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for cholesterol determination ( Thompson and Merola,1993 ). However, most of these methods were developed toquantify cholesterol content in blood serum, milk, lipid, orother lipid-based samples ( Fletouris et al., 1998 ; Russoet al., 2005 ; Kaneda et al., 1980 ; Punwar, 1975 ; Hwanget al., 2003 ; Galanos et al., 1964 ; Stein et al., 1991 ;

Cardenas et al., 1995 ; Abidi, 2004 ; Abidi, 2001 ; Lohningeret al., 1990 ).Due to the development of GC capillary columns with

high heat tolerance (280300 1 C) and increased resolution,the separation of free cholesterol became possible. Mostattention is now directed toward development of methodsto increase recovery efciency and stability ( Thompson andMerola, 1993 ). Because the direct saponication techniqueof the AOAC Ofcial Method 994.10 is relatively effective(Klatt et al., 1995 ; Adams et al., 1986 ), this study wasperformed to modify this AOAC procedure and toeliminate unnecessary steps to create a simple, precise,accurate, and productive method to quantify cholesterol inmeat products. The modied procedure was then testedwith the longissimus muscle (LM) from three divergentbreeds of cattle.

2. Materials and methods

2.1. Validation of cholesterol quantication technique

2.1.1. Standard reference material (SRM)Standard Reference Material 1546 (SRM-1546) was

purchased from National Institute of Standards andTechnology (NIST). The certied cholesterol content of

this material was 75 7 7.2mg/100 g, which was quantita-tively analyzed using an isotope dilution/gas chromato-graphy/mass spectrometry (ID/GC/MS) method developedat NIST (2004) . SRM was carefully mixed, frozen inliquid nitrogen, and homogenized to a powder to use forprocedure development.

2.1.2. Beef longissimus muscle samplesThree samples of beef LM with fat content determined to

be from 2.4% to 9.3% were used. Muscle samples werecollected between the 10th and 13th ribs, frozen immedi-ately using liquid nitrogen and stored in a 80 1 C freezerfor subsequent sample preparation. Frozen samples werelater trimmed of all external fat, leaving only white ecks of marbling within muscle bundles. Trimmed samples werechopped, homogenized to nely divided muscle powder,and stored at 80 1 C for subsequent analysis.

2.1.3. Standard materialsCholesterol standards were used at concentration of

0.0125, 0.025, 0.05, and 0.1 mg/mL to construct thestandard curve for cholesterol quantication. An internalstandard, 5 a -cholestane (Sigma-Aldrich, MO, USA), wasused as a correction factor to standardize injectionerrors. All standards were diluted in high-grade toluene(Sigma-Aldrich).

2.1.4. Experimental designTwo SRM-1546 samples (designated SRM-1546-1 and

SRM-1546-2) and three LM samples (designated LM-1,LM-2, and LM-3) were evaluated as nine sets of replicates.Samples used for the recovery test received 1 mg of freecholesterol (Sigma-Aldrich) added to each of the control

samples and were characterized with letter R.Six replicates were withdrawn per every set of samplesused for validation, with a sample size of 1 g being utilized.All samples were analyzed over four consecutive days. If more than one set of samples was prepared on the sameday, they were processed separately with at least a 6-hinterval in between. The extractable fat content of samplesvaried from 2.4% (LM-3) to 21.0% (SRM-1546).

2.1.5. Method accuracyThe accuracy of the method was determined using both

non-addition and addition of free cholesterol (Sigma-Aldrich) to sample matrices. For the non-addition of freecholesterol, two sets of SRM-1546 samples (SRM-1546-1and SRM-1546-2) were analyzed for comparison with thecertied concentration ( NIST, 2004 ). The other four sets of samples, designated SRM-1546R, LM-1R, LM-2R, andLM-3R, were used to determine the recovery performanceof the method. These four sets were all treated as unknownsamples and the actual identity was unknown to the analystuntil the data was collected.

2.1.6. Method repeatability precisionFive sets of SRM-1546 (SRM-1546-1 and SRM-1546-2,

of which data were previously collected) and LM

(LM-1, LM-2, and LM-3) samples were used to calcu-late the repeatability of the modied procedure usingcoefcient of variation (CV, %) of each data set tocompare with the expected precision, which is a functionof concentration (mass fraction, %), according to theAOAC Guidelines for Single Laboratory Validationof Chemical Methods for Dietary Supplements andBotanicals ( AOAC, 2002 ).

2.1.7. Method detection limitThe detection limit was determined using dilution of

standard solution. The cholesterol standard was dilutedand subjected to GC analysis until the responding signalwas twice as high as the noise signal and detectable at theretention time corresponding to that of free cholesterolstandard. The cholesterol detection limit was calculated asif the cholesterol amount was from 1 g of fresh tissue.

