lib 4396 melamine residues in catfish tissue

8/7/2019 LIB 4396 Melamine Residues in Catfish Tissue

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LIB 4396 Melamine Residues in Catfish

Tissue

Laboratory Information Bulletin

LIB No. 4396

Volume 23, May 2007

Determination of Melamine Residues in

Catfish Tissue by Triple Quadrupole

LC-MS-MS with HILICChromatography

Wendy C. Andersen, Sherri B. Turnipseed, Christine M. Karbiwnyk, and Mark R.

Madson

U.S. Food and Drug Administration, Animal Drugs Research Center, Denver, CO

The Laboratory Information Bulletin is a communication from the Division of Field

Science, Office of Regulatory Affairs, U.S. Food and Drug Administration for therapid 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 triple quadrupole liquid chromatography tandem mass spectrometry method is

presented for the quantitative determination and confirmation of melamine residues in

catfish. Catfish tissue was extracted with 50:50 acetonitrile:water and 1 N

hydrochloric acid and cleaned-up using Oasis MCX solid phase extraction

cartridges. Extracts were analyzed by LC-MS-MS with HILIC chromatography and

electrospray ionization in positive ion mode. The precursor ion for melamine is m/z

127. Two product ion transitions were monitored at m/z 85 and 68 for quantification

and confirmation. Catfish tissue was fortified at 10, 25, 50, 100, and 500 ng/g (ppb).

The average recovery of melamine from fortified samples (n = 17) was 76.3 % with

an RSD of 14.3 %.


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INTRODUCTION

Pet food, animal feed, wheat gluten, and other protein-based food commodities were

recently found to contain residues of melamine, an industrial chemical used in the

manufacturing of plastics, flame retardants, and other products. It is not approved by

the U.S. Food and Drug Administration for use in food or animal feed. Because

animals may eat food contaminated with melamine residues, there is a need for

analytical methods to determine melamine residues that may be present in animal

tissues. This Laboratory Information Bulletin presents a method for the determination

of trace levels of melamine in catfish muscle.

EXPERIMENTAL

Equipment and reagent sources have been provided for information and guidance.

Equivalent products may be substituted as appropriate.

Equipment

a. LC/MS/MS instrument: Thermo (San Jose, CA) TSQ Quantum triple

quadrupole mass spectrometer coupled to a Thermo Surveyor LC-MS pump

and autosampler. A metal needle sample kit was installed on the electrospray

source; the orientation of the spray to the orifice was set at the second notch

(approx 62 deg offset). XCaliber V.4 software was used to acquire and analyze

the data.

b. LC column - Atlantis HILIC Silica column, 3 m, 3.0 x 50 mm (P/N

186002017, Waters Corp., Milford, MA).c. Blender/homogenizer - RobotCoupe Blixer, homogenizer, 4 quart, model

RS1BX4V (RobotCoupe USA, Inc., Ridgeland, MS).

d. Vortex Mixer - Vortex Genie 2, (Scientific Industries, Bohemia, NY).

e. Centrifuge - refrigerated to 5 C, capable of accelerating 50 mL tubes to 4000

rpm (2730 rcf).

f. Evaporator - TurboVap LV nitrogen evaporator with thermostatted water

bath (P/N 103198, Zymark, Hopkinton, MA).

g. SPE Cartridge - Oasis MCX solid phase extraction columns, 6 mL, 150 mg,

(P/N 186000255, Waters Corp., Milford, MA).

h. Syringe filters - Acrodisc 13 mm syringe filter with 0.2 m nylon membrane

(P/N 4550T, Pall Life Sciences) with 1 mL disposable syringe (P/N 309602,Becton Dickinson, Franklin Lakes, NJ).

i. Centrifuge tubes - 50 mL disposable, conical, graduated, polypropylene tubes

with cap; 15 mL disposable, conical, graduated, polypropylene tubes with cap

(Falcon Blue Max, P/N 50 mL tubes 352070, 15 mL tubes 352097,

Becton Dickinson, Franklin Lakes, NJ).

j. Volumetric glassware and pipettors - 100.0 and 10.0 mL volumetric flasks,

class A; adjustable volume pipettors with disposable polypropylene tips - 10-

100 L (Eppendorf, Brinkmann Instruments, Inc., Westbury, NY), 200-1000

L (Ulster Scientific, Inc., New Paltz, NY), and 1-5 mL (Wheaton Science

Products, Millville, NJ).

k. Glassware and LC vials - disposable glass culture tubes (16x100 mm),disposable Pasteur pipettes; 2 mL glass LC vials with snap caps.


