Determination of olive oil 2-thiobarbituric acid reactive substances by parallel flow injection

Analytica Chimica Acta 417 (2000) 119–124

Determination of olive oil 2-thiobarbituric acid reactive substances by parallel flow injection Panayotis G. Nouros, Constantinos A. Georgiou∗ , Moschos G. Polissiou Chemistry Laboratory, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece Received 26 November 1999; received in revised form 7 April 2000; accepted 10 April 2000

Abstract A flow injection system incorporating multiple incubation coils is used for the automated determination of olive oil 2-thiobarbituric acid reactive substances (TBARS). The use of a laboratory-made parallel flow injection (PA-FI) analyser allows the simultaneous incubation of 10 samples. Olive oil samples are injected in a 2.0×10−2 M 2-thiobarbituric acid (TBA) stream in n-propanol and then acidified by merging with a 0.10 M trichloroacetic acid stream in n-propanol. The resulting reaction mixture is diverted to an incubation coil through a stream selection valve. All incubation coils, that are immersed in a water bath at 95±1◦ C, are loaded in 5 min and then the flow stops for 25 min. After sample incubation, the reaction mixture is cooled by passing through an ice bath and the reaction products are measured at 532 nm. The proposed PA-FI method achieves olive oil analysis without pretreatment and minimizes solvent consumption (1.9 ml of n-propanol, 2.7 mg of TBA and 16 mg of trichloroacetic acid per analysis). Precision was found to be better than 4.6% R.S.D. (n=10). The linear range was (0.4–10)×10−4 M (calculated as malondialdehyde (MDA)) and was suitable for olive oil analysis. The proposed method compares well with a manual method (relative difference 0–6.2% for the analysis of 30 olive oil samples). The analysis rate is 20 samples h−1 , while individual samples are incubated for 30 min. The PA-FI analyser developed allows automation of methods that require long incubation times without loss of sampling rate, overcoming the ‘one sample at a time’ disadvantage of FI. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Olive oil; Flow injection parallel analysis; Malondialdehyde; 2-Thiobarbituric acid reactive substances; Multichannel analyser; Incubation coils

1. Introduction The 2-thiobarbituric acid (TBA) test is an empirical method used for the assessment of lipid peroxidation. The test measures malondialdehyde (MDA) produced by the decomposition of polyunsaturated fatty acid (PUFA) peroxides [1]. Beyond MDA, other sample components [2,3] and products of the oxida∗ Corresponding author. Tel.: +301-5294-248; fax: +301-5294-265; http://www.aua.gr/georgiou. E-mail address: [email protected] (C.A. Georgiou)

tive decomposition of PUFAs [4] react with TBA. The term 2-thiobarbituric acid reactive substances (TBARS) has replaced the term MDA to indicate that the assay estimates lipid peroxidation and not MDA concentration. The amount of TBARS present in foods of vegetable or animal origin is a measure of oxidative rancidity [5–7]. TBARS values are also determined in serum and are increased in diseases causing lipid peroxidation [8]. The TBARS test is performed in strongly acidic medium, and the reaction mixture is heated at 80– 100◦ C for 30–60 min. The MDA–TBA adduct pro-

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duced absorbs at 532 nm and emits at 553 nm [9]. The amount of MDA measured, is the sum of the initially present MDA called ‘free’ MDA and the MDA produced by the decomposition of PUFA peroxides during the determination. This side reaction is catalysed by heavy metal ions [10] and the acids used [11,12]. In an effort to increase the precision, antioxidants, complexing agents and high concentrations of Fe3+ ions have been added in the reaction medium [13]. From the above, it is obvious that results obtained depend strongly on experimental parameters. To improve reproducibility, strict control of experimental parameters such as reaction timing and temperature should be achieved [14]. The flow injection technique allows reproducible timing of the analysis steps and is well suited for automation of assays based on fast reactions. However, for slow reactions the sampling rate is drastically decreased. This disadvantage is overcome by simultaneously incubating the samples in multiple incubation coils. This approach, named parallel flow injection (PA-FI) has not found many applications [15–17] in spite of the fact that many analytical procedures are based on slow reactions. Recently, a PA-FI analyser has been developed and used for the determination of olive oil peroxide [18] and iodine value [19]. Only two FI automated methods have been described so far for the determination of TBARS. Ikatsu et al. [20] developed a method for TBARS determination in plasma. In this method, two phase separators were used and reaction products were extracted in a

