液相色谱-质谱联用法测定人血浆双氢青蒿素浓度

目的:建立液相色谱-质谱联用法测定健康人血浆中双氢青蒿素浓度的方法。方法:以青蒿素为内标,血浆样品采用液-液萃取法处理。用电喷雾离子化和正离子多离子反应监测方式检测双氢青蒿素。结果:该方法双氢青蒿素线性范围为1.01~2020 ng.ml-1;定量下限为1.001±0.072 ng.ml-1;方法回收率在93.0%~98.2%;批内、批间变异系数均<10%。结论:该方法准确、灵敏、特异、简便,适用于健康人血浆双氢青蒿素浓度的测定。     

Validation of a sensitive LC/MS/MS method for simultaneous quantitation of flupentixol and melitracene in human plasma

A sensitive method has been developed and validated, using LC/ESI-MS/MS, for simultaneous quantitation of flupentixol and melitracen—antidepressant drugs, in human plasma. The quantitation of the target compounds was determined in a positive ion mode and multiple reaction monitoring (MRM). The method involved a repeated liquid–liquid extraction with diethyl ether and analytes were chromatographed on a C₈ chromatographic column by elution with acetonitrile–water–formic acid (36:64:1, v/v/v) and analyzed by tandem mass spectrometry. The method was validated over the concentration ranges of 26.1–2090 pg/ml for flupentixol and 0.206–4120 ng/ml for melitracen. The correlation coefficients of both analyst were >0.998 for six sets of calibration curves. The recovery was 60.9–75.1% for flupentixol, melitracen and internal standard. The lower limit of quantitation (LLOQ) detection was 26.1 pg/ml for flupentixol and 0.206 ng/ml for melitracen. Intra- and inter-day precision of the assay at three concentrations were 2.15–5.92% with accuracy of 97.6–103.0% for flupentixol and 0.5–6.36% with accuracy of 98.7–101.7% for melitracen. Stability of compounds was established in a battery of stability studies, i.e., bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for bioequivalence study of flupentixol and melitracen in healthy human male volunteers.     

A study of matrix effects on an LC/MS/MS assay for olanzapine and desmethyl olanzapine

The purpose of this research project was to investigate potential matrix effects of anticoagulant and lipemia on the response of olanzapine, desmethyl olanzapine, olanzapine-D₃ and desmethyl olanzapine-D₈ in an LC/MS/MS assay. Blank human serum and sodium heparin, sodium citrate, and K₃EDTA plasma with various degrees of lipemia were fortified with olanzapine, desmethyl olanzapine, olanzapine-D₃ and desmethyl olanzapine-D₈. Six replicates of each sample were extracted using Waters Oasis^® MCX cartridges and analyzed using electrospray LC/MS/MS. The analytes were separated on a Phenomenex LUNA phenyl hexyl, 2 mm×50 mm, 5 μm, analytical column and a gradient rising from 2 to 85% mobile phase B. Mobile phase A consisted of acetonitrile–ammonium acetate (20 mM) (52:48 v/v) and mobile phase B was formic acid–acetonitrile (0.1:100 v/v). Ion suppression was investigated through post column infusion experiments. The degree of lipemia of each sample, indicated by turbidity, was ranked into categories from least to greatest and used for statistical analyses. The results from analysis of variance testing indicated that lipemia, anticoagulant and their interaction significantly influenced mass spectral matrix effects and extraction matrix effects. Differential behavior between the analytes and labeled internal standards contributed to variability. The most significant source of variability however, was ion suppression due to co-eluting matrix components.     

HPLC/MS/MS for quantification of two types of neurotransmitters in rat brain and application: myocardial ischemia and protection of Sheng-Mai-San

Sheng-Mai-San (SMS), a traditional Chinese multiherbal formula, is widely used in clinic for the treatment of myocardial ischemia (MI) in China. Recently, it has been shown that the change of neurotransmitters in central nervous system is closely related to the pathogenesis of MI, whether SMS might affect the neurotransmitters at central nervous system in MI patients has not been studied yet. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of two types of neurotransmitters (neuropeptides and monoaminergic neurotransmitters) in rat brain tissue was developed. The instrument was operated under the multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in the positive ion mode. A good linear relationship with coefficients ≥0.99 was achieved over the concentration ranges of 10-1000ngmL(-1) for 5-hydroxytryptamine (5-HT) and norepinephrine (NE); 2-250ngmL(-1) for methionine-enkephalin (M-ENK) and leucine-enkephalin (L-ENK). Quantification limit for 5-HT and NE was 4.0ngmL(-1); and 2.0ngmL(-1) for M-ENK and L-ENK. The intra- and inter-day precision was less than 15% and accuracy was within ±15%. The analysis revealed significant reductions at the levels of 5-HT (p<0.01), NE (p<0.01), M-ENK (p<0.05) and L-ENK (p<0.01) in the MI group compared to the control group. These findings demonstrate that myocardial ischemia reduces the concentrations of 5-HT, NE, M-ENK and L-ENK in rat brains, while SMS shows protective effects on MI associated with reversing these four neurotransmitters to sham levels.     

