Over the past years, manufacturers have put a lot of effort in the traceability of raw materials used for the production of gelatin. Techniques such as PCR and ELISA are commonly used to analyze the species authenticity. However, false-positive and (false)-negative results, inconsistencies and low reactivity of commercially available kits have been observed with regard to these methods.
With our TrustGel™ method, gelatin manufacturers and their clients can now determine the purity of gelatin batches with regard to bovine and porcine constituents with a 0.05% limit of quantification. The large variability in commercial gelatins due to differences in raw material and processing was taken into account in the design of the validation study. Besides quantitative targets, our TrustGel™ method contains additional qualitative targets to obtain confirmation of the presence of the gelatin species of interest and to monitor the presence of other collagen types.
Our TrustGel™ method was developed according to the Triskelion general workflow for the development of quantitative protein LC-MS methods and utilizes stable isotope labeled internal standard (SIL IS) peptides to obtain acceptable and accountable method performance on the single sample level. Target selection was based on theory and on experiment. High resolution Orbitrap-MS data were used to select all the relevant targets, to confirm their sequences and to localize hydroxyproline residues (which differ only 0.04 Da in mass from (iso)leucine) to be able to synthesize equivalent SIL IS peptides. After target selection the method was transformed into a triple quadrupole UPLC-MS/MS method.
Calibration samples are prepared using pure reference gelatins and are subjected to the same sample preparation procedure as study samples. The method thus applies a combined internal standardization approach, which inherently corrects for analytical variations during the sample preparation. The validation experimental set-up was based on FDA and EMA guidelines for bioanalytical method validation and the validation study was performed according to Good Laboratory Practice, ensuring the scientific quality, data traceability and data integrity. Using our TrustGel™ method, species authenticity of gelatin can be demonstrated unambiguously with a 0.05% limit of quantification. The method will be expanded in the future.
Please click here to watch the video that explains the TrustGel™ method in more detail.