Pharmacokinetics of Nafamostat,a Potent Serine Protease Inhibitor,by a Novel LC-MS/MS Analysis

Nafamostat, a synthetic serine protease inhibitor, has been used for the treatment of inflammatory diseases such as pancreatitis. Recently, an increasing number of studies have shown the promising antiviral effects of nafamostat for the treatment of coronavirus disease-19 (COVID-19). This study aimed to develop a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis and to characterize the pharmacokinetics of nafamostat in rats. Nafamostat in the rat plasma was extracted by solid phase extraction, and ¹³C₆-nafamostat was used as an internal standard. The quantification limit of nafamostat in the rat plasma was 0.5 ng/mL. The LC-MS/MS method was fully validated and applied to characterize the pharmacokinetics of nafamostat in rats. Following intravenous injection (2 mg/kg), nafamostat in the plasma showed a multiexponential decline with an average elimination half-life (t_1/2) of 1.39 h. Following oral administration of nafamostat solutions (20 mg/kg) in 10% dimethyl sulfoxide (DMSO) and in 10% DMSO with 10% Tween 80, nafamostat was rapidly absorbed, and the average oral bioavailability was 0.95% and 1.59%, respectively. The LC-MS/MS method and the pharmacokinetic information of nafamostat could be helpful for the further preclinical and clinical studies of nafamostat.     

Preclinical Pharmacokinetics and Bioavailability of Oxypeucedanin in Rats after Single Intravenous and Oral Administration

Oxypeucedanin, a furanocoumarin extracted from many traditional Chinese herbal medicines, has a variety of pharmacological effects. However, the independent pharmacokinetic characteristics and bioavailability of this compound remains elusive. In this study, a rapid, sensitive, and selective method using ultra-high performance liquid chromatography–tandem mass spectrometry (UPLC/MS/MS) was developed for evaluating the intravenous and oral pharmacokinetics of oxypeucedanin. After intravenous administration of oxypeucedanin (2.5, 5, and 10 mg/kg), and intragastric administration of oxypeucedanin (20 mg/kg), blood samples were collected periodically from the tail vein. The plasma concentration-time curves were plotted, and the pharmacokinetic parameters were calculated using a non-compartmental model analysis. After intravenous administration of oxypeucedanin (single dosing at 2.5, 5, and 10 mg/kg) to rats, the pharmacokinetics fit the linear kinetics characteristics, which showed that some parameters including average elimination half-life (T_1/2Z of 0.61~0.66 h), mean residence time (MRT of 0.62~0.80 h), apparent volume of distribution (V_Z of 4.98~7.50 L/kg), and systemic clearance (CL_Z of 5.64~8.55 L/kg/h) are dose-independent and the area under concentration-time curve (AUC) increased in a dose-proportional manner. Single oral administration of oxypeucedanin (20 mg/kg) showed poor and slow absorption with the mean time to reach the peak concentration (T_max) of 3.38 h, MRT of 5.86 h, T_1/2Z of 2.94 h, and a mean absolute bioavailability of 10.26% in rats. These results provide critical information for a better understanding of the pharmacological effect of oxypeucedanin, which will facilitate its research and development.     

小儿苯巴比妥分散片在家犬体内药代动力学与生物利用度研究

报道了用高效液相色谱法测定小儿苯巴比妥分散片在家犬体内的药代动力学及生物利用度，为临床科学用药提供了依据。     

Pharmacokinetics,bioavailability and metabolism of scopoletin in dog by ultra‐high‐performance liquid chromatography combined with linear ion trap–Orbitrap tandem mass spectrometry

A highly sensitive and selective method based on ultra‐high‐performance liquid chromatography combined with linear ion trap–Orbitrap tandem mass spectrometry (UHPLC–LTQ–Orbitrap–MS) has been developed and validated for the determination of scopoletin in dog plasma. The analyte was extracted from plasma samples using acetonitrile and separated on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) with 0.05% ammonium hydroxide and acetonitrile as mobile phase. The developed method was linear over the concentration range of 1–500 ng/mL, with a correlation coefficient >0.9988. The intra‐ and inter‐day precisions (RSD) were <8.93% while the accuracy (RE) ranged from ?6.50 to 8.12%. Extraction recovery, matrix effect and stability for dog plasma samples were within the required limits. The validated method has been successfully applied to investigate the pharmacokinetics and metabolism of scopoletin in dog plasma after intravenous (1 mg/kg) and oral (10, 25, 50 mg/kg) administration. The results revealed that (a) scopoletin showed short elimination half‐life in dog; (b) its oral bioavailability was low (within the range of 5.69–7.08%); (c) scopoletin showed dose‐independent pharmacokinetic profiles in dog plasma over the dose range of 10–50 mg/kg; and (d) glucuronidation was the predominant metabolic pathway in dog.     

