Qualitative and quantitative characterization of secondary metabolites and carbohydrates in Bai-Hu-Tang using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and ultraperformance liquid chromatography coupled with photodiode array detector

Bai-Hu-Tang (BHT), a classic traditional Chinese medicine (TCM) formula used for clearing heat and promoting body fluid, consists of four traditional Chinese medicines, i.e., Gypsum Fibrosum (Shigao), Anemarrhenae Rhizoma (Zhimu), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (Zhigancao), and nonglutinous rice (Jingmi). The chemical composition of BHT still remains largely elusive thus far. To qualitatively and quantitatively characterize secondary metabolites and carbohydrates in BHT, here a combination of analytical approaches using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and ultraperformance liquid chromatography coupled with photodiode array detector was developed and validated. A total of 42 secondary metabolites in BHT were tentatively or definitely identified, of which 10 major chemicals were quantified by the extracting ion mode of quadrupole time-of-flight mass spectrometry. Meanwhile, polysaccharides, oligosaccharides, and monosaccharides in BHT were also characterized via sample pretreatment followed by sugar composition analysis. The quantitative results indicated that the determined chemicals accounted for 35.76% of the total extract of BHT, which demonstrated that the study could be instrumental in chemical dissection and quality control of BHT. The research deliverables not only laid the root for further chemical and biological evaluation of BHT, but also provided a comprehensive analytical strategy for chemical characterization of secondary metabolites and carbohydrates in traditional Chinese medicine formulas.     

Qualitative and quantitative analysis of traditional Chinese medicine Niu Huang Jie Du Pill using ultra performance liquid chromatography coupled with tunable UV detector and rapid resolution liquid chromatography coupled with time-of-flight tandem mass spectrometry

An ultra performance liquid chromatography coupled with tunable UV detector (UPLC-TUV) and rapid resolution liquid chromatography coupled with time-of-flight tandem mass spectrometry (RRLC-Q-TOF) method was developed for the quality assessment of Niu Huang Jie Du Pill (NHJDP), a commonly used traditional Chinese medicine (TCM). Ten compounds were simultaneously identified by electrospray ion mass spectrometry (ESI/MS) and comparison with reference standards and literature data. All of them were quantified by UPLC method. Baseline separation was achieved on an ODS-140HTP C₁₈ column (2.3 μm, 100 mm × 2.1 mm I.D.) with linear gradient elution of acetonitrile–0.1% formic acid. This developed method provides good linearity (r² > 0.9996), repeatability (RSD < 3.63%), intra- and inter-day precisions (RSD < 0.86%) with accuracies (97.88–101.56%) and recovery (98.88–101.92%) of 10 major constituents, namely baicalin, baicalein, wogonoside, wogonin, glycyrrhizic acid, liquiritin, rhein, emodin, chrysophanol and physcion. In addition, the principal component analysis (PCA) coupled with the UPLC fingerprint was applied to classify the NHJDP samples according to their manufacture corporation. This proposed method with high sensitivity and selectivity was successfully utilized to analyze 10 major bioactive compounds in 30 batches of NHJDPs, and the results demonstrate that this analytical method is simple and suitable for the original discrimination and quality control of this TCM.     

Qualitative and quantitative analysis of Fructus Corni using ultrasound assisted microwave extraction and high performance liquid chromatography coupled with diode array UV detection and time-of-flight mass spectrometry

The first successful combination of ultrasound assisted microwave extraction (UAME) with liquid chromatography analysis is described for the quality evaluation of Fructus Corni, a commonly used traditional Chinese medicine (TCM). Due to their multifarious biological activities, seven representative bioactive constituents (two phenolics and five iridoids) were chosen as targets for the quality assessment. The chromatographic separation was performed on a C18 Aq column with gradient elution using methanol and aqueous solution containing 0.2% acetic acid. The quantitative method developed was validated and successfully applied to determine the seven markers in 12 batches of Fructus Corni extract from various habitats. Significant variations were demonstrated in the contents of seven compounds. Further 13 components were tentatively identified by online TOF mass analysis.     

