In this study, two simple and accurate LC-MS/MS methods were firstly developed and validated to quantify EVT201, a new partial GABAA receptor agonist used for the treatment of insomnia, and its metabolites comprising M1, M2, M3, M4 and M6 in human urine. The analytes in urine samples were determined after simple dilution, and ideal chromatographic separations were obtained on C18 columns using gradient elution. The assays were performed in MRM mode on AB QTRAP 5500 tandem mass spectrometry (ESI+). The concentration ranges (ng/mL) of analytes in human urine were as follows: EVT201, 1.00 to 36.0; M1, 1.40 to 308; M2, 2.00 to 72.0; M3, 5.00 to 1100; M4, 2.00 to 300; and M6, 2.80 to 420. The methods were fully validated including selectivity, carryover, matrix effect, recovery, linearity, accuracy, precision, dilution integrity and stability, and acceptable criteria were obtained. The methods were successfully applied to a mass balance study of EVT201. The results showed that the total cumulative urinary excretion rate of EVT201 and its five metabolites was 74.25 +/- 6.50%, which suggested that EVT201 had high oral bioavailability, and urinary elimination was its major excretion pathway in human.

目的:建立快速、灵敏、可靠的LC-MS/MS法测定人血浆中甲基纳曲酮的浓度.方法:采用弱阳离子交换固相萃取柱进行样品预处理,以溴甲基纳曲酮-d3为内标,通过Ultra PFP Propyl柱(100mm×2.1 mm,5.0 μm)色谱柱进行分离,流动相为20 mmol·L-1乙酸铵水溶液(含1％甲酸)和乙腈,等度洗脱(15∶85,v/v),流速0.4 mL· min-1.通过电喷雾电离(ESI),以多反应监测(MRM)模式进行正离子监测,甲基纳曲酮和内标的离子对分别为m/z 356.3→284.2,m/z 359.3→287.1.结果:人血浆中甲基纳曲酮在0.1～ 120 ng·mL-1范围内线性关系良好(r＞0.99),定量下限为0.1 ng·mL-1;方法批内、批间精密度＜7.2％,准确度为94.5％ ～ 100.7％;甲基纳曲酮内标归一化的平均基质效应为99.1％,平均提取回收率为81.4％.该方法成功应用于12例健康受试者皮下注射0.075 mg· kg-1溴甲基纳曲酮注射液的药动学研究.结论:本研究建立的LC-MS/MS分析方法灵敏度高、快速且方法重现性好,满足生物分析的法规要求.

目的 建立测定人血浆中马尼地平浓度的液相色谱-串联质谱法.方法 200μL血浆样品经液液萃取后,使用Phenomenex Synergi 4μHydro-RP 80A色谱柱(150 mm×2.0 mm I.D.),以甲醇-5 mmol/L乙酸铵溶液(含0.3％ 乙酸)为流动相进行梯度洗脱.API 3200型三重四极杆串联质谱仪采用电喷雾离子源 、正离子方式检测 、多重反应监测(MRM)扫描,马尼地平和尼群地平的离子反应分别为质荷比(m/z)611.1→167.3和m/z 361.1→315.2.方法学验证内容包括选择性 、基质效应 、线性范围 、定量下限 、准确度与精密度 、提取回收率 、血浆/溶液样品稳定性.结果 马尼地平和内标尼群地平的保留时间分别约为2.47 min和3.03 min.马尼地平的线性范围为0.1～16.0 ng/mL,低中高浓度质控样品的批内 、相对标准偏差均小于15％,相对误差均在 ±15％ 以内,平均基质效应因子为(85.0±2.8)％,平均提取回收率为(89.2±4.8)％,血浆/溶液样品在不同保存条件下稳定性良好.结论 该方法灵敏耐用,可用于人血浆中马尼地平浓度的测定及马尼地平口服制剂的人体药代动力学研究.

