Accepted Manuscript Title: Validation of a confirmatory method of salbutamol in sheep hair by UPLC-MS/MS and its application to pharmacokinetic study Author: Suo Decheng Zhang Wei Zhang Yu Zhao Genlong Wang Ruigou Wang PeiLong Su Xiaoou PII: DOI: Reference:
S0731-7085(15)00232-0 http://dx.doi.org/doi:10.1016/j.jpba.2015.04.004 PBA 10039
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
31-1-2015 31-3-2015 2-4-2015
Please cite this article as: S. Decheng, Z.W.Z. Yu, Z. Genlong, W. Ruigou, W. PeiLong, S. Xiaoou, Validation of a confirmatory method of salbutamol in sheep hair by UPLCMS/MS and its application to pharmacokinetic study, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.04.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
2
A new method for determining salbutamol in animal hair was established.
3
The usefulness of different procedures for extraction of incurred hair
4
were evaluated.
5
The method was successfully applied to the pharmacokinetic study of
6
sheep hair.
us
cr
ip t
1
Ac ce p
te
d
M
an
7
Page 1 of 17
Validation of a confirmatory method of salbutamol in sheep hair by
8
UPLC-MS/MS and its application to pharmacokinetic study
9
Suo Decheng, Zhang Wei,Zhang Yu, Zhao Genlong, Wang Ruigou, Wang PeiLong, Su Xiaoou*
10
Institute of Quality Standards and Testing Technology for Agricultural Products, Chinese Academy of
11
Agricultural Science, Beijing 100081
12
*Corresponding author: Tel: +861082106307, Fax: +861082106580, E-mail: [email protected]
13
Abstract: A new method for determining salbutamol in hair of sheep by ultra-performance liquid
14
chromatography tandem mass spectrometry (UPLC-MS/MS) was established. Samples were extracted
15
with 0.1M of HCl solution. The mixture was heated to 60 °C in a water bath and kept at this
16
temperature for 4 h. The extracts were purified through SPE method and then dried with nitrogen.
17
Residues were redissolved in mobile phase. The target compound was determined by UPLC-MS/MS
18
with BEH-C18 column. The usefulness and feasibility of different treatment procedures of hair
19
containing salbutamol were evaluated. The range of linearity was 1–100 ng/g. The LOD was 0.3 ng/g,
20
and the LOQ was 1 ng/g. Recoveries were 89–106%, and coefficients of variation were 3.2–13.9%.
21
Pharmacokinetics of salbutamol was studied in healthy sheep after oral administration of 150 µg/kg
22
body weight salbutamol for 21 consecutive days. Salbutamol residues in hair were still detected after
23
21 days of administration.
24
Keys words: salbutamol, sheep hair, UPLC-MS/MS, pharmacokinetic study in sheep
28
Ac ce p
te
d
M
an
us
cr
ip t
7
29
efficiency in livestock by diverting nutrients from fat deposition to the production of muscle tissues
30
[4–6]. Given its potential adverse cardiovascular and central nervous system effects to consumers, SAL
31
was banned as feed additives for growth promotion in animals in China and the European Union [7, 8].
32
However, illegal use of SAL still occurs, which has already led to cases of potential risk for humans
33
after consumption of contaminated food.
25
26 27
1. Introduction
Salbutamol (SAL) [2-(Hydroxymethyl)-4-{1-hydroxy-2-[(2-methyl-2-propanyl)amino] ethyl}
phenol] , also known as albuterol, is a semi-selective β2-agonist commonly used in the therapy of asthma [1–3]. SAL is also used for the improvement of weight gain, carcass leanness, and feed
Page 2 of 17
34
The detection of veterinary drug residues is generally performed on urine and plasma samples taken from living animals as well as on tissue samples after slaughtering (muscle, liver, kidney, and fat).
36
However, multi-day studies performed in humans and rats with SAL have shown that urine and plasma
37
are matrixes unsuitable for detection of SAL abuse because of their low concentration and high
38
clearance rates after treatment discontinuation [9–12]. Hair is a suitable sample for veterinary drug
39
monitoring in livestock production because it provides drug stability and easy, simple, and fast sample
40
collection [13]. The study of cattle and human hair prove that hair is a good matrix to determine the
41
presence of β-agonist in living animal [14–18]. Previous research utilized clenbuterol. Moreover,
42
limited data are available regarding the depletion of SAL residues in hair. To the best of our knowledge,
43
only few report described the residue depletion of SAL in hair by enzyme-linked immunoassay and μ
44
FIA [19,20]. Although immunoassays for screening SAL residue in hairs have been reported, specific
45
and sensitive confirmatory methods for the identification and quantification of SAL in hair are required
46
by mass spectrometry.
