Validation of a sensitive LC/MS/MS method for simultaneous quantitation of flupentixol and melitracene in human plasma

A sensitive method has been developed and validated, using LC/ESI-MS/MS, for simultaneous quantitation of flupentixol and melitracen—antidepressant drugs, in human plasma. The quantitation of the target compounds was determined in a positive ion mode and multiple reaction monitoring (MRM). The method involved a repeated liquid–liquid extraction with diethyl ether and analytes were chromatographed on a C₈ chromatographic column by elution with acetonitrile–water–formic acid (36:64:1, v/v/v) and analyzed by tandem mass spectrometry. The method was validated over the concentration ranges of 26.1–2090 pg/ml for flupentixol and 0.206–4120 ng/ml for melitracen. The correlation coefficients of both analyst were >0.998 for six sets of calibration curves. The recovery was 60.9–75.1% for flupentixol, melitracen and internal standard. The lower limit of quantitation (LLOQ) detection was 26.1 pg/ml for flupentixol and 0.206 ng/ml for melitracen. Intra- and inter-day precision of the assay at three concentrations were 2.15–5.92% with accuracy of 97.6–103.0% for flupentixol and 0.5–6.36% with accuracy of 98.7–101.7% for melitracen. Stability of compounds was established in a battery of stability studies, i.e., bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for bioequivalence study of flupentixol and melitracen in healthy human male volunteers.     

Liquid-liquid extraction of strongly protein bound BMS-299897 from human plasma and cerebrospinal fluid, followed by high-performance liquid chromatography/tandem mass spectrometry

BMS-299897 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Liquid–liquid extraction (LLE), chromatographic/tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for the quantitation of BMS-299897 in human plasma and cerebrospinal fluid (CSF). Both methods utilized ¹³C₆-BMS-299897, the stable label isotope analog, as the internal standard. For the human plasma extraction method, two incubation steps were required after the addition of 5 mM ammonium acetate and the internal standard in acetonitrile to release the analyte bound to proteins prior to LLE with toluene. For the human CSF extraction method, after the addition of 0.5 N HCl and the internal standard, CSF samples were extracted with toluene and no incubation was required. The organic layers obtained from both extraction methods were removed and evaporated to dryness. The residues were reconstituted and injected into the LC/MS/MS system. Chromatographic separation was achieved isocratically on a MetaChem C18 Hypersil BDS column (2.0 mm × 50 mm, 3 μm). The mobile phase contained 10 mM ammonium acetate pH 5 and acetonitrile. Detection was by negative ion electrospray tandem mass spectrometry. The standard curves ranged from 1 to 1000 ng/ml for human plasma and 0.25–100 ng/ml for human CSF. Both standard curves were fitted to a 1/x weighted quadratic regression model. For both methods, the intra-assay precision was within 8.2% CV, the inter-assay precision was within 5.4% CV, and assay accuracy was within ±7.4% of the nominal values. The validation and sample analysis results demonstrated that both methods had acceptable precision and accuracy across the calibration ranges.     

Quantitative determination of apogossypol, a pro-apoptotic analog of gossypol, in mouse plasma using LC/MS/MS

A simple and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method based on internal standard quantitation using apigenin as the internal standard has been developed and validated for the analysis of the gossypol analog apogossypol, a pro-apoptotic compound, in mouse plasma. The methodology involves protein precipitation of plasma samples followed by LC/MS/MS analysis. Ascorbic acid was added to the spiking solutions and plasma samples to stabilize the easily oxidized compound. Separation of apogossypol and the internal standard from the plasma matrix was achieved using a C18 column with a gradient elution profile consisting of 5 mM ammonium acetate and methanol. The validated range of the method extended from 10 to 2000 ng/mL with accuracies of 85–115% and precision of <15%. The average recovery of apogossypol at three concentrations (50, 200 and 1000 ng/mL) assayed in triplicate using this methodology was determined to be 90.8 ± 12.9%. Recovery for the internal standard (apigenin) at a concentration of 500 ng/mL was found to be 99.9 ± 6.41%. Apogossypol concentrations of 50 ng/mL and above were found to be stable in extracted plasma for 24 h when stored at 25 °C. This method has been applied to the determination of apogossypol concentrations in plasma collected from mice given an IV dose of apogossypol.     

