Comparative analysis of 15 chemical constituents in Scutellaria baicalensis stem‐leaf from different regions in China by ultra‐high performance liquid chromatography with triple quadrupole tandem mass spectrometry

Scutellaria baicalensis is a traditional Chinese herbal medicine containing multiple components, which has been extensively used in clinics to treat epidemic febrile disease and hyperactivity cough. To get a deeper understanding about Scutellaria baicalensis stem‐leaf resources, we analyzed 15 chemical constituents in 35 batches of Scutellaria baicalensis stem‐leaf from eight regions in China. A rapid, simple, and sensitive method using ultra‐high performance liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry has been developed for the first time to simultaneously determine 15 chemical constituents (including phenolic acids and flavonoids) in Scutellaria baicalensis stem‐leaf. Sufficient separation of 15 target constituents was achieved on a Waters Acquity UPLC BEH C₁₈ (2.1 mm × 100 mm, 1.7 μm) column within 14 min under the optimized chromatographic conditions. The established method was validated and showed good linearity, precision, repeatability, stability, and recovery and was successfully applied for the simultaneous determination of the 15 chemical constituents in these samples. Hierarchical clustering analysis and principal components analysis were performed to estimate and classify these samples based on the contents of the 15 chemical constituents. This study provided theoretical basis and scientific evidence for the development and utilization of Scutellaria baicalensis stem‐leaf resources.     

Comparative analysis of the main bioactive components of Xin‐Sheng‐Hua granule and its single herbs by ultrahigh performance liquid chromatography with tandem mass spectrometry

Xin‐Sheng‐Hua granule, a representative formula for postpartum hemorrhage, has been used clinically to treat postpartum diseases. Its main bioactive components comprise aromatic acids, phthalides, alkaloids, flavonoids, and gingerols among others. To investigate the changes in main bioactive constituents in its seven single herbs before and after compatibility, a rapid, simple, and sensitive method was developed for comparative analysis of 27 main bioactive components by using ultrahigh‐performance liquid chromatography with triple quadrupole electrospray tandem mass spectrometry for the first time. The sufficient separation of 27 target constituents was achieved on a Thermo Scientific Hypersil GOLD column (100 mm × 3 mm, 1.9 μm) within 20 min under the optimized chromatographic conditions. Compared with the theoretical content, the observed content of each analyte showed remarkable differences in Xin‐Sheng‐Hua granule except thymine, p‐coumaric acid, senkyunolide I, senkyunolide H, and ligustilide; the total contents of 27 components increased significantly, and the content variation degrees for the different components were gingerols > flavonoids > aromatic acids > alkaloids > phthalides. The results could provide a good reference for the quality control of Xin‐Sheng‐Hua granule and might be helpful to interpret the drug interactions based on variation of bioactive components in formulae.     

Metabolites study on 5‐O‐methylvisammioside in vivo and in vitro by ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry

5‐O‐Methylvisammioside is one of major chromones of Radix Saposhnikoviae possessing definite pharmacological activities, but there are few reports with respect to the metabolism of 5‐O‐methylvisammioside. In this work, metabolites in vivo were explored in male Sprague‐Dawley rats and in vitro investigated on rat intestinal bacteria incubation model and were identified by using ultra high performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry. An online data acquisition method based on a multiple mass defect filter and dynamic background subtraction was developed to trace all probable metabolites. As a result, 26 metabolites in vivo (including 18, 15, 10, and 10 in rat urine, faece, bile, and blood) and 7 metabolites in vitro were characterized, respectively. Additionally, the main metabolic pathways in vivo and in vitro, including deglycosylation, deglycosylation + demethylation, deglycosylation + oxidation, N‐acetylation, and sulfate conjugation, were summarized by calculating the relative content of each metabolite. The obtained results significantly enriched our knowledge about 5‐O‐methylvisammioside metabolism and will lead to a better understanding of its safety and efficacy.     

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.     

