进出口动物源性食品中沃尼妙林及其同系物泰妙菌素残留量的测定方法_高效液相色谱串联质谱法_英文意见稿.doc

Foreword

Annex A, Annex B, Annex C, of is standard is informative annexes.
This standard was proposed by and is under the charge of China National Regulatory Commission for Certification and Accreditation.
The standard was drafted by Qin huangdao Entry-Exit Inspection and Quarantine Bureau of the People’s Republic of China.
The main drafters of this standard are Liu xiaomang et al.
This standard is a professional standard for entry-exit inspection and quarantine promulgated for the first time.

Determination of valnemulin and tiamulin Residues in foodstuffs of animal origin for import and export —LC-MS-MS method
1 Scope
This standard specifies the method of determination of valnemulin and tiamulin Residues in foodstuffs of animal origin for import and export.
This standard is applicable to the determination of valnemulin and tiamulin Residues in animal muscle, animal liver, fish ,egg and milk.
.
2 Normative reference
The following normative documents contain provisions which，through reference in this text，constitute provisions of this standard. For dated references，subsequent amendments to，or revisions of.,any of these publications do not apply. However protocol to agreements based on this standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below.For undated references，the latest edition of the normative document referred to applies.

GB/T 6682 Water for analytical laboratory use-Specification and test methods.
3 Principle
Valnemulin and tiamulin residues in the samples are extracted with acetonitrile, and cleaned by MCX solid phase extraction. Determined by LC/MS-MS(ESI+), quantified by external standard method.
4 Regents and materials
Unless otherwise specified ,all regents used are A.R. and “water” is deionized water.
4.1 Methanol：HPLC grade.
4.2 Acetonitrile: HPLC grade.
4.3 Formic acid: guarantee reagent.
4.4 n-Hexane.
4.5 Ammonia: 25%.
4.6 Sodium chloride.
4.7 anhydrous sodium sulfate: Ignite for 4 h at 650℃, cool to room temperature in desiccator and keep in a tightly closed container.
4.8 Formic acid-water（1+19，V:V): 5ml Formic acid(4.3) mix with 95ml water.
4.9 Ammonia-Methanol（1+19，V:V): 5ml Ammonia(4.5) mix with 95ml Methanol(4.1).
4.10 Acetonitrile-water（1+3，V:V): 25ml Acetonitrile(4.2) mix with 75ml water.
4.11 tandards: valnemulin, purity≥98%; tiamulin, purity≥98%
4.12 Valnemulin stock standard solutions(1.0mg/mL):Separately accurately weigh an adequate amount of valnemulin standards(4.11), dissolve in methanol and prepare a solution of 1.0mg/mL as the stock standard solutions. Solutions are stable for a year at –18 ℃.
4.13 tiamulin stock standard solutions(1.0mg/mL):Separately accurately weigh an adequate amount of tiamulin standards(4.11), dissolve in methanol and prepare a solution of 1.0mg/mL as the stock standard solutions. Solutions are stable for a year at –18 ℃.
4.14 Working standard solutions(1.0μg/ml): According to the requirement, pipette adequate amount of valnemulin stock standard solutions and tiamulin stock standard solutions, and dissolve in Acetonitrile-water(4.10) and prepare a solution of 1.0μg/mL. stored below 4℃ avoiding sunlight.
4.15 MCX solid phase extraction columns: 60mg, 3mL, or equivalent.
4.16 Membrane filter: 0.20μm.
5 Apparatus and equipment
5.1 LC-MS-MS: Equipped with ESI source.
5.2 Balance: 0.1mg and 0.01g sensitivity.
5.3 homogenizer:15 000r/min
5.4 shaker
5.5 Centrifuge:7 000r/min.
5.6 Rorary vacuum evaporator
5.7 Solid phase extraction.
5.8 Vacuum pump: Vacuum to 80 kPa.
6 Sample preparation and storage
5.1 animal muscle, animal liver, fish
All primary sample is reduced to 500g as the representative sample, which is blended and homogenized, and then divided into two equal portions. Each portion is placed in clean containers as the test sample, which is sealed and labeled. The test sample should be stored below in -18℃ avoiding sunlight. In the course of sample preparation, precautions should be taken to avoid contamination or any factors which may cause the change of residue content.
5.2 egg
All primary sample is reduced to 500g as the representative sample, which is shelled and homogenized, and then divided into two equal portions. Each portion is placed in clean containers as the test sample, which is sealed and labeled. The test sample should be stored below in 4℃ avoiding sunlight. In the course of sample preparation, precautions should be taken to avoid contamination or any factors which may cause the change of residue content.
5.3 milk
All primary sample is reduced to 500g as the representative sample, which is homogenized, and then divided into two equal portions. Each portion is placed in clean containers as the test sample, which is sealed and labeled. The test sample should be stored below in 4℃ avoiding sunlight. In the course of sample preparation, precautions should be taken to avoid contamination or any factors which may cause the change of residue content.