Determination of fentanyl in human plasma and fentanyl and norfentanyl in human urine using LC-MS/MS

Fentanyl, a potent analgesic drug, has traditionally been used intravenously in surgical or diagnostic operations. Formulations with fentanyl in oral transmucosal delivery system and in transdermal depot-patch have also been developed against breakthrough pain in cancer patients. In this report, LC–MS/MS methods to determine fentanyl in human plasma as well as fentanyl and its main metabolite, norfentanyl, in human urine are presented together with validation data. The validation ranges were 0.020–10.0 and 0.100–50.0 ng/ml for fentanyl in plasma and urine, respectively, and 0.102–153 ng/ml for norfentanyl in urine.

Liquid–liquid extraction of the compounds fentanyl, norfentanyl and the deuterated internal standards, fentanyl-d₅ and norfentanyl-d₅ from the matrixes was applied and separation was performed on a reversed phase YMC Pro C₁₈-column followed by MS/MS detection with electrospray in positive mode. The inter-assay precision (CV%) was better than 4.8% for fentanyl in plasma and 6.2% and 4.7% for fentanyl and norfentanyl, respectively, in urine.

The ruggedness of the methods, selectivity, recovery, effect of dilution and long-term stability of the analytes in plasma and urine were investigated. Effect of haemolysis and stability of fentanyl in blood samples were also studied.

The methods have been applied for the determination of fentanyl in plasma samples and fentanyl/norfentanyl in urine samples taken for pharmacokinetic evaluation after a single intra-venous (i.v.) dose of 75 μg fentanyl.     

Rapid and sensitive liquid chromatography-mass spectrometry method for determination of ropinirole in human plasma

A rapid and robust liquid chromatography–mass spectrometry (LC–MS/MS) method was developed for non-ergoline dopamine D₂-receptor agonist, ropinirole in human plasma using Es-citalopram oxalate as an internal standard. The method involves solid phase extraction from plasma, reversed-phase simple isocratic chromatographic conditions and mass spectrometric detection that enables a detection limit at picogram levels. The proposed method was validated with linear range of 20–1200 pg/ml. The extraction recoveries for ropinirole and internal standard were 90.45 and 65.42%, respectively. The R.S.D.% of intra-day and inter-day assay was lower than 15%. For its sensitivity and reliability, the proposed method is particularly suitable for pharmacokinetic studies.     

Determination of limonin in rat plasma by liquid chromatography-electrospray mass spectrometry

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for determination of limonin (LM) in rat plasma has been developed and validated. The method had advantages of a single liquid–liquid extraction with ether and high sensitivity. Analyses were conducted at a flow rate of 0.2 ml/min by a gradient elution. The detection utilized selected ion monitoring in the negative ion mode at m/z 460.00 and 423.15 for the deprotonated molecular ions of LM and the internal standard, respectively. The quantitation limit for LM in rat plasma was 1.0 ng/ml. The linearity was also excellent over the concentration range of 1.9–500 ng/ml of LM. The intra- and inter-day precision (relative standard deviation (R.S.D.%)) was lower than 10% and accuracy ranged from 90 to 110%, showing a good reproducibility. This developed method was successfully applied to analysis of LM in biological fluids.     

LC/MS/MS analysis of vesnarinone and its principal metabolites in plasma and urine

An LC/MS/MS assay was developed and successfully used to quantitate vesnarinone and its principal metabolites (OPC-8230, OPC-18136, and OPC-18137) in human plasma and urine. Samples were pre-treated with liquid–solid extraction followed by simultaneous monitoring of primary and daughter ions which were used for the identification and quantitation of the analytes on LC/MS/MS. This assay offers advantages of specificity, speed and greater sensitivity over the previously developed HPLC-UV assay. The lower limit of quantitation is 500 ng ml⁻¹ for vesnarinone and 20 ng ml⁻¹ for OPC-8230, OPC-18137, and OPC-18136 in plasma. Methodology is similar for the estimation of these analytes in urine with the lower limit of quantitation being 500 ng ml⁻¹ for vesnarinone and 100 ng ml⁻¹ for each metabolite. Ascorbic acid was added to stabilize the analytes from degradation. This LC/MS/MS method was developed to overcome many practical problems associated with the HPLC method. The LC/MS/MS method offers the flexibility of analyzing additional metabolites and changing the linearity range to accommodate the differences in linear range (200–10 000 ng ml⁻¹ for vesnarinone and 20–1000 for metabolites) for the analytes.     

