下面是一个FDA推荐的测定三聚氰胺的文章，用的是 VWR的多管振荡器
FDA最新三聚氰胺测定方法
来源：安徽省兽药饲料监察所
Determination of Melamine and Cyanuric Acid Residues
in Infant Formula using
$$lc-MS/MS
Sherri Turnipseed1, Christine Casey1, Cristina Nochetto2, David N. Heller2
1 Denver Laboratory, Office of Regulatory Affairs, Food and Drug Administration
2 Office of Research, Center for Veterinary Medicine, Food and Drug Administration
--------------------------------------------------------------------------------
The Laboratory Information Bulletin is a communication from the Division of Field Science, Office of Regulatory Affairs, U.S. Food and Drug Administration for the rapid dissemination of laboratory methods (or scientific regulatory information) which appear to solve a problem or improve an existing problem. In many cases, however, the report may not represent completed analytical work. The reader must assure, by appropriate validation procedures, that the reported methods or techniques are reliable and accurate for use as a regulatory method. Reference to any commercial materials, equipment, or process does not, in any way, constitute approval, endorsement, or recommendation by the U.S. Food and Drug Administration.
Abstract
A liquid chromatography triple quadrupole tandem mass spectrometry (
$$lc-MS/MS) method for residues of cyanuric acid (CYA) and melamine (MEL) developed by the Center for Veterinary Medicine (CVM) has been adapted for use with infant formula. This method consists of an initial extraction with 2.5% aqueous formic acid, followed by a series of filtration, centrifugation, and dilution steps. Both compounds are analyzed in the same chromatographic program using a zwitterionic HILIC
$$lc column. Electrospray ionization is used in both the negative ion (CYA) and positive ion (MEL) modes. Two selected reaction monitoring (SRM) transitions are monitored for both compounds. The amount of compounds present is determined with a calibration curve consisting of sample extracts from infant formula fortified from 0.25 to 5 µg/g that have been taken through the extraction procedure. The range of recovery from fortified infant samples (n =38) was 70-114 % (RSDs 4.5- 22.7 %), and 72-110% (RSDs 5.7-24.9%) for cyanuric acid and melamine, respectively. The limits of quantification and confirmation are 0.25 µg/g for both analytes in dry infant formula.
Introduction
In 2007, pet food, animal feed, wheat gluten, and other protein-based food commodities were found to contain residues of melamine and a related compound, cyanuric acid. Widespread pet illness and death was subsequently attributed to the formation of melamine-cyanurate crystals in the kidneys of these animals. At that time, several laboratories developed methods for these compounds in pet food, raw protein sources, and other animal feeds. Because animals may eat food contaminated with melamine residues, analytical methods to determine melamine residues in fish and animal tissues were also developed. Recently, there has been a problem with contamination of infant formula with melamine and cyanuric acid in China. This is suspected of causing illness to many children in that country.
A procedure has been evaluated for the simultaneous determination and confirmation of melamine and cyanuric acid in powdered milk-based infant formula. Results are reported to support the continued evaluation and application of this method. Concentrations are measured in the range of 0.25 to 5 µg/g (µg/g). Extracts at higher concentrations may be diluted into this range. The limits of quantification and confirmation are 0.25 µg/g for both analytes.
Summary of Procedure
Powdered samples (2 g) are extracted with 14 mL 2.5 % aqueous formic acid in a 50 mL polypropylene centrifuge tube. Extracts are centrifuged at 3750 g and then 1.4 mL of supernatant is transferred to a polypropylene Eppendorf tube and centrifuged at 16100 g (benchtop micro centrifuge). The extracts are filtered through PVDF syringe filters (0.2 micron pore) followed by dilution with acetonitrile and a second centrifugation at 16100 g. The extracts are analyzed by
$$lc-MS/MS. A zwitterionic HILIC column is used to separate melamine from cyanuric acid. Melamine is detected in positive ion mode and cyanuric acid is detected in negative ion mode. Analyte concentrations are ca
$$lculated using external standard calibration with a standard curve prepared in a pre-fortified control matrix which has been carried through the extraction procedure.
Standards and Reagents
Melamine (MEL). CAS #: 108-78-1. Sigma Chemical Company.
Cyanuric acid (CYA). CAS #: 504-19-8. Fisher Chemical Company or TCI America.
Acetonitrile (ACN.)
$$lc grade. Burdick & Jackson high purity or equivalent.
Formic acid. Reagent grade ≥ 95%. Sigma-Aldrich or Fisher Chemical Company.
Water.
$$lc grade, or purified by Millipore Milli-Q system to > 18 M-ohm resistivity, or equivalent.
Ammonium Formate. Sigma-Aldrich or Fisher. Purity > 97%.
Solutions:
0.1% Formic acid in water. Transfer 1 mL formic acid to 1 L graduated flask. Dilute to volume with
$$lc water.
Mobile Phase A. 0.1% Formic acid in Acetonitrile (5:95 v/v). Combine 50 mL of 0.1% formic acid in water with 950 mL ACN in a 1 L solvent bottle.
Mobile Phase B. 20 mM Ammonium Formate in Acetonitrile (50:50 v/v). Combine 500 mL of 20 mM ammonium formate and 500 mL of acetonitrile in a 1 L solvent bottle.
2.5% Formic acid in water. Transfer 25 mL formic acid to 1 L volumetric flask. Dilute to volume with
$$lc grade water.
20 mM Ammonium formate. Weigh 0.63 g of ammonium formate and dissolve in 0.5 L
$$lc grade water.
Equipment
Liquid chromatograph. Binary
$$lc pump is recommended for accurate mixing at low flow rate and rapid response to mobile phase gradient. (Waters, Agilent, Thermo Surveyor, or equivalent)
Liquid chromatography column. ZIC-HILIC, 2.1 x 150mm, 5µm, 200 Å, (Available in US from Nest Group #Q2712-152) (Merck Sequant)
Mass Spectrometer. Triple quadrupole capable of meeting system suitability. System used in the validation of infant formula was a Thermo (San Jose, CA) TSQ Quantum triple quadrupole mass spectrometer coupled to a Thermo Surveyor
$$lc-MS pump and autosampler.
Centrifuge. Capable of 4000 RPM with 50 mL tubes.
Microcentrifuge. Capable of 13,000 RPM with 1.5 or 2 mL tubes. (Eppendorf or equivalent)
Mixers and shakers. Single and multi tube vortex mixers (VWR), platform shaker.
Utrasonic bath. Including timer and heater
Centrifuge tubes. 50mL disposable polypropylene with caps, with graduations from 5 to 50 mL and 1.5 mL microcentrifuge tubes.
Syringe Filters. Polyvinylidene fluoride (PVDF), 13mm, 0.22um (Whatman Puradisc™)
Syringes. Three mL polypropylene.
我的社区
签到
