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EEGGEE UUNNIIVVEERR SSIITTYY

MASTER THESIS

DETERMINATION OF METAL CONTENTS OFHONEY, GRAPE SYRUP, VINEGAR AND FRUIT

JUICES PRODUCED IN TURKEY BY ICP-MSMETHOD

Levent ELBOL

Supervisors : Prof. Dr. F. Nil ERTA

Assist. Prof Dr. Hasan ERTA

Department of Chemistry

Code of Discipline : 405.03.01Date of Presentation : 04.12.2009

Bornova-ZMR2009

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EGE UNIVERSITYGRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES

(MASTER THESIS)

DETERMINATION OF METAL CONTENTS OF HONEY,GRAPE SYRUP, VINEGAR AND FRUIT JUICES PRODUCED

IN TURKEY BY ICP-MS METHOD

Levent ELBOL

Supervisors : Prof. Dr. F. Nil ERTA

Assist. Prof. Dr. Hasan ERTA

Department of Chemistry

Code of Discipline : 405.03.01Date of Presentation : 04.12.2009

Bornova-ZMR

2009


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Levent ELBOL tarafndan Yksek Lisans tezi olarak sunulan Trkiyede

retilen Bal, Pekmez, Sirke Ve Meyve Suyu rneklerinin Metal eriklerinin

ICP-MS le Analizlenerek ncelenmesi balkl bu alma E.. Lisansst

Eitim ve retim Ynetmelii ile E.. Fen Bilimleri Enstits Eitim ve

retim Ynergesinin ilgili hkmleri uyarnca tarafmzdan deerlendirilerek

savunmaya deer bulunmu ve 04.12.2009 tarihinde yaplan tez savunma

snavnda aday oybirlii/oyokluu ile baarl bulunmutur.

Jri yeleri: mza

Jri : Prof. Dr. F. Nil ERTA .................................

Bakan

Raportr : Prof. Dr. mran YKSEL .................................

ye

ye : Prof. Dr. Ali ELK .................................


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ZET

TRKYEDE RETLEN BAL, PEKMEZ, SRKE VE

MEYVE SUYU RNEKLERNN METAL ERKLERNN

ICP-MS LE ANALZLENEREK NCELENMES

ELBOL Levent

Yksek Lisans Tezi, Kimya Blm

Tez Yneticisi: Prof. Dr. Nil ERTA

Aralk 2009, 50 sayfa

Bu tezde Trkiye'de retilen sirke, pekmez, bal ve meyve suyu rneklerinin

metal iyonu ieriklerinin indktif elemi plazma-ktle spektrometresi (ICP-MS)

sistemi ile analizi amalanmtr. Bu tr eker ierikli gda maddelerine hile

amal katmlar yaplarak piyasaya arzedildii bilinen bir gerektir. Bu gdamaddelerinin kalitesinin yansra orjinalitesinin snanmasna ynelik varolan

gelitirilmi yntemlere seenek oluturabilecek doru ve duyarl yntem

gelitirilmesi konusunda almalar srmektedir.

Bu tez almasnn ana hedefi bu gda matrikslerinin metal ieriklerinin

gnmzn bu alanda en gelimi yntemi saylan ICP-MS sistemi ile

saptanmasdr. Bu amala rneklerin analize hazrlanmas, bozundurulmas,

lm, verilerin istatistiksel deerlendirilmesi ve sonularn dier bulgularla

kyaslanmas aamalarnda varolan altyap kullanlarak gerekli eksik malzemelerin

bu projeden temini ile bu amaca hzla ulalmas hedeflenmitir.

Pekmez, sirke, bal ve meyve suyu gibi gda maddelerine hile amal

katmlar yaplarak piyasaya arz edildii bilinen bir gerektir. Her ne kadar bu gda

maddelerinin safln tespit etmek iin gelitirilmi farkl yntemler olsa da bu

yntemler her zaman kesin sonu verememektedir. Bu nedenle bu gda maddeleri

iin gelitirilmi yntemlere alternatif ve destekleyici olabilmesi asndan


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ierdikleri metal bileimi tespit edilerek matrikslerin saflna ilikin yeni

parametreler oluturulmas amalanmaktadr. Ayn zamanda bir dier hedef de bu

maddelere evreden bulaabilen yada retim aamasnda prosesten

kaynaklanabilen metalik kirliliklerin saptanmasdr. Bu kontaminasyonlar iin

verilen maksimum tolerans snrlar ok dk olduundan analizde olduka duyar

ve tekrarlanabilir dolays ile de gvenilir tekniklerin gelitirilmesine gereksinim

vardr.

Gnmzde bal, pekmez, sirke ve meyve sularnn saflk analizlerinde genel

olarak izotop oranlar ktle spekktroskopisi yntemi kullanlmaktadr. Buna ek

olarak, eker bileenlerinin, refraktif indeks dedektrl yksek basn sv

kromatografisi HPLC- RI yntemi ile analizi, bal iin prolin ve polen analizi,meyve sular iin kat madde tayini gibi yntemler de kullanlmaktadr. Bu tez

almas kapsamnda bu analizlere ek olarak ICP-MS cihaznda bu gda

maddelerinin metal bileimleri tespit edilerek, bu maddelere zg yeni

parametrelerin oluturulmasna allacaktr. Bunun iin rnekler mikro dalga

bozundurma sistemi kullanlarak bozundurma ilemine tabi tutulacak ve bu yolla

analize hazrlanacaktr. Ardndan kalibrasyonu ve validasyonu yaplm olan ICP-

MS yntemi kullanlarak metal iyonlarnn deriimleri saptanacaktr. te yandan

her zaman analizine bavurulmayan kimileri insan vcudu iin esansiyel olan

metal iyonlarnn da analizi gerekletirilerek, bu tr eker ierikli gdalarn metal

ieriine farkl bir bak kazandrlacaktr.

Anahtar Szckler: Ar metal, bal, meyve suyu, ICP-MS, Mikrodalga ile

bozundurma


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ABSTRACT

DETERMINATION OF METAL CONTENTS OF HONEY,

GRAPE SYRUP, VINEGAR AND FRUIT JUICES PRODUCED IN

TURKEY BY ICP-MS METHOD

ELBOL Levent

Master Thesis in Chemistry

Supervisor: Prof. Dr. Nil ERTA

December 2009, 50 pages

In this study, to develop a method for revealing the adultery in food samples

namely fruit juices, honey, grape syrups and vinegar was aimed. For this purpose,

ICP-MS measurements were used to discriminate the real and fake samples. This

is a part of a main project together with the 13C measurements of these sugar

containing foods. Overall results will overlook the average composition of these

foods for deciding any adultery.

In the course of the study, the comparison of the sample preparation

techniques and validation of the methods was also accomplished. The method was

developed for food samples and overall data was evaluated to find a pattern to

help to discriminate the real and fake samples

It is known that honey, grape syrup, vinegar and fruit juices are exposed to

adulteration before marketing. Although there are a number of methods developed

for authencity of these products, reliable methods are always needed for accurate

analysis. Therefore, it was planned to develop an alternative of supportive method

to those methods already used. Metal content of these samples will be analyzed to

perceive the possibility of establishing another parameter specific to these sample

or to reveal the contamination of heavy metal impurities as well.The maximum


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tolerance limits are very low for these contaminations. So very sensitive,

repeatable and reliable methods should be developed.

Recently, the authencity of honey, grape syrup, vinegar and fruit juices are

usually searched by isotope ratio method and HPLC-RI method for sugarcontents. Besides, proline and pollen analysis for honey, brix analysis for fruit

juice are also required for reliable results. In the context of this thesis, in addition

to those developed methods for authenticity, the metal content of these samples

will be determined by means of ICP-MS method to perceive the possibility of

establishing another parameter specific to these sample or to reveal the

contamination of heavy metal impurities as well. The samples will be decomposed

in microwave digestion system before the analysis.Then the concentrations ofmetal ions will be determined with calibrated and validated ICP-MS method that

has a wide range.

Keywords: Heavy metal, honey, fruit juice, ICP-MS, microwave digestion


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ACKNOWLEDGMENT

I would like to present my gratitude to my supervisors, Prof. Dr. F. Nil

ERTA and. Do. Dr. Hasan ERTA for their precious suggestion, support, and

patience, and for enlightening me via their deep knowledge and experience. I also

thank to all of the members of the Ege University Center for Drug Research &

Development and Pharmacokinetic Applications Contract Research Organization

(ARGEFAR) especially to the assistant director Ercment KARASULU for

providing the precious conditions and support throughout my studies.

I would like to thank to my friend Bar GMTA who have

encouraged and helped me throughout this work.

I would like to thank and present my gratitude to my family for their,

understanding, support and patience.