Evaluation of the Pharmacokinetics of the Pancreastatin Inhibitor PSTi8 Peptide in Rats: Integration of In Vitro and In Vivo Findings

PSTi8 is a pancreastatin inhibitory peptide that is effective in the treatment of diabetic models. This study investigates the pharmacokinetic (PK) properties of PSTi8 in Sprague Dawley rats, for the first time. In vitro and in vivo PK studies were performed to evaluate the solubility, stability in plasma and liver microsomes, plasma protein binding, blood–plasma partitioning, bioavailability, dose proportionality, and gender difference in PK. Samples were analyzed using the validated LC-MS/MS method. The solubility of PSTi8 was found to be 9.30 and 25.75 mg/mL in simulated gastric and intestinal fluids, respectively. The protein binding of PSTi8 was estimated as >69% in rat plasma. PSTi8 showed high stability in rat plasma and liver microsomes and the blood–plasma partitioning was >2. The bioavailability of PSTi8 after intraperitoneal and subcutaneous administration was found to be 95.00 ± 12.15 and 78.47 ± 17.72%, respectively, in rats. PSTi8 showed non-linear PK in dose proportionality studies, and has no gender difference in the PK behavior in rats. The high bioavailability of PSTi8 can be due to high water solubility and plasma protein binding, low clearance and volume of distribution. Our in vitro and in vivo findings support the development of PSTi8 as an antidiabetic agent.     

Pharmacokinetics and bioavailability of gelsenicine in mice by UPLC–MS/MS

Gelsenicine is an indole alkaloid isolated from Gelsemium elegans Benth. In recent years, the role of G. elegans Benth preparations in anti‐tumor, analgesic, dilatation and dermatological treatment has attracted attention, and it has been applied clinically, but it is easy to cause poisoning with its use. An UPLC–MS/MS method was established to determine the gelsenicine in mouse blood, and the pharmacokinetics of gelsenicine after intravenous (0.1 mg/kg) and intragastric (0.5 and 1 mg/kg) administration was studied. Deltalin was used as internal standard; a UPLC BEH C₁₈ column was used for chromatographic separation. The mobile phase consisted of acetonitrile and 10 mmol/L ammonium acetate (0.1% formic acid) with a gradient elution flow rate of 0.4 mL/min. Multiple reaction monitoring mode was used for quantitative analysis of gelsenicine in electrospray ionization positive interface. Proteins from mouse blood were removed by acetonitrile precipitation. A validation of this method was performed in accordance with the US Food and Drug Administration guidelines. In the concentration range of 0.05–100 ng/mL, the gelsenicine in the mouse blood was linear (r > 0.995), and the lower limit of quantification was 0.05 ng/mL. In the mouse blood, the intra‐day precision RSD was <12%, the inter‐day precision RSD was <15%, the accuracy ranged from 89.8 to 112.3%, the average recovery was >76.8%, and the matrix effect was between 103.7 and 108.4%, which meet the pharmacokinetic research requirements of gelsenicine. The UPLC–MS/MS method is sensitive, rapid and selective, and has been successfully applied to the pharmacokinetic study of gelsenicine in mice. The absolute bioavailability of gelsenicine is 1.13%.     

Pharmacokinetics and metabolism of ulixertinib in rat by liquid chromatography combined with electrospray ionization tandem mass spectrometry

The purpose of this study was to develop and validate a simple and sensitive liquid chromatography tandem mass spectrometry method for the determination of ulixertinib in rat plasma. The plasma samples were precipitated with acetonitrile and then separated on a C₁₈ column with water containing 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.3 mL/min. Analytes were monitored on a TSQ Vantage triple quadrupole tandem mass spectrometer operated in positive electrospray ionization mode. Selected reaction monitoring transitions were m/z 433.1→262.1 for ulixertinib and m/z 450.1→260.1 for internal standard. The assay achieved good linearity over the concentration range of 0.1‐1000 ng/mL with correlation coefficient > 0.9991. The validated assay has been successfully applied to pharmacokinetic study of ulixertinib in rat after oral and intravenous administration. The results revealed that ulixertinib showed high exposure in rat plasma, low clearance, moderate oral bioavailability (45.13%), and dose‐independent pharmacokinetic profiles over the oral dose range of 1‐15 mg/kg. In addition, six metabolites from rat plasma and hepatocytes were detected and structurally identified by ultra‐high performance liquid chromatography combined with high‐resolution mass spectrometry. The metabolic pathways of ulixertinib referred to hydroxylation and dealkylation and glucuronidation.     