超高效液相色谱-四级杆-静电场轨道阱高分辨液质联用解析泰地罗新注射液中的有关物质

目的 本方法建立了超高效液相色谱-四级杆-静电场轨道阱高分辨质谱联用(UHPLC-Q Exactive)分析注射用泰地罗新中的有关物质。方法 采用(Zorbax SB-C18, 100mm×3.0mm, 1.8μm) 色谱柱,以0.1%三氟乙酸溶液(A)-0.1%三氟乙酸乙腈溶液(B)为流动相,0.3mL/min线性梯度洗脱分离;采用全扫描及自动触发二级质谱扫描的功能测定。采集有关物质的质谱母离子及子离子谱,并进行解析,推测有关物质的结构。结果 在所建立的条件下,泰地罗新及其有关物质之间分离良好,检测出12个有关物质,并对其进行结构解析。结论 建立的Q Exactive四级杆-静电场轨道阱高分辨质谱法能有效地分离分析泰地罗新及其有关物质,为注射用泰地罗新的质量控制和工艺优化提供了参考。     

Metabolomic approach to understand the acute and chronic hepatotoxicity of Veratrum nigrum extract in mice based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Veratrum nigrum L. (VN) is a poisonous traditional Chinese medicine herb present since thousands of years in China. Clinical studies have shown that VN has the ability to cause hepatotoxicity, which severely limits its clinical use. The mechanism of its hepatotoxicity has not been fully elucidated. The purpose of this study was to develop and characterize a model of acute and chronic hepatotoxicity induced by Veratrum nigrum L. extract (VNE) to understand the mechanism of liver tissue metabolomics approach using on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS). Mice were administered with VNE in the acute and chronic phases. Histopathologic inspections and biochemistry analysis disclosed severe liver damage after exposure to VNE. A partial least-squares discriminant analysis (PLS-DA) of the metabolomic profiles of rat liver tissues highlighted a number of metabolic disturbances induced by VNE, focusing on purine and pyrimidine metabolism, tryptophan metabolism, phospholipid metabolism, sphingolipid metabolism and fatty acid metabolism. These findings could well explain VNE-induced acute and chronic hepatotoxicity and reveal several potential biomarkers associated with this toxicity. This indicates that UHPLC-Q-TOFMS-based metabolomics approach demonstrated its feasibility and allowed a better understanding of VNE-induced liver toxicity dynamically.     

Quantitative determination and metabolic profiling of synthetic cathinone eutylone in vitro and in urine samples by liquid chromatography tandem quadrupole time-of-flight mass spectrometry

In Taiwan, synthetic cathinones are the most prevalent new psychoactive substances, and their use is growing continuously. Urine samples are currently analysed to determine drug abuse, but the metabolic profiles and metabolites of these compounds are not widely reported. Given that cases of eutylone abuse have been growing since 2020, this study established a method employing supported liquid extraction combined with liquid chromatography tandem quadrupole time-of-flight mass spectrometry to identify and quantify eutylone and its metabolites in urine samples. Method validation was performed, and eight authentic samples were analysed. Moreover, in vitro metabolism experiments were conducted, and metabolites were generated by incubating eutylone with human liver microsomes and cytosol. Metabolite characterisation was achieved by confirming the accurate mass and product ions in full MS/MS spectra. Five metabolites were identified in in vitro experiments; they resulted from eutylone N-dealkylation, β-ketone reduction, demethylenation, aliphatic hydroxylation and sequential demethylenation and O-methylation. The metabolic profile was obtained evaluating the metabolites at different incubation times: Demethylenation occurred first, followed by N-dealkylation, β-ketone reduction and aliphatic hydroxylation. Three additional metabolites were identified in authentic samples. Based on in vitro and in vivo evidence, we propose that the demethylenation and O-methylation metabolite, the β-ketone reduction metabolite, and the β-ketone reduction, demethylenation and O-methylation metabolite are the most appropriate biomarkers of eutylone consumption. Using these markers can help expand the eutylone detection window and provide information for toxicology research.     

A rapid method for the simultaneous determination of 11 saponins in Panax notoginseng using ultra performance liquid chromatography

A rapid ultra performance liquid chromatography coupled with photo diode array detection method (UPLC-PDA) was developed for the simultaneous determination of 11 saponins, namely notoginsenoside R1, ginsenoside Rg1, Re, Rf, Rb1, Rg2, Rc, Rb2, Rb3, Rd and Rg3 in Panax notoginseng. The analysis was performed on Acquity UPLC system with Acquity UPLC BEH C(18) column and gradient elution of water and acetonitrile in 12 min. The high correlation coefficient (r(2)>0.9968) values indicated good correlations between the investigated compounds' concentrations and their peak areas within the test ranges. The LOQ and LOD were lower to 0.2-2.4 and 0.1-1.8 ng on column, respectively. The overall intra- and inter-day variations (R.S.D.) of 11 saponins were lower than 3.1%. The developed method was successfully used for the analysis of saponins in P. notoginseng with overall recovery of 93.0-101.6% for the analytes. The results show that UPLC is a powerful tool for analysis of components in Chinese medicines.     

