Identification and characterization of supercritical fluid extracts of Rhizoma Chuanxiong by high performance liquid chromatography ion trap mass spectrometry

The main constituents, senkyunolide A, Z-ligustilide, neocnidilide, 3-butylphthalide, and ligustilide dimers, in supercritical CO₂ fluid extracts of Rhizoma Chuanxiong, a popular Chinese traditional medicine, have been identified and characterized by high performance liquid chromatography tandem mass spectrometry. Separations were carried out on an Agilent (ECLIPSE XDB) C18 analytical column by gradient elution with 0.25% acetic acid and methanol (containing 0.25% acetic acid). An Agilent 1100 series LC/MSD XCT system was operated under positive ESI and auto MS/MS modes for mass spectrometric analysis. Collision-induced dissociation (CID) fragmentations of these phthalides have been investigated and elucidated. Phthalides have primarily undergone two ESI CID pathways: side-chain cleavage with losses of alkenes and ring-opening with eliminations of H₂O followed by losses of CO. Direct neutral loss of CO has not been observed. Sodium adduct ions have demonstrated completely different CID pathways. __________ Translated from Journal Instrumental Analysis (in press, in Chinese)     

Approach based on high‐performance liquid chromatography fingerprint coupled with multivariate statistical analysis for the quality evaluation of Gastrodia Rhizoma

Gastrodia Rhizoma is a Traditional Chinese Medicine applied in the treatment of stroke, numbness of limb, headache and dizziness. However, its clinical effect is threatened by sulfur‐fumigation used in the process of storage. This article employs content determination coupled with high‐performance liquid chromatography fingerprint to investigate the effect of sulfur‐fumigation on Gastrodia Rhizoma so as to evaluate the quality of Gastrodia Rhizoma. The result was that most active ingredient in Gastrodia Rhizoma decreased after sulfur‐fumigation and the fingerprints analyzed by mathematical statistics between sulfur‐fumigated Gastrodia Rhizoma and unfumigated Gastrodia Rhizoma have substantial differences, which reveals that sulfur‐fumigation has a significant influence on the quality of Gastrodia Rhizoma. The conclusion of hierarchical clustering analysis, principal component analysis and partial least squares could validate each other, which implies that the method of mathematical statistics applied for assessing the quality of Gastrodia Rhizoma is effective and stable. The method not only affords a viable strategy for distinguishing Gastrodia Rhizoma whether sulfur‐fumigated or not and assessment of the quality of Gastrodia Rhizoma, but also provides a reference for other herbal medicine that suffers from sulfur‐fumigation.     

Ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry and chemometric analysis of licorice based on the simultaneous determination of saponins and flavonoids

Licorice is among the most popular herbal medicines and frequently used in traditional medicine, food products, and cosmetics. In China, only Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L. are officially used and are usually processed with honey prior to use. To maintain the quality of commercially available herbal products, a simple, rapid, and reliable ultra high performance liquid chromatography with triple quadrupole mass spectrometry was developed to investigate the major active constituents of commercially available licorice products. Nineteen components were accurately determined, including eight triterpenoid saponins, one triterpene, and ten flavonoids. Subsequently, multivariate statistical analysis methods were employed to further explore and interpret the experimental data. The results indicated that liquiritin apioside may be considered as a candidate index for the quality control of licorice as well as 18β‐glycyrrhizic acid and liquiritin. In addition, both 18β‐glycyrrhizic acid and licorice‐saponin G2 can be used for discrimination between crude and honey‐processed licorice. Furthermore, using 18β‐glycyrrhizic acid and liquiritin as markers, this work revealed that the quality of licorice products may have declined in recent years. This highlights the need for additional effort focused on good agricultural practice during the processing of licorice. In summary, this study provides a valuable reference for the quality assessment of licorice.     