Pharmacokinetics and bioavailability of ipatasertib in dog plasma using LC/MS/MS

A rapid, sensitive, and reliable liquid chromatography-tandem mass spectrometric method was developed to quantify ipatasertib in dog plasma. The dog plasma sample was deproteinated by using acetonitrile with ulixertinib as an internal standard followed by separation on a Spursil C₁₈-EP column with a gradient mobile phase comprising 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile. Positive ion electrospray was used, and multiple reaction monitoring transitions were m/z 458.2 > 387.2 for ipatasertib and m/z 433.1 > 262.1 for the internal standard. The developed method was validated with a linear range of 0.3–1500 ng/mL, and with correlation coefficient greater than 0.9989. The lower limit of quantification was 0.3 ng/mL. The intra- and inter-day precision ranged from 3.58 to 14.32%, whereas the intra- and inter-day accuracy was in the range of −2.50–13.25%. No carry-over and matrix effects were observed under the current conditions. The extraction recovery was demonstrated to be greater than 85.43%. Ipatasertib was stable during the storage, processing, and determination. The validated assay was further successfully applied to a pharmacokinetic study of ipatasertib in dogs after oral and intravenous administrations. The bioavailability of ipatasertib was determined to be 19.3%.     

Pharmacokinetics of pidotimod in broiler chickens by UHPLC–MS/MS after oral and intravenous administration

Pidotimod is widely used in children as an immune promoter but it has not been fully evaluated in animals. The pharmacokinetics of pidotimod and its oral bioavailability have not been described in broiler chickens. We developed a simple and sensitive UHPLC–MS/MS assay for rapid determination of pidotimod levels in chicken blood. Recoveries were nearly 100% and the coefficients of accuracy and precision were minimal. Healthy broiler chickens were given 10 mg/kg pidotimod either orally or intravenously. The oral pidotimod was rapidly absorbed (time to reach maximum concentration, 1.25 h) and rapidly eliminated (the mean residence time was 3.2 h). A noncompartmental analysis of the intravenous route indicated a mean plasma clearance of 2.2 L (h kg)⁻¹ with an estimated mean volume of distribution at steady state of 12.69 L/kg. The bioavailability of pidotimod after oral dosing was 27%.     

Development of HPLC with fluorescent detection using NBD‐F for the quantification of colistin sulfate in rat plasma and its pharmacokinetic applications

Colistin sulfate, composed of a mixture of colistin A sulfate (CLA) and colistin B sulfate (CLB), is available for treating life‐threatening infections caused by multidrug‐resistant Gram‐negative bacteria. In this study, the CLA and CLB were quantified separately. Colistin sulfate was extracted from rat plasma with a solid‐phase extraction C₁₈ cartridge and reacted with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), and the fluorescent derivatives were subjected to reversed‐phase high‐performance liquid chromatography analysis and used to investigate the pharmacokinetics of CLA and CLB in rat plasma. The recovery rates of CLA and CLB were 41.2 ± 4.4 and 45.5 ± 3.1%, respectively. The recovery rate calculated from the total area of CLA and CLB was 43.9 ± 3.6%. When 2 mm NBD‐F and 10 mm boric acid buffer (pH 9.5) were added to colistin sulfate, the highest recovery rate was obtained. The best heating time was 5 min at 60°C. The lower limits of quantification for CLA, CLB and the total area of CLA and CLB were 0.05, 0.05 and 0.1 μg/mL; the coefficients of variations were 13.5, 14.5 and 14.1%, respectively. This method was found to have acceptable linearity, precision and accuracy, and has been successfully applied to a pharmacokinetic study in rat plasma.     

