GB/T 5009.111-2003Determination of deoxynivalenol in food -- High performance liquid chromatographic method with immunoaffinity column clean-up (English PDF)

3.1 Reagents

3.1.1 acetonitrile (CH3CN). chromatography.

3.1.2 Methanol (CH3OH). chromatography.

3.1.3 n-hexane (C6H14).

3.1.4 Ammonia (NH3 · H2O).

3.1.5 formic acid (HCOOH).

3.1.6 Nitrogen (N2). purity >=99.9%.

3.2 Reagent preparation

3.2.1 acetonitrile - water (8416). amount of 160mL of water was added to 840mL of acetonitrile and mix.

3.2.2 acetonitrile saturated solution of hexane. amount of n-hexane in 200mL 250mL separatory funnel, adding a small amount of acetonitrile, shake vigorously A few minutes, standing layer, discarding the lower acetonitrile layer, that is, too.

3.2.3 Methanol - water (595). amount of 5mL of methanol was added to 95mL of water, and mix.

3.2.4 0.01% aqueous ammonia solution. Take 100µL aqueous ammonia was added to 1000mL of water, and mix (for ion source ESI- mode is used).

3.2.5 0.1% formic acid solution. Take 1mL formic acid is added to 1000mL of water, and mix (for ESI ion source mode is used).

3.3 Standard

3.3.1 deoxynivalenol bacteria enolase (DON, C15H20O6, CAS number. 51481-10-8). purity >=99%, or certified by the state and granted Standards Standards certificate.

3.3.2 3-acetyl deoxynivalenol bacteria enolase (3-ADON, C17H22O6, CAS number. 50722-38-8). purity >=99%, or by the State Certified and standard substance certificate.

3.3.3 15- acetyl deoxynivalenol bacteria enolase (15-ADON, C17H22O6, CAS number. 88337-96-6). purity >=99%, or by State Hotels certified and reference material reference material certificate.

3.3.4 13C15- deoxynivalenol fungi alcohol standard solution isotopes (13C-DON, 13C15H20O6). 25µg/mL, the purity of >=99%.

3.3.5 13C17-3- acetyl - deoxynivalenol fungi alcohol standard solution isotopes (13C-3-ADON, 13C17H22O6). 25µg/mL, purity >=99%.

3.4 Standard Solution

3.4.1 Standard stock solution (100µg/mL). Weigh DON, 3-ADON and 15-ADON1mg (accurate to 0.01mg), points Do not dissolved in acetonitrile and set the volume to 10mL. The solution was transferred to a vial, sealed and stored at -20 °C, valid for 1 year.

3.4.2 mixed standard working solution (10µg/mL). Imbibe 100µg/mLDON, 3-ADON and 15-ADON stock standard solution each 1.0mL in the same 10mL volumetric flask, add acetonitrile to volume. Sealed and stored at -20 °C, valid for six months.

3.4.3 Mixed isotope internal standard working solution (1µg/mL). Imbibe 13C15-DON and 13C17-3-ADON isotope internal standard (25µg/mL) each 1mL to 25mL same flask, add acetonitrile to volume. Sealed and stored at -20 °C, valid Six months.

3.4.4 standard series working solution. accurate Pipette the appropriate amount of mixed standard working solution and mixed isotope internal standard working solution with the initial mobile phase Formulated as 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 160ng/mL, 320ng/mL, mixed standard 640ng/mL of Series, in which the isotope internal standard at a concentration of 100ng/mL. Standard series solution at 4 °C preservation, valid 7d.

5.1 Sample Preparation

5.1.1 cereals and cereal products. taking at least 1kg sample was pulverized with a high-speed grinder and sieved to a particle size of less than 0.5mm ~ 1mm Aperture test sieve, mixed uniformly reduced to 100g, stored in a vial, sealed and stored for detection.

