den Boer Ethan, Koch Birgit C P, Huisman Ruud, de Jonge Robert
*Department of Clinical Chemistry; and †Hospital Pharmacy, University Medical Centre Rotterdam, The Netherlands.
Ther Drug Monit. 2014 Dec;36(6):819-23. doi: 10.1097/FTD.0000000000000085.
The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTX-PGs). We aimed to validate an immunoassay for the measurement of MTX-PG in erythrocytes.
Samples were analyzed by an adapted fluorescence polarization immune assay (FPIA) method on the FLx analyzer (Abbott). Cross-reactivity was determined in both plasma and erythrocyte pellet. In erythrocyte pellet, the imprecision, linearity, and lower limit of quantitation were determined. The method was compared with our in-house liquid chromatography tandem mass spectrometry (LC-MS/MS) method for total MTX-PG.
For the adapted FPIA method, a linear range of 25-1000 nmol/L (R = 0.993) was obtained for total MTX-PG in erythrocytes. A coefficient of variation of <17% for interday and <8% for intraday imprecision was found and average recovery was 91%. Lower limit of quantitation was determined at 50 nmol/L total MTX-PG with a coefficient of variation of 15%. There was no significant proportional bias of the FPIA assay compared with our in-house LC-MS/MS method, but a (nonsignificant) constant positive bias was present [FPIA = 1.00 (95% confidence interval: 0.60-1.95) × LC-MS/MS + 31.00 nmol/L (95% confidence interval: -11.83 to 61.00)]. Results could be very different for individual patients as reflected in the poor R of 0.419.
The FPIA method can be used to measure total MTX-PG in erythrocytes. Although there was no significant bias detected compared with the LC-MS/MS method, the FPIA method showed constant positive bias, probably because of interference from folates and MTX metabolites 2,4-diamino-N10-methylpteroic acid and 7-hydroxy-MTX. The correlation between both methods was average and resulted in large differences in individual patients, most likely because of problems during sample preparation.
叶酸拮抗剂甲氨蝶呤（MTX）是类风湿关节炎治疗的基础药物。MTX的治疗效果归因于细胞内MTX的水平，其在细胞内以多聚谷氨酸盐（MTX - PGs）形式存在。我们旨在验证一种用于测定红细胞中MTX - PG的免疫测定法。
采用适配的荧光偏振免疫测定（FPIA）方法在FLx分析仪（雅培公司）上对样本进行分析。在血浆和红细胞沉淀中测定交叉反应性。在红细胞沉淀中，测定不精密度、线性和定量下限。将该方法与我们实验室的液相色谱串联质谱（LC - MS/MS）法测定总MTX - PG进行比较。
对于适配的FPIA方法，红细胞中总MTX - PG的线性范围为25 - 1000 nmol/L（R = 0.993）。日间不精密度变异系数<17%，日内不精密度变异系数<8%，平均回收率为91%。总MTX - PG的定量下限测定为50 nmol/L，变异系数为15%。与我们实验室的LC - MS/MS方法相比，FPIA测定法没有显著的比例偏差，但存在（不显著的）恒定正偏差[FPIA = 1.00（95%置信区间：0.60 - 1.95）×LC - MS/MS + 31.00 nmol/L（95%置信区间： - 11.83至61.00）]。如较差的R值0.419所示，个体患者的结果可能差异很大。
FPIA方法可用于测定红细胞中的总MTX - PG。尽管与LC - MS/MS方法相比未检测到显著偏差，但FPIA方法显示出恒定正偏差，可能是由于叶酸和MTX代谢产物2,4 - 二氨基 - N10 - 甲基蝶酸及7 - 羟基 - MTX的干扰。两种方法之间的相关性一般，个体患者之间差异较大，很可能是由于样品制备过程中的问题。
