Determination of trace lead and cadmium in canned soft drinks in Syria.

INTRODUCTION: Soft drinks are highly consumed in Syria due to their preferable taste, advertisement, and lack of awareness about their harmful effects on the human body. Heavy-metal contamination is one of the top problems associated with the soft drinks industry. In this study, the levels of heavy metals (lead [Pb] and cadmium [Cd]) in carbonated and noncarbonated canned soft drinks in the Syrian market were investigated. The leaching of Pb and Cd in canned drinks was also investigated under different storage conditions.
MATERIALS AND METHODS: Soft drink samples were collected from the Damascus market. The samples were prepared using microwave digestion. All samples were analyzed using the developed and validated atomic absorption spectroscopy (AAS) method. RESULTS AND DISCUSSION: All studied samples at all stages of the study were free of Cd. The mean concentration of Pb ranged between 13.76 and 42.12 ppb. Our results showed that the levels of Cd and Pb were in the allowed limits according to Syrian Specification (1992/47) and the Food and Drug Administration (FDA) limits. There is no leaching of Pb and Cd in all studied samples under different storage conditions over 1 year of study.
CONCLUSION: The results of this study showed that all samples are following good manufacturing procedure (GMP) and safe to be consumed by costumers.

INTRODUCTION

Soft drinks can be described as nonalcoholic, sweetened, low-pH, water-based, often flavorful, colorful, and containing a quantity of fruit juice, fruit pulp, or other natural ingredients. These drinks are usually kept in metal cans, glass bottles, or tetra pack carton containers.[12] They are highly consumed due to their hydration effect, taste, and providing the body with the necessary amount of sugar and essential salts.[3] However, consuming high amounts of soft drinks could have adverse impacts on human health starting with increase the chance of tooth decay to obesity and metabolomic disease such as diabetics. Another adverse effect of soft drink consumption is the high possibility of consumer exposure to heavy-metal contamination. High levels of toxic heavy metals have been reported in soft drinks.[45678910] The sources of heavy metals in soft drinks include fruits, water, sweeteners, flavor agents, coloring dyes, or manufacturing procedures. Toxic heavy metals such as lead (Pb) and cadmium (Cd) have serious harmful effects on the human body. Pb is a well-known toxic metal causing acute and chronic toxicity by affecting the central nervous, hematopoietic, hepatic, and renal system producing serious disorders.[11] The main mechanism of Pb toxicity is the ability of Pb to deactivate antioxidant enzymes (especially those containing sulfhydryl groups), which cause an oxidative stress due to the inability of the body to readily detoxify the reactive intermediates or to repair the resulting damage.[12] In 2010, the Food and Drug Administration (FDA) confirmed that the presence of Pb in juices in percentage more than 50 ppb leads to serious health problems, so this value shall not be exceeded,[13] whereas the value of Pb should not exceed 0.025/kg body weight according to Syrian standards.[14]

Continuous exposure to Cd affects the kidney and could progress clinical renal complications, which may lead to renal failure. Cd toxicity is mainly due to its ability to replace other essential ions such as zinc, magnesium, and calcium in human enzymes leading to inactivation of these enzymes.[15] No safe value of Cd was recommended by the Syrian standard[14] or FDA[13] which means the soft drinks should be free of Cd, whereas the permissible value of Cd in soft drinks in the UK was 1 ppb.[16]

In Syria, soft drinks are highly consumed by all community components especially children.[17] Although many studies have shown that Middle East Area including Syria has high pollution with heavy metals including soil,[1819] fruits,[20] water,[212223] air,[24] and the Mediterranean Sea,[25] there are no studies showing the levels of heavy metals in the Syrian-marketed soft drinks. Thus, the aims of this study were to evaluate the concentrations of Pb and Cd in soft drinks available in the Syrian market and to compare their levels with the permitted levels published by regulatory authorities and other available studies in Middle East Area. For this purpose, a new accurate graphite furnace atomic absorption spectroscopy (GFAAS) method coupled to microwave digestion method was used.

MATERIALS AND METHODS

Carbonated and noncarbonated soft drink samples were collected from Damascus’s local market. To avoid the variation in sample storage conditions before metal analysis, all samples were chosen with less than 1-week manufacturing date. A total of 249 samples were collected containing different types of fruits, namely orange, apple, pineapple, mango, strawberry, cola, and red grapes as they are highly consumed. Two sources of samples were considered, which are local and nonlocal manufacturers. Table 1 shows the cods given to all types of samples in this study.

