Endogenous and exogenous testosterone levels after administration of deuterium-labelled testosterone propionate in hypogonadotropic hypogonadism

A gas chromatography-mass spectrometry-selected ion monitoring technique was employed to determine simultaneously the plasma concentrations of endogenous and exogenous testosterone in three patients with hypogonadotropic hypogonadism after a single i.m. dose of testosterone propionate-19,19,19-d3. The plasma levels of testosterone-19,19,19-d3 derived from testosterone propionate-19,19,19-d3 were maintained above the normal testosterone levels (greater than 4 ng/ml) for 48 h, while the plasma levels of endogenous testosterone changed little.     

Simultaneous measurements of endogenous and deuterium-labelled tracer variants of androstenedione and testosterone by capillary gas chromatography-mass spectrometry

A capillary gas chromatographic-mass spectrometric method for the simultaneous determination of androstenedione and testosterone in human plasma using [19,19,19-2H3]androstenedione and [19,19,19-2H3]testosterone as internal standards is described. For calculation of plasma androstenedione and testosterone, peak heights were measured by selected-ion monitoring of the molecular ions of the heptafluorobutyryl derivatives of androstenedione and [2H3]androstenedione (m/z 482 and 485) and of testosterone and [2H3]testosterone (m/z 680 and 683). The isotope dilution method needed no complex corrections for contributions and provides a sensitive and reliable technique with good accuracy, precision and reproducibility.     

Determination of plasma testosterone by mass fragmentography using testosterone-19-d3 as an internal standard. Comparison with radioimmunoassay

Analytical procedures for the measurement of testosterone by mass fragmentography (MF) using trideuterated testosterone (testosterone-19,19,19-d3) are described. For the calculation of plasma testosterone, peak height ratios were measured by MF performed on the molecular ions of the TFA derivative of testosterone (m/e 480) and testosterone-19,19,19-d3 (m/e 483). The sensitivity of the method was judged from the lower limit of detection of the mass spectrometer which was at 10 pg. For the measurement of the precision, the inter- and intra-assay coefficients of variation (C.V.) were calculated by using a pooled plasma sample; they were 3.15% and 1.79%, respectively. The specificity was investigated by the use of 5 alpha-dihydrotestosterone and the MF method was found to afford a highly selective technique. These results obtained by MF have been compared with the results obtained by a radioimmunoassay method.     

Determination of 20 alpha-hydroxy-9 beta,10 alpha-pregna-4,6-dien-3-one in plasma by selected ion monitoring

A selected ion monitoring (SIM) method has been devised for the determination of metabolites of dydrogesterone, 20 alpha-hydroxy-9 beta,10 alpha-pregna-4,6-dien-3-one (DHD) and DHD glucuronide, in plasma. Using testosterone as an internal standard (IS), DHD and IS were extracted with n-hexane and were purified by means of magnesium oxide column chromatography. The purified DHD and IS were converted to their diheptafluorobutyryl derivatives (DHD diHFB and testosterone diHFB) with heptafluorobutyric anhydride in acetone for analysis by SIM. SIM was carried out with a 2% OV-17 column (1 m) at 230 degrees C by monitoring the molecular ions of the derivatives (m/z 706 for DHD diHFB, m/z 680 for testosterone diHFB). DHD was determined from a calibration curve using a peak area method. The determination limit of the devised method was about 5 ng DHD per ml of plasma and the reproducibility was within +/- 6% of the coefficient of variation for 30 ng of DHD per ml of plasma or above.     

Electrospray collision-induced dissociation of testosterone and testosterone hydroxy analogs

