A rapid and sensitive liquid chromatography/tandem mass spectrometry method for determination of docetaxel in human plasma

A novel, rapid and sensitive isocratic liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for quantification of docetaxel in human plasma with paclitaxel as internal standard. The high sensitivity and specificity of MS/MS detection enabled the use of a small volume of plasma (0.05 mL) and a simple liquid-liquid extraction procedure. Furthermore, a very short run-time (3 min) fulfilled the need for monitoring plasma levels of docetaxel from large-scale clinical studies. The calibration curve for docetaxel was linear over the range 5-1000 ng/mL with coefficients of correlation >0.999 using only 0.05 mL plasma. The intra- and inter-day precisions (CV) of analysis were <7%, and accuracy ranged from 96 to 110%. The applicability of the method was demonstrated in a pharmacokinetic study of a 1-h infusion of docetaxel with dosages of 75 mg/m(2). Possible conjugated metabolites of docetaxel were not detected in patients' samples.     

Simple and sensitive quantification of bioactive peptides in biological matrices using liquid chromatography/selected reaction monitoring mass spectrometry coupled with trichloroacetic acid clean-up

A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method has been developed for the quantification of bioactive peptides in biological fluids. The method employs protein precipitation with 4% trichloroacetic acid (TCA) and selected reaction monitoring (SRM) using an immonium ion as the product ion. This method was applied to determine the synthetic parathyroid hormone (PTH) analog (MW 1721) in rat plasma and human hepcidin-25 (MW 2789) in human serum. TCA clean-up showed a sufficient recovery for peptides with a MW of less than 3000, and would be useful as a simple and rapid method because of direct injection of the supernatant without evaporation or dilution. In addition, TCA clean-up allowed us not only to reduce sample preparation time, but also to select an immonium ion as a product ion of SRM, which led to detection more sensitive than SRM using other types of product ions. The lower limits of quantitation (LLOQs) of the PTH analog and the human hepcidin-25 were 0.2 ng/mL and 5 ng/mL, respectively. This method was fully validated with acceptable linearity, intra- and inter-assay precisions, and accuracy. Furthermore, this simple and rapid method is applicable to pharmacokinetic studies.     

Rapid and sensitive liquid chromatography tandem mass spectrometry method for the quantification of ambroxol in human plasma

A sensitive, specific and rapid high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was described and validated for the quantification of ambroxol in human plasma using enalaprilat as the internal standard (IS). Chromatographic separation was performed on a Lichrospher CN column with a mobile phase of methanol and water (containing 0.1% formic acid) (70:30, v/v). The total run time was 5.0 min for each sample. The analytes was detected by mass spectrometry with electrospray ionization source in positive selected reaction monitoring mode. The precursor-fragment ion reaction for ambroxol was m/z 378.9 --> 263.8, and for IS was m/z 349.0 --> 205.9. The linearity was established over the concentration range of 1.56-400.00 ng/mL. The inter-day and the intra-day precisions were all within 10%. A simple protein precipitation with methanol was adopted for sample preparation. The extraction recoveries of ambroxol and IS were higher than 90.80%. The validated method was successfully applied in pharmacokinetic study after oral administration of 90 mg ambroxol to 24 healthy volunteers.     

A sensitive method for determination of salvianolic acid A in rat plasma using liquid chromatography/tandem mass spectrometry

Salvianolic acid A (SAA), a major effective constituent of Salvia miltiorrhizas, is widely used in traditional Chinese medicine. A sensitive rapid analytical method was established and validated for SAA in rat plasma, which was further applied to assess the pharmacokinetics of SAA in rats receiving a single oral dose of SAA. The method used liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode with chloramphenicol as the internal standard. A simple liquid-liquid extraction based on ethyl acetate was employed. The combination of a simple sample cleanup and short chromatographic run time (3 min) increased the throughput of the method substantially. The method was validated over the range 1.4-1000 ng/mL with a correlation coefficient >0.99. The lower limit of quantification was 1.4 ng/mL for SAA in plasma. Intra- and inter-day accuracies for SAA were 95-113 and 98-107%, and the inter-day precision was less than 12%. This method is more sensitive and faster than previous methods. After a single oral dose of 100 mg/kg of SAA, the mean peak plasma concentration (Cmax) of SAA was 318 ng/mL at 0.5 h, the area under the plasma concentration-time curve (AUC0-12 h) was 698 +/- 129 ng.h/mL, and the elimination half-life (T1/2) was 3.29 h.     

