Kalhorn T F, Thummel K E, Nelson S D, Slattery J T
Anal Biochem. 1985 Dec;151(2):343-7. doi: 10.1016/0003-2697(85)90185-x.
NADP+, NAD+, NADPH, and NADH were assayed by selective extraction and isocratic reversephase HPLC. Sample preparation involves freeze clamping and powdering liver under liquid nitrogen, extraction of dinucleotides with basic (reduced species) or acidic (oxidized species) cold ethanol, and injection onto the HPLC for quantitation at 340 nm (reduced) and 254 nm (oxidized). The mobile phase for the oxidized species is pH 5.25, 0.2 M ammonium phosphate/methanol, and for the reduced species is pH 6.0, 0.2 M ammonium phosphate/methanol/tributylamine. The method is linear over the range 0.016 to 2.0 nmol for the reduced species, and from 0.005 to 0.8 nmol for the oxidized pyridine dinucleotides. The recoveries were from 94.5% for NAD+ to 99.3% for NADPH, with standard deviations of approximately 2.5% for all species other than NADP+, which had a standard deviation of 10.4%. The coefficients of variation for repeated determinations of standards over 3 months were less than 4%.
通过选择性萃取和等度反相高效液相色谱法测定NADP⁺、NAD⁺、NADPH和NADH。样品制备包括在液氮下对肝脏进行冷冻钳夹并研磨成粉末，用碱性（还原型）或酸性（氧化型）冷乙醇萃取二核苷酸，然后注入高效液相色谱仪，在340nm（还原型）和254nm（氧化型）处进行定量分析。氧化型物质的流动相为pH 5.25的0.2M磷酸铵/甲醇，还原型物质的流动相为pH 6.0的0.2M磷酸铵/甲醇/三丁胺。该方法对于还原型物质在0.016至2.0 nmol范围内呈线性，对于氧化型吡啶二核苷酸在0.005至0.8 nmol范围内呈线性。回收率从NAD⁺的94.5%到NADPH的99.3%，除NADP⁺外所有物质的标准偏差约为2.5%，NADP⁺的标准偏差为10.4%。在3个月内对标准品进行重复测定的变异系数小于4%。