Bornova, 2009 Levent ELBOL


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CONTENTS

Page

ZET ................................................................................................................ V

ABSTRACT........................................................................................................VII

ACKNOWLEDGMENT ...................................................................................... X

LIST OF FIGURES ............................................................................................XV

LIST OF TABLES..........................................................................................XVIII

1.INTRODUCTION .............................................................................................. 1

1.1.Authenticity of Foods....................................................................................... 1

1.1.1.Fruit Juice...................................................................................................... 1

1.1.2.Honey............................................................................................................ 2

1.1.3.Grape Syrup .................................................................................................. 4

1.1.4.Vinegar.......................................................................................................... 5

1.2.Heavy Metals ................................................................................................... 6

1.3.Literature Survey on Determination of Heavy Metals in Foods...................... 7

1.4.The Aim of the Thesis.................................................................................... 10

1.5.ICP-MS .......................................................................................................... 10

1.5.1.Sample Introduction.................................................................................... 11


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CONTENTS (continue)

Page

1.5.2.Transfer of ions into Vacuum ..................................................................... 12

1.5.3.Ion Optics.................................................................................................... 12

1.5.4.Octopole Reaction System (ORS) .............................................................. 13

1.5.5.Plasma ......................................................................................................... 13

1.5.6.Plasma Generation ...................................................................................... 13

1.5.7.Advantage of Argon.................................................................................... 14

1.5.8.Elemental Analysis ..................................................................................... 15

1.6.Microwave Digestion..................................................................................... 15

1.6.1.Heating Mechanism .................................................................................... 16

1.6.2.Open Versus Closed Acid Digestion .......................................................... 17

1.7.Method Validation ........................................................................................ 18

2.EXPERIMENTAL............................................................................................ 21

2.1 Apparatus ....................................................................................................... 21

2.2 Chemicals and Reagents ................................................................................ 21

2.3 Analytical Procedures and ICP-MS Conditions ............................................ 21

3.RESULTS AND DISCUSSION ....................................................................... 23

3.1 Optimization Studies for Method Development ............................................ 23


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CONTENTS (continue)

Page

3.2 Validation Studies ..........................................................................................26

3.2.1 Linearity ......................................................................................................26

3.2.2 Repeatability ............................................................................................... 26

3.2.3 Sensitivity.................................................................................................... 27

3.2.4 Recovery ..................................................................................................... 27

3.2.5 Uncertainty .................................................................................................. 28

3.3 Comparison of thhe Developed Method with Direct Injection ......................29

3.4 Application of the Method to the Samles.......................................................30

3.4.1 Vinegar Results ...........................................................................................30

3.4.2 Grape Syrup Results....................................................................................30

3.4.3 Fruit Juice Results .......................................................................................33

3.4.4 Honey Results..............................................................................................45

4. CONCLUSION................................................................................................ 47

REFERENCES..................................................................................................... 48

CIRRICULUM VITAE........................................................................................ 50


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LIST OF FIGURES

Figure Page

1.1. Schematic representation of an ICP-MS ................................................11

1.2. Schematic of sample heating by microwaves.........................................17

1.3. Schematic representations of sample digestion systems .......................17

3.1. Typical ICP-MS results of Sc, V, Cr, Mn, Co, Ni, Cd, Cr, As, Mn, Cd,

In, Sb, Ba.....25

3.2. Typical ICP-MS results of Zn, Ge, Hg, Bi .............................................25

3.3. Typical ICP-MS results of Fe, Mg, K, Ca, Na .......................................25

3.4. Schematic representation of barium results of grape syrups, mulberry

and harnup syrups...................................................................................32

3.5. Schematic representation of iron results of grape syrups, mulberry and

harnup syrups..........................................................................................32

3.6. Schematic representation of manganese results of grape syrups,

mulberry and harnup syrups ................................................................32

3.7. Schematic representation of barium results of strawberry juices and

froudulent sample ...................................................................................35

3.8. Schematic representation of calcium results of strawberry juices and


3.9. Schematic representation of potassium results of strawberry juices and


3.10. Schematic representation of magnesium results of strawberry juices and



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LIST OF FIGURES (continue)

3.11. Schematic representation of sodium results of strawberry juices and

froudulent sample .................................................................................. 36

3.12. Schematic representation of zinc results of strawberry juices and


3.13. Schematic representation of cupper results of apricot juices and


3.14. Schematic representation of iron results of apricot juices and froudulentsample .................................................................................................... 39

3.15. Schematic representation of potassium results of apricot juices and


3.16. Schematic representation of magnesium results of apricot juices and


3.17. Schematic representation of manganese results of apricot juices and


3.18. Schematic representation of sodium results of apricot juices and


3.19. Schematic representation of cupper results of pomegranate juices and


3.20. Schematic representation of magnesium results of pomegranate juices

and froudulent sample............................................................................ 42

3.21. Schematic representation of zinc results of pomegranate juices and


3.22. Schematic representation of barium results of cherry juices and



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LIST OF FIGURES (continue)

3.23. Schematic representation of iron results of cherry juices and froudulent

sample.....................................................................................................44

3.24. Schematic representation of potassium results of cherry juices and


3.25. Schematic representation of sodium results of cherry juices and


3.26. Schematic representation of barium results of honey samples, sugaradded sample and froudulent sample .....................................................46

3.27. Schematic representation of calcium results of honey samples, sugar

added sample and froudulent sample .....................................................46

3.28. Schematic representation of cupper results of honey samples, sugar


3.29. Schematic representation of potassium results of honey samples, sugar



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LIST OF TABLES

Table Page

1.1. Limits for metal and sugar content of several fruit juices (mg/kg) (AIJN,

2007)......................................................................................................... 3

1.2. Typical honey composition in percent (% g/g)............................................. 3

1.3. Microwave digestion procedures of fruit juice samples ............................... 8

2.1. Microwave digestion procedure.................................................................. 22

2.2. Optimized conditions for ICP-MS system.................................................. 22

2.3. Used isotopes of analyzed elements............................................................ 22

3.1. The effect of sample weight on the analysis by comparing the metal content

of the fruit juice sample .............................................................................. 23

3.2. The comparison of the metal content of the fruit juice sample digested at

various temperatures................................................................................... 24

3.3. The comparison of the metal content of the fruit juice sample digested at

various time ................................................................................................ 24

3.4. Regression coefficients for metals studied from calibration curves obtained

with ICP-MS measurements ....................................................................... 26

3.5. Relative Standard Deviations of metals ...................................................... 27

3.6. LOD LOQ values of metals (g/L)............................................................. 27

3.7. Recovery percentages of the metals studied with ICP-MS......................... 28

3.8. Relative uncertainty values of metals ......................................................... 28


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LIST OF TABLES (continued)

Table Page

3.9. Comparison of the developed microwave digestion method with direct

injection.......................................................................................................29

3.10. Metal compositions of grape vinegar (GV) and falsified alcohol

vinegar(FAV) ..............................................................................................30

3.11. Metal compositions of grape syrups and mulberry syrup............................31

3.12. Metal compositions of pear juices ...............................................................33

3.13. Metal compositions of strawberry juices .....................................................34

3.14. Metal compositions of apple juices .............................................................37

3.15. Metal compositions of apricot juices ...........................................................38

3.16. Metal compositions of pomegranate juices .................................................41

3.17. Metal compositions of cherry juices............................................................43

3.18. Metal compositions of honey .....................................................................45


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H2SO4 Sulfuric Acid

He Helium

Hg Mercury

HCl Hydrochloric Acid

HNO3 Nitric Acid

ICP-AES Inductively Coupled Plasma- Atomic Emission Spectrometer

ICP-MS Inductively Coupled Plasma-Mass Spectrometer

ICP-OES Inductively Coupled Plasma- Optic Emission Spectrometer

IRMS Isotope Ratio Mass Spectrometer

K Potassium

LOD Limit of Detection

LOQ Limit of Quantification

Mg Magnesium

Mn Manganese

Na Sodium

Ni Nickel

OCTP Octopole

ORS Octopole Reaction System

ABBREVIATIONS (continued)


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Pb Lead

QP Quadrupole

RF Radio Frequency

RSD Relative Standard Deviation

S/C Spray Chamber

Se Selenium

Sb Antimony

TSE Turkish Standard Institute

USA United States of America

V Vanadium

W Watt

Zn Zinc


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1.INTRODUCTION

1.1Authenticity of Foods

Foods like honey, molasses, fruit juices and vinegar known that they arehealty foods. Many people consume these foods are just as healthy. Unfortunately,

they are all not healthy or original. In many countries of the world including our

country, this is known that many manufacturers make froud to these foods to earn

more. For a chemist, even though many analytical techniques have developed, it is

very hard to determine that it is natural or not as the competition between analyst

and falsifier is going on. For this reason, it is very important to develop new

techniques and aspects to detect forgery. Metal analysis is important to find

forgery as well as detecting non-essential metal in foods.

The compositions of various metals in different food types of various

countries have been the subject of many studies. Such data are not readily avaible

for most food in developing countries, such as Turkey. The objective of this thesis

is to provide a more detailed determination of the contents of the metals in fruit

juices, honey, vinegar and molases. Following sections give a brief explanation

about the composition of these materials.1.1.1 Fruit Juice

100% fruit juices are nutritious beverages that have been enjoyed by adults

and children for decades. 100% fruit juices can play an important role in a healthy

diet because they offer great taste and a variety of nutrients found naturally in

fruits. These juices are fat-free, nutrient-dense beverages that are rich in vitamins,

minerals and naturally occurring phytonutrients that contribute to good health.

