超高效液相色谱-四级杆/静电场轨道阱高分辨质谱法同时检测补中益气丸中毛蕊异黄酮葡萄糖苷、橙皮苷、甘草酸铵的含量

doi:10.3976/j.issn.1002-4026.2023.02.003

摘要/Abstract

摘要：

建立了一种超高效液相色谱-四级杆/静电场轨道阱高分辨质谱同时检测补中益气丸中毛蕊异黄酮葡萄糖苷、橙皮苷和甘草酸铵含量的方法。采用超高效液相色谱-四级杆/静电场轨道阱质谱,Hypersil Gold C₁₈型色谱柱,乙腈-0.1%甲酸水梯度洗脱,流速0.4 mL/min,柱温40 ℃,进样量1 μL;采用电喷雾离子源,正离子模式下,在质荷比100~1 500范围内全扫描,通过提取待测成分的精确质量数进行定量。结果表明,毛蕊异黄酮葡萄糖苷、橙皮苷、甘草酸铵线性范围分别为0.089 1~1.425 0 μg/mL、0.098 4~12.600 0 μg/mL、7.425 0~29.700 0 μg/mL(r≥0.999 3),检测限分别为5.57、4.10、5.80 ng/mL,定量限分别为22.26、12.30、23.20 ng/mL,精密度、重复性及样品稳定性(24 h)良好,加样回收率91%~105%(δ_RSD≤5.0%,n=6)。本方法简便快捷、特异性强、灵敏度高,可同时检测补中益气丸中毛蕊异黄酮葡萄糖苷、橙皮苷、甘草酸铵的含量。

关键词: 超高效液相色谱-四级杆/静电场轨道阱高分辨质谱, 补中益气丸, 毛蕊异黄酮葡萄糖苷, 橙皮苷, 甘草酸铵

Abstract:

In this study, we have developed a novel method for the simultaneous determination of calycosin 7-O-glucoside, hesperidin, and ammonium glycyrrhizinate in Buzhongyiqi Pills based on UPLC-Q/Orbitrap HRMS using Hypersil Gold C₁₈ chromatographic column. The gradient mobile phase comprised acetonile as well as water containing 0.1% formic acid. The flow rate, column temperature, and injection volume were 0.4 mL/min, 40 ℃, and 1 μL, respectively. Mass spectrometer was operated using an electrospray ionization source in the positive ion mode for detection. The scan range was 100~1 500 m/z. Quantification was performed by extracting the accurate mass of the target compounds.The linear ranges of calycosin 7-O-glucoside,hesperidin, ammonium glycyrrhizinate were 0.089 1~1.425 0 μg/mL,0.098 4~12.600 0 μg/mL, and 7.425 0~29.700 0 μg/mL (r≥0.999 3), respectively. Furthermore, the limits of detection were 5.57 ng/mL, 4.10 ng/mL, and 5.80 ng/mL, respectively, while the limits of quantitation were 22.26 ng/mL, 12.30 ng/mL, and 23.20 ng/mL, respectively. The results demonstrated good precision, repeatability, and sample stability. Spike recoveries were 91%~105%(δ_RSD≤5.0%,n=6). This method is simple, accurate, and highly sensitive, which is suitable for the simultaneous determination of calycosin 7-O-glucoside, hesperidin, and ammonium glycyrrhizinate in Buzhongyiqi Pills.

Key words: UPLC-Q/Orbitrap HRMS, Buzhongyiqi Pills, calycosin 7-O-glucoside, hesperidin, ammonium glycyrrhizinate

中图分类号:

R284.1

吴丹, 黄碧云, 王静, 刘龙. 超高效液相色谱-四级杆/静电场轨道阱高分辨质谱法同时检测补中益气丸中毛蕊异黄酮葡萄糖苷、橙皮苷、甘草酸铵的含量[J]. 山东科学, 2023, 36(2): 16-22.

WU Dan, HUANG Biyun, WANG Jing, LIU Long. Simultaneous determination of calycosin 7-O-glucoside, hesperidin, and ammonium glycyrrhizinate in Buzhongyiqi Pills via UPLC-Q/Orbitrap HRMS[J]. Shandong Science, 2023, 36(2): 16-22.

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名称	分子式	保留时间/min	理论质量数	实际定量质量数	质量偏差
毛蕊异黄酮葡萄糖苷	C₂₂H₂₂O₁₀	5.75	447.128 6[M+H⁺]	447.128 2	0.9×10^-6
橙皮苷	C₂₈H₃₄O₁₅	6.34	611.197 0[M+H⁺]	611.196 8	0.3×10^-6
甘草酸铵	C₄₂H₆₅NO₁₆	8.08	823.411 1[M-NH₃+H⁺]	823.410 6	0.6×10^-6

待测成分	回归方程	相关系数r	线性范围/(μg·mL^-1)
毛蕊异黄酮葡萄糖苷	Y=5.83×10⁶X+2.98×10⁶	0.999 6	0.09~1.43
橙皮苷	Y=1.81×10⁷X+2.57×10⁶	0.999 3	0.09~12.60
甘草酸铵	Y=3.46×10⁶X+2.46×10⁷	0.999 4	7.43~29.70

待测成分	称样量/g	样品中含量/μg	加入量/μg	检出量/μg	回收率/%	平均回收率/%	δ_RSD/%
	0.504	41.11	32.4	67.88	92.34
毛蕊异黄酮葡萄糖苷	0.501	40.87	41.3	75.28	91.61	96.32	4.61
	0.510	41.60	49.2	87.51	96.38
	0.504	78.48	62.0	128.10	91.19
橙皮苷	0.501	78.02	78.5	165.02	105.43	94.95	4.73
	0.510	79.42	93.8	162.23	93.66
	0.504	1 486.94	1 192.0	2 801.10	104.56
甘草酸铵	0.501	1 478.09	1 490.0	2 741.32	92.36	95.56	4.91
	0.510	1 504.64	1 785.0	3 358.03	102.08

批号	毛蕊异黄酮葡萄糖苷	橙皮苷	甘草酸铵	厂家
202201194	81.57	155.72	2 950.27	九芝堂股份有限公司
211004	51.20	1 050.96	2 991.62	仲景宛西制药股份有限公司
20220123	44.35	152.32	2 953.11	江西药都樟树制药有限公司