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。     

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.     

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。     

LC/MS/MS的多反应监测方法定量测定灯盏乙素

目的：建立一种可靠的灯盏乙素定量分析方法。方法：用三级四极串联质谱(MS/MS)作为HPLC的检测器,其中MS/MS使用了多反应监测(MRM)扫描方式。选择母→子离子对m/z -461→m/z -285作为MRM监测的离子对;HPLC流动相为100％甲醇,流速0.9 mL.min^-1,色谱柱Beckman ODS-1。以测定短葶飞蓬提取物的灯盏乙素含量为例,对此方法进行了应用。结果：灯盏乙素在短葶飞蓬提取物中含量为6.98%。方法线性范围20～160 ng.mL^-1 (γ=0.999);加入灯盏乙素标准品20,60和160 ng的加样回收率分别为：96.5%,97.4%和97.3%。检测限为1 ng,每个样品的分析时间为4 min。结论：此法灵敏、快速、准确,可应用于灯盏乙素的各种药剂、药代的研究。     

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.     

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.     

Rapid and simple LC‐MS/MS screening of 64 novel psychoactive substances using dried blood spots

The range of novel psychoactive substances (NPS) including phenethylamines, cathinones, piperazines, tryptamines, etc. is continuously growing. Therefore, fast and reliable screening methods for these compounds are essential and needed. The use of dried blood spots (DBS) for a fast straightforward approach helps to simplify and shorten sample preparation significantly. DBS were produced from 10 µl of whole blood and extracted offline with 500 µl methanol followed by evaporation and reconstitution in mobile phase. Reversed‐phase chromatographic separation and mass spectrometric detection (RP‐LC‐MS/MS) was achieved within a run time of 10 min. The screening method was validated by evaluating the following parameters: limit of detection (LOD), matrix effect, selectivity and specificity, extraction efficiency, and short‐term and long‐term stability. Furthermore, the method was applied to authentic samples and results were compared with those obtained with a validated whole blood method used for routine analysis of NPS. LOD was between 1 and 10 ng/ml. No interference from matrix compounds was observed. The method was proven to be specific and selective for the analytes, although with limitations for 3‐FMC/flephedrone and MDDMA/MDEA. Mean extraction efficiency was 84.6 %. All substances were stable in DBS for at least a week when cooled. Cooling was essential for the stability of cathinones. Prepared samples were stable for at least 3 days. Comparison to the validated whole blood method yielded similar results. DBS were shown to be useful in developing a rapid screening method for NPS with simplified sample preparation. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid and sensitive method for determination of veliparib (ABT-888), in human plasma,bone marrow cells and supernatant by using LC/MS/MS

A rapid and sensitive method was developed and validated using a liquid chromatographic method with tandem mass spectrometry detection (LC/MS/MS) for determination of veliparib (ABT-888) in plasma, bone marrow supernatant, and bone marrow cells. Sample preparation involved a single protein precipitation step by the addition of the sample with acetonitrile. Separation of veliparib and the internal standard, A620223.69, was achieved on a Atlantis™ dC₁₈ column (100 mm × 2.1 mm, 3 μm) column using a mobile phase consisting of acetonitrile–ammonium acetate (2 mM) containing formic acid (0.1%, v/v) using isocratic flow at 0.2 mL/min for 3 min. The analyte and internal standard were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 5–1000 nM. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods. This method was subsequently used to measure concentrations of veliparib in cancer patients receiving an oral daily dose of 10 mg with demonstration of drug accumulation in the marrow compartment and in the target leukemia bone marrow cells.     

Development and validation of a multi‐analyte LC‐MS/MS approach for quantification of neuroleptics in whole blood,plasma, and serum

Based on a similar approach for quantification of antidepressants, benzodiazepines, and z‐drugs, a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) multi‐analyte approach with simple liquid‐liquid extraction was extended for fast target screening and quantification of neuroleptics in whole blood, plasma, and serum. As this method is part of a multi‐analyte procedure for over 100 analytes from different drug classes and as the extracts were additionally used in the authors' laboratory for gas chromatography‐mass spectrometry (GC‐MS) analysis, one universal stable‐isotope‐labelled internal standard (SIL‐IS) was used to save time and resource. The method was validated with respect to international guidelines. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. Selectivity problems could not be observed, but matrix effects ranged from 68 to 211% in all samples. For the low quality control (QC), recovery ranged from 32 to 112%, process efficiency from 31 to 165% and for the high QC recovery from 42 to 141%, process efficiency from 29 to 154%. In addition statistical data evaluation of the variances of the recovery, matrix effects, and process efficiency data between whole blood vs. plasma, whole blood vs. serum, and plasma vs. serum were done. The presented LC‐MS/MS approach was applicable for selective detection of 33 neuroleptics as well as accurate and precise quantification of 25 neuroleptics in whole blood, 19 in plasma, and 17 in serum. More significant matrix effects (ME) for neuropletic drugs overall in plasma and serum as compared with whole blood were detected. Copyright © 2015 John Wiley & Sons, Ltd.     

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).     

