Extraction and analysis of carnitine and acylcarnitines by electrospray ionization tandem mass spectrometry directly from dried blood and plasma spots using a novel autosampler

Abstract

Acylcarnitines are routinely analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) both in clinical diagnostic and public health newborn screening laboratories from plasma and dried whole blood spots (DBS) on filter paper. The use of DBS as a convenient method of collecting and storing samples for subsequent analysis of various biomolecules is increasing, thus prompting the development of new devices to recover and quantify such analytes in an automated manner. Acylcarnitines were extracted directly from DBS using a novel autosampler that sequentially loads DBS cards into a pneumatic clamp and then pumps a fixed volume of solvent containing appropriate internal standards through a section of the DBS card directly into a triple quadrupole mass spectrometer via ESI. Plasma was first spiked with internal standard then spotted onto filter paper for analysis. Acylcarnitines were analyzed in DBS, and both free and total carnitine were assayed in dried plasma spots (DPS). Results using the new autosampling technique were of equal quality to those obtained by punching a 3-mm diameter disk from a DBS or DPS, then extracting and analyzing the target analytes from conventional 96-well microtiter plates, with far reduced time per sample. Recovery for most analytes was >60% and reproducibility was generally within 20% (CV). The simplicity and robustness of the DBS autosampler make it an attractive alternative to conventional methods of analyzing DBS specimens, thus saving time and labor costs, especially in high-throughput applications. Although the method as described is for direct infusion analysis, the autosampler is easily coupled to column hardware for applications requiring liquid chromatography/mass spectrometry (LC/MS).