超高效液相色谱-串联质谱法研究亚硫酸氢钠穿心莲内酯在大鼠体内排泄

目的: 建立超高效液相色谱-串联质谱法测定大鼠尿液和粪便中亚硫酸氢钠穿心莲内酯(ASB)含量,并用于研究大鼠静脉注射ASB在尿液和粪便中的排泄。 方法: 采用甲醇提取生物样品中的ASB,以脱水穿心莲内酯(DAG)为内标,在Hypersil Gold C₁₈色谱柱(50 mm× 2.1 mm,1.9 μm)上以流动相甲醇-水梯度洗脱,流速0.2 mL·min^-1,柱温35 ℃,分析时间6 min;在电喷雾离子化电离源上以选择反应监测方式进行负离子检测,用于定量分析的离子反应分别为m/z 413.2→287.2(ASB)和m/z 331.2→303.3(DAG)。大鼠自尾静脉注射80 mg·kg^-1 ASB,收集并测定给药后各时段的尿液和粪便中ASB浓度,计算各时段尿液和粪便中累计排泄率。 结果: 测定ASB的线性范围为50~5000 ng·mL^-1,方法定量下限为50 ng·mL^-1。日内和日间精密度(RSD)分别在11.2%和13.3%以内,准确度分别在85.8%~101.4% 和87.9%~97.5%,提取回收率为96.1%~98.3%,基质效应为96.2%~98.1%。大鼠72 h尿液中累计排泄率为16.9%±3.9%,粪便中为34.1%±18.4%。 结论: 本方法速度快、专属性好、准确度高,适用于大鼠尿液和粪便中ASB含量测定,可应用于ASB大鼠排泄研究。

Objective: To establish an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of andrographolide sodium bisulphite (ASB) in rat urine and feces,and investigate excretion of ASB in rats after intravenous administration. Methods: ASB was extracted from biological samples using methanol and separated on a Hypersil Gold C₁₈ column(50 mm× 2.1 mm,1.9 μm)with gradient mobile phase consisting of methanol and water at a flow rate of 0.2 mL·min^-1.Dehydroandrographolide (DAG) was adopted as an internal standard.Under negative electrospray ionization mode,reaction monitoring of the precursor-product ion transitions m/z 413.2→287.2 was selected for ASB and m/z 331.2→303.3 for DAG.Urine and feces were collected at different time points after intravenously admonition of with ASB at a single dose of 80 mg·kg^-1. Results: The linear calibration curve was obtained in the concentration range of 50-5000 ng·mL^-1 with a minimal quantification limit of 50 ng·mL^-1.Intra-and inter-day precision was less than 11.2% and 13.3%,respectively.Intra-and inter-day accuracy fell in the ranges of 85.8%-101.4% and 87.9%-97.5%,respectively.The recovery ranged from 96.1% to 98.3%,and the matrix effects were between 96.2% and 98.1%.The cumulative amounts of ASB excreted in urine and feces within 72 h as parent drug were 16.9%±3.9% and 34.1%±18.4% of administered dose,respectively. Conclusion: The method is rapid,specific and accurate,which is suitable for ASB quantification in urine and feces and can be applied to ASB excretion study in rats.

1 MO Mei-xia(莫美霞).Clinical application of Lianbizhi injection(莲必治注射液的临床应用概况).J TCM Univ Hunan(湖南中医学院学报),2005,25(3):63
2 YI Jun(易军),LI Xiao-fang(李晓芳),TANG Pei-lian(汤佩莲),et al.HPLC determination of andrographolide sodium bisulfite(HPLC测定亚硫酸氢钠穿心莲内酯).Chin J Pharm Anal(药物分析杂志),2006,26(8):1158
3 HU Zhong-hui(胡中慧),WANG Quan-jun(王全军),LIAO Ming-yang(廖明阳).Nephrotoxicity of andrographolide injection and its safe use.Adverse Drug React J (药物不良反应杂志),2009,11(1):28
4 HU Zhong-hui(胡中慧),WU Chun-qi(吴纯启),WANG Quan-jun(王全军),et al.Acute and long-term toxicity studies on the two kinds of Lianbizhi injectas in male SD rats(两种莲必治注射液急性和长期毒性研究).J Toxicol (毒理学杂志),2010,24(6):433
5 JIN Jing(金晶),ZHAO Zhong-xiang(赵钟祥),YAN Ju-jiao(晏菊娇),et al.A study of the pharmacokinetics of andrograpolide sodium bisulphite in Beagle dogs(亚硫酸氢钠穿心莲内酯在Beagle犬体内的药动学研究).Her Med(医药导报),2005,24(2):109
6 YANG Gen-yuan(杨根元),CHEN Wan-ping(陈婉萍),WANG Dan(王旦),et al.HPLC determination of Lianbizhi injection and Lianbizhi for injection(莲必治注射液及注射用莲必治的HPLC测定).Chin J Pharm(中国医药工业杂志),2004,35(3):168
7 FDA.Guidance for Industry:Bioanalytical Method Validation.May 2001