HPLC/MS/MS法测定人血浆中内源性尿嘧啶及二氢尿嘧啶

目的　为评价人体内二氢嘧啶脱氢酶的活性,建立高效液相色谱 串联质谱联用的分析方法,测定该酶的底物以及催化产物在血浆中的基础浓度。方法　血浆样品液 液萃取后,以纯水为流动相,以DiscoveryAmideC₁₆ 色谱柱初步分离,经气动辅助电喷雾离子源负离子化,在三级四极杆质量分析器上以多反应离子监测(MRM)方式检测尿嘧啶(MRM m/z 111.0→41.9)和二氢尿嘧啶(MRM m/z 113.0→42.0 )。结果　尿嘧啶和二氢尿嘧啶的最低定量浓度分别为0.5ng·mL^-1 和5ng·mL^-1 ;线性范围分别为0.5 - 100ng·mL^-1 和5 - 1000ng·mL^-1 ,精密度和准确度符合生物样品分析要求。结论　此法专属、灵敏、准确,适用于测定生物样本中内源性尿嘧啶和二氢尿嘧啶的基础浓度。     

液相色谱-串联质谱法同时测定人血浆中二甲双胍和格列吡嗪

建立了快速、灵敏的液相色谱-串联质谱法测定人血浆中的二甲双胍和格列吡嗪。血浆样品经0.3%甲酸-乙腈(v/v)沉淀蛋白后，以乙腈-水-甲酸(70∶30∶0.3，v/v/v)为流动相，流速为0.50 mL·min^-1。Zorbax Extend C₁₈柱分离，采用大气压化学电离源；以选择反应监测(SRM)方式进行正离子检测。用于定量分析的离子反应分别为m/z 130→m/z 60(二甲双胍)，m/z 446→m/z 321(格列吡嗪)和m/z 256→m/z 167(内标，苯海拉明)。测定血浆中二甲双胍的线性范围为2.00～2 000 ng·mL^-1， 定量下限为2.00 ng·mL^-1； 格列吡嗪的线性范围为1.00～1 000 ng·mL^-1， 定量下限为1.00 ng·mL^-1。该方法专属性好，灵敏度高，准确快捷，适用于二甲双胍和格列吡嗪的临床药代动力学研究。     

灯盏花素及其β-环糊精包合物在大鼠体内的药代动力学

目的建立测定大鼠血浆中灯盏乙素浓度的反相高效液相色谱法，研究灯盏花素及其β-环糊精包合物(灯盏花素-β-CD)大鼠灌胃后体内药代动力学行为。方法以甲醇-水-醋酸盐缓冲液为流动相，Shim-pack C₁₈为固定相；12只大鼠随机均分为2组，分别灌胃灯盏花素及其包合物后，检测血浆药物浓度。药时数据采用3P97药代计算程序处理。结果线性范围10-400 ng·mL^-1，方法回收率95.32%-98.81%；灯盏花素和包合物的C_max分别为(154±18) ng·mL^-1和(328±31) ng·mL^-1；AUC_0-12h分别为(710±126) ng·h·mL^-1和(1 093±200)ng·h·mL^-1，经t检验两者有极显著性差异(P<0.01)。结论该法准确、灵敏，适用于灯盏乙素血浆浓度的测定；制备的灯盏花素包合物与灯盏花素相比吸收显著增加。     

高灵敏度LC-MS/MS法测定犬血浆中的坦洛新

犬口服盐酸坦洛新控释片后血浆药物浓度C_max小于10 ng·mL^-1，需建立测定犬血浆中坦洛新的高灵敏度液相色谱-串联质谱法(LC-MS/MS)。血浆样品加入内标苯海拉明，用正己烷-二氯甲烷(2∶1)萃取后，反相C₁₈色谱柱分离，以甲醇-乙腈-甲酸铵(30∶40∶30，v/v/v)为流动相，流速为0.4 mL·min^-1。选用大气压化学离子化源(APCI)三重四极杆串联质谱仪，以选择反应监测方式进行检测，用于定量分析的离子反应分别为m/z 409→228(坦洛新)和m/z 256→167(苯海拉明)。坦洛新线性范围为0.02～50 ng·mL^-1，定量下限为0.02 ng·mL^-1。批内、批间精密度(RSD)均小于9.72%，准确度(RE)在-2.61%～8.82%。本方法灵敏度高，专属性强，用于犬口服盐酸坦洛新控释片后的药代动力学研究。     

