黑龙江医药
黑龍江醫藥
흑룡강의약
HEILONGJIANG MEDICAL JOURNAL
2014年
1期
1089-1093,1094
,共6页
高效液%相色谱%辅助%A检测试剂%盒辅酶A
高效液%相色譜%輔助%A檢測試劑%盒輔酶A
고효액%상색보%보조%A검측시제%합보매A
HPLC%Coenzyme%A Assay%Kit CoA
目的:通过比较多种辅酶A测定方法总结出适合注射用辅酶A的效价测定方法。方法:对多批注射用辅酶A制剂进行3种方法的效价测定,分别为《卫生部颁标准二部六册》上的磷酸转乙酰化酶法、高效液相色谱法及辅酶A测定试剂盒法,其中高效液相色谱法以磷酸盐缓冲液(pH7.0)-甲醇(90:10)为流动相,采用 Phenomenex C18色谱柱,流速1.0ml/min,259nm为检测波长同时利用二极管阵列检测器对200nm-400nm波段进行监视,并利用磷酸盐缓冲液(pH5.8)对样品进行溶解后进行测定。辅酶A试剂盒法是采用BioVision的Coenzyme A Assay Kit,BioVision公司开发了一种简单快捷的检测多种生物样本中辅酶A水平的方法,利用探针技术捕获游离的辅酶A,生成的产物在可见光波段有吸收下。最后对3种方法的结果进行比较。结果:高效液相色谱法中辅酶A在1U/ml-100U/ml的范围内,峰形及线形关系良好(r2=1),样品溶液室温放置长时间稳定。试剂盒法中辅酶A在每孔0.032U-3.2U范围内线形关系良好(r2=0.9976)。结论:高效液相色谱法与辅酶A测定试剂盒法要优于磷酸转乙酰化酶法。实现了室温条件下稳定测定多批注射用辅酶A,改良后的高效液相色谱法可以作为辅酶A效价测定的裁定方法,试剂盒法可以作为快速筛查与检验的方法。
目的:通過比較多種輔酶A測定方法總結齣適閤註射用輔酶A的效價測定方法。方法:對多批註射用輔酶A製劑進行3種方法的效價測定,分彆為《衛生部頒標準二部六冊》上的燐痠轉乙酰化酶法、高效液相色譜法及輔酶A測定試劑盒法,其中高效液相色譜法以燐痠鹽緩遲液(pH7.0)-甲醇(90:10)為流動相,採用 Phenomenex C18色譜柱,流速1.0ml/min,259nm為檢測波長同時利用二極管陣列檢測器對200nm-400nm波段進行鑑視,併利用燐痠鹽緩遲液(pH5.8)對樣品進行溶解後進行測定。輔酶A試劑盒法是採用BioVision的Coenzyme A Assay Kit,BioVision公司開髮瞭一種簡單快捷的檢測多種生物樣本中輔酶A水平的方法,利用探針技術捕穫遊離的輔酶A,生成的產物在可見光波段有吸收下。最後對3種方法的結果進行比較。結果:高效液相色譜法中輔酶A在1U/ml-100U/ml的範圍內,峰形及線形關繫良好(r2=1),樣品溶液室溫放置長時間穩定。試劑盒法中輔酶A在每孔0.032U-3.2U範圍內線形關繫良好(r2=0.9976)。結論:高效液相色譜法與輔酶A測定試劑盒法要優于燐痠轉乙酰化酶法。實現瞭室溫條件下穩定測定多批註射用輔酶A,改良後的高效液相色譜法可以作為輔酶A效價測定的裁定方法,試劑盒法可以作為快速篩查與檢驗的方法。
목적:통과비교다충보매A측정방법총결출괄합주사용보매A적효개측정방법。방법:대다비주사용보매A제제진행3충방법적효개측정,분별위《위생부반표준이부륙책》상적린산전을선화매법、고효액상색보법급보매A측정시제합법,기중고효액상색보법이린산염완충액(pH7.0)-갑순(90:10)위류동상,채용 Phenomenex C18색보주,류속1.0ml/min,259nm위검측파장동시이용이겁관진렬검측기대200nm-400nm파단진행감시,병이용린산염완충액(pH5.8)대양품진행용해후진행측정。보매A시제합법시채용BioVision적Coenzyme A Assay Kit,BioVision공사개발료일충간단쾌첩적검측다충생물양본중보매A수평적방법,이용탐침기술포획유리적보매A,생성적산물재가견광파단유흡수하。최후대3충방법적결과진행비교。결과:고효액상색보법중보매A재1U/ml-100U/ml적범위내,봉형급선형관계량호(r2=1),양품용액실온방치장시간은정。시제합법중보매A재매공0.032U-3.2U범위내선형관계량호(r2=0.9976)。결론:고효액상색보법여보매A측정시제합법요우우린산전을선화매법。실현료실온조건하은정측정다비주사용보매A,개량후적고효액상색보법가이작위보매A효개측정적재정방법,시제합법가이작위쾌속사사여검험적방법。
Objective:To summarize the assay method of coenzyme A for injection by the compare of multiple assay methods of coenzyme A.Methods:Assay the potency of coenzyme A for injection from different batches,the first method is the phosphotransacetylase method on Drug Specifications Promulgated by the Ministry of Public Health,P R China.Part 2,Vol 6,the others are HPLC and Coenzyme A Assay Kit.The HPLC method take the pH7.0 phosphate buffer-mathanol (90:10) as mobile phase at a flow rate of 1ml/min and Phenomenex C18 (250mm4.6mm,5m) as column.The detection wavelength was 259nm and oversee the spectrum and the chromatogram with Photodiode Array Detector from 200nm to 400nm.Detective samples after soluted by the pH5.8 phosphate buffer. The Coenzyme A Assay Kit from BioVision . BioVision has developed an easy, convenient assay to measure the CoA level in variety biolo gical samples. In the assay, free CoA is specifically utilized to generate products which react with OxiRed Probe to generate color.At last,compare the assay results by these methods.Results:In the HPLC experiment,the regression was linear over the concentration range of 1U/ml-100U/ml (r2=1),the sample solution keep stable at room temperature. In the Coenzyme A Assay Kit experiment ,the regression was linear over the concentration range of 0.032U/well-3.2U/well(r2=0.9976).Conclusion:The methods of HPLC and Coenzyme A Assay Kit were both better in potency assay than the phosphotransacetylase method.We can assay simultaneously couples of coenzyme A for injection at room temperature steadily,the HPLC method modified could be the referee method,the Coenzyme A Assay Kit could be the rapid method for screening and inspect.