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Updated FCC Developmental Melamine Quantitation (HPLC-UV)

April 2, 2007

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 l_max 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%.

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