Food Chemistry 145 (2014) 918926

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Review

Sample preparation: A critical step in the analysis of cholesterol oxidation products

Christiana A. Georgiou 1, Michalis S. Constantinou 1, Constantina P. Kapnissi-Christodoulou
Department of Chemistry, University of Cyprus, 1678 Nicosia, Cyprus

a r t i c l e

i n f o

a b s t r a c t
In recent years, cholesterol oxidation products (COPs) have drawn scientic interest, particularly due to their implications on human health. A big number of these compounds have been demonstrated to be cytotoxic, mutagenic, and carcinogenic. The main source of COPs is through diet, and particularly from the consumption of cholesterol-rich foods. This raises questions about the safety of consumers, and it suggests the necessity for the development of a sensitive and a reliable analytical method in order to identify and quantify these components in food samples. Sample preparation is a necessary step in the analysis of COPs in order to eliminate interferences and increase sensitivity. Numerous publications have, over the years, reported the use of different methods for the extraction and purication of COPs. However, no method has, so far, been established as a routine method for the analysis of COPs in foods. Therefore, it was considered important to overview different sample preparation procedures and evaluate the different preparative parameters, such as time of saponication, the type of organic solvents for fat extraction, the stationary phase in solid phase extraction, etc., according to recovery, precision and simplicity. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 4 March 2013 Received in revised form 28 June 2013 Accepted 28 August 2013 Available online 7 September 2013 Keywords: Cholesterol oxidation products Lipid extraction Sample preparation Saponication Solid phase extraction

Contents 1. 2. 3. 4. 5. Introduction . . . . . . . . Lipid extraction . . . . . Saponification . . . . . . . Solid phase extraction Conclusion . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918 920 921 923 925 925

1. Introduction Cholesterol is vulnerable to oxidation, leading to the formation of a variety of cholesterol oxidation products, the so-called COPs. In recent years, COPs have gained considerable attention due to their implications in human health (Clariana, Daz, Srraga, & GarcaRegueiro, 2011a). These compounds have been well documented for being potentially cytotoxic, mutagenic, carcinogenic, and they have been associated with the promotion of atherosclerosis (Brown & Jessup, 1999; Leonarduzzi, Sottero, & Poli, 2002; Lordan, Mackrill, & OBrien, 2009; Olkkonen, Baslas, & Nissil, 2012;
Corresponding author. Tel.: +357 22 892774; fax: +357 22 892801.
1

E-mail address: ckapni1@ucy.ac.cy (C.P. Kapnissi-Christodoulou). These authors contributed equally to this work.

Otaegui-Arrazola, Menendez-Carreo, Ansorena, & Astiasarn, 2010; Poli, Sottero, Gargiulo, & Leonarduzzi, 2009). It is thought that COPs modulate the structure and function of the cellular membrane and inhibit the activity of enzymes involved in cholesterol biosynthesis (Bielska, Schlesinger, Covey, & Ory1, 2012; Brown & Jessup, 2009; Gill, Chow, & Brown, 2008; Jusakul, Yongvanit, Loilome, Namwat, & Kuver, 2011; Olsen, Schlesinger, & Baker, 2009; Olsen, Schlesinger, Ory, & Baker, 2012). The main source of COPs is through diet, and particularly from the consumption of cholesterol-rich foods, such as meat, eggs and dairy products (Sampaio, Bastos, Soares, Queiroz, & Torres, 2006; Vicente & Torres, 2007). It is generally accepted that fresh foods contain very low levels of COPs (Cardenia, Rodriguez-Estrada, Baldacci, Savioli, & Lercker, 2012). However, inadequate storage, cooking and processing conditions tend to increase the degree of

0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.08.123

C.A. Georgiou et al. / Food Chemistry 145 (2014) 918926

919

Fig. 1. Structural formulas of the most important COPs and cholesterol.

cholesterol oxidation (Echarte, Ansorena, & Astiasarn, 2003; Hur, Park, & Joo, 2007; Olkkonen et al., 2012; Pignoli et al., 2009; Savage, Dutta, & Rodriguez-Estrada, 2002). This, therefore, raises questions about the safety of the consumers, and it suggests that it is imperative to develop a sensitive and a reliable analytical method in order to monitor these components in food samples (Yen, Inbaraj, Chien, & Chen 2010). The most common COPs found in several foodstuffs are demonstrated in Fig. 1. Gas chromatography (GC) and high performance liquid chromatography (HPLC) are the most widely used techniques for the analysis of COPs. The former can provide better resolution, but it requires a time-consuming derivatization of the analytes in order to enhance their volatility and thermal stability (Chen, Chien, Inbaraj, & Chen, 2012). HPLC, which is considered as the alternative method to GC, enables the analysis of these compounds in relatively low temperatures without the need for derivatization (Raith et al., 2005; Vicente, Sampaio, Ferrari, & Torres, 2012). Hence, it is a fast and a simple analytical methodology. The most common detection systems used for the qualitative and quantitative determination of COPs are spectrophotometric (UV), refractive index (RI), ame ionization (FI) and mass spectrometric (MS). Most of COPs absorb at low wavelengths (200215 nm region), where most organic compounds exhibit some UV absorbance. This, in turn, makes the UV detector less selective. In addition, a UV detector is not useful for the detection of some biological important oxysterols, such as 5,6-epoxy-cholesterol or cholestane-3b,5a,6b-triol, which do not possess adequate UV absorption characteristics (Saldanha, Sawaya, Eberlin, & Bragagnolo, 2006; Vicente et al. 2012). The use of MS and tandem MS/MS opens access to more selective detection, reducing matrix background and improving

prole with a low S/N ratio (Clariana et al., 2011; Saldanha et al., 2006). Prior to chromatographic determination and quantication of COPs, sample preparation is necessary to be employed in order to eliminate interferences and increase sensitivity. Although numerous research articles have, over the years, been published concerning extraction and purication of COPs from foodstuffs, no certied method is yet available. Thus, the development of a credible assay is an urgent problem that needs to be overcome (Chen et al., 2012; Janoszka, 2010; Sieber, 2005). This particular step of the analytical protocol is considered to be the most challenging for several reasons. The fact that COPs are part of the complex mixture of lipids, where they are mostly present in trace levels (ppm or ppb) indicating that the extensive workup and cleaning procedures before nal quantication are required. However, one major concern that needs special attention involves the possibility of generation of the so-called artifacts (Cardenia et al., 2012; Lozada-Castro, Gil-Diaz, Santos-Delgado, RubioBarroso, & Polo-Diez, 2011; Manini, Andreoli, Careri, Elviri, & Musci, 1998). Artifacts are COPs that are formed under analytical conditions, either due to cholesterol oxidation or due to the conversion of pre-existing COPs to others (Busch & King, 2009). The best known example is the degradation of 7-keto to cholesta-3,5dien-7-one in alkaline medium (Lozada-Castro et al., 2011). Some authors in order to limit artifact generation, recommended the use of antioxidants, such as butylated hydroxytoluene (BHT), during sample preparation (Janoszka, 2010; Du, Nam, & Ahn, 2001; Nam, Du, Jo, & Ahn, 2001). The general route in COPs analysis involves three major steps: extraction of lipids from the food matrix, hydrolysis of esteried

920

C.A. Georgiou et al. / Food Chemistry 145 (2014) 918926

sterols by saponication and subsequent enrichment of COPs with solid phase extraction (SPE) (Sieber, 2005; Ubhayasekera, Verleyen, & Dutta, 2004). However, this is not a standard procedure, as many studies use some modications (Table 1). The objective of this study is the extensive presentation of the main existing approaches of each analytical step mentioned above.