High-throughput quantitation of nefazodone and its metabolites in human plasma by high flow direct-injection LC-MS/MS

A rapid, selective and sensitive high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method coupled with high flow direct-injection on-line extraction has been developed and validated for the simultaneous quantitation of nefazodone and its three active metabolites, hydroxynefazodone, triazole-dione (BMS-180492) and m-chlorophyenylpiperazine (mCPP) in human plasma. The method utilized d₇-nefazodone, d₇-hydroxynefazodone, d₄-BMS-180492 and d₄-mCPP as internal standards (IS). The plasma samples were injected into the LC–MS/MS system after simply adding the internal standard solution and centrifuging. The required extraction and chromatographic separation of the analytes were achieved on an Oasis^® HLB column (on-line extraction column, 1 mm × 50 mm, 30 μm) and a conventional Luna C8 column (analytical column, 4.6 mm × 50 mm, 5 μm). Detection was by positive ion electrospray tandem mass spectrometry. The total analysis run time for each sample was 2 min, which included the time needed for on-line extraction, chromatographic separation and LC–MS/MS analysis. The assay was validated for each analyte and the concentrations ranged from 2.0 to 500 ng/ml for nefazodone, hydroxynefazodone and mCPP and from 4.0 to 1000 ng/ml for BMS-180492, respectively. The assay was used for the high-throughput sample analysis of thousands of pharmacokinetic study samples and was proven to be rapid, accurate, precise, sensitive, specific and rugged.     

Development and validation of a method for quantitative determination of valsartan in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of valsartan in human plasma was developed and validated. A 0.5 ml aliquot was extracted using solid-phase extraction in an Empore^® high performance extraction disk plate, universal resin 96-well format. The estimated calibration range of the method was 2–2000 ng/ml.

The method was fully validated with intra-day mean accuracy and precision of 94.8–107% and 2.19–5.40% and inter-day mean accuracy and precision of 93.5–105% and 1.87–5.67%, respectively. No significant loss of valsartan in processed samples was confirmed in processed samples for up to 24 h at 10 °C. Sample dilution up to 50-fold with blank human plasma provided acceptable analyses. No interference peaks or matrix effects were observed. No effect of QC sample location results was observed in a 96-well plate. This LC-MS/MS technique was found to improve quantitative determination of valsartan allowing its pharmacokinetic evaluation with clinically relevant doses.     

HPLC-MS/MS determination of a peptide conjugate prodrug of doxorubicin, and its active metabolites, leucine-doxorubicin and doxorubicin, in dog and rat plasma

A HPLC-MS/MS Electrospray (ESI) method was developed and validated to quantify a peptide conjugate prodrug of doxorubicin (Dox-Con) and its active metabolites leucine-doxorubicin (Leu-Dox) and doxorubicin (Dox) in dog and rat plasma. The analytes were extracted from plasma by solid-phase extraction on a Bond Elut® C8 cartridge and eluted with chloroform–methanol (2:1). Eluates were evaporated and reconstituted in acetonitrile–5 μM sodium trifluoroacetate in 0.1% aqueous formic acid (20:80) and injected onto a Waters Oasis® HLB column. Analytes were eluted from the column with a solvent gradient into the mass analyzer. The ions were quantified in the selected reaction-monitoring mode (SRM), using positive ions, on a triple quadrupole mass spectrometer. The lower limits of quantification for Dox-Con, Leu-Dox, and Dox in plasma, were approximately 5, 1 (dog)/6 (rat), and 0.5 ng/ml, respectively. Intra- and inter-assay accuracy (% of nominal concentration) and precision (%CV) for all analytes were within 15 and 16%, respectively.     

Simultaneous determination of SU5416 and its phase I and phase II metabolites in rat and dog plasma by LC/MS/MS

SU5416, Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone, is a cytostatic substance in development as an anti-angiogenic agent. SU5416 has several phase I and phase II metabolites including SU9838, SU6595, SU6689, 5′-hydroxy glucuronide of SU5416 and 5′-acyl glucuronide of SU5416. In order to support the preclinical studies, a liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) method for simultaneous determination of SU5416 and its metabolites in rat and dog plasma was developed. This method is fast, simple, sensitive (LOQ=2.0 ng/ml), reproducible and has a wide linear range (2.0–5000 ng/ml for SU5416, 2.0–2000 ng/ml for SU6689 and 2.0–1000 ng/ml for SU9838 and SU6595). This method was applied to rat and dog plasma samples obtained from pharmacokinetic and toxicokinetic studies.     

Quantitation of Armillarisin A in human plasma by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive LC–MS/MS method for quantifying Armillarisin A in human plasma after a single oral dose (40 mg) has been developed and validated. Sample preparation used liquid–liquid extraction with a mixture of diethyl ether–dichloromethane (60:40, v/v) in an acidic environment. The retention times of Armillarisin A and the internal standard, probenecid, were 1.63 and 1.78 min, respectively. The calibration curve was linear over the range 0.15–50 ng/mL with a limit of quantitation of 0.15 ng/mL. The coefficient of variation as a measure of intra- and inter-day precision was <9.3% and the accuracy was in the range 92.5–108.0%. The Armillarisin A concentration–time profile in human plasma was determined after an oral dose of a 40 mg tablet.     

