Article pubs.acs.org/JAFC
Development of a HS-SPME-GC-MS/MS Method for the Quantitation of Thymol and Carvacrol in Bovine Matrices and To Determine Residue Depletion in Milk and Tissues Sara Armorini,† James E. Yeatts,§ Keena A. E. Mullen,⊗ Sharon E. Mason,# Elmira Mehmeti,Δ Kevin L. Anderson,§ Steve P. Washburn,⊗ and Ronald E. Baynes*,§ †
Department of Veterinary Medical Sciences, University of Bologna, 40064 Ozzano dell’Emilia, Bologna, Italy Department of Population Health and Pathobiology and ⊗Department of Animal Science, North Carolina State University, Raleigh, North Carolina 27607, United States # Department of Biological Sciences, Campbell University, Buies Creek, North Carolina 27506, United States Δ Food Safety and Veterinary Institute, Tirana, Albania §
ABSTRACT: Thymol and carvacrol may be present in several phytoceutical products but there are no well-defined methods to measure these compounds in meat and milk from treated animals. U.S. regulatory authorities deem their presence as an adulteration of food. A rapid and sensitive HS-SPME-GC-MS/MS method was developed for the detection of thymol and carvacrol in bovine milk, plasma, liver, kidney, and fat. Inter- and intraday precision values were all less than 15.7 and 20.2% for thymol and carvacrol, respectively. The accuracy was in ranges of 69.9−111.8% for thymol and 74.0−119.2% for carvacrol. With the exception of fat tissue, stability studies showed that both compounds are stable over a 2 month period. A pilot pharmacokinetic study was conducted to evaluate the developed analytical method and to provide initial estimates of thymol and carvacrol depletion in plasma, milk, and several tissues. Treatment of lactating dairy cattle with phytoceutical products containing these substances resulted in low but measurable residue levels at 96 h for liver and 36 h for milk with very short apparent plasma and milk half-lives (0.99) in the concentration ranges of 0.0005−1 μg/mL for milk and plasma, 0.0005−0.1 μg/mL for liver and kidney, and 0.005−1.0 μg/mL for fat. The method showed an adequate sensitivity that was suitable for quantitation of the compounds in the bovine matrices evaluated. The LOQ was 0.001 μg/mL for all matrices except fat, which was 0.01 μg/mL. Accuracy and Precision. Tables 1 and 2 show the results of intraday and interday precision and accuracy of thymol and
intraday (n = 5) precision (RSD%)
accuracy (%)
precision (RSD%)
milk
0.001 0.05 0.5
105.6 96.7 101.4
3.8 3.6 2.7
109.6 85.3 95.0
5.24 13.6 8.0
plasma
0.001 0.05 0.5
105.1 69.9 94.6
2.6 3.3 3.2
109.1 81.7 100.6
5.9 4.6 3.1
liver
0.001 0.05 0.1
111.8 101.5 107.8
10.3 9.8 6.9
106.7 102.3 96.9
11.8 1.3 0.8
kidney
0.001 0.05 0.1
100.4 95.1 94.9
15.6 1.3 5.0
102.9 92.0 104.4
14.0 4.0 1.6
fat
0.01 0.05 0.5
80.9 86.0 90.1
15.2 15.7 7.8
110.3 96.5 98.8
13.9 8.1 10.4
matrix
accuracy (%)
precision (RSD%)
accuracy (%)
precision (RSD%)
milk
0.001 0.05 0.5
107.7 90.9 95.3
5.2 2.8 3.5
109.6 85.3 95.0
5.24 13.6 8.0
plasma
0.001 0.05 0.5
101.6 74 95.9
2.3 2.5 2.7
105 84.7 100.8
4.6 2.0 3.5
liver
0.001 0.05 0.1
119.2 100.3 110.6
12.4 9.5 7.3
103.8 100.4 99.1
10.0 4.9 1.3
kidney
0.001 0.05 0.1
112.2 95.7 93.7
7.9 1.3 5.1
97.0 94.0 103.5
9.8 6.2 2.5
fat
0.01 0.05 0.5
82.3 82.4 93.3
17.3 20.2 6.4
117.0 95.9 98.0
17.7 14.0 11.0
for plasma,