Chemical profile and difference analysis of four parts of Strobilanthes sarcorrhiza by ultrafast flow liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry and multivariate statistical analysis

Strobilanthes sarcorrhiza (CTS) is a medicinal plant with various pharmacological effects such as tonifying kidney and anti-inflammatory. However, the chemical composition and difference of its four parts (leaves, stems, rhizomes, and root tubers) have been rarely reported. In this study, ultrafast flow liquid chromatography coupled with quadrupole-time-of-flight MS was applied to analyze the chemical profile of CTS and identify 55 compounds, including terpenoids, phenylethanol glycosides, fatty acid derivatives, chain glycosides, flavonoid glycosides, and others. Among these compounds, 34 compounds were first identified in CTS. They were mainly terpenoids, phenylethanol glycosides, fatty acid derivatives, and so forth. Multivariate statistical analysis, such as principal component analysis and orthogonal partial least squares-discriminant analysis were also used to evaluate the difference in chemical compounds from the four parts of CTS. The results showed that phenylethanol glycosides were the main compounds of the underground parts, while terpenoids were the main compounds of the aboveground parts. This study revealed the chemical diversity and similarity of CTS and suggested that the rhizomes could be used as an alternative medicinal part to improve the resource utilization of CTS.     

Simultaneous determination of seven bioactive components in Guizhi Fuling capsule by microwave-assisted extraction combined with ultra performance liquid chromatography tandem mass spectrometry

A simple, rapid and reliable microwave-assisted extraction (MAE) combined with ultra performance liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of the seven bioactive constituents in Guizhi Fuling capsule (GFC), namely gallic acid, amygdalin, albiflorin, paeoniflorin, paeonol, cinnamic acid and pachymic acid, respectively. The operation of MAE optimised through orthogonal array design experiment was performed at 80°C for 10 min with methanol–water (70:30, v/v) as the extracting solvent. The method was validated including intra- and inter-day precision, repeatability and stability, with relative standard deviation less than 3.9%, 3.3%, 4.4% and 3.1%, respectively. All analytes showed the good linearity (r >0.999), and their average recoveries varied between 98.2% and 101.2%. The results indicated that this method was simple, effective and suitable for the quality control of GFC.     

Simultaneous determination of toxins in algae and water samples by high-performance liquid chromatography with triple quadrupole mass spectrometry

A facile method based on liquid chromatography coupled with electrospray ionization tandem triple quadrupole mass spectrometry working in selected reaction monitoring mode has been established to analyze toxins in the algae and water samples. Twelve types of toxins (anatoxin, cylindrospermopsin, dinophysistoxin-1, nodularin, okadaic acid, microcystins) were efficiently separated under optimized liquid chromatography coupled with mass spectrometry conditions in the selected reaction monitoring mode. Correlation coefficients of the calibration curves, all felt in the range of 0.9958-0.9998, indicated good linearity. The detection limits of toxins in this method were all lower than 0.20 ng/mL and the quantification limits were in the range from 0.04 to 0.60 ng/mL. Except for anatoxin, cylindrospermopsin, and nodularin, the other toxins' recoveries varied from 55.45 to 140.85%. And the relative standard deviations of interday and intraday precision were at 8.61% (n = 5). The high-performance liquid chromatography (HPLC)/electrospray ionization (ESI)-mass spectrometery (MS) method was also successfully applied to analyze the algae and water samples. Owing to its exclusive selectivity and excellent sensitivity, the developed method is a tool for comprehensive analyses of the 12 types of toxins at nanogram levels.     

Simultaneous determination of nine components in Anemarrhena asphodeloides by liquid chromatography-tandem mass spectrometry combined with chemometric techniques

A novel quantitative method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the important active constituents including four steroidal saponins, two xanthone glycosides, two isoflavonoids, and one anthraquinone in different parts of Anemarrhena asphodeloides from different habitats. Hierarchical clustering analysis and principal components analysis were performed to differentiate and classify the samples. The separation was performed on a C(18) column with acidified aqueous acetonitrile gradients. Quantification of the analytes was achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring scanning was employed with switching electrospray ion source polarity between positive and negative modes in a single run. The validation results of the method indicated that the method was simple, rapid, specific, and reliable. The results demonstrated that the quantitative difference in content of nine active compounds was useful not only for chemotaxonomy of many samples from different sources but also for the standardization and differentiation of many similar samples. Simultaneous quantification of bioactive components by HPLC-ESI-MS coupled with chemometric techniques would be a well-acceptable strategy to comprehensively control the quality of A. asphodeloides.     