Although GC-MS and LC-MS methods have been developed to identify SAL in different
48
samples [9, 20–23], to the best of our knowledge, no literature is available on the identification of SAL
49
in sheep hair. In this paper, a sensitive and reliable confirmatory method using ultra-performance liquid
50
chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed and validated for the
51
determination of SAL in sheep hair. The usefulness and feasibility of different procedures for the
52
57 58
were purchased from Fisher Technology Inc. (Muskegon, Michigan, USA).
59
β-glucuronidase/arylsulphatase was obtained from Merck (Darmstadt, Germany). Water was purified
60
using a Milli-Q Synthesis system from Millipore (Bedford, MA, USA). Bon Elut Plexa PCX (60 mg)
61
extraction cartridges were supplied by Agilent (Lake Forest, Illinois,USA). SAL was purchased from
62
Dr. Ehrenstorfer (Augsburg,Germany). SAL-D3, selected as IS, was purchased from Toronto Research
53 54 55 56
te
d
47
Ac ce p
M
an
us
cr
ip t
35
extraction and washing of hair containing SAL were evaluated. The proposed method was successfully applied to the pharmacokinetic study of sheep hair.
2. Experimental
2.1 Chemicals and Reagents
All reagents and solvents used were provided by Beijing Chemical company (Beijing, China)
unless otherwise mentioned. Liquid chromatography-grade methanol, acetonitrile, and formic acid
Page 3 of 17
63
Chemicals Inc. (North York, Canada). Stock solution of SAL and SAL-D3 was prepared in methanol at
64
a concentration of 1000 mg/L and was stored at −18 °C. This IS work solution of 50μg/L was diluted
65
from the stock solution by methanol.
66
2.2. Animal treatment and sample A total of 24 healthy sheep, weighing about 30 kg, were acclimated for 5 days under common
ip t
67
farm conditions. Specifically, 18 sheep received 150 µg/kg body weight SAL, which is a regular dose
69
as feed additive, corresponding to an oral administration of physiological saline solution containing 4
70
mg/mL SAL for 21 consecutive days. Six control animals did not receive any treatment. The hair
71
samples were collected at days 0, 7, 14, 21, 22, 23, 24, 26, 28, 31, 35, 38, and 42 after the first
72
administration and stored at 4 °C until analysis. The lengths of the collected hair segments were
73
typically from 1–2 cm up to about 6–7 cm.
74
2.3 Sample Treatment
an
us
cr
68
Hair was washed two times with 1% SDS solution, rinsed three times with purified water, and
76
dried in an oven at 50 °C. The dried hair was cut into small pieces (less than 5 mm) using scissors or an
77
oscillating mill (MM 400, Retsch Corp., Haan, Germany) and stored at 2−8 °C in a sealed glass
78
container until used.
79
2.4 Hair extraction
d te
Hair (100–500 mg) was placed in a centrifuge tube with 5 mL of 0.1 mol/L HCL solution after
86
Ac ce p
80
M
75
87
of methanol containing 5% ammonia hydrate. The eluate was evaporated to dryness under a stream of
88
nitrogen at 40 °C and reconstituted in 0.5 mL of 0.1% formic acid in water/acetonitrile (90:10, v/v).
89
Before UPLC-MS/MS analysis, each sample solution was passed through a 0.22 μm filter.
81 82 83 84 85
90 91 92
addition of 50 μL of IS work solution. The sample was briefly vortex-mixed and kept at 60 °C for 4 h. 2.5 Purifying procedure
After extraction, each sample was centrifuged at 8000 g for 5 min. The MCX SPE cartridges were
sequentially conditioned and equilibrated with 3.0 mL of methanol and 3.0 mL of water. Exactly 3 mL of the sample solution was loaded and passed through the cartridges at a low flow rate. The column was washed with 3.0 mL of water and 3.0 mL of methanol. Finally, the analyte was eluted with 3.0 mL
2.6 LC-MS/MS analysis Detection of SAL residues in hair samples was carried out with an Acquity UPLC equipped with a TQ mass spectrometer (Waters, Manchester, UK). Chromatographic separation was conducted with a
Page 4 of 17
BEH C18 column (150×1.0 mm i.d., 1.7 µm particles )( Waters, Milford, USA). The mobile phase was
94
constituted by solvent A (0.1% formic acid) and solvent B (acetonitrile). The flow rate was 0.3 mL/min
95
with linear gradient at the following conditions: 0–1 min 95% A, 1–4 min 55% A, and 4–5 min 95% A.
