A dried blood spot assay with HPLC–MS/MS for the determination of larotrectinib in mouse blood and its application to a pharmacokinetic study

Larotrectinib is a first-generation tropomyosin kinase inhibitor, approved for the treatment of solid tumors. In this paper, we present a validated dried blood spot (DBS) method for the quantitation of larotrectinib from mouse blood using HPLC–MS/MS, which was operated under multiple reaction monitoring mode. To the DBS disc cards, acidified methanol enriched with internal standard (IS; enasidenib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of larotrectinib and the IS was achieved on an Atlantis dC₁₈ column using 10 mm ammonium formate–acetonitrile (30:70, v/v) delivered at a flow-rate of 0.80 ml/min. Under these optimized conditions, the retention times of larotrectinib and the IS were ~0.93 and 1.37 min, respectively. The total run time was 2.50 min. Larotrectinib and the IS were analyzed using positive ion scan mode and parent–daughter mass to charge ion (m/z) transitions of 429.1 → 342.1 and 474.1 → 267.1, respectively, were used for the quantitation. The calibration range was 1.06–5,080 ng/ml. No matrix effect or carryover was observed. Hematocrit did not influence DBS larotrectinib concentrations. All of the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mouse pharmacokinetic study.     

A Study of Protein and Glycoprotein Syntheses in Pre-implantation Mouse Embryos Using Mini-2D-Electrophoresis, Video Densitometer Scanning and Computer-Image Analysis

In the present study, proteins and glycoproteins of mouse embryos at 2-cell,morula and blastocyst stages were analyzed.The techniques of ~(35)S-Met incorporation,ConA antiserum-precipitating ConA-binding proteins, mini-2D-electrophoresis, fluorography,video densitometer scanning and the computer-lmage system were used for analyses.Results of the investigations indicated that proteins and glycoproteins were synthesized by the embryos in a gradual increase manner from 2-cell to blastocyst. A relatively large amount of glycoproteins was synthesized during the morula and blastocyst stages.Two specific small glycoproteins respectively with molecular weights about 6500 and 9000 and PIs at 5.0 and 6.5 were apparently synthesized in the blastocyst but not in the 2-cell or the morula.     

Multivariate classification based on metal contents in human senile cataract lenses

Multivariate classification methods were used to evaluate data on the concentrations of eight metals in human senile lenses measured by atomic absorption spectrometry. Principal components analysis and hierarchical clustering separated senile cataract lenses, nuclei from cataract lenses, and normal lenses into three classes on the basis of the eight elements. Stepwise discriminant analysis was applied to give discriminant functions with five selected variables. Results provided by the linear learning machine method were also satisfactory; the k-nearest neighbour method was less useful.     

Dexamethasone delivery to posterior segment of the eye

Due to anatomic barriers and lacrimal drainage it is difficult to obtain therapeutic drug concentrations in the posterior part of the eye after topical drug administrations. Lipophilic cyclodextrins, such as randomly methylated β-cyclodextrin (RMβCD), are known to act both as solubilizers of water-insoluble drugs in aqueous solutions and as penetration enhancers that reduce the barrier function of lipophilic membranes. The purpose of this study was to investigate the effects of RMβCD on dexamethasone delivery from aqueous eye drop solution into rabbit eyes. Dexamethasone (0.5 and 1.5% w/v) drops (50 μl) were administered to the left eye of rabbits (n = 6) and the drug levels measured in different eye tissues 2 h after administration. In aqueous humor dexamethasone levels were 1,190 ± 110 and 1,670 ± 630 ng/g (mean ± SD) after administration of the 0.5 and 1.5% dexamethasone eye drops, respectively. In the retina the levels were 33 ± 7 and 66 ± 49 ng/g, and in optic nerve 41 ± 12 and 130 ± 50 ng/g, respectively. In a previous study the dexamethasone concentration in aqueous humor after topical administration of 1.3% (w/v) dexamethasone eye drops in aqueous 2-hydroxypropyl-β-cyclodextrin (HPβCD) solution was determined to be 320 ± 230 ng/g and 66 ± 20 ng/g after administration of Maxidex^® eye drops. Both the hydrophilic HPβCD and the lipophilic RMβCD enhance topical dexamethasone delivery into the eye, but of the two, the lipophilic RMβCD results in higher dexamethasone concentrations.     

