Background and Purpose: Ro 20-1724 improves cognition and attenuates oxidative
stress in the brain. However, the beneficial effects of Ro 20-1724 on Parkinson’s disease
(PD) remain unknown. Therefore, the present study is aimed to evaluate the effect of
Ro 20-1724 in the rotenone-induced PD model.
Experimental approach: In this study, cytotoxicity of Ro 20-1724 and cell viability
against rotenone were analysed via MTT assay in human neuroblastoma cell (SH
Sy5Y). Apoptotic markers and ROS were measured by flowcytometry, and pro
inflammatory cytokines were measured by ELISA. In in vivo PD model, muscle
coordination activity, locomotor activity, endogenous anti-oxidant enzyme level, and
Nissl staining were used to evaluate protection by Ro 20-1724 (500 µg/kg, i.p., for 21
days).
Key Results: Ro 20-1724 decreased rotenone-induced cytotoxicity in SH-Sy5Y cells.
Ro 20-1724 also decreased pro-apoptotic proteins, ROS production, and pro
inflammatory cytokines expression in rotenone-induced PD in SH-Sy5Y cells. In rats
treated with Ro 20-1724 significantly improved muscle coordination activity,
locomotor activity, and prevented both oxidative stress and dopaminergic neuronal loss.
Conclusions: Ro 20-1724 protected dopaminergic neurons from apoptosis and
neuroinflammation in PD model. Hence, Ro 20-1724 acts as a good drug candidate in
the treatment of PD.
Key words: Parkinson's disease, Rotenone, MTT Assay, ELISA, Locomotor activity.

Background: In middle-aged and elderly patients predisposed to gastrointestinal stromal tumors, amlodipine (AML) is often used to control blood pressure due to underlying conditions.

Aims：However, the mechanisms underlying the interaction between AML and the anti-tumor drug imatinib (IM) have not been thoroughly investigated.

Methods: Herein, we designed administration a single administration model and a continuous administration model in rats, respectively. Pharmacokinetic parameters and plasma metabolomics were analyzed. Additionally, network pharmacology, molecular dynamics simulations and traditional interaction cell experiments were performed later.

Results: The results indicated that both AUC and Cmax increased when IM and AML were combined. The bile acid pathway was identified as a common pathway for both drugs, and molecular dynamics simulations suggested some degree of self-assembly aggregation between IM and AML. The changes in membrane permeability and organic cation transporter (OCT1) expression confirmed that the interaction of the two drugs not only affected the resistance of the cell membrane, but also received OCT1 regulation.

Conclusions: This study provides important insights and theoretical support for enhancing the clinical efficacy of treatments for gastrointestinal stromal tumors with hypertension for the first time. We suggest that when the two drugs are used together in the clinic, the dosage should be payed more attention.

Keywords: Drug-drug interaction, pharmacokinetic, metabolomics, network pharmacology, molecular dynamics, imatinib

Background: Diabetes Mellitus (DM) is a chronic disease characterized by high blood glucose levels due to insufficient insulin production or ineffective insulin use. According to the International Diabetes Federation (IDF), in 2021, there were 537 million DM cases in adults aged 20-79 years, with a projected increase to 643 million by 2030. DM treatment focuses on medication and lifestyle changes, but research on anti-DM drug side effects remains limited in Indonesia.

Aims: to identify the characteristics of metformin side effects in Type 2 Diabetes Mellitus (T2DM) patients.

Methods: A descriptive study was conducted at Kramat Jati Community Health Center in February 2024. Primary data were collected through direct interviews using questionnaires with 83 T2DM patients who met the inclusion criteria.

Result: The majority of subjects were women 54/83 (65.1%) subjects. The largest age group was 51-60 years (37/83 subjects, 44.6%). Diagnosis duration was mostly 1-3 years and >3 years (35/83 subjects each, 42.2%). Metformin was the most commonly used drug, either as monotherapy (45 subjects, 54.2%) or combined with glimepiride (33 subjects, 39.8%), with the highest dose being 2 × 500 mg/day (45/83 subjects, 54.2%). The most reported side effects were metallic taste and bloating (9/83 subjects each, 10.8%), occurring mostly within six months (30/32 subjects, 93.8%).

Conclusions: in using Metformin for T2DM patients, the common side effects were metallic taste and bloating, mainly occurring within six months.

Key Word: Side effect, Metformin, Metallic taste, Bloating, anti diabetes Oral, Characteristic, Primary Health Care

Background: Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by sequence variations in the gene encoding for the CF transmembrane regulator (CFTR). Pharmacological therapies are mainly symptomatic. “CFTR modulators” have been developed to partially restore defective CFTR protein function.
Aims: Application of TDM to pediatric patients treated with ivacaftor/tezacaftor/elexacaftor combination.
Methods: Samples were collected from n=33 pediatric patients affected by cystic fibrosis with age comprised between 6-18 years. Ivacaftor (IVA), tezacaftor (TEZ) and elexacaftor (ELX) concentrations were simultaneously measured using LC-MS/MS system, in plasma samples collected before (T0) and 4 hours after (T4) a daily drug assumption. For IVA, the area under the curve 0-12 hours (AUC0-12) was calculated after estimating plasma concentration at 12 hours.
Results: For IVA, TEZ and ELX, median concentrations at T0 were 0.85 μg/mL, 1.39 μg/mL and 8.87 μg/mL, respectively. Median concentrations at T4 were 1.57 μg/mL for IVA, 5.02 μg/mL for TEZ and 20.14 μg/mL for ELX. Evaluation of AUC0-12h for IVA revealed a median drug exposure of 12.50 μg/mL*h. A positive linear correlation was found between Cmin and AUC0-24 values for IVA with a Spearman r correlation coefficient of 0.93 (95% CI=0.83-0.97, P<0.001).
Conclusions: Number of blood samples per patient may be reduced since exposure to IVA may be monitored by assessment of Cmin levels. TDM program could improve our knowledge on the sources of PK variability and the safety profile of these drugs in real life, facilitate a more cost-effective patients’ management.
Keywords: TDM, pediatric patients, Cystic fibrosis, LC-MS/MS

Background: DMDI is a Line chatbot designed to provide essential educational information on metformin for patients with Type 2 diabetes.

Aims: This research aimed to develop tools to educate patients and deliver comprehensive drug-related information.

Methods: The chatbot provides text and visual content on metformin tablets, including usage instructions, side effects, and actions for adverse reactions. Its effectiveness was evaluated by assessing user knowledge and satisfaction. Forty Type 2 diabetes patients at HRH Princess Maha Chakri Sirindhorn Medical Center, Nakhon Nayok, Thailand, participated in the study from November 1 to 30, 2024. Pre- and post-intervention tests (true/false questions) measured knowledge, and a semi-structured questionnaire assessed satisfaction. Volunteers were recruited via promotional posters. Participants completed a pre-assessment, received chatbot training, then completed a post-assessment and satisfaction survey. Data were analyzed using descriptive statistics and paired t-tests.

Results: The results demonstrated a statistically significant improvement in knowledge scores after using the Line chatbot and application (M = 13.6, SD = 0.90) compared to the scores prior to use (M = 9.03, SD = 1.72) (p = 0.000). User satisfaction with the application was rated at the highest level (M = 4.61, SD = 0.56).

Conclusions: This study successfully developed a Line chatbot prototype to educate Type 2 diabetes patients about metformin. A limitation was the focus on metformin, with other antidiabetic medications covered only by informational sheets due to budget constraints. Future research should expand this tool to include additional medications, improving diabetes management education.

Background: Unwanted drug reactions (Adverse Drug Reactions) are problems that occur unintentionally when administering drugs at therapeutic doses to humans. This undesirable drug reaction can also occur in the hospital. The pharmacy and therapy committee, one of whose tasks is to identify every unwanted drug event, needs to provide education and monitoring so that side effects do not harm patients.

Aims: To identify the incidence of unwanted drug reactions (ADR) in secondary health services and to understand the description of drug side effects that can occur.

Methods: This was a prospective, descriptive qualitative study, by providing education to health workers about the signs or symptoms of unwanted drug reactions and filling out the yellow sheet for reporting side effects of drugs which is submitted to the BPOM Pharmacovigilance Center. The data used is an existing data from a one-year period in 2023

Result: There were 36 patients experienced side effects from the drug. The incidence in children 8/36 (22%) and adults 30/36 (78%). The drug classes that caused the most side effects were NSAIDs (13/40) and cephalosporin antibiotics (11/40).The most incidents were skin: itching, bumps, redness 28/36 (78%). The treatment obtained in this cases were: observation without an additional therapy, antihistamines, corticosteroids or combination therapy.

Conclusion: Adverse Drug Reactions can occur in children and the elderly. From the Data NSAIDs and cephalosporin antibiotics are the two main classes of drugs that cause side effects.

Keywords: Adverse Drug Reactions, Pharmacovigillans, NSAID, Cephalosporin, descriptive qualitative study

Background: The availability of therapeutic drug monitoring (TDM) service is an invaluable resource for individualizing drug therapy for older Antiseizure medication (ASM). In resource-constrained setting, when allocation of funds demands distributive justice, it is imperative to rationalize need for TDM services.

Aims: This audit was intended to evaluate effectiveness of TDM services provided by our center for phenytoin, phenobarbitone or carbamazepine to make process and performance improvements.

Methods: The present study is an audit of TDM requisitions received by the Department of Pharmacology for antiepileptic drugs between Jan 2015 to June 2021. The study was conducted with approval from Institutional Ethics Committee (INT/IEC2017/673).

Results: In total, 934 samples were tested for TDM from 798 patients between January 2015 and June 2021.
Phenytoin was most common older ASM for which TDM was requested (59.8%), followed by phenobarbitone (27.4%) and carbamazepine (12.7%). Phenytoin is most common drug for which TDM requests were received from all age groups {adults (68.4%), children (64.7%) and infants (45.3%)} except the neonates. In 54.5% of instances, it was prescribed as monotherapy and in 15.5% as a part of combination ASM regimen. TDM requests received from 31.5% patients exhibited inadequate response. Out of these, nearly 50% of the patients were observed to have drug levels in the previously reported statistical therapeutic range for the drug.

Conclusion: TDM in our study was found to be instrumental in differentiating between drug toxicity and worsening of disease, especially in neonates, where the signs of meningitis would overlap with phenobarbitone toxicity signs.

Background:
Rare diseases have a low prevalence, limited medical expertise, scarce knowledge, inadequate care offerings, and restricted research, making patients the "orphans" of health systems. In KwaZulu-Natal (KZN), commonly reported rare diseases include dermatomyositis, inflammatory myositis, necrotizing myositis, neuromyelitis optica, and transverse myelitis. The KZN Provincial Pharmacy and Therapeutics Committee (PTC) approves nonformulary medicine requests for managing rare diseases; however, there is limited follow-up and research on the effectiveness of these treatments.

