58 November 2014Spinco Biotech

Application BriefFo

od S

afet

y

Analysis of Sodium Benzoate Preservative in Soft Drinks using Prominence-i HPLC

A rapid conventional HPLC method is described for the determination of food preservative - sodium benzoate in soft drinks. The method utilizes the new integrated Shimadzu HPLC Prominence-i LC-2030C.

In today’s world, the role of food additives becomes inevitable due to the increase in the production of treated and ready-to-eat convenience foods. Food additives are intentionally added to the food stuffs

and must be safe for lifetime consumption. They are used for the purpose of maintaining

or improving the quality, texture, consistency, appearance of the food

products. Food additives are classified on the basis of their functional use, in which the preservatives are most important additive and are used to prevent and retard the microbial spoilage of food. Preservatives are classified into two types; - Class I and Class II.

The Class I preservatives are common salt, sugar, glucose, dextrose, spices, vinegar, honey, etc. The Class II preservatives are nature identical / synthetic compounds like salts of benzoic acid, sorbic acids, lactic acid,

etc. The addition of Class I preservatives are not restricted

in food products, but the class II preservatives are Generally

Recognized As Safe (GRAS) and are listed in FDA GRAS food substances. It

is noted that, the benzoic acid and sodium benzoate may be used as antimicrobial and

flavouring agents in food products. The limit of benzoic acid / sodium benzoate as per FDA is 0.1% in food products.

Though the legislations are permitting these class II preservatives in food products, their use demands special care. In this context, a rapid and simple HPLC method has been developed and demonstrated in this article by IICMS using the new integrated Shimadzu

HPLC ‘Prominence-i’ LC-2030C to quantitatively determine the content of sodium benzoate in grape, lemon and orange flavoured soft drinks.

ExperimentalPreparation of Sodium benzoate standardPrepared the concentration of 100mg/L sodium benzoate in diluent and used as working standard.

Preparation of Sample solutionThe grape, lemon & orange flavoured soft drink samples with Class-II preservative were selected in this study. These soft drink samples were degassed thoroughly in ultrasonic bath for 20min. The sample aliquot of 0.5g of soft drink was transferred to 5mL volumetric flask & made up to the mark with diluent

and sonicated to mix well.

Results and DiscussionThe sodium benzoate standard solution and the simply diluted soft drink samples were injected directly into the Prominence-i LC-2030C chromatography system without any complex sample preparation and recorded the results. The retention time of sodium benzoate is observed about in 3.5min. The typical

Prominence-i Series

The New Industry Standard HPLCs

HPLC

Shimadzu “Prominence-i” evaluates the foodpreservative-sodium benzoate in soft drinks

Application News

A rapid conventional HPLC method is described for the determination of food preservative-sodium benzoate in soft drinks. The methodutilizes the new integrated Shimadzu HPLC Prominence-i LC-2030C.

In today’s world, the role of food additives becomes inevitable due to the increase in the production of treated and ready to eat conveniencefoods. Food additives are intentionally added to the food stuffs and must be safe for lifetime consumption. They are used for the purpose ofmaintaining or improving the quality, texture, consistency, appearance of the food products. Food additives are classified on the basis of theirfunctional use, in which the preservatives are most important additive and are used to prevent and retard the microbial spoilage of food.Preservatives are classified into two types; they are Class I and Class II.

The Class I preservatives are common salt, sugar, glucose, dextrose, spices, vinegar, honey, etc. The Class II preservatives are naturalidentical / synthetic compounds like salts of benzoic acid, sorbic acids, lactic acid, etc. The addition of Class I preservatives are not restrictedin food products, but the class II preservatives are Generally Recognized As Safe (GRAS) and are listed in FDA GRAS food substances. It isnoted that, the benzoic acid and sodium benzoate may be used as antimicrobial and flavouring agents in food products. The limit of benzoicacid / sodium benzoate as per FDA is 0.1% in food products.