2.1.8. Modied cholesterol analysis method The AOAC Ofcial Method 994.10, Cholesterol in

Foods, Direct Saponication-Gas ChromatographicMethod (First Action 1994) was used with the followingmodications of the saponication and extraction of free cholesterol, and was specically tested using raw beef LM samples. Sample weight was reduced to 1 g and allother chemicals, except anhydrous sodium sulfate, were

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decreased to 10% as compared to the amounts stipulated inthe original procedure. Meat samples were accuratelyweighed to 1.0000 g and placed into a 125-mL boilingask, followed by the addition of 2 mL of 50% potassiumhydroxide in water and 10mL of 95% ethanol. Themixture was stirred, boiled, and reuxed for 15 min, as

compared to 80 min in the original procedure. The boilingmixture was then cooled to room temperature (25 1 C) priorto the addition of 10 mL of high-grade toluene (Sigma-Aldrich). A 30-s mixing was required before the solutionwas transferred to a 250-mL (or 125-mL, if applicable)separatory funnel. At least ve washes of toluene extract toeliminate aqueous components were carried out. Except therst vigorous wash and vortex with 10 mL of 1.0 Npotassium hydroxide, the second mixture with 5 mL of 0.5 N potassium hydroxide and other three with 5 mLof distilled water were gently swirled. The amounts of washing solutions (1.0 N, 0.5 N KOH, and distilled water)were dramatically reduced due to the reduction of toluenesolvent used. For all washes, it was important that thetoluene layer be allowed to separate completely before theaqueous layer was discarded. The nal toluene layer, whichcould be cloudy, was poured into a 50-mL (25-mL if applicable) test tube containing about 57 g of anhydroussodium sulfate. The mixture of toluene and anhydroussodium sulfate was shaken so that all moisture associatedwith toluene could be removed. The toluene in test tubewas clear after shaking.

The 0.5 mL of crystal-clear toluene solution containingextracted cholesterol was mixed with 0.5 mL of internalstandard solution in a 2.0-mL vial before being subjected to

the GC system. There was no evaporation, reconstitution,and derivatization needed, as compared to the originalprocedure.

2.2. Cholesterol determination of longissimus muscle from purebred cattle

The cattle used in this study had been assembled,weighed, blocked by breeds, assigned to feedlot pens, andfed with the identical commercial nishing diet that wereisonitrogenous and isocaloric prior to harvest at acommercial processing plant in Amarillo, TX. After a 48-h chill, the carcasses were assigned a USDA Quality gradeand transferred to the Texas Tech University G.W DavisMeat Science Laboratory for further fabrication. Samplesof LM were obtained from 18 purebred steers whichconsisted of Angus (AN, n 5), Brahman (BR, n 4), andRomosinuano (RM, n 9) breeds. The LM samples werecollected between the 10th and 13th ribs, frozen immedi-ately in liquid nitrogen, and stored in a 80 1 C freezer forsample preparation.

Frozen LM were later trimmed of all external fat, whichleft only white ecks of marbling within muscle bundles.Trimmed samples were chopped, homogenized to nelydivided powder, and stored at 80 1 C for subsequentextraction and analysis.

The method for cholesterol quantication was modi-ed from the AOAC Ofcial Method 994.10 and validatedusing matrices of meat samples. The intramuscularfat content of LM was determined using the Soxhlettechnique.

2.2.1. Crude fat determinationCrude fat was determined using Soxhlet extraction

technique (AOAC Ofcial Method 991.36). About 45gof frozen muscle powder was placed in an aluminum panand accurately weighed. The sample was dried for 18 h at100110 1 C. After moisture data were collected, the driedsample was covered by layer of non-absorbent cotton, andthe pan was folded, weighed, and placed in Soxhletapparatus for 18 h. The sample pan was then dried for1 h at 100110 1 C before the dried weight was recorded.

2.3. Gas chromatographic analysis

The liberated cholesterol was quantied using theAgilent 6890N gas chromatographic system and the DB-17 capillary column (30m 0.250 mm 0.15 mm, AgilentTechnologies Inc., CA, USA). The DB-17 has mid-polarityand is suitable for analysis of free steroids. One microliter(1.0 mL) of analyte mixture was injected into GC systemwith split/splitless injector and ame ionization detector.The inlet temperature was 250 1 C and split ratio was 10:1.The carrier gas was helium at 2.5mL/min constant ow.The oven was programmed initially at 250 1 C, held for5 min, followed by increases of 5 1 C/min up to 260 1 C, andheld for 8 min. Total time for gas chromatographic

determination was 15 min. The detector was set at 3001

Cwith 200 mL/min airow, 80 mL/min hydrogen ow, and40 mL/min helium makeup ow.

