Simultaneous determination of TUG-891 and its metabolites in rat plasma using LC–HRMS with application to preclinical pharmacokinetic study

In this study, a simple and reliable LC–MS/MS method was first proposed for the simultaneous determination of TUG-891 and its metabolites TUG-891-alcohol, TUG-891-aldehyde, and TUG-891-acid in rat plasma. The analytes and fasiglifam (internal standard) were extracted from plasma samples with acetonitrile and separated using an Acquity BEH C₁₈ column (1.7 μm, 2.1 × 50 mm) with water containing 0.05% ammonium hydroxide and acetonitrile containing 0.05% ammonium hydroxide as the mobile phase. A Q-Exactive Orbitrap mass spectrometer in full-scan mode was used for mass detection, and the data analysis was obtained using a mass extraction window of 5 ppm. The calibration curves exhibited excellent linearity (correlation coefficient > 0.9981) in the concentration range of 0.5–1000 ng/mL. The lower limit of quantification was 0.5 ng/mL for all analytes. The intra- and inter-day precision was less than 11.31%, and the accuracy ranged from −11.50 to 9.50%. The extraction recovery of the analytes from rat plasma was greater than 82.31%, and no obvious matrix effect was found. The established method was further applied to the pharmacokinetic study of TUG-891, TUG-891-alcohol, TUG-891-aldehyde, and TUG-891-acid in rat after a single dose of 5-mg/kg treatment of TUG-891. The results demonstrated that TUG-891 was rapidly metabolized into its metabolites and the systemic exposures of the metabolites were much higher than those of TUG-891.     

Determination of rosamultin in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A specific and reliable LC–MS/MS method for the determination of rosamultin in rat plasma was validated. Plasma samples were prepared with protein precipitation method, and chromatographic separation was performed on a Thermo C₁₈ analytical column (4.6 mm × 50 mm, 3.0 μm). The mass spectrometry (MS) analysis was conducted in positive SRM mode for the transitions of m/z 673.2 → 511.1 for rosamultin and m/z 601.1 → 330.9 for IS. The method validation was conducted over the calibration range of 1.0–500 ng/mL with the precision ≤11.03% and accuracy within ±14.64%. The assay was applied to the pharmacokinetic study after oral administration of rosamultin at a dose of 20 mg/kg in rats.     

Simultaneous measurement of epinastine and its metabolite, 9,13b-dehydroepinastine,in human plasma by a newly developed ultra-performance liquid chromatography–tandem mass spectrometry and its application to pharmacokinetic studies

Epinastine is an antiallergic drug with high selectivity for histamine receptors. It has been reported that 9,13b-dehydroepinastine is present as a metabolite in vivo in humans, but there was little information about their pharmacokinetics (PKs) in humans. Although several analytical methods have been reported for epinastine analysis in different matrices, none are available for its metabolite. Therefore, the purpose of this study was to develop an analytical method to simultaneously measure epinastine and its metabolite, 9,13b-dehydroepinastine, in human plasma samples using an ultra-performance liquid chromatography–tandem mass spectrometer. Analytes were separated on a C₁₈ column. Quantification of this analysis was performed on a triple-quadrupole mass spectrometer. Chromatograms showed high sensitivity, selectivity, and resolution with no interference with plasma constituents. Calibration curves for both epinastine and 9,13b-dehydroepinastine in human plasma were 0.1–50 ng/ml and displayed excellent linearity with correlation coefficients (r²) >0.99. The developed analytical method satisfied the criteria of international guidance and was validated. The method could be successfully applied to pharmacokinetic studies of epinastine and, for the first time, the metabolite kinetics of epinastine to 9,13b-dehydroepinastine in humans after oral administration of 20 mg epinastine hydrochloride tablets. Our study is expected to be useful in future studies such as dosage settings and clinical pharmacotherapy.     