96
The injection volume was 10 µL. The TQ parameters used for the mass spectrometer were as follows:
97
ionization mode, electrospray positive ion mode; capillary voltage, 3.5 kV; cone voltage, 30 kV; source
98
temperature, 150 °C; desolvation temperature, 550 °C; cone gas (N2) flow rate, 30 L/h; dwell
99
time,0.25s and desolvation gas (N2) flow rate, 600 L/h. SAL and SAL-D3 were quantified by
100
monitoring one specific transition for each compound (Table 1). Multiple reaction monitoring
101
conditions were optimized with Intellistart Auto Tune Wizard Software by infusing solutions of pure
102
standards in methanol. All operations were controlled by Masslynx software version 4.1.
103
3. Results and Discussion
104
3.1 Method development
105
3.1.1 Washing and pre-treatment protocols
cr
us
an M
106
ip t
93
Given that animals can be exposed occasionally to environmental particles, urine, or feces containing chaff interferent as a consequence of authorized veterinary therapies, the hair samples were
108
routinely washed before analysis to remove lipids and other external contaminants. The washing
109
procedures generally described in forensic hair analysis include the use of EtOH, dichloromethane,
110
methylene chloride, 0.1% SDS, aqueous buffers with/out detergents (Tween 20), and water as
112 113 114 115 116 117
te
Ac ce p
111
d
107
additional rinse [13]. We evaluated five solutions (i.e., EtOH, dichloromethane, 0.1% SDS, aqueous buffers with Tween 20, and water) by determining the clearing degree of samples, lost of SAL in washing, repeatability of added SAL in hair, and ion suppression effects. Moreover, 0.1% SDS was adopted because it yielded more reproducible results and less ion suppression effects compared with other solutions.
3.1.2 Evaluation of Extraction Procedures by Hair Samples containing SAL Since the accumulation of organic drugs in hair was first described in the 1950s, four main
118
approaches were used to analyze drugs and to conduct extraction of analytes in hair (i.e., acid
119
extraction, alkaline extraction, enzymatic extraction, and organic solvent extraction) [13]. Previous
120
studies showed that blank hair spiked with drug reflects physiological conditions. However, this
121
finding is controversial and seems to depend on the nature of the drug. Given this criticism of spiked
Page 5 of 17
122
samples, the extraction efficiency of the most promising methods was further evaluated with positive
123
samples. The applicability of the different extraction strategies in positive samples should be evaluated
124
realistically (from animal experiments). Hair samples (100 mg) from sheeps that tested positive for SAL were extracted using five
126
different procedures. For the acid extraction (ACE), 5 mL of 0.1 mol/L HCL solution was added to
127
each hair sample, and the mixture was heated at 60 °C for 4 h. For the methanol extraction (ME), 5 mL
128
of methanol was added to each hair sample, and the mixture was kept at room temperature for 2 h. For
129
the alkaline extraction (ALE), 5 mL of 2 mol/L NaOH solution was added to each hair sample, and the
130
mixture was heated at 60 °C for 4 h. For the enzymatic extraction (EME), 5 mL of 25 mmol/L sodium
131
acetate buffer solution (pH 5.0) and 50 μL of β-glucuronidase/sulfatase were added to each hair sample
132
and were kept at 37 °C overnight (approximately 16 h). For the strong acid extraction (SACE), 5 mL of
133
1 M HCL solution was added to the hair samples, and the mixture was kept at 60 °C for 4 h.
cr
us
an
M
Fig 1 shows the result of different hair extraction procedures. The ALE method is commonly used in the analysis of agonist in hair [15, 18–20]. However, the drug recovery of this method is lower than
136
that of other methods. Alkaline extraction is a simple and rapid method that digests hair completely.