2.4. Data calculation and statistical analysis

Data obtained from chromatograms were calculatedusing the standard curve of the cholesterol standardsolutions. The ratios of standard peak areas to thecorresponding internal standard peak areas were plottedagainst standard concentrations, and the slope of standardcurve was calculated using method of least squares due to

the linear relationship. The internal standard concentrationwas the same in all standard and sample solutions.The resolution of column ( R s) provides a quantitative

measure of a columns ability to separate two analytes. Theresolution coefcient was calculated by the followingequation:

R s 2 tR ; cholesterol tR ; 5a -cholestaneW cholesterol W 5a -cholestane

,

where tR is the peak retention time, min and W is the peakwidth, min.

A resolution coefcient greater than 1.0 indicates theacceptable separation between two analytes ( Skoog et al.,1998 ).

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The recovery efciency ( R) was calculated using ratio of cholesterol content (mg/100 g) recovered to the cholesterolcontent (mg/100 g) added. The calculation was carried outusing the following equation:

R% C r 100 1C a ,

where C r is the cholesterol content recovered (mg/100 g),which was derived from subtraction of non-additionconcentration from the cholesterol-added concentrationand C a is the cholesterol content added, which wasexpressed in mg/100 g of sample.

The CV (%) of the data set was calculated and used asrelative standard deviation (RSD, %) as in the AOACGuidelines for Single Laboratory Validation of ChemicalMethods for Dietary Supplements and Botanicals ( AOAC,2002 ). To determine the repeatability precision, theHorwitz ratio (HORRAT), dened as ratio of determinedCV (%) to calculated CV (%) and presented in the StudyDirectors Manual of AOAC International ( AOAC, 2002 ),was calculated.

The differences in cholesterol concentrations of LMsamples from 18 purebred cattle were analyzed by one-wayanalysis variance ( P 0.05), using a completely rando-mized design with cattle breed serving as treatment. Therelationship between intramuscular fat content and cho-lesterol concentration was also determined using Pearsoncorrelation coefcient. The statistical analysis was accom-plished using the GLM, UNIVARIATE, and CORRprocedures of the Statistical Analysis System (SAS)Institute version 9.1. Means were separated using protected

t-test with LSMEANS/PDIFF option of PROC GLM(P 0.05).

3. Results

Free cholesterol without derivatization displayed a singlepeak at 10.06 min from injection ( Fig. 1 ). The smallestsample peak that could be determined was twice as high asthe noise signals and was equivalent to concentration of

0.00054 mg/mL solvent (0.54ppm) or 1.07 mg/100 g of fresh muscle. Increasing the injection temperature, oven-programmed temperature, and ow rate of carrier gasaccelerated the peak appearance (the retention times atother conditions not shown, P o 0.001). Despite theacceleration, the cholesterol peak was well separated from

the internal standard peak (5 a -cholestane at 5.15min)(Fig. 1 , Rs 25.9). The chromatograms were very cleanwithout any peak interfering with cholesterol or the 5 a -cholestane peaks ( Fig. 2 ). Hence, the results from this studyindicated that saponication by KOH/water resulted inremoval of all interferences in this matrix caused by fattyacid esters. Moreover, the peak ratio was highly linear tocholesterol standard concentration ( R2 0.99).

The method accuracy was proven by both the non-addi-tion ( Table 1 ) and the addition of free analyte ( Table 2 ).The analyses of SRM-1546 samples yielded concentrationswithin the expected range of the certied concentration

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Fig. 1. Typical chromatogram of free cholesterol determination by GC.

Fig. 2. Typical chromatogram of cholesterol separation with peak areareport embedded, using a longissimus muscle sample.

Table 1Method accuracy of a modied cholesterol determination procedure for

meat products determined by comparing analyzed concentrations of standard reference material to the certied concentrations without addingfree cholesterol

SRM Certiedconcentration(mg/100 g)

Recoveredconcentration(mg/100 g)

Recovery(%)

CV (%)

SRM-1546-1 a 77.12 7 3.03 102.83 3.75SRM-1546-2 b 757 7.2 75.57 7 4.03 100.76 5.08Average 76.35 7 2.12 101.80 3.34

Data represent recovery efciency.a SRM-1546-1: Standard Reference Material from the rst sample at

day 1 ( n 6).b SRM-1546-2: Standard Reference Material from the second sample at

day 2 ( n 6).