7 Procedure
7.1 Extraction
7.1.1 animal tissue, animal liver, fish
Weigh 2 g of test sample(accurate to 0.01 g) into a 50 mL plastic centrifuge tube, then add 5g anhydrous sodium sulfate(4.7) and 10 mL Acetonitrile into the centrifuge tube. Homogenize at 10 000 r/min for 2 min, then centrifuge at 4 000 r/min for 10 min. transfer the supernatant to a 100mL evaporated flask. extract again with 10 mL Acetonitrile. Combine the supernatants, evaporate to dryness at 40℃. The residue was dissolved with 5mL Formic acid-water(4.8), then it is ready for cleaned-up.
7.1.2 egg and milk
Weigh 5 g of test sample(accurate to 0.01 g) into a 50 mL plastic centrifuge tube, then add 2g Sodium chloride(4.6) and 10 mL Acetonitrile into the centrifuge tube. shake for 20 min, then centrifuge at 4 000 r/min for 10 min. Transfer 5mL supernatant to a evaporated flask, evaporate to dryness at 40℃. The residue was dissolved with 5mL Formic acid-water(4.8), then it is ready for cleaned-up.
7.2 Clean up
The MCX SPE cartridge(4.15) is conditioned with 3mL Ammonia-Methanol(4.9), 3mL methanol and 10 mL water in sequence. The above-mentioned extract solution is loaded, wash the cartridge with 3mL Formic acid-water(4.8), 3mL methanol and 3mL n-Hexane, discard the effluent. Dry the column with vacuum pump(5.5)for 10 minutes under 65 kPa Vacuum. Finally elute with 5mL Ammonia-Methanol(4.9) into test tube. Evaporate the elute solution to dryness under nitrogen at 40℃. Add accurately 1mL acetonitrile-water(4.10) to dissolve the residue. Filter with 0.2μm syringe filter and determination with LC/MS-MS.
7.3 Determination
7.3.1 HPLC operation conditions
a）Column: C18 1.7μm，50 mm×2.1 mm(i.d), or equivalent
b）Column temperature:35℃;
c）Injection volume:10μL;
d）Mobile phase:A: Formic acid-H2O (1+999)；B: Acetonitrile. Mobile phase and Flow rate rate see table 1
Table 1 Mobile phase and Flow rate
time/(min)
Flow rate /(µL /min)
A/%
B /%
0.00
1.1.1.1　 300
95
5
0.80
300
60
40
2.30
300
40
60
2.40
300
5
95
4.00
300
5
95

7.3.2 MS operation conditions
a) Ion source :ESI,
b) Scan mode:positive mode
c) Monitor mode: Multiple reaction monitoring(MRM)；
d) Other parameters are listed annex A.

