A separate assay of released and liposomal encapsulated eribulin in dog plasma by liquid chromatography with tandem mass spectrometry for its application to a pharmacokinetic study.

Eribulin has been used as a drug for the treatment of metastatic breast cancer and liposomal formulation of eribulin is under development to achieve a wider therapeutic index. It is important to separately determine released and encapsulated drugs in systemic circulation for liposomal drugs. In this study, a separate assay method was developed for the determination of released and total (encapsulated + released) eribulin concentrations in dog plasma by liquid chromatography with tandem mass spectrometry. The released eribulin in dog plasma was separated by ultrafiltration of plasma samples. Obtained plasma ultrafiltrate and untreated plasma samples recognized as released and total eribulin, respectively, were subjected to protein precipitation for extraction of eribulin. Eribulin was quantifiable from 0.1 ng/mL. Accuracy and precision of eribulin in both matrices were within ± 15 and 15%, respectively, indicating a robust assay. Released and total eribulin concentrations in plasma were determined after intravenous administration of the liposomal formulation of eribulin to dogs, resulting in minimal released eribulin in plasma. In conclusion, a robust method for released and total eribulin levels in dog plasma was developed and was successfully applied to a pharmacokinetic study in dogs to characterize the pharmacokinetic profiles.

Application of an electrochemiluminescence assay for quantification of E6011, an antifractalkine monoclonal antibody, to pharmacokinetic studies in monkeys and humans.

BACKGROUND: E6011, a humanized antifractalkine monoclonal antibody, is under development for the treatment of various inflammatory diseases, such as rheumatoid arthritis. A reproducible assay method has been developed for the determination of E6011 in monkey and human serum by electrochemiluminescence (ECL) assay. METHODS: E6011 in serum was captured by fractalkine and detected by ruthenium-labeled rabbit anti-E6011 Fab polyclonal antibodies for ECL detection. E6011 in serum was quantifiable from 0.02 and 0.1 µg/mL in monkey and human serum, respectively, with minimum required dilution of 500. The method was then validated in accordance with bioanalytical guidelines. RESULTS: Accuracy and precision of quality control samples at five concentrations in intra- and interbatch reproducibility demonstrated that relative error and relative standard deviation were within acceptable criteria. Recovery of E6011 was 92.9%-121.7% and 85.0%-109.3% in humans and monkeys. Dilution integrity, no prozone effects, and no impacts by antigen were also ensured. Parallelism was also confirmed using incurred clinical sample analysis. Various types of stability were assessed, which confirmed that E6011 in serum was stable for 367 and 735 days in monkey and human sera, respectively, under frozen conditions. CONCLUSION: The developed method was successfully applied supporting pharmacokinetic studies in monkeys and humans.

Minimal impact of hepatic and renal impairment on plasma protein binding of lenvatinib, and identification of its major plasma binding protein.

Plasma protein binding (PPB) can be different depending on the status of hepatic or renal functions. In this study, the PPB of lenvatinib was determined using equilibrium dialysis in plasma from healthy volunteers and from subjects with mild, moderate, or severe hepatic impairment or renal impairment. Plasma from these subjects, fortified with lenvatinib at four concentrations (20, 200, 500, or 1200 ng/ml), was dialysed against phosphate buffered saline (PBS), and then determinations of the total concentrations of lenvatinib in plasma and unbound concentrations in PBS were made. In addition, the binding of lenvatinib was determined in human serum albumin (HSA), α1 -acid glycoprotein (AAG), and human γ-globulin (HG) in order to identify major binding proteins in human plasma. The PPB of lenvatinib in subjects with HI or RI ranged from 97.5% to 98.2% in hepatic impairment and 98.0% to 98.4% in renal impairment, which was similar to that of healthy volunteers. The binding of lenvatinib to HSA, AAG, and HG was 96.6%-97.1%, 46.4%-69.9%, and 19.1%-23.9%, respectively. These findings suggest that lenvatinib mainly binds to HSA and neither renal nor hepatic impairment impacts the PPB of lenvatinib.

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.

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.

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.

Validation of a hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of gemcitabine in human plasma with tetrahydrouridine.

