Laser diode thermal desorption-positive mode atmospheric pressure chemical ionization tandem mass spectrometry for the ultra-fast quantification of a pharmaceutical compound in human plasma

An ultra-fast, reliable and sensitive analytical method enabling high-throughput quantitative analysis of pharmaceutical compounds in human plasma is described. The quantitative work was performed on one of our compound currently under clinical trial by employing a deuterated internal standard (IS). Plasma samples were treated on solid phase micro-extraction (SPME) plates prior their analysis by laser diode thermal desorption and atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD/APCI-MS/MS) in positive mode. The sample analysis run time was 10s as compared to the 7 min obtained for the validated LC-MS/MS method. The limit of quantification (LOQ) of the method was estimated at 1 ng/mL. The calibration graphs were linear with a regression coefficient R(2) > 0.997. The data of the partial validation show that LDTD/APCI-MS/MS assay was highly reproducible and selective. In addition, the deviations for intra and inter assay accuracy and precision data were within 15% at all quality control levels. The LDTD/APCI-MS/MS method was successfully applied to the analysis of clinical samples and the data obtained were consistent with those found with a validated LC-MS/MS assay. This work demonstrates that LDTD/APCI-MS/MS could be used for the ultra-fast and reliable quantitative analysis of pharmaceutical compounds in human plasma without using the separation step commonly associated with the LC-MS/MS assay.     

Liquid chromatographic-mass spectrometric method for quantitative determination of lisinopril in human plasma

A validated liquid chromatographic-mass spectrometric (LC/MS) method for the determination of lisinopril in human plasma is presented. Enalapril was used as an internal standard. After the addition of internal standard, solid phase extraction was used as a cleaning step. To separate lisinopril and enalapril from interfering endogenous plasma substances, the analysis was performed using column switching valve. The quantitative determination was performed using selected ion monitoring (+)-electrospray LC-MS. A combination of an acidic mobile phase and a reverse phase column was used. A precision in the linear range from 10 to 500.0 ng/mL plasma, absolute recovery of 91.69% for lisinopril and 90.26% for enalapril, stability for 3.5 months at -20 degrees C have been achieved. Limit of quantitation (LOQ) was 10 ng/mL while limit of detection (LOD) was about 1 ng/mL.     

LC-MS/MS 法测定人血浆中倍他米松

建立测定人血浆中倍他米松的LC-MS/MS方法。采用Venusil XBP C₈ (200 mm×3.9 mm ID, 5 μm)色谱柱，流动相为甲醇-水(含甲酸铵5 mmol·L^-1)(80∶20)，流速0.4 mL·min^-1；质谱仪离子源为电喷雾离子源(ESI)，正离子模式检测，监测离子为393.3→355.2(倍他米松)和361.3→343.2(泼尼松龙,内标)。血浆样本用乙酸乙酯处理。倍他米松在0.5~80.0 ng·mL^-1线性关系良好(r=0.999 2)， 血浆低、 中、 高3种浓度(1.0， 10.0， 60.0 ng·mL^-1)平均提取回收率为88.24%，定量限为0.5 ng·mL^-1。本方法操作简便、准确、灵敏，适用于复方倍他米松注射液人体药代动力学研究。     

Determination of norcantharidin in mouse tissues by liquid chromatography coupled to tandem mass spectrometry and its tissue distribution study

The purpose of this study is to determine the concentrations of norcantharidin (CAS NO: 5442-12-6) in mouse tissues and investigate its tissue distribution after intragastric administration of disodium norcantharidate solution. A highly sensitive and specific liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated, using ribavirin (CAS NO: 36791-04-5) as the internal standard (IS). Norcantharidin and IS were extracted from 0.3 mL tissue homogenates using protein precipitation with acetone under acid condition. The analyte was separated on a C18 reverse phase column and analyzed by MS/MS in the multiple reaction monitoring (MRM) mode using ESI with positive ionization, m/z 169→123 for norcantharidin and m/z 267→135 for IS. The developed method was validated over a linear range of concentrations 0.01~5 μg·mL - 1 in liver, lung, kidney, stomach, small intestine, uterus and testis, 0.005~0.5 μg·mL - 1 in heart, spleen and brain, the correlation coefficients (r2) were between 0.9918 and 0.9976. The tissue distribution study result was as follows: The AUC0-t of norcantharidin in tissues was in the order as follows: small intestine, stomach, uterus, kidney, testis, liver, lung, spleen, heart, brain.     

