Determination of neophytadiene in the subcutaneous fat of Iberian pigs from different feeding systems

El objetivo del trabajo se centró en determinar el conteni­ do de un hidrocarburo ramificado (neofitadieno) en la grasa subcutánea del cerdo ibérico, y su relación con el tipo de ali­ mentación recibida durante la etapa de cebo previa al sacrifi­ cio (en la fase final de cebo). Para ello, se estudiaron treinta y seis lotes de cerdos Ibéricos, pertenecientes a las cuatro categorías de calidad en base a la alimentación de los ani­ males establecidas en la Norma de Calidad (Bellota, Recebo, Campo y Cebo). Estas categorías se diferencian en el grado de aprovechamiento de los recursos naturales de la dehesa (bellotas y hierba) y en el nivel de sustitución de los mismos por piensos concentrados. Para el aislamiento e identificación de los hidrocarburos se utilizaron técnicas de SPE combinadas con GC­MS. Los lotes de Recebo y Bellota presentaron los mayores niveles de neofitadieno, seguidos por los lotes de Campo, y por último los lotes de Cebo. Se observó una gran variabilidad en los niveles de neofitadieno entre los lotes pertenecientes a una misma categoría de ali­ mentación, sobre todo en los lotes de Bellota, Recebo y Campo, por lo que es difícil una correcta clasificación de los animales en las tres categorías en base al contenido en neofitadieno. Sin embargo, la clasificación dentro de la cate­ goría de cebo se hace de forma correcta cuando los cerdos son producidos en condiciones intensivas.


INTRODUCTION
The unsaponifiable fraction of oils and fats contains small quantities of saturated and unsaturated hydrocarbons.These compounds normally occur in animal tissues and are derived mainly from plant origin, being important components of vegetable wax (PostBeittenmiller, 1996).For that reason, several authors have related the presence of specific hydrocarbons in animal tissues to the feed, mostly of plant origin, consumed by the animals (Tulliez and Bories, 1978;Tejeda et al., 2001b;Gamero et al., 2006).Hydrocarbons are inert in the digestive tract and are little modified during digestion and metabolism (Mayes et al., 1988;Rembold et al., 1989); however, they are absorbed at low doses by the mammalian small intestine (Tulliez and Bories, 1975) and the absortion decreases with the increase of chain length of the hydrocarbons (Mayes and Lamb, 1984).Several authors have described saturated, unsaturated and branched hydrocarbons in ruminant animal tissues (Bandurski and Nagi, 1975;Lintas et al., 1979).In monogastric animals, like the Iberian pig, nalkanes (parafinic hydrocarbons) and nalkenes, from nC 12 to nC 33 have been identified in subcutaneous (Tejeda et al., 1999;Gamero et al., 2006) and intramuscular (Tejeda et al., 2001a) fat, with the odd numbered carbon chains being the most abundant.Petrón et al. (2004) have also identified nalkanes and nalkenes in intramuscular fat from A. Hernández-MAtAMoros, e. González, J.M. GArcíA-cAsco And J.F. teJedA

Animal and diets
In this study a total of 725 Iberian pigs separated into 36 batches according to the feeding system during the final fattening period of pigs (Bellota, Recebo, Campo and Cebo) from November to March in three consecutive years (2008/2009, 2009/2010 and 2010/2011) was analyzed (see Table 1).A detailed description of these batches of animals can be found in a parallel paper by GarcíaCasco et al., 2013.After slaughtering by electric stunning and exsanguinations at different local slaughterhouses, backfat samples were taken 10 cm above the tail, were vacuum packaged and maintained at -20 °C until analysis.

Methods
The hydrocarbon fraction of subcutaneous fat was determined by a modification of the method of Tejeda et al. (2001b) and applied to intramuscular fat.This modification, described by Fernández (2007), involves the lipid extraction method, saponification time, SPE purification and gas chromatography conditions.

Lipid extraction
Samples of subcutaneous fat were trimmed and minced and lipids were extracted by heating in a microwave oven for 2 minutes at 550 W according to the method described by De Pedro et al. (1997).