A simple and reproducible bioanalytical method for the determination of gemcitabine in human plasma treated with tetrahydrouridine (THU) was developed and validated using a hydrophilic interaction ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). To prevent deamination of gemcitabine, blood was treated with THU, and the plasma samples obtained after centrifugation were used in this study. Gemcitabine and gemcitabine-(13)C, (15)N2 used as an internal standard, were extracted from human plasma treated with THU using a 96-well Hybrid SPE-Precipitation plate. Extracts were chromatographed on a hydrophilic interaction chromatography column with isocratic elution. Detection was performed using Quattro Premier with positive electrospray ionization multiple reaction monitoring mode. The standard curve ranged from 10 to 10,000 ng/mL without carryover. No significant interferences were detected in blank plasma and no interferences by 2'-2'-difluoro-2'-deoxyuridine, a metabolite of gemcitabine. Accuracy and precision in the intra-batch reproducibility study using quality control samples with three THU levels did not exceed ±5.4 and 7.3%, respectively, and the inter-batch reproducibility results also met the criteria. Stability of gemcitabine was ensured in whole blood and plasma as well as stability of THU in solutions. The UPLC-MS/MS method developed was successfully validated and can be applied for gemcitabine bioanalysis in clinical studies.

Pharmacokinetic profiles of methylcobalamin in rats after multiple administration routes by a simple LC-MS/MS assay with a small volume of plasma.

Methylcobalamin (MBL) is a vitamin B12 coenzyme and is effective for treating peripheral neuropathies. Little is known about pharmacokinetics (PK) of MBL in animals, we have developed a simple assay for MBL by using only 0.01 mL of plasma for PK of MBL in rats. Under minimal light exposure (<5 lx), MBL was extracted by a simple protein precipitation using methanol and detected by liquid chromatography with tandem mass spectrometry. MBL in rat plasma at 20-10,000 ng/mL was quantified using only 0.01 mL of plasma. Relative error and relative standard deviation met the acceptance criteria in reproducibility assessments, indicating the robustness of the assay. PK of MBL was evaluated after intravenous, intramuscular, and subcutaneous administration. PK of MBL was dose proportional at 5-20 mg/kg in both intramuscular and subcutaneous administrations. Bioavailability after the two dosing routes was complete (ca. 100 %). The incurred sample reanalysis also supported that the assay is robust. The established assay was successfully applied to PK studies in rats to find that MBL showed high bioavailability after intramuscular and subcutaneous administrations.

Significant increases in detection capability for assessment of organic anion transporting polypeptide-mediated drug-drug interaction in cynomolgus monkeys by modifying pretreatment of Coproporphyrin I and III, and glycochenodeoxycholate-3-sulfate in plasma.

BACKGROUND: Coproporphyrin I (CP-I), Coproporphyrin III (CP-III), and glycochenodeoxycholate-3-sulfate (GCDCA-S) act as endogenous substrates of Organic Anion Transporting Polypeptide (OATP) 1B and have been considered for application in OATP1B-mediated drug‒drug interaction (DDI) risk assessments. Prior assays of the endogenous OATP substrates might exhibit reduced DDI detection capability and possibly overlook low DDI risk. We pioneered a simultaneous assay of the three substrates in monkey plasma using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) and applied it to monkey studies to identify lower DDI risk. RESULTS: The methodology development indicated that precursors of CP-I/III were oxidized to form CP-I/III, diminishing the detection capability in DDI risk assessments. A precursor eliminated analytical (PEA) method was developed to eliminate the precursors through solid-phase extraction. This method aimed to prevent the oxidation of CP-I/III precursors by incorporating edaravone. For comparison, a precursor oxidized analytical (POA) method was also developed, wherein the precursors of CP-I/III were fully oxidized to CP-I/III. The PEA method achieved high sensitivity for CP-I/III and GCDCA-S, with lower quantification limits of 0.01 ng mL-1 and 0.5 ng mL-1, respectively. Both methods ensured that the validation parameters met the acceptance criteria. The two methods were applied to a monkey study, with CP-I/III showcasing notably enhanced DDI detection capabilities through the novel PEA method in comparison to the POA method. SIGNIFICANCE: This study's methodology has future implications for OATP-mediated DDI risk assessment using endogenous substrates. The novel PEA method can identify lower OATP-mediated DDI risks for drugs that the current methods cannot detect. Our method is likely applicable in clinical settings, and its utility should be assessed in clinical trials.

Nonclinical pharmacokinetics of E3112, a recombinant human hepatocyte growth factor, in rats and monkeys by a simple ELISA kit assay.