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 determination of the HIV-1 protease inhibitor indinavir in brain and testis of mice

A rapid and sensitive method for the determination of indinavir in mice brain and testis is described and validation data are provided. Indinavir and the internal standard (IS) amprenavir were isolated from homogenized tissue matrices using a mixed-mode solid-phase extraction (SPE) procedure and were then analyzed by reversed-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS). The mass spectrometer in the positive-ion multiple reaction monitoring mode used pairs of ions at m/z of 614.1/421.3 for indinavir and of 506.1/245.3 for IS. The calibration curves were linear over the range 0.0012-0.0390 micromol/kg for brain and 0.39-12.50 micromol/kg for testis. Linearity, repeatability and accuracy were validated. The applicability of the method was demonstrated by assessing indinavir in brain and testis of three mice dosed with intravenous bolus administration of indinavir (16.3 micromol/kg).     

A sensitive liquid chromatography and mass spectrometry method for the determination of posaconazole in human plasma

Posaconazole is a novel extended-spectrum triazole that has favorable in vitro, in vivo and clinical activity against a number of yeasts and moulds. Posaconazole is available as an oral suspension. The dosage found to result in monitored plasma levels that correlate with clinical evidence of good antifungal activity is 800 mg/day in divided doses. A liquid chromatographic/mass spectrometric method (LC-MS/MS) that can be used by clinicians wishing to quantitate, and thereby monitor, plasma levels of posaconazole in certain patients was validated. The method utilized semi-automated 96-well protein precipitation with gradient chromatographic separation of analytes using a Varian Polaris C-18A (2.0 mm x 50 mm, 5-microm particle size) column. The approximate retention time of posaconazole was 2.0 min. Analytes were detected by using tandem mass spectrometry. Sample introduction and ionization was performed by atmospheric pressure chemical ionization in the positive-ion mode. This method has been proven suitable for routine quantitation of posaconazole over the concentration range of 5.00-5000 ng/mL. Inter-run precision based on percent relative deviation for replicate quality controls was < or = 6.2%. Inter-run accuracy expressed as %DIFF was +/-4.0%. Posaconazole quality controls were stable in human plasma for up to five freeze-thaw cycles, when frozen at -20 degrees C for at least 105 days and when kept at room temperature for 24 h. The lower limit of quantitation was 5.00 ng/mL for a 100-microL sample aliquot. These data indicate that the LC-MS/MS method described is suitable for the rapid measurement of posaconazole over the concentration range of 5.00-5000 ng/mL.     

Forensic application and evaluation of a commercially available pregabalin immunoassay test in serum on an Olympus AU480

Misuse of pregabalin and its forensic relevance is steadily increasing. The aim of this study was to evaluate the usability of the commercially available ARKTM Pregabalin II Assay (ARK Diagnostics) for serum analysis of forensic samples. Overall, 156 samples were tested by both the immunoassay and a validated liquid chromatographic–tandem mass spectrometric (LC-MS/MS) method. Sensitivity was 100%, and specificity was 98.7% (n = 79 positive cases confirmed by LC-MS/MS in a range of 380–37,000 ng/mL). A good correlation (R² = 0.73) could also be shown between quantitative immunoassay and LC-MS/MS results. In conclusion, the assay shows excellent reliability for screening of forensic serum samples.     