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(76.35 7 2.12mg/100 g as compared to 75 7 7.2 mg/100 g;P 0.08). These results were produced with high precision(CV 3.34%) and there was no statistical difference(P 0.45) in cholesterol content among SRM-1546 sam-ples prepared on different days. The data of cholesterolrecovery tests ( Table 3 ) revealed high recovery perfor-

mance (97.97100.17%) with high precision (CV 3.19 4.58%) of the modied procedure. Acceptable recoveryefciency is 90108% for concentrations around 0.1% or100 mg/100g ( AOAC, 2002 ). There was, again, no sig-nicant difference in recovery efciency among the foursets of sample ( P 0.79), regardless of sample type(meat homogenate or LM), sources (different breeds of cattle). It was more interesting that the recovery efciencydid not vary although fat content of samples wassignicantly different (from 2.4% to 21.0%; P o 0.001).

The recovery efciency was analyzed to be independent of the fat content of the samples ( P 0.3).

The method stability was proven by its high repeatabilityprecision (CV 2.155.08%). The fact that Horwitzratios for all sample sets fell in the range of 0.52.0(HORRAT 0.711.73) highlighted the precision of the

modied procedure ( AOAC, 2002 ). Additionally, accord-ing to the CV (%) obtained ( Table 3 ), it was realized thatthe method performance on high-fat samples (SRM-1546,21.0% fat) had numerically higher coefcients of variationthan did the lower fat samples (LM samples, 2.49.3%).However, this phenomenon was not statistically analyzed.

The modied method for cholesterol quantication wasapplied to the analysis of cholesterol content of 18 LMsamples from Angus (AN, n 5), Brahman (BR, n 4),and Romosinuano (RM, n 9) steers ( Table 4 ). Choles-terol content (mg/100 g) in these LM samples was found tobe signicantly different among the three breeds(P 0.007). The cholesterol concentrations of LM samplesfrom Angus purebreds (70.25 mg/100g) was higher thanwas those of LM samples from Brahman and Romosinua-no purebreds (64.77 and 65.76 mg/100g; P 0.005 andP 0.006, respectively). The Brahman and RomosinuanoLM samples did not differ in cholesterol content

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Table 2Method accuracy of a modied cholesterol determination procedure formeat products determined by adding free cholesterol into unknownsamples and standard reference material

Sample Addedamount persample(mg)

Recovery (%) Acceptablerecovery a

(%)Range Mean CV, %

SRM1546-R b 1.00 96.68107.22 100.17 3.81

90108LM-1R c 1.00 96.76104.99 99.22 3.19LM-2R d 1.00 92.86105.50 98.59 4.58LM-3R e 1.00 94.48103.35 97.97 4.11

Data represent recovery efciency.a Acceptable recovery for concentration of 0.1% (AOAC, 2002).b SRM-1546-R: Recovery sample using SRM1546 (standard reference

material) sample from the rst sample at day 1 ( n 6). Samples weretreated as if their concentrations were unknown.

cLM-1R: Recovery sample using the rst longissimus muscle (LM)sample ( n 6).

d LM-2R: Recovery sample using the second LM sample ( n 6).eLM-3R: Recovery sample using the third LM sample ( n 6).

Table 3Precision of a modied cholesterol determination procedure in meat products determined by comparing experimentally determined coefcient of variation(CV, %) to calculated CV

Sample Fat (%) Cholesterolconcentration (%)

Calculated CV (%) a Determined CV (%) HORRAT b

SRM-1546-1 c 21.00 0.077 2.93 3.75 1.28SRM-1546-2 d 21.00 0.075 2.94 5.08 1.73LM-1 e 9.34 0.074 2.95 2.22 0.75LM-2 f 6.16 0.069 2.98 2.41 0.81LM-3 g 2.39 0.063 3.02 2.15 0.71

a Calculated CV: function of concentration: CV (%) [Concentration/100] 0.15 .b HORRAT: Horwitz ratio presented in the Study Directors Manual of AOAC International; dened as ratio of determined CV (%) to calculated CV

(%). The acceptable ratio falls into range of 0.52.0.cSRM-1546-1: Standard Reference Material from the rst sample at day 1 ( n 6).d SRM-1546-2: Standard Reference Material from the second sample at day 2 ( n 6).eLM-1: The rst longissimus muscle (LM) sample ( n 6).f LM-2: The second LM sample ( n 6).g LM-3: The third LM sample ( n 6).