Table 1

Samples coding

Taste	Noncarbonated drinks		Carbonated drinks
	Local company	Nonlocal company	Local company	Nonlocal company
Red grape	GA	GB	–	–
Mango	MA	MB	–	–
Pineapple	PA	PB	–	–
Orange	OA	OB	OY	OZ
Apple	AA	AB	AY	AZ
Strawberry	–	–	SY	SZ
Cola	–	–	CY	CZ
Sugar free cola	–	–	DY	DZ

All glasswares used in this study were of Grade A including volumetric flasks, beakers, and measuring cylinders. Glasswares were soaked in 5% nitric acid for 3 h followed by washing with deionized water, then dried before use.

Sample preparation

To obtain the highest recovery of trace elements, two methods of sample digestion were applied and compared in terms of recovery and repeatability. Those methods are microwave digestion and wet digestion methods.

Three samples of noncarbonated drinks R1, R2, and R3, and three samples of carbonated drinks M1, M2, and M3 were used for the comparison of the study.

Pb 10 µg/L was added to each sample, then the digestion method was applied and the obtained samples were analyzed and the recovery of each metal was calculated and compared using t test.

For microwave digestion method, sample preparation was carried out using microwave digestion system (Multiwave 3000, Anton Paar, Graz, Austria). 5g of each samples were weighted carefully and transferred into polytetrafluoroethylene (PTFE) vessels, and a mixture of 3 mL of nitric acid 65% and 2 mL of H2O2 30% was added. The thermal program applied for sample digestion is presented in Table 2.

Table 2

Microwave thermal program

Step	1	2	3
Temperature (°C)	160	190	–
Ramp (min)	10	10
Hold (s)	10	20	15

In the wet digestion method, a mixture of 65% nitric acid and 30% oxygen water (2:1) was used as a digestion solution. 5 mL of digestion solution was added to 5g of each sample and placed in a digestion vessel under a fume hood. The mixture was heated up to 130°C for 5 h until the digestion was completed.

Following completion of the digestion, the samples were left closed for 2 h until complete cooling to room temperature. The entire content of each vessel was filtered and transferred into a 25-mL volumetric flask and the volume was brought up to the mark using deionized water.

An adequate detection method of trace elemental analysis with its corresponding sample digestion/preparation is highly required for such study. Therefore, our study has been designed to develop, optimize, and validate a comprehensive process of analysis for determining toxic metals, namely Cd and Pb in soft drinks. Elemental analysis was performed using AAS (Model Zeenit 700p, Analytik Jena, Germany). Cd and Pb quantifications were performed by GFAAS. GFAAS conditions were studied and optimized for the highest recovery of Cd and Pb, as shown in Table 3.

Table 3

Graphite furnace atomic absorption spectrometry conditions

Metal	Wavelength (nm)	Ashing temperature (°C)	Atomization temperature (°C)
Pb	283.3	900	1500
Cd	228.8	800	1300

Pb = lead, Cd = cadmium

Primary stock standard solutions for each metal Cd and Pb with a concentration of 1000 ppb were prepared by weighing 1g of metal standard (1000ppm) into a 1-L volumetric flask and diluting it to the mark by deionized water. This stock solution was used for the preparation of standard solutions for calibration purposes by serial v/v dilution with deionized water.

Method development and validation were studied using different analytical parameters such as limit of detection (LOD), limit of quantification (LOQ), linearity (coefficient of correlation), recovery, and precision.[26]

Limit of detection and limit of quantification

LOD is the lowest concentration of analyte that can be detected and distinguished from the blank. The LOQ is the lowest concentration that can be detected with a suitable level of accuracy and precision.

Linearity

Linearity of an analytical method is the capability of the technique to obtain results that are directly proportional to the concentration of analyte in the sample (within the range). The calibration range was studied for low standard concentration in accordance with the aim of this study to detect trace concentrations of toxic metals. The criteria for the standard curve are the coefficient of correlation, which should be more than 0.990. In this study, calibration curve was initiated before each analysis of each element to check the linearity of the method.

Accuracy

The accuracy of measurement is the level of closeness with the actual value. Accuracy of the method was checked by measuring the recovery of two spiked Quality Control (QC) standard solutions in various concentrations prepared within the analytical working range for each element.

The preparations were performed by serial v/v dilution from the primary stock standards of 1000ppm for each element. The recovery percentages of the QC standards were calculated as follows:

Precision

The precision of measurement system or reproducibility is the degrees of variation of repeated measurements under unchanged conditions. The precision of the results was indicated from the values of standard deviation (SD) and relative standard deviation (RSD) of the QC measurements.[26] RSD is measured using the following equation:

RESULTS

Digestion method optimization

Table 4 shows the recovery and repeatability results of both digestion methods.

