Analysis of β-agonists in different biological matrices by liquid chromatography–tandem mass spectrometry

Analyte	Retention time (min)	Precursor ion (m/z)	Ion transition	Declustering potential	Collision energy (eV)
Brombuterol	4.2	366.8	213.9	56	43
			292.8		47
Clenbuterol	4.1	276.8	203.0	76	23
			132.1		47
Isoxsuprine	4.3	302.2	150.0	61	31
			107.0		37
			237.0		25
Mabuterol	4.3	310.9	217.0	20	35
			237.0		25
Mapenterol	4.5	324.9	217.0	146	33
Salbutamol	3.2	239.9	148.0	131	25
			166.1		19
Ractopamine	4.0	301.9	164.1	16	23
			136.1		31
Terbutaline	3.2	225.9	152.1	121	23
			125.1		35
Zilpaterol	3.1	261.9	185.0	26	35
			202.0		27
Clebuterol-D₉	4.1	285.9	204.0	31	25
Isoxsuprine -D₅	4.3	307.2	150.0	61	23
Mabuterol-D₉	4.3	319.9	238.0	76	25
Mapenterol-D₁₁	4.5	335.9	238.0	76	25
Ractopamine-D₆	4.0	308.0	168.1	56	23
Salbutamol-D₆	3.2	248.9	148.0	136	27

Table 2

Concentration levels used for validation of the β-agonist detection method in different matrices

Analyte	drinking water μg kg⁻¹	feed μg kg⁻¹	liver μg kg⁻¹	urine μg kg⁻¹	muscle μg kg⁻¹	lung μg kg⁻¹
Brombuterol	0.1; 0.2; 0.4; 1.6	2.5; 5.0; 10.0; 40.0	0.1; 0.2; 0.4; 1.6	0.1; 0.2; 0.4; 1.6	0.05; 0.1; 0.2; 0.8	0.1; 0.2; 0.4; 1.6
Clenbuterol	0.1; 0.2; 0.4; 1.6	2.5; 5.0; 10.0; 40.0	0.1; 0.2; 0.4; 1.6	0.1; 0.2; 0.4; 1.6	0.05; 0.1; 0.2; 0.8	0.1; 0.2; 0.4; 1.6
Isoxsuprine	0.25; 0.5; 1.0; 4.0	2.5; 5.0; 10.0; 40.0	0.25; 0.5; 1.0; 4.0	0.25; 0.5; 1.0; 4.0	0.25; 0.5; 1.0; 4.0	0.25; 0.5; 1.0; 4.0
Mabuterol	0.1; 0.2; 0.4; 1.6	2.5; 5.0; 10.0; 40.0	0.1; 0.2; 0.4; 1.6	0.1; 0.2; 0.4; 1.6	0.05; 0.1; 0.2; 0.8	0.1; 0.2; 0.4; 1.6
Mapenterol	0.1; 0.2; 0.4; 1.6	2.5; 5.0; 10.0; 40.0	0.1; 0.2; 0.4; 1.6	0.1; 0.2; 0.4; 1.6	0.05; 0.1; 0.2; 0.8	0.1; 0.2; 0.4; 1.6
Salbutamol	2.5; 5.0; 10.0; 40.0	2.5; 5.0; 10.0; 40.0	2.5; 5.0; 10.0; 40.0	0.5; 1.0; 2.0; 8.0	2.5; 5.0; 10.0; 40.0	0.5; 1.0; 2.0; 8.0
Ractopamine	0.5; 1.0; 2.0; 8.0	2.5; 5.0; 10.0; 40.0	0.5; 1.0; 2.0; 8.0	0.5; 1.0; 2.0; 8.0	0.5; 1.0; 2.0; 8.0	0.5; 1.0; 2.0; 8.0
Terbutaline	5.0; 10.0; 20.0; 80.0	2.5; 5.0; 10.0; 40.0	5.0; 10.0; 20.0; 80.0	1.5; 3.0; 6.0; 24.0	5.0; 10.0; 20.0; 80.0	1.5; 3.0; 6.0; 24.0
Zilpaterol	2.5; 5.0; 10.0; 40.0	2.5; 5.0; 10.0; 40.0	2.5; 5.0; 10.0; 40.0	0.5; 1.0; 2.0; 8.0	2.5; 5.0; 10.0; 40.0	0.5; 1.0; 2.0; 8.0