Measuring long-chain acyl-coenzyme A concentrations and enrichment using liquid chromatography/tandem mass spectrometry with selected reaction monitoring

Long-chain acyl-coenzymes A (acyl-CoAs) (LCACoA) are the activated forms of long-chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl-CoA ([U-(13) C]16-CoA) and oleoyl-CoA ([U-(13) C]18:1-CoA) using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile (ACN). The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate enrichment of palmitoyl-CoA ([U(-13)C]16-CoA) and oleoyl-CoA ([U(-13)C]18:1-CoA) using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupolemass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M+2+H](+) and [U(13)C]16-CoA and [U(13)C]18:1-CoA were monitored as [M+16+H](+) and [M+18+H](+), respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U(13) C]16-CoA and [U(13) C]18:1-CoA during a low dose intravenous infusion of [U(13) C]palmitate and [U(13) C]oleate in adults undergoing either a saline control experiment or an insulin/glucose infusion experiment. This technique should allow investigators to measure the trafficking of extracellular fatty acids to the intracellular LCACoA pool.     

Use of a variably sensitive selected reaction monitoring method to extend the linear dynamic range for quantitative high-performance liquid chromatography/tandem mass spectrometry

A method has been devised with the capacity to extend the linear dynamic range of a triple quadrupole mass spectrometer operated in the selected reaction monitoring (SRM) mode of analysis. This extended range experiment can be realized by simultaneously acquiring variably sensitive data, via collision energy adjustment, for the same precursor-to-product ion transition within a single SRM method. While this method can be applied universally to many different study types without any detrimental effect to the analysis or throughput, it was applied herein to acquire and quantify, within a single analysis, the concentrations of GSK-A in a multiple-dose rodent study, that previously required a dilution scheme. Using this methodology, the linear dynamic range of GSK-A was increased over traditional methods by nearly two orders of magnitude, from 2.00-10,000 ng/mL to 0.500-100,000 ng/mL.     

Sensitive and rapid liquid chromatography/tandem mass spectrometry assay for the quantification of amlodipine in human plasma

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of amlodipine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase C(18) column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 409/238 for amlodipine and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 50-10,000 pg/mL for amlodipine in human plasma. The lower limit of quantification was 50 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of amlodipine and the IS from spiked plasma samples were 74.7 +/- 4.6 and 72.1 +/- 2.0%, respectively. A run time of 1.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. The observed maximum plasma concentration (Cmax) of amlodipine (2.5 mg oral dose) was 1425 pg/mL, time to observed maximum plasma concentration (Tmax) was 8.1 h and elimination half-life (T(1/2)) was 50.1 h.     

Development of a sensitive and rapid liquid chromatography/tandem mass spectrometry method for the determination of apomorphine in canine plasma

A sensitive, rapid and specific quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of apomorphine (APO) in canine plasma. The analytes were prepared using one-step liquid-liquid extraction, and analyzed on a Waters Symmetry C(18) column interfaced with triple quadrupole tandem mass spectrometer. A mixture of methanol/0.1% formic acid in water (70: 30, v/v) was employed as the isocratic mobile phase. Positive electrospray ionization was utilized as the ionization source. The analyte and clenbuterol (internal standard) were both detected using multiple reaction monitoring (MRM) mode. The limit of detection (LOD) obtained was 0.03 ng/mL. The assay was linear over the concentration range of 0.1-100 ng/mL, and provided good precision (RSD) and good accuracy (RE). The analyte was stable by using antioxidants throughout the whole study. The experimental results show that LC/MS/MS is a rapid and sensitive method to analyze APO in plasma. Finally, the proposed method was successfully applied to a pharmacokinetic study of APO after intranasal administration of 0.5 mg apomorphine to 10 healthy beagle dogs.     

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.     

A liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of isoniazid and ethambutol in human plasma

Isoniazid and ethambutol are commonly used in various combination treatments for tuberculosis, and for this reason a rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for simultaneous quantification of these two drugs in human plasma. After a simple protein precipitation using methanol, the analytes and the internal standard metformin were chromatographed on a C18 column and detected by MS/MS. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The LC retention times for isoniazid and ethambutol were 2.46 and 2.27 min, respectively. The method was linear in the concentration range of 10.0-5000 ng/mL for each analyte using 100 microL plasma. The intra- and inter-day precisions, expressed as the relative standard deviation (RSD), were less than 5.7 and 6.4%, determined from QC samples for isoniazid and ethambutol, and the accuracies were within +/-2.1% and +/-4.5% in terms of relative error, respectively. The method was successfully employed in a pharmacokinetic study after oral administration of a multicomponent formulation containing 150 mg isoniazid, 500 mg ethambutol, 150 mg rifampicin and 250 mg pyrazinamide.     

A rapid and sensitive method for quantitation of nucleosides in human urine using liquid chromatography/mass spectrometry with direct urine injection

Oxidized nucleosides are biochemical markers for tumors, aging, and neurodegenerative diseases. However, during the last decade, the analytical methods for nucleosides by gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) with single-parameter detectors like electron-capture detection (ECD) have not been sufficiently rapid or reliable to detect nucleosides in urine and to analyze clinical samples. It has been reported (Dudley et al., Rapid Commun Mass Spectrom. 2000; 14: 1200) that liquid chromatography with electrospray mass spectrometry (LC/ESI-MS) is more specific and sensitive for analysis of nucleosides than HPLC with conventional detectors; however, this method required complex extraction steps. In the present work a direct LC/ESI-MS method for nucleosides without extraction of urine samples has been developed. Analysis of nucleosides using positive-ion mode with selected reaction monitoring effectively eliminated potential interferences from endogenous constituents of the urine. This highly selective and sensitive method made it possible to analyze urinary nucleosides with a lower limit of quantitation of 0.2 nmol/mL. The method has been validated, with both excellent linearity and reproducibility, in the calibration range from 0.2-400 nmol/mL. The correlation coefficients of the calibration curves were higher than 0.987. The coefficients of variation were in the range 0.03-14.92% (inter-day) and 0.54-14.39% (intra-day), respectively.     

A rapid and sensitive liquid chromatography/positive ion tandem mass spectrometry method for the determination of cimetropium in human plasma by liquid-liquid extraction

We have developed and validated a simple detection system with high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) for determining cimetropium levels in human plasma using scopolamine butyl bromide as an internal standard (I.S.). The acquisition was performed in the multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 357.9 > 103.1 for cimetropium and m/z 359.9 > 103.1 for butyl-scopolamine. The method involves a simple single-step liquid-liquid extraction with dichloromethane. The analyte was chromatographed on an YMC C18 reversed-phase chromatographic column by isocratic elution with 10 mM ammonium formate buffer-methanol (19:81, v/v; adjusted to pH 4.0 with formic acid). The results were linear over the studied range (0.2-100 ng ml(-1)), with r2 = 1.0000, and the total analysis time for each run was 2 min. Intra- and interassay precisions were 0.70-8.54% and 1.08-4.85%, respectively, and intra- and interassay accuracies were 97.56-108.23% and 97.48-103.91%, respectively. The lower limit of quantification (LLOQ) was 0.2 ng ml(-1). At this concentration, mean intra- and interassay precisions were 8.54% and 4.85%, respectively, and mean intra- and interassay accuracies were 97.56% and 98.91%, respectively. The mean recovery ranged from 62.71 +/- 4.06 to 64.23 +/- 2.32%. Cimetropium was found to be stable in plasma samples under typical storage and processing conditions. The devised assay was successfully applied to a pharmacokinetic study of cimetropium bromide administered as a single oral dose (150 mg) to healthy volunteers.     

