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,et al. 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. DOI: 10.12300/j.issn.1674-5817.2022.189.

Figures/Tables 7

Figure 1 Chromatogram of uric acid specificity verification
Note： 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 administration
Note：（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）

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