Aim:
This study investigates the potential effectiveness of nonformulary medicines, particularly rituximab-based therapies, approved by the KZN PTC for the management of rare diseases at tertiary hospitals in KZN since January 2019.

Methods:
A retrospective case-series study was conducted at Inkosi Albert Luthuli Central Hospital (IALCH). Patient records were reviewed for those who received rituximab-based therapy on a named-patient basis. Laboratory and imaging costs were extracted from the MediTech™ system and analysed according to hospital wards visited during the study period. The study was approved by the University of KwaZulu-Natal and other relevant authorities.

Results:
Rituximab treatment led to increased muscle strength and decreased creatinine kinase levels in patients with dermatomyositis, inflammatory myositis, and necrotizing myositis. In transverse myelitis, C-reactive protein levels significantly declined. Data on neuromyelitis optica were insufficient for assessment.

Conclusion:
The approval of rituximab for immunosuppression in these rare diseases was justified, as it demonstrated effectiveness in slowing disease progression.

Keywords:
Rituximab, rare diseases, immunosuppression, muscle strength, Kwa Zulu Natal

Background:To establish a simple, effective, accurate analysis method forTDM of levetiracetam in serum of patients with epilepsy. Meanwhile, an internal quality control method for TDM of levetiracetam aim to developed to guarantee the analytical quality of the assays.

Methods: HPLC-DAD method for the quantitative determination of levetiracetam has been developed and validated. Levetiracetam quality control products were self-made. The internal quality control results in our laboratory accompanying with TDM of levetiracetam were collected and retrospectively studied. The control charts for Levey-Jennings quality and Z-score were generated using Microsoft Excel. Westgard multi-rules were applied for quality assessment of TDM of levetiracetam.

Results: Calibration curves were linear over a levetiracetam concentration range of 1-80 μg/mL, with a correlation coefficient equal or greater than 0.997. The intra-day and inter-day coefficient of variations of low, medium, and high concentration of levetiracetam were 1.03-6.9%, 1.63-8.19%, respectively, while the extraction recovery rate were 63.72-83.85%, 67.15-83.72%, respectively. The accuracy ranged from 96.26% to 109.31%. Levery-Jennings and Z-score combined with Westgard multi-rules were used to evaluated 20 batches of quality control products and none of them were out of control.

Conclusion: This method is simple, effective and accurate for daily routine use in TDM of levetiracetam. The reliability and accuracy for levetiracetam measurement were improved though developing internal quality control method and the levetiracetam plasma concentration is helpful for further promotion the clinical rational use of the drug.

Key Words: Levetiracetam; therapeutic drug monitoring; HPLC-DAD; internal quality control; Westgard multi-rule theory

Background: The Cost-effectiveness of Therapeutic Drug Monitoring (TDM) in Psychiatric care is questionable

Aims: To review the cost-effectiveness of TDM in psychiatric care.

Methods: This review synthesizes findings from 12 studies exploring the cost-effectiveness of therapeutic drug monitoring (TDM) and related interventions in psychiatric care by using the Pubmed database 2000-2025.

Results: The studies collectively highlight the economic and clinical benefits of TDM and other tailored strategies for managing mental health conditions. Research on clozapine monitoring emphasizes its role in reducing adverse effects and healthcare costs for schizophrenia. Similarly, TDM of citalopram and atypical antipsychotics demonstrates improved therapeutic outcomes and economic efficiency. Studies on Alzheimer’s disease treatments, including cholinesterase inhibitors and the rivastigmine transdermal patch, underscore the cost-effectiveness of regionally tailored practices. Interventions like dementia care management and self-management education programs for epilepsy showcase the dual advantages of enhanced clinical outcomes and cost savings. Additionally, pharmaceutical care models in psychiatric hospitals and contingency management programs for heroin abstinence provide evidence for reducing healthcare costs while improving patient care. Research comparing long-acting and monthly formulations of antipsychotics, such as paliperidone palmitate, further supports the utility of cost-effective formulations in improving adherence and outcomes.

Conclusion: These findings collectively emphasize the importance of personalized, cost-effective strategies in optimizing psychiatric care and resource utilization.

Keywords: Cost-effectiveness, Comprehensive review, TDM in Psychiatric medications,

Background
Bacteriophages, viruses that kill bacteria, are tested in clinical trials as alternatives to antibiotics.
Bacteriophage purification is vital to ensure they ‘re free of bacterial endotoxins to prevent adverse events in therapy.
There is no gold-standard method for bacteriophage purification.
There are several methods for endotoxin detection and quantification.
Interleukin Receptor Associated Kinase 3 (IRAK3) is a negative regulator of inflammation.

Aims
Develop novel endotoxin quantification assay
Evaluate effectiveness of bacteriophage purification methods in removing endotoxins.
Determine whether bacteriophage morphotypes impact endotoxin purification efficacy.

Methods
Purification of bacteriophages using Triton X-100, CsCl ultracentrifugation, Pierce™ Endotoxin Removal Resin columns.
IL-6 & TNF-α cytokine responses from IRAK3 knockout THP-1 (human monocyte) cells used to measure endotoxin concentrations of crude and purified bacteriophages.

Results
Triton X-100 and CsCl were most effective in reducing cytokine production.
Biphasic standard curves of cytokine production by IRAK3 knockout THP-1 cells: IL-6 had better correlation with low endotoxin concentrations; TNF-α had better correlation with higher concentrations.
Endotoxin concentrations in bacteriophage preparations calculated from standard curves: Triton X-100 and CsCl were most effective in endotoxin removal. Bacteriophage morphology did not impact purification efficacy.
Triton X-100 resulted in lowest bacteriophage titre loss.

Conclusions
Triton-X 100 was effective at reducing endotoxin concentration and bacteriophage recovery.
Novel assay based on IRAK3 knockout THP-1 cells is robust method of predicting immune responses to bacteriophage preparations with wider range of endotoxin detection limits than commercial kits
Bacteriophage morphology didn’t influence efficacy of purification.

Keywords
Bacteriophages, endotoxins, immune response, IRAK3

Objective: To improve clinical treatment outcomes and reduce adverse reactions, a retrospective analysis was conducted on the treatment of schizophrenia, with the aim of providing a reference for clinical treatment.

Methods: Cases were retrospectively collected according to inclusion criteria, with basic data including patient age, gender, daily dose (D), days of medication, times of medication per day, drug concentration (C), concomitant medication, scale scores, and adverse reactions. Analysis was performed using Two-way ANOVA variance analysis, Kolmogorov-Smirnov test, correlation analysis, and multiple regression through Prism 8.2.1.4 and Excel software.

Results: Significant differences in amisulpride concentration were observed among patients of different ages and daily doses, with male patients having significantly lower concentrations than females (P<0.01), and females having significantly higher prolactin levels than males (P<0.01). Under the same dose and dosing interval, the number of adverse reactions and prolactin values in females were almost twice that of males. Correlation and multiple regression analysis showed that amisulpride concentration was positively correlated with C/D value (P<0.01) and adverse reactions (P<0.05). There was no clear correlation between amisulpride concentration and dosing frequency of once daily (Qd) or twice daily (Bid). However, the higher the concentration, the greater the number of adverse reactions, regardless of the dose.

Conclusion: Females are more likely to experience prolactin abnormalities when using amisulpride than males and should use it with caution. The incidence of adverse reactions to amisulpride is related to its concentration, and clinical monitoring of amisulpride concentration should be conducted regularly.

Background: Long-acting injectable (lai) antipsychotics provide improved adherence and reduced relapse rates. Paliperidone palmitate is available in different dosing intervals, including 1 month (pp1m), 3-month (pp3m), and 6-month (pp6m) formulations, allowing for individualized treatment approaches. While the transition from pp1m to pp3m is well-documented, real-world data on direct switching from pp3m to pp6m remain limited.

Aim: this study aims to describe paliperidone exposure in patients transitioning from pp3m to pp6m, providing data of a real-world setting.

Methods: Patients with a diagnosis of schizophrenia treated with lai paliperidone were enrolled at asl Città di Torino (Italy). Paliperidone was quantified at timing 0 (pp3m) and every month after switching to pp6m through liquid chromatography coupled with mass spectrometry. Trough concentrations were normalized considering the different doses.

Results: 8 patients were analzyed: 5 (62.5%) were males and median bmi was 28 (interquartile range, iqr, 24;35) Kg/m2. Median pp3m was 3.71 (IQR, 2.93; 6.90), pp6m1 5.05 (4.20; 1.13), pp6m2 5.31 (3.98; 8.81), pp6m3 4.48 (3.10; 7.23), pp6m4 3.93 (2.53; 7.82) and pp6m5 3.85 (2.36; 5.12) 10-8 mL-1. Pp6m5 exposure is similar to pp3m. Concerning different timings, a border-line statistical significance (p=0.062) was suggested. Bmi and age were not correlated with drug exposure.

Conclusions: For the first time, pp6m exposure was described, suggesting after 6 months patients reached similar durg concentrations compared to pp3m. This study supports favorable long-term outcomes of paliperidone every 6 months, but studies in larger cohorts of patients are required.

Keywords: paliperidone palmitate, long-term treatment, pharmacokinetics

This paper reviewed the innovative measures and achievements of the Department of Pharmacy of the Second Affiliated Hospital of Zhejiang University School of Medicine in the construction of therapeutic drug monitoring (TDM) platform and precision pharmacy practice. By integrating multi-disciplinary technologies such as drug gene testing, drug concentration monitoring and metabolomics analysis, the Department of Pharmacy of the Second School of Zhejiang University has established a complete TDM platform, covering concentration monitoring of more than 70 kinds of drugs in 12 categories and 11 kinds of gene testing items. On this basis, we pioneered a new model of pharmaceutical care with TDM resident pharmacists as the core. Through innovative measures such as optimizing report templates, developing information system modules, and constructing medical education models, we significantly improved the TDM awareness rate of medical staff and doctor-patient satisfaction, and reduced the incidence of adverse drug events. In addition, in cooperation with Zhejiang University, the Department of Pharmacy used big data and artificial intelligence technology to build a precision pharmacy information platform, and realized the in-depth mining of drug response rules and the optimization of individualized treatment plans. Through the "Mountain and Sea Project" and the construction of the medical Union, the Department of Pharmacy of the Second Hospital of Zhejiang University has also promoted TDM services to grass-roots medical institutions, helped the sinking of high-quality medical resources, and promoted the popularization of precision pharmaceutical services across the country.

Background: Leptin is a key hormone involved in energy homeostasis and metabolism, with genetic variations influencing its role in disease susceptibility. The leptin gene polymorphism rs7799039 (G2548A) has been implicated in metabolic disorders, including dyslipidemia and hypertension.