137
However, its main drawback is the occurrence of matrix interference for SAL. This phenomenon made
138
unequivocal and identification impossible. ME has the best recovery for added samples (data not
139
shown). However, ME cannot fully extract SAL in real hair because the binding ability of SAL to
140
145 146
in animal hair. Low acidic salvation avoids creating a complex matrix and protects SAL stability ,
147
enabling us to obtain a very pure extract, thus lowering the background chromatographic noise and
148
enhancing sensitivity.
149
3.2 Methods Validation
150
3.2.1 Linearity, LOD, and LOQ
141 142 143 144
151
te
d
135
Ac ce p
134
ip t
125
melanin is too strong for methanol to fully extract. Metabolites in urine and plasma were reported to conjugate with glucuronic acid and sulfate [22].
However, the contraction of ACE was more than that of EME, which could be caused by two reasons: conjugations with glucuronic acid and sulfate were degraded by HCL solutions or these conjugates did not enter hairs though plasma or urine. The difference between the results of the SACE and ACE methods was not statistically significant. However, in our opinion, ACE was the best treatment for SAL
Calibration curves were built by using samples spiked in the 1–100 ng/g range (n=3).and created
Page 6 of 17
152
after comparing the peak area ratio of the compounds to the corresponding IS. The linear equation was
153
Y=0.6291x+0.0715 with a correlation coefficient of 0.999.
154
The LOD was estimated to be 3 times the signal-noise ratio and was determined for all compounds by spiking non-contaminated hair with decreasing concentrations until equivalent response was
156
observed. The LOQ was estimated to be 10 times the signal-noise ratio and was determined for all
157
compounds by spiking non-contaminated hair with different concentrations until the concentration is
158
confidently determined and measured with acceptable imprecision (RSD ≤ 15%). The LOD and LOQ
159
were 0.3 and 1 ng/g, respectively.
160
3.2.3 Recovery and repeatability
cr
us
161
ip t
155
Recoveries were calculated by comparing the peak area ratio of the compounds to the corresponding contact IS in the sample（Table 2） Different blank samples were spiked with the
163
selected drug at 1, 10, 50, and 100 ng/g. Six replicates of each sample with different concentration were
164
analyzed, and the results are summarized in Table 2. The average recoveries of each compound ranged
165
from 87.0–106%. The imprecision was represented by the RSD, and these values are summarized, with
166
values of
S0731-7085(15)00232-0 http://dx.doi.org/doi:10.1016/j.jpba.2015.04.004 PBA 10039
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
31-1-2015 31-3-2015 2-4-2015
Please cite this article as: S. Decheng, Z.W.Z. Yu, Z. Genlong, W. Ruigou, W. PeiLong, S. Xiaoou, Validation of a confirmatory method of salbutamol in sheep hair by UPLCMS/MS and its application to pharmacokinetic study, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.04.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
2
A new method for determining salbutamol in animal hair was established.
3
The usefulness of different procedures for extraction of incurred hair
4
were evaluated.
5
The method was successfully applied to the pharmacokinetic study of
6
sheep hair.
us
cr
ip t
1
Ac ce p
te
d
M
an
7
Page 1 of 17
Validation of a confirmatory method of salbutamol in sheep hair by
8
UPLC-MS/MS and its application to pharmacokinetic study
9
Suo Decheng, Zhang Wei,Zhang Yu, Zhao Genlong, Wang Ruigou, Wang PeiLong, Su Xiaoou*
10
Institute of Quality Standards and Testing Technology for Agricultural Products, Chinese Academy of
11
Agricultural Science, Beijing 100081
12
*Corresponding author: Tel: +861082106307, Fax: +861082106580, E-mail: [email protected]
13
Abstract: A new method for determining salbutamol in hair of sheep by ultra-performance liquid
14
chromatography tandem mass spectrometry (UPLC-MS/MS) was established. Samples were extracted
15
with 0.1M of HCl solution. The mixture was heated to 60 °C in a water bath and kept at this
16
temperature for 4 h. The extracts were purified through SPE method and then dried with nitrogen.
17
Residues were redissolved in mobile phase. The target compound was determined by UPLC-MS/MS
18
with BEH-C18 column. The usefulness and feasibility of different treatment procedures of hair
19
containing salbutamol were evaluated. The range of linearity was 1–100 ng/g. The LOD was 0.3 ng/g,
20
and the LOQ was 1 ng/g. Recoveries were 89–106%, and coefficients of variation were 3.2–13.9%.
