Separation and Determination of Phenolic Antioxidants by HPLC with Surfactant/n-Propanol Mobile Phases

ngel Aparicio, Mar A a Paz San Andre s*, Soledad Vera

Departamento de Qu mica Anal tica, Facultad de Qu mica, Universidad de Alcala , 28871 Alcala de Henares, Madrid, Spain Ms received: September 21, 1999; accepted: January 28, 2000

Key Words: Antioxidants; propyl gallate; octyl gallate; dodecyl gallate; butylated hydroxyanisole; butylated hydroxytoluene; reverse-phase liquid chromatography Summary
The influence of cationic and anionic surfactants and short-chain alcohols in the mobile phase on the retention of five antioxidants has been studied. The solutes chosen were butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl, octyl, and dodecyl gallates (PG, OG, DG).The surfactants were hexadecyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), and n-propanol (PrOH) was the selected alcohol. A simple isocratic reversed-phase method for the antioxidant determination is proposed. Separation of five primary antioxidants takes 18 min with the mobile phase SDS 0.10 M/H3PO4 0.01 M/PrOH 30%. Variation of the percentage of alcohol in the mobile phase permits optimization of the retention times of the antioxidants. Detection limits in the pg range were obtained for the all solutes. The method was used to determine the antioxidants in olive oil at three different levels, giving mean recoveries close to 100% for all the solutes (BHA 102%, PG 99%, OG 99%, DG 99%) except BHT (84%).

phases in HPLC because slight variations in the amounts of the components may result in phase separation. In previous work we used mobile phases consisting of a surfactant with a high percentage of a short-chain alcohol, e.g. n-propanol and ethanol, for the separation of inorganic complexes and polycyclic aromatic hydrocarbons, in isocratic reversed-phase with very good results [36 39]. In this paper, we present a study of the influence of cationic and anionic surfactant/n-propanol/water systems as mobile phases in the determination of five synthetic antioxidants. The method is applied to their determination in olive oil. 2 Experimental 2.1 Reagents

1 Introduction Numerous publications address the determination of antioxidants added to oils, fats, or food products. Different types exist, but they are mainly phenolic substances, such as propyl, octyl, and dodecyl gallates (PG, OG, DG), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) [1, 2]. Several reviews on the analytical determination of antioxidants have been published [3 5], and the field has attracted particular interest in recent years [6 18]. High performance liquid chromatography (HPLC) is one of the techniques often used for the separation and determination of antioxidants [19 24]. In 1980, Armstrong and Henry [25] first demonstrated the usefulness of replacing organic modifiers in reversed-phase liquid chromatography with an aqueous micellar solution. Named micellar liquid chromatography (MLC), its popularity is demonstrated by the large number of published papers [26 31]. In many cases, the presence of an organic modifier, such as a short or medium chain alcohols, is necessary to reduce the retention times and to improve efficiency [32 34]. In pure aqueous surfactant solutions or in the presence of low concentrations of an organic modifier (a 15% v/v) normal micelles are present, but also aggregates of surfactant/water/ alcohol with the latter as dominant component; when the alcohol chain contains six or more carbon atoms, the aggregates formed are the reversed micelles; for short-chain alcohols it is not possible to distinguish the type of aggregate [35]. Reverse micelles present many problems as mobile

