Yuji Mano

Hematocrit-independent recovery is a key for bioanalysis using volumetric absorptive microsampling devices, Mitra™.

BACKGROUND A novel microsampling device, Mitra(TM), was evaluated for bioanalysis of E6005 and its O-desmethylated metabolite in human whole blood using an UPLC-MS. RESULTS A constant volume of blood samples was absorbed onto the tip of Mitra, the analytes were extracted by various solvents and then detected by UPLC-MS. Recovery of the analytes was high in acetonitrile-water (1:1, v/v) but was dependent on hematocrit (Hct) without sonication process, which led to biased accuracy at low and high Hcts. Inclusion of sonication process in extraction improved recovery at high Hct to yield acceptable accuracy across Hcts. CONCLUSION Optimization of extraction process to achieve high recovery regardless of Hct is critical in accurate bioanalysis via Mitra.

High-performance liquid chromatography–tandem mass spectrometry method for the determination of perampanel, a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist in human plasma

Perampanel (Fycompa(®)) is a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist registered for the adjunctive treatment of patients (≥12 years) with refractory partial onset seizures. In order to support clinical trials, as well as therapeutic drug monitoring, a sensitive bioanalytical method for the determination of perampanel concentrations in human plasma was established and validated using liquid chromatography with tandem mass spectrometry. Perampanel and an internal standard were extracted from human plasma (100 μL) by liquid extraction using methyl t-butyl ether, then evaporated and reconstituted. The chromatographic separation was conducted on a C8 column with isocratic elution at a flow rate of 0.2 mL/min. The established method showed linearity in the range 0.25-200 ng/mL with correlation coefficients of >0.99 that could be extended 10-fold as validated by dilution integrity analyses. No significant endogenous peaks were detected in the elution of analytes in blank human plasma and no significant matrix effect was observed. The intra- and inter-batch reproducibility analyses demonstrated accuracy and precision within the acceptance criteria. To check the impact of anti-epileptic drugs on the perampanel assay, accuracy, precision, and specificity were assessed in the presence of 14 anti-epileptic drugs. No anti-epileptic drugs at clinically relevant levels showed a significant impact on the perampanel assay.

A validated LC–MS/MS method of total and unbound lenvatinib quantification in human serum for protein binding studies by equilibrium dialysis

A sensitive method for the determination of total and unbound lenvatinib (Lenvima™), a novel tyrosine kinase inhibitor, in human serum was developed for protein binding studies using an equilibrium dialysis and liquid chromatography with tandem mass spectrometry. Serum samples (0.8 mL) were dialyzed against phosphate buffered saline (PBS) in dialyzer for 18 h at 37 °C to obtain dialysate and serum for unbound and total lenvatinib, respectively. After extraction by organic solvent, separation was achieved on a Symmetry Shield RP8 column with isocratic elution of 2 mM ammonium acetate (pH 4.0)-acetonitrile (3:2, v/v) at the flow rate of 0.2 mL/min. Detection was performed using API4000 with multiple reaction monitoring mode using positive electrospray ionization. The standard curve ranged from 0.0400 to 16.0 ng/mL and 0.0800 to 400 ng/mL as lenvatinib free base in PBS and serum, respectively. Accuracy and precision in the intra- and inter-batch reproducibility study were within the acceptance criteria. Various stability assessments including bench-top, freeze/thaw, processed samples, and frozen stability confirmed that lenvatinib was stable in serum and PBS. Application to in vivo protein binding studies in clinical studies was successfully performed and results showed that lenvatinib was highly protein bound in serum.

HPLC with fluorescence detection assay of perampanel, a novel AMPA receptor antagonist, in human plasma for clinical pharmacokinetic studies.

Perampanel (Fycompa®), a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥12 years) with refractory partial-onset seizures. To support therapeutic drug monitoring, a simple high-performance liquid chromatography (HPLC) assay with fluorescence detection was developed to determine perampanel concentrations in human plasma and validated to support clinical trials. Human plasma samples (1.0 mL) were processed by liquid extraction using diethyl ether, followed by chromatographic separation on a YMC Pack Pro C18 column (150 × 4.6 mm i.d., 5 µm) with isocratic elution of acetonitrile-water-acetic acid-sodium acetate (840:560:3:1.8, v/v/v/w) at a flow rate of 1.0 mL/min. Column eluent was monitored at excitation and emission wavelengths of 290 and 430 nm, respectively. The assay was linear (range 1.0-500 ng/mL) and this could be extended to 25 µg/mL by 50-fold dilution integrity. No endogenous peaks were detected in the elution of analytes in drug-free blank human plasma from six individuals and no interference was observed with co-medications tested. Intra- and inter-batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. Validation data demonstrated that our assay is simple, selective, reproducible and suitable for therapeutic drug monitoring of perampanel.