21
Pharmacokinetics of salbutamol was studied in healthy sheep after oral administration of 150 µg/kg
22
body weight salbutamol for 21 consecutive days. Salbutamol residues in hair were still detected after
23
21 days of administration.
24
Keys words: salbutamol, sheep hair, UPLC-MS/MS, pharmacokinetic study in sheep
28
Ac ce p
te
d
M
an
us
cr
ip t
7
29
efficiency in livestock by diverting nutrients from fat deposition to the production of muscle tissues
30
[4–6]. Given its potential adverse cardiovascular and central nervous system effects to consumers, SAL
31
was banned as feed additives for growth promotion in animals in China and the European Union [7, 8].
32
However, illegal use of SAL still occurs, which has already led to cases of potential risk for humans
33
after consumption of contaminated food.
25
26 27
1. Introduction
Salbutamol (SAL) [2-(Hydroxymethyl)-4-{1-hydroxy-2-[(2-methyl-2-propanyl)amino] ethyl}
phenol] , also known as albuterol, is a semi-selective β2-agonist commonly used in the therapy of asthma [1–3]. SAL is also used for the improvement of weight gain, carcass leanness, and feed
Page 2 of 17
34
The detection of veterinary drug residues is generally performed on urine and plasma samples taken from living animals as well as on tissue samples after slaughtering (muscle, liver, kidney, and fat).
36
However, multi-day studies performed in humans and rats with SAL have shown that urine and plasma
37
are matrixes unsuitable for detection of SAL abuse because of their low concentration and high
38
clearance rates after treatment discontinuation [9–12]. Hair is a suitable sample for veterinary drug
39
monitoring in livestock production because it provides drug stability and easy, simple, and fast sample
40
collection [13]. The study of cattle and human hair prove that hair is a good matrix to determine the
41
presence of β-agonist in living animal [14–18]. Previous research utilized clenbuterol. Moreover,
42
limited data are available regarding the depletion of SAL residues in hair. To the best of our knowledge,
43
only few report described the residue depletion of SAL in hair by enzyme-linked immunoassay and μ
44
FIA [19,20]. Although immunoassays for screening SAL residue in hairs have been reported, specific
45
and sensitive confirmatory methods for the identification and quantification of SAL in hair are required
46
by mass spectrometry.
Although GC-MS and LC-MS methods have been developed to identify SAL in different
48
samples [9, 20–23], to the best of our knowledge, no literature is available on the identification of SAL
49
in sheep hair. In this paper, a sensitive and reliable confirmatory method using ultra-performance liquid
50
chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed and validated for the
51
determination of SAL in sheep hair. The usefulness and feasibility of different procedures for the
52
57 58
were purchased from Fisher Technology Inc. (Muskegon, Michigan, USA).
59
β-glucuronidase/arylsulphatase was obtained from Merck (Darmstadt, Germany). Water was purified
60
using a Milli-Q Synthesis system from Millipore (Bedford, MA, USA). Bon Elut Plexa PCX (60 mg)
61
extraction cartridges were supplied by Agilent (Lake Forest, Illinois,USA). SAL was purchased from
62
Dr. Ehrenstorfer (Augsburg,Germany). SAL-D3, selected as IS, was purchased from Toronto Research
53 54 55 56
te
d
47
Ac ce p
M
an
us
cr
ip t
35
extraction and washing of hair containing SAL were evaluated. The proposed method was successfully applied to the pharmacokinetic study of sheep hair.
2. Experimental
2.1 Chemicals and Reagents
All reagents and solvents used were provided by Beijing Chemical company (Beijing, China)
unless otherwise mentioned. Liquid chromatography-grade methanol, acetonitrile, and formic acid
Page 3 of 17
63
Chemicals Inc. (North York, Canada). Stock solution of SAL and SAL-D3 was prepared in methanol at
64
a concentration of 1000 mg/L and was stored at −18 °C. This IS work solution of 50μg/L was diluted
65
from the stock solution by methanol.