A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry method for routine clinical monitoring of tacrolimus with the Waters Masstrak™ immmunosuppressant kit

Advances in mass spectrometry instruments have led to increased utilization of high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and it would be necessary to standardize blood quantification of immunosuppressant drugs. The aim of the study was to validate and assess the robustness of an LC-MS/MS method for quantification of tacrolimus in whole blood using the Waters Masstrak? Immunosuppressant Kit. After protein precipitation from whole blood samples, chromatographic separation was performed in 2 min. Detection was performed with a Waters Tandem Quadrupole MS Quattro Premier XE, operated in multiple-reaction monitoring in positive electrospray ionization mode. This method was validated and compared to Enzyme Multiplied Immunoassay Technique (EMIT) method in accordance with actual guidelines. The limit of quantification was 1.0 ng/mL and the calibration curve was linear to 27.6 ng/mL. Between-day and within-day trueness and precision were < 15% at three concentrations spanning the linear range. The EMIT assay showed an average positive bias of 28.3% compared with the LC-MS/MS. Internal and external quality control were always accepted and demonstrated the robustness of this method. In conclusion, we validated a rapid, simple and robust quantification of tacrolimus in blood samples with the Waters Masstrak? Immunosuppressant Kit.     

In vitro plasma protein binding determination of flunarizine using equilibrium dialysis and liquid chromatography-tandem mass spectrometry

A highly sensitive method was developed and validated for determining the free fraction of flunarizine in human plasma. Equilibrium dialysis was used for the separation of free (unbound) drug and liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used for quantitation. Post-dialysis plasma or buffer samples of 0.2 mL were extracted using a liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Supelco Supelcosil ABZ+Plus column, ionized using a positive ion atmospheric pressure electrospray ionization source, and analyzed using multiple reaction monitoring. The ion transitions monitored were m/z 405-->203 for flunarizine and m/z 409-->207 for flunarizine-d4 (internal standard, IS). The chromatographic run time was 3.5 min per injection, with retention times of 2.1 min for both flunarizine and IS. The calibration curve for flunarizine was linear over the concentration range of 0.25-2000 ng/mL (r(2)>0.9989) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v) with the lower limit of quantitation of 0.25 ng/mL. The inter-assay coefficient of variability (CV) for the quality control samples was less than 13.5%, and the inter-assay percent nominal was greater than 98.2%. In vitro protein binding of flunarizine was determined at concentrations of 5, 10 and 100 microg/mL using the validated method. Flunarizine was extensively bound to plasma protein with a 0.083+/-0.005% overall percent free drug in plasma and a CV value less than 7.8%. This validated method will be used for the ex vivo assessment of flunarizine protein binding in human plasma from a drug-drug interaction clinical study.     

Analytical performance of a new liquid chromatography/tandem mass spectrometric method for determination of everolimus concentrations in whole blood

The immunosuppressant everolimus was recently approved for prophylactic use in the United States, to prevent organ rejection in adult kidney transplant recipients. The currently accepted therapeutic range for everolimus is 3-8 ng/mL. Therapeutic drug monitoring (TDM) using predose EDTA whole blood samples is required to optimize dose. We describe a simple extraction method and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) to support routine TDM of everolimus. Samples were prepared by protein precipitation and filtration. The first quadrupole was set to select the ammonium adducts (Equation is included in full-text article.)of everolimus (m/z 975.62) and rapamycin-d3 (m/z 934.70), the internal standard. The second quadrupole was used as a collision chamber, and the third quadrupole was then used to select characteristic product ions of everolimus (m/z 908.50 and 890.50) and rapamycin-d3 (m/z 864.60 and 846.50). The method had an analytical measurement range of 2.0-150 ng/mL. Total imprecision, expressed as percent coefficient of variation (mean concentration), was 19.1% (3.3 ng/mL), 10.6% (5.9 ng/mL), 8.1% (19.2 ng/mL), 5.7% (25.8 ng/mL), and 9.1% (34.2 ng/mL). The new method was compared with 2 other everolimus methods also based on LC-MS/MS, with 64 residual patient specimens. Agreement, based on simple linear regression, was excellent. Method A comparison: y = 0.96x - 1.12 (r = 0.99), n = 20, 2.5-44.7 ng/mL. Method B comparison: y = 0.96x + 0.49 (r = 0.99), n = 44, 2.1-85.6 ng/mL. We conclude that this method could support TDM of everolimus for a wide range of clinical indications.     

