Introduction

Lipid oxidation which results in rancidity is the main cause of food deterioration and, in a course of off-odor formation, is also one of the main factors for food rejection by consumers (Kochhar 1996; Shahidi 1998). Although reactive oxygen species (ROS) influence many food constituents, such as proteins, carbohydrates, and vitamins, they play a key role in decreasing sensory quality of food in a result of lipid oxidation (Choe and Min 2006). It occurs in foods containing substantial amounts of fat, like oils, nuts, some dairy products, or meat, and also in these foods that contain only minor amounts of lipids. Lipid oxidation is a complex process where unsaturated fatty acids react with molecular oxygen via a free radical mechanism or in a photosensitized oxidation process. Hydroperoxides—the main, nonvolatile intermediates—decompose to an array of volatile compounds, including alkanes, alkenes, aldehydes, ketones, alcohols, esters, acids, and hydrocarbons, which influence food flavor, contributing to rancid, soapy, oily, and fishy notes, and also indicate the undergoing oxidation processes (Jeleń and Wasowicz 2012).

The main issues in the development of robust, reproducible, and reliable analytical method for lipid oxidation monitoring using volatile compounds as markers are as follows: (i) choice of proper compounds to monitor; (ii) method performance parameters—sensitivity, linearity, and reproducibility; (iii) investigation of matrix influence on the release of selected compounds; and (iv) simplicity and possibility of automation, to be used routinely in control labs and in oil processing plants.

Lipid oxidation in foods has been extensively researched, and many gas chromatographic methods were developed for monitoring this process. Static headspace (SHS), dynamic headspace (DHS), direct injection (DI), thermal desorption (TD), and headspace solid phase microextraction (HS-SPME) are the dominating techniques used in sample preparation for monitoring lipid oxidation (Cavalli et al. 2003; Dupuy et al. 1985; Jeleń et al. 2007; Kaykhaii and Rahmani 2007; Pastorelli et al. 2007; Cognat et al. 2012). Static headspace sampling, compared to other headspace analysis methods, may suffer relatively low sensitivity (Cavalli et al. 2003) and is usually used for determination of compounds present in mid ppb to high ppm levels. The sensitivity depends significantly on the type and volatility of the analyzed compound, detector used, and matrix in which the compound is analyzed. However, the main lipid oxidation products are often produced in concentrations that rationalize the use of static headspace sampling, which is one of the easiest to perform and automate methods for volatile compounds analysis.

Hexanal is a main product of linoleic acid oxidation via the 13-hydroperoxide, and it appears in many papers as a compound indicating lipid oxidation processes. It was used as an indicator of oxidative processes in biological systems (Frankel et al. 1989), to monitor lipid oxidation volatiles in products such as infant formulas (Garcia-Llatas et al. 2006), potato crisps (Kaykhaii and Rahmani 2007), wheat and buckwheat crackers (Mandić et al. 2013), and rapeseed oil (Gromadzka and Wardencki 2010). Muriel et al. (2007) used hexanal as a compound indicating oxidative stability in Iberian dry-cured loin, and it was proposed for evaluation of oxidative stability of meat and meat products (Shahidi et al. 1987). In nuts stored under UV, hexanal content increased in time (Pastorelli et al. 2007). The highest formation rates in the oxidation of olive oil were noted for nonanal and hexanal (Vichi et al. 2003). Hexanal content increased also during storage of potato chips (Sanches-Silva et al. 2004), infant formulas (Romeu-Nadal et al. 2004), and edible oils (Shahidi 1998).

The goal of this paper was to develop a simple and robust method, based on static headspace gas chromatography (SHS-GC), for the determination of hexanal as a lipid oxidation marker in fat-rich food matrices—potato chips, mayonnaise, and oil—with the focus on the specificity of particular matrix in this analysis.

Materials and Methods

Sample Preparation

Fresh rapeseed oil, mayonnaise (76.1 % fat), and potato chips (32 % fat) were purchased from a local supermarket for method development. Various oils and potato chips with different expiration dates were used in experiments to assess the levels of hexanal in these products. Fresh refined rapeseed oil was used also for standards preparation. Hexanal (99 % purity, GC) was supplied by Sigma-Aldrich (Poznań, Poland).

