Anal. Chem. 1993, 65, 2024-2028
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An Improved Protocol for Determining Ratios of Retinol-d4 to Retinol Isolated from Human Plasma Garry J. Handelman, Marjorie J. Haskell, A. Daniel Jones,?and Andrew J. Clifford‘ Department of Nutrition and Facility for Advanced Instrumentation, University of California, Davis, California 95616
An improved protocol for assessing vitamin A status in humans using stable isotope dilution is described. Human subjects were given an oral dose of retinyl-d* acetate, and blood specimens were collected after a period of pseudoequilibration. After the precipitation of proteins, the plasma was extracted with hexane and partitioned against acetonitrile. Retinol was isolated from the acetonitrile fraction using HPLC, and the retinol was converted to its tert-butyldimethylsilyl ether and analyzed by gas chromatography/mass spectrometry using selected-ion monitoring. The new method allows for more rapid isolation of retinol from plasma with fewer interferences than were seen in our earlier work. Significant improvements in the reliability, sensitivity, and precision of the method have been obtained in the present study, and these improvements should allow for the use of smaller doses of isotopically labeled vitamin A for evaluation of the vitamin A status of human populations.
INTRODUCTION Vitamin A (retinol) is a fat-soluble nutrient needed for growth, reproduction, cellular differentiation, normal visual function, immune response, and healthy epithelia.’ Vitamin A nutrition is important, particularly among deficient individuals, in the prevention of human diseases including c a n ~ e r .Methods ~~~ for assessing vitamin A status and associated complexitieswere recently reviewed.4 Because 9095% of the body’s vitamin A is stored in the liver, the size of the pool can be assessed from a liver biopsy? but this procedure can only be justified in special cases. Plasma retinol levels cannot be used alone to get an accurate assessment of human vitamin A nutritional status.6~7 Several alternative methods have been explored to determine vitamin A status, including isotope dilution using radioisotopes,&” administration of the unlabeled analog vitamin A2,12 and stable isotope dilution.l”l6 In the latter Facility for Advanced Instrumentation. (1)Frolik, C. A. In The Retinoids; Sporn, M., Roberts, A., Goodman, D., Eds.;Academic Press: Orlando, FL, 1984; Vol. 2, pp 177-208. (2)Goodman, D.S.N. Engl. J. Med. 1984,310,1023-1031. (3)Sporn, M. B.; Roberts, A. B. Cancer Res. 1983,73,3034-3039. (4)Underwood, B. A. J.Nutr. 1990,120,1459-1463. (5)Pitt, G. A. J. h o c . Nutr. SOC.1981,40,173-178. (6)Olson, J. A. J. Natl. Cancer Znst. 1984,73,1439-1444. (7)Pilch, S.M. J. Nutr. 1987,117,636-640. (8)Rietz, P.;Vuilleumeier, J. P.; Weber, F.; Wiss, 0.Ezperientia 1973, 29,168-170. (9)Green,M.H.; Green, J.B.;Lewie,K. C. J.Nutr. 1987,117,694-703. (10)Green, M. H.; Green, J. B.; Methods Enzymol. 1990,190,304-317. (ll)Sauberlich, H. E.;Hodges, R. E.; Wallace, D. L.; Kolder, H.; Canham, J. E.;Raica, N., Jr.; Lowry, L. K. Vitam. Horm. 1974,52,251275. (12)Tanumihardjo, S. A.; Olson, J. A. J. Nutr. 1988,118,598-603. (13)Bausch, J.; Rietz, P. Acta Vitamin. Enzymol. 1977,31,99-112. t
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protocol, a precise serving of retinyl-d4 acetate is ingested and allowed to reach a pseudoequilibrium with existing body stores over 7 days, and a blood specimen is drawn so that the ratio of labeled to unlabeled vitamin A can be determined. From this ratio, total body stores of vitamin A are calculated after assuming that 50% of the administered labeled vitamin A served is taken up by the 1 i ~ e r . lThe ~ term pseudoequilibrium is used because the subject continues to eat a normal diet which provides a continuous dilution of the vitamin A pool by unlabeled vitamin A; complete equilibrium between the labeled vitamin A and the unlabeled body stores of vitamin A is never achieved. The analytical protocol for measuring labeled and unlabeled vitamin A (retinol-& and retinol, respectively) in plasma specimens involves precipitation of plasma proteins with ethanol, extraction of total retinol with hexane, and isolation and purification of the extracted total retinol using HPLC. For best results, two HPLC separations were employed in series to isolate retinol from interfering substances, especially in lipemic specimens. Determination of the