stream of methyl-iso-butyl-ketone. One sample at a time was incubated in the FI manifold, so the sampling frequency was just 7 h−1 . Jørgensen and Sørensen [15] determined TBARS in aqueous meat extracts using a FI system based on a laboratory-made sampling and delay unit, constructed from two circular disks tightly fitted together. In this system, eight samples were simultaneously incubated. The optimized analytical conditions included a reaction time of 20 min at 60◦ C. In this way, a sampling rate of 18 h−1 was achieved. In this work, a laboratory-made PA-FI analyser consisting of 10 incubation coils is used for the determination of TBARS in olive oil. The aim of this study is the development of a completely automated method that eliminates sample pretreatment and minimizes solvent consumption. This work is a part of an on-going project in our laboratory on the development of automated methods of olive oil analysis [18,19,21,22].

2. Experimental 2.1. Apparatus The laboratory-made PA-FI analyzer depicted in Fig. 1 consists of an Ismatec IPC-8 peristaltic pump, a Milton Roy Spectronic-20 spectrophotometer equipped with a Helma 18 ␮l flow cell (1 cm path length), a Rheodyne 5011P injection valve, a Valco CD15-3110A ten position stream selection valve and an Advantech PCL-818 interface card for data

Fig. 1. Laboratory-made parallel flow injection analyser for the determination of 2-thiobarbituric acid reactive substances in olive oil. TBA: 2.0×10−2 M 2-thiobarbituric acid in n-propanol; CCl3 COOH: 0.10 M in n-propanol; L1 and L2 : 50 cm coils; IV: injection valve; D: spectrophotometer, λ=532 nm; SSV: ten position stream selection valve; C: 110 cm incubation coils; WB: water bath, 95±1◦ C; IB: ice bath; W: waste; a: digital control signals; b: analog data.

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acquisition and control. Valves were attached to pneumatic actuators and controlled through the Rheodyne 7163-033 and the Valco 41E1 solenoids. TEFLON tubing (0.8 mm i.d.) was used for the construction of incubation coils. The laboratory-made software package for data acquisition and control provided modules for FI experiments, data treatment (measurement of peak height and correction of drifting baseline) and data export to text files. The MS-DOS based software was developed in C language and provided for fast, up to 60 kHz data acquisition by using direct memory access (DMA) data transfer from the AD converter. Control of the pump (start and stop), injection valve (load, inject) and stream selection valve (select channel) was achieved through C language functions. An 80486 personal computer was used. The software developed has low system requirements and runs even on 8088 machines. To overcome problems due to bubble formation a C language function was incorporated in the data treatment module. After bubble detection, the function either replaces fault data points by fitting previous and next points to a third degree polynomial or marks the injection as unusable. To pump trichloroacetic acid, ismaprene® peristaltic pump tubes were used. 2.2. Reagents Trichloroacetic acid, n-propanol, TBA and 1,1,3,3tetraethoxypropane were purchased from Merck. n-Methyl-2-phenylindole was purchased from Sigma. The carrier stream was a 2.0×10−2 M TBA solution in n-propanol. This solution was prepared daily. A 0.10 M trichloroacetic acid stream in n-propanol was used to acidify the reaction mixture. To avoid bubble formation, the TBA and the trichloroacetic acid solutions were degassed by sonication for 15 min before use. 1,1,3,3-Tetraethoxypropane aqueous solutions are used for the standardization of manual procedures. For the PA-FI method, it was essential to prepare standards in olive oil as samples were injected without any pretreatment. Olive oils of TBARS values in the range of (0.05–30.0)×10−4 M (calculated as MDA) were prepared by weighing the appropriate amounts of two olive oils of 0.05×10−4 and 30.0×10−4 M TBARS value. Mixtures prepared in this way were standardized according to the method of Ke and Woyewoda [9]. These mixtures were stable for up to