All reagents were of analytical-reagent grade. Butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), propyl gallate (PG), and octyl gallate (OG) were obtained from Fluka (Buchs, Switzerland) and dodecyl gallate (DG) from Aldrich (Alcobendas, Madrid, Spain). The surfactants, sodium dodecyl sulfate (SDS) and hexadecyltrimethylammonium bromide (CTAB), n-propanol, and phosphoric acid were from Merck (Darmstadt, Germany). Stock solutions of the antioxidants were prepared by diluting the appropriate quantity in the mobile phase. Ultrapure water used was obtained from a Millipore Milli-Q system (Milford, USA). All the mobile phases were filtered (0.45 lm) and degassed in an ultrasonic bath. Ethanol, hexane, and petroleum ether were from Panreac (Barcelona, Spain). Acetonitrile and methanol were from Sharlau (Barcelona, Spain). 2.2 Apparatus A liquid chromatograph with a binary pump (Perkin-Elmer model 250), a programmable absorbance detector (Applied Biosystems model 785 A), and an injection valve (Rheodyne), with an injection volume of 20 lL, was used. The system is connected to a 486 personal computer running chromatographic software (Perkin-Elmer Turbochrom 4.0). A reversed-phase column (15063.9 mm i. d.; particle size 10 lm; Lichrosorb RP-18) (Sugelabor, Madrid, Spain) was used in all HPLC runs.
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Separation and Determination of Phenolic Antioxidants by HPLC

2.3 Procedure The mobile phase consisted of the surfactant SDS or CTAB, H3PO4 0.01 M for conversion of the phenolic antioxidants into the protonated form, and n-propanol. The mobile phases were prepared by weighing the necessary amounts of the surfactant (concentrations between 0.03 and 0.25 M), adding phosphoric acid for adjustment to pH = 2, and dissolving them in a mixture of n-propanol and ultrapure water at alcohol percentages from 20 to 50% (v/v). All mobile phases were filtered and degassed in an ultrasonic bath for 20 min before use. The variation of the retention factor, k, was studied as a function of surfactant concentration and n-propanol percentage. Detection was carried out by UV-visible spectrophotometry at the optimum wavelengths. These were: BHA 289 nm, BHT 277 nm, PG 272 nm, OG 272 nm, and DG 271 nm. The chromatogram of the mixture of the five antioxidants was recorded at 290 nm. 2.4 Sensitivity and Detection Limits The calibration method relating the height of the chromatographic peak with the injected quantity of analyte has been applied to determine linear range, sensitivity, and limits of detection. This was done using a mobile phase containing 0.1 M SDS, H3PO4, and 30% n-propanol (v/v). The amounts of antioxidant injected ranged from 3.6 ng to 27.0 lg BHA, from 110.2 ng to 154.2 lg BHT, from 63.7 pg to 5.1 ng PG, from 2.8 ng to 14.1 lg OG, and from 3.4 ng to 20.3 lg DG. The sensitivity is taken as the slope of the calibration curve and the detection limit was determined as the amount of analyte that yields a signal equal to three times the background noise measured as the height of the difference between the lower and higher values of the signal. 2.5 Determination of Antioxidants in Oil Samples The potential of the method for the determination of the five antioxidants in oil samples containing no preservatives was studied under the optimum experimental conditions. The oil sample (Koipe, Spain) was purchased in a local market. Different amounts of the antioxidants were added to 5 g of the samples. Two different extraction processes were carried out: for the antioxidant BHT, each sample was dissolved in 50 mL of hexane and the analyte was extracted with 5630 mL of acetonitrile for 3 min. The extracts were collected and acetonitrile was evaporated to dryness using a rotary evaporator on a water-bath at f 40 8C. The residue was dissolved in 10 mL methanol. An aliquot of this solution diluted in mobile phase was injected into the chromatographic system. For the other four antioxidants, each sample was dissolved in 25 mL of petroleum ether and the analytes were extracted with 3612.5 mL of EtOH (72%, v/v) for 3 min and with 30 mL
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for 1 min. The extracts were combined, filtered through a 0.45 lm glass fiber filter and diluted to 100 mL with the same EtOH (72%, v/v). An adequate aliquot diluted in mobile phase was injected into the chromatographic system. Five replicates were performed in each case. 3 Results and Discussion 3.1 Retention of the Antioxidants in the Chromatographic System The surfactant solubilizes the antioxidants in aqueous medium and n-propanol allows reduction of the retention times. Figure 1 shows the variation of retention factor, k, for the mobile phases containing CTAB as surfactant (0.03 to 0.25 M) and for the different percentages of n-propanol (20% to 50%, v/v). An increase in the surfactant concentration entails a decrease in retention time which is more marked at the lower concentrations of the surfactant. However, a mobile phase composition allowing separation of the five antioxidants was not found. Figure 2 shows a similar experiment but with SDS as surfactant. The behavior is the same as in CTAB, with respect to the variation of k with surfactant concentration and n-propanol percentage. However, in this case it was possible to find a mobile phase composition (SDS 0.1 M/H3PO4 0.01 M/PrOH 30% v/v), at which the separation of the five antioxidants is achieved in 18 min in isocratic elution mode, as shown in Figure 3 (k = 290 nm). Good resolution is observed in all cases, except for the peaks corresponding to OG and BHA which exhibited a resolution 0.91 calculated according to Snyder [42] for moderately overlapping bands. Based on the retention factor profile shown in Figure 2 the mobile phase composition can be modified to optimize the retention time of each solute, and to perform quantitative isocratic reversed-phase determinations on samples containing one or two antioxidants. Among the various possibilities of modifying the mobile phase composition exist, we chose to maintain a constant concentration of SDS (0.1 M) and to vary the percentage of n-propanol. 3.2 Analytical Characteristics of the Method The analytical characteristics of the method are summarized in Table 1 and Table 2. Several points warrant mention. The linear range obtained for each antioxidant is independent of the mobile phase and wavelength used, with very good correlation coefficients being obtained in all cases. With regard to the limits of detection, there are differences between the values obtained with the mobile phase of Table 1, and those optimized for individual solutes listed in Table 2. The main differences are for BHT, where the detection limit falls from 5.12 pg with a mobile phase composition
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Figure 1. Variation of the retention factor, k, with the CTAB concentration in the mobile phase in presence of: (1) PrOH 20% v/v; (2) PrOH 30% v/v; (3) PrOH 40% v/v; (4) PrOH 45% v/v, and (5) PrOH 50% v/v for 0 BHA, h BHT; * PG, g OG, and j DG. Flow: 1 mL N min 1; sample injected: 20 lL.

