Background: Due to rapid methotrexate (MTX) metabolism, particularly in patients receiving low doses (standard in rheumatoid arthritis (RA) patients), methotrexate polyglutamate (MTXPGs) serve as informative biomarkers of drug exposure. The MTXPGn(2-7) are predominantly present in red blood cells, and whole/capillary blood is the preferred matrix for MTXPG determination.

Aims: This study's primary objective was to quantify MTXPGs (following EMA guidelines) in Mitra™ samples collected from adults with RA taking MTX.

Methods: The Synergi-FUSION-RP column was maintained at 30⁰C, with a gradient flow of mobile phase (water/acetonitrile with 0.1% formic acid and 10mM NH₄HCO₃). A 20µL capillary blood sample was extracted using 0.1% formic acid enriched with MTXPG-d₅/d₇. The sample underwent purification with perchloric acid and was then neutralised with an equivalent amount of potassium acetate. The LC-MS/MS platform (Shimadzu-LC40/QTRAP-6500+) was employed for method optimisation and sample processing.

Results: The methodology was validated within the 0.1-100nM calibration range for each MTXPG. All precisions and accuracies were maintained within ±15% of their nominal values. The purification using perchloric acid exhibited the highest recovery; however, it required neutralisation due to significant matrix effects. Elevated levels of hematocrit (HT>60%) adversely affected the recovery of analytes from the VAMS sample. The MTXPGs remained stable in microsamples during storage for 10 days at room temperature (sunlight-protected).

Conclusions: The analytical technique has been optimised and validated in accordance with EMA standards. This assay could facilitate home-based monitoring of MTXPGs in capillary blood collected with the Mitra™ microsampling device.

Key Words: methotrexate, MTXPGs, LDMTX, rheumatoid arthritis, adherence

Background: Cytochrome P450 3A (CYP3A) metabolizes approximately 50% of human drugs, making CYP3A phenotyping crucial for personalized medicine. We developed the 6β-hydroxycortisol to cortisol concentration ratio (6β-OHF/F) in dried blood spots (DBS) as a minimally invasive CYP3A activity assessment. To apply this method clinically, establishing the 6β-OHF/F range in healthy individuals is necessary.

Aims: We aimed to investigate the normal range of the 6β-OHF/F ratio in DBS samples obtained from healthy participants.

Methods: 6β-OHF and cortisol concentrations in DBS were quantified using LC–MS/MS. The study population consisted of 37 healthy participants (21 men, aged 22–61 years, 16 women, aged 22–58 years). The study protocol was approved by the Tokyo University of Pharmacy and Life Sciences Human Subjects Review Board, and written informed consent was obtained from all participants.

Results: The measured concentrations of 6β-OHF and cortisol in four DBS spots were 0.0112–0.182 ng/80 μL and 1.05–7.77 ng/80 μL, respectively. The resulting 6β-OHF/F ratios in DBS from the 37 healthy participants ranged from 0.00627 to 0.0321, reflecting a 5-fold interindividual variability in CYP3A activity. The mean 6β-OHF/F ratio in women (0.0151 ± 0.0075) was higher than that in men (0.0138 ± 0.0034).

Conclusions: This minimally invasive index allows pre-treatment identification of patients with extremely low CYP3A activity, enabling optimized initial drug dosing and reduced adverse reaction risk. We propose a 6β-OHF/F value of <0.005 in DBS to identify patients requiring reduced doses of CYP3A-metabolized drugs.

Keywords: biomarker, dried blood spots, 6β-hydroxycortisol/cortisol ratio, CYP3A, phenotyping

Background
Although drug abuse has increased substantially over the last decade to reach the level of a national crisis in the United States, little progress in drug of abuse monitoring and treatment has been made. Recently, dried blood spots (DBS) have emerged as a promising alternative that do not have the drawbacks of IV blood samples. DBS are collected after a finger prick, thus, eliminating the need for a phlebotomist, however, DBS are currently not yet used for toxicology screening.