Application of volumetric absorptive microsampling device for quantification of tacrolimus in human blood as a model drug of high blood cell partition

&NA; Volumetric absorptive microsampling device (VAMS) was evaluated for bioanalysis of tacrolimus, which was used as a model drug with high blood cell partition. Aliquots of blood (ca. 10 &mgr;L) with different hematocrits and fortified with tacrolimus were wicked up by VAMS and tacrolimus was extracted with a methanol‐water mixture (1:1, v/v) via sonication. After chromatography on an AQUITY UPLC HSS T3 column (100 × 2.1 i.d., mm, 1.8 &mgr;m), tacrolimus and the internal standard ascomycin, were detected in the positive ion mode with electrospray ionization by monitoring of transitions m/z 826.6 → 616.4 and m/z 814.6 → 604.0, respectively. An assay method to quantify tacrolimus from 1 to 250 ng/mL in whole blood was qualified by ensuring that linearity, selectivity, intra‐ and inter‐batch reproducibility, and stability were within the acceptance criteria. Consistent and high extraction recovery of tacrolimus was ensured from blood with low‐ (20%), mid‐ (45%), and high‐hematocrit (65%) levels with minimal matrix effects. Apparent instability at ambient temperature or 4 °C possibly due to reduced recovery suggests that tacrolimus in VAMS should be stored at −25 °C until assay. Potential reduced recovery over time from VAMS should be taken into consideration in method optimization. Graphical abstract Figure. No caption available. HighlightsA method to quantify tacrolimus in human blood using volumetric absorptive microsampling devices (VAMS) was established.The method can quantify tacrolimus from 1 ng/mL using only 10 &mgr;L of blood.The impact of hematocrit on the quantitation of tacrolimus, a model drug of high blood cell partition, was evaluated.A hematocrit‐independent assay was achieved by using 50% methanol as an extraction solvent.Tacrolimus wicked up by VAMS should be stored frozen at −25 °C until assay.

An inter-laboratory cross validation study for the determination of perampanel in human plasma by liquid chromatography assays.

For sample assay to support global clinical studies of perampanel, a novel AMPA receptor antagonist, six chromatographic assay methods in human plasma were developed and fully validated at each laboratory using liquid chromatography with tandem mass spectrometry (LC-MS/MS) or LC with fluorescence detection (LC-FL). In this study, samples fortified with known perampanel concentrations were assayed at six laboratories to find whether assay data are comparable. Perampanel was extracted by protein precipitation or liquid-liquid extraction, chromatographed on a reverse-phase column then detected by MS/MS or FL to achieve the limit of quantification of 0.25 or 1 ng/mL. Cross-validation samples at four concentrations prepared at a central laboratory were determined at six laboratories and the mean accuracy at each concentration was within ±15% except the low concentration at one laboratory (relative error -17.4%), suggesting that plasma concentrations of perampanel in clinical trials can be compared across laboratories.

Validation of a UPLC-MS/MS method for the simultaneous determination of E6005, a phosphodiesterase 4 inhibitor, and its metabolite in human plasma.

E6005, a novel phosphodiesterase 4 inhibitor, is currently under clinical development for the treatment of atopic dermatitis. As ER-392710 (M11), a hydrolyzed metabolite, is a main metabolite, a simultaneous assay method for quantification of E6005 and M11 in human plasma has been developed and validated using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). E6005, M11, and each deuterium-labeled compound used as internal standard were extracted from 100μL human plasma by solid phase extraction then chromatographed on an Acquity UPLC BEH C18 column (100mm×2.1mm i.d., 1.7μm) under gradient elution. The analytes were detected by selected reaction monitoring in the positive ion mode with the mass transition of m/z 473.1/163.0 and m/z 459.1/149.0 for E6005 and M11, respectively. E6005 and M11 were quantifiable ranging from 1 to 200ng/mL with no carryover. Accuracy and precision in intra- and inter-batch reproducibility assays were within the acceptance criteria recommended by the regulatory bioanalytical guidelines. Various stability assessments including possible conversion of E6005 to M11 were thoroughly performed to demonstrate the stability of E6005 and M11 in human blood and plasma. The method was successfully applied to support clinical trials.