Though the legislations are permitting these class II preservatives in food products, their use demands special care. In this context, a rapid andsimple HPLC method has been developed and demonstrated in this article by IICMS using the new integrated Shimadzu HPLC ‘Prominence-i’ LC-2030C to quantitatively determine the content of sodium benzoate in grape, lemon and orange flavoured soft drinks.

Experimental

Preparation of Sodium benzoate standard

O ONa

Prepared the concentration of 100mg/L sodium benzoate indiluent and used as working standard

Preparation of Sample solutionThe grape, lemon & orange flavoured soft drink samples withClass-II preservative were selected in this study. These softdrink samples were degassed thoroughly in ultrasonic bath for20min. The sample aliquot of 0.5g of soft drink was transferredto 5mL volumetric flask & made up to the mark with diluent and

Results and Discussion

The sodium benzoate standard solution and the simplydiluted soft drink samples were injected directly into theProminence-i LC-2030C chromatography system withoutany complex sample preparation and recorded theresults The retention time of sodium benzoate is

Fig 1: Structure of Sodium Benzoate

3.0

mV(x100)Ch1 (230nm)

to 5mL volumetric flask & made up to the mark with diluent andsonicated to mix well.

results. The retention time of sodium benzoate isobserved about in 3.5min. The typical HPLCchromatogram of Sodium benzoate standard 100mg/Lconcentration is shown below:

Table 1: Instrument and Analytical Conditions

HPLC Prominence-i LC-2030C

Column C-6 Phenyl Hexyl (50x4.6mm, 3µ)

HPLC Chromatogram of Sodium Benzoate standard 100mg/L

3.55

min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

0.0

1.0

2.0Workstation Lab solution Version 5.62 P

Flow Rate 0.7mL/min (Isocratic)

Mobile Phase10mM Ammonium formate, pH adjusted to 3.0 with dilute formic acid : Acetonitrile (75:25)

Diluent Water : Acetonitrile (75:25) The method is tested for the HPLC validation parameters andWavelength 230nm

Injection Volume 5 µL Run Time 7.00 minutes

found to be precise, linear and accurate. The %RSD of arearesponse for the six replicate sodium benzoate standardinjections is found to be 0.25%. The area response is tabulatedin Table 2 and the corresponding overlaid standardchromatogram is shown in Fig.2.

HPLC

Shimadzu “Prominence-i” evaluates the foodpreservative-sodium benzoate in soft drinks

Application News

A rapid conventional HPLC method is described for the determination of food preservative-sodium benzoate in soft drinks. The methodutilizes the new integrated Shimadzu HPLC Prominence-i LC-2030C.

In today’s world, the role of food additives becomes inevitable due to the increase in the production of treated and ready to eat conveniencefoods. Food additives are intentionally added to the food stuffs and must be safe for lifetime consumption. They are used for the purpose ofmaintaining or improving the quality, texture, consistency, appearance of the food products. Food additives are classified on the basis of theirfunctional use, in which the preservatives are most important additive and are used to prevent and retard the microbial spoilage of food.Preservatives are classified into two types; they are Class I and Class II.

The Class I preservatives are common salt, sugar, glucose, dextrose, spices, vinegar, honey, etc. The Class II preservatives are naturalidentical / synthetic compounds like salts of benzoic acid, sorbic acids, lactic acid, etc. The addition of Class I preservatives are not restrictedin food products, but the class II preservatives are Generally Recognized As Safe (GRAS) and are listed in FDA GRAS food substances. It isnoted that, the benzoic acid and sodium benzoate may be used as antimicrobial and flavouring agents in food products. The limit of benzoicacid / sodium benzoate as per FDA is 0.1% in food products.

Though the legislations are permitting these class II preservatives in food products, their use demands special care. In this context, a rapid andsimple HPLC method has been developed and demonstrated in this article by IICMS using the new integrated Shimadzu HPLC ‘Prominence-i’ LC-2030C to quantitatively determine the content of sodium benzoate in grape, lemon and orange flavoured soft drinks.

