GB 29697-2013Determination of Diazepam and Methaqualone residues in animal derived food by Gas Chromatography-Mass Spectrometric method (English PDF)

6.1 Preparation of the sample

Fresh or thawed take appropriate blank or test tissue, minced, and homogenized.

--- the test sample taken after homogenization, as the feed try.

--- blank sample taken after homogenization, as a blank sample.

--- blank sample taken after homogenization, adding a suitable concentration of the standard working solution, is added as a blank sample.

Save 6.2 sample

Or less at -20 °C.

Determination Step 7

7.1 Extract

Sample Weigh 2g ± 0.02g, a centrifuge tube, acetonitrile was added 20mL, ultrasonic water bath for 5min. 4000r/min centrifugal 10min, the the supernatant in the heart-shaped flask. At 50 deg.] C and rotary evaporated to near 5mL, 7.5 mL of n-hexane was added, mixing was allowed to stand, the supernatant was discarded. Take the lower liquid at 50 °C nitrogen purge to 1mL, water was added 4mL, mixing spare.

7.2 Purification

C18 column successively with methanol and 3mL 3mL water activation, taking stock solution through the column, washed with 50% aqueous methanol with 3mL, 70% A 3mL aqueous alcohol solution, collecting eluate, blown dry with nitrogen at 50 °C, the residue was dissolved with 200µL of acetonitrile, gas chromatography - mass spectrometry Determination.

7.3 Preparation of standard curve

The precise amount of 10µg/mL working standard solutions amount, diluted with methanol, and formulated to a concentration of 1,10,50,100,500 1000ng/mL standard solution series, gas chromatography - mass spectrometry. Peak area was measured to the vertical axis, the corresponding concentration of the standard solution Degree abscissa, the standard curve. Seeking regression equation and correlation coefficient.

7.4 Determination

7.4.1 Reference Gas chromatography conditions

7.4.1.1 Gas Chromatographic Column. DB-5MS column (30m × 0.25mm, diameter 0.25µm), or equivalent person.

7.4.1.2 Carrier gas. helium.

7.4.1.3 Carrier gas flow rate. 0.9mL/min.

7.4.1.4 Inlet temperature. 290 °C.

7.4.1.5 Column temperature. initial temperature 150 °C, holding 1min, 10 °C/min temperature programmed to 290 °C, maintained 4min.

7.4.1.6 Injection volume. 1µL, constant current mode splitless.

7.4.2 Mass Reference Conditions

7.4.2.1 electron impact ionization (EI).

7.4.2.2 electron impact energy. 70eV.

7.4.2.3 Ion source temperature. 230 °C.

7.4.2.4 Quadrupole temperature. 150 °C.

7.4.2.5 Solvent delay. 9min.

7.4.2.6 Detection Mode. selected ion detection.

7.4.2.7 qualifier ion. diazepam of m/z283, m/z256, m/z241, m/z221; methaqualone of m/z250, m/z235, m/z217, m/z91.

7.4.2.8 quantitatively ion. diazepam of m/z256; methaqualone of m/z235.

7.4.3 Assay

7.4.3.1 Qualitative determination

The drug content in the sample solution, the volume of selected peak area close to the standard working solution and the like injections of the sample solution and the like. By air Relative retention time and MS-SIM common characterization. Retention time of the sample solution and standard pharmaceutical test substance relative deviation is not 1%, and the difference in the relative abundance of the selected ion which is less than 20%. Standard solution of diazepam and methaqualone, blank and blank samples Selected ion chromatogram of the sample is added in Appendix A.

7.4.3.2 Quantitative determination

Working standard solutions were taken and the corresponding amount of the sample solution concentration, single-point calibration for multi-point calibration, chromatographic peak area. standard and diazepam solution and sample solution methaqualone response value should be in the linear range of the detection instrument, sample solution injection process parameters should be inserted Standard working solution.

7.5 Blank test

But without addition of the sample, the same steps employed in parallel operation.

8 and the result of the calculation expression Try feed for diazepam and methaqualone residues according to formula (1).

Ai × cS × 5 × V

AS × m

Where.

--- for the respective X-diazepam try and feed residues methaqualone, micrograms per kilogram (µg/kg);

AI --- respective sample solution methaqualone and diazepam peak area;

--- the AS standard solution diazepam respective peak area and methaqualone;

cS --- respective methaqualone and diazepam concentration, in units of nanograms per milliliter (ng/mL) in standard solution;

V --- volume of the sample solution, in milliliters (mL);

m --- try supply feed mass in grams (g).

Note. The blank value should be subtracted from the results, expressed as the arithmetic mean of the measurement result measured parallel to three significant figures.

9.1 Sensitivity

The detection limit of the method was 0.5µg/kg, the limit of quantitation was 1µg/kg.

9.2 Accuracy

This method of adding 1µg/kg ~ 50µg/kg on recovery levels of 70% to 120%.

9.3 Precision

The relative standard deviation of the method <=15%, inter-assay relative standard deviation <=20%.

Appendix A

Chromatogram

Figure A.1 methaqualone and diazepam selected ion chromatogram of the standard solution (10µg/L)

Figure A.2 in pig muscle selected ion chromatogram of blank sample

Figure A.3 is added in pig muscle blank diazepam, methaqualone selected ion chromatogram of the sample (1µg/kg)