1650 Yu et al.: Journal of AOAC International Vol. 94, No. 5, 2011
VETERINARY DRUG RESIDUES
Development of a Simple Liquid Chromatography-Tandem Mass Spectrometry Method for Multiresidue Determination of Antifungal Drugs in Chicken Tissues Ying Yu Beijing Center for Disease Control and Prevention, Beijing 100013, People’s Republic of China Beijing University of Chemical Technology, College of Science, Institute of Applied Chemistry, Beijing 100029, People’s Republic of China Jing Zhang Beijing Center for Disease Control and Prevention, Beijing 100013, People’s Republic of China Bing Shao1 Beijing Center for Disease Control and Prevention, Beijing 100013, People’s Republic of China Nanchang University, School of Life Sciences and Food Engineering, Nanchang 330031, People’s Republic of China Yongning Wu China Center for Disease Control and Prevention, Institute of Nutrition and Food Safety, Beijing 100085, People’s Republic of China Hejun Duan Beijing Center for Disease Control and Prevention, Beijing 100013, People’s Republic of China Hongtao Liu Beijing University of Chemical Technology, College of Science, Institute of Applied Chemistry, Beijing 100029, People’s Republic of China A method involving LC coupled with MS/MS (LC/MS/MS) was designed for simultaneous quantification of 10 antifungal drugs (voriconazole, griseofulvin, clotrimazole, bifonazole, econazole, ketoconazole, itraconazole, miconazole, terconazole, and fluconazole) in the liver and muscles of chickens. Homogenized tissue samples were extracted with acetonitrile and subsequently underwent freezing-delipidation. A Waters Acquity Ultra Performance LC BEH C18 column was used to separate the analytes, coupled with MS/MS using an electrospray ionization source. The accuracy of the method was confirmed with a mean recovery of 71–121%, and acceptable coefficients of variation (4–23%, n = 6). The detection capability of these compounds in two different matrixes was 0.50–2.82 µg/kg. This method can be applied for the screening and confirmation of target antifungal drugs in chicken tissues.
L
ivestock husbandry is associated with various pharmaceuticals used on a large scale to prevent or control microbial infection, increase meat
Received June 23, 2010. Accepted by JB February 10, 2011. 1 Corresponding author’s e-mail:
[email protected] DOI: 10.5740/jaoacint.10-244
production, and reduce cost. One of the most important classes of drugs is the antifungal agent group, commonly used in veterinary husbandry to treat various diseases caused by Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans (1). Based on their chemical structure, antifungal drugs can be classified as polyene antibiotics (e.g., amphotericin B, nystatin), fluorinated pyrimidine (5-fluorocytosine), azoles (miconazole, terconazole, and fluconazole) and allylamine derivatives (griseofulvin). Azoles and griseofulvin are widely applied for treating avian mycoses (2). Their inappropriate or illicit administration in veterinary husbandry may lead to highlevel residues of these drugs in edible tissues. It has been reported that azoles can induce adverse effects due to the inhibition of steroid synthesis in mammals (3), whereas griseofulvin is neurotoxic, hepatotoxic, and carcinogenic (4). Antifungal resistance is also a growing concern in birds and mammals, including humans (5, 6). In view of such potential harm to the health of consumers, sample detection and market surveillance are, therefore, important. There are many LC-based methods for determination of antifungal drugs in biosamples, including LC coupled with UV (4–20) or fluorescence detection (3, 21) and the LC/MS technique (22, 23). Most of these methods can be used to reliably quantify one or two drugs in plasma or serum (3, 7–12, 21). In the past few years, antifungal drugs have presented additional environmental and human safety concerns because pharmaceuticals and personal care products are increasingly being discarded
Yu et al.: Journal of AOAC International Vol. 94, No. 5, 2011 1651
Table 1. Instrument conditions for antifungal drug analyses LC conditions
Mobile phase
Fluconazole
Other 9 analytes
A: Water
A: Water
B: Acetonitrile Gradient list
MS conditions
Time (min) 0
2.5
2.6
B: Methanol 4.5
4.6
Time (min) 0
4.0
Total flow
0.3 mL/min
Column
Acquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm)
Ionization mode
ESI–
ESI+
Capillary voltage
3.0 kV
2.2 kV
Desolvation gas
550 L/h
Source temperature
100°C
Desolvation gas temperature
350°C
Multiplier voltage
650 V
RF lens 1
2.4
2.8
Aperture
0.5
0.8
RF lens 2
0.5 Ultra-high purity argon
Pressure of collision chamber
3.2 × 10–3 mbar
Entrance voltage
–1
Exit voltage
2
Experimental Apparatus (a) Homogenizer.—B-400 (Büchi, Postfach, Flawil, Switzerland). (b) Electronic balance.—BS110S (Sartorius Inc., Goettingen, Lower Sachsen, Germany).
