Why dried blood spots are an ideal tool for CYP1A2 phenotyping

Background and Objective: Dried blood spot (DBS) sampling has gained wide interest in bioanalysis during the last decade and has already been successfully applied in pharmacokinetic and phenotyping studies. However, all of the available phenotyping studies used small datasets and did not include a systematic evaluation of DBS-specific parameters. The latter is important since several of these factors still challenge the breakthrough of DBS in routine practice. In this study, caffeine and paraxanthine are determined in capillary DBS, venous DBS, whole blood and plasma for cytochrome P450 (CYP) 1A2 phenotyping. The aim of this study was to explore the usefulness of DBS as a tool for CYP1A2 phenotyping. Methods: A CYP1A2 phenotyping study was conducted in 73 healthy volunteers who received a 150 mg oral dose of caffeine. Six hours post-administration, caffeine and paraxanthine concentrations and paraxanthine:caffeine molar concentration ratios, i.e., the actual CYP1A2 phenotyping indices, were determined in capillary DBS (obtained by non-volumetric application, direct from the fingertip), venous DBS, whole blood, and plasma. Furthermore, the impact of DBS-specific parameters, including hematocrit, volume spotted, and punch location, was evaluated. Results: Concentrations of caffeine and paraxanthine in capillary DBS were, respectively, on average 12.7 and 13.8 % lower than those in venous DBS and 31.5 and 33.1 % lower than those in plasma. While these differences were statistically significant (p = 0.053). This ratio also alleviated the impact of hematocrit and volume spotted. Conclusions: Using the largest DBS-based phenotyping study to date, we have demonstrated that CYP1A2 phenotyping in capillary DBS is a valid and convenient alternative for the classical plasma-based approach. Additionally, we have provided an objective basis as to why DBS are an ideal tool for CYP1A2 phenotyping

Determination of gamma-hydroxybutyric acid in dried blood spots using a simple GC-MS method with direct 'on spot' derivatization

The objective of this study was the development of an accurate and sensitive method for the determination of gamma-hydroxybutyric acid (GHB) in dried whole blood samples using a GC-MS method. The complete procedure was optimized, with special attention for the sample pre-treatment, and validated. Therefore, dried blood spots (DBS) of only 50 µl were prepared and, after addition of internal standard GHB-d6, directly derivatized using 100 µl of a freshly prepared mixture of trifluoroacetic acid anhydride (TFAA) and heptafluorobutanol (HFB-OH) (2:1). The derivatized extract was injected into a gas chromatograph coupled to a mass spectrometer (GC-MS), operating in the electron impact mode (EI), with a total run time of 12.3 min. Method validation included the evaluation of linearity, precision, accuracy, sensitivity, selectivity and stability. A weighting factor of 1/x2 was chosen and acceptable intra-batch precision, inter-batch precision and accuracy were seen. The linear calibration curve ranged from 2 to 100 µg/ml, with a limit of detection of 1 µg/ml. Our procedure, utilizing the novel approach of direct “on spot” derivatization, followed by analysis with GC-MS, proved to be reliable, fast and applicable in routine toxicology

Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: Influence of ionization type, sample preparation, and biofluid

AbstractThe purpose of the present work was to evaluate the synergistic effect of ionization type, sample preparation technique, and bio-fluid on the presence of matrix effect in quantitative liquid chromatography (LC)-MS/MS analysis of illicit drugs by post-column infusion experiments with morphine (10-μg/mL solution). Three bio-fluids (urine, oral fluid, and plasma) were pretreated with four sample preparation procedures [direct injection, dilution, protein precipitation, solid-phase extraction (SPE)] and analyzed by both LC-electrospray ionization (ESI)-MS/MS and LC-atmospheric pressure chemical ionization (APCI)-MS/MS. Our results indicated that both ionization types showed matrix effect, but ESI was more susceptible than APCI. Sample preparation could reduce (clean up) or magnify (pre-concentrate) matrix effect. Residual matrix components were specific to each bio-fluid and interfered at different time points in the chromatogram. We evaluated matrix effect in an early stage of method development and combined optimal ionization type and sample preparation technique for each bio-fluid. Simple dilution of urine was sufficient to allow for the analysis of the analytes of interest by LC-APCI-MS/MS. Acetonitrile protein precipitation provided both sample clean up and concentration for oral fluid analysis, while SPE was necessary for extensive clean up of plasma prior to LC-APCI-MS/MS