Simultaneous determination of six active components by a single standard to determine multicomponents combined with fingerprint analysis for the quality control of Rhizoma Chuanxiong

To control the quality of Rhizoma Chuanxiong, a method based on high‐performance liquid chromatography method coupled with diode array detection was developed for the quantitative analysis of six active ingredients using a single standard to determine multi‐components and chemical fingerprint analysis for the first time. The separation was performed on an Agilent Zorbax SB‐C₁₈ column by gradient elution with methanol and aqueous phase (containing 0.5% glacial acetic acid) at a flow rate of 1.0 mL/min. The UV wavelength was set at 274 nm. This assay was fully validated with respect to precision, repeatability, and accuracy. All calibration curves showed good linearity (R²> 0.9994) within test ranges. The limit of detection and limit of quantification were lower than 0.01 and 0.03 μg/mL, respectively. The relative standard deviation for repeatability and the intermediate precision of six analytes were less than 1.6 and 2.5%, respectively, the overall recovery was 96.1–103.1%. In addition, fingerprint chromatography using hierarchical clustering analysis and similarity analysis was performed to differentiate and classify the samples. The method described here could provide a more comprehensive and reasonable scientific assessment of the quality of Rhizoma Chuanxiong. Therefore, the strategy is feasible, credible, and is easily and effectively adapted for evaluating the quality control of Rhizoma Chuanxiong.     

固相萃取-超高效液相色谱-三重四极杆质谱法同时测定不同水体中的6种雌激素

建立了固相萃取-超高效液相色谱-三重四极杆质谱（SPE-UPLC-MS/MS）联用技术同时测定不同水体中6种雌激素（雌三醇、17-β-雌二醇、17- α-雌二醇、雌酮、炔雌醇、己烯雌酚）的分析方法。样品经HLB固相萃取柱提取和净化后经BEH C18色谱柱分离,采用MS/MS多反应监测模式（MRM）进行分析。采用内标法定量,以雌三醇-D3、17-β-雌二醇-D2、己烯雌酚-D8为内标。当6种雌激素的质量浓度在1.0~100 μg/L线性范围内时,所得回归方程的相关系数（r）均不小于0.9982;方法检出限为0.27~0.45 ng/L,定量限为1.08~1.78 ng/L;在高、中、低3个添加水平下的回收率为68.3%~97.4%,相对标准偏差（RSD）小于15%。该方法灵敏、准确,检测范围广,分析速度快,适用于地表水、废水、饮用水源水及生活用水等不同水体中6种雌激素的同时检测。     

Determination of sparstolonin B by ultra‐high performance liquid chromatography coupled with triple quadrupole mass spectrometry: application to pharmacokinetic study of sparstolonin B in rat plasma

Sparstolonin B (SsnB), a spontaneous isocoumarin compound isolated from the tuber of Scirpus yagara Ohwi. (Cyperaceae), possesses potent anti‐inflammatory and antitumor activity. In the present study, a rapid and simple UHPLC/MS/MS method for determination of SsnB in rat plasma was developed and validated. Plasma samples were pretreated by liquid–liquid extraction with ethyl acetate containing rhein as an internal standard and separated on a C₁₈ column at 35 °C, with a gradient mobile phase consisting of acetonitrile and water containing 0.2% (v/v) formic acid within 2.1 min. MS/MS detection was accomplished in multiple reaction monitoring mode with negative electrospray ionization. The precursor–product ion transitions were m/z 266.9 [M–H]^? → m/z 211.0 for SsnB and m/z 283.2 [M–H]^? → m/z 239.0 for IS. The intra‐ and inter‐day precision (RSD) was <8.98% and the accuracy (RE) ranged from ?7.40 to 4.50%. The extraction recoveries ranged from 96.28 to 97.30%. The pharmacokinetic parameters were calculated using Win Nonlin53 software. The absolute bioavailability of SsnB was estimated to be 6.98%. The proposed method was successfully applied to a pharmacokinetic study of SsnB in rats after intravenous administration with a dose of 0.5 mg/kg and oral administration at a dose of 5 mg/kg. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of Chuanxiong Rhizoma substrate on production of ligustrazine in endophytic Bacillus subtilis by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Ligustrazine was the active ingredient of the traditional Chinese medicine Chuanxiong Rhizoma. However, the content of ligustrazine is very low. We proposed a hypothesis that ligustrazine was produced by the mutual effects between endophytic Bacillus subtilis and the Ligusticum chuanxiong Hort. This study aimed to explore whether the endophytic B. subtilis LB5 could make use of Chuanxiong Rhizoma fermentation matrix to produce ligustrazine and clarify the mechanisms of action preliminarily. Ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry analysis showed the content of ligustrazine in Chuanxiong Rhizoma was below the detection limit (0.1 ng/mL), while B. subtilis LB5 produced ligustrazine at the yield of 1.0268 mg/mL in the Chuanxiong Rhizoma‐ammonium sulfate fermentation medium. In the fermented matrix, the reducing sugar had a significant reduction from 12.034 to 2.424 mg/mL, and rough protein content increased from 2.239 to 4.361 mg/mL. Acetoin, the biosynthetic precursor of ligustrazine, was generated in the Chuanxiong Rhizoma‐Ammonium sulfate (151.2 mg/mL) fermentation medium. This result showed that the endophytic bacteria B. subtilis LB5 metabolized Chuanxiong Rhizoma via secreted protein to consume the sugar in Chuanxiong Rhizoma to produce a considerable amount of ligustrazine. Collectively, our preliminary research suggested that ligustrazine was the interaction product of endophyte, but not the secondary metabolite of Chuanxiong Rhizoma itself.     