66
2.2. Animal treatment and sample A total of 24 healthy sheep, weighing about 30 kg, were acclimated for 5 days under common
ip t
67
farm conditions. Specifically, 18 sheep received 150 µg/kg body weight SAL, which is a regular dose
69
as feed additive, corresponding to an oral administration of physiological saline solution containing 4
70
mg/mL SAL for 21 consecutive days. Six control animals did not receive any treatment. The hair
71
samples were collected at days 0, 7, 14, 21, 22, 23, 24, 26, 28, 31, 35, 38, and 42 after the first
72
administration and stored at 4 °C until analysis. The lengths of the collected hair segments were
73
typically from 1–2 cm up to about 6–7 cm.
74
2.3 Sample Treatment
an
us
cr
68
Hair was washed two times with 1% SDS solution, rinsed three times with purified water, and
76
dried in an oven at 50 °C. The dried hair was cut into small pieces (less than 5 mm) using scissors or an
77
oscillating mill (MM 400, Retsch Corp., Haan, Germany) and stored at 2−8 °C in a sealed glass
78
container until used.
79
2.4 Hair extraction
d te
Hair (100–500 mg) was placed in a centrifuge tube with 5 mL of 0.1 mol/L HCL solution after
86
Ac ce p
80
M
75
87
of methanol containing 5% ammonia hydrate. The eluate was evaporated to dryness under a stream of
88
nitrogen at 40 °C and reconstituted in 0.5 mL of 0.1% formic acid in water/acetonitrile (90:10, v/v).
89
Before UPLC-MS/MS analysis, each sample solution was passed through a 0.22 μm filter.
81 82 83 84 85
90 91 92
addition of 50 μL of IS work solution. The sample was briefly vortex-mixed and kept at 60 °C for 4 h. 2.5 Purifying procedure
After extraction, each sample was centrifuged at 8000 g for 5 min. The MCX SPE cartridges were
sequentially conditioned and equilibrated with 3.0 mL of methanol and 3.0 mL of water. Exactly 3 mL of the sample solution was loaded and passed through the cartridges at a low flow rate. The column was washed with 3.0 mL of water and 3.0 mL of methanol. Finally, the analyte was eluted with 3.0 mL
2.6 LC-MS/MS analysis Detection of SAL residues in hair samples was carried out with an Acquity UPLC equipped with a TQ mass spectrometer (Waters, Manchester, UK). Chromatographic separation was conducted with a
Page 4 of 17
BEH C18 column (150×1.0 mm i.d., 1.7 µm particles )( Waters, Milford, USA). The mobile phase was
94
constituted by solvent A (0.1% formic acid) and solvent B (acetonitrile). The flow rate was 0.3 mL/min
95
with linear gradient at the following conditions: 0–1 min 95% A, 1–4 min 55% A, and 4–5 min 95% A.
96
The injection volume was 10 µL. The TQ parameters used for the mass spectrometer were as follows:
97
ionization mode, electrospray positive ion mode; capillary voltage, 3.5 kV; cone voltage, 30 kV; source
98
temperature, 150 °C; desolvation temperature, 550 °C; cone gas (N2) flow rate, 30 L/h; dwell
99
time,0.25s and desolvation gas (N2) flow rate, 600 L/h. SAL and SAL-D3 were quantified by
100
monitoring one specific transition for each compound (Table 1). Multiple reaction monitoring
101
conditions were optimized with Intellistart Auto Tune Wizard Software by infusing solutions of pure
102
standards in methanol. All operations were controlled by Masslynx software version 4.1.
103
3. Results and Discussion
104
3.1 Method development
105
3.1.1 Washing and pre-treatment protocols
cr
us
an M
106
ip t
93
Given that animals can be exposed occasionally to environmental particles, urine, or feces containing chaff interferent as a consequence of authorized veterinary therapies, the hair samples were
108
routinely washed before analysis to remove lipids and other external contaminants. The washing
109
procedures generally described in forensic hair analysis include the use of EtOH, dichloromethane,
110
methylene chloride, 0.1% SDS, aqueous buffers with/out detergents (Tween 20), and water as
112 113 114 115 116 117
te
Ac ce p
111
d
107
additional rinse [13]. We evaluated five solutions (i.e., EtOH, dichloromethane, 0.1% SDS, aqueous buffers with Tween 20, and water) by determining the clearing degree of samples, lost of SAL in washing, repeatability of added SAL in hair, and ion suppression effects. Moreover, 0.1% SDS was adopted because it yielded more reproducible results and less ion suppression effects compared with other solutions.
