Exploring dried blood spot sampling technique for simultaneous quantification of antiretrovirals: lamivudine, stavudine and nevirapine in a rodent pharmacokinetic study.

A high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method for the simultaneous quantification of lamivudine, stavudine and nevirapine was developed and validated in dried blood spot (DBS) cards. The analytes were separated using an isocratic mobile phase on a reverse phase column and analyzed by MS/MS in the MRM mode using the respective [M + H]⁺ ions, m/z 230-112 for lamivudine, m/z 225-127 for stavudine, m/z 267-226 for nevirapine, m/z 383-337 for zidovudine (IS). The lower limit of quantification was 1 ng/mL for both lamivudine and stavudine and 10 ng/mL for nevirapine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The method was successfully applied to quantify them in a rat pharmacokinetic study in whole blood, plasma and DBS cards after a single oral co-administration at the dose of 10, 2 and 13 mg/kg for lamivudine, stavudine and nevirapine, respectively, to male Wistar rats. Following oral administration the pharmacokinetic results in all the matrices are in close agreement. Thus accomplishment of this method would facilitate the ease of collection of clinical samples on DBS cards for lamivudine, stavudine and nevirapine during human clinical trials and therapeutic drug monitoring.

LC-ESI-MS/MS method for quantification of ambrisentan in plasma and application to rat pharmacokinetic study.

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of ambrisentan in plasma. The analyte and the internal standard (armodafinil) were extracted from plasma by acetonitrile precipitation and they were separated on a reversed-phase C(18) column with a gradient program. The MS acquisition was performed with multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 379-347 for ambrisentan and m/z 274-167 for the IS. The assay exhibited a linear dynamic range of 1-2000 ng/mL for ambrisentan in plasma. Acceptable precision (<10%) and accuracy (100 ± 8%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify ambrisentan concentrations in a rodent pharmacokinetic study after a single oral administration of ambrisentan at 2.5 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C(max) ; 1197 ± 179 ng/mL) was achieved at 1.0 ± 0.9 h (T(max) ), and the area under the curve (AUC) was 6013 ± 997 ng h/mL. Therefore, development of such a simple and sensitive method in rat plasma should translate into a method for ambrisentan in human plasma for clinical trials.

Pharmacokinetic profiling of efavirenz-emtricitabine-tenofovir fixed dose combination in pregnant and non-pregnant rats.

During pregnancy, the disposition of various drugs is altered due to changes in physiological condition, maternal gastrointestinal absorption, gastric secretion and motility. A fixed dose combination of antiretrovirals is commonly prescribed for the treatment of HIV infection. There is a need to understand the pharmacokinetics and placental transfer of efavirenz-emtricitabine-tenofovir in fixed dose combination during pregnancy. The pharmacokinetics and placental transfer of efavirenz-emtricitabine-tenofovir fixed dose combination was evaluated in timed pregnant and non-pregnant Sprague-Dawley rats at 30, 10, 15 mg/kg p.o., respectively. The plasma, placental tissue, amniotic fluid and fetal tissue concentrations were measured using high performance liquid chromatography combined with tandem mass spectrometric detector (LC-MS/MS). To summarize, the pharmacokinetic profile of efavirenz remained similar in the pregnant and non-pregnant rats. However, a considerable difference in the pharmacokinetics of emtricitabine and tenofovir was observed in pregnant and non-pregnant rats. Efavirenz and emtricitabine showed appreciable placental, amniotic fluid and fetal exposure compared with tenofovir. The present study suggests that a profound impact on antiretroviral pharmacokinetics was observed during pregnancy and there is a need to monitor the exposure levels of each drug when administered as a fixed dose combination during pregnancy. Further studies to explore the pharmacokinetic parameters of fixed dose antiretrovirals during the preclinical stage in a timed-pregnancy rat model are required. Such studies can help in the development of safe and effective medications with a reduced risk of perinatal transmission of HIV-1 infection.