HPLC-MS/MS法测定人体色甘酸钠血浆浓度及其药代动力学研究

采用HPLC-MS/MS法测定人血浆中色甘酸钠浓度，进行其滴鼻液和鼻用喷雾剂的药代动力学研究并评价其生物等效性。采用高效液相分离系统，流动相为乙酸铵-甲醇(含50%乙腈)(15∶85)，固定相为AGT Venusil XBP C₁₈(250 mm×4.6 mm ID， 5 μm)色谱柱。采用质谱检测系统， ESI离子源， 正离子模式， 多级反应监测(MRM)方式， m/z 469→263.1(色甘酸钠)， m/z 447.2→327.1(内标，普伐他汀钠)。在0.3～20 ng·mL^-1色甘酸钠血药浓度呈线性关系， 定量限为0.3 ng·mL^-1， 回收率在94.1%以上， 日内日间的RSD均小于14.3%。单剂量给药色甘酸钠鼻用喷雾剂或滴鼻液， 其药代动力学参数T_1/2分别为(1.82±0.54)和(1.59±0.52) h； T_max分别为(0.47±0.12)和(0.44±0.15) h； C_max分别为(9.79±4.66)和(10.88±4.05) ng·mL^-1， AUC_{0-5 h}分别为(11.52±3.46)和(12.63±4.23) ng·mL^-1·h。色甘酸钠鼻用喷雾剂相对生物利用度F_r为(93.6±13.8)%。本法灵敏度高， 适用于色甘酸钠治疗药物监测及其药代动力学和生物利用度研究。     

溴吡斯的明血药浓度的HPLC-MS/MS测定及其在人体的药动学研究

目的 建立HPLC-MS/MS测定人血浆中溴吡斯的明的药物浓度,并研究溴吡斯的明片在健康人体内的药动学。方法 采用HPLC-MS/MS检测法,用乙腈沉淀蛋白法处理血浆样本。采用Shimadzu Shim-Pack VP-ODS色谱柱(150 mm×2.0 mm,5 μm);流动相为甲醇-水(含5 mmol·L^-1醋酸铵和0.1%甲酸),梯度洗脱;流速：0.2 mL·min^-1;采用三重四级杆质谱仪,电喷雾离子源,正离子模式,选择性离子检测,溴吡斯的明和内标新斯的明的检测离子分别为m/z 181.1→72.2,m/z 223.1→208.1。20名健康受试者口服溴吡斯的明片60 mg后,在不同时间点采集血浆样品,采用建立的HPLC-MS/MS条件测定溴吡斯的明血药浓度,计算药动学参数。结果 溴吡斯的明血药浓度在0.5~100 ng·min^-1内样品峰面积比与浓度线性关系良好(r=0.996 9),绝对回收率,日内、日间精密度,冷冻、冻融、室温稳定性均符合生物样品分析要求。20名健康受试者口服溴吡斯的明片60 mg 后的主要药动学参数如下：C_max为(43.26±10.33)ng·mL^-1;T_max为(1.77±0.48)h;t_1/2为(5.11±0.96)h;AUC_0-t为(204.8±64.93)ng·h·mL^-1;AUC_0-∞为(212.42±68.31)ng·h·mL^-1。结论 该方法简单、准确、灵敏,适用于溴吡斯的明血药浓度测定和药动学研究。     