Aims: This study aims to explore the association between rs7799039 of Leptin gene and dyslipidemia and hypertension, which are related to induced cardiovascular disease.

Methods: The rs7799039 polymorphism was genotyped using Real-Time PCR, and clinical outcome data, including lipid profiles and blood pressure measurements, were recorded.

Results: The results reported that patients carrying the homozygous variant (AA) related significantly to dyslipidemia and hypertension (P-value = 0.032, 0.008, respectively).

Conclusions: Evidence suggests that genetic variations in leptin signaling may contribute to lipid metabolism disturbances and blood pressure regulation, potentially leading to an increased risk of cardiovascular diseases.

Key Words: Leptin gene, Metabolic syndrome, Dyslipidemia, Hypertension

Backround: Vitamin K2 (VK2) is a fat-soluble vitamin which plays an irreplaceable role in bone mineralization, and soft tissue calcification. In the process of calcification of soft tissues and bone mineralization, matrix Gla proteins (MGP) and osteocalcin are crucial. VK2 functions as a cofactor of gamma-glutamyl carboxylase, an enzyme which activates vitamin K-dependent proteins.

Aims: The aim of this study was to assess the effect of VK2 on ectopic calcifications, with special regard to the rotator cuff calcification in patients with calcific tendinitis.

Methods: 60 subjects with confirmed diagnosis of calcific tendinitis were supplemented with 120 g/day of VK2 (30 patients) or placebo (30 patients) for 6 months. The subjects were invited periodically every 3 months for blood collection (ALP, creatinine, Ca, P, 25-OH vitamin D, VK2, MGP, dp-uc MGP), X-ray study and clinical examination.

Results: Calcifications disappeared completely or were significantly reduced in 90% of patients supplemented with VK2. After three months of VK2 supplementation we observed significantly increased levels of VK2 (p ˂ 0.001). The VK2 median concentration after three months was 1.830 ng/mL in comparison with pre-treatment levels 0.274 ng/mL. Calcifications were reduced in 20% of patients supplemented with placebo. The median VK2 concentration before the treatment was 0.500 ng/mL in comparison with median (0.418 ng/mL) after three months of supplementation.

Conclusions: According to our research VK2 supplementation significantly accelerates the resorption of calcifications in the rotator cuff tendon and muscle tissue.

Keywords: calcific tendinitis, dp-ucMGP, LC-MS, MGP, MK-7, vitamin K2

Introduction: Inappropriate and unsupervised topical oral corticosteroid use can cause significant dermatologic side effects. We report a case of a 38-year-old female with atopy who developed acneiform eruptions and striae after prolonged misuse of these medications.

Case Presentation: A 38-year-old female with a history of atopy presented with acneiform eruptions and striae. These developed after a year of using topical oral corticosteroids (including 8 mg Methylprednisolone and 0.5 mg Dexamethasone) and 2% Ketoconazole cream to self-treat red patches. The initial lesions, appearing on the chest, were pinhead-sized, reddish, and accompanied by a burning sensation. Lesions increased daily, with itching worsening at night. She also had a history of gastritis, allergies, and asthma. Physical examination revealed multiple discrete miliary-sized monomorphic erythematous maculopapular eruptions on the chest, thighs, back, and left femoral region, with an erythematous plaque on the left brachial region.
Treatment involved stopping corticosteroids and addressing potential complications with antibiotics (100 mg Doxycycline) and topical therapies (3 gr of Clindamycin combined with 110 ml of Acne Feldin as a topical solution). Acneiform eruptions improved, but striae persisted, likely due to decreased collagen synthesis, increased extracellular matrix degradation, epidermal thinning, oxidative stress, and vascular dysfunction.

Conclusion: This case highlights the risks of unsupervised topical oral corticosteroid use, especially in atopic patients. Clinicians should educate patients about proper use and potential side effects, including long-term skin damage, to prevent such complications.

Keywords: Oral Corticosteroids, Adverse Drug Reactions, Acneiform Eruptions, Striae

Background: Secukinumab has been approved for the treatment of psoriasis. Off-label high-dose regimens has been used for certain patients. Therapeutic drug monitoring can assist in adjusting treatment plans.

Aims: This study aimed to determine the therapeutic threshold of secukinumab and evaluate the influence of patient characteristics on secukinumab concentrations in patients with psoriasis .

Methods: Patients with psoriasis (n=36) treated with secukinumab during the maintenance therapy were enrolled in this cross-sectional study. Disease activity was determined using the Psoriasis Area and Severity Index (PASI) with an optimal response (OR) defined as ≤ 2. Secukinumab trough concentrations were measured using an in-house developed sandwich ELISA method.

Results: The median secukinumab concentrations in patients with OR were significantly higher than in those without (35.08 vs 25.94 μg/mL, P<0.05). Secukinumab levels greater than 35.43 µg/mL were associated with OR (sensitivity of 50.00%, specificity of 89.89%, AUC=0.70, and P<0.05). Multivariate logistic regression showed that secukinumab concentrations was significantly associated with OR (OR 1.02; 95% CI 1.01-1.12; P=0.04). Linear mixed-effects analysis indicated that increase in body weight and decrease in albumin were associated with decrease in secukinumab concentrations. IL-17A levels showed no correlation with PASI score or secukinumab concentrations.

Conclusions: Secukinumab concentrations over 35.43 µg/mL were associated with OR in patients with psoriasis . When determining the treatment dose, attention may be paid to patients with higher body weight and lower albumin levels in order to prevent underdosing.

Keywords: secukinumab; therapeutic drug monitoring; ELISA; psoriasis;IL-17A

Background:
Diabetes and its complications are driven by chronic inflammation and oxidative stress. High mobility group box-1 (HMGB-1), a nuclear protein released during cellular stress, plays a key role in triggering inflammatory pathways through its interactions with the receptor for advanced glycation end-products (RAGE) and toll-like receptors (TLRs). These pathways contribute to the development of diabetic complications, including nephropathy, retinopathy, and neuropathy. Targeting HMGB-1 could offer novel therapeutic strategies for mitigating diabetes-related complications.

Aim:
Investigate the role of HMGB-1 in diabetes complications, highlighting its potential as a pharmacological target to improve patient outcomes.

Methods:
A systematic review of literature published between 2013 and 2024 was conducted using databases such as PubMed, Scopus, and Web of Science. Studies evaluating HMGB-1’s mechanisms, its role in diabetes complications, and potential pharmacological interventions were analysed.

Results:
HMGB-1 overexpression correlates with increased inflammation and tissue damage in diabetic patients. Preclinical studies show that inhibiting HMGB-1 or blocking its receptors can reduce inflammatory responses, oxidative stress, and fibrosis, thereby preventing or slowing the progression of diabetic complications. Several experimental HMGB-1 inhibitors, including glycyrrhizin and gabexate mesylate, have demonstrated promising effects in early studies.

Conclusion:
HMGB-1 is a key mediator of inflammation in diabetes complications, making it a promising pharmacological target. Further research is needed to develop HMGB-1 inhibitors for clinical use and to assess their long-term safety and efficacy in diabetic patients.

Keywords:
HMGB-1, diabetes complications, inflammation, pharmacological target, oxidative stress, RAGE.

(Backgroud) The interactions between co-administered enteral nutrients (ENs) and drugs reportedly affect the absorption of the respective drugs. However, the gastrointestinal absorption of Valproate (VPA), an antiepileptic drug used worldwide, co-administered with liquid ENs has not been clarified.

(Aims) This study measured the plasma VPA level in rats after oral co-administration of VPA with liquid ENs and its components

(Methods) VPA 100 mg/kg, dissolved in different ENs (F2α, Racol NF, Ensure Liquid, and Renalen LP) or other components (dextrin, sucrose, degraded guar gum and indigestible), were administered to 7–8-week-old male Sprague Dawley rats. Blood samples were collected from the cannula inserted into the right jugular vein, 0.083, 0.25, 0.5, 0.75, 1, 1.5, and 2 h after VPA administration, then the plasma VPA concentration was measured using high-performance liquid chromatography. The control group was simultaneously administered with water.

(Results) The VPA area under the plasma concentration-time curve from 0 to 2 h (AUC0→2h) was significantly lower for the group co-administered with Renalen LP than that of the control group. Furthermore, dextrin and indigestible dextrin especially contained in the Renalen LP also decreased VPA AUC0→2h; however, other liquid ENs and components did not affect the VPA pharmacokinetics.

(Conclusions) Co-administration with Renalen LP decreased the gastrointestinal absorption of VPA. Moreover, dextrin and indigestible dextrin in Renalen LP likely contributed to the reduced absorption. Therefore, the composition of liquid ENs may affect the absorption of VPA differently, and these findings would guide the future selection of liquid ENs for orally administered VPA.

Background: Benzopyran derivatives play a crucial role in various research fields, including medicinal chemistry, due to their diverse biological activities. These compounds have shown potential in managing diabetes by inhibiting α-amylase, a key enzyme in carbohydrate metabolism. In this study, a series of benzopyran-3-carbonyl derivatives were synthesized and evaluated for their antidiabetic potential using in-silico and in-vivo methods.

Aims: This study aimed to investigate the antidiabetic efficacy of novel benzopyran-3-carbonyl derivatives through molecular docking studies and biological evaluations, including α-amylase inhibition and antihyperglycemic activity in streptozotocin (STZ)-induced diabetic rats.

Methods: A molecular docking study was conducted using the α-amylase protein (PDB ID: 1OSE) to screen 60 benzopyran-3-carbonyl derivatives. The derivatives were synthesized using 3,4-diaminobenzoic acid as the starting material. α-Amylase inhibitory activity was assessed at 100 µg/mL, and IC50 values were determined. In-vivo antihyperglycemic activity was evaluated in STZ-induced diabetic rats by measuring blood glucose levels and area under the curve (AUC) analysis.

Results: The docking study identified D1, D4, D10, D12, D13, D16, and D19 as promising inhibitors of α-amylase. Among them, D10 exhibited the highest α-amylase inhibition (24.67%) with an IC50 value of 72.55 µg/mL. In-vivo studies showed a moderate reduction in blood glucose levels, with a significant decrease of 155 mg/dL over 24 hours compared to the standard drug acarbose.

Conclusions: Benzopyran-3-carbonyl derivatives demonstrated potential α-amylase inhibitory activity but exhibited moderate antihyperglycemic effects in STZ-induced diabetic rats. Further structural modifications may enhance their therapeutic efficacy.

Key Words: Benzopyran, molecular docking, α-amylase inhibition, antidiabetic activity, streptozotocin-induced diabetes.