relative amounts of retinol-& and retinol in the purified total retinol isolate has been accomplished with ambient temperature on-column injection gas chromatography (GC) coupled to mass spectrometric detection using selected-ion monitoring (SIM).16J6 During these analyses, retinol was found to undergo varying amounts of dehydration on exposed silica sites and, primarily, in mass spectrometer ion sources. This reactivity resulted in varying ratios of the intensities of the molecular ion (M+,mlz 286 for retinol and 290 for retinol-d4 to [M - HzOI+,mlz 268 and 272, respectively),with the most reliable analytical results obtained from the ratio of peak areas for [M - HzOl+. Also, variations in the ratio of integrated areas A272/Aza were attributed to changing amounts of [M - CHsl+from unlabeled retinol that contributed to the response measured at mlz 272 due to the presence of naturally abundant heavy isotopes such as I3C. These considerations provided the driving force to improve the precision of the method. The previous protocol is improved here by developing a simpler one-step HPLC isolation procedure, and converting retinol to a stable derivative which avoids the need for oncolumn injection is more compatible with automated injection and reduces variability in the mass spectrum. The new protocol significantly improves the sensitivity and precision in determining ratios of retinoLd4lretinol in plasma samples, and these improvements in turn will allow for administration of smaller doses of labeled vitamin A and the possibility of performing detailed studies of vitamin A pharmacokinetics in humans. (14)Clifford, A. J.; Jones, A. D.; Tondeur, Y.;Furr, H. C.; Bergen, H. R.; Olson, J. A.; h o c . 34th Annu. Conf. Mass Spectrom. Allied Top. 1986,49,327-328. (15)Clifford, A. J.; Jones, A. D.; Furr, H. C. Methods Enzymol. 1990, 189,94-104. (16)Furr, H. C.; Amedee, M. 0.; Clifford, A. J.; Bergen, H., 111; Jones, A. D.; Anderson, D. P.; Olson, J. A. Am. J. Clin. Nutr. 1989,49,713-716. (17)Rietz, P.; Wiss, 0.; Weber, F. Vitam. Horm. 1974,32,237-249. 0 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
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EXPERIMENTAL SECTION A healthy 52-year-old human volunteer consumed 20 mg of purified all-trans retinyl-10,19,19,19-d4 acetate (Cambridge Isotopes, Cambridge, MA), in 1mL of olive oil before a breakfast of coffee,with cream ( -56 g),and a bran muffin. Blood specimens were drawn into 10-mLglass tubes containingKs-EDTAat several time points during the first day, and every few days thereafter for several months. Blood specimens were stored at 3 OC for -4 h, plasma was separated by centrifugation, and 1-mL aliquots of plasma were transferred to glass vials and stored at -20 "C. 1. Isolation of Retinol from Plasma. Aliquots of 1 mL of frozen plasma are thawed and transferred to 8-mL glass test tubes with Teflon-lined screw caps. Care should be taken to avoid contamination of the samples by plasticizers (from soft plastics) and alkanes from sources such as parafilm. Absolute ethanol (1mL) is added,and the tube is vortexed for 30 s. HPLCgrade hexane (4 mL) is added, and the tube is again vortexed for 30 s. Retinol is extracted into the hexane (upper layer), which is separated by centrifugation, and transferred to another 8-mL glass test tube. The hexane extract is evaporated under a stream of N2 at 40 OC. HPLC-grade acetonitrile (1mL) is added to the residue, and the tube is vortexed for 10 s. This step extracts the retinol but leaves behind much of the nonpolar lipid which has poor solubility in acetonitrile. The acetonitrile solution is transferred to another glaestube and evaporated to dryness under Nz. The residue is dissolved in 70 pL of methanol and loaded onto a 15cm long X 0.46 cm i.d. AdsorbosphereHS ODS HPLC column (AlltechAssociates, Deerfield, IL) with a 3-pm particle size. The isocratic mobilephase is acetonitrile:methanoka"onium acetate (85150.01, v:v:w) at a flow rate of 1mL/min. The absorbance of the eluent is monitored at 325 nm, and the retinol peak is collectedin a 2-mL glass vial which contains 10pL of 0.1 % BHT (in hexane). A broad zone of column eluent from 20 s ahead of to 20 s after the retinol peak (total volume -700 pL) is collected to ensure that both isotopomers of retinol are quantitatively recovered. After three samples have been purified on the HPLC, the column must be washed with the mobile phase diluted with an equal volume of 2-propanol for 20 min. This removes cholesteroland other lipids of intermediatepolarity which would accumulate and eventually elute from the column and contaminate subsequent samples. The HPLC column is then equilibrated for 15min with the initial mobile phase before additional samples can be purified. 