A novel approach to therapeutic drug monitoring of Ciclosporin in pediatric renal transplant recipients using volumetric absorptive microsampling (VAMS) - Teaching old dog new tricks.

BACKGROUND AND AIMS: Ciclosporin (CSA) is an immunosuppressive agent that requires therapeutic drug monitoring (TDM). High partitioning in erythrocytes indicates that whole blood (WB) is a suitable matrix for CSA determination. Alternative sampling strategies, such as volumetric absorptive microsampling (VAMS), are novel possibilities for blood collection during TDM for various analytes, including immunosuppressants. This technique is attractive for vulnerable pediatric patients, including home-based self-sampling, remote therapy, and adherence control. MATERIALS AND METHODS: This study aimed to develop and validate a new method for CSA determination based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of WB and VAMS samples. Additionally, these methods were applied for CSA determination in clinical samples from pediatric transplant recipients. A strong point of this study is the assessment of an external proficiency testing scheme. RESULTS: Both methods were successfully validated within the 1 - 2000 ng/mL calibration range, with LOD 0.5 and 1 ng/mL for WB and VAMS methods, respectively. All the validation parameters fulfilled the international acceptance criteria for bioanalytical methods. Cross-validation confirmed the interchangeability of the LC-MS/MS method developed in this study. CONCLUSION: This study developed and validated novel methods for CSA determination in whole blood and VAMS using LC-MS/MS. Clinical validation and proficiency testing confirmed their utility in routine clinical practice.

The relationship between 6-thioguanine levels and remission outcomes in children with autoimmune hepatitis. Single center experience.

Aim of the study: The treatment of autoimmune hepatitis (AIH) is based on steroids and azathioprine (AZA). AZA is a pro-drug which is converted among others into 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP). The aim of the study was to determine the relationship between the AZA active metabolite 6-TG and both the biochemical and histological remission outcomes. Material and methods: The authors conducted a retrospective analysis of a single chart review. The sample size consisted of 44 pediatric patients with AIH. Biochemical remission was defined as an alanine aminotransferase (ALT) level below 40 U/l and histological remission was defined as a situation when the control biopsy revealed inflammation grade G1 (or lower) in the Batts-Ludwig score. Statistical analysis was applied to assess the difference in remission outcomes in patients with different levels of 6-TG. Results: In the benchmark variant of our statistical analysis, we found that the correlation between 6-TG and ALT in the sample was not statistically significant. Moreover, the difference between the mean levels of ALT in the populations in and without remission was not statistically significant (the p-value of the t-test was 0.16). Conclusions: Our results tend to support the claim that there is no statistically significant relationship between 6-TG concentration and remission (both biochemical and histological) in pediatric patients with AIH.

Therapeutic drug monitoring of mycophenolic acid (MPA) using volumetric absorptive microsampling (VAMS) in pediatric renal transplant recipients: ultra-high-performance liquid chromatography-tandem mass spectrometry analytical method development, cross-validation, and clinical application.

BACKGROUND: Mycophenolic acid (MPA) is widely used in posttransplant pharmacotherapy for pediatric patients after renal transplantation. Volumetric absorptive microsampling (VAMS) is a recent approach for sample collection, particularly during therapeutic drug monitoring (TDM). The recommended matrix for MPA determination is plasma (PL), and conversion between capillary-blood VAMS samples and PL concentrations is required for the appropriate interpretation of the results. METHODS: This study aimed to validate and develop a UHPLC-MS/MS method for MPA quantification in whole blood (WB), PL, and VAMS samples, with cross and clinical validation based on regression calculations. Methods were validated in the 0.10-15 µg/mL range for trough MPA concentration measurement according to the European Medicines Agency (EMA) guidelines. Fifty pediatric patients treated with MPA after renal transplantation were included in this study. PL and WB samples were obtained via venipuncture, whereas VAMS samples were collected after the fingerstick. The conversion from VAMSMPA to PLMPA concentration was performed using formulas based on hematocrit values and a regression model. RESULTS: LC-MS/MS methods were successfully developed and validated according to EMA guidelines. The cross-correlation between the methods was evaluated using Passing-Bablok regression, Bland-Altman bias plots, and predictive performance calculations. Clinical validation of the developed method was successfully performed, and the formula based on regression was successfully validated for VAMSMPA to PLMPA concentration and confirmed on an independent group of samples. CONCLUSIONS: This study is the first development of a triple matrix-based LC-MS/MS method for MPA determination in the pediatric population after renal transplantation. For the first time, the developed methods were cross-validated with routinely used HPLC-DAD protocol.

