Anal. Chem. lQQ2,84, 1077-1087
1077
AC RESEARCH
Monitoring Cholesterol Autoxidation Processes Using Multideuteriated Cholesterol Bruce A. Wasilchuk,+P. W. Le Quesne, and Paul Vouros* Department of Chemistry and Barnett Institute of Chemical Analysis, Northeastern University, Boston, Massachusetts 02115
Recent experimentalevidence indicates a relationship between the presence of a-epoxycholesterol and skin c a n ~ e r In . ~ addition, the a-epoxycholesterolcan function as a direct-acting m ~ t a g e n . ~A? number ~ of products of the autoxidation of cholesterol are known to be active in the suppression of sterol synthesis and HMG-CoA reductme activity." Choleatanetriol, which was found to be present in freeze-dried pork when improperly stored,1° is considered one of the most toxic cholesterol oxidation products."J2 In addition both a-epoxycholesterol and cholestanetriol have been shown to inhibit DNA ~ynthesis.~ The toxicological evidence clearly demonstrates the detrimental biological effects which cholesterol oxidation products can exhibit. The dietary intake of these compounds is, therefore, of concern since cholesterol-rich foods such as beef, liver, and egg products are currently subjected to proINTRODUCTION cessing conditionswhich can effectively promote the oxidation Medical evidence derived from animal experimentation and of ch~lesterol.'~-~~ histological studies suggests the strong possibility that choGiven the toxicological evidence and the propensity for lesterol oxidation products ("cholesterol oxides"), and not cholesterol oxidation during food processing, the need for necessarily cholesterol, 1, may play a significant role in arterial accurate and reliable analytical methodology to isolate and wall injury and lesion development resulting in the onset of quantify a variety of cholesterol oxidation products in foods atherosclerosis in Studies involving the intraveis clearly evident. Unfortunately, this has proved to be a nous injection of specific cholesterol oxides such as 5a-chodifficult task. In developing analytical methodology for lestane-3@,5a,6@-triol (cholestanetriol),5cholestene-3P,25-diol (25-hydroxycholesterol),and 5,6a-epoxy-5a-cholestan-3~-01 cholesterol oxide determinations three basic problems must be considered: the large excess of cholesterol in relation to (a-epoxycholesterol)resulted in characteristic patterns of cell the amount of cholesterol oxides in the sample,16the chemical death in rabbit aortas and pulmonary arteries as early as 24 similarity of cholesterol to its oxidation products,17and the h after administrations2 In addition, dietary intake of choformation of artifacts arising from the oxidation of cholesterol lestanetriol a t the 0.1% level has resulted in the formation during the analytical procedure." While the first two probof aortal lesions resembling those found naturally in man.4 lems can be effectively solved through judicious choice of analytical methodology, it is the last that continues to pose a major challenge to analysts. It is well-known that cholesterol can oxidize under relatively mild conditions to produce a wide range of oxidation products.17 Many of these oxides can be produced quite readily by exposure of cholesterol to air at room temperature1a-20or HO by the action of heatla or irradiation.21 In view of the ease of cholesterol oxidation it is not surprising that harsh ana1 Cholesterol l a ZHg-Cholesterol lytical manipulations such as hot alkali ~aponification,'~J~ and purification by brominati~n/debromination'~ can introduce In addition to their demonstrated atherogenicity, cholesterol cholesterol oxide artifacts into the analysis and, therefore, oxides have also exhibited other detrimental biological effects. uncertainty into the analytical results. Relatively recent findings, however, suggest that these same artifacts may also 'Present addreas: CIBA-GEIGY Corp., Old Mill Rd.,Suffen, NY 10901. appear during some of the more routine and seemingly inDeuterium-labeled cholesterol Is used to monitor for artlfactuaWy produced cholesterol oxklatlon products durlng analyskr. [*H,&holesterol, labeled on the slde chaln, Is added to the sample lmmedlately upon Isolatlon, and the ratlos of labeled to unlabeled oxldes and of labeled to unlabeled cholesterol are monltored by caplllary gas chromatography-mass spectrometry. The analytlcal methodology Involves an lnltlal solvent extractlon followed by slllca gel LC and reversedphase HPLC to Isolate and concentrate the oxide fraction. The feaslblllty of the technlque for analysis of cholesterol oxldes In foods and blologlcal samples at the part per mllllon level with an accuracy of better than f5% Is demonstrated.
0003-2700/92/0364-1077$03.00/00 1992 American Chemical Society
1078
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
nocuous analytical manipulations. The purity and stability of solvents used during analysis have been shown to be critical to minimizing artifacts during analysis. It has been demonstrated that ethyl ether containing peroxide impurities may promote cholesterol degradation leading to the formation of a variety of oxidation products.22 In addition, cholesterol-containing samples exposed to preservatives such as alcohol or formaldehyde should also be avoided since primary products of cholesterol oxidation (cholesterol hydroperoxides) have been detected in human tissues fixed in f~rmaldehyde.'~In order to avoid such solvent-related oxidations of cholesterol, rigorous purification of all solvents during analysis and frequent changing of solvents in TLC developing tanks are n e ~ e s s a r y . ' ~ , ~ ~ The interaction of cholesterol with adsorption media (Le., silica gel, silicic acid) is another important factor in the formation of cholesterol oxide artifacts during analytical manipulation~,~ presumably -~~ due to enhanced exposure of the layer of adsorbed cholesterol to air. For example, it was found that the use of adsorbing agents such as Kieselgel (Merck, Darmstadt) and especially silicic acid utilized for column chromatography may lead, under certain conditions, to the degradation of cholesterol to produce: 7-ketocholesterol, 3,5-cholestadien-7-one, and the epimeric 7-hydroxycholesterols.22It has been demonstrated that cholesterol adsorbed on silica gel and exposed to air at room temperature may oxidize in very short time periods. Rather than taking several days, as noted in one detectable quantities of cholesterol oxide artifacts have also been discovered on TLC plates within minutes after d e ~ e l o p m e n t . ~ ~ The care taken in the handling of cholesterol-rich samples during analysis can also be a source of cholesterol oxide artifacts. One study26 demonstrated that the levels of 25hydroxycholesterolin aortal tissue samples varied according to the analytical conditions utilized. It was shown that the level of 25-hydroxycholesterolwas directly related to the extent of exposure of the sample to light and air during analysis, suggesting autoxidation as its origin.26 It is obvious that among the problems faced by researchers in the area of cholesterol oxide analysis, the problem of the formation of cholesterol oxide artifacts during extraction, isolation, separation, and assay is perhaps the most serious. The development of accurate and reliable analytical methodology for the determination of cholesterol oxidation products is, therefore, necessary. Basically, two approaches can be taken to accomplish this goal. The first involves modifying the analytical procedure either by incorporating the use of antioxidants or by manipulating the physical parameters, such that the spontaneous oxidation of cholesterol does not occur. It has been demonstrated, however, that this approach is not always effe~tive.'~"The use of EDTAB as a metal complexing agent and butylated hydr~xytoluene~~ as an antioxidant have been attempted previously to eliminate artifact formation. Although the levels of oxide artifacts did diminish in these studies, artifact formation was not completely eliminated. Careful control of the physical parameters during an analysis has also been attempted in order to eliminate oxide artifact formation. Several studiesl3Rhave appeared in which samples have been kept under a nitrogen atmosphere, in the dark, and at reduced temperatures a t all times. Despite these precautions, and rigorous deoxygenation of laboratory solvents, complete prevention of oxidation during analysis was not achieved. Further studies on antioxidants and procedures for the inhibition of oxidation are apparently needed. A second approach to eliminating the problem of oxidative artifah is to determine the extent of cholesterol oxide artifact formation during analysis and then apply an appropriate correction to the analytical data. This approach, if it could
be conveniently applied to existing methodology with a minimum of modification to that methodology, would be highly desirable. This is, therefore, the approach adopted in this work to investigate the problem of cholesterol oxide artifact interferences by GC-MS. It should be noted that a related approach, relying on the use of tritiated cholesterol and HPLC to correct for such artifacts was published by Kudo, et al.,29since the completion of the study reported here.