8.1
0.5
Collision gas
into the environment (24–26). An LC/MS method that can simultaneously detect nine drug residues, including three antifungals, in wastewater was reported recently (25). Analytical methods for the determination of nystatin in rabbit tissues and amphotericin B in chicken livers were established using LC-UV by Groll et al. (19) and Echevarría et al. (20), respectively. However, few studies have focused on the analysis of residues of azoles in food of animal origin. Azole antifungal drugs are not listed in European Regulation 37/2010/EU (27) or in the U.S. Food and Drug Administration Code of Federal Regulations Title 21 (28). Many azoles are frequently used in poultry and other birds (2, 29), however, and clotrimazole, miconazole, and griseofulvin are authorized for veterinary purposes in China (30). Many antifungal drugs also might be used illicitly during poultry breeding, so developing a comprehensive detection method is necessary for surveillance. The aim of the present study was to develop a comprehensive method for the simultaneous determination of 10 antifungal drugs (including azoles and griseofulvin) in the liver and muscles of chickens.
8.0
(c) Centrifuge tubes.—50 mL (Code 2343-050, Asahi Glass Co. Ltd, Tokyo, Japan). (d) Centrifuge tubes.—1.5 mL (MCT-150-C, Axygen Scientific, Union City, CA). (e) Glass vials.—40 mL (La-Pha-Pack, Langerwerde, Germany). (f) Vortex mixer.—SA8 (Bibby Sterilin Ltd, Stone, UK). (g) Centrifuge.—Allegra™ X-22R (Beckman Inc., Fullerton, CA). (h) Nitrogen evaporator.—N-Evap™ 116 (Organomation Associates Inc., Berlin, MA). (i) LC system.—Acquity™-Ultra Performance LC (Waters, Milford, MA). (j) MS/MS system.—Micromass Quattro Ultima™ Pt, controlled by MassLynx Ver. 4.1 software (Manchester, New Hampshire, UK). (k) LC column.—BEH C18, 2.1 × 100 mm, 1.7 μm (Waters). (l) Water purification system.—Millipore-Elix-QE-QG (Millipore Inc., Billerica, MA). (m) Muffle furnace.—L 9/11/B 170 (Nabertherm Industrial Furnaces Ltd, Lilienthal, Bremen, Germany). Reagents (a) Methanol.—HPLC grade (Dima Technology Inc., Richmond Hill, VA). (b) Acetonitrile.—HPLC grade (Dima Technology Inc.). (c) Acetone.—HPLC grade (Dima Technology Inc.).
1652 Yu et al.: Journal of AOAC International Vol. 94, No. 5, 2011 Table 2. LC/MS/MS acquisition parameters for the determination of antifungal drugs Analyte Griseofulvin
Transition
Cone voltage, V Collision energy, eV
353.0 to 165.1
a
Retention time, min
Internal standard
44
17 17
2.35 ± 0.03
Voriconazole-d3
350.1 to 281.1a 350.1 to 127.2
35
14 27
2.54 ± 0.02
Voriconazole-d3
Voriconazole-d3
352.9 to 283.9
35
14
2.53 ± 0.02
Ketoconazole
531.1 to 112.1a 531.1 to 82.4
50
36 36
3.66 ± 0.02
353.0 to 285.1 Voriconazole
Ketoconazole-d8
Ketoconazole-d8
538.9 to 119.1
50
36
3.65 ± 0.04
Clotrimazole
277.7 to 166.3a 277.7 to 242.2
40
16 18
3.94 ± 0.02
Clotrimazole-d5
282.7 to 169.4
40
16
3.95 ± 0.02
Bifonazole
a
311.1 to 243.1 311.1 to 228.0
48
10 29
3.95 ± 0.02
—
Itraconazole
705.3 to 392.0a
58
32
4.04 ± 0.02
Itraconazole-d5
705.3 to 432.0
Clotrimazole-d5
31
Itraconazole-d5
710.9 to 397.0
58
32
4.04 ± 0.02
Econazole
381.3 to 125.1a 381.3 to 193.3
35
22 16
4.27 ± 0.01
—
Miconazole
417.1 to 160.9a 417.1 to 229.0
61
24 17
4.59 ± 0.02
—
Terconazole
531.9 to 219.2a
39
30 25
4.46 ± 0.06
Ketoconazole-d8 Fluconazole-d4
531.9 to 277.1 Fluconazole
305.1 to 191.0a 305.1 to 129.9
35
11 20
1.76 ± 0.01
Fluconazole-d4
308.9 to 194.0
35
11
1.76 ± 0.01
a
Ion was used for quantification.
