Determination of Bis(Chloromethyl)Ether in Laboratory Indoor Air

Quantitative analysis of cholesterol by gas chromatography and gas chromatography/mass spectrometry

Monica Culea, Gh. Ciurdaru, Lidia Luncian, Mihaela Apetri, Laura Fromondi, R. Podea Natex S.R.L., 3400 Cluj-Napoca, P.O.Box 374, Romania

Abstract A method for quantitative analysis of cholesterol in egg yolk and tissues is presented. The internal standard was diosgenin, added 5mg/g of egg yolk or 1mg/g of tissue. The extraction was performed in dichloroethane:methanol in an optimization process of the amount of sample, extractant solvent volume, extraction temperature, extraction time. The extraction was followed by methylation of fatty acids and gas chromatographic and gas chromatography- mass spectrometric analysis. The extract was derivatized with methanol HCl 1M, at 60oC, 1h for fatty acids. A HP-5 capillary column, 30mx0.32mm, 0.25µm film thickness, in a temperature program: 70oC( 1 min) to 100oC with a rate of 20oC per minute, then 30o C per minute to 310 oC (10min) was used. The method was validated showing good analytical parameters. The method was applied for monitoring the cholesterol level in egg yolk or tissues for testing different functional food diet to chicken.

Keywords: cholesterol, gas chromatography; gas chromatography-mass spectrometry; egg yolk; tissue;

Introduction Cholesterol, indispensable substance to our body present in our cell membrane, is healthy to be intake in low quantities by food. It is present especially in egg yolk, meat and dairy products. In the last years many articles show the health benefits of different ingredients contained in foods. The influence of some of these functional foods could be observed in the variation of cholesterol level in biological matrices [1]. Several methods have been used to quantitate total cholesterol in multicomponent foods; eggs [2-6], meat [7-9], dairy products. Methods usually involve an organic extraction and a quantitative technique such as gas chromatography (GC) or liquid chromatography (LC). The organic extract is saponificated or not and the cholesterol sample is derivatized or not before GC analysis. Many collaborative studies are made for the optimization of the experimental design approach for obtaining the best recoveries, low solvent consumption and reduced extraction times [2-10]. The aim of this paper is to present a rapid method for cholesterol quantitation in egg yolk and tissues. Cholesterol was analysed without derivatization. The recovery of our method was compared with the recovery obtained by other methods [2]. Saponification was replaced by methylation for monitoring the total cholesterol and fatty acids levels too. The analytical technique chosen is gas chromatography (GC) with flame ionization detection (FID) and gas chromatography - mass spectrometry (GC/MS) for identification of the separated compounds.

Experimental Apparatus A Hewlett Packard GC 5890 couplet with a MS engine 5989B in the EI mode was used for compounds identification. The GC was equipped with an HP-5MS capillary column 30mx0.25mm diameter, 0,25µm film thickness, in the temperature program: 70 oC for 1 min, then increased to 100oC with a rate of 20 oC /min, followed by a rate of 30oC/min to 310oC(10min) at a helium flow rate of 1ml/min. A Hewlett Packard 6890 gas chromatograph was equipped with a HP-5 fused silica capillary column, 30mx0.32mm, 0.25µm film-thickness with the same temperature program. Deactivation by treating the injector glass liner with 5% dimethyldichlorosilane in toluene gave a higher sensitivity. The GC/MS interface line and the ion source were maintained at 200oC, and quadrupol analyser at 100 oC. Electron energy was 70eV and electron emission 300µA.

Reagents and chemicals Solvents (methanol, ethanol, methylen chloride, 1,2dichloroethane, purchased from Chimopar (Bucharest, Romania) were purified by distillation, when necessary. Acetyl chloride was purchased from Carlo Erba (Milano,Italy). Cholesterol (99% pure) and diosgenin (99% pure) were purchased from Sigma-Aldrich ( Deisenhofen, Germany).

Procedure Six aliquots containing 50, 100, 150, 200, 250 and 300 µg cholesterol and the same quantity of 80 µg of the internal standard diosgen were prepared. 3 µl were injected two times from each alliquot by using the autosampler injector to obtain a regression curve for egg cholesterol determination in the range 0-300 mg. The relative integrated areas were plotted against the injected amount as the calibration curve. For tissue cholesterol analysis the chosen range was 0-70mg; seven aliquots of 10, 20, 30, 40, 50, 60 and 70 µg cholesterol and the same quantity of 100 µg of the internal standard diosgen were prepared, then injected twice by using the autosampler injector. The relative integrated areas were plotted against the injected amount as the calibration curve. The weight of boiled and dried egg yolk corresponding to 1g of boiled egg yolk ( or 1 g well mixed tissue) was placed in a 10 ml screw cap glass centrifuge tube with 5 ml 1,2 dichloroethane :methanol (1:1). The extraction was performed at 50oC, 30 minutes, agitating well every 5 minutes. The extract was cooled, centrifuged and the supernatant was removed. 0.5 ml were transferred was methylated with 0.5 ml methanol: acetyl chloride (10:1,v/v) at 60oC for 1h. The reagent was removed under nitrogen. 0.5 ml 1,2dichloroethane and 5mg diosgenin/g in egg yolk or 1 mg diosgenin/g in tissues were added. Three extracts were made and each sample and injected twice.

Experimental design 10 replicates egg yolk were analyzed by our method. 3 replicated dried egg yolk were analyzed by our method and by saponification [2] to compare the recovery obtained by the two methods.