Quantification of urapidil, α-1-adrenoreceptor antagonist, in plasma by LC-MS/MS: validation and application to pharmacokinetic studies.

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of urapidil in plasma. Following liquid-liquid extraction, the analyte was 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 388 to 205 for urapidil and m/z 452 to 344 for the internal standard. The assay exhibited a linear dynamic range of 0.1-500 ng/mL for urapidil in plasma. Acceptable precision (<7%) and accuracy (100 ± 8%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify urapidil concentrations in a preclinical pharmacokinetic study after a single oral administration of urapidil at 3 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C(max); 616 ± 73 ng/mL) was achieved at 0.5 h (T(max)) and area under curve (AUC(0-24)) was 1841 ± 308 ng h/mL. The half-life (t(1/2)) and clearance (Cl) were 2.47 ± 0.4 h and 1660 ± 276 mL/h/kg, respectively. Moreover, it is plausible that the assay method in rat plasma would facilitate the adaptability of urapidil quantification in human plasma for clinical trials.

Quantification of methyllycaconitine, selective α7 nicotinic receptor antagonist, in rodent plasma and brain tissue by liquid chromatography tandem mass spectrometry--application to neuropharmacokinetics of methyllycaconitine in rats.

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of methyllycaconitine (MLA) in rat plasma and brain tissue. Following acetonitrile protein precipitation, the analyte was separated using a gradient mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 683-216 for MLA and m/z 260-116 for the internal standard. The assay exhibited a linear dynamic range of 0.5-250 ng/mL for MLA in rat plasma and brain tissue. The lower limit of quantification was 0.5 ng/mL. Acceptable precision (<12%) and accuracy (100 ± 6%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify MLA concentrations in a rodent pharmacokinetic and brain penetration study.

Simultaneous quantification of a non-nucleoside reverse transcriptase inhibitor efavirenz, a nucleoside reverse transcriptase inhibitor emtricitabine and a nucleotide reverse transcriptase inhibitor tenofovir in plasma by liquid chromatography positive ion electrospray tandem mass spectrometry.

A high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method for the simultaneous quantification of efavirenz, emtricitabine and tenofovir was developed and validated with 100 microL human plasma. Following solid-phase extraction, the analytes were 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 316 to 168 for efavirenz, m/z 248-130 for emtricitabine and m/z 288-176 for tenofovir, m/z 482-258 for rosuvastatin (IS), m/z 260-116 for propranolol (IS). The method exhibited a 100-fold linear dynamic range for all the three analytes in human plasma (20-2000, 2-200 and 20-2000 ng/mL for efavirenz, emtricitabine and tenofovir respectively). The lower limit of quantification was 2 ng/mL for emtricitabine and 20 ng/mL for both efavirenz and tenofovir with a relative standard deviation of less than 11%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 4 min for each sample made it possible to analyze more than 250 human plasma samples per day. The method is precise and sensitive enough for its intended purpose. The method is also successfully applied to quantify efavirenz, emtricitabine and tenofovir concentrations in a rodent pharmacokinetic study.

A sensitive and selective quantification of catecholamine neurotransmitters in rat microdialysates by pre-column dansyl chloride derivatization using liquid chromatography-tandem mass spectrometry.

A rapid and sensitive liquid chromatography tandem mass spectrometry method for simultaneous quantification of catecholamine neurotransmitters in microdialysates was developed. The catecholamine neurotransmitters dopamine (DA) and norepinephrine (NE) were pre-column derivatized with dansyl chloride and analyzed. A gradient elution method was used to separate the analytes from the interferences on an Agilent Poroshell 120 EC-C18 outer porous micro particulate column. The method was robust and sensitive to determine with the lower limit of quantification value of 0.068pmol/mL and 0.059pmol/mL for DA and NE, respectively. It has acceptable precision and accuracy for concentrations over the standard curve range. The method was successfully applied for simultaneous quantitation of DA and NE in the prefrontal cortex (PFC) dialysates of rats obtained from a microdialysis study dosed with vehicle and atomoxetine through intra peritoneal (i.p.) route at a dose of 3mg/kg to monitor the change in extracellular concentrations. Thus, accomplishment of this method would facilitate the neurochemical monitoring for discovery of new chemical entities targeted for the treatment of attention deficit hyperactivity disorder (ADHD).