2. Lipid extraction Lipids exist in biological matrices mainly in two major forms; they are present as droplets in storage tissues or they can be constituents of cell membranes (Christie, 1993; Farese & Walther, 2009; Guardiola, Dutta, Codony, & Savage, 2002; Reue, 2011). In each case, they are not only closely associated with each other, but they interact with non-lipid material, such as proteins, via hydrophobic and Van der waals forces, hydrogen bonding and electrostatic interactions, as well (Christie, 1993; Jensen, & Mouritsen, 2004). For this reason, the isolation of liposoluble compounds from foodstuff, including COPs, is not easy, and thus, the use of a method capable to provide total extraction of lipids is necessary for a reliable analysis. In principle, the quantitative extraction of lipids from tissues can be achieved with the use of an ideal solvent, which could simultaneously dissolve the lipids and disrupt the polar and hydrophobic interactions between them and other tissue matrix (Iverson, Lang, & Cooper, 2001; Guardiola et al., 2002). Therefore, the use of a single apolar organic solvent, such as hexane, is unsuitable. To meet this requirement, a fairly polar solvent or mixture must be employed (Christie, 1993; Iverson et al. 2001). Folch, Lees, and Stanley (1957) were among the rst to recognize this aspect, and they developed a method using a mixture of chloroform/methanol (2:1, v/v) for the lipid extraction. Regarding the proper amount of sample and solvents, they suggested that tissue should be homogenized with chloroform/methanol (2:1, v/v) to a nal volume 20 times the volume of the tissue sample. Due to the toxicity of chloroform, alternative solvent systems have been developed. A method described by Hara and Radin involved the use of n-hexane/2-propanol (3:2, v/v) mixture as the lipid extractant, while Maxwells method reported the use of dichloromethane and methanol (Hara & Radin, 1978; Maxwell, Mondimore, & Tobias, 1986). Apart from a re-development of Hara and Radins method, these two procedures have not found wide application in COPs analysis (Ferioli, Caboni, & Dutta, 2008; Ferioli, Dutta, & Caboni, 2010; Ubhayasekera, Tres, Cobony, & Dutta, 2010). In brief, Folchs method is by far the most widely used method, as it continues to be considered the most efcient and reliable technique for the exhaustive isolation of lipids from various tissues
Table 1 Different pathways of sample preparation in COPs analysis. Matrix Porcine patties Dry-cured shoulder Pig feet meat and skin Tea-leaf eggs Cooked ham, Serrano ham, minced beef, and soft cheese Minced beef Pickled mackerel Pates and pork sausage Deep-fried pork rinds, dried beef and sun-dried shrimp
*

(Grau, Cobony, Grimpa, Baucells, & Guardiola 2001; Janoszka, 2010; Mazalli & Bragagnolo, 2007; Mazalli & Bragagnolo, 2009; ski, & Koczak, 2006; Sampaio et al. 2006). InevitaObara, Obiedzin bly, the use of a quite polar solvent system will lead to co-extraction of non-lipid material; hence, Folch et al. suggested an appropriate washing step in order to remove these contaminants before proceeding to the next step of the analytical protocol, which, in our case, it is the purication of COPs. This step is essential in order to partition the system into two phases; the lower organic layer containing all of the tissue lipids and the upper one consisting of non-lipid co-extracted contaminants. However, one important parameter, which can inuence the quantitative recovery of the more polar lipids, such as COPs, is the amount of water or salt solution that is added after sample homogenization with chloroform/methanol (2:1, v/v). According to the original paper, the proposed volume ratio of chloroform/ methanol/water must be exactly 8:4:3. These proportions should be constant; otherwise, selective losses of these lipids, into upper phase, may occur (Christie, 1993; Guardiola et al. 2002). Nevertheless, it seems that this critical aspect is often overlooked in some studies (Chen, Lu, Chien, & Chen, 2010; Lee, Chien, & Chen 2006; Lee, Chien, & Chen 2008). Several slightly modied versions of Folchs method also exist, with the version proposed by Boselli, Velazco, Caboni, and Lercker, (2001) being the most known to COP research society. Particularly, the samples were homogenized with chloroform/methanol (1:1, v/v), and they were kept at 60 C in order to maximize extraction recovery yield. The mixture was re-homogenized with an equal amount of chloroform, and then, it was passed through lter paper to eliminate the solid residue, which mainly consisted of proteins. Subsequently, the ltrate, which contained the lipids accompanied by non-lipid substances, was mixed with 1 M KCl solution and was left overnight at 4 C for phase separation. The lower phase, freed from non-lipid substances, was collected and dried with a vacuum evaporator for further purication of COPs (Boselli et al., 2005, Bonoli, Caboni, Rodriguez-Estrada, & Lercker, 2007; Soto-Rodriguez et al., 2008; Boselli, Rodriguez-Estrada, Fedrizzi, & Caboni, 2009; Verardo et al., 2010; Bosselli, RodriguezEstrada, Ferioli, Caboni, & Lercker, 2010; Cardenia et al., 2011; Pignoli et al. 2009). In our knowledge, no studies were performed by the authors to clarify whether the temperature could induce artifact formation. The second most known lipid extraction procedure was developed by Bligh and Dyer (Bligh & Dyer, 1959). This method is still used as an alternative to Folchs method, but to a lesser extent in COPs analysis (Broncano, Petrn, Parra, & Timn, 2009; Clariana & Garca-Regueiro, 2011b; Clariana et al., 2011a; Petrn,

Lipid extraction* Chloroform/methanol (1:2,v/v) Methanol/chloroform/water (2:1:1, v/v) Chloroform/hexane (2:1,v/v) Chloroform/methanol (2:1,v/v) Chloroform n-Hexane/i-propanol (3:2,v/v) Dichloromethane/methanol (2:1,v/v) Chloroform/methanol (2:1,v/v) Chloroform/methanol (1:1,v/v)

Purication Si-SPE & NH2-SPE Si-SPE & NH2-SPE Si-SPE Si-SPE Si-SPE Cold saponication (RT, 1 M KOH in EtOH) Cold saponication (RT, with 1 M KOH in EtOH) Cold saponication (RT, 2 N NaOH in MeOH) Cold saponication (RT, 1 M KOH in MeOH) 18 h, with 1820 h, 12 h, with 20 h, with

Enrichment

References Rodrguez-Carpena et al. (2012) Clariana et al. (2011a) Chen et al., (2011) Chen et al. (2010) Lozada-Castro et al. (2011)

2-foldSiSPE Si-SPE

Ferioli et al. (2008) Ubhayasekera, Jayasinghe, Ekanayake, and Paresh (2012) Derewiaka and Obiedzinski (2010) Soto-Rodriguez et al. (2008)

NH2-SPE

Lipid extraction referred to the rst solvent system used for sample homogenization.