Chromatograms of blank and spiked lung matrices with β-agonists at 0.2 μg kg⁻¹ for clenbuterol, mabuterol, mapenterol, brombuterol, and at 1.0 μg kg⁻¹ for salbutamol and zilpaterol, ractopamine, isoxsuprine, and terbutaline

Table 3

Validation report for β-agonists

Matrix	Repeatability (RSD_r,%)	Within-lab reproducibility (RSD_wR,%)	CCα (μg kg−¹)	CCβ (μg kg⁻¹)	Apparent recovery (%)	Concentration range (ng/mL)	Matrix effect (%)	Expanded uncertainty (μg kg⁻¹)
					Clenbuterol
drinking water	4.1 ± 3.8	4.5 ± 4.0	0.10	0.11	103.9 ± 4.3	0.1–1.6	4.3 ± 3.9%	0.20 ± 0.02
feed	6.2 ± 4.6	5.6 ± 6.1	2.50	2.70	104.5 ± 6.8	2.5–40.0	5.0 ± 3.9%	5.00 ± 1.14
liver	4.3 ± 4.1	5.1 ± 4.7	0.10	0.12	103.5 ± 6.2	0.1–1.6	6.1 ± 3.2%	0.20 ± 0.06
urine	4.6 ± 3.7	5.3 ± 4.4	0.10	0.11	102.6 ± 5.1	0.1–1.6	5.6 ± 2.7%	0.20 ± 0.04
muscle	5.9 ± 6.5	4.5 ± 7.6	0.05	0.06	109.1 ± 4.4	0.1–1.6	4.3 ± 6.5%	0.10 ± 0.02
lung	4.3 ± 3.8	4.8 ± 4.6	0.10	0.11	96.1 ± 4.9	0.1–1.6	5.3 ± 4.9%	0.20 ± 0.05
					Mabuterol
drinking water	3.7 ± 3.6	4.7 ± 3.9	0.10	0.11	99.5 ± 4.8	0.1–1.6	4.0 ± 3.5%	0.20 ± 0.03
feed	5.6 ± 5.4	6.7 ± 5.6	2.50	2.62	106.2 ± 7.8	2.5–40.0	6.0 ± 3.3%	5.00 ± 1.25
liver	5.9 ± 5.8	7.3 ± 6.3	0.10	0.12	95.4 ± 7.3	0.1–1.6	6.3 ± 5.2%	0.20 ± 0.05
urine	5.2 ± 4.9	6.3 ± 5.1	0.10	0.11	103.0 ± 4.1	0.1–1.6	5.4 ± 4.9%	0.20 ± 0.04
muscle	6.3 ± 4.1	6.4 ± 5.0	0.05	0.06	95.9 ± 6.0	0.05–0.8	5.8 ± 5.0%	0.10 ± 0.02
lung	5.9 ± 3.9	6.6 ± 5.6	0.10	0.11	93.2 ± 4.6	0.1–1.6	6.3 ± 4.6%	0.20 ± 0.04
					Mapenterol
drinking water	3.2 ± 3.4	4.1 ± 4.0	0.10	0.12	98.9 ± 4.8	0.1–1.6	4.0 ± 2.0%	0.10 ± 0.02
feed	5.4 ± 4.6	5.6 ± 7.1	2.50	2.61	105.1 ± 5.6	2.5–40.0	5.1 ± 2.9%	5.00 ± 1.14
liver	5.4 ± 5.0	6.4 ± 5.2	0.10	0.12	93.3 ± 5.6	0.1–1.6	5.4 ± 4.9%	0.20 ± 0.04
urine	5.3 ± 4.5	5.4 ± 4.9	0.20	0.12	102.0 ± 5.5	0.1–1.6	4.9 ± 5.1%	0.20 ± 0.04
muscle	4.1 ± 3.4	5.0 ± 4.2	0.05	0.06	97.0 ± 5.3	0.05–0.8	5.5 ± 4.1%	0.10 ± 0.02
lung	3.9 ± 3.7	5.4 ± 4.8	0.10	0.11	100.3 ± 6.1	0.1–1.6	4.8 ± 5.2%	0.20 ± 0.05
					Brombuterol