Complications with the gas chromatographic analysis of steroids prompted the use of alternative techniques for their identification. High-performance liquid chromatography/mass spectrometry with atmospheric pressure ionization allowed the collection of data for structural identification of these compounds. The objective of this study was to investigate the up-front collision-induced dissociation (UFCID) electrospray ionization (ESI) mass spectra of testosterone and monohydroxylated testosterones. The positive ion UFCID ESI mass spectrum of testosterone showed three significant ions at m/z 97, 109 and 123. The relative abundance of these ions in the UFCID ESI mass spectra of monohydroxylated testosterones varied with the position of the hydroxy group. Statistical data allowed the prediction of hydroxy group position on testosterone by evaluation of the relative abundance of the m/z 97, 109, 121 and 123 ions. Data from the ESI mass spectral analysis of testosterone in a deuterated solvent and from the analysis of cholestenone and 4-androstene-3 beta, 17 beta-diol indicated that the initial ionization of testosterone occurred at the 3-one position. CID parent ion monitoring analyses of the m/z 97, 109 and 123 ions indicated that each resulted from different fragmentation mechanisms and originated directly from the [M + H]+ parent ion. The elemental composition of these fragment ions is proposed based on evidence gathered from the CID analysis of the pseudo-molecular ions of [1,2-2H2]-, [2,2,4,6,6-2H5]-, [6,7-2H2]-, [7-2H]-, [19,19,19-2H3]- and [3,4-13C2]testosterone. The structure and a possible mechanism of formation of the m/z 109 and 123 ions is presented. The results of this study advance the understanding of the mechanisms of collision-induced fragmentation of ions.     

Determination of 2-methoxyestradiol in human plasma, using liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay was developed for the quantitative determination of 2-methoxyestradiol (2ME2) in human plasma. Sample pretreatment involved liquid-liquid extraction with ethyl acetate of 0.3-mL aliquots of plasma spiked with the internal standard, deuterated 2ME2 (2ME2-d5). Separation was achieved on a Zorbax Eclipse C18 column (2.1 x 50 mm, i.d., 5 microm) at room temperature using a gradient elution with methanol and water at a flow rate of 0.25 mL/min. Detection was performed using atmospheric pressure chemical ionization MS/MS by monitoring the ion transitions from m/z 303.1 --> 136.8 (2ME2) and m/z 308.1 --> 138.8 (2ME2-d5). Calibration curves were linear in the concentration range of 1-100 ng/mL. The accuracy and precision values, obtained from three different sets of quality control samples analyzed in quintuplicate on four separate occasions, ranged from 105-108% and from 3.62-5.68%, respectively. This assay was subsequently used for the determination of 2ME2 concentration in plasma of a patient with cancer after a single oral administration of 2ME2 at a dose of 2200 mg.     

Determination of testosterone in human hair by gas chromatography-selected ion monitoring mass spectrometry.

A method for the determination of testosterone in human hair by gas chromatography-mass spectrometry using d3-testosterone as internal standard is described. Our method consisted of alkaline digestion, fast liquid-liquid extraction, LH-20 chromatography and derivatization with heptafluorobutyric anhydride. Quantification was achieved by selected ion monitoring of m/z 680 (testosterone) and m/z 683 (d3-testosterone). Our method needed no complex corrections for isotope contributions. The procedure provided a sensitive and specific technique with good accuracy and precision. For the first time, testosterone has been quantified by gas chromatography-mass spectrometry in human hair. The concentrations (median, range, ng g-1 hair) reflected a significant (p = 0.05; t-test) sex difference with 2.7 ng g-1 (2.5-4.2) in male and 1.7 ng g-1 (1.0-3.4) in female hair.     

Sensitive liquid chromatography tandem mass spectrometry method for the quantification of Quetiapine in plasma

A sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of quetiapine in rat plasma. Following liquid-liquid extraction, the analyte was separated using a gradient mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]+ ions, m/z 384 to m/z 221 for quetiapine and m/z 327 to m/z 270 for the internal standard. The assay exhibited a linear dynamic range of 0.25-500 ng/mL for quetiapine in rat plasma. The lower limit of quantification was 0.25 ng/mL with a relative standard deviation of less than 7%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated method was successfully used to analyze rat plasma samples for application in pre-clinical pharmacokinetic studies. This method in rodent plasma could be adapted for quetiapine assay in human plasma.     

Sensitive sulphur-specific detection of omeprazole metabolites in rat urine by high-performance liquid chromatography/inductively coupled plasma mass spectrometry

The use of high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS) with sulphur-specific detection was investigated as a method for obtaining metabolite profiles for the drug omeprazole administered as a 1:1 mixture of (32)S- and (34)S-labelled material. Analysis based on the monitoring of the chromatographic eluent at either m/z 32 or 34 was not successful due to insufficient sensitivity caused by interferences from polyatomic ions. However, reaction of sulphur with oxygen in the hexapole collision cell, combined with monitoring at m/z 48 (for (32)S) or m/z 50 (for (34)S), provided a facile method for metabolite profiling. Detection of m/z 48 was superior in sensitivity to detection of m/z 50.     