7.3.3 LC-MS/MS determination
7.3.3.1 Qualitation determination
The qualitative ions for analyst include one precursor ion and two product ions at least. Under the same determination conditions, The ratio of the chromatographic retention time of the analyte to that of the external standard, i.e. the relative retention time of the analyte, shall correspond to that of the calibration solution at a tolerance of ± 2.5 %.The relative intensities of the detected ions of analyst, shall correspond to those of the calibration standard at comparable concentrations ,within the tolerances shown in table2, then the corresponding analyte must be present in the sample.
Table 2 Maximum permitted tolerances for relative ion intensities while confirmation

Relative ion intensities K
K＞50%
20%<K<50%
10%of the analyte. Quantify the sample solution by standard working curve .The responses of the analyte in the standard working solution and the sample solution should be within the linear range of the instrument detection . Quantified by external standard method. The MRM chromatogram of valnemulin and tiamulin are shown in Fig.B.1of annex B.
7.4 Blank test
The operation of the blank test is the same as that described in the method of determination but
with the omission of sample addition.

7.5 Calculation and expression of result
Calculate the results according to formula ( 1):
……………………………… (1)
where
X- the content of compound in the test sample, µg/kg;
A- the peak area of compound derivative in the sample solution,
A0－the peak area of compound derivative in the blank test;
c－the concentration of compound in the mixed standard working solution, ng / mL；
V－the final volume of sample solution, mL；
AS－the peak area of compound derivative in the mixed standard working solution;
m－the corresponding mass of test sample in the final sample solution, g。

8 Limit of determination and recovery
8.1 Limit of quantification
The limits of quantification of this method are 5.0 μg/kg respectively for valnemulin and tiamulin.
8.2 Recovery
The results of recoveries were showed on table C. 1 of annex C.

Annex A
(Informative annex)
LC-MS/MS conditions

LC-MS/MS conditions
a) Ion source :ESI,
b) Scan mode:positive mode
c) Monitor mode: Multiple reaction monitoring(MRM)；
d) Ionspray voltage：5400 V；
e) Curtain gas: 3.5 L/min;
f) Nebulizer gas: 6 L/min;
g) Auxiliary gas: 8 L/min；
h) Auxiliary gas temp：550℃；
k) Quality ions , quantity ions, Dwell time, retention time, Declustering potential and collision energy see table A.1.
table A.1.- MRM condition

compound
Quality ions/(m/z)
Quantity ions/(m/z)
Dwell time/(ms)
retention time/min
Declustering potential/(V)
Collision energy/(V)
tiamulin
494.30/192.10
494.30/192.10
80
1.98
234
25
494.30/119.00
38
valnemulin
565.30/263.10
565.30/263.10
80
2.09
100
25
565.30/285.20
29

1) Non-commercial statement; the equipments and their type AB SCIEX QTRAP 5500 involved in the standard method are not related to commercial aims, and the analysts are encouraged to equipments of different corporation or different type.
Annex B
（Informative annex）
MRM chromatogram of the standard

The MRM chromatogram of valnemulin and tiamulin standard see figure B.1.
tiamulin
valnemulin
图

Fig B.1.- MRM chromatogram of valnemulin and tiamulin

Annex C
（Informative annex）
The result of recovery in different matrix

The result of recovery in different matrix see table C.1.

Table C.1.-The data of recovery (n=10)
Compound

Chicken
Fish
Liver
Egg
Milk
Spiking level
/
(μg/kg)
Average recovery/%
Spiking level
/
(μg/kg)
Average recovery/%
Spiking level
/
(μg/kg)
Average recovery/%
Spiking level
/
(μg/kg)
Average recovery/%
Spiking level
/
(μg/kg)
Average recovery/%
Valnemulin
5.0
91.4
5.0
91.0
5.0
89.5
5.0
86.9
5.0
80.0
20.0
94.0
20.0
93.2
20.0
90.5
20.0
89.2
20.0
82.0
100.0
93.4
100.0
92.5
100.0
92.2
100.0
90.4
100.0
86.2
Tiamulin
5.0
92.2
5.0
91.2
5.0
87.5
5.0
89.2
5.0
79.9
20.0
93.5
20.0
93.5
20.0
89.5
20.0
92.5
20.0
80.3
100.0
94.0
100.0
93.2
100.0
90.5
100.0
92.6
100.0
87.0

9