UPLC-MS/MS测定大鼠血浆中香附烯酮和α-香附酮浓度及其药动学研究

目的 建立UPLC-MS/MS测定大鼠血浆中香附烯酮和α-香附酮的方法，并研究其药动学。方法 采用Phenomennex C₁₈(150 mm×2.0 mm，3 μm)色谱柱，以乙腈-水为流动相进行梯度洗脱，柱温30 ℃，进样量1 μL，蛇床子素为内标，采用电喷雾离子源，正离子模式，香附烯酮m/z为219.1/135.1，α-香附酮m/z为219.1/111.0，蛇床子素m/z为245.0/123.0。检测大鼠血浆中香附烯酮、α-香附酮的浓度，应用DAS 2.0软件拟合主要的药动学参数。结果 香附烯酮在10~500 ng·mL^-1内线性关系良好(r=0.991 0)，α-香附酮在2.5~300 ng·mL^-1内线性关系良好(r=0.994 1)，日内精密度RSD＜9.45%，日间精密度RSD＜9.09%，加样回收率＞86.79%。SD大鼠灌胃给予香附挥发油提取物(20 mg·kg^-1)后，香附烯酮和α-香附酮C_max、AUC_0-∞、MRT_(0-∞)分别为(8 862.59±1 106.81)ng·L^-1，(7 060.94±774.25)ng·L^-1·h，(3.21±0.72)h和(934.69±106.81)ng·L^-1，(792.26±74.52)ng·L^-1·h，(4.94±0.82)h。结论 建立的方法能够快速、准确测定血浆中香附烯酮和α-香附酮的浓度，可用于香附烯酮和α-香附酮在大鼠体内的药动学研究。     

HPLC/MS法研究左旋黄皮酰胺及其代谢物在Beagle犬血浆中的药代动力学HPLC/MS法研究左旋黄皮酰胺及其代谢物在Beagle犬血浆中的药代动力学

目的用HPLC/MS法研究左旋黄皮酰胺［(-)-clau］及其代谢物6-羟基-黄皮酰胺(6-OH-clau)在Beagle犬血浆中的药代动力学过程。方法Beagle犬灌胃左旋黄皮酰胺30 mg·kg^-1，采集静脉血样，血浆经乙酸乙酯萃取分离后，用HPLC/MS选择性正离子检测内标(格列吡嗪，［M+H］⁺，m/z 446)法测定左旋黄皮酰胺(［M+H］⁺，m/z 298)及6-羟基-黄皮酰胺(［M+H-H₂O］⁺，m/z 296)的浓度，以甲醇-水-冰醋酸(60∶40∶0.8)为流动相，流速1.0 mL·min^-1。用3P97软件计算药代动力学参数。结果左旋黄皮酰胺和6-羟基-黄皮酰胺分别在1.0～200 ng·mL^-1和0.2～40.0 ng·mL^-1线性关系良好(r>0.999)，萃取回收率均大于85%。原药及其代谢物的体内过程均符合二室模型；左旋黄皮酰胺及6-羟基-黄皮酰胺的C_max分别为(21±10) ng·mL^-1和(3.9±2.2) ng·mL^-1；T_max分别为(0.8±0.5) h和(1.3±0.5) h；T_1/2α分别为(0.9±0.6) h和(1.4±0.6) h；T_1/2β分别为(19±23) h和(13±12) h；AUC_{0-24 h}分别为(69±14) h·ng·mL^-1和(12±7) h·ng·mL^-1。结论Beagle犬灌胃左旋黄皮酰胺后迅速吸收，血药浓度一相消除很快，但末端消除较慢；其代谢物6-羟基-黄皮酰胺血药浓度经时过程与左旋黄皮酰胺相似，但血药浓度相对较小。     

UPLC-MS/MS测定血浆中伊伐布雷定及其活性代谢产物的含量

目的 建立超高效液相色谱-串联质谱法测定人血浆中伊伐布雷定及其活性代谢产物(N-去甲伊伐布雷定)的含量。方法 选用Waters Acquity BEH C₁₈(50 mm×2.1 mm,1.7 μm)色谱柱,流动相为0.1%甲酸水溶液(A)-乙腈(B),梯度洗脱;流速为0.4 mL·min^-1;电喷雾离子源,多反应监测。伊伐布雷定：[M+H]⁺,m/z 469.3→177.2,N-去甲伊伐布雷定：[M+H]⁺,m/z 455.2→262.2,卡马西平：[M+H]⁺,m/z 237.1→194.2。结果 伊伐布雷定线性范围为0.2~100 ng·mL^-1(r=0.998 1),N-去甲伊伐布雷定线性范围为0.05~25 ng·mL^-1(r=0.993 1);两者日间、日内精密度均<15%,方法回收率>90%,稳定性较好。结论 该方法快速、灵敏、重复性好,适用于血浆中伊伐布雷定及其代谢产物含量测定。     

