Screening method for the simultaneous determination of allantoin and uric acid from dried blood spots.

Uric acid and its oxidation product allantoin are excellent biomarkers of oxidative stress in humans. Currently, there are high requirements not only for tests monitoring oxidative stress but also for screening laboratory tests in general. The highest demand is imposed on the simplest sampling, easy transport of the sample, and the shortest possible analysis time. The possible solution how to fulfil the requirements is sampling by dried blood spot technique with subsequent HPLC-MS/MS analysis. A fast, sensitive, and reliable HPLC-MS/MS method for the simultaneous determination of uric acid and allantoin from dried blood spots using stable isotopically labelled analogs as internal standards was developed. The separation took place in the reversed phase within 3 min, with protein precipitation and extraction in a one-step procedure. The analytical parameters of the method were satisfactory with an excellent linear range. The presented method was used to determine allantoin and uric acid levels in dried blood spot samples from 100 healthy volunteer donors. The median uric acid concentration in the cohort was 239.3 µmol/L and the median allantoin concentration was 5.6 µmol/L. The presented analytical protocol and method are suitable for screening and monitoring allantoin and uric acid levels as biomarkers of oxidative stress in clinical practice.

Dried blood spot as an alternative sample for screening of fatty acid, amino acid, and keto acid metabolism in humans.

Dried blood spot (DBS) is a simple and noninvasive sample collection technique allowing self-collection at home. It can be used as an alternative sample for the screening of metabolism in humans because changes in the levels of some fatty acids (FAs), amino acids (AAs), and keto acids (KAs) can be associated with metabolic disorders (e.g., diabetes mellitus). In this study, we optimized three different methods that are sensitive enough for the determination of the aforementioned analytes from a small volume of biological material in DBS. A total of 20 AAs, 5 KAs, and 24 FAs were determined. This sampling technique was applied to prepare samples from 60 individuals by a finger prick. The samples were analyzed using chromatographic methods, and acquired data were statistically evaluated. Even though most analytes were higher in men, only five AAs, three KAs, and eight FAs showed significant gender dependency (α = 0.05). Asparagine, serine, and α- and γ-linolenic acids showed significant age dependency (α = 0.05). Most statistically significant correlations were positive and were found within one category. This work shows that because of many benefits, the DBS sample could be a good alternative to whole blood sample collection for the screening of metabolism in humans, in general, or in individualized medicine. The chromatographic methods can be used in future research, for example, to set the reference range or plasma-correction factors (various aspects such as age or gender should be considered).

Determination of selected α-keto acids in dried blood samples using HPLC with fluorescence detection.

The determination of α-keto acids derived from amino acids is currently the most reliable approach for the diagnosis of some congenital metabolic diseases. An HPLC method for the simultaneous measurement of selected α-keto acids in dried blood samples has been developed and evaluated. Blood spot samples from a group of healthy blood donors were collected onto #903 Specimen Collection Paper. Prior the separation, the α-keto acids were derivatized with 1,2-diamino-4,5-dimethoxybenzene to the corresponding 3-substituted-6,7-dimethoxy-2(1 H)-quinoxalinol derivatives. For the separation, a reverse-phase column LichroCart 125-4, Purospher RP-18e, 5 µm, was used. The mixture of 25% ACN in deionized water (mobile phase A) and 100% ACN (mobile phase B) were used for a gradient elution of α-keto acids derivatives. Analytical performance of this method is satisfactory for all α-keto acids. The intra-assay and inter-assay coefficients were below 10% and recoveries were close to 100%. We have developed relatively simple, rapid, selective and sufficiently sensitive HPLC method with fluorescence detection for the determination of selected α-keto acids in dried blood samples. The presented method is suitable for clinical testing purposes.