Qualitative and quantitative analysis of the major constituents in Acorus tatarinowii Schott by HPLC/ESI‐QTOF‐MS/MS

Acorus tatarinowii Schott (ATS) is a well‐known traditional Chinese medicine (TCM) for the treatment of epilepsy, amnesia and insomnia. In this study, a methodology utilizing high‐performance liquid chromatography (HPLC) coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐QTOF‐MS/MS) was established for the separation and structural identification of the major chemical constituents in ATS for the first time. Overall, 46 major constituents including flavonoid glycosides, phenylpropane derivatives, amides and lignans were identified or tentatively characterized. Seven major constituents, including four phenylpropane derivatives and three lignans, were further quantified as marker substances, which showed good linearity within the test ranges. These results indicated that the developed quantitative method was linear, sensitive, and precise for quality control of ATS. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of the chemical constituents of the different processed products of Anemarrhena asphodeloides Rhizomes by high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this work, high‐performance liquid chromatography (HPLC) coupled with a hybrid quadrupole time of‐flight mass spectrometry (Q‐TOF‐MS/MS) was used to study chemical compositions of different processed products of Rhizoma Anemarrhenae (RA). A Grace Alltima^TM C₁₈ column (250 × 4.6 mm, 5 µm) was used for separation. Mobile phase consisted of 0.1% formic acid and acetonitrile, using gradient elution. ESI‐MS data was acquired in both positive and negative mode. The experiment was established on the basis of a series of reference substances (two xanthone and seven saponins) to qualitatively identify the chemical compounds of different processed products of RA by MS analysis. There was no difference in the type of chemical constituents between different processed products of RA. A total of 25 compounds were identified, including four xanthones, 21 steroidal saponins and eight pairs of isomers. Copyright © 2015 John Wiley & Sons, Ltd.     

Fragmentation patterns study of iridoid glycosides in Fructus Gardeniae by HPLC‐Q/TOF‐MS/MS

Iridoid glycosides (IGs), the major constituents in Fructus Gardeniae, have demonstrated various pharmacological activities, but there is no systematic chemical profile of IGs in Fructus Gardeniae in the published literature until now. Therefore, it is imperative that a rapid and sensitive high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐Q/TOF‐MS/MS) method is established for comprehensive characterization of IGs in Fructus Gardeniae. Firstly, the fragmentation patterns of six known IGs were investigated and proposed and further concluded the diagnostic fragment ions and characteristic fragmentation pathways. Then, based on the summarized fragmentation patterns and the known compounds in the literatures, the other IGs in Fructus Gardeniae were identified successively. As a result, a total of 20 IGs were identified, of which three pairs of epimers were structurally characterized and differentiated. More importantly, one compound, the isoshanzhiside methyl ester, was tentatively identified as a new compound. The results of this study demonstrate the superiority of HPLC‐MS with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of the multiple groups of constituents in Fructus Gardeniae. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of chemical constituents in Rhodiola Crenulate by high‐performance liquid chromatography coupled with Fourier‐transform ion cyclotron resonance mass spectrometer (HPLC‐FT‐ICR MS)

In this work, an approach using high‐performance liquid chromatography coupled with diode‐array detection and Fourier‐transform ion cyclotron resonance mass spectrometer (HPLC‐FT‐ICR MS) for the identification and profiling of chemical constituents in Rhodiola crenulata was developed for the first time. The chromatographic separation was achieved on an Inertsil ODS‐3 column (150 mm × 4.6 mm,3 µm) using a gradient elution program, and the detection was performed on a Bruker Solarix 7.0 T mass spectrometer equipped with electrospray ionization source in both positive and negative modes. Under the optimized conditions, a total of 48 chemical compounds, including 26 alcohols and their glycosides, 12 flavonoids and their glycosides, 5 flavanols and gallic acid derivatives, 4 organic acids and 1 cyanogenic glycoside were identified or tentatively characterized. The results indicated that the developed HPLC‐FT‐ICR MS method with ultra‐high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents in R. crenulata. And it provides a helpful chemical basis for further research on R. crenulata. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous determination of six constituents in Mahuang Fuzi Xixin by UPLC-PDA–MS/MS

Mahuang Fuzi Xixin (MFX), a classic recipe in traditional Chinese medicine, belongs to an exterior-relieving formula. For quality control of the MFX products, qualitative analysis using ultra-high performance liquid chromatography with diode-array detector–tandem mass spectrometry (UPLC-PDA–MS/MS) was undertaken. Six compounds from the MFX were simultaneously detected. Among them, astragalin and kaempferol 3-rutinoside were quantified through the UPLC–MS/MS method, while asarinin, sesamin, kakuol and methyleugenol were quantified through the UPLC-PDA method. This method can be applied to the quantitative determination of the six compounds in the MFX.     