Comparison of triple quadrupole mass spectrometry and Orbitrap high‐resolution mass spectrometry in ultrahigh performance liquid chromatography for the determination of veterinary drugs in sewage: benefits and drawbacks

This paper presents a comparison of triple quadrupole tandem mass spectrometry (MS/MS) and Orbitrap high‐resolution mass spectrometry (HRMS) combined to ultrahigh performance liquid chromatography for the determination of glucocorticoids and polyether ionophores in sewage, in order to show the major benefits and drawbacks for each mass spectrometry analyser. Overall, HRMS measurements have enhanced performance in terms of confirmatory capabilities than MS/MS measurements. Moreover, similar limits of quantification, limits of detection, linear range and repeatability for glucocorticoids with both the MS/MS and HRMS methods were compared, but in the case of polyether ionophores, slightly better limits of detection and limits of quantification were obtained with the HRMS method because of the high sensitivity obtained when diagnostic ions are used for quantification instead of selected reaction monitoring transitions for these compounds. The two methods have been applied to the analysis of several influent and effluent sewage samples from sewage treatment plants located in the Tarragona region (Catalonia, Spain), showing an excellent correlation between the two methods. Copyright © 2014 John Wiley & Sons, Ltd.     

超高效液相色谱-三重四极杆质谱法同时检测人尿中12种单羟基多环芳烃代谢物

建立了高效、准确的同时测定人体尿样中多种多环芳烃代谢物的方法。取10.0 mL尿液,酶解,经固相萃取净化浓缩、0.2 μm滤膜过滤后,以Acquity UPLC^®HSS T3（100 mm×2.1 mm,1.8 μm）为分析柱,甲醇和水为流动相,采用负离子电喷雾多反应监测模式检测样品中多环芳烃代谢物的含量。12种多环芳烃代谢物在0.04~20.0 μg/L范围内线性关系良好,相关系数>0.99,方法检出限为0.01~0.41 μg/L,平均回收率为80.0%~105%,批内RSD为1.21%~9.12%,批间RSD为4.43%~19.7%。应用该方法对淮河流域某区域的100份人体尿样进行了检测,多环芳烃代谢物的检出率为98%。该方法操作简单,灵敏度高,选择性好,可同时检测尿中12种羟基多环芳烃代谢物。     

Multi-mycotoxin analysis in eggs using a QuEChERS-based extraction procedure and ultra-high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry

A reliable and rapid method has been developed for the determination of 10 mycotoxins (beauvericin, enniatin A, A1, B1, citrinin, aflatoxin B1, B2, G1, G2 and ochratoxin A) in eggs at trace levels. Ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) has been used for the analysis of these compounds in less than 7 min. Mycotoxins have been extracted from egg samples using a QuEChERS-based extraction procedure (Quick, Easy, Cheap, Effective, Rugged and Safe) without applying any further clean-up step. Extraction, chromatographic and detection conditions were optimised in order to increase sample throughput and sensitivity. Matrix-matched calibration was used for quantification. Blank samples were fortified at 10, 25, 50 and 100 μg kg(-1), and recoveries ranged from 70% to 110%, except for ochratoxin A and aflatoxin G1 at 10 μg kg(-1), and aflatoxin G2 at 50 μg kg(-1). Relative standard deviations were lower than 25% in all the cases. Limits of detection ranged from 0.5 μg kg(-1) (for aflatoxins B1, B2 and G1) to 5 μg kg(-1) (for enniatin A, citrinin and ochratoxin A) and limits of quantification ranged from 1 μg kg(-1) (for aflatoxins B1, B2 and G1) to 10 μg kg(-1) (for enniatin A, citrinin and ochratoxin A). Seven samples were analyzed and aflatoxins B1, B2, G1, G2, and beauvericin were detected at trace levels.     

