Qualitative and quantitative analysis of major constituents from Dazhu Hongjingtian capsule by UPLC/Q‐TOF‐MS/MS combined with UPLC/QQQ‐MS/MS

In this work, a sensitive and efficient method was established and validated for qualitative and quantitative analysis of major bioactive constituents in Dazhu Hongjingtian capsule by liquid chromatography tandem mass spectrometry. A total of 32 compounds were tentatively identified using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Furthermore, 12 constituents, namely gallic acid, 3,4‐dihydroxybenzoic acid, salidroside, p‐ coumaric acid‐4‐O ‐β ‐d ‐glucopyranoside, bergeninum, 4‐hydroxybenzoic acid, 4‐hydroxyphenylacetic acid, syringate, 6′′‐O ‐galloylsalidroside, rhodiosin, rhodionin and kaempferol‐7‐O ‐α ‐l ‐rhamnoside, were simultaneously quantified by the developed ultra‐performance liquid chromatography coupled with a triple quadrupole mass spectrometry method in 9 min. All of them were analyzed on an Agilent ZorBax SB‐C₁₈ column (3.0 × 100 mm, 1.8 μm) with linear gradient elution of methanol–0.1% formic acid water. The proposed method was applied to analyze three batches of samples with acceptable linearity (R , 0.9979–0.9997), precision (RSD, 1.3–4.7%), repeatability (RSD, 1.7–4.9%), stability (RSD, 2.2–4.9%) and recovery (RSD, 0.6–4.4%) of the 12 compounds. As a result, the analytical method possessing high throughput and sensitivity is suitable for the quality control of Dazhu Hongjingtian capsule.     

Quantitative determination of 15 bioactive triterpenoid saponins in different parts of Acanthopanax henryi by HPLC with charged aerosol detection and confirmation by LC–ESI‐TOF‐MS

Triterpenoid saponins are difficult to analyze using high‐performance liquid chromatography coupled to UV/vis spectrophotometry due to their lack of chromophores. This study describes the first analytical method for the determination of 15 triterpenoid saponins from the leaves, stems, root bark, and fruits of Acanthopanax henryi, using a high‐performance liquid chromatography with charged aerosol detection coupled with electrospray ionization mass spectrometry method. The separation was carried out on a Kinetex XB‐C18 column with an acetonitrile/water gradient as the mobile phase, followed by charged aerosol detection. The operating conditions of charged aerosol detection were set at 24 kPa for nitrogen pressure and 100 pA for the detection range. Liquid chromatography with electrospray ionization mass spectrometry is described for the identification of compounds in plant samples. The electrospray ionization mass spectrometry method involved the use of the [M + Na]⁺ and [M + NH₄]⁺ ions for compounds 1 – 15 in the positive ion mode with an extracted ion chromatogram. The developed method was fully validated in terms of linearity, sensitivity, precision, repeatability, and recovery, then subsequently applied to evaluate the quality of A. henryi.     

Quality assessment of traditional Chinese medicine herb couple by high‐performance liquid chromatography and mass spectrometry combined with chemometrics

This study was designed to develop a simple, specific and reliable method to overall analyze the chemical constituents in clematidis radix et rhizome/notopterygii rhizome et radix herb couple using high‐performance liquid chromatography coupled with tandem mass spectrometry and multiple chemometric analysis. First, the separation and qualitative analysis of herb couple was achieved on an Agilent Zorbax Eclipse Plus C₁₈ column (250 mm × 4.6 mm, 5 μm), and 69 compounds were unambiguously or tentatively identified. Moreover, in quantitative analysis, eight ingredients including six coumarins and two triterpenoid sapogenins were quantified by high‐performance liquid chromatography coupled with tandem mass spectrometry. In terms of good linearity (r² ≥ 0.9995) with a relatively wide concentration range, recovery (85.40–102.50%) and repeatability (0.99–4.45%), the validation results suggested the proposed method was reliable, and successfully used to analyze ten batches of herb couple samples. Then, hierarchical cluster analysis and principal component analysis were used to classify samples and search significant ingredients. The results showed that ten batches of herb couple samples were classified into three groups, and six compounds were found for its better quality control.     

