LC/MS/MS与GC/MS法分析鉴定小鼠尿中沙美特罗的代谢产物

目的:鉴定沙美特罗在小鼠尿中的主要代谢产物.方法:ig给药后,收集小鼠尿液,经固相提取,葡萄糖醛酸酶水解,进行LC/MS/MS分析和硅烷化后进行GC/MS分析同时分离鉴定沙美特罗代谢产物.结果和结论:在给药后尿样中发现沙美特罗原型和4种代谢产物M1～M4,其结构推测为19-羟基沙美特罗(M1)、2-羰基沙美特罗(M2)、19-羰基沙美特罗(M3)和19-羟基-8-甲氧基沙美特罗(M4).     

System suitability in bioanalytical LC/MS/MS

System suitability is widely recognized as a critical component of bioanalysis. This paper discusses a generic system suitability test that monitors instrument performance throughout a run when used for liquid chromatography tandem mass spectrometry (LC/MS/MS) in bioanalysis. This system suitability process is designed to ensure that the LC/MS/MS system is performing in a manner that leads to the production of accurate and reproducible data that can be submitted with confidence to regulatory agencies. This process contains tests for signal stability, carryover, and instrument response. This approach is integrated throughout an analytical run and has been used in the analysis of over 25,000 batches of clinical samples. Two case studies are presented in which quality control samples and standards meet all acceptance criteria (based on Standard Operating Procedures and the Food and Drug Administration's recommendations for bioanalytical method validation) but failed the proposed system suitability test, and thus were rejected. In these case studies, the concentrations of a significant number of clinical samples (over 35%) were affected, resulting in changes of more than 15% when the samples were reanalyzed. These data indicate that the poor performance of an LC/MS/MS system could adversely affect the calculated concentrations of unknown samples even though the results for quality control samples appear to be acceptable.     

LC/MS/MS法测定比格犬血浆中埃克替尼及其在药动学研究中的应用

目的:建立灵敏、快速的液相色谱-串联质谱(LC/MS/MS)法测定比格犬血浆中埃克替尼,并用于药动学研究。方法:健康比格犬32只,随机分成4组后,静脉(10mg/kg)或灌胃(10,20和40mg/kg)给予埃克替尼。采用LC/MS/MS法测定血药浓度,并计算出药动学参数。结果:测定埃克替尼的线性范围是0.5～10000ng/mL,日内和日间精密度(RSD)均小于10。静脉给药后AUC0-t为(27.3±15.3)μg.mL-1.h。灌胃给药后AUC0-t分别为(7.47±3.30)、(23.5±11.5)、(54.5±24.9)μg.mL-1.h,埃克替尼在犬体内的绝对生物利用度是27.4。结论:该分析方法选择性好、灵敏度高、操作简便,并成功应用于埃克替尼的比格犬药动学研究。     

LC/ESI/MS method development for the analysis of hepatotoxic cyclic peptide microcystins in animal tissues.

Microcystins (MCYSTs) are a family of related cyclic heptapeptides produced by several genera and species of blue-green algae (cyanobacteria). MCYSTs are potent and specific inhibitors of the serine threonine family of protein phosphatases, especially PP1 and PP2A. MCYSTs inhibit a liver's protein phosphatase by forming a covalent linkage between MCYSTs' Mdha residue and the phosphatase's cysteine residue. Due to the covalent linkage, analysis of MCYSTs in animal tissues has been limited to determination of unbound MCYST concentration. The MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) oxidation procedure allows for the detection of total MCYST burden by releasing the carboxylic acid MMPB from MCYST's Adda amino acid. An internal standard 4-phenylbutyric acid (4PB) accounts for losses during the method. LC/MS conditions were developed using a ThermoFinnigan LCQDuo ion trap in negative electrospray ionization (ESI). Since both compounds produce the [M-H](-) ion, analysis occurs in selected ion monitoring (SIM) mode for both MMPB (m/z 207.1) and 4PB (m/z 163.1). Complete oxidation of MCYST-LR in liver tissues occurs in 3h. A solid phase extraction (SPE) cartridge removes MMPB and 4PB from the oxidant solution. The process efficiency for the SPE procedure is only 51.3%; however, suppression experiments indicate a 41.8% loss in signal strength due to matrix interferences. Therefore, the extraction efficiency for the SDB-XC cartridge procedure is 93.1%. This research has been successful in developing an LC/MS method for the analysis of total MCYST burden in animal tissues.     

