journal of food and drug analysis 25 (2017) 254e259

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Original Article

Fast and simple method for semiquantitative determination

of calcium propionate in bread samples
Chutima Matayatsuk Phechkrajang a,b,*, Surin Yooyong a
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand b Center of
Excellence for Innovation in Drug Design and Discovery, Faculty of Pharmacy, Mahidol University,
Bangkok, Thailand

articleinfo Calcium propionate has been widely used as a preservative in bakery and in bread. It
is sometimes not carefully used, or a high concentration is added to preserve
products. High consumption of calcium propionate can lead to several health
Article history: problems. This study aims to develop a fast and simple semiquantitative method
Received 20 January based on color complex formation for the determination of calcium propionate in a
2015 bread sample. A redebrown complex was obtained from the reaction of ferric
Received in revised form ammonium sulfate and propionate anion. The product was rapidly formed and easily
15 March 2016 observed with the concentration of propionate anion >0.4 mg/mL. A high-
Accepted 23 March 2016 performance liquid chromatography (HPLC) method was also developed and
Available online 2 June validated for comparison. Twenty-two bread samples from three markets near
2016 Bangkok were randomly selected and assayed for calcium propionate using the above

two developed methods. The results showed that 19/22 samples contained calcium
propionate >2000 mg/kg. The results of the complex formation method agreed with
Keywords: the HPLC method.
bread Copyright © 2016, Food and Drug Administration, Taiwan. Published by Elsevier
preservative
Taiwan
calcium
LLC. This is an open access article under the CC BY-NC-ND license (http://
propionate
colorimetry creativecommons.org/licenses/by-nc-nd/4.0/).
abstract

“ ”
allocated an acceptable daily intake as not specified or
1. Introduction “ ”
not limited [1,2]. However, the fact that acceptable daily
In the countryside areas of Thailand, some health intake is not specified does not mean that unlimited intake
problems come from dietary foods. Local food is acceptable. It could, in principle, be allowed for use in
manufacturers do not usually know about good foods in general with no limitation other than being in
manufacturing practices, as well as country food laws. accordance with good manufacturing practices. The
Calcium propionate has been widely used as a United States Food and Drug Administration
preservative in bakery and in bread. It has a potency for recommended that the daily intake of calcium propionate
preventive bacteria and fungi. Calcium propionate is for adults is 1 mg/kg/d [3]. If 1 kg of food was taken per
day for a man of 50 kg body weight, the amount of

* Corresponding author. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhaya Road, Rajathevee, Bangkok
10400, Thailand.
E-mail address: [email protected] (C.M. Phechkrajang). http://dx.doi.org/10.1016/j.jfda.2016.03.013
1021-9498/Copyright © 2016, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
calcium propionate in the food should not exceed 2000
listed as E number 282 in the Codex Alimentarius and is mg/kg.
 journal of food and drug analysis 25 (2017) 254e259 255

