Limitation standard of toxic aconitines in Aconitum proprietary Chinese medicines using on-line extraction electrospray ionization mass spectrometry

Development of rapid analytical methods and establishment of toxic component limitation standards are of great importance in quality control of traditional Chinese medicine. Herein, an on-line extraction electrospray ionization mass spectrometry (oEESI-MS) coupled with a novel whole process integral quantification strategy was developed and applied to direct determination of nine key aconitine-type alkaloids in 20 Aconitum proprietary Chinese medicines (APCMs). Multi-type dosage forms (e.g., tablets, capsules, pills, granules, and liquid preparation) of APCM could be determined directly with excellent versatility. The strategy has the characteristics of high throughput, good tolerance of matrix interference, small amount of sample (∼0.5 mg) and reagent (∼240 μL) consumption, and short analysis time for single sample (<15 min). The results were proved to be credible by high performance liquid chromatography−mass spectrometry (LC–MS) and electrospray ionization mass spectrometry, respectively. Moreover, the limitation standard for the toxic aconitines in 20 APCMs was established based on the holistic weight toxicity (HWT) evaluation and the Chinese Pharmacopoeia severally, and turned out that HWT-based toxicity evaluation results were closer to the real clinical applications. Hence, a more accurate and reliable APCM toxicity limitation was established and expected to play an important guiding role in clinics. The current study extended the power of ambient MS as a method for the direct quantification of molecules in complex samples, which is commonly required in pharmaceutical analysis, food safety control, public security, and many other disciplines.     

Simultaneous determination of quetiapine and three metabolites in human plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry

AIM: To develop a high performance liquid chromatography-electrospray mass spectrometry (HPLC-MS/ESI) method for simultaneous determination of quetiapine and its sulfoxide-, 7-hydroxy-, 7-hydroxy-N-dealkyl-metabo-lites in human plasma. METHODS: The HPLC separation of the compounds was performed on a Kromasil C18, (5 urn, 4.6 mm×150 mm) column, using water (formic acid: 1.70 mmol/L, ammonium acetate: 5.8 mmol/L)-acetoni trile (65:35) as mobile phase, with a flow-rate of 0.95 mL/min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and detected in the selected ion recording (SIR) mode. The samples were extracted using solid-phase extraction columns. RESULTS: The calibration curves were linear in the ranges of 10-2000 μg/L for quetiapine, 1-200 μg/L for its metabolites, respectively. The average extraction recoveries for all the four samples were above 85 %.     

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.     

高效液相色谱-串联质谱法测定血浆中黄豆苷元的浓度

目的:建立高效液相色谱-串联质谱联用(LC-MS-MS)的方法测定人血浆内黄豆苷元的浓度,并应用于药动学研究和生物等效性评价。方法:以木犀草素为内标,甲醇(含0．2%乙酸)为蛋白沉淀剂,采用Merck LiChroCART C_(18)色谱柱分离,通过LC-MS-MS电喷雾离子源(ESI),以选择反应监测(SRM)方式进行检测,离子极性监测为负离子,用于定量分析的离子分别为黄豆苷元m／z 252．9,木犀草素m／z 284．8。结果:血浆中的杂质不干扰黄豆苷元和木犀草素的测定,线性范围为0．1004～80．32μg·L~(-1)(r=0．994 0),血浆中黄豆苷元的绝对回收率大于80%,浓度为0．4016,4．016和40．16μg·L~(-1)的QC样品的批内和批间精密度RSD均小于10%。结论:该方法简便准确、灵敏度高,可以用于黄豆苷元的人体药动学研究和生物等效性评价。     

高效液相色谱-串联质谱法测定血浆中熊去氧胆酸的浓度

目的：建立高效液相色谱-质谱联用的方法测定人血浆内的熊去氧胆酸浓度的方法。方法：采用Shimadzu VP-ODS(4．6 mm×150 mm,5μm)色谱柱；柱温25℃；流动相为(A)乙腈-水(含5 mmol·L~(-1)的乙酸铵)(35：65,V/V),(B)乙情；梯度程序为0～2min,A：B=94：6；2～4．5min,A：B= 80：20：4．5～7min,A：B=94：6；流速为1．0 mL·min~(-1)；通过液相串联质谱,电喷雾离子源(ESI),以选择反应监测(SRM)方式进行检测；离子极性监测负离子(-)；检测离子为熊去氧胆酸m/z 391．1 [M-H]~-→391．1[M-H]~-,盐酸西替利嗪(内标)m/z 387．1[M-H]~-→387．1[M-H]~-。结果：熊去氧胆酸的最低定量限为40．144μg·L~(-1),线性范围为40．14～12 043μg·L~(-1)(r=0．995)。结论：该方法简便、灵敏度高,可以用来进行熊去氧胆酸的人体药动学和生物等效性研究。     