Objective To develop a rapid and sensitive liquid chromatography-tandem mass spectrometric(LC-MS/MS) method for the determination of valproic acid in human plasma.Methods After treating human plasma sample by acetonitrile protein precipitation method,the analytes were separated on a Shimpack VP-ODS analytical column(150 mm×2.0 mm I.D,5 μm) with the mobile phase of methanol and 5 mmol/L ammonium acetate (55∶45,v/v)at a flow rate of 0.4 mL/min.Detection was carried out by adopting the multiple reaction monitoring(MRM) scanning mode in the API3200 triple quadrupole tandem mass spectrometer,electrospray ionization source,negative ion mode,selected monitoring ionic reactions were m/z 142.9→m/z 142.9(valproic acid) and m/z 179.0→m/z 179.0(1-sulfonic acid).Results Valproic acid and internal standard 1-sulfonic acid retention time were 3.03 min and 2.38 min respectively.The plasma valproic acid linear range was 0.800-80.0 μg/mL(r>0.99) with the lower limit of quantitation(LLOQ) 0.800 μg/mL.The intra-and inter-batch relative standard deviations(RSD) were both less than 15%,and the relative errors(RE) were within ±15%.The mean extraction recovery rate was(84.1±2.4)%,and the mean matrix effect factor was(104.3±2.0)%.In the stability study,valproic acid was found to be stable in plasma under various storage conditions.Conclusion This method is suitable for the determination of valproic acid in human plasma and human pharmacokinetic study of valproic acid semisodium sustained release tablet.

OBJECTIVE To study the bioequivalence of domestic and imported moxifloxacin hydrochloride tablets under fasting and fed conditions.METHODS Two formulations,randomized,two-period crossover clinical trials were performed.Twenty four healthy male volunteers were given a single oral dose of 400 mg of domestic or imported moxifloxacin hydrochloride tablets under fasting and fed conditions,respectively.Moxifloxacin in plasma were determined by a HPLC-FLD method.By means of WinNonlin Ver 6.4 software,pharmacokinetic parameters were calculated using a non compartmental mode.The 90％ confidence intervals (90％ CIs) of the test-to-reference geometric mean ratio (GMR) of AUC and Cmax were calculated to assess the bioequivalence.RESULTS The 90％ CIs of the GMR of AUC0-t,AUC0-∞ and Cmax between the test and reference drug fell within the limit ranges of 80.00％～125.00％ under fasting and fed conditions,respectively.Cmax decreased and tmax delayed,AUC and t1/2 of moxifloxacin were basically the same under fed condition compared to the fasting conditions.CONCLUSION The domestic moxifloxacin tablets are proved to be bioequivalent to imported moxifloxacin hydrochloride tablets both under fasting and fed conditions.

生物药剂学分类系统将药物按溶解度和渗透性分为4类,在指导新药研发和剂型设计、预测药物体内体外相关性、进行生物豁免研究等方面发挥了非常重要的作用.本文综述了基于生物药剂学分类系统的口服固体速释制剂生物豁免所需溶解度、渗透性和溶出度的接受标准和测定方法,并分类描述了进行过生物豁免研究的药物及其有关资料;建议我国尽快开展和接受生物豁免.

Efonidipine hydrochloride is a new generation dihydropyridine Ca2+ channel blocker designed to inhibit both T-type and L-type Ca2+ channels. Efonidipine possesses a chiral carbon and is clinically administered as a racemate. In the present study, an enantioselective and sensitive LC-MS/MS method of determining efonidipine enantiomers in human plasma was developed and validated to characterize the stereoselective pharmacokinetics. Plasma samples were processed by liquid-liquid extraction (LLE). Chiral separation was optimized on a CHIRALPAK (R) ID column using an isocratic mobile phase of acetonitrile/water (60:40, v/v). Detection was using MS in multiple reaction monitoring (MRM) mode, using the transitions of m/z 6323 -> 91.1 for efonidipine enantiomers, and m/z 493.3 -> 117.2 for cilnidipine (internal standard). The calibration curves were linear over 0.100-20.0 ng/mL for each enantiomer. The lower limit of quantification (LLOQ) for each enantiomer was established at 0.100 ng/mL. Intra- and inter-day precisions were less than 12.1% for each enantiomer in terms of relative standard deviation (RSD), and accuracies were between -5.0% and 5.0% in terms of relative error (RE) for each enantiomer. No chiral inversion was observed during sample storage, preparation procedure and analysis. The validated method was successfully applied to a stereoselective pharmacokinetic study of efonidipine in healthy subjects after oral administration of 40 mg (20 mg x 2) efonidipine hydrochloride tablets. (C) 2016 Elsevier B.V. All rights reserved.

