A method for the simultaneous determination of the contents of five new preservatives (ethylhexylglycerin, 1,2-hexanediol, 1,2-pentanediol, 1,3-octanediol, and 1,2-octanediol) in oral care toothpaste by high performance liquid chromatography (HPLC) was established. Methanol-water (volume ratio 7∶3) was used as the extraction reagent for toothpaste. The samples were subjected to ultrasonic treatment at 40 ℃ for 20 min (power 300 W), followed by centrifugation at 10 000 r/min for 10 min. The supernatant was filtered through a 0.22 μm organic filter membrane. The samples were purified using an Oasis HLB solid-phase extraction column. Analysis was performed on an Agilent 1260 Infinity HPLC system equipped with an Agilent G1315D diode array detector. An acid-resistant Agilent ZORBAX Eclipse Plus C18 chromatographic column was selected, with the column temperature set at 35 ℃. The mobile phase consisted of water (containing 0.1% formic acid) and methanol. The detection wavelength was 210 nm. The results show that the intraday relative standard deviation (RSD) of the method is all less than 4%, and the interday RSD is all less than 5%, indicating good precision of the method. Linear regression analysis reveals that the linear range of the five new preservatives is 0.1-200 μg/mL, with the correlation coefficients (r) ranging from 0.999 1 to 0.999 5, limits of detection (LOD) from 0.05 to 0.08 μg/mL, and limits of quantification (LOQ) from 0.15 to 0.25 μg/mL, demonstrating a good linear relationship and high applicability of the method. The spiked recoveries of the five new preservatives range from 95.56% to 104.94%, with all RSD values below 5% (1.63%-4.45%). This method is applied to determine the contents of the five new preservatives in 32 types of toothpaste. Ethylhexylglycerin is detected in 4 types of toothpaste, and 1,2-hexanediol is detected in 2 types of toothpaste. In conclusion, this method exhibits good precision, applicability, and accuracy for the simultaneous determination of the five new preservatives in toothpaste.