In Thailand, there were notifications of the ministry method. For the HPLC assay, the filtrate was further
of Public Health of Thailand announced in 1984 and diluted 10 times with a mobile phase before injection.
1989. The allowed concentration of calcium propionate
in food according to those announcements was 0.2%. 2.4. Testing for calcium propionate
Although oral calcium propionate might not cause
severe toxicity, long term health effects have been Some 1.0 mL of 0.6 mg/mL calcium propionate standard
reported. Propionate preservatives can contribute to or solution was added to a 1.0-mL bread sample preparation.
cause hyperactivity [4], visual hallucinations [5], The color of the reaction was monitored after a few drops
irritability, restlessness, inattention, and sleep of ferric ammonium sulfate test solution (TS) was added.
disturbance in some children [6e8]. Some researchers The color of the reaction tube was compared with the
found that brief infusions of propionic acid in rats standard preparation tube and blank tube. Standard
produced short bouts of behavioral, i.e., hyperactivity, preparation was performed by adding a few drops of ferric
object fixation, social impairments and other effects such ammonium sulfate TS to 2.0 mL of 0.4 mg/mL of calcium
as seizures, similar to those seen in autistic spectrum propionate. The blank tube was performed by adding a
disorders [9,10]. few drops of ferric ammonium sulfate TS to 2.0 mL of
Food safety is a key factor of good health. deionized water. A redebrown color complex was formed
Unfortunately, some preservatives are illegally added or indicating the presence of calcium propionate. For the
exceed regulation limits in foodstuffs. Several analytical sample containing calcium propionate < 2000 mg/kg, the
methods have been developed for determination of these color was weaker than that of the standard tube, but
illegally used preservatives including food contaminants stronger than that of the blank tube.
[11e13]. For propionic acid, the reported quantitative
determination methods were gas chromatography [14] 2.5. HPLC condition
and gas chromatography/mass spectrometry [15,16].
Although these methods have been accepted for their Quantitative determination of calcium propionate in bread
accurate and precise results, but they still have some samples was employed using Shimadzu model LC-10 AD
drawbacks such as requiring expensive instruments and (Shimadzu Corporation, Kyoto, Japan) equipped with an
well-trained analysts. Moreover, toxic organic solvent SPD10A UV detector. A symmetry C18 HPLC column
waste was also produced. In this study, a simple and (4.6 mm
cheap method of calcium propionate investigation in 150mm,5mm)wasutilized.Amixtureof0.05%phosphoricaci
bread products based on the colorimetric method was d and methanol with the ratio of 90 and 10 by volume was
developed. The developed colorimetric method was the successively mobile phase. The flow rate was 1.0
successfully applied to determine calcium propionate in mL/min and the presence of calcium propionate was
bread products and the results were in agreement with
monitored at 210 nm. 2.6. HPLC method validation
high-performance liquid chromatography (HPLC). This
indicated the accuracy of the colorimetric method. Method validation parameters, i.e., linearity, accuracy
precision, and specificity were evaluated. Linearity was
performed by using a series concentration of standard
2. Materials and methods solutions in the range of 25.0e250.0 mg/mL. The
concentrations (X-axis) were then plotted with their
2.1. Preparation of standard solution of corresponding peak areas (Y-axis). Linear least square
calcium propionate regression was used to calculate the correlation coefficient
(r).
About 200 mg of calcium propionate was accurately Accuracy of the HPLC method was studied with
weighted into a 100-mL volumetric flask, then dissolved standard addition approach. Three concentrations, low,
and diluted with deionized water. Further dilution of this middle, and high, of standard calcium propionate covering
stock solution was employed with deionized water to the linearity range were spiked into equal amounts of
obtain a standard solution of 0.6 mg/mL and 0.4 mg/mL. bread samples. Three replicates were performed for each
concentration level. Accuracy of the method was
2.2. Preparation of ferric ammonium sulfate expressed as recovery percent of standard found from the
test solution sample.
Precision was evaluated with three concentrations of
About 8.0 g of ferric ammonium sulfate was dissolved the desiredcompoundcoveringlinearityrange and three
and made up to 100 mL with deionized water. replicate samples for each concentration. Precision was
expressed as relative standard deviation percent (% RSD)
2.3. Bread sample preparation of replicate samples of each concentration.
Specificity of the HPLC method was obtained by
A sample was cut into small pieces and 10 g weighed comparing the chromatograms of the sample with the
accurately into a 250-mL beaker. Some 100 mL of standard and standard spiked-sample. Retention times of
deionized water was added, and the mixture was left calcium propionate in the sample and standard spiked-
standing for 2 hours before filtering. The filtrate was sample might equal that of the calcium propionate
used to test for calcium propionate in the colorimetric standard.
 journal of food and drug analysis 25 (2017) 254e259

256

Results and discussion

3.1. Semiquantitative determination of calcium
propionate

From the results, it could be seen that the redebrown

complex can be formed from the reaction of ferric ions
and propionate ions with the complex ratio of 1:1. The
ratio of complex formation was already proved by the
mole-ratio method which was described by Yoe and Jones
[17]. The chemical reaction used in this study was similar
to that which occurs between ferric ions and thiocyanate
’
ions in Volhard s method as displayedin Eq.(1) [18].
Therefore, thechemicalreaction between ferric and
propionate ions was proposed as in Eq. (2).

