Quantification of Uric Acid of Rat Serum by Liquid Chromatography-ultraviolet Detection and Its Comparison Study

doi:10.12300/j.issn.1674-5817.2022.189

Abstract

Abstract:

Objective To establish a more accurate and sensitive liquid chromatography-ultraviolet (LC-UV) method for the determination of uric acid in rat serum, and compare the results with those of commercial kits, providing a new method for the accurate determination of uric acid in the rat hyperuricemia model induced by potassium oxonate. Methods A hyperuricemia model was established by intraperitoneal injection of potassium oxonate (300 mg/kg) into SPF-grade male SD rats, and the control group was administered an equal amount of 0.5% sodium carboxymethylcellulose solution. Blood samples were collected from the posterior orbital venous plexus and centrifuged to obtain serum samples. After precipitation with 0.1% trifluoroacetic acid-acetonitrile (containing the internal standard 3,4-dihydroxybenzylamine hydrobromide), the supernatant was injected for analysis. Uric acid was separated on a Waters XBridge HILIC column (150 mm×4.6 mm, 3.5 μm) using acetonitrile (containing 0.5% formic acid and 2 mmol/mL ammonium formate) as the organic phase and methanol solution (methanol∶water=1∶1, containing 0.5% formic acid with 2 mmol/L ammonium formate) as the aqueous phase for isocratic elution and detection at 290 nm. Serum samples treated with activated carbon were used as substitute matrices for the methodological verification. Serum uric acid levels in rats with potassium oxonate-induced hyperuricemia were measured using the established LC-UV method and commercially available kits (uricase and phosphotungstic acid methods), and the accuracies of the three methods were compared. Results Serum uric acid showed a good linear relationship (R>0.999) at mass concentration of 10–200 μg/mL in rats, the lower limit of quantification was 10 μg/mL, the accuracy ranged from -2.17% to 2.21%, the intra-batch precision ranged from 0.52% to 1.95%, the inter-batch precision ranged from 3.04% to 4.90%, and the extraction recovery ranged from 83.12% to 89.91%. In the rat model, the results obtained using the commercially available phosphotungstic acid method kit were significantly higher than those of the LC-UV method, and those obtained using the commercially available uricase method kit were significantly lower than those of the LC-UV method, but the LC-UV method showed the best recovery of the spiked sample (95.90%–99.96%). Conclusion The LC-UV method developed in this study can determine the concentration of uric acid in rat serum with higher accuracy than commercially available kits and is recommended for the determination of serum uric acid in the rat model of hyperuricemia induced by potassium oxonate.

Key words: Uric acid, Liquid chromatography, Internal standard method, Hyperuricemia, Rats

CLC Number:

R-332

Ziyin XIA, Yuanyuan CHAI, Yunxia XU, Qinwei YU, Xin HUANG, Luyong ZHANG, Zhenzhou JIANG. Quantification of Uric Acid of Rat Serum by Liquid Chromatography-ultraviolet Detection and Its Comparison Study[J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 314-322.

Figures/Tables 7

Figure 1 Chromatogram of uric acid specificity verificationNote： A， 10 μg/mL uric acid standard solution； B， 50 μg/mL DHBA （3，4-dihydroxybenzylamine hydrobromide ） standard solution； C， Blank biomatrix； D， Lower limit of quantitation sample； E， Rat serum （without internal standard） in the control group （intraperitoneal injection of an equal amount of 0.5% CMC-Na solution）； F， Rat serum in the control group.

