高效液相色谱法测定全血中加替沙星

HPLC Determination of Gatifloxacin in Whole Blood

提出了用环丙沙星为内标及用荧光检测的加替沙星(GXTC)的高效液相色谱测定方法。全血样品中GXTC用pH7.0的磷酸盐缓冲溶液萃取并经固相萃取小柱净化,用甲酸及甲醇(4+96)混合溶液作淋洗液淋洗萃取小柱,所得淋出液用吹氮法蒸干,残渣溶于1.0 mL甲醇中,分取10.0μL溶液供高效液相色谱测定。测定中用甲醇、乙腈及甲酸(2+998)溶液三者以15比15比70的体积比混合后作为流动相,在Cloversil C18柱(150 mm×3.0 mm,5μm)上进行分离。荧光检测激发波长为288 nm,发射波长为478 nm。加替沙星在15.0～240.0μg·L-1范围内呈线性,方法回收率在97.0%～102.0%之间,日内相对标准偏差(n=6)小于6.0%,方法的测定限(10S/N)为15.0μg·L-1。

A method of HPLC determination of gatifloxacin （GXTC） in whole blood with fluorescence detection and using ciprofloxacin （CPXC） as internal standard was proposed. GXTC was extracted from the blood sample with PBS of pH 7.0 and purified by SPE microcolumn. A mixed solution of formic acid and methanol （4 to 96 by vol. ） was used as eluant, the eluate was dried by N2-blowing and the residue taken up with 1. 0 mL of methanol, from which 10. 0 μL was alquoted for HPLC determination. The cloversil C18 column （150 mm×3. 0 mm, 5 μm） was used as the stationary phase, and a mixture of methanol, acetonitrile and formic acid solution （2＋998）, mixed in the ratio of 15 to 15 to 70 by volume, was used as the mobile phase. FI rescence detections were made at the wavelengths of （λex） 288 nm and （λem） 478 nm. Linear relationship between values of ratio of peak areas of GTXC/CPXC and mass concentration of GTXC was obtained in the range of 15.0-240. 0μg · L^-1. Lower limit of determination （10S/N） of the method was found to be 15.0μg·L^-1. Values of recovery found at different concentration levels were in the range of 97.0%- 102.0% within-day precision was tested, and values of RSD＇s （n=6） found were less than 6.0%.