Pharmacokinetics and bioavailability study of ginsenoside Rk1 in rat by liquid chromatography/electrospray ionization tandem mass spectrometry

Ginsenoside Rk1 (Rk1) exhibited various potent biological activities. However, its pharmacokinetic profile in vivo remains unclear. In the present study, a simple and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for determination of Rk1 in rat plasma and applied in a pharmacokinetic study. The sample was precipitated with acetonitrile and separated on a Zorbax Eclipse XDB C18 column (50 × 2.1 mm, 1.8 μm). The mobile phase was composed of 0.1% formic acid in water and acetonitrile at a flow rate of 0.4 mL/min. Rk1 and internal standard (ginsenoside Rg3) were quantitatively monitored with precursor‐to‐product ion transitions of m/z 765.4 → 441.5 and m/z 783.5 → 621.4, respectively. The assay was linear over the concentration range of 5–1000 ng/mL (r > 0.99) with the LLOQ of 5 ng/mL. Other parameters including intra‐ and inter‐day precision and accuracy, extraction recovery and matrix effect were within the acceptable limits. The analyte was stable under the tested storage conditions. The validated method has been successfully applied to a pharmacokinetic study of Rk1 in rat plasma after intravenous (5 mg/kg) and oral (25 mg/kg, 50 mg/kg) administration. After oral administration, Rk1 could be detected in blood at 30 min and reached the highest concentration at 4.29~4.57 h. Our results demonstrated that Rk1 showed low clearance, moderate half‐life (3.09–3.40 h) and low bioavailability (2.87–4.23%). The study will provide information for the further application of Rk1.     

Pharmacokinetics and tissue distribution of coptisine in rats after oral administration by liquid chromatography–mass spectrometry

Coptisine, one of the main components isolated from Coptidis rhizoma, has been reported to have many beneficial pharmacological effects including anti‐inflammatory, anti‐hypercholesterolemia, neuroprotective and cardioprotective properties. However, to date the information related to the in vivo pharmacokinetics (PK) of coptisine is very limited. The purposes of our study are to establish a fast and sensitive quantification method of coptisine using liquid chromatography–mass spectrometry (LC–MS) and evaluate the PK profile of coptisine in rats. The calibration curve for coptisine was linear from 0.78 to 50 ng/mL. After single‐dose oral administration of coptisine, the mean peak plasma concentration values for groups treated with 30, 75 and 150 mg/kg doses ranged from 44.15 to 66.89 ng/mL, and the mean area under the concentration–time curve values ranged from 63.24 to 87.97 mg/L h. The absolute bioavailability was calculated to range from 1.87 to 0.52%. Coptisine remained in all analyzed samples at low concentrations after oral administration of 30 mg/kg.     

The Discovery of Actinospene,a New Polyene Macrolide with Broad Activity against Plant Fungal Pathogens and Pathogenic Yeasts

Phytopathogenic fungi infect crops, presenting a worldwide threat to agriculture. Polyene macrolides are one of the most effective antifungal agents applied in human therapy and crop protection. In this study, we found a cryptic polyene biosynthetic gene cluster in Actinokineospora spheciospongiae by genome mining. Then, this gene cluster was activated via varying fermentation conditions, leading to the discovery of new polyene actinospene (1), which was subsequently isolated and its structure determined through spectroscopic techniques including UV, HR-MS, and NMR. The absolute configuration was confirmed by comparing the calculated and experimental electronic circular dichroism (ECD) spectra. Unlike known polyene macrolides, actinospene (1) demonstrated more versatile post-assembling decorations including two epoxide groups and an unusual isobutenyl side chain. In bioassays, actinospene (1) showed a broad spectrum of antifungal activity against several plant fungal pathogens as well as pathogenic yeasts with minimum inhibitory concentrations ranging between 2 and 10 μg/mL.     

Chitosan Encapsulated Meloxicam Nanoparticles for Sustained Drug Delivery Applications: Preparation,Characterization, and Pharmacokinetics in Wistar Rats

