Liquid chromatography/tandem mass spectrometry for the determination of carbamazepine and its main metabolite in rat plasma utilizing an automated blood sampling system

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of carbamazepine and its main metabolite carbamazepine 10,11-epoxide in rat plasma is described. The method consists of a liquid-liquid extraction procedure and electrospray LC/MS/MS analysis. The chromatographic separation was achieved within 5 min using a C(8) (150 mm x 2.1mm) 5 microm column with a mobile phase composed of water/acetonitrile/acetic acid (69.5:30:0.5, v/v/v) at a flow rate of 0.4 ml/min. D(10)-carbamazepine is used as the internal standard for all compounds. Analytes were determined by electrospray ionization tandem mass spectrometry in the positive ion mode using selected reaction monitoring (SRM). Carbamazepine was monitored by scanning m/z 237-->194, carbamazepine 10,11-epoxide by m/z 253-->210 and d(10)-carbamazepine by m/z 247-->204. The lower limit of quantitation (LLOQ) is 5 ng/ml for each analyte, based on 0.1 ml aliquots of rat plasma. The extraction recovery of analytes from rat plasma was over 87%. Intra-day and inter-day assay coefficients of variations were in the range of 2.6-9.5 and 4.0-9.6%, respectively. Linearity is observed over the range of 5-2000 ng/ml. This method was used for pharmacokinetic studies of carbamazepine and carbamazepine 10,11-epoxide in response to two different blood sampling techniques (i.e., manual sampling versus automated sampling) in the rat. Several differences between the two sampling techniques suggest that the method of blood collection needs to be considered in the evaluation of pharmacokinetic data.     

Comparative pharmacokinetic studies of peimine and peiminine in rat plasma by LC-MS-MS after oral administration of Fritillaria thunbergii Miq. and Fritillaria thunbergii Miq. - Glycyrrhiza uralensis Fisch. couple extract

A sensitive LC-MS-MS method has been successfully applied to pharmacokinetic study of peimine and peiminine in rat plasma after oral administration of Fritillaria thunbergii Miq. exact and Fritillaria thunbergii Miq. - Glycyrrhiza uralensis Fisch. couple extract. The results indicated that plasma profiles of peimine and peiminine confirmed to two-compartment open model with weighting function of 1/C2 for data fitting and parameter estimation and the utilization with Glycyrrhiza uralensis Fisch. could decrease C(max) and prolong MRT(0-infinity) and t1/2 of peimine remarkly with the bioavailability of peimine remained practically unchanged. Meanwhile, the concentration of peimine in rat plasma was more stable. Nevertheless, there were no significant differences among all calculated parameters of peiminine.     

HPLC-ELSD测定黄氏响声丸中贝母素甲和贝母素乙的含量

目的：建立黄氏响声丸中贝母素甲和贝母素乙的含量测定方法。方法：采用HPLC法,色谱柱：Agilent ZORBAX SB—C18柱（4．6mm×250mm,5μm）,流动相为乙腈-水-二乙胺（65：35：0．03）,流速为1．0mL·min^-1,柱温为30℃;SOFTA-300SELSD型蒸发光散射检测器（漂移管温度85℃,雾化室温度50℃）。结果：贝母素甲进样量在0．5056—10．1120μg范围内,贝母素乙进样量在0．4106～8．2120μg范围内,其进样量的自然对数与峰面积积分值的自然对数有良好的线性关系（r〉0．9990）,贝母素甲和贝母素乙平均加样回收率分别为97．69％和97．39％,RSD分别为1．61％和1．37％。结论：该方法稳定、重复性好,结果准确,可作为黄氏响声丸中贝母素甲和贝母素乙的含量测定方法。     

Sensitive bioassay for the simultaneous determination of pseudoephedrine and cetirizine in human plasma by liquid-chromatography-ion trap spectrometry

