. The recovery rates of each alkaloid ranged from 97.1 to 102.9%. The method was successfully applied for quantitative analysis of nine alkaloids in ten batches of crude and vinegar-processed Corydalis turtschaninovii. Second, the analgesic effects of crude and vinegar-processed Corydalis turtschaninovii were evaluated in mice. Third, principle component analysis, canonical correlation analysis, and partial least squares regression were used to analysis the relationship between the contents of nine major alkaloids and the analgesic effect of different crude and vinegar-processed samples. Tetrahydropalmatine, coptisine, and dehydrocorydaline have a close positive correlation with the analgesic effect.

A biochemometrics strategy for tracing diuretic components of crude and processed Alisma orientale based on quantitative determination and pharmacological evaluation.

We proposed a biochemometrics strategy for tracing diuretic components of herbs based on quantitative determination and pharmacological evaluation. First, a sensitive and robust liquid chromatography coupled with tandem mass spectrometry approach was established for simultaneous quantification of six major triterpenoids in crude and salt-processed Alisma orientale. The separation of triterpenoids was achieved on a BEH C18 column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid. Six major triterpenoids were detected by multiple reaction monitoring in the negative ion mode. Glycyrrhetinic acid was used as the internal standard. The approach showed good linearity. Intra- and inter-day precisions were all within 2.9%. The recovery rates of each triterpenoid ranged from 97.9% to 103.2%. The approach was then successfully employed for quantitative analysis of six triterpenoids in ten batches of crude and salt-processed A. orientale. Second, the diuretic effects of crude and salt-processed A. orientale were evaluated in mice. Third, principal component analysis and canonical correlation analysis were used to uncover the relationship between the contents of six major triterpenoids and the diuretic effect of different crude and salt-processed samples. Alisol B, alisol F, and alisol A have a close positive correlation with the diuretic effect.

Determination of major components from Radix Achyranthes bidentate using ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry and an evaluation of their anti-osteoporosis effect in vitro.

Ecdysterone and saponins are the most characteristic components of Radix Achyranthes bidentate, which acts on the human body to promote collagen synthesis and stimulates cell growth. However, the relationship between these components and the differentiation of MC3T3-E1 osteoblastic cells is unknown. We developed a rapid ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry method for direct determination of one ecdysterone and four saponins in crude and salt-processed Radix Achyranthes bidentate. The method was interrogated in terms of linearity, intra- and inter-day precision, repeatability, stability and recovery. The method was linear within the concentration ranges of 0.003-336 µg/mL for ß-ecdysterone, 0.0035-130 µg/mL for 25S-inokosterone, 0.004-423 µg/mL for ginsenoside Ro, 0.0036-66 µg/mL for chikusetsusaponin IV and 0.0044-111 µg/mL for chikusetsusaponin IVa. The intra- and inter-day precisions were all within 2.7%. The standard addition method determined recovery rates for each component (98.7-102.5%). The method was successfully applied to simultaneously quantify five components in ten batches of crude and salt-processed Radix Achyranthes bidentate. Subsequently, the examination of these extracts on the differentiation of MC3T3-E1 osteoblastic cells were carried out. Finally, the relationships between the contents of five components and their anti-osteoporosis effect were investigated by using canonical correlation analysis.

Nine components pharmacokinetic study of rat plasma after oral administration raw and prepared Semen Cassiae in normal and acute liver injury rats.

In China, Semen Cassiae has long been used to protect liver, brighten eyes, and relieve constipation. Prepared Semen Cassiae is produced from raw Semen Cassiae by processing, the two forms of Semen Cassiae have different clinical applications. Pathological state is an important factor affecting the efficacy of drugs, the pharmacokinetic behavior of drugs could be significantly changed when people or animal were under different pathological state. To clarify the effect of processing mechanism and pathological state for pharmacokinetic behavior, the pharmacokinetics of nine components of raw and prepared Semen Cassiae under normal and acute liver injury rats were examined. The results showed that the bimodal phenomenon appeared on the plasma concentration-time profiles of obtusin, emodin, chrysophanol, aloe emodin and rhein. The Tmax of aurantio-obtusin, obtusin, chrysoobtusin, emodin, chrysophanol, aloe emodin, physcion in normal groups administrated prepared Semen Cassiae were shorter than those administrated raw Semen Cassiae. For the AUC0-t , aurantio-obtusin, obtusin, chrysoobtusin, chrysophanol, aloe emodin and physcione in model groups administrated prepared Semen Cassiae were significantly higher than other groups, unlike above components, rhein had poor absorption in model groups. The study would be useful for further studies on pharmacokinetics and clinical application of raw and prepared Semen Cassiae.