Development and validation of an ultra performance liquid chromatography-tandem mass spectrometry method for the quantification of daptomycin in human plasma

A rapid, simple and accurate analytical method based on ultra performance liquid chromatography (UPLC) combined with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a hybrid q TOF instrument has been developed and fully validated for the quantification of daptomycin (DPT) in human plasma. The samples were analyzed after simple pretreatment involving protein precipitation, while chromatographic separation of DPT and the internal standard (reserpine) was achieved on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution with 0.1% aqueous formic acid (FA) and acetonitrile with 0.1% FA (with DPT eluting at 2.60 min). The method presented good fit (r > 0.999) over the quantification range of 0.01-10 μg mL⁻¹ with the lower limit of quantitation (LLOQ) being 0.01 μg mL⁻¹ of human plasma for DPT. The intra- and inter-day precision, measured as % relative standard deviation, was less than 11% for DPT. The validation results showed that the developed method demonstrated adequate selectivity, sensitivity, precision and accuracy and therefore was successfully applied to the analysis of clinical samples following intravenous (iv) administration of 5.4 mg kg⁻¹ DPT to patients suffering from post-traumatic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA). The developed methodology is the first report of an accurate mass tandem MS method for the analysis of this potent antibiotic in human plasma and can be used to further study pharmacokinetic, bioequivalence and even metabolic aspects related to this drug.     

An optimized procedure for metabonomic analysis of rat liver tissue using gas chromatography/time-of-flight mass spectrometry

In this paper, we present a tissue metabonomic method with an optimized extraction procedure followed by instrumental analysis with gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) and spectral data analysis with multivariate statistics. Metabolite extractions were carried out using three solvents: chloroform, methanol, and water, with design of experiment (DOE) theory and multivariate statistical analysis. A two-step metabolite extraction procedure was optimized using a mixed solvent of chloroform–methanol–water (1:2:1, v/v/v) and then followed by methanol alone. This approach was subsequently validated using standard compounds and liver tissues. Calibration curves were obtained in the range of 0.50–125.0 μg/mL for standards and 0.02–0.25 g/mL acceptable for liver tissue samples. For most of the metabolites investigated, relative standard deviations (RSD) were below 10% within a day (reproducibility) and below 15% within a week (stability). Rat liver tissues of carbon tetrachloride-induced acute liver injury models (n = 10) and healthy control rats (n = 10) were analyzed which demonstrated the applicability of the developed procedure for the tissue metabonomic study.     

大鼠血浆中冬青素A的LC-MS测定法及其药动学研究(英文)

冬青素A系中药毛冬青的主要成分之一, 本文建立了液相色谱质谱法(LC-MS)研究冬青素A在大鼠体内的药动学特征。色谱分离采用C18柱, 甲醇-5 mM 醋酸铵(80:20, v/v) 为流动相质谱检测采用ESI源, 负离子检测, 冬青素A的检测离子为m/z 501.1→501.1,地高辛(内标) 的检测离子为m/z779.4→779.4。大鼠血浆加入磷酸溶液以乙酸乙酯提取, 分取有机层以氮气流吹干, 流动相复溶后进行LC-MS分析。方法学评价表明该法定量限为1.05 ng/mL, 在1.05-525.5 ng/mL范围内线性关系良好。日内和日间变异均小于10%, 提取回收率大于80%。采用建立的LC-MS法进行了大鼠单剂量口服冬青素A后, 其在大鼠体内的药动学研究, 获得了主要的药动学参数。     

UPLC法测定拉莫三嗪和奥卡西平活性代谢物血清药物浓度

目的建立超高效液相色谱(UPLC)同时测定人血清中拉莫三嗪(Lamotrigine,LTG)和奥卡西平活性代谢物10-羟基卡马西平(Monohydroxy-carbazepine,MHD)浓度的方法。方法色谱柱为Waters ACQUITY UPLCTM BEH C_(18)柱(2.1 mm×50 mm,1.7μm),柱温30℃,流动相:乙腈-10 mmol/L乙酸铵(20∶80;甲酸调pH=4),流速:0.2 mL/min,进样量2μL,检测波长240 nm。结果血清中内源性物质对样本测定无干扰,LTG和M HD分别在1~40μg/mL和1.25~80μg/mL浓度范围内线性关系良好,相关系数r分别是0.999 8和0.999 7。本方法两个化合物准确度RE值在±10%范围内,批内和批间精密度RSD值均<10%,LTG回收率73%~80%,M HD回收率89%~97%。结论本法操作简便、专属性强、灵敏度高、重现性好,符合生物样品的分析要求,适用于LTG和奥卡西平活性代谢物MHD的浓度监测。     

Quantitative determination of doxorubicin in the exosomes of A549/MCF-7 cancer cells and human plasma using ultra performance liquid chromatography-tandem mass spectrometry

