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.

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.

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.

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 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.

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.

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.

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.

Quantitative and qualitative application of a novel capillary microsampling device, Microsampling Wing™ (MSW), using antiepileptic drugs in rats.

A novel microsampling device, namely, the Microsampling Wing™ (MSW), was evaluated using three anti-epileptic drugs (AEDs): carbamazepine, lamotrigine, and phenytoin. A simultaneous assay method of the three AEDs was developed and qualified via liquid chromatography with tandem mass spectrometry. Using 2.8 µL plasma, the three AEDs were quantifiable from 1 or 2 ng/mL. According to the intra-assay reproducibility assessment and additional validation parameters, the established method is reproducible. To apply the device to a pharmacokinetic (PK) study in rats, a cocktail of the three AEDs was orally administered to rats. Whole blood samples were serially collected using the MSW device and a glass capillary from the tail vein, and plasma samples (each 2.8 µL) from each device were assayed to compare PK parameters. The PK parameters of the three AEDs were similar between the two devices. A metabolite identification study was also conducted after oral administration of carbamazepine to rats. At least seven metabolites were detected in plasma, and the major metabolite was carbamazepine 10,11-epoxide, which is in accordance with the reported results. These findings suggest that the MSW device is a useful microsampling device for PK and metabolite identification studies.

A sensitive bioanalytical assay for methylcobalamin, an endogenous and light-labile substance, in human plasma by liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study.

Methylcobalamin (mecobalamin) is a vitamin B12 coenzyme and is effective for the treatment of peripheral neuropathies. As mecobalamin is an endogenous substance in human plasma at low levels and is light-labile, a sensitive assay method has been developed by using liquid chromatography with tandem mass spectrometry with particular care taken for light exposure. Stability tests performed under light showed that mecobalamin was stable in plasma in amber tubes only when exposed to <10 lx light, and thus, all the assays used for the method validation and the in-study sample assays should be performed under red light with minimal light exposure. Mecobalamin and its stable isotope, which was used as an internal standard, were extracted from 0.1 mL plasma by protein precipitation with methanol and were quantifiable in a range from 0.05 to 20 ng/mL. The reproducibility assessment showed acceptable accuracy and precision (≤15 %). Mecobalamin was stable in plasma at room temperature for 21 h when exposed to <10 lx light in amber tubes and for 205 days when stored frozen. The validated method was successfully applied to an in-study sample assay by performing a clinical pharmacokinetic study of mecobalamin, in which a successful incurred sample reanalysis was performed.

Establishment of a simultaneous assay for lemborexant, a novel dual orexin receptor antagonist, and its three metabolites, and its application to a clinical protein binding study.

A simultaneous assay for the determination of lemborexant and three metabolites (M4, M9, and M10) in human plasma and phosphate buffered saline (PBS) was developed and validated using liquid chromatography with tandem mass spectrometry, in support of plasma protein binding (PPB) studies. The analytes were extracted from plasma and PBS by solid phase extraction and then chromatographed on a reversed phase C18 column to ensure peak separation of three metabolites with the same mass transition. The analytes and the corresponding deuterated substances used as an IS were detected in the positive ion mode by multiple reaction monitoring. Lemborexant and three metabolites were quantifiable from 4 and 300 pg/mL in PBS and plasma, respectively, without any carryover. Extraction recovery was almost complete without matrix effects. The accuracy and precision in the intra- and inter-assay reproducibility were within the criteria as well as in the dilution integrity. Stability of the four analytes was ensured to cover duration of equilibrium dialysis and storage of samples. The established method was applied to an ex vivo PPB study in 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.

Application of a Volumetric Absorptive Microsampling Device to a Pharmacokinetic Study of Tacrolimus in Rats: Comparison with Wet Blood and Plasma.

BACKGROUND AND OBJECTIVES: Volumetric absorptive microsampling (VAMS) devices are useful for sampling a smaller volume of blood from rodents in the preclinical setting. In the present study, we evaluated the proof of concept of a VAMS device by comparing the pharmacokinetic data of tacrolimus in rats among dried blood in VAMS, wet blood, and plasma. METHODS: Tacrolimus was administered orally, to rats, at a dose of 10 mg/kg. Only 10 µL aliquots of blood were absorbed by VAMS devices at designated time points. Tacrolimus was extracted with a methanol-water mixture (1:1, v/v) via sonication. Tacrolimus levels in wet blood (10 µL) and plasma (10 µL) were quantified after protein precipitation. RESULTS: Tacrolimus in VAMS devices was quantifiable from 0.2 ng/mL using high-performance liquid chromatography with tandem mass spectrometer. Accuracy and precision were within the acceptance criteria. Bland-Altman plots showed that tacrolimus concentrations in VAMS devices were similar to those in wet blood, regardless of tacrolimus levels. On the other hand, tacrolimus levels in plasma were different from those in VAMS devices, especially at lower concentrations, likely due to partition of tacrolimus to blood cells. However, pharmacokinetic parameters were comparable among the three matrices. CONCLUSIONS: Collectively, these findings suggest that the VAMS device can be a useful device for pharmacokinetic studies in rats.

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.

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±â€¯15.3% and percentage bias for clinical study samples was within±â€¯11.6%. These findings suggest that lenvatinib concentrations in human plasma can be compared across laboratories and clinical studies.

LC-MS-MS Determination of Oxcarbazepine and an Active Metabolite in Human Plasma for Clinical Application.

A bioanalytical method for the simultaneous determination of oxcarbazepine (OXC) and its pharmacologically active metabolite, 10, 11-dihydro-10-hydroxycarbamazepine (HOXC), in human plasma was developed using a high-performance liquid chromatography with tandem mass spectrometry. After protein precipitation by acetonitrile, the analytes (OXC and HOXC) and a stable-labeled isotope of OXC as an internal standard (IS) were chromatographed on a Synergi Hydro-RP column (2.0 mm × 50 mm, 4 µm) with a gradient elution at a flow rate 0.5 mL/min. Detection was performed in electrospray ionization in the positive mode by monitoring the selected ion transitions at m/z 253.1 → 180.2, m/z 255.1 → 192.2 and m/z 257.2 → 184.2 for OXC, HOXC and the IS, respectively. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve ranged from 0.02 to 10 µg/mL for OXC and 0.1-50 µg/mL for HOXC using only 0.05 mL of plasma. No interferences were detected in blank plasma and hemolyzed plasma did not have any impacts on the assay. Accuracy and precision in the intra- and inter-batch reproducibility were within 15%. Neither cross-analytes inter-conversion nor matrix effects were observed. The method was successfully applied to determine plasma concentrations of OXC and HOXC to support a clinical study.

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.

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 µL 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 200 ng/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 160 days. The DBS method was applied to determine E6005 and M11 concentrations in blood samples supporting a pediatric clinical trial.