Background:
Hypertension is one of the leading risk factors for morbidity and mortality. Guidelines recommend lifetime antihypertensive drug therapy to reduce organ damage. Despite lifestyle modifications and pharmacological treatment, a large percent of patients fail to achieve satisfactory blood pressure control - so-called resistant or uncontrolled hypertension. Possible causes include poor adherence and pharmacokinetic variability, both of which can be evaluated objectively with serum drug concentrations.
Valsartan is an angiotensin II receptor antagonist (ARB) and is one of the most prescribed antihypertensives in Norway. Few studies have described the interindividual pharmacokinetic variability of valsartan, and its blood pressure lowering effect, although limited data suggests that high variability for valsartan.

Aims:
The main aim of the study was to study the pharmacokinetic variability of valsartan in a large patient population with controlled and uncontrolled hypertension.

Methods:
Data was collected between 2017 and 2022 as part of the Individualized blood pressure treatment study (IDA), a national hypertension project. Patients prescribed two or more antihypertensive drugs, one of which was valsartan, were studied. Patient parameters included age, sex, BMI, hip-waist ratio, prescribed medication history, measured serum levels of valsartan, additional biomarkers, blood sample time, blood pressure levels (office and ambulatory), heart rate and cytochrome genotype.

Results:
Pharmacokinetic variability for N=1147 hypertensive patients prescribed with valsartan, and the effect of multiple covariates on this variability, will be illustrated. Further results pending.

Conclusions:
The study illustrated the pharmacokinetic variability of valsartan based on serum concentration measurement in a large patient population.

Keywords: hypertension, valsartan, TDM

Background:
Aging significantly impacts drug pharmacokinetics (PK) and pharmacodynamics (PD), altering the safety and efficacy of chronically administered medications in geriatric patients. Physiological changes, including decreased renal and hepatic function, altered body composition, and receptor sensitivity variations, can influence drug metabolism, clearance, and therapeutic response. Understanding these age-related modifications is crucial for optimizing pharmacotherapy in geriatric populations.

Aim:
Investigate how aging influences the PK and PD of chronically administered medicines, identify associated risks, and discuss strategies to enhance drug therapy in geriatric patients.

Methods:
A comprehensive literature review was conducted using peer-reviewed studies published between 2014 and 2024. Databases such as PubMed, Scopus, and Web of Science were searched for relevant articles focusing on age-related PK and PD changes, drug dosing strategies, and adverse drug reactions (ADRs) in elderly patients.

Results:
Aging leads to reduced drug clearance due to hepatic and renal impairment, increased drug half-life, and enhanced drug sensitivity, particularly with cardiovascular and central nervous system agents. Lipophilic drugs exhibit prolonged distribution, while hydrophilic drugs demonstrate higher plasma concentrations. The risk of ADRs escalates with polypharmacy, necessitating careful medication selection and dose adjustments.

Conclusion:
Age-related physiological changes profoundly impact drug therapy in geriatric patients, increasing the likelihood of ADRs. Personalized pharmacotherapy, therapeutic drug monitoring, and deprescribing strategies are essential for optimizing treatment outcomes and minimizing risks in geriatric populations.

Keywords:
Aging, pharmacokinetics, pharmacodynamics, geriatrics, adverse drug reactions, polypharmacy.

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: 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: Apalutamide is used in the treatment of castration-resistant prostate cancer. A simple, specific, selective, and effective liquid chromatography tandem mass spectrometry method for quantifying apalutamide and its active metabolite concentration in human plasma was developed and validated according to the FDA and EMA validation guidelines.

Methods: 24 patients diagnosed with NM-CRPC were recruited. Blood samples were drawn after 4 weeks’ administration with Apalutamide at a dose of 180 mg once daily to ensure steady therapeutic blood levels were achieved. Apalutamide and N-desmethyl apalutamide were analyzed by quantitative liquid chromatography tandem mass spectrometry to identify the concentrations among individuals and the effect on the baseline level of PSA and adverse events.

Results: The linear range, precision, accuracy, matrix effect, recovery, carryover, and stability were appropriate according to regulatory agencies. The apalutamide blood concentration range of the 24 patients was 0.517–7.27 µg/mL, and the median value was 4.92 µg/mL. The N-desmethyl apalutamide blood concentration range was 1.78–8.32 µg/mL, and the median value was 5.71 µg/mL. The median serum PSA level decreased from 61.03 (range 0.57–885.93) ng/mL at baseline to 0.970 (range 0.01–47.9) ng/mL at week 4.

Conclusions: Therapeutic drug monitoring can help evaluate the individual differences between patients taking apalutamide. A dose of 180 mg could reduce baseline PSA level significantly (p < 0.05), and the incidence of skin rash was less compared to a dose of 240mg.

Key Words: Apalutamide, N-desmethyl apalutamide, Therapeutic drug monitoring, LC-MS/MS, Castration-resistant prostate cancer, Human plasma

Background: Dexmedetomidine (DEX) is a multipotent and highly selective agonist of α₂-adrenergic receptor, commonly utilized for sedation and analgesia in neonatal intensive care units (NICUs). It provides effective sedation with minimal respiratory depression and neuroprotective properties. Nevertheless, the pharmacokinetics of DEX in neonates are highly variable, and optimal dosing remains uncertain. Therapeutic drug monitoring (TDM) could support personalized dosing by ensuring appropriate drug exposure and minimizing the risk of adverse effects.

Aims: The study aimed to develop and validate a sensitive and specific LC-MS/MS method for the quantification of DEX in plasma, whole blood, and VAMS-collected capillary blood to support TDM in neonates.

Methods: Sample preparation involved protein precipitation using an acetonitrile-methanol mixture containing an internal standard (DEX-D4). Chromatographic separation was achieved on a C18 column using a gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Detection was performed in MRM mode on a triple quadrupole mass spectrometer with ESI in positive mode. The monitored transitions were m/z 205 → 99 (DEX-D4) and 201 → 95 (DEX).

Results: The method showed linearity in the range of 0.5–50 ng/mL (R² = 0.995) with high precision and accuracy. Stability was confirmed for 5 days at 4°C. The validated method demonstrated reliable performance across all matrices, supporting its use in neonatal TDM.

Conclusions: The developed LC-MS/MS method provides a reliable tool for DEX quantification in neonatal TDM, enabling individualized dosing and improved therapeutic outcomes.

Keywords: dexmedetomidine, LC-MS/MS, VAMS, TDM, neonatal sedation, pharmacokinetics

Background: Brivaracetam belongs to the third generation of antiepileptic drugs and is a structural analogue of levetiracetam but has higher lipophilicity. Interindividual variability necessitates therapeutic drug monitoring, especially when used in polytherapy and in patients with altered pharmacokinetics. Currently, brivaracetam measurement relies solely on complex chromatographic methods, limiting their availability in many laboratories.

Aim: of this study is to develop and validate an HPLC method with a diode array detector (HPLC-DAD) that is sufficiently sensitive and suitable for therapeutic monitoring of brivaracetam.

Methods: The HPLC method was validated according to CLSI and ICH guidelines. Preparation of serum samples was performed by liquid–liquid extraction using ethyl acetate:hexane (1:1) with chloramphenicol as internal standard. Separation was achieved using a 5µm 4.6 x 250mm C18 Shim-pack GIST column with the mobile phase consisted of a 50 mM phosphate buffer (pH=4.5) and organic mixture of acetonitrile and methanol (8/3; v/v). Brivaracetam was quantified at 210 nm.

Results: The developed HPLC-DAD method is linear over a concentration range of 0.4-20 µmol/L. Repeatability showed a relative standard deviation lower than 6.6% and intermediate precision lower than 6,7%, except for the value at the lower limit of quantification (0.4 µmol/L) which was 12.6 %. Bias was lower than 8 %. The method has satisfactory specificity and greenness.

Conclusions: The validation parameters were within the acceptance criteria of the ICH guidelines. The developed method is simple, reproducible, more sensitive than other published HPLC method and suitable for the therapeutic drug monitoring of brivaracetam.

Background: Approximately 1-2% of patients undergoing hip or knee arthroplasty will encounter a periprosthetic joint infection (PJI). Presently, treatment involves revision surgeries alongside long-term antibiotic therapy. Although optimal dosing is very important, there remains insufficient understanding regarding the relationship between plasma concentrations and target-site concentrations of the antibiotics.

Aims: Our aim was to develop a novel ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique for quantifying two frequently prescribed antibiotics, vancomycin and clindamycin, in synovial tissue and bone samples.

Method: Method validation was performed based on the FDA and EMA guidelines.

Results: The method was found linear for vancomycin from 0.8 to 25 µg/g and for clindamycin from 0.5 to 25 µg/g in bone and from 1 to 25 µg/g for both compounds in synovial tissue (r2 >0.990). The results of the validation for precision, accuracy and carry-over were all within acceptance range. All clindamycin extracts showed to be stable in the autosampler for bone and synovial tissue samples after 24 hours. The vancomycin extracts were stable for synovial tissue after 24 hours but not for extracts of bone samples.

Conclusions: A quantification method for vancomycin and clindamycin in synovial tissue and bone using UPLC-MS/MS was successfully developed and validated. To our knowledge, this is the first validated method for quantifying vancomycin and clindamycin in synovial tissue and bone using UPLC-MS/MS. This method has already been applied in a clinical research study.

Key words: antibiotics, periprosthetic joint infection, synovial tissue and bone, chromatography, mass spectrometry

Background: Delta 9-tetrahydrocannabinol (Δ9-THC) metabolises to a glucuronide conjugate, 11-nor-9-carboxyΔ9-THC (Δ9-THCCOOH), which is detected in urine specimens. Two isobaric compounds, Δ8-THCCOOH and a carboxylic acid metabolite of cannabidiol (7-CBDCOOH), are potential interferents in mass spectrometry analytical methods and are increasingly apparent in toxicology cases due to internet-availability of Δ8-THC products and increases in the medicinal use of cannabidiol in Australia, respectively.

Aims: To assess the specificity of GCMS and LCMSMS methods for the confirmation of Δ9-THCCOOH in the presence of these isobaric compounds.

Method: Reference material for Δ8-THCCOOH and 7-CBDCOOH were spiked, alone and with Δ9-THCCOOH, into blank urine. All specimens were analysed with GCMS method as well as an alternative LCMSMS method. Authentic urine samples suspected of containing an isobaric interferent were also assessed.

Results: Δ8-THCCOOH was insufficiently chromatographically resolved using the GCMS method from Δ9-THCCOOH. This resulted in the spiked Δ8-THCCOOH samples failing the qualifier ion ratio criterion (>30%) for one of the ion pairs, as did the suspected authentic urines. Baseline resolution was able to be achieved with an increased chromatographic run-time, which demonstrated that Δ8-THCCOOH was the interferent in the authentic specimens. 7-CBDCOOH did not produce a signal for the selected ions. The LCMSMS method was similar, with insufficient chromatographic separation and failure of qualifier ion ratio criterion (>30%).

