Analysis of Antioxidants in Bakery Products following the GB 5009.32-2016 Method

Dean Duan, Senior Scientist
Merck R&D APAC Lab, Shanghai, China

Abstract

The Discovery^® HS C18 column was used to separate the 9 antioxidants propyl gallate (PG), 1-(2,4,5-trihydroxyphenyl)-1-bunanone (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), butylated hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxybenzyl alcohol (Ionox-100), octyl gallate (OG), butylated hydroxytoluene (BHT) and dodecyl/lauryl gallate (DG) in pastries following the Chinese national standard GB 5009.32-2016 method for determination of 9 antioxidants in food. The here-developed method using a Discovery^® HS C18 column fulfills the system suitability criteria of the GB 5009.32-2016 method and exceeds the sensitivity criteria.

Section Overview

• Introduction
• Experimental
• Results & Discussion
• Conclusion
• References

Introduction

Food antioxidants are additives that prevent or delay the oxidative deterioration of food to improve its stability and extend its storage period. Within the permitted levels for normal consumption, these antioxidants do not pose a health risk. However, individuals who are allergic or have asthma may develop hypersensitive reactions over time.¹ The permissible limits for antioxidants vary for different foods in the different countries.²

The Chinese national standard GB 5009.32-2016 method³ describes a C18 HPLC column for the antioxidant measurement. Following this, a Discovery^® HS C18 column was used for the here described analysis of 9 antioxidants (Figure 1), propyl gallate (PG), 1-(2,4,5-trihydroxyphenyl)-1-bunanone (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), butylated hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxybenzyl alcohol (Ionox-100), octyl gallate (OG), butylated hydroxytoluene (BHT) and dodecyl/lauryl gallate (DG), in a bread roll sample.

Figure 1. Structures of food antioxidant compounds investigated in this study.

Experimental

Standard Preparation

• Antioxidants stock solution (1 mg/mL each): Weigh 10 mg of each of the 9 antioxidants into a 10 mL brown volumetric flask. Add ~8 mL of acetonitrile and sonicate for 5 minutes. Top-up to mark with acetonitrile and mix well.
• Standard solutions for external calibration: Dilute stock solution to 1, 2, 5, 10, 20, 50, 100 and 200 µg/mL with acetonitrile.

Sample Preparation

Saturated solvent solutions: Take 200 mL of acetonitrile and 200 mL hexane into a 500 mL separating funnel. Cap and shake the funnel vigorously for 1 minute and allow it to stand for 5 minutes. Separate the solvents by draining the lower layer (hexane saturated acetonitrile) into a beaker followed by the upper layer (acetonitrile saturated hexane) into another beaker.

• Cut the pastry sample into smaller pieces and grind it into a paste or powder.
• Weigh 1 g (accurate to 0.01 g) of sample into a 50 mL centrifuge tube. For spiked samples, add appropriate quantities of the mixed antioxidants stock solution to the sample and vortex for 1 minute.
• Add 5 mL of a hexane solution saturated with acetonitrile to the sample, vortex for 1 minute, and stand for 10 minutes.
• Add 5 mL of saturated sodium chloride solution to the above.
• Add 5 mL of acetonitrile solution saturated with hexane to the above. Vortex for 2 minutes and centrifuge at 3000 rpm for 5 minutes. Transfer the acetonitrile layer (middle layer) to a 150 mL distillation flask.
• Repeat extraction twice with a 5 mL acetonitrile solution saturated with hexane. Collect all extracts into the same distillation flask.
• Evaporate the extract to dryness using a rotary evaporator under nitrogen in a 40 °C water bath.
• Dissolve the residue in 2 mL of acetonitrile. Filter through a 0.22 μm membrane before HPLC analysis (Table 1).