(d) Ultra-pure water.—Made by a Milli-Q Water Purification System (Millipore Corp., Bedford, MA). (e) Sodium sulfate (anhydrous).—Analytical grade (Chemical Reagent Beijing Co., Ltd, Beijing, China). Baked at 600°C for approximately 3 h in the muffle furnace before use. (f) Standards.—Voriconazole [purity 99.5%, Toronto Research Chemicals (TRC) Inc., North York, ON, Canada]; griseofulvin (purity >95%, Sigma–Aldrich, St. Louis, MO); clotrimazole [purity 99%, National Institute for the Control of Pharmaceutical and Biological Products (nicpbp), Beijing, China]; bifonazole (purity >98%, NICPBP); econazole (purity 99.0%, Dr. Ehrenstorfer GmbH, Augsburg, Bayern, Germany); ketoconazole (purity 97%, NICPBP); miconazole (purity 99%, NICPBP); itraconazole (purity 99%, NICPBP); terconazole [purity 100.0%, United States Pharmacopeia (USP), Rockville, MD]; and fluconazole (purity 100.0%, USP). (g) Isotope-labeled internal standards (IS).—All the isotope-labeled standards were purchased from TRC: voriconazole-d3 (purity 99.5%); clotrimazole-d5 (purity 99.5%); ketoconazole-d8 (purity 99.5%); itraconazole-d5 (purity 99.5%); fluconazole-d4 (purity 99.5%).
Preparation of Standard Solutions (a) Stock standard solutions.—Individual stock standard solution (1 mg/mL) was prepared by dissolving 10 mg of each analyte in 10 mL methanol, as well as dissolving 1 mg of the isotope-labeled IS in 1 mL methanol. (b) Intermediate stock standard.—The intermediate stock standard was 10 mg/L, made by adding 100 µL of each stock solution to an individual 10 mL volume flask and diluting to the required volume with methanol. (c) Working mixed standard solutions.—Working solutions at serial concentrations (1.0, 2.0, 5.0, 10.0, 20.0, and 50.0 µg/L) were obtained by combining aliquots of intermediate stock solutions followed by subsequent dilution with methanol. (d) Working IS solution.—The working IS solution was 100 µg/L, made by combining 100 µL of each intermediate stock solution of the IS in an individual 10 mL flask and diluting to the required volume with methanol. All the standard solutions were stored in brown glass
Yu et al.: Journal of AOAC International Vol. 94, No. 5, 2011 1653
Figure 1. LC/MS/MS chromatograms of antifungal drugs in a spiked chicken liver sample (spiking level: 10 μg/kg, left) and a blank sample (right).
bottles. Stock standards were stable for ≥6 months at –20°C, whereas working solutions were prepared every week. Sample Preparation, Extraction, and Cleanup Samples of liver and muscle from chickens were bought in Beijing supermarkets, verified as being free of drugs,
and used as blank samples for method development and validation. Tissue samples were homogenized. Aliquots of 2.0 g samples were transferred into 50 mL centrifuge tubes and spiked with 200 µL working IS solution. For the recovery test and preparation of matrix-fortified calibration standards, an appropriate amount of working
1654 Yu et al.: Journal of AOAC International Vol. 94, No. 5, 2011 Table 3. Signal suppressiona of analytes in chicken liver extracts using different treatment procedures Acetonitrile extracted, %
Acetonitrile extraction followed by purification using an NH2 cartridge, %
Acetonitrile extraction followed by freezing filtration, %
Griseofulvin
77
0
0
Voriconazole
74
0
45
Ketoconazole
79
36
0
Clotrimazole
76
0
49
Bifonazole
98
57
86
Itraconazole
95
88
75
Econazole
95
75
83
Miconazole
94
91
56
Terconazole
96
29
68
Fluconazole
86
10
0
Analyte
a
Signal suppression (%) = (1 – Sm/S0) × 100%, where Sm is the slope of the matrix-matched standard curve and S0 is the slope of the pure standard curve.