3.1.2 Evaluation of Extraction Procedures by Hair Samples containing SAL Since the accumulation of organic drugs in hair was first described in the 1950s, four main
118
approaches were used to analyze drugs and to conduct extraction of analytes in hair (i.e., acid
119
extraction, alkaline extraction, enzymatic extraction, and organic solvent extraction) [13]. Previous
120
studies showed that blank hair spiked with drug reflects physiological conditions. However, this
121
finding is controversial and seems to depend on the nature of the drug. Given this criticism of spiked
Page 5 of 17
122
samples, the extraction efficiency of the most promising methods was further evaluated with positive
123
samples. The applicability of the different extraction strategies in positive samples should be evaluated
124
realistically (from animal experiments). Hair samples (100 mg) from sheeps that tested positive for SAL were extracted using five
126
different procedures. For the acid extraction (ACE), 5 mL of 0.1 mol/L HCL solution was added to
127
each hair sample, and the mixture was heated at 60 °C for 4 h. For the methanol extraction (ME), 5 mL
128
of methanol was added to each hair sample, and the mixture was kept at room temperature for 2 h. For
129
the alkaline extraction (ALE), 5 mL of 2 mol/L NaOH solution was added to each hair sample, and the
130
mixture was heated at 60 °C for 4 h. For the enzymatic extraction (EME), 5 mL of 25 mmol/L sodium
131
acetate buffer solution (pH 5.0) and 50 μL of β-glucuronidase/sulfatase were added to each hair sample
132
and were kept at 37 °C overnight (approximately 16 h). For the strong acid extraction (SACE), 5 mL of
133
1 M HCL solution was added to the hair samples, and the mixture was kept at 60 °C for 4 h.
cr
us
an
M
Fig 1 shows the result of different hair extraction procedures. The ALE method is commonly used in the analysis of agonist in hair [15, 18–20]. However, the drug recovery of this method is lower than
136
that of other methods. Alkaline extraction is a simple and rapid method that digests hair completely.
137
However, its main drawback is the occurrence of matrix interference for SAL. This phenomenon made
138
unequivocal and identification impossible. ME has the best recovery for added samples (data not
139
shown). However, ME cannot fully extract SAL in real hair because the binding ability of SAL to
140
145 146
in animal hair. Low acidic salvation avoids creating a complex matrix and protects SAL stability ,
147
enabling us to obtain a very pure extract, thus lowering the background chromatographic noise and
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enhancing sensitivity.
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3.2 Methods Validation
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3.2.1 Linearity, LOD, and LOQ
141 142 143 144
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te
d
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Ac ce p
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ip t
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melanin is too strong for methanol to fully extract. Metabolites in urine and plasma were reported to conjugate with glucuronic acid and sulfate [22].
However, the contraction of ACE was more than that of EME, which could be caused by two reasons: conjugations with glucuronic acid and sulfate were degraded by HCL solutions or these conjugates did not enter hairs though plasma or urine. The difference between the results of the SACE and ACE methods was not statistically significant. However, in our opinion, ACE was the best treatment for SAL
Calibration curves were built by using samples spiked in the 1–100 ng/g range (n=3).and created
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after comparing the peak area ratio of the compounds to the corresponding IS. The linear equation was
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Y=0.6291x+0.0715 with a correlation coefficient of 0.999.
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The LOD was estimated to be 3 times the signal-noise ratio and was determined for all compounds by spiking non-contaminated hair with decreasing concentrations until equivalent response was
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observed. The LOQ was estimated to be 10 times the signal-noise ratio and was determined for all
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compounds by spiking non-contaminated hair with different concentrations until the concentration is
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confidently determined and measured with acceptable imprecision (RSD ≤ 15%). The LOD and LOQ
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were 0.3 and 1 ng/g, respectively.
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3.2.3 Recovery and repeatability
cr
us
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ip t
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Recoveries were calculated by comparing the peak area ratio of the compounds to the corresponding contact IS in the sample（Table 2） Different blank samples were spiked with the
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selected drug at 1, 10, 50, and 100 ng/g. Six replicates of each sample with different concentration were
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analyzed, and the results are summarized in Table 2. The average recoveries of each compound ranged
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from 87.0–106%. The imprecision was represented by the RSD, and these values are summarized, with
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values of