HPLC-UV Analysis

Table 1. HPLC conditions used for antioxidant analysis

LC Conditions
Column:	Discovery® HS C18, 5 μm, 25 cm × 4.6 mm I.D. (568523-U)
Mobile phase:	[A] 0.5% Formic acid in water; [B] methanol
Gradient:	Time (min)	A%	B%
	0.00	50.0	50.0
	5.00	50.0	50.0
	15.00	20.0	80.0
	20.00	20.0	80.0
	25.00	10.0	90.0
	27.00	50.0	50.0
	30.00	50.0	50.0
Flow rate:	1.0 mL/min
Pressure:	2550 psi
Column temp.:	35 °C
Detector:	UV, 280 nm
Injection:	10 µL
Sample:	Acetonitrile extract and standards

Method Suitability, System Suitability, Calibration

Acceptance Criteria as described in GB 5009.32-2016

• Linear correlation coefficient: R² > 0.99
• Method recovery: 80 to 120%
• LOQ <20 mg/kg

 

Results & Discussion

Chromatographic results for a standard injection, a pastry sample and a spike pastry sample are displayed in the Figures 2-4. The specificity/retention times of the 9 antioxidants is shown in Table 2. The calibration curve for propyl gallate (PG) is displayed as example in the Figure 5. Chromatographic data of the pastry sample spiked with 9 antioxidants at 20 mg/kg (Figure 4) is shown in Table 3. The repeatability (n=5) for a standard solution at 20 µg/mL each compound was between 0.19 and 0.51 %RSD (Table 4). The external calibration linearity data from 1 to 200 µg/mL showed R² values >0.999 (Table 5). The % recovery stayed withing the required 80-120% range (Table 6). Table 7 compares the LOD and LOQ values of this method and the GB 5009.32-2016 method, showing the developed method exceeds the criteria. The determination of antioxidants with an actual bread roll sample sourced from a local store is shown in Table 8. In the investigated unspiked sample a BHA content of 4.82 mg/kg was determined.

Figure 2. Mixed standard solution of 9 antioxidants, 20 μg/mL. For Peak IDs see Table 2.

Table 2. Chromatographic data of the 9 antioxidants standard solution at 20 μg/mL

Peak ID	Compound	Abbreviation	Retention Time (min)	RRT	Resolution	Tailing Factor
1	Propyl gallate	PG	5.9	1.00	--	0.86
2	1-(2,4,5-Trihydroxyphenyl)-1-bunanone	THBP	9.0	1.53	10.7	0.97
3	tert-Butylhydroquinone	TBHQ	9.6	1.63	2.1	0.97
4	Nordihydroguaiaretic acid	NDGA	15.2	2.59	27.9	0.96
5	Butylated hydroxyanisole	BHA	16.2	2.75	6.7	0.94
6	3,5-Di-tert-butyl-4-hydroxybenzyl alcohol	Ionox-100	18.1	3.08	14.2	0.94
7	Octyl gallate	OG	19.0	3.23	6.5	0.96
8	Butylated hydroxytoluene	BHT	28.0	4.76	51.7	0.93
9	Dodecyl gallate	DG	28.4	4.82	1.8	0.94

Figure 3. Chromatogram of an unspiked pastry sample (bread roll).

Figure 4. Spiked pastry sample (bread roll) with 9 antioxidants at 20 mg/kg.

Table 3. Chromatographic data of pastry sample spiked with the 9 antioxidants at 20 mg/kg

Peaks	Compound	Retention Time (min)	Resolution	Tailing Factor
1	PG	5.9	5.5	0.87
2	THBP	9.0	6.0	0.95
3	TBHQ	9.5	2.1	0.95
4	NDGA	15.2	27.6	0.95
5	BHA	16.2	5.8	0.95
6	Ionox-100	18.1	12.4	0.92
7	OG	19.0	6.3	0.94
8	BHT	27.9	17.0	0.92
9	DG	28.3	1.7	0.93

Calibration Data

Calibration in the range of 1-200 µg/mL with 8 standard solutions provided linear calibration curves with R² values between 0.99958 and 0.99996 (Table 5) meeting the method requirement of R² > 0.99. As example, the curve for PG is given in Figure 5.

Table 5. External calibration linearity of 9 antioxidants, 8 calibrants each (except BHT with 7 calibrants)

Compound	Range (µg/mL)	Linear equation	R2
PG	1 - 200	y = 24,922.61009x - 11,621.71451	0.99991
THBP	1 - 200	y = 37,473.03828x - 50,168.98139	0.99969
TBHQ	1 - 200	y = 8,268.98088x - 6,319.57287	0.99994
NDGA	1 - 200	y = 11,848.51640x - 11,774.29529	0.99958
BHA	1 - 200	y = 7,790.82192x - 4,247.73808	0.99992
Ionox-100	1 - 200	y = 2,896.25484x - 2,062.23462	0.99980
OG	1 - 200	y = 20,953.38489x - 15,011.41737	0.99979
BHT	2 - 200	y = 4,156.59672x - 3,457.70460	0.99988
DG	1 - 200	y = 16,748.24953x - 4,498.60232	0.99996

Figure 5. Calibration curve of PG shown as example.