液相色谱-电喷雾串联质谱法测定人血浆中班布特罗:在药代动力学研究中的应用

目的　建立液相色谱 串联质谱法测定人血浆中班布特罗浓度,研究中国受试者口服该药的动力学特点。方法　血浆样品经液 液萃取后,采用液相色谱电喷雾串联质谱法以选择离子反应监测(SRM)方式进行检测。结果班布特罗的线性范围为0.05 - 4.0ng·mL^-1 ,最低定量浓度为0.05ng·mL^-1 ,该法的日内及日间精密度(RSD)小于8% ,准确度(RE)在±9%范围内。18名中国健康受试者单剂量口服班布特罗10 mg后,主要药动学参数T_max,C_max, T_1/2和AUC_0-t分别为(2.3±1.3)h ,(3.95±2.20 )ng·mL^-1 ,(11.4±6.1)h和(26.85±11.77)ng·h·mL^-1 。结论　该法灵敏度高,操作简便、快速,适用于临床药代动力学研究     

小型猪血浆中二丙酸倍他米松及其代谢物倍他米松的LC-MS/MS同时测定方法的建立及在药动学研究中的应用

目的：建立同时测定小型猪血浆中二丙酸倍他米松及其代谢物倍他米松的LC-MS/MS法,并研究小型猪皮肤外用二丙酸倍他米松乳膏后,二丙酸倍他米松及倍他米松的药动学特征。方法：血浆样品经酸化后以乙醚-环己烷（4∶1）提取,LC-MS/MS分析,以Hedera ODS-2（150 mm×2.1 mm,5μm）为分析柱,流动相为5 mmol·L-1醋酸铵水溶液（含0.1%乙酸）–甲醇,梯度洗脱。二丙酸倍他米松、倍他米松及内标布地奈德的监测离子对分别为m/z 563.2（[M＋CH3COO]-）→483.1、m/z 451.2（[M＋CH3COO]-）→361.0和m/z 489.3（[M＋CH3COO]-）→357.1。对小型猪外用二丙酸倍他米松乳膏后其血浆中二丙酸倍他米松及代谢物倍他米松的浓度进行测定,并计算主要药动学参数。结果：二丙酸倍他米松血药浓度在26.85~644.4 ng·L-1范围内线性关系良好,倍他米松血药浓度在10.62~637.2 ng·L-1范围内线性关系良好。结论：本方法专属性强、简便、灵敏,可用于血浆样本中二丙酸倍他米松及其代谢物倍他米松的测定及药动学研究。     

Pharmacokinetics of gastrodin and its metabolite p-hydroxybenzyl alcohol in rat blood, brain and bile by microdialysis coupled to LC-MS/MS

Gastrodin is a pharmacologically active substance isolated from Gastrodia elata Blume with sedation, anti-convulsion and anti-epilepsy activities. A rapid and sensitive liquid chromatography technique coupled to tandem mass spectrometry (LC-MS/MS) system was developed to determine gastrodin and its metabolite p-hydroxybenzyl alcohol (HBA) in rat blood, brain and bile collected using microdialysis technique. The analytes were separated using a reversed phase column (4.6 mm x 150 mm, 5 microm). The mobile phase for column separation was 30% methanol with a flow rate of 0.6 mL/min. As a post-column addition, 1% ammonium hydroxide solution (in methanol) was additionally pumped via a T-connection using a chromatographic pump (BAS PM-80, USA) at a flow rate of 0.2 mL/min after the column separation. A LC-MS/MS system equipped with a negative electrospray ionization (ESI) source in multiple reaction monitoring (MRM) mode was used to monitor m/z 285.0-->122.9 and m/z 123.0-->105.0 transitions for gastrodin and HBA, respectively. The lower limit of quantification (LLoQ) for gastrodin and HBA were 0.5 and 2 ng/mL, respectively. The calibration curves were linear over the range of 0.5-5,000 ng/mL and 2-1,000 ng/mL for gastrodin and HBA with a coefficient of determination >0.995, respectively. This selective and sensitive method is useful for the determination of gastrodin and HBA and in the pharmacokinetic studies of these compounds.     

