Determination of some antibiotic residues (tetracycline, oxytetracycline and penicillin-G) in beef sold for public consumption at Dukem and Bishoftu (Debre Zeyit) towns, central Ethiopia by LC/MS/MS

Figures & data

Table 1. Maximum residue limits for some common antibiotic drugs

Approved drugs	EU MRLs^a (µg/kg)	CAC MRLs^b (µg/kg)	U.S. Tolerance^c (µg/kg)	ADI^d
Penicillin G	50 (meat)	50 (meat)	50 (meat)	<30µg/person/day
Tetracyclines	100(muscle) 300 (liver) 600 (kidney)	200 (muscle) 600 (liver) 1200 (kidney)	2000 (muscle) 6000 (liver) 12000 (kidney)	0–30µg/kg bw

^aThe values of the MRLs were taken from the Commission Regulation (EU) No 37/2010.

^{b & d}The values of the MRLs and ADI were taken from the Codex CAC/MRL No. 02/2018.

^cThe values of the tolerance were collected from Baynes et al. (2016).

Figure 1. Total ion chromatogram (TIC) of blank sample extract.

Figure 2. TIC from single injections of standard extract of each analyte.

Table 2. Optimum mass parameters for each monitored ions in the analytical work

Analyte	RT (min)	Precursor ion [M+H]^+	Product ions [m/z]	Fragmentor (V)	Collisionenergy (eV)	Cell accelerator voltage
Oxytetracycline	7.75 ± 0.001	461.2	426.2	120	16	7
			443.4	120	8	7
Tetracycline	8.514 ± 0.001	445.1	410.2	120	15	7
			154.0	120	16	7
Penicillin G	13.42 ± 0.002	335.1	176.0	110	13	4
			160.1	110	5	4

RT, retention time; CE, collision energy; CAV, cell accelerator voltage.

Table 3. System suitability parameters of the analytical method

System suitability
	${\bar{\mathit{t}}}_{\mathit{R}}\pm \mathbf{\%}\mathit{R}\mathit{S}\mathit{D}$	$\bar{x}\pm \mathbf{\%}\mathit{R}\mathit{S}\mathit{D}$	Column Efficiency (N)*	Symmetry factor	Resolution** (Rs)
Oxytetracycline	7.75 ± 0.0002	307648 ± 2.12	72225	1.72	5.65
Tetracycline	8.51 ± 0.029	639224 ± 1.49	49113	1.70	2.20
Penicillin G	13.42 ± 0.000	10567 ± 0.98	560793	2.10	43.7

${\bar{\mathit{t}}}_{\mathit{R}}$ = Mean retention time; $\bar{x}$ = mean response signal; *Maximum value; **Minimum value.

*$N=5.54(t_R/w_h{)}^2$; **$R_s=1.18\left(t_{R2}-t_{R1}\right)/\left(w_{h1}+w_{h2}\right)$

Figure 3. TIC of (a) blank sample and (b) sample spiked with standard mix at MRLs.

Figure 4. Extracted ion chromatograms of blank sample showing no interfering peaks.

Figure 5. Positive control sample spiked with standard mix of the analytes at MRLs.

Note: In the figure (a) corresponds to extracted ion chromatogram for the quantifier; (b) represents overlaid chromatograms of the qualifier and quantifier ions with their ion ratio and (c) depicts the mass spectrum for the MRM transitions.

Table 4. Calibration data for OTC, TTC and Pen G residue analysis in Beef muscle

	Cal. Line (y = ax+b)
Analyte		Linearity range	Batch	Slope (a)	Y-intercept (b)	Coefficient of determination (R^2)
OTC		50–300 µg/kg	1	742	10760	0.978
			2	693	−12221	0.993
			3	661	7106	0.991
			Average	698.49	1881.8	0.9909
				y = 698.49x + 1881.8 R^2 = 0.9909
TTC		50–300 µg/kg	1	1463	−2091	0.982
			2	1398	−23178	0.993
			3	1450	−1282	0.991
			Average	1436.9	−8850.2	0.9919
				y = 1436.9x − 8850.2 R^2 = 0.9918
Pen G		25–150 µg/kg	1	48	408	0.973
			2	45	−121	0.988
			3	40	279.6	0.987
			Average	44.369	188.8	0.9934
				y = 44.369x + 188.8 R^2 = 0.9934

Table 5. Precision, accuracy, decision limits (CCα) and detection capabilities (CCβ)