表面加强激光解析电离-飞行时间-质谱技术在急性重症胰腺炎预后相关蛋白质谱检测中的应用

目的 应用蛋白芯片表面加强激光解析电离-飞行时间-质谱(SELDI-TOF-MS)技术检测急性重症胰腺炎不同预后相关蛋白质谱,探讨急性重症胰腺炎轻重程度及预后评估的新方法.方法 用SELDI-TOF-MS方法检测浙江省人民医院2008年3月至2010年10月23例急性重症胰腺炎患者的蛋白质谱,按是否发生器官功能衰竭、胰腺脓肿、腹内压异常、死亡将全部患者分组进行蛋白指纹图谱比较.结果 蛋白质峰谱特征显示,在1094 u、2751 u、5904 u处并发胰腺脓肿(13例)的重症胰腺炎患者峰值(13.21±3.73,45.62±10.31,48.37±9.24)明显高于未并发胰腺脓肿组(70例)(4.33±1.79,8.87±3.21,4.45±1.59),差异有统计学意义,均P<0.05.在635 u并发器官功能衰竭组(11例)峰值(8.56±3.21)明显低于未并发器官功能衰竭组(12例)(37.82±12.65);而在4103 u、4187 u处并发器官功能衰竭组(21.63±8.23.9.81±2.32)峰值则高于未并发器官功能衰竭组(3.32±1.29,1.14±0.49).在4173 u处并发腹内压增高组(10例)峰值(8.94±3.58)明显高于无腹内压增高组(13例)(1.97±0.73);而在5635 u处并发腹内压增高组峰值(0.62±0.23)则低于无腹内压增高组(15.78±6.34).2例死亡患者蛋白指纹表现为血清蛋白全面合成功能减退,峰值降低.结论 蛋白指纹图谱技术可筛选出有意义的生物标记蛋白,为急性重症胰腺炎并发症的早期诊断与治疗提供了新的路径,对急性重症胰腺炎预后评估具有重要意义.

Abstract：

Objective To analyze the serum proteomic pattern in different severe acute pancreatitis complication by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) for evaluating the stage and prognosis of severe acute pancreatitis. Methods Serum samples were collected from 23 severe a-cute pancreatitis. Serum from organ nonfunction, pancreatic abscess, intra-abdominal hypertension and death were profiled using WCX Proteinchip and analyzed by mass spectrometry. Results Protein peak in the spectrum characteristics showed that at 1094u, 2751u, 5904u, the peak value of concurrent pancreas abscess pancreatitis [13.21 ± 3.73, 45.62±10.31, 48.37±9.24] were significantly higher than those in none-pancreas abscess group [4.33 ± 1.79, 8. 87 ±3.21, 4.45 ±1.59] (P<0.05). At 635u, the peak value of concurrent organ failure group(8.56 ± 3.21) was obviously lower than those in none-organ failure group(37.82 ± 12.65). At 4103u and 4187u, the peak value of in complicated organ failure group [21.63 ±8.23, 9.81±2.32] were higher than those in none-organ failure group [3.32±1.29, 1.14 ±0.49], At4173u, the peak value of concurrent increased intraabdominal pressure group(8.94 ±3.58) was significantly higher than that in none-intraabdominal pressure group(1.97 ±0. 73) , while at 5635u the peak value of concurrent increased intraabdominal pressure groups ( 0. 62 ± 0. 23 ) was lower than that without intraabdominal pressure group (15.78 ±6.34). Two cases died. Protein fingerprints showed that overall serum protein composite function dropped and the peak value reduced. Conclusion Proteomic technology can significantly identify novel significant biomarkers in the serum, which provides a new way to diagnose and treat the different complication of severe acute pancreatitis early and has clinical significance in evaluating the prognosis of severe acute pancreatitis.     

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.     

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.     

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.     