A study of matrix effects on an LC/MS/MS assay for olanzapine and desmethyl olanzapine

The purpose of this research project was to investigate potential matrix effects of anticoagulant and lipemia on the response of olanzapine, desmethyl olanzapine, olanzapine-D₃ and desmethyl olanzapine-D₈ in an LC/MS/MS assay. Blank human serum and sodium heparin, sodium citrate, and K₃EDTA plasma with various degrees of lipemia were fortified with olanzapine, desmethyl olanzapine, olanzapine-D₃ and desmethyl olanzapine-D₈. Six replicates of each sample were extracted using Waters Oasis^® MCX cartridges and analyzed using electrospray LC/MS/MS. The analytes were separated on a Phenomenex LUNA phenyl hexyl, 2 mm×50 mm, 5 μm, analytical column and a gradient rising from 2 to 85% mobile phase B. Mobile phase A consisted of acetonitrile–ammonium acetate (20 mM) (52:48 v/v) and mobile phase B was formic acid–acetonitrile (0.1:100 v/v). Ion suppression was investigated through post column infusion experiments. The degree of lipemia of each sample, indicated by turbidity, was ranked into categories from least to greatest and used for statistical analyses. The results from analysis of variance testing indicated that lipemia, anticoagulant and their interaction significantly influenced mass spectral matrix effects and extraction matrix effects. Differential behavior between the analytes and labeled internal standards contributed to variability. The most significant source of variability however, was ion suppression due to co-eluting matrix components.     

Simultaneous determination of lithospermic acid B and its three metabolites by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the simultaneous determination of lithospermic acid B and its three main O-methylated metabolites in rat serum with silibinin as the internal standard. The calibration curves for LSB, and the three metabolites were linear over the ranges of 16–4096, and 8–2048 ng/ml, respectively, with coefficients of correlation >0.998. For LSB, the intra-assay coefficient of variance (CV) was less than 9.3% and the inter-assay CV was less than 8.9%. The inter-assay mean accuracy was between 92.8% and 104.7%. For the three metabolites, the intra-assay CV was less than 8.7% and the inter-assay CV was less than 9.9%. The inter-assay mean accuracy was between 92.5% and 107.9%. This quantitation method was successfully applied to a pharmacokinetic study of LSB in rats. Also, a total recovery of 5.2%was found in bile after oral administration.     

高效液相色谱-串联质谱法同时测定人血浆中瑞格列奈和二甲双胍及其在人体药动学中的应用

目的:建立高效液相色谱-串联质谱法(HPLC-MS/MS)同时测定人血浆中瑞格列奈和二甲双胍浓度的方法,并进行人体药动学研究。方法:以乙腈沉淀蛋白处理血浆样品,替米沙坦做内标,采用Kromasil 60-5CN色谱柱(2.1 mm×100 mm, 5 μm),流动相为20 mmol·L^-1醋酸铵水溶液(含0.2%甲酸)-乙腈(45:55);质谱采用电喷雾离子化-多反应离子监测(ESI-MRM)。并测定12名健康志愿者单剂量口服瑞格列奈盐酸二甲双胍复方片(2 mg:500 mg)后瑞格列奈和二甲双胍的血浆浓度。结果:瑞格列奈和二甲双胍分别在0.05~50 μg·L^-1、2~2 000 μg·L^-1范围内线性良好,定量下限分别为0.05 μg·L^-1和2 μg·L^-1,日内日间精密度均小于8%,提取回收率分别为77.32%~80.65%和78.98%~82.46%。瑞格列奈和二甲双胍的主要药动学参数C_max分别为(29.50±9.57),(1 167±197.77)μg·L^-1;t_max分别为(0.83±0.39),(1.88±0.64)h;t_1/2分别为(1.11±0.38),(4.31±0.86)h;AUC_0→∞分别为(56.90±20.31),(7 407.32±1 653.25)μg·L^-1·h^-1。结论:建立的分析方法灵敏、准确可靠,样品处理快速、简便,适用于瑞格列奈/盐酸二甲双胍复方片人体药动学和生物等效性研究。     

A direct determination of thymidine triphosphate concentrations without dephosphorylation in peripheral blood mononuclear cells by LC/MS/MS

A rapid, sensitive and specific analytical method with minimal sample preparation for the measurement of thymidine triphosphate (TTP) in peripheral blood mononuclear cells (PBMC) by LC/MS/MS has been developed. PBMC were separated from whole blood or buffy coat. The analyte and internal standard were extracted from PBMC with 70% methanol (pH 7.2). These extracts after centrifugation were directly injected onto LC/MS/MS without need for any further sample preparation. The calibration curve was linear over the range 0.8–800 ng/ml. Mean inter- and intra-assay coefficients of variation (CVs) over the range of the standard curve were less than 10%. The overall recovery of TTP was 103.5%.     

