组分百日咳抗原中间产物中氨基酸含量的邻苯二甲醛柱前衍生化RP-HPLC检测

Determination of amino acids in fractional pertussis antigen intermediates by RP-HPLC with pre-column derivatization of O-phthalaldehyde

目的建立邻苯二甲醛(O-phthalaldehyde,OPA)柱前衍生化反相高效液相色谱法(reversed phase high performance liquid chromatography,RP-HPLC)并验证,以检测百日咳抗原中间产物中氨基酸的含量。方法采用OPA柱前衍生化RP-HPLC检测百日咳抗原中间产物百日咳毒素脱毒液中L-天冬氨酸钠盐、丝状血凝素脱毒液中甘氨酸及黏附素脱毒液中L-赖氨酸的含量,并对该方法的专属性、系统适用性、线性、准确度、耐用性、检测限和定量限进行验证。结果OPA柱前衍生化RP-HPLC检测配置溶液,对照品和供试品溶液可见对应的氨基酸色谱峰,供试品溶液的保留时间和峰面积相对标准偏差均小于2.0%。氨基酸浓度在5~100μg/ml时,浓度与峰面积线性关系良好,检测氨基酸样品的加标回收率均在80%~120%之间。用磺基水杨酸和盐酸稀释剂处理供试品后,2种稀释剂检测值之间的相对误差在±5%范围内。定量限为5μg/ml,检测限为2μg/ml。结论成功建立了检测百日咳抗原中间产物中氨基酸含量的OPA柱前衍生化RP-HPLC检测方法,该方法的专属性、系统适用性、线性、准确度、耐用性良好。

ObjectiveTo establish and verify the reversed phase high performance liquid chromatography(RP-HPLC)with pre-column derivatization of O-phthalaldehyde(OPA)method to detect the amino acids contents in pertussis antigen intermediates.Methods L-aspartic acid sodium salt content in pertussis toxin detoxification solution,glycine content in filamentous hemagglutinin detoxification solution and L-lysine content in adhesin detoxification fluid of pertussis antigen intermediates were detected by RP-HPLC with pre-column derivatization of OPA.The specificity,system applicability,linearity,accuracy,durability,limit of detection and quantification of this method were verified.ResultsWhen configuration solutions were detected by RP-HPLC with pre-column derivatization of OPA,the corresponding amino acid peaks were observed in control and test solutions,and the relative standard deviations of retention time and peak area of the test solutions were less than 2.0%.When the amino acid concentration was in the range of 5-100μg/ml,the concentration showed a good linear relationship with the peak area.The recoveries of amino acid samples were 80%-120%.After the test material was treated with sulfosalicylic acid and hydrochloric acid diluent,the relative errors between the detection values of 2 diluents were within±5%.The limit of quantification was 5μg/ml and the limit of detection was 2μg/ml.ConclusionAn RP-HPLC method with pre-column derivatization of OPA for the determination of amino acid content in pertussis antigen intermediates is successfully established with good specificity,system applicability,linearity,accuracy and durability.