Table of Contents

ACRONYM...............................................................................................................................................3
1. SUMMARY (Summarize the research proposal ).................................................................................4
2. BACKGROUND AND JUSTIFICATION...........................................................................................6
2.1-Introduction.....................................................................................................................................6
2.2 Background......................................................................................................................................8
2.3 Justification of the study / Rationale for the Study/.......................................................................11
3. OBJECTIVES OF THE RESEARCH PROJECT...............................................................................15
3.1 General Objective..........................................................................................................................15
3.2 Specific Objectives........................................................................................................................15
3.3-Hypothesis.....................................................................................................................................15
4. MATERIALS AND METHODS.........................................................................................................24
4.1. Study Site......................................................................................................................................24
4.2. Study Design, and Study Population............................................................................................24
4.3. Study Populations.........................................................................................................................25
4.4. Sample size determination............................................................................................................26
4.5. Data collection through Questionnaire.........................................................................................27
4.6. Data management.........................................................................................................................34
4.7. Data quality assurance..................................................................................................................34
4.8. Data Analysis................................................................................................................................35
5. ETHICAL APPROVAL......................................................................................................................37
6. IMPLEMENTAION OF THE PROJECT BY PHASE.......................................................................38
7. Cost of the project with complete budget breakdown.........................................................................39
8. BENEFITS OF THE STUDY RESULTS...........................................................................................40
9. FACILITIES AVAILABLE FOR THE STUDY (MAJOR FACILITIES).........................................41
10. REFERENCES..................................................................................................................................42

1. Summary

There are several methods for determination of sodium benzoate and other
preservatives in soft drinks and fruit juices but much of this methods highly
costly and time consuming, so rapid and reliable method for identification
and quantification of sodium benzoate in different soft drinks and Fruit
Juices is a procedure, in which high performance liquid chromatography
technique is used. The aim of this study is to determine the sodium benzoate
level in packed soft drinks and fruit juice samples consumed in the city of
Addis Ababa.

The HPLC determination of sodium benzoate will be performed on a

reversed-phase C18 column by an isocratic elution with 1.0 M acetic acid in
75% (v/v) Methanol and adjusting the pH to 3 by adding successive small
amounts of a saturated solution of sodium acetate, and UV detection at 255
nm. The method will be developed, fully validated with the required
analytical parameters.

2. Background and Justification

2.1. Introduction

Food additives have become increasingly important in modern food technology

[1]. The demand for new, tasty, convenient and nutritious foods continued to
increase the early days of processed foods and beverages. It is estimated that over
2500 different additives are currently being used in food products [2]. Food
additives are used for various purposes, including preservation, coloring, and
sweetening. The preservatives are added to stop or delay nutritional losses due to
microbiological, enzymatic or chemical changes of foods and to prolong the shelf
life and quality of foods. Benzoic acid and its salts such as sodium, potassium and
calcium benzoates are normally preferred and used as food preservatives. They are
normally represented by E- numbers such as E210-benzoic acid, E211- sodium
benzoate, E212- potassium benzoate and E213- calcium benzoate. Although
sodium benzoate is generally recognized as safe (GRAS), short-term exposure to
sodium benzoate can irritate the eyes, skin and the respiratory tract. And long-term
exposure or repeated exposure may cause skin sensitization.

Children are at higher risks as they have high quantity intake per kg body weight
with different dietary patterns and food preferences. In fact carbonated water-based
flavored drinks, soft drinks and fruit drinks are major contributors to the benzoic
acid exposure in teenagers because of their high consumption levels of these
products. Adverse effects include asthma, urticaria, metabolic acidosis,
convulsions and so on [3, 4]. The development of allergic reactions to benzoates in
humans, such as urticaria, nonimmunological contact urticaria and asthma, has
been reported in some studies [5-11].

Sodium benzoate is a permitted food additive by the international laws in

restrictive amounts for processed foods and non alcohol, but their content must be
declared and must not exceed the established limits by legislation.

2.2. Background

The analytical determination of these preservatives is not only important for

quality assurance purposes but also for consumer interest and protection. The most
common analytical method for the determination of Benzoic acid (BA) or Sodium
benzoate has been reversed-phase HPLC [12,13], although other analytical
methods such as Capillary Electrophoresis [14], Spectrophotometry [15], HPLC
[16-21] and SPME-HPLC [22] have also been reported. These methods achieved
good sensitivity; however, they also involve many steps, which can result in loss of
the analytes during sample preparation.

