Cociglio M, Hillaire-Buys D, Alric C
Laboratoire de Pharmacologie Clinique, Centre Hospitalier Universitaire, Höpital Saint-Charles, Montpellier, France.
J Chromatogr B Biomed Appl. 1995 Dec 1;674(1):101-10. doi: 10.1016/0378-4347(95)00301-x.
A high-performance liquid chromatographic (HPLC) method was designed to meet analytical and metrological requirements for routine blood level monitoring of methotrexate (MTX) and its main metabolite 7-hydroxymethotrexate (7OMTX). The metabolite, unavailable as a pure substance, was measured by reference to MTX calibration according to their respective ultraviolet absorbances. Acetonitrile deproteinization and chloroform clean-up provided plasma samples devoid of long-retained contaminants. The precision of the HPLC measurements, reproducibility of clean-up recovery, matrix effects and linearity were assessed by analysis of variance and linear regression in an appropriate experimental design, within a range from 0.205 to 16.7 mg/l of MTX and from 0.084 to 6.83 mg/l of 7OMTX. The clean-up recovery from plasma was 88% for MTX and 72% for 7OMTX, owing to retention on the protein precipitate. The assay was linear, the measurement precision was 3.3% for MTX and 6.2% for 7OMTX and the clean-up reproducibility was 4% for MTX and 3.6% for 7OMTX. By reference to automated fluorescence polarization immunoassay, the HPLC method resulted in plasma MTX values 10% lower, probably owing to the higher specificity of HPLC. Unsystematically sequenced plasma samples from 35 children following 24-h MTX infusions provided estimated half-decay times of 16 and 19 h for MTX and 7OMTX, respectively, and 7OMTX:MTX concentration ratios of 7 at 48 h and of 5 at 72 h from starting infusions.
设计了一种高效液相色谱（HPLC）方法，以满足甲氨蝶呤（MTX）及其主要代谢物7-羟基甲氨蝶呤（7OMTX）常规血药浓度监测的分析和计量要求。该代谢物没有纯品，根据其各自的紫外吸光度，通过参照MTX校准物进行测定。乙腈去蛋白和氯仿净化处理可提供不含长期残留污染物的血浆样品。在MTX浓度范围为0.205至16.7mg/l、7OMTX浓度范围为0.084至6.83mg/l内，通过适当实验设计中的方差分析和线性回归，评估了HPLC测量的精密度、净化回收率的重现性、基质效应和线性。由于保留在蛋白质沉淀上，MTX从血浆中的净化回收率为88%，7OMTX为72%。该测定呈线性，MTX的测量精密度为3.3%，7OMTX为6.2%，MTX的净化重现性为4%，7OMTX为3.6%。与自动荧光偏振免疫测定法相比，HPLC法测得的血浆MTX值低10%，这可能是由于HPLC具有更高的特异性。对35名儿童在输注MTX 24小时后采集的非系统排序血浆样本进行分析，结果显示MTX和7OMTX的估计半衰期分别为16小时和19小时，从开始输注起48小时时7OMTX与MTX的浓度比为7，72小时时为5。