Qualitative analysis of Psoraleae Fructus by HPLC‐DAD/TOF‐MS fingerprint and quantitative analysis of multiple components by single marker

A variety of bioactive substances may account for the recognized efficacy and wide clinical application of Psoraleae Fructus in China. A high‐performance liquid chromatography–diode array detector (HPLC‐DAD) fingerprint method was developed to present the comprehensive phytochemical profile of the crude drug. Thirteen major compounds were separated and identified by HPLC coupled with time‐of‐flight mass spectrometry (HPLC/TOF‐MS), namely psoralenoside (PO), isopsoralenoside (IPO), psoralen (PS), isopsoralen (IPS), neobavaisoflavone (NBF), bavachin (BC), corylin (CN), bavachromene (BCM), psoralidin (PD), isobavachalcone (IBC), bacachinin (BCN), corylifol A (CA) and bakuchiol (BK). Then quantitative analysis of multiple components by single marker (QAMS) was applied in content determination of PO, IPO, PS, IPS, BC, IBC, BCN, CA and BK, with NBF as the internal standard. The calculation results indicated no significant difference from the traditional external standard method (p > 0.05, RSD < 2.62%), suggesting that QAMS is a reliable and convenient method for content determination of multiple chemical compositions, especially when there is a shortage of reference substances. In conclusion, simultaneous qualitative and quantitative analysis of Psoraleae Fructus may be fulfilled through the newly proposed method of QAMS combined with HPLC‐DAD/TOF‐MS fingerprint.     

Rapid identification of primary constituents in parotoid gland secretions of the Australian cane toad using HPLC/MS‐Q‐TOF

Toad parotoid gland secretion or toad venom has in recent years been increasingly shown to possess potentially beneficial pharmacological effects; this speculation has drawn much interest centred on elucidating the chemical basis of its multimodal effects. For this purpose, we explored the use of a rapid and accurate analysis method for systemic investigation of the parotoid gland chemistry, when extracted from Australian cane toads. Full‐scan data of cane toad venom extract was acquired using high‐performance liquid chromatography coupled with a hybrid quadrupole–time of flight mass spectrometry system (HPLC/MS‐Q‐TOF), with multiple ionization sources (ESI and APCI) in positive and negative mixed modes. By measuring the exact mass differences between the theoretical and measured mass of each assumed compound, we confirmed the presence of 12 key constituents. The present results demonstrate that the use of HPLC/MS‐Q‐TOF with multiple ionization sources delivers exemplary selectivity and sensitivity, allowing for the rapid and accurate identification of constituents within cane toad venom. This paves the way for this technique to be used in future routine screening of components within the genus Bufo and for key analytes too, then reliably assessed for any purported beneficial (clinic) properties. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification and determination of the major constituents in Kai‐Xin‐San by UPLC‐Q/TOF MS and UFLC‐MS/MS method

In order to have overall chemical material information of Kai‐Xin‐San (KXS), the reliable ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometer (UHPLC–Q‐TOF‐MS) and ultra‐fast liquid chromatography mass spectrometer (UFLC‐MS/MS) methods were developed for the identification and determination of the major constituents in KXS. Moreover, the UHPLC–Q‐TOF‐MS method was also applied to screen for multiple absorbed components in rat plasma after oral administration of KXS. The UHPLC–Q‐TOF‐MS method was achieved on Agilent 6520 Q‐TOF mass and operated in the negative ion mode. Good separation was performed on a ZORBAX Eclipse Plus C18 column with a gradient elution at a flow rate of 0.2 ml/min. A total of 92 compounds in KXS were identified or tentatively characterized based on their exact molecular weights, fragmentation patterns, and literature data. A total of 26 compounds including 23 prototype components and three metabolites were identified in rat plasma after oral administration of KXS. Then, 16 major bioactive constituents were chosen as the benchmark substances to evaluate the quality of KXS. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple‐reaction monitoring mode(MRM). The analysis was completed with a gradient elution at a flow rate of 0.4 ml/min within 35 min. The simple and fast method was validated and showed good linearity, precision, and recovery. Furthermore, the method was successful applied for the determination of 16 compounds in KXS. All results would provide essential data for identification and quality control of active chemical constituents in KXS. Copyright © 2016 John Wiley & Sons, Ltd.     