Saponification
Two grams of extracted lipids were saponified by refluxing for 15 min at 100 °C with 70 mL of 15% KOH in ethanol (w/v).The warm solution was transferred to a separating funnel, 70 mL of distilled water were added and the unsaponifiable fraction was extracted with 70 mL of hexane.The organic drycured hams.Other studies have focused on the analysis of the branched hydrocarbon fraction in Iberian pig adipose tissues.In this sense, Tejeda et al. (2001b) identified neophytadiene, a diterpenoid hydrocarbon that belongs to the group of compounds known as phytanes, in intramuscular lipids from B. femoris muscle and Petrón et al. (2005) in intramuscular lipids from drycured ham.
The analysis of the hydrocarbons in the unsaponifiable fraction is difficult as they are present at low concentrations (Tejeda et al., 1999), usually below the limit of detection of the analytical methods (Gamero et al., 2006).Chromatographic techniques are adequate to separate and quantify these compounds.Some authors (Bernardini et al., 1982;Tejeda et al., 1999) use thin layer chromatography (TLC) to isolate nalkanes and nalkenes.The use of solid phase extraction (SPE) technique, instead of TLC methods, as concentration step allows a substantial reduction in analysis time.Then, gas chromatography coupled to mass spectrometry (GCMS) can be applied to identify and quantify hydrocarbons (Tejeda et al., 2001a;Petrón et al., 2004).More recently, an offline combination of HPLC and gas chromatography multistage mass spectrometry (GCMS 3 ) has been used to detect and quantify the hydrocarbon fraction (Narváez Rivas et al., 2008).Moreover, purge and trap GC MS combined with chemometrical tools have been applied to the authentication of the fattening systems of Iberian pigs (NarváezRivas et al., 2010 and2011).
The analysis of the hydrocarbon fraction is of great interest since it could contribute to differentiating among the types of diet given to the Iberian pigs previous to slaughter.Spanish legislation classifies Iberian pig products into different categories depending on the feeding regime and production system involved (BOE, 2007): Bellota (i.e.freerange animals fed on acorn and pasture), Recebo (i.e.animals fed on acorns and pasture supplemented with concentrate feeds in an outdoor system), Campo (i.e.animals fed on concentrate feeds in an outdoor system) and Cebo (i.e.animals fed on concentrate feeds in an intensive system).The Bellota category is the highest priced in the market, followed by Recebo, Campo and then by Cebo.Therefore, different methods, based on chemical analysis of different hydrocarbon fraction compounds, as nalkanes (Tejeda et al., 2001a;Petrón et al., 2004), nalkenes (VieraAlcaide et al., 2009), neophytadiene (Tejeda et al., 2001b;Petrón et al., 2005), entkaurene (NarváezRivas et al., 2008) or volatile compounds (NarváezRivas et al., 2010 and2011) have been carried out to differentiate among the products obtained from these fattening systems.
The aim of the present study was to determine the content of a branched hydrocarbon of the unsaponifiable fraction (neophytadiene) from subcutaneous fat, and its relationship with the management feeding system of Iberian pigs according with the four categories (Bellota, Recebo, Campo and Cebo) as defined by the Spanish legislation.The relative response factor was close to the unit.Results were expressed as peak area × 100/ internal standard peak area (relative area units).

Statistical analysis
The results were subjected to analysis by a KruscalWallis test for k samples, which is the nonparametric equivalence of one way ANOVA.An individual animal was the experimental unit for analysis of all data.The model used involved the category of feeding system.MannWhitney post hoc tests were used for comparison among treatments.This statistical procedure was carried out using software Statistical Package for the Social Sciences (SPSS) package (v.19).

Identification of hydrocarbons
Figure 1 shows the GCMS chromatogram of the hydrocarbon fraction isolated from subcutaneous layer was washed three times with 50 mL of distilled water, then dried over anhydrous sodium sulphate and subsequently concentrated to 2 mL.

Solid phase extraction (SPE)
The concentrate was transferred onto an amino propyl 500 mg minicolum using 30 mL of hexane for hydrocarbon elution.After evaporating to dryness under vacuum, the residue was dissolved in 1 mL of hexane, and 50 µL were taken for the gas chromatographic analysis.

Identification and quantitative determination of hydrocarbons
Hydrocarbons were identified and quantified on an Agilent 6890 chromatograph, using a Hewlett Packard fused silica capillary column (12 m × 0.2 mm i.d.) equipped with an Agilent 5973 mass selective detector.The oven program was from 100 to 196 °C at 6 °C min -1 , and from 196 °C to 280 °C at 30 °C min -1 , finally the oven was held at 280 °C for 15 min.Total analysis time was 33.8 min.Injector temperature was 260 °C, and the GCMS transfer line temperature was held at 280 °C.The split ratio was 1:25, inlet pressure 14 psi, and the injected volume was 2 µL.The mass spectrometer operated in the electron impact mode with an electron energy of 70 eV, a multiplier voltage of Figure 1 GCMS chromatograms of hydrocarbons identified in subcutaneous fat.1: dodecene (nC 12:1 ); 2: dodecane (nC 12 ); 3: tridecane (nC 13 ); 4: tetradecene (nC 14:1 ); 5: tetradecane (nC 14 ); 6:pentadecane (nC 15 ); 7: hexadecene (nC  is a branched hydrocarbon belonging to the group of compounds known as phytanes, and is abundant in plant cuticular waxes (Lintas et al., 1979).Other authors, using HPLC technique for the purification of the hydrocarbon fraction, followed by GCMS 2 , suggest that this compound is an isomeric form of neophytadiene (Gamero et al., 2006).Previous papers reported the presence of higher levels of neophytadiene in pasture, while this compound was not present in the acorns or concentrate consumed by Iberian pigs during the fattening period.Therefore, neophytadiene can be used as a key parameter to differentiate among types of diet given to the animals previous to slaughter (Tejeda et al., 2001b).These authors found a clear effect of feeding regime of the Iberian pig on the concentration of neophytadiene in the intramuscular lipids, with higher contents in intramuscular lipid from pigs raised extensively with acorn and pasture for a longer period of time.In contrast, neophytadiene was not identified in Iberian pigs fed on an intensive system with a concentrated diet (Tejeda et al., 2001b, Petrón et al., 2005).