液相色谱-质谱联用法测定人参皂苷Re在健康人血浆的浓度

目的建立液相色谱-质谱联用法测定人参皂苷Re在健康人血浆中浓 度的方法。方法血浆样品用固相萃取法处理。用电喷雾离子化和正离子多 离子反应监测方式检测人参皂苷Re。结果该方法人参皂苷Re线性范围为 1．05～1 050 ng·mL-1;定量下限为1．05 ng·mL-1;方法回收率在99．3％～ 104．3％;日内、日间变异系数(RSD)均<15％。结论该法准确、灵敏、特异, 适用于健康人血浆人参皂苷Re浓度测定。     

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。     

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).     

HPLC／MS／MS测定恒河猴血浆中纳曲酮及其代谢物纳曲醇的浓度

目的:建立高效液相色谱-质谱联用法测定恒河猴血浆中纳曲酮及其代谢物纳曲醇的浓度。方法:液相色谱:色谱柱为Nucleocil ODS(5μm,50 mm×2.0 mm),流动相为乙腈-甲醇-水-冰醋酸(20:20:60:1),流速为0.2 mL·min-1;质谱:离子源为Turbo Ionspray源,采用多反应监测(MRM)方式进行检测,m／z342.3→324.3(纳曲酮)、m／z 344.3→326.6(纳曲醇,代谢产物)、m／z 328.4→310.4(纳洛酮,内标)。样品经有机相提取纯化后进样。结果:纳曲酮和纳曲醇的线性范围分别为0.098-150 ng·mL-1和0.781-25 ng·mL-1,萃取回收率均大于75％,日内误差(RSD)均小于9.3％,日间误差(RSD)均小于12％。结论:本方法准确、快速、灵敏、专属性好,适用于动物试验及临床上测定血浆中纳曲酮及其代谢物纳曲醇的浓度和药代动力学研究。     

Validated HPLC-MS/MS method for determination of quetiapine in human plasma

A validated, highly sensitive and selective high-pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitative determination of quetiapine (QUE) in human Na2EDTA plasma with mass spectrometry (MS) detection. Clozapine (CLO) was employed as an internal standard. Samples were extracted using solid phase extraction (SPE). Oasis HLB cartridges and the concentration of quetiapine was determined by isocratic HPLC-MS/MS. The SRM mode was used for MS/MS detection. The method was validated over a concentration range of 1.0-382.2 ng/mL. Inter- and intra-day precision and accuracy of the proposed method were characterized by relative standard deviation (R.S.D.) and the percentage of deviation, respectively; both were lower than 8%. The developed method was employed in the pharmacokinetic study of quetiapine.     

液相色谱-串联质谱法测定苯妥英在健康人体的血药浓度并研究其生物等效性

目的建立快速、灵敏的液相色谱-串联质谱法测定健康人血浆中的苯妥英(抗癫痫药),并进行生物等效性研究。方法血浆样品50μL经液- 液萃取后,以甲醇-水-甲酸(90:10:0．2)为流动相,Zorbax SB-C18柱分离; 样品经大气压化学电离源(APCI)正离子化后,通过三重四极杆串联质谱仪, 用选择反应监测(SRM)对苯妥英(m／z 253→m／z 182)和柳胺酚(m／z 230→ m／z 121,内标)进行测定。用此法测定了20名受试者单剂量口服受试和参比制剂后苯妥英的血药浓度。结果线性范围为2．5—3 000 ng·mL-1,定量下限为2．5 ng·mL-1;日内、日问精密度(RSD)均<7．0％,准确度(RE)在 -0．5％～2．3％。2种制剂的Cmax、AUC0-t均无显著性差异。结论该法专属、灵敏、快速,适用于复方制剂中苯妥英的生物等效性评价。     