EXPERIMENTAL SECTION Materials. Acetonitrile, ethyl acetate, methanol, chloroform, and water were "Baker Analyzed" HPLC grade (Doe and Ingalls Co., Medford, MA). Reagentrgrade acetone (EM Science, Cherry Hill, NJ) was used in drying all laboratory glassware. HPLC-grade acetone, used in sample handling, was obtained from Aldrich Chemical Co. (Milwaukee, WI). 2-Propanolwas HPLC grade from Fisher Scientific (Fairlawn, NJ). Pyridine used in silylation reactions was silylation grade (Pierce Chemical Co., Rockford, IL). The silylating reagents, N,O-bis(trimethylsily1)trifluoroacetamide (BSTFA), BSTFA plus 1% trimethylchlorosilane (TMCS),and N,O-bis(trimethylsily1)acetamide(BSA) were used as received from Pierce Chemical Co. (Rockford, IL). Silica gel, 100-200 mesh (Fisher Scientific Co., Medford, MA), was prewashed in ethyl acetate/methanol(955)and then vacuum filtered to remove most of the solvent. The silica gel was then dried at 100 O C overnight in a vacuum oven (Model 5830, National Applicance Co., Portland, OR). After drying, the silica gel was allowed to cool to room temperature in a desiccator partially filled with anhydrous calcium sulfate (W. A. Hammond Drierite Co., Xenia, OH). Steroid standards were purchased from the following sources: cholestanetriol from Research Plus Laboratories Inc. (Denville, NJ); cholesterol, 25-hydroxycholestero1, 7-ketocholesterol, 6ketocholestanol, and a-epoxycholesterol from Steraloids, Inc. (Wilton, NH).fl-Epoxycholesterolwaa obtained from the College of Pharmacy, Northeastern University (Boston, MA). 7aHydroxycholesteroland 78-hydroxycholestero1were synthesized by reduction of 7-ketocholesterolmfollowed by HPLC purification?'. Both a-epoxycholesteroland fl-epoxycholesterolcontained major impurities necessitating extensive purification by HPLC.31 Selected standards were purified by HPLC as needed. Subsequent analysis by capillary GC indicated a purity greater than 99%. The sample that was utilized in the analysis was a powdered liver extract (Pioneer Specialty Foods, Fargo, ND) which is available as a dietary supplement from local health food stores. Instrumentation. The high-performance liquid chromatographic (HPLC) system utilized consisted of two Gilson (Middletown, WI) Model 302 HPLC pumps connected to a Model 802 manometric module and a Model 811 dynamic mixer. Injections were made utilizing a Rheodyne (Cotati, CA) Model 7125 six-port injection valve equipped with either a 100- or 200-pL injection loop. A Waters Associates (Milford,MA) Model R-401 Differential Refractometer or a Gilson (Middletown, WI) Model HM Holochrome W detector was used for detection of eluting components. A Linear Instruments Corp. (Irvine, CA) Model 486 two-channel strip chart recorder and a Hewlett-Packard (Palo Alto,CA) Model 3392A reporting integrator were used for the recording and integration of chromatographicdata. The entire HPLC system was interfaced to and controlled by an Apple (Cupertino,CA) I1 Plus personal computer. Mobile-phase flow rates and compositions were controlled by the Apple Computer utilizing Model 702, version 1.2 Gradient Manager software (copyright 1981, Gilson International, Middletown, WI). Gas chromatography-mass spectrometry (GC-MS) was performed on a Finnigan-MAT (San Jose, CA) Model 4021B quadrupole mass spectrometer interfaced to a Hewlett-Packard (Palo Alto, CA) Model 5890 GC equipped with a capillary on-column injector. Samples were injected into the GC dissolved in BSA. One to two microliters were introduced directly onto the capillary column utilizing a syringe fitted with a narrow-bore, 19-cm fused silica needle (J&W Scientific, Folsom, CA) at an injection temperature of 50 "C. Helium was used as carrier gas at a room temperature flow rate of 1.6 mL/min. A 60-m fused silica capillary column (0.32-mm i.d., 0.25 pm, DB-1) (J&W Scientific, Folsom, CA) was utilized in all GC and GC-MS analyses. The column
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
temperature was programmed from 50 "C to 290 OC at 30 OC/min and then held at 290 "C for 30 min. The GC-MS interface was constructed in our laboratory and consisted of a heated box enclosing a stainleas steel transfer line?I The fused silica capillary column was fed through the heated transfer line interface and positioned at the entrance to the m w spectrometer source block. The mass spectrometer source temperature was set to 290 OC, and the interface temperature was set to ca. 300 "C. Mass spectra during GC-MS analyses were obtained by ammonia chemical ionization (positive ion mode) at a source pressure of 0.3 Torr. Data acquisition and manipulation were carried out with a Finnigan Incos data system, version 3.1. The computer system consisted of a Data General (Westboro, MA) Nova 3 minicomputer, a Model ST-2222 10 megabyte Wangco (LosAngeles, CA) disk drive, a Tektronix (Beaverton, OR) Model 4010 terminal, and a Versatec (Santa Clara, CA) Model 800-A electrostatic printer / plotter. The determination of the isotopic enrichment of nonadeuteriated cholesterol was performed on a Nuclide (State College, PA) 12-90-G,single-focusing, magnetic sedor mass spectrometer. The instrument was interfaced to an IBM XT-personalcomputer system, utilizing Technivent (St.Louis,MO) PCMAG version 2.14 (copyright 1987) software to control data acquisition and manipulation. The cholesterol samples were analyzed as their corresponding TMS ethers by direct insertion probe utilizing electron impact ionization. The ion source temperature was set to 200 "C, the electron accelerating potential was 15 eV, and the ion accelerating potential was 4.5 kV. A 500-W mercury arc lamp was used for ultraviolet (UV) irradiation of samples and standards during oxidation. The lamp was powered by a Model P-510-D power supply (George W. Gates and Co., Long Island, NY) and the radiation focused with a double-focusing plano-convex, 35 mm, fused quaTtz lens assembly (Oriel Corp., Stamford, CT). The lamp was cooled by a 4.5-in. diameter, 115 V (0.004 hp) fan during operation. Homogenization of sample powders was carried out utilizing a Potter-Elvehjem Tissue Grinder with Teflon pestle (Fisher Scientific, Pitteburgh, PA). The pestle (21 cm X 12-mm 0.d.) was connected to a Model K41 variable-speed electric stirrer (Tri-R Instrumente, Rockville Center, NY). Methods. Silylation of Laboratory Glassware. All laboratory glassware was subjected to vapor-phase silanization as described by F e n i m ~ r in e ~order ~ to minimize analyte losses due to surface adsorption on glass. Synthesis of [2H9]Cholesterol.The details for the synthesis of the internal monitor were described previ0usly.3~Briefly, the procedure involved initial exchange of the hydrogens a to the (starting carbonyl group of 3@-acetoxy-26-norcholest-5-en-25-one material) in [O-2Hl]methanol. Grignard reaction with C2H3Mgp produced 25-hydroxy[2Hs]cholesterolwhich was reacetylated in the 3-position to protect the OH group. The 25-hydroxy group was replaced with Br by reaction with PBr3 Debromination was carried out in h e y nickel in the presence of deuterium gas, and, after washing in 10% Na02H in 2H20,a 1:l mixture of [2Hg]cholesterol and [2H7]desmosterolwas obtained. The two compounds were separated and collected by reversed-phase preparative HPLC. The isotopically labeled cholesterol, la, which was synthesized for use in this study consisted of nine deuteriums incorporated into the sterol side chain ( [24,24,25,26,26,26,27,27,27-2Hg]cholesterol). Isotopic enrichment calculations revealed that the 6.8% I8, [2Hg]cholesterolsample contained 66.8% 2Hs,24.0% % 2H7,and 2.3% 2H6incorporation. No significant amounts of producta with lower deuterium content and no unlabeled analogue were present in the final product. Cholesterol Content in Liver. The determination of the amount of cholesterol in powdered liver samples was accomplished by reversed-phase HPLC utilizing the same Gilson HPLC system as described in the instrumental section. The HPLC column was a preparative Zorbax CIS column (25 cm X 9.4-mm i.d., 5-pm particle size) (Dupont, Wilmington, DE) connected to a guard column consisting of a Guard-Pak Precolumn Module (Waters Associates,Milford, MA) containing a p-Bondapak C18precolumn insert (Waters Associates). Methanol (HPLC grade) was used as the isocratic mobile phase at a flow rate of 1.0 mL/min. Eluting
1079
