Volumetric absorptive microsampling as an alternative tool for therapeutic drug monitoring of first-generation anti-epileptic drugs

Dosage adjustment of anti-epileptic drugs by therapeutic drug monitoring (TDM) is very useful, especially for the first-generation anti-epileptic drugs (AEDs). Microsampling-the collection of small volumes of blood-is increasingly considered a valuable alternative to conventional venous sampling for TDM. Volumetric absorptive microsampling (VAMS) allows accurate and precise collection of a fixed volume of blood, eliminating the volumetric blood hematocrit bias coupled to conventional dried blood spot collection. The aim of this study was to develop and validate an LC-MS/MS method for the determination and quantification of four anti-epileptic drugs (carbamazepine, valproic acid, phenobarbital, and phenytoin) and one active metabolite (carbamazepine-10,11-epoxide) in samples collected by VAMS. The method was fully validated based on international guidelines. Precision (%RSD) was below 10%, while, with a single exception, accuracy (%bias) met the acceptance criteria. Neither carry-over nor unacceptable interferences were observed, the method being able to distinguish between the isomers oxcarbazepine and carbamazepine-10,11-epoxide. All compounds were stable in VAMS samples for at least 1 month when stored at room temperature, 4 A degrees C, and - 20 A degrees C and for at least 1 week when stored at 60 A degrees C. Internal standard-corrected matrix effects were below 10%, with %RSDs below 4%. High (> 85%) recovery values were obtained and the effect of the hematocrit on the recovery was overall limited. Successful application on external quality control materials and on left-over patient samples demonstrated the validity and applicability of the developed procedure

Is the hematocrit still an issue in quantitative dried blood spot analysis?

Hematocrit-related issues remain a major barrier for (regulatory) acceptance of the classical dried blood spot (DBS) analysis in the bioanalytical and clinical field. Lately, many attempts to cope with these issues have been made. Throughout this review, an overview is provided on new strategies that try to cope with this hematocrit effect (going from avoiding to minimizing), on methods estimating a DBS volume, and on methods estimating or measuring the hematocrit of a DBS. Although many successful strategies have been put forward, a combination of different technologies still provides the most complete solution. Therefore, further efforts and the availability of a straightforward guideline for analytical and clinical method validation should help to overcome the hurdles still associated with DBS sampling. (C) 2018 Elsevier B.V. All rights reserved

Evaluation of the Performance and Hematocrit Independence of the HemaPEN as a Volumetric Dried Blood Spot Collection Device

Dried blood spots (DBS) are often used as a less invasive alternative to venous blood sampling. Despite its numerous advantages, the use of conventional DBS suffers from the hematocrit (hct) effect when analyzing a subpunch. This effect could be avoided by using hct-independent sampling devices, of which the hemaPEN is a recent example. This device collects the blood via four integrated 2.74 μL microcapillaries, each depositing the blood on a prepunched paper disc. In this study, we evaluated the technical performance of the hemaPEN devices, using an extensive bioanalytical validation and application on authentic patient samples. An LC-MS/MS method quantifying caffeine and its metabolite paraxanthine in dried whole blood (using the hemaPEN device) was fully validated, meeting all preset acceptance criteria. A comparative analysis of 91 authentic patient samples (hct range: 0.17–0.53) of hemaPEN, 3 mm DBS subpunches, and whole blood revealed a limited hct dependence (≤7% concentration difference over a 0.20–0.50 hct range) for the hemaPEN devices, which we could not attribute to the analytical procedure. Using conventional partial-punch DBS (3 mm punches), concentration differences of ≥25% over this hct range were found. The hemaPEN showed to be robust to the effects of blood sample volume, device lot, analytical operator, and storage stability. The technical performance of the hemaPEN when dealing with patients having a high hct and in cases where a large blood drop is present should be further investigated. Based on the successful validation and application on patient samples, we conclude that the hemaPEN device shows good potential for the volumetric collection of DBS.S.D. would like to thank the Research Foundation-Flanders (FWO) for granting her a PhD fellowship (application number:11F3119N). L.P.-G. would like to thank the Consellería de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, for her predoctoral (ED481A-2018/059) contractS