超高效液相色谱-串联质谱法测定动物肌肉组织和鸡蛋中残留的11种甾体激素类药物

建立了采用超高效液相色谱-串联质谱(UPLC-MS/MS)同时测定猪、牛、羊和鸡肌肉组织及鸡蛋中睾酮、甲基睾酮、黄体酮、群勃龙、勃地龙、诺龙、美雄酮、司坦唑醇、丙酸诺龙、丙酸睾酮及苯丙酸诺龙等11种甾体激素多残留的分析方法。试样在碱性条件下用叔丁基甲醚提取,冷冻离心脱脂净化,以乙腈和甲酸水溶液为流动相,梯度洗脱,反相液相色谱分离。采用电喷雾离子化、多反应监测方式(MRM),对11种甾体激素同时进行定性定量测定。动物肌肉和鲜蛋中睾酮、甲基睾酮、勃地龙、美雄酮及司坦唑醇的检出限为0.3 μg/kg,群勃龙、诺龙、黄体酮、丙酸诺龙、丙酸睾酮及苯丙酸诺龙的检出限为0.4 μg/kg。在动物组织及鸡蛋中添加1,2及10 μg/kg 水平的药物回收试验中,睾酮、甲基睾酮、勃地龙、美雄酮及司坦唑醇的回收率均在62.3%～105%之间,相对标准偏差为0.5%～15%;群勃龙、诺龙、黄体酮、丙酸诺龙、丙酸睾酮及苯丙酸诺龙的回收率大于50.0%,相对标准偏差小于16%。11种甾体激素在1～100 μg/L范围内,线性关系良好,相关系数都大于0.99。该方法的样品前处理简单、快速,测定灵敏、准确,选择性好,可满足动物源食品中甾体激素类药物多残留的同时测定。     

超高效液相色谱-串联质谱法测定食用植物油中胆固醇含量

提出了食用植物油中胆固醇的超高效液相色谱-串联质谱测定方法。食用植物油经皂化后用石油醚-乙醚(1+1)溶液提取,以Waters ACQUITY UPLC BEH C18色谱柱(50mm×2.1mm,5μm)为分离柱,以甲酸-甲醇(0.1+99.9)溶液为流动相,以2,2,3,4,4,6-d6胆固醇为内标,采用大气压化学电离源在多反应监测负离子模式下进行测定,胆固醇和内标的定量离子对分别为m/z369.2/146.9,369.2/160.9和375.2/166.5。胆固醇在0.1～5mg·L-1范围内呈线性,测定下限(10S/N)为0.02ng。在3个浓度水平上对方法做回收试验,测得回收率在102%～110%之间。     

超高效液相色谱-质谱/质谱联用法测定人血浆中的辛伐他汀

建立了超高效液相色谱-质谱/质谱联用法(UPLC-MS/MS)测定人血浆中辛伐他汀的浓度。血浆样品经乙醚-正己烷-异丙醇(体积比为80∶20∶3)提取,以洛伐他汀为内标,采用ACQUITY UPLCTM BEH C18柱(50 mm×2.1 mm,1.7 μm)分离,以乙腈-10 mmol/L乙酸铵水溶液(体积比为85∶15)为流动相,流速为0.25 mL/min,通过电喷雾离子化,采用多反应监测(MRM)方式进行正离子检测。线性范围为0.051～20.4 ng/mL,日内及日间测定的相对标准偏差不高于10%,平均回收率为91.6%。方法灵敏度高,分析速度快,操作简便,适用于辛伐他汀药物动力学和生物等效性研究。     