Study on chemical constituents and fingerprints of Phellodendron amurense Rupr. based on ultra-performance liquid chromatography–quadrupole–time-of-flight–mass spectrometry

The chemical constituents from Phellodendron amurense Rupr. were characterized systematically by ultra-performance liquid chromatography—quadrupole–time-of-flight–mass spectrometry method for collecting mass spectrometry data, and the fingerprints method was established, providing reference for its quality control. The chromatographic column was ACQUITY UPLC BEH-C₁₈ (100 mm×2.1 mm, 1.7 μm). The mobile phase was acetonitrile-0.1% formic acid aqueous solution and the compounds from P. amurense Rupr. were identified by Qualitative Analysis 10.0 software, reference substance, retention time, mass spectrometry fragmentation pattern and database retrieval. Meanwhile, liquid chromatography–mass spectrometry fingerprint methods of P. amurense Rupr. and Phellodendron chinense Schneid. were established by using the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine (2012 edition), and the differences were analyzed by multivariate statistical analysis methods. A total of 105 compounds were identified, including 102 alkaloids, two phenolic acids, and one lactone compound. Liquid chromatography–mass spectrometry fingerprint method was established with ideal precision, stability and repeatability, and 12 quality differential markers were recognized between the above two herbs. Liquid chromatography–mass spectrometry method can be used for qualitative analysis of the constituents of Phellodendron amurense Rupr., providing reference for clarifying the material basis and promoting the clinical precision medication and quality evaluation of P. amurense Rupr.     

Rapid characterization of the chemical constituents of Cortex Fraxini by homogenate extraction followed by UHPLC coupled with Fourier transform ion cyclotron resonance mass spectrometry and GC–MS

Cortex Fraxini is an important traditional Chinese medicine. In this work, a rapid and reliable homogenate extraction method was applied for the fast extraction for Cortex Fraxini, and the method was optimized by response surface methodology. Ultra high performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry and gas chromatography with mass spectrometry were established for the separation and characterization of the constituents of Cortex Fraxini. Liquid chromatography separation was conducted on a C₁₈ column (150 mm × 2.1 mm, 1.8 μm), and gas chromatography separation was performed on a capillary with a 5% phenyl‐methylpolysiloxane stationary phase (30 m × 0.25 mm × 0.25 mm) by injection of silylated samples. According to the results, 33 chemical compounds were characterized by liquid chromatography with mass spectrometry, and 11 chemical compounds were characterized by gas chromatography with mass spectrometry, and coumarins were the major components characterized by both gas chromatography with mass spectrometry and liquid chromatography with mass spectrometry. The proposed homogenate extraction was an efficient and rapid method, and coumarins, phenylethanoid glycosides, iridoid glycosides, phenylpropanoids, and lignans were the main constituents of Cortex Fraxini. This work laid the foundation for further study of Cortex Fraxini and will be helpful for the rapid extraction and characterization of ingredients in other traditional Chinese medicines.     

Separation and identification of phenolic compounds in Bidens pilosa L. by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A validated method based on ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was established to separate and identify phenolic compounds in Bidens pilosa L. Mass spectrometry experiments were performed both in positive and negative ion modes. A total of 35 compounds were detected, and 26 phenolic compounds were unequivocally identified or tentatively assigned based on retention time, maximum UV absorption, molecular formula, and fragments. The ultra high performance liquid chromatography method was validated and showed good linearity (R² ≧ 0.9996) over the test range. The limits of detection and quantification were above 0.072 and 0.162 μg/mL, respectively. The relative standard deviations of intraday and interday precision were below 0.3 and 1.6%, respectively.     

Simultaneous detection of eight active components in Radix Tinosporae by ultra high performance liquid chromatography coupled with electrospray tandem mass spectrometry

A rapid and sensitive ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous determination of eight major active components (magnoflorine, menisperine, 20‐hydroxyecdysone, cepharanthine, columbamine, jatrorrhizine, columbin, and palmatine) in Radix Tinosporae. The separation was performed on an InterSustainSwift C₁₈ column (1.9 μm, 2.1 id × 100 mm) at 40 °C with a gradient elution. A mixture of acetonitrile and methanol (v/v = 1:1) and ammonium acetate buffer (25 mmol/L ammonium acetate with 0.2% formic acid) were used as mobile phases, and the flow rate was set at 0.4 mL/min. The recovery was tested in real samples and calculated to be 86.97–111.28%, and all the compounds showed good linearity (r > 0.998) in relatively wide concentration ranges. The developed method was applied to the determination of eight active compounds in real herb samples, which were collected from four different places. It has been demonstrated that the proposed method has great potential for the quality control of the traditional Chinese medicine Radix Tinosporae.     

