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Title: [Determination of donkey skin ingredients in Asini Corii Colla by ultra-high performance liquid chromatography-tandem mass spectrometry].
Author: Gong L, Shi F, Su S, Xie Q, Xian R, Hang B, Zhao Y.
Journal: Se Pu; 2021 Nov; 39(11):1255-1260. PubMed ID: 34677021.
Abstract:
In recent years, due to the shortage of donkey skin resources, the price of Asini Corii Colla has seen a rapid increase. Consequently, fake gelatin prepared from horse, mules, pig, and cow skin has appeared in the market, resulting in unreliable quality of Asini Corii Colla. Therefore, there is an urgent need to develop an efficient and accurate method for improving the quality of Asini Corii Colla. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to determine the donkey skin components in Asini Corii Colla. Accordingly, 0. l g of the evenly mixed sample was weighed and placed in a 50 mL volumetric flask; then, 1% ammonium bicarbonate solution was added to dissolve the sample, and the solution was diluted to the scale. Precisely 1.00 mL of the solution was extracted into a 5 mL volumetric flask, followed by the addition of 1.0 mL trypsin solution and 100 μL mixed internal standard working solution. This mixture was diluted to the scale using 1% ammonium bicarbonate solution, shaken, and placed in an incubator for 16 h to induce enzymolysis at a constant temperature of 37 ℃. The mixture was subsequently removed from the incubator, cooled to ambient temperature, filtered through a 0.22 μm membrane, and analyzed by LC-MS. Separation was performed on an UPLC system with a BEH C18 column (100 mm×2.1 mm, 2.5 μm) under gradient elution using acetonitrile containing 0.1% (v/v) formic acid (B) and water containing 0.1% (v/v) formic acid (A) as the mobile phases at a flow rate of 0.3 mL/min. The column temperature was 30 ℃, and the sample size was 2 μL. The gradient elution conditions are: 0-1 min, 10%B; 1-5 min, 10%B-30%B; 5-5.1 min, 30%B-70%B; 5.1-7 min, 70%B; 7-7.1 min, 70%B-10%B; 7.1-10 min, 10%B. The marker peptides were determined in positive electrospray ionization (ESI ⁺) and multiple reaction monitoring (MRM) modes using the isotopic internal standard method. The optimized enzymolysis conditions were as follows: enzymolysis temperature, 37 ℃; enzymolysis time, 16 h; and amount of enzyme, 1 mL. The two marker peptides showed good linearities in the range of 50 to 1250 mg/L; the correlation coefficients (r) were greater than 0.996, and the limits of quantitation (S/N=10) were 20 mg/kg. At spiked levels of 300 mg/kg, 600 mg/kg, and 900 mg/kg, the average recovery ratios of the two marker peptides were 103.2% to 108.3%, while the relative standard deviations (RSDs) of 1.0%-3.0%. This method was favorable for testing actual samples. Asini Corii Colla from 29 production companies was detected by this method, and the sum contents of the two marker peptides was different because the production process and raw materials were different. The sum contents of the samples were 0.096% to 0.180% with an average of 0.151%. The developed method is simple, reliable, and reproducible, and it is suitable for detecting the donkey hide components Asini Corii Colla. 近年来由于驴皮资源短缺,阿胶价格大幅度上涨,市场上出现了大量以马、骡、猪、牛皮等熬制而成的假胶,导致阿胶质量的参差不齐,严重扰乱了市场,急需高效准确的检测方法提升阿胶品质。该研究采用超高效液相色谱-串联质谱技术,建立了阿胶中驴皮源成分的检测方法。样品加水溶解后,于37 ℃下经胰蛋白酶酶解,产生驴源性特征肽段,以0.1%甲酸乙腈溶液和0.1%甲酸水溶液作为流动相进行梯度洗脱,单次分析时间10 min,在电喷雾正离子(ESI⁺)模式下进行多反应监测(MRM),同位素内标法定量。驴源多肽A1、A2在50~1250 μg/L范围内线性关系良好,相关系数(r)均大于0.996,方法定量限(S/N=10)为20 mg/kg,在300、600、900 mg/kg 3个添加水平上驴源多肽A1、A2的回收率范围为103.2%~108.3%,各加标水平平行测定结果的相对标准偏差(RSD)为1.0%~3.0%,完全能够满足实际样品检测需求。对29批不同生产企业的阿胶进行测定,结果表明,不同企业生产的阿胶中驴源多肽A1、A2的含量之和存在差异,含量为0.096%~0.180%,平均值为0.151%,提示驴源多肽A1、A2含量较低的阿胶生产厂家应注重皮源质量,改进生产工艺,以提升产品质量。该方法操作简便,结果可靠,重现性好,可用于阿胶中驴皮源成分的测定。

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