Development of an indirect competitive enzyme-linked immunosorbent assay for detecting flunixin and 5-hydroxyflunixin residues in bovine muscle and milk

Figures & data

Figure 1. Chemical structures of flunixin and 5-hydroxyflunixin.

Table 1. The MRLs set by different countries for flunixin residues in foods of animal origin.

Analyte	US	EU	Canada	Japan	China
Flunixin in bovine muscle (μg/kg)	25	20	20	20	–
5-Hydroxyflunixin in milk (μg/L)	–	40	6	60	40

- : No MRLs were set for target analytes.

Table 2. Optimisation of dilutions for coating antigen and antibody using checkerboard titration. The data shown in the table are absorbance values.

Antibody^a dilution	Flunixin concentration (ng/mL)	Coating antigen^b dilution
		1000	2 000	4000	8000
1 000	0	2.943	2.735	2.238	1.749
	10	1.611	1.437	1.313	0.907
2 000	0	2.387	1.872	1.382	1.108
	10	0.807	0.916	0.731	0.612
4 000	0	1.245	1.165	0.823	0.447
	10	0.674	0.714	0.411	0.215
8 000	0	0.965	0.759	0.571	0.438
	10	0.446	0.315	0.317	0.212

^aThe concentration of antibody was 2.4 mg/mL.

^bThe concentration of coating antigen was 3.6 mg/mL.

Figure 2. Effects of pH (A), ionic strength (B) of assay buffer, and acetonitrile tolerance (C) on the assay performance of icELISA.

Figure 3. Calibration curves of icELISA for flunixin and 5-hydroxyflunixin residue analysis. The data are average values of triplicate samples (average ± SD).

Table 3. IC₅₀ and cross-reactivity values using 5-hydroxyflunixin as a standard.

Analyte	Chemical Structure	IC50 (ng/mL)	Cross-Reactivity (%)
5-Hydroxyflunixin		0.29	100
Flunixin		1.43	20.3
Aspirin		>200	<0.1
Paracetamol		>200	<0.1
Ibuprofen		>200	<0.1
Celecoxib		>200	<0.1
Naproxen		>200	<0.1
Nabumetone		>200	<0.1
Diclofenac acid		>200	<0.1
Indometacin		>200	<0.1
Nimesulide		>200	<0.1

Figure 4. Evaluation of matrix effects of bovine muscle (A) and milk (B) on the calibration curves. Extract efficiency (C) from acidified incurred samples were calculated as the percentage of results obtained with the assay buffer.

Table 4. Average recovery and CV values for flunixin and 5-hydroxyflunixin detection in bovine muscle and milk (n = 3).

Analyte	Food matrix	Spiked concentration	Intra-assay		Inter-assay
			Recovery (%)	CVs (%)	Recovery (%)	CVs (%)
Flunixin	Bovine muscle	5^a	83	8.9	88	9.3
		10^a	87	5.8	94	10.2
		20^a	92	7.6	91	9.8
5-Hydroxyflunixin	Milk	2^b	81	8.5	85	11.3
		5^b	86	7.4	105	8.3
		10^b	97	6.8	92	9.6

^aThe unit of the spiked concentration is μg/kg.

^bThe unit of the spiked concentration is μg/L.

Figure 5. Correlation between icELISA and LC-MS/MS in detecting flunixin residues in bovine muscle (A) and 5-hydroxyflunixin residues in milk (B). The data are average values of triplicate samples (average ± SD).