Aims
It was the aim to develop and validate a DBS-based toxicology screening/ therapeutic drug monitoring strategy. The assay developed during the present thesis includes 135 drugs of abuse and pain drugs including key metabolites.

Methods
The validation followed applicable FDA and Clinical Laboratory Standards Institute guidelines. For the drugs for which the assay worked, matrix effects and autosampler stability were also established. Transport stability at increased temperature and moisture still needs to be established.

Results
94 drugs across 18 drug classes worked well on the platform with clinical assay criteria, 27 will work as a semi-quantitative screening assay while 13 drugs do not work on the current platform.

Conclusions
The resulted validated assay has great potential to become commercialized with endless possibilities from forensic testing to therapeutic drug monitoring purposes. The fact that it is done meeting regulatory requirements also helps it become a marketable product.

Keywords
Toxicology, VAMS, Alternative Sample Strategy, Validation, LC-MSMS

Background: (Val)ganciclovir is the first-choice agent for treating congenital cytomegalovirus infection, particularly in neonates. Due to potential toxic side effects and the physiological challenges associated with neonates, therapeutic drug monitoring with dosing adjustments is essential to ensure the safety and efficacy of pharmacotherapy.

Aims: This study aimed to evaluate the pharmacokinetics of VGCV using the microsampling technique in neonates with cCMV infection.

Methods: For GCV monitoring, the previously validated HILIC-LC-MS/MS method was employed. Simultaneously with GCV, creatinine levels were determined in a single analytical run. Mitra™ volumetric-absorptive microsamples were collected at the following time points: C₀ (30 minutes before drug administration) and at C₂, C₄, C₈, and C₁₂ (after oral administration of Valcyte®). The patients received a dose of 16 mg/kg every 12 hours. In this preliminary study, 10 neonates were included.

Results: The mean AUC value was approximately 56.67±24.88 mgxh/L, with more than 70% of patients achieving the target AUC(0-24) value within the 40-60 mgxh/L range. Interestingly, clinical manifestations of side effects were not observed. For patients with toxic or subtherapeutic AUC levels, the clinical pharmacist suggested a dosing adjustment for Valcyte®. The accuracy of sampling using Mitra™ was greater than 95%, and the evaluated sample stability was established at a minimum of 2 weeks under ambient conditions (RT).

Conclusions: The Mitra™ VAMS is a suitable tool for multi-point individual drug exposure and pharmacokinetic evaluation of VGCV in neonates treated in hospitals and at home (after appropriate training of caregivers).

Key Words: (val)ganciclovir, CMV, AUC, VAMS, neonates, LC-MS/MS, TDM

Background and Aims: Oxazolidinones such as linezolid and tedizolid are active against methicillin-resistant Staphylococcus aureus infections. Thrombocytopenia caused by linezolid is a concern, and therapeutic drug monitoring (TDM) has been reported to help prevent this condition. Although plasma concentrations provide valuable information for treatment decisions, plasma sample collection can be burdensome for both patients and healthcare providers. Therefore, there is an interest in saliva as an alternative to TDM.

Methods: To establish a monitoring method using saliva, we collected saliva and plasma samples from rats after linezolid or tedizolid administration. Subsequently, a prospective study was conducted to evaluate the potential clinical application of TDM for linezolid and tedizolid using saliva. Patients who received linezolid or tedizolid were enrolled in the study. We analyzed the concentration profiles of linezolid and tedizolid in saliva and examined their correlation with plasma concentrations.

Results: The saliva/plasma (S/P) concentration ratio of linezolid in rats and patients were 0.66 and 1.03, respectively. The S/P ratio of tedizolid in rats and patients were 0.05 and 0.33, respectively. Strong correlations were found between the saliva and plasma concentrations of linezolid (r=0.945 in rats, r=0.83 in patients) and tedizolid (r=0.964 in rats).

Conclusions: Considering the correlation between saliva and plasma concentrations of linezolid and tedizolid as well as the characteristics of saliva, our results suggest that saliva is a useful matrix for TDM. Notably, the concentrations of linezolid in the plasma and saliva of patients were similar, indicating its potential applicability in clinical settings.