Conclusion: Δ8-THCCOOH should be assessed as a part of validation of Δ9-THCCOOH confirmation methods. Adjustments to the established methods was able to identify this interferent in authentic urine samples.

Key words: Δ9-THC, Δ9-THCCOOH, Δ8-THCCOOH, 7-CBDCOOH

Background: Tasurgratinib is a novel fibroblast growth factor receptor inhibitor that has recently been approved for the treatment of patients with unresectable biliary tract cancer with FGFR2 gene fusions or rearrangements. In vitro studies have demonstrated that a dealkylated metabolite, M2, exhibits comparable pharmacological activity to tasurgratinib.

Aims: To evaluate the pharmacokinetic (PK) profiles of tasurgratinib and M2 in humans, it is necessary to develop a sensitive and robust assay. The objective is to establish and apply a simultaneous assay using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) to PK sample assays in support of clinical trials.

Methods: The analytes were extracted from 0.1 mL of plasma by protein precipitation and separated on a reverse-phase analytical column utilizing an at-column dilution configuration. The analytes were detected by selected reaction monitoring in electrospray ionization in the positive ion mode. The validation study assessed the following parameters: Linearity, selectivity, carryover, reproducibility (accuracy and precision), dilution integrity, extraction recovery, matrix effects, and stability. The plasma concentrations of tasurgratinib and M2 following an oral dose of tasurgratinib at 35 mg were determined. In order to guarantee the reproducibility of the assay, incurred sample reanalysis (ISR) was conducted.

Results: Tasurgratinib and M2 were quantifiable from 0.025 ng/mL, with accuracy and precision falling within the accepted range. The PK of tasurgratinib and M2 exhibited comparable systemic exposures, and the ISR results satisfied the acceptance criteria.

Conclusions: An established LC-MS/MS assay was validated and successfully applied to a clinical PK study.

Keywords: Tasurgratinib, Metabolite, Pharmacokinetics

Background: Asthma, a chronic respiratory disorder characterized by airway inflammation, bronchoconstriction, and hyperresponsiveness, poses a significant global health challenge. Given the widespread use of bronchodilators in asthma management, ensuring the precision and safety of therapeutic regimens is critical.

Aims: To develop an analytical tool capable of quantifying four bronchodilator drugs in human plasma, namely, theophylline, diprophylline, formoterol, and salbutamol.

Methods: We established a high-performance liquid chromatography (HPLC) method with ultraviolet detection (HPLC-UV) to quantify these drugs. The plasma concentration ranges were categorized as follows: theophylline (0.691–103.575 μg/mL), diprophylline (0.612–122.4 μg/mL), formoterol (0.537–107.4 μg/mL), and salbutamol (0.7675–153.5 μg/mL). The method employed a straightforward extraction protocol and gradient elution on a C18 column for separation.

Results: The HPLC-UV method achieved rapid quantification of all four drugs in under 18 minutes per run, demonstrating exceptional linearity (correlation coefficients >0.999). It exhibited high specificity, with no interference from plasma components, and sensitivity sufficient to guide dose adjustments. Reproducibility was confirmed by extraction recovery rates and intra-/inter-day precision, both consistently below 5% for all analytes.

Conclusions: This analytical tool stands as a valuable asset in the optimization of therapeutic strategies for individuals with asthma, contributing to the enhancement of treatment efficacy in asthma management.

Keywords: Asthma, High-Performance Liquid Chromatography, Therapeutic Drug Monitoring, plasma, Bronchodilator Drugs

Background:Accurate calibration of analytical methods is crucial for ensuring the reliability of quantitative results in clinical laboratories. Calibration frequency and the number of calibrators used can significantly impact the performance of quality controls (QCs) and overall assay stability.

Aims:The objective of this study is to evaluate the stability of calibration curves for various analytes, including azoles, beta-lactams, and immunosuppressants, and to assess the suitability of different calibration frequencies and internal standards for optimal QC performance.

Methods:A comprehensive review of in-house QC data over six months was performed. We compared the stability of calibration curves using QCs with weekly and monthly calibration methods. Additionally, we analyzed QCs from single curves for azoles, beta-lactams, and immunosuppressants. Different numbers of calibrators (6, 3, and 2(1)) were examined across 10 runs to evaluate their impact on QC performance. We also assessed the use of different deuterated internal standards within the same run to evaluate matrix effects and calibration curve performance.

Results:The maximum bias using 3 calibrators (compared to 6 calibrators) was 1.9% and using 2 calibrators was 3.4%, and generally much lower. Repat azole assay referring to previous calibration showed stability for one or more weeks provided no change in instrument conditions.

Conclusions:This study emphasizes the importance of calibration frequency and the number of calibrators in maintaining laboratory assay accuracy. It recommends a balanced calibration strategy considering cost, precision, and stability, highlighting deuterated internal standards to reduce matrix effects in patient samples, which enhances laboratory efficiency and improves patient diagnoses.

Mass Spectrometry, calibration, QCs

Introduction: In the past decade, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a crucial analytical tool in clinical chemistry laboratories. However, the high cost of these machines often limits their routine use. This research demonstrates how a consolidated LC-MS/MS approach can replace multiple HPLC methods, thereby streamlining turnaround times, reducing costs, and conserving resources.

Methods: To enhance the analysis of 29 drug assays, our laboratory implemented a mass spectrometry strategy that combined these into four efficient workflows. Each workflow underwent stringent validation to ensure reliability, focusing on parameters such as linearity, trueness, precision, specificity, and matrix effects. A comprehensive method comparison was conducted against traditional HPLC protocols to evaluate performance metrics. The effectiveness of the new methodologies was further confirmed through participation in external quality assessments (EQA).

Results: The new methods demonstrated high efficiency, with run times ranging from 1 to 4 minutes. This combined approach resulted in significant cost savings by reducing both instrument and scientist time. Improved service delivery was noted from two assays running multiple times per week, significantly enhancing turnaround times in comparison to weekly HPLC methods.

Discussion: This innovative strategy enhances analytical efficiency and the quality of results while optimizing service delivery. Furthermore, the refined methodology is adaptable for various applications, meeting different laboratory requirements. These results highlight the potential of this integrated approach to transform laboratory practices and elevate analytical capabilities across multiple domains.

Mass Spectrometry, LC-MS/MS, Efficiency, Cost effective, Multiplexing

Background: Mycophenolic acid (MPA) is a widely used immunosuppressant in transplant recipients, primarily administered as mycophenolate mofetil (MMF). Despite its clinical efficacy, MPA exhibits significant inter- and intra-individual pharmacokinetic variability, necessitating therapeutic drug monitoring (TDM). The primary metabolite, mycophenolic acid glucuronide (MPAG), contributes to the drug’s pharmacokinetics but lacks pharmacological activity.

Aim: Study aimed to develop and validate a simple, reliable, and cost-effective high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for simultaneous quantification of MPA and MPAG in human plasma.

Methods: The method employed a C18 analytical column and an isocratic mobile phase consisting of 20mM phosphate buffer (pH 2.3-2.4) and acetonitrile (62:38, v/v). Carbamazepine was used as an internal standard. Sample preparation involved protein precipitation with acetonitrile. The retention times for MPAG, MPA, and the internal standard were 1.5, 7.2, and 3.2 minutes, respectively.

Results: Linearity was established over the concentration range of 0.39-50 mg/L for MPA and 1.56-400 mg/L for MPAG (R² > 0.999). The method demonstrated acceptable within-day and between-day precision (CV% < 15%) and accuracy (> 94%). Absolute recoveries of MPA and MPAG were above 97%, with no significant matrix effects.

Conclusion: This validated HPLC-UV method is suitable for routine TDM of MPA and MPAG in clinical settings. It provides an alternative to LC-MS/MS, offering a cost-effective, simple, and robust approach for monitoring MPA therapy in transplant patients. The method ensures high sensitivity, specificity, and reproducibility, facilitating individualized dose adjustments to optimize therapeutic outcomes.

Keywords: Mycophenolic acid, MPAG, HPLC-UV, therapeutic drug monitoring, pharmacokinetics, immunosuppressant

Background
Intraperitoneal administration of cisplatin provides superior pharmacological effects against peritoneal metastatic cancer cells compared to intravenous administration. However, analyzing cisplatin levels in peritoneal fluid using high-performance liquid chromatography (HPLC) is challenging due to cisplatin’s low light absorption and the complex peritoneal fluid matrix, which can interfere with detection.

Objectives
This study aims to develop and validate a simple, accurate, and reproducible HPLC-UV method for quantifying cisplatin levels in peritoneal fluid samples.

Methods
Cisplatin concentrations were measured using a fully validated HPLC-UV method. Peritoneal fluid (50 μL) was diluted 20-fold with 0.9% saline. A 100 μL aliquot of the diluted sample was mixed with 25 μL of 5% sodium diethyldithiocarbamate, followed by vigorous mixing and incubation at 45°C for 10 minutes in the dark. The reaction was terminated with 200 μL of cold acetonitrile and centrifuged at 4°C, 14,000 rpm for 10 minutes. A 50 μL supernatant aliquot was injected into the HPLC-UV system. Analytes were eluted under gradient conditions at a 1.4 mL/min flow rate and detected at 254 nm.

Results
The total analysis time per sample was 23 min. The method demonstrated no endogenous interference, a linear range of 1-40 μg/mL (r2 = 0.999), and high accuracy (97.1-104.4%). Intra- and inter-day precision and accuracy were within acceptable limits [99.6% (10.15%CV) and 96.1% (9.72%CV)]. Recovery was 97.9-101.9%.

Conclusions
This fast and simple HPLC-UV method reliably quantifies cisplatin in peritoneal fluid and can be used to monitor therapeutic drugs in cancer patients receiving intraperitoneal cisplatin.

Keywords: Cisplatin, Peritoneal Fluid, HPLC

Background: As the clinical and forensic communities adopt oral fluid testing for ease of collection and on-site evaluations, the need to detect a broad range of analytes with sufficient sensitivity becomes paramount. Meeting the LOQ targets outlined by SAMHSA demands robust extraction methods and high-performance analytical instrumentation.

Aims: Confirm and quantitate 31 drugs in oral fluid using a 4.5-minute LC-MS method.

Methods: Calibration levels, 0.5 to 200ng/mL, were made in human oral fluid. Calibrators and 10 case study samples were diluted with a preserving buffer and spiked with the internal standards. 500µL of sample were extracted using DPX SPE tips. Analytes were separated on a Vanquish™ Horizon UHPLC system. Data was acquired on the Stellar™ mass spectrometer using targeted-MS2 (tMS2) mode with an inclusion list for the 31 target drugs. Optimized HCD or CID fragmentation was used to provide unique and high-intensity fragments.

