OBJECTIVE  To establish an HPLC fingerprint of Periplaneta americana and determine the content of nine amino acids utilizing quantitative analysis of multi-components with a single-marker(QAMS) method, thereby comprehensively evaluating the quality differences of amino acids in Periplaneta americana.

METHODS  The samples were hydrolyzed in 6 mol∙L⁻¹ hydrochloric acid solution at 120 ℃ for 6 h, and then derivatized with phenylisothiocyanate(PITC) in a 40 ℃ water bath for 1 h. The analysis of the derivatized products was carried out using a Kromasil 100-5-C₁₈ column(4.6 mm×250 mm, 5 μm), with 0.1 mol∙L⁻¹ sodium acetate solution(pH 6.5±0.05)-acetonitrile(97∶3) as mobile phase A and acetonitrile-water(4∶1) as mobile phase B. The gradient elution was performed at a flow rate of 0.8 mL∙min⁻¹, with a detection wavelength of 254 nm and a column temperature of 40 ℃. The injection volume was 2 μL. The fingerprint chromatograms were evaluated for perform similarity evaluation, cluster analysis(CA), principal component analysis(PCA), and orthogonal partial least squares discriminant analysis(OPLS-DA). The relative correction factors(f) of the internal reference alanine to glycine, arginine, threonine, tyrosine, valine, isoleucine, leucine and phenylalanine were established and their durability was investigated. The contents of 9 amino acids in 16 batches of Periplaneta americana were determined by external standard method(ESM) and QAMS, respectively. The accuracy of the QAMS method was verified by independent sample t-test.

RESULTS  The amino acid fingerprint of 16 batches of Periplaneta americana were analyzed using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2004A edition), SPSS 27.0, and SIMCA 14.1. A total of 17 common peaks were identified, with 15 known components clarified. The similarity among the 16 batches ranged from 0.888 to 0.982, indicating relatively good quality consistency. Both CA and PCA classified the samples into three major groups, suggesting certain differences in quality among samples from different origins. OPLS-DA identified eight major quality-differentiating markers, including proline, lysine, valine, isoleucine, threonine, leucine, alanine and common peak g. In terms of content determination, the specificity, linearity(r≥0.9999), precision, repeatability, and stability of nine components were all satisfactory. The average recoveries ranged from 98.70% to 101.93%, with relative standard deviations(RSD) between 1.08% and 2.24%. For the validation of QAMS method, the relative correction factors(f) of alanine with respect to eight amino acids were determined as follows: glycine 0.8338, arginine 1.9641, threonine 1.4489, tyrosine 1.8306, valine 1.2767, isoleucine 1.3816, leucine 1.4082, and phenylalanine 1.6542. The t-test results showed no statistically significant difference between the content determination results obtained by the ESM and the QAMS method.

CONCLUSION  The HPLC fingerprint combined with QAMS method established in this study is simple to operate, highly specific, and exhibits good reproducibility. It can effectively evaluate the amino acid composition in Periplaneta americana samples, thereby laying a theoretical foundation for the quality control of amino acid components in Periplaneta americana crude drugs and their extracts.