Identification and determination of the major constituents in traditional Chinese medicinal plant Polygonum multiflorum thunb by HPLC coupled with PAD and ESI/MS

An HPLC-PAD-MS method has been developed for the qualitative and quantitative analysis of the major chemical constituents in Polygonum multiflorum Thunb. Chromatographic separation was conducted on an Alltima C18 column using water:acetonitrile:acetic acid as the mobile phase. Altogether nine compounds of various classes, including stilbene glucosides, anthraquinone glucosides and anthraquinone derivates, were identified by online ESI/MS. Their identities were ascertained by comparison with data derived from the literature and/or standard compounds. Five components were quantified by HPLC-PAD and the method was fully validated. All the linear regressions were acquired with R2 > 0.99 and the quantification limits (with a signal-to-noise ratio of 3) ranged from 0.63 and 1.57 ng. Repeatability was evaluated by intra- and inter-day assays and RSD value was within 2.47%. Recovery studies for the quantified compounds were found to be within the range 96.32-102.53% with RSD less than 2.35%. The overall procedure is rapid and reproducible and is considered suitable for qualitative and quantitative analysis of a large number of samples.     

Development of an LC‐MS method for simultaneous quantitation of amentoflavone and biapigenin,the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood

Introduction – Biflavones of Hypericum perforatum L. are bioactive compounds used in the treatment of inflammation and depression. Determination of amentoflavone and biapigenin from blood is challenging owing to their similar structures and low concentrations. Objective – To develop a rapid, sensitive and accurate method based on liquid‐phase extraction followed by high‐performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC‐ESI‐MS) for quantification of biflavones in human plasma. Methodology – After extraction from blood, the analytes were subjected to HPLC with an XTerra® MS C₁₈ column and a binary mobile phase consisting of 2% formic acid in water and acetonitrile under isocratic elution conditions, with ESI‐MS detection in the negative ion mode and multiple reaction monitoring (MRM). Results – Both calibration curves showed good linearity within the concentration range 1–500 ng/mL. Limits of detection (S/N = 3) were 0.1 ng for pure substances and the limits of quantitation (S/N = 5) were 1.0 ng/mL from analyte‐spiked serum. The grand mean recovery was 90% from several subsamples of each biflavone. The imprecision (RSD) of peak areas was between 5% (intraday) and 10% (interday) for high concentrations (250 ng/mL) and between 10% (intraday) and 15% (interday) for low concentrations (1 ng/mL). Inaccuracy of the mean was less than 20% at the lower limit of quantitation. Conclusion – The developed and validated method for determination of biflavones from human plasma was effectively applied to pharmacokinetic studies of 13 probands and preliminary results indicate biphasic concentration–time curves. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of nepetalactones in catnip (Nepeta cataria L.) by HPLC coupled with ultraviolet and mass spectrometric detection

Nepetalactones, the major chemical components of catnip (Nepeta cataria L.), were analysed by reversed-phase HPLC coupled with UV and MS detection. Two major nepetalactones, Z,E-nepetalactone and E,Z-nepetalactone, were successfully identified and quantified. The linearity range for Z,E-nepetalactone was determined as 0.00655-0.655 mg/mL with a correlation coefficient of 0.9999, and the linearity range of E,Z-nepetalactone was found to be 0.00228-0.456 mg/mL with a correlation coefficient of 0.9999, under UV detection at 228 nm. The linearity ranges were from 0.00164 to 0.0328 mg/mL, with a correlation coefficient of 0.9999, for ZE-nepetalactone and 0.00114-0.0228 mg/mL, with a correlation coefficient of 0.9999, for E,Z-nepetalactone by MS detection with selected ion monitoring of ion peak m/z 167. The MS detection was found to be more sensitive than UV detection and this method was validated as simple, reliable and sensitive for catnip nepetalactone analysis. This method can be used for identification and fingerprinting of catnip products.     

High-performance liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry for analysis of Angelica sinensis

An HPLC-PAD-API/MS method for analysing the chemical constituents of Angelica sinensis (A. sinensis) has been developed. ESI and APCI spectra, in both positive ion (PI) and negative ion (NI) modes, provided very useful information concerning the molecular weights of detected compounds. By comparing the retention times, UV spectra, mass spectra and molecular weights of detected compounds with those published in literature, 15 constituents of A. sinensis could be tentatively identified. This technique involving combined MS information may provide an objective, reliable and rapid analytical method for the quality control and database research of traditional Chinese medicines.     

Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry

A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts.     

中药降脂方与阿托伐他汀代谢性相互作用的体外研究

目的：本研究旨在评估降脂中药复方2（Fang-2）及其6个饮片（虎杖、泽泻、甘草、苍术、厚朴和夏枯草）的水提物对肝脏细胞色素CYP3A4的抑制作用,及对阿托伐他汀的代谢性药物相互作用。方法：（1）应用中药标准化制备技术,制备降脂中药复方2及其6个饮片的水提物;采用超速离心法制备肝微粒体。（2）评价降脂中药复方2及其饮片对CYP3A4抑制作用。（3）评价降脂中药复方2及其饮片的水提物对阿托伐他汀的代谢的影响。结果：（1）体外实验的结果提示复方2的水提物显著地抑制了微粒体CYP3A4的活性,其IC50值为9．884mg／mL。虎杖水提物对微粒体CYP3A4的活性具有显著的抑制作用,其IC50值为0．5491mg／mL。（2）阿托伐他汀是首过代谢CYP3A4的底物,在肝脏微粒体中大于70％的母体被代谢,其体外清除率Clint和半数清除时间分别为41．10mL／mg／min和60．43分钟。降脂中药复方2水提物与肝微粒体孵育后,阿托伐他汀的首过代谢显著减慢。降脂中药复方2及其6个饮片水提取物对CYP3A4的抑制强度与避免阿托伐他汀首过代谢的潜力成正比。结论：本文首次应用体外方法研究了在临床上共同使用与PCI术后的阿托伐他汀和降脂中药复方2之间的代谢性相互作用,揭示了后者及其饮片通过抑制CYP3A4改善了前者的强烈的首过代谢,从而可能优化临床治疗方案。     

Protective effect of polygoni cuspidati radix and emodin on <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> cytotoxicity and infection

Vibrio vulnificus, a good model organism of bacterial septicemia, causes fatal septicemia manifesting a fulminating course and a high mortality rate within days. In order to identify new natural substances preventing V. vulnificus infection, a plant library was screened for inhibiting cytotoxicity to host cells by using Trypan blue staining and LDH assay. We found that Polygoni Cuspidati Radix potently suppressed the acute death of HeLa and RAW264.7 cells in a dose dependent manner. Further studies revealed that Polygoni Cuspidati Radix inhibited V. vulnificus growth and survival in HI broth and seawater, respectively. We confirmed that Polygoni Cuspidati Radix contained high level of emodin by thin layer chromatography (TLC). Emodin showed direct antibacterial activity against V. vulnificus. In addition, emodin prevented the morphologic damages and acute death of HeLa cells caused from V. vulnificus. The safety of Polygoni Cuspidati Radix and emodin to host cells was confirmed by MTT assay. Polygoni Cuspidati Radix and emodin protected mice from V. vulnificus infection.     

Analysis of sesquiterpene lactones in Eupatorium lindleyanum by HPLC‐PDA‐ESI‐MS/MS

Introduction – The aerial part Eupatorium lindleyanum is commonly used as an antipyretic and detoxicant clinically in traditional Chinese medicine. Our previous research showed that germacrane sesquiterpene lactones were its main active constituents, so the development of rapid and accurate methods for the identification of the sesquiterpene lactones is of great significance. Objective – To develop an HPLC‐PDA‐ESI‐MS/MS method capable for simple and rapid analysis of germacrane sesquiterpene lactones in the aerial part E. lindleyanum. Methodology – High‐performance liquid chromatography‐photodiode array detection‐electrospray ionization‐tandem mass spectrometry was used to analyze germacrane sesquiterpene lactones of Eupatorium lindleyanum. The fragmentation behavior of germacrane sesquiterpene lactones in a Micromass Q/TOF Mass Spectrometer was discussed, and 9 germacrane sesquiterpene lactones were identified by comparison of their characteristic data of HPLC and MS analyses with those obtained from reference compounds. Results – The investigated germacrane sesquiterpene lactones were identified as eupalinolides C (1), 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐14‐hydroxy‐costunolide (2), eupalinolides A (3), eupalinolides B (4), eupalinolides E (5), 3β‐acetoxy‐8β‐(4′‐oxo‐tigloyloxy)‐14‐hydroxy‐heliangolide (6), 3β‐acetoxy‐8β‐(4′‐oxo‐ tigloyloxy)‐14‐hydroxy‐costunolide (7), hiyodorilactone B (8), and 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐ costunolide (9). Compounds 6, 7 and 9 were reported for the first time. Conclusion – HPLC‐PDA‐ESI‐MS/MS provides a new powerful approach to identify germacrane sesquiterpene lactones in E. lindleyanum rapidly and accurately. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of potato glycoalkaloids using high-pressure liquid chromatography-electrospray ionisation/mass spectrometry