Quantitative determination of bilobetin in rat plasma by HPLC–MS/MS and its application to a pharmacokinetic study

Although bilobetin, a biflavone isolated from the leaves of Ginkgo biloba, represents a variety of pharmacological activities, to date there have been no validated determination methods for bilobetin in biological samples. Thus, we developed a liquid chromatographic method using a tandem mass spectrometry for the determination of bilobetin in rat plasma. After protein precipitation with acetonitrile including diclofenac (internal standard), the analytes were chromatographed on a reversed-phased column with a mobile phase of purified water and acetonitrile (3:7, v/v, including 0.1% formic acid). The ion transitions of the precursor to the product ion were principally deprotonated ions [M − H]⁻ at m/z 551.2 → 519.2 for bilobetin and 296.1 → 251.7 for the IS. The accuracy and precision of the assay were in accordance with US Food and Drug Administration regulations for the validation of bioanalytical methods. This analytical method was successfully applied to monitor plasma concentrations of bilobetin over time following intravenous administration in rats.     

Simultaneous determination of novel β-lactamase inhibitor WCK 4234 and Meropenem in dog plasma by LC–MS/MS and its application to preclinical pharmacokinetic study

A precise and accurate liquid chromatography–tandem mass spectrometric (LC–MS/MS) bioanalytical method has been developed and validated for the simultaneous quantification of WCK 4234 and meropenem (MEM) in dog plasma. Protein precipitation using acetonitrile was employed as a sample preparation approach. Cefepime was used as an internal standard. The developed method was selective, sensitive (limit of quantification, 0.075 μg/ml for both drugs), accurate (recovery > 90%), precise (CV < 10%) and linear (r² ≥ 0.99, concentration range 0.075–120 μg/ml for both analytes). The developed method was successfully applied for the determination of both drugs in plasma to assess the pharmacokinetics in beagle dogs. WCK 4234 + MEM in a 1:1 ratio at 15 + 15 and 30 + 30 mg/kg doses were administered by the intravenous route. The mean plasma concentration and area under the concentration–time curve of WCK 4234 ranged from 38.3 to 77.4 μg/ml and from 47.8 to 77.1 μg h/ml, respectively, and the values for MEM ranged from 52.2 to 115.3 μg/ml and 70.5 to 133.6 μg h/ml respectively. The elimination half-life of WCK 4234 and MEM was around 0.8 h.     

LC–MS/MS assay for the quantification of foretinib in rat plasma and its application to preclinical pharmacokinetic study

A simple and sensitive ultra-high-performance liquid chromatography tandem mass spectrometric method was developed and validated for the determination of foretinib in rat plasma. The analyte and internal standard were extracted from the bio-samples with acetonitrile and then separated on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) using 0.1% formic acid aqueous and acetonitrile as mobile phase, at a flow rate of 0.4 ml/min. The mass detection was performed in positive selected reaction monitoring mode with precursor-to-product transitions at m/z 317.1 > 128.1 for foretinib and m/z 502.2 > 323.1 for internal standard. The assay was demonstrated to be linear in the concentration range of 0.5–1000 ng/ml, with correlation coefficient >0.999. The mean extraction recovery of foretinib from rat plasma was within the range of 84.55–88.09%, while the matrix effect was in the range of 88.56–99.21%. The intra- and inter-day precisions were <12.95% and the accuracy ranged from −7.55 to 8.57%. Foretinib was stable in rat plasma under the tested storage conditions. The validated assay was successfully applied to the pharmacokinetic study of foretinib in the rats. The results revealed that foretinib showed moderate elimination half-life, low clearance and dose-independent pharmacokinetic profiles inrats.     

Determination of ginsenoside Rh3 in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

Ginsenoside Rh3 (GRh3) is a bacterial metabolite of ginsenoside Rg5, which is the main component of hot-processed ginseng. A simple, efficient and sensitive method was developed and validated for the determination of GRh3 in rat plasma by LC–tandem mass spectrometry. After protein precipitation with methanol/acetonitrile (1:1, vol/vol) using propranolol as the internal standard, the target analytes were separated on an XDB C₁₈ column, with methanol containing 0.1% formic acid and water containing 0.1% formic acid used as mobile phases for gradient elution. Mass spectrometry was performed in electrospray ion source–positive ion mode and multiple reaction monitoring mode, monitoring the transitions m/z 622.5 → 425.5 and m/z 260.1 → 116.1 for GRh3 and internal standard, respectively. The concentration range of GRh3 was 20–20,000 ng/mL and the correlation coefficient (r²) was greater than 0.99. The accuracy error and relative standard deviation were below 15%. The extraction recovery and matrix effect were 74.2% to 78.7% and 96.9% to 108.4%, respectively. Under different conditions, GRh3 was stable in the range of 1.8%–8.7%. This method has been successfully applied to study the pharmacokinetics of GRh3 with an oral dose of 10.0 mg/kg and an intravenous dose of 2.0 mg/kg in rats, respectively. The absolute bioavailability of GRh3 was 37.6%.     