氯吡格雷在人血浆浓度的液质联用测定法

目的建立测定人血浆中氯吡格雷(抗心肌及抗脑梗死药)血药浓度的HPLC/MS/MS法。方法血浆样品用叔丁基甲醚提取,内标为噻氯匹定;色谱柱用X-Terra MS C_(18)(2.1 mm×50 mm,5μm),柱温30℃,流动相:乙腈-水(含10 mmol·L~(-1)醋酸铵,调pH为4.0)为70:30,流量为0.3 mL·min~(-1);质谱用ESI离子源,定量分析的离子反应分别为m/z 322→z 212 (氯吡格雷),m/z 264→m/z 154(内标噻氯匹定)。结果氯吡格雷的线性范围为10×10~4～1.2×10~4pg·mL~(-1)(γ=0.9993),日内、日间RSD均小于10%,提取回收率约为80%。结论该方法快速、准确、灵敏度高,可用于氯吡格雷血药浓度测定。     

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.     

A sensitive LC/MS/MS bioanalysis assay of orally administered lipoic acid in rat blood and brain tissue

A sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) bioanalytical method was developed and validated to analyze lipoic acid (LA) in rat blood and brain samples. Ten mobile phase combinations were investigated during method development. Mobile phase combination of 0.1% acetic acid (pH 4 adjusted with ammonia solution)/acetonitrile was most optimum in terms of sensitivity and peak shape of LA and the internal standard, valproic acid. Sample extraction method was explored using liquid–liquid extraction and protein precipitation methods. Protein precipitation yielded the highest recovery of the analytes from blood and brain ranging from 92 to 115%. The lower limit of quantitation (LLOQ) of LA was 0.1 ng/mL (0.485 nM) in both blood and brain while on-column lower limit of detection (LLOD) was 0.03 pg. The precision (% R.S.D.) ranged from 1.49 to 26.39% and 1.49 to 10.89% for intra- and inter-day assays, respectively. The accuracy ranged from 91.2 to 116.17% for intra-day assay and 102.68 to 114.33% for inter-day assay.     

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法测定辛伐他汀血浆浓度及其在健康人体内的药物动力学研究

目的:建立人体血浆中辛伐他汀的LC/MS/MS测定方法,并研究辛伐他汀片在男性健康志愿者体内的药物动力学行为,评价其生物利用度和生物等效性。方法:采用两制剂双周期自身对照试验设计。18名男性健康志愿者随机交叉服用单剂量辛伐他汀试验片剂和参比片剂20mg,采用液相色谱-串联质谱(LC/MS/MS)分析方法测定血浆辛伐他汀的浓度。采用DAS2.0程序计算药物动力学参数和相对生物利用度,并进行等效性评价。结果:测定单剂量口服20mg辛伐他汀参比片剂和试验片剂的AUC_((0→24))分别为(14.90±5.86)和(14.37±4.94)ng·h·ml~(-1),AUC_((0→∞))分别为(15.62±6.29)和(14.78±5.02 )ng·h·ml~(-1);C_(max)分别为(4.54±2.11)和(4.00±1.34)ng·ml~(-1);T_(max)分别为(1.75±0.79)和(1.39±0.65)h。以AUC_((0→24))与AUC_((0→∞))计算相对生物利用度分别为(108.0±52.7)%和(106.4±52.5)%。结论:该法准确灵敏,测得的数据可靠,统计分析表明两种制剂生物等效。     

HPLC荧光和质谱两种检测器测定大鼠血浆及组织中的表阿霉素

目的 建立测定大鼠血浆及组织中表阿霉素浓度的方法.方法 采用RP-HPLC荧光和质谱两种检测器,Lima C18(2)分析柱(150 mm×4.6 mm,5μm),流动相为0.01 mol·L-1乙酸铵-乙腈(60:40,甲酸调pH3.5),流速0.6 ml·min-1,样品在碱性条件下,用乙酸乙酯漩涡混合提取浓集后进样,λEx=450 am,λEm=530 nm,质谱以MRM模式测定,内标法定量.结果 荧光检测标准曲线在2.44～2.50×103μg·L-1有良好线性,定量限2.44μg·L-1,日内RSD小于5.0%,日间RSD小于8.6%,方法回收率99%～113%,萃取回收率86.8%～89.7%;质谱检测标准曲线在0.49～2.00×103μg·L-1有良好线性,定量限0.49μg·L-1,日内RSD小于6.5%,日间RSD小于8.7%,方法回收率98～5%.结论 所建方法快速简便、灵敏准确,适用于血浆及组织中表阿霉素的测定及药物动力学研究.     

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.