Feasibility of following up gamma-hydroxybutyric acid concentrations in sodium oxybate (Xyrem®)-treated narcoleptic patients using dried blood spot sampling at home : an exploratory study

Background: Gamma-hydroxybutyric acid (GHB), well known as a party drug, especially in Europe, is also legally used (sodium oxybate, Xyrem (R)) to treat a rare sleep disorder, narcolepsy with cataplexy. This exploratory study was set up to measure GHB concentrations in dried blood spots (DBS) collected by narcoleptic patients treated with sodium oxybate. Intra- and inter-individual variation in clinical effects following sodium oxybate administration has been reported. The use of DBS as a sampling technique, which is stated to be easy and convenient, may provide a better insight into GHB concentrations following sodium oxybate intake in a real-life setting. Objective The aim was twofold: evaluation of the applicability of a recently developed DBS-based gas chromatography mass spectrometry (GC MS) method, and of the feasibility of the sampling technique in an ambulant setting. Methods: Seven narcoleptic patients being treated with sodium oxybate at the Department for Respiratory Diseases of Ghent University Hospital were asked to collect DBS approximately 20 min after the first sodium oxybate (Xyrem (R); UCB Pharma Ltd, Brussels, Belgium) intake on a maximum of 7 consecutive days. Using an automatic lancet, patients pricked their fingertip and, after wiping off the first drop of blood, subsequent drops were collected on a DBS card. The DBS cards were sent to the laboratory by regular mail and, before analysis, were visually inspected to record DBS quality (large enough, symmetrically spread on the filter paper with even colouration on both sides of the filter paper). Results: Of the seven patients, three patients succeeded to collect five series of DBS, one patient decided to cease participation because of nausea, one was lost during follow-up and two patients started falling asleep almost immediately after the intake of sodium oxybate. Analysing the DBS in duplicate resulted in acceptable within-DBS card precision. DBS with acceptable quality were obtained by patients without supervision. Conclusion: Our results demonstrate the acceptable precision of the complete procedure, from sampling at home to quantitative analysis in the laboratory. Given the intra-and inter-individual variability in clinical effects seen with sodium oxybate, the easy adaptation of DBS sampling opens the possibility of following up GHB concentrations in patients in real-life settings in future studies

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

Potassium-based algorithm allows correction for the hematocrit bias in quantitative analysis of caffeine and its major metabolite in dried blood spots

Although dried blood spot (DBS) sampling is increasingly receiving interest as a potential alternative to traditional blood sampling, the impact of hematocrit (Hct) on DBS results is limiting its final breakthrough in routine bioanalysis. To predict the Hct of a given DBS, potassium (K+) proved to be a reliable marker. The aim of this study was to evaluate whether application of an algorithm, based upon predicted Hct or K+ concentrations as such, allowed correction for the Hct bias. Using validated LC-MS/MS methods, caffeine, chosen as a model compound, was determined in whole blood and corresponding DBS samples with a broad Hct range (0.18-0.47). A reference subset (n = 50) was used to generate an algorithm based on K+ concentrations in DBS. Application of the developed algorithm on an independent test set (n = 50) alleviated the assay bias, especially at lower Hct values. Before correction, differences between DBS and whole blood concentrations ranged from -29.1 to 21.1 %. The mean difference, as obtained by Bland-Altman comparison, was -6.6 % (95 % confidence interval (CI), -9.7 to -3.4 %). After application of the algorithm, differences between corrected and whole blood concentrations lay between -19.9 and 13.9 % with a mean difference of -2.1 % (95 % CI, -4.5 to 0.3 %). The same algorithm was applied to a separate compound, paraxanthine, which was determined in 103 samples (Hct range, 0.17-0.47), yielding similar results. In conclusion, a K+-based algorithm allows correction for the Hct bias in the quantitative analysis of caffeine and its metabolite paraxanthine