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1 month when stored at room temperature in sealed bottles under nitrogen. 2.3. Analyser operation — timing sequence The following steps are involved in the analysis: 1. Loading of the sample loop (40 ␮l). During this time an incubation coil (C, Fig. 1) is washed. A load-wash time of 20 s was allocated for this step. 2. Sample is injected in the TBA stream. The TBA stream then merges with the trichloroacetic acid stream. Mixing is achieved through the use of a 50 cm coil (L1 , Fig. 1). Then, the reaction mixture is diverted through the stream selection valve (SSV, Fig. 1) to the incubation coil that is immersed in a water-bath thermostated at 95±1◦ C. Then the SSV turns to the next incubation coil. A forward time of 10 s was allocated for this step. 3. Steps 1 and 2 are repeated 10 times, so all 10 incubation coils are loaded after 5 min. Then, while samples are incubated for 25 min (stop time), the pump stops. In this way reagent consumption is minimized. 4. The pump starts. The next sample (eleventh) is loaded in the sample loop while the first sample is driven to the detector through a 50 cm coil (L2 , Fig. 1) immersed in an ice bath. Then, the measured sample is washed out. A load-wash time of 20 s was allocated for this step. Steps 2–4 are repeated until all samples are analyzed.

3. Results and discussion 3.1. Optimization FI is well suited for the automation of wet chemistry methods based on homogeneous aqueous solutions. However, when the sample and the carrier are prepared in distinct organic solvents, difficulties arise from refractive index [23], density and viscosity [24] gradients formed during sample dispersion in the carrier stream. Previous work conducted in our laboratory [18,19,21] proved that n-propanol as solvent provides a homogenous medium for olive oil analysis by FI. Trichloroacetic acid was preferred over acetic acid, that is used in some versions of the manual procedure, as lower concentrations are sufficient for the acidifica-

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Table 1 Effect of incubation time on the characteristics of the calibration grapha Incubation time (min)

Slope±S.D. (A×M−1 ×10−1 )

Intercept±S.D. (A×102 )

r

Linear range (M×104 )

10 20 30 40 60

57.6±0.8 126±1 178±4 291±5 597±2

−0.2±0.4 −0.3±0.5 −1±2 4±2 7±5

0.9996 0.9999 0.9991 0.9997 0.997

2–30 0.8–15 0.4–10 0.1–6 0.05–3

a

Absorbance (A) vs. TBARS value, n=8.

tion of the reaction mixture. When we tried acetic acid we had to use a glacial acetic acid stream to achieve the acidity required for the reaction. This stream did not mix readily with the n-propanol stream used in the TBA carrier, resulting in poor precision. The effect of trichloroacetic acid concentration was studied in the range of 0.01–0.2 M indicating that concentrations not less than 0.10 M should be used. The TBA–MDA reaction rate increases with incubation temperature so the reaction mixture is heated up to 100◦ C in manual procedures. To achieve high sensitivity in the PA-FI method, a high incubation temperature should be used. The incubation temperature chosen was 95±1◦ C as the boiling point of n-propanol is 97.4◦ C. The effect of incubation time on the calibration curve is shown in Table 1. The calibration curve slope increases with increasing incubation time as the reaction proceeds and more colored products accumulate. The incubation time chosen was 30 min as the linear range of (0.4–10)×10−4 M was found to be adequate for olive oil analysis. By increasing the incubation time, the linear range can be shifted to lower concentrations. Using this incubation time a sampling rate of 20 samples h−1 was achieved. In an attempt to shorten the incubation time and achieve higher sampling rates we evaluated the recently introduced reagent n-methyl-2-phenylindole [25,26] that is used as an alternative to TBA. This assay format utilizes hydrochloric acid and produces a colored product that absorbs at 586 nm. To use this reagent in a monophasic assay for olive oil analysis we had to replace hydrochloric by trichloroacetic acid. This study was conducted by mixing 500 mg of olive oil with 5.0 ml of a 2.0×10−2 M solution of the reagent in n-propanol containing 0.10 M trichloroacetic acid. The original TBARS assay was also performed in parallel and the absorbance was measured at 15 min