超高效液相色谱-串联质谱法测定兔血浆中的丝裂霉素C

建立了采用超高效液相色谱-串联质谱测定兔血浆中丝裂霉素C的方法。以兔空白血浆为基质,通过添加标准溶液的方法配制含丝裂霉素C和内标物曲安奈德的样品,选用乙酸乙酯为提取溶剂,液-液萃取法处理血浆样品。采用Hypersil Gold C18分析柱(50 mm×2.1 mm, 1.9 μm),流动相为甲醇-0.1%甲酸水溶液(90:10, v/v),等度洗脱,流速0.2 mL/min,柱温35 ℃,在3 min内实现了快速分离。采用电喷雾正离子(ESI+)模式电离,选择反应监测(SRM)模式检测,以曲安奈德作为内标物进行定量。用于监测的定量离子对分别为丝裂霉素C m/z 335.2→242.2和曲安奈德m/z 435.2→397.3/415.2,用基质匹配标准溶液法进行定量。结果表明: 兔血浆中丝裂霉素C的质量浓度在1～1000 μg/L范围内线性关系良好(r=0.9978,权重系数(weighting): 1/x2);血浆中丝裂霉素C的检出限(信噪比为3)为0.2 μg/L;其平均回收率为85%～ 115%;日内及日间的相对标准偏差(RSDs)均小于15%,满足生物样品检测的要求。该方法可用于兔气管外壁给药后的血浆样品中丝裂霉素C的检测。本方法选择性强、灵敏度高、操作简便快速、重现性好,适用于丝裂霉素C药代动力学等方面的研究。     

High-performance liquid chromatography/tandem mass spectrometry method for the determination of arbidol in human plasma

An HPLC/MS/MS method for the determination of arbidol in human plasma was developed. Arbidol and internal standard (loratadine) were extracted from alkaline plasma with tert-butyl methyl ether and analyzed on a Zorbax SB C18 column (30 x 2.1 mm id, 3.5 microm particle size). The detection was by monitoring arbidol at m/z 479.1 --> 434.1 and the internal standard at m/z 383.2 --> 337.2. The method was validated according to U.S. Food and Drug Administration guidelines. The calibration curve was linear over the range of 0.5-500 ng/mL using a 100 microL sample volume. The intraday and interday precisions were less than 6.5%, and acceptable values were obtained for accuracy, recovery, and sensitivity. The developed method was selective, simple, sensitive, and easily applicable.     

Determination of glycyrrhizin in dog plasma by liquid chromatography-mass spectrometry and its application in pharmacokinetic studies

A sensitive liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method was established and validated for the determination of glycyrrhizin in dog plasma. After treatment with methanol to precipitate proteins, plasma samples were analyzed on a reversed-phase C18 (ODS) column with a mobile phase of methanol:1% formic acid solution (75:25, v/v). MS determination was performed using negative electrospray ionization (negative ESI) in the selected ion monitoring mode. Glycyrrhizin was monitored at the m/z 821 channel and internal standard (gliquidone) at the m/z 526 channel. The calibration curve was linear over the range from 0.05 μg mL(-1) to 10 μg mL(-1) with a correlation coefficient above 0.99. This method was successfully applied to the pharmacokinetic studies in beagle dogs. The absolute bioavailability of glycyrrhizin in beagle dogs was 3.24%.     

Determination and pharmacokinetics of manidipine in human plasma by HPLC/ESIMS

A sensitive HPLC/ESIMS method was established for the determination of manidipine in human plasma and pharmacokinetics study. After basified plasma with ammonia, manidipine and the internal standard (IS) (felodipine) were extracted with n-hexane and separated on a Hypersil ODS2 column with a mobile phase of methanol-5 mm ammonium acetate solution containing 0.1% acetic acid (85:15, v/v). MS determination was performed by electrospray ionization in the selected ion monitoring mode. Manidipine was monitored at m/z 611.4 and IS at m/z 384. The assay had a calibration range from 0.2 to 20 ng/mL and a lower limit of quantification of 0.1 ng/mL. The method has been successfully applied to the pharmacokinetic study in healthy volunteers.     

Simultaneous quantitation of HIV-protease inhibitors ritonavir, lopinavir and indinavir in human plasma by UPLC-ESI-MS-MS

A selective, sensitive and high-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been developed and validated for the quantification of HIV-protease inhibitors ritonavir (RTV), lopinavir (LPV) and indinavir (IDV) in human plasma. Sample clean-up involved protein precipitation of both drugs and fluconazole used as internal standard from 100 μL human plasma. All the analytes were chromatographically separated on a Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm particle size) analytical column using 0.1% formic acid and methanol (40:60, v/v) as the mobile phase. The parent → product ion transitions for ritonavir (m/z 721.40→ 296.10), lopinavir (m/z 629.40→ 447.40) and indinavir (m/z 614.4→ 421.0) IS (m/z 307.10 → 220.10) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was validated over the concentration range of 30-15,000 ng/mL for LPV and IDV and 3-1500 ng/mL for RTV. The method was successfully applied to a pilot bioequivalence study in 36 healthy human subjects after oral administration of lopinavir 200 mg and ritonavir 50 mg tablet formulation under fasting conditions.     