compounds were detected by UV at 212 nm (0.2 range). Cholesterol peak areas were obtained by on-line integration. A series of four calibration solutions of cholesterol in acetone was prepared, spanning the range from 0.25 to 10 pg/pL. A total of 20 pL of each solution was injected onto the reversed-phase HPLC column, utilizing a 20-pL sampling loop. Each calibration point was analyzed in duplicate. The average cholesterol peak area for each calibration point was plotted against the cholesterol concentration to obtain a cholesterol calibration curve. Lipids were isolated from the powdered liver samples by chloroform extraction. Fifteen milliliters of degassed chloroform were added to the sample and homogenized for 1min. The sample was then vacuum filtered and washed with chloroform. The combined extract and washings were evaporated to dryness then transferred to a 3.0-mL silanized glass vial in 100 pL of acetone (HPLC grade). Twenty microliters of this solution was injected onto the HPLC and the resulting cholesterol peak integrated. The average cholesterol peak area from two injections of the liver sample was fitted to the previously constructed cholesterol calibration curve to give the amount of cholesterol per milligram of powdered liver. Powdered Liver Analysis. After determination of the cholesterol level in the powdered liver, the deuteriated cholesterol probe was applied to the actual sample analysis. Approximately 250 mg of the powdered liver was accurately measured out then quantitatively transferred to a silanized 25-mL test tube. The appropriate amount of [%Is]cholesterolneeded for 250 mg of the liver sample was dissolved in 200 pL of HPLC-grade acetone and purified by reversed-phase HPLC utilizing the same Zorbax CIS column as described in the previous section. The deuteriated cholesterol sample was injected onto the HPLC column utilizing a 200-pL sampling loop. Methanol (HPLC grade, degassed) was used as the mobile phase at a flow rate of 2.0 mL/min. A refractive index detector was used to detect eluting compounds. The purified [2Hg]cholesterolcollected from the HPLC column was added immediately to the liver sample. Subsequently, 5.0 pg of the 6-ketocholestanol internal standard was also added to the liver sample. The spiked sample was then homogenized for 30 s. The solvents present in the sample after spiking were removed by evaporation under a gentle stream of nitrogen at room temperature. The resulting solid sample was placed in a refrigerator for approximately24 h at 0 "C in order to allow the spiked deuteriated cholesterolto attain the same chemical environment as the sample's endogenous cholesterol. Lipids were next isolated from the liver sample by chloroform extraction as described above. The sample was evaporated to dryness then dissolved in 1.0 mL of degassed ethyl acetate/methanol (955). Residual water and polar organic compounds were removed from the liver extract by silica gel liquid chromatography (LC). Activated silica gel was packed into a glass column (50 cm X 1.75cm id.) fitted with a glass frit and Teflon stopcock to a height of 4 cm and then equilibrated in ethyl acetate/methanol(95:5). One milliliter of the sample in ethyl acetate/methanol(955) was placed on the head of the column followed by two, 1-mL rinses of the sample flask (using the same solvent). The sample was eluted from the column using 70 mL of ethyl acetate/methanol (955). Following collection, the eluate was reduced in volume by rotary evaporation and transferred to a 3.0-mL silanized glass vial equipped with a Teflon-linedcap. The sample was evaporated to dryness under a stream of nitrogen at room temperature and dissolved in 200 pL of HPLC-grade acetone. Cholesterol oxides were isolated from the sample solution by preparative reversed-phase HPLC. The HPLC column was a i.d., 10-pm particle size) p-Bondapak C18column (30 cm X 7.8" (Waters Associates, Milford, MA) connected to a guard column. Acetonitrile was used as the isocratic mobile phase at a flow rate of 3.0 mL/min. In order to establish the oxide collection points for the subsequent sample fractionation, a solution of 100 pg of each sterol (including cholestanetriol, 6-ketocholestanol, pepoxycholesterol, a-epoxycholesterol,and cholesterol) in 200 pL of HPLC-grade acetone was first injected by loop onto the reversed-phase column. Collection of the sample oxide fraction was begun just prior to the elution of the cholestanetriol and ended with the trailing edge of the a-epoxycholesterol. The sample was then injected onto the column in 200 pL of HPLC-grade acetone utilizing a 200-pL sampling loop. The collected oxide fraction
1080
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
was reduced in volume and then stored in 3.0-mL amber-colored glass vials equipped with Teflon-lined caps. The samples were derivatized to form the trimethylsilylethers of the sterols before analysis by GC-MS. Residual acetonitrile was removed from the sample using a stream of nitrogen. The sample was next dissolved in two drops of silylation-gradepyridine and then reacted with BSTFA, 1% TMCS (0.5 mL) for approximately 24 h at room temperature. Following derivatization, the sample was evaporated to dryness under nitrogen and dissolved in 100 pL of BSA. A 1-2-pL aliquot of this concentrate was introduced into the GC-MS by direct on-column injection. For the purpose of quantification of both labeled and unlabeled oxide componenta, a seven-point calibration curve using 6-ketocholestanol as internal standard was constructed for each oxide of interest. The calibration curves consisted of one blank data point and six data points obtained from standard solutions in the range of 0.05-20 pg. Relative Rates of Cholesterol Oxidation. Labeled and unlabeled cholesterol, in a 1:l or 3:l ratio (2Ho/2Hs),were weighed out separately (2 mg total) and mixed together in a 3.0-mL silanized glass vial. The cholesterol mixture was dissolved in 100 pL of HPLC-grade acetone for subsequent HPLC purification using a p-Bondapak semipreparative CI8 column (7.8 mm X 30 cm) as described earlier. The mobile phase was deoxygenated by helium purging for 30 min prior to uae. The purified cholesterol fraction was collected in a silanized, 250-mL round-bottom flask which was rotary evaporated to dryness. After cooling the sample flask to room temperature, 1.0 mL of HPLC-grade ethyl acetate was pipetted into the flask to dissolve the purified cholesterol mixture. Approximately 100 pL of this solution was withdrawn, utilizing a 1.0-mL graduated pipette and deposited in a 3.0-mL silanized,amber-coloredglass vial equipped with a Teflon-lined cap. The cholesterol aliquot was evaporated to dryness under nitrogen and then derivatized with BSTFA, 1%TMCS. This cholesterol aliquot was obtained in order to establish the initial [2Ho]/[2Hs]cholesterolratio prior to oxidation. The remainder of the purified cholesterol solution was rotary evaporated to dryness. An aqueous dispersion of the mixture of [2Ho]-and [2Hs]cholesterol was prepared by dissolving in approximately 10 mL of warm acetone to which was added ca.60 mL of tap water. The acetone was removed by rotary evaporation to give an aqueous dispersion of cholesterolwhich was slightly cloudy in appearance. The cholesterol dispersion was transferred to a silanized 150-mL beaker and topped up to 80 mL with tap water. The dispersed cholesterol sample was heated to 60-70 “C, purged with oxygen, and irradiated with UV radiation from a mercury arc lamp, situated approximately 15 cm above the top of the cholesterol dispersion, for approximately 2 h. Cholesterol and its oxidation products were extracted from the aqueous phase using chloroform, transferred to a 3.0-mL silanized glass vial, and then evaporated to dryness. The oxidized sample was then dissolved in 100 pL of acetone for subsequentHPLC separation to remove cholesterol. After collection, the oxide fraction was evaporated to dryness, transferred to a 3.0-mL silanized glass vial and then derivatized with BSTFA, 1% TMCS. Following derivatization, the excess silylating reagent was evaporated from the oxide fraction under a stream of nitrogen and then dissolved in 100 pL of BSA for subsequent GC-MS analysis. In order to determine the 2Ho/%Is oxide peak area ratios by GC-MS, the mass spectrometer was scanned from mf z 365 to m / z 417 (52 u)in 0.25 s and then from m / z 455 to m f z 498 (43 u) in 0.20 s. In order to generate 25-hydroxycholestero1from cholesterol, a 1:l ratio of cholesterol and [2Hs]cholesterolwas measured out and purified as described previously. The purified cholesterol fraction was collected and then transferred to a 150-mL silanized beaker. The solvent was evaporated under a gentle stream of nitrogen so that the crystalline cholesterol mixture coated the bottom of the beaker. The sample beaker was placed on a Model PC-351 hot plate/stirrer (Corning Glass Works, Coming, NY)and heated to a temperature of 90-100 “C. A Model UVSL-25 multiband UV lamp (Ultra-VioletProducts, San Gabriel, CA) was placed directly above the mouth of the beaker (ca. 5 in. from the sample) and allowed to irradiate the sample at a wavelength of 254 nm for 5 h. After reaction, the oxidized cholesterol mixture was transferred to a 3.0-mL silanized glass vial for subsequent