LC/MS检定前列舒乐胶囊中的淫羊藿苷的含量

目的 建立前列舒胶囊中淫羊藿苷含量LC/MS测定方法。方法 采用LC/MS测定前列舒乐胶囊中淫羊藿苷的含量,并比较其与HPLC法的优劣。结果LC/MS测定淫羊藿苷0.05～0.50 mg范围内线性关系良好,与标准方法比较相对偏差小于2%,符合药品标准的规定。结论 LC/MS检测前列舒乐胶囊中的淫羊藿苷方法简便,快速,灵敏度高,可以进行定量分析。     

LC/MS/MS analysis of vesnarinone and its principal metabolites in plasma and urine

An LC/MS/MS assay was developed and successfully used to quantitate vesnarinone and its principal metabolites (OPC-8230, OPC-18136, and OPC-18137) in human plasma and urine. Samples were pre-treated with liquid–solid extraction followed by simultaneous monitoring of primary and daughter ions which were used for the identification and quantitation of the analytes on LC/MS/MS. This assay offers advantages of specificity, speed and greater sensitivity over the previously developed HPLC-UV assay. The lower limit of quantitation is 500 ng ml⁻¹ for vesnarinone and 20 ng ml⁻¹ for OPC-8230, OPC-18137, and OPC-18136 in plasma. Methodology is similar for the estimation of these analytes in urine with the lower limit of quantitation being 500 ng ml⁻¹ for vesnarinone and 100 ng ml⁻¹ for each metabolite. Ascorbic acid was added to stabilize the analytes from degradation. This LC/MS/MS method was developed to overcome many practical problems associated with the HPLC method. The LC/MS/MS method offers the flexibility of analyzing additional metabolites and changing the linearity range to accommodate the differences in linear range (200–10 000 ng ml⁻¹ for vesnarinone and 20–1000 for metabolites) for the analytes.     

高效液相色谱-质谱联用测定人血浆中辛伐他汀浓度

目的建立测定人血浆中辛伐他汀浓度的方法。方法血浆样品中加入内标,用乙醚提取,浓缩后采用高效液相色谱-质谱联用进行测定。结果血浆样品中辛伐他汀线性范围为0.1～20ng·mL-1(r=0.9999);萃取回收率为94.3%。结论本方法灵敏度高、专属性强、重现性好、准确,可用于辛伐他汀片人体药动学及生物等效性研究。     

A direct determination of thymidine triphosphate concentrations without dephosphorylation in peripheral blood mononuclear cells by LC/MS/MS

A rapid, sensitive and specific analytical method with minimal sample preparation for the measurement of thymidine triphosphate (TTP) in peripheral blood mononuclear cells (PBMC) by LC/MS/MS has been developed. PBMC were separated from whole blood or buffy coat. The analyte and internal standard were extracted from PBMC with 70% methanol (pH 7.2). These extracts after centrifugation were directly injected onto LC/MS/MS without need for any further sample preparation. The calibration curve was linear over the range 0.8–800 ng/ml. Mean inter- and intra-assay coefficients of variation (CVs) over the range of the standard curve were less than 10%. The overall recovery of TTP was 103.5%.     

LC／MS／MS法测定人血中硫普罗宁的浓度及药物动力学研究

目的建立LC／MS／MS法测定人血中硫普罗宁浓度的方法,并研究其在健康男性受试者体内的药物动力学。方法采用LC／MS／MS（ESI源）测定硫普罗宁的血浆浓度,计算药物动力学参数。结果硫普罗宁线性范围为25．0-5000ng·mL^-1,定量下限为25．0ng·mL^-1日内、日间精密度（RSD）均〈15％,准确度（RE）在15％以内。应用本法测得20名健康男性受试者口服200mg硫普罗宁胶囊后主要药代动力学参数为：tmax,为（4．20±1．01）h,t1／2为（5．61±4．42）h,Cmax为（4456±2447）ng·mL^-1,AU C0-24h为（20566±9902）ng·mL^-1,Ke为（0．173±0．094）h^-1。结论该法操作简便、快速、灵敏,可用于测定血浆中硫普罗宁浓度。     