2. Derivatization of Retinol Isolated from Plasma. The HPLC eluant containing purified retinol and retinol-d4 is evaporated to dryness, and 20 pL of N-methyl-N-(tert-butyldimethylsily1)trifluoroacetamide (MTBSTFA, Pierce, Rockford, IL) is added. The vial is sealed tightly (with a Teflon-lined cap) and heated at 125OC for 60min to form the tert-butyldimethylsilyl (tBDMS) derivatives. Derivatized samples are stored at 4 "C in the dark and are stable for at least 3 months as long as contamination by moisture is avoided. 3. Measuring Retinol-dd and Retinol by GC/MS. A 2-pL aliquot of the derivatized retinol isotopomers is loaded onto a DB-1 fused silica capillary column (15 m long X 0.25 mm i.d., 0.10 pm f i thickness; J&W Scientific,Folsom, CA) using a 30-s purge delay and a splitless injector set at 285 "C to minimize accumulation of lipid in the injector. GC conditions include a temperature program from 150to 250 OC at 25 OC/min, using helium as carrier gas at a linear velocity of 35 cm/s. The direct interface transfer line between the gas chromatograph and the mass spectrometer is held at 285 OC. Retinol isotopomers elute at about 5.2 min at a column temperature of about 250 OC, with the retinol-d4 derivative eluting 1 s earlier than the retinol derivative. Full-scan mass spectra and SIM are performed with a Trio-2 quadrupole mass spectrometer (VG Masslab, Altrincham, UK). A source temperature of 200 OC and 70 eV electron ionization were employed throughout this study. SIM is carried out by monitoring fragment ions of the tBDMS derivatives at m/z 255 (retinol) and m/z 259 (retinol-d,); these fragments are formed by cleavage of the derivatives between C-14 and C-15 (see Figure 1).In the SIM program, the sampling interval for each fragment is set at 50 ms, followed by a 40-ms delay before sampling on the
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Flgure 1. Electron ionization (70 eV) spectra of tBDMS-retinol and of tBDMS-retinoCd4. Molecular ions appear at mlz 400 and 404 for tBDMS-retinol and for tBDMS-retinokd4, panels A and B, respectively. Fragment ions monitored for quantitative analysis appear at mlz 255 and 259 for tBDMS-retinol and for tBDMS-retlnoC& respectively.
alternate channel. The longdelay time allowsthe signalamplifier to settle between channels. 4. Calibrationand Mass Spectrometer Standardization. The retinol-d4and retinol used to prepare the standard calibrating mixtures was purified by isocratic HPLC on a 15 cm long X 0.46 cm i.d. Adsorbosphere NH2 HPLC column (Alltech Associates, Deerfield, IL) with a 3-pm particle size using a hexane:ethanol (95:5, v:v) mobile phase. Stock solutions of purified retinol-d, and retinol were individually prepared in hexane and were standardized based upon their extinction coefficients of 51 OOO M-l cm-l at 325 nm and molecular weights of 290 and 286 for retinol-d4and retinolrespectively. Allliquid transfers were made with positive displacement pipettors with Teflon-tipped plungers and glass tips. A set of calibrating standard mixtures of retinol-d4and retinol were prepared by adding 20 pg of retinol to 20.00,3.33,1.00,0.33, and 0.00 pg of retinol-d4toprovide retinol-ddretinolweight ratios and0.0000,respectively. In contrast of 0.500,0.167,0.0500,0.0167 to the use of deuterated analogs as internal standards for quantitation, the isotope dilution protocol yields plasma samples with somewhat constant concentrations of unlabeled retinol but variable concentrations of retinol-dd. The standard mixtures were derivatized as described above and stored at 4 OC in the derivatizingreagent. This set of standards was analyzed by GC/ MS using SIM, and integrated areas of the fragment ions m / z 255 (retinol) and mlz 259 (retinol-dd) were calculated. After subtraction of the area ratio obtained for a standard containing no retinol-d4, a linear regression equation was calculated between retinol-ddretinol concentration ratios (r-variable)and corrected ratios of integrated areas for m/z 259 and 255 ( A d A , ; y-variable). The corrected y-variable values (for individual specimens)were substituted into the regression equation to solve for the concentration ratios.