Detection of buffalo mozzarella adulteration by an ultra‐high performance liquid chromatography tandem mass spectrometry methodology

Over the past years, LC–MS‐based approaches have gained a growing interest in food analysis by using different platforms and methodologies. In particular, enhanced selectivity and sensitivity of multiple reaction monitoring (MRM) scan function offer powerful capabilities in detecting and quantifying specific analytes within complex mixtures such as food matrices. The MRM approach, traditionally applied in biomedical research, is particularly suitable for the detection of food adulteration and for the verification of authenticity to assure food safety and quality, both recognized as top priorities by the European Union Commission. Increasingly stringent legislation ensure products safety along every step ‘from farm to fork’, especially for traditional foods designed with the Protected Designation of Origin certification. Therefore, there is a growing demand of new methodologies for defining food authenticity in order to preserve their unique traits against frauds. In this work, an ultra performance liquid chromatopgraphy‐electrospray ionization‐tandem mass spectrometry (MS/MS) methodology based on MRM has been developed for the sensitive and selective detection of buffalo mozzarella adulteration. The targeted quantitative analysis was performed by monitoring specific transitions of the phosphorylated β‐casein f33‐48 peptide, identified as a novel species‐specific proteotypic marker. The high sensitivity of MRM‐based MS and the wide dynamic range of triple quadrupole spectrometers have proved to be a valuable tool for the analysis of food matrices such as dairy products, thus offering new opportunities for monitoring food quality and adulterations. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid analysis and characterization of multiple constituents of corn silk aqueous extract using ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

Corn silk is a well‐known traditional Chinese medicine that has been widely used for its antidiabetic, antioxidant, antihyperlipidemic, and other effects in China for thousands of years. Numerous studies have revealed that corn silk contains multiple bioactive constituents that are beneficial for human health. However, the constituents of corn silk in vivo remain ambiguous. In this study, high‐throughput ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry technology using multivariate statistical analysis was established to systematically investigate the constituents migrating into blood from corn silk aqueous extract. As a result, 76 compounds were identified, including caffeic acid and ten of its derivatives, (E)‐p‐coumaric acid and two of its derivatives, ferulic acid and four of its derivatives, and five flavones. Among the identified constituents, 21 constituents, including nine prototype components and 12 metabolites derived from eight components, were characterized in sequence. Based on the significance of the results, the applied approach was powerful for the accurate determination and rapid screening of bioactive components from corn silk aqueous extract. The obtained results are valuable for the in‐depth understanding and further pharmacological study of corn silk aqueous extract.     

Multiconstituent identification in root,branch, and leaf extracts of Juglans mandshurica using ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

As a traditional medicinal plant, Juglans mandshurica has been used for the treatment of cancer. Different organs of this plant showed anti‐tumor activity in clinic and laboratory. Comparative identification of constituents in different plant organs is essential for investigation of the relationship between chemical constituents and pharmacological activities. For this aim, the roots, branches, and leaves of J. mandshurica were extracted with 50% v/v methanol and then subjected to ultra‐high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry analysis conducted under low and high energy. As a result, we have to date identified 111 compounds consisting of 56 tannins, 29 flavonoids, 13 organic acids, 8 naphthalene derivatives, and 5 anthracenes. Five compounds, namely, diquercetin trihydroxy‐truxinoyl‐glucoside, two quercetin kaempferol dihydroxy‐truxinoyl‐glucosides, syringoyl‐tri‐galloyl‐O‐glucose, and dihydroxy‐naphthalene syringoyl‐glucoside, were tentatively identified as new compounds. Of the compounds identified, 76 were found in the root extract, 67 in the branch extract, and 37 in the leaf extract. Only six compounds including four organic acids and two tannins were found in all three extracts. We developed a rapid and sensitive ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry approach to identify multiple constituents of complex extracts without separation and ion selection. The results presented provide useful information on further research of the bioactive compounds of J. mandshurica .     