A liquid chromatography-ion trap mass spectrometry coupled with electrospray ionization (HPLC-ESI-ion trap mass spectrometry) method for simultaneous determination of cetirizine and pseudoephedrine in human plasma is presented. Chromatographic separation was performed on a Hypurity C18 column (Thermo Hypersil-Keystone 2.1 mm x 150 mm, 5 microm, USA), The mobile phase was composed of 65% methanol and 35% water (contained 0.1% formic acid, 10 mM ammonium formate), which was run with a flow-rate of 0.2 ml/min at 40 degrees C. Quantitation was achieved by monitoring the product ions at m/z 166-->m/z 148 (pseudoephedrine), m/z 389.9-->m/z 201.1 (cetirizine), m/z 264-->m/z 246 (tramadol, IS). The calibration curve of pseudoephedrine and cetirizine was established with standard solutions. The limit of detection for pseudoephedrine and cetirizine each was 5 ng/ml. This simplified analytical method is sensitive, specific and accurate enough for simultaneous determination of pseudoephedrine and cetirizine in human plasma and is successfully applied to the pharmacokinetic study of pseudoephedrine and cetirizine.     

液相色谱-串联质谱法测定犬血浆中布地奈德

建立液相色谱-串联质谱法测定犬血浆中布地奈德。血浆样品碱化后，经乙酸乙酯液-液萃取，以乙腈-5 mmol·L^-1醋酸铵(60∶40，v/v)为流动相，Capcell Pak C₁₈ MG柱分离；采用电喷雾电离源，以多反应监测(MRM)方式进行负离子检测，用于定量分析的离子反应分别为m/z 489→m/z 357(布地奈德)和m/z 493→m/z 413(内标，曲安奈德)。测定血浆中布地奈德方法的线性范围为25.0～2 000 pg·mL^-1，定量下限为25.0 pg·mL^-1，日内、日间精密度(RSD)均小于15%，准确度(RE)在-8.1%～-1.7%。应用本法研究6只比格犬单次和多次给予布地奈德缓释胶囊9 mg后的药代动力学结果显示：单次给药后T_max为(3.5±3.3) h，C_max为(786±498) pg·mL^-1；多次给药后C_max为(2 142±1 515) pg·mL^-1。该法选择性强、灵敏度高、操作简便，适用于布地奈德缓释制剂的药代动力学研究。     

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/质谱联用法测定人血浆中莫沙必利

建立测定人血浆中莫沙必利的高效液相色谱-质谱/质谱联用法。取血浆样品经液-液萃取后，以乙腈为有机相，0.3%甲酸水溶液为水相，采用梯度洗脱的方式，用C₁₈柱分离，通过电喷雾离子化，以多反应监测(MRM)方式进行正离子检测。莫沙必利线性范围为0.17~68.00 ng·mL^-1，定量下限为0.17 ng·mL^-1，每个样品测试时间仅2.8 min，日内、日间精密度(RSD)均小于13%，准确度(RE)在±6.3%范围内。应用此法研究了20名志愿者单剂量口服枸橼酸莫沙必利片后的药代动力学特点。该方法、灵敏、准确、快速，适用于莫沙必利的药代动力学及生物等效性研究。     

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。     

Liquid chromatographic-tandem mass spectrometric method for the simultaneous quantitation of telmisartan and hydrochlorothiazide in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the simultaneous determination of telmisartan and hydrochlorothiazide in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether-dichloromethane (60:40, v/v). The analytes and internal standard, probenecid, were separated on a Venusil XBP-C(8) column using gradient elution with acetonitrile-10 mM ammonium acetate-formic acid at a flow rate of 1.2 mL/min. Detection was by electrospray negative ionization mass spectrometry using multiple reaction monitoring of the transitions at m/z 513.0-->469.4 for telmisartan, m/z 295.9-->268.9 for hydrochlorothiazide and m/z 283.9-->239.9 for probenecid. For both analytes, the method was linear in the range 1.00-600 ng/mL with intra- and inter-day precision (as relative standard deviation) 相似文献   

Determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in monkey and dog plasma by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

A specific and simultaneous assay of morphine, morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) in monkey and dog plasma has been developed. These methods are based on rapid isolation using solid phase extraction cartridge, and high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometric (MSMS) detection. Analytes were separated on a semi-micro ODS column in acetonitrile-formic (or acetic) acid mixed solution. The selected reaction monitoring for assay in monkey and dog plasma, as precursor-->product ion combinations of m/z 286-->286 for morphine, m/z 462-->286 for glucuronides and m/z 312-->312 for internal standard (IS, nalorphine) were used. The linearity of morphine, M-3-G and M-6-G was confirmed in the concentration range of 0.5-50, 25-2500, 2.5-250 ng/ml in monkey plasma, 0.5-100, 25-5000, 2.5-500 ng/ml in dog plasma, respectively. The precision of this assay method, expressed as CV, was less than 15% over the entire concentration range with adequate assay accuracy. Therefore, the HPLC-ESI-MSMS method is useful for the determination of morphine, M-3-G and M-6-G with sufficient sensitivity and specificity in pharmacokinetic studies.     

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 clozapine and its N-desmethyl and N-oxide metabolites in plasma by liquid chromatography/electrospray tandem mass spectrometry and its application to plasma level monitoring in schizophrenic patients

A liquid chromatography tandem mass spectrometry (LC-MS-MS) assay method for the simultaneous determination of clozapine and its N-desmethyl (norclozapine) and N-oxide metabolites in human plasma is described. The compounds were extracted from plasma by a single step liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C-18 column, ionized using positive ion atmospheric pressure electrospray ionization method by a TurboIonspray source and analyzed using multiple reaction monitoring mode. The ion transitions monitored were m/z 327 --> m/z 270 for clozapine, m/z 313 --> m/z 192 for norclozapine, m/z 343 --> m/z 256 for clozapine-N-oxide and m/z 421--> m/z 201 for internal standard. The standard curves of clozapine, norclozapine and clozapine-N-oxide were linear over the range of 1 ng/ml to 1000 ng/ml when 0.5 ml of plasma was used for the analysis (r(2) >0.998). Three pooled plasma samples collected from patients who were treated with clozapine were used as long-term quality control samples to check the validity of spiked standard curve samples made at various times. The intra- and inter-assay variations for the spiked standard curve and quality control samples were less than 14%. These variations for the long-term patient quality control samples were less than 11%. The LC-MS-MS assay for simultaneous determination of clozapine, norclozapine and clozapine-N-oxide reported here is highly specific, sensitive, accurate and rapid. This method is currently being used for the plasma level monitoring of clozapine and its N-desmethyl and N-oxide metabolites in patients treated with clozapine. The plasma levels of clozapine, norclozapine and clozapine-N-oxide varied widely within and among patients. The data revealed that the norclozapine and clozapine N-oxide metabolites were present at about 58%+/-14% and 17%+/-6% of clozapine concentrations in plasma, respectively.     

LC/MS/MS法测定血浆中左羟丙哌嗪浓度及其药代动力学

目的建立测定血浆中左羟丙哌嗪的液相色谱-串联质谱法，考察左羟丙哌嗪在中国健康志愿者体内的药代动力学行为。方法血浆样品经液-液提取后，进行色谱分离，在三重四极杆串联质谱仪上，以多重反应离子监测(MRM)方式进行定量分析，用于监测的离子为m/z 237 → m/z 120（左羟丙哌嗪）和m/z 288 → m/z 58(佐米曲普坦，内标)。结果左羟丙哌嗪的最低定量浓度为0.25 μg·L^-1，线性范围为0.25-500.0 μg·L^-1，精密度与准确度符合生物样品分析要求。结论该法操作简便、快速、灵敏度高。可检测出健康志愿者po左羟丙哌嗪60 mg,其24 h后的血药浓度，适于临床药代动力学研究。     