intervals for 2 h. For the incubation temperature of 65◦ C used we found that the two assay formats have the same sensitivity and reaction rate. As shown in Table 1, the sensitivity of the PA-FI method for 30 min incubation time is 100-folds lower than that of the manual method (molecular extinction coefficient: 1.9×105 M−1 cm−1 [9]). This is due to the lower incubation time allowed (30 min versus 45 min) and the sample dilution due to dispersion in the PA-FI analyser. However, the sensitivity is adequate for the analysis of various olive oil samples. The reaction rate and the analytical signal increases by increasing the TBA concentration. In our experiments, a 2.0×10−2 M TBA solution was chosen as more concentrated solutions ((3.0–6.0)×10−2 M) precipitated within 8 h rendering them unsuitable for the PA-FI method. As stated in Section 1, the TBARS assay variant utilized in this work (without separation of the ‘free’ MDA) can be used for the estimation of olive oil peroxidation and not olive oil MDA content. It is interesting to note that the reaction proceeds during the course of heating, as more PUFA peroxides decompose resulting in an increase of the colored products. This is clearly shown in Fig. 2 where the reaction was followed for 3 h. It should be stated here that the reaction of aqueous standard solutions is completed within 45 min. 3.2. Method evaluation Precision was evaluated by replicate analyses of olive oil samples of 1.51×10−4 , 3.99×10−4 and 8.93×10−4 M TBARS values. Relative standard deviations (R.S.D.) were found to be 4.6, 3.5 and 2.8% (n=10), respectively. It should be noted that the R.S.D. values determined contain the variance due to differences in the flow paths lengths of the 10 incuba-

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Table 2 Results of the analysis of olive oil samples Sample no.

Fig. 2. Absorption spectra of a 10% v/v olive oil dissolved in n-propanol containing 2.0×10−2 M 2-thiobarbituric acid and 0.10 M trichloroacetic acid after heating at 65◦ C for (a) 30, (b) 45, (c) 60, (d) 90, (e) 120 and (f) 180 min.

tion coils. Another variant of the PA-FI technique that uses flow reversals eliminating differences in flow path lengths has been developed in our laboratory [18]. This variant could not be implemented in this work due to the use of the cooling coil (L2 , Fig. 1). To evaluate the proposed method in olive oil analysis, 30 olive oil samples were analyzed and the results were compared with those acquired through the method of Ke and Woyewoda [9]. Results are presented in Table 2. Relative differences for TBARS values determined using the proposed and the literature method ranged from 0 to 6.2%, the overall mean difference being 2.3%. The correlation of the results obtained by the two methods is described by a linear regression line with a slope of 0.991±0.008, an intercept of 0.02±0.04 and a correlation coefficient r=0.999, demonstrating the equivalence of the two methods.

4. Conclusions Olive oil samples are directly injected in the PA-FI analyser without any pretreatment. Results obtained using the proposed method are in accordance with those obtained using a manual procedure. The proposed method requires just 1.9 ml of n-propanol, 2.7 mg of TBA and 16 mg of trichloroacetic acetic acid per analysis minimizing the big amounts of solvents used in manual procedures and eliminates

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Determined olive oil TBARS value (M×104 )

Relative difference (%)

FI method

Literature method

0.86 1.43 1.47 1.89 1.94 1.96 2.23 2.29 2.34 2.42 2.43 2.48 2.67 2.82 2.87 2.99 3.12 3.44 4.04 4.15 4.30 4.47 4.78 5.18 5.34 6.92 7.26 8.85 8.95 10.2

0.81 1.37 1.51 1.83 2.01 2.09 2.12 2.27 2.33 2.43 2.49 2.52 2.64 2.78 2.86 3.03 3.30 3.33 3.99 4.09 4.25 4.47 4.75 5.27 5.39 6.75 7.09 8.93 9.16 9.88

6.2 4.4 −2.6 3.3 −3.5 −6.2 5.2 0.9 0.4 −0.4 −2.4 −1.6 1.1 1.4 0.3 −1.3 −5.4 3.3 1.2 1.5 1.2 0.0 0.6 −1.7 −0.9 2.5 2.4 −0.9 −2.3 3.2 Mean

2.3

the use of chloroform that is banned in a number of countries. The use of multiple incubation coils in the PA-FI method permits an incubation time of 30 min while achieving a sampling rate of 20 samples h−1 . In this way the ‘one sample at a time’ disadvantage of FI is eliminated.

Acknowledgements Support from the Research Committee of the Agricultural University of Athens through a research grant is gratefully acknowledged.

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