Table 4

Recovery and repeatability results of both digestion methods

		e primary stock standards of 1000ppm			Wet digestion method
Sample	Added value (µg/L)	Measured conc. (µg/L) (±SD)	%R	RSD	Measured conc. (µg/L) (±SD)	%R	RSD
R1	10	10.03(±0.08)	100.34	0.79	10.42 (±0.11)	104.24	1.06
R2	10	9.89 (±0.09)	98.92	0.91	10.43 (±0.12)	104.27	1.18
R3	10	9.87 (±0.08)	98.70	0.85	10.43(±0.11)	104.28	1.08
M1	10	10.13(±0.09)	101.28	0.89	10.23 (±0.10)	102.28	1.02
M2	10	9.97 (±0.07)	99.70	0.74	10.22 (±0.11)	102.17	1.12
M3	10	10.06(±0.08)	100.56	0.84	10.12(±0.12)	101.20	1.17

SD = standard deviation, RSD = relative standard deviation

Recovery percentages of Pb for wet digestion and microwave digestion were varied between 101.20%–104.28% and 98.70%–101.28%, respectively. Statistically the application of Student’s t test indicated that there were no significant differences between the recovery results for both methods of all elements for wet digestion on a hot plate and microwave digestion at a 95% confidence level. However, lesser SD was observed by the microwave digestion method showing significantly higher repeatability. In addition, microwave digestion is a time-saver method as compared to the wet method. Therefore, the microwave digestion method was applied for entire sample preparation in our study.

Development and validation of atomic absorption spectroscopy analytical method

To obtain accurate results, the method of analysis of both elements was developed and validated for the low standard range. The sensitivity of the method was optimized to detect and quantify very low concentrations of Pb and Cd. Therefore, the LOD was 0.43 and 0.147 ppb for Pb and Cd, respectively, whereas the LOQ was 1.29 and 0.44 ppb for Pb and Cd, respectively. With such low LOQ, the linear range for Pb was set to the range of 0.5–45 ppb for Pb and 0.1–5 ppb for Cd. Figure 1A and B shows the linear standard curve for Pb and Cd, respectively.

Figure 1

Linear standard curve: (A) lead (Pb) and (B) cadmium (Cd)

Both metals showed an excellent coefficient of determinations using GFAAS and low values of LOD and LOQ which showed the sensitivity of the method. Table 5 summarizes the typical parameters of the calibration curve such as linear ranges, R2 coefficient of determination, standard concentration, and absorbance for Cd and Pb measured by the developed GFAAS methods.

Table 5

Linear ranges, R2, standard concentration and absorbance for cadmium and lead measured by graphite furnace atomic absorption spectrometry

Metal	Standard concentration (µg/L)	Absorbance	Coefficient of determination (R2)
Cd	0.1	0.0094	0.997
	0.5	0.0453
	1	0.1113
	2	0.2463
	4	0.4381
	5	05151
Pb	0.5	0.00329	0.999
	2	0.0131
	5	0.0237
	15	0.0424
	30	0.0834
	45	0.113

Pb = lead, Cd = cadmium

Accuracy and precision

Accuracy was studied by the recovery calculation of three QC samples, whereas precision was measured by calculating the RSD% value for repeated measurement of QC samples. The results are presented in Table 6.

Table 6

Accuracy and precision results

Metal	Sample ID	Prepared conc. (µg/L)	Measured conc. (µg/L) (±SD)	% Recovery	% Precision
Pb	QC 1	5	4.99 (±0.04)	99.92	0.76
	QC 2	10	9.87 (± 0.08)	98.70	0.81
	QC 3	30	30.06 (±0.25)	100.2	0.83
Cd	QC 1	0.5	0.51 (±0.01)	102	1.9
	QC 2	1	0.995 (±0.05)	99.5	0.4
	QC 3	3	2.99 (±0.02)	99.7	0.72

Pb = lead, Cd = cadmium, SD = standard deviation

The results obtained for Cd and Pb analysis by GFAAS in QC samples showed excellent levels of accuracy and precision within the acceptable requirements specified by International Council for Harmonization (ICH) guidelines.[26]

The developed methods were used for the analysis of Pb and Cd in carbonated and noncarbonated soft drinks in the Syrian market and the leaching of both metals in canned soft drinks under different storage conditions. The results showed the absence of Cd in all studied samples, which are in accordance with Syrian and World health regulations. However, Pb was found in all studied samples in concentrations that ranged between 13.76 and 42.12 ppb. Pb concentrations are presented in Table 7.

Table 7

Lead concentrations found in canned soft drinks

Noncarbonated drinks		Carbonated drinks
Local company	Nonlocal company	Local company	Nonlocal company
GA	28.89	OY	17.63
MA	24.16	AY	15.83
PA	21.25	SY	18.49
OA	21.85	CY	16.45
AA	32.72	DY	16.84
GB	28.44	OZ	16.11
MB	24.16	AZ	15.75
PB	21.03	SZ	16.25
OB	22.42	CZ	15.67
AB	33.51	DZ	13.66

Heavy-metal leaching in canned soft drinks was studied over 1 year under two different storage conditions. Cd did not appear in all studied samples under both storage conditions, whereas Pb was presented in all studied samples. Table 8 shows the levels of Pb in studied samples over the study period.