高灵敏度LC/MS/MS法同时测定人血浆中麻黄碱和氯苯那敏

麻黄碱是存在于草麻黄和木贼麻黄等植物中的生物碱,属拟肾上腺素类药物,包括互为差向异构体的麻黄碱和伪麻黄碱两种活性不同的成分;氯苯那敏为丙胺类组胺H1-受体拮抗剂,作用较强,用量少,中枢抑制作用小,为常用抗过敏药,临床上可用于缓解各种感冒症状,与麻黄碱联合应用,用于缓解支气管哮喘与慢性喘息性支气管炎所致支气管痉挛.     

HPLC-ELSD法测定浙贝母中主要生物碱的含量

目的利用HPLC-ELSD测定贝母类药材中的贝母素甲和贝母素乙。方法XTerra RP18色谱柱(150 mm×3.9 mm ID, 5 μm)，流动相为乙腈-10 mmol·L^-1 NH₄HCO₃(浓氨水调至pH 10.10)=A∶B系统，梯度洗脱，以ELSD检测。结果贝母素甲在1.09-4.36 μg，贝母素乙在1.04-4.16 μg与峰面积的对数值呈线性关系，回收率分别为98.96%(n=4)，RSD 1.01%；98.40%(n=4)，RSD 2.63%。结论本方法能够很好地测定浙贝母药材中的主要生物碱贝母素甲、贝母素乙，可用于浙贝母药材的质量控制。     

浙贝母碱和去氢浙贝母碱的镇咳镇静作用

浙贝母是百合科植物浙贝(Fritillaria verticillata Willd var.thunbergii Bak)的鳞茎,历来用于镇咳祛痰,但用现代方法研究浙贝母镇咳作用的报告甚少,且未得出一致的结果。为此,我们用化学、机械、电刺激等方法观察了浙贝母所含主要生物碱浙贝母碱(peimine,p)与去氢浙贝母碱(peiminine,PE)对小鼠、豚鼠和猫的镇咳作用,同时还观察了对中枢神经系统的作用,结果如下。     

Development of a sensitive bioanalytical method for determination of PNU-83757 in rat,monkey and human plasma: from LC-UV to LC-MS/MS

To support pre-clinical pharmacokinetic/toxicokinetic (PK/TK) evaluation, a sensitive bioanalytical method for determination of N-cyano-N'-(tert-pentyl)-N"-(3-pyridinyl) guanidine (PNU-83757), in rat and monkey plasma was required. Although the UV response of PNU-83757 was quite decent and the extracts using solid phase extraction (SPE) were very selective and concentrated, the best limit of quantitation (LOQ) achieved was 0.4 ng ml(-1) using 0.5 ml plasma for extraction and 2 ng ml(-1) using 0.1 ml plasma for extraction, which was insufficient for PK/TK evaluation at lower doses. When using liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometric detection (LC-APCI-MS/MS, positive ions) and SPE, a LOQ of 0.045 ng ml(-1) for PNU-83757 was reached. Quantitation was accomplished using the precursor --> product ion combinations of m/z 232 --> 162 for PNU-83757 and m/z 236 --> 166 for the internal standard, [2H(4)]PNU-83757, in the multiple reaction monitoring mode. This method has been successfully utilized for PK/TK evaluation in pre-clinical studies and proved to have sufficient sensitivity to determine plasma concentrations for a dose level as low as 1 microg kg(-1) day(-1) in the rat and monkey. Further improvement of this method by using electrospray mass spectrometric detection (LC-ESI-MS/MS, positive ions) and automated membrane SPE, gave an LOQ of 0.008 ng ml(-1), and allowed analysis of large numbers of samples to support clinical PK studies in microg dose levels.     