Hexanal stock solutions (1 g/L) were prepared in fresh refined rapeseed oil or water (depending on the experiment) and diluted with oil or water respectively for quantitation purposes. The influence of the sample weight and water addition on the extraction efficiency of hexanal in potato chips and mayonnaise samples was estimated by testing solid samples weighting 1, 5, and 9 g, spiked with 25 μg/g of hexanal versus wetted samples of the same weight with the addition of 9, 5, and 1 mL of water, respectively. Sample weight effects were compared as the differences between hexanal peak areas.

For method development, potato chip samples were ground (frozen in liquid nitrogen prior to grinding) with a laboratory grinder. Finely powdered potato chips were mixed with ultrapure water in headspace vials in proportions provided above. Samples were then spiked with hexanal to the final concentration of 25 mg/L. The vials were then capped with aluminum caps with silicone-PTFE septa and mixed for 3 min using Vortex. Mayonnaise was prepared the same way as potato chips (except freezing and grinding). Rapeseed oil (10 mL) was transferred into a headspace vial, then spiked with hexanal to the final concentration of 5 mg/L, capped, and vortexed for 3 min. Water samples containing various hexanal concentrations were prepared similarly to rapeseed oil samples using hexanal-in-water stock solution. Calibration curves were prepared using ten hexanal concentration levels in the range of 0.1–200 mg/L for all the samples.

Chromatographic Analysis

Analyses were run on Agilent Technologies 7890A gas chromatograph coupled to Agilent Technologies 7697A static headspace sampler (Agilent Technologies, Wilmington, DE). Twenty-milliliter headspace vials were used in all experiments. The headspace sampler was connected to the split/splitless port of GC. A polar Zebron ZBWax column (60 m × 530 μm × 1.0 μm) by Phenomenex was used for separation. Samples were injected in a 1:5 split mode at 140 °C (inlet pressure, 4.43 psi). Oven temperature was programmed from 40 °C (5 min) to 100 °C at 10 °C/min. Helium was used as a carrier gas at 7 mL/min, constant flow. GC was equipped with a FID detector (temperature, 240 °C; H2 flow, 30 mL/min; airflow, 300 mL/min; auxillary gas (N2), 15 ml/min). Temperature and time parameters for vial incubation were different for each examined matrix: For analysis of hexanal in water, oven temperature was 75 °C, loop temperature was 85 °C, transfer line temperature was 95 °C, and equilibrium time was 20 min; for oils, 90 °C, 100 °C, 110 °C, and 30 min, respectively; for potato chips, 60 °C, 70 °C, 80 °C, and 40 min, respectively; and for mayonnaise, 90 °C, 100 °C, 110 °C, and 40 min, respectively.

Results and Discussion

Static Headspace Sample Transfer Parameters

Injection using static headspace samplers involves some steps to develop a method, depending on a headspace sampler. In simple syringe-type autosamplers, syringe temperature and plunger speed are the parameters to look at, whereas loop-based autosamplers, as the one used, require more options in sample introduction to consider. Table 1 summarizes tested methods to facilitate the most efficient hexanal extraction from oil. Method 2 characterized by injection time of 1 min, fill to pressure option (5 psi), rate of loop fill of 20, loop fill (default), final pressure 1.717 psi, venting loop option (yes), and equilibrium of 0.05 was chosen. Before experiments on sample transfer parameters, different shake intensities (levels 1–3) were tested providing for shake level 1, 2, and 3 hexanal peak areas of 6543 pA*s (±242), 6598 pA*s (±252), and 6891 pA*s (±187), respectively. Therefore, in all consecutive tests, shake level 3 was used.

Table 1 Headspace sampler extraction/injection parameters for hexanal determination in rapeseed oil
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Effect of Sample Size on Extraction Efficiency

To determine how sample size (weight) of chips and mayonnaise influences peak areas to 1, 5, and 9 g of these products, 9, 5, and 1 mL of water were added and samples were vortexed for 3 min prior to incubation in headspace oven. The intention of water addition to the solid or emulsified samples was to influence partition coefficients between sample and headspace in the vial and promote migration of hexanal into headspace. As can be seen on Fig. 1, the highest peak responses were provided for 1 g of chips/mayonnaise with 9 mL of water added. Addition of increasing amount water for both matrices provided higher responses as the different solubility of hexanal in oil and water favored partition from water to headspace. In the analysis of test samples, these parameters were used.