Table 4Cholesterol concentration as determined by a modied cholesterolprocedure in longissimus muscle samples from Angus ( n 5), Brahman(n 4), and Romosinuano ( n 9) in relation to intramuscular (i.m.) fatcontent

Breed Cholesterol content(mg/100 g) Fat (%) Fatnesscorrelation

Angus ( n 5) 70.25a 7.00 r 0.90,P o 0.001Brahman ( n 4) 64.77b 3.56

Romosinuano ( n 9) 65.76b 3.53

Within column of cholesterol content, means without common letter (a, b)differ ( P o 0.05). Pearson coefcient of correlation is signicant if P o 0.05.


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(P 0.52). Finally, the cholesterol content of LM samplesincreased with an increase in intramuscular fat due toits signicant positive correlation to the muscle fatness(r 0.9, P o 0.001).

4. Discussion

The method for cholesterol quantication in multi-component foods was adopted in 1976 ( AOAC, 1996a, b ;Punwar, 1976 ). This method involved lipid extraction,saponication, extraction of unsaponied matter withbenzene, derivatization, and GC determination with5a -cholestane as the internal standard. Cholesterol recov-ery from this method fell into the range of 85.891.4% withCV (%) of 12.514.4% for several types of food samples(Punwar, 1976 ). It has been conrmed that saponicationor hydrolyzation is a very critical step when determiningtotal cholesterol ( Hwang et al., 2003 ; Rodriguez-Palmeroet al., 1994 ; Kaneda et al., 1980 ; Naeemi et al., 1995 ; Klattet al., 1995 ). Saponication not only liberates cholesterol,but also puries it from other components such as fattyacids. Despite the fact that lipids in samples were formerlyextracted before cholesterol liberation was performed, VanElswyk et al. (1991) concluded that direct saponicationwas the most accurate method to release free cholesterol.This conclusion was previously stated by Adams et al.(1986) when these authors evaluated direct saponicationusing ethanolic KOH to prepare meat sample, and theysuggested that the lipid extraction step could be eliminated.Their data showed excellent repeatability (CV 1.74%)and recovery (99.8%), which was rarely seen in the current

studies. Direct saponication in their study yielded higherrecovery efciency than did other methods, even theAOAC Ofcial Method 43.235. The saponication techni-que can be performed with KOH in either water ( AOAC,1996a, b ; Klatt et al., 1995 ), or alcohol ( Fenton, 1992 ;Hwang et al., 2003 ). In this study, the saponication usingKOH in water with addition of ethanol was found to be avery effective mean to remove all fatty acids in the form of soaps in the aqueous phase, making them separable duringextraction and purication. Slightly different from thisstudy, De La Huerga and Sherrick (1972) concluded thatthe saponication procedure of Abell et al. (1952) was mostsuitable by using 0.33 or 0.5 M KOH solution in ethanol.However, their conclusion was based on the test of serumcholesterol, not food samples or materials with highprotein content (LM samples: 2021% protein, data notshown) or complex cell structure like muscle tissue. Incontrast, to saponify food samples, other studies usedaqueous KOH with signicant addition of ethanol ( Indyk,1990 ; Beyer and Jensen, 1989 ; Patton et al., 1990 ; Kovacset al., 1979 ; Van Elswyk et al., 1991 ; Kaneda et al., 1980 ).Especially, Indyk (1990) and Van Elswyk et al. (1991) usedthe same KOH-in-water (50%)/ethanol ratio (1:5) as didthis study, which resulted in good separation and recovery;while the ratio reported by Kovacs et al. (1979) was slightlydifferent (1:4). Although Fenton (1992) reported some

peaks of free fatty acid, it was interesting that no additionalpeaks overlapped the cholesterol and 5 a -cholestane peaksin this study. Hwang et al. (2003) suggested the use of KOH/methanol saponication and BF3 methylation toeliminate fatty acid interferences together with ether toextract cholesterol; however, the chromatogram in their

study showed some signicant peaks other than those of cholesterol and the internal standard. The existence of those unnecessary peaks limits the ability to quicken peakappearance (by increasing carrier gas velocity or tempera-ture over the optimum value for theoretical plate number)due to possible peak overlap caused by a decrease innumber of theoretical plates and resolution ( Skoog et al.,1998 ).