Simultaneous determination of eight bioactive components in Marsdenia tenacissima extract in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

As a traditional Chinese medicine, Marsdenia tenacissima (Roxb.) Wight et Arn. plays an indispensable role in clinical practice owing to its specific efficacy in treating malignant tumors, leukocythemia, cystitis and asthma. This study aimed to establish a novel and scientific LC–MS/MS approach to simultaneously determine tenacissoside B, H, G and I, caffeic acid, cryptochlorogenic acid, chlorogenic acid and neochlorogenic acid from M. tenacissima extract within the rat plasma samples. Digoxin was used as the internal reference. All determinations were carried out using the Eclipse Plus C₁₈ column, and water (containing 0.1% formic acid) was used as the mobile phase A, while acetonitrile was the mobile phase B for gradient elution. The UPLC methods were validated, including calibration curves, accuracy, precision, stability and recovery of the total eight analytes, in accordance with the requirements for biopharmaceutical analysis. Moreover, the proposed approach was also used in comprehensive pharmacokinetic research on those eight analytes in rats following M. tenacissima extract gavage. According to the pharmacokinetic parameters, tenacissoside B, I, H and G are the long-acting and primary bioactive constituents in M. tenacissima extract, with long mean residence times and high concentrations. Our findings shed light on the absorption mechanism and provide significant information for the clinical application of M. tenacissima.     

Determination of mitragynine in plasma with solid-phase extraction and rapid HPLC–UV analysis, and its application to a pharmacokinetic study in rat

A new solid phase extraction method for rapid high performance liquid chromatography–UV determination of mitragynine in plasma has been developed. Optimal separation was achieved with an isocratic mobile phase consisting of acetonitrile–ammonium acetate buffer, 50 mM at pH 5.0 (50:50, v/v). The method had limits of detection and quantification of 0.025 and 0.050 μg/mL, respectively. The method was accurate and precise for the quantitative analysis of mitragynine in human and rat plasma with within-day and between-day accuracies between 84.0 and 109.6%, and their precision values were between 1.7 and 16.8%. Additional advantages over known methods are related to the solid phase extraction technique for sample preparation which yields a clean chromatogram, a short total analysis time, requires a smaller amount of plasma samples and has good assay sensitivity for bioanalytical application. The method was successfully applied in pharmacokinetic and stability studies of mitragynine. In the present study, mitragynine was found to be fairly stable during storage and sample preparation. The present study showed for the first time the detailed pharmacokinetic profiles of mitragynine. Following intravenous administration, mitragynine demonstrated a biphasic elimination from plasma. Oral absorption of the drug was slow, prolonged and was incomplete, with a calculated absolute oral bioavailability value of 3.03%. The variations observed in previous pharmacokinetic studies after oral administration of mitragynine could be attributed to its poor bioavailability rather than to the differences in assay method, metabolic saturation or mitragynine dose.     

LC–MS/MS method for determination of kansuinine a in rat plasma and its application to a rat pharmacokinetic study

Kansuinine A is a macrocyclic jatrophane diterpene isolated from the plant Euphorbia kansui Liou. It exhibits many pharmacological activities including cytoxic, antitumor, antiallergic and proinflammatory effects. In the present study, a simple and sensitive LC–MS/MS method was established and validated for the determination of kansuinine A in rat plasma. After methanol-mediated protein precipitation, chromatographic separation was achieved on an Acquity BEH C₁₈ column (2.1 × 100 mm, 1.7 μm) using acetonitrile and 0.1% formic acid in water as mobile phase by gradient elution. Kansuinine A and IS were quantified in negative multiple reaction monitoring mode with ion transitions at m/z 731.1–693.2 for kansuinine A and m/z 723.2–623.1 for IS. The method showed excellent linearity over the range 1–500 ng/ml. The intra- and inter-day precisions (relative standard deviation) were 2.13–4.28 and 3.83–7.67%, respectively, whereas accuracy (relative error) ranged from −4.17 to 3.73%. The extraction recovery, stability and matrix effect met the requirement of the regulations issued by the US Food and Drug Administration. The validated method was successfully applied to the pre-clinical pharmacokinetic study of kansuinine A in rats after oral administration (20 mg/kg) and intravenous administration (2 mg/kg). This study provides valuable reference for the further study of E. kansui liou, especially for the drug development and clinical application of kansuinine A.     

