Determination of Melamine and Cyanuric acid in some Thesis

2017

Determination of Melamine and Cyanuric acid in some

food products using Liquid Chromatography with

Tandem Mass Spectrometry

Thesis Submitted

By

Tawfeek Mohamed Ahmed Mohamed Khedr

B. Sc. in Chemistry

Faculty of Science, Ain Shams University

2010

In the Partial Fulfillment for the Requirement of the

Master Degree in Chemistry

Chemistry Department, Faculty of Science

Ain Shams University

Under Supervision of

Prof.Dr. Mohamed Mahmoud Mohamed Abo-Aly

Professor, Department of inorganic Chemistry, Faculty of

science, Ain shams University

Dr. Emad Ramadan Mohamed Attallah

Quality Manager, Central Laboratory of Residue Analysis

of Pesticides and Heavy Metals in Food, Agricultural

Research Center

2017

APPROVAL SHEET

Determination of Melamine and Cyanuric acid in some

food products using Liquid Chromatography with

Tandem Mass Spectrometry

Thesis Submitted

By


In the Partial Fulfillment for the Requirement of the

Master Degree in Chemistry

The master submitted by the student / Tawfeek Mohamed

Ahmed Mohamed Khedr

Supervisors' approval:


Professor, Department of inorganic Chemistry, Faculty of science,

Ain shams University

Signature: ……………….……

Prof.Dr. Emad Ramadan Mohamed Attallah

Quality Manager, Central Laboratory of Residue Analysis of

Pesticides and Heavy Metals in Food, Agricultural Research Center

Signature: ………………………

Head of Chemistry Department

Prof Dr. Ibrahim Hosseini Ali Badr

Acknowledgment

I express my thanks to the graceful and merciful God for helping me

in this thesis. I wish to express my deep gratitude and thanks to

Prof Dr. Mohamed Abo-Aly, Prof of in-organic and analytical

chemistry, Department of Chemistry department, Faculty of Science,

Ain Shams University, Cairo, Egypt for his supervision, continuous

encouragement and valuable comments throughout the work and for

revising this thesis.

I also wish to express deep gratitude and thanks to Dr. Emad R.

Attala, Researcher at Central Lab of Residue Analysis of Pesticides

and Heavy Metals in Food, for his great effort and assistance

through this study and for revising this thesis. Thanks and

appreciation to all the staff members and technicians of Central

Laboratory of Residue Analysis of Pesticides and Heavy Metals in

Foods. My special thanks to my father, my mother, and my brothers

for giving me inspiration, confidence and patience throughout this

study.


Abstract

Name: Tawfeek Mohamed Ahmed Mohamed Khedr

Title of the thesis: Determination of Melamine and

Cyanuric acid in some food products using Liquid

Chromatography with Tandem Mass Spectrometry.

Position: Chemist

Degree: M.Sc., Faculty of science, Ain Shams University.

Simple, rapid and sensitive method for determination of

melamine and cyanuric acid in baby milk and liquid milk

was developed using LC-MS/MS. Extraction with 100 mM

ammonium acetate buffer at pH 4 was selected based on the

resulted accepted recovery and precision. The Sample was

extracted with ammonium acetate buffer, followed by

centrifugation; the supernatant was diluted with

acetonitrile, and re-centrifuged. The extract was filtered and

injected into LC-MS/MS system. Different parameters (e.g.

declusting potential and collision energy) were optimized

using Sciex 4000 QT in electron spray ionization positive

and negative modes. Due to LC-MS/MS signal

suppression/enhancement, determination of melamine and

cyanuric acid was based on matrix matched standard

calculation. The method was validated at different

fortification levels for liquid milk and baby milk. Recovery

values of melamine ranged from 72% to 97% with RSD%

less than 5%. Recovery values for cyanuric acid ranged

from 83 and 112% with RSD% less than 8.6%. Limit of

quantification of 0.05 mg/kg and 0.01 mg/kg was obtained

for melamine for baby milk and liquid milk, respectively.

For Cyanuric acid Limit of quantifications were 0. 5 mg/kg,

0. 1 mg/kg for baby milk and liquid milk, respectively. The

method performance was confirmed by participating in

different proficiency tests from FAPAS, UK and EURL-

SRM, Germany, and accepted Z-scores were obtained.

