Quantitative determination of tetracyclines in medicated feed for food-producing animals by HPLC–DAD

Figures & data

Table 1. Validation parameters studied for three target analytes (OTC, CTC, DC).

Validation parameter	n samples for each fortification level	Fortification levels (mg kg^−1)
Linearity range (r^2 > 0.99)	3	1–2–5–10–20
Targeted LOQ (mg kg^−1)	6	40
Trueness (recovery %)	6	40–200–500–1000
Intra-day precision	6	40–200–500–1000
Inter-day precision	18 (6 × 3 days)	40–200–500
Uncertainty	6	40–200–500–1000

Figure 1. (a) Chromatogram of blank sample and (b) chromatogram of the fortified sample at the concentration of 5 mg kg⁻¹. Distinctive peaks corresponding to each analyte are labelled accordingly.

Table 2. Validation parameters studied for trueness, repeatability and intra-laboratory reproducibility calculations for three target analytes (OTC, CTC, DC).

	Trueness (n = 6)				Repeatability (n = 6)			Intra-laboratory reproducibility (n = 18)
	Fortification level (mg kg^−1)	Mean recovery (%)	SD	CV (%)	Mean concentration (mg kg^−1)	SD	CV (%)	Mean concentration (mg kg^−1)	SD	CV (%)
OTC	40	110	0.5	0.4	49.4	0.2	0.4	49.5	0.2	0.5
	200	101	1.0	1.0	225.6	2.2	1.0	226.4	1.7	0.8
	500	111	0.7	0.6	624.0	3.7	0.6	613.1	12.6	2.0
	1000	110	0.9	0.8	1231.8	9.8	0.8	–	–	–
CTC	40	112	2.4	2.1	47.3	1.0	2.1	47.0	1.0	2.1
	200	100	0.8	0.8	211.5	1.4	0.7	213.3	3.6	1.8
	500	106	1.9	1.8	556.8	9.9	1.8	553.7	9.2	1.7
	1000	107	0.5	0.5	1127.0	5.5	0.5	–	–	–
DC	40	110	2.4	2.2	49.7	1.0	2.1	49.9	0.6	1.3
	200	101	0.5	0.5	227.1	1.1	0.5	227.4	1.8	0.8
	500	106	1.6	1.5	600.2	8.8	1.5	601.9	8.1	1.3
	1000	108	0.6	0.6	1222.7	6.9	0.6	–	–	–

Table 3. Evaluated uncertainty parameters.

Analyte	Maximum extended relative uncertainty U(ȳ)	Degree of freedom (ν)	Coverage factor (k)
OTC	15^a	5^a	2.00^a
	14.8^b	5^b	2.00^b
CTC	16.2^a	5^a	2.00^a
	15.7^b	5^b	2.00^b
DC	15.6^a	5^a	2.00^a
	15.2^a	5^b	2.00^b

Validation range for all analytes was 40–1000 mg kg⁻¹.

^a In single.

^b In double.

Table 4. Non-compliant tetracycline concentrations in analysed feed samples.

Year (n^a)	Medicated feed target species	Active substance	Declared dosage (mg kg^−1)	Quantified dosage (mg kg^−1 ± U(ȳr))
2021 (n = 34)	Swine	DC	400	275 ± 21
	Swine	CTC	800	278 ± 25
2022 (n = 30)	Swine	CTC	560	544 ± 88^b
		DC	Not declared	57 ± 9
2023 (n = 39)	Swine	DC	320	174 ± 26
	Swine	DC	480	352 ± 53
	Swine	CTC	2000	114 ± 18
	Swine	CTC	1000	677 ± 105
	Swine	CTC	2000	1207 ± 188
	Fish	OTC	7500	3890 ± 576

^aNumber of analysed samples during the specified year.

^b The declared dosage of chlortetracycline is within the 10% tolerance and is considered compliant.

Data availability statement

The data that support the findings of this study are available from the corresponding author, EB, upon reasonable request.