HPLC purification. The oxide fraction collected from the HPLC purification was evaporated to dryness then derivatized as described previously. Test of Deuteriuted Cholesterol Quuntitatwn Scheme. In these experiments a “synthetic”sample was prepared by first oxidizing a mixture of labeled and unlabeled cholesterol. The resulting mixture of labeled and unlabeled oxides was subsequentlyspiked with known quantities of cholesterol oxide standards and 6ketocholestanol (internal standard). In this instance, the spiked oxides represented “endogenous” components. Those oxides formed during the oxidation of cholesterol represent the ~ “endogenous” “artifacts”. The goal, therefore,was to q u a n t the oxides in the presence of interfering oxide “artifacts” by utilizing the deuteriated cholesterol probe and its associated correction scheme. A total of four quantitation tesb was carried out. The first such test involved the use of only 7-ketocholesterol and a-epoxycholesterolas spiked “endogenous”oxides. Subsequent quantification testa, employed a larger variety of cholesterol oxidation products as spiked “endogenous”oxides. The experimentaldetails for testing the deuteriated cholesterol quantitative accuracy are similar to those described previously for the determination of the relative rates of cholesteroloxidation. Labeled and unlabeled cholesterol were weighed out in approximately a 1:l ratio and purified by reversed-phase HPLC (pBondapak CIS). An aliquot of the purified cholesterol solution was removed, derivatized, and analyzed by GC-MS so as to establish the initial %/%&cholesterolratio prior to oxidation. The remainder of the purified cholesterol solution was oxidized as an aqueous suspension as described previously but was heated to only 50 O C for 2 h. Following oxidation, the aqueous suspension was spiked with known quantities of cholesterol oxide standards and 6-ketocholestanol (the internal standard) and then extracted from the aqueous phase using chloroform. The oxides were collected by reversed-phase HPLC, derivatized then analyzed by GC-MS. Oxidation of Powdered Liver. A total of 3.5 g of powdered liver was weighed out and quantitativelytransferred to a large silanized 25-mL test tube. Due to inhomogeneities in the liver powder, the total 3.5-g sample was first extracted into chloroform before it was divided into seven separate and equal parts. The powder was initially extracted with degassed chloroform, homogenized, and then filtered as described previously. The filtrate was collected in a silanized 500-mL filter flask which was packed in ice. This precaution was taken to minimize cholesterol oxidation before addition of the deuteriated cholesterol monitor. The solids left on the filter paper were subsequently washed with chloroform and combined with the initial filtrate. The extracted liver powder was transferred to a silanized 250-mL round-bottom flask and rotary evaporated to dryness. A total of 6.3 mg of deuteriated cholesterol was measured out and dissolved in 400 pL of HPLC-grade acetone. The labeled cholesterol was purified by HPLC using two, 200-pL injections. The purified cholesterol fraction was introduced into the extracted liver sample as it eluted from the HPLC column. To guard against oxidation at this stage the flask containing the liver extract was packed in ice. After rotary evaporation, the spiked liver sample was allowed to reach room temperature and then dissolved in 70.0 mL of degassed chloroform. Six 10-mL aliquota were pipetted from this solution and placed in separate silanized round-bottom flasks. Mild Conditions. Three of the above aliquots were spiked with 7.0 pg of the 6-ketocholestanolinternal standard and stored overnight at 0 “C. The three samples were analyzed by the established procedure. However, the conditions during the analysis were modified so as to preclude the autoxidation of cholesterol. All solvents used during the analpis were rigorously deoxygenated by helium purging for 30 min. During LC and HPLC separation, the sample receiving flasks were packed in ice and the eluting sterol fraction collected under a nitrogen atmosphere. All rotary evaporations were conducted at low temperature (ca. 30-40 “C) and under a nitrogen atmosphere. Evaporation of small quantities of solvent was accomplished at room temperature by sweeping nitrogen across the sample. In addition, all three analyses were conducted in semidarkness. The resulting oxide solutions were converted to their trimethylsilyl ether derivatives as described previously.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
1081
Table I. Major Ions Obtained by Ammonia Chemical Ionization for Cholesterol Oxidation Products of Interesta
compound cholesterol 7a-hydroxycholesterol 7~-hydroxycholestero1 @-epoxycholesterol a-epoxycholesterol 25-hydroxycholesterol 6-ketocholestanol 7-ketocholesterol
(M + H)'
(M
+ H)' 18
-
(M + NHJ' 90 386 (66)
475 (74) 475 (9) 475 (28) 473 (100)
457 (33) 457 (16)
402 (3) 402 (15)
ammonia CI peaks (M + H)' - (M + H)' 90 18 - 90 369 (100) 457 (100) 457 (100) 385 (90) 385 (100) 385 (10) 385 (100) 383 (11)
367 (100 367 (58) 367 (100) 367 (11)
(M + H)' 90 - 90
(M + NHI)' 90 - 33
367 (68) 367 (79)
369 (51) 369 (21) 369 (7)
Relative intensities are given in parentheses. All oxides were completely converted to their corresponding TMS derivatives with the exception of 25-hydroxycholestero1which formed the mono-TMS derivative (carbon no. 3).
Harsh Conditions. The remaining three liver aliquots were oxidized as aqueous suspensions in the manner described previously in order to generate cholesterol oxide "artifacts". Each sample was heated to 50 OC, purged with oxygen, and irradiated with W radiation for 1h. After oxidation each sample suspension was spiked with 7.0 wg of the 6-ketocholestanolintemal standard and stored overnight at 0 "C. The aqueous samples were rotary evaporated to dryness through the addition of 1-propanol so as to form the binary azeotrope (bp 88 OC).% The oxidized liver samples were analyzed according to the established procedure without any modifications. Calibration curves for analysis of both the "harsh" and "mild" samples were prepared as described previously. Mass Spectral Measurements. During quantitative mass spectral analysis two major sources of error were identified which affected significantlythe results obtained. First, the mass spectra of the cholesterol oxideTMS ethers examined in this work were found to exhibit occasional relative peak intensity variations under positive ammonia chemical ionization conditions, presumably due to changes in the mass spectrometer source conditions with time. As a result, quantification of individual oxides based on the signal intensity of a single mass spectral peak led to a systematic error in the final quantitative determination. The second source of error was associated with the extent of deuterium incorporation in the [2H9]cholesterolmolecule. It was shown previously that the deuteriated cholesterol synthesized contained approximately 66% of the 'H,-labeled species. The most intense isotope in the mass spectral peaks of the labeled oxides is, therefore,the *Hgspecies. Since calibration curves were constructed using unlabeled oxides, quantification of the labeled peak could conoxides based on the most intense isotope ('He) ceivably introduce a 33% error into the quantitative result. This occurs since 33% of the labeled oxides would not be accounted for due to their distribution among the less intense isotopes ('Hb-'H8). In order to circumvent these problems, all of the major mass spectral peaks for each oxide (see Table I), including both labeled and unlabeled species and their associated isotope peaks, were scanned during analysis. This was accomplished by implementing a multiple ion detection scheme. From scans 1-350, two separate m m intervals were scanned. The fiist was from m/z 367 to m/z 378 in 0.24 s. The second was from m/z 457 to m/z 468 in 0.24 s. This mass range was utilized to detect labeled and unlabeled 7a-hydroxycholesterol. After scan 350, the two mass-scan intervals were automatically increased so as to include the epoxycholesterol diastereoisomers as well as 7~-hydroxycholestero1.The new scan intervals were, therefore, m/z 367 to m/z 396 (0.24 s) and m/z 457 to m/z 486 (0.24 8). These m w intervals were scanned until scan 750 was reached at which point the mass spectrometer data system automatically altered the scan intervals and times to m/z 367-mlz 404 (0.28 s) and m/z 473-m/z 484 (0.14 8 ) . These mass intervals were utilized to detect 6-ketocholestanol and 7-ketocholesterol. Upon reaching scan 850, the data system was programmed to automatically terminate data acquisition. The final quantification of labeled and unlabeled oxides was accomplished by first summing the isotopes for each major peak in the mass spectrum of the particular oxide. These isotope summationswere then added together to produce a reconstructed
Table 11. Mass Spectral Peaks Summed for Labeled and Unlabeled Sterols labeled masses"
compound cholesterol
7a-hydroxycholesteroland 7~-hydroxycholesterol p-epoxycholesterol and a-epoxycholesterol
375-380 392-397 373-378 463-468 373-380 391-396 463-468
(378) (395) (376) (466)
(376, 378)
(394) (466) 481-486 (484)
6-ketocholestanol
7-ketocholesterol
389-394 (392)
unlabeled masseso 369-371 (369) 386-388 (386) 367-369 (367) 457-459 (457) 367-371 (367,369) 385-387 (385) 457-459 (457) 475-477 (475) 367-371 (367, 369) 385-387 (385) 402-404 (402) 475-477 (475) 383-385 (383) 473-475 (473)
479-484 (482) "Massin parentheses is the nominal mass of the most intense isotope. Table 111. GC-MS Calibration Curve Data for the Cholesterol Oxidation Products of Interest (R*= Correlation Coefficient) compound
slope
7a-hydroxycholestero1 7~-hydroxycholestero1 a-epoxycholesterol &epoxycholesterol 7-ketocholesterol
1.548 1.559 1.527 1.250 1.696
Y intercept
R2
0.185
0.9992 0.9979 0.9993 0.9967 0.9994
-0.484
-0.228 -0.589 -0.197
total ion plot for each oxide component. Table II shows the massea that were summed for each labeled and unlabeled sterol. The reconstructed total ion plots were integrated for subsequent fitting to the unlabeled oxide calibration curves. The GC-MS calibration plot data which were obtained utilizing the above peak summations are given in Table 111.