Experimental

Preparation of Sodium benzoate standard

O ONa

Prepared the concentration of 100mg/L sodium benzoate indiluent and used as working standard

Preparation of Sample solutionThe grape, lemon & orange flavoured soft drink samples withClass-II preservative were selected in this study. These softdrink samples were degassed thoroughly in ultrasonic bath for20min. The sample aliquot of 0.5g of soft drink was transferredto 5mL volumetric flask & made up to the mark with diluent and

Results and Discussion

The sodium benzoate standard solution and the simplydiluted soft drink samples were injected directly into theProminence-i LC-2030C chromatography system withoutany complex sample preparation and recorded theresults The retention time of sodium benzoate is

Fig 1: Structure of Sodium Benzoate

3.0

mV(x100)Ch1 (230nm)

to 5mL volumetric flask & made up to the mark with diluent andsonicated to mix well.

results. The retention time of sodium benzoate isobserved about in 3.5min. The typical HPLCchromatogram of Sodium benzoate standard 100mg/Lconcentration is shown below:

Table 1: Instrument and Analytical Conditions

HPLC Prominence-i LC-2030C

Column C-6 Phenyl Hexyl (50x4.6mm, 3µ)

HPLC Chromatogram of Sodium Benzoate standard 100mg/L

3.55

min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

0.0

1.0

2.0Workstation Lab solution Version 5.62 P

Flow Rate 0.7mL/min (Isocratic)

Mobile Phase10mM Ammonium formate, pH adjusted to 3.0 with dilute formic acid : Acetonitrile (75:25)

Diluent Water : Acetonitrile (75:25) The method is tested for the HPLC validation parameters andWavelength 230nm

Injection Volume 5 µL Run Time 7.00 minutes

found to be precise, linear and accurate. The %RSD of arearesponse for the six replicate sodium benzoate standardinjections is found to be 0.25%. The area response is tabulatedin Table 2 and the corresponding overlaid standardchromatogram is shown in Fig.2.

Fig 1: Structure of Sodium Benzoate

November 2014 59Spinco Biotech

Application News

Table 2: Area response of Sodium Benzoate 100mg/L standard

System Precision DataSodium benzoate standard (100mg/L)

Injection RT (min) Area Response

The method is found to be linear between 2.0mg/L to 200mg/L ofsodium benzoate standard. The linearity graph was plotted betweenthe concentration and the area response of sodium benzoate. Thelinearity coefficient was found to be 1.0000.

Injection RT (min) Area Response1 3.55 29704352 3.55 29638293 3.55 29640314 3.55 29610785 3.55 29558796 3.56 2948774

Average 3.55 2960671STDEV 0 0041 7500 2104 3000000

4000000

5000000

6000000

7000000

Are

a R

esp

on

se

Linearity Graph - Sodium Benzoate

500000

750000

uV

Data6:LC_1109013.lcd UV:230nmData5:LC_1109012.lcd UV:230nmData4:LC_1109011.lcd UV:230nmData3:LC_1109010.lcd UV:230nmData2:LC_1109009.lcd UV:230nmData1:LC_1109008.lcd UV:230nm

% RSD = 0.25%

STDEV 0.0041 7500.2104%RSD 0.11 0.25

System Precision

Fig. 4 Linearity Graph of Sodium Benzoate

0

1000000

2000000

3000000

0.00 50.00 100.00 150.00 200.00 250.00

A

Conc.(ppm)

Correlation coef. (r)= 1.0000

Fig 2: Overlaid HPLC chromatogram of Sodium benzoate Standard 100mg/L (6 Replicate Injections)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

0

250000

g y p

The accuracy of the method is tested by spiking the sodiumbenzoate standard at 50%, 100%, 150% & QL level in grapeflavoured soft drink and found the recovery result between 96.92%to 99.64%.