HPLC‐Q‐TOF‐MS/MS for analysis of major chemical constituents of Yinchen–Zhizi herb pair extract

The Yinchen–Zhizi herb pair (YZHP) consists of Herba Artemisiae Scopariae (Yinchen in Chinese) and Fructus Gardeniae (Zhizi in Chinese), and is mainly used to treat icteric hepatitis, itching skin and eczema. However, the bioactive constituents responsible for the pharmacological effects of YZHP are still unclear to date. In this work, a rapid and sensitive method was established to comprehensively study the constituents in YZHP extract by HPLC‐Q‐TOF MS/MS. The analysis was performed on an HPLC system equipped with an Agilent poroshell 120 EC‐C₁₈ column (100 × 2.1 mm, 2.7 mm) working in a gradient elution program coupled to quadrupole‐time‐of‐flight mass spectrometry operating in the negative ion mode. As a result, a total of 46 compounds including 17 from Herba Artemisiae Scopariae and 36 from Fructus Gardeniae were detected and tentatively identified in YZHP extract by comparing the retention time and mass spectrometry and retrieving the reference literature. More importantly, a series of constituents, such as many iridoid glycosides, were reported for the first time in this formula. The HPLC‐Q‐TOF MS/MS method was developed and utilized successfully to identify the major constituents in YZHP extract and would be helpful for further metabolism and pharmacology research on YZHP. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Identification of chemical constituents and rat metabolites of Kangxianling granule by HPLC‐Q‐TOF‐MS/MS

A high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass tandem mass spectrometry method was established to characterize the chemical constituents of Kangxianling granule (KXL), a traditional Chinese medicine formula, and the metabolic profile in rat urine and plasma after oral administration of KXL. A total of 27 compounds in KXL extract and 13 prototype compounds with 12 metabolites in rat urine and plasma were identified. Among the 27 detected compounds, 15 were identified by comparing the retention time and MS data with that of reference compounds and the other 12 compounds were tentatively assigned based on the MS data and reference literature. The main prototype components absorbed in rat were amygdalin, salvianolic acid B, tanshinones and anthraquinones. Hydroxylation, glucuronidation and sulfation were the principal metabolic pathways in rat. The results revealed that the 25 compounds identified in rat urine and plasma were the potential active ingredients of KXL, which provides helpful chemical information for further study of the pharmacology mechanism of KXL. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of the constituents in rat plasma after oral administration of Shexiang Baoxin pill by HPLC‐ESI‐MS/MS

A valid method using liquid chromatography coupled with electrospray ionization (ESI) and ion trap mass spectrometry was established for the study of the absorbed components in rat plasma after oral administration of a traditional Chinese medicine (TCM) Shexiang Baoxin pill. The plasma was deproteinated by adding methanol prior to liquid chromatography, in which separation was carried out on a Symmetry C₁₈ column (5 µm, 250 × 4.6 mm). A linear gradient with 0.5% formic acid–water–acetonitrile was used as mobile phase. Mass spectra were acquired in both negative and positive modes. Twenty‐one components including 17 components from Shexiang Baoxin pill and four metabolites were observed from a comprehensive analysis of the chromatography of Shexiang Baoxin pill, controlled plasma and dosed plasma. All of the 17 prototype compounds and three of the metabolites were identified by comparing their retention behaviors and MS and MS/MS spectra with reference compounds and literature data. This study developed an integrated method for screening the bioactive constituents in plasma after oral adminstration of Chinese herbal medicine and provided helpful chemical information for further pharmacology and active mechanism research on TCM. Copyright © 2009 John Wiley & Sons, Ltd.     

HPLC/QTOF‐MS/MS application to investigate phenolic constituents from Ficus pandurata H. aerial roots

Ficus pandurata H. aerial roots are used as a traditional Chinese medicine for the treatment of uarthritis, indigestion and hyperuricemia. However, the bioactive constituents responsible for the pharmacological effects of F. pandurata H. are unclear. A simple and efficient HPLC/QTOF‐MS/MS (high‐performance liquid chromatography/electrospray ionization with quadrupole time‐of‐flight tandem mass spectrometry) method was established to detect and identify active constituents in the n‐butanol extract of F. pandurata H. aerial roots. Chemical constituents were separated and investigated by HPLC/QTOF‐MS/MS in the negative‐ion mode. Thirty‐seven compounds, including hydroxycinnamic acid derivatives, hydroxybenzoic acid derivatives, hydroquinone glycosides, flavonoid glycosides, etc., were identified or tentatively characterized in the n‐butanol extract of F. pandurata H. aerial roots by comparing the UV spectra, accurate mass spectra and fragmentation pathways and retrieving the reference literatures. Moreover, the flavonoid trisaccharides and hydroxybenzoic acid derivatives were tentatively characterized in F. pandurata H. for the first time. The analytical tool used here is very valuable in the rapid separation and identification of the multiple and minor constituents in the n‐butanol extract of F. pandurata H. aerial roots. Copyright © 2014 John Wiley & Sons, Ltd.     

