聚山梨酯80又名吐温80, 为一种亲水型非离子表面活性剂, 是食品、 保健品和药品中常用的辅料, 作为增溶剂和澄清剂广泛用于中药注射剂。 近年来, 不良反应的发生使得聚山梨酯80的质量和应用愈加受到重视, 有研究认为其加入可能引起注射剂不良反应增加。 为避免超量使用, 有必要对该辅料的投料加以严格控制。 中药注射剂中聚山梨酯80的含量测定是当下研究的热点和难点, 可以通过分光光度法、 分子排阻-蒸发光散射检测法(SEC-ELSD)、 液质联用法(LC-MS)直接测定, 也可以水解后法经液相色谱-紫外检测法(HPLC-UV)或气相色谱法(GC)间接测定。 但由于聚山梨酯80为聚氧乙烯聚合数目不同的混合物、 不同厂家生产的聚山梨酯80化学组分及比例存在较大差异, 难以采用统一的转换公式或对照品准确定量。 此外, 中药注射剂的复杂基质造成的假阳性干扰也对定量提出了挑战。 为解决以上问题, 以生脉注射液为例, 提出基于吸收系数的中药注射剂中聚山梨酯80含量测定新方法。 优化检测波长、 显色剂种类、 液液萃取过程振荡和静置时间, 在6个不同品牌仪器上测得聚山梨酯80-硫氰酸钴配合物的吸收系数(E1cm1%)为104.23, 相对标准偏差(RSD)为2.08%。 生脉注射液稀释10倍后, 精密量取供试品溶液1.0 mL, 精密加入硫氰酸钴溶液10 mL, 二氯甲烷20 mL, 涡旋振荡3 min。 将混合液移至分液漏斗中, 静置30 min, 取下层二氯甲烷液, 将前1 mL弃去, 接收约15 mL, 在320 nm处测定吸光度, 再根据Lambert-Beer定律, 利用获得的吸收系数计算得到聚山梨酯80的含量。 方法阴性无干扰, 精密度和重复性相对标准偏差均低于3%, 平均回收率为98.42%。 为进一步验证方法的准确性, 分别采用吸收系数法和标准曲线法测定了2个厂家的10批生脉注射液, 并与实际投料量比较。 配对 t检验结果表明, 当置信度为95%时, 两种方法无显著性差异, 吸收系数法测得结果与企业生产中聚山梨酯的实际投料量也无显著性差异。 研究采用前人未采用的、 灵敏度更高的320 nm为检测波长, 显著降低了基质干扰, 克服了中药注射剂中聚山梨酯80测定结果与实际投料量难以吻合的问题。 吸收系数法无需使用对照品, 亦不用制备标准曲线, 可为中药注射剂中聚山梨酯80的检查标准提供切实可行的解决方案。 所建方法灵敏、 准确、 快速、 简便, 为含聚山梨酯80制剂的质量控制提供了关键常数及新的思路。

Polysorbate 80, also known as Tween 80, is an amphipathic non-ionic surfactant commonly used as excipient in the food, personal care and pharmaceutical industries. Particularly, it has been widely used in Traditional Chinese Medicine (TCM) injections to enhance solubility and clarity. In recent years, more attentions have benn paid to the quality and application of polysorbate 80 due to incidence of adverse reactions. Some declared that adding of polysorbate 80 might increase the side effects. To avoid abuse of this excipient, it is essential to control the levels of polysorbate 80 in therapeutic formulations within a target level. Assay of polysorbate 80 in TCM injections has become a hot and difficult issue nowadays. Content of polysorbate 80 in preparations could be quantified directly by spectrophotometry, size-exclusion chromatography coupled with evaporative light scattering detector (SEC-ELSD) or liquid chromatography combined with mass spectrometry (LC-MS) method. Acid hydrolysis followed by high performance liquid chromatography coupled with ultraviolet detector (HPLC-UV) or gas chromatography (GC) determination was also used as an indirect measurement of polysorbate 80. But it's hard to find a unified conversion formula or reference standard for accurate quantification owing to the fact that the surfactant is a complex mixtures of oligomers, whose composition and ratio of chemical components vary by different manufacturers. In addition, it becomes a challenge to measure polysorbate 80 in TCM injections without false-positive interference due to the complex matrix of the preparation. Taking Shengmai Injection as example, a novel method for determination of polysorbate 80 in TCM injections was proposed based on absorption coefficient, which offered a solution for the above problems. After optimization of detection wavelength, chromogenic reagent and of the shaking and standing time of the liquid-liquid extraction, absorption coefficient (E1cm1%) of cobalt thiocyanate complex of polysorbate 80 was obtained from 6 different bands of instruments as 104.23 with RSD of 2.08%. Shengmai Injection was first diluted by 10 times. To 1.0 mL of the test solution, 10 and 20 mL of dichloromethane were added accurately to form a polysorbate/thiocyanate complex, followed by cyclotron oscillation for 3 minutes. The mixture was transferred into a separating funnel and was stranded for 30 minutes. The first 1 mL of the lower layer was discarded and the next 15 mL was collected. Then the absorbance was measured at 320 nm. Finally the content of polysorbate 80 was calculated by using the previously obtained absorption coefficient based on Lambert-Beer law. With no interference from the negative sample, the established method provided precision and reproducibility (% relative standard deviation) less than 3% and accuracy (% spike recovery) of 98.42%. To further validate the accuracy of the method, 10 batches of Shengmai Injection from 2 manufacturers were determined by both the absorption coefficient method and the standard curve method, and the results were compared with the actual feeding content of polysorbate 80 in the sample. Results of paired t test indicated that there were no significance difference between the results obtained by two methods, or between the results of the absorption coefficient method and the actual feeding content of polysorbate 80 ( p>0.05). The investigation selected 320 nm as the detection wavelength to improve the sensitivity, which was never adopted in previous work. In this way, remarkable decrease of matrix interference was achieved, thus the problem of misfit determination results and the actual feeding content of polysorbate 80 was solved. With no need for reference standard or standard curve, the search offered a feasible tool for test standard of polysorbate 80 in TCM injection. All in all, the proposed method was sensitive, accurate, rapid and simple, which can provide key constants and new ideas for quality control of preparations containing polysorbate 80.