A liquid chromatography-tandem mass spectrometry approach for study the tissue distributions of five components of crude and salt-processed Radix Achyranthes in rats.

This study developed a robust and reliable approach using liquid chromatography- tandem mass spectrometry for the simultaneous determination of five saponins in rat tissues: ß-ecdysterone, chikusetsusaponin IV, ginsenoside Ro, 25S-inokosterone and chikusetsusaponin IVa. This is the first report on a comparative tissue distribution study of crude and salt-processed Radix Achyranthes in rats. After one-step protein precipitation by acetonitrile, the tissue samples were sent to LC-MS/MS for multiple reaction monitoring. The retention times of the five saponins and internal standard were 1.77, 3.14, 3.01, 1.83, 3.26 and 4.77 min. The standard curves showed good linear regression (r2 > 0.9991) in the range of 10.3-1562.5 ng/mL. The intra- and inter-day accuracy and precision were within 15% of the nominal concentration. The recoveries of the five saponins were 92.0-99.9%. Finally, this approach was successfully applied to tissue distribution analysis of the five saponins after oral administration of crude and salt-processed Radix Achyranthes in rats. The largest concentration of the five saponins was observed in kidney after salt-processing, which indicated that processing could enhance the bioavailability.

A sensitive UPLC-MS/MS method for simultaneous determination of polyphenols in rat plasma: Application to a pharmacokinetic study of dispensing granules and standard decoction of Cinnamomum cassia twigs.

This study established a rapid and reliable approach using liquid chromatography-tandem mass spectrometry for the simultaneous determination of cinnamic acid, vanillic acid and protocatechuic acid in rat plasma. This is the first report on a comparative pharmacokinetic study of dispensing granules and standard decoction of Cinnamomum cassia twigs in rats. After liquid-liquid extraction by ethyl acetate, the plasma samples were subjected to LC-MS/MS for multiple reaction monitoring. The standard curves showed good linear regression (r2 > 0.9991) in the range of 10.0-16000 ng/mL. The intra- and inter-day accuracy and precision were found to be within 15% of the nominal concentration. The recoveries of the three phenolics ranged from 88.7 to 105.7%. Finally, this approach was successfully applied to pharmacokinetic analysis of the three phenolics after oral administration of standard decoction and dispensing granules of C. cassia twigs in rats. Although the values of AUC0-t of vanillic acid and protocatechuic acid in standard decoction group were larger than those of the dispensing granule group, no significant difference was observed for the two groups. Of note, the elimination rates of vanillic acid were slower in the standard decoction group than the dispensing granule group.

A reliable LC-MS/MS method for the quantification of five bioactive saponins of crude and processed Bupleurum scorzonerifolium in rat plasma and its application to a pharmacokinetic study.

A simple and reliable liquid chromatography-mass spectrometry (LC-MS) method was developed for simultaneous determination of saikosaponin A, saikosaponin B1 , saikosaponin C, saikosaponin D and saikosaponin F in rat plasma using glycyrrhetinic acid as an internal standard (IS). The separation was operated on a Waters BEH C18 column. The mobile phases of gradient elution consisted of acetonitrile (A) and 0.1% aqueous acetic acid (B). The mass spectrometric detection was accomplished in multiple reaction monitoring mode. The five saponins displayed good linearity (r2 > 0.9996). The lower limits of quantitation of saikosaponin A, saikosaponin B1 , saikosaponin C, saikosaponin D and saikosaponin F were determined to be 2.9, 2.3, 3.5, 2.9 and 3.1 ng/mL, respectively. Moreover, the intra- and inter-day precisions of the five saponins showed an RSD within 2.96%, whereas the accuracy (RE) ranged from -2.28 to 2.78%. Finally, the developed method was fully validated and applied to a comparative pharmacokinetic study of the five bioactive saponins in rats following oral administration of crude and vinegar-processed Bupleurum scorzonerifolium.

Ultra-high-performance liquid chromatography with tandem mass spectrometry method for determination of four compounds in rat plasma after oral administration of Xanthii fructus and stir-fried Xanthii fructus extracts.