基质辅助激光解析电离飞行时间质谱检测耐甲氧西林金黄色葡萄球菌δ-毒素的应用

目的 本研究采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)检测δ-毒素,评估其在耐甲氧西林金黄色葡萄球菌(MRSA)的分型和毒力表达中的作用。方法 使用Bruker microflex MALDI-TOF仪器,采集2000~20000Da质量范围内,以正线性模式采集图谱,使用仪器配套的MALDI Biotyper 2.0软件分析MRSA菌株的原始图谱,产生的峰列表直接使用flexAnalysis、clinProTools3.0软件分析。结果 本研究中共检测83株MRSA,共有39(47.0%)株MRSA检出(3005±5)m/z信号峰,其中HA-MRSA 19(22.9%)株,CA-MRSA 20(26.5%)株,P=0.766,两者之间无显著性差异;33(39.8%)株MRSA检出(3035±5)m/z信号峰,其中HA-MRSA 9 (10.8%)株,CA-MRSA 24(28.9%)株,P=0.003,两者之间有显著性差异;11(13.%)株MRSA既未检出(3005±5)m/z信号峰也未检出(3035±5)m/z信号峰,全部为HA-MRSA菌株。spa分型中检出(3005±5)m/z信号峰,15(18.1%)株为t062型,8株为t015(9.6%)型,4株为t030(4.8%)株,P=0,有显著性差异;检出(3035±5)m/z信号峰,31(37.3%)株为t437型,2(2.4%)株t8660型,P=0,有显著性差异;(3035±5)m/z作为spa t437型特征信号ROC曲线下面积0.89,P=0。11株未检出δ-毒素,6株分离自骨关节标本,3株分离自呼吸道标本,1株分离自慢性溃疡的分泌物标本,1株分离自血液;在血平板的菌落特征,6株MRSA菌落形态发生改变,5株菌落形态正常。结论 MALDI-TOF MS使用常规方法即可快速检测MRSA的δ-毒素,其质谱峰为(3005±5)m/z和(3035±5)m/z两种;(3035±5)m/z是δ-毒素的同基因变异体(HldG10S)的质谱峰,该峰可快速鉴别spa t437型;不产生δ-毒素的菌株是agr调控系统失调的表现,与慢性感染、小菌落形成、无明显β-溶血有关。     

Quantification of three beta‐lactam antibiotics in breast milk and human plasma by hydrophilic interaction liquid chromatography/positive‐ion electrospray ionization mass spectrometry

The use of cephalosporins during breast feeding raises several issues, including the risk of drug exposure through breast milk for the infant. In this paper, a hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI‐MS) was developed for the quantitation of cefuroxime, cefoxitin, and cefazolin in breast milk and human plasma. The assay was based on the use of small sample size, 25 μL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the HILIC/ESI‐MS system. All analytes and the internal standard, alfuzosin, were separated by using a ZIC®‐HILIC analytical column (150.0 × 2.1 mm i.d., particle size 3.5 µm, 200 Å) with isocratic elution. The mobile phase was composed of a 6% 12.5 mM ammonium acetate water solution in acetonitrile and pumped at a flow rate of 0.25 mL min^‐1. The assay was linear over a concentration range of 0.2 to 5 µg mL^‐1 and 0.4 to 20 µg mL^‐1 for all the analytes in breast milk and in human plasma, respectively. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 12 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application of HILIC in the analysis of antibiotics in breast milk and human plasma and it can be used to support a wide range of clinical studies. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantitative analysis of R-84760, a selective kappa-opioid receptor agonist, in plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A sensitive method for monitoring R-84760, a selective κ-opioid receptor agonist, in plasma using liquid chromatography with electrospray ionization tandem mass spectrometry was explored. R-84760 and internal standard (I.S., d₈-R-84760) were extracted from human or various animal plasmas with ethyl acetate. The analysis was performed by the selected reaction monitoring method, and the precursor-product combinations of m/z 399–328 for R-84760 and m/z 407–328 for I.S. were chosen for quantification. The calibration curve was linear in the range 5–500 pg/ml, and the limit of quantification was 5 pg/ml using 1 ml of human plasma. Pharmacokinetic studies of R-84760 in rats, dogs, and monkeys were performed by this method. The plasma concentration of unchanged form after administration at a trace dosage amount was able to be monitored. Interspecies correlations of pharmacokinetic parameters obtained in animals were utilized to estimate pharmacokinetic behavior in humans. The results showed that it is possible to perform pharmacokinetic studies on R-84760 by this quantitative analysis.     

