Development of a barley reference material for gluten analysis.

Celiac disease (CD) can be triggered in susceptible individuals by the consumption of gluten, a complex storage protein mixture present in wheat, rye and barley. There is no specific reference material (RM) available for barley and this leads to inaccurate quantitation of barley gluten in supposedly gluten-free foods. Therefore, the aim was to select representative barley cultivars to establish a new barley RM. The relative protein composition of the 35 barley cultivars averaged 25% albumins and globulins, 11% d-hordeins, 19% C-hordeins, and 45% B/γ-hordeins. The mean gluten and protein content was 7.2 g/100 g and 11.2 g/100 g, respectively. The prolamin/glutelin ratio (1:1) commonly used in ELISAs to calculate the gluten content was found to be inappropriate for barley (1.6 ± 0.6). Eight cultivars suitable as potential RMs were selected to ensure a typical barley protein composition and improve food safety for CD patients.

Characterization of rye flours and their potential as reference material for gluten analysis.

The safety of gluten-free products relies on accurate gluten analysis, most commonly using ELISA. These test kits are calibrated to gliadins or wheat gluten, because there is no reference material (RM) for rye. Our aim was to select representative samples out of 32 rye cultivars for use as RM. All cultivars were characterized by RP-HPLC, gel permeation HPLC and R5 and G12 ELISA. The protein and gluten content ranged from 5.5 to 11.2 g/100 g and 3.0 to 7.8 g/100 g, respectively. The average protein distribution was 40% albumins/globulins, 23% γ-75k-secalins, 17% γ-40k-secalins, 14% ω-secalins and 6% high-molecular-weight-secalins. The mean prolamin/glutelin ratio was 4.4 for rye and this translates to an estimated conversion factor from rye prolamins to gluten of 1.2, instead of the usual factor of 2. Seven cultivars were selected for RM production based on cluster analysis, geographical origin and availability to comprehensively cover the diversity of rye.

Ion exchange in supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS): Application for polar and ionic drugs and metabolites in forensic and anti-doping analysis.

Supercritical fluid chromatography (SFC) is an orthogonal technique to UHPLC. In recent years, SFC has demonstrated potential for use in the analysis of a broad variety of analytes of different polarities, if modifiers and additives are utilized as additional mobile phase constituents. However, to date, little research has been carried out on ion-exchange separation of highly polar and ionic analytes using SFC. The objective of this work was to investigate the elution characteristics of polar compounds using SFC combined with tandem mass spectrometry. Highly polar and even ionic drugs and metabolites, with a diversity of functional groups such as gamma-hydroxybutyrate (GHB), gamma-butyrolactone, GHB-glucuronide, ethyl sulfate, ethyl glucuronide as well as meldonium and gamma-butyrobetaine, were selected for the study. To investigate the chromatographic behavior of the solutes using SFC, a systematic chromatographic method development workflow including a basic validation in human urine was implemented. To ensure the best selectivity, columns with different stationary phase chemistries (silica, NH2, CN, SCX, Diol, EP and amide) were screened. Furthermore, different modifier compositions were evaluated, including pure methanol and methanol with various additives (ammonium acetate or ammonium formate, water, and/or ammonia or formic acid in varying amounts). Trends in retention time shifts were investigated by the systematic variation of gradients, backpressure and column compartment temperature. The quality and reliability of the method has been tested in the course of a basic validation by evaluation of selectivity, linearity, limit of identification, limit of quantification, carry-over, precision, and matrix effect. The highest chromatographic selectivity (especially with respect to the separation of the critical pair of zwitterionic meldonium and γ-butyrobetaine) and suitable peak shapes for all target analytes were obtained using the strong cation exchange column. We observed that increasing buffer concentrations improved the peak shape. Ion-exchange stationary phase and polar additives (buffers, bases, acids and water) mixed with organic modifiers enabled separation of small ionic molecules using SFC. Hence, SFC further demonstrated its wide applicability in small molecule analysis, and may be considered an alternative separation technique to improve the separation of polar analytes.