液相色谱串联质谱法测定人血清中硝苯地平的浓度

目的建立液相色谱串联质谱法测定人血清中硝苯地平浓度。方法血清样品用甲醇沉淀蛋白,内标为尼群地平,色谱柱为Kromasil C18(4.6mm×150mm,5μm),柱温为30℃,流动相为甲醇-水(90:10,V/V),流速为0.5ml·min-1。质谱采用ESI离子源负离子检测,定量分析的离子对为:m/z345.1/122.0(硝苯地平),m/z359.2/122.0(内标尼群地平)。结果硝苯地平在0.5μg·L-1～50μg·L-1范围内线性关系良好(r=0.9987),批内批间RSD均小于15%,提取回收率为64.06%～77.10%。结论该方法快速,灵敏,准确,可用于硝苯地平的临床药动学研究。     

Simultaneous determination of desloratadine and its active metabolite 3-hydroxydesloratadine in human plasma by LC/MS/MS and its application to pharmacokinetics and bioequivalence

A rapid and simple liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method was developed and validated for the simultaneous determination of desloratadine and its active metabolite 3-hydroxydesloratadine concentrations in human plasma. After liquid-liquid extraction with ethyl ether for sample preparation, the chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mm x 2.0mm, 5 microm, Shiseido). [(2)H(4)]desloratadine and [(2)H(4)]3-OH desloratadine were used as internal standards. A mobile phase consisted of 5mM ammonium formate in water, methanol and acetonitrile (50:30:20). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. A quadratic regression (weighted 1/concentration) gave the best fit for calibration curves over the concentration range 0.05-10 ng/mL for both desloratadine and 3-OH desloratadine. The method was shown to be accurate, rapid and sufficiently sensitive to be successfully applied to a pharmacokinetic and bioequivalent study.     

Development and validation for high selective quantitative determination of metformin in human plasma by cation exchanging with normal-phase LC/MS/MS

An assay based on cation exchange solid-phase extraction and liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed for the quantitative determination of metformin in human plasma. The analytical method consists of cation exchange solid-phase extraction (VersaPlate CBA) without any further evaporation/dissolution steps and cation exchange-based HPLC separation (Capcell Pak SCX column) with a normal-phase gradient system followed by semi-micro LC/MS/MS in positive ion selected reaction monitoring mode using electrospray ionization. The method exhibited excellent performance in terms of selectivity, robustness, short run time (7 min/sample) and simplicity of sample preparation.

The calibration range was 10–1000 ng/ml with 0.2 ml of plasma. Intra- and inter-day mean accuracies were within the ranges of 100.3–105.0% and 101.2–105.3%, respectively. Intra- and inter-day precisions were within the ranges of 0.8–1.9% and 1.5–8.6%, respectively. Mean absolute recovery was 67.0% for metformin. No apparent loss of metformin after extraction was observed in an autosampler at 10 °C for 24 h. Dilution of metformin by blank human plasma up to 20-fold was tested and revealed no impact on the results of determination. Furthermore, the method exhibited high selectivity, since no effect on metformin analysis was observed on comparison of samples with or without nateglinide and other agents in plasma. Results obtained with the method were also comparable to a published LC–UV method on cross-validation.

This method can be applied to various clinical pharmacokinetic studies of metformin.     

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.     

Stereoselective analysis of carvedilol in human plasma using HPLC/MS/MS after chiral derivatization

A relatively high-throughput high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method using a chiral derivatization reagent was developed for the quantitative determination of carvedilol enantiomers in human plasma. S-carvedilol and R-carvedilol are extracted from human plasma by protein precipitation using acetonitrile containing racemic [(2)H(5)]-carvedilol as an internal standard. Extracts are then derivatized with 2,3,4,6-tetra-O-acetyl-beta-glucopyranosyl isothiocyanate (GITC) and analysed using HPLC-MS/MS with a TurboIonspray (TIS) interface and selected reaction monitoring. Using 150microL of plasma, the method was validated over a concentration range of 0.2-200ng/mL. The maximum within-run precision observed in a three run quality control was 8.2% for S-carvedilol and 6.7% for R-carvedilol, respectively. The maximum percentage bias observed at all quality control sample concentrations was 9.4% for S-carvedilol and 11.6% for R-carvedilol, respectively. The HPLC-MS/MS method was also compared with a previously developed high-performance LC/fluorescence method by analysing 25 samples containing racemic carvedilol. Based on results obtained, these two methods were found to be equivalent. However, compared with LC/fluorescence method, HPLC-MS/MS method is more sensitive, uses less plasma, and also employs a less time-consuming sample preparation process.     

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.     

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。     

A direct LC/MS/MS method for the determination of ciclopirox penetration across human nail plate in in vitro penetration studies

Due to severe chelating effect caused by N-hydroxylpyridone group of ciclopirox, there is no published direct HPLC or LC/MS/MS method for the determination of ciclopirox in any in vitro or in vivo matrix. Instead, the time-consuming pre-column derivatization methods have been adapted for indirect analysis of ciclopirox. After overcoming the chelating problem by using K₂EDTA coated tubes, a direct, sensitive and high-throughput LC/MS/MS method was successfully developed and validated to determine the amount of ciclopirox that penetrated across the nail plate during in vitro nail penetration studies. The method involved adding a chemical analog, chloridazon as internal standard (IS) in K₂EDTA coated tubes, mixing IS with ciclopirox in a 96-well plate and then proceeding to LC/MS/MS analysis. The MS/MS was selected to monitor m/z 208.0 → 135.8 and 221.8 → 77.0 for ciclopirox and IS, respectively, using positive electrospray ionization. The method was validated over a concentration range of 8–256 ng/mL, yielding calibration curves with correlation coefficients greater than 0.9991 with a lower limit of quantitation (LLOQ) of 8 ng/mL. The assay precision and accuracy were evaluated using quality control (QC) samples at three concentration levels. Analyzed concentrations ranged from 101% to 113% of their respective nominal concentration levels with coefficients of variation (CV) below 10.6%. The average recovery of ciclopirox from nail matrix was 101%.