Phytonutrients are compounds in fruits, vegetables and other plants that

researchers find have disease preventative and disease fighting properties.

The determination of juice authenticity is exceedingly complex and requires

cooperative efforts of well-informed, dedicated individuals from many disciplines

and involves many analytical methodologies (K.W. Barnes, 1999). Metal

determinations can resolve many issues and are typically performed to answer

three questions. First, how much of a nutrient metal (mineral) is present and is the

product labeled properly?


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Next, is the product what it claims to be and does it comply with trade laws?

Have authentic products been used, or has the product been adulterated? Finally,

is the product wholesome and safe to eat, is it contaminated, or has tampering

occurred?

To promote and maintain fair competition and commercial viability of fruit

juices, European Fruit Juice Association (AIJN) serves for over 40 years. AIJN

has been the representative association of the fruit juice industry in the E.U. It

represents the industry from the fruit processors to the packers of the consumer

products. One of the main activities of AIJN is the development of instruments

such as the reference guidelines, codes of practice, position papers, etc. for the

benefit of the whole fruit juice industry. These instruments complement the fruitjuice legislation. AIJN developed two very important tools for the industry; first

one is The Code of Practice for evaluation of fruit and vegetable juices which sets

absolute quality requirements and criteria for the evaluation of identity and

authenticity of 20 different fruit juices. The second one is The European Quality

Control System which aims at maintaining the good and healthy image of fruit

juice products and ensuring fair competition in the single European market.

More recently AIJN developed a guideline for the interpretation of the fruitjuice directive 2001/112 EC, a guideline for restoration aroma, a traceability

guideline as well as a revision of its hygiene Code. Turkey is an affiliated member

of AIJN and Turkish Fruit Juice Industry Association (MEYED) facilitates on this

subject (meyed.com.tr). According to AIJN, metal contents of several fruit juices

were reported and given in Table 1.1 (AIJN, 2008).

1.1.2. Honey

Honey possesses valuable nourishing, healing and prophylactic properties.

These properties result from its chemical composition (Przybylowski et al., 2005).

Honey production starts valuable nourishing, healing and prophylactic properties

which are produce by Hymenoptera collected from bees. Collected nourishes are

changed in body of bees and stores in comb eyes for maturation, as a result honey

bee that is dense and desert (Demirezen, 2005).


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Table 1.1 Limits for metal and sugar content of several fruit juices (mg/kg) (AIJN, 2007)

Metal Apple Apricot Pomegranate Strawberry Pear Cherry

As 0.1 0.1 0.1 0.1 0.1 0.1

Ca 30-120 85-200 50-120 80-300 35-130 80-240

Cd 0.05 0.05 0.05 0.05 0.05 0.05

Cu 5.0 5.0 5.0 5.0 5.0 5.0

Fe 5.0 5.0 5.0 5.0 5.0 5.0

Hg 0.01 0.01 0.01 0.01 0.01 0.01

K 900-1500 2000-4000 1300-3000 1000-2300 1000-2000 1600-3500

Mg 40-75 65-130 20-110 70-170 45-95 80-200

Na Max 30 Max 35 Max 30 Max 30 Max 30 Max 30

Pb 0.05 0.05 0.05 0.05 0.05 0.05

Sn 1.0 1.0 1.0 1.0 1.0 1.0

Zn 5.0 5.0 5.0 5.0 5.0 5.0F/G 2.0-3.3 0.4-1.0 1.0-1.2 1.0-1.3 Min 2.5 0.7-0.9

13C -27/ -24 - - - - -

Brix 11.2 11.2 15.0 13.5 11.9 7.0

Honey is a semi liquid product, which contains a complex mixture of

carbohydrates, mainly glucose and fructose; other sugars are present at trace

levels, depending on floral origin. Moreover, organic acids, lactones, amino acids,

minerals, vitamins, enzymes, pollen wax and pigments are present. Honey isproduced either from many flowers or from single flower pollens.

The quality criteria for honey are described as its contents. Typical honey

percent combination is given in Table 1.2. These contents are; acidity, sugar

component ratio, mineral content, diastase activity, hydroxymethylfurfural

content, prolin (amino acid). Thus, analytical methods have to overcome all the

honey matrix effects.

Table 1.2 Typical honey composition in percent (% g/g)

Content % Content %

Water 17 Acid 0.57

Fructose 38 Proteins 0.26

Glucose 31 Ash 0.17

Sucrose 1 Others* 12

*maltose, alkaloids, tannins, enzymes, vitamins, pollens


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Honey can named as natural with these analyses but this is not enough for

its quality. As food stuff used for healing purposes, honey must be free ofobjectionable contents. It should contain only small amounts of pollutants, such as

trace metals. Analysis of honey for trace elements content is necessary in food

quality control.

In addition, bee honey has been used as monitors of a variety of

environmental contaminants, including heavy metals, low level radioactivity and

pesticides. Heavy metals have an important function for environmental pollution.

Experiments carried out in Polandshow that, large amounts of heavy metals were

found in honeys from hives located near extra urban crossroad and steelworks

(Tuzen, 2005).The climate and rich vegetation in Turkey provide a very suitable

environment for apiculture which is in a state of expansion. Turkey was the third

largest country with 3,686,000 hives in 1993, following Russia and USA. The

production of honey was 59.207 tons in 1995 and increased to 80.000 tons in

1997. Recently, both international and Turkish studies have drawn attention to the

occurrence of the metal contents of honey (Tuzen, 2005).

1.1.3. Grape Syrups

Grape syrups are widely produced and consumed in Turkey. Searched for

adultery in food can be focused on grape syrups as they are potentially available

for adultery. On the other hand, molasses is a viscous byproduct of the processing

of sugar cane or sugar beets into sugar. The quality of molasses depends on the

maturity of the sugar cane or sugar beet, the amount of sugar extracted, and the

method of extraction. Sweet sorghum syrup is known in some parts of the United

States as molasses, though it is not true molasses.

Recently, adulteration of grape syrups is increasingly being recognized as a

problem in Turkey. Grape syrups are produced in two forms; solid and liquid.

Solid grape syrup, also called as Zile, is produced by simply adding starch, egg

white, powdered sugar, honey, milk powder and ripe raisin concentrate to grape

syrup and it is not considered for adulteration.


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According to regulations of Turkish Standard Institute (TS-3792), liquid

type should be produced only from fruit extract and should not contain any

additive material including Zile (TSE, 2008). In order to reduce cost, it is

however, easily and usually adulterated with cheaper carbohydrates such as

sucrose, high-fructose corn syrup and glucose syrup, which may be regarded as

rather harmless.

Furthermore, grape syrup is sometimes illegally mixed with second grade

fruit juice concentrates such as mulberry, fig and carob bean and often treated

with citric acid as preservative and caramel as coloring matter (imek et al,

2002).

1.1.4. Vinegar

Vinegar is an acidic liquid processed from the fermentation of ethanol in a

process that yields its key ingredient, acetic acid. It also may come in a diluted

form. The acetic acid concentration typically ranges from 4 to 8% by volume for

table vinegar (typically 5%) and higher concentrations for pickling (up to 18%).

Natural vinegars also contain small amounts of tartaric acid, citric acid, and other

acids. Vinegar has been used since ancient times and is an important element in

European, Asian, and other cuisines.

Vinegar is made from the oxidation of ethanol by acetic acid bacteria in

wine, cider, beer, fermented fruit juice, or nearly any other liquid containing

alcohol. Commercial vinegar is produced either by fast or slow fermentation

processes. Slow methods generally are used with traditional vinegars and

fermentation proceeds slowly over the course of weeks or months. The longer

fermentation period allows for the accumulation of a nontoxic slime composed of

acetic acid bacteria and soluble cellulose, known as the mother of vinegar.

The fraud in vinegar production is made by adding dilute acetic acid. This

can be easily detected by IRMS technique. But, if the vinegar made by ethyl

alcohol, it is important to determine acetic acid with IRMS. Therefore, metal

spectrum of natural vinegar will guide us to determine whether vinegar is made by

ethyl alcohol or diluted acetic acid.


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1.2. Heavy Metals

Trace metals are important in daily diets, because of their essential

nutritious value and possible harmful effects. Trace metals are classified into three

classes based on their effects on life. The essential nutritive metals are Co, Cu, Fe,I, Mn and Zn. However, elements such as Cu and Zn have emetic action when

ingested in higher amounts. The non-nutritive non-toxic metals which are not

harmful when present in amounts not exceeding 100 ppm include Al, B, Cr, Ni

and Sn. However, the increasing chromium intake calls for concern. The non-

nutritive toxic metals which are known to have deleterious effects even at

amounts below 100 ppm are As, Sb, Cd, F, Pb, Hg and Se. For example, arsenic

exposure induces cardiovascular diseases, developmental abnormalities,neurologic and neurobehavioral disorders, diabetes, hearing loss, hematologic

disorders and various types of cancer .