5.1.2 Alcohol. Take bulk wine at least 1L, for bags, bottles and other packaging samples taken at least three packaging (or the same batch number), all the liquid Body with a sample container after homogenous mixing machine, reduced to 100g (mL) stored in the vial, sealed and stored for detection. Including two Wine samples of carbon monoxide before you use the refrigerator at 4 °C for 30min, filtered before use or ultrasonic degassing.

5.1.3 soy sauce, vinegar, soy sauce and butter products. taking at least 1L samples for bags, bottles and other packaging samples taken at least three packaging (or the same batch Number), after all the liquid sample in a container with a homogenizer mix, reduced to 100g (mL) stored in the vial, sealed for Detection.

5.2 Sample Extraction

5.2.1 cereal and its products. Weigh 2g (accurate to 0.01g) sample was 50mL centrifuge tube was added 400µL mixed isotope internal standard After shaking the working fluid mixed stand 30min. Added 20.0mL of acetonitrile - water (8416), placed in an ultrasonic/or a vortex shaker Ultrasonic or oscillation 20min. 10000r/min centrifugal 5min, the supernatant was collected A spare in a clean container.

5.2.2 Alcohol. Weigh 5g (accurate to 0.01g) sample was 50mL centrifuge tube was added 200µL mixed isotope internal standard working solution vibrator After mixing swing standing 30min, with acetonitrile dilute to 10mL, mix, placed in an ultrasonic/Vortex shaker or ultrasonic or oscillation 20min. 10000r/min centrifugal 5min, the supernatant was collected B alternate in a clean container.

5.2.3 soy sauce, vinegar, soy sauce and butter products. Weigh 2g (accurate to 0.01g) sample was 50mL centrifuge tube, mixed with bits added 400µL Su internal standard working solution After shaking the mixing stand 30min. Added 20.0mL of acetonitrile - water (8416), placed in an ultrasonic/Vortex or shaker or ultrasonic oscillations 20min. 10000r/min centrifugal 5min, the supernatant was collected C alternate in a clean container.

5.3 Sample purification

NOTE. The following sample purification process, according to the actual situation, choose one of the methods can be.

5.3.1 Universal solid-phase extraction (SPE)

A take 5mL supernatant or supernatant or supernatant B C a 50mL centrifuge tube, 10mL of acetonitrile was added a saturated solution of n-hexane Liquid vortex mixing 2min, after 5000r/min centrifugal 2min, discard the layer of n-hexane at 40 °C ~ 50 °C under dry nitrogen, was added 4mL the residue was dissolved sufficient water to be purified.

The SPE is connected to the solid phase extraction, washed with methanol and 3mL 3mL water activated balance. The above-mentioned water 4mL reconstituted the liquid column to control the flow rate drops 1 to 2 drops per second. With 3mL water, 1mL5% methanol - aqueous solution followed by rinsing thoroughly drained after the column. use 4mL methanol, after collecting all the eluent at 40 °C ~ 50 °C under dry nitrogen. The initial mobile phase was added 1.0mL residue, Vortex 10s, with 0.22µm filter membrane in an injection vial until injection.

5.3.2 DONs special solid phase purification column purification

A glass tube take 8mL supernatant or supernatant or supernatant B C DONs dedicated to solid phase clean-up columns will fill the purification column Feeding tube inserted into a glass tube and slowly push the filler tube to purify liquid precipitation, pipette 5mL decontamination liquid at 40 °C ~ 50 °C under dry nitrogen. plus the initial mobile phase into 1.0mL residue was vortexed 10s, with 0.22µm filter membrane in an injection vial, until injection.

Note. The operational aspects of the use of different vendors DONs special type of solid-phase purification column, in the sample, purification may be slightly different, can be carried out in accordance with the specification requirements operating.

5.3.3 Immunoaffinity column purification

In advance of recovering cryopreserved immunoaffinity column to room temperature.