In this study, a simple method that provides accurate results for sodium benzoate in
foods is presented. The method uses a simple filtration and dilution for sample
preparation followed by detection and determination by HPLC with UV detection.

2.3. Justification of the study

A simple analytical tool to determine preservatives in packed soft drinks and fruit
juices will be developed based on HPLC. The method will be useful for regulatory
laboratories and manufacturers of such food items in Ethiopia and elsewhere.

The base line level of sodium benzoate in soft drinks and fruit juices in Addis
Ababa and by extension in Ethiopia will be known. The health risks will be
also assessed.

Results will be presented in scientific conferences and published in academic

Journals. Awareness creation using mass media like radio, television and
newspaper may be used.

3. Objectives of the Research Project

 3.1. General Objective

 To quantify level of sodium benzoate in packed soft drinks and fruit

juices.
 To develop an analytical method by HPLC for sodium benzoate
determination in packed soft drinks and fruit juices.
 To validate the analytical methods developed using HPLC.

3.2. Specific Objectives

 To infer the daily uptake level of sodium benzoate by consuming soft

drinks.
 To know the safety of soft drinks and fruit juices with respect to
sodium benzoate level.
 To infer the daily uptake level of sodium benzoate by consuming fruit
juices.

4. Materials and Methods

4.1. Study Site

The study will be conducted in Addis Ababa from March 2019 to June 2019. The
city has 10 sub cities each having different number of woredas. As a capital city,
Addis Ababa is a major trade center for packed fruit juices and soft drinks with
many super markets and shops that sell packed fruit juice and soft drinks.

Samples will be collected from seven randomly selected representative sub cities
and analyzed at EPHI Food Science and Nutrition laboratory.

4.2. Study Design, and Study Population

Packed fruit juices and soft drink samples will be collected from super markets and
shops in Addis Ababa city.

A total of 100 samples (60 packed soft drinks with four soft drinks and 40 packed
fruit juices) from ten sub cities will be collected. This was achieved by collecting
duplicate samples for each type of fruit juice and soft drinks. In this study variety
of imported packed fruit juice types will be included.

A wide range of super market and shops will be covered in order to ensure that the
survey is representative of the supply of the products in Addis Ababa.

4.3. Data Collection

The data collection will be made by laboratory experiment, the laboratory based
experiment involves filtration, identification and quantification of sodium benzoate
from packed fruit juices and soft drink samples using HPLC instrument.

5. Implementation of the Project by Phase

Phase1. Duration: One Month

Description: Selecting the appropriate mobile solvent composition, that
can selectively separate the analyte from the matrix, and to optimize to get separate
and clear peak of the understudy analyte.

Phase2. Duration: One Month

Description: Trying to get the best straight line that fits for the calibration
curve using different concentration level. And full method validation will be
carried out.

Phase3. Duration: One Month

Description: To select areas from all sub city of Addis Ababa for sample
collection in order to get best representative sample for analysis. After selection of
areas, buying, transporting to the laboratory and putting in a refrigerator till
analysis is to done.

Phase4. Duration: One Month

Description: Real analysis of the different samples will be done by the
HPLC instrument found in the department laboratory. The obtained results will be
analysed and write up of the study will be completed.

6. Cost of the project with complete budget breakdown

 COST OF THE PROJECT
6.1. PERSONNEL COSTS (salaries, per diem, etc.)
NAME Qualification Salary/month Duration of
in ETB work Total

TOTAL

6.2. Equipment

TYPE OF EQUIPMENT ESTIMATED COST IN

ETH. BIRR

TOTAL

6.3 TRANSPORT COST

SPECIFICATION OF TRANSPORT (Fuel, tickets, etc.)
LOCAL To collect samples from different areas of Addis Ababa Estimated

FOREIGN______________________________ Cost in ETB

TOTAL COST 15000 15000

____________________
____________________

6.4. MISCELLANEOUS (Information, stationery, Estimated Cost

publications, etc.). 5000
Total 5000

6.5. SUPPLIES (chemicals, reagents, etc.). Estimated Cost

Total 46000 46000

6.6. GRAND TOTAL (by phase) PHASE Amount in ETB

Phase I 1 20000
Phase II 2 50000
Phase III 3 20000
Phase IV 4 10000

7. Benefits of the study result

The result of the study can be used as a source for regulatory bodies for their
work to determine maximum permitted level of sodium benzoate in packed
soft drinks and fruit juices in Ethiopia.