Xanthii fructus (XF), the fruit of Xanthium sibiricum Patr., is a traditional Chinese materia medica commonly used to treat allergic rhinitis and other rhinitis diseases. To uncover the mechanism of the stir-frying process and its effect on the pharmacokinetic behavior of active compounds in model rats, four active compounds-chlorogenic acid, 4-caffeoylquinic acid, 1,5-O-dicaffeoylquinic acid and apigenin-were selected based on previous spectrum-effect experiments. High performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-QqQ-MS) technology, an accurate and feasible method, was applied to measure the concentration of these four compounds in rat plasma. This validated method can accurately measure the concentration of each compound at each sampling point of rat plasma. This validated method shows good linearity, extraction recoveries, matrix effects, intra- and inter-day precision and stabilities. Compared with the XF group, the maximum plasma concentration (Cmax ) value of 1,5-O-dicaffeoylquinic acid decreased remarkably (p < 0.05) after oral administration of stir-fried Xanthii fructus (SXF) extract, while the other compounds showed no significant difference. The mean residence time value of chlorogenic acid (p < 0.05) and 1,5-O-dicaffeoylquinic acid (p<0.01) after oral administration of SXF extraction demonstrated significant differences compared with the XF group, while the other two compounds showed no statistical difference, indicating that the stir-frying process prolonged the effect time and delayed the removal time of chlorogenic acid and 1,5-O-dicaffeoylquinic acid. The values of the area under the plasma concentration-time curve from zero to the last quantifiable time-point, the area under the plasma concentration-time curve from zero to infinity, the time to maximum concentration and the elimination half-life of four compounds in the SXF group showed no statistically significant difference from the XF group. From this data, we speculated that the stir-frying process can not only keep the absorption of 4-caffeoylquinic acid and apigenin, but also increase the effect time of chlorogenic acid and 1,5-O-dicaffeoylquinic acid, which could be the mechanism underlying the stir-frying process enhancing the effects of XF.

Comparative Study on Pharmacokinetics of Four Active Compounds in Rat Plasma after Oral Administration of Raw and Wine Processed Chuanxiong Rhizoma.

In Chinese medicine, the effect of promoting blood circulation and removing stasis could be enhanced after Chuanxiong Rhizoma is processed by wine. However, the relevant mechanism remains unclear. In this manuscript, a rapid and sensitive quantification method employing ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established and validated to simultaneously determine butylidenephthalide, ligustilide, senkyunolide A and ferulic acid in rat plasma after oral administration of raw Chuanxiong Rhizoma (RCR) and wine-processed Chuanxiong Rhizoma (WCR) respectively. All analytes were extracted from plasma by proteins precipitation with methanol. Chromatographic separation was carried out on a Hypersil GOLD C18 column by using a gradient mobile phase system of acetonitrile and water with 0.01% formic acid, the flow rate was 0.3 mL/min. For exact mass detecting, quick switching mode was used, positive and negative ions could be detected in one injection. The pharmacokinetic profiles of four components in the two groups were evaluated and compared. The results showed that, compared to the RCR group, the Vd and AUC0→t values of four active compounds were increased and decreased respectively in WCR group, which revealed the effect of wine processing to Chuanxiong Rhizoma: the stronger the effect, the wider the distribution.

Development of an analytical strategy to identify and classify the global chemical constituents of Ziziphi Spinosae Semen by using UHPLC with quadrupole time-of-flight mass spectrometry combined with multiple data-processing approaches.

According to traditional Chinese medical theory, Ziziphi Spinosae Semen needs to be stir-fried before clinical application for its sedative-hypnotic effect enhancement. A rapid and comprehensive analysis strategy of ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry and multiple data analysis platforms was developed for the efficient and sensitive identification of components in crude and parched Ziziphi Spinosae Semen to explore the composition changes that happen during the stir-frying process. Both positive and negative ion modes were applied for mass spectrometry detection, and 40 components were identified from crude and parched Ziziphi Spinosae Semen, respectively. Principal component analysis and t-test were applied to find differences between crude and parched Ziziphi Spinosae Semen. As a result, Ziziphi Spinosae Semen samples could be clearly divided into two groups according to their processing methods, and 19 key markers that contributed to the classification significantly (P < 0.05) were found. This kind of change in contents of components might be responsible for the recommended clinical application of parched Ziziphi Spinosae Semen.

Quality evaluation of raw and processed Crataegi Fructus by color measurement and fingerprint analysis.

Crataegi Fructus and its processed products have been used as a traditional medicine for a long time, and numerous active components are responsible for their curative effects. However, a comprehensive and fast method for the quality control of its processed products is still lacking. In this study, two analytical methods based on color measurements and fingerprint analysis are established. In the color measurements, the color values of the peel and flesh of Crataegi Fructus were evaluated spectrophotometrically. Based on the results, a color reference range was established using percentiles, and the standard color difference value was established using the median color values. Then, the color values of Crataegi Fructus and its processed products were analyzed using Bayes linear discriminant analysis and mathematical functions were built in order to predict the degree of processing. Moreover, high-performance liquid chromatography fingerprint analysis was performed on a Hibar C18 column, and a high-performance liquid chromatography fingerprint pattern was obtained, from which nine peaks were identified. Chemometric methods were successfully applied to differentiate raw and processed Crataegi Fructus.