超高效液相色谱-三重四极杆质谱法同时测定美术颜料中33种游离态初级芳香胺

建立了超高效液相色谱-三重四极杆质谱法(UPLC-MS/MS)同时测定水粉画颜料、油画颜料、丙烯画颜料等美术颜料中33种初级芳香胺(PAAs)的检测方法。样品中的初级芳香胺用乙腈提取,经离心分离、氮吹浓缩后,以甲醇-水(1:9, v/v)定容至2 mL, 0.22 μm膜过滤后上机检测。采用BEH Phenyl柱(100 mm×2.1 mm, 1.7 μm),以含0.07%(v/v)甲酸的甲醇溶液-水(1:9, v/v)为流动相,梯度洗脱分离,UPLC-MS/MS多反应监测模式(MRM)检测,同位素内标法定量。方法优化了色谱分离条件、质谱碎裂电压、碰撞能量等,并考察了提取时间、提取溶剂、浓缩方式等对回收率的影响。33种初级芳香胺的方法检出限为5~50 μg/kg,定量限为15~150 μg/kg, 3种不同基质样品在3个添加水平的平均回收率为70.1%~115.8%,相对标准偏差(RSD)为2.1%~15%。本方法操作简便、快速、准确、灵敏度高,能满足相关测定的要求。     

A novel strategy for target profiling analysis of bioactive phenylethanoid glycosides in Plantago medicinal plants using ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry

Phenylethanoid glycosides are a group of phenolic compounds with diverse biological activities such as hypotensive, diuretic, and hypoglycemic effects. In this study, a target profiling analysis approach using ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry (MS) was established on the basis of parent ion scanning for m/z 161, the characteristic product ion for phenylethanoid glycosides. It was successfully employed to discriminate the chemical composition of phenylethanoid glycosides between Plantaginis Herba and Plantaginis Semen, two medicinal parts of Plantago plants, which are widely used as herbal medicine in China. Totally, 34 phenylethanoid glycosides were characterized and tentatively identified by their retention times, MS, and tandem quadrupole MS (MS/MS) data. Combined with chemometrics analysis of principal component analysis and orthogonal projection to latent structural discriminate analysis, eight of them, especially acteoside and plantamajoside, were picked out and contributed to the chemical distinction between Plantaginis Herba and Plantaginis Semen, which might be responsible for the differences in diuretic and hypotensive effects between the two medicinal parts. This new approach for target profiling provides not only a novel idea for specific analysis of active chemical constituents in the same type, but also a promising and reference method for quality evaluation of traditional Chinese medicines.     

Chinese hamster ovary‐sphingomyelin synthase2 biospecific extraction and liquid chromatography with tandem mass spectrometry analysis for the prediction of bioactive components of Rhizoma Polygoni Cuspidati

A novel strategy for predicting bioactive components in traditional Chinese medicines using Chinese hamster ovary‐sphingomyelin synthase2 (CHO‐SMS₂) cell biospecific extraction and high‐performance liquid chromatography with diode array detection and tandem mass spectrometry analysis was proposed. The hypothesis is that when cells are incubated with the extract of traditional Chinese medicines, the potential bioactive components in the traditional Chinese medicines should selectively combine with the cells, while the cell‐combining components would be detectable in the extract of denatured cells. The identities of the cell‐combining components could be determined by liquid chromatography with tandem mass spectrometry. Using the proposed approach, the potential bioactive components of Rhizoma Polygoni Cuspidati, a commonly used traditional Chinese medicine for atherosclerosis, were detected and identified. Eight compounds in the extract of Rhizoma Polygoni Cuspidati were detected as the components selectively combined with CHO‐SMS₂ cells, which is a stable cell line that highly expresses sphingomyelin synthases, it was found that piceid, resveratrol, emodin‐8‐β‐d‐ glucoside, physcion‐8‐β‐d‐ glucoside, emodin, physcion, 3,5,4‘‐trihydroxystilbene‐3‐O‐(6“‐galloyl)‐glucoside, and emodin‐1‐O‐glucoside combined specifically with CHO‐SMS₂ cells. The results indicate that the proposed approach may be applied to predict the bioactive candidates in traditional Chinese medicines.     

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.