Development and full validation of an HPLC methodology to quantify atorvastatin and curcumin after their intranasal co‐delivery to mice

There is increasing interest in atorvastatin and curcumin owing to their potential anticancer activity. A new, accurate and sensitive HPLC method was developed, for the first time, to simultaneously quantify atorvastatin and curcumin in mouse plasma and brain, liver, lung and spleen tissues following protein precipitation sample preparation. The chromatographic separation was achieved in 13 min on a C₁₈ column, at 35°C, using a mobile phase composed of acetonitrile–methanol–2% (v/v) acetic acid (37.5:2.5:60, v/v/v) at a flow rate of 1.0 mL/min. The detection of analytes and internal standard was carried out at 247, 425 and 250 nm, respectively. According to international guidelines, the method was shown to be selective, with lower limits of quantification ranging from 10 to 500 ng/mL for curcumin, and from 100 to 600 ng/mL for atorvastatin, linear over a wide concentration range (r² ≥ 0.9971) and with acceptable accuracy (bias ± 12.29%) and precision (coefficient of variation ≤13.15%). The analytes were reproducibly recovered at a percentage >81.10% and demonstrated to be stable under various experimental conditions in all biological matrices. This method can be easily applied to in vivo biodistribution studies related to the intranasal administration of atorvastatin and curcumin, separately or simultaneously.     

Application of a sensitive and specific LC‐MS/MS method for determination of eriodictyol‐8‐C‐β‐d‐glucopyranoside in rat plasma for a bioavailability study

This study is the first to detail the development and validation of a rapid, sensitive and specific LC‐ESI‐MS/MS method for the determination of eriodictyol‐8‐C‐β‐d ‐glucopyranoside (EG) in rat plasma. A simple protein precipitation method was used for plasma sample preparation. Chromatographic separation was successfully achieved on an Agilent Zorbax XDB C₁₈ column (2.1 × 50 mm, 3.5 µm) using a step gradient program with the mobile phase of 0.1% formic acid aqueous solution and acetonitrile with 0.1% formic acid. EG and the internal standard (IS) were detected using an electrospray negative ionization mass spectrometry in the multiple reaction monitoring mode. This method demonstrated good linearity and did not show any endogenous interference with the active compound and IS peaks. The lower limit of quantification of EG was 0.20 ng/mL in 50 μL rat plasma. The average recoveries of EG and IS from rat plasma were both above 80%. The inter‐day precisions (relative standard deviation) of EG determined over 5 days were all within 15%. The present method was successfully applied to a quantification and bioavailability study of EG in rats after intravenous and oral administration. The oral absolute bioavailability of EG in rats was estimated to be 7.71 ± 1.52%. Copyright © 2014 John Wiley & Sons, Ltd.     

Stereoselective analysis of flecainide enantiomers using reversed‐phase liquid chromatography for assessing CYP2D6 activity

Stereoselective analyses of flecainide enantiomers were performed using reversed‐phase high‐performance liquid chromatography (HPLC) equipped with a polysaccharide‐based chiral column (Chiralpak AS‐RH) and fluorescence detector. Excitation and emission wavelengths were set at 300 and 370 nm, respectively. Flecainide enantiomers in serum and urine were extracted using diethyl ether. The mobile phase solution, comprising 0.1 m potassium hexafluorophosphate and acetonitrile (65:35, v/v), was pumped at a flow rate of 0.5 mL/min. The recoveries of flecainide enantiomers were greater than 94%, with the coefficients of variation (CVs) <6%. The calibration curves of flecainide enantiomers in serum and urine were linear in the concentration range 5–500 ng/mL and 0.75–15 µg/mL (r > 0.999), respectively. CVs in intra‐day and inter‐day assays were 1.8–5.8 and 3.4–7.5%, respectively. In a pharmacokinetic study, the ratios of (S)‐ to (R)‐flecainide (S/R ratio) in the area under the curve and the amount of flecainide enantiomers excreted in urine were lower in a subject carrying CYP2D6*10/*10 than in subjects carrying CYP2D6*1/*2. The S/R ratio of trough serum flecainide concentration ranged from 0.79 to 1.16 in patients receiving oral flecainide. The present HPLC method can be used to assess hepatic flecainide metabolism in a pharmacokinetic study and therapeutic drug monitoring. Copyright © 2014 John Wiley & Sons, Ltd.     