Keywords: Oxazolidinones, saliva, alternative sampling

Background: Dried blood spot (DBS) sampling is considered an alternative sampling strategy for TDM in patients receiving OPAT.

Aims: To provide insights in the barriers and facilitators of DBS sampling for OPAT patients.

Methods: A qualitative study was conducted in patients performing DBS sampling for TDM of vancomycin and creatinine during OPAT. Patients were interviewed using a semi-structured interview guide based on the theoretical domains framework and consolidated framework for implementation research. The results were transcribed verbatim and analysed inductively by two researchers according to thematic analysis.

Results: 15 barriers and 15 facilitators were classified in four domains: information, technique, process and patient. The overall view on DBS sampling was very positive. Patients mentioned the added value of DBS sampling for healthcare and society in terms of health care personnel and time. Involvement and control over their own healthcare was also mentioned as a positive attribute of DBS sampling. Facilitators included training including the information to warm the hands, DBS properties (simple and quick to perform, easy integration in routine, time- and cost saving) and previous experience with finger prick. The patient’s sense of responsibility is crucial for DBS success. Barriers included insufficient information on the background of DBS sampling, the limitation of assays available with DBS sampling, posting and mail delivery and aversion towards needles.

Conclusions: This study addressed barriers and facilitators of DBS sampling for TDM during OPAT and provides multiple advices for proper DBS implementation.

Key Words: OPAT, TDM, dried blood spot sampling, vancomycin, qualitative analysis

Background: Creatinine (CRE) is the most popular renal function biomarker, and venous plasma/serum collection is necessary to monitor it in routine clinical practice. Our centre successfully introduced home-based self-sampling for immunosuppressant monitoring, and optimizing the methodology for additional determination of CRE in a single sample seems highly valuable.

Aims: The study aimed to evaluate the EMA's approach to determining creatinine (an endogenous molecule) in qDBS (Capitainer B) and VAMS (Mitra) samples collected from pediatric renal transplant recipients.

Methods: During HILIC-based (Luna chromatographic column from Phenomenex), LC-MS/MS (6500TQ AB Sciex) method optimization and validation, the surrogate followed approaches were evaluated: surrogate analyte, surrogate matrix, the background subtraction approach and the standard addition protocols. The extraction of CRE (or surrogate) was achieved using a water/methanol medium, while protein was precipitated using a zinc sulphate/acetonitrile mixture.

Results: The methodology has been evaluated in the 0.1 - 40 mg/L calibration range. The accuracies and precision were within nominal values. Despite high ion suppression, sample dilution must be done before analysis. Signal-to-noise ratios were lower than 20% for each evaluated approach. The ¹³C-CRE compensated matrix effects were more satisfactory than d₅-CRE.

Conclusions: The surrogate analyte approach (d₃-CRE) produced the most reliable results among those proposed by the EMA in the ICH M10 bioanalytical validation guidelines. The optimized method may be used to determine creatinine in VAMS and qDBS samples concomitantly with immunosuppressants, but it requires two different analytical runs.

Key Words: creatinine, renal transplantation, LC-MS/MS, surrogate analyte, VAMS, qDBS

Background: Soft-mist inhalers (SMIs) are used to treat respiratory diseases. Because SMI has a long drug mist emission time of approximately 1.5 seconds, some patients are unable to keep inhaling during drug mist emission. However, information regarding the impact of improper use on pulmonary drug deposition is lacking. Our previous study demonstrated that a particle emission monitoring can be used to estimate pulmonary drug deposition via dry powder inhaler [Hatazoe S, AAPS PharmSciTech, 2024].

Aims: This study aimed to evaluate whether the particle emission monitoring can be used to estimate pulmonary drug deposition via SMI.

Methods: Spiriva® Respimat® was selected as the SMI model. Pulmonary deposition of inhaled tiotropium was evaluated using an Andersen cascade impactor, an in vitro aerodynamic particle size analyzer. The inhalation flow rate and particle emission profile were measured using an inhalation flow meter and a photo reflection method, respectively. Inhalation performance was characterized by the fine particle fraction deposited on the whole lung (FPF-WL; aerodynamic dimeter < 4.7 μm).