In cancer therapy, exosomes efflux enhances resistance of cancer cells toward anticancer agents through mediating the transport of anticancer drugs outside the cells. In this study, a rapid, simple and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of Doxorubicin (DOX) in exosomes of cancer cells and human plasma using Ketotifen as an internal standard (IS). Plasma samples spiked with DOX and two cancer cell lines (A549 & MCF-7) were incubated with different concentrations of DOX and IS. The analytes were then extracted with methanol after protein precipitation and the chromatographic separation was carried out using a C18 column, with a mixture of acetonitrile–water- formic acid (85:15:0.1%, v/v/v) as mobile phase. Multiple reaction monitoring (MRM) was utilized to monitor the protonated precursor to product ion transitions of m/z 544.25?>?397.16 and m/z 310.08?>?96.97 for the quantification of DOX and IS, respectively. The method was linear over ranges of 1–1000?ng/mL for DOX in plasma and 2–1000?ng/mL for DOX in exosome samples. The lower limit of quantification of this method was 1?ng/mL, 2?ng/mL and 2?ng/mL in human plasma, A549 & MCF-7 cells respectively. Intra- and inter day precision of all quality control concentrations were less than 10.33% and the accuracy values ranged from ?4.82 to 12.60%. The optimized UPLC-MS/MS method proved to be fast, specific, simple and highly sensitive and was successfully applied for the estimation of DOX in the exosomes of cancer cells and plasma.     

Comparison of fast liquid chromatography/tandem mass spectrometric methods for simultaneous determination of cladribine and clofarabine in mouse plasma

Several fast high performance liquid chromatography/atmospheric pressure ionization/tandem mass spectrometric (HPLC-API/MS/MS) methods were evaluated for the simultaneous determination of cladribine and clofarabine in mouse plasma samples. The chemical separation for analytes under reversed-phase conditions were achieved by using either ultra-performance liquid chromatography (UPLC) or micro-column HPLC coupled to either a quadrupole linear ion trap mass spectrometer (QTrap MS) or a triple quadrupole mass spectrometer. Atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI) interfaces in the positive mode were employed prior to mass spectrometric detection. The effects of various dopant solvents on the APPI sensitivities of analytes and the internal standard were investigated. The matrix ionization suppression potential for the test compounds in plasma samples on fast HPLC-MS/MS methods was examined by a post-column infusion technique. In this work, these proposed approaches were successfully employed to determine the concentrations of cladribine and clofarabine in mouse plasma in the low ng/ml region. The mouse plasma levels of all analytes obtained by these fast HPLC-MS/MS methods were compared and found to be well correlated in terms of analytical accuracy.     

Analysis of tolvaptan and its metabolites in sports drug testing by high‐performance liquid chromatography coupled to tandem mass spectrometry

Tolvaptan is prohibited by the World Anti‐doping Agency (WADA) under class S5 – Diuretics and masking agents. Less than 1% of the administrated dose is excreted by humans in urine. Knowledge concerning the metabolism in humans, and especially the excretion of metabolites in human urine, is limited. An analysis method based on the dilute‐and‐shoot approach using high‐performance liquid chromatography coupled to tandem mass spectrometry (HPLC‐MS/MS) for detection was developed and validated. Ion transitions, which are part of this method, can easily be included in already existing screening methods used in routine doping analysis for the detection of diuretics. After administration of a single dose of tolvaptan to one male subject, low concentrations of the drug itself could be detected in urine samples over a time period of 24 h. In addition, hydroxyl metabolites of tolvaptan and one carboxyl metabolite with a cleaved benzazepine ring system were identified. These metabolites showed detection times of up to 150 h. An inclusion of these metabolites in the methods used in doping control analysis seems therefore to be of value. Copyright © 2016 John Wiley & Sons, Ltd.     

Ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) for time-dependent profiling of raw and steamed Panax notoginseng

The metabolic profiles of Panax notoginseng and its associated therapeutic values are critically affected by the duration of steaming. The time-dependent steaming effect of P. notoginseng is not well-characterized and there is also no official guideline on its duration of steaming. In this paper, a UHPLC/TOFMS-based metabolomic platform was developed for the qualitative profiling of multiparametric metabolic changes of raw P. notoginseng during the steaming process. Our method was successful in discriminating the differentially processed herbs. Both the unsupervised principal component analysis (PCA) score plot (R²X = 0.664, Q² (cum) = 0.622, and PCs = 2) and the supervised partial least square-data analysis (PLS-DA) model (R²X = 0.708, R²Y = 0.461, and Q²Y = 0.271) demonstrated strong classification and clear trajectory patterns with regard to the duration of steaming. The PLS-DA model was validated for its robustness via a prediction set, confirming that the UHPLC/TOFMS metabolic profiles of the raw and differentially steamed P. notoginseng samples were highly reproducible. Based on our method, the minimum durations of steaming for the maximum production of bioactive ginsenosides such as Rg3 and Rh2 were also predicted. Our novel time-dependent metabolic profiling approach represents the paradigm shift in the quality control of P. notoginseng products.     

