fda三聚氰胺的检验方法.doc

GC-MS Screen for the Presence of Melamine
(Adapted from FDA/ORA Forensic Chemistry Center SOP T015)
Revised April 10,
PURPOSE:
This procedure provides a general guide for the sample preparation and analysis of wheat gluten and pet food matrices for melamine using gas chromatography/mass spectrometry.  This procedure is designed to screen the TMS derivative of a methanol extract of the sample and it incorporates instrumental parameters which permit 10.5 minute run times.
SCOPE:
This procedure is applicable to dry wheat gluten, dry pet food, and wet “cuts and gravy” type pet food.  This procedure involves extractions in methanol and therefore preselects compounds which are soluble or partially soluble in methanol.  Due to solubility limitations of melamine in methanol, this method is intended to be a qualitative screen only.
RESPONSIBILITY:
It is the responsibility of the analyst to note any modifications to this procedure in the worksheet.
DEFINITIONS AND ACRONYMS:
TMS – trimethylsilyl
SAFETY CONSIDERATIONS:
Accepted safety measures should be employed when working with chemicals and pressurized gases.
EQUIPMENT:
Agilent 5975 GC-MS system equipped with a 30m DB-5MS column (or equivalent)
Reacti-Vap – Evaporating/Heating Module
REAGENTS:
Methanol: HPLC grade
Pyridine: Certified A.C.S.
BSTFA with 1% TMCS: bis(trimethylsilyl)trifluoroacetamide with 1%
Trimethylchlorosilane (e.g. Sylon BFT, Supelco)

QC ELEMENTS:
A blank should be run at the onset of each analysis and then randomly throughout the analysis if there is suspicion of carryover.  A fortified sample (see Part C) should be analyzed with each set of samples to demonstrate effective system performance.
PROCEDURES (PROCESS DETAILS):
This procedure may be used with the GC operating in either the splitless or split (1:20) mode.  Generally, the splitless mode is advisable for samples where more sensitivity is required.   The split mode is suitable where sample availability is not limited and the recommended amount can be prepared (~0.5 g sample preparations).
A. Methanol Extract (Underivatized Extract):
Unknown Wheat Gluten/Pet Food Samples:  Transfer approximately 0.500 g of the sample into a 20 ml scintillation vial and add 5 ml methanol.  Vortex briefly to mix, then sonicate for 10 minutes.  Centrifuge for approximately 6 minutes at 4500 rpm and then filter the liquid extract into a GC vial using a 0.45 µm nylon filter disc.  If sample is limited, reduce extraction volumes and sample weights proportionately and analyze in the splitless mode.  Duplicate preparations are recommended.
B. Trimethylsilyl (TMS) derivatives:
Melamine is detected as the 3-TMS derivative with a nominal molecular weight of 342 amu.  The retention time of the 3-TMS derivative is approximately 7.1 minutes in the GC-MS chromatogram (see Figures 1-4).
Blanks: Transfer 40µL of methanol used to extract the sample to an autosampler vial.  Evaporate to dryness under a stream of dry air using a Reacti-Vap module set at approximately 70°C.  Add 200 µl pyridine and 200 µl BSTFA, vortex briefly to mix, and incubate at approximately 70°C for 30 minutes. 
Known Samples: In general, if working with a known concentration of analyte (e.g. standards), appropriate dilutions should be made with pyridine/BSTFA to achieve an on column concentration no greater than 100 µg/mL.  After calculating suitable dilutions, transfer an appropriate amount of the standard solution to an autosampler vial.  Evaporate to dryness under a stream of dry air using a Reacti-Vap module set at approximately 70°C.  Add 200 µl pyridine and 200 µl BSTFA, vortex briefly to mix, and incubate at approximately 70°C for 30 minutes. 
Unknown Samples: Transfer 40 µl of the methanol extract prepared for analysis in section A into an autosampler vial.  Evaporate to dryness under a stream of dry air using a Reacti-Vap module set at approximately 70°C.  Add 200 µl pyridine and 200 µl BSTFA, vortex briefly to mix, and incubate at approximately 70°C for 30 minutes.
C. Sample Fortification:
This method was successfully applied to detect melamine in wet and dry pet food composites (Figure 2) and dry wheat glutens (Figure 3).  Based on HPLC-UV results, this method was able to detect melamine levels down to 0.01% (by weight) in official FDA samples using ~0.500 g samples with TMS derivatization and splitless injection. 
Uncontaminated wheat gluten and pet food composites were spiked with various levels of melamine and taken through the method using ~0.500 g samples and splitless injection.  The method successfully detected melamine (as the 3TMS derivative) at 14 micrograms/g in the wheat gluten and at 7 micrograms/g in the pet food composite.  At low spike levels, extracted ion chromatograms were used to verify the presence of four major ions of the melamine-TMS mass spectrum (m/z=342, 327, 171, and 99).  Sample fortification experiments to demonstrate successful detection of melamine in the specific matrix of interest should be performed by the individual laboratories.
D. Standard Preparation:
Melamine is sparingly soluble in methanol and slightly more soluble in methanol/water mixtures.  Up to ~1.5 mg/mL of melamine standard can be dissolved in a 50/50 mixture of methanol/water with sonication for 30 minutes.  If more concentrated solutions of melamine are desired, dimethyl sulfoxide (DMSO) may be used as a primary solvent followed by methanol or acetonitrile for serial dilutions.
Note: Due to the presence of water in the standard solution, dry down times for the   melamine standard on the Reacti-Vap will be increased.
E. Instrument Parameters:
INSTRUMENT:  
5975 Agilent GC 6890N Series with 7683B Series autosampler  
DETECTOR: 
Agilent Mass Selective Detector (MSD) model 5975i
SOFTWARE: 
Enhanced Chemstation version D.02.00.275; Wiley 7th Edition and NIST 02 MS Library
COLUMN: 
J&W Scientific DB-5MS 5% Phenyl Methyl Siloxane with 10M Duraguard
Part #19091S-433, Serial # US4937937H
ID: 0.25mm      Film Thickness: 0.25 microns      Length: 30 meters (nominal) 
CARRIER GAS: 
Helium
Flow Rate: 1.3 ml/min
Avg. Linear Velocity: 35 cm/sec
Pressure: 17.5 psi (initial)
Mode: constant flow 
INJECTION:

