Objective Leather fiber-based materials are rich in collagen protein, which have unique morphological and structural characteristics, so they are highly sought after in the market. Currently, some countries and regions, such as the United States, the United Kingdom and the European Union, have implemented some regulations on such materials, particularly regarding their leather fibers content. Thus, the collagen content of such products has become a critical indicator which is concerned by both manufacturers and brand owners, and it is also closely linked to consumer rights and fair market competition. However, the applied existing test methods (chemical dissolution and nitrogen content (kjeldahl)) are susceptible because of some interference factors, such as dissolution conditions, sample substances et.al., which may affect the accuracy of the determination results. Therefore, an analytical method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed in this study, aiming to achieve a rapid and accurate determination of collagen content in leather fiber-based materials.

Methods Firstly, the method quantified the content of L-hydroxyproline (L-HYP) as a characteristic amino acid in collagen by HPLC-MS/MS with the external standard method. The samples were hydrolyzed with 6 mol/L hydrochloric acid solution to convert collagen into its characteristic amino acid L-hydroxyproline (L-HYP). Through optimization, the HPLC-MS/MS chromatogram showed good peak shape, and indicated a high purity of the target compound of L-HYP which was well separated with other substances under the conditions of HILIC column and 0.1% formic acid mobile phase. Then, some hydrolysis conditions of the materials including temperature, time, and the dosage of 6 mol/L hydrochloric acid were optimized through single-factor experiments. The optimal conditions were determined as 90 ℃, 22 h, and 5 mL of 6 mol/L hydrochloric acid. The actual collagen content in the material was calculated based on the L-HYP concentration measured via external standard calibration, incorporating its conversion factor from L-HYP collagen to combined with the material's moisture content.

Results The results indicate that L-HYP exhibited a linear dependence between signal and the concentration range of 5.3-530.0 ng/mL, described by the equation y = 2290x-5408, with a correlation coefficient (R²) of 0.9995. Using blank spiked with standard for determination, the signal-to-noise ratio at 5.3 ng/mL was 354.9 (＞10), yielding the method's limit of quantification of 0.01%. At low, medium, and high spiked concentrations (5.30, 10.60, and 53.00 ng/mL), the average spiked recovery rate ranged from 93.34% to 98.89%, with the relative standard deviation (RSD) was 3.44% to 4.96%, indicating good accuracy and precision of this method. The method was applied to analyze 12 commercially available leather fiber-based materials, including top-layer leather, coated and coated split leather and regenerated leather. Their collagen contents were determined by ranging from 29.2% to 83.1%, with significant differences in the content among different kinds of materials. Some top-layer leather showed the highest collagen content, followed by coated and coated split leather, while regenerated leather showed the lowest collagen content. The analytical results of this method were highly consistent with the actual manufacturing processes, verifying its effectiveness and reliability in practical applications.

Conclusion The present method is an effective technical optional approach for determining the collagen content in leather fiber-based materials because of its non-derivatization step, easy operation, minimal interference, rapid analysis, and reliable accurate results, which could meet the needs of manufacturers and brand owners and provide a reference for quality control of leather fiber-based materials.