Results: Limits of quantitation (LOQ) were determined for the 31 drugs as low as 0.5ng/mL. LOQ was defined as the back calculated concentration where %difference and %RSD were less than 20%. All 31 drugs were confirmed with retention times and MS2 ion ratios. The ability to use HCD or CID fragmentation allowed for optimizing each compound based on most favorable energies and fragments.

Conclusions: This fast, quantitative Stellar mass spectrometry method detected and confirmed 31 drugs of abuse in oral fluid. A complete workflow was presented with LOQ’s as low as 0.25ng/mL up to 1,000ng/mL.

Key Words: Drugs of Abuse, Mass Spectrometry, Oral Fluid, Toxicology

Background: Bisphenols, widely used in plastics, epoxy resins, and paper coatings, are endocrine-disrupting chemicals associated with an increased risk of cancer and metabolic disorders.

Aims: This study aimed to develop and validate a liquid chromatography-mass spectrometry (LC-MS) method for detecting BPA, bisphenol S (BPS), and bisphenol F (BPF) in human saliva.

Methods: Saliva samples were spiked with bisphenols and extracted using acetonitrile. An internal standard and ammonium acetate (10 mM) were added, followed by vortexing and centrifugation (3000 rpm,15 min). The supernatant was filtered (0.22 µm nylon syringe filter) and injected into the LC-MS system. Analytes were separated under gradient conditions at a flow rate of 0.4 mL/min and detected at m/z 227.05>212.05 (BPA), 249.0>108.0 (BPS), and 199.10>105.05 (BPF).

Results: The method demonstrated high accuracy (92.7-105.8%) with a linear calibration range of 0.01-100 ng/mL (r2 = 0.995). The lower limits of quantification were 0.01 ng/mL for BPS and BPF, and 0.5 ng/mL for BPA. Accuracy at 0.03, 30, and 70 ng/ml was 102.15 (11.85 %CV), 98.28 (3.23 %CV), and 96.52 (5.39 %CV) for BPS and 107.26 (13.27 %CV), 95.05 (5.37 %CV), and 92.79 (7.98 %CV) for BPF. BPA showed accuracy values of 100.29 (6.87 %CV), 100.71 (5.06 %CV), and 100 (4.73 %CV) at 1.5, 30, and 70 ng/ml, respectively. Matrix effects were 90-116.4% (1-16.5 %CV).

Conclusions: This validated method offers a simple and reliable approach for simultaneously measuring bisphenols exposure in saliva, making it a valuable tool for biomonitoring environmental exposure.

Keyword: Bisphenol, LC-MS, Saliva

Background: Immunosuppressive therapy with tacrolimus (TAC), cyclosporine (CsA), sirolimus (SIR), and everolimus (EVE) requires therapeutic drug monitoring (TDM) due to its narrow therapeutic range. Thus, reliable analytical methods are essential for high-quality results. The QuEChERS method has shown excellent recovery rates for various analytes, serving as a viable alternative to traditional extraction methods for determining IMDs.

Aims: The study aimed to develop and validate a novel, robust, and rapid LC-MS/MS method for determining immunosuppressants in the whole blood of pediatric liver transplant recipients.

Methods: During method development, the various QuEChERS mixtures were tested, supported by acetonitrile and/or methanol. The Luna PhenylHexyl column was used for chromatographic separation with mass spectrometry detection in MRM mode with a QTRAP6500+ device. The method was applied to TDM of clinical samples from pediatric patients after liver transplantation.

Results: The MgSO4, NaCl, di-/trisodium citrate with C18e sorbent composition was employed as the final QuEChERS mixture. Acetonitrile was selected as the suitable solvent, and extraction was facilitated by cold-induced phase-extraction. The method has been successfully validated within the 0.5–60 ng/mL (TAC, SIR, EVE) and 1–2000 ng/mL (CsA) calibration ranges. The mean intra- and interday accuracies and precision (<±15% and <10%, respectively) met the acceptance criteria. The matrix effect factors observed for the method were more satisfactory than those of a previously validated method based only on simple protein precipitation.

Conclusions: The QuEChERS method is distinguished by its high-recovery extraction of immunosuppressants from whole blood for LC-MS/MS analysis.

Key Words: QuEChERS, immunosuppressants, LC-MS/MS, TDM, liver transplantation

Background: Cephalosporins (CEPs) are bactericidal β-lactam antibiotics commonly used for prophylaxis and antimicrobial treatment in children following surgeries, sepsis/septic shock, or SIRS. Their pharmacokinetics are complex, particularly in critically ill paediatric patients, and the drug concentration is crucial for PK/PD evaluation, as is routine TDM.

Aims: This study aimed to optimise and validate a novel LC-MS/MS-based assay for determining six CEPs (cefazolin, cefotaxime, ceftazidime, cefuroxime, ceftriaxone, and cefepime) in capillary blood collected using the Mitra™ microsampling device.

Methods: The Kinetex-F5 column from Phenomenex was employed for CEPs separation in LC-MS/MS analysis (IVDR system/Citrine-6500+). Simple protein precipitations were facilitated by a 2-propanol/acetonitrile mixture with isotope-labelled internal standards corresponding to the analytes. The EMA, FDA, and IATDMCT guidelines were applied for validation purposes. The 10μL Mitra™ VAMS device was used to sample paediatric patients hospitalised in intensive care units at The Children's Memorial Health Institute in Warsaw, Poland.

Results: The method was successfully validated within the 0.1-50mg/L calibration range for each CEP. The accuracy and precision were within the acceptance ranges proposed by the EMA. The hematocrit effect did not influence the extraction/recovery of each analyte. The stability of the dried matrix in Mitra™ samples was established as 7 days at RT. The quantitative and qualitative matrix effects were not significant for each analysed CEPs.

Conclusions: The successfully validated method (alongside a previously validated method for the unbound fraction of CEP quantification) has been employed for PK/PD-monitoring of CEPs (fT%>4xMIC) in the AntiSepsis study.

Key Words: cephalosporins, β-lactams, sepsis, microsampling, paediatrics

Background: Flucloxacillin, a beta-lactam (BL) antibiotic, is commonly used to treat Gram-positive infections. To optimize dosing, TDM is performed by measuring total flucloxacillin plasma concentrations, subsequently estimating unbound concentrations, using a fixed free fraction (fu, 5% ). However, hypoalbuminemia, common in ICU patients, alters the equilibrium between bound and unbound drug, making total concentrations unreliable.

Aims: Validate and implement an in-house method to measure unbound flucloxacillin concentrations. 

Method: Methods for quantifying bound and unbound flucloxacillin concentrations in plasma were validated using an Ultimate 3000 with a Q-Exactive hybrid quadrupole-Orbitrap (UHPLC-HRMS). Total and unbound concentrations were measured in 75 samples from 46 patients (ICU, surgery and internal medicine). Ultrafiltration was performed using Centrifree 30 kDa devices (incubation and centrifugation at 37°C, 1900g, 30 minutes). Therapeutic ranges were 50-100 mg/L (total) and 2-20 mg/L (unbound).

Results: Mean (range) total and unbound flucloxacillin concentrations were 61.9 mg/L (3.76-154 mg/L) and 8,86 mg/L (0.218-28.1 mg/L), respectively (fu 14.0% (4.74% - 40.3%)). Mean albumin concentration was 28.3 g/L (19.8-45.0 g/L). When classified into subtherapeutic, therapeutic and toxic concentrations, total concentrations resulted in 25, 51 and 2 samples, respectively, while unbound concentrations identified 12, 58 and 8 samples in these categories. In 21 cases, total concentration resulted in a different classification than unbound concentration, potentially leading to inaccurate dose adjustments. 

Conclusion: For highly protein-bound drugs, like flucloxacillin, total concentrations do not provide reliable guidance for dose adaptations in these patients. Unbound concentrations should be measured using a standardized protocol. 

Key Words: unbound concentration, ultrafiltration, hypoalbuminemia, flucloxacillin

Background:
Difficult-to-treat gram-negative bacteria (DT-GNB) infections pose challenges in antibiotic therapy due to antimicrobial resistance, requiring TDM to optimise antibiotic dosing. LC-MS/MS is a gold-standard technique for antibiotic quantification, but cross-validation between laboratories is essential to ensure reproducibility and clinical implementation. This study evaluates the cross-site transferability of an in-house LC-MS/MS assay for routine TDM in Singapore.

Aims:
To assess the inter-laboratory performance of an LC-MS/MS assay for 11 key antibiotics by cross-validating between a research laboratory and the SGH Antibiotic TDM service laboratory.

Methods:
The LC-MS/MS assay, which includes common β-lactams, β-lactam/β-lactamase inhibitors, fosfomycin, tigecycline, and levofloxacin, was evaluated using calibration curves, quality control (QC) samples, and patient samples analysed at both sites. Linearity, intra- and inter-laboratory accuracy and precision (A&P) were evaluated. Acceptance criteria followed European Medicine Agency (EMA) guidelines (2011), requiring QC accuracy within 15% and at least 67% of the repeats to show a measurement difference within 20% of the mean.

Results:
Calibration curves (n=3) for all antibiotics showed excellent linearity (R²≥0.997), and QC (n=3) A&P were within 15% in both laboratories. A total of 56 out of 60 antibiotic measurements (93.3%) in 24 patients' plasma samples achieved the acceptance criteria (% concentration difference: ±0.11-19.8%).

Conclusions:
Cross-validation of our in-house antibiotic assay met EMA acceptance criteria, indicating that the method is reproducible at different laboratory sites, supporting the clinical implementation of antibiotic TDM in Singapore.

Background: Therapeutic drug monitoring interpretation for antifungal in central India is resource limited. iMARC AIIMS Bhopal caters antifungal management and TDM Bioassay.

Aims: Analysis of iMARC TDM database of routine reports for plasma range of voriconazole and posaconazole.
Objectives
a. Monitor clinico-mycological characteristics of patients indicated for routine TDM voriconazole or posaconazole.
b. Evaluate the target plasma concentration range of each azole routinely tested voriconazole or posaconazole for three years.

Methods: Prospective hospital based cross-sectional study. Data extraction and analysis conducted from database of routine patient TDM reports of iMARC. Further enrolled TDM results in study, bedside patient record monitoring was done

Results:
Total enrolled sample requisition for voriconazole 36/53 and posaconazole 17/53.
Trough samples predominant compared to random sample TDM requests, voriconazole 28/44 (0.6%), posaconazole 16/44 (0.36%). Random voriconazole 8/9 (0.89%), posaconazole 1/9 (0.1%). Mean voriconazole trough concentration 3.85ug/ml, mean random voriconazole concentration 6.8ug/ml. Mean posaconazole trough concentration 0.56 ug/ml. All posaconazole concentration subtherapeutic in mucormycosis, attributed and empty stomach. Voriconazole prescribed in invasive aspergillosis had subtherapeutic level 8/28 (0.28%) due to simultaneous prescription of few interfering medications.