Figure 2. Variation of the retention factor, k, with the SDS concentration in the mobile phase in presence of: (1) PrOH 20% v/v; (2) PrOH 30% v/v; (3) PrOH 40% v/v; (4) PrOH 45% v/v and (5) PrOH 50% v/v for 0 BHA, h BHT; * PG, g OG, and j DG. Flow: 1 mL N min 1; sample injected: 20 lL.

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Separation and Determination of Phenolic Antioxidants by HPLC

Table 3. Determination of antioxidants in oil samples. The conditions are the same of the Table 2. Antioxidant Spiked, mg N kg 1 54.1 198.3 432.6 88.1 198.3 440.7 29.7 212.2 424.4 56.5 197.6 395.3 67.7 203.1 473.8 Found, mg N kg 1 l SDa) 57.2 l 0.9 207.8 l 2.9 416.3 l 9.4 75.4 l 1.3 165.5 l 2.7 352.1 l 12.9 30.0 l 0.5 202.0 l 1.6 426.9 l 9.9 56.0 l 0.5 182.7 l 2.7 415.1 l 8.5 64.6 l 1.9 214.7 l 6.2 463.3 l 7.4 % Recovery l SDa) 105.7 l 1.6 104.8 l 1.4 96.2 l 2.2 85.5 l 1.4 83.4 l 1.3 83.0 l 3.5 100.9 l 1.8 95.3 l 0.7 100.6 l 2.3 99.1 l 0.9 92.5 l 1.4 105.0 l 2.1 95.7 l 1.8 105.7 l 3.0 97.9 l 1.8

BHA

BHT

PG

OG

DG

a)

SD is the standard deviation, n = 5.

