4.6 Article

Standard addition method (SAM) in LC-MS/MS to quantify gluten-derived metabolites in urine samples

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DOI: 10.1016/j.jpba.2023.115416

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Gluten free diet monitoring; Celiac disease; Bioanalytical methods validation; Endogenous quantification; Standard addition method (SAM); LC-MS; MS

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A strict adherence to a gluten-free diet is crucial for celiac patients to alleviate symptoms and improve quality of life. This study developed and validated a method based on the standard addition methodology to detect and quantify two metabolites related to gluten intake in urine samples. The method, which required less than 1 mL of urine per sample, showed potential threshold values for discriminating between a gluten-free diet and a gluten-rich diet.
A tight adherence to a gluten-free diet (GFD), the most effective treatment currently available for celiac disease, is important to reduce symptoms, avoid nutritional deficiencies and improve quality of life in celiac patients. The development of analytical methods allowing detecting gluten exposure due to occasional or involuntary food transgressions could represent a useful tool to monitor patient habits and conditions and prevent long-term complications. The aim of this work was to develop and validate an approach based on the standard addition methodology (SAM) for the detection and quantification of two main metabolites of alkylresorcinols, 3,5-dihy-droxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA), whose presence in urine samples is related to the intake of gluten-containing foods. Analytically, the method consisted of a protein precipitation step followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. The chromatographic method involved the use of a hydrophilic interaction liquid chromatography (HILIC) in a direct phase approach; LC-MS/MS analyses were performed in selected reaction monitoring (SRM) mode. Manipulation and instrumental errors were normalised using stable isotopic standards (ISs). The SAM approach here described requires less than 1 mL of urine per sample, thus greatly reducing the sample volume needed. Noteworthy, despite the small cohort of samples analysed, our data allowed to identify a potential threshold value, around 200 ng/mL for DHBA and 400 ng/mL for DHPPA, to discriminate between a GFD and a gluten rich diet (GRD).

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