Determination of Synthetic Food Colorants in Food Products on Local Markets of Gaza Strip

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Determination of Synthetic Food Colorants in Food Products on Local Markets of Gaza Strip Al-Azhar University–Gaza Deanship of Postgraduate Studies Faculty of Pharmacy Master of Pharmaceutical Sciences Determination of Synthetic Food Colorants in Food Products on Local Markets of Gaza Strip By Asma Salama Mohammed Abu Laila Supervisors Dr. Mohammed K. Shbair Dr. Mai A. Ramadan Assist. Prof. of Toxicology Assist. Prof. of Pharmaceutical Chemistry Faculty of pharmacy, Faculty of pharmacy, Al-Azhar University-Gaza Al-Azhar University-Gaza A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master in Pharmaceutical Sciences 2019 Declaration I declare that the thesis titled “Determination of Synthetic Food Colorants in Food Products on Local Markets of Gaza Strip” submitted for the degree of master of pharmaceutical sciences, is the result of my own research work and the work provided in this thesis, unless otherwise referenced, is the researcher's own work, and has never been submitted elsewhere for any other degree qualifications nor for any academic titles, nor for any other academic or publishing institutions. I affirm that I will be completely responsible in academic and legal terms if this work proves the opposite. Signature: Asma S. M. Abu laila Date: 11/07/2019. i Dedication To the soul of my father & my mother To my Family To my Friends To everyone who encouraged and supported me Asma S. M. Abu Laila ii Acknowledgment Foremost, all my prayers and thankfulness are to Allah who granted to me life, power and courage to facilitate this work. I would like to express my profound gratitude and deep regard to my supervisors, Dr. Mohammed Shbair and Dr. Mai Ramadan for their improving suggestions, advices, guidance, patience, and constant encouragement throughout this thesis, so I ask Allah to reward them on my behalf. I extend my appreciation to Al-Azhar University, Gaza, Faculty of Pharmacy and to all the academic staff in it, especially Dr. Usama Abu Mohsen. Thanks to The German Academic Exchange Service (DAAD) for HPLC device donation to Al-Azhar University, which was used in the study. Great thanks extended to Mr. Ziad Abu Zayed, Drug and Toxicology Analysis Centre, Al-Azhar University, Gaza, for the valuable information provided by him. I am grateful for his cooperation during experimental part. Great Thanks to Mr. Mohammed Matar for his assistance in providing chemicals and required materials for experimental part. Deeply thanks to Arab German pharmaceutical company, Ltd for assistance in providing reference colorants: tartarazine, sunset yellow, ponceau 4R and carmosine. Thanks are also due to Ms. Elham Abu Waked for help. My deepest love and thanks to my family. Words cannot express how grateful I am to my sisters, Rayqa, Amal, Sameera, Nora, Tagreed, Azhar, Sabreen and Tshreen, to my brother Ahmed and his family for supporting me. My sincere thanks to my colleagues, pharmacy staff members and partner in working field in Ministry of Health, especially in Abu Yousef El Najar Hospital. Last, but not least, my appreciation goes to all my friends for their encouragement. iii Abstract Introduction: Synthetic food colorants are found in many food products such as drinks, brightly colored candies and yogurt to improve the appearance of the food items and drinks. Their application in food industry is strictly controlled by regulation and acceptable daily intake (ADI) values as a result of toxicity studies. Aim: To determine the concentrations of mostly abundant synthetic colorants (tartrazine, sunset yellow, ponceau 4R and carmosine) in food products available on local markets of Gaza-strip and to compare them with ADI. Methodology: 18 samples (9 concentrated juices, 6 soda drinks and 3 fruit juices) containing tartrazine, sunset yellow, ponceau 4R and carmosine either separately or mixture were collected from markets randomly. Reversed-phase high performance liquid chromatography (RP-HPLC) method with C18-column was applied using mobile phase acetonitrile (ACN) : acetate buffer pH 5 (15 : 85) to optimize separation of tartrazine, sunset yellow and ponceau 4R and using mobile phase acetonitrile (ACN) : acetate buffer pH 5 (30 : 70) to optimize separation of carmosine within available instrument and materials in our laboratories. The detection was by UV-detector at λ max for each colorants. The purity of reference material was determined using a standard material. The method was validated according to international conference on harmonization (ICH) guidelines. Samples of food items were prepared by simple dilution and degassing pretreatment then analyzed by HPLC-coupled with UV-detector. The results were compared with ADI per colorants. Results: Three colorants (tartrazine, sunset yellow and ponceau 4R) were