Dried Plasma Spots and Oral Fluid as Alternative Matrices for Therapeutic Drug Monitoring of Busulfan: Analytical Method Development and Clinical Evaluation

Abstract

Background: Busulfan (BU) is an alkylating agent with a narrow therapeutic index and high intraindividual pharmacokinetic variability used in conditioning therapy for hematopoietic stem cell transplantation. Monitoring BU exposure during high-dose conditioning regimens is recommended and positively impacts outcomes. We aimed to develop, validate, and apply a ultra-high-performance liquid chromatography-mass spectrometry (MS)/MS assay to measure BU concentrations in oral fluid and dried plasma spots (DPS) as alternative matrices to plasma. Methods:We prepared plasma and oral fluid samples by protein precipitation and DPS after liquid extraction. We analyzed extracts using an LC-MS/MS system with an Acquity HSS T3 column in the positive electrospray ionization mode. The method was validated and applied to 79 paired plasma and oral fluid samples from 7 patients on BU conditioning treatment. DPS were prepared by pipetting plasma onto Whatman 903 paper. The correlation between BU in plasma, oral fluid, and DPS samples was evaluated.Results: Run time was 4.0 minutes. The assay was linear at 50-5000 ng mL 21 (r . 0.99), precise (1.9%-5.3% oral fluid and 1.8%-5.9% DPS), and accurate (98.1%-108.9% oral fluid and 93%-103.1% DPS). BU was stable in DPS at 238C for 24 hours. BU levels in oral fluid (r = 0.927) and DPS (r = 0.982) were significantly correlated with plasma. Despite the good correlation, we found a wide variation between oral fluid and plasma levels. The area under curves (AUCs) calculated with oral fluid concentrations were 79.1%-167.1% of plasma AUCs. Bland-Altman plots found a better agreement for DPS, with AUCs estimated from corrected DPS levels at 83.1%-114.1% of plasma values. Conclusions:We developed and validated a simple and fast ultrahigh-performance liquid chromatography-MS/MS assay to measure BU in oral fluid and DPS. The results do not support the use of oral fluid as a matrix for routine therapeutic drug monitoring of BU. The AUC estimated from BU measurements in DPS was comparable to that in plasma, supporting the use of DPS in BU therapeutic drug monitoring as an alternative matrix, with adequate short-term stability and logistic advantages.

Genetic individual identification from dried urine spots: A complementary tool to drug monitoring and anti‐doping testing

“…[1][2][3] The most important, as well as the most studied and widely applied DMS, [4][5][6][7] is the dried blood spots (DBS) technique, described for the first time by Guthrie and Susi in 1963 and used in neonatal screening for the diagnosis of congenital phenylketonuria. 8 In the following years, the sampling of biological matrices on paper supports was used on multiple body fluids, such as plasma (DPS), 9,10 urine (DUS), 11,12 saliva, 13 tears, 14 breast milk, 15 synovial fluid, 16 cerebrospinal fluid (CSF), 17 and amniotic fluid. 18 This diffusion is well explained by highlighting the numerous advantages that the use of DMS for laboratory analysis entails: ease of sampling, especially in case of blood; simplicity in transport and storage, being suitable to be kept at room temperature even for long periods of time; improvement in the quality of life of subjects suffering from chronic diseases who must undergo periodical analyses; and suitability to a multitude of different analytical approaches.…”

Alternative Sampling Strategies in Therapeutic Drug Monitoring: Microsampling Growing Toward Maturity

“…15 This Special Issue also nicely illustrates several drug classes for whom TDM and particularly microsampling-assisted TDM are important to help guide therapy: immunosuppressants, anticonvulsants, antibiotics, and anticancer drugs. [8][9][10][11][22][23][24] The review by Moorthy et al in this Special Issue emphasizes the key advantage associated with microsampling, that is, the fact that it only requires the collection of minimal amounts of blood. This renders it a well-suited, patient-centric approach for pharmacokinetic studies in children where the amount of blood that can be collected may be limited.…”

“…As is evident from the contributions of Granzotto et al and Mukai et al, the use of dried microsamples may facilitate sampling and aid logistics. 23,24 This has been exemplified for busulfan and a set of tyrosine kinase inhibitors, which were determined from dried plasma spots generated by application on filter paper. Such approaches that use dried samples aid in transportation and improve the stability of the samples, which can be of special relevance to developing countries, where specialized transportation of samples from a clinical (eg, transplantation) site to an analytical site performing the actual TDM may not be easily available.…”