Heavy metals may enter the human body through food, water, air, or

absorption through the skin when they come in contact with humans in agriculture

and in manufacturing, pharmaceutical, industrial or residential settings. Food is a

major source of human exposure to metals.

Potential sources of human exposure include consumer products and

industrial waste as well as the working environment. Cumulative poisoning occurs

due to ingestion of food containing metals such as lead and arsenic over a long

period. Some metals have detrimental effects on the quality or nutritive value of

food. For example, copper tend to destroy vitamin C in fruit products (Williams et

al, 2007). Heavy metals become toxic when they are not metabolized by the body

and they accumulate in the soft tissues.

The metabolism of the toxic metal may be similar to metabolically related

essential ones. Such is the case with effects of Pb and Ca in the central nervous

system and Pb, Fe and Zn in heme metabolism.

Human cells that are involved in the transport of metals such as gastro-

intestinal, liver or renal tubular cells are particularly susceptible to toxicity.

Factors such as age, diet, interactions and exposure to other toxic metals influence

the toxicity levels of metals in humans. Children and the elderly are believed to be

more susceptible to toxicity from metallic exposure.


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Trace metals are present in foods in amounts below 50 ppm and have some

toxicological or nutritional significance. While some inorganic elements such as

Na, K, Ca, P are essential for man, elements like Pb, Cd, Hg, As are found to

cause deleterious effects even in low levels of 1050 ppm (Williams et al,2007).

Hence, determination of both major and trace levels of metal contents in food is

important for both food safety and nutritional considerations.

The compositions of various metals in different food types have been the

subject of many studies (Iwegbue et al, 2008). Such data are readily available for

most food in developing countries.

1.3. Literature Survey on Determination of Heavy Metals in Foods

Recently, numerous instrumental methods are used to determine heavy

metals in many kinds of foods, mainly spectroscopic methods including

inductively coupled plasma with mass detector (ICP-MS), and optic emission

(ICP-OES) and Graphite Furnace Atomic Absorption Spectroscopy (GFAAS).

Changes in the quality of bee honey are also caused by the contamination

with micro-polluting agents, toxic to consumers. The honey used in a study was

harvested from beehives situated in an area where ecological unbalances induced

by the non-ferrous metal industry through pollution. For this purpose, 5.0 g of

honey was placed in a small container and heated until turned into caramel and

then placed on the flame and burned further until the sample stops smoking. The

container is then placed in an electric oven and heated to 700oC until calcinated

for 3 hours. The resulted ash is cooled to room temperature and then dissolved in

5 ml solution of nitric acid (1:6). The solution is then heated until evaporated to

half its volume prior to the quantization with AAS. The amounts of Pb, Cd and Zn

contained in the samples have been determined using an AAS. The results

suggested that honey could be used to detect contaminating agents from the

environment (Bratu, et al. 2005).

In a study carried out in our country, bee honey samples collected from 16

stations around Kayseri were weighted ca. 2.5 g and incinerated at 450oC and

then, dissolved in nitric acid. The contents of cadmium, lead, nickel, zinc and

copper in the samples were determined by ICP-OES. The results have revealed

that mean intake of heavy metals is generally tolerable (Demirezen et al, 2005).


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In another study, the trace metal contents in 15 different honey samples

collected from different farms in Middle Anatolia were determined by GFAAS

after microwave digestion. The contents of trace metals were found to be in the

range of 1.0~5.2 g/g, 0.25~1.10 g/g, 0.18~1.21 g/g, 1.1~24.2 g/g,17.6~32.1

g/kg and 10.9~21.2 g/kg for Fe, Cu, Mn, Zn, Pb and Cd, respectively (Tzen,

2005).

In another study carried out with vinegar, after weighting (0.5-1.0 g) and

mixing with 10 mL of nitric acid, samples were digested first at 50oC for 2-3

hours and then, at 90oC to dryness. Cooled digests were dissolved in 1 M nitric

acid and then, analyzed for their lead concentration by GFAAS or ICP-MS

(Ndungu et al, 2004).It is known that fruit juices contain trace metals. Therefore, the trace metal

contribution of juices should be considered; however, literature survey has

revealed that less attention was paid to their determination in fruit juices. In a

study with fruit juices, the samples were weighed about 10.0 g into the Teflon

PFA digestion vessels and 10 mL ultrapure nitric acid and 2 mL ultrapure

concentrated H2SO4 was added. Then, microwave digestion procedure given in

Table 1.3 was applied. Finally, the vessels were cooled for 5 minutes and dilutedto 100 mL with ultrapure water (K.W. Barnes, 1999).

Table 1.3 Microwave digestion procesures of fruit juice samples

Parameter Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

Power (%) 10 20 0 15 0

Power (watt) 51 141 0 96 0

Pressure (psi) 20 50 20 80 0

Run Time (min) 2 5 2 15 5

Canned fruit juice samples were stored at almost idential conditions similar

to shops. In a study with canned fruit juices, 300 mL of the liquid samples was

heated in evaporating dish on a regulated hot plate. The caramelous mass was

formed in most cases was than digested with a mixture of perchloric and nitric

acid. The digest was diluted to 25 mL mark using 1 M nitric acid. The sample

solutions were subsequently analyzed for the metals using a GFAAS equipped

with D2 background correction devices (Chukwujindu et al, 2008).


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A multi-metal Standard solution of nine metals was prepared to give the

following concentrations: Cu 1.0 ppm, Zn 0.2 ppm, Ni 0.2 ppm, Cr 0.2 ppm, Mn

0.05 ppm, Pd 0.5 ppm, Cd 0.5 ppm, Co 0.2 ppm, Sn 0.2 ppm. In fruit juices, large

particles were first removed by centrifuging and interference from sugar was

compensated for by using standard method of additions. Fruit juices were

centrifuged for 15 min at 4,500 rpm to obtain pulp free liquid. Aliquots 0, 1, 2 and

3 ml of the multi-metal standard solution were added to 100 ml volumetric flasks

containing 3, 2, 1 and 0 ml of distilled water respectively and diluted to 100 ml

with clear juice. The prepared samples were aspirated directly into the atomic

absorption spectrophotometer. Carbonated beverages were analysed after the

removal of carbon (IV) oxide by aeration. Four 100 ml aliquot of carbonated

drinks were pipetted into 250 ml beakers containing 0, 1, 2 and 3 ml of multi-

metal standard solution respectively. After heating on a hot plate until the volume

was reduced to 75 ml, the samples were cooled and diluted to 100 ml and the

prepared samples were aspirated directly into the spectrophotometer (Williams et

al, 2007).

The fruit samples were cleaned, peeled (if necessary) and washed to obtain

edible parts prior to analysis, then homogenized. Samples were weighed (10-50 g)

in quartz crucibles, dried at 105C for 24 hours and subsequently ashed in a

muffle furnace at 400C. The juice samples (100 ml) were poured into quartz

crucibles and evaporated to dry residue at 100C, then ashed in a muffle furnace

like the fruit samples. Ash was dissolved in 1mol/l nitric acid and filled up in 50

ml volumetric flasks to the mark by the same acid. The content of Pb and Cd in

the mineralised sample was determined after extraction of the complexes with

APDC (1-pyrrolidindithiocarbamate ammonium) to MIBK (methyl-

isobuthylketon) phase using the flame atomic absorption spectrometry (F-AAS)

method. The content of Zn and Cu in the diluted sample solutions was determined

by the same method . All the instrumental conditions applied for metal

determinations were set in accordance to the general recommendations

(wavelengths for Pb, Cd, Zn and Cu: 283.3 nm, 228.8 nm, 213.9 nm and 324.8

nm, respectively) (Krejpcio et al, 2004).


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1.4. The aim of the Thesis

In this study, it was planned to develop a method for revealing the adultery

in food samples namely fruit juices, honey, grape syrups and vinegar. For this

purpose, ICP-MS measurements were used to find a pattern to help todiscriminate the real and fake samples. This is a part of a main project together

with the 13C measurements of these sugar containing foods. Overall results will

overlook the average composition of these foods for deciding any adultery.

In the course of the study, the comparison of the sample preparation

techniques and validation of the methods was also planned. Next section describes

the fundamentals of the analytical methods used in the thesis study.

1.5. ICP-MS

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is an analytical

technique used for elemental determinations. The technique was commercially

introduced in 1983 and has gained general acceptance in many types of

laboratories. ICP-MS has many advantages over other elemental analysis

techniques such as atomic absorption and optical emission spectrometry,

including ICP Atomic Emission Spectroscopy (ICP-AES), including: detection

limits for most elements equal to or better than those obtained by GFAAS, higher

throughput than GFAAS, the ability to handle both simple and complex matrices

with a minimum of matrix interferences due to the high-temperature of the ICP

source, superior detection capability than ICP-AES with the same sample

throughput and finally, the ability to obtain isotopic information.