目的:建立测定人血浆中谷氨酸和精氨酸的方法,以应用于人体药代动力学研究.方法:血浆样本经与丹酰氯衍生化反应后,选用Synergi 4μ Hydro-RP 80A柱(150 mm ×2.0 mm,4μm),以甲醇-10 mmol·L-1乙酸铵(含0.1％甲酸)(50∶ 50)为流动相,流速0.3 mL·min-1,采用API3200型三重四极杆串联质谱仪的多重反应监测(MRM)扫描方式进行监测,电喷雾离子化源,正离子方式,选择监测离子反应分别为m/z 381.0--170.2(谷氨酸丹酰氯衍生化产物)、m/z 408.0--170.3(精氨酸丹酰氯衍生化产物)和m/z 353.0 →157.1(内标D-高丝氨酸丹酰氯衍生化产物).结果:谷氨酸、精氨酸和内标D-高丝氨酸的丹酰氯衍生化产物的保留时间分别为4.62、3.21和4.39 min;血浆中谷氨酸和精氨酸的线性范围均为3.00～300 μg·mL-1(r＞0.99),定量下限(LLOQ)均为3.00 μg·mL-;批内、批间精密度(RSD)均小于15％;准确度(RE)均在±15％的范围以内;谷氨酸和精氨酸平均提取回收率分别为(85.6±3.3)％和(79.9±3.9)％;平均基质效应因子分别为(116.3±4.9)％和(102.4±7.3)％;稳定性试验中,在各种贮存条件下血浆中谷氨酸和精氨酸均较稳定.结论:该方法适用于谷氨酸精氨酸注射液人体药代动力学研究.

ObjectiveTo develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of cefdinir in human plasma and evaluate the bioequivalence of its two granules.MethodsA single oral dose of 100 mg cefdinir test and reference granules were given to 24 healthy male volunteers in a randomized, two-way crossway study. The concentrations of cefdinir in plasma were determined by LC-MS/MS, and the pharmacokinetic parameters were calculated by WinNonlin 6.3 Software, and the bioequivalence of the two preparations was evaluated.ResultsThe linear range for determining cefdinir in plasma samples was 10.0 — 2000 ng/mL(r= 0.999 7), with the lower limit of quantification (LLOQ) of 10.0 ng/mL. The intra- and inter-batch relative standard deviation (RSD) was lower than 6.0%, and relative error (RE) was within ± 4.0%. Degradation degree of cefdinir in human plasma was increased under the room temperature storage with the extension of storage time. The main pharmacokinetic parameters of test and reference preparations were as follows: AUC0-t were (6238.22 ± 1993.74) and (6331.35 ± 1850.42) ng·h/mL; AUC0-∞ were (6 343.68 ± 2070.73) and (6429.76 ± 1901.33) ng·h/mL;Cmax were (1290 ± 391) and (1330 ± 384) ng/mL;tmax were 2.50 (2.00 — 4.50) h and 2.50 (2.00 — 3.50) h;t1/2 were (1.61 ± 0.17) and (1.61 ± 0.17) h, respectively. The geometric mean ratios (GMRs) for AUC0-t, AUC0-∞, andCmax were 97.88% (90% CI, 91.75% — 104.42%), 97.95% (90% CI, 91.76% — 104.56%) and 96.75% (90% CI, 90.43% — 103.52%), respectively.ConclusionThe established method is rapid, sensitive, accurate, selective, reliable, and sufficient for the determination of cefdinir in human plasma, andthe two preparations are bioequivalent.