RESULTS AND DISCUSSION The use of isotope dilution approaches to assess vitamin A nutritional status requires analytical methods capable of
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ANALYTICAL CHEMISTRY, VOL. 85, NO. 15, AUGUST 1, 1993 100 1 8o
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Figure 2. Selected ton chromatograms of t6DMS-retinol (mlr 255) and tBDMS-retinoi-d4 (m/z 259) from extract of a plasma sample containing about 4 % retinol-d4.
reliably measuring the ratios of labeled to unlabeled retinol from a small volume of plasma. In these experiments retinyld4 acetate is administered orally to individuals because this retinoid is stable (relative to retinol) and easily prepared in high purity. Based on earlier studies,16 administration of a 20-mg dose of retinyl-d4 acetate to individuals with average weight of 70 kg is expected to yield plasma where the ratio of retinol-ddretinol would lie between 0.01 and 0.10 after pseudoequilibration. 1. GCIMS Analysis of tBDMS Derivatives of Retinol andRetinol-d4. The tBDMS ethers of retinol and retinol-d4 elute as sharp peaks at a retention time of -5.2 min, with peak widths (at 112 height) of 3 s. The GCIMS analyses by SIM of derivatives from extracts of 10separate 1-mLaliquots of plasma taken from a single pool, labeled with -4 96 retinold4, are shown in Figure 2. Due to the thorough sample preparation, the chromatograms for mlz 255 and 259 are remarkably free of peaks from interfering substances and could be chromatographed using a steep 25 "Clmin temperature gradient to minimize analysis times. Fragment ions in the electron ionization spectra of tBDMS ethers of retinol-d4 and retinol (Figure 1) are similar to E1 spectra reported for the trimethylsilyl ether of retinol,18which also exhibits a major fragment due to cleavage at C-15. In addition to the molecular ions which appear at mlz 404 and 400, respectively, major fragment ions are present at mlz 259 and 255, which correspond to loss of C4Hg-Si(CH&-O-CH2* from the molecular ions. These are the most abundant ions in the spectrum that are unambiguous in demonstrating the presence or absence of the deuterium label, and all quantitative analyses in the current research have been performed by monitoring these two ions. Unlike tBDMS derivatives of most compounds whose mass spectra exhibit an abundant peak at EM CdHgl+ and virtually no molecular ion, the tBDMS ether of retinol does not yield significant amounts of [M- C&]+. 2. Optimum Derivatization Conditions. Incubating retinol with derivatizing reagent (MTBSTFA) at 80 OC for 30 min resulted in a poor yield of tBDMS-retinol as determined by GC with flame ionization detection. It is
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(18) Vecchi, V. M.; Vetter, W.; Walther, W.; Jermetad, S. F.; Schutt, G.W.; Helu. Cham. Acta 1967,50, 1243-1249.
0.1 0.2 0.3 0.4 Ratio of retinol-d4/retinol
0.5
Flgure 3. Representative caiibratlon curve, generated from analysis of standard mixtures of ret1n0l-d~and retinol, made by adding a Wed amount of retinol to varying amounts of retlnoCd4 as descrlbed in the text. Standard solutions were derivatizedand analyzed by integrating the peak areas at mlz 259 for retinoCd4and at mlz 255 for retinol.