C.A. Georgiou et al. / Food Chemistry 145 (2014) 918926

921

Garcia-Regueiro, Martin, Muriel, & Antequera, 2003; RodriguezCarpena, Morcuende, Petrn, & Estevez, 2012). As mentioned earlier, the water content withheld in the tissue is taken into account as a ternary component of the extraction system. Initially, the sample, containing endogenous water, is homogenized with a mixture of chloroform/methanol (1:2, v/v), resulting in the formation of a monophasic solution. After that, further homogenization takes place with chloroform and water, leading to a biphasic system. The addition of chloroform is necessary in order to ensure the complete extraction of lipids. The upper phase is discarded, while the chloroform layer is concentrated for the isolation of COPs. The suggested proportions for a sample, containing 80% endogenous water, after water and chloroform addition, is 2:2:1.8 for chloroform, methanol and water, respectively. In cases, where the moisture content is less than 80%, it is necessary either to add water or reduce solvent volumes in order to meet the requirements described above (Bligh and Dyer, 1959). Although there are very few reports available regarding the comparison between different extraction methods, they have clearly demonstrated that the existing ones are not totally equivalent in their extraction efciency, and therefore, they must be evaluated prior to application. Chen et al. (2010) evaluated different solvent systems in order to enhance COPs extraction efciency in egg samples. Various solvents, including hexane, methanol, ethyl acetate, acetone and chloroform were examined either separately or combined. The mixture of chloroform/methanol (2:1 v/v) proved to be the most suitable because it minimized the amount of impurities in eggs. In contrast, Chen et al. (2012) followed the same procedure for the analysis of pig feet samples, and they observed that this system was less appropriate as it led to coextraction of interfering compounds. Better results were obtained when chloroform/ hexane (2:1, v/v) was adopted. These two studies strengthened the view, which suggests that the selection of a particular solvent system is affected by the nature of the food matrix. Even though the use of a single solvent for lipid extraction is rare, Lozada-Castro et al. (2011), comparing different sample preparation procedures, indicated that the use of pure chloroform was the optimum choice. Moreover, the authors validated the method in terms of extraction time (6, 12, 18 h) and solvent volume (40, 70, 100 ml). The evaluation data demonstrated that long extraction time resulted in better recoveries, while the volumes of 70 and 100 ml provided comparable results. In a recent study, Georgiou & Kapnissi-Christodoulou, 2013 compared and evaluated two extraction solvent systems, chloroform/methanol (2:1 v/v) and nhexane/2-propanol (3:2 v/v). The former proved to be superior with a very good recovery value (106 8%), while the latter system provided a dramatic decrease of recovery (20 4%). One possible explanation was the quite apolar nature of solvents that were used in fat extraction (Georgiou & Kapnissi-Christodoulou, 2013). This observation was in agreement with Dionisi et al., who also noted that Folchs method generated the least percentage of artifacts compared to Radins and Maxwells (Dionisi, Golay, Aeschlimann, & Fay, 1998).

3. Saponication Purication is the most critical step of the analytical procedure, since it demands selective isolation of the COPs from lipid fraction (Ruiz-Gutierrez & Perez-Camino, 2000). This clean-up step has been challenging for some decades since COPs are present in minor quantities in several foodstuffs (Tai, Chen, & Chen, 1999). If we also consider that more than 95% of dietary fat is in the form of triacylglycerols and the remaining part consists of phospholipids, esteried and free cholesterol, partial glycerides, free fatty acids and lipid-soluble vitamins, their isolation from this complex lipid

mixture is even more difcult (Ulberth and Rossler, 1998). An efcient purication procedure should, therefore, remove COPs from the bulk of lipids, as these compounds, if they contaminate the isolated COP containing fraction can interfere with COPs quantication (Cardenia et al. 2012; Chen et al. 2010). Saponication and/or SPE are usually employed in order to increase their relative concentration prior to chromatographic analysis. Saponication (alkaline hydrolysis) plays two important roles in the context of COPs analysis. Firstly, it removes the predominating acylglycerols of the crude lipid extract by converting them into water-soluble soaps and free glycerol, and secondly, it hydrolyzes cholesterol esters. In the saponication procedure, lipid extract is incubated in methanolic or ethanolic solution of NaOH or KOH for a period of time, and this is followed by liquid/liquid extraction for the concentration of the non-saponiable fraction (Guardiola et al. 2002). Much of the research regarding saponication has focused on artifact generation. The temperature of incubation and the alkalinity concentration were recognized as two critical parameters, which resulted in artifact formation by degrading unstable COPs, e.g. 7-ketocholesterol (Fig. 2) (Busch & King, 2009). In a recent study, Busch et al. investigated the stability of 7ketocholesterol in solution at various temperatures during saponication. Particularly, 7-keto was subjected to 1 M methanolic KOH at 37 C for 18 h or 45 C for 3 h, and the recovery results were compared to that obtained under control conditions at 24 C for 18 h. At temperatures greater than 24 C, the recovery of 7-keto was only 53% (37 C) and 49% (45 C) in relation to the control, suggesting sensibility at high temperatures. These ndings also indicated that the temperature of saponication affected at a greater extent the degradation in respect to the time of incubation. The same group reported further work on the stability of 7-keto in different alkaline conditions (3.6 M KOH for 3 h and 1 M KOH for 18 at 24 C). When the sample was treated with a stronger alkaline solution, the recovery dropped to 71%, revealing the liability of 7-keto (Busch & King, 2010). These observations are in agreement with a corresponding study performed by Park, Guardiola, Park, and Addis (1996) who suggested that 7-keto was sensitive to minor changes in temperature and it was also inuenced by the strength of the alkaline reagents that were used. However, it should be pointed out that the use of saponication is not prohibitive. As suggested by many researchers, if saponication is conducted at room temperature under mild conditions (1 M KOH), it can be considered safe with minimal artifact generation (Tai et al. 1999). For instance, Park et al. (1996) reported that careful implementation of cold saponication led to 97% recovery of 7keto, and thus, the degradation was practically negligible. Considering the above, many saponication protocols have been created at these conditions (Ferioli et al. 2008; Soto-Rodriguez et al. 2008; Cardenia et al. 2012; Baggio & Bragagnolo, 2006; Baggio, Miguel, & Bragagnolo, 2005; Bonoli et al. 2007; Morales-Aizpurua & Tenuta-Filho, 2005). In a method comparison involving three different saponication methods, the criteria for the selection of the optimum one were the recovery data obtained for each COP at three concentration levels (5, 10 and 20 lg) (Ubhayasekera et al. 2004). Methods A and B, were performed at room temperature for 18 h, but they differed

Fig. 2. Degradation of 7-keto during hot saponication.