drinking water	3.1 ± 3.3	4.2 ± 4.1	0.10	0.12	95.4 ± 4.3	0.1–1.6	4.0 ± 3.6%	0.20 ± 0.05
feed	5.6 ± 4.1	6.2 ± 4.0	2.50	2.67	103.2 ± 6.8	2.5–40.0	5.3 ± 3.9%	5.00 ± 1.24
liver	4.9 ± 4.7	5.3 ± 4.9	0.10	0.11	96.5 ± 6.8	0.1–1.6	6.1 ± 4.8%	0.20 ± 0.05
urine	4.5 ± 3.9	5.1 ± 4.1	0.10	0.11	99.6 ± 5.2	0.1–1.6	5.1 ± 4.6%	0.20 ± 0.04
muscle	4.6 ± 4.7	5.4 ± 4.8	0.05	0.06	105.4 ± 5.4	0.05–0.8	4.5 ± 5.3%	0.10 ± 0.03
lung	4.9 ± 3.5	5.4 ± 4.6	0.10	0.12	98.2 ± 4.9	0.1–1.6	5.3 ± 3.6%	0.20 ± 0.05
					Ractopamine
drinking water	3.1 ± 3.7	4.6 ± 3.6	0.50	0.57	103.5 ± 3.9	0.5–8.0	3.9 ± 4.1%	1.00 ± 0.23
feed	4.4 ± 3.1	5.6 ± 4.6	2.50	2.56	102.3 ± 5.2	2.5–40.0	6.0 ± 3.2%	5.00 ± 1.35
liver	4.0 ± 4.3	4.6 ± 4.9	0.50	0.52	103.6 ± 6.8	0.5–8.0	5.3 ± 6.0%	1.00 ± 0.26
urine	4.2 ± 3.8	5.3 ± 4.2	0.50	0.52	102.8 ± 5.1	0.5–8.0	5.8 ± 5.3%	1.00 ± 0.23
muscle	4.6 ± 4.9	5.4 ± 5.3	0.50	0.52	105.0 ± 4.7	0.5–8.0	6.5 ± 5.7%	1.00 ± 0.16
lung	4.9 ± 4.5	5.4 ± 4.9	0.50	0.56	103.8 ± 4.3	0.5–8.0	5.5 ± 3.9%	1.00 ± 0.26
					Salbutamol
drinking water	4.2 ± 2.4	5.4 ± 4.0	2.50	2.54	104.9 ± 4.8	2.5–40.0	5.3 ± 3.1%	5.00 ± 1.25
feed	5.9 ± 4.6	6.5 ± 6.6	2.50	2.72	105.1 ± 6.9	2.5–40.0	6.0 ± 4.1%	5.00 ± 1.31
liver	5.3 ± 4.7	7.4 ± 5.2	2.50	2.61	93.3 ± 5.6	2.5–40.0	6.5 ± 5.4%	5.00 ± 1.45
urine	4.9 ± 4.8	6.4 ± 4.9	0.50	0.52	97.0 ± 6.3	0.5–8.0	5.4 ± 5.5%	1.00 ± 0.25
muscle	5.2 ± 3.3	5.7 ± 4.3	2.50	2.61	105.3 ± 4.6	2.5–40.0	6.5 ± 6.5%	5.00 ± 1.16
lung	4.9 ± 4.5	5.0 ± 4.6	0.50	0.52	101.5 ± 5.1	0.5–8.0	4.8 ± 5.6%	1.00 ± 0.33
					Zilpaterol
drinking water	3.2 ± 2.4	4.1 ± 3.0	2.50	2.58	105.4 ± 4.8	2.5–40.0	3.0 ± 2.1%	5.00 ± 1.19
feed	4.1 ± 4.5	5.9 ± 4.6	2.50	2.73	104.5 ± 6.3	2.5–40.0	5.3 ± 3.9%	5.00 ± 1.40
liver	6.4 ± 5.7	7.7 ± 6.2	2.50	2.79	107.3 ± 5.6	2.5–40.0	6.3 ± 6.3%	5.00 ± 1.52
urine	7.1 ± 4.3	8.0 ± 4.9	0.50	0.56	100.3 ± 6.8	0.5–8.0	5.3 ± 4.6%	1.00 ± 0.23
muscle	5.1 ± 4.3	5.9 ± 4.8	2.50	2.69	112.0 ± 6.1	2.5–40.0	7.3 ± 5.5%	5.00 ± 1.64
lung	4.0 ± 3.5	8.2 ± 7.6	0.50	0.56	106.4 ± 5.3	0.5–8.0	7.0 ± 4.3%	1.00 ± 0.45
					Terbutaline