Neophytadiene subcutaneous fat contents
Table 1 shows the neophytadiene contents in the subcutaneous fat from the different batches of Iberian pigs which were studied based on feeding systems (Bellota, Recebo, Campo and Cebo).Fourteen batches of Iberian pigs raised on the Bellota system were studied, which showed levels of neophytadiene ranging from 2.4 to 15.4 and a mean value of 6.8±3.1 relative area units (expressed as peak area × 100/internal standard peak area).In both Recebo and Campo systems eight batches of animals were studied with contents of neophytadiene from 3.4 to 17.4, and 0.8 to 9.4, being 7.6±6.4and 3.8±2.4 the mean of the batches, respectively.Finally, six batches were analyzed in the Cebo category, where neophytadiene varied between 0.0 and 0.9, with 0.3±0.2relative area units as the mean value of the group.The results showed a great variability between batches for a same category of feeding system, mainly in the Bellota, Recebo and Campo systems, which are feeding systems based on extensive or semiextensive rearing conditions, with free access of the pigs to pasture.Regarding the Cebo system, neophytadiene was not identified in GCMS chromatograms in four batches of animals studied, which could be explained by the fact that pigs are maintained in intensive conditions throughout the production period.However, two lots of animals presented a low neophytadiene content, which can be explained because the pigs were raised with pasture in semiextensive conditions before the end of fattening in intensive conditions.Neophytadiene is the most abundant hydrocarbon in pasture, but is not present in acorn and concentrate feeds (Tejeda et al., 2001b), thus the presence of neophytadiene in subcutaneous fat is associated with the consumption of pasture.In this sense, Tejeda et fat.The peaks of chromatograms were identified comparing the GCMS spectra with those from the National Institute of Standard and Technology (NIST) libraries.Both nalkanes and nalkenes were identified, being nalkenes of even chain between nC 12:1 and nC 20:1 the most abundant, followed by the correspondent nalkanes, in accordance with the results obtained by Gamero et al. (2006) in subcutaneous fat, and by Petrón et al. (2004) in intramuscular lipids from Iberian drycured ham.Hydrocarbons are preferentially concentrated in subcutaneous adipose tissues, followed by perirrenal and intramuscular fat (Lintas et al., 1979).Evennumbered (enca) and oddnumbered carbon atoms (onca) nalkane in the nC 12 to nC 33 range were present in the unsaponifiable fraction of subcutaneous fat.However, odd chain nalkenes were not identified in chromatograms, in agreement with previous bibliography on the Iberian pig (Petrón et al., 2004 and2005;Gamero et al., 2006).Some other peaks were observed in the chromatograms and were identified as branched hydrocarbons, as squalene and neophytadiene, among others.The mass spectrum obtained when analyzing the peak at retention time 15.31 (N) was (except for the molecular ion, only fragments with relative abundances greater than 15% are given): m/z 53 (15%), 55 (70%), 56 (20%), 57 (74%), 67 (57%), 68 (100%), 69 (66%), 70 (16%), 71 (38%), 79 (19%), 81 (58%), 82 (72%), 83 (48%), 95 (85%), 96 (27%), 97 (34%), 109 (27%), 111 (17%), 123 (52%), 124 (20%), 278 (3% M+).The rest of the branched hydrocarbons corresponded to those having a linear or cyclical chain with one or more branched methyl groups, which could explain, together with their low concentrations, that several of these compounds remain unidentified.In the literature, several authors have reported the same problems in identifying some of the branched hydrocarbons in many animal tissues (Bastic et al., 1989;Berdagué and García, 1990;García et al., 1991;Petrón et al., 2005).The existence of branched hydrocarbons in bovine (Lintas et al., 1979) and pig (Bastic et al., 1989;Bernardini et al., 1982) tissues have been reported.Berdagué and García (1990) analyzed volatiles in drycured hams and found a number of branched hydrocarbons, while branched hydrocarbons of low molecular weight were isolated from Iberian drycured ham by García et al. (1991) andRuiz et al. (1999).Tejeda et al. (1999) also found these compounds in adipose tissue from Iberian drycured ham.Squalene is the major branched hydrocarbon identified in chromatograms, in agreement with previous studies on the Iberian pig (Tejeda et al., 2001b;Petrón et al., 2005;Gamero et al., 2006).However, squalene presence in adipose tissues is mainly an intermediary product in cholesterol synthesis (Stryer, 1990), which explains the absence of differences in concentration of this compound in adipose tissues of Iberian pigs due to feeding system (Tejeda et al., 2001b;Petrón et al., 2005).Neophytadiene (3methylene7,11,15trimethylhexadecen1ene) A. Hernández-MAtAMoros, e. González, J.M. GArcíA-cAsco And J.F. teJedA was consumed by the first category compared to second one.Therefore, the great variability observed in this study between batches from a same feeding category could be directly related with the environmental variability of the «dehesa» ecosystem.Several authors have evidenced the variations between montanera seasons in different years of study in the same geographical area and between distinct geographical areas, which demonstrates that the extensive rearing system is a dynamic and variable element (GeaIzquierdo et al., 2006;Tejerina et al., 2010).These dynamic changes could be the explanation for the variability in the tissue composition of Iberian pigs and therefore on the quality of meat products from Iberian pigs reared according to this unique freerange system.
Table 2 shows the comparison of neophytadiene content among the four categories of feeding systems studied (KruskalWallis test: H (3, N=730)=362.5; p=0.000).No significant differences were observed between the Bellota and Recebo al. (2001b) reported that the neophytadiene content in intramuscular lipids from B. femoris m. from pigs raised extensively for a long period of time is higher than in pigs raised for a short period with acorn and pasture, presumably because more pasture Histograms of relative frequencies of neophytadiene contents (relative area units expressed as peak area × 100/internal standard peak area) in subcutaneous fat from Iberian pigs from the four categories of feeding systems studied (Bellota, Recebo, Campo and Cebo).
batches.However, animals raised in these two range systems presented significantly higher (p<0.001)contents of this branched hydrocarbon than Campo and Cebo Iberian pigs.Moreover, Cebo batches exhibited a significantly lower neophytadiene content, compared to the other three categories.These results are in agreement with previous works by Tejeda et al. (2001b) and Petrón et al. (2006) when comparing Iberian pigs raised according to Cebo and Montanera systems, in intramuscular lipids from fresh and drycured products, respectively.
Figure 2 represents the distribution of the samples in a relative frequency histogram for neophytadiene content for the different categories of feeding system studied.The results showed that when neophytadiene is not present in subcutaneous fat samples, they can only belong to Iberian pigs produced in intensive conditions without the consumption of pasture (Cebo category).When neophytadiene content is present in small amounts (between 0.0 and 1.3 relative area units), only 2% and 1% of the animals are classified as Bellota and Recebo categories, respectively; 33% of samples are classified as Campo; and finally, 96% of samples are classified as Cebo.