Results and discussion Method validation Linearity in the range 0-300 mg cholesterol gave a good correlation coefficient of r=0.998 and in the range 0-70 mg cholesterol of r=0.999. The regression curve obtained for the two ranges were: y=0.02x - 0.02 (Fig. 1) and y= 0.01x - 0.01 ; precision of the method measured in the aliquots containing 50, 60 and 200 µg cholesterol gave R.S.D lower than 2% (n=9) and for egg yolk control 6.5% (n=10); accuracy showed better than 4 %; (Table 1) recovery gave 95% (Table 2); L.O.D was lower than 5ng at a signal-to-noise ratio of 10. The chromatogram of separation of the compounds studied is presented in Fig.2. The results of the precision, accuracy and recovery studies are presented in Tables 1 and 2. The average values are resulted from three extraction procedures and two injections of each extract. Precision and day-to-day reproducibility measured for 50 and 60 µg standards (n=6) showed relative standard deviation values bellow 4%. The mean values for the recoveries found for each method studied are compared. The best values for recovery were found for dried egg yolk (Table 2). Significant test studies showed no significant differences at the 0.05 level between the two methods (Table 3). The mass spectra fot cholesterol and diosgenin are shown in Fig.3. Diosgenin was a mixture of two isomers with the same mass spectrum and closed retention times.

Conclusions This paper presents a method of cholesterol determination in egg yolk and tissues. The comparison between two extraction methods gave similar recovery results without significant differences of the mean values of cholesterol levels. The method was validated showing good analytical parameters: linearity in the range 0-300 mg cholesterol gave a good correlation coefficient of r=0.999; precision of the method gave R.S.D. 6.5% (n=10 in egg yolk; accuracy showed R.S.D. better than 3 %; recovery gave 95% comparable with the recoveries obtained with other methods (99%) [2]; L.O.D was lower then 5ng. The boiled and dried egg showed better analytical parameters. The methods are suitable for the determination of cholesterol amount in food and biological matrices. The method is simple, rapid, accurate and suitable for cholesterol level variation study for testing the benefits of functional food diet.

The author thank the management, Director and colleagues, S. C. Natex s.r.l., for their co-operation in conducting this study.

References 1. C. M. Hasler, “Functional foods: Their role in diseases prevention and heath promotion” Food Tech. 52( 11) 1998, 63-70. 2. J J.D.Beyer, F.X. Milani, M. J. Dutelle, R.L. Bradley , “Gas Chromatographic determination of cholesterol in egg products”J. Assoc. Off. Anal. Chem. 72( 5) 1979, 746- 748. 3. M. T. Rodriguez- Estrada, M. F. Caboni, A. Costa, G. Lercker, “ Gas chromatographic analysis of cholesterol oxidation products on a thermostable medium polarity capillary column”, J. High Resolut. Chromatogr. 21(9) 1998, 509-512. 4. G. Pasin, G. M. Smith, M. O’Mahony, “Rapid determination of total cholesterol in egg using commercial diagnostic cholesterol reagent”, Food Chem. 61(1-2) 1998, 255-259. 5. M. S. Lai, J. I. Gray, M. E. Zabik, “Evaluation of solid-phase extraction and gas chromatography for determination of cholesterol oxidation products in spray-dried whole egg”, J. Agric. Food Chem. 43(5) 1995, 1122-1126. 6. F. Guardiola, R. Codony, M. Rafecas, J. Boatella, “Selective gas-chromatographic determination of cholesterol in eggs”, J. Am. Oil Chem Soc. 71(8) 1994, 867-871. 7. M. Fenton, J. S. Sim., “Determination of egg yolk cholesterol content by on-column capillary gas chromatography”, J. Chromatogr. 540 (1-2) 1991, 323-329. 8. E. D. Naeemi, N. Ahmad, T. K. Al-Sharrah, M. Behbahani, “Rapid and simple method for determination of cholesterol in processed food”, J. AOAC Int. 78(6) 1995, 1522-1525. 9. K. Duckett, D. G. Wagner, “ Effect of Laidlomycin proprionate on bovine longissimus muscle fatty acid and cholesterol content”, J. Agric. Food Chem. 45, 1997, 1725-1728. 10. M. Van. Boven, P. Daenens, K. Maes, M. Cokelaere, “ Content and composition of free sterol free fatty alcohols in jojoba oil”, J. Agric. Food. Chem. 45, 1997, 1180-1184.

Table 1 Precision and accuracy characterization for cholesterol in egg yolk (n=9) ______Cholesterol(mg) precision accuracy added measured R.S.D. (%) R.S.D. (%) ______200 201.85 0.04 0.92 50 48.27 1.39 3.49 60 59.36 1.16 2.71 ______R.S.D.= relative standard deviation

Table 2 Comparative recovery mean values for cholesterol in egg yolk (n=3) ______Procedure S M US USM (%) (%) (%) (%) ______boiled and dried 99.8 95.54 70.40 95.36 boiled 94.06 91.6

______S = saponification[2]; M=methyilation(ours); US=unsaponification; USM=unsaponification and methylation

Table 3 Comparative mean value for cholesterol by two different methods (n=3) ______Method Cholesterol (mg) S. D. R.S.D. M 193.41 6.58 3.40 S 209.56 1.25 0.59 ______M = methylation; S = saponification; S. D. : standard deviation; R.S.D.: relative standard deviation

Figures legend

Fig. 1. Correlation curve for egg yolk cholesterol calculation Fig. 2 The separation chromatogram of lipids in egg yolk. Fatty acids methyl esters (FAME) of: myristic acid (C14:0); palmitoleic acid (C16:1); palmitic acid C16:0); oleic acid (C18:1); linoleic acid (C18:2) ;stearic acid (C18:0); arachidonic acid( C20:4); Fig. 3 Diosgenin (M=414) and cholesterol (M=386) mass spectra