Sensitive liquid chromatography/mass spectrometry assay for the quantification of azithromycin in human plasma

A simple, rapid, sensitive and specific liquid chromatography/electrospray ionization mass spectrometry method was developed and validated to quantify azithromycin in human plasma, using erythromycin as the internal standard (IS). A simple sample preparation method of protein precipitation with methanol was employed. Methanol, acetonitrile and water (12:30:58, v/v/v) were used as the isocratic mobile phase, with 0.1% formic acid and 0.1% ammonium acetate in water. Selected ion monitoring was specific for azithromycin and erythromycin. The assay was linear over the concentration range 4.69-600 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9994 to 0.9998. The intra- and inter-day precisions, calculated from quality control samples, were less than 8.24%. The method was employed in a pharmacokinetic study after oral administration of 500 mg azithromycin dispersible tablet to 20 healthy volunteers.     

A highly sensitive liquid chromatography tandem mass spectrometry method for simultaneous quantification of midazolam, 1'-hydroxymidazolam and 4-hydroxymidazolam in human plasma

A highly sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of midazolam and its major metabolites 1'-hydroxymidazolam and 4-hydroxymidazolam in human plasma was developed and validated. Stable isotope-labeled midazolam-D(4) and 1'-hydroxymidazolam-D(4) were used as internal standards. Compounds were extracted from 0.5 mL plasma by liquid-liquid extraction with ethyl acetate-heptane (1:4). Chromatography was achieved using a Sunfire C(18) column. The mobile phase was a gradient with 10 m m formic acid in Milli-Q water and methanol at a flow rate of 0.3 mL/min. Total run time was 10 min. Detection was performed using a tandem mass spectrometer with positive electrospray ionization. Calibration curves were linear over the range of 0.10-50.0 ng/mL for midazolam and 0.025-25.0 ng/mL for both metabolites. For all compounds the lower limit of quantification was 0.10 ng/mL. Imprecision was assessed according to the NCCLS EP5-T guideline and was below 10% for all compounds. Mean recoveries were between 94 and 109% for midazolam and its metabolites. The validated method was successfully applied in a pharmacokinetic study investigating in vivo CYP3A-activity in a large cohort of renal allograft recipients using sub-therapeutic doses of midazolam as a drug-probe.     

Rapid and simple liquid chromatography tandem mass spectrometry method for the quantification of zidovudine in rat plasma

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of zidovudine in rat plasma. Following solid-phase extraction, the analytes were separated using an isocratic 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 268/127 for zidovudine and m/z 230/112 for the internal standard. The method exhibited a linear dynamic range of 5-500 ng/mL for zidovudine in rat plasma. The lower limit of quantification was 5 ng/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 1.5 min for each sample made it possible to analyze more than 400 plasma samples per day. The validated method was applied for pharmacokinetic studies of the novel drug delivery systems of zidovudine in rats.     

Rapid and sensitive liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of yonkenafil and its major metabolites in rat plasma

Yonkenafil is a promising drug for treatment of male erectile dysfunction. Previous studies showed that the piperazine‐N,N’‐deethylation metabolite, piperazine‐N‐deethylation metabolite, and piperazine‐N‐deethylation‐N,N’‐deethylation metabolite were the major metabolites of yonkenafil after extensive metabolism. We developed a sensitive and selective method for the simultaneous quantification of yonkenafil and its major metabolites using high‐throughput liquid chromatography with tandem mass spectrometry. Analytes and internal standard were extracted from a small quantity of plasma (50 μL) using liquid–liquid extraction with diethyl ether/dichloromethane (60:40, v/v), and the baseline separation was achieved on Zorbax SB‐C₁₈ column using ammonia/water/methanol (0.2:20:80, v/v/v) as the mobile phase. The assay was performed with an electrospray positive ionization mass spectrometry through the multiple‐reaction monitoring mode within 2 min. Calibration curve of the method was linear within the range of 1.00–1000 ng/mL for all the analytes with the intra‐ and interday precisions of 4.0–5.2 and 4.0–5.3% for yonkenafil, 3.1–4.9 and 3.1–5.2% for the piperazine‐N,N’‐deethylation metabolite, 4.8–6.8 and 4.8–7.3% for the piperazine‐N‐deethylation metabolite, and 2.9–6.1 and 5.4–6.3% for the piperazine‐N‐deethylation‐N,N’‐deethylation metabolite, respectively. The recoveries were above 90% with low matrix effects. The validated assay was successfully applied to support a preclinical pharmacokinetic study in six rats using a single oral dose of yonkenafil (8 mg/kg).