HPLC-MS／MS法测定大鼠血浆中兰索拉唑及其代谢产物的浓度

目的：建立大鼠血浆中兰索拉唑及其代谢产物5-羟基兰索拉唑、兰索拉唑砜的HPLC-MS／MS测定方法。方法：色谱条件：色谱柱：Diamonsil C18柱（150mm×2．1mm,5um）;流动相：乙腈-水（合0．01％甲酸及2mmol·L-1的醋酸铵（43：57,V／V）;流速：0．3ml·min-1;柱温：40℃;进样量10ul。质谱条件：电喷雾离子源（ESI）,以多反应监测离子方式测定兰索拉唑及其代谢产物,选择性监测质荷比（m／z）为368．0／163．9（兰索拉唑）,384．1／179．9（5-羟基兰索拉唑）,383．9／115．9（兰索拉唑砜）,326．0／280．1（内标奥美拉唑）。样品用乙腈沉淀蛋白处理。结果：兰索拉唑、5-羟基兰索拉唑、兰索拉唑砜的线性范围分别为11．40～4560．00,1．26～504．00,1．24～496．00ng·ml-1;定量下限分别为11．40,1．26,1．24ng·ml-1;批内、批间精密度RSD均〈15％。结论：该方法灵敏、准确、快速、专属性好,适用于兰索拉唑及其代谢产物在大鼠体内的药代动力学研究。     

LC/MS联用法测定人血浆中右美沙芬的浓度及其药动学研究

目的:建立以液/质联用法测定人血浆中右美沙芬浓度的方法,并研究其在健康人体的药动学。方法:以曲马多为内标,血浆样品经液-液萃取后,采用Zorbax Extend-C18色谱柱进行分离,通过Q TRAPTM四极杆-线性离子阱质谱仪,以多反应检测方式进行测定。结果:右美沙芬的线性范围为0.05~10.0ng.mL-1(r=0.999 5);平均相对回收率在97.7%~99.5%之间;日内、日间精密度的RSD均小于6.8%,右美沙芬的定量下限为0.05ng.mL-1。结论:本法快速、简便、准确、灵敏,适用于右美沙芬的人体药动学研究。     

液相色谱-质谱联用法测定大鼠血浆中木通皂苷D的浓度

目的建立液相色谱-质谱联用法（LC-MS/MS）测定大鼠血浆中木通皂苷D的浓度。方法选用XDB-C18色谱柱,以5 mmol/L乙酸铵水溶液（含0.1%甲酸）-甲醇溶液为流动相,采用梯度洗脱进行分离。样本经甲醇沉淀后进样,选用3200QTRAP型质谱仪的多重反应监测扫描方式进行检测。结果木通皂苷D线性范围为10～1000 ng/ml,最低定量限为10ng/ml。准确度与精密度结果显示方法日间、日内变异均小于15%,相对误差为-2.8%～4.6%,低、中、高3个浓度提取回收率为95.3%～108.1%。结论本研究所建立的方法快速、灵敏、专属性强、重现性好,可用于大鼠血浆中木通皂苷D浓度的测定和药代动力学研究。     

Determination of fentanyl in human plasma by head-space solid-phase microextraction and gas chromatography-mass spectrometry

A head-space solid-phase microextraction (HS-SPME) method coupled to GC–MS was developed to extract fentanyl from human plasma. The protein binding was reduced by acidification and, eventually, the sample was deproteinized with trichloroacetic acid. The parameters influencing adsorption (extraction time, temperature, pH and salt addition) and desorption (desorption time and temperature) of the analyte on the fibre were investigated and validated for method development. The developed method proved to be rapid, simple, easy and inexpensive and offers high sensitivity and reproducibility. Linear range was obtained from 0.1 ng/ml to 2 μg/ml. The limit of detection was 0.03 ng/ml while an inter-day precision of less than 5% (n = 15) could be achieved. The method has been applied for the determination of fentanyl in plasma samples after application of 50 μg/h Duragesic fentanyl patch.