Repeatability

The repeatability for the 9 analytes was between 0.19 to 0.51 %RSD (n=5) for the 20 µg/mL standard solution (Table 5).

Table 4. Repeatability (n=5) of 9 antioxidants standard solution at 20 µg/mL

Compound	Mean Area	Std Dev	% RSD
PG	486621	1405	0.29
THBP	726323	3197	0.44
TBHQ	156435	796	0.51
NDGA	228485	745	0.33
BHA	152404	307	0.20
Ionox-100	56983	108	0.19
OG	411556	1551	0.38
BHT	80597	308	0.38
DG	325426	1323	0.41

Recovery Data    

For spiked samples (20 µg/kg) for the 9 analytes, recovery ranged from 77.2 to 115.4% and %RSD for triplicate measurement from 1.40 to 4.12 % (Table 6). Regarding the method criteria for recovery (80 to 120%) only TBHQ showed with 77.2% a recovery slightly below the limit.

Table 6. % recovery for 9 antioxidants upon cleanup extraction for a bread roll sample spiked at 20 mg/kg

Compound	Recovery (%)	% RSD (n=3)
PG	101.1	2.30
THBP	107.9	1.40
TBHQ	77.2	2.80
NDGA	98.0	0.55
BHA	115.4	2.00
Ionox-100	106.6	1.90
OG	90.8	3.30
BHT	85.7	4.12
DG	80.8	2.02

Method Sensitivity

The method showed good sensitivities exceeding the requirement of the GB method (Table 7). Determined LODs were <1.1 mg/kg and LOQs of <3.07 mg/kg, exceeding the methods criteria of 20 mg/kg.

Table 7. LOD and LOQ of method against GB method requirements

	Developed Method		GB requirement
Compound	LOD (mg/kg)	LOQ (mg/kg)	LOD (mg/kg)	LOQ (mg/kg)
PG	0.93	2.80	<2	<20
THBP	1.01	3.07	<4	<20
TBHQ	0.55	1.68	<10	<20
NDGA	0.46	1.39	<4	<20
BHA	0.35	1.07	<10	<20
Ionox-100	0.68	2.06	<20	<20
OG	0.33	1.00	<2	<20
BHT	0.80	2.42	<4	<20
DG	0.48	1.44	<10	<20

Sample measurement

For the investigated unspiked pastry sample only BHA was detected at 4.82 mg/kg (Table 8).

Table 8. Results of antioxidants detected in the unspiked pastry sample

Compound	Amount (mg/kg)
PG	<LOD
THBP	<LOD
TBHQ	<LOD
NDGA	<LOD
BHA	4.82
Ionox-100	<LOD
OG	<LOD
BHT	<LOD
DG	<LOD

Conclusion

The Discovery^® HS C18 column could separate the 9 antioxidants in the pastry matrix efficiently with excellent peak shapes. The shown method meets the acceptance criteria of GB 5009.32-2016 regarding recovery (except for TBHQ) and exceeds the sensitivity requirement with lower LOD and LOQ values.

See more applications for Food & Beverage testing.

References

1. Food Safety Focus: Should We Worry about Preservatives and Antioxidants in Food – Or Should We Not?. [Internet]. Centre for Food Safety, The Government of the Hong Kong Special Administrative Region.[cited 31 Dec 2022]. Available from: https://www.cfs.gov.hk/english/multimedia/multimedia_pub/multimedia_pub_fsf_199_02

2. WHO Food Additives Series: 42. [Internet]. WHO INCHEM. International Programme on Chemical Safety– Safety Evaluation of Certain Food Additives. .[cited 31 Dec 1998]. Available from: https://www.inchem.org/documents/jecfa/jecmono/v042je24.htm

3. Determination of nine antioxidants. [Internet]. GB 5009.32-2016 .[cited 31 Dec 2022]. Available from: https://www.gbstandards.org/China_standard_english.asp?code=GB%205009.232-2016