922

C.A. Georgiou et al. / Food Chemistry 145 (2014) 918926

from each other, mainly on the type of solvent used in alkaline hydrolysis. In the former method, 95% ethanol was used, while in the latter, 95% methanol in water was used. In method C, the sample was incubated in ethanolic solution at 60 C for 1 h. Method A demonstrated the highest recoveries at all levels of spiked COPs. This indicated that this procedure was the most efcient for the COPs isolation from tallow. Therefore, the solvent of the alkaline solution can even affect the nal concentration. As expected, hot saponication provided the lowest recovery of 7-keto when compared to the other two methods. This enhanced the above notion that the degradation of COP occurred under thermal conditions. On the other hand, many investigators, in order to circumvent the problem of artifact generation, replaced the saponication step with SPE in order to isolate and enrich COPs. SPE is a faster and a milder technique in relative to saponication (Chen et al. 2010; Chen et al. 2012; Ulberth and Rossler, 1998). Purication by SPE will be discussed later. Direct saponication of the food matrix, without prior lipid isolation, is a viable option for sample preparation (Mariutti, Nogueira, & Bragagnolo, 2008; Mazalli, Sawaya, Eberlin, & Bragagnolo 2006; Saldanha, Benassi, & Bragagnolo, 2008; Saldanha & Bragagnolo, 2007; Saldanha et al. 2006). In a study by Saldanha et al. (2006), sample preparation was carried out using direct saponication, followed by extraction of the unsaponiable material. The investigation of different saponication factors, including percentage of KOH in water (2050%), volume of ethyl alcohol (26 mL) and time of saponication (1224 h), demonstrated that these parameters play a crucial role for reliable nal quantication. Total dissolution of the sample and prevention of emulsion were achieved by using a solution of 50% KOH in water added to 6 mL of ethyl alcohol for 22 h. Furthermore, they examined the type of

solvent (n-hexane and diethyl ether) and the number of extractions (three, four and ve) of the nonsaponiable matter in order to optimize the methodology of saponication. N-hexane was chosen as the ideal extraction solvent over diethyl ether due to higher recoveries. The analysis data of samples, which underwent four and ve successive extractions, indicated no signicant differences; hence, four extractions were selected. Similar results were obtained by Mariutti et al. (2008), who supported that hexane was the most appropriate extraction solvent, since fewer interference peaks were observed. One interesting study, which was cited in numerous research articles and reviews, was carried out by Dionisi et al. (1998). They compared four of the major extraction procedures used for COPs analysis in milk powders. Three of them involved a preliminary fat extraction (Folchs, Radins and Maxwells method) followed by a saponication step, while the last one involved a direct saponication step. Each method performance was evaluated according to the following four variables: standard deviation, coefcient of variation, artifact generation, and recovery. The direct method demonstrated the best compromise among the four alternatives, because it provided good repeatability and the lowest artifact formation. Furthermore, this procedure was superior to others in terms of time analysis and solvent quantity. However, the application of direct saponication in more complex matrices may be unsuitable (Busch & King, 2009). Lozada-Castro et al. (2011) observed that direct saponication, which was applied on cooked ham, produced emulsions that were difcult to handle. In addition, Georgiou & Kapnissi-Christodoulou, 2013 reported that the use of this method in meat samples provided chromatograms with interfering peaks, making the identication of 22R-hydroxycholesterol impossible.

Fig. 3. SPE elution systems examined by Guardiola et al. (1995) for the enrichment of COPs. ( COP extraction solvent).

C.A. Georgiou et al. / Food Chemistry 145 (2014) 918926 Table 2 The most frequently used SPE protocols with schematic representation.

923

Morgan et al. 1989 / Lai et al. 1995 a. b. c. d. 10 mL n-hexane/diethyl ether (95:5) 25 mL n-hexane/diethyl ether (90:10) 15 mL n-hexane/diethyl ether (80:20) 10 mL acetone or 5 mL acetone

References Chen et al. 2012, LozadaCastro et al. 2011; Chen et al. 2010; Lee et al. 2008; Lee et al 2006; Thurner et al. 2007. References Ubhayasekera et al. 2012; Ubhayasekera et al. 2010; Varleyen et al. 2003. References Cardenia et al. 2011; Boselli et al. 2010; Verardo et al. 2010; Boselli et al. 2009; References Rodriguez-Carpena et al. 2012; Clariana et al. 2011a; Broncano et al. 2009; Petron et al. 2003

a b
c d

Larkeson et al. 2000 / Ubhayasekera et al. 2004 a. b. d. 2 mL n-hexane/diethyl ether (75:25) 3 mL n-hexane/diethyl ether (60:40) 4 mL acetone or 4 mL acetone/methanol (60:40)

a, b, c d COPs

apolar lipids & cholesterol

Rose-Sallin et al. 1995 e. f. g. 6 mL n-hexane/ethyl acetate (95:5) 10 mL n-hexane/ethyl acetate (90:10) 10 mL acetone

e f
g

Ulberth et al. 1998 a. b. c. d. f. g.

10 mL n-hexane/diethyl ether (95:5) 30 mL n-hexane/diethyl ether (90:10) 10 mL n-hexane/diethyl ether (80:20) 10 mL methanol/acetone (60:20) & re-dissolved in n-hexane/ethyl acetate (90:10) 15 mL n-hexane/ethyl acetate (90:10) 10 mL acetone

e, f g COPs

apolar lipids & cholesterol

Ulberth, Morgan and Lai methods are applied for purication of the lipid extract while the others for the enrichment of the unsaponiable matter.

It is worth to mention that some authors proposed the replacement of cold saponication by transesterication, a method that uses milder conditions, in order to minimize the possibility of degradation of vulnerable COPs by prolonged contact with alkali. Nonetheless, there is a lack of studies performed by the use of this alternative procedure for the isolation of COPs from food matrices (Bodin & Diczfalusy, 2002; Caldern-Santiago, Peralbo-Molina, Priego-Capote, Luque, & de Castro, 2012; Ubhayasekera et al., 2004). As a conclusion, these studies underlined that the development of an analytical method involving saponication, is necessary to be evaluated in terms of all saponication factors including time, concentration of KOH, temperature and sample weight. The monitoring of artifact generation during preparation requires considerable attention in order to guarantee reliable results.