A rapid, sensitive and accurate ICP-MS method using alternate analyte-free matrix for calibration standards preparation and a rapid direct dilution procedure for sample preparation was developed and validated for the quantification of exogenous strontium (Sr) from the drug in human serum. Serum was prepared by direct dilution (1:29, v/v) in an acidic solution consisting of nitric acid (0.1%) and germanium (Ge) added as internal standard (IS), to obtain simple and high-throughput preparation procedure with minimized matrix effect, and good repeatability. ICP-MS analysis was performed using collision cell technology (CCT) mode. Alternate matrix method by using distilled water as an alternate analyte-free matrix for the preparation of calibration standards (CS) was used to avoid the influence of endogenous Sr in serum on the quantification. The method was validated in terms of selectivity, carry-over, matrix effects, lower limit of quantification (LLOQ), linearity, precision and accuracy, and stability. Instrumental linearity was verified in the range of 1.00-500ng/mL, corresponding to a concentration range of 0.0300-15.0μg/mL in 50μL sample of serum matrix and alternate matrix. Intra- and inter-day precision as relative standard deviation (RSD) were less than 8.0% and accuracy as relative error (RE) was within ±3.0%. The method allowed a high sample throughput, and was sensitive and accurate enough for a pilot bioequivalence study in healthy male Chinese subjects following single oral administration of two strontium ranelate formulations containing 2g strontium ranelate.

建立实时荧光定量PCR方法检测CYP1A2* 1F基因多态性,并探讨CYP1A2*1F基因多态性对阿戈美拉汀药代动力学的影响.该方法采用扩增阻滞突变系统(ARMS)结合TaqMan探针技术(ARMS-TaqMan法)同时检测野生和突变两个反应体系,根据野生引物循环阈值(Ctw)与突变引物循环阈值(Ctm)的差值绝对值(ACt)来判断CYP1A2* 1F的基因型.同时进行口服阿戈美拉汀片25mg的药代动力学试验,采用液相色谱-串联质谱法测定血浆中阿戈美拉汀浓度,采用WinNonlin 6.3软件处理血浆中药物浓度-时间数据,非房室模型方法计算药代动力学参数Tmax、Cmax、AUC0→t、AUC0→∞和T1/2.结果表明,在55名健康受试者中,CYP1A2 * 1F野生型共12例(21.8％),其△Ct值为9.89±0.56;杂合型22例(40.0％),其△Ct值为0.41±0.14;纯合突变型21例(38.2％),其△Ct值为6.85±0.32.统计分析显示,CYP1A2* 1F 3种基因型组间各药代动力学参数比较皆无显著性差异(P＞0.05),但所建立的用于CYP1A2* 1F基因多态性检测的ARMS-TaqMan法具有准确、操作简单、高通量等优势,便于进一步筛查基因多态性对阿戈美拉汀药代动力学的影响,适合临床应用.本方法检测原理可用于各种单碱基突变率的检测,具有广泛的应用前景.

Efonidipine hydrochloride is a new generation dihydropyridine calcium channel blocker designed to inhibit both T-type and L-type calcium channels. For the first time, a simple and robust LC-MS/MS method was developed for the determination of efonidipine in human plasma over the range of 0.100-20.0ng/mL. Efonidipine was extracted from plasma by an LLE procedure, separated by LC and detected by MS/MS in positive mode ESI. The method was validated for selectivity, carryover, sensitivity, extraction recovery, matrix effects, linearity, accuracy and precision, dilution integrity and stability studies. The calibration curves were linear over 0.100-20.0ng/mL (r≥0.9980). The lower limit of quantification (LLOQ) was established at 0.100ng/mL. Intra- and inter-day precisions (LLOQ, low-QC, mid-QC, high-QC and ultra-high QC) were less than 12.5% in terms of relative standard deviation (RSD), and accuracies were between -5.0% and 5.0% in terms of relative error (RE). Matrix effect was acceptable (105.6-110.2%) and extraction recovery was reproducible (85.8-91.3%, RSD≤10.0%). Efonidipine was stable in the investigated conditions. The method was applied to the pharmacokinetics of efonidipine in human subject.