The cleanness of resultant chromatogram was causednot only by saponication but also by extraction andpurication. Cholesterol with very low polarity in saponi-ed mixture had to be extracted using a solvent that couldmix well in a waterethanol environment and create ahomogeneous-phase extraction. Other studies used eitherether ( Hwang et al., 2003 ) or hexane ( Fenton, 1992 ; Fentonand Sim, 1991 ) to extract cholesterol, claiming that noextra clean-up steps required. Their data showed that therecovery efciencies were comparable to those from thisstudy. The use of ether as a solvent, however, resulted inthe formation of peroxides that could cause degradation of sterols ( Fenton, 1992 ). Indyk (1990) and Fenton and Sim(1991) extracted cholesterol only once with hexane andreported that it was sufcient for an accurate recovery.However, Patton et al. (1990) , Kovacs et al., (1979) , andAl-Hasani et al. (1990, 1993) all had to use multiple hexane

extractions to obtain adequate recoveries. Lognay et al.(1989) showed a need for ve diethyl ether extractions toobtain quantitative recovery of cholesterol. In this study,these solvents, especially hexane, were also evaluated and itwas found to require at least double extractions, similar tofatty acid extraction using hexane ( Li and Watkins, 2003 ),to obtain over 90% recovery (data not shown). Thisphenomenon was probably due to their extremely lowpolarity, which results in their exceptional exclusion fromwater-based saponied mixture. The multiple-extractionprocess requires evaporation of the solvent and reconstitu-tion of dried residue, creating additional preparation steps.Hence, single extraction using toluene was chosen due to itsdesirable solvent properties and relatively low toxicity(Li and Watkins, 2003 ). The 10-mL toluene extraction wasfound to be appropriate due to high recoveries of themethod ( Table 2 ), which were very dependent of solventnature. Oles et al. (1990) studied some factors affecting therecovery of cholesterol from various food matrices: type of alcohol, extraction solvent, use of antioxidants, time andtemperature of hydrolysis; and found that toluene extrac-tion resulted in signicantly higher recoveries than amixture of hexanediethyl ether (85:15, v/v). This resulttogether with the ones in this study, again, conrmed thesuitability of toluene in extracting cholesterol. Occasionallyhowever, an emulsion may form which can be remedied by

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use of anhydrous sodium sulfate, which can absorbmoisture and dry the solvent. With adoption from theAOAC 994.10 Ofcial Method, the saponication andwashing techniques were found to be very effective ineliminating all unrecognizable peaks that might interferewith the peaks of cholesterol and 5 a -cholestane, allowing

an increase in analyzing speed without jeopardizingseparation quality.The decrease of the sample size from 5 to 10 g (to obtain

at least 1 g of fat) to 1 g was proven to be time and cost-effective without experiencing any uncertainty of methodperformance. The modication signicantly reduced 90%of the costs of chemicals and solvent use, as compared tothe AOAC Ofcial Method 994.10 ( AOAC, 1996a, b ).Total time used in this method was 3045 min per sample,as compared to at least 7080min for saponication only,and extra time for drying, reconstitution, and derivatiza-tion in the original method ( AOAC, 1996a, b ). Thistimesaving would lead to very high productivity anddecreased errors related to method complication. Thechromatographic separation of free cholesterol withoutderivatization to trimethylsilyl ethers has been studied anddocumented in recent years due to the development of capillary GC columns with high resolution and heattolerance ( Lee et al., 1998 ; Fenton, 1992 ; Wu et al., 1997 ;Hwang et al., 2003 ; Thompson and Merola, 1993 ;Al-Hasani et al., 1993 ). These studies have found thatcholesterol was not required to be transformed to morevolatile derivatives, such as silylated form to be quantita-tively detected and quantied ( Hwang et al., 2003 ). Theresults from those studied were very comparable with the

traditional derivatization techniques. As with the currentstudy, although detector response for cholesterol wasslightly lower than its silylated ethers ( Hwang et al.,2003 ), the method sensitivity still allowed for quantitativedetection of cholesterol content in meat samples.

In a collaborative study, the AOAC 994.10 method hadan average repeatability of 4.81% and cholesterol recoveryof 100.03% when performed with NIST egg powder ( Klattet al., 1995 ). Accordingly, the modication used in thisstudy compared favorably with its original method with anaverage repeatability and recovery efciency of 3.12% and98.99%, respectively. The decrease in saponication time(from at least 70 to 15 min) did not produce any negativeeffect on recovery efciency. The decision to decreasesaponication time was made because the large amount of cholesterol in muscle is in free form, hydrogen-bonded ornon-covalently bonded, and packed into the cell membranerather than in esteried form with fatty acids ( Karp, 2005 ).The utilization of aqueous KOH with addition of alcoholinstead of alcoholic KOH as used in many studiespreviously mentioned suggested that the presence of waterplayed some role in hydrolyzing cell membrane andreleasing cholesterol. Hence, using aqueous KOH withlater addition of ethanol was suitable for hydrolyzingfresh meat samples. Hwang et al. (2003) suggested usingmethylation as an additional treatment after saponication

to hydrolyze ester bonds between cholesterol and fattyacids. However, this technique might be only useful forlipid samples such as oil, extracted fat, of which esterlinkages are much more predominant than of muscle-basedsamples, which contain more free cholesterol, a majorcomponent of cell membrane.