LC-MS/MS法测定人血浆中加巴喷丁的浓度及其药动学研究

建立液相色谱串联质谱(LC-MS/MS)法测定人血浆中加巴喷丁的浓度并将其应用于人体药动学研究。取血浆样品经甲醇沉淀蛋白后,以甲醇0.2%甲酸水溶液(80∶20)为流动相,用Inertsil ODS-3 C18柱(50 mm×2.1 mm ID,3μm)分离,采用电喷雾离子源,以多反应监测(MRM)方式进行正离子检测,定量分析的离子反应分别为m/z 172→m/z 154(加巴喷丁)和m/z 130→m/z 71(内标二甲双胍)。加巴喷丁线性范围为40.8～8.16×103 ng.mL 1,定量限为40.8 ng.mL 1,每个样品测试时间仅2.2 min,日内、日间精密度(RSD)均小于12%,准确度(RE)在±6.4%范围内。应用此法研究了20名健康志愿者单剂量口服加巴喷丁胶囊600 mg后的药动学特点。该方法快速、专属、灵敏、适用性强,可应用于加巴喷丁的人体药动学研究。     

Simultaneous determination of peimine and peiminine in rat plasma by LC-MS/MS and its application in the pharmacokinetic study

To establish an LC-MS/MS method for simultaneous determination of peimine and peiminine in rat plasma after oral and intravenous administration of Fritillaria thunbergii Miq. extract, the pharmacokinetic parameters were calculated as well. Peimine, peiminine and internal standard carbamazepine were extracted from plasma with liquid-liquid extraction by ethyl acetate, then separated on a Luna C18 column by using acetonitrile-water containing 10 mmol x L(-1) ammonium formate (35:65), as mobile phase. The electrospray ionization (ESI) source was applied and operated in positive ion mode. Peimine was detected at m/z 432.4 --> 414.4, peiminine at m/z 430.4 --> 412.4 and carbamazepine (IS) at 237.1 --> 194.2. The linear calibration curves were obtained at the concentration range of 0.8-800 ng x mL(-1) for peimine and peiminine. The extraction recoveries were 94.1%-105.3% and 85.8%-98.6%, respectively. The precisions, accuracy and stability of the analytes meet the requirements. The method was shown to be effective, convenient, and suitable for simultaneous pharmacokinetic study of peimine and peiminine in rat.     

LC-MS determination and bioavailability study of imidapril hydrochloride after the oral administration of imidapril tablets in human volunteers

The purpose of the present study was to develop a standard protocol for imidapril hydrochloride bioequivalence testing. For this reason, a specific LC-MS method was developed and validated for the determination of imidapril in human plasma. A solid-phase extraction cartridge, Sep-pak C18, was used to extract imidapril and ramipril (an internal standard) from deproteinized plasma. The compounds were separated using a XTerra MS C18 column (3.5 microm, 2.1 x 150 mm) and acetonitrile-0.1% formic acid (67:33, v/v) adjusted to pH 2.4 by 2 mmol/L ammonium formic acid, as mobile phase at 0.3 mL/min. Imidapril was detected as m/z 406 at a retention time of ca. 2.3 min, and ramipril as m/z 417 at ca. 3.6 min. The described method showed acceptable specificity, linearity from 0.5 to 100 ng/mL, precision (expressed as a relative standard deviation of less than 15%), accuracy, and stability. The plasma concentration-versus-time curves of eight healthy male volunteers administered a single dose of imidapril (10 mg), gave an AUC12hr of imidapril of 121.48 +/- 35.81 ng mL(-1) h, and Cmax and Tmax values of 32.59 +/- 9.76 ng/mL and 1.75 +/- 0.27 h. The developed method should be useful for the determination of imidapril in plasma with sufficient sensitivity and specificity in bioequivalence study.     