LC-MS方法研究氢溴酸高乌甲素在小鼠体内的药代动力学(英文)

本文建立了一种快速、灵敏的LC-MS法用于检测小鼠血浆中的高乌甲素浓度。采用ESI源和多反应监测(MRM)的方式进行检测,所选用的高乌甲素和内标延胡索乙素的反应离子对分别为m/z 585→535和m/z 356→m/z 192。该方法在3.0～2 000.0 ng·mL-1浓度内线性关系良好,定量下限为3.0 ng·mL-1,日内和日间精密度(RSD)均小于9.9%,准确度(RE)在±4.8%之内。氢溴酸高乌甲素分别以1.0、2.0和4.0 mg·kg-1单剂量静脉注射给予小鼠后,t1/2分别为0.47、0.48和0.49 h,AUC0-t分别为55.5、110.5和402.9 ng·h·mL-1。实验结果表明,氢溴酸高乌甲素单剂量静脉注射给予小鼠后,在低剂量(1.0～2.0 mg·kg-1)范围内其药动学行为符合线性动力学特征,当给药剂量(2.0 mg·kg-1)增大至4.0 mg·kg-1时,AUC增加至约4倍,而Vz和CL却显著降低,呈现非线性动力学特征,可能与高浓度下药物血浆蛋白结合率的降低有关。     

高通量甲基衍生化固相萃取LC-MS/MS法定量测定人血浆内的米诺膦酸

采用氘代米诺膦酸为内标,建立了高通量衍生化固相萃取LC-MS/MS的定量方法检测人血浆中的米诺膦酸(1)。将血浆样品300μl上样于弱阴离子交换96孔固相萃取板上,配合正压装置在pH 7.0避光条件下与三甲基硅烷化重氮甲烷(TMS-DAM)发生衍生化反应,洗脱液于烘箱70℃反应1 h使衍生化完全。以Eclipse XDB苯基柱为色谱柱,流动相为乙腈∶含0.15%甲酸10 mmol/L乙酸铵溶液,线性梯度洗脱。采用电喷雾离子源(ESI),正离子模式,以多反应监测(MRM)扫描方式进行监测,用于定量分析的离子对为m/z 393.2→m/z 267.1(五甲基米诺膦酸)和m/z 397.1→m/z271.2(D4-五甲基米诺膦酸)。本法线性范围为10~1 500 pg/ml,批内RSD为2.6%~7.5%,批间RSD为2.5%~7.9%;本法提取回收率为89.7%~96.8%,基质效应为96.9%~102.3%。本方法灵敏、快速、高通量,可用于人血浆中1的含量测定和临床研究。     

LC-MS/MS法测定人血浆中西酞普兰及其在制剂生物等效性中的应用

A sensitive and selective LC-MS/MS method for determination of citalopram in human plasma was established to study the bioequivalence of different formulations containing citalopram. The samples were simply pretreated by protein precipitation using acetonitrile, and then analyzed on a Zorbax Extend C₈column. The mobile phase consisted of acetonitrile-water-formic acid (60∶40∶0.2), at a flow-rate of 0.5 mL·min^-1. A Thermo Finnigan TSQ Quantum Ultra tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring using the precursor to product ion combinations of m/z325 → m/z109 and m/z265 → m/z167 was performed to quantify citalopram and the internal standard, respectively. The pharmacokinetic parameters of citalopram in different formulations were calculated by non-compartment model. The linear calibration curves were obtained in the concentration range of 0.10-100 μg·L^-1. The lower limit of quantification was 0.10 μg·L^-1. The intra- and inter-day relative standard deviation (RSD) over the entire concentration range was less than 5.2%. Accuracy determined at three concentrations (0.25, 8.00 and 90.0 μg·L^-1 for citalopram) ranged from -4.7% to 1.3%. Each plasma sample was chromatographed within 3.0 min. The method was successfully used in bioequivalence study of citalopram in human plasma after oral administration of 20 mg citalopram. Calculated with AUC_{1-120 h}, the bioavailability of two formulations was (102.1±10.9)%. The method is rapid, selective, robust and is proved to be suitable for bioequivalence evaluation of different formulations containing citalopram.