4. Solid phase extraction The majority of published articles report the use of SPE for COPs enrichment. SPE can be applied either directly to the crude lipid extract or to the non-saponiables for further purication (Guardiola et al., 2002). In regard to the polarity of different constituents of the lipid fraction, cholesterol esters and triacylglycerols are the least polar, phospholipids are the most polar and cholesterol and its oxidation products are in between (Ulberth and Rossler, 1998). Since SPE exploits differences in the polarity of interfering compounds and analytes, adequate separation is achieved by stepwise elution with solvents of increasing polarity (Busch and King, 2010). Thereby, enrichment of COPs can be successfully accomplished with a suitable choice of solvents. Generally, the purication with SPE is achieved by use of the following idea; retention of COPs on the stationary phase (usually polar) after loading the sample, removal of the interfering neutral lipids (cholesterol ester and triacylglycerols) by washing the column with an apolar solvent system and elution of the retained cholesterol derivatives with a medium polarity solvent. Phospholipids, which are the most polar

compounds, are strongly retained on the column (Caldern-Santiago et al. 2012). Usually, a chromatographic purication is preferred over saponication. (Chen et al.2010). Except from being simple, low-cost and non time-consuming, it minimizes artifact generation and/or breakdown of COPs since the sample is not subjected to any contact with hot or cold alkaline solution. Another important advantage is the capability of removing, to a large extent, cholesterol, which can interfere with the detection and quantication of COPs (Clariana et al., 2011; Guardiola, Codony, Rafecas, & Boatella, 1995). However, it should be mentioned that without previous saponication, the total amount of COPs in a food sample can be underestimated. This can happen because, under certain oxidation conditions, autoxidation of cholesterol fatty acid esters is faster than that of free cholesterol, and consequently, a signicant amount of COPs may exist as fatty acid esters, which cannot be quantied (Angulo, Romera, Ramirez, & Gil 1997). Therefore, saponication is recommended in order to avoid the analytical error mentioned above. Several SPE methods have been developed for selective isolation of COPs prior to chromatographic analysis. Many types of sorbents have been recommended with silica (Si) and aminopropyl (NH2) being the two predominant options, either alone or in combination. On the other hand, apolar sorbents like octadecyl-modied silica (ODS) have not found widespread use (Clariana et al., 2011; Ulberth and Rossler, 1998). There are also a few cases where silicic acid home-made columns were constructed for the purication of COPs (Nam et al., 2001). In this part of the review, different SPE methods are summarized with more emphasis given on those that are most frequently used, until nowadays. Guardiola et al. (1995) compared four different elution systems in order to nd the one that is the most effective in regard to recovery. The most effective provides maximum recovery of ve COPs from Si-SPE cartridge (7b-OH, a-CE, triol, 7-keto and 25-OH), and it simultaneously minimizes cholesterol recovery in oxysterol fraction (Fig. 3). Elution systems I and II suffered from low recoveries of

924

C.A. Georgiou et al. / Food Chemistry 145 (2014) 918926

Fig. 4. Work-up diagram of the 3-fold SPE procedure proposed by Janoskza 2010 for the recovery of COPs from the lipid extract (CHCl3: chloroform; DE: diethyl ether; HE: nhexane; IPA: 2-propanol; MeOH: methanol).

COPs, especially in the case of triol, which is the most polar COP. In this case, triol was not recovered at all, due to the low polarity of the extraction solvent. In contrast, systems III (a modied version of Morgan & Armstrong, 1989)) and IV, provided the highest recoveries of the different COPs due to an increase in polarity of the last solvent used for COPs elution (Morgan & Armstrong, 1989). As shown in Fig. 3, the two latter sequences also applied an additional washing step with n-hexane/diethyl ether (80:20, v/v) prior to nal isolation. This solvent mixture was essential for the optimum elimination of cholesterol from oxysterol fraction. System IV was nally considered as the optimum SPE method chosen because of better precision. However, Guardiolas SPE-method has not found a widespread use. In contrast, the original elution sequence suggested by Morgan et al. (1989) is frequently employed for the purication of lipid extract. Likewise, Larkeson, Dutta, and Hansson (2000) enriched COPs further from the non-saponiable fraction of minced meat products by a Si-SPE. Nevertheless, the elution proportions were entirely different, with a signicant reduction of solvent volumes, in relation to those mentioned above (Table 2) (Larkeson et al., 2000; Verleyen et al., 2003). Rose-Sallin, Huggett, Bosset, Tabacchi, and Fay (1995) described a clean-up procedure of the unsaponiables, which were obtained after direct saponication of milk powder, by using an NH2-SPE. The dried extract was dissolved in 1 mL of hexane/ethyl acetate (95:5 v/v) and was loaded into an NH2 cartridge, which was previously activated with 3 mL of hexane. The residual apolar compounds together with cholesterol were eliminated by washing the cartridge with the solvent sequence demonstrated in Table 2. The COPs fraction was eluted with acetone (Rose-Sallin, Huggett, Bosset, Tabacchi, & Fay, 1995). This method was widely used as a very efcient way to clean-up and extract COPs from the unsaponiables of several matrices. In a recent study performed by Caldern-Santiago et al. (2012), an NH2-SPE was also employed for COPs concentration from milk after saponication of the lipid extract. Particularly, the obtained unsaponiable fraction was reconstituted in 500 lL chloroform and was applied into the cartridge, which was preconditioned by two consecutive washing steps with n-hexane. After the elimination of apolar substances with hexane, different proportions of acetone, n-hexane and ethyl acetate were examined in order to

achieve the optimum elution of COPs. A mixture of n-hexane/ethyl acetate (50:50, v/v) was nally proven to be the best. In a comprehensive study, Ulberth and Rossler (1998) evaluated the efciency of seven SPE methods in terms of sample capacity, purity of the extract, and recovery of COPs from spiked milk fat. The variables, in this study, were not only the solvent eluent systems, but also the packing material of the cartridge. SPE clean-up protocol using NH2 sorbent, provided efcient removal of triglycerides, while it was incapable of eliminating cholesterol and/or partial glycerides from COPs extract. On the other hand, C18 cartridge led to greater contamination of COPs fraction. Same results with the latter were observed when a silica cartridge was used with n-hexane/diethyl ether mixtures at ratios of 8:2 and 1:1, respectively. However, the use of a silica cartridge in combination with the elution system of Morgan et al., resulted in the cleanest extract. In addition, they proposed a combination of the optimum Si-SPE followed by an NH2-SPE in order to reduce contaminants to a further degree. This conjugated method proved to be the most suitable for the removal of matrix components, and, at the same time, it provided high recovery of COPs. The main drawback was the low recovery of triol during NH2-SPE. The method mentioned above was also used by Petrn et al. (2003) and Rodriguez-Carpena, Morcuende, Petrn, and Estevez (2012) with little modications in the sample volume (10 mg extracted lipids instead of 500 mg). Another SPE combination study was proposed by Ferioli et al. (2008). COPs were enriched from unsaponiable material by use of two successive Si-SPE. The procedure was repeated twice in order to achieve a better purication of COPs-containing fraction from apolar materials and cholesterol. Particularly, each cartridge was pre-equilibrated with 3 ml of n-hexane, and after fat loading, it was washed with 3 ml of n-hexane/diethyl ether (3:1, v/v) and 3 ml of n-hexane/diethyl ether (3:2, v/v). The rst two fractions were discarded, and COPs were eluted with acetone/methanol (3:2, v/v). Janoszka (2010) adapted a multistage SPE-clean up procedure, based on the method described by Regueiro and Maraschiello (1997), in order to concentrate COPs from the lipid extract (Regueiro & Maraschiello, 1997). This unique threefold SPE procedure involved the isolation of sterols from the lipid fraction by using a

C.A. Georgiou et al. / Food Chemistry 145 (2014) 918926

925

combination of SPE mega elut columns prior to nal extraction of oxysterols. COPs were further puried with a Si-SPE column (500 mg) as illustrated in Fig. 4.