Figure 2 Standard curve of uric acid

Table 1 Intra- and inter-batch accuracy and precision, extraction recovery and stability test of uric acid determination

尿酸质量浓度 ρ/(μg·mL^-1)	批内准确度和精密度 Intra-A&P/% (n = 5)		批间准确度和精密度 Inter-A&P/% (n = 15)		提取回收率 Extraction recovery/% (n =5)			稳定性 Stability (RE)/%
尿酸质量浓度 ρ/(μg·mL^-1)	RE	RSD	RE	RSD	$x ¯ ± s$	RSD		4 °C (n =5)		FTC (n = 5)	RT (n = 3)	-20 °C (n = 3)	-80 °C (n = 3)
10	-1.99	2.42	-3.67	5.07
25	-2.17	1.95	-3.68	4.90	89.91±4.55	5.06	-3.12		-2.19		-11.47	-1.09	-9.51
100	0.74	1.54	1.15	3.04	84.42±1.61	1.90	-2.48		-2.23		-6.85	-5.01	-14.51
160	2.21	0.52	-0.56	3.67	83.12±5.63	6.77	-5.46		-4.57		-8.41	-6.74	-12.76

Table 1 Intra- and inter-batch accuracy and precision, extraction recovery and stability test of uric acid determination

尿酸质量浓度 ρ/(μg·mL^-1)	批内准确度和精密度 Intra-A&P/% (n = 5)		批间准确度和精密度 Inter-A&P/% (n = 15)		提取回收率 Extraction recovery/% (n =5)			稳定性 Stability (RE)/%
尿酸质量浓度 ρ/(μg·mL^-1)	RE	RSD	RE	RSD	$x ¯ ± s$	RSD		4 °C (n =5)		FTC (n = 5)	RT (n = 3)	-20 °C (n = 3)	-80 °C (n = 3)
10	-1.99	2.42	-3.67	5.07
25	-2.17	1.95	-3.68	4.90	89.91±4.55	5.06	-3.12		-2.19		-11.47	-1.09	-9.51
100	0.74	1.54	1.15	3.04	84.42±1.61	1.90	-2.48		-2.23		-6.85	-5.01	-14.51
160	2.21	0.52	-0.56	3.67	83.12±5.63	6.77	-5.46		-4.57		-8.41	-6.74	-12.76

Figure 3 Determination of serum uric acid concentrations in rats before and 1 h after administrationNote：（A） LC-UV method was used to detect the serum uric acid concentration of rats in the two groups before and 1 h after administration (CON, control group administered 0.5% CMC-Na solution; HUA, hyperuricemia group administered 300 mg/kg potassium oxyzate). Compared with the CON group, ???P<0.00 1; Compared to before administration, ###P<0.001. n = 6). (B-C) Serum uric acid concentration was measured using three methods before and 1 h after administration (LC-UV, liquid chromatography-ultraviolet; Kit-PTA, phosphotungstic acid method Kit; Kit-enzyme, uricase method Kit. Compared with LC-UV, ^P<0.05， ^^P<0.01， ^^^P<0.00 1. n = 6）.

Table 2 Comparison of the spiked recovery of LC-UV method and commercially available kits

取样时间/h Time/h	加样质量浓度/(μg·mL^-1) ρ/(μg·mL^-1)	加样回收率/% Recovery of spiked samples/%
取样时间/h Time/h	加样质量浓度/(μg·mL^-1) ρ/(μg·mL^-1)	LC-UV	磷钨酸法 Kit-PTA	尿酸酶法 Kit-enzyme
0^a	10	99.97±6.86	118.83±21.22	60.05±11.2
	25	95.90±4.54	117.90±8.48	84.31±4.31
1^a	10	98.18±3.23	125.00±5.34	27.37±8.62
	25	99.64±0.99	112.96±3.85	43.55±5.38

Table 3 Comparison of liquid chromatography-ultraviolet (LC-UV) method with phosphotungstic acid method and uricase method for determining serum uric acid

检测方法

Method

原理

Principle

特点

Features

局限性

Limitations

磷钨酸法

Phosphotungstic

acid method

尿酸在碱性溶液中与磷钨酸反应，生成尿囊素、二氧化碳和钨蓝，钨蓝的生成量与尿酸含量成正比

操作简单，检测时间短，成本低，可用于自动化分析

特异性差，准确度低，检测结果易受血清中其他还原性物质影响

液相色谱-紫外检测法

LC-UV

溶于流动相中的各组分经过固定相时，由于与固定相发生作用的大小不同，导致其保留时间不同

专属性强，准确度高，线性范围广，色谱柱可反复使用

成本较高、检测时间较长

尿酸酶法

Uricase method

尿酸在尿酸酶的作用下生成尿囊素、二氧化碳和过氧化氢，过氧化氢与显色剂反应显色

操作简单，检测时间短，成本低，可用于自动化分析

尿酸酶的活性易被血清中氧嗪酸钾抑制，准确度低

Figure 4 Structures of uric acid （A） and 3，4-dihydroxy-benzylamine hydrobromide （DHBA）（B）