Modernization of Chinese herbal compound and the high performance liquid chromatography tandem mass spectrometry (HPLC-MS)

Chinese herbal compound is playing an important role on curing human diseases.And it has been a trend that Chinese herbal compound is being used all over the world in 21 century.However,our Chinese herbal compound is facing serious challenge for the lack of canonical system of quality criterion for Chinese herbal compound so it has been a urgent problem to set up the quality control standards and reveal therapeutic basis of Chinese herbal compound.In order to give full play to the advantages of Chinese herbal compound,modern scientific and technological is used to research of Chinese herbal compound,especially the high performance liquid chromatography tandem mass spectrometry(HPLC-MS),because it is high sensitive,rapid,and obtain more information.It is very necessary that HPLC-MS is uesed to elucidate the effective components of basic substances of Chinese Herbal Compound,and endow traditional Chinese medicine with modern scientific connotation.     

Comparison of capillary electrophoresis and high performance liquid chromatography for determination of flavonoids in Achillea millefolium

Flavonoids represent an important bioactive component in Achillea millefolium. The comparison of the most commonly used analytical methods for the identification and quantification of flavonoids, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC), is presented. The methods were optimized and validated. Using a 20 mM borate buffer with 30% (v/v) of methanol (pH 9.3) in the CE analysis and a gradient elution with water-acetonitrile mobile phase in the HPLC analysis, sufficient separation of the analytes was achieved. A relatively high injection volume in the CE analysis (30 mbar x 30s) enabled low limit of detection (LOD) (0.3-0.7 mg/L). Repeatability of both methods was acceptable (relative standard deviation of peak area were <6%). Additionally, the amount of flavonoids in a real sample of the dried herbal drug was determined.     

Identification and quantification of 32 bioactive compounds in Lonicera species by high performance liquid chromatography coupled with time-of-flight mass spectrometry

Flos Lonicerae, referred to the flower buds of several medicinal Lonicera species, is a commonly used traditional Chinese herbal medicine. A multi-component-assay quality control method, using high performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (HPLC-ESI/TOF MS), has been developed for the simultaneous identification and quantification of 32 bioactive compounds in Flos Lonicerae. The limits of detection (LOD) and quantification (LOQ) were in the range of 0.002–0.089 and 0.006–0.355 μg/ml, respectively. All calibration curves showed good linear regression (r² ≥ 0.99) within the test ranges. The overall intra- and inter-day precisions of analytes were less than 3.47% for peak area and 0.38% for retention time. The recoveries were from 85.4% to 101.6%. The validated method was applied to assay of 32 compounds in 8 medicinal Lonicera species. Furthermore, six unknown chromatographic peaks were tentatively characterized. It was demonstrated that the HPLC-ESI/TOF MS method was suitable for quality control of Lonicera species, owing to the advantages of accurate mass analysis, resolving power, enhanced selectivity and high sensitivity.     

Metabolic urinary profiling of alcohol hepatotoxicity and intervention effects of Yin Chen Hao Tang in rats using ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry

This paper was designed to study metabonomic characters of the hepatotoxicity induced by alcohol and the intervention effects of Yin Chen Hao Tang (YCHT), a classic traditional Chinese medicine formula for treatment of jaundice and liver disorders in China. Urinary samples from control, alcohol- and YCHT-treated rats were analyzed by ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) in positive ionization mode. The total ion chromatograms obtained from the control, alcohol- and YCHT-treated rats were easily distinguishable using a multivariate statistical analysis method such as the principal components analysis (PCA). The greatest difference in metabolic profiling was observed from alcohol-treated rats compared with the control and YCHT-treated rats. The positive ions m/z 664.3126 (9.00 min) was elevated in urine of alcohol-treated rats, whereas, ions m/z 155.3547 (10.96 min) and 708.2932 (9.01 min) were at a lower concentration compared with that in urine of control rats, however, these ions did not indicate a statistical difference between control rats and YCHT-treated rats. The ion m/z 664.3126 was found to correspond to ceramide (d18:1/25:0), providing further support for an involvement of the sphingomyelin signaling pathway in alcohol hepatotoxicity and the intervention effects of YCHT.