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Reagents and Standards

a. Melamine (MEL) was obtained from Sigma-Aldrich (99+% purity, FW

126.12, CAS 108-78-01, cat. no. 240818, St. Louis, MO).

b. Solvents - high purity chromatographic and spectrophotometric grade

acetonitrile and methanol were used. Dichloromethane was liquidchromatographic grade. All water used was deionized and purified to 18.2

Mcm (Millipore, Bedford, MA).

c. Hydrochloric acid - concentrated, ACS grade, (CAS 7647-01-0, EMD

Chemicals, Inc., Gibbstown, NJ). Solutions with concentrations 1.0 M and 0.1

M of HCl in water are used in this method.

d. Ammonium hydroxide - concentrated, ACS grade, (CAS 1336-21-6, EMD

Chemicals, Inc., Gibbstown, NJ). This was used to prepare solutions with

concentrations of 2 % NH4OH in water (v:v) (for standards) and 5 % NH4OH

in methanol (v:v).

e. Ammonium formate - reagent grade (CAS 540-69-2, Sigma-Aldrich, St.

Louis, MO). This was used to prepare a 20 mM solution in water.

f. Extraction solution - 50:50 (v:v) acetonitrile:water.

g. Mobile phase - The mobile phase solvents were acetonitrile and 20mM

ammonium formate buffer. These were used in the LC gradient elution

program. Additionally, a solution containing 95:5 (v:v)

acetonitrile:ammonium formate (20mM) was used to prepare calibration

standards and reconstitute extracts.

Standard Solutions

Stock solution: 10.00 mg of melamine (MEL) was added to a 100.0 mL volumetricflask and diluted to mark with a solution of 2 % ammonium hydroxide in water (v:v)

to produce a stock solution with a concentration of 100.0 g/mL. The solution was

sonicated for 20-30 minutes until crystals of melamine were no longer visible.

Intermediate solutions: A 10.0 g/mL standard was prepared by adding a 1.0 mL

aliquot of the MEL stock to a 10.0 mL volumetric flask and diluting to the mark with

water. A 1.0 g/mL standard was prepared by adding a 1.0 mL aliquot of the MEL

stock to a 100.0 mL volumetric flask and diluting to the mark with water.

Calibration standards: Calibration curves were constructed from calibration standards

of melamine with concentrations of 10, 25, 50, 100, 500, 1000, and 2000 ng/mL.These calibrants were prepared daily using appropriate aliquots of the 1 and 10 g/mL

melamine intermediate solutions diluted to 5 mL with 95:5 acetonitrile:ammonium

formate mobile phase.

Control Tissues

Control tissues were fillets of fresh or frozen, aquacultured catfish that were obtained

from a local market, from the FDA Gulf Coast Seafood Laboratory (GCSL), and from

the FDA Center for Veterinary Medicine (CVM).

Tissue preparation


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acetonitrile:ammonium formate (20 mM), vortex mixed for 10 seconds, and filtered

through a 0.2 m nylon syringe filter into a glass LC vial.

Several end spikes (ES) were evaluated to determine the loss of instrumental

sensitivity due to ion suppression and to confirm that solvent standards, rather than

matrix standards, could be used in this method. For these, control tissue was extractedaccording to the procedure above, but before the final dried extract was reconstituted

in mobile phase, an appropriate aliquot of standard was added to the culture tube.

Acetonitrile:ammonium formate (20 mM) (95:5) was then added to produce a final

volume of 1.0 mL. For example:

10 ng/g ES - 10 L of 1 g/mL standard plus 975 L of mobile phase





These samples were then vortex mixed for 10 seconds and syringe-filtered into a glass

LC vial.

LC-MS-MS Analysis

The LC-MS-MS electrospray conditions were optimized by tuning with a 10 ng/L

solution of MEL flowing into the mass spectrometer with a syringe pump at 10

L/min while 350 L/min of 75:25 acetontrile:ammonium formate buffer (20 mM)

was added via a T-union. The combined stream was introduced into the electrospray

interface. Selected reaction monitoring (SRM) MS/MS was performed on theprotonated molecular ion for MEL using the following general parameters: Source

voltage = 5 kV; Capillary temperature = 270 C; Sheath gas (nitrogen) = 14

(arbitrary) units; Auxiliary gas (nitrogen) = 0 (arbitrary) units; Q1 peak width = 0.7

amu; Q3 peak width = 0.7 amu; Collision gas = 1.5 torr Argon; peak width = 1 amu,

and scan time = 0.5 sec. Source collision induced dissociation was set to 20 V.

It should be noted that the following scan events and mobile phase gradient for the

LC-MS-MS were set up in this method validation to detect not only melamine, butalso ammeline (ANE), ammelide (ADE), and cyanuric acid (CYA). As will be

described below, the extraction described herein was only suitable for the

determination of melamine. For the first 1.3 minutes after sample injection, a negativeion scan event was included (m/z 12885) to detect cyanuric acid, but this compound

was not evaluated at this time. For the rest of the chromatographic run, three positive

ion scan events with two SRM transitions each were monitored according to the

following chart:

CompoundRetention Time

(min)

Precursor Ion

(m/z)

Quantitative

SRM transition

(m/z)

(Collision

Energy)

Confirmatory

SRM transition

(m/z)

(Collision Energy)

MEL 2.7 127 85 (7) 68 (23)


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ANE 3.1 128 86 (5) 69 (26)

ADE 2.0 129 87 (14) 70 (27)

The LC-program for the three amine compounds was based on one developed at

Procter and Gamble. A Waters Atlantis HILIC silica column was used with anacetonitrile:ammonium formate buffer (20 mM) gradient. The mobile phase

composition started out at 95 % acetonitrile and decreased (linearly) to 50 %

acetonitrile over 5 minutes. The mobile phase was then returned to 95 % acetonitrile

between 5 and 7 minutes, and the column was re-equilibrated for 5 minutes. The flow

rate was 350 L/min. The column was kept in an insulated compartment, but the

temperature was not controlled. The injection volume was 10 L and the needle was

flushed with 400 L of 50:50 water:methanol between samples. The LC flow was

diverted to waste for the first 0.14 minutes and again at 6 minutes.