Method development and validation for simultaneous determination of ebastine and its active metabolite carebastine in human plasma by liquid chromatography–tandem mass spectrometry and its application to a clinical pharmacokinetic study in healthy Chinese volunteers

A simple LC–tandem mass spectrometry (MS/MS) method to determine ebastine and carebastine (active metabolite) in human plasma was developed and validated. Analytes and internal standards were precipitated by protein precipitation and separated on Synergi Hydro-RP 80A column (4 μm, 50 mm × 2.0 mm; Phenomenex) by gradient elution with mobile phase A comprising 0.1% formic acid in 5 mm ammonium acetate (NH₄Ac) and B comprising 100% methanol at a flow rate 0.4 mL/min. Ions were detected in positive multiple reaction monitoring mode, and they exhibited linearity over concentration range 0.01–8.0 and 1.00–300 ng/mL for ebastine and carebastine, respectively. A clinical pharmacokinetic study was conducted in healthy Chinese volunteers under fasting and fed conditions after a single oral administration of 10 mg ebastine. The maximum plasma concentration (C_max), time to C_max (T_max) and elimination half-life for ebastine were 0.679 ± 0.762 ng/mL, 1.67 ± 1.43 h and 7.86 ± 6.18 h, respectively, whereas these for carebastine were 143 ± 68.4 ng/mL, 5.00 ± 2.00 h and 17.4 ± 4.97 h, respectively under fasting conditions; the corresponding values under fed conditions were 4.13 ± 2.53 ng/mL, 3.18 ± 1.09 h and 21.6 ± 7.77 h for ebastine and 176 ± 68.4 ng/mL, 6.14 ± 2.0 h and 20.0 ± 4.97 h for carebastine.     

Quantification of α- and β-amyrin in rat plasma by gas chromatography-mass spectrometry: application to preclinical pharmacokinetic study

α- and β-Amyrins are naturally occurring triterpenes with a wide range of biological activities. In this study, a reliable GC-MS method was developed and validated for the quantification of α- and β-amyrins in rat plasma. The calibration curves were linear (R(2) > 0.996) with a limit of quantification of 1.0 ng ml(-1) for both α- and β-amyrins. The precision and repeatability of this method was good as the relative standard deviation were 12% or less. The absolute recovery ranged from 71% to 89%, while the analytical recovery ranged from 95% to 99%. The pharmacokinetic profiles of α- and β-amyrins in rats were subsequently investigated in Sprague-Dawley rats. β-Amyrin was administered intravenously and also orally in two forms, namely, as a suspension of the pure compound and the crude plant extract. α-Amyrin was administered orally as a suspension of the crude plant extract. β-Amyrin had a very long terminal elimination half-life (t(1/2λz) = 610 ± 179 min) and extremely slow clearance (Cl = 2.04 ± 0.24 ml min(-1) kg(-1)). The absolute oral bioavailability of β-amyrin in the crude plant extract was about fourfold higher than that in the suspension of pure form (3.83% vs 0.86%). When given in crude plant extract, both α- and β-amyrins had a similar dose normalized C(max). This reliable GC-MS method will enable further pharmacokinetic investigations of α- and β-amyrins.     

