高效液相色谱法分析甲氨蝶呤及多聚谷氨酸甲氨蝶呤

Determination of methotrexate and methotrexate polyglutamates with HPLC

目的：建立高效液相色谱法测定甲氨蝶呤及多聚谷氨酸甲氨蝶呤的方法。方法：待测样品加入10％三氯醋酸溶液沉淀蛋白，旋涡混合、离心，取上清液进样。XTerraTM RP18（3．9mm×150mm，5μm）色谱柱，流动相为甲醇和磷酸盐缓冲液（7．5mmol·L^-1，pH7．3）梯度洗脱；流速1mL·min^-1；样品温度15℃；柱温30℃；紫外检测器，检测波长309nm。结果：五聚谷氨酸甲氨蝶呤、四聚谷氨酸甲氨蝶呤、三聚谷氨酸甲氨蝶呤、二聚谷氨酸甲氨蝶呤、甲氨蝶呤的保留时间分别为3．194，4．147，5．488，6．738，8．063min;线性范围1．5625～400μmol·L^-1，甲氨蝶呤及多聚谷氨酸甲氨蝶呤的回收率为89．01％～105．54％（RSD〈C5％）；最低检测浓度0．01μmol·L^-1（S／N〉3），9min内可完成5种组分的分析。结论：该方法灵敏度高，结果准确，分析快速、简便。

OBJECTIVE To establish an HPLC method for the determining the methotrexate （MTX） and methotrexate polyglutamates （MTXPG）. METHODS Add 10% trichloroacetic acid to the samples and vortex mixed,the protein in the samples was deposited. After centrifugal separation,the ascending clear layer was determined by HPLC with UV （309 nm） detection. All of the compounds were isolated from XTerra^TM RP18 5 μm,3.9 mm× 150 mm column with mobile phase of methanol and 7. 5 mmol· L^-1 sodium phosphate （pH 7. 3）, gradient elution with a flow rate of 1.0 mL·min^-1. The sample temperature was 15 ℃. The column temperature was 30 ℃. RESULTS The retention time of MTXPG3, MTXPG4, MTXPG3, MTXPG2, MTX was 3. 194, 4. 147, 5. 488, 6. 738, 8. 063 min; The linearity range of calibration curves of MTXPG5-2, — MTX was 1. 562 5 -400 μmol· L^-1 ;The recovery of MTXPG3-2, ＆ MTX was 89. 01% - 105.54% （RSD〈5%）;The limit of detection was 0. 01 μmol·L^-1 （S/N〉3） ;The whole operation can be finished in less than 9 minutes. CONCLUSION This method is sensitive, rapid, accurate and simple.