Simultaneous and rapid determination of gefitinib,erlotinib and afatinib plasma levels using liquid chromatography/tandem mass spectrometry in patients with non‐small‐cell lung cancer

A simultaneous, selective, sensitive and rapid liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of gefitinib, erlotinib and afatinib in 250 μL samples of human blood plasma. Diluted plasma samples were extracted using a liquid‐phase extraction procedure with tert‐butyl methyl ether. The three drugs were separated by high‐performance liquid chromatography using a C₁₈ column and an isocratic mobile phase running at a flow rate of 0.2 mL/min for 5 min. The drugs were detected using a tandem mass spectrometer with electrospray ionization using imatinib as an internal standard. Calibration curves were generated over the linear concentration range of 0.05–100 nm in plasma with a lower limit of quantification of 0.01 or 0.05 nm for all compounds. Finally, the validated method was applied to a clinical pharmacokinetic study in patients with nonsmall‐cell lung cancer (NSCLC) following the oral administration of afatinib. These results indicate that this method is suitable for assessing the risks and benefits of chemotherapy in patients with NSCLC and is useful for therapeutic drug monitoring for NSCLC treatment. As far as we know, this is the first report on LC‐MS/MS method for the simultaneous quantification of NSCLC tyrosine kinase inhibitor plasma concentrations including afatinib. Copyright © 2015 John Wiley & Sons, Ltd.     

Flexible and powerful strategy for qualitative and quantitative analysis of valepotriates in Valeriana jatamansi Jones using high‐performance liquid chromatography with linear ion trap Orbitrap mass spectrometry

Valeriana jatamansi Jones is an important medicinal plant and its quality is closely related to its region of origin. In the current study, we utilized a flexible and powerful strategy for comprehensive evaluation of the quality diversity for 15 regions in China. The method was based on a hybrid linear ion trap‐Orbitrap mass spectrometry platform. For structure characterization, fragmentation patterns were detected by analyzing a series of standard compounds using data dependent multistage mass spectrometry acquisition. A fragment ion database for valepotriates was established, and the acquired data were high throughput filtered by fragment ion search for compound identification. For quantitative purposes, we normalized the mass spectrometry data of 15 samples using SIEVE 2.0 and the differences in composition were analyzed using principal component analysis combined with hierarchical clustering analysis. The results identified a total of 92 compounds from Valeriana jatamansi Jones. Samples from Dali, Kunming, and Baoshan have better qualities and concentrations of the main active constituents. To verify our strategy, we compared the valtrate, acevaltrate, and baldrinal contents using high‐performance liquid chromatography with diode array detector. We developed and validated a comprehensive qualitative and quantitative analytical method to achieve quality control of Valeriana jatamansi Jones.     

Chemical constituents of Meconopsis horridula and their simultaneous quantification by high‐performance liquid chromatography coupled with tandem mass spectrometry

Meconopsis horridula Hook.f. Thoms has been used as a traditional Tibetan medicine to clear away heat, relieve pain, and mobilize static blood. In this study, a reliable method based on high‐performance liquid chromatography with diode array detection and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was established for the identification of components in this herb. A total of 40 compounds (including 17 flavonoids, 15 alkaloids, and eight phenylpropanoids) were identified or tentatively identified. Among them, 17 components were identified in the herb for the first time. Compound 39 appears to be a novel compound, which is confirmed as 3‐(kaempferol‐8‐yl)‐2,3‐epoxyflavanone by NMR spectroscopy and mass spectrometry. Moreover, seven major constituents were simultaneously quantified by the developed high‐performance liquid chromatography with tandem triple‐quadrupole mass spectrometry method. The quantitative method was validated and quality parameters were established. The study provides a comprehensive approach for understanding this herbal medicine.     