Evaluation of the Capitainer-B microfluidic device as a new hematocrit-independent alternative for dried blood spot collection

The hematocrit-bias still remains one of the most discussed issues when it comes to dried blood spot (DBS) analysis. Therefore, many attempts to cope with this issue have been made, among which the development of novel sampling tools such as the Capitainer-B (further referred to as MF (microfluidic)-DBS) devices. These are designed to allow a straightforward absorption of a fixed volume (13.5 mu L) of blood by a preperforated paper disc, which can be analyzed afterward. The aim of this study was to evaluate the potential of these devices to nullify the hematocrit-based area bias and to investigate whether the amount of blood applied has an influence on the device performance. An LC-MS/MS method for the quantification of caffeine and paraxanthine in MF-DBS was fully validated, meeting all preset acceptance criteria. In a next step, using a set of 133 authentic, venous patient samples with a hematocrit range of 18.8-55.0, concentrations of both compounds in MF-DBS were compared to those in corresponding partial-punch pipetted DBS (PI-DBS) and liquid blood samples. When compared to blood as a reference, the concentrations obtained in MF-DBS were not affected by a bias in function of the evaluated hematocrit, in contrast to those obtained from partial-punch PI-DBS. Furthermore, analysis of samples resulting from spiking different volumes of whole blood at different hematocrit levels, revealed that the amount of blood applied at the device inlet has no influence on the performance of the devices. Therefore, it can be concluded from this study, being the first in which the impact of the hematocrit and the applied volume is evaluated by analyzing authentic, venous patient samples, that MF-DBS devices effectively assist in eliminating the hematocrit-based area bias, independently from the applied blood volume

Dried blood microsampling-based therapeutic drug monitoring of antiepileptic drugs in children with nodding syndrome and epilepsy in Uganda and the Democratic Republic of the Congo

Nodding syndrome is a highly debilitating, generalized seizure disorder, affecting children in subregions of sub-Saharan Africa. Despite numerous efforts to uncover the etiology, the exact cause of this syndrome still remains obscure. Therefore, to date, patients only receive symptomatic care, including the administration of first-generation antiepileptic drugs for seizure control. As data on the efficacy of drugs within this population are completely lacking, the aim of this study was to explore how therapeutic drug monitoring could help to understand the differential response to therapy. Considering the challenging environment in which sampling had to be performed (remote areas, devoid of electricity, running water, etc), dried blood matrices [ie, dried blood spots (DBSs)] and volumetric absorptive microsampling (VAMS) were considered fit-for-purpose. In addition, owing to the similarities between the syndrome and other forms of epilepsy, samples originating from patients suffering from (onchocerciasis-associated) epilepsy were included. In total, 68 patients with Nodding syndrome from Uganda, 58 Ugandan patients with epilepsy, and 137 patients with onchocerciasis-associated epilepsy from the Democratic Republic of the Congo were included. VAMS samples and DBS were analyzed using validated methods, involving manual extraction or fully automated extraction, respectively, before quantification using liquid chromatography coupled with tandem mass spectrometry. Analysis revealed that serum concentrations (calculated from DBS) within the respective reference ranges were attained in only 52.9% of the 68 Nodding syndrome patients treated with valproic acid, in 21.4% of the 56 Ugandan epilepsy patients treated with carbamazepine, and in 65.7% of the 137 onchocerciasis-associated epilepsy patients from the Democratic Republic of the Congo treated with phenobarbital. In all other instances, concentrations were subtherapeutic. Furthermore, on comparing DBS with VAMS concentrations, an inexplicable overestimation was observed in the latter. Finally, no obvious link could be observed between the obtained drug concentrations and the number of seizures experienced during the last month before sampling, elaborating the fact that the level of improvement in some patients cannot simply be linked to reaching therapeutic concentrations