Determination of mangiferin in rat plasma by liquid–liquid extraction with UPLC–MS/MS

An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method employing electrospray ionization (ESI) has been developed for the determination of mangiferin in rat plasma using diphenhydramine as the internal standard (IS). Liquid–liquid extraction (LLE) was used for sample preparation and the analysis was achieved with gradient elution on C₁₈ reversed phase column. The method was validated over the concentration range 0.02–5.0 μg/mL for oral administration and 0.4–100 μg/mL for intravenous administration. The intra-day and inter-day precision of mangiferin expressed as RSD < 15% and the accuracy (RE) did not exceed 15%. This validated method is a novel technique for sample preparation and quantitation, which was successfully applied to estimate the bioavailability of mangiferin.     

Enantioselective determination of sibutramine and its active metabolites in human plasma

Although racemic sibutramine has been widely used for the treatment of obesity, its enantioselective detection method has not been elucidated in human plasma. In this report we introduce a validated analytical method for the determination of sibutramine and its two active metabolites, desmethylsibutramines using LC–MS/MS. R- and S-isomers of those compounds in human plasma were extracted using diethyl ether–hexane (4:1, v/v) followed by an addition of NaOH solution. After removing the organic layer, the residue was reconstituted in the mobile phase 10 mM ammonium acetate solution adjusted to pH 4.0 with acetic acid–acetonitrile (94:6, v/v). Both isomers in the extract were separated on a Chiralcel AGP chiral stationary-phase column and were quantified in a tandem mass spectrometry. The assay method was in accordance with FDA regulations for the validation of bioanalytical methods. This method was successfully used to profile the plasma concentrations of the stereoisomers of sibutramine and its two active metabolites with time in healthy volunteers.     

A rapid and sensitive LC–ESI-MS/MS method for detection and quantitation of methylprednisolone and methylprednisolone acetate in rat plasma after intra-articular administration

A rapid, sensitive and specific liquid chromatography–electrospray-tandem mass spectrometric (LC–ESI-MS/MS) method for the simultaneous detection and quantitation of methylprednisolone acetate (MPA) and methylprednisolone (MP) in rat plasma, using a triple-stage quadrupole, has been developed and validated.     

A direct LC/MS/MS method for the determination of ciclopirox penetration across human nail plate in in vitro penetration studies

Due to severe chelating effect caused by N-hydroxylpyridone group of ciclopirox, there is no published direct HPLC or LC/MS/MS method for the determination of ciclopirox in any in vitro or in vivo matrix. Instead, the time-consuming pre-column derivatization methods have been adapted for indirect analysis of ciclopirox. After overcoming the chelating problem by using K₂EDTA coated tubes, a direct, sensitive and high-throughput LC/MS/MS method was successfully developed and validated to determine the amount of ciclopirox that penetrated across the nail plate during in vitro nail penetration studies. The method involved adding a chemical analog, chloridazon as internal standard (IS) in K₂EDTA coated tubes, mixing IS with ciclopirox in a 96-well plate and then proceeding to LC/MS/MS analysis. The MS/MS was selected to monitor m/z 208.0 → 135.8 and 221.8 → 77.0 for ciclopirox and IS, respectively, using positive electrospray ionization. The method was validated over a concentration range of 8–256 ng/mL, yielding calibration curves with correlation coefficients greater than 0.9991 with a lower limit of quantitation (LLOQ) of 8 ng/mL. The assay precision and accuracy were evaluated using quality control (QC) samples at three concentration levels. Analyzed concentrations ranged from 101% to 113% of their respective nominal concentration levels with coefficients of variation (CV) below 10.6%. The average recovery of ciclopirox from nail matrix was 101%.     

An LC–MS/MS method for the simultaneous determination of chlorogenic acid,forsythiaside A and baicalin in rat plasma and its application to pharmacokinetic study of Shuang-huang-lian in rats

An LC–MS/MS method was developed for the simultaneous determination of chlorogenic acid, forsythiaside A and baicalin, three major ingredients in Shuang-huang-lian preparations, in rat plasma. Following extraction by methanol–ethyl acetate–trifluoroacetic acid (49:49:2, v/v/v), the extracted analytes were separated on a reverse phase C12 column using a gradient mobile phase system of acetonitrile–water containing 0.1% formic acid. The limits of quantification were between 1.0 and 2.1 ng/mL, the precision was <7% and the accuracy was between 94% and 107%. The validated method was applied to a comparative pharmacokinetic study in rats after administration of Shuang-huang-lian solutions via intravenous, peroral or intratracheal routes. The results showed that the three chemical markers were more rapidly and thoroughly absorbed following pulmonary delivery as compared with peroral administration.     

