Development of a Quantitative UPLC-ESI/MS Method for the Simultaneous Determination of the Chitin and Protein Content in Insects

Abstract In a context where the commercial and nutritional interest in insect chitin is always increasing, an accurate and precise method to quantify this biopolymer, especially in food/feed, is required. In addition, quantification of insect crude protein through nitrogen determination is normally overestimated due to the presence of chitin. In this work, for the first time, an RP-UPLC-ESI/MS method for the simultaneous quantification in insects of chitin, as glucosamine (GlcN), and protein, as total amino acids, is presented. The method is based on acid hydrolysis and derivatization of amino acids and GlcN with the AccQ Tag reagent. Method was optimized and validated in terms of linearity, LOD and LOQ, intraday and inter-day repeatability, and accuracy. A hydrolysed commercial chitin was selected as reference standard for calibration. The instrumental LOD and LOQ correspond respectively to a concentration of 0.00068 mM and 0.00204 mM. The intraday precision satisfied the Horwitz ratio. Data from inter-day precision showed the necessity to perform the analysis within 1 week utilizing standard calibration solutions freshly prepared. A matrix effect was observed, which suggested the necessity to use an internal calibration curve or to work in a particular concentration range of GlcN. The chitin and protein content in black soldier fly ( Hermetia illucens ) and lesser mealworm ( Alphitobius diaperinus ) were found in agreement with results obtained by independent methods. The optimized method was also tested on two different commercial food supplements, suggesting its applicability on a wide range of matrices. This newly developed method proved to be simple, more accurate, and faster if compared to methods which separately analyse chitin and protein content.