necessary to incubate the samples at 125 "C for 60 min to obtain quantitative derivatization of retinol with this reagent (and disappearance of underivatized retinol from the chromatograms). To test the stability of tBDMS-retinol, total retinol was extracted from a plasma pool containing -4 96 retinoLd4 and derivatized. Aliquots of derivatized material were analyzed immediately, after storage in a glass vial for 2 months at 4 "C, and again after an additional 2 weeks at 20 OC. Neither the absolute peak areas nor the ratios of retinol-d4to retinol changed measurably over time, demonstrating that neither degradation nor exchange of deuterium label had occurred during storage. Because of this stability, derivatized calibration standards can be stored for months at 4 "C and analyzed with different batches of plasma samples. In addition to facilitating storage, use of these stable derivatives eliminates requirementsfor special handling and is compatible with automated analysis. The tBDMS derivative proved to be capable of withstanding injector temperatures of 285 "C without signs of decomposition or loss of signal in the MS. Stability at such temperaturesproved to be important because at lower injector temperatures (5200 "C) nonvolatile substances in plasma samples accumulate within the injector and leave deposits which degrade the quality of separation for subsequent samples. No degradationof the chromatographic peak widths or shapes was observed while the injector was held at 285 "C. 3. Method Calibration. As measured from the integrated areas for mlz 259 and 255, the molar relative response of retinol-d4 was unexpectedly found to be about 0.70 of the response of retinol, as seen in the slope of the calibration curve in Figure 3. Since the purities and concentrations of standard solutions of both retinol and retinol-d4 were verified by HPLC and UV-visible spectra and extinction coefficients, this phenomenon is probably due to a secondary kinetic isotope effect upon fragmentation rates. The ratio of molar response factors can, however, vary between 0.65 and 0.8, depending on the tuning of the mass spectrometer and other instrumental conditions. Therefore, to obtain optimal accuracy, a calibration curve should be generatedwith each set of samples,and new calibration curves should be generated whenever changes are made in instrument tuning.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
For a blank plasma sample (no r e t i n o l 4 present), the observed (AUdA255) ratio obtained with the VG Trio-2 quadrupole is typically 0.004, instead of the theoretical value of 0.0002 (based on natural abundances). This appears to be the limit obtainable with this quadrupole analyzer, and it is attributed either to background signal (possiblystray photons) that passes through the analyzer throughout the entire mass range of the instrument or to the incomplete mass resolution by the quadrupole analyzer. Much smaller errors were observed in previous studies of free retinol on a VG ZAB magnetic sector instrument.'s An essential part of the calibration procedure is the analysis of the standard containing 20 rg of retinol + 0.00 r g ofretinold4. The ratio A269lAm for this standard is then subtracted from values of Aag/Am determined for all other calibrating standards. This is particularly important when plasma specimens contain 1 5 7% retinol-d4as is anticipated for studies of retinol elimination kinetics or when vitamin A pools are large. Preliminary evaluation of the ability of a singly pumped bench-top GC/MS (Hewlett-Packard MSD Model 5971) to perform isotope dilution analyses of retinol in plasma yielded a poorer signal to noise (S/N) ratio than we obtained on the differentially pumped VG Trio-2 instrument. Isolation of retinol from larger plasma sample volumes may be needed for analysis with less sensitive mass spectrometers. These calibration curves,generated once a week for several months, have independently given excellent correlation coefficients (r2 2 0.9991, indicating consistent linearity over the range of retinol-ddretinol from 0.0016 to 0.5. 4. Precision of the Method and Detection Limit for Retinol-d4. A pooled plasma sample was made from blood drawn 10 days after a human volunteer ingested 20 mg of retinyl-d4 acetate. At this time - 4 % of the total retinol in plasma is retinol-d4. Ten separate 1-mL aliquots of this plasma pool were extracted, derivatized, and analyzed on four separate occasions, over a 2-month period. On two of those occasions, the derivatized sample was analyzed once, while on the other two occasions quadruplicate injections of derivatized extracts were made to determine the magnitude of instrumental variability and of variability inherent in the extraction and isolation procedure. Ratios of retinold4 to unlabeled retinol were calculated based upon calibration curves generated the same day as the analysis of plasma extracts. The coefficient of variation between separately extracted and isolated plasma samplesfrom the pool described above (CV = SD/mean X 100)was4.6% (Table I). The withinday CV for individual injections of four separately extracted plasma samples was 3.9 % The S/N ratio for m/z 259 from 1mL of plasma with 1% retinol-d4is typically 10/1. Therefore, the detection limit for retinol-d4is -1.2 ng or -0.2% of the total retinol in a 1-mLsample of typical plasma. These results demonstrate that our improved method is sensitive and reproducible with an overall CV of 45% at levels of enrichments expected at pseudoequilibration following administration of a 20-mg dose of retinyl acetate-d4.