An ICP-MS combines a high-temperature ICP (Inductively Coupled

Plasma) source with a mass spectrometer. The ICP source converts the atoms of

the elements in the sample to ions. The ICP-MS instrument employs plasma as the

ionization source and a mass spectrometer analyzer to detect the ions produced. It

can simultaneously measure most elements in the periodic table and determine

analyte concentration down to the sub nanogram-per-liter (ng/L) or part-per

trillion (ppt) levels. It can perform qualitative, semi quantitative, and quantitative

analysis, and since it employs a mass analyzer, it can also measure isotopic ratios.

Figure 1.1 shows the main components of the ICP-MS system.


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Figure 1.1 Schematic representation of an ICP-MS (Agilent Technologies).

ICP-MS has many applications in food science and is used in the analysis of

a wide range of samples in the plough to plate food chain. Samples analyzed by

plasma spectrometry can vary from the relatively simple and well defined, for

example a single fruit or vegetable, through to complex, highly processed whole

meals, diets, digesta, excreta or other biological samples. The establishment of

routine automated analytical methods using ICP-MS have permitted multi-

element measurements of most elements in the Periodic Table (S. J. Hill, 2007).

1.5.1 Sample introduction

The first step in analysis is the introduction of the sample. This has been

achieved in ICP-MS through a variety of means. The most common method is the

use of a nebulizer. This is a device which converts liquids into an aerosol, and that

aerosol can then be swept into the plasma to create the ions. Nebulizers work best

with simple liquid samples. However, there have been instances of their use withmore complex materials like slurry.

Many varieties of nebulizers have been coupled to ICP-MS, including

pneumatic, cross-flow, Babington, ultrasonic, and desolvating types. The aerosol

generated is often treated to limit it to only smallest droplets, commonly by means

of a double pass or cyclonic spray chamber. Use of auto samplers makes this

easier and faster.


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1.5.2 Transfer of ions into vacuum

The carrier gas (usually argon) is sent through the central channel and into

the very hot plasma. The sample is then exposed to radio frequency which

converts the gas into the plasma. The high temperature of the plasma is sufficientto cause a very large portion of the sample to form ions. This fraction of

ionization can approach 100% for some elements (e.g. sodium), but this is

dependent on the ionization potential.

A fraction of the formed ions passes through a ~1mm hole (sampler cone)

and then a ~0.4mm hole (skimmer cone). The purpose of which is to allow a

vacuum that is required by the mass spectrometer.

The vacuum is created and maintained by a series of pumps. The first stage

is usually based on a roughing pump, most commonly a standard rotary vane

pump. This removes most of the gas and typically reaches a pressure of around

133 Pa. Later stages have their vacuum generated by more powerful vacuum

systems, most often turbomolecular pumps. Older instruments may have used oil

diffusion pumps for high vacuum regions.

1.5.3 Ion optics

Before mass separation, a beam of positive ions has to be extracted from the

plasma and focused into the mass-analyzer. It is important to separate the ions

from UV photons, energetic neutrals and from any solid particles that may have

been carried into the instrument from the ICP. Traditionally, ICP-MS instruments

have used transmitting ion lens arrangements for this purpose. Examples include

the Einzel lens, the Barrel lens and Omega Lens. Another approach is to use ion

guides (quadrupoles, hexapoles, or octopoles) to guide the ions into mass analyzer

along a path away from the trajectory of photons or neutral particles. The primary

role of the ion lenses is to transfer and focus the ions efficiently into the mass

filter. Recently, a compound or multi lens ion optic, for efficient ion focusing

across, was introduced into the mass spectrometer in the sampling/scimming

process. If these reach the detector, the background noise will increase, and

detection limits will suffer. Off-axis omega lens on the instrument eliminates

neutral species, ensuring very low backgrounds, enabling sub-ppt detection limits

for most elements.


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1.5.4 Octopole Reaction System (ORS)

The ORS features an off-axis reaction cell, which effectively removes

spectral interferences in most complex sample matrices. In order to analyze the

complicated and heterogeneous samples that can cause problems with

conventional instruments, the ORS-ICP-MS has been designed specifically to

handle high matrices and elements suffering from significant Ar-based (plasma

based) interferences such as Fe, Se, As etc. The ORS consists of an octopole ion

guide, mounted off-axis to minimize random background levels, inside a cell that

can be pressurized with a reaction gas (usually H2 or He).

Difficult polyatomic interferences such as Ar2, ArCl and MAr are

dissociated by collisions with the reaction gas within the cell, enabling otherwise

interfering analytes to be determined. Because the ORS employs simple reaction

gases, side reactions that would create new, unpredictable interferences are

eliminated, and the ORS can be operated in passive mode without a mass-filter.

1.5.5 Plasma

The plasma used in an ICP-MS is made by ionizing argon gas (Ar Ar+ +

e-). The energy required for this reaction is obtained by pulsing an electrical

current in wires that surround the argon gas. A complete description of plasma

generation is given in the following section. After injecting the sample, the

plasma's extreme temperature causes the sample to separate into individual atoms

(atomization). Next, the plasma ionizes these atoms (M M+ + e-) so that they

can be detected by the mass spectrometer.

1.5.6 Plasma Generation

An inductively coupled plasma (ICP) is sustained in a torch that consists of

three concentric tubes, usually made of quartz. The end of this torch is placed

inside an induction coil supplied with a radio-frequency electric current. A flow of

argon gas (usually 14 to 18 L/min) is introduced between the two outermost tubes

of the torch and an electrical spark is applied for a short time to introduce free

electrons into the gas stream. These electrons interact with the radio-frequency

magnetic field of the induction coil and are accelerated first in one direction, then


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the other, as the field changes at high frequency (usually 27.12 million cycles per

second).

The accelerated electrons collide with argon atoms, and sometimes a

collision causes an argon atom to part with one of its electrons. The releasedelectron is in turn accelerated by the rapidly-changing magnetic field. The process

continues until the rate of release of new electrons in collisions is balanced by the

rate of recombination of electrons with argon ions (atoms that have lost an

electron). This produces a fireball that consists mostly of argon atoms with a

rather small fraction of free electrons and argon ions.

1.5.7 Advantage of Argon

Making the plasma from argon has several advantages. First, argon is

abundant and therefore cheaper than other noble gases. Argon also has a higher

first ionization potential than all other elements except He, F, and Ne. Because of

this high ionization energy, the reaction (Ar+ + e- Ar) is more energetically

favorable than the reaction (M+ + e- M). This ensures that the sample remains

ionized (as M+) so that the mass spectrometer can detect it.

Argon can be purchased for use with the ICP-MS in either a refrigerated

liquid or a gas form. However it is important to note that whichever form of argon

purchased, it should have a guaranteed purity of 99.9% Argon at a minimum. It is

important to determine which type of argon will be best suited for the specific

situation. Liquid argon is typically cheaper and can be stored in a greater quantity

as opposed to the gas form, which is more expensive and takes up more tank

space.

If the instrument will be in an environment where it will get infrequent use,

then buying argon in the gas state will be most appropriate as it will be more than

enough to suit smaller run times and will remain stable for longer periods of time,

whereas liquid argon will suffer loss to the environment due to venting of the tank

when stored over extended time frames. However if ICP-MS will be used

routinely, then going with liquid argon will be the most suitable. If there are to be

multiple ICP-MS instruments running for long periods of time, then it will most

likely be beneficial for the laboratory to install a bulk or micro bulk argon tank

which will be maintained by a gas supply company, thus eliminating the need to


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change out tanks frequently as well as minimizing loss of argon that is left over in

each used tank as well as down time for tank changeover.

1.5.8 Elemental analysis

The ICP-MS allows determination of elements with atomic mass ranges 7 to

250. This encompasses Li to U. Some masses are prohibited such as 40 due to the

abundance of argon in the sample. Other blocked regions may include mass 80

(due to the argon dimer), and mass 56 (due to ArO), the latter of which greatly

hinders Fe analysis unless the instrumentation is fitted with a reaction chamber.

A typical ICP-MS will be able to detect in the region of ng/L to 10 or 100

mg/L or around 8 orders of magnitude of concentration units. Unlike atomic

absorption spectroscopy, which can only measure a single element at a time ICP-

MS has the capability to scan for all elements simultaneously. This allows rapid

sample processing.

Although ICP-MS and ICP-AES are powerful techniques, care is still

needed to ensure adequate quality control when performing measurements over a

wide concentration range and a thorough evaluation of the accuracy and precision

may be required for each element. Consequently, it can take considerable effort to

develop robust methods which account for all possible matrix and any associated

interferences while still being able to take advantage of the speed and sensitivity

offered by the approach.

Additionally, large amounts of data can be generated (including isotopic

information in ICP-MS) which require careful processing and such procedures can

be very time-consuming. Regardless of the type of matrix, analysis of the samples

most frequently encountered in food science can be considered in a number of

discrete stages, namely sample collection and storage, preparation, pre-treatment,

quantification, quality control and reporting (S. J. Hill, 2007).