RESULTS AND DISCUSSION Isotopically labeled cholesterol was utilized in this work in order to determine whether the analytical procedure produces artifacta in the sample being analyzed. The method employed involves the addition of labeled cholesterol to the sample at the initial stage of the analytical procedure. Early in the analysis a cholesterol aliquot is obtained in order to establish the unlabeled to labeled cholesterol ratio in the sample. The basic premise of the method is that, if oxidation of cholesterol occurs during the procedure, the spiked labeled cholesterol will also be oxidized concurrently with the sample's endogenous cholesterol to produce labeled oxides. If, however, cholesterol is not oxidized during analysis the labeled cholesterol will remain intact. As illustrated in Figure 1 (which assumes the addition of an amount of labeled cholesterol equal
1082
ANALYTICAL CHEMISTRY, VOL. 04, NO. 10, MAY 15, 1992 'H.-CHOLESTEROL
I
SAMPLE
Cholesterol
1
ilA 'Hg
'
-on
2
''
OH
3
S-Cholestene-36, 7.-diol (7~-Hydrokycholesterol)
5-Cholestene-36, 76-diol (7o-H~drokycholesterol)
ANALYTICAL PROCEDURE
I
2Ho
5
A 4
5 . 6a-Epoxy-S~-Cholestan-3a-oi (mEpoxycholesterol)
GC-MS ANALYSIS OF OXIDES
'
5,6e-Epoky-S8-Cholestan.36-ol (p-Epokycholesterol)
HO0
6 (Ai
(8)
S-Cholestene-3~,25-diol (25-Hydroqcholesterol)
3a-Hydroxy-5.Cholesien -7-one (7-Ketocholesterol)
(C)
Flgun 1. General scheme outllnlng the use of deuterlated cholesterol
to detect the formation of cholesterol oxide artifacts during analysis.
to that of the cholesterol present in the sample), this type of approach can produce three distinctly different results. If only an unlabeled oxide component is deteded in the final analysis, this indicates that the observed oxide is an endogenous component of the sample and is not formed as an artifact during analysis (case A). Case B shows a situation where both a labeled and an unlabeled oxide component are present. Since the unlabeled to labeled oxide ratio is the same as the unlabeled to labeled cholesterol ratio, this indicates that the observed unlabeled oxide is formed entirely as an artifact during analysis. Case C shows a situation where, once again, both a labeled and an unlabeled oxide component are present. However, in this case, the unlabeled to labeled oxide ratio is quite different from the unlabeled to labeled cholesterol ratio. This indicates that a fraction of the unlabeled oxide component is formed as an artifact during analysis and that the remaining portion is endogenous to the sample. A fourth possible scenario (not shown in Figure l), namely the detection of only labeled oxides has also been suggested. While in principle this is possible, its occurrence would be of no practical utility as this would be encountered only in samples containing no cholesterol oxidation products or, for that matter, no cholesterol at all. One goal of this study is to demonstrate that the formation of cholesterol oxide artifacts during analytical manipulations can be readily detected through the use of the deuteriated cholesterol probe. In addition, it should be possible to use the information obtained from the labeled cholesterol probe to correct the analytical data for the presence of these procedurally derived artifacts. It will be demonstrated that the labeled to unlabeled cholesterol ratio can be used in conjunction with that of labeled to unlabeled oxides in order to provide such a correction. The application of the deuteriated cholesterol correction should, therefore, provide a more realistic quantitative assessment of those cholesterol oxidation products endogenous to the sample. The cholesterol oxidation products of interest are shown in Figure 2. The IUPAC names for these cholesterol derivatives are also given in Figure 2; however, the more common names given in parentheses, will be utilized to refer to them here.
/li.-;*
HO
8
H
OH
50-Cholestane-35, SabB-triol (Cholesmnetriol)
O
W
0
9
3oHydroxy-5aCholestan -6-one (6-Ketocholestanol)
-re 2. Cholesterol oxldatlon products of Interest, common names gken In parentheses.
The site of labeling on the cholesterol molecule was of critical concem since the possibility existed that labeling on the cholesterol ring system may introduce undesirable isotope effects during the oxidation of labeled and unlabeled cholesterol and during the mass spectral analysis of labeled and unlabeled oxides. This latter effect has been demonstrated by an earlier study which showed severe isotope effects during the mass spectral analysis of the trimethyhilyl (TMS) ethers of cholesterol and hexadeuteriated [2,2,3,4,4,6-2H6]cho1esterol (labeled on the ring system).3s Since most of the pertinent maas spectral fragmentations in cholesterol and its oxidation products arise from cleavages associated with the A and B rings and their substituents,3l the mass spectral analyses of most of the sterol TMS ethers of interest should not be influenced by the isotopic enrichment of the sterol side chain. These considerations certainly hold for electron impact (EI) induced fragmentations but may also be of concern for chemical ionization (CI) in NH3 reagent gas which we opted to use here. In the latter case, many of the fragment ions used for quantitation are formed by sequential eliminations of trimethylsilanol and/or HzO. Any hydrogen abstraction associated with these processeg is more likely to involve the ring system than the side chain. In addition to the mass spectral considerations, side-chain labeling should also minimize isotope effects during the oxidation of cholesterol since the bulk of the oxides of interest are derived from reactions on the A and B rings of the steroid skeleton (see Figure 2). One possible exception, however, is 25-hydroxycholestero1 in which the 2bposition is the site of oxidative attack. This p a r t i c k topic will be discussed in more detail later. In view of these considerations, a side chain labeled derivative of cholesterol was the preferred candidate to use as a quantitative probe of cholesterol oxide artifact formation.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
1083
Table IV. Comparison of the Initial Unlabeled to Labeled Cholesterol Ratio to the Final Unlabeled to Labeled Oxide Ratios for 1:l and 3:l Cholesterol Oxidations (Each Ratio Represents the Average of Three Injections, RSD = Relative Standard Deviation) 21-&,/2H9peak area ratios (&RSD)by GC-MS trial no. 1 (1:l 2 (1:l 1 (3:l 2 (3:l
ratio) ratio) ratio) ratio)
initial cholesterol ratio
&epoxycholesterol
error, %
a-epoxycholesterol
error, %
7-ketocholesterol
error, %
1.41 (*3.4%) 1.26 (*3.2%) 3.05 (&0.84%) 3.17 (&1.9%)
1.49 (*0.64%) 1.29 (*3.1%) 3.26 (*0.84%) 3.74 (*2.7%)
5.6 2.4 6.8 17.9
1.39 (&1.4%) 1.35 (&1.5%) 2.93 (&2.6%) 3.61 (&4.1%)
1.4 7.1 3.9 13.9
1.52 (*0.63%) 1.26 (*1.6%) 3.23 (&0.97%) 3.23 (*1.9%)
7.8 0.16 5.9 1.9
Relative Rates of Cholesterol Oxidation. The thesis of utilizing isotopically labeled cholesterol to monitor changes in a sample's endogenous cholesterol during analytical manipulations necessitates that both the labeled and unlabeled compounds behave in an identical manner during analysis. To illustrate this, consider a situation where the labeled cholesterol is oxidized preferentially over that of the unlabeled cholesterol in the sample. In this case the f i i analyais would reveal the presence of labeled oxide components. This result might lead to the erroneous conclusion that artifacts had been formed in the sample during analysis. This would, therefore, generate a large negative error in the final result due to overcorrection of the quantitative data. The opposite situation would be equally detrimental. If the unlabeled cholesterol is oxidized preferentially over that of its labeled counterpart, the final analysis would reveal no labeled components. This could lead to the erroneous conclusion that no artifacts had been formed during analysis. A large positive error would, therefore, arise in the final quantification since no correction for artifacts would have been applied to the data. Thus, in order to confirm the validity of the proposed approach, it was first necessary to establish that the relative rates of oxidation of labeled and unlabeled cholesterol were identical. To assess this, mixtures of labeled and unlabeled cholesterol were oxidized. The ratios of (21-b/W9)oxidea formed were calculated and compared to the initial ratio of unlabeled to labeled (W,,/2HS) cholesterol before oxidation. Agreement between these two ratios would be essential in order to establish the feasibility of the method. A total of four cholesterol oxidations was performed and the produds analyzed by GC-MS. In two of these, the initial unlabeled to labeled cholesterol ratio was approximately 1:l. In the other two, the initial unlabeled to labeled cholesterol ratio was approximately 3:l. Figure 3 shows the GC-MS trace of the sample resulting from the oxidation of an approximately 1:l mixture of [2Ho]/[2H,]cholesterol. Three major products are formed during this oxidation: 8-epoxycholesterol,a-epoxycholesterol, and 7-ketocholesterol. Note that this experiment produces a doublet peak for each compound of interest in the GC-MS trace, The first peak of each doublet corresponds to the labeled component, the second peak to the unlabeled component. The cholesterol peaks in the GC-MS trace were obtained from the cholesterol aliquot removed just after purification (see Figure 1)and spiked into the finaloxide fraction. The ratio of labeled to unlabeled cholesterol, therefore, represents the initial [2Ho]/[2Hg]cholester~l ratio prior to oxidation. In order to explore the possibility of using smaller amounts of deuteriated cholesterol in the sample analysis, the above oxidation experiment was repeated using an approximately 3:l unlabeled-to-labeled cholesterol ratio. A comparison of the cholesterol and oxide peak area ratios obtained by GC-MS analyses for both the 1:l and 3:l cholesterol oxidations is summarized in Table IV. As is evident from Table IV there is very good agreement between the initial unlabeled-to-labeled cholesterol ratio and
E
1
I
I u)
I
n
1
M
'
35
rime (min.) 3. capillary GCMs (RIC) trace of a i:i[+~,,]/[*~~ctw~atero~ oxklatbn: 1, cholesterol;5, @poxychokterd; 4, a+poxycholesterd: 7, 7-ketocholesterol:A, labeled component: B, unlabeled component.