Paraxanthine/caffeine concentration ratios in hair: an alternative for plasma-based phenotyping of cytochrome P450 1A2?

Background and Objective: Although metabolite-to-parent drug concentration ratios in hair have been suggested as a possible tool to study the metabolism of drugs in a non-invasive way, no studies are available that evaluated this in a systematic way. Cytochrome P450 (CYP) 1A2 is a drug-metabolizing enzyme characterized by large inter-individual differences in its activity. The standard approach for CYP1A2 phenotyping is to determine the paraxanthine/caffeine ratio in plasma at a fixed timepoint after intake of a dose of the CYP1A2 substrate caffeine. The aim of this study was to evaluate whether paraxanthine/caffeine ratios measured in hair samples reflect the plasma-based CYP1A2 phenotype. Methods: Caffeine and paraxanthine concentrations were measured in proximal 3 cm segments of hair samples from 60 healthy volunteers and resulting paraxanthine/caffeine ratios were correlated with CYP1A2 phenotyping indices in plasma. Results: Paraxanthine/caffeine ratios in hair ranged from 0.12 to 0.93 (median 0.41); corresponding ratios in plasma ranged from 0.09 to 0.95 (median 0.40). A statistically significant correlation was found between ratios in hair and plasma (r = 0.41, p = 0.0011). However, large deviations between ratios in both matrices were found in individual cases. Although the influence of several factors on paraxanthine/caffeine ratios and hair-plasma deviations was investigated, no evident factors explaining the observed variability could be identified. Conclusion: The variability between hair and plasma ratios complicates the interpretation of hair paraxanthine/caffeine ratios on an individual basis and, therefore, compromises their practical usefulness as alternative CYP1A2 phenotyping matrix

Does volumetric absorptive microsampling eliminate the hematocrit bias for caffeine and paraxanthine in dried blood samples? : a comparative study

Volumetric absorptive microsampling (VAMS) is a novel sampling technique that allows the straightforward collection of an accurate volume of blood (approximately 10 mu L) from a drop or pool of blood by dipping an absorbent polymeric tip into it. The resulting blood microsample is dried and analyzed as a whole. The aim of this study was to evaluate the potential of VAMS to overcome the hematocrit bias, an important issue in the analysis of dried blood microsamples. An LC-MS/MS method for analysis of the model compounds caffeine and paraxanthine in VAMS samples was fully validated and fulfilled all pre-established criteria. In conjunction with previously validated procedures for dried blood spots (DBS) and blood, this allowed us to set up a meticulous comparative study in which both compounds were determined in over 80 corresponding VAMS, DBS and liquid whole blood samples. These originated from authentic human patient samples, covering a wide hematocrit range (0.21-0.50). By calculating the differences with reference whole blood concentrations, we found that analyte concentrations in VAMS samples were not affected by a bias that changed over the evaluated hematocrit range, in contrast to DBS results. However, VAMS concentrations tend to overestimate whole blood concentrations, as a consistent positive bias was observed. A different behavior of VAMS samples prepared from incurred and spiked blood, combined with a somewhat reduced recovery of caffeine and paraxanthine from VAMS tips at high hematocrit values, an effect that was not observed for DBS using a very similar extraction procedure, was found to be at the basis of the observed VAMS-whole blood deviations. Based on this study, being the first in which the validity and robustness of VAMS is evaluated by analyzing incurred human samples, it can be concluded that VAMS effectively assists in eliminating the effect of hematocrit