Determination of meloxicam in human plasma by ultra-high-performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to detect meloxicam in human plasma. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source was used in positive ion mode. Protein precipitation with acetonitrile was used for sample preparation. Meloxicam and ¹³C₆-meloxicam internal standard were analyzed on an Acquity CSH C₁₈ column with a mobile phase of acetonitrile and water in 0.1% formic acid using a gradient program for separation. The retention time of meloxicam was 1.1 min and the total run time was only 2.0 min. Detection was performed in multiple reaction monitoring mode using an electrospray ionization source with optimized mass spectrometry parameters. The calibration curves were linear in the range 10.0–3.00 × 10³ ng/ml (r ≥ 0.99). The within-run and between-run RSDs were ≤14.8%. The within-run and between-run REs ranged from −4.6 to 10.7%. There was no significant matrix effect, and the recovery rate was high. This method was fully validated, including reinjection reproducibility in human plasma. The method was applied to the pharmacokinetic study. All of the incurred sample reanalysis methods met the criteria.     

A rapid UHPLC–MS/MS method for the quantification of ARQ531 in rat plasma: Validation and its application to a pharmacokinetic study

A simple and sensitive ultra-high performance liquid chromatography–tandem mass spectrometric (UHPLC–MS/MS) method was developed and validated for the determination of ARQ531, a Bruton’s tyrosine kinase inhibitor in rat plasma. After protein precipitation with acetonitrile, the samples were separated on a UPLC BEH C₁₈ column with 0.1% formic acid in water and acetonitrile as mobile phase at a flow rate of 0.4 ml/min. The mass detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring with precursor-to-product ion transitions of m/z 479.1 > 365.1 and m/z 441.2 > 138.1 for ARQ531 and internal standard, respectively. Good linearity (correlation coefficient > 0.9988) was achieved over the concentration range of 0.5–1,000 ng/ml and the lower limit of quantitation was 0.5 ng/ml. The accuracy ranged from −13.50 to 11.35% and the precision was <8.87%. The extraction recovery was >85.56%. ARQ531 was demonstrated to be stable under the tested conditions. The validated method was further applied to a pharmacokinetic study of ARQ531 in rats after intravenous (1 mg/kg) and oral (1, 3 and 10 mg/kg) administration. The results demonstrated that ARQ531 displayed linear pharmacokinetic profiles over the oral dose range of 1–10 mg/kg and good oral bioavailability (>50%).     

LC–MS/MS analysis of puerarin and 18β-glycyrrhetinic acid in human plasma after oral administration of Samso-eum and its application to pharmacokinetic study

The aim of this study was to confirm pharmacokinetic screening of multiple components in healthy Korean subjects after oral administration of Samso-eum and perform quantitation of active components in the human plasma. Thirteen potential bioactive components [puerarin (PRR), daidzin, nodakenin, ginsenoside Rb1, 18β-glycyrrhetinic acid (18β-GTA), 6-shogaol, naringin, glycyrrhizin, hesperidin, platycodin D, naringenin, hesperetin, and 6-gingerol] were screened based on literature. The results showed that three analytes (daidzin, naringenin, and hesperetin) were detected in trace amounts. In addition, PRR and 18β-GTA were detected in human plasma after the oral administration of Samso-eum. In this study, a liquid chromatography–electrospray ionization-tandem mass spectrometry method was validated for the simultaneous determination of PRR and 18β-GTA in human plasma. This was the first study to evaluate pharmacokinetics of PRR and 18β-GTA after the usual oral dose of Samso-eum (30 g containing 102.48 mg PRR, 48.18 mg glycyrrhizin) in human subjects.     

Determination of the immunostimulatory drug—glucosoaminyl-muramyl-dipeptide—in human plasma using HPLC–MS/MS and its application to a pharmacokinetic study

GMDP (glucosoaminyl-muramyl-dipeptide), a synthetic analog of the peptidoglycan fragment of the bacterial cell wall, is an active component of the immunomodulatory drug Licopid. But the pharmacokinetic parameters of GMDP in humans after oral administration have not been investigated yet. The present study aimed at developing and validating a sensitive LC–MS/MS method for the analysis of GMDP in human plasma. The sample was prepared by solid-phase extraction using Strata-X 33 μm polymeric reversed-phase 60 mg/3 mL cartridges Phenomenex (Torrance, CA, USA). The analytes were separated using an Acquity UPLC BEN C18 column, 1.7 μm 2.1 × 50 mm Waters (Milford, USA). GMDP and internal standard growth hormone releasing peptide-2 (pralmorelin) were ionized in positive electrospray ionization mode and detected in multiple reaction monitoring mode. The developed method was validated within a linear range of 50–3000 pg/mL for GMDP. Accuracy for all analytes, given as the deviation between the nominal and measured concentration and assay variability , ranged from 1.61 to 3.02% and from 0.89 to 1.79%, respectively, for both within- and between-run variabilities. The developed and validated HPLC–MS/MS method was successfully used to obtain the plasma pharmacokinetic profiles of GMDP distribution in human plasma.     

