柱前衍生高效液相色谱法测定盐酸四环素原料药中微量甲醛含量

Determination of Trace Formaldehyde in Tetracycline Hydrochloride Raw Material Medicine by Precolumn Derivatization HPLC

目的建立测定盐酸四环素中微量甲醛含量的柱前衍生化-高效液相色谱法。方法衍生化试剂为2,4-二硝基苯肼乙腈溶液乙酸钠/冰乙酸缓冲液（p H=5）等体积混合,色谱柱为Phenomenex Hyperclone BDS C18柱（250 mm×4.6 mm,5μm）,流动相为乙腈-水（45∶55,V/V）等度洗脱,柱温为35℃,检测波长为355 nm,流速为1.0 m L/min,进样量为20μL。结果甲醛质量浓度在0.012 13~0.485 20μg/m L范围内与峰面积线性关系良好（r=0.999 9）;柱前衍生化反应获得的衍生物较稳定,建立的方法精密度高、重复性好,检测限（LOD）和定量限（LOQ）分别为0.012 1μg/m L和0.036 4μg/m L;甲醛的平均加样回收率为96.57%,RSD为4.18%;两个不同厂家盐酸四环素原料药中游离甲醛相对百分含量分别为（4.75±0.036）×10-6%和（4.78±0.031）×10-6%。结论该方法简便、可行,适用于盐酸四环素原料药中游离甲醛的含量测定及质量评价。

Objective To establish a precolumn derivatization high performance liquid chromatography（ HPLC） method for determining the trace formaldehyde in tetracycline hydrochloride raw material medicine. Methods The derivatization reagent was the isovolumetric mixture of 2,4- dinitrophenylhydrazine acetonitrile solution,sodium acetate / glacial acetic acid buffer solution（ p H = 5）. The chromatographic column was the Phenomenex Hyperclone BDS C18 column（ 250 mm × 4. 6 mm,5 μm）,the column temperature was at 35 ℃ with acetonitrile- water（ 45 ∶ 55,V / V） as the mobile phase,the flow rate was 1. 0 m L / min and the detection wavelength was 355 nm,the isocratic elution was adopted; the sample size was 20 μL. Results The mass concentration of formaldehyde in the range of 0. 012 13-0. 485 20 μg / m L showed a good linear relation with the peak area（ r = 0. 999 9）; the derivative obtained by the precolumn derivatization reaction was stable,the established method had high precision and good reproducibility. The limit of detection（ LOD） and limit of quantification（ LOQ） were 0. 012 1 μg / m L and 0. 036 4 μg / m L,respectively; the average recovery rate was 96. 57%,RSD = 4. 18%;the content of free formaldehyde in tetracycline hydrochloride from different manufacturers was（ 4. 75 ± 0. 036） × 10- 6 % and（ 4. 78 ± 0. 031） × 10-6% respectively. Conclusion This method is simple,feasible and suitable for the content determination and quality evaluation of formaldehyde in tetracycline hydrochloride raw material medicine.