Determination of chiglitazar, a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) agonist, in human plasma by liquid chromatography-tandem mass spectrometry

Chiglitazar is a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) agonist. A LC-MS/MS method for the determination of chiglitazar was developed and validated. The assay used 0.2 mL of plasma. 90% acetonitrile containing internal standard was used for protein precipitation. The mobile phase contained 70/30 (v/v) of methanol and water at a flow rate of 0.25 mL/min. Detection was by negative ion electrospray tandem mass spectrometry on a Sciex API 3000. The standard curve, which ranged from 2 to 1500 ng/mL, was fitted to a 1/x weighted quadratic regression model. The validation results demonstrated that the method was sensitive, rapid, selective and robust and provided satisfactory precision and accuracy. The method has been successfully used for the analysis of clinical samples in pharmacokinetic studies of chiglitazar.     

液相质谱串联法同时测定舒尼替尼及其活性代谢产物SU12662在BABL/c裸鼠血浆中的浓度及其药物动力学研究(英文)

本研究建立并验证了一种灵敏、快速、简单的液质联用方法,用于同时测定BABL/c裸鼠血浆中舒尼替尼及其活性代谢产物SU12662的药物浓度。血浆样品采用蛋白沉淀方法处理,并使用帕唑帕尼作为内标。采用C18反相柱进行分离,流动相为10 mM甲酸胺–乙腈(65:35,v/v,pH 3.25),流速0.5 m L/min。所有化合物均采用电喷雾电离源,正离子方式检测。舒尼替尼及SU12662的最低定量下限均为0.5 ng/m L,线性范围均为0.5–1000 ng/m L(r>0.99)。该方法对舒尼替尼及SU12662的测定均具有良好准确度以及可靠的日内、日间精密度,方法稳定性良好,无明显基质效应。此方法成功用于BABL/c裸鼠口服20 mg/kg舒尼替尼的药物代谢动力学研究。     

Simultaneous determination of dextromethorphan and dextrophan in rat plasma by LC-MS/MS and its application to a pharmacokinetic study

A highly selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantitating dextromethorphan (DXM) and its metabolite dextrophan (DXO) in rat plasma using pirfenidone as an internal standard. Protein precipitation with acetonitrile was employed for the sample preparation. Chromatographic separation was achieved on a SB-C18 column at 25 degrees C, with a gradient elution programme of which acetonitrile-0.1% formic acid in water as mobile phase. The flow rate was 0.4 mL/min. Detection is carried out by multiple reaction monitoring (MRM) on a ion-trap LC-MS/MS system with an electrospray ionization interface. The assay is linear over the range 1-500 ng/mL for DXM and 1-250 ng/mL for DXO, with a lower limit of quantitation of 1 ng/mL for both. Intra- and inter-day precision of the assay were less than 9.80% and the accuracy were in the range 96.35-106.39%. The developed method was successfully applied to analyze the drug in samples of rat plasma for pharmacokinetic study.     

Simultaneous determination of zidovudine and lamivudine in human serum using HPLC with tandem mass spectrometry

A method employing high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS) has been developed and validated for the simultaneous determination of clinically relevant levels of zidovudine (AZT) and lamivudine (3TC) in human serum. The method incorporates a fully automated ultrafiltration sample preparation step that replaces the solid-phase extraction step typically used for HPLC with UV detection. The calibration range of the dual-analyte LC-MS/MS method is 2.5–2500 and 2.5–5000 ng ml⁻¹ for AZT and 3TC, respectively, using 0.25 ml of human serum. The lower limit of quantification was 2.5 ng ml⁻¹ for each analyte, with a chromatographic run time of approximately 6 min. Overall accuracy, expressed as bias, and inter- and intra-assay precision are <±7 and <10% for AZT, and <±5 and <12.1% for 3TC over the full concentration ranges. A cross-validation study demonstrated that the LC-MS/MS method afforded equivalent results to established methods consisting of a radioimmuno-assay for AZT and an HPLC-UV method for 3TC. Moreover, the LC-MS/MS was more sensitive, allowed markedly higher-throughput, and required smaller sample volumes (for 3TC only). The validated method has been used to support post-marketing clinical studies for Combivir™ — a combination tablet containing AZT and 3TC.     