It will create awareness among the society about the health risks associated
with preservatives in packed foods and drinks.

8. Facilities available for the study

The study will be conducted at Food science and nutrition laboratory. HPLC
(High performance Liquid Chromatography) equipment with UV Visible
spectroscopy detector are available.

Glass wares, analytical balances as well as reversed phase columns for the
experiments are also present in the department lab.

9. References

1. Rahman, M. A., Sultan, M. Z., Rahman, M. S. and Rashid, M. A.2015. Food

adulteration: A serious public health concern in Bangladesh. Bangladesh
Pharm. J. 18, 1-7.

2. Branen, A.L. and Haggerty, R.J. 2002. Introduction to food additives. (In
Branen, A.L., Davidson, P.M., Salminen, S. & Thorngate III, J.H., eds. Food
additives. 2nd ed. New York: Marcel Dekker. pp. 1-9.
3. Tfouni, S.A.V. and Toledo,C.F. 2002. Determination of benzoic and sorbic
acids in Brazilian foods. Food control. 13, 117-123.

4. WHO (1996). Toxicological Evaluation of Certain Food Additives. Prepared

by the 46th Meeting of the Joint FAO/WHO Expert Committee on Food
Additives (JECFA).

5. Hannuksela, M. and Haahtela, T. 1987. Hypersensitivity reactions to food

additives. Allergy 42, 561-575.

6. Juhlin, L. 1981. Recurrent urticaria: clinical investigation of 330 patients.

Brit. J. Dermatol.104, 369-381.

7. Juhlin, L., Michaelsson, G., and Zeterstrom, O. 1972. Urticaria and asthma
induced by food and drug additives in patients with aspirin hypersensitivity.
J. Allergy Clin. Immunol. 50, 92.

8. Lahti, A., V€a€an€anen, A., Kokkonen, E. L., and Hannuksela, M. 1987.

Acetylsalicyclic acid nonimmunologic contact urticaria. Cont. Dermatitis,
16, 133–135.

9. Michaelsson, G.and Juhlin, L. 1973. Urticaria induced by preservatives and

dye additives in foods and drugs. Brit. J. Dermatol. 88, 525-532.

10. Rademaker, M., and Forsyth, A. 1989. Contact dermatitis in children. Cont.
Dermatitis, 20, 104-107.
11.Safford, R. J., Basketter, D. A., Allenby, C. F., & Goodwin, B. F. J. 1990.
Immediate contact reactions to chemicals in the fragrance mix and a study of
the quenching action of eugenol. Brit. J. Dermatol.123, 595-606.

12. Saad, B., Bari, M.F., Saleh, M.I., Ahmad, K., and Talib, M.K., 2005. J.
Chromatogr. A, 1073, 393.

13. Theron, M., Rykerslues, F.J., 2011. Oraganic acid and food preservation,
the academic division.

14. Tang, Y., Wu, M., 2007. Food Chem., 103, 243.

15. Hofer, K., and Jenewein, D. 2000. Food Technol., 211, 72.

16. Ferreira, I.M., Mendes, E., Brito, P., and Ferreira, M.A., 2000. Food Res.
Intl., 33. 113.

17. Pylypiw, H.M., Grether, M.T. 2000. J. Chromatogr. A, 883, 299-304.

18. Chen, Q.C., and Wang, J. 2001. J. Chromatogr. A, 973, 299.

19. Tfouni, S.A., and Toledo, M.C. 2002. Food Control, 13, 117.

20. Chinnici, F., Spinabelli, U., Riponi, C., and Amati, A., 2005. J. Food Comp.
Anal., 18, 121.

21. Kucukcetin, A., Sik, B., and Demir, M., 2008. Aras. Makal, 33, 159.

22. Wen, Y., Wang, Y., and Fng, Y.Q., 2007. Anal. Bio. Anal Chem., 388,
1779.