E3112 is a recombinant human hepatocyte growth factor which is under development for the treatment of acute liver failure. Pharmacokinetics (PK) evaluation in experimental animals is important and thus a simple assay for the determination of E3112 in rat and monkey serum has been validated using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. E3112 in rat and monkey serum was quantifiable from 0.313 ng/mL to 15.0 ng/mL without prozone effects. Dilution integrity enabled accurate assay up to 500,000-fold dilution. Accuracy and precision were within the acceptance criteria. PK of E3112 was investigated after intravenous administration to rats and monkeys. PK of E3112 was similar between male and female animals in both species. Nonlinear PK of E3112 was observed in rats after intravenous bolus dose at 1-100 mg/kg while nonlinear PK was not significant in monkeys after intravenous infusion at 0.5-25 mg/kg. These findings suggest that the assay of E3112 in serum using a commercially available ELISA kit was validated and successfully applied to PK studies in rats and monkeys.

A sensitive assay for the determination of E7090, a novel selective inhibitor of fibroblast growth factor receptors, and its metabolite in human plasma by UPLC-MS/MS with at-column dilution.

E7090, a novel fibroblast growth factor receptors inhibitor, is currently under clinical development for the treatment of patients with solid tumors. The previous assay was insufficient in detection sensitivity for E7090 and high exposure of a dealkylated metabolite, M2, was noted in a clinical trial at low doses. Thus, a sensitive assay for the simultaneous determination of E7090 and M2 in human plasma has been developed using ultra-performance liquid chromatography with tandem mass spectrometer (UPLC-MS/MS). E7090 and M2 were extracted from 0.1 mL of plasma by protein precipitation and chromatographed on a reverse phase column utilizing at-column dilution which enables larger volume sample injection to the UPLC-MS/MS. E7090 and M2 were quantifiable from 0.025 ng/mL, which was 40-fold higher sensitivity than the previous assay. Accuracy as relative error and precision as relative standard deviation were within ± 15% and 15%, respectively, ensuring the reproducibility of the assay. The developed assay method was applied to a clinical trial of E7090, and plasma concentrations of E7090 and M2 were quantifiable up to 144 h postdose. These results indicated that the developed more sensitive assay was reproducible and was successfully applied to a clinical trial of E7090.

Development of the Complement C5 Assay by LC-MS/MS in Monkey Serum and Comparison with Enzyme-Linked Immunosorbent Assay.

Complement C5 (C5) is the key component for the complement activation pathway, which is important for innate immunity, and inhibition of C5 is considered to be effective in antibody-mediated rejection in organ transplantation. Thus determination of C5 levels in systemic circulation is a simple way to understand efficacy of drugs that aim to inhibit C5 production. We have developed a simple liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay for C5 in cynomolgus monkey serum. C5 in monkey serum was subjected to tryptic digestion, and two signature peptides, DSSVPNTGTAR and LQGTLPVEAR, were assayed by LC-MS/MS with electrospray ionization in the positive ion mode. Assay reproducibility in serum samples was evaluated, and the assay was applied to the C5 assay in monkey serum after administration of C5 siRNA encapsulated in lipid nanoparticles to monkeys. The time profiles of C5 after administration of C5 siRNA were comparable between the two signature peptides by LC-MS/MS and were also similar to those by an enzyme-linked immunosorbent assay using an assay kit. These findings suggest that the established LC-MS/MS assay of C5 is reliable to determine C5 levels in monkey serum.

Generic UPLC-MS/MS and Gyrolab assays with blood microsampling for pharmacokinetic assessments of therapeutic antibodies in mice.

Serial blood sampling from one animal is useful to understand relationship between pharmacokinetics (PK) and pharmacological or toxicological events in individual animals. To assess its feasibility in mice, two therapeutic antibodies were used to evaluate impacts by different blood sampling methods, sampling sites, and assay platforms on PK. Denosumab and Panitumumab were intravenously administered to mice and only 0.05 mL of blood sample per point was collected from jugular vein or tail vein. Blood samples were collected serially from a mouse or collected by traditional composite sampling from each mouse. Plasma concentrations of the two drugs were assayed by a generic ligand binding assay using Gyrolab or by a generic ultra-performance liquid chromatography with tandem mass spectrometry. The two assay platforms showed acceptable accuracy and precision and gave comparable PK parameters of the drugs, suggesting that both assays were successfully applied to the PK assessments. Comparable results in the PK profiles were noted between serial and composite blood samplings and differences in the two sampling sites did not impact PK. These findings suggest that microsampling combined with generic assays is useful to assess PK profiles of therapeutic antibodies in mice.

An electrochemiluminescence assay for quantification of Denileukin Diftitox and its anti-drug antibodies in rat serum.