Fig. 1
figure 1

Effect of mayonnaise (a) and chip (b) sample weight on hexanal extraction efficiency (hexanal peak area [pA*s]). All samples were 10 g: sample weight 1 means 1 g of mayonnaise (or chips) + 9 mL of water, sample weight 5 is 5 g of sample + 5 mL of water, and sample weight 9 means 9 g of sample + 1 mL of water

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Effect of Temperature on Hexanal Extraction Efficiency

Figure 2 shows all four matrices, which were thermostated for 20 min at three different temperatures—60, 75, and 90 °C. As the increase of temperature influences partition of compounds of high K values into the headspace, the hexanal peak area was different at temperatures tested. Comparing matrices with high fat content, they usually required higher extraction temperatures. For rapeseed oil, the increase of temperature caused increased concentration of hexanal in headspace, and the biggest peak area was noted for 90 °C, as the viscosity of oil decreases with temperature increase. For water (Fig. 2d), the highest tested temperature did not result in the highest amount of extracted hexanal, as the water pressure became an important factor in partition of hexanal into headspace at 90 °C. The mayonnaise showed similar trend as oil, despite the proportions of oil/water. For potato chips, the responses for examined temperatures were comparable, and at 90 °C, the amount of extracted hexanal decreased, similarly as in water. Comparing mayonnaise and chips, for the same amounts of food products, the oil favors migration of hexanal into headspace compared to solid potato chips matrix.

Fig. 2
figure 2

Effect of vial equilibration temperature on the peak area of hexanal in various matrices: a oil, b potato chips, c mayonnaise, and d water

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Effect of Time on Hexanal Extraction Efficiency

As static headspace is an equilibrium method, the optimal extraction time should be chosen, long enough to facilitate the highest and constant analyte responses and at the same time to avoid the possible sample decomposition due to prolonged heating. When time extraction profiles were compared in a range of 10–40 min (Fig. 3), the matrices differed in terms of extraction profiles: A slight peak area increase was noted for mayonnaise and potato chips, whereas for oil and especially water, prolonged heating resulted in the decrease of hexanal peak areas. Usually, higher temperatures and longer equilibrium times result in higher peak areas of extracted compounds. Mandić et al. (2013) used incubation time of 10 min at 90 °C for five aliphatic, saturated aldehydes in wheat and buckwheat crackers, whereas Gromadzka and Wardencki (2010) used 20-min extraction at 80 °C to isolate volatile aldehydes from rapeseed oil using static headspace method. When comparing different methods for extraction of olive oil volatiles, Cavalli et al. (2003) used loop transfer autosampler for determination of olive oil volatiles in their work and used 110 °C oven temperature and 120-min equilibration time in static headspace method, which was the least sensitive of examined methods in terms of number of compounds extracted. They also indicated that such harsh equilibration conditions could induce thermal oxidation of virgin olive oil. For infant formulas, Garcia-Llatas et al. (2006) used much lower extraction temperature for headspace SPME (37 °C) to avoid a possible formation of artifacts.

Fig. 3
figure 3

Effect of vial equilibration time on peak areas of hexanal in various matrices: a rapeseed oil, b potato chips, c mayonnaise, and d water

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Examined matrices were of very different physical characteristics. They differed in their structures influencing flavor compound release, and it can be supposed that apart from partition coefficients, the resistance to mass transfer was a major factor, which determined rate and degree of flavor release. Resistance to mass transfer determines the rate at which equilibrium is reached, whereas partition coefficient determines the degree of flavor release. In emulsified systems, the distribution of hexanal involves three phases (oil, water, and the interface), so it is different than in one phase systems (oil or water) (Jacobsen et al. 1999). Also, factors influencing oxidation in emulsions are far more complex (Waraho et al. 2011).