Pharmacokinetics and metabolism study of veratramine in mice after oral administration using LC‐MS/MS

A simple and sensitive high‐performance liquid chromatography coupled with hybrid triple quadrupole–linear ion trap mass spectrometry (Q‐trap‐MS) method was developed and validated for the determination of veratramine, the major bioactive and neurotoxic component in Veratrum nigrum L. Veratramine and the internal standard (IS) were separated with a Waters Symmetry C₁₈ column and eluted with a gradient mobile phase system containing acetonitrile and 0.1% aqueous formic acid. The analysis was performed by using positive electrospray ionization mode with multiple reaction monitoring (MRM). Transition ions of m/z 410.2 → 295.2 for veratramine and m/z 426.1 → 113.8 for the IS were monitored. The method was validated with a good linearity in the range of 1–1000 ng/mL and lower limit of quantification of 1 ng/mL. The precision (CV) of intra‐ and inter‐day ranged from 3.92 to 7.29%, while the accuracy (bias) intra‐ and inter‐day were between ?4.78 and 1.65%. The recovery, stability and matrix effect were within the acceptable ranges. Five metabolites of veratramine, including four hydroxylated and one sulfated metabolites, were tentatively identified using predictive MRM–information dependent acquisition–enhanced product ion mode (predictive MRM‐IDA‐EPI). The developed method was successfully applied to the pharmacokinetic and metabolic study of veratramine in mice after oral administration of veratramine. Copyright © 2016 John Wiley & Sons, Ltd.     

Simple HPLC‐UV for the quantification of a new leishmanicidal candidate (E)‐1‐4(trifluoromethyl) benzylidene)‐5‐(2‐4‐dichlorozoyl) carbonylhydrazine (LASSBio‐1736) in rat plasma for pharmacokinetics assessment

In this study, a sensitive HPLC‐UV assay was developed and validated for the determination of LASSBio‐1736 in rat plasma with sodium diclofenac as internal standard (IS). Liquid–liquid extraction using acetonitrile was employed to extract LASSBio‐1736 and IS from 100 μL of plasma previously basified with NaOH 0.1 M. Chromatographic separation was carried on Waters Spherisorb^®S5 ODS2 C₁₈ column (150 × 4.6 mm, 5 μm) using an isocratic mobile phase composed by water with triethylamine 0.3% (pH 4), methanol and acetonitrile grade (45:15:40, v/v/v) at a flow rate of 1 mL/min. Both LASSBio‐1736 and IS were eluted at 4.2 and 5 min, respectively, with a total run time of 8 min only. The lower limit of quantification was 0.2 μg/mL and linearity between 0.2 and 4 μg/mL was obtained, with an R² > 0.99. The accuracy of the method was >90.5%. The relative standard deviations intra and interday were <6.19 and <7.83%, respectively. The method showed the sensitivity, linearity, precision, accuracy and selectivity required to quantify LASSBio‐1736 in preclinical pharmacokinetic studies according to the criteria established by the US Food and Drug Administration and European Medicines Agency. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous determination of paeoniflorin from total glucosides of paeony in Sprague–Dawley rats and spontaneously hypertensive rats by high‐performance liquid chromatography–tandem mass spectrometry: in vivo and in vitro studies

Paeoniflorin is a well‐known monoterpene glucoside in the herbal drug that exhibits a number of biological activities. The pharmacokinetic characteristics of paeoniflorin from total glucosides of paeony in spontaneously hypertensive rats (SHR) are still unclear. It is essential to investigate the in vivo and in vitro pharmacokinetic differences of paeoniflorin from total glucosides of paeony in Sprague–Dawley (SD) and SHR. The in vivo pharmacokinetic data were analyzed using DAS 2.0 software and the in vitro metabolic characteristics were measured using rat hepatic microsomes. The concentration of paeoniflorin in biological samples was determined using high‐performance liquid chromatography–electrospray ionization tandem mass spectrometry method, which showed good precision and stability. The plasma concentration–time profiles of paeoniflorin following oral administration of total glucosides of paeony showed a single peak and there were significant differences in the mean values of AUC_(0–t), AUC_(0–∞), CL_z/F and T_max between SD and SHR (p < 0.05). The metabolic rate of paeoniflorin from total glucosides of paeony was slower in SHR than in SD rats (p < 0.05). The results might be useful in further applications of paeoniflorin and total glucosides of paeony. Copyright © 2016 John Wiley & Sons, Ltd.     

