丹磺酰氯柱前衍生化高效液相色谱法测定骨肽注射液中组胺含量方法的验证

Validation of HPLC with Dansyl Chloride Pre-column Derivatization for the Determination of Histamine in Ossotide Injection

目的建立和验证丹磺酰氯柱前衍生化高效液相色谱法测定骨肽注射液中组胺含量的检测分析方法。方法将骨肽注射液经三氯乙酸沉淀后,取上清液以丹磺酰氯丙酮溶液为衍生化试剂,衍生化后进行高效液相色谱测定。色谱条件为:色谱柱采用C_(18)柱(250 mm×4.6 mm,5μm),以乙腈为流动相梯度洗脱,柱温:30℃,检测波长:254 nm。方法构建后对此方法进行方法学考察(标准曲线、检出限、加标回收率、专属性、重复性、耐用性)。结果进样量在1.018ng~203.6ng范围内峰面积与进样量呈良好的线性关系,相关系数r=0.999,检出限为1.27ng·mL^(-1),加标平均回收率为93.06%,重复性RSD值小于1.0%,专属性及耐用性均符合要求。结论该方法准确可靠、灵敏度高,重复性好,可用于测定骨肽注射液中的组胺含量。

OBJECTIVE To establish and validate a method to determine histamine content in ossotide injection.METHODS The supernatant derivasation with dansylchloride was performed after ossotide injection precipitated by Trichloroacetic acid,the histamine was determined by a gradient elution HPLC method on a C_(18) column(250 mm×4.6 mm,5μm)with a mobile phase consisting of acetonitrile and water and detection at 254 nm.The method was validated in terms of linearity,precision,accuracy,recovery and durability.RESULTS The method showed excellent linearity in the range 1.018-203.6 ng,and the resulting correlation coefficients for histamine in ossotide injection range were 0.999,a limit of detection of 1.27 ng·mL^(-1) could be achieved,mean recovery rate was 93.06%.The RSD of histamine content in six test samples was less than 1.0%,and the recovery and durability meet the requirements.CONCLUSION The method is accurate,reliable,sensitive and reproducible.It could be applied in the detection of histamine in ossotide injection.