Results: The particle emission monitoring device successfully visualized the differences in timing between inhalation and drug mist emission. FPF-WL via SMI was increased by less difference in the timing between inhalation and drug mist emission and longer inhalation duration time. An inhalation duration shorter than 1.2 seconds reduced pulmonary drug deposition to 25% of the maximum.

Conclusions: Particle emission monitoring is applicable for the prediction of pulmonary drug deposition via SMI as well as dry powder inhalers.

Keywords: alternative sampling strategy, soft-mist inhaler, pulmonary drug delivery

Background: Direct oral anticoagulants (DOACs), such as rivaroxaban (RVX), apixaban (APX), dabigatran (DAB), and edoxaban (EDX), are commonly used in clinical practice to prevent blood clots and strokes. However, they are characterised by a wide therapeutic range, and monitoring these medications may be beneficial for specific populations, including children, elderly patients with renal function difficulties, and those with obesity.

Aims: This study aimed to develop and validate a novel LC-MS/MS method for determining EDX, RVX, APX (and its metabolite, O-desmethylapixaban), and DAB in Mitra™ and SEP10® microsampling devices.

Methods: The in vitro/ex vivo methods have been optimised and validated using the LC-MS/MS system QTRAP6500+ from AB Sciex. An Agilent column C18-XDB was used with a 0.5 mL/min mobile phase flow in gradient mode. Various solvents were employed to extract dried matrices, supported by sonication and cold-induced phase extraction.

Results: Each analyte validated the LC-MS/MS method in the 0.5 - 500 ng/mL calibration range. The methanol/water mixture was used to extract analytes, while acidified acetonitrile was used for protein precipitation. The stable isotope-labelled IS satisfactorily compensated for the matrix effects for each analyte. All acceptance criteria proposed by the EMA and FDA were fulfilled, and analyte recovery did not affect the hematocrit effect (>95%).

Conclusions: The method for determining DOACs was successfully validated in accordance with EMA and FDA guidelines. Moreover, the technique has been standardised through four rounds of IPT. The assay will be implemented for PK-monitoring in paediatric patients undergoing anticoagulant treatment.

Key Words: apixaban, rivaroxaban, dabigatran, edoxaban, microsampling

Background: Multidrug-resistant tuberculosis (MDR-TB) remains a major global health challenge, due to increasing resistance rates and few well-tolerated anti-TB drugs. Linezolid, a key MDR-TB drug, causes haematological and neurological adverse events with prolonged treatment or higher doses. Therapeutic drug monitoring (TDM) is recommended to optimise dosing, but traditional plasma-based TDM is invasive and resource-intensive, limiting its use particularly in high-burden low-resource settings.

Aims: To investigate the feasibility of using saliva as an alternative for linezolid TDM in MDR-TB.

Methods: Adults receiving linezolid for MDR-TB for ≥7 consecutive days had blood and saliva samples collected at 0, 2, and 5 hours post-dose at steady-state (ACTRN12620000681954). Plasma and saliva linezolid concentrations were quantified using liquid chromatography–mass spectrometry. Saliva-plasma correlations were compared via Passing-Bablok regression, Bland-Altman analysis and Pearson's correlation test.

Results: Of the 17 patients, 76% were male, with a mean age of 46 years and weight of 49 kg. A total of 102 saliva and plasma linezolid concentrations were analysed as 54 concentrations were below the limit of quantification. Saliva concentrations were, on average, 1.34 times higher (95% CI, 0.08–2.61) than in plasma. There was good agreement between saliva and plasma concentrations (r = 0.90; P < 0.05) with only 1.9% of concentrations falling outside the 95% limits of agreement.

Conclusion: Saliva linezolid concentrations are strongly correlated with those in plasma, suggesting saliva is a potential non-invasive alternative matrix for linezolid TDM to minimise toxicity and improve MDR-TB treatment outcomes.

Keywords: Linezolid, pharmacokinetic, therapeutic drug monitoring, saliva.