Selected Method Performance Parameters

To determine the usefulness of method of hexanal determination in tested matrices, calibration curves were determined for a concentration range of 1–200 mg/L(kg) for the majority of samples (Table 2). Calibration curve equations are provided together with the R 2 values, which indicate very good linearity in a broad concentration range. The sensitivity (calibration curve slope) for hexanal determination in rapeseed oil is much higher than that for mayonnaise and chips. Comparing it to water, differences related to hexanal solubility and release from the matrix become evident, influencing calibration curve slopes. Limit of detection (LOD) and limit of quantitation (LOQ) values can be calculated in a number of ways, which are listed in validation guidelines (Boque and Vander Heyden 2009; Huber 2010; ICH 2005), and often, their direct comparison is complex. We decided to provide LOD as a concentration that generates hexanal peak of signal-to-noise ratio (S/N) of 2. LOQ was estimated as 3 LOD (Table 2). For all matrices spiked with hexanal to achieve a concentration of 1 mg/L, repeatability was measured as relative standard deviation (%) for five samples consecutively run in a single sequence, and the intermediate precision was calculated from spiked samples run in triplicate in 3 consecutive days. Static headspace autosamplers usually provide very good precision in analysis of especially homogenous samples. In testing precision with spiked samples, the main source of error is the sample preparation process itself. However, precision of described method was comparable to that of other methods, where hexanal was determined in similar food matrices.

Table 2 Selected parameters for static headspace gas chromatography method for hexanal determination in various matrices
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In a work of Mandić et al. (2013) where static headspace was used for determination of aldehydes in crackers, the repeatability (%RSD) of method for peak areas ranged from 1.13 % (pentanal) to 3.86 % (hexanal). LOD for hexanal was 0.010 μg measuring a signal to noise, which is several times lower than that achieved using the described method after recalculation for amounts that enter GC. Sanches-Silva et al. (2004) evaluated the suitability of SPME-GC/MS and HPLC for the determination of hexanal in potato crisps. They used ground and mixed with water (1:9) crisps, from which hexanal was extracted using SPME and achieved 1 ng/mL LOD compared to 9 ng/mL for HPLC method with derivatization with 2,4-DNPH. The repeatability for SPME-GC/MS method was 7.56 %. Romeu-Nadal et al. (2004) obtained a detection limit for hexanal in infant formulas equal to 19.60 ng, and repeatability (as %RSD) was 2.29. Using liquid-phase microextraction (LPME) to isolate hexanal from potato crisps, followed by GC-FID, Kaykhaii and Rahmani (2007) achieved LOD of 100 ng/L with repeatability (% RSD) of 3.9. The above described methods (SPME, LPME) that unify in one extraction and concentration steps allow determination of analytes in concentrations lower than static headspace, in which a fixed volume of gas phase is transferred into gas chromatograph with amount of analyte in it defined by its partition coefficient. It could be clearly observed comparing LODs of developed method with cited references in which pre-concentration techniques were used.

Application of the Methods to the Determination of Hexanal in Oils and Potato Chips

The developed method was applied to the screening of hexanal contents in samples of oils and potato chips of different shelf lives. Typical chromatogram of oil is shown in Fig. 4. In all analyzed samples, hexanal peak was well resolved from other peaks. The amount of hexanal in tested samples (Table 3) varied significantly, ranging from 0.45 mg/kg for fresh (before expiration date) refined oils to 8.86 mg/kg for cold-pressed sunflower oil stored for 4 years in refrigerator. The range of hexanal in potato chips was from not detected to 6.90 mg/kg. As all the potato chips samples had similar expiration dates, the differences in hexanal content may indicate the different quality of fats used for chip preparation and/or effectiveness of antioxidants used.

Fig. 4
figure 4

Chromatogram of volatile compounds isolated from cold pressed sunflower oil (sample 3 in Table 3) with hexanal peak marked

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Table 3 Hexanal content in analyzed oils and chips
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Conclusions

The presented method of quantitation of hexanal in different matrices allows its quantitation in a broad range of concentrations. Hexanal is a good indicator of lipid oxidation processes in foods, and its odor thresholds vary substantially for different matrices, and it is estimated at 0.012 mg/kg in water, whereas for oil, it is 0.32 or 0.075 mg/kg depending whether it is perceived orthonasally or retronasally (Beliz et al. 2009). Therefore, the developed method for hexanal determination in oils and foods rich in fat provides sufficient sensitivity, comparable to odor thresholds for oil matrices. The developed method is characterized by ease of sample preparation and automation.