The limit of detection and quantitation are derived from the residualstandard deviation & slope obtained from the linearity data. Thepredicted concentration of LOQ was prepared and proved for its

Table 3: The content of sodium benzoate in six replicatepreparation of grape flavoured soft drink

The grape flavoured soft drink was taken for this validation study.The %RSD for the content of sodium benzoate for the six replicatepreparation of grape flavoured soft drink was found to be 1.05%.The data and overlaid HPLC chromatograms are shown in Table 3and Fig.3 respectively.

Table 4: QL level Precision data

QL Level Precision data

SODIUM BENZOATE (2 17 QL l l t ti )

predicted concentration of LOQ was prepared and proved for itsprecision and accuracy. The %RSD for the six replicate injections isfound to be 0.28%. The area response is tabulated in table 4 and thecorresponding overlaid chromatograms are shown in Fig. 5

Sample Batch No.No. of

Preparation

Content of Sodium

Benzoate (% w/w)

Limit (% w/w)

Soft drink grape SD-001

1 0.0170

000%

2 0.0170

3 0.0170

SODIUM BENZOATE (2.17ppm, QL level concentration)

Injection Area response

1 62022

2 61808

3 62197

4 61727

5 62007

175000uV

Data3:LC 1109053 lcd UV:230nmData2:LC_1109052.lcd UV:230nmData1:LC_1109051.lcd UV:230nm

grape flavoured

SD 001

0.104 0.0171

5 0.0174

6 0.0172

% RSD = 1.05%

Sodium Benzoate

6 61827

Average 61931

STDEV 174.5699

%RSD 0.28%

15000

20000

uV

D t 6 LC 1209024 l d UV 230Data5:LC_1209023.lcd UV:230nmData4:LC_1209022.lcd UV:230nmData3:LC_1209021.lcd UV:230nmData2:LC_1209020.lcd UV:230nmData1:LC_1209019.lcd UV:230nm Sodium Benzoate

at QL level

% RSD =0 28%

0

25000

50000

75000

100000

125000

150000

Data6:LC_1109056.lcd UV:230nmData5:LC_1109055.lcd UV:230nmData4:LC_1109054.lcd UV:230nmData3:LC_1109053.lcd UV:230nm

% RSD =1.05%

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

0

5000

10000

15000 Data6:LC_1209024.lcd UV:230nm % RSD =0.28%

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

Fig 3: Overlaid HPLC chromatogram of sodium benzoate in grape flavoured soft drink (6 Replicate preparations)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

Fig. 5: Overlaid HPLC chromatogram of Sodium Benzoate at Quantitation Level(6 Replicate Injections)

Application News

Table 2: Area response of Sodium Benzoate 100mg/L standard

System Precision DataSodium benzoate standard (100mg/L)

Injection RT (min) Area Response

The method is found to be linear between 2.0mg/L to 200mg/L ofsodium benzoate standard. The linearity graph was plotted betweenthe concentration and the area response of sodium benzoate. Thelinearity coefficient was found to be 1.0000.

Injection RT (min) Area Response1 3.55 29704352 3.55 29638293 3.55 29640314 3.55 29610785 3.55 29558796 3.56 2948774

Average 3.55 2960671STDEV 0 0041 7500 2104 3000000

4000000

5000000

6000000

7000000

Are

a R

esp

on

se

Linearity Graph - Sodium Benzoate

500000

750000

uV

Data6:LC_1109013.lcd UV:230nmData5:LC_1109012.lcd UV:230nmData4:LC_1109011.lcd UV:230nmData3:LC_1109010.lcd UV:230nmData2:LC_1109009.lcd UV:230nmData1:LC_1109008.lcd UV:230nm

% RSD = 0.25%

STDEV 0.0041 7500.2104%RSD 0.11 0.25

System Precision

Fig. 4 Linearity Graph of Sodium Benzoate

0

1000000

2000000

3000000

0.00 50.00 100.00 150.00 200.00 250.00

A

Conc.(ppm)

Correlation coef. (r)= 1.0000

Fig 2: Overlaid HPLC chromatogram of Sodium benzoate Standard 100mg/L (6 Replicate Injections)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

0

250000

g y p

The accuracy of the method is tested by spiking the sodiumbenzoate standard at 50%, 100%, 150% & QL level in grapeflavoured soft drink and found the recovery result between 96.92%to 99.64%.