A method for quantifying two toxic glycoalkaloids, alpha-solanine and alpha-chaconine, in potato (Solanum tuberosum) tuber tissue was developed using HPLC-electrospray ionisation (ESI)/MS. Potato samples were extracted with 5% aqueous acetic acid, and the extracts were subjected directly to HPLC-ESI/MS after filtration. By determining the intensities of the protonated molecules of alpha-solanine (m/z 868) and alpha-chaconine (m/z 852) using selected ion monitoring (positive ion mode), a sensitive assay was attained with detection limits of 38 and 14 ppb for the two glycoalkaloids, respectively. The high sensitivity and selectivity of MS detection effectively reduced the time of analysis thus enabling a high throughput assay of glycoalkaloids in potato tubers.     

Determination of saponins in the kernel cake of Balanites aegyptiaca by HPLC-ESI/MS

The kernel cake produced from Balanites aegyptiaca fruit of Israeli origin was analysed for its saponin constituents using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The HPLC was equipped with a reversed-phase C18 column and a refractive index detector (RID), and elution was isocratic with methanol and water (70:30). The MS system was equipped with electrospray ionisation (ESI). Nine compounds were chromatographically separated, their masses were determined in the negative ion mode and subsequent fragmentation of each component was carried out. From the nine components, six saponins with molecular masses of 1196, 1064, 1210, 1224, 1078 and 1046 Da were identified, with the compound of mass 1210 Da being the main saponin (ca. 36%). Saponins with masses of 1224 and 1046 Da have not been previously reported in B. aegyptiaca. In all saponins, diosgenin was found to be the sole aglycone. This study shows that HPLC-ESI/MS is a quick and reliable technique for characterizing the saponins from kernel cake of B. aegyptiaca.     

Validated Method for the Quantification of Atractylenolide III in Different Processed Products of Rhizoma Atractylodes Macrocephalae

Introduction – Rhizoma Atractylodes Macrocephalae (RAM) contains several sesquiterpene compounds including atractylenolide III (AO‐III). This bioactive compound may be used as a chemical marker for the quality control of different processed RAM products. Objective – To develop and validate an RP‐HPLC method for the quantitative determination of AO‐III in RAM and in a variety of processed RAM products. Methodology – HPLC was carried out using a Kromssil C₁₈ RP‐column eluted with methanol–water (70:30) at a flow rate of 1.0 mL/min and with UV detection at 220 nm. Full validation was performed using standard methods. Results – The linear range of AO‐III was 5–50 µg/mL; the regression equation was y = 10210x + 11194 (r = 0.9994) and the average recovery was 101.08% (RSD = 0.98%). The detection and quantification limits for AO‐III were determined to be 0.005 and 0.018 µg/mL at signal‐to‐noise ratios of approximately 3:1 and 10:1, respectively. Conclusion – The described HPLC method is appropriate for quality assurance and differentiation of AO‐III in RAM and different processed products.     

UHPLC/HR‐ESI‐MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti‐lipase Activities’ Evaluation

This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti‐lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and β‐carotene‐linoleic acid bleaching assays) while anti‐lipase activity was evaluated using 4‐methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR‐ESI‐MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N‐caffeoyltyramine, lyciumide A, N‐dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds.     

