Wu Xiao, Huang Yue, Sun Jinghan, Wen Yongqing, Qin Feng, Zhao Longshan, Xiong Zhili
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning Province 110016, PR China.
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning Province 110016, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Jan 1;1072:40-49. doi: 10.1016/j.jchromb.2017.10.005. Epub 2017 Oct 6.
A HILIC-UHPLC-MS/MS untargeted urinary metabonomic method combined with quantitative analysis of five potential polar biomarkers in rat urine was developed and validated, to further understand the anti-osteoporosis effect of Gushudan(GSD) and its mechanism on prednisolone-induced osteoporosis(OP) rats in this study. The metabolites were separated and identified on Waters BEH HILIC (2.1mm×100mm, 1.7μm) column using the Waters ACQUITY™ ultra performance liquid chromatography system (Waters Corporation, Milford, USA) coupled with a Micromass Quattro Micro™ API mass spectrometer (Waters Corp, Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers. Primary potential polar biomarkers including creatinine, taurine, betaine, hypoxanthine and cytosine, which were related to energy metabolism, lipid metabolism and amino acid metabolism, were found in the untargeted metabonomic research. Moreover, these targeted biomarkers were further separated and quantified in multiple-reaction monitoring (MRM) with positive ionization mode, using tinidazole as internal standard (I.S.). Good linearities (r>0.99) were obtained for all the analytes with the low limit of quantification from 1.00 to 12.8μg/mL. The relative standard deviation (RSD) of the intra-day and inter-day precisions were within 15.0% and the accuracy ranged from -14.3% to 13.5%. The recovery was more than 85.0%. And the validated method was successfully applied to investigate the urine samples of the control group, prednisolone-induced osteoporosis model group and Gushudan-treatment group in rats. Compared to the control group, the level of creatinine, taurine, betaine, hypoxanthine and cytosine in the model group revealed a significant decrease trend (p<0.05), while the Gushudan-treatment group showed no statistically differences by an independent sample t-test. This paper provided a better understanding of the therapeutic effect and mechanism of GSD on prednisolone-induced osteoporosis rats.
本研究建立并验证了一种亲水相互作用超高效液相色谱-串联质谱（HILIC-UHPLC-MS/MS）非靶向尿代谢组学方法，并对大鼠尿液中五种潜在极性生物标志物进行定量分析，以进一步了解骨疏丹（GSD）对泼尼松龙诱导的骨质疏松症（OP）大鼠的抗骨质疏松作用及其机制。代谢产物在Waters BEH HILIC（2.1mm×100mm，1.7μm）色谱柱上分离和鉴定，采用Waters ACQUITY™超高效液相色谱系统（美国沃特世公司，米尔福德）与Micromass Quattro Micro™ API质谱仪（美国马萨诸塞州米尔福德沃特世公司）联用。主成分分析（PCA）用于识别潜在生物标志物。在非靶向代谢组学研究中发现了主要的潜在极性生物标志物，包括肌酐、牛磺酸、甜菜碱、次黄嘌呤和胞嘧啶，它们与能量代谢、脂质代谢和氨基酸代谢有关。此外，这些靶向生物标志物在多反应监测（MRM）中以正离子模式进一步分离和定量，使用替硝唑作为内标（I.S.）。所有分析物均获得良好的线性关系（r>0.99），定量下限为1.00至12.8μg/mL。日内和日间精密度的相对标准偏差（RSD）在15.0%以内，准确度范围为-14.3%至13.5%。回收率超过85.0%。该验证方法成功应用于研究大鼠对照组、泼尼松龙诱导的骨质疏松模型组和骨疏丹治疗组的尿液样本。与对照组相比，模型组中肌酐、牛磺酸、甜菜碱、次黄嘌呤和胞嘧啶的水平呈现显著下降趋势（p<0.05），而骨疏丹治疗组经独立样本t检验无统计学差异。本文为骨疏丹对泼尼松龙诱导的骨质疏松大鼠的治疗作用和机制提供了更好的理解。