The limit of detection and quantitation are derived from the residualstandard deviation & slope obtained from the linearity data. Thepredicted concentration of LOQ was prepared and proved for its

Table 3: The content of sodium benzoate in six replicatepreparation of grape flavoured soft drink

The grape flavoured soft drink was taken for this validation study.The %RSD for the content of sodium benzoate for the six replicatepreparation of grape flavoured soft drink was found to be 1.05%.The data and overlaid HPLC chromatograms are shown in Table 3and Fig.3 respectively.

Table 4: QL level Precision data

QL Level Precision data

SODIUM BENZOATE (2 17 QL l l t ti )

predicted concentration of LOQ was prepared and proved for itsprecision and accuracy. The %RSD for the six replicate injections isfound to be 0.28%. The area response is tabulated in table 4 and thecorresponding overlaid chromatograms are shown in Fig. 5

Sample Batch No.No. of

Preparation

Content of Sodium

Benzoate (% w/w)

Limit (% w/w)

Soft drink grape SD-001

1 0.0170

000%

2 0.0170

3 0.0170

SODIUM BENZOATE (2.17ppm, QL level concentration)

Injection Area response

1 62022

2 61808

3 62197

4 61727

5 62007

175000uV

Data3:LC 1109053 lcd UV:230nmData2:LC_1109052.lcd UV:230nmData1:LC_1109051.lcd UV:230nm

grape flavoured

SD 001

0.104 0.0171

5 0.0174

6 0.0172

% RSD = 1.05%

Sodium Benzoate

6 61827

Average 61931

STDEV 174.5699

%RSD 0.28%

15000

20000

uV

D t 6 LC 1209024 l d UV 230Data5:LC_1209023.lcd UV:230nmData4:LC_1209022.lcd UV:230nmData3:LC_1209021.lcd UV:230nmData2:LC_1209020.lcd UV:230nmData1:LC_1209019.lcd UV:230nm Sodium Benzoate

at QL level

% RSD =0 28%

0

25000

50000

75000

100000

125000

150000

Data6:LC_1109056.lcd UV:230nmData5:LC_1109055.lcd UV:230nmData4:LC_1109054.lcd UV:230nmData3:LC_1109053.lcd UV:230nm

% RSD =1.05%

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

0

5000

10000

15000 Data6:LC_1209024.lcd UV:230nm % RSD =0.28%

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

Fig 3: Overlaid HPLC chromatogram of sodium benzoate in grape flavoured soft drink (6 Replicate preparations)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

Fig. 5: Overlaid HPLC chromatogram of Sodium Benzoate at Quantitation Level(6 Replicate Injections)

grape flavoured soft drink was found to be 1.05%. The data and overlaid HPLC chromatograms are shown in Table 3 and Fig.3 respectively.

The method is found to be linear between 2.0mg/L to 200mg/L of sodium benzoate standard. The linearity graph was plotted between the concentration and the area response of sodium benzoate. The linearity

Application Brief

Food

Saf

ety

HPLC

Shimadzu “Prominence-i” evaluates the foodpreservative-sodium benzoate in soft drinks

Application News

A rapid conventional HPLC method is described for the determination of food preservative-sodium benzoate in soft drinks. The methodutilizes the new integrated Shimadzu HPLC Prominence-i LC-2030C.

In today’s world, the role of food additives becomes inevitable due to the increase in the production of treated and ready to eat conveniencefoods. Food additives are intentionally added to the food stuffs and must be safe for lifetime consumption. They are used for the purpose ofmaintaining or improving the quality, texture, consistency, appearance of the food products. Food additives are classified on the basis of theirfunctional use, in which the preservatives are most important additive and are used to prevent and retard the microbial spoilage of food.Preservatives are classified into two types; they are Class I and Class II.