drinking water	5.2 ± 4.4	6.0 ± 4.5	5.00	5.34	106.9 ± 3.8	5.0–80.0	5.1 ± 4.6%	10.00 ± 3.60
feed	6.2 ± 3.6	7.6 ± 3.9	2.50	2.70	107.5 ± 7.1	2.5–40.0	6.6 ± 4.3%	5.00 ± 1.72
liver	4.2 ± 4.7	6.1 ± 5.7	5.00	5.25	93.3 ± 5.1	5.0–80.0	5.7 ± 6.1%	10.00 ± 3.12
urine	6.1 ± 4.2	7.3 ± 5.0	1.50	1.54	102.0 ± 6.5	1.5–24.0	8.8 ± 4.7%	3.00 ± 0.86
muscle	3.4 ± 3.2	4.2 ± 3.6	5.00	5.63	105.3 ± 3.8	5.0–80.0	3.5 ± 3.5%	10.00 ± 4.1
lung	5.2 ± 3.5	6.0 ± 4.0	1.50	1.64	96.3 ± 5.1	1.5–24.0	6.3 ± 6.2%	3.00 ± 0.82
					Isoxsuprine
drinking water	3.6 ± 4.3	4.4 ± 5.1	0.25	0.27	102.9 ± 4.9	0.25–4.0	3.2 ± 2.3%	0.50 ± 0.09
feed	3.6 ± 3.7	4.6 ± 4.0	2.50	2.59	104.5 ± 2.8	2.5–40.0	5.0 ± 4.9%	5.00 ± 1.19
liver	3.8 ± 4.0	4.9 ± 5.2	0.25	0.26	102.3 ± 4.3	0.25–4.0	4.6 ± 4.5%	0.50 ± 0.11
urine	4.9 ± 3.8	5.4 ± 4.5	0.25	0.26	106.2 ± 4.6	0.25–4.0	5.3 ± 4.0%	0.50 ± 0.12
muscle	3.6 ± 3.8	4.3 ± 4.1	0.25	0.26	98.0 ± 4.3	0.25–4.0	4.5 ± 3.5%	0.50 ± 0.08
lung	3.6 ± 4.5	5.8 ± 5.6	0.25	0.27	108.0 ± 6.2	0.25–4.0	4.3 ± 5.0%	0.50 ± 0.12

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DOI: https://doi.org/10.2478/jvetres-2021-0058 | Journal eISSN: 2450-8608 (formerly 2300-3235)

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Language: English

Page range: 469 - 475

Submitted on: Apr 19, 2021

Accepted on: Oct 20, 2021

Published on: Nov 20, 2021

Published by: National Veterinary Research Institute in Pulawy

In partnership with: Paradigm Publishing Services

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© 2021 Tomasz Śniegocki, Bartosz Sell, Andrzej Posyniak, published by National Veterinary Research Institute in Pulawy
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Volume 65 (2021): Issue 4 (December 2021)

Analysis of β-agonists in different biological matrices by liquid chromatography–tandem mass spectrometry

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Table 1

Table 2

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Table 3

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