5. Conclusion In the light of the above, the sample preparation constitutes the most crucial step in COPs analysis. Even though in recent years numerous techniques of extraction and purication have been published, the choice between them remains a problem. The general conclusion of the current review is that all sample preparation parameters (i.e., type of solvents for the lipid extraction, the time of saponication and the elution systems or type of sorbents of SPE) must be evaluated prior to application. This aspect is essential in order to achieve an efcient recovery of COPs from the food matrix and to provide the most reliable results. The existence of several modied versions demonstrated that even minor changes, during the procedure, can inuence the nal quantication of COPs. Emphasis should also be given on artifact formation. This is a very important parameter that is usually omitted, leading to inaccurate data. In addition, interlaboratory comparisons of evaluation and quantitation data can be performed in order to improve the methodologies, eliminate the analytical errors, and consequently, develop a reliable and certied methodology for the analysis of COPs in food samples. References
Angulo, A. J., Romera, J. M., Ramirez, M., & Gil, A. (1997). Determination of Cholesterol Oxides in Dairy Products. Effect f Storage Conditions. Journal of Agricultural and Food Chemistry, 45, 43184323. Baggio, S. R., & Bragagnolo, N. (2006). Cholesterol oxide, cholesterol, total lipid and fatty acid contents in processed meat products during storage. LWT, 39, 513520. Baggio, S. R., Miguel, A. M. R., & Bragagnolo, N. (2005). Simultaneous determination of cholesterol oxides, cholesterol and fatty acids in processed turkey meat products. Food Chemistry, 89, 475484. Bielska, A. A., Schlesinger, P., Covey, D. F., & Ory1, D. S. (2012). Oxysterols as nongenomic regulators of cholesterol homeostasis. Trends in Endocrinology and Metabolism, 23, 99106. Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purication. Canadian Journal of Biochemistry and Physiology, 37, 911917. Bodin, K., & Diczfalusy, U. (2002). Analysis of cholesterol oxidation products in plasma, tissues and food. European Journal of Lipid Science and Technology, 104, 435439. Bonoli, M., Caboni, M. F., Rodriguez-Estrada, M. T., & Lercker, G. (2007). Effect of feeding fat sources on the quality and composition of lipids of precooked readyto-eat fried chicken patties. Food Chemistry, 101, 13271337. Boselli, E., Caboni, M. F., Rodriguez-Estrada, M. T., Toschi, T. G., Daniel, M., & Lercker, G. (2005). Photoxidation of cholesterol and lipids of turkey meat during storage under commercial retail conditions. Food Chemistry, 91, 705713. Boselli, E., Rodriguez-Estrada, M. T., Fedrizzi, G., & Caboni, M. F. (2009). Cholesterol photosensitized oxidation of beef meat under standard and modied atmosphere at retail conditions. Meat Science, 81, 224229. Boselli, E., Velazco, V., Caboni, M. F., & Lercker, G. (2001). Pressurized liquid extraction of lipids for the determination of oxysterols in egg-containing food. Journal of Chromatography A, 917, 239244. Bosselli, E., Rodriguez-Estrada, M. T., Ferioli, F., Caboni, M. F., & Lercker, G. (2010). Cholesterol photosensitized oxidation of horse meat slices stored under different packaging lms. Meat Science, 85, 500505. Broncano, J. M., Petrn, M. J., Parra, V., & Timn, M. L. (2009). Effect of different cooking methods on lipid oxidation and formation of free cholesterol oxidation products (COPs) in Latissimus dorsi muscle of Iberian pigs. Meat Science, 83, 431437. Brown, A. J., & Jessup, W. (1999). Oxysterols and atherosclerosis. Atherosclerosis, 142, 128. Brown, A. J., & Jessup, W. (2009). Oxysterols: Sources, cellular storage and metabolism, and new insights into their roles in cholesterol homeostasis. Molecular Aspects of Medicine, 30, 111122. Busch, T. P., & King, A. J. (2009). Artifact generation and monitoring in analysis of cholesterol oxide products. Analytical Biochemistry, 388, 114. Busch, T. P., & King, A. J. (2010). Stability of cholesterol, 7-ketocholesterol and bsitosterol during saponication: Ramications for artifact monitoring of sterol oxide products. Journal of the American Oil Chemists Society, 87, 955962.