Determination of steroidal glycosides in Yucca gloriosa flowers by LC/MS/MS

An high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) method, was developed for the quantitative analysis of the steroidal glycosides occurring in Yucca gloriosa flowers. The HPLC experiments were performed by means of an octadecyl-modified reversed-phase C-18 column and a binary mobile phase system under gradient elution conditions. The fragmentation patterns of steroidal saponins were analyzed by ESI–MSⁿ in positive ion mode and a specific multiple reaction monitoring MS/MS detection was developed for their quantitative determination. The described method provides high sensitivity and specificity for quantitative determination of the steroidal glycosides in Y. gloriosa flowers. Quantification was performed against an external calibration line obtained using each pure steroidal glycoside. Short- and long-term repeatabilities of the methods were better than 3 and 6%, respectively. The method was validated according to EMEA guidelines and applied to real samples.     

Chemical investigation on Sijunzi decoction and its two major herbs Panax ginseng and Glycyrrhiza uralensis by LC/MS/MS

Sijunzi decoction consists of Panax ginseng, Poria cocos, Atractylodes macrocephala and Glycyrrhiza uralensis. High performance liquid chromatography coupled with tandem mass spectrometry (LC/MSⁿ) was applied to identify and characterize three types of active components, ginsenoside (from P. ginseng), flavonoid and triterpenoid (from G. uralensis) in Sijunzi decoction. Spectra of MS and MS/MS from [M + Na]⁺ ions of ginsenosides were acquired and interpreted for their identification. Fragmentations with losing masses of 194 or 176 Da were the characteristic ions of triterpenoids in the MS/MS analysis. A characteristic fragment ion of the aglycon moiety at m/z 257 from source collision-induced dissociation was observed for flavonoid. LC/MS was also applied for the comparison of relative peak area of major active components between Sijunzi decoction and the single herb extracts. The concentration ratios of major active components detected in the individual herbs of P. ginseng and G. uralensis were found different from those in Sijunzi decoction. The experimental data indicated that the decocting process could result in the difference in the amount of active components.     

Quantitative determination of apogossypol, a pro-apoptotic analog of gossypol, in mouse plasma using LC/MS/MS

A simple and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method based on internal standard quantitation using apigenin as the internal standard has been developed and validated for the analysis of the gossypol analog apogossypol, a pro-apoptotic compound, in mouse plasma. The methodology involves protein precipitation of plasma samples followed by LC/MS/MS analysis. Ascorbic acid was added to the spiking solutions and plasma samples to stabilize the easily oxidized compound. Separation of apogossypol and the internal standard from the plasma matrix was achieved using a C18 column with a gradient elution profile consisting of 5 mM ammonium acetate and methanol. The validated range of the method extended from 10 to 2000 ng/mL with accuracies of 85–115% and precision of <15%. The average recovery of apogossypol at three concentrations (50, 200 and 1000 ng/mL) assayed in triplicate using this methodology was determined to be 90.8 ± 12.9%. Recovery for the internal standard (apigenin) at a concentration of 500 ng/mL was found to be 99.9 ± 6.41%. Apogossypol concentrations of 50 ng/mL and above were found to be stable in extracted plasma for 24 h when stored at 25 °C. This method has been applied to the determination of apogossypol concentrations in plasma collected from mice given an IV dose of apogossypol.     

Structural elucidation of a process-related impurity in ezetimibe by LC/MS/MS and NMR

A major process-related impurity associated with the synthesis of ezetimibe was detected by LC–MS. The isolated impurity was found not to have been previously reported. Based on LC/MS/MS studies and accurate mass data, the structure of that impurity was proposed to be 2-(4-hydroxybenzyl)-N,5-bis-(4-fluorophenyl)-5-hydroxypentanamide. The postulated structure was unambiguously confirmed with the help of the NMR and IR analyses of a synthetically obtained sample. The chemical shift of the labile proton of that new entity was assigned by a 2D-NOESY NMR experiment. A rationalization for the formation of this impurity is provided.     

