市售龟甲胶质量分析研究

Quality Analysis of Glue of Tortoise Plastronin Market

目的分析市售龟甲胶质量状况,研究建立龟甲胶中L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸4个氨基酸的定性和定量分析方法。方法样品以6mol.L-1盐酸于沸水浴水解1h,蒸干,甲醇溶解,以苯酚-0.5%硼砂溶液(4∶1)为展开剂,硅胶G为固定相,茚三酮试液为显色剂进行TLC分析;样品以6mol.L-1盐酸于150℃水解1h,以异硫氰酸苯酯(PITC)为衍生化试剂衍生后进行HPLC分析,采用Phenomenex Luna C18色谱柱(4.6mm×250mm,5μm),柱温为43℃;流动相A为乙腈-0.1mol.L-1醋酸钠(7∶93),流动相B为乙腈-水(4∶1),梯度洗脱,流速为1.0mL.min-1;检测波长为254nm,进行HPLC测定分析。应用等离子发射光谱仪对市售龟甲胶样品进行铅、镉、砷、汞、铜和铬元素的测定分析。结果 TLC能较好地鉴别出龟甲胶中的L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸。HPLC测定L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸的线性范围分别为0.01727～0.3454μg(r=1.0000),0.03536～0.7072μg(r=1.0000),0.01508～0.3016μg(r=0.9998),0.01868～0.3736μg(r=1.0000);加样回收率(n=6)分别为97.0%(RSD=2.4%),97.3%(RSD=1.8%),94.7%(RSD=2.2%),96.8%(RSD=1.9%)。测定样品中铅、镉、砷、汞、铜和铬元素的含量,发现部分样品铬元素含量较高。结论所建立的L-羟脯氨酸、甘氨酸、丙氨酸和L-脯氨酸的定性和定量方法准确、可靠,具有良好的重复性和稳定性,可用于龟甲胶中这4种氨基酸的检测。市售龟甲胶样品中铬元素含量普遍较高,应加强对该元素的检测控制。

Objective To develop TLC and HPLC methods for the determination of 4 amino acids （L- hydroxyproline, glycine, alanine, L-proline） in glue of tortoise plastron and analyze the quality of glue of tortoise plastron in market. Method The sample hydrolyzed for 1 hour was detected by TLC using phenol carbolic acid-0.5% borax solution （4 ： 1） as mobile phase, silica gel G as stationary phase, triketohydrindene as colour reagents. The sample hydrolyzed for 1 hour at 150℃, derived with phenyl siothiocyanate （PITC） was detected by HPLC. The HPLC detection was performed on a Phenomenex Luna C18 column （4.6 mm×250 mm, 5 μm）, with gradient elution of acetonitrile- 0. 1 mol · L-1 sodium acetate （7 ： 93） （A） and acetonitrile-water （4 ： 1） （B） at flow rate of 1.0 mL ·min-1, and the detection wavelength was 254 nm. Samples were digested with microwave digestion method and determined by inductively coupled plasma-atomic emission spectrometry （ICP-AES） method. Results L- Hydroxyproline, glycine, alanine, L-proline had good linearity in the ranges of 0. 01727-0. 3454 lzg （r= 1. 0000）, 0. 03536-0. 7072 μg （r=1. 0000）, 0. 01508-0. 3016μg （r=0. 9998）, 0. 01868-0. 3736 μg （r=1. 0000）, respectively. The average recoveries were 97.0% （RSD= 2.4%）, 97.3% （RSD= 1.8%）, 94.7% （RSD=2.2%）, 96.8% （RSD=1. 9%, respectively. The contents of PB, Cd, As, Hg, Cu and Cr in ten batches of glue of tortoise plastron from market were determined, and the contents of Cr in some batches were high. Conclusion The established method is accurate with high repeatability, which is helpful for the determination of L-hydroxyproline, glycine, alanine, L-proline, 4 amino acides in glue of tortoise plastron. The content of Cr in ten batches of glue of tortoise plastron in market is high which suggest us to pay more attention on it.