Wyss R, Bucheli F
Pharmaceuticals Division, F. Hoffmann-La Roche Ltd, PRPK, Basel, Switzerland.
J Chromatogr B Biomed Sci Appl. 1997 Oct 24;700(1-2):31-47. doi: 10.1016/s0378-4347(97)00303-4.
A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 microm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250x4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3-100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%-94.4% and the mean inter-assay precision was 2.8%-3.2% (range 0.3-100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at -20 degrees C for 4.3 months and at -80 degrees C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.
建立了一种采用自动柱切换的高灵敏度高效液相色谱法，用于同时测定人、食蟹猴、兔、大鼠和小鼠血浆样品中13 - 顺式维甲酸（异维甲酸）、全反式维甲酸（维甲酸）及其4 - 氧代代谢物的内源性水平。向0.4 ml血浆中加入含内标阿维A的乙醇（1.5 ml）进行去蛋白处理。离心后，将1.4 ml上清液直接注入填充有LiChrospher 100 RP - 18（5微米）的预柱。进样时使用1.25%醋酸铵和醋酸 - 乙醇（8:2，v/v）作为流动相，并在线加入1%醋酸铵和2%醋酸 - 乙醇（102:4，v/v）以降低进样溶液的洗脱强度。预柱反冲清洗后，保留的组分以反冲模式转移至分析柱，通过梯度洗脱进行分离，并在360 nm处检测。使用两根联用的Superspher 100 RP - 18封端柱（均为250×4 mm）进行分离，流动相由乙腈 - 水 - 10%醋酸铵 - 醋酸组成：（A）600:300:60:10（v/v/v/v），（B）950:20:5:20（v/v/v/v），（C）990:5:0:5（v/v/v/v）。该方法至少在0.3 - 100 ng/ml范围内呈线性，定量限为0.3 ng/ml。人血浆的平均回收率为93.2% - 94.4%，批间精密度平均为2.8% - 3.2%（范围0.3 - 100 ng/ml）。动物血浆也得到了类似结果。发现所有研究物种的血浆中的分析物在 - 20℃下储存4.3个月以及在 - 80℃下储存至少9个月时是稳定的。在此温度下，人血浆样品甚至可稳定保存2年。该方法成功应用于来自临床和毒代动力学研究的6000多份人血浆样品和1000多份动物血浆样品。对照患者和孕妇中测定的内源性水平与志愿者已发表的数据相似。