Conclusion: Resource limited setting without HPLC or LCMS may not deter from antifungal stewardship and optimal patient with conventional bioassay.

Background
Interest in Therapeutic Drug Monitoring (TDM) of antibiotics has grown, yet it remains underutilized in clinical practice. Rapid, accurate, and user-friendly diagnostic assays are essential for routine implementation.

Aims
The study assessed comparability of TDM results between a new automated assay under development at Roche Diagnostics and the routinely used Chromsystems method at the Medical University of Vienna (MUV).

Methods
Human blood samples of intensive care units obtained for TDM of meropenem, piperacillin and linezolid were routinely analyzed at the MUV using a High Performance Liquid Chromatography (HPLC) assay. Residual material was then remeasured on a prototype of the Cobas i601 analyzer at Roche Diagnostics, which recently received CE-mark. The High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLC-MS/MS) based assays are still under development. Weighted Deming regression and Pearsons’s R were used for method comparison.

Results
Meropenem samples (n=90) showed mean concentrations (range) of 25.3 µg/ml (1.3 – 78.6) vs. 25.6 µg/ml (1.3 – 72.0) for Cobas i601 and MUV results, respectively, r=.989. Piperacillin samples (n=112) showed mean concentrations (range) of 46.5 µg/ml (0.3 – 181.0) vs. 49.1 µg/ml (0.3 – 175.0) for Cobas i601 and MUV results, respectively, r=.991. Linezolid samples (n=90) showed mean concentrations (range) of 7.0 µg/ml (0.2 – 22.7) vs. 7.6 µg/ml (0.2 – 24.6) for Cobas i601 and MUV measurements, respectively, r=.997.

Conclusions
Cobas i601 analyzer and MUV measurements demonstrate a high level of agreement supporting the transferability of results between the two assays.

Keywords
Therapeutic Drug Monitoring, Intensive Care, Antibiotic Stewardship

Background: Sildenafil (SIL) is one of the essential drugs in pulmonary arterial hypertension (PAH). A considerable percentage of ineffective treatment in PAH may be related to subtherapeutic dosage or non-adherence.

Aims: The aim of the study was to develop a simple analytical method enabling the determination of SIL in plasma of PAH patients.

Methods: An isocratic HPLC-UV system with a manual injector was applied. The separation of SIL and internal standard gallopamil (GAL) was performed on Supelcosil LC-CN column (150x4.6 mm, 5 μm) at ambient temperature. The mobile phase: CH3CN:H2O:0.5M KH2PO4:85% H3PO4 (172:324.2:3.7:0.1, v/v) was pumped at a flow rate of 1.8 mL/min. Diisopropyl ether (3 mL) was used for 3-min liquid-liquid extraction from 0.4 mL plasma sample buffered with 0.5M glycine. The sample was then re-extracted into 0.01 M HCl. The analytical wavelength was 290 nm.

Results: SIL and GAL were eluted at retention times of 3.4 and 7.3 min, respectively. The extraction yield was: 88.5% for SIL and 84.7% for GAL. The method was calibrated in the range: 2-500 ng/mL. For intraassay, the inaccuracy ranged from -18.1% to +1.0% and imprecision ranged 2.6-6.1% while for interassay, the inaccuracy was from 9.6% to +13.0% and the imprecision ranged 3.9-9.9%. The method has been successfully used in pilot study in 66 PAH patients, enabling SIL monitoring at steady-state.

Conclusions: The method can be used for both therapeutic drug monitoring (TDM) and adherence studies as an economical alternative to the LC-MS/MS technique.

Keywords: HPLC-UV, Sildenafil, Adherence, Pulmonary Arterial Hypertension

Background: Ketamine (KET) is widely used for rapid and effective anesthesia, particularly in Neonatal Intensive Care Units (NICUs). As an NMDA receptor antagonist, it provides strong analgesic and sedative effects without causing respiratory depression. Despite its clinical benefits, the optimal dosing regimen remains unclear, and individual pharmacokinetic variability significantly impacts both efficacy and the risk of toxicity. Therapeutic drug monitoring (TDM) of ketamine and its metabolite, norketamine (NOR), can facilitate personalized dosing by ensuring target concentrations are achieved. Measurements in whole blood, plasma, and capillary blood (VAMS) offer a promising approach for optimizing ketamine dosing in neonates.

Amis: This study aimed to develop and validate a sensitive, specific, and accurate method for simultaneous quantification of KET and NOR in various biological matrices.

Methods: Protein precipitation was performed using an acetonitrile-methanol mixture containing the internal standard (KET-D4), and the supernatant was directly injected into the chromatographic system. Separation was achieved with gradient elution using 0.1% formic acid in water and 0.1% formic acid in acetonitrile on a C18 column. Detection was conducted in MRM mode on a triple quadrupole mass spectrometer with ESI in positive mode. Transitions m/z 242 → 183 (KET-D4), 238 → 179 (KET), and 224 → 125 (NOR) were monitored.

Result: The method demonstrated linearity from 2–500 ng/mL (R² = 0.989) with high precision and accuracy. Stability was satisfactory for 7 days at 4°C.

Conclusion: This method is a reliable and practical tool for improving pain management in neonates.

Keywords: analgesics, LC-MS/MS, VAMS, TDM

Abstract
Background
Tigecycline (TIG), a broad-spectrum glycylcycline antibiotic, is commonly used for treating multidrug-resistant infections. To facilitate therapeutic drug monitoring and pharmacokinetic studies, a robust and fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of tigecycline in human plasma.

Objectives
This study aimed to develop and validate an LC-MS/MS method for the quantification of TIG in human plasma.

Methods
Plasma samples were spiked with TIG and an internal standard, then extracted using 0.1% trifluoroacetic acid in acetonitrile, followed by vortexing and centrifugation at 12,000 rpm for 10 minutes. Chromatographic separation was achieved on an ACE® C18 column (3 × 100 mm, 3 µm) using 0.1% formic acid in acetonitrile and 0.1% formic acid in water as mobile phases at a flow rate of 0.4 mL/min. The analyte was quantified by multiple reaction monitoring in positive mode.

Results
The method demonstrated high accuracy with a linear calibration range of 0.05–2.5 mg/L (r² = 0.997). The intra- and inter-day relative standard deviations of tigecycline were less than 15%, and the accuracy ranged from 87.50% to 111.20%.

Conclusions
This study successfully established a simple, rapid, and highly sensitive method for detecting tigecycline plasma concentrations. The method can be applied to therapeutic drug monitoring.

Keyword: Tigecycline, LC-MS/MS

Background: Diquat is one of the most commonly utilized herbicides, and cases of diquat poisoning are frequently reported. The rapid detection of diquat is essential for effective clinical diagnosis and treatment.

Aims: This study aims to establish a simple, rapid and reliable method for the identification and quantification of diquat in urine using liquid chromatography-tandem mass spectrometry.

Methods: Samples were prepared by the centrifugation and dilution with water. Then, diquat was separated using a hydrophilic chromatography BEH Z-HILIC column, with a gradient elution consisting of 100 mM ammonium formate in water (pH 3.0, adjusted with formic acid) and acetonitrile in a total run time of 8 min. Mass spectrometric analysis was conducted using electrospray ionization in positive-ion multiple reaction monitoring mode. The precursor ion and the two product ions of diquat were 183.0, 157.1 and 130.4.

Results: The method demonstrated satisfactory validation requirements, with a good linear relationship in the range of 250-1000 ng/mL(r2>0.995). Furthermore, this method was applied to analyze a urine sample obtained from Emergency Department of the Guizhou Provincial People’s Hospital, which was identified diquat poisoning with a measured urine concentration of 813 ug/mL.

Conclusions: A simple, rapid and reliable method of the determination of diquat was developed and successfully applied for diquat detection in urine samples. This study provided a novel method for the clinical detection of diquat poisoning.

Keywords: liquid chromatography-tandem mass spectrometry, BEH Z-HILIC column, urine, diquat, pesticides.

Background: High-resolution tandem mass spectrometry (HR-MS/MS) offers unmatched specificity due to superior mass accuracy, combined with broad linearity and high scanning speed in the latest instrumentation. In this study, we present a robust HR-MS/MS method for the sensitive detection of 27 psychoactive compounds in blood and blood collected on volumetric absorptive microsampling (VAMS) devices.

Aims: Accurate confirmation of compound presence is essential for targeted and quantitative toxicology analysis.

Methods: Sample preparation involved internal standard spiking, protein precipitation, extraction with acetonitrile: methanol (1:1, 0.1% formic acid), nitrogen evaporation, and reconstitution in the LC mobile phase. Quantitative analysis was performed using reversed-phase chromatography on an ExionAC LC system coupled with a ZenoTOF 7600 (SCIEX), operating in positive electrospray ionization mode with scheduled MRMhr acquisition. Compound confirmation was based on a multi-criteria approach, emphasizing MS/MS spectral matching, precursor and quantifier ion mass defect, isotope pattern, and retention time.

Results: Transitioning from a QQQ to HR-MS/MS improved data interpretation, particularly at low analyte concentrations, minimizing matrix interferences and false positives. A multi-level confirmation scoring system was developed, assigning weighted contributions to five parameters, with a 60% cut-off ensuring reliable compound identification. The method was validated for blood and VAMS samples, demonstrating excellent linearity (R ≥ 0.995), precision (%CV ≤ 15%), accuracy (80–120%), and recovery (%CV ≤ 15%). The lower limit of quantification (LLOQ) ranged from 0.001–0.05 ng/ml.

Conclusions: This HR-MS/MS method provides high sensitivity, specificity, and robustness, enabling accurate quantification of psychoactive compounds in forensic applications.

Key Words: HR-MS/MS, psychoactive compounds, blood, VAMS

Background: Methotrexate (MTX) is used at high doses (HDMTX, 1,000-33,000 mg/m² by prolonged i.v. infusion) with leucovorin (LV) rescue for a variety of cancers. HDMTX safety is improved by monitoring serum creatinine and MTX levels during renal clearance of MTX, which can initially exceed 1000 µmol/L. Delayed clearance can cause acute kidney injury, further delaying clearance and severe toxicity. MTX is monitored at 12-24h intervals until it drops to a safe level for discharge, ≤0.05 µmol/L. MTX levels during clearance also inform LV dosing. Accurate measurement of MTX, especially at 0.050 μmol/L is an important clinical tool.