Pipette accurately 5mL supernatant A or B supernatant or supernatant C, at 40 °C ~ 50 °C under dry nitrogen, was added 2mL water sufficient the residue was dissolved, to be immunoaffinity column within the original liquid flow after doing the above sample solution was transferred to a glass syringe barrel. The air pressure injection pump and glass Emitter connected to adjust the dripping speed, control sample solution with 1 drop per second flow rate through the immunoaffinity column, until the air into the affinity column. use 5mLPBS buffered saline solution and 5mL water leaching has immunoaffinity column at a flow rate of approximately 1 drop to 2 drops per second, until the air and into the pro-Note the discarded all effluent, drained cartridge.

Accurate added 2mL methanol affinity column, under the control of one drop per second, the speed drops to collect all the eluent to a test tube, at 50 °C Firmly eluent blown with nitrogen to nearly dry, add 1.0mL initial phase flow, vortex 30s dissolve the residue, 0.22µm filter membrane, the filtrate was collected in an injection vial to prepare injections.

Note. Use different vendors immunoaffinity column, the sample in the sample, operational leaching and elution may be slightly different, should be carried out in accordance with specification requirements operating.

5.4 Liquid chromatography - tandem mass spectrometry reference conditions (refer to one of these methods can be based on the actual situation)

5.4.1 Ion Source mode. ESI

Liquid chromatography - mass spectrometry reference conditions are listed below.

a) LC Column. C18 column (column length 100mm, column diameter 2.1mm; particle size 1.7µm), or equivalent person;

b) Mobile phase. A phase. 0.1% formic acid; B phase. 0.1% formic acid - acetonitrile;

c) gradient. 2% B (0min ~ 0.8min), 24% B (3.0min ~ 4.0min), 100% B (6.0min ~ 6.9min), 2% B (6.9min ~ 7.0min);

d) flow rate. 0.35mL/min;

e) Column temperature. 40 °C;

f) Injection volume. 10µL;

g) Capillary voltage. 3.5kV; cone voltage. 30V; desolvation temperature. 350 °C; desolvation gas flow rate. 900L/h;

h) deoxynivalenol bacteria enolase and acetylated derivatives thereof MS conditions refer to Table 1.

Table 1 deoxynivalenol bacteria enolase and Acetyl Derivatives Mass reference conditions (ESI)

Compound

name

Parent ion

Cone voltage

Quantitative ion

Collision voltage

Qualifier ion

Collision voltage

DON 297 20 249 10 203 16 3-ADON 339 17 231 13 203 13 15-ADON 339 18 137 9 321 7 13C-DON 312 20 263 10 245 16 13C-3-ADON 356 17 245 13 - -Note. 3-ADON and 15-ADON calculated as isomers, 15-ADON optional 13C-3-ADON isotope internal standard as appropriate quantification.

5.4.2 Ion Source mode. ESI-

Liquid chromatography - mass spectrometry reference conditions are listed below.

a) LC Column. C18 column (column length 100mm, column diameter 2.1mm; particle size 1.7µm), or equivalent person;

b) Mobile phase. A phase. 0.01% aqueous ammonia solution; phase B. acetonitrile;

c) gradient. 2% B (0min ~ 0.8min), 24% B (3.0min ~ 4.0min), 100% B (6.0min ~ 6.9min), 2% B (6.9min ~ 7.0min);

d) flow rate. 0.35mL/min;

e) Column temperature. 40 °C;

f) Injection volume. 10µL;

g) capillary voltage. 2.5kV; cone voltage. 45V; desolvation temperature. 500 °C; desolvation gas flow rate. 900L/h;

h) deoxynivalenol bacteria enolase and acetylated derivatives thereof MS conditions refer to Table 2.

Table 2 deoxynivalenol bacteria enolase and Acetyl Derivatives Mass reference conditions (ESI-)

Compound

name

Parent ion

Cone voltage

Quantitative ion

Collision voltage

Qualifier ion

Collision voltage

DON 295 14 265 12 138 18 3-ADON 337 12 307 10 173 12 15-ADON 337 12 150 12 219 12 13C-DON 310 16 279 12 145 14 13C-3-ADON 354 18 323 14 230 18 Note. 3-ADON and 15-ADON calculated as isomers, 15-ADON optional 13C-3-ADON isotope internal standard as appropriate quantification.