Caldern-Santiago, M., Peralbo-Molina, ., Priego-Capote, F., Luque, Dolores, & de Castro, M. (2012). Cholesterol oxidation products in milk: Processing formation and determination. European Journal of Lipids Science and Technology, 114, 687694. Cardenia, V., Rodriguez-Estrada, M. T., Baldacci, E., Savioli, S., & Lercker, G. (2012). Analysis of cholesterol oxidation products by fast gas chromatography/mass spectrometry. Journal of Separation Science, 35, 424430. Cardenia, V., Rodriguez-Estrada, M. T., Cumella, F., Sardi, L., Casa, C. D., & Lercker, G. (2011a). Oxidative stability of pork meat lipids as related to high-oleic sunower oil and vitamin E diet supplementation and storage conditions. Meat Science, 88, 271279. Chen, Y. C., Chien, J. T., Inbaraj, B. S., & Chen, B. H. (2012). Formation and inhibition of cholesterol oxidation products during marinating of pig feet. Journal of Agricultural and Food Chemistry, 60, 173179. Chen, L. J., Lu, Y. F., Chien, J. T., & Chen, B. H. (2010). Formation and inhibition of cholesterol oxidation products in tea-leaf eggs during marinating. Journal of Agricultural and Food Chemistry, 58, 1046710474. Christie, W. W. (1993). Preparation of lipid extracts from tissues. Advances in Lipid Methodology, 2, 195213. Clariana, M., Daz, I., Srraga, C., & Garca-Regueiro, J. (2011). Comparison of the determination of eight cholesterol oxides in drycured shoulder by GCFID, GCMS, and GC tandem mass spectrometry. Food Analytical Methods, 4, 465474. Clariana, M., & Garca-Regueiro, J. A. (2011). Effect of high pressure processing on cholesterol oxidation products in vacuum packaged sliced dry-cured ham. Food and Chemical Toxicology, 49, 14681471. Derewiaka, D., & Obiedzinski, M. (2010). Cholesterol oxides content in selected animal products determined by GC-MS. European Journal of Lipid Science and Technology, 112, 11301137. Dionisi, F., Golay, P. A., Aeschlimann, J. M., & Fay, L. B. (1998). Determination of cholesterol oxidation products in milk powders: methods comparison and validation. Journal of Agricultural and Food Chemistry, 46, 22272233. Du, M., Nam, K. C., & Ahn, D. U. (2001). Cholesterol and lipid oxidation products in cooked meat as affected by raw-meat packaging and irradiation and by cookedmeat packaging and storage time. Journal of Food Science, 66, 13961401. Echarte, M., Ansorena, D., & Astiasarn, I. (2003). Consequences of microwave heating and frying on the lipid fraction of chicken and beef patties. Journal of Agricultural and Food Chemistry, 51, 59415945. Farese, R. V., & Walther, T. C. (2009). Lipid Droplets Finally Get a Little R-E-S-P-E-CT. Cell, 139, 855860. Ferioli, F., Caboni, M. F., & Dutta, P. C. (2008). Evaluation of cholesterol and lipid oxidation in raw and cooked minced beef stored under oxygen-enriched atmosphere. Meat Science, 80, 681685. Ferioli, F., Dutta, P. C., & Caboni, M. F. (2010). Cholesterol and lipid oxidation in raw and pan-fried minced beef stored under aerobic packaging. Journal of the Science of Food and Agriculture, 90, 10501055. Folch, J., Lees, M., & Stanley, G. H. S. (1957). A simple method for the isolation and purication of total lipides from animal tissues. Journal of Biological Chemistry, 226, 497509. Georgiou, C. A., & Kapnissi-Christodoulou, C. P. (2013). Qualitative and quantitative determination of COPs in Cypriot meat samples using HPLC. Determination of the most effective sample preparation procedure. Journal of Chromatographic Science, 51, 286291. Gill, S., Chow, R., & Brown, A. J. (2008). Sterol regulators of cholesterol homeostasis and beyond: The oxysterol hypothesis revisited and revised. Progress in Lipid Research, 47, 391404. Grau, A., Cobony, R., Grimpa, S., Baucells, M. D., & Guardiola, F. (2001). Cholesterol oxidation in frozen dark chicken meat: inuence of dietary fat source, and atocopherol and ascorbic acid supplementation. Meat Science, 57, 197208. Guardiola, F., Codony, R., Rafecas, M., & Boatella, J. (1995). Comparison of three methods for the determination of oxysterols in spray-dried egg. Journal of Chromatography A, 705, 289304. Guardiola, F., Dutta, P. C., Codony, R., & Savage, G. P. (2002). Cholesterol and Phytosterol Oxidation Products Analysis, Occurrence, and Biological Effects. AOCS Press, (Chapter 3). Hara, A., & Radin, N. S. (1978). Lipid extraction of tissues with a low toxicity solvent. Analytical Biochemistry, 90, 420426. Hur, S. J., Park, G. B., & Joo, S. T. (2007). Formation of cholesterol oxidation products (COPs) in animal products. Food Control, 18, 939947. Iverson, S. J., Lang, S. L. C., & Cooper, M. H. (2001). Comparison of the Bligh and Dyer and Folch Methods for Total Lipid Determination in a Broad Range of Marine Tissue. Lipids, 36, 12831287. Janoszka, B. (2010). 7-ketocholesterol and 7-hydroxycholesterol in pork meat and its gravy thermally treated without additives and in the presence of onion and garlic. Meat Science, 86, 976984. Jensen, M. ., & Mouritsen, O. G. (2004a). Lipids do inuence protein function - the hydrophobic matching hypothesis revisited. Biochimica et Biophysica Acta, 1666, 205226. Jusakul, A., Yongvanit, P., Loilome, W., Namwat, N., & Kuver, R. (2011). Mechanisms of oxysterol-induced carcinogenesis. Lipids in Health and Disease, 2011(10), 4451. Larkeson, B., Dutta, P. C., & Hansson, I. (2000). Effects of frying and storage on cholesterol oxidation in minced meat products. Journal of the American Oil Chemists Society, 77, 675680.

926

C.A. Georgiou et al. / Food Chemistry 145 (2014) 918926 Regueiro, J. A. G., & Maraschiello, C. (1997). Procedure for the determination of eight cholesterol oxides in poultry meat using on-column and solvent venting capillary gas chromatography. Journal of Chromatography A, 764, 279293. Reue, K. (2011). A Thematic Review Series: Lipid droplet storage and metabolism: from yeast to man. Journal of Lipid Research, 52, 18651868. Rodriguez-Carpena, J.-G., Morcuende, D., Petrn, M. J., & Estevez, M. (2012). Inhibition of cholesterol oxidation products (COPs) formation in emulsied porcine patties by phenolic-rich avocado (Persea americana Mill.) extracts. Journal of Agricultural and Food Chemistry, 60, 22242230. Rose-Sallin, C., Huggett, A. C., Bosset, J. O., Tabacchi, R., & Fay, L. B. (1995). Quantication of cholesterol oxidation products in milk powders using [2H7] cholesterol to monitor cholesterol autoxidation artifacts. Journal of Agricultural and Food Chemistry, 43, 935941. Ruiz-Gutierrez, V., & Perez-Camino, M. C. (2000). Update on solid-phase extraction for the analysis of lipid classes and related compounds. Journal of Chromatography A, 885, 321341. Saldanha, T., Benassi, M. T., & Bragagnolo, N. (2008). Fatty acid contents evolution and cholesterol oxides formation in Brazilian sardines (Sardinella brasiliensis) as a result of frozen storage followed by grilling. LWT, 41, 13011309. Saldanha, T., & Bragagnolo, N. (2007). Cholesterol oxidation is increased and PUFA decreased by frozen storage and grilling of atlantic hake llets (Merluccius hubbsi). Lipids, 42, 671678. Saldanha, T., Sawaya, A. C. H. F., Eberlin, M. N., & Bragagnolo, N. (2006). HPLC separation and determination of 12 cholesterol oxidation products in sh: Comparative study of RI, UV, and APCI-MS detectors. Journal of Agricultural and Food Chemistry, 54, 41074113. Sampaio, G. R., Bastos, D. H. M., Soares, R. A. M., Queiroz, Y. S., & Torres, E. A. F. S. (2006). Fatty acids and cholesterol oxidation in salted and dried shrimp. Food Chemistry, 95, 344351. Savage, G. P., Dutta, P. C., & Rodriguez-Estrada, M. T. (2002). Cholesterol oxides: their occurrence and methods to prevent their generation in foods. Asia Pasic Journal of Clinical Nutrition, 11, 7278. Sieber, R. (2005). Oxidised cholesterol in milk and dairy products. International Dairy Journal, 15, 191206. Soto-Rodriguez, I., Campillo-Velazquez, J. P., Ortega-Martinez, J., Rodriguez-Estrada, M. T., Lercker, G., & Garcia, S. H. (2008). Cholesterol oxidation in traditional Mexican dried and deep-fried food products. Journal of Food Composition and Analysis, 21, 489495. Tai, C.-Y., Chen, Y. C., & Chen, B. H. (1999). Analysis, formation and inhibition of cholesterol oxidation products in foods: An overview (Part I). Journal of Food and Drug Analysis, 7, 243257. Ubhayasekera, J. K. A. S., Jayasinghe, P., Ekanayake, S., & Paresh, C. D. (2012). High cholesterol oxidation in pickled mackerel (Rastrelliger kanagurta) from Sri Lanka. European Journal of Lipid Science and Technology, 114, 695700. Ubhayasekera, S. J. K. A., Tres, A., Cobony, R., & Dutta, P. C. (2010). Effects of different levels of trans fatty acids and oxidised lipids in diet on cholesterol and cholesterol oxidation products formation in rabbit. Food Chemistry, 121, 11981202. Ubhayasekera, S. J. K. A., Verleyen, T., & Dutta, P. C. (2004). Evaluation of GC and GCMS methods for the analysis of cholesterol oxidation products. Food Chemistry, 84, 149157. Ulberth, F., & Rossler, D. (1998). Comparison of solid phase extraction methods for the cleanup of cholesterol oxidation products. Journal of Agricultural and Food Chemistry, 46, 26342637. Verardo, V., Pasini, F., Iafelice, G., Messia, M. C., Marconi, E., & Caboni, M. F. (2010). Inuence of storage conditions on cholesterol oxidation in dried egg pasta. Journal of Agricultural and Food Chemistry, 58, 35863590. Verleyen, T., Dutta, P. C., Verhe, R., Dewettinck, K., Huyghebaert, A., & Greyt, D. W. (2003). Cholesterol oxidation in tallow during processing. Food Chemistry, 83, 185188. Vicente, S. J. V., Sampaio, G. R., Ferrari, C. K. B., & Torres, E. A. F. S. (2012). Oxidation of cholesterol in foods and its importance for human health. Food Reviews International, 28, 4770. Vicente, S. J. V., & Torres, E. A. F. S. (2007). Formation of four cholesterol oxidation products and loss of free lipids, cholesterol and water in beef hamburgers as a function of thermal processing. Food Control, 18, 6368. Yen, T. Y., Inbaraj, B. S., Chien, L. T., & Chen, B. H. (2010). Gas chromatography-mass spectrometry determination of conjugated linoleic acids and cholesterol oxides and their stability in a model system. Analytical Biochemistry, 400, 130138.