Evaluation and quantitative analysis of 11 compounds in Morinda officinalis using ultra‐high performance liquid chromatography and photodiode array detection coupled with chemometrics

Morinda officinalis (Rubiaceae) is a traditional Chinese medicine widely used for the treatment of impotence and osteoporosis in clinical therapy. In the present study, a rapid and simple ultra‐high performance liquid chromatography with photodiode array detection method was developed and validated for the simultaneous determination of 11 bioactive compounds in M. officinalis . This assay method was validated with respect to linearity (R ²  > 0.9991), precision, repeatability, limit of detection, limit of quantification, and accuracy (with observed recovery rates between 94.21 and 100.38%). The quantitative results revealed significant differences in the concentrations of the selected compounds. Additionally, chemometric methods, including hierarchical clustering analysis, principal component analysis, and partial least‐squares discriminate analysis, were applied to compare and sort the 25 batches of M. officinalis samples based on the quantitative data of the analytes. All of the samples were clearly divided into two groups: the Hainan samples were successfully discriminated from the samples from other origins. Simultaneous determination of multiple compounds using the proposed method combined with chemometrics could be a viable strategy to compare and evaluate the quality of M. officinalis .     

Determination of the transformation of ginsenosides in Ginseng Radix et Rhizoma during decoction with water using ultra‐fast liquid chromatography coupled with tandem mass spectrometry

This study was conducted to determine the variations of ginsenosides in Ginseng Radix et Rhizoma when using different preparation solvents and explore the major factors for changes. With an established ultra‐fast liquid chromatography coupled with tandem mass spectrometry method which could quantify 52 ginsenosides, the extraction differences were characterized and compared using different solvents (water, 70% aqueous ethanol, and ethanol). Subsequently, a series of aqueous solutions with different pH were prepared to test the influence of pH to the changes of ginsenosides. Meanwhile, acetic acid and aspartic acid were used to verify whether the reaction had a relationship with the kind of acids. After refluxing with water, not only highly polar ginsenosides were extracted, some less polar ginsenosides such as ginsenoside Rg₃, Rg₅, Rk₁, and Rh₂ occurred or increased rapidly. Further experiments indicated that less polar ginsenosides were easier to generate at low pH values, and the reaction was only related to pH other than what kind of acids were used. It is the first time to elaborate the contents variation of 52 ginsenosides when using different extraction methods. The results indicated that decoction with water could enhance the transformation of highly polar ginsenosides to less polar ginsenosides and the process was pH dependent.     

Characterization of the multiple components of Acanthopanax Senticosus stem by ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

Acanthopanax Senticosus Harms. has been used widely in traditional Chinese medicine for the treatment of chronic bronchitis, neurasthenia, hypertension and ischemic heart disease. However, the in vivo constituents of the stem of Acanthopanax Senticosus remain unknown. In this paper, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry and the MarkerLynx^TM software combined with multiple data processing approach were used to study the constituents in vitro and in vivo. The aqueous extract from the Acanthopanax Senticosus stem and the compositions in rat serum after intragastric administration were completely analyzed. Consequently, 115 compounds in the aqueous extract from Acanthopanax Senticosus stem and 41 compounds absorbed into blood were characterized. Of the 115 compounds in vitro, 54 were reported for first time, including sinapyl alcohol, sinapyl alcohol diglucoside, and 1‐O‐sinapoyl‐β‐d ‐glucose. In the 41 compounds in vivo, 7 were prototype components and 34 were metabolites which were from 21 components of aqueous extract from Acanthopanax Senticosus stem, and the metabolic pathways of the metabolites were elucidated for first time. The results narrowed the range of screening the active components and provided a basis for the study of action mechanism and pharmacology.     

Identification of lipophilic components in Citri Reticulatae Pericarpium cultivars by supercritical CO2 fluid extraction with ultra‐high‐performance liquid chromatography–Q Exactive Orbitrap tandem mass spectrometry

