Ann-Sofie Ingels

Dried blood spots in toxicology: from the cradle to the grave?

About a century after its first described application by Ivar Bang, the potential of sampling via dried blood spots (DBS) as an alternative for classical venous blood sampling is increasingly recognized. Perhaps best known is the use of DBS in newborn screening programs, ignited by the hallmark paper by Guthrie and Susi half a century ago. However, it is only recently that both academia and industry have recognized the many advantages that DBS sampling may offer for bioanalytical purposes, as reflected by the strong increase in published reports during the last few years. Currently, major DBS applications include newborn screening for metabolic disorders, epidemiological surveys (e.g. HIV monitoring), therapeutic drug monitoring (TDM), as well as toxicology. In this review, we provide a comprehensive overview of the distinct subdisciplines of toxicology for which DBS sampling has been applied. DBS sampling for toxicological evaluation has been performed from birth until autopsy, aiming at the assessment of therapeutic drugs, drugs of abuse, environmental contaminants, toxins, as well as (trace) elements, with applications situated in fields as toxicokinetics, epidemiology and environmental and forensic toxicology. We discuss the strengths and limitations of DBS in the different subdisciplines and provide future prospects for the use of this promising sampling technique in toxicology.

Screening and confirmation methods for GHB determination in biological fluids

The purpose of this review is to provide a comprehensive overview of reported methods for screening and confirmation of the low-molecular-weight compound and drug of abuse gamma-hydroxybutyric acid (GHB) in biological fluids. The polarity of the compound, its endogenous presence, its rapid metabolism after ingestion, and its instability during storage (de novo formation and interconversion between GHB and its lactone form gamma-butyrolactone) are challenges for the analyst and for interpretation of a positive result. First, possible screening procedures for GHB are discussed, including colorimetric, enzymatic, and chromatography-based procedures. Confirmation methods for clinical and forensic cases mostly involve gas chromatography (coupled to mass spectrometry), although liquid chromatography and capillary zone electrophoresis have also been used. Before injection, sample-preparation techniques include (a combination of) liquid–liquid, solid-phase, or headspace extraction, and chemical modification of the polar compound. Also simple “dilute-and-shoot” may be sufficient for urine or serum. Advantages, limitations, and trends are discussed.

Do capillary dried blood spot concentrations of gamma‐hydroxybutyric acid mirror those in venous blood? A comparative study

Gamma-hydroxybutyric acid (GHB) is a well-known illicit club and date-rape drug. Dried blood spot (DBS) sampling is a promising alternative for classical venous sampling in cases of (suspected) GHB intoxication since it allows rapid sampling, which is of interest for the extensively metabolized GHB. However, there is limited data if -and how- capillary DBS concentrations correlate with venous concentrations. We conducted a comparative study in 50 patients with suspected GHB intoxication, to determine and to correlate GHB concentrations in venous DBS (vDBS) and capillary DBS (cDBS). This is the first study that evaluates in a large cohort the correlation between capillary and venous concentrations of an illicit drug in real-life samples. Of the 50 paired samples, 7 were excluded: the vDBS concentration was below the LLOQ of 2 µg/mL in 3 cases and 4 samples were excluded after visual inspection of the DBS. Bland-Altman analysis revealed a mean % difference of -2.8% between cDBS and vDBS concentrations, with the zero value included in the 95% confidence interval of the mean difference in GHB concentration. A paired sample t-test confirmed this observation (p = 0.17). Also the requirement for incurred sample reproducibility was fulfilled: for more than two-thirds of the samples the concentrations obtained in cDBS and those in vDBS were within 20% of their mean. Since equivalent concentrations were observed in cDBS and vDBS, blood obtained by fingerprick can be considered a valid alternative for venous blood for GHB determination.

Quantitation of gamma-hydroxybutyric acid in dried blood spots in newborn screening.

When reporting on new methods for the detection of analytes in biological matrices, it is important to make adequate referral to the existing state-of-the-art ones. We feel that the article by Forni et al. [1] has failed in doing so. This paper cites neither existing publications on the use of dried blood spots (DBS) for gamma-hydroxybutyric acid (GHB) quantitation [2,3], nor recent relevant similar LC– MS/MS procedures for GHB detection in biofluids [4,5]. Moreover, based upon the reported limited validation data, we consider it premature to state that this article provides data for true quantitation of GHB in DBS (as stated in the title). Given the absence of referral to a lower limit of quantitation (LLOQ), one can assume that the LLOQ corresponds to the lowest point of the calibration curve, i.e. 8 nM. Importantly, intraand interday imprecision and bias were not reported at this level. In this context, it is not clear how quantitative data were obtained for over 1000 data points that lie below this presumed LLOQ, which, per definition, is a limit below which no quantitation should be performed. Hence, rather than providing a mean GHB concentration, reporting a median value would be recommendable in this case. In addition, the calibration line has been extended through the origin, which is contraindicated in quantitative bioanalysis; and, for the value of 4851 nM, it is not clear whether this has been obtained by extrapolating the calibration curve or by diluting the sample (no dilution integrity experiment is discussed). Last but not least (as can readily be deduced from the preparation of the calibrators), all reported concentrations (nM levels) are incorrect: nM should be replaced by μM throughout the article (as well as throughout this letter). In conclusion, while we agree that the authors have provided evidence suggesting that their method may be used for semiquantitative screening purposes (to screen for outliers, using a cut-off value), it cannot be concluded from the presented data that the method is capable of truly quantitatively determining endogenous GHB concentrations.