The Class I preservatives are common salt, sugar, glucose, dextrose, spices, vinegar, honey, etc. The Class II preservatives are naturalidentical / synthetic compounds like salts of benzoic acid, sorbic acids, lactic acid, etc. The addition of Class I preservatives are not restrictedin food products, but the class II preservatives are Generally Recognized As Safe (GRAS) and are listed in FDA GRAS food substances. It isnoted that, the benzoic acid and sodium benzoate may be used as antimicrobial and flavouring agents in food products. The limit of benzoicacid / sodium benzoate as per FDA is 0.1% in food products.

Though the legislations are permitting these class II preservatives in food products, their use demands special care. In this context, a rapid andsimple HPLC method has been developed and demonstrated in this article by IICMS using the new integrated Shimadzu HPLC ‘Prominence-i’ LC-2030C to quantitatively determine the content of sodium benzoate in grape, lemon and orange flavoured soft drinks.

Experimental

Preparation of Sodium benzoate standard

O ONa

Prepared the concentration of 100mg/L sodium benzoate indiluent and used as working standard

Preparation of Sample solutionThe grape, lemon & orange flavoured soft drink samples withClass-II preservative were selected in this study. These softdrink samples were degassed thoroughly in ultrasonic bath for20min. The sample aliquot of 0.5g of soft drink was transferredto 5mL volumetric flask & made up to the mark with diluent and

Results and Discussion

The sodium benzoate standard solution and the simplydiluted soft drink samples were injected directly into theProminence-i LC-2030C chromatography system withoutany complex sample preparation and recorded theresults The retention time of sodium benzoate is

Fig 1: Structure of Sodium Benzoate

3.0

mV(x100)Ch1 (230nm)

to 5mL volumetric flask & made up to the mark with diluent andsonicated to mix well.

results. The retention time of sodium benzoate isobserved about in 3.5min. The typical HPLCchromatogram of Sodium benzoate standard 100mg/Lconcentration is shown below:

Table 1: Instrument and Analytical Conditions

HPLC Prominence-i LC-2030C

Column C-6 Phenyl Hexyl (50x4.6mm, 3µ)

HPLC Chromatogram of Sodium Benzoate standard 100mg/L

3.55

min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

0.0

1.0

2.0Workstation Lab solution Version 5.62 P

Flow Rate 0.7mL/min (Isocratic)

Mobile Phase10mM Ammonium formate, pH adjusted to 3.0 with dilute formic acid : Acetonitrile (75:25)

Diluent Water : Acetonitrile (75:25) The method is tested for the HPLC validation parameters andWavelength 230nm

Injection Volume 5 µL Run Time 7.00 minutes

found to be precise, linear and accurate. The %RSD of arearesponse for the six replicate sodium benzoate standardinjections is found to be 0.25%. The area response is tabulatedin Table 2 and the corresponding overlaid standardchromatogram is shown in Fig.2.

HPLC chromatogram of sodium benzoate standard 100mg/L concentration is shown below:

The method is tested for the HPLC validation parameters and found to be precise, linear and accurate. The %RSD of area response for the six replicate sodium benzoate standard injections is found to be 0.25%. The area response is tabulated

Application News

Table 2: Area response of Sodium Benzoate 100mg/L standard

System Precision DataSodium benzoate standard (100mg/L)

Injection RT (min) Area Response

The method is found to be linear between 2.0mg/L to 200mg/L ofsodium benzoate standard. The linearity graph was plotted betweenthe concentration and the area response of sodium benzoate. Thelinearity coefficient was found to be 1.0000.

