Evaluation of dried blood spots as an alternative sampling strategy for 5-fluorouracil monitoring: From method development to clinical application.

Therapeutic drug monitoring (TDM) of 5-Fluorouracil (5-FU) is strongly recommended because of its large inter-individual pharmacokinetic variability, narrow therapeutic window, and incidence of toxicity. However, there are several factors that limit the application of TDM in clinical settings. Considering the intrinsic advantages of dried microsamples, such as minimally invasive sampling, analyte stability, and cost-effective logistics, this study aimed to develop a method for the determination of 5-FU in dried blood spots (DBS) using ultra-high liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and to evaluate its clinical application. Sample preparation was based on an aqueous extraction followed by protein precipitation. Separation was performed in an Acquity UPLC® HSS C18 (150 ×2.1 mm, 1.8 µm), and the mobile phases were water and acetonitrile with 0.5% acetic acid. The total run time was 5.5 min. The method was linear from 100 to 2000 ng/mL, precise (maximum CV% of 7.5%), and accurate (98.3-115.4%). The average recovery was 70%. Blood hematocrit had a minimal impact on the assay. DBS samples were stable for 21 days at 4, 25, and 45 °C. A total of 40 paired samples of plasma, capillary DBS, and venous DBS were analyzed. Median 5-FU concentrations were 444.7, 637.0, and 499.7 ng/mL for plasma, capillary DBS, and venous DBS, respectively. Capillary and plasma concentrations were significantly correlated (r > 0.90), but there was a lack of agreement between the methods, as capillary DBS levels were on average 146% of plasma. Venous DBS corresponded to 110% of the measured plasma concentrations, with a strong correlation (r > 0.97) and agreement between the methods. Our study is the first to report the use of DBS samples to quantify 5-FU. Further studies are needed to establish whether capillary samples can replace plasma.

Analytical and clinical validation of a dried blood spot assay for the determination of paclitaxel using high-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: Paclitaxel (PCT) is a chemotherapeutic drug widely used for the treatment of several types of tumors, and its use is associated with severe adverse events, mainly neurologic and hematopoietic toxicities. The relation between systemic exposure and clinical response to PCT was previously described, making paclitaxel a potential candidate for therapeutic drug monitoring (TDM). The use of dried blood spot (DBS) sampling could allow complex sampling schedules required for TDM of PCT. The aim of this study was to develop and validate an LC-MS/MS assay for the quantification of PCT in DBS. METHODS: PCT was extracted from one 8 mm DBS punch with a mixture of methanol and acetonitrile, followed by chromatographic separation in a Kinetex C18 (50 × 4.6 mm, 2.6 µm) column. Detection was performed in a 5500-QTRAP® mass spectrometer, with a run time of 2.3 min. RESULTS: The assay was linear in the range of 2.5 to 400 ng mL-1. Precision (CV%) and accuracy at the concentration levels of 7.5, 40 and 150 ng mL-1 were 1.69-4.9% and 106.25 to 109.92%, respectively. PCT was stable for 21 days at 25 and 45 °C. The method was applied to DBS samples obtained from 34 patients under PCT chemotherapy. The use of a simple correction factor, derived from the correlation between PCT concentrations in plasma and DBS in this set of patients, allowed unbiased estimation of PCT plasma concentrations from DBS measurements, with similar clinical decisions using either plasma or DBS measurements. CONCLUSIONS: DBS testing of PCT concentrations represents a promising alternative for the dissemination of PCT dose individualization.

Determination of irinotecan and its metabolite SN-38 in dried blood spots using high-performance liquid-chromatography with fluorescence detection.

Irinotecan (IRI) is an antineoplastic drug widely used for the treatment of colorectal and advanced pancreatic cancer. Despite its clinical utility, the clinical use of IRI is associated with potentially severe hematopoietic and gastrointestinal toxicities. The quantification of IRI and its active metabolite SN-38 in dried blood spots (DBS) may be an alternative to individualize the drug dose through a minimally invasive and easy collection method. The aim of this study was to develop and validate a simple and fast HPLC-FL assay for simultaneous IRI and SN-38 measurement in DBS, with adequate analytical performance for clinical use. The method employs liquid extraction of one 8mm disk of whole blood, followed by separation in a reversed phase Eclipse Plus C8 column (150×4.6mm, 5µm). Detection was performed with a fluorescence detector, with excitation wavelength of 370 and emission of 420 for IRI and 540nm for SN-38 and internal standard (camptothecin). Total analytical run time was 17min. Mobile phase was a mixture of 0.1M phosphate buffer pH 4.0 and acetonitrile (80:20, v/v), at 1mLmin-1. The assay was linear in the range 10-3,000ngmL-1 and from 0.5 to 300ngmL-1 for IRI and SN-38, respectively. Precision assays presented CV% of 2.71-5.65 and 2.15-10.07 for IRI and SN-38, respectively, and accuracy in the range of 94.26-100.93 and 94.24-99.33%. IRI and SN-38 were stable at 25 and 42°C for 14days in DBS samples. The method was applied to DBS samples obtained from fingerpicks from 19 volunteers receiving IRI in single or combined chemotherapy regimens, collected 1 and 24h after beginning of the infusion. The estimated plasma concentration of IRI and SN-38 in sample collected 1h after star of infusion had 16 of 19 values within the ±20% range of the measured plasma concentrations. On the other hand, predictions of IRI and SN-38 plasma concentrations from DBS measurements obtained 24h after the beginning of the infusion were poor. AUC of IRI that was calculated using plasma and DBS-estimated concentrations, with a high correlation (r=0.918). The method presented suitable characteristics for the clinical use. However, translation of IRI and SN-38 DBS to plasma concentrations is challenging due to the compound's variable plasma/blood partition.