Ion‐pairing high‐performance liquid chromatography‐mass spectrometry of impurities and reduction products of sulphonated azodyes

An HPLC separation method with triethylammonium acetate mobile phase additive developed for the analysis of impurities in polysulphonated azo dyes provides good separation selectivity and compatibility with electrospray ionisation (ESI) mass spectrometry. The negative‐ion ESI mass spectra containing only peaks of deprotonated molecules [M–H]^– for monosulphonic acids, [M–xH]^x–, and sodiated adducts [M–(x + y)H + yNa]^x– for polysulphonic acids allow easy molecular mass determination of unknown impurities. Based on the knowledge of the molecular masses and of the fragment ions in the MS/MS spectra, probable structures of trace impurities in commercial dye samples are proposed. To assist in the interpretation of the mass spectra of complex polysulphonated azodyes, additional information can be obtained after chemical reduction of azodyes to aromatic amines. The structures of the non‐sulphonated reduction products can be determined by reversed‐phase HPLC/MS with positive‐ion atmospheric pressure chemical ionisation and of the sulphonated products by ion‐pairing HPLC/MS with negative‐ion ESI.     

Quality assessment of Cortex Phellodendri by high‐performance liquid chromatography coupled with electrospray ionization mass spectrometry

A simple method based on liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (LC‐DAD‐ESI‐MS) was developed for the quality assessment of Cortex Phellodendri (CP), which was mainly derived from two species of Phellodendron chinense Schneid and Phellodendron amurense Rupr. Total 41 compounds, including 14 phenols, 24 alkaloids and three liminoidal triterpenes were identified or tentatively characterized from the 75% methanol extract of CP samples by online ESI‐MSⁿ fragmentation and UV spectra analysis. Among them, two phenols and six alkaloids were simultaneously quantified using HPLC‐DAD method. The validated HPLC‐DAD method showed a good linearity, precision, repeatability and accuracy for the quantification of eight marker compounds. Furthermore, the plausible fragmentation pathway of the representative compounds were proposed in the present study. The differences of the chemical constituents content and the comprehensive HPLC profiles between the two CP species using LC‐DAD‐ESI‐MS method are reported for the first time, indicating that the CP drugs from different resources should be used separately in the clinic. Copyright © 2009 John Wiley & Sons, Ltd.     

新型除草剂及其杂质的LC-MS和GC-MS分析

High performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) have been utilized to analyze the synthesized 2-(2-arylaminomethylphenoxy)pyrimidine derivatives, which are a new kind of environmentally benign herbicides and have passed the temporary pesticide registration. The identification of main product and impurities has been achieved according to the UV and mass spectra. Moreover, one impurity, introduced by the raw material in the last step of the synthetic route, was identified by GC-MS analysis. It can be concluded that the combination of chromatography and mass spectrometry, including LC-MS and GC-MS, provided a vital tool of the pesticide science.     

Identification of very long chain fatty acids by atmospheric pressure chemical ionization liquid chromatography‐mass spectroscopy from green alga Chlorella kessleri

A method is described for the enrichment of very long chain fatty acids (VLCFAs) from total fatty acids of heterotrophically cultivated green freshwater alga Chlorella kessleri and their identification as picolinyl esters by means of liquid chromatography‐mass spectrometry with atmospheric pressure chemical ionization (LC‐MS with APCI). The method is based on the use of preparative reversed phase HPLC of hundred‐milligram amounts and their subsequent identification by microbore APCI LC‐MS. A combination of these two techniques was used to identify unusual VLCFAs up to C₄₇, both saturated and monounsaturated, with two positional isomers (ω‐9 and ω‐26).     

Quantitative determination of dipyridamole in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

Dipyridamole is a classic platelet inhibitor which has been a key medicine in clinical therapy of thrombosis and cerebrovascular disease. A rapid, selective and convenient method using high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) was developed for determination of dipyridamole in human plasma. After protein precipitation of 200 μL plasma with methanol, dipyridamole and diazepam (internal standard) were chromatographed on an Ultimate? XB‐C₁₈ (50 × 2.1 mm i.d, 3 μ) column with the mobile phase consisting of methanol–ammonium acetate (5 mM ; 80 : 20, v/v) at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization source (ESI⁺). The retention times of dipyridamole and diazepam were 1.4 and 1.2 min, respectively. The method was validated over a concentration range of 0.0180–4.50 μg/mL (r² ≥ 0.99) with a lower limit of quantitation (LLOQ) of 0.0180 μg/mL for dipyridamole. The intra‐ and inter‐day precisions (RSD) of the assay at all three QC levels were 1.6–12.7% with an accuracy (RE) of ?4.3–1.9%, which meets the requirements of the FDA guidance. The HPLC‐MS/MS method herein described was proved to be suitable for pharmacokinetic study of sustained‐release dipyridamole tablet in volunteers after oral administration. Copyright © 2009 John Wiley & Sons, Ltd.