Gypenosides (Gps) are the major bioactive components in Gynostemma species. They include neutral Gps and acidic malonylgypenosides (MGps). MGps are abundant in Gynostemma species and can be transformed into corresponding Gps via extraction, concentration, and drying. If only the Gps were quantified and MGps were ignored, the quality of Gynostemma species would be underestimated. This study aimed to develop a sample preparation method involving demalonylation and ultrahigh-performance liquid chromatography–charged aerosol detector (UHPLC-CAD) analysis to determine the contents of gypenoside XLIX (Gp XLIX) and gypenoside A (Gp A). First, the optimized ultrasonic extraction method was established to extract G. longipes powder ultrasonically. Then, the extracted solution was put into a closed container (centrifuge tube) and heated in a water bath at 95 °C. Then, MGps were converted into corresponding Gps. The proposed preparation method was compared with the other three methods, including water bath reflux heating, alkali hydrolysis, and extraction of heated powder, and was shown to exhibit higher conversion and better convenience. Subsequently, an UHPLC-CAD method was established and validated. Gp XLIX and Gp A showed excellent linear correlations between 15.55 and 248.8 μg/mL and 24.10–385.5 μg/mL, respectively (R² > 0.999). The limit of detection was 1.40 ng (Gp XLIX) and 2.41 ng (Gp A), and the limit of quantification was 7.77 ng and 14.46 ng, respectively. The relative standard deviation for precision, stability, and repeatability was 0.63–3.15%. The average recovery of Gp XLIX and Gp A was 98.97% and 98.23%, respectively. The established method was applied for determining Gp XLIX and Gp A contents in wild or cultivated G. longipes samples collected from the Qinba Mountains area. The contents of Gp XLIX and Gp A were 5.16–23.02 mg/g and 15.78–54.55 mg/g, respectively. Conclusively, the proposed sample preparation and analysis method could be used for the quality control and evaluation of G. longipes.

绞股蓝皂苷 (Gps) 是绞股蓝属植物中的主要生物活性成分。它们包括中性 Gps 和酸性丙二酰绞股蓝皂甙 (MGps)。MGps在绞股蓝属植物中含量丰富，通过提取、浓缩、干燥可转化为相应的Gps。如果只量化Gps而忽略MGps，绞股蓝的质量就会被低估。本研究旨在开发一种涉及脱丙二酰化和超高效液相色谱-荷电气溶胶检测器 (UHPLC-CAD) 分析的样品制备方法，以确定绞股蓝皂苷 XLIX (Gp XLIX) 和绞股蓝皂苷 A (Gp A) 的含量。首先，建立了优化的超声提取方法提取龙舌兰超声波粉末。然后，将提取液放入密闭容器（离心管）中，在95℃水浴中加热。然后，MGps被转换成相应的Gps。将所提出的制备方法与水浴回流加热、碱水解、加热粉末提取等其他三种方法进行了比较，显示出更高的转化率和更好的便利性。随后，建立并验证了 UHPLC-CAD 方法。Gp XLIX 和 Gp A 分别在 15.55 和 248.8 μg/mL 和 24.10–385.5 μg/mL 之间显示出极好的线性相关性 ( R ²> 0.999）。检测限为 1.40 ng (Gp XLIX) 和 2.41 ng (Gp A)，定量限分别为 7.77 ng 和 14.46 ng。精密度、稳定性和重复性的相对标准偏差为 0.63–3.15%。Gp XLIX 和 Gp A 的平均回收率分别为 98.97% 和 98.23%。将所建立的方法用于测定采自秦巴山区的野生或栽培长鳍龙胆样品中Gp XLIX 和Gp A 的含量。Gp XLIX 和 Gp A 的含量分别为 5.16-23.02 mg/g 和 15.78-54.55 mg/g。综上所述，所提出的样品制备和分析方法可用于龙舌兰的质量控制和评价。