a 1.5 mL Eppendorf tube and stored at –20°C overnight. It was then centrifuged at 15 000 rpm for 5 min at 4°C; aliquots of the supernatant were injected into the LC/MS/MS system.
mixed standard was spiked. An appropriate amount of anhydrous sodium sulfate (approximately 6–10 g) was added until free-flowing powder could be obtained after vortex mixing. A 10 mL volume of acetonitrile was added to the mixture, which was vortex-mixed for 2 min and then sonicated for 10 min at 40°C. After cooling to room temperature, the mixture was centrifuged at 10 000 rpm (7286 × g) for 10 min at 4°C. The supernatant was decanted into a 40 mL glass vial, and residues were extracted again with 10 mL acetonitrile. The supernatants were combined and evaporated to dryness under a gentle stream of nitrogen at 40°C. The residues were reconstituted with 1 mL methanol containing 0.1% (v/v) formic acid. The resulting solution was transferred into
LC/MS/MS Analyses Liquid chromatographic separation was done on a Waters Acquity Ultra Performance LC system, based on separation by an Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm) at 40°C. The injection volume was 5 μL. Sample extracts were analyzed by a Micromass Quattro Ultima Pt Mass Spectrometer equipped with an electrospray ionization (ESI) interface in selected reaction monitoring
Table 4. Slopes of neat standard calibration curves and different matrix-fortified calibration curves Chicken muscle Analyte
Internal standard
Standard
S1
Griseofulvin
Voriconazole-d3
0.224
Voriconazole
Voriconazole-d3
Ketoconazole
Ketoconazole-d8
Clotrimazole Bifonazole
Itraconazole-d5
a
Chicken liver
S2
S3
S1
S2
S3
RSD, %
0.351
0.225
0.297
0.259
0.253
0.352
19
0.733
0.843
0.663
0.759
0.789
0.755
0.852
8
0.309
0.331
0.324
0.376
0.23
0.321
0.319
14
Clotrimazole-d5
0.855
0.729
0.637
0.838
0.767
0.826
0.851
10
Clotrimazole-d5
5.539
3.879
5.277
8.894
10.548
5.884
5.118
37
8.125
15.099
21.745
13.531
24.701
12.143
13.917
37
Itraconazole
Itraconazole-d5
1.287
1.528
1.233
1.479
1.234
1.618
1.774
14
Econazole
Clotrimazole-d5
29.648
14.621
24.883
20.587
28.876
35.149
16.388
31
Itraconazole-d5
70.968
50.873
101.93
93.354
75.968
72.111
33.241
33
Miconazole
Clotrimazole-d5
5.762
7.374
1.897
5.169
13.334
17.328
19.128
66
Itraconazole-d5
14.575
22.677
7.981
14.498
21.126
35.129
30.530
46
Terconazole
Ketoconazole-d8
20.715
22.056
26.643
27.096
26.129
25.179
32.975
16
Fluconazole
Fluconazole-d4
0.187
0.170
0.152
0.163
0.171
0.168
0.169
6
a
S1, S2, and S3 indicate different tissue samples.