Methyllycaconitine: a non-radiolabeled ligand for mapping α7 neuronal nicotinic acetylcholine receptors - in vivo target localization and biodistribution in rat brain.

INTRODUCTION: Reduction of cerebral cortical and hippocampal α7 neuronal nicotinic acetylcholine receptor (nAChR) density was observed in the Alzheimer's disease (AD) and other neurodegenerative diseases. Mapping the subtypes of nAChRs with selective ligand by viable, quick and consistent method in preclinical drug discovery may lead to rapid development of more effective therapeutic agents. The objective of this study was to evaluate the use of methyllycaconitine (MLA) in non-radiolabeled form for mapping α7 nAChRs in rat brain. METHODS: MLA pharmacokinetic and brain penetration properties were assessed in male Wistar rats. The tracer properties of MLA were evaluated in rat brain by dose and time dependent differential regional distribution studies. Target specificity was validated after blocking with potent α7 nAChR agonists ABBF, PNU282987 and nicotine. High performance liquid chromatography combined with triple quad mass spectral detector (LC-MS/MS) was used to measure the plasma and brain tissue concentrations of MLA. RESULTS: MLA has shown rapid brain uptake followed by a 3-5 fold higher specific binding in regions containing the α7 nAChRs (hypothalamus - 1.60 ng/g), when compared to non-specific regions (striatum - 0.53 ng/g, hippocampus - 0.46 ng/g, midbrain - 0.37 ng/g, frontal cortex - 0.35 ng/g and cerebellum - 0.30 ng/g). Pretreatment with potent α7 nAChR agonists significantly blocked the MLA uptake in hypothalamus. The non-radiolabeled MLA binding to brain region was comparable with the α7 mRNA localization and receptor distribution reported for [(3)H] MLA in rat brain. DISCUSSION: The rat pharmacokinetic, brain penetration and differential brain regional distribution features favor that MLA is suitable to use in preclinical stage for mapping α7 nAChRs. Hence, this approach can be employed as an essential tool for quicker development of novel selective ligand to map variation in the α7 receptor densities, as well as to evaluate potential new chemical entities targeting neurodegenerative diseases.

Quantification of cinacalcet by LC-MS/MS using liquid-liquid extraction from 50 µL of plasma.

A simple and economical high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of cinacalcet in plasma. Following liquid-liquid extraction, the analyte was separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]⁺ ions, m/z 358-155 for cinacalcet and m/z 310-148 for the internal standard. The assay exhibited a linear dynamic range of 0.1-200 ng/mL for cinacalcet in plasma. Acceptable precision (<10%) and accuracy (100±5%) were obtained for concentrations over the standard curve range. A run time of 3.5 min for each sample made it possible to analyze more than 250 samples per day. The method was successfully applied to quantify cinacalcet concentrations in a preclinical pharmacokinetic study after a single oral administration of cinacalcet at 10 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C(max); 160±56 ng/mL) was achieved at 1.0 h (T(max)), area under the curve (AUC) and half-life (t(½)) were 949±257 ng h/mL and 3.58±0.4 h, respectively.

Liquid chromatography-tandem mass spectrometry method for the quantification of dimebon in rat plasma and brain tissue.