.
5. Suitability of the Method for Pharmacokinetic Studies. The retinol-ddretinol concentration ratios in blood
plasma specimens drawn at various times during a 10-day period following the ingestion of 20 mg of retiny14 acetate are shown in Figure 4. The expected rise, subsequent fall, and final plateau in the retinol-&retinol concentration ratios provide the data for characterizing the kinetics of appearance and disappearance of serum retinol in human subjects. From plots of this nature, the point of maximum labeling, the halflife for appearance, and the half-life for disappearance of retinold4 in humans can be evaluated in a manner analogous
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Table I. Isotope Dilution Analyees of Retinol and Retinol-& in 10 Separate Aliquots of a Pooled Plasma Sample day sample description retinol-ddretinolb 1 2
3
4
plasma aliquot 1' plasma aliquot 2 plasma aliquot 3 plasma aliquot 4 plasma aliquot 5 plasma aliquot 6 plasma aliquot 7 plasma aliquot 8 plasma aliquot 9 plasma aliquot 10 mean ratio (n = 10)
0.0431 0.0388 0.0410 0.0409 0.0428 0.0457 0.0404 0.0418 0.0426 0.0407 0.0418 4.5 9%
cv
'Ten separate aliquota of plasma from a common pool were extracted, purified, and derivatized separatelyand were analyzed on four different dates. Calculated ratios of retinol-ddretinolwere determinedfor each set of pointa using calibrationcurves generated with each day's samples.
.
.
.
to radioisotope studies of retinol kinetics in rodents.gJ9 Data generated using the new protocol should be useful for future investigations and modeling of retinol kinetics in humans.
CONCLUSIONS The aims of our ongoing studies in vitamin A nutrition require that a small amount of retinol-d4be quantified relative to a much larger amount of unlabeled retinol. Given our desire to administer small doses (10-20 mg) of labeled vitamin A, it was anticipated that such doses would yield as little as 1% labeling of the plasma retinol after equilibration with the whole-body pool, and the potential use of the method for pharmacokinetic studies of retinol over long periods requires precisionadequate to measure even lower levels of enrichment. Determination of the ratio of retinol-ddretinol in this range is a challenging problem that is further complicated by the need for sufficient analytical precision and accuracy to distinguish unhealthy, healthy, and marginally healthy nutritional status. Previous GC/MS analytical protocols in this laboratory utilized underivatized retinol and ambient-temperature oncolumn injection GC/MS.16Js The tBDMS-retinol derivatives (19)Lewis,K.C.;Green, M. H.; Underwood,B.A. J. Nutr. lSSl,111, 1136-1144.
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used in the new protocol elute as sharper peaks than underivatized retinol with improved S/N ratios and decreased probabilities of coeluting interferences. The resulting improvements in precision (CV = 4.5% at -4% retinol-dr) mean that smaller amounts of tBDMS-retinol derivatives can be more reliably measured, allowing for the use of smaller doses of retinyl-dr acetate than were possible in our earlier studies. The procedure for retinol isolation is efficient yet easy to perform, yielding extracts sufficiently clean so that steep temperature programs can be used. In our hands, 4-6 samples/h can be analyzed by GUMS, and splitless injection allowsfor greater ease in using automated injection for analysis of large numbers of samples. This GUMS method can also be readily applied to the estimation of the whole-body pools of retinol as related to the presence of various disease states in both affluent and malnourished populations. Following the time course of the ratio of retinol-ddretinol (asin Figure 4) allows detailed kinetic studies of vitamin A metabolism to be performed in humans and should prove useful in studies of disorders in vitamin A
uptake and metabolism. The availability of deuterated carotenoidsnow makes it poeeible for studies of the conversion of carotenoids to retinoids in humans using this method as well.
ACKNOWLEDGMENTS The authors are grateful to Professors Harold Furr and James Olson for valuable comments and discussions. This research has been supported by the National Institutes of Health (Grant R01-DK-43098).
Author Supplied Registry Numbers: MTBSTFA, 77377-52-7;retinol, 68-26-8; retinyl acetate, 127-47-9;butylated-hydroxytoluene, 128-37-0; acetonitrile, 75-05-8; ammonium acetate, 631-61-8. RECEIVEDfor review December 29, 1992. Accepted April 2, 1993.