1.6 Microwave Digestion

Microwave ovens began to find widespread use in chemical laboratories in

the late 1980s. The use of laboratory microwave units has become increasingly

popular because of the significant improvement in chemical reaction rates that are

possible using microwave radiation. The aim of all digestion methods is totransfer the element(s) of interest into the final solution quantitatively and


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efficiently, preferably with total decomposition of the bulk matrix and removal of

potentially interfering species (S. J. Hill, 2007).

As a rule, acid digestion procedures are employed for the determination of

elements in solids subsequent to sampling and mechanical sample preparation inorder to completely transfer the analytes into solution so that they can be

introduced into the determination step (e.g., ICP-AES, ICP-MS, AAS or

polarography) in liquid form.

The goal of every digestion process is therefore the complete solution of the

analytes and the complete decomposition of the solid (matrix) while avoiding loss

or contamination of the analyte. A typical microwave acid digestion can be

completed in a matter of minutes, whereas the same conventional hot platedigestion can take hours. Microwave digestion usually involves placing a sample

in an acid solution and heating to high temperatures and pressures. These extreme

conditions will dissolve most materials, but is potentially hazardous. The goals of

microwave digestion techniques are; complete solution of the elements, complete

decomposition of the matrix, avoiding losses and contamination, reduction of

handling and process times.

For technicians, there is an additional need to ensure that the digestion is

safe, reproducible and simple, that is, that it can be performed without excessive

manual effort. Since sample preparation typically also consumes the largest share

of task time, this process also has economic significance.

1.6.1 Heating Mechanism

Liquids heat by two mechanism: dipole rotation and ionic conduction. Polar

molecules will tend to align their dipole moments with the microwave electric

field. Because the field is changing constantly, the molecules are rotated back and

forth, which causes them collide with other nearby molecules. Ions in solution

will tend to migrate in the presence of a microwave electric field. This migration

causes the ions to collide with other molecules. Heat is generated when molecules

or ions collide with nearby molecules or ions (Dean, 2004).


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Figure 1.2 Schematic of sample heating by microwaves

1.6.2 Open versus Closed Acid Digestion

Open acid digestions are performed either with a reflux system or in a

beaker on a laboratory hot plate. Common to both methods is the temperature

limitation as a consequence of the solutions boiling point and the risk of

contaminants from the air. Volatile elements such as mercury may be lost during

the digestion times of, typically 2-15 hours.

The temperature limitation can be overcome by working with a closed

pressure vessel. This rise in pressure results in a dramatic increase in the reaction

kinetics allowing acid digestions to be carried out in a matter of hours or, if

microwave heating is employed, in 20-40 minutes. However, this also makes it

clear that the temperature represents what is actually the most significant reaction

parameter. It is the ultimate determinant of the digestion quality, but also results

in a pressure increase in the vessel and therefore in a potential safety hazard.

Therefore, the pressure must ultimately also be considered.

(a) Hot plate (b) Closed vessel microwave digestionFigure 1.3 Schematic representations of sample digestion systems


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The advantage of closed procedure in comparison with open digestion in a

recycling device or with the traditional hot plate lies in the significantly higher

working temperatures which can be achieved. While operating temperatures in

open systems are limited by the boiling point of the acid solution, closed digestion

vessels typically allow temperatures in the range of 200-260oC to be reached. This

results in a dramatic increase in the reaction kinetics, allowing digestions to be

carried out in hours.

However, this also makes it clear that the temperature represents what is

actually the most significant reaction parameter. It is the ultimate determinant of

the digestion quality, but also result in pressure increase in the vessel and

therefore in potential safety hazard. Therefore, the pressure must ultimately alsobe considered.

1.7 Method Validation

Methods validation is the process of demonstrating that analytical

procedures are suitable for their intended use. The methods validation process for

analytical procedures begins with the planned and systematic collection by the

applicant of the validation data to support analytical procedures (Bliesner, 2006).

Fundamental terms of validation are given below;

Linearity: The linearity of an analytical method is its ability to elicit test

results that are directly proportional to the concentration of analytes in samples

within a given range or proportional by means of well-defined mathematical

transformations. Linearity may be demonstrated directly on the test substance (by

dilution of a standard stock solution) and/or by using separate weighing of

synthetic mixtures of the test product components, using the proposed procedure.

Linearity is determined by a series of 3 to 6 injections of 5 or more

standards whose concentrations span 80120 percent of the expected

concentration range. The response should be directly proportional to the

concentrations of the analytes or proportional by means of a well-defined

mathematical calculation. A linear regression equation applied to the results

should have an intercept not significantly different from 0. If a significant nonzero

intercept is obtained, it should be demonstrated that this has no effect on the

accuracy of the method.


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Repeatability is the variation experienced by a single analyst on a single

instrument. Repeatability does not distinguish between variation from the

instrument or system alone and from the sample preparation process. During the

validation, repeatability is performed by analyzing multiple replicates of an assay

composite sample by using the analytical method.

Limits of Detection and Quantification: The detection limit (DL) or limit

of detection (LOD) is the point at which a measured value is larger than the

uncertainty associated with it. It is the lowest concentration of analyte in a sample

that can be detected but not necessarily quantified. The limit of detection is

frequently confused with the sensitivity of the method. The sensitivity of an

analytical method is the capability of the method to discriminate small differencesin concentration or mass of the test analyte. In practical terms, sensitivity is the

slope of the calibration curve that is obtained by plotting the response against the

analyte concentration or mass.

The quantization limit (QL) or limit of quantization (LOQ) of an individual

analytical procedure is the lowest amount of analyte in a sample that can be

quantitatively determined with suitable precision and accuracy. The quantization

limit is a parameter of quantitative assays for low concentrations of compounds insample matrices and is used particularly for the determination of impurities. It is

usually expressed as the concentration of analyte in the sample.

The LOD and LOQ were determined on the basis of signal-to-noise ratio,

and formulated below where cminis the minimum concentration of the calibration

series, S (b): The signal count of the blank sample, S (s): The signal count of the

sample that known concentration;

)()(3 min

sSbScLOD =

)(

)(10 minsS

bScLOQ

=

Accuracy and Recovery:The accuracy of an analytical method is the extent

to which test results generated by the method and the true value agree. Accuracy

can also be described as the closeness of agreement between the value that is

adopted, either as a conventional, true or accepted reference value, and the valuefound.


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The true value for accuracy assessment can be obtained in several ways.

One alternative is to compare the results of the method with results from an

established reference method. This approach assumes that the uncertainty of the

reference method is known. Secondly, accuracy can be assessed by analyzing a

sample with known concentrations (e.g., a control sample or certified reference

material) and comparing the measured value with the true value as supplied with

the material. If certified reference materials or control samples are not available, a

blank sample matrix of interest can be spiked with a known concentration by

weight or volume.

After extraction of the analyte from the matrix and injection into the

analytical instrument, its recovery can be determined by comparing the responseof the extract with the response of the reference material dissolved in a pure

solvent. Because this accuracy assessment measures the effectiveness of sample

preparation, care should be taken to mimic the actual sample preparation as

closely as possible. If validated correctly, the recovery factor determined for

different concentrations can be used to correct the final results.

Uncertainty: In metrology, measurement uncertainty describes a region

about an observed value of a physical quantity, also called a measurand, which islikely to enclose the true value of that quantity. Uncertainty of a method

associated with the result of a measurement that characterizes the dispersion of the

values that could reasonably be attributed to the measurand. Assessing and

reporting measurement uncertainty is fundamental in chemistry.

In practice the uncertainty on the result may arise from many possible

sources, including examples such as incomplete definition, sampling, matrix

effects and interferences, environmental conditions, uncertainties of weights andvolumetric equipment, reference values, approximations and assumptions

incorporated in the measurement method and procedure, and random variation.

The measurement uncertainty tells us what size the measurement error mightbe.

The basis for the evaluation is a measurement and statistical approach, where the

different uncertainty sources are estimated and combined into a single value.

Basis for the estimation of measurement uncertainty is the existing knowledge.

Existing experimental data should be used quality control charts, validation,interlaboratory comparisons, CRM etc.


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EXPERIMENTAL

2.1 Apparatus

Agilent 7500ce ICP-MS system was used throughout the experiments.

Samples were digested in microwave oven (CEM Mars 5) prior to the analysis.

2.2 Chemicals and Reagents

Nitric acid (ultrapure) was supplied from Fluka. Water was obtained using a

USF Purelab and Millipore Elix & Rios Systems combined Milli-Q Synthesis

System, including UV radiation and ultra filtration units.

Standard solution of Fe, K, Ca, Na, Mg, Ag, As, Ba, Cd, Co, Cr, Cu, Hg,

Mn, Ni, Pb, Sb, Se, Tl, V, Zn (environmental calibration standard and multielement calibration standard) was purchased from Agilent. Internal standard mix

(Li, Sc, Y, Ge, In) was also purchased from Agilent. Tin standard was purchased

from High Purity Standard and boron standard solution puchased from JT Baker.

Stock solutions of elements prepared in 5% of HNO3 and 2% of HCl with

deionized water. Standard solutions were prepared weekly by diluting stock

solutions in 2% of nitric acid and 1% of HCl.