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of dimebon in rat plasma and brain tissue. Following liquid-liquid extraction, the analyte was separated using a gradient mobile phase on a reversed phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 320-277 for dimebon and m/z 407-100 for the internal standard. The assay exhibited a linear dynamic range of 0.25-250 ng/mL for dimebon in rat plasma and brain tissue. Acceptable precision (<11%) and accuracy (100+/-7%) were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze more than 250 samples per day. The method was successfully applied to quantify dimebon concentrations in a rodent pharmacokinetic study. Moreover, it can be believed that the assay method in rat plasma would facilitate the ease of adaptability of dimebon quantification in human plasma for clinical trials.

Liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry method for the quantification of pregabalin in human plasma.

A sensitive high-performance liquid chromatography positive ion atmospheric pressure chemical ionization tandem mass spectrometry method was developed and validated for the quantification of pregabalin in human plasma. Following liquid-liquid extraction, the analyte was 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 160-142 for pregabalin and m/z 482-258 for the internal standard. The assay exhibited a linear dynamic range of 1-10,000ng/mL for pregabalin in human plasma. The lower limit of quantification was 1ng/mL with a relative standard deviation of less than 11.4%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 4.0min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic studies.

Liquid chromatography tandem mass spectrometry method for the quantification of clonidine with LLOQ of 10 pg/mL in human plasma.

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of clonidine in human plasma. Following liquid-liquid 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 230 to 44 for clonidine and m/z 254 to 44 for the internal standard. The assay exhibited a linear dynamic range of 10-2000 pg/mL for clonidine in human plasma. The lower limit of quantification was 10 pg/mL with a relative standard deviation of less than 6.8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method was successfully used to analyze human plasma samples for application in pharmacokinetic studies.

Sensitive liquid chromatography tandem mass spectrometry method for the quantification of sitagliptin, a DPP-4 inhibitor, in human plasma using liquid-liquid extraction.

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of sitagliptin, a DPP-4 inhibitor, in human plasma. Following liquid-liquid 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 408-235 for sitagliptin and m/z 310-148 for the internal standard. The assay exhibited a linear dynamic range of 0.1-250 ng/mL for sitagliptin in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 6%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic studies.

Quantification of fexofenadine in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry using mosapride as internal standard.

A rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of fexofenadine in human plasma using mosapride as internal standard. 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 502/466 for fexofenadine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 1-500 ng/mL for fexofenadine in human plasma. The lower limit of quantification was 1 ng/mL with a relative standard deviation of less than 5% for fexofenadine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 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.

Quantification of pseudoephedrine in human plasma by LC-MS/MS using mosapride as internal standard.

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of pseudoephedrine in human plasma using mosapride as internal standard. 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 166/148 for pseuoephedrine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 2-1000 ng/mL pseudoephedrine in human plasma. The lower limit of quantification was 2 ng/mL with a relative standard deviation of less than 9% for pseudoephedrine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 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.

High-throughput quantification of perindopril in human plasma by liquid chromatography/tandem mass spectrometry: application to a bioequivalence study.

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of perindopril in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 369/172 for perindopril and m/z 417/234 for the internal standard. The method exhibited a linear dynamic range of 0.1-100 ng/mL for perindopril in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 6.1%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 450 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability and bioequivalence studies.

Simultaneous quantification of fexofenadine and pseudoephedrine in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: method development, validation and application to a clinical study.

To support the pharmacokinetic and bioavailability study of a once-daily fexofenadine/pseudoephedrine combination, a high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) method for the simultaneous quantification of fexofenadine and pseudoephedrine was developed and validated with 500 microL human plasma using mosapride as an internal standard (IS). Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 502/466 for fexofenadine, m/z 166/148 for pseuoephedrine and m/z 422/198 for the IS. The method exhibited linear dynamic ranges of 1-500 ng/mL and 2-1000 ng/mL for fexofenadine and pseudoephedrine, respectively, in human plasma. The lower limits of quantification were 1 and 2 ng/mL with a relative standard deviation of less than 10% for fexofenadine and pseudoephedrine, respectively. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time was 2 min and more than 400 human plasma samples could be analyzed in one day by running the system overnight. The method is precise and sensitive enough for its intended purpose.