液相色谱-串联质谱法测定人血浆中盐酸舍曲林浓度

目的：建立测定人血浆中盐酸舍曲林浓度的液相色谱-串联质谱法。方法：以替米沙坦为内标,内标法定量。流动相：乙腈-10mmol·L^-1乙酸铵-1%甲酸（70：30：0.1）;质谱采用离子喷雾离子化源,扫描方式为多重反应监测（MRM）,用于定量分析的离子反应分别为m/z306.3→m/z159.1（舍曲林）和m/z515.2→m/z276.1（替米沙坦）。结果：舍曲林和替米沙坦的保留时间分别为2.22min和2.44min;舍曲林的线性范围为0.5～50.0ng·mL^-1,r=0.9992,回归方程：Y=0.0021＋0.0217X,最低检测浓度为0.5ng·mL^-1;提取回收率在82%～90%范围内;日内相对标准差〈4%,日间精密度〈13%。结论：此法适合人体血浆盐酸舍曲林浓度的监测及生物利用度研究,结果准确、可靠。     

Quality control of Chinese medicinal preparations LC/ESI(+)/MS/MS analyses of saikosaponins-a and -c as markers of Bupleuri radix samples

We have used LC/ion trap tandem MS analysis to determine saikosaponin-a and -c as target markers in crude 70% methanol extracts from three different species of Bupleuri radix and the 10 most-popular Chinese medicinal preparations containing "Chaihu" (B. radix) without any clean-up. The optimal ionization characteristics were obtained when using positive-ion electrospray ionization (ESI) with 50 microM sodium acetate as an additive in the mobile phase. We observed good linearity over the range from 0.02 to 2 microg/ml for saikosaponin-a and from 0.02 to 1 microg/ml for saikosaponin-c. The intra-day precisions varied between 3.3 and 8.8% for saikosaponin-a and 0.3 and 11.1% for saikosaponin-c. The limits of detection were 0.01 microg/ml for both markers. The recoveries of saikosaponin-a and -c from the extract of a medicinal preparation sample (Chai-Hu-Ching-Gan-Tang, No. 13 in the table of section Analysis on actual samples) were 97 and 100%, respectively, at a 1 microg/ml spiking concentration of each marker. The highest concentrations of saikosaponin-a and -c among the three B. radixes were found in B. kaoi Liu Chao & Chuang (10.1 mg/g) and in B. falcatum (3.4 mg/g), respectively. The amounts of these saikosaponins in the 10 Chinese medicinal preparations ranged between 0.11 and 1.22 mg/g for saikosaponin-a and between 0.01 and 0.33 mg/g for saikosaponin-c.     

氘代内标液质串联法测定血浆中沙丁胺醇的浓度

目的:建立液相-质谱串联法测定人血浆中的沙丁胺醇含量.方法:血浆离心后过玻璃纤维滤膜,经酶解加入氘代沙丁胺醇内标溶液,用C18小柱净化后以3%的氨水甲醇溶液对Oasis MCX小柱进行洗脱,吹干,以0.1%甲酸水溶液-甲醇溶液(95∶5)溶解残余物,用液相质谱串联法测定,以0.1%甲酸乙腈溶液和0.1%甲酸溶液为流动相梯度洗脱.结果:沙丁胺醇在0.25～10.00 μg·kg-1线性关系良好,检出限0.1μg·kg-1.结论:本法灵敏准确、重现性与特异性强、干扰少,可用于人体内沙丁胺醇药动学与生物利用度研究.     