Injection Type:  Splitless; or Split (1:20)
Injection Volume: 1µl
Injector Temperature: 250ºC
Splitless Injection Purge Flow:  10.0 ml/min at 2.0 minutes 
GC PARAMETERS:

Start Temperature: 75ºC; Hold: 1.0 min
Ramp Rate: 30ºC/min
Final Temperature: 300ºC; Hold: 2.0 min
Transfer Line Temperature 280ºC
Run Time:  10.5 minutes 
MS PARAMETERS:

Mass Range: 40-450 amu
Scan Mode: Full (Electron Ionization)
Filament Delay:  4.2 min (splitless); 3.8 min (split)
Threshold: 100
MS Quad: 150ºC
MS Source: 230ºC 
F. Peak Identification:
The mass spectrum of the 3-TMS derivative of melamine (Figure 1) exhibits the molecular ion at m/z=342, a methyl group loss at m/z=327, and significant ions at m/z=171, 99, and 73.  The standard of melamine should be prepared (and TMS-derivatized) and analyzed along with the sample preps on the same day to confirm the identification.  Figures 2a and 2b are examples of a GC-MS chromatogram of melamine contaminated pet food.  Additional peaks present in the TIC are consistent with TMS derivatives of phosphate and various sugars.  Figure 3 is an example of a GC-MS chromatogram of melamine contaminated wheat gluten.

Figure 1

Figure 2a

Figure 2b

 Figure 3

Web page updated by mdt - April 11, , 10:26 AM ET

Updated FCC Developmental Melamine Quantitation (HPLC-UV)
April 2,

Sample Preparation/Extraction:
Wheat gluten: Ground in a Retsch ZM 100 centrifugal rotor mill (ring sieve 0.5 mm).
Moist pet food: Moist chunks and gravy were blended to the consistency of a gritty pudding prior to sampling.
Samples weighed into glass scintillation vials. Extract using 50:50 acetonitrile:water.
Procedure: Add indicated volume of extraction solvent (see note below) 1. Cap and vortex thoroughly, get aggressive with this step (critical due to slow dissolution rate of melamine). Sonicate 30 minutes. Filter portion of extract through 2-stage GMF-nylon (0.45 m m) filters. Dilute filtered extract 250  ml extract + 750  ml solvent 2 to maintain solubility of matrix components.
1 Wheat gluten: Extract in proportion 0.1 g to 10 ml. For melamine contents above 2% w/w, extract in proportion 0.05 g to 15 ml.
1 Moist pet food: Extract in proportion 2.0 – 2.5 g to 10 ml.
2 Solvent for final dilution may be water or 0.1 N HCl. Final dilution is necessary for compatibility with ion-pairing chromatography. We have observed some differences in behavior between the wheat gluten and pet food samples with respect to maintaining solubility during final dilution (these are most likely matrix components which fall out). 0.1 N HCl seems to help maintain solubility for final dilution of the wheat glutens.
HPLC-UV Operating Parameters:
Column: Zorbax Rx C8 (retention is too high on C18 column)
Buffer: 10 mM citric acid, 10 mM sodium octane sulfonate, adjusted to pH 3.0
Mobile phase: 85:15 buffer:acetonitrile
Flow rate: 1.0 ml/min.
Injection volume: 10 ml
Column thermostat: 40 oC (column thermostatting is necessary for ion-pair separations)
Detection wavelength: 240 nm
Spectral collection: 200 – 400 nm (look for lmax near 236 nm)
Retention time: 4.2 - 4.3 min.
Run time: 10 min.
Standard Preparation:
Stock standard was prepared in 76:24 acetonitrile: water. A check stock standard was prepared in 60:40 acetonitrile: water. Dilutions were made in either water or 0.1N HCl giving equivalent calibration curves.
Figures of Merit:
Linear range: established from 1.0 – 400 mg/ml; calibration range 1.0 – 200 mg/ml used for most of work
Reproducibility: duplicate preparations agree within 0.1 - 5% relative basis
Spike/recovery (based on spiking solid melamine powder into test matrix prior to extraction): 90 -110%.
Web page updated by mdt - April 3, , 9:08 AM ET