Denileukin Diftitox (DD), comprising fragments of diphtheria toxin (DT) and interleukin-2 (IL2), was developed for the treatment of lymphoma and has been approved for marketing in Japan. Toxicological evaluation including pharmacokinetics and immunogenicity in preclinical animals is important for drug development and thus the assays of DD and anti-drug antibody (ADA) were developed by electrochemiluminescence (ECL) detection. For the DD assay, ruthenium-labeled anti-DT Ab and biotinylated anti-IL2 Ab were mixed with serum samples and the mixture was captured by streptavidin-coated wells for ECL detection. For the ADA assay, signals of immuno-complex of biotinylated DD, ruthenium-labeled DD, and ADA, bound to streptavidin plate were determined. DD was quantifiable from 10 ng/mL. Accuracy and precision of quality control samples were within ±20% and 20%, respectively, and stability of DD in rat serum was successfully assessed. Precision of positive control samples of ADA was within the acceptance criteria and cut point values for ADA detection in the screening and confirmatory assay were determined by statistical analysis. Drug-induced ADA was detected by screening assay followed by confirmatory assay. The developed method was successfully applied to assess pharmacokinetics and immunogenicity to support toxicity studies in rats.

Quantification of anti-drug antibodies against E6011, an anti-fractalkine monoclonal antibody, in monkey and human serum, by an electrochemiluminescence assay.

E6011, a humanized anti-fractalkine monoclonal antibody, is under development for the treatment of various inflammatory diseases, such as rheumatoid arthritis. Therapeutic antibodies may induce production of anti-drug antibodies (ADA) that may deteriorate efficacy and/or enhance immunogenic reaction. It is important to have an ADA assay to understand the characteristics of biotherapeutics under development. A simple and reproducible assay has thus been developed for the determination of ADA against E6011 in monkey and human serum by electrochemiluminescence (ECL) detection. An immune-complex of biotinylated E6011, ADA, and ruthenium-labeled E6011 was attached to avidin-coated wells for ECL signal detection. Screening and confirmatory cutpoints were determined to judge negative or positive ADA. Sensitivity of ADA was 1.61 and 1.34 ng/mL in monkey and human serum, respectively. Accuracy and precision of the assay were within ±20% and 20%, respectively. Drug tolerance of the assay in monkey and human sera was ensured up to 100 and 1000 µg/mL E6011 at the surrogate ADA levels of 1 and 4 µg/mL, respectively. The developed assay was successfully applied to ADA quantification in monkeys and humans in support of immunogenicity assessments.

Cross-species comparison in nonclinical pharmacokinetics of lenvatinib by a simple HPLC with ultraviolet detection.

Lenvatinib (Lenvima) is a tyrosine kinase inhibitor on the market and has been used for the treatment of various types of cancer. It is important to understand differences in pharmacokinetics (PK) between nonclinical animals and humans, and thus, we evaluated PK of lenvatinib in mice, rats, dogs, and monkeys. A simple assay for lenvatinib was developed by high performance liquid chromatography with ultraviolet detection and validated in accordance with the bioanalytical guidelines. Lenvatinib was quantifiable at 5-100,000 ng/mL using 50 µL of plasma. Accuracy and precision in the intra- and inter-batch reproducibility were within the acceptance criteria, indicating a robust assay. Lenvatinib was intravenously or orally administered to mice, rats, dogs, and monkeys to fully characterize the cross-species PK. Total clearance and volume of distribution were relatively low and bioavailability of lenvatinib was approximately 64-78% in all the species tested. PK of lenvatinib in mice and rats after oral dose was almost linear at the doses ranging from 3 to 30 mg/kg. An empirical allometric scaling successfully predicted oral systemic exposure of lenvatinib in humans. Collectively, PK profiles of lenvatinib in nonclinical animals were well characterized and were useful for PK prediction in humans.

A Simple UPLC-MS/MS Assay of Rifampin in a Small Volume of Human Plasma.

Rifampin (RIF) is a typical cytochrome P450 (CYP) 3A inducer and inhibitor of organic anion transporting polypeptide (OATP) 1B1 to assess drug-drug interaction (DDI) via CYP3A or OATP1B1 in clinical settings. To ensure sufficient exposure of RIF in DDI studies, it is important to determine plasma RIF concentrations. In this study, we developed a simple RIF assay in a small volume of human plasma by ultraperformance liquid chromatography with tandem mass spectrometry. RIF in 0.02 mL of plasma was extracted using protein precipitation and separated on a reverse phase column under gradient elution of three mobile phases, where the mobile phase C containing 1% formic acid was exclusively used to reduce the carryover of RIF. RIF and the internal standard were detected by multiple reaction monitoring in positive-ion electrospray ionization. RIF was quantifiable at 0.025-10 µg/mL without the carryover issue. The intra- and inter-run assays confirmed the reproducibility of the assay. Stability assessments ensured that RIF in human plasma was stable for 6 h at room temperature and for 409 days at -15 °C or below. The assay was successfully applied to a pharmacokinetic study with successful incurred sample reanalysis.