CONCLUSIONS
The presence of branched hydrocarbons, mainly neophytadiene, in the subcutaneous fat from Iberian pigs is related to their rearing system during the fattening period prior to slaughter.However, the levels of neophytadiene are variable among batches of the same category of feeding, as a consequence of the great variability of the "dehesa" system in different geographical areas and different years.Therefore, it is not possible to differentiate between Iberian pigs from Bellota and Recebo categories.The correct classification of pigs according to feeding systems is better in Campo and Cebo categories.Only Iberian pigs produced in intensive conditions throughout the production period were correctly assigned in all cases to the Cebo category.
in the subcutaneous fat of iberian pigs from Different feeDing systems

Table 1 Neophytadiene contents (relative area units expressed as peak area × 100/internal standard peak area) from subcutaneous fat from the different batches of Iberian pigs fed on Bellota, Recebo, Campo and Cebo systems
Determination of neophytaDiene in the subcutaneous fat of iberian pigs from Different feeDing systems .Hernández-MAtAMoros, e. González, J.M. GArcíA-cAsco And J.F. teJedA 1756 V and collected data at 1 scan/s over a m/z range from 40 to 300.Spectra were compared with those of the standards and spectra from the NIST (National Institute of Standards and Technology) library.Quantitative determination was performed by adding an appropriate amount of the internal standard (nnonadecane, nC 19 ) to the final extract.
a Standard deviation.bCoefficient of variation.A