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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
Tawfeek Mohamed Ahmed Mohamed Khedr
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:
Prof.Dr. Mohamed Mahmoud Mohamed Abo-Aly
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.
Tawfeek Mohamed Ahmed Mohamed Khedr
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:
Prof.Dr. Mohamed Mahmoud Mohamed Abo-Aly
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
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