HPLC-ELSD法测定化学药物成盐离子含量准确性的影响因素探讨

目的：探讨用混合模式离子交换柱建立HPLC-ELSD法测定化学药物成盐离子含量准确性的影响因素。 方法：采用新型混合模式离子交换柱Acclaim Trinity P1(3.0 mm×100 mm，5 μm)，以30 mmol·L^-1醋酸铵缓冲液(取醋酸铵4.59 g，加水1900 mL使溶解，用冰醋酸调节pH至5.2，加入乙腈100 mL)-乙腈(50∶ 50，v/v)为流动相，流速0.3 mL·min^-1，柱温25 ℃；以蒸发光散射检测器检测，漂移管温度90 ℃，氮气作为载气，流速2.6 L·min^-1。建立HPLC-ELSD法测定奥美拉唑钠中钠离子含量并进行方法学验证；同时选择奥美拉唑钠、头孢曲松钠、盐酸博宁霉素，分别代表药物分子在色谱过程中不保留、强保留和不洗脱3种状态，进行准确度、精密度试验，以数据统计软件SPSS 19.0进行统计分析，考察药物分子洗脱状态对成盐离子含量准确性(准确度、精密度)的影响。 结果：钠离子浓度在1.94~77.48 μg·mL^-1范围内与峰面积呈二项式相关关系(r=0.9999)；加样回收率(n=3)分别为104.5%、101.7%、102.6%，RSD分别为1.3%、3.7%、2.2%；奥美拉唑钠中钠离子平均含量(n=8)为6.23%，与理论钠离子含量(6.24%)无显著性差异(t-检验，α=0.05)；药物分子洗脱状态对钠离子测定的中间精密度、重复性结果以及氯离子测定的重复性有影响(ANOVA分析，α=0.05)，药物分子很难洗脱以及不洗脱将导致精密度偏低。 结论：采用新型混合模式离子交换柱， 用HPLC-ELSD法测定化学药物中成盐离子含量时，方法通用性好，专属性强，线性范围宽，耐用性好，但药物分子很难洗脱或者不洗脱在色谱填料上将导致精密度偏低。

Objective :To investigate the factors affecting the reliability of HPLC-ELSD method in content determination of salt-forming ions in chemical active pharmaceutical ingredients. Methods: The content of sodium in omeprazole sodium chemical reference standard was analyzed using a new HPLC-ELSD method.The HPLC system was equipped with a new type of mixed-mode ion exchange column Acclaim Trinity P1(3.0 mm×100 mm,5 μm)and an ELSD detector using a mixture of 30 mmol·L^-1ammonium acetate buffer(4.59 g of ammonium acetate dissolved in 1900 mL of water;pH was adjusted to 5.2 with glacial acetic acid;supplemented with 100 mL of acetonitrile)-acetonitrile(50∶ 50,v/v)as mobile phase and the flow rate was 0.3 mL·min^-1.The column temperature was 25 ℃.The temperature of drift tube in the evaporating light scattering detector was 90 ℃.Nitrogen gas was used as carrier gas and the flow rate was 2.6 L per minute.The HPLC-ELSD method was validated adequately.Three typical chemical active pharmaceutical ingredients i.e.omeprazole sodium,ceftrixone sodium and boningmycin hydrochloride were grouped into three typical elution status of unreserved,very strongly reserved and unable to elute.The data of precision and accuracy from method validation of omeprazole sodium,ceftrixone sodium and boningmycin hydrochloride were analyzed by the statistical software SPSS 19.0 edition to investigate the effect of the elution status of active ingredient on the accuracy and precision of the method. Results: The concentration of sodium was binomially related with the peak area from 1.94 μg·mL^-1to 77.48 μg·mL^-1.The spiked recoveries(n=3)were 104.5%,101.7%,102.6% and RSDs were 1.3%,3.7%,2.2%,respectively.The measured sodium content(n=8)of omeprazole sodium was averaged as 6.23% and showed no significant difference from the theoretical value of 6.24%(t-test,α=0.05),which revealed good accuracy independent of elution status.However,the elution status of active ingredients had significant effect on intermediate precision,repeatability of sodium content and repeatability of chloride content(ANOVA analysis,α=0.05).And the status of active ingredients which were very strongly reserved or unable to elute led to slight reduction in precision. Conclusion :Using a new type of mix-mode ion-exchange column,the HPLC-ELSD methods for assaying the content of salt-forming ions in chemical active pharmaceuticals are generic,specific,linear,accurate,moderately precise and robustly independent of elution status.However,the status of active ingredients which are very strongly reserved or unable to elute led to slight reduction in precision.