For therapeutic drug monitoring in remote settings, dried blood spots (DBS) are particularly advantageous, as blood sample collection and handling is uncomplicated. The aim of this study was to develop and validate an automated extraction method for the analysis of nevirapine, efavirenz and lopinavir in DBS samples. Automated extraction was performed with methanol : water (70 : 30 v/v), using a DBS-MS 500 autosampler coupled to a liquid chromatography tandem mass spectrometry system. The autosampler used digital images of each DBS to position the extraction head, sprayed 10 μl of internal standard onto each DBS and extracted a 4-mm disc (Ø) from the centre of each spot by unilateral flow using 25-μl extraction solvent. The analytes were baseline separated on a pentafluorophenyl column and analysed by using electrospray ionization with multiple reaction monitoring in positive polarity mode for nevirapine and lopinavir and in negative mode for efavirenz. The method was linear between 10 and 10 000 ng/ml for all analytes. Automated sample extraction resulted in consistent recoveries (nevirapine: 70 ± 6%, efavirenz: 63 ± 11% and lopinavir: 60 ± 10%) and matrix effects between different donors and concentration levels. Intra-day and inter-day accuracy and precision deviations were ≤15%. Manual and automated extractions of DBS samples collected within the framework of an adherence assessment study in rural Tanzania showed good agreements with deviations of less than 10%. Our study highlights that therapeutic drug monitoring samples obtained in the resource-constrained setting of rural Africa can be reliably determined by automated extraction of DBS. Overall, automatization improved method sensitivity and facilitates analysis of large sample numbers. Copyright © 2017 John Wiley & Sons, Ltd.
The impact of the hematocrit (HCT) on the dried blood spot (DBS)’s spreading area is one of the most important hurdles, which prevents the full acceptance of quantitative microsampling strategies. Several destructive and non-destructive strategies to assess the HCT from a DBS post-sampling have been presented. Unfortunately, the current methods are either labor-intensive, require a complicated algorithm or are not automatable. Here, we present a novel setup that permits the fully automated reflectance analysis to measure the HCT from a DBS. The underlying principle is based on the concept of the non-destructive single-wavelength HCT measurement. The novel module was embedded within the DBS-MS 500 platform to enable high-throughput analysis of HCT values in combination with automated DBS extraction. The novel setup was assessed and optimized for the probe to card distance, stability, anti-coagulant, spotting volume, scan number, calibration variability, accuracy and precision. It showed excellent inter-day (≤3.7%) and intra-day (≤1.16%) precision as well as high accuracy when analyzing authentic samples (101% ± 7% [range: 87–127%]). Besides, the simple and straightforward application of an HCT correction for DBS was demonstrated during a pharmacokinetic study with diclofenac involving three subjects. Thereby, the sample’s HCT and the HCT impact on the analyte were assessed and compensated. In conclusion, the novel setup enables quantitative analysis of non-volumetric samples in an automated fashion without compromising the concept of cost-effective, minimally invasive sampling.
Phosphatidylethanol (PEth) in human blood samples is a marker for alcohol usage. Typically, PEth is detected by reversed-phase liquid chromatography coupled with negative ion tandem mass spectrometry, investigating the fatty acyl anions released from the precursor ion upon collision-induced dissociation (CID). It has been established that in other classes of asymmetric glycerophospholipids, the unimolecular fragmentation upon CID is biased depending on the relative position (known as sn-position) of each fatty acyl chain on the glycerol backbone. As such, the use of product ions in selected-reaction-monitoring (SRM) transitions could be prone to variability if more than one regioisomer is present in either the reference materials or the sample. Here, we have investigated the regioisomeric purity of three reference materials supplied by different vendors, labeled as PEth 16:0/18:1. Using CID coupled with ozone-induced dissociation, the regioisomeric purity (% 16:0 at sn-1) was determined to be 76, 80 and 99%. The parallel investigation of the negative ion CID mass spectra of standards revealed differences in product ion ratios for both fatty acyl chain product ions and ketene neutral loss product ions. Furthermore, investigation of the product ion abundances in CID spectra of PEth within authentic blood samples appears to indicate a limited natural variation in isomer populations between samples, with the cannonical, PEth 16:0/18:1 (16:0 at sn-1) predominant in all cases. Different reference material isomer distributions led to variation in fully automated quantification of PEth in 56 authentic dried blood spot (DBS) samples when a single quantifier ion was used. Our results suggest caution in ensuring that the regioisomeric compositions of reference materials are well-matched with those of the authentic blood samples.
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