Juices, vinegars, grape syrups and honey samples were collected fromvaious locations of Turkey and their 13C and sugar component analysis were

performed before their heavy metal analysis done. The samples known with their

natural origin were analyzed as their trace metal content. The fraud samples of

apple juice and pear juice could not be encountered. So their fraudulent samples

were prepared by adding fructose syrup and then analysed.

2.3 Analytical procedures and ICP-MS conditions

Sample preparation procedures are given below.

Step 1: Weight 0.3 g juice into Teflon PFA vessels.

Step 2: Add 5 mL HNO3 to the sample and cap the vessel.

Step 3: Digest following the procedure juice 1 in Table 2.1

Step 4: Cool for approximately 5 minutes and vent vessels.

Step 5: Transfer the samples into clean, acid washed volumetric flasks and dilute

to 30 mL with 18 M, distilled, ultrapure water.

Step 6: Transfer the samples into clean polyethylene bottles.


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Table 2.1. Microwave digestion procedure

Heating Program: Ramp to Temperature

Stage Max.Power (W)

% Power Ramp(min)

Pressure(psi)

Temperature(oC)

Hold (min)

1 600* 100 30:00 N.A 210 5:00* Power should be adjusted up or down with respect to the number of vessels. General guidelinesare as follows: 1-2 vessels (300 W), 3-6 vessels (600 W), 7 or more vessels (1200 W).

The optimized ICP-MS conditions are given in Table 2.2.

Table 2.2 Optimized conditions for ICP-MS system

Plasma conditions Ion Lenses Octopole Parametres

RF Power 1500 W Extract 1 -54.9 V Octp RF 180 V

RF Matching 1.8 V Extract 2 -120 V OCTP Bias -6 V

Sample Depth 8 mm Omega Bias -30 V Reaction Cell

Carrier Gas 0.91 L/min Omega Lens 0.2 V Reaction Mode On

Make up Gas 0.17 L/min Cell Entrance -20 V H2 Gas 0 mL/min

Nebulizer Pump 0.08 rps QP focus 5 V He Gas 1 mL/min

S/C temperature 2 oC Cell Exit -28 V Ar Gas 15 mL/min

Isotopes of each element used in method validation studies and sample

analysis are listed in Table 2.3.

Table 2.3 Used isotopes of analyzed elements

Metal isotope Metal isotope Metal isotope

As 75 Cu 63 Ni 60

B 11 Fe 56 Pb 208

Ba 137 Hg 202 Sb 121

Ca 40 K 39 Se 82

Cd 111 Mg 24 Sn 118

Co 59 Mn 55 V 51

Cr 53 Na 23 Zn 66


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3. RESULTS AND DISCUSSION

3.1. Optimization Studies for Method Development

The optimization of the method was considered as mainly two parts; first

part is the optimization of sample preparation step and second part is the

optimization of the detection system, i.e. ICP-MS. In the first part the parameters

related with the microwave digestion system were considered namely the mass of

the sample, the volume of nitric acid and digestion temperature and time. The

parameters were tested by using CRM of fruit juice. As no reference material is

available for honey and grape syrup, the optimized conditions were also applied

for these types of samples.

Initial studies were conducted by adopting the sample weights

recommended in the manual however; the use of 2.5 g of sample mixed with 10

mL of nitric acid has resulted in a severe inner pressure problem which causes

vessel deterioration. Therefore, the sample weight was reduced to 0.3 g and mixed

with 3 mL of nitric acid for safer digestion which also gives consistent values with

former procedure. Table 3.1 summarizes the results obtained with ICP-MS

procedure given in Experimental Section.

Table 3.1 The effect of sample weight on the analysis by comparing the

metal content of the fruit juice sample

Certificated value (Fapas-T0776)

Cd(ng/mL)

Fe(g/mL)

Sn(g/mL)

Pb(ng/mL)

63.928.1 10.02.2 100.716.1 43.619.2

Sampleweight

Nitricacid

volume(mL)

Determined value

2.5 10 68.1 9.71 106.4 54.1

1.5 10 69.0 10.1 92.8 47.4

1.0 7 60.8 10.3 102.1 44.4

0.5 5 64.5 9.82 103.1 44.6

0.3 3 67.0 9.96 99.5 43.9

As can be followed from the table above, 0.3 g of sample can be used safely

without compromising and this amount was chosen for further studies. Other

parameters were digestion temperature and time. The effect of digestion

temperature can be seen in Table 3.2.


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Table 3.2 The comparison of the metal content of the fruit juice sample

digested at various temperatures

Certificated value(Fapas-T0776)

Cd(ng/mL) Fe(g/mL) Sn(g/mL) Pb(ng/mL)

63.928.1 10.02.2 100.716.1 43.619.2

Temp.oC

Determinedvalue

190 59.8 9.4 96.2 49.6

200 60.8 10.3 102.1 44.4

210 64.4 10.3 99.6 46.1

According to above results, digestion procedure at three different

temperature values have resulted similar metal ion contents without statistically

significant difference however, 210oC was selected for better digestion. The effect

of digestion time can be seen in Table 3.3.

Table 3.3 The comparison of the metal content of the fruit juice sample

digested at various time

Certificated value(Fapas-T0776)

Cd(ng/mL)

Fe(g/mL)

Sn(g/mL)

Pb(ng/mL)

63.928.1 10.02.2 100.716.1 43.619.2

Ramp

(min)

Hold

(min)

Determinedvalue

10:00 10:00 52.5 8.26 86.8 33.6

20:00 5:00 60.9 9.4 88.7 41.7

30:00 5:00 64.1 10.3 100.1 44.2

As can be seen from table the most accurate values obtained with 30 minute

ramping time and 5 minute hold time.

The second stage was ICP-MS parameters. An optimum mass detector

condition was conducted by using a tune solution that includes Li, Y, Ce, Tl and

Co. Medium concentration (10 ng/mL) of tune solution has been injected to mass

detector. In addition, during the optimization process the nebulizer flow, tourch

position, spray chamber temperature and ion lenses were optimized. The

optimized ICP-MS conditions were given in Experimental Section.

Typical ICP-MS results of Sc, V, Cr, Mn, Co, Ni, Cd, Cr, As, Mn, Cd, In,

Sb, Ba are given in Figure 3.1. The system is used in helium mode for this


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analysis. Zn, Ge, Hg, Bi are determined in no-gas mode and results are given in

Figure 3.2. Fe, Mg, K, Ca, Na can be detected in alkaline method and results are

given in Figure 3.3. These different methods were chosen according to the

manual.

Figure 3.1. Typical ICP-MS results of Sc, V, Cr, Mn, Co, Ni, Cd, Cr, As, Mn, Cd, In, Sb,Ba

Figure 3.2. Typical ICP-MS results of Zn, Ge, Hg, Bi

Figure 3.3. Typical ICP-MS results of Fe, Mg, K, Ca, Na


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3.2. Validation Studies

Under the optimized conditions given above, the validation of the method

was accomplished by determining the linearity, repeatability, reproducibility,

accuracy and the detection and quantification limits of the method. Uncertainty ofthe measurements was also calculated.

3.2.1 Linearity

For calibration studies, 17 metals studied were divided into two groups. The

first group includes Na, K, Fe, Mg and Ca and the calibration range is 0.100 to

2.500 mg/kg. The second group includes As, B, Ba, Cr, Cd, Co, Cu, Hg, Mn, Mo,

Ni, Sb, Se, Sn, Pb, V and Zn and the calibration range is 1.00 to 25.00 g/kg. The

MS response was found linear in these concentration ranges. The calibration

standards were prepared in acid mixture containing 2% HNO3 and 0.1% HCl.

The linearity of the metod was assessed with five standard injections to ICP-

MS as three replicates. Linearity was verified using the value of regression

coefficient, R2. The calibration curves and equations with regression coefficient

are given Table 3.4.

Table 3.4 Regression coefficients for metals studied from calibration curves

obtained with ICP-MS measurements

Metal R2 Metal R2 Metal R2

As 0.9998 Cu 0.9988 Ni 0.9999

B 0.9997 Fe 0.9999 Pb 0.9995

Ba 0.9997 Hg 0.9983 Sb 0.9998

Ca 0.9998 K 0.9999 Se 0.9995

Cd 0.9998 Mg 0.9999 Sn 0.9999

Co 0.9996 Mn 0.9994 V 0.9997

Cr 0.9997 Na 0.9999 Zn 0.9996

3.2.2. Repeatability

The injection repeatability was calculated by five replicated injections of

1.00 mg/L for the first group and 5.00 g/L for the second group standard

samples. Injection repeatability for individual components as RSD % can be seen

in Table 3.5.


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Table 3.5 Relative standard deviations of metals.