Yu et al.: Journal of AOAC International Vol. 94, No. 5, 2011 1655
Table 5. CCα and CCβ of the analytes in samples of chicken liver and chicken muscle Chicken liver
calculate the S/N at the time window in which the analyte is expected, then 3 times the S/N to be used as CCα. CCβ = CCα + 1.64 SD20 representative samples
Chicken muscle
Analyte
CCα, µg/kg
CCβ, µg/kg
CCα, µg/kg
CCβ, µg/kg
Griseofulvin
0.58
2.32
1.56
2.82
Voriconazole
0.45
1.25
0.26
0.68
Ketoconazole
0.51
1.44
0.47
1.07
Clotrimazole
0.14
2.11
0.46
1.61
Bifonazole
0.05
0.95
0.14
0.79
Itraconazole
0.94
2.10
0.76
1.83
Econazole
0.07
1.10
0.10
0.50
Miconazole
0.16
1.11
0.28
0.82
Terconazole
0.54
2.10
1.12
2.45
Fluconazole
0.33
1.41
0.34
0.89
mode. Nitrogen (purity, 99.9%) was the desolvation gas. LC and MS parameters are summarized in Table 1. The compound-dependent cone voltage and collision energy are shown in Table 2. Calculations Calibration curves for the target compounds (except for bifonazole, miconazole, and econazole) were obtained by carrying out a linear regression analysis on the ratio of standard solution areas to IS areas versus analyte concentrations from 1 to 50 μg/L with 20 μg/L IS. Quantification of bifonazole, miconazole, and econazole was based on matrix-fortified curves ranging from 1 to 25 μg/kg. Evaluation of the matrix effect was conducted using the strategy applied by Matuszewski et al. (31) with slight modification. Signal suppression was defined as 1 minus the ratio between the slope of the matrix-matched standard curves and the slope of the standard-solution curves, then multiplied by 100 to obtain a percentage. Absolute recoveries were used to evaluate the efficiency of sample preparation, and were calculated as the slope of matrix-fortified standard curves divided by the slopes of matrix-matched standard curves, and then multiplied by 100 to obtain a percentage. According to the literature, the zero-tolerance principle could be applied for drugs not subjected to legislation regarding maximum residue limits (32–34). According to European Commission Decision 2002/657/EC (35), the decision limit (CCα) and detection capability (CCβ) for nonpermitted substances were determined as follows: CCα = 3S/N20 representative blank samples analyzing 20 blank tissue samples per matrix to be able to
spiked at CCα level
Twenty control blank samples (liver and muscle) were analyzed to determine the specificity of the method by looking for interfering peaks within a 2.5% margin of the relative retention time of each compound. Results and Discussion LC/MS/MS Following the gradient profile shown in Table 1, the mobile phase compositions (i.e., water–methanol, water–acetonitrile, water containing 0.1% formic acid–methanol, and water containing 0.1% formic acid–acetonitrile) were optimized to achieve maximal sensitivity. Results indicated that higher sensitivity and good chromatographic behavior could be achieved under the ESI positive mode if water–methanol was used. Water–acetonitrile was more desirable for the ESI negative mode. MS tuning of the various analytes was done by flow+ injecting a standard into the mass spectrometer. [M+H] was the most abundant ion for most analytes, except – fluconazole and clotrimazole. [M-H] was found to be the dominant ion for fluconazole. The most prominent ion of clotrimazole was m/z 277.7, which originated from the insource collision-induced dissociation, and corresponded + to [M-C3H2N2] . These ions were chosen as precursor ions; product ions were subsequently optimized (Table 2). The boldfaced transition stated in Table 2 is selected for quantification, whereas the second, less-sensitive one is used for confirmation. For the IS, only one transition was monitored. Chromatograms of target compounds are given in Figure 1, which shows the reconstructed ion chromatograms of a blank sample and a spiked sample of chicken liver. This indicated good selectivity, because there were no interferences at the elution time of the target analytes. Sample Preparation The chicken liver matrix was used as the typical case for the development of the pretreatment method because of its complexity. Octanol–water partition coefficients TM in EPI SuiteTM (logKow; calculated by KOWWIN software (Ver. 3.11; U.S. Environmental ProtectionAgency, http://www.epa.gov/exposure/pubs/episuite.htm) for the 10 target drugs ranged from 0.58 (fluconazole) to 5.86 (miconazole), demonstrating a wide range of polarity. Methanol, acetonitrile, and acetone, thus, were initially used as extractants for screening purposes. Ten milliliters
1656 Yu et al.: Journal of AOAC International Vol. 94, No. 5, 2011 Table 6. Accuracy and precision of 10 target compounds from fortified processed chicken liver and chicken muscle
Analyte Griseofulvin
Voriconazole
Ketoconazole
Clotrimazole
Bifonazole
Itraconazole
Econazole
Miconazole
Terconazole
Fluconazole
