柱前衍生化RP-HPLC法同时测定多肽类药物中7个生物胺的含量  

Simultaneous determination of seven kinds of biogenic amines in polypeptide drugs by RP-HPLC with pre-column derivatization

作　　者：陈小英 王嫦鹤 刘雪峰 刘海静[1,2] CHEN Xiao-ying;WANG Chang-he;LIU Xue-feng;LIU Hai-jing(Shaanxi University of Chinese Medicine,Xianyang 712046,China;Shaanxi Institute for Food and Drug Control,Xi’an 710065,China)

机构地区：[1]陕西中医药大学,咸阳712046 [2]陕西省食品药品检验研究院,西安710065

出　　处：《药物分析杂志》2022年第4期643-652,共10页Chinese Journal of Pharmaceutical Analysis

基　　金：国家科技重大专项重大新药创制(2017Z09101001);陕西省创新能力支撑计划(2020PT-041)。

摘　　要：目的:建立柱前衍生化RP-HPLC法测定注射用胸腺肽、骨肽注射液、鲑降钙素注射液、注射用谷胱甘肽、转移因子口服液和转移因子胶囊中色胺、2-苯乙胺、腐胺、尸胺、组胺、酪胺、亚精胺7个生物胺含量。方法:样品经0.1mol·L^(-1)盐酸溶液溶解,以丹磺酰氯为衍生化试剂,用反相高效液相色谱法测定。采用C_(18)色谱柱(250 mm×4.6 mm,5μm),以含0.1%醋酸的0.01 mol·L^(-1)醋酸铵溶液(A)-乙腈(B)为流动相,梯度洗脱(0~21 min,70%A→50%A;21~25 min,50%A→30%A;25~60 min,30%A;60~70 min,30%A→70%A),流速1.0 mL·min^(-1),检测波长254 nm,柱温35℃。结果:色胺、2-苯乙胺、尸胺、酪胺和亚精胺的线性范围均为0.1~50 mg·L^(-1)(r>0.999),腐胺的线性范围为0.06~31.91 mg·L^(-1)(r>0.999),组胺的线性范围为0.10~26.12 mg·L^(-1)(r>0.999);平均回收率在73.6%~104.0%。注射用胸腺肽检出的生物胺种类和含量最高,转移因子制剂其次,骨肽注射液中只检出组胺,鲑降钙素注射液和注射用谷胱甘肽均未检出生物胺。注射用胸腺肽检出腐胺、尸胺、组胺、酪胺和亚精胺。其中,注射用胸腺肽A企业样品中组胺和腐胺含量较高,分别约为0.113%、0.159%;B企业样品中尸胺、酪胺和亚精胺含量较高,分别约为0.373%、0.363%和0.090%;转移因子口服液和转移因子胶囊检出有腐胺、尸胺、酪胺和亚精胺。结论:该方法准确可靠,灵敏度高,重复性好,易于操作,可用于多肽类药物中生物胺的检测。Objective:To develop a method for the determination of seven kinds of biogenic amines,including tryptamine,2-phenylethylamine,putrescine,cadaverine,histamine,tyramine and spermidine in thymopolypeptides for injection,ossotide injection,salmon calcitonin injection,glutathione for injection,transfer factor oral solution and transfer factor capsules by RP-HPLC with pre-column derivatization.Methods:Samples were dissolved in 0.1 mol·L^(-1)hydrochloric acid solution and determined by RP-HPLC with dansyl chloride asderivatization reagent.The experiment was performed on a C_(18)column(250 mm×4.6 mm,5μm)with gradient elution(0-21 min,70%A→50%A;21-25 min,50%A→30%A;25-60 min,30%A;60-70 min,30%A→70%A)using 0.01 mol·L^(-1)ammonium acetate solution containing 0.1%aceticacid(A)-acetonitrile(B)as the mobile phase at a flow rate of 1.0 mL·min^(-1).The detection wavelength was 254 nm.The column temperature was 35℃.Results:The calibration curves were linear in the rangs of 0.1-50 mg·L^(-1)(r>0.999)for tryptamine,2-phenylethylamine,cadaverine,tyramine and spermidine,0.06-31.91 mg·L^(-1)(r>0.999)for putrescine,0.1-26.12 mg·L^(-1)(r>0.999)for histamine.The average recoveries varied from 73.6%to 104.0%.The type and content of biogenic amines were the highest in thymopolypeptides for injection,followed by transfer factor preparation.Only histamine was detected in ossotide injection,and no biogenic amines were detected in salmon calcitonin injection and glutathione for injection.Putrescine,cadaverine,histamine,tyramine and spermidine were found in thymopolypeptides for injection.The highest contents of histamine,putrescine in the thymopolypeptides for injection of enterprise A were about 0.113%and 0.159%,respectively.The contents of cadaverine,tyramine and spermidine in enterprise B were higher,about 0.373%,0.363%and 0.090%,respectively.Putrescine,cadaverine,tyramine and spermidine were detected in transfer factor oral solution and transfer factor capsules.Conclusion:The method is accurate and reliable,sensitive,reproduc

关 键 词：生物胺 柱前衍生化 反相高效液相色谱法 多肽药物 注射用胸腺肽 骨肽注射液 转移因子口服液 丹磺酰氯 

分 类 号：R917[医药卫生—药物分析学]