Meloxicam (MLX) is currently used in the therapeutic management of both acute and chronic inflammatory disorders such as pain, injuries, osteoarthritis, and rheumatoid arthritis in both humans and animals. Gastrointestinal toxicity and occasional renal toxicity were observed in patients taking it for a long-term period. Meloxicam’s late attainment of peak plasma concentration results in a slow onset of action. The goal of the current study was to prepare and characterize chitosan encapsulated meloxicam nanoparticles (CEMNPs) with high bioavailability and less gastro intestinal toxicity in order to prevent such issues. The size of the prepared CEMNPs was approximately 110–220 nm with a zetapotential of +39.9 mV and polydispersity index of 0.268, suggesting that they were uniformly dispersed nanoparticles. The FTIR and UV-Vis spectroscopy have confirmed the presence of MLX in the prepared CEMNPs. The pharmacokinetics have been studied with three groups of male Wistar rats receiving either of the treatments, viz., 4 mg·kg⁻¹ of MLX and 1 or 4 mg·kg⁻¹ of CEMNPs. Plasma samples were collected until 48 h post administration, and concentrations of MLX were quantified by using reverse (C₁₈) phase HPLC. Non-compartmental analysis was applied to determine pharmacokinetic variables. Upon oral administration, the maximum concentration (C_max) was reached in 4 h for CEMNPs and 6 h for MLX. The mean area under the plasma MLX concentration-time curve from ‘zero’ to infinity (AUC_0–∞), half-life (t_1/2β), and mean resident time (MRT) of 1 mg·kg⁻¹ of CEMNPs was 1.4-, 2-, and 1.8-fold greater than 4 mg·kg⁻¹ of MLX. The prepared CEMNPs demonstrated quicker absorption and prolonged release along with a significant improvement in the bioavailability of MLX, paving a prospective path for the development of drugs with enhanced bioavailability with less side effects.     

Comparative Pharmacokinetic Study of Forchlorfenuron in Adult and Juvenile Rats

Forchlorfenuron (CPPU) is a plant growth regulator extensively used in agriculture. However, studies on CPPU pharmacokinetics are lacking. We established and validated a rapid, sensitive, and accurate liquid chromatography–mass spectrometry method for CPPU detection in rat plasma. CPPU pharmacokinetics was evaluated in adult and juvenile rats orally treated with 10, 30, and 90 mg/kg of the compound. The area under the plasma drug concentration–time curve from 0 to 24 h (AUC), at the final time point sampled (AUC_0–t), and the maximum drug concentration of CPPU increased in a dose-dependent manner. The pharmacokinetic parameters AUC_0–t and absolute bioavailability were higher in the juvenile rats than in adult rats. The mean residence time and AUC_0–t of juvenile rats in the gavage groups, except for the 10 mg/kg dose, were significantly higher in comparison to those observed for adult rats (p < 0.001). The plasma clearance of CPPU in juvenile rats was slightly lower than that in the adult rats. Taken together, juvenile rats were more sensitive to CPPU than adult rats, which indicates potential safety risks of CPPU in minors.     

Epipyrone A,a Broad-Spectrum Antifungal Compound Produced by Epicoccum nigrum ICMP 19927

We have isolated a filamentous fungus that actively secretes a pigmented exudate when growing on agar plates. The fungus was identified as being a strain of Epicoccum nigrum. The fungal exudate presented strong antifungal activity against both yeasts and filamentous fungi, and inhibited the germination of fungal spores. The chemical characterization of the exudate showed that the pigmented molecule presenting antifungal activity is the disalt of epipyrone A—a water-soluble polyene metabolite with a molecular mass of 612.29 and maximal UV–Vis absorbance at 428 nm. This antifungal compound showed excellent stability to different temperatures and neutral to alkaline pH.     

Eco-Friendly UPLC–MS/MS Method for Determination of a Fostamatinib Metabolite,Tamatinib, in Plasma: Pharmacokinetic Application in Rats

Fostamatinib is a prodrug of the active metabolite tamatinib, which is a spleen tyrosine kinase (Syk) inhibitor used in the treatment of primary chronic adult immune thrombocytopenia and rheumatoid arthritis. A highly sensitive, rapid, reliable, and green method was developed and validated using ultra-performance liquid chromatography and tandem mass spectrometry (UPLC–MS/MS) for quantification of tamatinib in rat plasma. Ibrutinib was used as internal standard and liquid–liquid extraction was applied using tert-butyl methyl ether. The analyte was separated on an Acquity^TM CSH C₁₈ (2.1 mm × 100 mm, 1.7 µm) column using mobile phase consisting of 10 mM ammonium acetate and acetonitrile (10:90) and the flow rate was 0.25 mL/min. Electrospray ionization (ESI) was carried out in positive mode. Quantitation of tamatinib and the IS was performed using multiple reaction monitoring mode with precursor-to-product transitions of m/z 471.1 > 122.0 and m/z 441.1 > 84.0, respectively. The calibration range was 0.1–1000.0 ng/mL and the linearity of the method was ≥0.997. The developed method greenness was investigated. All principal parameters for the method, including linearity, accuracy, precision, recovery, and stability, were within acceptable ranges. Tamatinib pharmacokinetic study in rats was successfully carried out using the developed method.     