Sensitive and rapid simultaneous quantitation of leucovorin and its major active metabolite 5-methyl-tetrahydrofolate in human plasma using a liquid chromatography coupled with triple quadruple mass spectrometry

Bioanalysis of an endogenous compound such as leucovorin is never an easy task on a liquid chromatography tandem mass spectrometer (LC–MSMS). Unless it is necessary, regulatory guidance discourages working with surrogate matrices for calibration curve standard preparation. Herein, a selective and sensitive liquid chromatography–tandem mass spectrometry method for simultaneous determination of leucovorin and 5-methyl tetrahydrofolic acid in human plasma was developed and validated. Stable labeled internal standards, i.e. leucovorin D₄ and 5- methyl tetrahydrofolic acid ¹³C₅, were used as internal standards to track and compensate the parent compounds during processing and extraction from plasma. The method involves a rapid solid-phase extraction from plasma followed by reverse-phase gradient chromatography and mass spectrometry detection with a total run time of 5 min. The method was developed and validated from 5 to 2,202 ng/ml for leucovorin and from 5 to 1,300 ng/ml for 5-methyl tetrahydrofolic acid. The mean recoveries for leucovorin and 5-methyl tetrahydrofolic acid were 100.4 and 100.9% respectively. The validated method enabled the simultaneous analysis of leucovorin and 5-methyl tetrahydrofolic acid in samples from clinical pharmacokinetic studies of leucovorin. The peak concentrations of leucovorin and 5-methyl tetrahydrofolic acid were 651–883 and 518–635 ng/ml, respectively, in fasted and fed conditions. The terminal half-life values for leucovorin and 5-methyl tetrahydrofolic acid were 9.3–10.5 and 9.2–17.6 h, respectively.     

Simultaneous quantitative analysis of nine constituents in six Chinese medicinal materials from Citrus genus by high‐performance liquid chromatography and high‐resolution mass spectrometry combined with chemometric methods

In this study, we aim to determine the chemical constituents of six Chinese medicinal materials from the Citrus genus using high‐performance liquid chromatography and high‐resolution mass spectrometry. Eight flavonoids and one coumarin were identified and further quantified as marker substances by high‐performance liquid chromatography method. The separation was performed on an Agilent TC‐C₁₈ column with 0.1% formic acid and acetonitrile as the mobile phase under gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, intra‐ and inter‐day precision and repeatability, limit of detection, limit of quantitation, and recovery. It was subsequently applied to evaluate the quality of 103 batches of the Chinese medicinal materials from the Citrus genus. In addition, the principal constituent analysis was used to compare the samples of different species from the Citrus genus leading to successful classification of the samples in accordance with their origins. It was found that the contents of nine constituents varied greatly in different ripening stages and varieties of the samples from the Citrus genus. In addition, neoeriocitrin and 5,7‐dimethoxycoumarin were determined as two unique constituents of ‘Zhiqiao’ and ‘Foshou’, respectively. In conclusion, this study provides a chemical basis for quality control of Chinese medicinal materials from the Citrus genus.     

Qualitative and quantitative analysis of Alpiniae Oxyphyllae Fructus by high-performance liquid chromatography coupled to Fourier transform-ion cyclotron resonance mass spectrometry

Alpiniae Oxyphyllae Fructus, as a homology of medicine and food, has been widely used in China for thousands of years. However, the existing qualitative and quantitative methods are difficult to evaluate the quality of Alpiniae Oxyphyllae Fructus samples from multiple sources. In this paper, an high-performance liquid chromatography fingerprint was established for assessing the quality of Alpiniae Oxyphyllae Fructus from different areas. Then, high-performance liquid chromatography was coupled to Fourier transform-ion cyclotron resonance mass spectrometry for characterization of the chemical compositions in Alpiniae Oxyphyllae Fructus. In fingerprint analysis, 54 common peaks were confirmed and six chromatographic peaks of them were identified. The similarity of 14 samples from different areas was between 0.990 and 1.000. Moreover, a total of 30 chemical components were characterized by high-performance liquid chromatography coupled to Fourier transform-ion cyclotron resonance mass spectrometry method, six compounds of which were decisively identified. Finally, the content of nootkatone was determined by high-performance liquid chromatography. In conclusion, the methods used in this study are efficient for qualitative and quantitative analysis of Alpiniae Oxyphyllae Fructus. Also, these methods can be used to control the quality of other traditional Chinese medicines.     