Effect of piperine on the bioavailability and pharmacokinetics of rosmarinic acid in rat plasma using UPLC-MS/MS.

1. The purpose of the present study was to investigate the effect of piperine (PP) on the pharmacokinetics of rosmarinic acid (RA) in rat plasma and to determine whether PP could enhance the oral bioavailability of RA via inhibition of its glucuronidation. 2. The pharmacokinetic profiles of RA between oral administration of RA (50 mg/kg) alone and in combination with different oral dose PP (20, 40, 60, and 80 mg/kg) to rats were investigated via a validated UPLC/MS/MS method. 3. The AUC and Cmax of RA were significantly increased in combination with different dose PP dose dependently, especially in the presence of 60 and 80 mg/kg PP (p < 0.01). The relative bioavailability of RA in the presence of 20, 40, 60, and 80 mg/kg PP was 1.24-, 1.32-, 2.02-, and 2.26-folds higher, respectively, compared with the control group given RA alone. Compared with RA, the pharmacokinetic modulations of RA glucuronide were even more apparent, and the glucuronidation of RA was remarkedly inhibited. 4. This study demonstrated that PP significantly improved the in vivo bioavailability of RA partly attributing to the inhibition of gut and hepatic metabolism enzymes of RA.

A metabolomics research based on UHPLC-ESI-Q-TOF-MS coupled with metabolic pathway analysis: Treatment effects of stir-frying Xanthii Fructus on allergic rhinitis in mice model.

Xanthii Fructus (XF), a well-known herb in traditional Chinese medicine, has been frequently used for the treatment of allergic rhinitis in the clinic. Its therapeutic metabolic mechanism, however, remains undetermined. In this work, a metabolomics research coupled with metabolic pathway analysis has been employed to screen out the potential mechanism in its effects on allergic rhinitis. Specifically, mouse serum samples containing XF were analyzed based on ultra-high performance liquid chromatography equipped with electrospray ionization quadruple time-of-flight mass spectrometry detection (UHPLC-ESI-Q-TOF-MS) in both positive and negative polarity. In addition, the raw data gained from UHPLC-ESI-Q-TOF-MS were processed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) in order to discover remarkable metabolites. Twenty-seven potential biomarkers in mouse serum were filtered from free databases like HMDB. Interestingly, this study filtered the potential metabolic pathways including glycerophospholipid metabolism and branch-chain amino acid metabolism. We hope that this paper will provide a feasible strategy for revealing the therapeutic mechanism of XF in allergic rhinitis mice model.

[Problems and solutions in modern research of traditional Chinese herbal pieces processing technology].

Chinese medicine processing is the main feature that distinguishes traditional Chinese medicine from natural medicine and plant medicine, and is the main feature in clinical medication of traditional Chinese medicine. The research of Chinese medicine processing technology is an important link to realize standardization and standardization of Chinese herbal pieces, with urgent need to attract high attention. At present, there are still many problems in the research of processing technology of Chinese herbal pieces, mainly including inconsistent processing technology, large differences in process technology parameters, and unstable production technology of Chinese herbal pieces, resulting in uncontrollable quality of Chinese herbal pieces and affecting the clinical efficacy of Chinese medicine. This paper focused on the establishment of a unified standard processing technology, and put forward the countermeasures for the processing technology of Chinese medicine based on a comprehensive analysis of the current situations of the processing technology of Chinese herbal pieces, with significance for guiding the establishment of a standardized processing technology of Chinese medicine.

UHPLC-MS/MS quantification combined with chemometrics for the comparative analysis of different batches of raw and wine-processed Dipsacus asper.

A rapid and sensitive ultra-high performance liquid chromatography with tandem mass spectrometry approach was established for the simultaneous determination of 4-caffeoylquinic acid, loganic acid, chlorogenic acid, loganin, 3,5-dicaffeoylquinic acid, dipsacoside B, asperosaponin VI, and sweroside in raw and wine-processed Dipsacus asper. Chloramphenicol and glycyrrhetinic acid were employed as internal standards. The proposed approach was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. Intra- and interassay variability for all analytes were 2.8-4.9 and 1.7-4.8%, respectively. The standard addition method determined recovery rates for each analytes (96.8-104.6%). In addition, the developed approach was applied to 20 batches of raw and wine-processed samples of Dipsacus asper. Principle component analysis and partial least squares-discriminate analysis revealed a clear separation between the raw group and wine-processed group. After wine-processing, the contents of loganic acid, chlorogenic acid, dipsacoside B, and asperosaponin VI were upregulated, while the contents of 3,5-dicaffeoylquinic acid, 4-caffeoylquinic acid, loganin, and sweroside were downregulated. Our results demonstrated that ultra-high performance liquid chromatography with tandem mass spectrometry quantification combined with chemometrics is a viable method for quality evaluation of the raw Dipsacus asper and its wine-processed products.

