Development and Validation of a High-Performance Liquid Chromatography With Ultraviolet Detection Method to Facilitate Therapeutic Monitoring of Teicoplanin Using Dried Blood Spots.

BACKGROUND: In neonatal and pediatric intensive care units, Gram-positive infections are a significant cause of morbidity and mortality. The increase in infections caused by methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococci have led to the increased use of glycopeptides, which treat invasive infections caused by Gram-positive organisms, particularly those resistant to beta-lactam antibiotics. Teicoplanin has bacteriostatic activity against Gram-positive bacteria, but its pharmacokinetics in children is highly variable, with most children failing to reach target levels at the recommended dose. This study aimed to develop a cost-effective method for determining concentrations using dried blood spot (DBS). METHODS: A method to determine the concentrations of teicoplanin in 20 µL blood or plasma using the Whatman 903 Protein Saver filter was evaluated. High-performance liquid chromatography with ultraviolet detection high-performance liquid chromatography with ultraviolet/vis was used, with internal standard ketoconazole. In addition, a method to quantify teicoplanin using 50 µL of liquid plasma was established to compare the results with the values obtained by DBS and dried plasma methods. RESULTS: The method was successfully developed and validated for 20 µL DBS. Furthermore, 50 µL of plasma was used to quantify teicoplanin with a lower limit of quantification of 10 mg/L. Precision and accuracy ranged from 2.3% to 10.7% and 95%-114.2%, respectively. A consistent factor (1.15) was used to calculate teicoplanin plasma concentrations from whole blood, indicating the reliability of the DBS method for therapeutic drug monitoring of teicoplanin. CONCLUSIONS: A simple, reliable, and cost-effective method using high-performance liquid chromatography with ultraviolet/vis was established to determine pediatric teicoplanin concentrations in both small plasma sample volumes and whole blood using DBS, and an accurate correlation factor for estimating teicoplanin plasma concentrations from DBS was identified. This method is suitable for the use in pediatrics.

Development and validation of a bioanalytical method for the quantification of methotrexate from serum and capillary blood using volumetric absorptive microsampling (VAMS) and on-line solid phase extraction (SPE) LC-MS.

High-dose methotrexate is part of the polychemotherapy protocols for the treatment of Acute lymphoblastic leukaemia (ALL) with therapeutic drug monitoring (TDM) to adjust leucovorin rescue. An immunoassay is commonly used to analyse serum samples collected via venous blood sampling. However, immunoassays cannot distinguish between the parent drug and its metabolites. Besides, the blood volume required by venous blood sampling is high. Therefore, the aim of this project was to develop a fast, simple, reliable and cost-efficient micro sampling bioanalytical method using capillary blood to minimize the harm of children and to analyse both methotrexate and its metabolites. To achieve this aim, a LC-MS method with on-line solid phase extraction (SPE) for the simultaneous detection of methotrexate and its metabolites from capillary blood using volumetric-absorptive-microsampling (VAMS) technology was developed and fully validated. Besides, the method was also validated and modified for serum samples to compare the results with the immunoassay. A single-quadrupole MS detector was used for detection. Through the use of on-line SPE technology, a lower limit of quantitation of 0.03 µM for MTX and 7-OH-MTX and of 0.05 µM for DAMPA from a 10 µL capillary blood sample was achieved. The accuracy is between 90.0 and 104% and the precision between 4.7 and 12% for methotrexate and its metabolites, respectively. Because of the cross reactivity of the immunoassay a cross-validation was not successful. Besides, a correlation factor of 0.46 for MTX between plasma and whole-blood was found. A fast, simple, reliable and cost-efficient extraction and analysis LC-MS method could be developed and validated, which is applicable in ambulatory and clinical care.

Development and validation of a bioanalytical method for the quantification of axitinib from plasma and capillary blood using volumetric absorptive microsampling (VAMS) and on-line solid phase extraction (SPE) LC-MS.

For therapeutic drug monitoring (TDM) of axitinib, the new volumetric absorption microsampling technology (VAMS™) was applied to obtain capillary blood samples in an ambulatory setting and the results were compared to plasma samples as the gold standard. On-line solid phase extraction (SPE) applying a Turboflow HTLC Cyclone™ 1.0 × 500 mm column was used to reduce costs and working time. For the analytical separation, a Kinetex 2.6 µm C18 100 Å, 100 × 3.0 mm column with a flow rate of 0.3 mL/min in gradient mode was utilised. The mobile phase consisted of acetonitrile, water and formic acid (A: 05:95:0.1 v/v and B: 95:05:0.1 v/v). For the detection, a single-quadrupole MS detector was used. Through the use of on-line SPE technology, it is possible to reach a LLOQ of 0.5 µg/L from a 10 µL capillary blood sample. For lower concentrations, a MS/MS-detector coupled with the same chromatographic system was applied reaching a LLOQ of 0.04 µg/L. This newly developed method was validated with both detectors at different calibration ranges for plasma and capillary blood as matrix. The precision of the within- and between-runs was within a range of 0.6-7.8% and 1.8 - 14% CV, respectively, while the accuracy was within a range of 81.2-115% and 87.7-116%, respectively. A reliable, simple, less personnel-intensive and cost-efficient extraction and analysis LC-MS and LC-MS/MS method could be developed and validated, which is applicable in ambulatory and clinical care.

Developing a Nationwide Infrastructure for Therapeutic Drug Monitoring of Targeted Oral Anticancer Drugs: The ON-TARGET Study Protocol.

Exposure-efficacy and/or exposure-toxicity relationships have been identified for up to 80% of oral anticancer drugs (OADs). Usually, OADs are administered at fixed doses despite their high interindividual pharmacokinetic variability resulting in large differences in drug exposure. Consequently, a substantial proportion of patients receive a suboptimal dose. Therapeutic Drug Monitoring (TDM), i.e., dosing based on measured drug concentrations, may be used to improve treatment outcomes. The prospective, multicenter, non-interventional ON-TARGET study (DRKS00025325) aims to investigate the potential of routine TDM to reduce adverse drug reactions in renal cell carcinoma patients receiving axitinib or cabozantinib. Furthermore, the feasibility of using volumetric absorptive microsampling (VAMS), a minimally invasive and easy to handle blood sampling technique, for sample collection is examined. During routine visits, blood samples are collected and sent to bioanalytical laboratories. Venous and VAMS blood samples are collected in the first study phase to facilitate home-based capillary blood sampling in the second study phase. Within one week, the drug plasma concentrations are measured, interpreted, and reported back to the physician. Patients report their drug intake and toxicity using PRO-CTCAE-based questionnaires in dedicated diaries. Ultimately, the ON-TARGET study aims to develop a nationwide infrastructure for TDM for oral anticancer drugs.