Glucose and glycerol concentrations and their tracer enrichment measurements using liquid chromatography tandem mass spectrometry

The present study describes a new liquid chromatography tandem mass spectrometry method for high‐throughput quantification of glucose and glycerol in human plasma using stable isotopically labeled internal standards and is suitable for simultaneous measurements of glucose and glycerol enrichments in connection to in vivo metabolic studies investigating glucose turnover and lipolytic rate. Moreover, in order to keep up with this new fast analysis, simple derivatization procedures have been developed. Prior to analysis, glucose and glycerol were derivatized using benzoyl chloride in order to form benzoylated derivatives via new simplified fast procedures. For glucose, two internal standards were evaluated, [U‐¹³C₆]glucose and [U‐¹³C₆, D₇]glucose, and for glycerol, [U‐¹³C₃, D₈]glycerol was used. The method was validated by means of calibration curves, quality control samples, and plasma samples spiked with [6,6‐D₂]glucose, [U‐¹³C₆]glucose, and [1,1,2,3,3‐D₅]glycerol in order to test accuracy, precision, and recovery of the method. Moreover, post preparative and freeze‐thaw sample stability were tested. The correlation of calibration curves for the glucose concentration were r² = 0.9998 for [U‐¹³C₆]glucose and r² = 0.9996 for [U‐¹³C₆, D₇]glucose, and r² = 0.9995 for the glycerol concentration. Interday accuracy for glucose using [U‐¹³C₆]glucose and glycerol determined in spiked plasma were respectively 103.5% and 106.0%, and the coefficients of variation were 2.0% and 9.7%, respectively. After derivatization, plasma samples were stable for at least 14 days. In conclusion, we have developed and validated a novel, accurate, and sensitive high‐throughput liquid chromatography tandem mass spectrometry method for simultaneous determination of glucose and glycerol concentrations and enrichment of infused tracers most commonly used in human metabolic kinetic studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Quality consistency evaluation of commercial transfer factor injections by chromatographic fingerprint combined with multivariate statistical analysis

The current quality control methods relying mainly on chromogenic reaction can hardly ensure the quality and safety of the biochemical drug with complex chemical composition. Therefore, a chromatographic fingerprint method was developed for the quality evaluation of a multicomponent biochemical drug, transfer factor injection. High‐performance liquid chromatography fingerprint was measured by using a C₁₈ column (250 × 4.6 mm, 5 µm) with a mobile phase composed of 0.1% trifluoroacetic acid–water and 0.085% trifluoroacetic acid–acetonitrile under gradient elution. The developed method was validated and was subsequently applied to 57 batches of commercial products which were sampled by National Drug Assessment Program. High‐resolution mass spectrometry analysis was performed on characteristic peaks of fingerprints, and a series of amino acids, nucleosides, and deoxynucleosides were identified. In the fingerprint assessments, principal component analysis and Hotelling T² analysis yielded the best results. The results generally indicated that there was a significant difference among products of batch‐to‐batch or from different manufacturers. Abnormal samples and its discriminatory components were also explored. In summary, the established fingerprinting method with multivariate statistical analysis could offer an efficient, reliable, and practical approach for quality consistency evaluation of transfer factor injection, providing a reference for the quality control of other multicomponent biochemical drugs.     

Online background cleanup followed by high‐performance liquid chromatography with tandem mass spectrometry for the analysis of perfluorinated compounds in human blood

In this study, a novel method for the analysis of perfluorinated compounds in whole blood has been developed and validated. The method was developed by using a conventional reversed‐phase C₁₈ column as a trapping column for the elimination of background contamination and high‐performance liquid chromatography with tandem mass spectrometry for the target compounds analysis. The trapping column provided fast online separation of the background contamination of perfluorinated compounds. In this developed method, the limits of detection for different perfluorinated compounds ranged from 0.06 to 0.14 ng/mL. It is notable that the limit of detection (0.07 ng/mL) for perfluorooctanoic acid was improved significantly after the elimination of background contamination. The method was also validated in terms of sensitivity, accuracy, and precision. The recoveries ranged from 66.8 to 111.9%, with relative standard deviations from 2.1 to 15.3%. Our preliminary data suggest that the novel method based on trapping column cleanup followed by high‐performance liquid chromatography with tandem mass spectrometry could be applied in studies on the human exposure to perfluorinated compounds.     