小儿感冒茶的薄层鉴别和定量测定研究

目的为制定小儿感冒茶(山银花、板蓝根、重楼)的质量控制方法。方法用TCL鉴别山银花、板蓝根、重楼。用高效液相色谱法测定小儿感冒茶中绿原酸的含量。结果通过方法学考察,绿原酸检测浓度在0.244~1.952μg范围内与峰面积积分值呈良好的线性关系,加样回收率为99.22%,RSD%=0.92(n=2)。结论实验应用方法简便准确、重现性好、精密度高。所建质量标准可用于小儿感冒茶的质量控制。     

HPLC coupled on-line to ESI-MS and a DPPH-based assay for the rapid identification of anti-oxidants in Butea superba

A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed in order to combine separation, activity determination and structural identification of anti-oxidants in complex mixtures in one run. The sample was separated by HPLC and the eluate split into two flows. The major portion was fed into an electrospray ionisation MS/MS system, while the minor part was mixed with a free radical, 2,2'-diphenyl-1-picrylhydrazyl (DPPH), and the reaction determined spectrophotometrically. The negative peaks, which indicated the presence of anti-oxidant activity, were monitored by measuring the decrease in absorbance at 517 nm. The developed method was successfully applied to the identification of anti-oxidant compounds in a fraction, obtained by solid-phase extraction, of an extract of a Thai medicinal plant, Butea superba Roxb. The anti-oxidant compounds were separated and identified as procyanidin B2, (-)-epicatechin and procyanidin B5.     

Definitive screening design enables optimization of LC–ESI–MS/MS parameters in proteomics

In proteomics, more than 100,000 peptides are generated from the digestion of human cell lysates. Proteome samples have a broad dynamic range in protein abundance; therefore, it is critical to optimize various parameters of LC–ESI–MS/MS to comprehensively identify these peptides. However, there are many parameters for LC–ESI–MS/MS analysis. In this study, we applied definitive screening design to simultaneously optimize 14 parameters in the operation of monolithic capillary LC–ESI–MS/MS to increase the number of identified proteins and/or the average peak area of MS1. The simultaneous optimization enabled the determination of two-factor interactions between LC and MS. Finally, we found two parameter sets of monolithic capillary LC–ESI–MS/MS that increased the number of identified proteins by 8.1% or the average peak area of MS1 by 67%. The definitive screening design would be highly useful for high-throughput analysis of the best parameter set in LC–ESI–MS/MS systems.     

Separation of Organoarsenicals by Means of Zwitterionic Hydrophilic Interaction Chromatography (ZIC®‐HILIC) and Parallel ICP‐MS/ESI‐MS Detection

An analytical scheme was developed for the separation and detection of organoarsenicals using a zwitterionic stationary phase of hydrophilic interaction chromatography (ZIC^®‐HILIC) coupled in parallel to electrospray ionization mass spectrometry (ESI‐MS) and to inductively coupled plasma mass spectroscopy (ICP‐MS). The optimization of separation and detection for organoarsenicals was mainly focused on the influence of the percentage of acetonitrile (MeCN) used as a major component of the mobile phase. Isocratic and gradient elution was applied by varying the MeCN percentage from 78 % to 70 % MeCN and 22 % to 30 % of an aqueous solution of ammonium acetate (125 mM NH₄Ac; pH 8.3) on a ZIC^®‐HILIC column (150 × 2.1 mm id, 3.5 μm), to allow for the separation and successful detection of nine organoarsenicals (i.e., 3‐nitro‐4‐hydroxyphenylarsonic acid (roxarsone, Rox), phenylarsonic acid (PAA), p‐arsanilic acid (p‐ASA), phenylarsine oxide (PAO), dimethylarsinate (DMA), methylarsonate (MMA), arsenobetaine (AsB), arsenocholine (AsC) and trimethylarsine oxide (TMAO)) within 45 min. All analytes were prepared in the mobile phase. The flow rate of the mobile phase, the splitting ratio between ICP‐MS and ESI‐MS detection, and the oxygen addition were adapted to ensure that there appeared a stably burning inductively coupled plasma. Furthermore, the analytical method was evaluated by the identification and quantification of AsB in the reference material DORM‐2 (dogfish muscle) resulting in a 95‐% recovery with respect to the AsB concentration in the extract.     