Pharmacokinetics,bioavailability and tissue distribution studies of rhodojaponin III in mice using QTRAP LC–MS/MS

Rhodojaponin III is a bioactive diterpenoid isolated from the medicinal plant Rhododendron molle G. Don. Quantitative analysis of rhodojaponin III was challenging and the pharmacokinetics of oral rhodojaponin III remained to be investigated. Here, a rapid and sensitive liquid chromatography tandem mass spectrometric (LC–MS/MS) method was developed and validated. The calibration curve was linear over the concentration range of 1–200 ng/mL (r = 0.992). The method was further validated following internationally approved guidelines and all the issues including intra‐ and inter‐day precision, accuracy, carryover, extraction recovery, matrix effects and stability met the recommended limits. The method was then applied to study the pharmacokinetics of rhodojaponin III in mice after intravenous (0.06 mg/kg) or oral (0.24 mg/kg) administration. The results showed that rhodojaponin III had fast oral absorption (time to peak concentration, 0.08 h) and good oral bioavailability (73.6%). In addition, rhodojaponin III was quickly eliminated after it was intravenously or orally administered, with half‐life values of 0.19 and 0.76 h, respectively. After oral administration, it was widely distributed in tissues including kidney, lung, heart, spleen and thymus, but had extremely low concentrations in liver and brain. The data presented in this study is beneficial for the further study of rhodojaponin III.     

Determination of 3-hydroxy pterocarpan, a novel osteogenic compound in rat plasma by liquid chromatography-tandem mass spectrometry: application to pharmacokinetics study

A rapid, sensitive and selective LC‐MS/MS method for the quantitative analysis of 3‐hydroxy pterocarpan (S006‐1709) in female rat plasma has been developed and validated. A Discovery RP₁₈ column was used for the chromatographic elution using acetonitrile and 0.1% acetic acid in water as mobile phase (80:20 v/v) at the flow rate of 0.5 mL/min. MS/MS analysis was performed using a triple quadrupole mass spectrometer with electrospray ionization in negative ion mode using biochanin as an internal standard (IS). Extraction of S006‐1709 and IS from rat plasma was done by liquid–liquid extraction method using diethyl ether. The LC‐MS/MS method was sensitive with 1.95 ng/mL as the limit of detection and 3.9 ng/mL as the lower limit of quantification. The method was linear in the concentration range of 3.9–1000 ng/mL. The percentage bias for intraday and interday accuracy was not greater than 4.2 and the %RSD for intraday and interday precision was not greater than 13.2. The recoveries of S006‐1709 and IS were 73.9–79.3 and 85.7%, respectively. S006‐1709 was found to be stable in various stability studies. The validated LC‐MS/MS method was successfully applied for the oral pharmacokinetics study of S006‐1709 at 10 mg/kg in female Sprague–Dawley rats. Copyright © 2010 John Wiley & Sons, Ltd.     

A fast, sensitive and simple method for mirtazapine quantification in human plasma by HPLC-ESI-MS/MS. Application to a comparative bioavailability study

In the present study a simple, fast, sensitive and robust method to quantify mirtazapine in human plasma using quetiapine as the internal standard (IS) is described. The analyte and the IS were extracted from human plasma by a simple protein precipitation with methanol and were analyzed by high‐performance liquid chromatography coupled to an electrospray tandem triple quadrupole mass spectrometer (HPLC‐ESI‐MS/MS). Chromatography was performed isocratically on a C₁₈, 5 µm analytical column and the run time was 1.8 min. The lower limit of quantitation was 0.5 ng/mL and a linear calibration curve over the range 0.5–150 ng/mL was obtained, showing acceptable accuracy and precision. This analytical method was applied in a relative bioavailability study in order to compare a test mirtazapine 30 mg single‐dose formulation vs a reference formulation in 31 volunteers of both sexes. The study was conducted in an open randomized two‐period crossover design and with a 14 day washout period. Since the 90% confidence interval for C_max, AUC_last and AUC_0–inf were within the 80–125% interval proposed by the Food and Drug Administration and ANVISA (Brazilian Health Surveillance Agency), it was concluded that mirtazapine 30 mg/dose is bioequivalent to the reference formulation, according to both the rate and extent of absorption. Copyright © 2012 John Wiley & Sons, Ltd.