A validated UPLC-MS/MS assay of E7090, a novel selective inhibitor of fibroblast growth factor receptors, in human plasma and urine.

E7090, a novel fibroblast growth factor receptors inhibitor, is currently under clinical development for the treatment of patients with solid tumors. Assays for the determination of E7090 concentrations in human plasma and urine have been developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to evaluate pharmacokinetic profiles of E7090. E7090 and a deuterated labeled internal standard (IS) were extracted from 50 µL of plasma by protein precipitation. In quantification of E7090 in urine, 50 µL of urine samples fortified with 15 µL of ethanol (10:3, v/v) to minimize nonspecific binding of E7090 to urine containers were subjected to the assay without extraction. E7090 and the IS were separated by chromatography on a reverse phase column and were detected by selected reaction monitoring in the positive ion mode. The lower limit of quantification was set at 1 ng/mL and E7090 was quantifiable from 1 to 3000 ng/mL in plasma and urine. Accuracy and precision were measured during the reproducibility assessments and were within ± 7.0% and 9.1%, respectively, in plasma and within ± 7.0% and 5.8%, respectively, in urine, indicating sufficient reproducibility. The validated methods were successfully applied to the quantification of E7090 in human plasma and urine to support a Phase-1 clinical trial.

Liposome-Encapsulated Eribulin Shows Enhanced Antitumor Activity over Eribulin for Combination Therapy with Anti-PD-1 Antibody.

Eribulin is a microtubule dynamics inhibitor with tumor microenvironment modulation activity such as vascular remodeling activity. Here, we investigated antitumor and immunomodulatory activities of eribulin and its liposomal formulation (eribulin-LF) as monotherapies or in combination with anti-programmed death 1 (PD-1) Ab. The antitumor activity of eribulin or eribulin-LF as monotherapy or in combination with anti-PD-1 Ab was examined in a P-glycoprotein-knockout 4T1 model. Eribulin and eribulin-LF showed stronger antitumor activity in immunocompetent mice compared with immunodeficient mice, indicating that they have immunomodulatory activity that underlies its antitumor activity. Combination therapy of eribulin and eribulin-LF with anti-PD-1 Ab showed antitumor activity, and the combination activity of eribulin-LF with anti-PD-1 Ab was observed at a lower dose and longer interval of administration compared with that using eribulin. To examine the immunomodulatory activity of eribulin and eribulin-LF and its underlying mechanisms, we performed flow cytometry, IHC, and gene expression profiling. IHC and flow cytometry revealed that eribulin-LF increased microvessel density and intratumoral populations of cytotoxic T cells and natural killer cells rather than eribulin. Gene expression profiling demonstrated that eribulin-LF induces IFNγ signaling. Furthermore, IHC also showed that eribulin-LF increased infiltration of CD8-positive cells together with increased CD31-positive cells. Eribulin-LF also increased ICAM-1 expression, which is essential for lymphocyte adhesion to vascular endothelial cells. In conclusion, eribulin showed combination antitumor activity with anti-PD-1 Ab via immunomodulation due to its vascular remodeling activity, and the liposomal formulation showed improved antitumor activity over the standard formulation.

A simple UPLC-MS/MS assay with a core-shell column for the determination of exemestane in human plasma for clinical application.

Exemestane is one of the aromatase inhibitors and has been used to treat breast cancer by lowering estrogen levels. Accurate quantification of exemestane is important to set an optimal dose, and thus a simple assay for exemestane is developed by ultra-performance liquid chromatography with tandem mass spectrometer. Exemestane was extracted from human plasma samples (100 µL) by simple protein precipitation with acetonitrile/methanol (1/1, v/v). Interference peaks observed close to the elution of exemestane led us to use a core shell column for higher selectivity instead of totally porous columns. The extracts were chromatographed on CORTECS UPLC C18, under a gradient elution at a flow rate of 0.25 mL/min and detected in the selected reaction monitoring. Validation parameters were assessed in accordance with the bioanalytical guidelines using quality control samples. Exemestane in human plasma was quantifiable from 0.5 to 50 ng/mL with high extraction recovery and minimal matrix effects. Hemolyzed or hyperlipemic plasma did not impact the exemestane assay. Exemestane was stable in human plasma for 392 days at -15°C or below. The developed assay was robust and successfully applied to quantifying exemestane concentrations in human plasma to support a clinical trial.