超高效液相色谱-三重四极杆质谱法测定心脏组织中腺苷含量

doi:10.3724/SP.J.1123.2023.09016

摘要/Abstract

摘要：

采用超高效液相色谱-三重四极杆质谱(UPLC-MS/MS)建立了一种灵敏度高、特异性好,且能够快速准确检测心脏组织中腺苷含量的方法。将样品溶于含10 μmol/L稳定剂2-羟基-3-壬基腺嘌呤盐酸盐(EHNA)的1 mL超纯水中,低温研磨2 min, 60 Hz冰水浴超声萃取40 min。以甲醇和5 mmol/L醋酸铵溶液为流动相,流速为0.4 mL/min,柱温为40 ℃,进样量为3 μL,采用电喷雾离子源(ESI)正离子切换多反应监测(MRM)模式对心脏组织中的腺苷进行定性定量分析,以标准曲线外标法准确定量。结果显示,在检测范围内线性关系拟合较优,线性范围为0.1~160 ng/mL,相关系数(r²)为0.9930,检出限(LOD)为0.03 ng/mL,定量限(LOQ)为0.1 ng/mL;腺苷在鼠心脏组织低、中、高3个水平的加标回收率分别为113.6%、96.3%、102.9%,日内精密度为1.7%~8.4%,日间精密度为2.6%~7.4%。相关性和一致性分析结果表明,UPLC-MS/MS法和双抗体夹心法测量结果存在正偏倚,两种方法腺苷检测结果呈极显著的正相关(P<0.0001);采用该方法随机测定17份小鼠和17份大鼠心脏样品,小鼠和大鼠心脏组织中腺苷的含量范围为3.25~8.78 mg/kg和10.24~15.19 mg/kg,腺苷的平均含量分别为5.37 mg/kg和12.60 mg/kg。本研究为心脏组织腺苷含量的测定提供了一种简单、精确、高效的检测方法,可为临床研究和疾病诊断提供重要的技术支持。

关键词: 超高效液相色谱-三重四极杆质谱, 腺苷, 心脏组织

Abstract:

A method based on ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) was developed and validated for the rapid and accurate determination of adenosine (Ado) in cardiac tissues with high sensitivity and specificity. The samples were dissolved in 1 mL of ultrapure water containing 10 μmol/L 2-hydroxy-3-nonyladenine hydrochloride (EHNA) as a stabilizer, ground at low temperature for 2 min, and then ultrasonically extracted at 60 Hz in an ice-water bath for 40 min. Methanol and 5 mmol/L ammonium acetate solution were used as the mobile phases under a flow rate of 0.4 mL/min, a column temperature of 40 ℃ and an injection volume of 3 μL. The Ado in cardiac tissue was qualitatively and quantitatively analyzed by electrospray ionization (ESI) positive-ion-switching in multiple reaction monitoring (MRM) mode. A solvent standard curve and the external standard method were used for the accurate quantification of Ado. The results showed that the matrix effect of Ado in cardiac tissue was very low. A good linear relationship was obtained in the range of 0.1-160 ng/mL, and the correlation coefficient (r²) was 0.9930. The limits of detection (LOD) and quantification (LOQ) were 0.03 and 0.1 ng/mL, respectively. The spiked recoveries of Ado in murine cardiac tissue were 113.6%, 96.3%, and 102.9% at three spiked levels of low, medium, and high, respectively. The intra-day repeatability (RSDs) were 1.7%-8.4%, and the inter-day reproducibility (RSDs) were 2.6%-7.4%. Based on the correlation and consistency results, a positive bias was observed between the proposed UPLC-MS/MS method and the double-antibody sandwich method. Moreover, the Ado contents detected by these two methods were significantly positively correlated (P<0.0001). Cardiac tissue samples were collected from 17 mice and 17 rats and detected in our laboratory. The content ranges of Ado in the cardiac tissues of mice and rats determined by the developed UPLC-MS/MS method were 3.25-8.78 mg/kg and 10.24-15.19 mg/kg, respectively (average adenosine contents: 5.37 and 12.60 mg/kg, respectively). The developed method is simple, accurate, sensitive, and it is suitable for the determination of Ado in cardiac tissues. It also provides important technical support for cardiac clinical research and disease diagnosis.