Fe3þ(aq) þ SCNe(aq) / Fe(SCNe)2þ(aq)

(color soluble complex)

Fe3þ(aq) þ CH3CH2COOe(aq) / Fe(CH3CH2COOe)2þ(aq)

(redebrown soluble complex) (2)

The reaction was clearly observed for calcium

propionate equal to or higher than 0.4 mg/mL. To
confirm the limit of detection (LOD) of the reaction, a
graph of probability of identification (POI) (Y-axis) and
calcium propionate concentration (X-axis) was
constructed [19]. Series concentrations of calcium
propionate, 0.0e1.0 mg/mL, 10 replicates for each
concentration, were prepared and tested with ferric
ammonium sulfate TS. Then, the POI of each
concentration was calculated using the following
equation: POI ¼ number of positive results/10.
 journal of food and drug analysis 25 (2017) 254e259 257

The concentration corresponding to POI ¼ 0.95 was

set as the LOD [19]. As shown in Figure 1, POI equal to
0.95 or 95% positive results was observed when the

3.3. Determination of calcium propionate in bread

Figure 1 e Graph of probability of identification and calcium propionate sample by colorimetric method
concentration indicating the limit of detection of calcium propionate by
colorimetric method. The colorimetric method described in this study is a semi-
3.2. Bread sample preparation

Ten grams of a bread sample was cut into small pieces and soaked in 100 mL
of deionized water. Because of the bulk of the bread, < 100 mL deionized
water was not enough to soak the sample very well. If 10 g of the sample
containing 0.2% of calcium propionate was dissolved in 100 mL deionized
water, the resulted solution would contained 0.2 mg/mL of calcium
propionate which was lower than LOD of the method (0.4 mg/mL).
Therefore, a standard solution of calcium propionate at a concentration of 0.2
mg/mL was added to the sample solution to increase the concentration up to
the LOD level.

quantitative approach.
In general,qualitative analysis

Figure 2 e Linearity graph of the developed high-performance liquid chromatography (HPLC) method for calcium propionate.
concentration of calcium propionate in the solution was
up to 0.4 mg/mL.
 journal of food and drug analysis 25 (2017) 254e259

establishes the chemical identity of the species in the concentration was lower than the LOD of the developed
sample, while quantitative analysis determines the relative colorimetric method that was 0.4 mg/mL. To reach
amounts of these species in numerical results. detection ability, standard calcium propionate was added
Semiquantitative analysis is between qualitative and to the sample solution to increase the final concentration
quantitative methods. It can provide the detection of an up to 0.4 mg/mL.
analyte presence including the approximate quantity by In the testing procedure, 1.0 mL of sample solution
comparing with a standard at allowance concentration. (the expected concentration was 0.2 mg/mL) was added
For the semiquantitation method of calcium with 1.0 mL of standard solution 0.6 mg/mL. Therefore,
propionate used in this study, the reaction can precisely the final concentration of this solution was about 0.4
detect the amount of calcium propionate equal to or mg/mL. A few drops of ferric ammonium sulfate reagent
greater than 0.4 mg/ mL. However, the allowance limit was added and the formation of a redebrown complex was
of calcium propionate in bread is 0.2% or 2000 mg/kg. If observed. The reaction of 2.0 mL of standard solution 0.4
a 10-g of sample was used in the assay, at least 100 mL mg/mL was performed for comparison. Some 2.0 mL
of deionized water was needed to soak the sample and deionized water was dropped with a few drops of ferric
dissolve the calcium propionate inside. It was impossible ammoniumsulfate reagent and used as a negative control.
to enrich the concentration by using deionized water < For interpretation, if the color of the redebrown complex
100 mL because of the bulky bread sample. If calcium of the sample was stronger than that of the standard, it
propionate wasusedin breadup tothelimit of indicated that the amount of calcium propi-
2000mg/kgand onate in the sample was > 0.2% or 2000 mg/kg and vice