Chicken liver Chicken muscle Spiking level, Accuracy, Precision, Accuracy, Precision, µg/kg % % % % 3
121
23
112
20
6
107
17
110
22
12
104
16
106
19
1.5
98
18
109
21
3
93
17
105
20
6
89
15
99
8
1.5
104
19
98
16
3
106
20
93
14
6
114
18
107
14
3
113
15
88
13 12
6
99
15
90
12
120
14
91
4
1.5
71
14
88
20
3
88
13
76
8
6
90
12
89
9
3
82
12
95
17
6
97
12
108
14
12
101
11
117
7
1.5
84
15
109
21
3
78
10
98
18
6
77
9
115
15
1.5
71
13
76
14
3
82
12
78
14
6
88
9
87
13
3
91
15
112
20
6
90
11
106
8
12
102
11
113
10
1.5
91
8
92
9
3
105
5
105
9
6
106
5
96
6
of acetonitrile, methanol, or acetone were added to 2 g aliquots of chicken liver with analytes spiked at 10 μg/kg. As a result, average recoveries for most target compounds at this concentration for a single extraction were 76–95, 55–93, and 14–84% for acetonitrile, methanol, and acetone, respectively. A second extraction with 10 mL acetonitrile was found to increase the recovery by 10%, while a third 10 mL extraction did not result in any significant improvement in recovery (50% of analytes, such as itraconazole, clotrimazole, and miconazole, were extracted into hexane due to their low polarity. We also carried out this experiment using fortified chicken liver extracts. The result was similar to that of the pure standard solution; thus, hexane was not suitable for the purification procedure. SPE is frequently applied for concentration and purification in the detection of trace-level analytes in complex matrixes (including food and environmental samples). In the present study, two RP SPE cartridges (C18 and HLB) and a normal-phase NH2 SPE cartridge were examined with respect to the matrix effect and recovery. For the former two cartridges, the condition solution was 6 mL methanol and 6 mL water; the eluting solution was 6 mL methanol. As well as for the NH2 cartridge, 6 mL methanol and 6 mL acetonitrile were used as the condition and elution solutions, respectively. The final extracts looked cleaner after SPE; however, several analytes such as fluconazole and terconazole were weakly retained on the C18 and HLB cartridges and resulted in poor recoveries (70%; Table 3). Freezing delipidation has been found to be an effective process to eliminate triacylglycerols and phospholipids, which are major lipids in meat tissue (36). This approach was assessed in the present study according to the difference in freezing points between lipids and antifungal drugs. After being frozen overnight at –20°C, the final extract solution exhibited significantly lower ion signal suppression with virtually no change in the recoveries of target compounds. In view of its simplicity and ability to provide a cleaner extract, the freezing-defattening step was included in the procedure despite the fact that it significantly increases the time for sample preparation. Method Validation Isotope dilution is a favorable method to compensate for the loss of target compounds in sample preparation and the ion suppression of the mass spectrometer. However, because it is difficult to obtain all isotopic standards for the
Yu et al.: Journal of AOAC International Vol. 94, No. 5, 2011 1657
Table 7. Intraday and interday reproducibility of 10 antifungal drugs in chicken liver Spiking level, 3.0 µg/kg Analyte
Spiking level, 6.0 µg/kg
Intraday precision, %
Interday precision, %
Intraday precision, %
Interday precision, %
Griseofulvin
19
21
16
19
Voriconazole
19
18
18
18
Ketoconazole
18
19
16
19
Clotrimazole
14
18
14
15
Bifonazole
15
20
14
15
Itraconazole
16
19
13
17
Econazole
13
19
13
15
Miconazole
16
20
11
13
Terconazole
18
19
13
14
Fluconazole
5
7
5
6
target drugs in multiresidual analyses, several compounds were sometimes analyzed with one isotopic standard. The standard curves in methanol and in different matrixes were compared with appropriately screened ISs. The slopes (Table 4) of most analytes in methanol and in different matrixes after cleanup were comparable RSDs ≤19% for seven of 10 compounds, indicating that the choice of ISs was appropriate. The ISs for bifonazole, econazole, and miconazole were not suitable because the RSDs of slopes were >30%. Thus, matrix-fortified curves were used to quantify these three drugs. Good linearity was obtained for all analytes, with correlation coefficients of r >0.995. CCα and CCβ of the analytes using this method were determined as described in the Experimental section, and ranged from 0.07 to 1.56 µg/kg and from 0.50 to 2.82 µg/kg, respectively (Table 5). The accuracy of the method was confirmed with a mean recovery evaluated by the spiked blank samples at three concentrations, with each condition carried out in six replicates. Five tissue pools were used in the test. The accuracy of the procedure ranged between 71 and 121% (Table 6), and the precision was represented by the RSD percentage at each fortification level for each compound; these values, summarized in Table 6, were