Key words: ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS), adenosine (Ado), cardiac tissue

中图分类号:

O658

朱佐银, 郭文博, 赵含珂, 王杰, 杨俊花, 周新丽. 超高效液相色谱-三重四极杆质谱法测定心脏组织中腺苷含量[J]. 色谱, 2024, 42(4): 345-351.

ZHU Zuoyin, GUO Wenbo, ZHAO Hanke, WANG Jie, YANG Junhua, ZHOU Xinli. Determination of adenosine content in heart tissue by ultra performance liquid chromatography-triple quadrupole mass spectrometry[J]. Chinese Journal of Chromatography, 2024, 42(4): 345-351.

图/表 7

表 1 腺苷的保留时间和质谱参数

Table 1 Retention time and MS parameters of adenosine (Ado)

Sample	t_R/min	Precursor ion (m/z)	Product ions (m/z)	Cone voltage/V	Collision energies/V
Ado	3.01	268.129	119.098^*/136.133	28	40/16

图 1 腺苷在不同色谱柱和流动相条件下的MRM色谱图

Fig. 1 MRM chromatograms of Ado using different columns and mobile phases a. column: Waters XBridge BEH C18 (100 mm×3 mm, 2.5 μm); mobile phase: 5 mmol/L ammonium acetate-methanol; b. column: Waters Acquity UPLC BEH C18 (100 mm×2.1 mm, 1.7 μm); mobile phase: 5 mmol/L ammonium acetate-methanol; c. column: Waters XBridge BEH C18 (100 mm×3 mm, 2.5 μm); mobile phase: 0.1% formic acid aqueous solution-methanol.

图 2 采用不同提取剂时腺苷的MRM色谱图

Fig. 2 MRM chromatograms of Ado with different extraction solvents a. water; b. acetonitrile-water (20∶80, v/v); c. acetonitrile-water (80∶20, v/v); d. acetonitrile; e. 70% perchloric acid aqueous solution.

图 3 心脏组织中腺苷的含量水平

Fig. 3 Content levels of Ado in the heart tissue

图 4 两种腺苷测定方法的相关性检验

Fig. 4 Correlation test between two methods for Ado determination

图 5 两种腺苷测定方法的一致性检验

Fig. 5 Consistency test between two methods for Ado determination LOA: limits of agreement.

表 2 本文所建立的方法与文献报道的检测方法对比

Table 2 Comparison of the method developed in this study with assays reported in the publication

Method	Samples	LOD	Ref.
Electrochemical method	plasma and serum	1.0 mmol/L (0.27 ng/mL)	[14]
Electrochemical method	plasma	8.3 mmol/L (2.22 ng/mL)	[15]
Electrochemical method	in vivo	0.1 mmol/L (0.026 ng/mL)	[16]
Hydrogel aptasensor	serum and urine	0.09 mmol/L (0.024 ng/mL)	[17]
LC-UV	health foods	1000 ng/mL	[18]
UPLC-MS/MS	cordyceps products	0.01 ng/mL	[19]
HILIC-MS/MS	serum and urine	0.01 mmol/L (0.026 ng/mL)	[24]
UPLC-MS/MS	heart tissue	0.03 ng/mL	this work