Rapid and simultaneous measurement of midazolam, 1'-hydroxymidazolam and digoxin by liquid chromatography/tandem mass spectrometry: application to an in vivo study to simultaneously measure P-glycoprotein and cytochrome P450 3A activity

In order to simultaneously determine in vivo P-glycoprotein (P-gp) and Cytochrome P450 3A (CYP3A) activity, a new, rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to simultaneously determine midazolam (MDZ, as CYP3A substrate), 1'-hydroxymidazolam (1'-OHMDZ) and digoxin (DG, as P-gp substrate) in rat plasma using digitoxin as the internal standard (IS). After a single step liquid-liquid extraction with tert-butyl methyl ether/dichloromethane (75:25, v/v), analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). Chromatographic separation was performed on an XTerra MS C18 column (50mm×2.1mm, i.d. 3.5μm). The MS/MS detection was conducted by monitoring the fragmentation of 326.05 → 244.00 (m/z) for MDZ, 342.02 →168.01 (m/z) for 1'-OHMDZ, 798.33 → 651.36(m/z) for DG and 782.67 → 635.24 (m/z) for IS. The method had a chromatographic running time of 3min and linear calibration curves over the concentrations of 2-400ng/mL for MDZ and 1'-OHMDZ and 0.5-100ng/mL for DG. The recoveries of the method were 86.8-96.3% for MDZ, 84.6-86.4% for 1'-OH MDZ, and 81.7-85.1% for DG. The lower limit of quantification (LLOQ) of the method was 2ng/mL for MDZ and 1'-OHMDZ and 0.5ng/mL for DG. The intra- and inter-batch precision were less than 15% for all quality control samples at concentrations of 5, 50 and 320ng/mL for MDZ and 1'-OHMDZ and 1, 10 and 80ng/mL for DG. The validated LC-MS/MS method has been successfully used to analyze the concentrations of MDZ, 1'-OH MDZ and DG in rat plasma for simultaneous measurement of in vivo P-gp and CYP 3A activity.     

Simultaneous determination of amoxicillin and ambroxol in human plasma by LC-MS/MS: validation and application to pharmacokinetic study

A rapid, simple and sensitive LC-MS/MS method was developed for simultaneous determination of amoxicillin and ambroxol in human plasma using clenbuterol as internal standard (IS). The plasma samples were subjected to a simple protein precipitation with methanol. Separation was achieved on a Lichrospher C(18) column (150 mm x 4.6mm ID, dp 5 microm) using methanol (containing 0.2% of formic acid) and water (containing 0.2% of formic acid) as a mobile phase by gradient elution at a flow rate of 1.0 mL/min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 365.9-->348.9 (amoxicillin), m/z 378.9-->263.6 (ambroxol) and m/z 277.0-->203.0 (IS). Calibration curves were linear in the concentration range of 5-20,000 ng/mL for amoxicillin, and 1-200 ng/mL for ambroxol, with the intra- and inter-run precisions of <9% and the accuracies of 100+/-7%. The method has been validated and applied to pharmacokinetic studies of compound amoxicillin and ambroxol hydrochloride tablets in healthy Chinese volunteers.     

高效液相色谱-质谱联用测定家兔体内可乐定血药浓度

目的用HPLC-MS测定家兔体内可乐定血药浓度,研究可乐定在家兔体内的药代动力学行为。方法 色谱仪为Waters Alliance 2790,色谱柱为XTerra C18(150 mm×2.1 mm ID,5 μm)。流动相为乙腈-碳酸氢铵水溶液,梯度洗脱;质谱仪为Micromass ZQ-4000仪,电喷雾电离源,选择离子监测(SIM)质荷比(m/z)为230。结果本方法回收率较高,重现性好。线性范围为1～80 μg·L^-1,最低检测浓度为0.05 μg·L^-1。主要药代动力学参数Cmax,AUC0-t和tmax分别为(27±9) μg·L^-1,(5 352±1 121) μg·L^-1,(79±17) h。结论本法灵敏度高,选择性强,具有较高的精密度和准确度。透皮贴剂中的可乐定可以平稳地透过家兔皮肤,稳定地控释达7 d。