References 21

1	孙琳, 王桂侠, 郭蔚莹. 高尿酸血症研究进展[J]. 中国老年学杂志, 2017, 37(4):1034-1038. DOI: 10.3969/j.issn.1005-9202.2017.04.112 .
	SUN L, WANG G X, GUO W Y. Research progress of hyperuricemia[J]. Chin J Gerontol, 2017, 37(4):1034-1038. DOI: 10.3969/j.issn.1005-9202.2017.04.112 .
2	FATHALLAH-SHAYKH S A, CRAMER M T. Uric acid and the kidney[J]. Pediatr Nephrol, 2014, 29(6):999-1008. DOI: 10.1007/s00467-013-2549-x .
3	XU C F, YU C H, XU L, et al. High serum uric acid increases the risk for nonalcoholic fatty liver disease: a prospective observational study[J]. PLoS One, 2010, 5(7): e11578. DOI: 10.1371/journal.pone.0011578 .
4	LIU J, XU C F, YING L M, et al. Relationship of serum uric acid level with non-alcoholic fatty liver disease and its inflammation progression in non-obese adults[J]. Hepatol Res, 2017, 47(3): E104-E112. DOI: 10.1111/hepr.12734 .
5	PERTICONE M, TRIPEPI G, MAIO R, et al. Risk reclassification ability of uric acid for cardiovascular outcomes in essential hypertension[J]. Int J Cardiol, 2017, 243:473-478. DOI: 10.1016/j.ijcard.2017.05.051 .
6	PRASAD M, MATTESON E L, HERRMANN J, et al. Uric acid is associated with inflammation, coronary microvascular dysfunction, and adverse outcomes in postmenopausal women[J]. Hypertension, 2017, 69(2):236-242. DOI: 10.1161/HYPERTENSIONAHA.116.08436 .
7	KUBOTA Y, MCADAMS-DEMARCO M, FOLSOM A R. Serum uric acid, gout, and venous thromboembolism: the atherosclerosis risk in communities study[J]. Thromb Res, 2016, 144:144-148. DOI: 10.1016/j.thromres.2016.06.020 .
8	KANBAY M, JENSEN T, SOLAK Y, et al. Uric acid in metabolic syndrome: from an innocent bystander to a central player[J]. Eur J Intern Med, 2016, 29:3-8. DOI: 10.1016/j.ejim.2015.11.026 .
9	SHARAF EL DIN U A A, SALEM M M, ABDULAZIM D O. Uric acid in the pathogenesis of metabolic, renal, and cardiovascular diseases: a review[J]. J Adv Res, 2017, 8(5):537-548. DOI: 10.1016/j.jare.2016.11.004 .
10	BORGHI C, AGABITI-ROSEI E, JOHNSON R J, et al. Hyperuricaemia and gout in cardiovascular, metabolic and kidney disease[J]. Eur J Intern Med, 2020, 80:1-11. DOI: 10.1016/j.ejim.2020.07.006 .
11	周子正, 徐琳, 高建东. 高尿酸血症动物模型研究进展[J]. 医学综述, 2020, 26(8): 1462-1466. DOI: 10.3969/j.issn.1006-2084.2020.08.002 .
	ZHOU Z Z, XU L, GAO J D. Research progress of hyperuricemia animal model[J]. Med Recapitul, 2020, 26(8): 1462-1466. DOI: 10.3969/j.issn.1006-2084.2020.08.002 .