参考文献

[1]	Ren S X.[MS Dissertation]. Xinxiang: Henan Normal University, 2017: 1
[1]	任韶霞.[硕士学位论文]. 新乡: 河南师范大学, 2017: 1
[2]	Wu M D, Liu X Y, Feng J N, et al. Chinese Journal of Applied Physiology, 2022, 38(1): 79
[2]	吴明达, 刘雪莹, 冯剑南, 等. 中国应用生理学杂志, 2022, 38(1): 79
[3]	Lü C Y, Zhang J Q, Tang Y, et al. Chinese Journal of Ultrasound in Medicine, 2020, 36(9): 805
[3]	吕创业, 张建琴, 唐颖, 等. 中国超声医学杂志, 2020, 36(9): 805
[4]	Yuan H M, Xue Z L, Yang X P, et al. Bulletin of Fermentation Science and Technology, 2019, 48(3): 150
[4]	袁红梅, 薛正莲, 杨心萍, 等. 发酵科技通讯, 2019, 48(3): 150
[5]	Cunha R A. J Neurochem, 2016, 139(6): 1019
[6]	Borea P A, Gessi S, Merighi S, et al. Trends Pharmacol Sci, 2016, 37(6): 419
[7]	Li X Y, Zhang Z M, Li W. World Latest Medicine Information, 2018, 18(48): 28
[7]	李翔云, 张泽明, 李薇. 世界最新医学信息文摘, 2018, 18(48): 28
[8]	Hao M L. Drug Standards of China, 2003, 4(6): 20
[8]	郝美玲.中国药品标准, 2003, 4(6): 20
[9]	Hu Y F, Huang Z Q, Li J F. Journal of Analytical Science, 2016, 32(6): 795
[9]	胡月芳, 黄志强, 李金芳. 分析科学学报, 2016, 32(6): 795
[10]	Sui Y, Guo T, Sun Y, et al. Chinese Journal of Hospital Pharmacy, 2005, 25(3): 232
[10]	隋因, 郭涛, 孙沂, 等. 中国医院药学杂志, 2005, 25(3): 232
[11]	El Nour K M, Mersal G. Int J Electrochem Sci, 2021, 16(9): 1
[12]	Yang Z X, Zhou Y S, Liao X J. Chinese Journal of Modern Drug Application, 2009, 3(3): 13
[12]	杨振新, 周燕双, 廖晓嘉. 中国现代药物应用, 2009, 3(3): 13
[13]	Gao Y L, Li Y, Zhang H X, et al. Strait Pharmaceutical Journal, 2009, 21(11): 67
[13]	高云玲, 李晔, 张河溪, 等. 海峡药学, 2009, 21(11): 67
[14]	Cao S, Zhao H, Chen K, et al. An Electrochemical Aptasensor Based on Multi-walled Carbon Nanotubes Loaded with PtCu Nanoparticles as Signal Label for Ultrasensitive Detection of Adenosine. (2023-06-15) [2023-09-15]. https://pubmed.ncbi.nlm.nih.gov/37121659
[15]	Yang H M, Hu P Y, Tang J, et al. J Solid State Chem, 2021, 25(48): 1383
[16]	Zhang D, Ma J J, Meng X W, et al. Anal Chim Acta, 2019, 1076: 55
[17]	Shen P Y, Jang K, Cai Z Y, et al. Microchim Acta, 2022, 189(11): 418
[18]	Zhang K H,. and Veterinary Medicine, Gansu Animal Husbandry 2017, 47(8): 27
[18]	张开慧. 甘肃畜牧兽医, 2017, 47(8): 27
[19]	Wang G Y. Brand Standardization, 2022(1): 90
[19]	王冠宇.品牌与标准化, 2022(1): 90
[20]	Hu S, Yuan M, Ge W J, et al. China Food Safety Magazine, 2021(26): 59
[20]	胡莎, 袁梦, 葛文静, 等. 食品安全导刊, 2021(26): 59
[21]	Lofgren L, Pehrsson S, Hagglund G, et al. PLoS One, 2018, 13(10): 1
[22]	Huszar E, Barat E, Kollai M. Chromatographia, 1996, 42: 318
[23]	Xu L, Huang X J, Luo X, et al. Lishizhen Medicine and Materia Medica Research, 2021, 32(4): 984
[23]	许莉, 黄晓婧, 罗霄, 等. 时珍国医国药, 2021, 32(4): 984
[24]	Zhang X X, Hu Y Q, Hong X J, et al. J Chromatogr B, 2022, 1209: 123428