Quantitative data was obtained from the area counts of the chromatographic peak

observed with the SRM of the m/z 12785 selective ion monitoring transition usingQuanBrowser software and the ICIS integration program. The melamine found in

each fortified sample was calculated from the peak areas of this transition using a

calibration curve generated from melamine standards with concentrations ranging

from 10 to 2000 ng/mL (ppb) in mobile phase. For confirmation, peak area counts

from the m/ z12785 and m/z 12768 SRM transitions were generated with the

ICIS algorithm in the QualBrowser software program. Gaussian smoothing function

of five points was applied. Relative abundances were calculated from these peak areas

and compared to contemporary standards.

RESULTS AND DISCUSSIONMelamine was extracted from fortified catfish with an average recovery of 76.3 %

(14.3 % RSD, n = 17) over the concentration range 10 to 500 ng/g (ppb), as

summarized in Table 1. The observed relative abundance data for the two transitions

is summarized in Table 2, and selected SRM chromatograms are presented in Figure

1. Melamine was confirmed in all spikes. The method was validated using control

tissue from three different sources. The controls were skinless catfish fillets obtained

from: a local market, the FDA Gulf Coast Seafood Laboratory, and FDA's Center for

Veterinary Medicine. The recovery of melamine from 100 ng/g fortified tissue was

comparable among the three sources of tissue with recoveries of 69.3 % for GCSL

catfish, 75.5 % for CVM catfish, and 73.4, 72.9, and 74.4 % for three samples ofmarket catfish.

In the control catfish tissue, a small peak was observed at the retention time for

melamine at a level that could not be quantified. The signal-to-noise ratio of this peak

was not greater than 3:1. Melamine was not confirmed in any of the control tissues.

Melamine was not detected in the method blank.

The standard curve for solvent-based external standards was linear over the range

from 10 to 2000 ng/g. Two calibration curves were prepared for two different days of

analysis. One was over the range 10 to 100 ng/mL with r2 = 0.9972, and the other was

over the range 25 to 2000 ng/mL with r2 = 0.9997. The end spike samples analyzed inconjunction with the fortified samples had recoveries ranging from 87.6 to 97.8 %


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indicating that ion suppression was not significant. The use of solvent-based standards

is justified for this method. An internal standard was not used in this method.

In addition to melamine, tissues were also fortified with ammeline (ANE) and

ammelide (ADE) (standards were prepared similarly to those for MEL). However, the

recovery of ANE and ADE from fortified tissues was low. Approximately 15 % of theANE residue was recovered from the tissue at the 100 ng/g fortification level, and 32

% was recovered at the 500 ng/g fortification level. ANE was not detected at the 50

ng/g fortification level. ADE was only 3 % recovered at the 500 ng/g fortification

level, and could not be detected at the lower levels. The current extraction procedure

is not acceptable for these compounds.

Table 1 - Recovery of Melamine from Fortified Tissue and Negative Controls

Sample

Average %

Recovery

% RSD (n)Control Not Confirmed (n = 4)

Method Blank Not Detected (n = 1)

10 ng/g Fortified Catfish 90.3 5.2 % (n = 3)





Table 2 - Relative Abundance Data

Day Samples

% Relative Abundance

m/z 12768 transition relative to the

m/z 12785 transition

Day 1

Solvent Standards (Average, n= 7) 76.5

500 ng/g spikes 76.1, 75.3, 76.9

100 ng/g spikes 75.4, 75.5, 75.0, 84.4, 74.9

50 ng/g spikes 79.1, 78.6, 77.7End Spikes (500, 100, 50 ng/g) 76.6, 77.3, 78.5, 77.9, 79.9

Day 2

Standards (Average, n= 6) 73.9

25 ng/g spikes 72.5, 82.7, 83.0

10 ng/g spikes 73.3, 81.5, 82.5

End Spikes (25, 10 ng/g) 80.1, 80.7


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Figure 1 - SRM chromatograms for control catfish, catfish fortified with 10 ng/g of

melamine and catfish fortified with 50 ng/g of melamine.


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REFERENCES

1. "LC-MS/MS Method for the Analysis of Melamine in Porcine Meat Tissue"

California Health and Food Safety Laboratory, University of California at

Davis http://www.cahfs.ucdavis.edu1, posted 4/30/2007.
http://www.cahfs.ucdavis.edu/http://www.cahfs.ucdavis.edu/

lib 4396 melamine residues in catfish tissue

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