Characterization of homoisoflavonoids in different cultivation regions of Ophiopogon japonicus and related antioxidant activity

Homoisoflavonoids were identified in Ophiopogon japonicus of two cultivation regions in China: Hang-maidong (grown in Zhejiang province) and Chuan-maidong (grown in Sichuan province). Liquid chromatography (LC), coupled with electrospray ionization (ESI) tandem mass spectrometry (MS), was developed to analyze homoisoflavonoids in these two areas. Based on LC–MS/MS data, 24 homoisoflavonoid compounds—19 in Hang-maidong and 17 in Chuan-maidong—were identified or tentatively characterized. The homoisoflavonoids in the two regions were similar in chemical profile, but distinctive in their combination and ratio. Methylophiopogonanone A and methylophiopogonanone B were the major contributors to the total homoisoflavonoid content. Hang-maidong had a higher total homoisoflavonoid content and better DPPH (2,2-diphenyl-1-picryl-hydrazyl) radical-scavenging activity than that of Chuan-maidong. The total phenolic content showed no significant difference between the two regions. This study allows a clear chemical differentiation of Hang-maidong from Chuan-maidong.     

A sensitive LC/MS/MS bioanalysis assay of orally administered lipoic acid in rat blood and brain tissue

A sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) bioanalytical method was developed and validated to analyze lipoic acid (LA) in rat blood and brain samples. Ten mobile phase combinations were investigated during method development. Mobile phase combination of 0.1% acetic acid (pH 4 adjusted with ammonia solution)/acetonitrile was most optimum in terms of sensitivity and peak shape of LA and the internal standard, valproic acid. Sample extraction method was explored using liquid–liquid extraction and protein precipitation methods. Protein precipitation yielded the highest recovery of the analytes from blood and brain ranging from 92 to 115%. The lower limit of quantitation (LLOQ) of LA was 0.1 ng/mL (0.485 nM) in both blood and brain while on-column lower limit of detection (LLOD) was 0.03 pg. The precision (% R.S.D.) ranged from 1.49 to 26.39% and 1.49 to 10.89% for intra- and inter-day assays, respectively. The accuracy ranged from 91.2 to 116.17% for intra-day assay and 102.68 to 114.33% for inter-day assay.     

Simultaneous characterization and quantitation of 11 coumarins in Radix Angelicae Dahuricae by high performance liquid chromatography with electrospray tandem mass spectrometry

A high performance liquid chromatography with electrospray tandem mass spectrometry (HPLC–ESI-MS/MS) method has been developed to characterize and quantify 11 coumarin compounds in Radix Angelicae Dahuricae simultaneously. By using this HPLC–ESI-MS/MS method, all 11 coumarins were separated and determined within 10 min. These coumarins were detected by ESI⁺ ionization method and quantified by multiple reaction monitor (MRM). The linear regressions were acquired with r² > 0.995, respectively. The precision was evaluated by intra- and inter-day tests, and relative standard deviation (R.S.D.) values were reported within the range of 1.14–4.42% and 0.37–4.00%. The recovery studies for the quantified compounds were observed over the range of 92.1–105.6% with R.S.D. values less than 4.55%. It demonstrated that the method developed was successfully applied for identification and quantification of 11 coumarins in Radix Angelicae Dahuricae. The results showed that the contents of coumarins in Radix Angelicae Dahuricae were processed differently and varied significantly.     

Quantification of the major metabolites of bromhexine in human plasma using RRLC–MS/MS and its application to pharmacokinetics

(E)-4-hydroxydemethylbromhexine (E-4-HDMB) and (E)-3-hydroxydemethylbromhexine (E-3-HDMB) were found as major metabolites, while (Z)-4-hydroxydemethylbromhexine and (Z)-3-hydroxydemethylbromhexine as minor metabolites of bromhexine in human plasma. These compounds were identified in comparison with synthetic authentic samples. A sensitive and selective rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS) method was developed to quantify the concentration of bromhexine and its two major metabolites (E-4-HDMB and E-3-HDMB) in human plasma. Following solid phase extraction, the analytes were separated on a Zorbax 1.8 μm particle size reversed-phase C₁₈ column, using a gradient elution program with solvents consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in 5 mM ammonium acetate at a flow rate of 0.7 mL/min. Detection was carried out with an Agilent 6460 triple-quadrupole mass spectrometer operated with an electrospray ionization source mode operated in the positive ion mode. The recovery of bromhexine, E-4-HDMB, E-3-HDMB, and internal standard (IS) was 63.1–70.9%, 60.5–68.4%, 57.0–63.5%, and 87.8%, respectively. The matrix factors of bromhexine, E-4-HDMB, E-3-HDMB, and IS were 89.9–96.7%, 89.6–94.8%, 90.4–91.4%, and 103%, respectively. After an oral administration of 8.0 mg bromhexine to five healthy male subjects, AUC_0–24 h values of bromhexine, E-4-HDMB, and E-3-HDMB were found to be 93.5 ± 31.9, 34.0 ± 14.5, and 15.8 ± 6.89 ng h/mL, respectively; while C_max values were 24.6 ± 5.16, 3.11 ± 1.13, and 5.36 ± 2.55 ng/mL, respectively. Plasma concentration of bromhexine, E-4-HDMB, and E-3-HDMB declined with t_1/2 which gave 3.6 ± 0.5, 8.4 ± 2.7, and 6.4 ± 2.5 h, respectively.