Simultaneous determination of sutetinib and its active metabolite sutetinib N-oxide in human plasma by liquid chromatography–tandem mass spectrometry: Evaluation of plasma stability

From the point of view of drug efficacy and safety, pharmacokinetic profiles of both In this work, a sensitive and reliable liquid chromatographic–tandem mass spectrometric method was established for simultaneous determination of sutetinib and N-oxide metabolite (SNO) in human plasma and further applied to a pharmacokinetic study. Analytes were extracted from plasma samples (100 μl) via acetonitrile-induced protein precipitation and separated on a C₁₈ column using ammonium acetate with ammonium hydroxide and acetonitrile as the mobile phase. Positive electrospray ionization was carried out through multiple reaction monitoring with transitions of m/z 440.2 → 367.1 and 446.2 → 367.1 for sutetinib and SNO, respectively. The method was linear within the concentration range of 0.5–100 ng/ml for both analytes. The precision, accuracy, selectivity, recovery and matrix effect of this method all met the requirements of bioanalytical guidance. In addition, a plasma stability assessment demonstrated unexpected results. Sutetinib was prone to form covalent conjugates with plasma albumin in vitro. The degree of covalent binding increased with increasing temperature, resulting in a significant decrease in its plasma concentrations. However, SNO could not easily bind with albumin owing to steric hindrance or electronegativity. Furthermore, sutetinib and SNO remained stable when blood and plasma samples were kept on wet ice. The validated method was successfully employed for the pharmacokinetic evaluation of sutetinib in patients with advanced malignant solid tumors.     

A MSALL quantitative method for the determination of Poloxamer 188 in rat plasma by UHPLC–Q-TOF/MS and its application to pharmacokinetic study

Poloxamer (PL)188 is a commonly used pharmaceutical excipient with unique physicochemical properties. In this study, an MS^ALL quantitative method for the determination of PL188 in rat plasma by UHPLC–Q-TOF/MS was developed and validated. PL188 was analyzed on PLRP-S reversed-phase column (50 × 4.6 mm, 8 μm, 1,000 Å) with mobile phase 0.1% formic acid–water and 0.1% formic acid in acetonitrile–isopropanol (2:3, v/v). The liner range was 0.1–10.0 μg/ml. A pharmacokinetic study was performed on rats at a dose of 5 mg/kg by intravenous injection. The pharmacokinetic parameters of intravenous injection were as follows: half-life was 2.0 ± 1.1 h, volume of distribution was 5.1 ± 3.2 L/kg, area under the concentration–time curve was 3.0 ± 0.6 μg/L h and clearance was 1.7 ± 0.3 L/h/kg. The results indicated that PL188 could be rapidly distributed to tissues with a high clearance rate. This study can provide a good reference for the further study of PL188.     

Development and validation of novel LC–MS/MS method for determination of Lusutrombopag in rat plasma and its application to pharmacokinetic studies

A novel, simple and sensitive method for the determination of Lusutrombopag in rat plasma using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated. The determination was performed on an API4000 triple quadrupole mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]⁺ ions m/z 593.1 → 272.3 for Lusutrombopag. The limit of detection was 0.5 ng/mL, and the lower limit of quantification was 2.0 ng/mL in rat plasma. Good linearity was obtained over the range of 2.0–150.0 ng/mL and the correlation coefficient was found to be 0.9998. The intra and inter-day precisions were found to be 3.8–6.9% and 6.8–10.5%, respectively. The intra and inter-day accuracy derived from QC samples was found to be 2.5–4.9% and 5.5–7.2%, respectively. The analyte was stable under various conditions (at room temperature, during freeze-thaw, in the autosampler and under deep-freeze conditions). The F-test and t-test at 95% confidence level were subjected on data for statistical analysis. The developed method was successfully applied to the pharmacokinetic study in rats.