the final unlabeled-to-labeled oxide ratios for the 1:l cholesterol oxidation. In most cases less than 6% difference exists between the two ratios. The GC-MS results for the 3:l cholesterol oxidation experiment are also given in Table IV. It is evident that the cholesterol and oxide ratios do not agree as well as in the 1:l cholesterol oxidation experiment. This may be due to adsorptive losses of the smaller amount of labeled component in the GC-MS interface. These results suggest that it is preferable to spike the sample with an amount of labeled cholesterol approximately equal to the amount of unlabeled cholesterol initially present in the sample. The excellent agreement between the cholesterol and oxide ratios indicates the absence of a primary isotope effect during the oxidation of labeled and unlabeled cholesterol. This is expected given the distance separating the site of oxidative attack from the site of isotopic labeling. The situation is quite different, however, for the formation of 25-hydroxycholestero1 since generation of this oxide involves oxidative attack at the 25-position of cholesterol. Because of the presence of a deuterium atom in the 25-position of the molecule, cleavage of this C-D bond to produce a radical site would be expected to occur more slowly than cleavage of the corresponding C-H bond in unlabeled cholesterol due to a primary isotope effect. In order to test this hypothesis, a 1:l mixture of labeled and unlabeled cholesterol was oxidized under slightly different conditions so as to favor the formation of 25-hydroxycholesterol (see Experimental Section). The GGMS trace obtained from thisoxidation experiment is shown in Figure 4. Doublet peaks, indicating the presence of labeled and unlabeled components, are seen for 7a-hydroxycho1estero1,cholesterol, 78hydroxycholesterol, the epoxycholesteroldiastereoisomers,and the 7-ketocholesterol. Note that two peaks, 6 and 6', are observed in Figure 4 for 25-hydroxycholestero1. They correspond to the mono-TMS and.di-TMS derivatives of unlabeled
1084
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992 6
1
I
Table V. GC-MS Quantitative Results for the Initial Oxide Spiking Experiment Employing 7-Ketocholesteroland a-Epoxycholesterol (the Average of Three Replicate Determinations Is Reported, RSD = Relative Standard Deviation)
7 B
GC-MS,
actual,
compound
7-ketocholesterol
pg
4.99 10.01
a-epoxycholesterol
pg
(kRSD)
error, %
5.04 (*2.9%) 9.22 (*4.2%)
1.0 7.9
Table VI. Quantitative GC-MS Results of the Spiking Experiment Employing All Oxides of Interest (n = 3) (RSD = Relative Standard Deviation)
Time (min.)
Flewe 4. Capillary OCMS (RIC) trace of a 1:l [*H0]/[*H9]cholesteroi oxidation favoring the formation of 25-hydroxycholestero1. 2, 7ahydroxycholesterol; 1, cholesterol; 3, 7&hydroxycholesteroi; 5, pepoxychoiesterol; 4, a-epoxycholesterol; 6, 25-hydroxycholesterolmono-TMS; 7, 7ketocholesterol;6', 25-hydroxychdesterol-dI-TMS; A, labeled component: B, unlabeled component.
compound
actual, pg
GC-MS, pg (kRSD)
error, 70
7a-hydroxycholesterol 78-hydroxycholesterol j3-epoxycholesterol a-epoxycholesterol 7-ketocholesterol
8.00 8.00 4.99 8.01 8.00
7.84 (f2.3%) 8.11 (*3.0%) 5.36 (*2.7%) 7.94 (*2.3%) 8.21 (*2.4%)
2.0 1.4 7.4 0.9 2.6
Since both labeled and unlabeled cholesterol are oxidized a t the same rate, eq 1can be written UC/LC = UOA/LOA
(1)
The term on the left represents the unlabeled to labeled cholesterol ratio and the term on the right represents the resulting unlabeled to labeled oxide artifact ratio. This equation can be rearranged to yield UOA = (UC/LC)(LO,)
Flgure 5. Deuterlated cholesterol correction scheme. LC, labeled cholesterol; UC, unlabeled cholesterol; LOA,labeled oxMe artlfacts; UOA, unlabeled oxide artifacts; UOT, total unlabeled oxides in the sample; UOs, unlabeled oxides endogenous to the sample (desired quantity).
25-hydroxycholestero1,respectively. Since the success of the deuteriated cholesterol probe depends on the identical behavior of both labeled and unlabeled cholesterol during oxidation, it is obvious that cholesterol deuteriated on the 25position cannot be used as a probe to detect the presence of 25-hydroxycholestero1artifacts formed during analysis. A possible alternative would be the use of 13C-labeledcholesterol or, of course, a labeled cholesterol bearing no deuterium on the 25-position. Such an internal monitor containing seven deuterium atoms can be prepared by hydrogenation of [2H,]desmosteroI which is formed in the last step of our synthetic scheme (see the Experimental Section). QuantificationTesting. It is clear from the earlier discussion that the levels of deuteriated oxides observed in the GC-MS determination reflect the levels of cholesterol oxide artifacts formed. In view of this, it should be possible to subtract the artifact contribution represented by the labeled oxides, from the unlabeled oxide peak intensities. Unfortunately, this subtraction cannot be appIied directly to the unlabeled oxide peak intensities since the amounts of labeled and unlabeled oxide artifacts will depend on the [2Ho]/ [2Hg]cholesterolratio in the sample. Therefore, in order to use the deuteriated cholesterol probe to correct the analytical data, the labeled and unlabeled oxide intensities as well as the [2Ho]/ [2Hg]cholester~l ratio must be considered in the calculation. The variables utilized in the deuteriated cholesterol correction scheme are illustrated in Figure 5. The method of applying the correction is as follows:
(2)
If the amount of labeled oxide artifacts (LOA)produced during analysis and the total amount of unlabeled oxides in the sample (UOT)are calculated using the 6-ketocholestanol internal standard, then eq 2 can be utilized to calculate the artifact contribution to the unlabeled oxide signal (UOA) shown in Figure 5. The last step in the correction procedure simply involves subtraction of the calculated unlabeled artifact amount (UOA) from the total unlabeled oxide amount (UOT) to give the unlabeled oxides endogenous to the sample (UOs). In order to test the validity of the deuteriated cholesterol correction scheme, a series of oxide spiking experiments was performed. The GC-MS trace for the initial deuteriated cholesterol quantification test, which employed spiking of only the a-epoxycholesterol and 7-ketocholesterol, is shown in Figure 6. A cursory inspection of the chromatographic trace in Figure 6 serves to demonstrate the general utility of the deuteriated cholesterol probe. For those oxidation products that were not spiked, Le., 7a-hydroxycholesterol, 70hydroxycholesterol, and 0-epoxycholesterol, the unlabeled/ labeled oxide ratios agree well with the initial unlabeled/labeled cholesterol ratio. However, for those oxidation products that were spiked (a-epoxycholesterol and 7-ketocholesterol) and, therefore, represent the presence of "endogenous" oxides, the final unlabeled/labeled oxide ratios differ substantially from the initial cholesterol ratio. The similarity of the cholesterol and oxide ratios can, therefore, be utilized as a rapid method for detecting the presence of oxide artifacts in a sample. The quantitative data obtained from this experiment utilizing the deuteriated correction scheme are given in Table V. Very good agreement is indicated between the actual amounts spiked and the amounts calculated after correcting the data for the presence of artifacts. Quantitative data for additional cholesterol oxides were generated by spiking the "synthetic sample" with the 7hydroxycholesterol epimers, the epoxycholesterol diastereoisomers and the 7-ketocholesterol. The GC-MS trace obtained
ANALYTICAL
7 A
1
T i c (min.)
Figure 6. GC-MS (RIC) trace of lnltlal oxide spiklng experlment. A, labeled oxlde component: B, unlabeled oxide component: 2, 7ahydroxycholesterol: 1, cholesterol; 3, 7@-hydroxycholesteroI;5, &epoxycholesterol: 4, a-epoxychoiesterol: 9, Bketocholestanol; 7, 7ketocholesterol.
7
2 8
9
Time (min.)