Ion scan of each compound, multiple reaction monitoring (MRM) ion channel FIG see Figure B.1 ~ Figure B.8.

5.5 Qualitative Determination

The target compound in the sample peak retention time compared with the corresponding standard chromatographic retention time, the range of variation should be ± 2.5% within.

MS qualifier ion for each compound should appear, including at least a parent ion and two ions, and detect the same batch of the same compound, the relative abundance of the two sub-target in the sample ion compound concentration equivalent ratio compared to the standard solution, which is not allowable deviation

Table 3 exceeds the range specified.

The maximum permissible relative ion abundances Table 3 Qualitative deviations Relative ion abundances > 50% 20% ~ 50% 10% ~ 20% <=10% Permissible relative deviation of ± 20% ± 25% ± 30% ± 50%

5.6 Standard curve

Under 5.4 liquid chromatography tandem mass spectrometry analysis conditions, the standard solution series from low to high concentration injection testing to DON, 3-Peak area ratio ADON and 15-ADON peaks corresponding to each of the internal standard peak - concentration plotted standard curve regression equation, which Linear correlation coefficient should be greater than 0.99.

5.7 Determination of the sample solution

Take the test solution injection treatment was 5.2, 5.3, calculate the mass concentration of the target substance in the test solution of the internal standard method, the sample is calculated by 6 Content of the analyte. Analyte response test solution should be within the linear range of the standard curve over the linear range should be appropriate to reduce the amount of sampling After the re-determination.

5.8 Blank test

Except that no sample addition, according to the steps 5.3 and 5.4 of the blank experiment. Confirm contain interfering substances should not be tested components.

10.1 Reagents

10.1.1 methanol (CH3OH). chromatography.

10.1.2 acetonitrile (CH3CN). chromatography.

10.1.3 polyethylene glycol [molecular weight of 8000, HO (CH2CH2O) nH].

10.1.4 Sodium chloride (NaCl).

10.1.5 disodium hydrogen phosphate (Na2HPO4).

10.1.6 potassium dihydrogen phosphate (KH2PO4).

10.1.7 potassium chloride (KCl).

10.1.8 hydrochloric acid (HCl).

10.2 Reagent preparation

10.2.1 phosphate buffer solution (hereinafter referred to as PBS). Weigh 8.00g of sodium chloride, 1.20 g of disodium hydrogenphosphate, potassium dihydrogenphosphate 0.20 g, 0.20g potassium chloride, dissolved with 900mL of water, adjusted to pH 7.0 with hydrochloric acid, water volume to 1000mL.

10.2.2 methanol - water (2080). amount of 200mL of methanol was added to 800mL of water, and mix.

10.2.3 acetonitrile - water (1090). Measure 100mL acetonitrile was added to 900mL of water, and mix.

10.3 Standards

Deoxynivalenol bacteria enolase (C15H20O6, CAS number. 51481-10-8). purity >=99%, or granted by the National Certification and Standards Standard Substance certificate.

10.4 Standard Solution

10.4.1 Standard stock solution (100µg/mL). Weigh deoxynivalenol bacteria enol 1mg (accurate to 0.01mg), dissolved in acetonitrile and Volume to 10mL. The solution was transferred to a vial, sealed and stored at -20 °C, valid for 1 year.

10.4.2 series of standard working solution. accurate Pipette the appropriate amount deoxynivalenol fungi alcohol standard stock solution was diluted with an initial phase flow, preparation to 100ng/mL, 200ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, 5000ng/mL working solution of the standard series, 4 °C preservation, valid 7d.

12.1 Sample Preparation

With 5.1.

12.2 Sample Extraction

12.2.1 cereal and its products. Weigh 25g (accurate to...