To systematically identify the lipophilic constituents of Citri Reticulatae Pericarpium from different cultivars, supercritical CO₂ fluid extraction and ultra‐high‐performance liquid chromatography–Q Exactive Orbitrap tandem mass spectrometry were integrated for the component analysis of 18 batches of Citri Reticulatae Pericarpium from 12 cultivars for the first time. A total of 57 components from the supercritical CO₂ fluid extracts were demonstrably or tentatively identified by the obtained parent peaks, fragment peaks, and retention times. In total, two flavonoids, six organic acids, nine coumarins, three aldehydes, seven esters, three terpenes, one limonoid, and five other compounds were detected for the first time; notably, coumarin components have not yet been reported in Citri Reticulatae Pericarpium. Furthermore, the extract constituents differed between cultivars. In particular, organic acids were more abundant in Citrus reticulata “Chachi” than in other cultivars, and pterostilbene was exclusively found in Citrus reticulata “Yichangju”. The results showed that a greater variety of compounds in Citri Reticulatae Pericarpium could be extracted by supercritical CO₂ fluid extraction and detected by ultra‐high‐performance liquid chromatography–Q Exactive Orbitrap tandem mass spectrometry. This study provides a more scientific basis for further analysis of the pharmacological activity and quality of Citri Reticulatae Pericarpium components from different cultivars.     

Simultaneous determination of ferulic acid,paeoniflorin, and albiflorin in rat plasma by ultra‐high performance liquid chromatography with tandem mass spectrometry: Application to a pharmacokinetic study of Danggui‐Shaoyao‐San

A rapid, selective, and sensitive ultra‐high performance liquid chromatography‐tandem mass spectrometry method was developed for simultaneous determination of ferulic acid, paeoniflorin, and albiflorin, the major active constituents of Danggui‐Shaoyao‐San, in rat plasma using geniposide as the internal standard. The plasma samples were processed by protein precipitation with acetonitrile, and then separated on a Shim‐Pack XR‐ODS C₁₈ column (75 mm × 3.0 mm, 2.2 μm) using gradient elution program with a mobile phase consisting of 0.1% aqueous formic acid and acetonitrile at a flow rate of 0.4 mL/min. The detection was achieved on a 3200 QTRAP mass spectrometer equipped with electrospray ionization source in negative ionization mode. Quantification was performed using multiple reaction monitoring mode by monitoring the fragmentation of m/z 192.9→134.0 for ferulic acid, m/z 525.0→120.9 for paeoniflorin, m/z 525.2→121.0 for albiflorin, and m/z 433.1→225.1 for the internal standard, respectively. The calibration curve was linear in the range of 5–2500 ng/mL for all the three analytes (r ≥ 0.9972) with the lower limit of quantitation of 5 ng/mL. The intraday and interday precisions were below 12.1% for all the analytes in terms of relative standard deviation, and the accuracy was within ±11.5% in terms of relative error. The extraction recovery, matrix effect and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of ferulic acid, paeoniflorin, and albiflorin after oral administration of Danggui‐Shaoyao‐San to rats.     

Ultra high performance liquid chromatography coupled with electrospray ionization/quadrupole time‐of‐flight mass spectrometry for the rapid analysis of constituents in the traditional Chinese medicine formula Wu Ji Bai Feng Pill

An ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry method in both positive and negative ion modes was established in order to comprehensively investigate the major constituents in Wu Ji Bai Feng Pill. Briefly, a Waters ACQUITY UPLC HSS C₁₈ column was used to separate the aqueous extract of Wu Ji Bai Feng Pill. A total of 0.1% formic acid in acetonitrile and 0.1% aqueous formic acid v/v were used as the mobile phase. All analytes were determined using quadrupole time‐of‐flight mass spectrometry with electrospray ionization source in positive and negative ion modes. At length, a total of 173 components including flavones and their glycosides, monoterpene glycosides, triterpene saponins, phenethylalchohol glycosides, iridoid glycosides, phthalides, tanshinones, phenolic acids, sesquiterpenoids and cyclopeptides were identified or tentatively characterized in Wu Ji Bai Feng Pill in an analysis of 16.0 min based on the accurate mass and tandem mass spectrometry behaviors. The developed method is rapid and highly sensitive to characterize the chemical constituents of Wu Ji Bai Feng Pill, which could not only be used for chemical standardization and quality control of Wu Ji Bai Feng Pill, but also be helpful for further study in vivo metabolism of Wu Ji Bai Feng Pill.     

