Norris R L, Thomas M J, Craswell P W
J Chromatogr. 1986 Aug 22;381(1):53-61. doi: 10.1016/s0378-4347(00)83564-1.
The technique of dual-wavelength monitoring was used to verify the purity of high-performance liquid chromatographic (HPLC) peaks quantified as 25-hydroxyergocalciferol and 25-hydroxycholecalciferol. The data obtained show the need for a second HPLC step prior to quantitation. Potential inaccuracy arising from inadvertent collection of radio-labelled decomposition products was assessed. Between-day coefficients of variation were 7.3, 5.0 and 3.6%, respectively for 11.3 (n = 12), 17.1 (n = 14), and 32.9 (n = 8) ng/ml of 25-hydroxycholecalciferol. For 25-hydroxyergocalciferol, these values were 6.4 and 3.8% for 11.1 (n = 12) and 20.1 (n = 8) ng/ml concentrations, respectively. Comparison of total 25-hydroxycalciferol with a competitive protein binding assay was made. The comparison produced a correlation coefficient (r) of 0.94 and a relationship of y = 1.03x + 3.3. Four of the samples contained more than 10 ng/ml of 25-hydroxyergocalciferol and the results are consistent with the reported 100% cross-reactivity of the competitive binding protein method for 25-hydroxyergocalciferol and 25-hydroxycholecalciferol. A simple regeneration procedure is also described which enables Sep-Pak C18 cartridges to be reused up to eighteen times. Samples may be stored at -18 degrees C for upto several months before assay and either serum or plasma may be used.
采用双波长监测技术来验证高效液相色谱（HPLC）中定量为25-羟基麦角钙化醇和25-羟基胆钙化醇的峰的纯度。所获得的数据表明在定量之前需要进行第二步HPLC分析。评估了因意外收集放射性标记分解产物而产生的潜在不准确性。对于11.3（n = 12）、17.1（n = 14）和32.9（n = 8）ng/ml的25-羟基胆钙化醇，日间变异系数分别为7.3%、5.0%和3.6%。对于25-羟基麦角钙化醇，浓度为11.1（n = 12）和20.1（n = 8）ng/ml时，这些值分别为6.4%和3.8%。将总25-羟基钙化醇与竞争性蛋白结合测定法进行了比较。比较得出相关系数（r）为0.94，关系为y = 1.03x + 3.3。其中四个样品含有超过10 ng/ml的25-羟基麦角钙化醇，结果与报道的竞争性结合蛋白法对25-羟基麦角钙化醇和25-羟基胆钙化醇的100%交叉反应性一致。还描述了一种简单的再生程序，可使Sep-Pak C18柱重复使用多达18次。样品在分析前可在-18℃下储存长达数月，血清或血浆均可使用。