Table 8

Lead concentrations in canned soft drinks stored under two different conditions

Storage conditions		Controlled temperature					Noncontrolled temperature
Period (month)	0	3	6	9	12	0	3	6	9	12
GA	28.89	29.18	27.75	28.76	29.36	28.89	29.19	28.56	31.22	30.89
MA	24.16	24.17	24.67	23.88	24.08	24.16	24.71	24.34	23.59	25.45
PA	21.25	21.15	21.21	21.54	20.90	21.25	21.57	21.93	22.39	23.38
OA	21.85	21.97	21.22	22.56	22.28	22.85	21.95	22.15	22.25	22.91
AA	32.72	32.45	32.76	33.18	31.71	32.72	32.58	32.73	32.72	32.86
YA	15.84	16.23	15.82	16.17	15.68	15.84	15.41	15.71	15.43	15.89
YC	16.32	16.66	16.50	16.08	15.61	16.32	16.58	16.82	17.46	16.89

DISCUSSION

Sample preparation technique has a vital role in the analysis of heavy metals due to the importance of cleanup and good recovery. In our study, we compared two methods that are the wet digestion method and the microwave digestion method. We found that the microwave method is faster, safer, and better in terms of recovery and repeatability. Many methods have been used for sample preparation including dry ashing method,[727] wet digestion method,[5] and microwave digestion method.[6] The microwave digestion method is preferable to the wet digestion method due to many factors, such as it minimizes acid amount, reduces metal loss, reduces time consumption, and is better for operator health.[28] On the contrary, many instruments and techniques were used for the analysis of heavy metals in soft drinks including AAS,[45,7] inductively coupled plasma mass spectrometry (ICP-MS),[29] and inductively coupled plasma optical emission spectrometry (ICP-OES).[630] In our study, we have used GFAAS technique due to its ability to detect low traces of metals, suitability for Pb and Cd analysis, and relatively low cost of operation.

In our study, Cd concentration was below the detection limit in all samples all over the study. This is in consistent with the Syrian Standard No. (1992/47),[14] which specified that drinks should be free of Cd. This result is also in agreement with other studies,[4] as Cd was reported to be below the detection limit in all studied samples in the Nigerian market. However, another Nigerian study[10] reported the presence of Cd in some fruit juice drinks. This may be because samples studied were juice samples containing 80% of natural fruit juice, which is one of the main sources of heavy-metal contamination,[10] whereas in our study the natural fruit juice content does not exceed 30% maximum. The absence of Cd in all samples indicates the good manufacturing procedure (GMP) compliance and ensures a nonpolluted source of water, fruit, and other ingredient used in soft drink manufacturing.

Pb has been found in all studied samples in concentrations ranged between 13.76 and 42.12, which is below the permitted limit specified in Syrian Standard No. (1992/47)[14] and FDA standards.[13] The study also showed a higher Pb concentration in noncarbonated soft drinks compared to carbonated drinks. The possible sources of Pb in soft drinks are the fruits, water, contamination during the manufacturing process, and soft drink minor ingredients such as flavors, sweeteners, and coloring dyes. Fruits are the main source of contamination in noncarbonated soft drinks as they represent 30% of their content. The fruit could be contaminated by heavy metals due to many reasons that involve contaminated soil, water, fertilizer, and pesticides. Fruits contamination with a high content of heavy metals has been reported in the Middle East[20] as well as soil contamination.[18] High heavy metal levels were also reported in river and groundwater[212223] and fertilizers.[31] Our results were in accordance with many studies performed in the Middle East and reported low Pb concentrations in soft drinks using other techniques such as ICP-OES[6] and ICP-MS.[29]

Our study also found no significant leaching of heavy metals in canned soft drinks over 1 year of storage under controlled and noncontrolled conditions. This result is in accordance with other recent studies, which shows the compatibility of used cans for soft drinks manufacturing.

CONCLUSION

In conclusion, soft drinks in the Syrian market are safe for human consumption as they are not contaminated by heavy metals especially Cd and Pb. However, although the Pb concentrations are in the permissible limits, we recommend using free of contamination sources for the production of soft drinks. We advise costumers to reduce daily soft drink consumption as heavy metals could accumulate in their body over time and cause harmful effects and diseases. Also, soft drinks contain high sugar content, which lead to obesity and diabetics over noncontrolled consumption.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.