The simultaneous determination of coumarins in Angelica gigas root by high performance liquid chromatography-diode array detector coupled with electrospray ionization/mass spectrometry

An high performance liquid chromatography-diode array detector coupled with electrospray ionization/mass spectrometry (HPLC-DAD/MS) based method has been developed for the simultaneous determination of nine coumarin compounds, nodakenin (1), peucedanone (2), marmesin (3), decursinol (4), 7-hydroxy-6-(2R-hydroxy-3-methylbut-3-enyl)coumarin (5), demethylsuberosin (6), decursin (7), decursinol angelate (8) and isoimperatorin (9) in the Korean medicinal herb, Cham-Dang-Gui, the dried root of Angelica gigas (Umbelliferae). The methanol extracts were analyzed by HPLC using a reversed-phase C18 column (5 microm, 4.5 mm x 250 mm) using a gradient acetonitrile-water solvent system at a flow rate of 1.0 ml/min. The analysis of six coumarins (1, 3, 4 and 6-8) with DAD was done at 330 nm and showed excellent linearity (r(2)=0.998-0.999) in a range of 0.2-250 microg/ml for all the compounds. The average recoveries (n=3) were between 96.5% and 110.8%. Identification of each peak was also discussed with the electrospray ionization multi-stage tandem mass spectroscopy (ESI-MS(n)). The amount of these coumarin compounds was evaluated in A. gigas samples. Meanwhile, three coumarins (2, 5 and 9) could not been quantified by DAD because these peaks were overlapped with others. Determination of these compounds could be successfully accomplished with the HPLC-ESI/MS in selected ion monitoring/selected reaction monitoring mode.     

High performance liquid chromatography (HPLC) fingerprints and primary structure identification of corn peptides by HPLC-diode array detection and HPLC-electrospray ionization tandem mass spectrometry

Corn peptides (CPs) are reported to have many biological functions, such as facilitating alcohol metabolism, antioxidation, antitumor, antihypertension, and hepatoprotection. To develop a method for quality control, the high-performance liquid chromatography (HPLC) system was applied. Twenty-eight common peaks were found in all the CPs of corn samples from Enshi, China, based on which, a fingerprinting chromatogram was established for use in quality control in future research. Subsequently, the major chemical constituents of these common peaks were identified respectively using the HPLC-diode-array detection electrospray ionization tandem mass spectrometry (DAD-ESI-MS/MS) system, and 48 peptide fractions were determined ultimately. This was the first time for the majority of these peptides to be reported, and many of them contained amino acids of glutamine (Q), L and A, which might play an important role in the exhibition of the bioactivities of CPs. Many peptides had a similar primary structure to the peptides which had been proven to be bioactive such as facilitating alcohol metabolism, scavenging free radicals, and inhibiting lipid peroxidation. This systematical analysis of the primary structure of CPs facilitated subsequent studies on the relationship between the structures and functions, and could accelerate holistic research on CPs.     

Quantitative analysis of R-84760, a selective κ-opioid receptor agonist, in plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A sensitive method for monitoring R-84760, a selective κ-opioid receptor agonist, in plasma using liquid chromatography with electrospray ionization tandem mass spectrometry was explored. R-84760 and internal standard (I.S., d₈-R-84760) were extracted from human or various animal plasmas with ethyl acetate. The analysis was performed by the selected reaction monitoring method, and the precursor-product combinations of m/z 399–328 for R-84760 and m/z 407–328 for I.S. were chosen for quantification. The calibration curve was linear in the range 5–500 pg/ml, and the limit of quantification was 5 pg/ml using 1 ml of human plasma. Pharmacokinetic studies of R-84760 in rats, dogs, and monkeys were performed by this method. The plasma concentration of unchanged form after administration at a trace dosage amount was able to be monitored. Interspecies correlations of pharmacokinetic parameters obtained in animals were utilized to estimate pharmacokinetic behavior in humans. The results showed that it is possible to perform pharmacokinetic studies on R-84760 by this quantitative analysis.     

