INTRODUCTION:
Food preservatives play a vital role in the
modern food industry and generally use for maintaining food quality and its
characteristics as well as promoting food safety and preventing nutritional losses from chemical alterations
and preserving the products during their shelf life which prevent or inhibit the growth of bacteria, fungi
and yeasts as well as preventing spoilage and decay. Food preservatives have been
the subject of interest among consumers, health professionals, commercial and
industrial agencies, alike because they are widely consumed in the diet by most
segments of the population and can exert
adverse health effects, especially for children and pregnant women. Food
preservatives must be in allowable safety limits, which must not exceed the maximum allowed
concentration of sodium benzoate and potassium sorbate 0.1% and 0.2% respectively1.
There are many Analytical methods used in
qualitative and quantitative detection of sodium benzoate and potassium sorbate
in foods such as UV–Visible spectrophotometry, HPLC methods, gas
chromatography, capillary electrophoresis and polarography2.
Benzoate was determined in soft drinks and fruit juices, lemon juice and soy
sauce by using ion chromatography (IC)3. Methyl ethyl and
propyl paraben have been identified in different beverages using HPLC coupled
with UV detector and DAD detector4-5. Gas chromatography and
solid-phase extraction were used to identify some preservatives such as benzoic
acid and sorbic acid, as well as methyl and ethyl and propyl paraben in soft
drinks, several sauces and jams6-7. Benzoic acid and sorbic
acid were identified in soft drinks using capillary electrophoresis with UV and
visible detector8. Benzoic acid and sorbic acid and esters of
Para-hydroxy benzoate (Methyl – propyl) were identified in the juices, soft
drinks, cheese and meat products by using RP- HPLC with UV detector, mass
spectroscopy detector (MS) and photo diode
array detector9-10-11-12.
Benzoic acid and sorbic acid were identified in Chinese meat using gas
chromatography with flame ionization detector, The level of sorbic acid and
benzoic acid in these samples ranged from <10 to 2,130 ppm and <10 to 933
ppm respectively13. Sodium benzoate was identified in
different beverage samples commercially available in Riyadh local markets using
spectrophotometry14.As well as benzoic acid levels were
identified in the milk produced in China using HPLC with photo diode array
detector15.
MATERIAL AND METHODS:
Sampling models:
Different brands of tomato sauce, soft drinks,
beverages and energy drinks were selected for this study.
Apparatus:
Analytical separation was carried out on a (Knauer)
HPLC with detector (UV Spectral Photometer S-2500), (Purospher ® STAR) RP-18
columns dimensions (250 mm × 4.6 mm) -5μm (Merck), Jet Stream column Oven,
ultrasonic bath (Daihan), analytical balance 0.1 mg (Pricesa), Germany digital
pipettes (Isolab). The solvents and materials were used analytical grade: HPLC
water and methanol (Isolab), sodium benzoate, potassium sorbate, ammonium
acetate and caffeine (Merck), HPLC glacial acetic acid (Panreac).
Samples preparation:
Five batches from each food product (tomato sauce, soft
drink, beverages and energy drink) were studied. Liquid samples were prepared by
transferring 5.0 ml of sample into 25 mL volumetric flask, the volume was
completed to 25 mL using HPLC-grade water. Semi-solid samples (tomato sauce)
were prepared by mixing 10 g of the sample with 50 ml of HPLC-grade water, then
the mixture transferred into 100 mL volumetric flask, the volume was completed
to 100 mL using HPLC-grade water and placed in the ultrasonic bath for 15
minutes then the solution was filtered and transferred 12.5 ml of liquid
filtrate (after neglecting the first 10 ml), 1 ml of internal standard
solution to 25 ml volumetric flask, the volume was completed to 25 mL using
HPLC-grade water. Final sample solution filtered through a 0.45 µm and degassed
by ultrasonication and then the samples were injected in the chromatograph.
Standard preparation:
Standards stock solutions of both salts sodium benzoate and potassium sorbate were prepared at 1000 mg/L with HPLC-grade
water. Combine 2, 4, 6, 8, and 10 mL of each stock standard into 100 mL volumetric flasks
and dilute to final volume with
HPLC-grade water. The concentration range of the standard curves was 0.02 - 0.10 mg/ml
(Fig.1).
Mobile phase preparation:
The mobile phase consisted of 25% ammonium
acetate buffer and 75% HPLC-grade methanol, (ammonium acetate buffer was
prepared by weighing 3.8 g ammonium acetate and dissolving it in 1 L HPLC-grade water). Mobile phase pH was adjusted to 4.4 by glacial acetic acid. All mobile phase
were filtered through a 0.45µm membrane filter and degassed by ultrasonication.