Figure 7. GC-MS (RIC) trace of splklng experiment employing all oxides of Interest. A, labeled component: B, unlabeled component: 2, 7ahydroxycholesterol:3, 7&hydroxycholesterol; 5, fl-epoxycholesterol: 4, cy-epoxycholesterol; 9, Bketocholestanol(internal standard): 7, 7-ketocholesterol.
from this experiment is shown in Figure 7 and the data s u m marized in Table VI. Once again, very good agreement (generally within 5%) is obtained between the actual amounts spiked and the amounts calculated utilizing the deuteriated cholesterol correction scheme. One further factor considered in the development of the quantitative analytical scheme concerned the [2Ho]/i2H91cholesterol ratio. It was found that the cholesterol ratio used in the above correction scheme had to be determined after the analytical workup since a purified cholesterol fraction could only be obtained after HPLC, the last step in the analytical workup (see the Experimental Section). In view of this, it was necessary to demonstrate that the [2Ho]/ [2Hg]cholesterolratio prior to oxidation was indeed equal to the final [2Ho]/[2H9]cholesterolratio after oxidation, so that this final ratio could be used successfully in the deuteriated cholesterol correction scheme. The average of four determinations of the cholesterol ratio prior to oxidation gave a
CHEMISTRY, VOL. 64, NO.
io, MAY 15, 1992 ioas
value of 1.395 f 0.010. Subsequently, the average of four determinations for the cholesterol ratio after oxidation gave 1.384 0.014. A comparison between the two ratios reveals less than a 0.8% difference between these two average values. The standard deviations given for each average ratio indicate that both values lie well within experimental error of each other. It was concluded, therefore, that the final [2Ho]/ [2Hg]cholesterolratio could be used successfully in the deuteriated cholesterol correction scheme. It should be noted, however, that this equality between the initial and final cholesterol ratios will only be valid as long as side-chain oxidations of cholesterol during the analysis are kept to a minimum. As was demonstrated for the 25-hydroxycholesterol, production of side chain labeled oxides results in isotope effeds giving rise to the preferential formation of the unlabeled oxide over that of the labeled analogue. This would, therefore, deplete the amount of unlabeled cholesterol faster than the labeled cholesterol and introduce a systematic error into the final [2Ho]/[2Hg]cholesterolratio determination. Liver Sample Analysis. A powdered liver extract was chosen to test the validity of the deuteriated cholesterol probe since previous studies, involving cholesterol oxide analysis of a variety of consumable items, showed that similar products contain high levels (13-70 pg/g) of cholesterol oxidation products.36 In addition, initial GC screening of a variety of powdered food samples revealed that this liver extract contained substantial quantities (10-20 pg/g) of several cholesterol oxides of interest including 7au-hydroxycholesterol, 7~-hydroxycholesterol, 8-epoxycholesterol,a-epoxycholesterol, and 7-keto~holesterol.~~ Prior to cholesterol oxide analysis of the liver extract utilizing the deuteriated cholesterol probe, an initial determination of the approximate quantity of cholesterol in the sample was performed. This quantification was necessary since this result had a bearing on the amount of deuteriated cholesterol eventually spiked into the powdered liver sample. It may be recalled that a ratio in the range from 1:l to 3:l unlabeled to labeled cholesterol proved satisfactory for the detection of cholesterol oxide artifacts. The cholesterol content of the powdered liver sample was found to be 1.55 pg/mg sample. Accordingly, 0.4 mg of the deuteriated cholesterol was added to 250 mg of the powdered liver in order to obtain an approximate 1:l ratio between the unlabeled and labeled cholesterol in the sample. GC-MS analysis of the spiked liver extract showed the presence of several cholesterol oxidation products including 7cy-hydroxycho1estero1,7B-hydroxycholestero1, B-epoxycholesterol, a-epoxycholesterol, and 7-ketocholesterol. However, all of the oxide peaks observed corresponded to the unlabeled oxides. Even at lower attenuation, no labeled oxide components were detected by GC-MS suggesting that autoxidation of cholesterol did not occur during the analysis of this sample. A possible explanation is that the presence of natural antioxidants in the powdered liver sample may have protected cholesterol from oxidation during the analysis. This is reasonable since it has been shown that some biological samples contain traces of antioxidants which may protect sensitive
molecule^.^^-^^ The initial purpose of the powdered liver analysis in conjunction with labeled cholesterol was to evaluate the ability of the deuteriated cholesterol probe to correct for the presence of cholesterol oxide artifacts in an actual sample matrix. Since no artifacts were formed in this sample under the analytical conditions utilized this evaluation was not possible. However, by modifying the analytical conditions slightly it was possible to evaluate the deuteriated cholesterol probe in a sample matrix. This was accomplished by analyzing two identical powdered liver samples. The first sample was analyzed under
1088
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992 3
1
3
5
5
B
7
i
a
J l LW
Time (&.)
Flgwe 8. GC-MS (RIC) trace of powdered llver exlract analyzed under mild conditlons. 2, 7ahydroxycholesterol; 3, 7~-hydroxycholesterol; 5, jhpoxycholesterol; 4, a-epoxycholesterol; 9, 6-ketocholestanol (internal standard); 7, 7-ketocholesterol.
Table VII. GC-MS Quantitative Results of “Harsh”and ‘Mild” Powdered Liver Samples (n = 3) (RSD = Relative Standard Deviation) compound 7a-hydroxycholesterol 7~-hydroxycholesterol @-epoxycholesterol a-epoxycholesterol 7-ketocholesterol
mild, pg (*RSD)
5.94 (*2.8%) 8.16 (*3.3%) 5.64 (*3.9%) 0.45 (f2.2%) 4.67 (*2.6%)
harsh, pg (IRSD)
error, %
6.16 (*4.2%) 7.96 (A3.1%) 6.05 (*6.6%) 0.51 (&2.5%) 4.63 (*4.0%)
3.8 -2.4 7.3 15.4 -0.90
conditions designed to minimize oxidation in order to insure that no artifacts could be formed. The levels of cholesterol oxides detected under these (mild) conditionswere, therefore, representative of the oxides endogenous to the sample. An identical liver sample, in conjunction with the deuteriated cholesterol probe, was analyzed under a different set of conditions in which deliberate steps were taken to oxidize the cholesterol in the sample. Since the endogenous levels of oxides in both samples should be constant, the results obtained from the second analysis (harsh conditions), after applying the deuteriated cholesterol correction scheme, should agree with the results of the first analysis (mild conditions). Obviously, barring any systematic error, the level of agreement between these two results will be directly related to the effectiveness of the deuteriated cholesterol monitor to correct for artifacts in the sample matrix. As indicated in the experimental section, the addition of the 6-ketocholestanol internal standard was performed after extraction of the liver powder. Therefore,the possibility exists that the absolute quantification of the observed oxide levels may be inaccurate. It should be noted, however, that in order to establish the utility of the deuteriated cholesterol probe during sample analysis a comparison between the cholesterol oxide levels in the “harsh” and “mild” samples must be made. This relative quantification will, therefore, be independent of sample losses during the extraction process since both sets of samples were extracted simultaneouslyand under identical conditions. The GC-MS traces obtained from the analyses of the powdered liver samples under both “mild” and ‘harsh” conditions are shown in Figures 8 and 9, respectively and summarized in Table W. Note that no deuteriated oxide artifacts are present in the sample analyzed under ‘mild” conditions. As is evident from the results of the GC-MS analyses for the
harsh conditions. A, labeled components; B, unlabeled components; 2, 7a-hydroxycholesteroI;3, 7j%ydroxycholesterol; 5, &epoxyd.lols sterol; 4, a+poxycholesterol; 9, &ketocholestanol (Internal standard); 7, 7-ketocholesterol. two seta of samples given in Table VII, very good agreement between “harsh” and “mild” samples is obtained. The close agreement between “harsh” and “mild” samples analyzed by GC-MS, proves that use of the deuteriated cholesterol monitor can effectively correct the analytical data for the presence of artifacts even in an actual sample environment.
