Simultaneous quantification of seven B vitamins from wheat grains using UHPLC-MS/MS.

B-group vitamins are important micronutrients for maintaining human health; nevertheless, B vitamin deficiency is a globally widespread issue. Thus, it is relevant to accurately assess the B-vitamin content in staple crop products such as wheat grains. Here, we developed a multi-enzyme extraction method allowing accurate quantification of seven B vitamins in wheat using ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Free forms of thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), biotin (B7) and folates (B9) were determined with recoveries ranging from 81 to 118% and accuracy below 15% bias. The precision was below 20% relative standard deviation and the internal standards adequately compensated for matrix effects. The method was applied to determine the B vitamin stabilities in wheat grains stored at different temperatures and periods. The results provide an important basis in future studies aiming at understanding nutritional availability of B vitamins.

Application of non-contact hematocrit prediction technologies to overcome hematocrit effects on immunosuppressant quantification from dried blood spots.

Fully automated dried blood spot (DBS) analysis for therapeutic drug monitoring (TDM) of the immunosuppressants tacrolimus, sirolimus, everolimus and cyclosporin A suffers from a so-called hematocrit (hct) effect. This effect is related to the analysis of a partial DBS punch and extractability differences imposed by blood with different hcts. As this is intrinsic to automated DBS analysis, this poses a serious drawback for accurate immunosuppressant quantification. Knowledge of a sample's hct allows to correct the derived immunosuppressant concentrations for this effect. Unfortunately, when using the DBS approach for sampling at patients' homes, this hct will typically not be available. The aim of this study was to investigate the validity of a correction algorithm during fully automated DBS analysis of immunosuppressants, based on knowledge of the DBS' hct, obtained via two distinct non-contact hematocrit prediction strategies, using either near-infrared (NIR) or ultra-violet/visible (UV/VIS) spectroscopy. For tacrolimus, sirolimus, everolimus, and cyclosporin A, 48, 47, 58 and 48 paired venous whole blood and venous DBS patient samples were collected, respectively, and analyzed using an automated DBS-MS 500 HCT extraction unit coupled to a liquid chromatography tandem mass spectrometry system. Additionally, for all 201 samples the hct of the DBS was predicted based on NIR and UV/VIS spectroscopy. For tacrolimus and cyclosporin A, both hct prediction strategies allowed for adequate correction of the hct effect. Also for sirolimus and everolimus the results greatly improved after hct correction, although a hct bias remained for sirolimus and for everolimus a slightly significant hct effect was observed after NIR- and UV/VIS-based correction. Application of both hct prediction strategies ensured that clinical acceptance limits (i.e. ≥ 80% of the samples within 20% difference compared to whole blood) were met for all analytes. In conclusion, we demonstrated that non-contact hct prediction strategies, applied in tandem with fully automated DBS analysis, can be used to adequately correct immunosuppressant concentrations, yielding a good agreement with whole blood.

Application of a Volumetric Absorptive Microsampling (VAMS)-Based Method for the Determination of Paracetamol and Four of its Metabolites as a Tool for Pharmacokinetic Studies in Obese and Non-Obese Patients.

BACKGROUND: The pharmacokinetic (PK) profile of a drug is influenced by several factors, which can lead to a suboptimal dosing regimen in specific patient populations. As obesity becomes increasingly prevalent, it is important that optimized dosing schemes are available for these patients. To set up such dosing schemes, PK studies should be performed in this population. Regarding paracetamol (acetaminophen [APAP]), obese patients would benefit from a tailored dosing scheme, as both the volume of distribution and metabolism are increased compared with non-obese patients. This includes metabolism by cytochrome P450 2E1, which is involved in APAP-associated hepatotoxicity. To decrease the burden for patients in these PK studies, finger-prick sampling could be used. OBJECTIVE: The aim of this study was to compare the quantitative determination of APAP and four metabolites in different blood-based matrices and to determine if capillary dried blood samples, collected directly following finger-prick, could serve as a tool to investigate APAP PK in obese and non-obese patients. METHODS: In this study, we performed a clinical validation of methods for the determination of APAP and four of its metabolites (APAP-glucuronide, APAP-sulfate, APAP-mercapturate, and APAP-cysteine) in blood, plasma, and dried blood. The latter was obtained by volumetric absorptive microsampling (VAMS), either starting from the venous blood or collected directly following a finger-prick. Results were compared between the different matrices and, in addition, blood:plasma (B:P) ratios were determined for the different analytes. RESULTS: Liquid and dried venous blood results were in good agreement. Furthermore, differences between capillary (finger-prick) and venous VAMS blood samples remained limited for most analytes. However, for APAP-cysteine, caution should be paid to the interpretation of concentrations in (dried) blood. With the exception of APAP, concentrations were higher in plasma compared with blood, with B:P ratios ranging between 0.52 and 0.65. A time-dependent change in median B:P ratio was observed for APAP and APAP-cysteine. Additionally, a time-dependent trend was seen for APAP, as well as for APAP-glucuronide and APAP-mercapturate, for the distribution between capillary and venous blood. CONCLUSIONS: We demonstrated that finger-prick sampling is a viable alternative to conventional venous blood sampling to investigate the PK of APAP and its metabolites in obese and non-obese patients.

In-depth evaluation of automated non-contact reflectance-based hematocrit prediction of dried blood spots.

Dried blood spot(s) (DBS) microsampling has increasingly attracted interest as a patient-centric alternative to conventional blood withdrawal. Despite the many advantages associated with DBS sampling, its widespread use in clinical practice is still hampered, which is mainly caused by the hematocrit (Hct) effect. One approach to cope with this issue is the Hct prediction of DBS using ultraviolet-visible (UV-Vis) spectroscopy. Recently, a UV-Vis-based Hct prediction module has been incorporated into the automated CAMAG® DBS-MS 500 HCT system. However, although a proof-of-principle yielded promising results, there is no formal in-depth evaluation of the performance of this module. Hence, it remained to be established to what extent automated Hct prediction of DBS via this module can universally be applied and generates acceptable results. Using authentic patient samples, we set up and validated a calibration model and evaluated whether this could serve as a 'generic' calibration model for different, independent Hct prediction modules. A quadratic calibration curve with 1/x2 weighting was established. The bias, intra-day and total precision were below 0.025 L L-1, 2.2% and 2.7%, respectively. Additionally, the influence of storage and the robustness of the method was evaluated. Moreover, a lab-lab comparison of the performance of the Hct module of two independently operated instruments demonstrated that the validated model can be used as a generic calibration model. Finally, application of the method to venous DBS (n = 48) prepared from patient samples in the context of therapeutic drug monitoring of tacrolimus revealed a good concordance between the actual (i.e. Sysmex-based) and UV-Vis-based predicted Hct.