Metabolite analysis of human fecal water by gas chromatography/mass spectrometry with ethyl chloroformate derivatization

Fecal water is a complex mixture of various metabolites with a wide range of physicochemical properties and boiling points. The analytical method developed here provides a qualitative and quantitative gas chromatography/mass spectrometry (GC/MS) analysis, with high sensitivity and efficiency, coupled with derivatization of ethyl chloroformate in aqueous medium. The water/ethanol/pyridine ratio was optimized to 12:6:1, and a two-step derivatization with an initial pH regulation of 0.1 M sodium bicarbonate was developed. The deionized water exhibited better extraction efficiency for fecal water compounds than did acidified and alkalized water. Furthermore, more amino acids were extracted from frozen fecal samples than from fresh samples based on multivariate statistical analysis and univariate statistical validation on GC/MS data. Method validation by 34 reference standards and fecal water samples showed a correlation coefficient higher than 0.99 for each of the standards, and the limit of detection (LOD) was from 10 to 500 pg on-column for most of the standards. The analytical equipment exhibited excellent repeatability, with the relative standard deviation (RSD) lower than 4% for standards and lower than 7% for fecal water. The derivatization method also demonstrated good repeatability, with the RSD lower than 6.4% for standards (except 3,4-dihydroxyphenylacetic acid) and lower than 10% for fecal water (except dicarboxylic acids). The qualitative means by searching the electron impact (EI) mass spectral database, chemical ionization (CI) mass spectra validation, and reference standards comparison totally identified and structurally confirmed 73 compounds, and the fecal water compounds of healthy humans were also quantified. This protocol shows a promising application in metabolome analysis based on human fecal water samples.     

Determination of urinary phytoestrogens by HPLC-MS/MS: a comparison of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI)

A comparison of the analytical performance of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) for the quantitative determination of six urinary phytoestrogens (daidzein, O-desmethylangolensin, equol, enterodiol, enterolactone and genistein) by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is presented here. Both APCI and ESI were suitable for the analysis of these compounds; however, ESI did improve measurement imprecision and sensitivity in certain cases. Method imprecision (between-run coefficients of variation [CVs] from duplicate analysis of three quality control [QC] urine pools across 20 runs) was 5.6-12% for ESI, as opposed to 5.3-30% for APCI. At low concentrations (3-60 ng/mL, analyte dependent) imprecision was lower with ESI, whereas both techniques were generally commensurate at high concentrations (200-1000 ng/mL, analyte dependent). Method accuracy (spiked analyte recovery from the QC pools) was comparable between techniques: 86-114% for ESI; 95-105% for APCI. Limits of detection (LODs) were equivalent or better with ESI compared to APCI, with the most significant LOD improvement observed for equol (ESI: 0.3 ng/mL; APCI: 2.7 ng/mL). This translated into a substantial increase in equol detection frequency (% of sample results above LOD) within a random patient sample subset (98% for ESI, compared to 81% for APCI, n=378). Correlation (Pearson) and agreement (Deming regression, Bland-Altman bias) between ESI and APCI results in the patient subset was better in cases where imprecision and sensitivity was similar for both techniques (daidzein, enterolactone, genistein: r=0.993-0.998; slope=0.98-1.03; bias=-4.2 to -0.8%); correlation and/or agreement was poorer for analytes, where APCI imprecision and sensitivity were inferior (equol, O-desmethylangolensin, enterodiol). Baring significant factors arising from differences in ionization source design, these observations suggest that ESI is more appropriate for urinary biomonitoring of these compounds by LC-MS/MS.     

Determination of 5-aminoimidazole-4-carboxamide in human plasma by ion-pair extraction and LC-MS/MS

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was established for the determination of 5-aminoimidazole-4-carboxamide (AICA) in human plasma. The method included a solvent extraction of AICA as an ion pair with 1-pentanesulfonate ion and a separation on a Hypersil ODS2 column with the mobile phase of methanol-water (68:32, v/v). Determination was performed using an electrospray ionization source in positive ion mode (ESI(+)). Multiple reaction monitoring (MRM) was utilized for the detection monitoring m/z at 127-->110 for AICA, and 172-->128 for IS. The calibration curve was linear within a range from 20 to 2000 ng/mL and the limit of quantity for AICA in plasma was 20 ng/mL. RSD of intra-assay and inter-assay were no more than 5.90% and 5.65%.