Methods: The ARK™ Methotrexate II Assay is a recombinant antibody based homogeneous enzyme immunoassay for the quantitative determination of methotrexate in human serum or plasma on automated clinical chemistry analyzers with an analytical measurement interval (AMI) of 0.030 to 1.30 µmol/L and reportable range up to 1,200 µmol/L by dilution. Performance was demonstrated on the Beckman AU680 analyzer using CLSI evaluation protocols.

Results: Within-lab precision (%CV) at control levels from 0.070 to 0.800 µmol/L was ≤3.0%; Limit of Quantitation (LoQ) was 0.030 μmol/L; deviations from linearity <8% over the AMI. Results for manual and on-instrument dilutions of supplemented samples up to 1200 µmol/L fell within 10% of nominal values. Passing Bablok analysis of the results for 90 patient specimens of the assay vs (LC-MS/MS) was 1.0278 + 0.0000 (r2 = 0.977).

Conclusion: The FDA-cleared ARK™ Methotrexate II Assay demonstrated excellent performance over an expanded AMI of 0.030 to 1.30.

Key words: Methotrexate, Immunoassay

Background: Linezolid, an antibiotic, is known to be metabolized in the liver, but the specific role of its metabolites, particularly PNU-142300, in causing adverse effects such as myelosuppression has not been fully established, especially in patients with renal dysfunction.

Aims: To develop a rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the simultaneous determination of linezolid and its metabolite, PNU-142300, in human serum. This would enable the investigation of their concentrations and their potential link to myelosuppression in patients with renal insufficiency.

Methods: The method involves preparing human serum samples using acetonitrile for protein precipitation, followed by separation on a Waters Acquity UPLC BEH C18 column with gradient elution. Detection is performed using UPLC-MS/MS with multiple reaction monitoring (MRM) for linezolid, PNU-142300, and tedizolid (internal standard, IS).

Results: The method demonstrates good linearity, precision, accuracy, and stability for quantifying linezolid and its metabolite in serum.

Conclusions: A fast, accurate UPLC-MS/MS assay was developed to measure linezolid and its metabolite PNU-142300 in serum. With a quick 3-minute analysis and simple sample preparation, this method is ideal for routine therapeutic drug monitoring. The study also found that linezolid-induced myelosuppression is more common in patients with renal impairment, and the PNU-142300 to linezolid ratio may reduce this toxicity.

Key Words: Linezolid, PNU-142300, myelosuppression, human serum, UPLC-MS/MS

Background: Ketamine, a nonbarbiturate and rapid-acting dissociative anesthetic is FDA cleared for use in human and veterinary surgical procedures. Structurally and pharmacologically similar to phencyclidine (PCP) it is less potent, has a faster onset and shorter duration of action. Recently it has attracted attention due to its utility as an adjunctive, opioid-sparing analgesic with sustained antidepressant effects, in patients with acute suicidal ideation or behavior. Besides medical use ketamine is commonly abused as a recreational drug because of its ability to induce an altered state of consciousness. First generation immunoassays to detect ketamine suffered from cross reactivity yielding false positives, necessitating highly specific assays to circumvent difficulties encountered in laboratories performing urine drug screens.

Methods: The performance characteristics of ARK Ketamine II assay, including precision, spiked recovery, specificity were evaluated on the Beckman Coulter AU680 automated clinical analyzer employing two reagents in a homogeneous immunoassay format.

Results: The assay demonstrated acceptable precision, with no overlap at 50 ng/mL cutoff using ± 50% controls, and at 100 ng/mL cutoff using ± 25% controls. Recovery was between 103.7% and 106.1% of the spiked ketamine from 20.0 to 500.0 ng/mL. The cross-reactivity to several structurally similar derivatives was minimal. Human urine samples (N=223) were confirmed negative by both immunoassay and LC-MS/MS, additionally for positive patient samples (N=50), ARK Ketamine II assay showed 100 % sensitivity and 100 % specificity for both cutoffs.

Conclusion: The ARK Ketamine II Assay provides a highly specific, sensitive, rapid, and reliable measurement of ketamine in human urine.

Background: Hyaluronan (HA) is a repeating disaccharide structure of N-acetyl-D-glucosamine and D-glucuronic acid and widely exists at various molecular weights. It has been reported that most of an orally administered HA, regardless of its molecular weight, is degraded by intestinal microflora into HA oligosaccharides of less than octa saccharides and absorbed. However, there will not be a method for simultaneous determination.

Aims: We focused on HA 8-mer or less and aimed to establish a simultaneous quantification method of LC/MS-MS with a simple pretreatment to clarify the absorption properties of orally administered HA oligosaccharides.

Methods: First, carboxy groups of HA oligosaccharides were derivatized with a condensing reagent and Girard’s reagent P. After pretreatment by solid-phase extraction using hydrophilic interaction, peaks of HA 2, 4, 6 and 8-mer were detected by LC/MS-MS. Validation studies were conducted for this method and plasma concentrations of HA were determined after oral administration of HA formulation to rats.

Results: We confirmed by an MS scan that the derivatized HA oligosaccharides could be analyzed by the newly developed quantitative method and we identified the peak of derivatized HA oligosaccharides. The MS/MS conditions were optimized, and a calibration curve was generated. Plasma concentrations of HA 4, 6 and 8-mer were determined after oral administration of HA formulation.

Conclusions: The calibration curve showed good linearity for HA 4, 6, and 8-mer. This method is valid and enables measurement of plasma concentrations of HA oligosaccharides.

Key Words: hyaluronan; absorption; oligosaccharides; LC/MS-MS; validation; simultaneous determination.

Therapeutic drug monitoring (TDM) for kidney transplant patients on Tacrolimus and Everolimus has become more efficient with recent advancements in Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) automation, allowing simultaneous testing.

This study evaluates two methods for quantifying Tacrolimus and Everolimus: LC-MS/MS and immunoassay. The quantification of both drugs was performed utilizing the fully automated Clinical Laboratory Automated Module (CLAM) integrated with the Shimadzu LC-MS/MS system, in addition to conventional immunoassay methods. For Tacrolimus, Chemiluminescence Immunoassay (CMIA) was employed, while Everolimus was tested in batches using Particle Enhanced Turbidimetric Inhibition Immunoassay (PETINIA).

Blood samples from kidney transplant patients were collected at the Department of Nephrology, Hospital Kuala Lumpur, between Aug to Nov 2021. Measurement was obtained using a commercially available kit by Alsachim DOSIMMUNE™ for automated immunosuppressant analysis. The results were compared using Passing-Bablok regression and Bland-Altman analysis.

The Passing-Bablok regression analysis showed systematic biases in Tacrolimus (y = 0.508 + 0.812x) and Everolimus (y = -0.739 + 1.370x). Spearman correlation coefficients were 0.978 for Tacrolimus and 0.622 for Everolimus (P < 0.0001). Bland-Altman analysis revealed a mean bias of -0.7 for Tacrolimus (limits: -2.1 to 0.8) and 0.8 for Everolimus (limits: -3.6 to 5.2). Proportional bias and outliers at higher concentrations suggest measurement inconsistencies. While general agreement exists, further validation and calibration are needed for optimal clinical and analytical use.

The study supports adopting the Shimadzu CLAM-LC-MS/MS system for more accurate and reliable TDM with simultaneous reporting, pending further validation.

Therapeutic Drug Monitoring (TDM), LC-MS/MS, Kidney Transplant, Immunosuppressants, Automation, Method Comparison

Background: Linezolid (LZD), used to treat infections caused by multidrug-resistant Gram-positive cocci, has been associated with concentration-dependent risk of thrombocytopenia. The metabolite PNU-142586, derived from LZD, is suspected to contribute to this toxicity. However, the pharmacokinetics of PNU-142586 remain unclear.

Aims: This study aimed to characterize the pharmacokinetics of LZD and PNU-142586 following a single intravenous dose of LZD.

Methods: Six healthy adult subjects received a single 600 mg intravenous infusion of LZD. Blood samples were collected for up to 10 hours after administration. LZD and PNU-142586 concentrations were measured using high-performance liquid chromatography. Pharmacokinetic parameters were estimated using non-compartmental analysis. This study was approved by the research ethics review committees of Toyama University Hospital and Nihon University School of Pharmacy (R2020147, R23A-005).

Results: LZD exhibited the maximum concentration (Cmax) of 20.4 mg/L and the area under the concentration-time curve from 0 to 10 hours (AUC0-10) of 102.6 mg·h/L. PNU-142586 exhibited Cmax of 1.6 mg/L, AUC0-10 of 109.0 mg·h/L, and time to Cmax (Tmax) of 4.5 hours. The coefficient of variation (CV) for AUC0-10 was 6.1% for LZD and 19.4% for PNU-142586. The formation rate constant for PNU-142586 was 0.40 /h.

Conclusions: The pharmacokinetics of LZD were consistent with previous reports. The higher CV of PNU-142586 AUC0-10 indicates greater inter-individual variability compared to LZD. The Tmax and formation rate constant of PNU-142586 was relatively consistent among subjects. This study provides the first detailed characterization of the formation rate of PNU-142586 from LZD following intravenous administration.

Keywords: linezolid, PNU-142586, metabolite, pharmacokinetics.

Background: No study has yet explored whether there are differences between the extended infusion (EI) and continuous infusion (CI) dosing regimens in achieving pharmacokinetic/pharmacodynamic (PK/PD) targets and clinical bacteriological effectiveness for imipenem.
Aims: The study aimed to compare the PK/PD parameters of imipenem administered via EI (administered continuously for 2h) versus CI, and to compare the target PK/PD indices.
Methods: Forty critically ill ICU patients, who were administered imipenem either as a targeted or empirical treatment, were divided into two groups: the EI group (1.5g/d [0.5g, q8h] [n=5], 2.0g/d [0.5g, q6h] [n=8], 3.0g/d [1g, q8h] [n=7]) and the CI group (1.5g/d [n=5], 2.0g/d [n=8], 3.0g/d [n=7]), based on their daily dosage. Pharmacokinetic parameters for imipenem were calculated using the Winnonlin software. The primary outcome was to compare the attainment of PK/PD targets.
Results: There were no significant differences in PK parameters between the CI and EI groups (P＞0.05). Overall target attainment of PK/PD indices was higher with CI compared to EI. When the PKPD target value is set to %fT>MIC, with MIC≥4, there is a significant difference between the two groups (75% vs. 100%; P=0.024). When the PKPD target value is %fT>4MIC, with MIC≥1, there is a significant difference between the two groups %fT>4×MIC (75% vs. 100%; P=0.024).
Conclusions: Even though the stability of imipenem limits its use for continuous infusion, continuous infusion of imipenem can better achieve PKPD targets for critically ill patients or pathogens with higher MICs.
Keywords：Imipenem, extended infusion, Continuous Infusion, PK/PD