Screening and analysis of potentially active components in Shenxiong glucose injection using UHPLC coupled with photodiode array detection and MS/MS

Shenxiong glucose injection, a pharmaceutical preparation containing a water extract of the roots of Salvia miltiorrhizae and ligustrazine hydrochloride, is widely used in clinical to treat cardiovascular diseases in China. The chemical components of the water extract have been reported and the cardioprotective effects of the injection have been evaluated. However, the chemical constituents of the injection and their correlations with its pharmacological effects have not been established. In this study, 13 chemical constituents of the injection have been identified or characterized by ultra‐high performance liquid chromatography with diode array detection and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry. Besides, the potentially active compounds of this preparation that directly act on cardiac cells have been screened by cell extraction and ultra high performance liquid chromatography targeted multiple reaction monitoring. As a result, eight potentially active compounds, danshensu ( 1 ), ligustrazine hydrochloride ( 4 ), salvianolic acid I/H ( 7 ), lithospermic acid ( 8 ), salvianolic acid D ( 9 ), rosmarinic acid ( 10 ), salvianolic acid B ( 12 ), and salvianolic acid C ( 13 ), were obtained and structurally characterized from the 11 target compounds used for screening. The liquid chromatography with quadrupole time‐of‐flight mass spectrometry and liquid chromatography with multiple reaction monitoring tandem mass spectrometry combination method has demonstrated its potency for the screening, detection, and structural identification of bioactive compounds in a complex matrix.     

Classification of the medicinal plants of the genus Atractylodes using high‐performance liquid chromatography with diode array and tandem mass spectrometry detection combined with multivariate statistical analysis

Analytical methods using high‐performance liquid chromatography with diode array and tandem mass spectrometry detection were developed for the discrimination of the rhizomes of four Atractylodes medicinal plants: A. japonica, A. macrocephala, A. chinensis, and A. lancea. A quantitative study was performed, selecting five bioactive components, including atractylenolide I, II, III, eudesma‐4(14),7(11)‐dien‐8‐one and atractylodin, on twenty‐six Atractylodes samples of various origins. Sample extraction was optimized to sonication with 80% methanol for 40 min at room temperature. High‐performance liquid chromatography with diode array detection was established using a C₁₈ column with a water/acetonitrile gradient system at a flow rate of 1.0 mL/min, and the detection wavelength was set at 236 nm. Liquid chromatography with tandem mass spectrometry was applied to certify the reliability of the quantitative results. The developed methods were validated by ensuring specificity, linearity, limit of quantification, accuracy, precision, recovery, robustness, and stability. Results showed that cangzhu contained higher amounts of atractylenolide I and atractylodin than baizhu, and especially atractylodin contents showed the greatest variation between baizhu and cangzhu. Multivariate statistical analysis, such as principal component analysis and hierarchical cluster analysis, were also employed for further classification of the Atractylodes plants. The established method was suitable for quality control of the Atractylodes plants.     

Analysis of bioactive components and multi‐component pharmacokinetics of saponins from the leaves of Panax notoginseng in rat plasma after oral administration by LC–MS/MS

In this study, simple ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass tandem mass spectrometry is used to characterize the absorbed components in rat plasma after the oral administration of saponins from the leaves of Panax notoginseng. Seventeen prototype compounds are structurally characterized. Furthermore, a simple and sensitive liquid chromatography with tandem mass spectrometry method is also used for the simultaneous determination of notoginsenoside Fc, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb3, ginsenoside Rd, and notoginsenoside Fe in rat plasma within 5 min. After n‐butanol mediated liquid–liquid extraction, all analytes were separated on a C₁₈ column and monitored in negative ion mode. Linearity, sensitivity, intra‐ and inter‐assay precision, accuracy, recovery, matrix effect, and stability were all within acceptable ranges. The validated liquid chromatography with tandem mass spectrometry method is successfully applied to the pharmacokinetic study of saponins from the leaves of Panax notoginseng in rats after oral administration. The results suggest that notoginsenoside Fc and ginsenoside Rb3 showed relatively higher exposure compared with other saponins. All saponins showed a long duration in plasma with a t_1/2 longer than 15 h, except notoginsenoside Fe (t_1/2 = 2.78 h). This study provides important information about the metabolism of saponins from the leaves of Panax notoginseng, which is useful for completely understanding its mechanism of action.