Determination of sodium cromoglycate in human plasma by liquid chromatography with tandem mass

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of sodium cromoglycate (SCG) in human plasma after a nasal dose of 10.4 mg sodium cromoglycate nasal spray, using pravastatin sodium as the internal standard. The method was validated over a linear range of 0.300-20.0 ng/mL. SCG and I.S. were extracted from 1.0 mL of heparinized plasma by C(18) solid-phase extraction cartridges using methanol as eluting solvent. The dried residue was reconstituted with 100 microL of mobile phase, and 10 microL was injected onto the LC-MS/MS system. Chromatographic separation was achieved on a C(18) column (250 x 4.6 mm i.d., 5 microm particle size) with a mobile phase of methanol-acetonitrile-water (containing 2 mmol/L ammonium acetate; 42.5:42.5:15, v/v/v) at a flow rate of 0.4 mL/min. The analytes were detected with a triple quad LC-MS/MS using ESI with positive ionization. Ions monitored in the multiple reaction monitoring mode were m/z 469.0 (precursor ion) to m/z 245.0 (product ion) for SCG and m/z 447.2 (precursor ion) to m/z327.1 (product ion) for pravastatin sodium (internal standard) The average recovery of SCG from human plasma was 94.88% and the lower limit of quantitation was 0.3 ng/mL. Results from a 3-day validation study demonstrated excellent precision and accuracy across the calibration range of 0.3-20 ng/mL. The method was successfully applied to the pharmacokinetic study of SCG in healthy Chinese volunteers. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Selective and sensitive determination of bis(7)-tacrine, a high erythrocyte binding acetylcholinesterase inhibitor, in rat plasma by high-performance liquid chromatography-tandem mass spectrometry

The current study aims to develop a specific and sensitive LC-MS/MS method for determination of bis(7)-tacrine (B7T) in rat plasma. A 100 microL plasma sample was extracted with ethyl acetate. B7T and the internal standard (IS), pimozide, in the samples were then analyzed with LC-MS/MS in positive electrospray ionization condition. Chromatographic separation of B7T and IS was achieved in a C(18) reversed-phase HPLC column (150 x 2.1 mm i.d.) by isocratic elution with a mobile phase consisting of 0.05% formic acid in water and acetonitrile (1:1, v/v) at a flow rate of 0.35 mL/min. Multiple-reaction monitoring (MRM) mode was employed to measure the ion transitions: m/z 247 to 197 for B7T and m/z 462 to m/z 328 for IS, respectively. The method was linear over the studied ranges of 100-5000 and 10-100 ng/mL. The intra-day and inter-day variations of the analysis were less than 6.8% with standard errors less than 9.0%. The detection limit of B7T in rat plasma was 1 ng/mL. The developed method was successfully applied to the pharmacokinetic study of B7T after intravenous administration of 1 mg/kg B7T and further proved to be readily utilized for determination of B7T in rat plasma samples.     

Rapid and highly sensitive liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantitation of buspirone in human plasma: application to a pharmacokinetic study

A rapid and sensitive method for the quantitation of buspirone in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was developed. Plasma samples were treated by liquid-liquid extraction with methyl tert-butyl ether (MTBE). The chromatographic separation was performed isocratically on a reversed-phase Shiseido C18 column (50 mm x 2.0 mm, 3 microm) with a mobile phase of acetonitrile/0.1% acetic acid (1:1, v/v). The acquisition was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 386 --> 122 for buspirone and m/z 409 --> 238 for amlodipine (the internal standard). The method was validated to determine its specificity, recovery, limit of quantitation, accuracy and precision. The lower limit of quantitation was 0.02 ng/mL with a relative standard deviation of less than 10%. The present method provides an accurate, precise and sensitive tool for buspirone and was successfully applied to a pharmacokinetic study in eight subjects.