药动学参数的个体内变异系数≥30％的药物制剂为高变异药品,高变异药品的生物等效性研究困扰着众多药学工作者.本研究参考FDA和EMA的相关指导原则和国外相关文献,结合我们已经完成的高变异药品的生物等效性试验,探讨高变异药品及其参比制剂校正的平均生物等效性试验的方法.结果表明采用参比制剂校正的、半重复或全重复、交叉试验设计进行平均生物等效性试验,可避免纳入过多受试者参加试验,降低试验成本,提高临床试验的可操作性.

生物等效性试验是药品研发和评价的一个重要内容.生物等效性试验首选药动学研究的方法,药动学研究则必然涉及到生物分析,因此生物分析对于生物等效性试验具有举足轻重的作用,其结果的可靠性将直接影响生物等效性试验的质量.但我国目前却没有完整、独立的生物分析方面的指导原则.基于上述原因,我们根据国内外的相关指导原则,并尽可能结合我们的实际工作,探讨生物等效性试验中生物分析需注意的关键问题.

The gender differences in pharmacokinetics of a combination tablet of niacin extended-release/simvastatin were evaluated in healthy Chinese volunteers. Thirty-six healthy male and female volunteers were enrolled in the study receiving a single oral dose of niacin extended-release/simvastatin 1,000/20 mg. The results indicated that the systemic exposure of simvastatin hydroxy acid and the total urine excretion of niacin were significantly higher for females compared with those for males, and the T max of niacin in plasma was significantly shorter for males than that for females. There were no significant differences in the systemic exposure of simvastatin, niacin, and NUA in plasma between males and females.

目的 建立测定人尿液中替米沙坦的LC-MS/MS方法(液相色谱-串联质谱法),并应用于中国健康受试者口服替米沙坦后的尿液样本分析,以探究中国人群中替米沙坦的尿液排泄情况.方法 尿液样本分别未经酶解及酶解处理后,采用液液萃取法提取后进样测定,未经酶解处理所得为尿液中替米沙坦原形,经酶解处理所得为尿液中替米沙坦总量,而两者之差即为以葡萄糖醛酸结合物形式存在的替米沙坦.结果 替米沙坦尿药浓度在1.00～500.00 ng/mL范围内线性关系良好,定量下限为1.00 ng/mL;相对误差(RE)在±15％以内,批内相对标准偏差(RSD)、批间RSD均小于15％.中国健康受试者尿液中以替米沙坦葡萄糖醛酸结合物形式存在的替米沙坦占尿液总排泄量的98％左右,替米沙坦经由尿液排泄的数量不到给药剂量的1％.结论 实验建立的LC-MS/MS方法专属准确、简单灵敏、分析速度快,可满足尿液未经酶解及酶解后替米沙坦测定的要求.中国人群的替米沙坦尿液排泄情况与西方人群并无差异.

The detection of drug metabolites, especially for minor metabolites, continues to be a challenge because of the complexity of biological samples. Imperatorin (IMP) is an active natural furocoumarin component originating from many traditional Chinese herbal medicines and is expected to be pursued as a new vasorelaxant agent. In the present study, a generic and efficient approach was developed for the in vivo screening and identification of IMP metabolites using liquid chromatography-Triple TOF mass spectrometry. In this approach, a novel on-line data acquisition method mutiple mass defect filter (MMDF) combined with dynamic background subtraction was developed to trace all probable urinary metabolites of IMP. Comparing with the traditionally intensity-dependent data acquisition method, MMDF method could give the information of low-level metabolites masked by background noise and endogenous components. Thus, the minor metabolites in complex biological matrices could be detected. Then, the sensitive and specific multiple data-mining techniques extracted ion chromatography, mass defect filter, product ion filter, and neutral loss filter were used for the discovery of IMP metabolites. Based on the proposed strategy, 44 phase I and 7 phase II metabolites were identified in rat urine after oral administration of IMP. The results indicated that oxidization was the main metabolic pathway and that different oxidized substituent positions had a significant influence on the fragmentation of the metabolites. Two types of characteristic ions at m/z 203 and 219 can be observed in the MS/MS spectra. This is the first study of IMP metabolism in vivo. The interpretation of the MS/MS spectra of these metabolites and the proposed metabolite pathway provide essential data for further pharmacological studies of other linear-type furocoumarins.