12	王璟. 检测血尿酸的方法比较及进展[J]. 糖尿病临床, 2014, 8(8):362-363. DOI: 10.3969/j.issn.1672-7851.2014.08.005 .
	WANG J. Comparison and progress of methods for detecting serum uric acid[J]. Clin J Diabetes World, 2014, 8(8):362-363. DOI: 10.3969/j.issn.1672-7851.2014.08.005 .
13	WANG Q W, WEN X, KONG J M. Recent progress on uric acid detection: a review[J]. Crit Rev Anal Chem, 2020, 50(4):359-375. DOI: 10.1080/10408347.2019.1637711 .
14	GUMUSTAS M, KURBANOGLU S, USLU B, et al. UPLC versus HPLC on drug analysis: advantageous, applications and their validation parameters[J]. Chromatographia, 2013, 76(21):1365-1427. DOI: 10.1007/s10337-013-2477-8 .
15	WEN S S, ZHANG Z X, CHEN X P, et al. An improved UPLC method for determining uric acid in rat serum and comparison study with commercial colorimetric kits[J]. Acta Chromatogr, 2019, 31(3):201-205. DOI: 10.1556/1326.2018.00449 .
16	江民川, 赵阳, 马芳芳, 等. 尿酸检测方法的研究进展[J]. 化工科技, 2021, 29(3):75-79. DOI: 10.3969/j.issn.1008-0511.2021.03.014
	JIANG M C, ZHAO Y, MA F F, et al. Research progress on uric acid detection methods[J]. Sci Technol Chem Ind, 2021, 29(3):75-79. DOI: 10.3969/j.issn.1008-0511.2021.03.014 .
17	ZHAO J X. A simple, rapid and reliable high performance liquid chromatography method for the simultaneous determination of creatinine and uric acid in plasma and urine[J]. Anal Methods, 2013, 5(23):6781-6787. DOI: 10.1039/C3AY41061G .
18	BUSZEWSKI B, NOGA S. Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique[J]. Anal Bioanal Chem, 2012, 402(1):231-247. DOI: 10.1007/s00216-011-5308-5 .
19	张海晨, 李水军, 孙贺伟, 等. 液相色谱－串联质谱法测定尿酸及与常规检测方法的比较[J]. 检验医学, 2015, 30(5):422-426. DOI: 10.3969/j.issn.1673-8640.2015.05.004 .
	ZHANG H C, LI S J, SUN H W, et al. Liquid chromatography-tandem mass spectrometry for uric acid and its comparison with clinical routine determination method[J]. Lab Med, 2015, 30(5):422-426. DOI: 10.3969/j.issn.1673-8640.2015.05.004 .
20	刘旭圆, 商倩, 李川, 等. HPLC法用于小鼠高尿酸模型中血尿酸测定及相关药物评价[J]. 药物评价研究, 2017, 40(3):319-323. DOI: 10.7501/j.issn.1674-6376.2017.03.006 .
	LIU X Y, SHANG Q, LI C, et al. HPLC method for determination of uric acid in plasma of hyperuricemia model mice[J]. Drug Eval Res, 2017, 40(3):319-323. DOI: 10.7501/j.issn.1674-6376.2017.03.006 .
21	MCCALLEY D V. Managing the column equilibration time in hydrophilic interaction chromatography[J]. J Chromatogr A, 2020, 1612:460655. DOI: 10.1016/j.chroma.2019.460655 .