Therapeutic Drug Monitoring of Tacrolimus Based on Volumetric Absorptive Microsampling Technique (VAMS) in Renal Transplant Pediatric Recipients-LC-MS/MS Method Development, Hematocrit Effect Evaluation, and Clinical Application.

Tacrolimus (TAC) is post-transplant pharmacotherapy's most widely used immunosuppressant. In routine clinical practice, frequent uncomfortable venipuncture is necessary for whole-blood (WB) collection to check trough TAC levels. Volumetric absorptive microsampling (VAMS) is an alternative strategy to WB collection. In this study, we aimed to validate and develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for TAC quantification in WB and VAMS samples. After extraction with water and protein precipitation, the samples were directly analyzed using LC-MS/MS. Whole-blood and VAMS capillary-blood samples were collected from 50 patients treated with TAC during the follow-up visits. The cross-correlation between the developed methods was evaluated using Passing-Bablok regression and a Bland-Altman bias plot. The matrix effect (ME) and carry-over were insignificant for both scenarios. There was a high correlation between the processes and no significant clinical deviation. LC-MS/MS methods were successfully developed and validated in the 0.5-60 ng/mL calibration range. This study demonstrated and confirmed the utility of VAMS-based TAC monitoring in the pediatric population. This is the first study to directly develop and validate the VAMS LC-MS/MS method for evaluating the hematocrit effect in the pediatric population. The statistical correlation between immunochemical and VAMS-based methods was satisfactory.

Cefazolin prophylaxis in children undergoing cardiac surgery with the use of cardiopulmonary bypass-is the dosing correct?

OBJECTIVES: To determine the recommended concentrations of cefazolin to be used for antibiotic prophylaxis during paediatric cardiac surgery with extracorporeal circulation (ECC). METHODS: Twenty paediatric patients undergoing cardiac surgery with ECC and cefazolin antibiotic prophylaxis were included in the study. Blood samples for measurement of total cefazolin plasma concentration were collected at the following measurement time points: directly after skin incision, 15 min after ECC start, 5 min after ECC cessation and at skin closure. The target concentration was set for ≥40 mg/l, which corresponded to ≥8 mg/l of unbound cefazolin concentration. RESULTS: The median total cefazolin plasma concentrations at the measurement time points were 62.8, 67.7, 45.8 and 34.2 mg/l, respectively, and target concentrations were achieved in 90%, 85%, 65% and 40% of children, respectively. Among patients who received ≥30 mg of cefazolin per 100 ml of ECC priming, target concentrations after ECC cessation were reached in 80% of patients, while in those with <30 mg cefazolin per 100 ml in 20% of patients (P = 0.031). CONCLUSIONS: Direct extrapolation of antibiotic prophylaxis recommendations from adults to children may result in suboptimal antibiotic concentrations. An additional cefazolin dose to ECC priming appears necessary and the dosing should be based on ECC priming volume rather than on the weight of the patient.

The comparison between light-scattering detectors based on LED and photodiode for immunoprecipitation assays of transferrin and ferritin.

