GB/T 21315-2007 English PDF (GBT21315-2007)

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GB/T 21315-2007: Determination of penicillin residues in foodstuffs of animal origin -- LC-MS/MS method
GB/T 21315-2007
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 67.120
X 04
Determination of penicillins residues in foodstuffs of
animal origin - LC-MS/MS method
ISSUED ON: OCTOBER 29, 2007
IMPLEMENTED ON: APRIL 01, 2008
Issued by: General Administration of Quality Supervision, Inspection and
Quarantine of the PRC;
Standardization Administration of the PRC.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative references ... 4 
3 Principles ... 4 
4 Reagents and materials ... 4 
5 Instruments ... 6 
6 Sample preparation and storage ... 6 
7 Determination procedures ... 6 
8 Calculation and expression of results ... 9 
9 Limit of quantitation and recovery rate ... 10 
Appendix A (Informative) Reference mass spectrometry conditions for mass
spectrometry/mass spectrometry determination ... 11 
Appendix B (Informative) Reconstructed ion chromatogram of quantitative ion
pairs of 11 penicillins standards ... 13 
Appendix C (Informative) Addition recovery rate ... 15 
Determination of penicillins residues in foodstuffs of
animal origin - LC-MS/MS method
1 Scope
This Standard specifies the determination and confirmation of LC-MS/MS
method for penicillins residues in foodstuffs of animal origin.
This Standard applies to the determination of 11 penicillins residues of
amoxicyllin, ampicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, gpenicillin,
penicillin, azlocillin, methicillin, phenethicillin in pig muscles, pig liver, pig
kidneys, milk, and eggs.
2 Normative references
The following documents contain provisions which, through reference in this
Standard, constitute provisions of this Standard. For the dated references, their
subsequent amendments (excluding corrections) or revisions do not apply to
this Standard. However, the parties who enter into agreement based on this
Standard are encouraged to investigate whether the latest editions of these
documents are applicable. For undated reference documents, the latest
editions apply to this Standard.
GB/T 6682-1992 Water for analytical laboratory use - Specification and test
methods (neq ISO 3696:1987)
3 Principles
The penicillins residues in the sample are extracted using acetonitrile-water
solution. After the extract is concentrated, USE a buffer solution to dissolve and
a solid-phase extraction cartridge to purify. After the eluent is blow-dried by
nitrogen, USE liquid chromatography-mass spectrometry/mass spectrometry to
determine; USE external standard method to quantify.
4 Reagents and materials
Unless otherwise stated, the reagents used are of analytical pure; and the water
is the Grade 1 water specified in GB/T 6682-1992.
WEIGH about 5 g (accurate to 0.01 g) of sample in a 50 mL centrifuge tube;
ADD 15 mL of acetonitrile-water solution (4.9); homogenize for 30 s;
CENTRIFUGE at 4000 r/min for 5 min; TRANSFER the supernatant to a 50 mL
centrifuge tube. TAKE another centrifuge tube; ADD 10 mL of acetonitrile-water
solution (4.9); WASH the homogenizer head. USE a glass rod to mash the
precipitate in the centrifuge tube; ADD the above-mentioned washing
homogenizer head solution; oscillate on a vortex mixer for 1 min; CENTRIFUGE
at 4000 r/min for 5 min. The supernatant is combined into a 50 mL centrifuge
tube. The head is washed repeatedly using 10 mL of acetonitrile-water solution
(4.9) and extracted once. The supernatant is combined into a 50 mL centrifuge
tube. USE acetonitrile-water solution (4.9) to dilute to 40 mL. Accurately
PIPETTE 20 mL into a 100 mL heart-shaped bottle.
7.1.2 Milk sample
WEIGH 10 g (accurate to 0.01 g) of sample in a 50 mL centrifuge tube; ADD 20
mL of acetonitrile (4.9); homogeneous extraction for 30 s; CENTRIFUGE at
4000 r/min for 5 min; TRANSFER the supernatant to a 50 mL centrifuge tube.
TAKE another centrifuge tube; ADD 10 mL of acetonitrile-water solution (4.9);
WASH the homogenizer head. USE a glass rod to mash the precipitate in the
centrifuge tube; ADD the above-mentioned washing homogenizer head solution;
oscillate on a vortex mixer for 1 min; CENTRIFUGE at 4000 r/min for 5 min.
The supernatant is combined into a 50 mL centrifuge tube. The head is washed
repeatedly using 10 mL of acetonitrile-water solution (4.9) and extracted once.
The supernatant is combined into a 50 mL centrifuge tube. USE acetonitrile-
water solution (4.9) to dilute to 50 mL. Accurately PIPETTE 25 mL into a 100
mL heart-shaped bottle.
The heart-shaped bottle is evaporated on a rotary evaporator (37 °C water bath)
to remove acetonitrile (Easy-to-foam sample can be added with 4 mL of
saturated sodium chloride solution).
7.2 Purification
Immediately ADD 25 mL of phosphate buffer solution (4.11) to the heart-shaped
bottle from which acetonitrile has been removed; vortex-mix for 1 min; USE 0.1
mol/L sodium hydroxide to adjust the pH to 8.5. At a rate of 1 mL/min, PASS
the pretreated solid-phase extraction cartridge; first USE 2 mL of phosphate
buffer solution (4.11) to rinse twice; then USE 1 mL of ultrapure water to rinse;
and USE 3 mL of acetonitrile to elute (The speed is controlled at 1 mL/min).
The eluent, at 45 °C, is blown dry with nitrogen. USE 0.025 mol/L phosphate
buffer solution (4.12) to dilute to 1 mL. After passing through a 0.45 μm filter
membrane, immediately USE liquid chromatograph-mass spectrometer/mass
spectrometer to determine.
reference retention time of 11 penicillins is approximately: amoxicyllin 8.5 min,
ampicillin 12.2 min, azlocillin 16.5 min, methicillin 16.8 min, gpenicillin 18.1 min,
penicillin 19.4 min, oxacillin 20.3 min, phenethicillin 20.5 min, cloxacillin 21.5
min, nafcillin 22.3 min, dicloxacillin 23.5 min. The reconstructed ion
chromatogram of the quantitative ion pair of penicillins standard solutions is
shown in Figure B.1.
7.3.4 Qualitative determination
According to the above conditions, the sample is determined; and a standard
working curve is established. If the retention time of the compound mass
chromatographic peak in the sample, compared with the standard solution, is
within the allowable deviation of ±2.5%; the signal-to-noise ratio of the
reconstructed ion chromatographic peak of the qualitative ion pair of the
compound under test is greater than or equal to 3 (S/N≥3); the signal-to-noise
ratio of the reconstructed ion chromatographic peak of the quantitative ion pair
is greater than or equal to 10 (S/N≥10); and the relative abundance of the
qualitative ion pair, compared with the standard solution of the equivalent
concentration, has a deviation not exceeding the requirements of Table 2; then
it can judged that there is the corresponding target compound in the sample.
Table 2 -- Maximum allowable deviation of relative ion abundance during
qualitative confirmation
7.3.5 Quantitative determination
According to the external standard method, USE the standard working curve to
perform quantitative determination.
7.3.6 Blank test
Except that no sample is added, all the operation procedures mentioned above
are followed.
8 Calculation and expression of results
USE chromatographic data processor or formula (1) to calculate the penicillins
residues in the sample. The calculation result needs to deduct the blank value:
Relative ion abundance
Allowable relative deviation