Quantitative determination of opioids in whole blood using fully automated dried blood spot desorption coupled to on‐line SPE‐LC‐MS/MS

Abstract

Opioids are well known, widely used painkillers. Increased stability of opioids in the dried blood spot (DBS) matrix compared to blood/plasma has been described. Other benefits provided by DBS techniques include point-of-care collection, less invasive micro sampling, more economical shipment, and convenient storage. Current methodology for analysis of micro whole blood samples for opioids is limited to the classical DBS workflow, including tedious manual punching of the DBS cards followed by extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis. The goal of this study was to develop and validate a fully automated on-line sample preparation procedure for the analysis of DBS micro samples relevant to the detection of opioids in finger prick blood. To this end, automated flow-through elution of DBS cards was followed by on-line solid-phase extraction (SPE) and analysis by LC-MS/MS. Selective, sensitive, accurate, and reproducible quantitation of five representative opioids in human blood at sub-therapeutic, therapeutic, and toxic levels was achieved. The range of reliable response (R(2) ≥0.997) was 1 to 500 ng/mL whole blood for morphine, codeine, oxycodone, hydrocodone; and 0.1 to 50 ng/mL for fentanyl. Inter-day, intra-day, and matrix inter-lot accuracy and precision was less than 15% (even at lower limits of quantitation (LLOQ) level). The method was successfully used to measure hydrocodone and its major metabolite norhydrocodone in incurred human samples. Our data support the enormous potential of DBS sampling and automated analysis for monitoring opioids as well as other pharmaceuticals in both anti-doping and pain management regimens.

Quantitation of fentanyl analogs in dried blood spots by flow-through desorption coupled to online solid phase extraction tandem mass spectrometry

“…These sorbents included Oasis ® HLB (Waters, Milford, MA), which was previously used for these compounds 41 Resin SH, C2-SE, CN-SE, C8-SE, C18 HD, Resin GP, MM anion, and MM cation (HySphere™) (Spark, Emmen, Netherlands). The C18 HD cartridge, which has been used previously with this system for similar compounds 25 gave the highest response with baseline resolution from matrix peaks for all analytes, and thus was selected for further development.…”

“…20,22,23 The automated desorption of DBS has been applied to multiple analytes with mass spectrometric analysis. 15,17,20,24,25 More specifically, fentanyl has been quantitated from DBS using automated desorption but required separate SPE and LC separation. 25 Fentanyl and two analogs have also been detected from DBS using manual extraction with HPLC-MS/MS.…”

Opening the toolbox of alternative sampling strategies in clinical routine: A key-role for (LC-)MS/MS

“…For most applications, the limited amount of material, combined with the required sensitivity, imposes the need for a dedicated LC-MS/MS configuration. Recent progress in this field includes the set-up of hands-off online systems (see also section 5.1) 64 . When considering the cut off for driving under the influence of drugs, alcohol and medicines (DRUID), for most analytes procedures have been described that are able to achieve the required sensitivity when starting from a ≤6.4-mm DBS punch or when starting from 10 µl or less dried blood 63 .…”

“…The way the elution is performed depends on the type of analyzer: the DBS Autosampler TM and the Sample Card and Prep system TM both employ flow through desorption, whilst in the DBS-MS 500 system extraction solvent is guided horizontally through the DBS during a surface sealed extraction, after which the extract is guided into a sample loop [119][120][121] . After elution, the extract can be subjected to on-line sample clean-up and/or separation on an LC column or even direct injection into the MS, depending on the chosen configuration 8,64,[119][120][121] . Importantly, using automated DBS analyzers, the entire DBS extract is introduced into the (LC-)MS/MS system, thereby increasing method sensitivity, since in off-line approaches only part of the extract is injected into the analyzing system.…”

Novas estratégias de preparo de amostras complexas para a determinação de sulfonamidas

“…As colunas de SPE são tipicamente empacotadas com partículas (apresentando, por exemplo, uma funcionalidade de fase reversa (RP)), ou são colunas monolíticas orgânica (94,102). As colunas empacotadas tem restrições de tamanho (partículas comuns não se "encaixam" em colunas extremamente estreitas), e também pode-se considerar tipicamente bem adequado para pequenas moléculas (25).…”