versa. 3.4. HPLC method development and validation

Table 1 e Accuracy and precision of the developed highperformance
liquid chromatography (HPLC) method.
Calcium propionate in bread could be separated from the
Concentration levels (mg/mL) % Recovery (± % RSD, n ¼ sample matrix by using successive HPLC conditions as
50 102.3 ± 3.3 100 102.4 ± 3.4 described above with a retention time of 5.4 minutes. The
200 106.5 ± 6.0 developed method was then validated for the intended
purpose. Linear equation was Y ¼ 674.7X e 7226 with
RSD relative standard deviation. correlation
¼
dissolved completely in deionized water 100 mL, the
concentration would be 0.2 mg/mL. Unfortunately, this

Figure 3 e Chromatograms showing the specificity of the high-performance liquid chromatography (HPLC) method for
calcium propionate. Chromatograms of calcium propionate (A) in a bread sample, (B) standard calcium propionate
spiked sample, and (C) standard calcium propionate. Chromatographic conditions: the column was a Symmetry C18
 journal of food and drug analysis 25 (2017) 254e259 259

HPLC column (4.6 mm £ 150 mm., i.d.,5 mm); the flow rate was 1.0 mL/min; the mobile phase was methanol and
0.05% phosphoric acid (10:90, v/v); the injection volume was 20 mL; UV detection occurred at 210 nm; and the run time
was 9 minutes.
258

coefficient (r) >0.99 (Figure 2). Recovery percent values

of the standard found from standard spiked-samples
ranging from 102.3% to 106.5% indicated the accuracy of
the developed HPLC method. Precision of the method was
acceptable when the % RSD values of replicate samples
were < 6.0%. Method validation results are summarized in
Table 1. From linearity, accuracy, and precision results,
the range of the developed HPLC method could be also
claimed at 50.0e200.0 mg/mL. In addition, the developed
HPLC method had specificity for monitoring of calcium
propionate in bread samples. As seen from Figure 3,
retention times of calcium propionate in samples and
standard spiked-samples were the same as the retention
time of the authentic compound. Moreover, the peak area
of the standard spiked-sample was in agreement with the
summation of calcium propionate peaks in standard and
sample chromatograms.

3.5. Sample determination

Twenty-two bread products were randomly sampled from

samples
three areas outside Bangkok. Manufacturing information HPLC high-performance liquid chromatography.
such as mailing address, manufacturing date, expiration ¼
date, and food compositions of several samples were not From the results shown in Table 2, it could be seen that
presented. Amounts of calcium propionate in these the results obtained from colorimetry were in agreement
samples were determined by the developed colorimetry with the HPLC method. It was also noticed that 19/22
and HPLC methods. samples were found to have calcium propionate higher
than the recommended concentration of 2000 mg/kg.
In summary, a simple, fast, and cheap colorimetry
Table 2 e Assay results of calcium propionate in bread samplesmethod was successfully developed and can be used as a
obtained from the developed colorimetric method and high- screening method for calcium propionate in bread
performance liquid chromatography (HPLC) method. samples. A simple HPLC method was also developed
and validated. Twenty-two bread samples were sampled
Sample Source Calcium from three areas near Bangkok, and the amounts of
Calcium propionate
No. No. propionate from calcium propionate in these samples were determined
from HPLC
method colorimetric (mg/kg) method using the developed colorimetry and HPLC methods.
Determination results obtained from both methods were
< 2000 > 2000 mg/kg similar. From these results, it was seen that the
mg/kg developed colorimetry method was applicable as a
screening method for calcium propionate in bread
products.

Conflicts of interest

All authors declare no conflicts of interest.

Acknowledgments

This research was supported by the health promotion

project of the Faculty of Pharmacy Mahidol University
(project number 53-00-1253/0331-11) and Center of
Excellence for Innovation in Drug Design and
Discovery, Faculty of Pharmacy, Mahidol University.
 journal of food and drug analysis 25 (2017) 254e259

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