Chromatographic conditions:
Isocratic mobile phase consisted of a
mixture of acetic buffer (pH=4.4) and methanol in the ratio 25:75 (v/v).
Purospher ® STAR RP-18, 250mm×4.6 mm-5µm column, flow rate of 1.2 mL/min,
variable UV detector was set at 235 nm, column temperature at 40°C and injected sample volume was 20µL.
Fig.1. Linearity graphs of Na benzoate, and K sorbate
RESULTS AND DISCUSSION:
Determination standard solutions for sodium benzoate
and potassium sorbate:
It was separated and identified the components of a
mixture consisting of sodium benzoate and potassium sorbate with caffeine as
internal standard (0.04 mg/ml) at five different standard concentrations, and
every concentration was injected five times, (Fig.2).
Figure 2: Chromatogram of standard solutions under optimum
conditions. Peaks: 1- caffeine, 2- Na benzoate, 3- K sorbate.
Fig.3 Effect of mobile phase pH on the retention
times of 1-K sorbate, 2-Na benzoate.
Effect of mobile phase pH on the retention times tR
of sodium benzoate and potassium sorbate:
The effect of mobile phase pH acidified by acetic acid
in range (PH = 3.0-7.5) was studied by increasing the pH value due to
deprotonation the negative charge of both sodium benzoate and potassium sorbate
increased and drawing the curve representing the relation between the retention
times of both in terms of pH, the relation tR = f (pH) using the
same mobile phase component ratios and the injected quantity. It was shown that
the ionization suppression was impossible for both preservative with decrease
in pH, and this required the work at constant pH, using a convenient buffer in
the range between 3.5- 4.5 to perform an accuracy mixture separation, Fig.3.
Effect of changing temperature on the separation of
potassium sorbate and sodium benzoate:
the effects of the column temperature on the degree of
separation peaks for both potassium sorbate and sodium benzoate in the range
between 25°C and 50 °C was studied and has been shown that the separation of
potassium sorbate and sodium benzoate at 25 C was complete, but accompanied with very long analysis time and Band broadening, while at 50 °C was noted the interference of the two peaks, which necessitated selection of temperature 40°C, where the completed separation with a suitable analysis time does not exceed 6 minutes.
Fig.4
Peaks resolution for K sorbate and Na benzoate in relation to methanol
percentage.
Peaks resolution for potassium sorbate and sodium
benzoate Rs:
Resolution Rs of potassium sorbate and sodium benzoate
peaks was Identified, by changing the ratios of mobile phase components
(methanol and acetate buffer) with constant mobile phase pH (pH=4.4), It was
concluded that the best ratio for the mobile phase components (25:75) and the
best completed separation for all peaks begin at Rs = 1.4 (Fig.4).
Separation of real food samples:
The chromatographic separation was performed
for sodium benzoate and (or) potassium sorbate in (tomato sauce, soft drink,
beverages and energy drink) using the precedent chromatographic conditions.
The total analysis time was less than 6 min with good resolution, peak shapes
and minimal tailing. It was determined sodium benzoate and potassium sorbate in
all samples without any interference, peaks were completely separated and the
resolutions were more than (1.4). The peaks of sodium benzoate and potassium
sorbate in the samples were identified by comparing the retention time with
that of the standards, as well as Correlation coefficients and linear equations
of peak areas and standard concentrations were used for the quantitative
analysis, as it is shown in five representative samples figures (5-8).
Fig.5: Chromatogram of tomato Sauce ''Saha'' sample.
1- caffeine, 2- sodium benzoate. sorbate.
Fig.6: Chromatogram of beverage ''Original'' sample.1- caffeine, 2- Na benzoate, 3-K
Fig.7:Chromatogram of beverage sample. ''Mirinda
Orange'' 1- caffeine, 2- Na benzoate, 3- K sorbate.
Fig.8: Chromatogram of soft drink sample ''7up''1-
caffeine, 2- Na benzoate.
CONCLUSION:
The HPLC method with UV spectrophotometric
detection on a 5µm Purospher® STAR RP-18 column was developed for the
determination sodium benzoate and potassium sorbate in food samples, using
caffeine as internal standard. The total analysis time was less than 6 min.
Proposed HPLC method is rapid, direct, accurate and precise for simultaneous
determination of SB, PS in food samples and the matrix components do not
interfere with the determination of preservatives in all samples.
The accuracy of the method was confirmed
with an average recovery ranging between 93.8 and 98.7%, 95.6 and 99% of sodium
benzoate and potassium sorbate respectively.