Study of organic acids in Schisandrae Chinensis Fructus after vinegar processing.

The ripened fruit of Schisandrae Chinensis Fructus has unique medical properties in Chinese medicine. It is commonly used after vinegar steaming. Vinegar steaming changes the color of Schisandrae Chinensis Fructus from red to black and enhances its acidic and astringent properties. Lignans are the well-investigated components in this herb. However, Schisandrae Chinensis Fructus is acidic in the theory of Chinese medicine, and whether vinegar processing changes its organic acid components remains largely unknown. In this study, the organic acids in this herb were derived by the method of methyl esterification, and further analyzed by gas chromatography with mass spectrometry. A total of 39 organic acid compounds were identified. Interestingly, Schisandrae Chinensis Fructus after vinegar processing showed a significant increase in the content of levulinic acid as compared to the unprocessed ones. Pharmacological experiments demonstrated that levulinic acid inhibited the contractility of isolated intestine and had an inhibitory effect on the excessive hyperfunction of small intestinal propulsion. Moreover, the extracts of vinegar-processed Schisandrae Chinensis Fructus had a stronger inhibitory on the excessive hyperfunction of small intestinal propulsion than that of unprocessed ones. Taken together, this study offers novel insight into the effect of Schisandrae Chinensis Fructus after vinegar processing.

Qualitative analysis of multiple compounds in raw and prepared Semen Cassiae coupled with multiple statistical strategies.

In China, Semen Cassiae is used clinically to improve eyesight, relieve constipation, and to treat headache and dizziness. Prepared Semen Cassiae is obtained by stir-frying raw Semen Cassiae until it turned dark brown, micro dilatancy, and overflow aroma. After processing, the therapeutic effects change-the purgation effect is alleviated and the hepatoprotective effect is enhanced. To explore the changes in chemical compositions of Semen Cassiae after processing and clarify the material basis of the changed therapeutic effects, an ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry coupled with automated data analysis software and statistical strategy was developed. As a result, 53 compounds in raw Semen Cassiae and 43 compounds in prepared Semen Cassiae were found, a total of 55 chemical compounds were identified. Principle component analysis and t-test were processed by Markerview 1.2.1 software. Finally, 39 peaks were found to be the main contributors to the significant difference (p < 0.05) between raw and prepared Semen Cassiae. Compared with raw Semen Cassiae, 19 peaks showed a higher intensity in prepared Semen Cassiae, while the contents of 20 compounds in prepared Semen Cassiae were lower, most of which belonged to naphthopyrones glycosides and anthraquinone glycosides.

A UPLC-MS/MS approach for simultaneous determination of eight flavonoids in rat plasma, and its application to pharmacokinetic studies of Fu-Zhu-Jiang-Tang tablet in rats.

The purpose of this study is to establish and validate a UPLC-MS/MS approach to determine eight flavonoids in biological samples and apply the method to pharmacokinetic study of Fu-Zhu-Jiang-Tang tablet. A Waters BEH C18 UPLC column was employed with methanol/0.1% formic acid-water as mobile phases. The mass analysis was carried out in a triple quadrupole mass spectrometer using multiple reaction monitoring with negative scan mode. A one-step protein precipitation by methanol was used to extract the analytes from blood. Eight major flavonoids were selected as markers. Our results showed that calibration curves for 3'-hydroxypuerarin, mirificin, puerarin, 3'-methoxypuerarin, daidzin, rutin, astragalin and daidzein displayed good linear regression (r2 > 0.9986). The intra-day and inter-day precisions (RSD) of the eight flavonoids at high, medium and low levels were <8.03% and the bias of the accuracies ranged from -5.20 to 6.75%.The extraction recoveries of the eight flavonoids were from 91.4 to 100.5% and the matrix effects ranged from 89.8 to 103.8%. The validated approach was successfully applied to a pharmacokinetic study in Sprague-Dawley rats after oral administration of FZJT tablet. Double peaks were emerged in curves of mean plasma concentration for 3'-methoxypuerarin, which was reported for the first time.