Injection RT (min) Area Response1 3.55 29704352 3.55 29638293 3.55 29640314 3.55 29610785 3.55 29558796 3.56 2948774

Average 3.55 2960671STDEV 0 0041 7500 2104 3000000

4000000

5000000

6000000

7000000

Are

a R

esp

on

se

Linearity Graph - Sodium Benzoate

500000

750000

uV

Data6:LC_1109013.lcd UV:230nmData5:LC_1109012.lcd UV:230nmData4:LC_1109011.lcd UV:230nmData3:LC_1109010.lcd UV:230nmData2:LC_1109009.lcd UV:230nmData1:LC_1109008.lcd UV:230nm

% RSD = 0.25%

STDEV 0.0041 7500.2104%RSD 0.11 0.25

System Precision

Fig. 4 Linearity Graph of Sodium Benzoate

0

1000000

2000000

3000000

0.00 50.00 100.00 150.00 200.00 250.00

A

Conc.(ppm)

Correlation coef. (r)= 1.0000

Fig 2: Overlaid HPLC chromatogram of Sodium benzoate Standard 100mg/L (6 Replicate Injections)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

0

250000

g y p

The accuracy of the method is tested by spiking the sodiumbenzoate standard at 50%, 100%, 150% & QL level in grapeflavoured soft drink and found the recovery result between 96.92%to 99.64%.

The limit of detection and quantitation are derived from the residualstandard deviation & slope obtained from the linearity data. Thepredicted concentration of LOQ was prepared and proved for its

Table 3: The content of sodium benzoate in six replicatepreparation of grape flavoured soft drink

The grape flavoured soft drink was taken for this validation study.The %RSD for the content of sodium benzoate for the six replicatepreparation of grape flavoured soft drink was found to be 1.05%.The data and overlaid HPLC chromatograms are shown in Table 3and Fig.3 respectively.

Table 4: QL level Precision data

QL Level Precision data

SODIUM BENZOATE (2 17 QL l l t ti )

predicted concentration of LOQ was prepared and proved for itsprecision and accuracy. The %RSD for the six replicate injections isfound to be 0.28%. The area response is tabulated in table 4 and thecorresponding overlaid chromatograms are shown in Fig. 5

Sample Batch No.No. of

Preparation

Content of Sodium

Benzoate (% w/w)

Limit (% w/w)

Soft drink grape SD-001

1 0.0170

000%

2 0.0170

3 0.0170

SODIUM BENZOATE (2.17ppm, QL level concentration)

Injection Area response

1 62022

2 61808

3 62197

4 61727

5 62007

175000uV

Data3:LC 1109053 lcd UV:230nmData2:LC_1109052.lcd UV:230nmData1:LC_1109051.lcd UV:230nm

grape flavoured

SD 001

0.104 0.0171

5 0.0174

6 0.0172

% RSD = 1.05%

Sodium Benzoate

6 61827

Average 61931

STDEV 174.5699

%RSD 0.28%

15000

20000

uV

D t 6 LC 1209024 l d UV 230Data5:LC_1209023.lcd UV:230nmData4:LC_1209022.lcd UV:230nmData3:LC_1209021.lcd UV:230nmData2:LC_1209020.lcd UV:230nmData1:LC_1209019.lcd UV:230nm Sodium Benzoate

at QL level

% RSD =0 28%

0

25000

50000

75000

100000

125000

150000

Data6:LC_1109056.lcd UV:230nmData5:LC_1109055.lcd UV:230nmData4:LC_1109054.lcd UV:230nmData3:LC_1109053.lcd UV:230nm

% RSD =1.05%

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

0

5000

10000

15000 Data6:LC_1209024.lcd UV:230nm % RSD =0.28%

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

Fig 3: Overlaid HPLC chromatogram of sodium benzoate in grape flavoured soft drink (6 Replicate preparations)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

Fig. 5: Overlaid HPLC chromatogram of Sodium Benzoate at Quantitation Level(6 Replicate Injections)

in Table 2 and the corresponding overlaid standard chromatogram is shown in Fig.2.

The grape flavoured soft drink was taken for this validation study. The %RSD for the content of sodium benzoate for the six replicate preparation of