Metal RSD Metal RSD Metal RSD

As 1.2 Cu 1.6 Ni 1.5

B 2.6 Fe 0.5 Pb 3.2

Ba 3.1 Hg 3.7 Sb 2.3

Ca 0.4 K 4.6 Se 1.9

Cd 4.0 Mg 0.6 Sn 2.4

Co 2.1 Mn 2.2 V 2.0

Cr 3.0 Na 0.2 Zn 4.9

3.2.3. Sensitivity

The detection limits (LOD) and quantification limits (LOQ) were

determined on the basis of signal-to-noise ratio, LOQ and LOD for selected

metals for fruit juices matrices are listed in Table 3.6.

Table 3.6 LOD and LOQ values of metals (g/L)

Metal LOD LOQ Metal LOD LOQ Metal LOD LOQ

As


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3.2.5. Uncertainty

The uncertainty of measurement was calculated from quality control charts

of each element. The relative uncertainties are given in Table 3.8.

Table 3.7 Recovery percentages of the metals studied with ICP-MS

MetalLow Conc.

%Average Conc.

%High Conc.

%As 100 88 99

B 95 97 91

Ba 100 112 110

Ca 119 89 103

Cd 107 76 104

Co 93 77 98

Cr 95 80 105

Cu 110 103 101Fe 85 87 75

Hg 87 100 101

K 92 96 103

Mg 87 93 79

Mn 97 103 105

Na 91 81 81

Ni 97 88 104

Pb 103 92 98

Sb 96 103 106

Se 100 79 89Sn 98 86 93

V 100 87 117

Zn 86 91 84

Table.3.8 Relative uncertainty values of metals

Metal Uncertainty Metal Uncertainty Metal Uncertainty

As 0.08 Cu 0.25 Ni 0.32

B 0.24 Fe 0.23 Pb 0.16

Ba 0.34 Hg 0.22 Sb 0.27

Ca 0.40 K 0.32 Se 0.09

Cd 0.26 Mg 0.43 Sn 0.27

Co 0.16 Mn 0.66 V 0.16

Cr 0.16 Na 0.39 Zn 0.29


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3.3. Comparison of the Developed Method with Direct Injection

The method developed so far including microwave digestion and subsequent

ICP-MS measurements was compared with direct injection of simply diluted fruit

juices to the ICP-MS system. The table below shows the results of cherry andapricot juices with microwave digestion and direct injection to the ICP-MS

system.

Table 3.9 Comparison of the developed microwave digestion method with direct injection (mg/kg)

MetalMicrowave

(cherry)

Directinjection(cherry)

Microwave(appricot)

Directinjection(appricot)

As 0.072 0.022 0.035 0.006

B 16.22 10.95 9.98 8.71Ba 1.389 0.667 0.963 0.781

Ca 499.0 257.2 6743 365.5

Cd 0.017 0.002 0.010 0.001

Co 0.039 0.022 0.030 0.020

Cr 0.194 0.078 0.238 0.071

Cu 0.411 0.091 1.668 0.137

Fe 17.11 10.94 19.26 9.988

Hg 0.028 < LOQ 0.020 0.003

K 4418 2292 7290 4275

Mg 487.2 349.6 39.8 27.07

Mn 2.456 1.644 1.78 0.157

Na 132.2 48.83 86.49 68.95

Ni 0.756 0.456 0.973 0.127

Pb < LOD < LOD 0.020 0.020

Sb 0.067 0.004 0.025 0.004

Se 0.167 0.033 0.162 0.010

V 0.100 0.022 0.056 0.056Zn 3.583 2.156 3.448 0.963

It is evident from the table that there is a big gap between the results of two

methods. In the light of the data given above it can be clearly stated that digestion

method is required for and more reliable determination of the selected metal ions

in fruit juices. Further sections present the results obtained for several samples

including original and fake foodstuff.


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3.4. Application of the Method to the Samples

3.4.1. Vinegar Results

The metal compositions of vinegar made of grape and falsified samples

were given in Table 3.10.

Table 3.10 Metal compositions of grape vinegar (GV) and falsified alcohol vinegar (FAV) (mg/kg)

Metal GV FAV Metal GV FAV

As 0.012 0.017 Mg 83.12 13.04

Ba < LOD < LOD Mn 0.613 0.046

Ca 27.34 9.243 Na 187.9 7.738

Cd < LOD 0.005 Ni 0.019 0.021

Co < LOQ 0.040 Pb < LOD < LOD

Cr < LOD 0.007 Sb < LOD 0.007

Cu < LOD < LOD Se < LOD < LOD

Fe 2.750 0.559 V 0.050 0.023

Hg < LOD < LOD Zn < LOD < LOD

K 1281 347

Comparing to the results obtained for real and fake samples it can be clearly

said that the potassium, iron, calsium, magnesium, sodium and manganese valuesof grape vinegar is much more than its fake substitute. Therefore, the metal

composition of vinegar has a potential for establishing another parameter to detect

any fraud.

3.4.2. Grape Syrup Results

The metal compositions of nine grape syrups and a mulberry syrup are

given in Table 3.11. As can be seen from the table, similar results were obtained

for real and fake samples. Therefore it is hard to classify grape syrups according

to their metal compositions. But calcium, iron and potassium amounts of grape

syrup, harnup syrup and mulberry are substantially different from each other

indicating a possibility to distinguish the original samples.


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Table 3.11 Metal compositions of grape syrups, harnup and mulberry syrup (mg/kg)

As B Cd Co Cr Hg Ni Pb Sb V

Grape1 0.006 8.555 < LOD 0.031 < LOD < LOQ 0.367 < LOD 0.012 < LOD

Grape2 0.031 6.134 0.003 0.022 0.060 0.006 0.344 0.035 0.005 0.041

Grape3 0.075 8.146 0.003 0.026 0.114 0.006 0.246 0.008 0.004 0.056

Grape4 0.024 15.64 0.001 0.135 < LOD 0.048 0.472 0.026 0.006 0.003

Grape5 0.201 95.38 0.004 0.092 < LOD 0.009 0.472 0.025 0.021 < LOD

Grape6 0.189 46.33 0.003 0.089 < LOD 0.028 0.467 0.024 0.016 < LOD

Grape7 0.020 4.072 0.001 0.031 < LOD 0.014 0.217 0.008 0.004 < LOD

Grape8 0.021 8.156 0.003 0.038 < LOD < LOQ 0.346 0.025 0.012 < LOD

Grape9 0.019 11.14 0.001 0.025 < LOD 0.009 0.397 0.011 0.004 < LOD

Mean 0.065 22.62 0.002 0.054 0.019 < LOQ 0.370 0.018 0.009 0.011

Mullberry 0.114 91.95 0.002 0.057 0.007 < LOQ 0.671 0.045 0.011 < LOD

Harnup 0.062 45.71 0.003 0.037 < LOD 0.011 0.522 0.039 0.011 < LOD

Continued

Ba Ca Cu Fe K Mg Mn Na Se Zn

Grape1 0.396 373 0.452 16.95 4648 119.3 2.282 618.9 < LOD 0.888

Grape2 0.268 231 0.380 11.87 2841 114.6 1.702 401.1 0.018 0.902

Grape3 0.275 141 0.477 7.48 13050 1491 3.114 297.1 < LOD 0.338

Grape4 0.376 254 3.761 15.61 5068 498.6 3.362 475.5 0.020 2.916

Grape5 0.954 197 1.008 9.47 7237 271.5 3.380 155.7 0.008 1.226

Grape6 0.953 509 0.979 14.88 7085 427.3 3.352 996.1 0.005 1.193

Grape7 0.279 204 2.069 6.18 2229 113.3 1.003 683.9 0.006 0.508

Grape8 0.467 251 1.015 13.45 5168 496 3.626 568 0.006 2.246

Grape9 0.879 306 0.867 8.17 6158 298.1 3.915 601 0.011 1.216

Mean 0.539 274 1.223 11.56 4831 323.3 2.86 533 0.008 1.270Mullberry 0.918 130 0.394 35.71 11450 483 4.169 386 0.015 1.330

Harnup 1.151 352 2.329 28.91 8928 387.6 7.426 332.2 0.017 4.936

As you can see from the table above, the iron, barium and manganese

results of harnup and mulberry molasses are different from the values of grape

syrups. Figure 3.3, 3.4 and 3.5 gives the schematic representation of results above.

The upper and lower limits show the standard deviations of nine grape syrups.


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Figure 3.4. Schematic representation of barium results of grape syrups, mulberry andharnup syrups.

Figure 3.5. Schematic representation of iron results of grape syrups, mulberry and harnupsyrups

Figure 3.6. Schematic representation of manganese results of grape syrups, mulberry andharnup syrups


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3.4.3. Fruit Juice Results

The metal composition of pear juices are given in Table 3.12.

Table 3.12. Metal compositions of pear juices (mg/kg)

As B Cd Co Cr Hg Ni Pb Sb V

pear1 0.032 2.35 0.001 0.011 < LOQ < LOD 0.027 0.002 0.880 0.066

pear2 0.082 2.35 0.001 0.009 < LOQ 0.001 0.044 0.030 1.613 < LOQ

pear3 < LOQ 1.04 0.001 0.009 0.020 0.001 0.036 0.015 < LOD 0.004

pe

ms alakalı 2

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