Of most concern was that of lower repeatabilityprecision associated with high-fat samples. Most fattyacids in the sample were converted to potassium salts orsoap, which could act as emulsifying agents and prevent thecomplete separation of toluene from the rest of themixture. As observed, the solution cloudiness seemed toappear in samples with higher fat content, especially inSRM-1546 samples. Fenton and Sim (1991) also reportedthat fat content could affect the extraction of cholesterol.By increasing the amount of soybean oil added to chole-sterol standard (for 0170mg), these authors observed theefciency of rst extraction to be decreased from 98.2% to94.1%. However, this study showed that higher fat contentonly caused an increased variation in cholesterol determi-nation while the average recovery efciency was notinuenced due to no signicant difference in recoveriesamong samples ( P 0.79). Therefore, the samples with fatcontents of 920% should be processed with special care toavoid the emulsication. Additionally, as cholesterolcontent begins to approach the detection limit, someanalyte enrichment techniques such as increasing samplesize or solvent evaporation and reconstitution should beperformed to obtain sufcient cholesterol in nal solutionprior to GC analysis.

The validated method was used to quantify cholesterol in

LM samples from cattle. The signicantly fatter Angus LMsamples had signicantly higher cholesterol content(70.25mg/100g) as compared with Brahman and Romosi-nuano ( P 0.005, 0.006, respectively). The cholesterolcontent was also found to be positively correlated tomuscle fatness. Generally, cholesterol content in meat isnot necessarily increased with fatness because a largeamount of cholesterol is found in cell membranes as thefree form ( Karp, 2005 ). However, cholesterol is also stored(e.g. in cytosol) in esteried form with fatty acids,especially with long-chained fatty acids ( Kvilekval et al.,1994 ; Xie et al. , 2002 ; Lewis-Barned et al., 2000 ).Consequently, it is possible that the stored cholesterolbecomes more signicant when fat content is increasedbecause it was hypothesized and proven that fatty acids,especially some unsaturated fatty acids, regulate cholester-ol esterication and secretion ( Xie et al., 2002 ). Xie et al.(2002) demonstrated that unsaturated fatty acids drove theesterication reaction and enhanced lipoprotein cholesterolsecretion by the liver under conditions where cholesterolbalance across this organ was constant.

Regarding the cholesterol level in beef muscle, Padreet al. (2006) reported the lower cholesterol content(45.745.8 mg/100g) in LM of bulls and steers nishingin pasture systems as opposed to traditional nishing;however, the intramuscular fat content was also much

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lower (1.713.38%) than that in LM from this study. Theyalso found no difference in cholesterol content amonganimals despite the variation in fatness. Rule et al. (1997)emphasizes that breed, nutrition, and gender do not affectcholesterol concentration of bovine skeletal muscle. Theyalso suggested that changes in cholesterol content in muscle

might require marked changes in structure of muscle cellsassociated with a marked redistribution of membrane fattyacids. Additionally, Rule et al. (2002) reported slightlyhigher cholesterol content of beef LM (5253 mg/100 g)than those previously mentioned in Padre et al. (2006)study, but their i.m. fat content was not reported.However, by investigating lipid characteristics of long-issimus thoracis between Angus and Wagyu steers fed tothe US and Japanese endpoints, Chung et al. (2006)reported very similar levels of cholesterol in longissimusthoracis from Angus (7278 mg/100 g) as compared to LMsamples from Angus in this study. They also indicated apositive, but weak, relationship between cholesterol con-tent in muscle and intramuscular fatness. Interestingly,Rhee et al. (1982) found that cholesterol concentrationswere directly related to the USDA marbling scores.Cholesterol was increased from 51.77 to 64.74 mg/100 gwith an increase in marbling scores from practically devoid(2.73% i.m. fat) to moderately abundant (12.08% i.m. fat).The results from studies previously mentioned indicated anunpredictable relationship between fatness and cholesterol.It might be hypothesized that genetic differences betweenAngus and the other two breeds possibly cause a signicantinuence on cell structure, especially of adipocytes, whichcould result in higher cholesterol levels. However, the

relationships among cholesterol level and quantity, size,and structure of adipose and muscle cells are not wellunderstood and need further study.