[1]	Liya ZHAO, Liju NI, Caiqin ZHANG, Jianping TANG, Yangzheng YAO, Yanyan NIE, Xiaoxue GU, Ying ZHAO. Establishing a Genetic Detection Protocol of Single Nucleotide Polymorphisms Panels in Inbred Rats Based on Multiplex PCR-LDR [J]. Laboratory Animal and Comparative Medicine, 2023, 43(5): 548-558.
[2]	Lingzhi YU, Jianyun XIE, Liping FENG, Xiaofeng WEI. Establishment of Fluorescence qPCR Method for Detection of Staphylococcus Aureus and Its Application in Feces Detection of Rats and Mice [J]. Laboratory Animal and Comparative Medicine, 2023, 43(5): 566-573.
[3]	Ying TAN, Wenping LIAO, Qilong GAO, Yong LI, Xinhui SHI, Jingkun WANG. Physiological Indexes and Histopathology Analysis of Sodium Iodate-Induced Retinitis Pigmentosa in Rats [J]. Laboratory Animal and Comparative Medicine, 2023, 43(2): 124-135.
[4]	Jian GE, Jingfen SUN, Yongjie WU. Taurine Has no Protective Effect on Rat Corneal Endothelial Cells Injured by Benzalkonium Chloride [J]. Laboratory Animal and Comparative Medicine, 2023, 43(1): 39-43.
[5]	Qin XU, Yan NI, Wenhui SHI, Jianying LI, Jiangwei LIU, Hongqiong ZHAO, Xinming XU. Analysis on Ileum and Colon Microflora of SPF Male SD Rats based on High-throughput Sequencing [J]. Laboratory Animal and Comparative Medicine, 2023, 43(1): 53-60.
[6]	Bin WU, Xu WANG, Dongxu FU, Yujun ZHU, Jinlu HUANG, Shunxing ZHU. Effect of the Traditional Chinese Medicine Shuganjieyu Formula on Constipation Type Irritable Bowel Syndrome and Brain-gut Axis in Rats [J]. Laboratory Animal and Comparative Medicine, 2022, 42(6): 551-559.
[7]	Chen GAO, Chunling FAN, Yurong LI, Wenjuan PEI, Caiping GUAN. Changes in Expression of Monocarboxylate Transporters in the Rat Cerebral Cortex after Exercise-induced Fatigue Under Simulated High-altitude Hypoxia and its Significance [J]. Laboratory Animal and Comparative Medicine, 2022, 42(5): 384-392.
[8]	Bingxin XU, Kaijian FAN, Tingyu WANG, Huijin CHEN. Effect of Dexamethasone on Cartilage Degeneration in Rats with Collagen-induced Arthritis [J]. Laboratory Animal and Comparative Medicine, 2022, 42(5): 416-422.
[9]	Huiyan QIN, Huafeng CHEN, Hui YANG, Hailan LUO, Weizhong FU, Qingbo LI, Jiehong ZHANG. The Capacity of Silkworm Cocoon Water to Mitigate the Level of Oxidative Stress in Aged Rats [J]. Laboratory Animal and Comparative Medicine, 2022, 42(5): 393-400.
[10]	Xiaorui ZHANG, Jing CAO, Qianqian WU, Jijun LIU, Guoyuan CHEN, Baojin WU. Effects of Probucol Formulations on Mesenteric Lymphatic Trans-port Efficiency and Pharmacokinetics in Rats [J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 275-283.
[11]	Sijia ZHAO, Xinyu HE, Quan JING, Lin MA, Chunlan GUO, Kuo WAN. Evaluation of Pain in Acute Pulpitis Hyperalgesia Model Rats [J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 333-341.
[12]	Yiru WANG, Xiaoying JIANG, Ruoxi DONG, Yibin PAN, Xianghui HAN, Yongqing CAO. Modified Method for Inducing Acute Intestinal Fibrosis in Rats Using 2,4,6-Trinitrobenzene Sulfonic Acid [J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 284-293.
[13]	Xiaorui ZHANG, Jing CAO, Qianqian WU, Kang KANG, Guoyuan CHEN, Baojin WU. A Preliminary Method for Continuous Drainage of Mesenteric Lymph Fluid in Rats [J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 267-274.
[14]	Dingshan FENG, Yeyu HUANG, Xiaoxin ZHANG, Aiqin WU, Zhan WANG, Linliang SU. Effects of Storage Time on Electrolyte Content and pH Value in Rat Serum Samples [J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 301-305.
[15]	Xiao LU, Lin ZHANG, Hui JI, Shanxiang JIANG. Efficacy of DZ1462, a Novel Sodium-phosphate Transporter Inhibitor, on 5/6 Nephrectomy-induced Hyperphosphatemia Model Rats [J]. Laboratory Animal and Comparative Medicine, 2022, 42(3): 187-193.