Comparison of plasma pharmacokinetics of Tanreqing solution between intratracheal aerosolization and intravenous injection in rats

A rapid ultra high performance liquid chromatography tandem mass spectrometry method was developed for the simultaneous analysis of baicalin, oroxylin A‐7‐O‐β‐d ‐glucoronide and chlorogenic acid in rats plasma, and applied to comparison of pharmacokinetics of Tanreqing solution between intratracheal aerosolization and intravenous injection. Results of the analytical method validation assay showed high sensitivity, accuracy and suitable recovery. Results of pharmacokinetics showed similar decline phases for baicalin, oroxylin A‐7‐O‐β‐d ‐glucoronide and chlorogenic acid in two different delivery routes. The half‐lives of intratracheal aerosolization and intravenous injection were 0.90 and 1.22 h for baicalin, 0.47 and 0.17 h for oroxylin A‐7‐O‐β‐d ‐glucoronide and 0.22 and 0.13 h for chlorogenic acid, and this implies that compounds were retained in the lung for a relatively short time. This study was the first to provide important pharmacokinetics information for traditional Chinese medicine delivery to the lung.     

Validated liquid chromatography–tandem mass spectrometry method for simultaneous quantitation of bendamustine and copanlisib in mouse plasma: Application to a pharmacokinetic study in mice

In this study, we report the development and validation of an LC–tandem mass spectrometry method for the simultaneous quantitation of bendamustine and copanlisib in mouse plasma as per the US FDA regulatory guidelines. The sample processing involves extraction of bendamustine and copanlisib along with internal standard (IS; warfarin) from 50 μL mouse plasma using a liquid–liquid extraction method. The chromatographic separation of bendamustine, copanlisib and the IS was achieved on an Atlantis dC₁₈ column using an isocratic mobile phase (5 mM ammonium acetate:methanol, 20:80 v/v). Bendamustine, copanlisib and the IS eluted at 0.88, 1.39 and 0.74 min, respectively, with a total run time of 2.5 min. The calibration curve ranged from 3.99–2996 and 4.33–3248 ng/mL for bendamustine and copanlisib, respectively. Inter- and intra-day precision and accuracy, stability in processed samples and upon storage, dilution integrity and incurred sample reanalysis were investigated for both the analytes. The intra- and inter-day precisions were in the ranges of 2.01%–5.05% and 2.74%–6.13% and 1.98%–7.64 and 8.62%–9.04% for bendamustine and copanlisib, respectively. Stability studies showed that both analytes were stable on bench top for 6 h, in auto-sampler for 24 and at −80°C for 30 days. The validated method was successfully applied to a pharmacokinetic study in mice.     

Stability of Antimicrobial Drug Molecules in Different Gravitational and Radiation Conditions in View of Applications during Outer Space Missions

The evolution of different antimicrobial drugs in terrestrial, microgravity and hypergravity conditions is presented within this review, in connection with their implementation during human space exploration. Drug stability is of utmost importance for applications in outer space. Instabilities may be radiation-induced or micro-/hypergravity produced. The antimicrobial agents used in space may have diminished effects not only due to the microgravity-induced weakened immune response of astronauts, but also due to the gravity and radiation-altered pathogens. In this context, the paper provides schemes and procedures to find reliable ways of fighting multiple drug resistance acquired by microorganisms. It shows that the role of multipurpose medicines modified at the molecular scale by optical methods in long-term space missions should be considered in more detail. Solutions to maintain drug stability, even in extreme environmental conditions, are also discussed, such as those that would be encountered during long-duration space exploratory missions. While the microgravity conditions may not be avoided in space, the suggested approaches deal with the radiation-induced modifications in humans, bacteria and medicines onboard, which may be fought by novel pharmaceutical formulation strategies along with radioprotective packaging and storage.     

Pharmacokinetic analysis of plasma bicyclol by liquid chromatography–tandem mass spectrometry

Bicyclol is a synthetic drug widely used to treat chronic hepatitis B. This study aimed to develop a selective, sensitive and high‐throughput liquid chromatography–tandem mass spectrometric method for the detection of bicyclol in human plasma. Bicyclol was detected using a multiple reaction monitoring mode, with ammonium adduct ions (m/z 408.2) as the precursor ion and the [M‐CH₃]⁺ ion (m/z 373.1) subjected to demethylation as the product ion. Chromatographic separation was achieved using a Zobax Eclipse XDB‐C₁₈ column with a gradient elution and a mobile phase of 2 mm ammonium formate and acetonitrile. Bicyclol was extracted from plasma matrix by precipitation. A linear detection response was obtained for bicyclol ranging from 0.500 to 240 ng/mL, and the lower limit of quantification was 0.500 ng/mL. The intra‐ and inter‐day precisions were all ≤7.4%, and the accuracies were within ±6.0%. The extraction recovery was >95.9%, and the matrix effects were between 96.0% and 108%. Bicyclol was found to be unstable in human plasma at room temperature, but the degradation was minimized by conducting sample collection and preparation in an ice bath. The validated method was successfully applied to investigate the pharmacokinetics of bicyclol tablets in six healthy Chinese volunteers.