LC/MS/MS法测定人血浆中愈创木酚甘油醚浓度及药动学研究

目的:建立以高效液相色谱-质谱联用法测定人血浆中愈创木酚甘油醚浓度的方法,并研究愈创木酚甘油醚片的人体药动学。方法:碱化的血浆药品经乙酸乙酯萃取后,以甲醇-1%甲酸(70:30)为流动相,对乙酰氨基酚为内标,采用Aquasil C18柱分离。通过液相色谱-串联质谱联用仪,以选择反应监测方式进行检测,定量分析的离子反应分别为m/z199→m/z125(愈创木酚甘油醚)和m/z152→m/z110(对乙酰氨基酚)。结果:愈创木酚甘油醚检测浓度在5·0～2500ng·mL-1范围内线性关系良好(r=0·9953),定量下限为5·0ng·mL-1。20名受试者口服愈创木酚甘油醚片0·2g后的主要药动学参数Cmax为(754·6±190·5)ng·mL-1、t1/2为(0·97±0·12)h、tmax为(0·63±0·22)h、AUC0～t为(1435·8±441·9)ng·h·mL-1、AUC0～∞为(1444·9±449·3)ng·h·mL-1。结论:本方法简便、快速、灵敏,可用于愈创木酚甘油醚的临床药动学研究。     

Chemical investigation on Sijunzi decoction and its two major herbs Panax ginseng and Glycyrrhiza uralensis by LC/MS/MS

Sijunzi decoction consists of Panax ginseng, Poria cocos, Atractylodes macrocephala and Glycyrrhiza uralensis. High performance liquid chromatography coupled with tandem mass spectrometry (LC/MSⁿ) was applied to identify and characterize three types of active components, ginsenoside (from P. ginseng), flavonoid and triterpenoid (from G. uralensis) in Sijunzi decoction. Spectra of MS and MS/MS from [M + Na]⁺ ions of ginsenosides were acquired and interpreted for their identification. Fragmentations with losing masses of 194 or 176 Da were the characteristic ions of triterpenoids in the MS/MS analysis. A characteristic fragment ion of the aglycon moiety at m/z 257 from source collision-induced dissociation was observed for flavonoid. LC/MS was also applied for the comparison of relative peak area of major active components between Sijunzi decoction and the single herb extracts. The concentration ratios of major active components detected in the individual herbs of P. ginseng and G. uralensis were found different from those in Sijunzi decoction. The experimental data indicated that the decocting process could result in the difference in the amount of active components.     

A sensitive human bone assay for quantitation of tigecycline using LC/MS/MS

Tigecycline (Tygacil,Wyeth) is a first-in-class, broad spectrum antibiotic with activity against multiple-resistant organisms. In order to address the unexpectedly low tigecycline concentrations in human bone samples analyzed using a LC/MS/MS method developed elsewhere, we have developed and validated a new and sensitive human bone assay for the quantitation of tigecycline using LC/MS/MS. The new method utilizes the addition of a stabilizing agent to the human bone sample, homogenization of human bone in a strong acidic-methanol extraction solvent, centrifugation of the bone suspension, separation by liquid chromatography, and detection of tigecycline by mass spectrometry. Linearity was demonstrated over the concentration range from 50 to 20,000 ng/g using a 0.1g human bone sample. The intra- and inter-day accuracy of the assay was within 100+/-15%, and the corresponding precision (CV) was <15%. The stability of tigecycline was evaluated and shown to be acceptable under the assay conditions. The extraction recovery of tigecycline with the current method was 79% when using radio-labeled rat bone samples as a substitute for human bone samples. Twenty-four human bone samples collected previously from volunteers without infections who had elective orthopedic surgery after receiving a single dose of tigecycline were re-analyzed using the current validated method. Tigecycline concentrations in these samples ranged from 238 to 794 ng/g with a mean value 9 times higher than the mean concentration previously reported. The data demonstrated that the current method has significantly higher extraction efficiency than the previously reported method.     

LC/MS联用法测定人血浆中右美沙芬的浓度及其药动学研究

目的:建立以液/质联用法测定人血浆中右美沙芬浓度的方法,并研究其在健康人体的药动学。方法:以曲马多为内标,血浆样品经液-液萃取后,采用Zorbax Extend-C18色谱柱进行分离,通过Q TRAPTM四极杆-线性离子阱质谱仪,以多反应检测方式进行测定。结果:右美沙芬的线性范围为0.05~10.0ng.mL-1(r=0.999 5);平均相对回收率在97.7%~99.5%之间;日内、日间精密度的RSD均小于6.8%,右美沙芬的定量下限为0.05ng.mL-1。结论:本法快速、简便、准确、灵敏,适用于右美沙芬的人体药动学研究。