According to the results of this study,
although both preservatives are added to different foodstuffs in a wide range
of concentrations, none of the samples presented levels below the range of
antimicrobial activity2.
Potassium sorbate and sodium benzoate was separated and
identified directly without preextraction in different soft drink and beverages
samples.
The identified amount of preservatives in
tomato sauce samples was in allowable safety limits and less than the
authorized amounts, as well as a difference in amounts between different
brands, While the beverages samples was in allowable safety limits and
authorized amounts.
In samples contain both potassium sorbate
and sodium benzoate together, the added amounts of those preservative were in
allowable safety limits and did not exceed the authorized limits.
ACKNOWLEDGMENTS:
This work was financially and technically supported by
the Ministry of High Education though the University of Aleppo, Faculty of
Science, Department of Chemistry in cooperation with Faculty of Technology
Engineering, Department of Technology of Foodstuff in University of Aleppo,
SYRIA.
REFERENCES:
1. SMITH G.; SHUM L.H.; Food Additives Data
Book First Edition Blackwell Science Ltd., Iowa,Usa.740-755(2003)
2. MICHAEL P.; DAVIDSON J.; SOFOS
N.;BRANENA.L.; Antimicrobials In Food, Food Science and Technology ; 143,11-85(2005).
3. Determination of Benzoate in Liquid Food
Products By Reagent-Free Ion Chromatography, Application Note 165, www.dionex.com
.
4. KRISTINA K.; BIRUTE P.; AUDRIUS P.; Isocratic
Hplc Determination Of Preservatives in Beverages, Vilnius University
,Naugarduko 24, LT Vilnius Lithuania(2006)
5. Survey Of Preservatives In Soft Drinks -Food Survey
Information Sheets on the http://www.food.gov.uk/science/surveillance/,No.
84/05(2005).
6. LILI W A.;XIAO Z.;YIPING W.;WEI W.; Simultaneous
Determination of Preservatives In Soft Drinks, Yogurts and Sauces By A Novel
Solid-Phase Extraction Element And Thermal Desorption-Gas Chromatography, Analytica
Chimica Acta, 577, 62–67(2006)
7. GONZA´LEZ M.;. GALLEGO M.;. VALCA´RCEL M.;
Simultaneous Gas Chromatographic Determination Of Food Preservatives Following
Solid-Phase Extraction, Journal Of Chromatography A, 823,
321–329 (1998)
8. RICHARD A. F.; ELIZABETH L. I.;NICOLO D.;
JENNIFER M. A.; HARRY E. N.; Development Of A Capillary Electrophoresis Method For The Simultaneous
Analysis Of Artificial Sweeteners, Preservatives And Colours In Soft Drinks,
Journal Of Chromatography A,876, 213–220(2000)
9. HYOUNG S.L.; RUSSELL.L.; ROUSEFF and JAMES
F.F.; Determination Of Food Preservatives In Orange Juice By Reversed Phase Liquid
Chromatography, Journal Of Food Science, 51(3), 568–570(1986)
10. XIU Q L.; FENG Z.;YAN S.;WEI Y.;XIAO
G,C.;YAN F.;JERRY Z.; Accurate Screening For Synthetic Preservatives In Beverage
Using High Performance Liquid Chromatography With Time-Of-Flight
Mass Spectrometry, Analytica
Chimica Acta, 608,165–177(2008)
11. TOUNI S.A.V.; TOLEDO M.C.F..; Determination
Of Benzoic And
Sorbic Acids In Brazilian Food, Food Control, 13,117–123,(2002)
12. ALI.M. S.;. ASSOC J; Determination Of
Benzoic Acid, Sorbic Acid, And Methyl, Ethyl, Propyl, and Butyl Parabens By HPLC, Off. Anal.
Chem., 68, 488-492(1985)
13. LOL.Y.;WU C.C.;JANK S.J.; CHIOU S.T.;HWANG D.F.;
Simultaneous Gas Chromatography Analysis of Preservatives in Chinese
Traditional Meat Products, Journal of Food And Drug Analysis, 9(4),
215-219(2001)
14. ALGHAMDI A. H.;ALGHAMDI A. F.;ALWARTHANA.A.,
Determination Of Content Levels Of Some Food Additives in Beverages
Consumed in Riyadh City, J.King Saud Univ,18(2), 99-109(2005)
15. PING Q, HONG H.; XIAOYAN L.; Assessment Of
Benzoic Acid Levels In Milk In China, Food Control, 20,414
418(2009)