5. Conclusion

The modications, which were made to AOAC OfcialMethod 994.10, revealed a new cholesterol quanticationprocedure that was validated to be efcient and reliable.The new procedure was not only faster, but also more cost-effective. The procedure, which was previously outlined,allowed a quick and quantitative detection of cholesterolconcentration in fresh meat samples. The results from thisstudy were comparable to most recent studies in precisionand accuracy, which were shown to be quantitativelyapplicable. The cholesterol contents determined weresimilar to those from other studies and to certiedreference materials. The fact that the SRM is a meathomogenate of pork and chicken products blendedtogether in a commercial process ( NIST, 2004 ) indicatesthat the modied method was a precise and accuratedetermination of the true cholesterol content of not onlyfresh meat samples but also variety of meat products.However, further validation is recommended and can beuseful for an application of this modied method in othertypes of meat samples.

References

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Abidi, S.L., 2001. Chromatographic analysis of plant sterols in foods and

vegetable oils. Journal of Chromatography A 935, 173201.Abidi, S.L., 2004. Capillary electrochromatography of sterols and related

steryl esters derived from vegetable oils. Journal of Chromatography A1059, 173201.

Adams, M.L., Sullivan, D.M., Smith, R.L., Richter, E.F., 1986.Evaluation of direct saponication method for determination of cholesterol in meats. Journal of Association of Ofcial AnalyticalChemists 69, 844846.

Al-Hasani, S.M., Shabany, H., Hlavac, J., 1990. Rapid determination of cholesterol in selected frozen foods. Journal of Association of OfcialAnalytical Chemists 73 (5), 817820.

Al-Hasani, S.M., Hlavac, J., Carpenter, M.W., 1993. Rapid determinationof cholesterol in single and multi-component prepared foods. Journalof AOAC International 74 (4), 902906.

AOAC International, 1996a. AOAC Ofcial Method 994.10: Cholesterol

in FoodsDirect SaponicationGas Chromatographic Method.Association of Ofcial Analytical Chemists, Arlington, VA.

AOAC International, 1996b. AOAC Ofcial Method 976.26: Cholesterolin Multi-component FoodsGas Chromatographic Method. Associa-tion of Ofcial Analytical Chemists, Arlington, VA.

AOAC International, 2002. AOAC Guidelines for Single LaboratoryValidation of Chemical Methods for Dietary Supplements andBotanicals. Association of Ofcial Analytical Chemists, Arlington,VA.

Beyer, R.S., Jensen, L.S., 1989. Overestimation of the cholesterol contentof eggs. Journal of Agricultural and Food Chemistry 37, 917920.

Cardenas, M.S., Ballesteros, E., Gallego, M., Valcarcel, M., 1995.Automatic gas chromatographic determination of the high-densitylipoprotein cholesterol and total cholesterol in serum. Journal of Chromatography B: Biomedical Sciences and Applications 672 (1),

716.Chung, K.Y., Lunt, D.K., Choi, C.B., Chae, S.H., Rhoades, R.D.,

Adams, T.H., Booren, B., Smith, S.B., 2006. Lipid characteristics of subcutaneous adipose tissue and M. longissimus thoracis of Angus andWagyu steers fed to US and Japanese endpoints. Meat Science 73,432441.

De La Huerga, J., Sherrick, J.C., 1972. The measurement of cholesterol inserum: reference method. Annals of Clinical and Laboratory Science 2(5), 360366.

FDA, 1993. Food Labeling: Nutrient Content Claims, General Principles,Petitions, Denition of Terms. Denitions of Nutrient Content Claimsfor the Fat, Fatty Acid, and Cholesterol Content of Food (FR Doc.92-31504, pp 2302-2426. Docket No. 91N-0394 and 94N-O153,CDER 199302. Retrieved 2008-03-08: http://www.fda.gov/OHRMS/DOCKETS/98fr/1993-92-31504.pdf ).

Fenton, M., 1992. Chromatographic separation of cholesterol in foods.Journal of Chromatography A 624, 369388.

Fenton, M., Sim, J.S., 1991. Determination of egg yolk cholesterol contentby on-column capillary gas chromatography. Journal of Chromato-graphy A 540, 323330.

Fletouris, D.J., Botsoglou, N.A., Psomas, I.E., Mantis, A.I., 1998. Rapiddetermination of cholesterol in milk and milk products by directsaponication and capillary gas chromatography. Journal of DairyScience 81, 28332840.

Galanos, D.S., A vazis, G.A.M., Kapoulas, V.M., 1964. A simple methodfor the determination of serum glycerides, free cholesterol, andcholesterol esters using a binary solvent system. Journal of LipidResearch 5, 242244.

Hwang, B.S., Wang, J.T., Choong, Y.M., 2003. A simplied method forthe quantication of total cholesterol in lipids using gas chromato-graphy. Journal of Food Composition and Analysis 16, 169178.

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