HPLC测定脱氧核苷酸钠的含量和有关物质

Determination of the Content and Related Substances of Sodium Deoxyribonucleotide by HPLC

关键词:  脱氧核苷酸钠  脱氧胞嘧啶核苷酸  脱氧腺嘌呤核苷酸  脱氧胸腺嘧啶核苷酸  脱氧鸟嘌呤核苷酸  高效液相色谱法  含量测定  有关物质

Key words:  sodium deoxyribonucleotide  2’-deoxycytidine 5’-monophosphate  2’-deoxyadenosine 5’-monophosphate  2’-deoxythymidine 5’-monophosphate  2’-deoxyguanosine 5’-monophosphate  HPLC  assay  related substances

摘要:

目的 HPLC测定脱氧核苷酸钠的含量和有关物质方法的建立。方法 色谱柱为Agilent Zorbax SAX(4.6 mm×250 mm，5 μm)，流动相为0.05 mol·L^-1磷酸盐缓冲液(取磷酸二氢钾6.80 g加水800 mL，用磷酸调节pH值至3.3，加水稀释至1 000 mL)，流速0.5 mL·min^-1，检测波长258 nm。结果 脱氧核苷酸钠中脱氧胞嘧啶核苷酸(d-CMP)、脱氧腺嘌呤核苷酸(d-AMP)、脱氧胸腺嘧啶核苷酸(d-TMP)、脱氧鸟嘌呤核苷酸(d-GMP) 4种单核苷酸的进样量分别在0.017 6~3.382 7 μg、0.019 6~ 3.758 0 μg、0.014 8~2.847 7 μg和0.018 0~3.452 8 μg内与峰面积线性关系良好(r均为1.000)；d-CMP、d-AMP、d-TMP、d-GMP的平均回收率分别为96.73%，97.51%，97.98%，98.39%；RSD分别为0.17%，0.39%，1.32%，0.32%(n=9)；精密度、重复性良好。结论 采用本方法主峰与杂质峰的分离度好，结果准确可靠，灵敏度高，为本品质量标准的修订和提高提供了基础。

Abstract:

OBJECTIVE To establish a method for determining the content and related substances of sodium deoxyribonucleotide by HPLC. METHODS The Agilent Zorbax SAX(4.6 mm×250 mm, 5 μm) was used, the mobile phase was 0.05 mol·L^-1 potassium dihydrogen phosphate solution (adjust pH to 3.3 by phosphoric acid) at the flow rate of 0.5 mL·min^-1, the detective wavelength was 258 nm. RESULTS The calibration curves showed good linearity of 2’-deoxycytidine 5’-monophosphate (d-CMP), 2’-deoxyadenosine 5’-monophosphate (d-AMP) , 2’-deoxythymidine 5’-monophosphate (d-TMP), 2’-deoxyguanosine 5’-monophosphate (d-GMP) over the range of 0.017 6~3.382 7 μg, 0.019 6~3.758 0 μg, 0.014 8~2.847 7 μg and 0.018 0~3.452 8 μg, r=1.000. The average recovery rates of d-CMP, d-AMP, d-TMP and d-GMP were 96.73%, 97.51%, 97.98% and 98.39%, RSD were 0.17%, 0.39%, 1.32% and 0.32%(n=9). CONCLUSION The established method has excellent resolution for the related substances, and it is accurate and sensitive for testing the content and related substances of sodium deoxyribonucleotide which can be used for control of the production.