Novel aspects of quantitation of immunogenic wheat gluten peptides by liquid chromatography-mass spectrometry/mass spectrometry

A novel, specific and sensitive non-immunological liquid chromatography-mass spectrometry (LC-MS) based assay has been developed to detect and quantify trace levels of wheat gluten in food and consumer products. Detection and quantification of dietary gluten is important, because gluten is a principle trigger of a variety of immune diseases including food allergies and intolerances. One such disease, celiac sprue, can cause intestinal inflammation and enteropathy in patients who are exposed to dietary gluten. At present, immunochemistry is the leading analytical method for gluten detection in food. Consequently, enzyme-linked immunosorbent assays (ELISAs), such as the sandwich or competitive type assays, are the only commercially available methods to ensure that food and consumer products are accurately labeled as gluten-free. The availability of a comprehensive, fast and economic alternative to the immunological ELISA may also facilitate research towards the development of new drugs, therapies and food processing technologies to aid patients with gluten intolerances and for gluten-free labeling and certification purposes. LC-MS is an effective and efficient analytical technique for the study of cereal grain proteins and to quantify trace levels of targeted dietary gluten peptides in complex matrices. Initial efforts in this area afforded the unambiguous identification and structural characterization of six unique physiologically relevant wheat gluten peptides. This paper describes the development and optimization of an LC-MS/MS method that attempts to provide the best possible accuracy and sensitivity for the quantitative detection of trace levels of these six peptides in various food and consumer products. The overall performance of this method was evaluated using native cereal grains. Experimental results demonstrated that this method is capable of detecting and quantifying select target peptides in food over a range from 10pg/mg to 100ng/mg (corresponding to approximately 0.01-100ppm). Limits of detection (LOD) and quantification (LOQ) for the six target peptides were determined to range from 1 to 30pg/mg and 10-100pg/mg respectively. Reproducibility of the assay was demonstrated by evaluation of calibration data as well as data collected from the analysis of quality control standards over a period of four consecutive days. The average coefficient of determination (R(2)) for each peptide was consistently found to be >0.995 with residuals ranging from approximately 80% to 110%. Spike recovery data for each peptide in various matrices was evaluated at a concentration level near the approximate LOQ for each, as well as at higher concentration levels (30 and 60ng/mg). The average range of accuracy of detection for all peptides at the lower concentration level was determined to be 90% (+/-11), while accuracy at the 30 and 60ng/mg levels was 98% (+/-5%) and 98% (+/-3%), respectively. The usefulness and capabilities of this method are presented in a practical application to prospectively screen a variety of common commercially available (native and processed) gluten-containing and gluten-free foods and products.