Validation of an ultra-performance liquid chromatography–tandem mass spectrometry method for the determination of flecainide in human plasma and its clinical application

A simple and reproducible bioanalytical method for the determination of flecainide in human plasma was developed and validated using an ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) to obtain higher sensitivity than the current available methods. After simple protein precipitation, flecainide and a stable isotope-labeled internal standard (IS) were chromatographed on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) with isocratic elution of mobile phase consisting of 45% methanol containing 0.1% formic acid at a flow rate 0.25 mL/min. Detection was performed in positive electrospray ionization by monitoring the selected ion transitions at m/z 415.4/301.1 for flecainide and m/z 419.4/305.1 for the IS. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve was linear from 2.5 to 1000 ng/mL using 0.1 mL of plasma. No significant interferences were detected in blank human plasma. Accuracy and precision in the intra- and inter-batch reproducibility study were within acceptance criteria. Neither hemolysis effects nor matrix effects were observed. The UPLC-MS/MS method developed was successfully applied to determine plasma flecainide concentrations to support clinical studies and incurred sample reanalysis also ensured the reproducibility of the method.

Method validation studies and an inter-laboratory cross validation study of lenvatinib assay in human plasma using LC-MS/MS

Cross validation studies for bioanalytical methods are important to ensure that assay data from all study sites where sample analysis is performed can be compared throughout clinical trials. To support global clinical studies of lenvatinib, a novel multi-targeted tyrosine kinase inhibitor, seven bioanalytical methods by liquid chromatography with tandem mass spectrometry (LC-MS/MS) were developed at five laboratories. In this study, methods were initially validated at each laboratory according to bioanalytical guidelines. For subsequent inter-laboratory cross validation, quality control (QC) samples and clinical study samples with blinded lenvatinib concentrations were assayed to confirm comparable assay data. Lenvatinib and an internal standard were extracted by protein precipitation, liquid-liquid extraction, or solid phase extraction and then detected in positive ion electrospray mode by multiple reaction monitoring using LC-MS/MS. The assay method developed at each laboratory was successfully validated with parameters within the acceptance criteria recommended by the guidelines. In the cross validation study, accuracy of QC samples was within± 15.3% and percentage bias for clinical study samples was within± 11.6%. These findings suggest that lenvatinib concentrations in human plasma can be compared across laboratories and clinical studies.

A dried blood spot assay with UPLC–MS/MS for the simultaneous determination of E6005, a phosphodiesterase 4 inhibitor, and its metabolite in human blood

HIGHLIGHTSThe dried blood spot (DBS) assay has been developed and validated for E6005 and its metabolite (M11) in human blood.E6005 and M11 in DBS were quantifiable from 1ng/mL as free base using only 25&mgr;L blood.Typical validation parameters were within the acceptance criteria recommended by bioanalytical guidelines.DBS‐associated specific validation parameters, including impacts by blood volume and hematocrit, were also evaluated.Stability assessments showed that E6005 and M11 were stable for 160days at ambient temperature, and below −15°C. ABSTRACT E6005, a novel phosphodiesterase 4 inhibitor, is currently under clinical development for the treatment of atopic dermatitis. To support pediatric clinical trials, the dried blood spot assay for simultaneous determination of E6005 and its main metabolite, ER‐392710 (M11), has been developed using ultra‐performance liquid chromatography with tandem mass spectrometry. E6005 and M11, in 25&mgr;L blood spotted onto FTA™ DMPK‐C cards, were extracted by water/acetonitrile (1:1, v/v), and then chromatographed on a reversed phase column under gradient elution. The mass transitions, m/z 473.1→163.0 for E6005 and m/z 459.1→149.0 for M11, with corresponding stable isotope internal standard, m/z 477.2→167.0, and m/z 463.2→153.0, were monitored. E6005 and M11 were quantifiable from 1 to 200ng/mL as free base. Accuracy and precision of the two analytes in the intra‐ and inter‐batch reproducibility were within ±8.0% and 15.7%, respectively. Extraction recoveries of the analytes were 73% or more and hematocrit ranging from 26.9% to 51.8% did not impact the analytes’ accuracy. Various stability assessments, including possible conversion of E6005 to M11, were thoroughly performed, and bench‐top stability was ensured up to 160days. The DBS method was applied to determine E6005 and M11 concentrations in blood samples supporting a pediatric clinical trial.