efficiently separated within 8 minutes. Carmosine was eluted within 4 minutes. λ max was 429, 483, 507 and 516 nm for tartrazine, sunset yellow, ponceau 4R and carmosine respectively. The method was linear ( R2 > 0.999) within a range 1-100 µg/ml for colorants. The assay was accurate (SD < 2%). Intra- and inter-day precision showed % RSD less than 2 % . The method was specific, thus no interference from possible ingredients in food stuffs was recorded. Consumption of one drink containing sunset yellow available in the markets of Gaza-strip can exceed ADI for a child weighed 15 Kg and consumption of one drink containing carmosine or sunset yellow provides a child weighed 15 Kg 40-85 % of ADI per colorant. Conclusion: RP-HPLC assay was applied successfully for analysis of synthetic colorants in some food items. A consumption of only one drink in some cases can exceed ADI and in another cases provides a high percent of ADI, thus measurements of synthetic colorants must be taken by responsible authorities to safeguard the health of children. Key word: Tartrazine, Sunset yellow, Ponceau 4R, Carmosine, HPLC, ADI, Food items. iv الملخص باللغة العربية المقدمة: ملونات الطعام اﻻصطناعية موجودة في العديد من المواد الغذائية مثل المشروبات, الحلوى الملونة الزاهية و الزبادي لتحسين مظهر المواد الغذائية والمشروبات. استخدامها في صناعة المواد الغذائية يتم التحكم فيها من خﻻل التنظيم وقيم اﻻستهﻻك اليومي المقبولة كنتيجة لدراسات السمية. الهدف: تحديد تراكيز الملونات اﻻصطناعية اﻷكثر وفرة غالبا )التارترازين, السانسيت اﻷصفر, البونسيو فور آر والكارموزين( في المنتجات الغذائية المتاحة في اﻷسواق المحلية لقطاع غزة ومقارنتها مع قيم اﻻستهﻻك اليومي المقبولة. المنهجية: تم جمع 18 عينة )9 عصائر مركزة , 6 مشروبات صودا و 3 عصائر فواكه( المحتوية على التارترازين, السانسيت اﻷصفر, البونسيو فور آر والكارموزين إما بشكل منفصل أو خليط من اﻷسواق بشكل عشوائي. تم تطبيق طريقة كروماتوجرافيا السائلة عالية اﻷداء ذات المرحلة العكسية بعمود السي18 باستخدام سائل الخروج أسيتونيتريل : أسيتات بفر باﻻس الهيدروجيني 5 (15 :85) لتحسين فصل التارترازين , السانسيت اﻷصفر و البونسيو فور آر واستخدام سائل الخروج أسيتونيتريل : أسيتات بفر باﻻس الهيدروجيني 5 (30 :70) لتحسين فصل الكارموزين من خﻻل المواد واﻷدوات المتوفرة في مختبراتنا. تم الكشف عن λ ماكس لكل الملونات بواسطة كاشف اﻷشعة فوق البنفسجية. تم تحديد درجة نقاوة المواد المرجعية باستخدام مادة قياسية. تم التحقق من صحة هذه الطريقة وفقًا ﻹرشادات المؤتمر الدولي حول التنسيق. تم تحضير عينات من المواد الغذائية عن طريق المعالجة المسبقة التخفيف البسيط وإزالة الغازات ثم تحليلها بواسطة كروماتوجرافيا السائلة عالية اﻷداء المرتبط بكاشف اﻷشعة فوق البنفسجية. وتمت مقارنة النتائج مع قيم اﻻستهﻻك اليومي المقبولة لكل ملونات. النتائج: ثﻻث ملونات )التارترازين, السانسيت اﻷصفر و البونسيو فور آر( تم فصلها بكفاءة في غضون 8 دقائق. تم خروج الكارموزين في غضون 4 دقائق. λ ماكس كانت 429, 483, 507 و 516 نانوميتر للتارترازين, السانسيت اﻷصفر, البونسيو فور آر والكارموزين على التوالي. الطريقة أظهرت عﻻقة خطية )معامل اﻻرتباط < 0.999( في حدود تراكيز الملونات -1 100 ميكروغرام / مليليتر. الفحص كان دقيق (اﻻنحراف المعياري > 2% ). الدقة بنسبة اﻻختﻻف خﻻل اليوم وبين اﻻيام اظهرت اﻻنحراف المعياري النسبي > 2%. الطريقة كانت محددة , بحيث ﻻ يوجد أي تداخل تم تسجيله للمكونات المحتملة اﻻخرى في المواد الغذائية. استهﻻك مشروب واحد يحتوي على السانسيت اﻷصفر متاح في اسواق قطاع غزة يمكن أن يتجاوز قيم اﻻستهﻻك اليومي المقبولة لطفل يزن 15 كجم و استهﻻك مشروب واحد يحتوي على الكارموزين أو السانسيت اﻷصفر لطفل يزن 15 كجم يصل إلى حد 40-85 ٪ من قيم اﻻستهﻻك اليومي المقبولة لكل ملون. الخﻻصة: تم تطبيق اختبار كروماتوجرافيا السائلة عالية اﻷداء ذات المرحلة العكسية بنجاح لتحليل الملونات اﻻصطناعية في بعض المواد الغذائية. استهﻻك مشروب واحد فقط في بعض الحاﻻت يمكن أن يتجاوز قيم اﻻستهﻻك اليومي المقبولة وفي حاﻻت أخرى يصل الى نسبة عالية من قيم اﻻستهﻻك اليومي المقبولة, وبالتالي يجب أن يتم اتخاذ المقاييس للملونات اﻻصطناعية من قبل السلطات المسؤولة للحفاظ على صحة اﻷطفال. الكلمات الدالة: التارترازين, السانسيت اﻷصفر, البونسيو فور آر , الكارموزين , كروماتوجرافيا السائلة عالية اﻷداء , قيم اﻻستهﻻك اليومي المقبولة , المواد الغذائية. v List of contents Declaration ...................................................................................................................................... i Dedication ...................................................................................................................................... ii Acknowledgment .......................................................................................................................... iii Abstract ......................................................................................................................................... iv v ............................................................................................................................ الملخص باللغة العربية List of contents .............................................................................................................................
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