CONCLUSIONS This work has demonstrated that the deuteriated cholesterol monitor can be used effectively to detect the presence of cholesterol oxide artifacts. In a sample containing endogenous oxides, these artifacta are manifested as a series of characteristic peak doublets. The first peak of each doublet corresponds to the labeled oxide artifacts, the second peak correspondsto a summation of the unlabeled artifacts formed and the unlabeled oxides endogenous to the sample. The success of the deuteriated cholesterol monitor to detect oxide artifah easentially stems from this characteristic peak doublet pattern. Given the excellent chromatographic separation of the labeled and unlabeled analogues, it was also reasonable to explore the feasibility of GC-FID alone for the quantitative assessment of artifacts. While the GC-FID yielded excellent results for standard samples, it failed to perform an acceptable analysis of the liver sample due to the poor selectivity of the FID as opposed to the MS detector. As shown in previous studies, the insidious nature of cholesterol oxide artifact formation during analysis makes it extremely difficult to eliminate this problem entirely. The most popular approach to dealing with this problem seems to be in the use of antioxidants added to the sample just prior to analysis. However, several studies have shown that even with the use of antioxidants, cholesterol oxidation during analysis was only reduced, but not eliminated.27p40In spite of these findings it would be most advantageous to have an antioxidant system which is 100% effective and which does not interfere with the finalquantification of cholesterol oxides. To this end, the deuteriated cholesterol monitor can be used in conjunction with various antioxidant systems and analytical methods in order to evaluate the effectiveness of the antioxidant in eliminatingcholesterol autoxidation during analysis and the capacity of the analytical method to form cholesterol oxide artifacts. In addition to artifact detection, it has been demonstrated that the deuteriated cholesterol monitor can be used effectively
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
to quantify endogenous oxides in the presence of oxide artifacts. This is accomplished by essentially subtracting out the artifact contribution to the endogenous oxide signal. This ability to correct the analytical data for artifacts provides an alternative method of dealing with the problem of procedurally derived cholesterol oxide artifacts. The utility of this approach can be manifested in two ways. If it is discovered that oxide artifacts are produced during a given analysis, the analyst is then faced with the task of modifying the analytical methodology so as to preclude artifact formation. Due to the extreme ease of cholesterol autoxidation during analysis, this, obviously, can be a lengthy and difficult process. By utilizing the deuteriated cholesterol monitor to correct the analytical data, the analyst can obtain the desired quantification of endogenous oxides without having to implement extensive modifications of the analytical methodology. If it is not possible to modify the existing analytical method then implementation of the deuteriated cholesterol monitor and its associated correction scheme may be the only recourse. As pointed out, however, selection of the isotopically labeled internal probe should be made with due consideration of the site of deuterium incorporation. As the example of 25hydroxycholesterol indicates, problems may arise if the site of deuterium labeling coincides with that of oxidative attack. It should be noted that the technique of utilizing an isotopically labeled compound to monitor the decomposition or alteration of sensitive compounds during analysis or storage need not be limited specifically to cholesterol. Any compound suspected of undergoing degradation during handling should be amenable to this type of analytical methodology providing, of course, that a suitably labeled tracer can be found. The general applicability of the labeled tracer is strengthened by the fact that "...there is still an unawareness of the care with which biological material must be handled to avoid artificial oxidation of sensitive sterols and unsaturated lipids".17 Until such an awareness is achieved there will be a definite need for the type of analytical methodology developed herein. This becomes particularly crucial with the increased demand for trace level analyses where artifact formation will always be of concern.
ACKNOWLEDGMENT This work was supported, in part, by a grant from the Council for Tobacco Research and funds from a Biomedical Research Support Grant (RR07143). This is contribution No. 519 from the Barnett Institute. Registry No. Cholesterol, 57-88-5; %,-cholesterol, 62743-62-8; 7a-hydroxycholesterol,566-26-7; 7@-hydroxycholesterol,566-27-8; P-epoxycholesterol, 4025-59-6; a-epoxycholesterol, 1250-95-9; 25-hydroxycholeaterol,2140-46-7;6-ketocholestanol, 1175-06-0; 7-ketocholesterol, 566-28-9.
REFERENCES (1) Imal. H.: Werthessen, N. T.; Taylor, C. 6.; Lee, K. T. Arch. Pethd. Leb. Med. 1978, 100, 565-572. (2) Imai, H., Werthessen, N. T., Subramanyam, V., LeQuesne, P. W., Soloway, A. H. and Kanlsawa, M., Science, 1980. 207, 651.
1087
Taylor, C. B., Peng, S.-K., Werthessen, N. T., Them, P. and Lee, K. T., Am. J . CM.Nutr., 1979, 32, 40-57. Cook, R. P. and MacDougall, J. D. B., C .J. Exp. Pethol., 1888, 49, 265-271
Sevanin, A. and Peterson, A. R., Froc. Net/. Aced. Scl., U.S.A. 1984, 81, 4198-4202. Peng, S.-K., Tham, P., Taylor, C. 6. and Mikkelson, B., Am. J. Clln. Nm., 1979, 32(5), 1033-42. Kandtush, A. A. Autoxidation In Food and Blologlcai Systems: Simic, M. G., Karel. M., Ed.; Plenum Press, New York, 1980 p 589. Park, P. S. W. and Addis, P. 6.. J. Food Scl., 1987, 52, 1500. Addis, P. B., Fd. (3". Toxic., 1988, 2 4 , 1021. Park, P. S. W. and Addis. P., J. Assoc. Anal. Chem., 1989, 66. 1632-34. Mlseler, S. R., Wasilchuk, 6. A., and Merrkt, C., Jr., J. Food. Scl.. 1985, 50, 595-598, 646. Flscher, K. H.; &osch, W. Ana!ys& of Oxychoksterols In Foods;Lipid Oxidn.: BIOI. Food Chem.pects. Contrib. LIPID Forum/SIK Symp., Meeting Date 1985, 125-34. Ed. Marcuse, Reinhard, Scand. Forum Llpid Res. Technol.: Ooeteborg, Swed. Fhrocchlaro, E. T., and Rlchardson, T.. J. Food. protect., 1983. 46, 9 17-925. Koopman, 6. J., V a n W e n , J. C.. Wolthers, 6. 6. and Vanderpas, J. 6.. J. ChrOmarOgr., 1987, 416, 1-13. Smith, L. L. U w k f e r o l Autoxkletbn; Plenum Press: New York, 1981. Smith, L. L., Mathews, W. C., Prlce, J. C., Bachman, R. C. and Reynolds. 6.. J. ChrameW.. 1987, 27, 187. Smith, L. L., Teng, J. i..Kuiig, M. J. and Hill, F. L. J. Og. chem., 1973, 38, 1763. Weher, N. D.. Noomnont, P.; and Felmelster, A., J. LlpidRes., 1972, 13. .. 252. Bergstrom, S. and Samuelson. 6. The Autoxidathm of Cholesterol. I n AuYOxkletkn end antbxklents; Lundberg, W. 0.. Ed.; John Wlby 8 Sons. Inc.: New York; Chapter 6. p 233. Claude, J. R. and Beaumont, J. L., J. ChrOmetOgr. 1988. 2 1 , 189-201. Horvath, C.. J. chr4merogr. 1988, 22, 52-59. cohen,A.. Netlonal Bvssu of Standards TechnbalNote, 457; Schaffer, R. Ed., U.S. Dept. Commerce: Washington City. D. C., 1968. pp 73-90. ShepkO. 1. L. fJhP@Y, Memods end Tedmiqws; Zlef, M., SpdghtS, R., Ed.; Marcel Dekker, Inc.: New York, 1972 pp 193-204. V a n k , J. E. and Smith. L. L. Bloahsmlsby, 1987, 6 . 3269-3278. Meeker, 0. and Unruh, J., Jr., J. A m . Ofl Chem/sts Soc.. 1986, 63, 767-77 1. Danlelsson, H., Acta. Chem. Sand., 1960, 14, 846-860. Kudo, K., Emmons. G. T.. Casserly, E. W., Via, D. P., Smith, L. C., Pyrek, JSt. and Schroepfer, 0. J.. J. Lipld Res., 1989, 30, 1097-1 111. Chicoye, E., Powrie W. D. and Fennema, O., LlpMs, 1968, 3 , 551-556. Wasilchuk, 6. A., Ph.D. Dissertation. Northeastern University. Boston, MA, 1989. Fenlmore, D. C.. Davis, C. M., Whitford J. H. and Hanlngton, C. A.. Anal. Chem., 1978, 48, 2289-2290. Wasilchuk, 6. A., Felbush. P., LeQuesne P. W. and Vouros, P., N e m l Product chemrstty I I I ; Rahman. A., LeQuesne, P. W., Eds.: SpringerVerlag: Berlin, 1988. pp 275-287. Gordon, A. J. and Ford, R. A. The Chemists Companion;John Wlley 6. Sons: New York, 1972 pp 24-30. Wolthers, 6. G., Hlndrlks. F. R., Musklet F. A. J. and Goen, A., Clln. Chlmlca Acta. 1980, 103, 305-315. Park, S. W. and Addis. P. B., J. Food Science. 1985, 50, 1437. Pennock, I . F.. Gertrude N., and Mahler. H. R., Blochem. J., 1962. 85, 530. Mlllls 0. C. and Randall, H. P., J. B&/. Chem., 1958, 232, 589. Mlllls, 0. C.. Arch. Blochem. Blophys., 1980, 86. 1. Brooks, C. J. W., American Oil Chemists Society, annual meeting, Phlladelphla, PA, 1985.
RECEIVED for review October 22,1991. Accepted February 10, 1992.