Analyte	Spiked Conc. (µg/kg)	Amount recovered (µg/kg)	Repeatability (%RSD)	Within lab reproducibility (%RSD)	%Recovery	CCα (µg/kg)	CCβ (µg/kg)
OTC	50	49.43	5.41	16.40	98.86	109	118
	100	98.70	11.14	11.45	98.70
	150	147.34	3.36	5.23	98.23
TTC	50	55.67	4.30	5.52	111.34	109.6	119
	100	111.05	1.83	3.76	111.05
	150	147.83	3.46	6.07	98.55
Pen G	25	24.5	10.73	14.95	98.01	55.9	61.8
	50	50.64	4.36	4.74	101.27
	75	73.14	8.04	11.37	97.52

Table 6. Retention times acquired for OTC, TTC and Pen G reference standards

	Average Reference retention times (Rt) and Acceptance limits (ALs)
	Oxytetracycline (461.2→426.2, 443.4)		Tetracycline (445.1→410.2, 154.1)		Penicillin G (335.1→176.0, 160.1)
Run	Rt	ALs (±0.1 absolute)	Rt	ALs (±0.1 absolute)	Rt	ALs (±0.1 absolute)
Batch 1	7.759	7.659–7.859	8.522	8.422–8.622	13.421	13.321–13.521
Batch 2	7.755	7.655–7.855	8.519	8.419–8.619	13.419	13.319–13.519
Batch 3	7.749	7.649–7.849	8.508	8.408–8.608	13.421	13.321–13.521
Mean of all	7.754	7.654–7.854	8.516	8.416–8.616	13.420	13.32–13.52

Table 7. Relative ion ratios (IR) recorded for OTC, TTC and Pen G reference standards

Average Reference ion ratios (IR) and relative Acceptance limits (ALs)
Run	Oxytetracycline (461.2→426.2, 443.4)		Tetracycline (445.1→410.2, 154.1)		Penicillin G (335.1→176.0, 160.1)
	Reference Ion ratio	ALs (±25%)	Reference Ion ratio	ALs (±25%)	Reference Ion ratio	ALs (±20%)
Batch 1	31.56	23.67–39.45	23.46	17.60–29.33	90.67	72.54–108.8
Batch 2	31.18	23.39–38.98	23.75	17.81–29.67	90.72	72.58–108.86
Batch 3	32.28	24.21–40.35	25.05	18.79–31.31	91.20	72.96–109.44
Mean	31.67	23.75–39.59	24.09	18.07–30.11	90.86	72.69–109.03

Table 8. Oxytetracycline (OTC), Tetracycline (TC), and Pencillin G (PNG) residue levels in muscle samples in the study areas

Determined residue contents (µg/kg) by sample locations
Dukem				Bishoftu
Sample ID	OTC	TTC	PNG	Sample ID	OTC	TTC	PNG
Sample 1–5	ND*	ND	ND	Sample 51	18.18	ND	ND
Sample 6	24.8	ND	ND	Sample 52–62	ND	ND	ND
Sample 7	ND	ND	ND	Sample 63	18.41	ND	ND
Sample 8	21.0	ND	ND	Sample 64–69	ND	ND	ND
Sample 9–10	ND	ND	ND	Sample 70	19.73	ND	ND
Sample 11	26.1	9.35	ND	Sample 71–84	ND	ND	ND
Sample 12	18.9	ND	ND	Sample 85	7.63	ND	ND
Sample 13	ND	ND	ND	Sample 86–100	ND	ND	ND
Sample 14	23.4	ND	ND
Sample 15–20	ND	ND	ND
Sample 21	19.7	ND	ND
Sample 22–50	ND	ND	ND
Range (µg/kg)	18.9–26.1	-	-		7.63–19.73	-	-
Mean (µg/kg)	22.32	-	-		15.9875	-	-
SD	2.90	-	-		5.613	-	-
$ts/\sqrt{n}$	2.38	-	-		13.2	-	-
95% CI of mean value (µg/kg)	22.32±2.38	-	-		15.99±13.2	-	-
No. of samples (N)	50	50	50		50	50	50
% samples with residues	6 (12%)	1 (2%)	-		4 (8%)	-	-

Figure 6. Extracted ion chromatograms (EIC) of blank sample.

Figure 7. MRM of spiked sample with the three analytes (OTC, TTC and Pen G) at 100 μg/kg.

Commission Regulation (EU) No 37/2010. (2009, December 22). On pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. (OJL 15/1, 20.1.2010, pp. 1–72).

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