Combination of FASP and fully automated 2D‐LC‐MS/MS allows in‐depth proteomic characterization of mouse zymogen granules

Zymogen granule (ZG) constituents play important roles in pancreatic injury and disease. In previous studies, proteomic analyses with rat zymogen granules were separated by two‐dimensional gel electrophoresis or one‐dimensional SDS–PAGE, followed by in‐gel tryptic digestion. In order to overcome the disadvantage of in‐gel digestion and to carry out further in‐depth proteomic analysis of the zymogen granules, in this study, by combining a filter‐aided sample preparation method and fully automated 2D‐LC‐MS/MS technique, 800 ZG proteins were identified with at least two unique peptides for each protein, 75% of which have not been previously reported. The identified proteins revealed broad diversity in protein identity and function. This is the largest dataset of ZG proteome, and also the first dataset of the mouse ZG proteome, which may help elucidate on the molecular architecture of ZGs and their functions. Copyright © 2012 John Wiley & Sons, Ltd.     

Validated HPLC method for simultaneous quantification of mutant IDH1/2 inhibitors (enasidenib,ivosidenib and vorasidenib) in mouse plasma: Application to a pharmacokinetic study

Isocitrate dehydrogenase (IDH) inhibitors comprise a novel class of anticancer drugs, which are approved to treat acute myeloid leukemia patients having mutations on IDH1/2. We report the development and validation of a high‐performance liquid chromatography (HPLC) method for the simultaneous quantitation of IDH inhibitors, namely enasidenib (EDB), ivosidenib (IDB) and vorasidenib (VDB), in mouse plasma as per the US Food and Drug Administration regulatory guidelines. The method involves extraction of EDB, IDB and VDB along with internal standard (IS; phenacetin) from mouse plasma (100 μl) using a simple protein precipitation process. The chromatographic analysis was performed on an HPLC system using a gradient mobile phase (comprising 10 mm ammonium acetate and acetonitrile in a flow‐gradient) and an X‐Terra Phenyl column. The UV detection wave length was set at λ_max 265 nm. EDB, IDB, VDB and the IS eluted at 7.36, 8.60, 9.50 and 5.12 min, respectively, with a total run time of 10 min. The calibration curve was linear over a concentration range of 0.20–12.5 μg/ml for EDB and 0.50–12.5 μg/ml for IDB and VDB (r² = ≥0.998 for all of the analytes). Validation results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.     

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.     

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.     

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.     

Quantitative analysis of enasidenib in dried blood spots of mouse blood using an increased‐sensitivity LC–MS/MS method: Application to a pharmacokinetic study

A simple, sensitive and rapid assay method has been developed and validated as per regulatory guidelines for the estimation of enasidenib on mouse dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The method employs liquid extraction of enasidenib from DBS disks of mouse whole blood followed by chromatographic separation using 0.2% formic acid–acetonitrile (25:75, v/v) at a flow rate of 1.0 mL/min on an Atlantis dC₁₈ column with a total run time of 2.0 min. The MS/MS ion transitions monitored were m/z 474.0 → 267.1 for enasidenib and m/z 309.2 → 251.3 for the internal standard (warfarin). The assay was linear in the range of 1.01 – 3044 ng/mL. The within‐run and between‐run precisions were in the range of 3.18 – 9.06 and 4.66 – 8.69%, respectively. Stability studies showed that enasidenib was stable on DBS cards for 1 month. This novel method has been applied to analyze the DBS samples of enasidenib obtained from a pharmacokinetic study in mice.