Rapid identification of chemical compositions in callicarpa kwangtungensis Chun by ultra‐high‐performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high‐resolution accurate mass spectrometry

Callicarpa kwangtungensis Chun is a traditional Chinese medicine that has various therapeutic effects. Despite its wide use in Chinese medicine, the study is still quite limited, especially its chemical compositions. In this research, an ultra‐high‐pressure liquid chromatography coupled with Q Exactive hybrid quadrupole‐orbitrap high‐resolution accurate mass spectrometry tandem mass spectrometry method was utilized to analyze its chemical compositions for the first time. As a result, a total of 124 compounds, including 20 phenylethanoid glycosides, 31 flavonoids, 36 organic acids, 26 terpenoids and 11 phenols, were identified or tentatively characterized in 30 min. Among them, 49 compounds, including 5 phenylethanoid glycosides, 12 flavonoids, 16 organic acids, 12 terpenoids, and 4 phenols, were identified in Callicarpa kwangtungensis Chun for the first time. Besides, the fragmentation pathways were also discussed. This research established a rapid and reliable method to analyze the chemical compositions of complicated herb without the process of isolation, and provide abundant information on the chemical material basis for further bioactivity and quality control studies.     

Simultaneous determination of multiple active components in rat plasma using ultra‐fast liquid chromatography with tandem mass spectrometry and application to a comparative pharmacokinetic study after oral administration of Suan‐Zao‐Ren decoction and Suan‐Zao‐Ren granule

Suan‐Zao‐Ren decoction has been used to treat insomnia for many years. In this work, a rapid and sensitive ultra‐fast liquid chromatography with tandem mass spectrometry method was first developed and fully validated for the simultaneous quantification of seven main active components, spinosin, mangiferin, neomangiferin, ferulic acid, liquiritin, isoliquiritin, and liquiritin apioside in rat plasma. The method was also successfully applied to compare the pharmacokinetics of these active ingredients after oral administration of Suan‐Zao‐Ren decoction and Suan‐Zao‐Ren granule. The separation was achieved on a Venusil MP C₁₈ column and the detection was conducted by the multiple reaction monitoring mode using negative ion mode. Each calibration curve had good linearity over a wide concentration range. The precision of intra‐ and interday were all within 15%, and the extraction recoveries at different analyte concentrations were all above 82.0%. The established method was successfully applied to compare the pharmacokinetic profiles of the analytes between Suan‐Zao‐Ren decoction and Suan‐Zao‐Ren granule groups. The results indicated that all the analytes had similar mean concentration‐time curves trend between two groups. No significant differences were observed in pharmacokinetic parameters of mangiferin, while the others had significant differences.     

Rapid simultaneous determination of twelve major components in Pien Tze Huang by ultra‐performance liquid chromatography coupled with triple quadrupole mass spectrometry

An efficient method using ultra‐performance LC coupled with triple quadrupole MS was developed for the rapid determination of 12 major active components in Pien Tze Huang (PZH), a well‐known traditional Chinese formula. Chromatographic separation was achieved on a Waters XBridge BEH RP18 column (50 mm × 2.1 mm id, 1.7 μm) with a gradient mobile phase (A: 0.1% aqueous formic acid and B: acetonitrile with 0.1% formic acid) at a flow rate of 0.8 mL/min. The chromatographic peaks of 12 components were identified by comparing their retention time and MS data with the related reference compounds. Multiple‐reaction monitoring was employed for the quantitative analysis. Ten batches of PZH were analyzed with a good linear regression relationship (r, 0.9987–0.9995), intraday precisions (RSD, 2.05–4.80%), interday precisions (RSD, 1.99–4.98%), repeatability (RSD, 2.21–4.20%), stability (RSD, 3.52–4.81%), and recovery (95.63–104.80%). By using this established method, the present study offered highly sensitive, specific, and speedy determination of 12 major components, which promoted the quality control investigation of PZH greatly.     