Key words: Melamine, Cyanuric acid, Baby milk, LC-

MS/MS.

Supervisors' approval:


Professor, Department of inorganic Chemistry, Faculty of

science, Ain shams University

Signature: ……………….……

Dr. Emad Ramadan Mohamed Attallah

Quality Manager, Central Laboratory of Residue Analysis of

Pesticides and Heavy Metals in Food, Agricultural Research Center

Signature: ………………………

Head of Chemistry Department

Prof Dr. Ibrahim Hosseini Ali Badr

Aim of work

The present study aimed to introduce a sensitive and simple

method for the determination of melamine residue in baby

milk and liquid milk that can be used for the routine

determination of melamine and cyanuric acid residue in

different milk samples and also cover the maximum residue

limits of melamine and cyanuric acid in Egypt.

So the plan of work was to:

1- Optimization of analytical method for determination of

Melamine and cyanuric acid in some food products

using Liquid Chromatography with Tandem Mass

spectrometry (LC-MS/MS).

2- Validation of the analytical method for determination

of Melamine and cyanuric acid in some food products.

3- Monitoring the Melamine and cyanuric acid in some

food products in Egyptian local markets.

List of abbreviations

APCI: Atmospheric Pressure Chemical Ionization

CE: Collision Energy

CPS: count per second

CV: Coefficient of variation

CXP: Collision Exit Potential

CYA: Cyanuric acid

CYR: Cyromazine

DAD: Diode array

DIW: De-Ionized Water

DP: Declustering Potential

EFSA: European Food Safety Authority

EP: Exit Potential

EPA: Environmental Protection Agency of United States

ESI: Electron Spray Ionization

EU: European Union

EURL-SRM: European Union reference Laboratory for

Single Residue Methods

FAO: Food and Agriculture Organization

FAPAS: Food Analysis Performance Assessment Scheme

FDA: Food and Drug Administration

g: gravity acceleration

GC/MS/MS: Gas Chromatography-Tandem Mass

Spectrometry

GC-MS: Gas Chromatography–Mass spectrometry

HILIC: Hydrophilic Interaction Chromatography

HPLC-UV: High Performance Liquid Chromatography

with Ultraviolet detection

IUPAC: International Union of Pure and Applied

Chemistry

LC-MS/MS: Liquid Chromatography Tandem Mass

Spectrometry

LOD: limit of Detection

LOQ: Limit of Quantitation

mg/kg: Milligram per Kilogram

MLN: Melamine

MRL: Maximum Residue Limit

MRM: Multiple Reactions Monitoring

ppm: Part Per Million

PT: Proficiency Test

RPC: Reversed Phase liquid Chromatography

RSD: Relative Standard Deviation

SIM: Single Ion Monitoring

SML: Specific migration limit

SPE: Solid Phase Extraction

SRM: Single Reaction Monitoring

TFHA: Tri-decafluoroheptanoic Acid

UN: United Nation

UPLC–MS/MS: Ultra-performance liquid chromatography

–tandem mass spectrometry

WHO: World Health Organization

MISPE: Molecularly imprinted solid-phase extraction

Contents

Approval Sheet…………………………………………….

Acknowledgment ……...……………………………..…...

Abstract ……...…………………………………………..

Aim of Study……………………………………………..

List of abbreviations………………………………………

Publications………………………………………………

Table of contents ……………………………………….....

List of figures …………………………………………...

List of tables ……………………………………….........

1 Introduction ................................................................ 1

1.1 Melamine ................................................................. 1

1.1.1 Sources of melamine contamination .............. 2

1.1.2 Physical and chemical properties .................. 2

1.1.3 Melamine applications ................................... 2

1.2 Cyanuric acid ........................................................... 4

1.2.1 Sources of cyanuric acid contamination ........ 4

1.2.2 Physical and chemical properties .................. 4

1.2.3 Cyanuric acid applications ............................. 6

1.3 Metabolism of melamine and cyanuric acid ........... 6

Documents

Determination of Melamine and Cyanuric acid in some Thesis