超高效液相色谱法测定男性精浆中生物胺含量的研究

Ultra-performance liquid chromatography for determination of the contents of biogenic amines in the seminal plasma

目的:建立一种超高效液相色谱测定人精浆中腐胺、亚精胺、精胺3种生物胺含量的方法。方法:精浆样品用5%三氯乙酸提取,丹磺酰氯柱前衍生化,采用C18(2.1×50 mm,1.7μm)色谱柱,以水-乙腈为流动相梯度洗脱,流速为0.3 ml/min,检测波长为245 nm,柱温:35℃;进样量3.0μl。测定了捐精志愿者52例(正常组)和无精子症患者23例(无精子症组)精浆中3种生物胺的含量,与精液常规参数进行相关性分析。结果:在上述色谱条件下,3种生物胺在呈良好的线性关系(r≥0.999),最低检出限为0.03~0.08μg/ml;仪器精密度较好RSD≤0.72%,平均回收率为89.74%~108.87%。正常组精浆中生物胺含量[腐胺(8.19±7.85) mg/ml,亚精胺(77.30±32.58) mg/ml,精胺(246.44±83.99) mg/ml]显著高于无精子症组[腐胺(2.43±1.38) mg/ml,亚精胺(31.99±16.21) mg/ml,精胺(166.15±79.28) mg/ml]。正常组和无精子症组中精胺与亚精胺、亚精胺与腐胺的含量均存在显著相关性(P﹤0.05)。正常组精浆3种生物胺含量与精液参数没有相关性。结论:建立的方法灵敏度高、重现性好、出峰时间短,可以快速准确检测男性精浆中生物胺含量。

Objective: To establish an ultra-performance liquid chromatography(UPLC) method for determination of the contents of the three biogenic amines putacine, spermidine and spermine in human seminal plasma. Methods: Seminal plasma samples were extracted with 5% trichloroacetic acid and processed by pre-column derivatization with dansyl chloride. Chromatographic separation was performed with a C18(2.1×50 mm,1.7 μm) chromatographic column using water and acetonitrile for mobile-phase gradient elution at a flow rate of 0.3 ml/min, a detection wavelength of 245 nm, a column temperature of 35℃ and an injection volume was 3.0 μl. The contents of putacine, spermidine and spermine in the seminal plasma of 52 healthy sperm donors(the normal group) and 23 azoospermia patients(the AS group) were measured, and their correlation with routine semen parameters were analyzed. Results: The three biogenic amines showed a good linearity(r ≥ 0.999), with a lower detection limit of 0.03-0.08 μg/ml. The relative standard deviation(RSD) of precision was ≤ 0.72% and the average recovery rate was 79.74%-108.87%. The normal group, compared with the AS patients, showed significantly higher contents of putrescine([8.19 ± 7.85] vs [2.43 ± 1.38] mg/ml, P < 0.05), spermidine([77.30 ± 32.58] vs [31.99 ± 16.21] mg/ml, P < 0.05) and spermine([246.44 ± 83.99] vs [166.15 ± 79.28] mg/ml, P < 0.05). However, the contents of the three biogenic amines in the seminal plasma exhibited no significant correlation with the routine semen parameters in the normal group. Conclusion: The ultra-performance liquid chromatography method we established, with the advantages of high sensitivity and reproducibility and short peak-time, can quickly and accurately determine the contents of biogenic amines in the seminal plasma.