E-nose, HS-SPME-GC-MS and untargeted metabolomics reveal effects on 60Co gamma irradiation of Neng-An-Jun-Ning Pills: Changes in odor and chemical compositions.

Tibetan medicinal preparations are mainly composed of crude medicinal powder, which are susceptible to microbial contamination. Due to its high efficiency and low-treatment characteristics, ⁶⁰Co gamma irradiation is currently the most commonly used sterilization method for Chinese medicinal materials and their preparations. This study investigates the impact of ⁶⁰Co gamma irradiation on the Neng-An-Jun-Ning Pill (NJP), a Tibetan medicinal preparation rich in aromatic herbs. The research aims to evaluate the impact of ⁶⁰Co gamma irradiation on the odor, volatile chemical compounds, and main active ingredients of NJP due to irradiation changes. The aim is to evaluate the effects of various irradiation doses on the odor, volatile compounds, and key active ingredients of NJP, while establishing an appropriate dose through microbial limit testing Untargeted metabolomics was used to evaluate the changes in compound composition of this formula preparation under this dose. NJP samples were irradiated with ⁶⁰Co gamma at doses of 3, 6, 10, 15, and 20 kGy. The results of the E-nose (Electronic nose) analysis showed that sensors S1 and S8 demonstrated high sensitivity to the odor of NJP. Meanwhile, HS-SPME-GC-MS (Headspace solid-phase microextraction-gas chromatography- mass spectrometry) identified 50 volatile compounds, predominantly alkenes, esters, and phenolic compounds. In irradiated samples, alkene and phenolic content increased while ester content decreased. GC-MS results showed that Calamenene and Methyl Tetradecanoate only exist in irradiated groups, which may serve as indicator compounds to determine whether the product has undergone irradiation. Analysis of data from all experimental groups led to the identification of 18 differential volatile compounds. HPLC analysis showed that at 15 and 20 kGy, hydroxysafflor yellow A and ellagic acid decreased significantly, while eugenol content increased. Therefore, the overall results showed the optimal ⁶⁰Co gamma irradiation dose for NJP was 6 kGy, considering sterility compliance (dose ≥6 kGy), minimal odor, and major active ingredients' impact at medium doses, and adherence to irradiation guidelines. Under 6 kGy irradiation, UPLC-Q Exactive MS (Ultra-high-performance liquid chromatography coupled with quadrupole - Exactive mass spectrometry) detected 2084 compounds, mainly terpenoids and flavonoids. Through chemometrics analysis, 616 differential compounds were identified. This study provides a theoretical basis for the irradiation processing and quality control of Tibetan medicinal preparations such as NJP, and also serves as a valuable reference for the development of irradiation technology in Tibetan medicine and traditional Chinese medicine.