Undoubtedly, light-emitting diodes (LEDs) and photodiodes (PDs) are indispensable optoelectronic devices in modern analytical chemistry. LEDs can serve as either light emitters or detectors, thus being an alternative to the most popular detection systems consisted of PD. In this contribution, a comparison between LED-LED and LED-PD detectors, operating in turbidimetric and nephelometric modes, has been carried out for immunoprecipitation detection of transferrin and ferritin. The greatest emphasis was placed on the study of detectors responses under different measurement conditions including current powering an emitter, amplification gain in the case of PD as detector or the construction of detection cells designed for the Multicommutated Flow Analysis (MCFA). The assumption was to obtain the fully-mechanized system with simple but efficient detection system to enable the determination of iron-binding proteins occurring at different concentration ranges in human body. As a result, the optimized arrangements of LED-LED and LED-PD setups were characterized by similar analytical characteristics, enabling the determination of transferrin with the detection limit (LOD) of 0.2 mg/L and RSDs of 2.8-4.8% for LED-LED, and LOD of 0.1 mg/L and RSDs of 0.9-3.6% for LED-PD. In the case of ferritin detection, only the response of the LED-PD detector was statistically distinguishable in the range of 130-198 µg/L of protein with recorded analytical signal change of 20 mV value. The addition of polymer for signal enhancement provided the increase of response range to 107-253 µg/L, enabling the developed system for detection of pathological serum ferritin levels.

Microwave-assisted preparation, structural characterization, lipophilicity, and anti-cancer assay of some hydroxycoumarin derivatives.

ABSTRACT: A new series of hydroxycoumarin derivatives has been synthesized using conventional synthesis. The syntheses were accelerated by microwave assistance. Yields in both cases were comparable (59-69 %). The structures were established by 1H and 13C NMR spectroscopy and high-resolution mass spectrometry. Five compounds (5-hydroxy-4,7-dimethylcoumarin, 6-acetyl-5-hydroxy-4,7-dimethylcoumarin, 4-(cyanomethoxy)chromen-2-one, 5-(cyanomethoxy)-4,7-dimethylchromen-2-one, and 6-acetyl-5-(cyanomethoxy)-4,7-dimethylchromen-2-one) were assayed for anti-cancer activity. For all presented coumarin derivatives, lipophilicity was measured using reversed-phase TLC in different eluent systems with standardization. In addition, the crystal structure of 6-acetyl-5-hydroxy-4,7-dimethylcoumarin has been solved by X-ray structure analysis of single crystals.

Synthesis, structural studies and biological activity of new Cu(II) complexes with acetyl derivatives of 7-hydroxy-4-methylcoumarin.

The new Cu(II) complexes with 6-acetyl-7-hydroxy-4-methylcoumarin (HL1) and 8-acetyl-7-hydroxy-4-methylcoumarin (HL2) have been obtained by the electrochemical method. The density functional theory calculations and X-ray absorption spectroscopy techniques have been used to geometrically describe a series of new compounds. The studies have been focused on the coordination mode of the hydroxy ligands to the metallic centre. The complexes, Cu(HL1)2 and Cu(HL2)2⋅0.5H2O, have flat square geometry with oxygen atoms in the first coordination sphere. Two bidentate anionic coumarins are bonded to the metal cation via the acetyl and deprotonated hydroxyl O atoms. Biological activity, including microbiological and cytotoxic, has been evaluated and found to be enhanced in comparison with the parent ligands. Moreover, the Cu(II) complex with 8-acetyl-7-hydroxy-4-methylcoumarin shows similar antifungal activity as commercially used fluconazole.

Synthesis and anticancer activity of 7-hydroxycoumarinyl gallates.

BACKGROUND: The search for anti-cancer agents includes naturally occurring substances and theirs modifications. Therefore we invented and designed compounds that represent fused derivatives of gallic acid with coumarins. METHODS: As a result, a series of 8 novel esters of gallic acid and 7-hydroxycoumarins were synthesized and evaluated for anticancer activity. The structures of the compounds were established by IR, (1)H, (13)C NMR and HR MS spectra. The esters were assayed for antiproliferative activity against human leukemia HL-60 and prostate cancer DU145 cell lines. The activity of novel esters was evaluated by cell viability assays as well as by analysis of cell cycle and cell death mechanism. RESULTS: The esters were found to be of similar or higher activity than gallic acid. No pronounced harmful effect was observed in non-cancer cells. CONCLUSIONS: The novel compounds represent an excellent starting point for the further optimization and the design of therapeutically effective anti-cancerous drugs.