Figure 3. Chromatogram corresponding to the separation of the antioxidants, with the retention times (min). PG (1.68); OG (3.44); BHA (4.09); DG (6.30) and BHT (16.08). Mobile phase: SDS 0.10 M/H3PO4 0.01 M/PrOH 30% v/v. Flow: 1 mL N min 1, amount injected 20 lL. Table 1. Analytical characteristics of the antioxidant determination by HPLC with a mobile phase: SDS 0.1 M/H3PO4 0.01 M/PrOH 30% v/v, k = 290 nm. Flow: 1 mL min 1. Sample injected: 20 lL. Antioxidant Linear range Slope (mV/ ng N mL1) 0.6375 0.0974 3.5788 1.4077 0.7563 r D. L.a), pg

SDS 0.10 M/H3PO4 0.01 M/PrOH 30% (v/v). The low detection limits found in the mobile phases, SDS 0.10 M/H3PO4 0.01 M/PrOH x% v/v indicate that the proposed method offers very good chromatographic performance. 3.3 Determination of Antioxidants in Olive Oil The antioxidants were determined in a sample of olive oil spiked with three different amounts of the analytes. The amounts added were below and above the levels issued by the European Union [41]. The analyses were carried out using best mobile phase composition for each antioxidant as listed in Table 2. Table 3 shows the amounts of antioxidant added and found, as well as the percentage recovery, as the mean values of five replicates for the three levels of addition. Except for BHT, where the mean recovery is 84%, the average recovery for the other solutes is close to 100%: BHA 102%, PG 99%, OG 99% and DG 99%.

BHA BHT PG OG DG
a)

3.6 ng 27.0 lg 110.2 ng 154.2 lg 63.7 pg 5.1 lg 2.8 ng 14.1 lg 3.4 ng 20.3 lg

0.9999 0.9999 0.9999 0.9999 0.9999

0.67 5.12 0.14 0.31 0.33

D.L. is the detection limit.

of SDS 0.10 M/H3PO4 0.01 M/PrOH 30% (v/v) to 1.40 pg in presence of n-propanol 50%, and for propyl gallate, where the smallest value is found in the separation phase

Table 2. Analytical characteristics of the antioxidant HPLC determination with the optimum mobile phase for each antioxidant: SDS 0.1 M/H3PO4 0.01 M and different percentages of PrOH in each case. Flow: 1 mL N min 1. Sample injected: 20 lL. Antioxidant % PrOH (v/v) k, nm tr, min Linear Range Slopea) l SD (mv/ng1 N mL) 1.05 l 0.03 0.35 l 0.01 2.2 l 0.1 1.07 l 0.04 0.83 l 0.01 r D.L.b), pg

BHA BHT PG OG DG
a) b)

45 50 20 30 45

289 277 272 272 271

2.37 5.55 2.70 3.44 3.53

3.6 ng 27.0 lg 110.2 ng 154.2 lg 63.7 pg 5.1 lg 2.8 ng 14.1 lg 3.4 ng 20.3 lg

0.9999 0.9995 0.9996 0.9998 0.9995

0.41 1.42 0.23 0.40 0.30

Mean value for 4 determinations. D.L. is the detection limit.

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The recoveries are similar to those obtained for another samples, such as corn oil, biscuits, soups, margarine, or several oil types, by various methods [7, 9, 12], and superior to those obtained by liquid chromatography [24] using modification of the Gertz and Herrmann method [42], i. e. extraction with acetonitrile-isopropanol (50 : 50, v/v) giving very low recoveries. 4 Conclusions HPLC determination of antioxidants with mobile phases containing a surfactant, an alcohol such as n-propanol, and the acid H3PO4 gives very good analytical results. SDS gives better results than CTAB (which is more toxic than SDS). Separation of the five antioxidants studied is possible in 18 min with a mobile phase composition SDS 0.10 M/H3PO4 0.01 M/PrOH 30% v/v in isocratic reverse-phase. Under these conditions, the n-propanol content can be modified to optimize the retention time for each antioxidant. Independently of the n-propanol percentage in the mobile phase, the analyses show a good linear range, and sensitivities and limits of detection in the pg range. The method has been used to determine antioxidants in olive oil at three concentration levels. The percentage recoveries are very close to 100% all cases except BHT. References
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