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。     

Quantitative determination of lysophosphatidic acids (LPAs) in human saliva and gingival crevicular fluid (GCF) by LC-MS/MS

Lysophosphatidic acid (LPA) is a phospholipid mediator that plays multiple cellular functions by acting through G protein-coupled LPA receptors. LPAs are known to be key mediators in inflammation, and several lines of evidence suggest a role for LPAs in inflammatory periodontal diseases. A simple and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify LPA species (LPA 18:0, LPA 16:0, LPA 18:1 and LPA 20:4) in human saliva and gingival crevicular fluid (GCF). LPA 17:0 was used as an internal standard and the LPA species were extracted from saliva by liquid-liquid extraction using butanol. Chromatography was performed using a Macherey-Nagel NUCLEODUR^® C8 Gravity Column (125 mm × 2.0 mm ID) with a mixture of methanol/water: 75/25 (v/v) containing 0.5% formic acid and 5 mM ammonium formate (mobile phase A) and methanol/water: 99/0.5 (v/v) containing 0.5% formic acid and 5 mM ammonium formate (mobile phase B) at a flow rate of 0.5 mL/min. LPAs were detected by a linear ion trap-triple quadrupole mass spectrometer with a total run time of 8.5 min. The limit of quantification (LOQ) in saliva was 1 ng/mL for all LPA species and the method was validated over the range of 1-200 ng/mL. The method was validated in GCF over the ranges of 10-500 ng/mL for LPA 18:0 and LPA 16:0, and 5-500 ng/mL for LPA 18:1 and LPA 20:4. This sensitive LC-MS/MS assay was successfully applied to obtain quantitative data of individual LPA levels from control subjects and patients with various periodontal diseases. All four LPA species were consistently elevated in samples obtained from periodontal diseases, which supports a role of LPAs in the pathogenesis of periodontal diseases.     

A liquid chromatography–tandem mass spectrometry method for the quantification of PAC-1 in rat plasma

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on an ACQUITY UPLC™ BEH C₁₈ column, with acetonitrile and water (39:61 (v/v) both containing 0.1% formic acid) as mobile phase at a flow rate of 0.20 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The calibration curve was linear over the range of 10–1500 ng/mL (r > 0.99). The LOQ was evaluated to be 0.3 ng/mL. The method described herein is sensitive, selective and faster than other existing method, and was successfully applied to the pharmacokinetic study and gender difference investigation of PAC-1 after oral administration in rats.     

Determination of pinostrobin in rat plasma by LC-MS/MS: application to pharmacokinetics

A rapid and sensitive method for the determination of pinostrobin in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the first time. Isoliquiritigenin was used as an internal standard in rat plasma. Chromatographic separation was performed on an HiQ Sil C₁₈ column with isocratic elution at a flow rate of 1 mL/min. The mobile phase consisted of water and methanol (9:91, v/v) containing 0.1% formic acid. The quantification limit was 10 ng/mL within a linear range of 10-1000 ng/mL (R = 0.9984). The intra- and inter-day assay precision ranged from 3.8-5.3% to 3.2-5.2%, respectively, and the intra- and inter-day assay accuracy was between 93.2-95.1% and 95.5-104.3%, respectively. Our results indicated that the LC-MS/MS method is effective for pharmacokinetic study of pinostrobin in rat plasma.     

LC-MS/MS法测定大鼠血浆中吴茱萸碱的浓度及其药代动力学研究

目的：建立用于测定吴茱萸碱血药浓度的液相色谱-串联质谱联用分析方法,并研究吴茱萸碱在大鼠体内的药代动力学。方法：6只大鼠灌胃给药吴茱萸碱100mg/kg,眼底取血,LC-MS/MS法测定血药浓度,并用DAS药代动力学程序拟合计算药代动力学参数。结果：吴茱萸碱浓度在0.2～50ng/mL内,线性关系良好（r^2=0.9997）。提取回收率96.12%～99.46%,日内、日间RSD分别为4.61%～13.51%和5.65%～11.49%。主要药代动力学参数为：Cmax=（5.3±1.5）ng/mL;tmax=（22±8）min;t1/2=（451±176）min。结论：建立的LC-MS/MS方法专属性强,灵敏度高,可用于吴茱萸碱的体内定量分析。