Liquid chromatography electrospray ionization tandem mass spectrometry（LC/ESI-MS/MS） method for quantitative estimation of moxifloxacin in human plasma

A rapid and sensitive liquid chromatography tandem mass spectrometry（LC-MS/MS） method has been developed and validated for the determination of moxifloxacin（MOXI） in human plasma. After a simple protein precipitation using acetonitrile, the post treatment samples were analysed on a C18 column interfaced with a Triple Quadropole Tandem Mass Spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of 0.1% formic acid–acetonitrile（60：40, v/v）. Ciprofloxacin（CIPRO） was used as an internal standard. The analyte and internal standard（CIPRO） were monitored in the multiple reaction monitoring mode（MRM）. The mass transition ion-pair has been followed as m/z 402→358.2 for MOXI and 332→288.1 for CIPRO. The method was linear in the concentration range of 25–5000 ng/mL. The lower limit of quantification was 25 ng/mL. The intra- and inter-day precision（relative standard deviation） and accuracy（relative error） values were within 12.4%. Each plasma sample was analyzed within 3 min.     

LC-MS/MS法同时测定人血浆中氯吡格雷及其羧酸代谢物的浓度

目的建立快速测定人血浆中氯吡格雷及其羧酸代谢物SR26334含量的LC-MS/MS法。方法乙醚-正己烷(4:1,V:V)2次液-液提取法(中性和酸化条件下),采用Teknokroma C_(18)色谱柱,以那格列奈和吡格列酮为内标,同时测定血浆中氯吡格雷和SR26334的浓度。流动相:甲醇-0.1%甲酸(80:20,V:V);流速:0.2mL·min~(-1);以多反应离子监测方式检测:氯吡格雷[M+H]~+,m/z 322.1→212.1;那格列奈[M+H]~+,m/z 318.3→166.2;氯吡格雷羧酸代谢物SR26334[M+H]~+,m/z 308.1→q98.1;吡格列酮[M+H]~+,m/z 357.2→134.2。结果氯吡格雷和内标那格列奈的保留时间分别在4.4和3.7min,SR26334和内标吡格列酮的保留时间分别在1.3和1.7min,氯吡格雷的线性范围为5～5000ng·L~(-1);SR26334的线性范围为20～2500μg·L~(-1)。提取回收率大于75%,方法回收率大于90%,日内、日间RSD小于10%(n=5)。结论本方法简便快速,适用于氧吡格雷制剂的新药临床研究和临床长期治疗病人血药浓度的常规监测。     

Metabolomic study of myocardial ischemia and intervention effects of Compound Danshen Tablets in rats using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Myocardial ischemia (MI) is a worldwide epidemic. Compound Danshen Tablets (CDTs), an herbal compound preparation, are widely used to treat MI in China. In this study, we aimed to explore novel biomarkers to increase the understanding of MI and investigate therapeutic mechanisms of CDT by using a metabolomic approach. Plasma extracts from sham, MI model, CDT- and western medicines (isosorbide dinitrate, verapamil, propranolol, captopril, and trimethazine)-treated rats were analyzed by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF-MS). The orthogonal partial least square (OPLS) model was built to find metabolites expressed in significantly different amounts between MI and sham rats. Meanwhile, partial least squares discriminant analysis (PLS-DA) was used to investigate CDT's protective effects. The results showed that CDT presented protective effects on MI by reversing potential biomarkers to sham levels, especially for the four metabolites in the pathway of purine metabolism (hypoxanthine, xanthine, inosine and allantoin).     

Metabolism of GTI-2040, a phosphorothioate oligonucleotide antisense, using ion-pair reversed phase high performance liquid chromatography (HPLC) coupled with electrospray ion-trap mass spectrometry

GTI-2040 is a 20-mer phosphorothioate oligonucleotide, which is complementary to the messenger ribonucleic acid (mRNA) of the R2 subunit of ribonucleotide reductase. This study characterized both the in vivo and in vitro metabolism of GTI-2040. A highly specific ion-pair reversed-phase electrospray ionization (IP-RP-ESI) liquid chromatography-mass spectrometry (LC-MS) method was used for the identification of GTI-2040 and metabolites from a variety of biological samples including exonuclease enzyme solutions, plasma, urine, mouse liver/kidney homogenates, and human liver microsomes. Progressively chain-shortened metabolites truncated from the 3' terminal of GTI-2040 were detected in all of the evaluated biological samples. GTI-2040 was found to be a good substrate for 3' but not 5' exonuclease. While the pattern of n-1 chain-shortened 3'-exonucleolytic degradation was similar in the mouse liver and kidney homogenates, the latter was found to contain a larger number of shortenmers, the kidneys appeared to possess higher enzymatic reactivity toward GTI-2040. Thus, metabolism of GTI-2040 was found to occur in a variety of biological samples, mainly mediated by the 3' exonuclease.