Metabolic profiling comparison of isovitexin in normal and kidney stone model rats by ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry

Isovitexin, a bioactive flavonoid constituent isolated from Desmodii Styracifolii, is considered an adjuvant for antiurolithiasis diseases. In this study, an ultra‐high‐performance liquid chromatography coupled with hybrid triple quadruple time‐of‐flight mass spectrometry method was developed to characterize and compare the metabolic profiling of isovitexin experimented on normal and kidney stone model rats. The comparative research indicated that 28 metabolites (18 phase I and 10 phase II) in normal rats and 33 metabolites (20 phase I and 13 phase II) in kidney stone model rats were initially identified. The results of relative quantitative determination reflected that the contents of metabolites produced by deglycosylation, reduction, and isomerization in kidney stone model rats were greater than those in healthy rats. Instead, the levels of oxidative and dehydrogenated metabolites in normal groups were higher than those in kidney stone model groups. The results of this study are valuable and important for understanding the metabolic process of isovitexin in clinical application, and especially the metabolism study in kidney stone model rats could provide a beneficial reference for the further search of effective substances associated with the treatment of kidney stones.     

Rapid simultaneous quantification of fructooligosaccharides in Morinda officianalis by ultra‐high performance liquid chromatography

Many Chinese herbal medicines with tonifying effects contain high levels of inulin fructooligosaccharides. These herbal medicines have high development and utilization value because of their effects against dementia, depression, and oxidative stress; on improving learning and memory ability; and on enhancing immunity. In this study, a method was developed for the separation and simultaneous quantitation of fructose, glucose, sucrose, and ten inulin fructooligosaccharides by ultra‐high‐performance liquid chromatography with evaporative light scattering detection within 10 min. Separation was performed on an Amide column with gradient elution. The calibration curves for the 13 constituents showed good linearity (R² > 0.9991). The limits of detection and quantification were 10.78–33.44 and 35.94–124.81 μg/mL, respectively, and the recoveries ranged from 98.90 to 103.67%. This method was successfully used to quantify the 13 constituents in the Chinese herbal medicine Morinda officinalis. The contents of the ten inulin fructooligosaccharides ranged from 56.28 to 60.71%. This method is accurate, rapid and simple and can be used for quantitative analysis in the quality control of herbal medicines and functional foods.     

Simultaneous determination of eight bioactive constituents of Zhi‐Zi‐Hou‐Po decoction in rat plasma by ultra high performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study

Zhi‐Zi‐Hou‐Po Decoction, consisting of Gardenia jasminoides Ellis, Magnolia officinalis Rehd. et Wils., and Citrus aurantium L, is a classical Traditional Chinese Medicine formula for the treatment of depression. In order to make good and rational use of this formula in the future, a sensitive, selective, and reliable ultra high performance liquid chromatography with tandem mass spectrometry method was developed for simultaneous determination of two iridoid glycosides (geniposide and genipin gentiobioside), two lignans (honokiol and magnolol), four flavonoid glycosides (isonaringin, naringin, hesperidin, and neohesperidin), the major bioactive constituents of Zhi‐Zi‐Hou‐Po Decoction, in rat plasma using paeoniflorin as internal standard. Plasma samples were pretreated by a simple protein precipitation with acetonitrile. Chromatographic separation was performed on a shim‐pack XR‐ODS C₁₈ column (75 × 3.0 mm, 2.2 µm) using gradient elution with mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.5 mL/min. Mass spectrometric detection was conducted on a 3200 QTRAP mass spectrometry equipped with electrospray ionization source in negative ionization mode. Quantification was performed using multiple reactions monitoring mode. Calibration curves exhibited good linearity (r > 0.9947) over a wide concentration range for all analytes, and the lower limits of quantification were 10, 5, 1, 5, 1, 5, 1, and 5 ng/mL for geniposide, genipin gentiobioside, honokiol, magnolol, isonaringin, naringin, hesperidin, and neohesperidin, respectively. The intraday and interday precisions at three quality control levels were less than 12.3% and the accuracies ranged from ?11.2 to 10.7%. Extraction recovery, matrix effect, and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of the eight analytes after oral administration of Zhi‐Zi‐Hou‐Po decoction to rats.