A sensitive and selective LC-MS/MS method for the determination of agomelatine in human plasma was developed and validated. After simple liquid-liquid extraction, the analytes were separated on a Zorbax SB-C18 column (150 × 2.1 mm i.d., 5 µm) with an isocratic mobile phase consisting of 5 mm ammonium acetate solution (containing 0.1% formic acid) and methanol (30:70, v/v) at a flow-rate of 0.3 mL/min. The MS acquisition was performed in multiple reaction monitoring mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.3 and m/z 285.2 → 193.2 for agomelatine and internal standard, respectively. The methods were validated for selectivity, carry-over, matrix effects, calibration curves, accuracy and precision, extraction recoveries, dilution integrity and stability. The validated method was successfully applied to a pharmacokinetic study of agomelatine in Chinese volunteers following a single oral dose of 25 mg agomelatine tablet.

Objective:To evaluate the effect of glucose on the bioavailability of metformin hydrochloride tablets.Methods:Twenty healthy male subjects were given a single oral dose of 500 mg metformin hydrochloride tablet in fasting condition with warm boiled water or 20% glucose aqueous solution.Venous blood glucose levels from pre-dosing to 4 h post-dosing were monitored by hexokinase method using automatic biochemical analyzer.Plasma concentrations of metformin up to 24 h post-dosing were determined by LC-MS/MS.The pharmacokinetic parameters were calculated by DAS software.The relative bioavailability(F) of metformin hydrochloride tablet given with 20% glucose solution compared with that administered with warm boiled water was calculated,and bioequivalence was investigated to evaluate the effect of glucose on bioavailability of metformin hydrochloride tablets.Results:Venous blood glucose level of healthy subjects significantly decreased at 1.5~4 h post-dosing of metformin hydrochloride tablets in fasting condition with warm boiled water,while significantly increased after being administered with 20% glucose solution.The pharmacokinetic parameters of metformin hydrochloride tablets administered with warm boiled water and 20% glucose solution were as follows:Tmax(2.20±0.94)and(2.08±1.02) h,Cmax(1.00±0.34)and(0.960±0.339) ng·mL-1,t1/2(3.25±0.62)and(3.18±0.74) h,AUC0~t(5.72±1.50)and(5.58±1.35) ng·h·mL-1,and AUC0~∞(5.77±1.51)and(5.64±1.34) ng·h·mL-1,respectively.Compared with being administered with warm boiled water,the F0~t and F0~∞ of metformin hydrochloride tablets administered with 20% glucose solution were(100.2±22.3)% and(100.4±22.1)%,respectively,which indicated that the two administration ways were bioequivalent.Conclusion:The bioavailability of metformin hydrochloride tablets is not affected by glucose.In order to prevent hypoglycemic reaction,it is suggested that oral antidiabetic drugs including metformin hydrochloride be administered with glucose solution in bioequivalence studies,and glucose solution be given to subjects within a certain period after dosing.

The objective of the present study was to develop a practical strategy for the identification of metabolites following the in vivo metabolism and in vitro microbial biotransformation of isoimperatorin using liquid chromatography hybrid triple quadrupole-linear ion trap mass spectrometry (LC/QTRAP-MS) and liquid chromatography time of flight mass spectrometry (LC/TOF-MS). As a result, 19 metabolites were characterised in rat urine, plasma, bile and faeces after the oral administration of isoimperatorin and 13 products were identified in the sample from the transformation. Four metabolites were prepared by in vitro microbial biotransformation, and one was confirmed to be a novel compound. The side chain of isoimperatorin was found to be the primary metabolic site that underwent oxidation metabolism both in vivo and in vitro, and the metabolism of isoimperatorin in vivo and in vitro has good correlation. This is the first study of the metabolism of isoimperatorin in vivo.