Lee, H.-W., Chien, J.-T., & Chen, B.-T. (2006). Formation of cholesterol oxidation products in marinated foods during heating. Journal of Agricultural and Food Chemistry, 54, 48734879. Lee, H. W., Chien, J. T., & Chen, B. H. (2008). Inhibition of cholesterol oxidation in marinated foods as affected by antioxidants during heating. Food Chemistry, 108, 234244. Leonarduzzi, G., Sottero, B., & Poli, G. (2002). Oxidized products of cholesterol: dietary and metabolic origin, and proatherosclerotic effects (review). Journal of Nutritional Biochemistry, 13, 700710. Lordan, S., Mackrill, J. J., & OBrien, N. M. (2009). Oxysterols and mechanisms of apoptotic signaling: implications in the pathology of degenerative diseases. Journal of Nutritional Biochemistry, 20, 321336. Lozada-Castro, J. J., Gil-Diaz, M., Santos-Delgado, M. J., Rubio-Barroso, S., & PoloDiez, L. M. (2011). Effect of electron-beam irradiation on cholesterol oxide formation in different ready-to-eat foods. Innovative Food Science and Emerging Technologies, 12, 519525. Manini, P., Andreoli, R., Careri, M., Elviri, L., & Musci, M. (1998). Atmospheric pressure chemical ionization liquid chromatography/mass spectrometry in cholesterol oxide determination and characterization. Rapid Communications in Mass Spectrometry, 12, 883889. Mariutti, L. R. B., Nogueira, G. C., & Bragagnolo, N. (2008). Optimization and validation of analytical conditions for cholesterol oxides extraction in chicken meat using response surface methodology. Journal of Agricultural and Food Chemistry, 56, 29132918. Maxwell, R. J., Mondimore, D., & Tobias, J. (1986). Rapid method for the quantitative extraction and simultaneous class separation of milk lipids. Journal of Dairy Science, 69, 321325. Mazalli, M. R., & Bragagnolo, N. (2007). Effect of storage on cholesterol oxide formation and fatty acid alterations in egg powder. Journal of Agricultural and Food Chemistry, 55, 27432748. Mazalli, M. R., & Bragagnolo, N. (2009). Increase of cholesterol oxidation and decrease of PUFA as a result of thermal processing and storage in eggs enriched with n-3 fatty acids. Journal of Agricultural and Food Chemistry, 57, 50285034. Mazalli, M. R., Sawaya, A. C. H. F., Eberlin, M. N., & Bragagnolo, N. (2006). HPLC method for quantication and characterization of cholesterol and its oxidation products in eggs. Lipids, 41, 615622. Morales-Aizpurua, I. C., & Tenuta-Filho, A. (2005). Oxidation of cholesterol in mayonnaise during storage. Food Chemistry, 89, 611615. Morgana, J. N., & Armstrong, D. J. (1989). Wide-bore capillary gas chromatographic method for quantication of cholesterol oxidation products in egg yolk powder. Journal of Food Science, 54, 427430. Nam, K. C., Du, M., Jo, C., & Ahn, D. U. (2001). Cholesterol oxidation products in irradiated raw meat with different packaging and storage time. Meat Science, 58, 431435. ski, M., & Koczak, T. (2006). The effect of water activity on Obara, A., Obiedzin cholesterol oxidation in spray- and freeze-dried egg powders. Food Chemistry, 95, 173179. Olkkonen, V. M., Baslas, O., & Nissil, E. (2012). Oxysterols and Their Cellular Effectors. Biomolecules, 2, 76103. Olsen, B. N., Schlesinger, P. H., & Baker, N. A. (2009). Perturbations of membrane structure by cholesterol and cholesterol derivatives are determined by sterol orientation. Journal of American Chemical Society, 131, 48544865. Olsen, B. N., Schlesinger, P. H., Ory, D. S., & Baker, N. A. (2012). Side-chain oxysterols: From cells to membranes to molecules. Biochimica et Biophysica Acta, 1818, 330336. Otaegui-Arrazola, A., Menendez-Carreo, M., Ansorena, D., & Astiasarn, I. (2010). Oxysterols: A world to explore. Food and Chemical Toxicology, 48, 32893303. Park, W. T., Guardiola, F., Park, S. H., & Addis, P. B. (1996). Kinetic evaluation of 3bhydroxycholest-5-en-7-one (7-ketocholesterol) stability during saponication. Journal of the American Oil Chemists Society, 73, 623629. Petrn, M. J., Garcia-Regueiro, J. A., Martin, L., Muriel, E., & Antequera, T. (2003). Identication and quantication of cholesterol and cholesterol oxidation products in different types of Iberian hams. Journal of Agricultural and Food Chemistry, 51, 57865791. Pignoli, G., Rodriguez-Estrada, M. T., Mandrioli, M., Barbanti, L., Rizzi, L., & Lercker, G. (2009). Effects of different rearing and feeding systems on lipid oxidation and antioxidant capacity of freeze-dried egg yolks. Journal of Agricultural and Food Chemistry, 57, 1151711527. Poli, G., Sottero, B., Gargiulo, S., & Leonarduzzi, G. (2009). Cholesterol oxidation products in the vascular remodeling due to atherosclerosis. Molecular Aspects of Medicine, 30, 180189. Raith, K., Brenner, C., Farwanah, H., Mller, G., Eder, K., & Neubert, R. H. H. (2005). A new LC/APCI-MS method for the determination of cholesterol oxidation products in food. Journal of Chromatography A, 1067, 207211.