Anal. Chem. 1988, 60, 1929-1933
1929
Determination of Fat-soluble Vitamins in Oil Matrices by Multidimensional High-Performance Liquid Chromatography Jeanice M. Brown-Thomas,* Azza A. Moustafa,' S t e p h e n A. Wise, a n d Willie E. May Organic Analytical Research Division, Center for Analytical Chemistry, National Bureau of Standards, Gaithers burg, Maryland 20899
Multidlmenslonal hlgh-performance liquid chromatographlc procedures for the determlnatlon of selected fat-soluble vitamins in foodlike oil matrices are presented. An on-line liquid chromatographic procedure, which was developed for the certification of selected fat-soluble vitamins in Standard Reference Material (SRM) 1563, Cholesterol and Fat-Soluble Vitamins in Coconut Oil, utiilzes gel permeatlon chromatography (GPC) and normal-phase high-performance liquid chromatography (HPLC) to quantify retinyl acetate and ergocalciferol. The on-line GPC/normaCphase HPLC procedure was followed by a reversed-phase HPLC step to quantify dl-a-tocopheryl acetate In the fortifled coconut oil. Agreement Is within 7% for the concentrations of the vltamlns in the fortlfled coconut oil SRM as determined by using the muitldlmenslonal liquld chromatographlc procedures and the amounts of the various constltuents added to the natural material. The method provides rapid analysis time, minimal sample handling, and analyte specificity. A modlfied procedure has also been used for the determlnatlon of a-tocopherol in SRM 1588, Organlcs In Cod Liver Oil.
The need for accurate methods for the determination of vitamins in food and foodlike matrices is rapidly increasing. Traditional methods of vitamin assay have required that each vitamin be determined individually by using widely differing physical, chemical, and biological methods. However, the methods, including colorimetry, fluorometry, spectrophotometry, and titrimetry, may not provide accurate and precise results for the food matrices tested (1). Therefore, for validating the entire measurement procedure, analytical methods should be developed that have built-in quality control steps (2). These steps include the use of internal standards, reference compounds of known purity for each nutrient for establishment of instrumental response factors, and when available, Standard Reference Materials (SRMs) of matrices similar to the food sample being measured. In this work, two multidimensional high-performance liquid chromatographic (HPLC) procedures are described, which were developed for determination of selected fat-soluble vitamins in a fortified coconut oil SRM (SRM 1563) and a cod liver oil SRM (SRM 1588). The first procedure uses gel permeation chromatography (GPC) to eliminate the bulk of the lipid material from the oil matrix, followed by normalphase HPLC on a polar chemically bonded phase. Vitamin A as retinyl acetate and ergocalciferol (vitamin D2)are simultaneously determined in the oil by using this on-line GPC/normal-phase HPLC procedure. The second procedure consists of on-line GPC/normal-phase HPLC followed by an off-line reversed-phase HPLC step. This procedure was used to quantify vitamin E as dl-a-tocopheryl acetate in the for-
* Author to whom correspondence should be addressed.
Permanent address: Faculty of Pharmacy, Cairo University, Cairo, Egypt.
tified coconut oil and the natural concentration of a-tocopherol in the cod liver oil SRM. Landen et al. (3, 4 ) and others (5-8) have used off-line HPLC methods to isolate fat-soluble vitamins from food matrices. The major advantage of our approach is the on-line capability, which minimizes sample handling and manipulations and provides added selectivity due to the coupling of multiple independent chromatographic mechanisms through use of different columns sequentially. Other advantages include a rapid analysis time, and the elimination of laborious sample preparation steps, such as tedious extraction steps, to cleanup foodlike samples with high lipid content. EXPERIMENTAL S E C T I O N Apparatus. The liquid chromatograph consisted of a single-piston solvent delivery system and gradient elution capability with a loop-type (fixed 26-pL volume) sample injector, a programmable wavelength ultraviolet (UV)-visible detector, and a dual channel remote printer/plotter data system. The HPLC columns used for the vitamin determinations were (1)a 10-pm polystyrene/divinylbenzene gel column (50 A, 60 X 0.77 cm i.d., Polymer Laboratories, Inc., Amherst, MA), (2)a semipreparative aminocyano column (25 X 0.94 cm i.d., Partisil PAC, Whatman, Clifton, NJ), (3) a semipreparativeaminosilane column (27 X 0.94 cm i.d., pBondapak NH2,Waters Associates, Milford, MA), and (4) a 5-pm polymeric CI8 column (25 X 0.45 cm i.d., Vydac TP, The Separation Group, Hesperia, CA). The on-line multidimensional HPLC system consisted of two columns, a gel column followed by a semipreparative normal-phase column (amine or aminocyano) arranged with a valve so that the second column could be switched in or out of the solvent flow. Mobile Phases. All organic solvents used were HPLC grade. The solvents used as the mobile phases for the multidimensional normal-phase HPLC system were hexane, methylene chloride, and methyl tert-butyl ether. Methanol, propanol, butanol, and water were used in various solvent compositions for the reversed-phase HPLC systems. The specific solvent compositions used in each of the HPLC systems are described later. Standard Solutions. Standard solutions of dl-a-tocopheryl acetate (5-10 mg/100 mL, Eastman Kodak Co., Rochester, NY), retinyl acetate (2 mg/100 mL, Sigma Chemical Co., St. Louis, MO), ergocalciferol (1mg/100 mL; Sigma Chemical Co., St. Louis, MO), and 2-methyl-2-(4,8,12-trimethyltridecyl)-6-chromanol (tocol) (10-14 mg/100 mL, Hoffmann-La Roche, Basel, Switzerland) were prepared with hexane (E.M. Science, Cherry Hill, NJ). Standard solutions of a-tocopherol were prepared in both hexane and methanol (both obtained from E.M. Science) at 6 mg/100 mL and 10 mg/100 mL, respectively. Each standard solution was run prior to sample analysis for peak identification (based on retention time) and for obtaining detector response factors of the analytes. The purities of the retinyl acetate, dla-tocopheryl acetate, and ergocalciferol were determined to be 87%, 96%, and 98%, respectively (9). The purities were determined by comparing the absorbances of dilute solutions with literature values for the absorptivities of the compounds. Because certain impurities absorb at the wavelengths at which the absorbances had been measured, potential errors from such interferences were compensated for by performing liquid chromatographic separations whereby the proportion of that absorbance due to the substance of interest was determined. The purity of ergocalciferol was also determined by differential scanning calorimetry (DSC).
This article not subject to US. Copyright. Published 1988 by the American Chemical Society
1930
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15,
Coconut Oil Sample. The coconut oil used in SRM 1563 was obtained from ICN Biochemicals, Inc., Cleveland, OH. The SRM consists of a natural (SRM 1563-1) and fortified (SRM 1563-2) coconut oil. The fortified oil contains added amounts of cholesterol, dl-a-tocopheryl acetate, retinyl acetate, and ergocalciferol. The oil also contains 2,6-di-tert-butyl-4-methylphenol (BHT) to minimize oxidation. To prepare the oil sample for multidimensional HPLC analysis, we diluted an aliquot of approximately 0.7 g of the fortified oil with 2-3mL of tocol in HPLC-grade hexane (14 mg/100 mL). Cod Liver Oil Sample. The cod liver oil, a Norwegian pharmaceutical-grade oil, was obtained from Dr. Karlheinz Ballschmiter, University of Ulm, Federal Republic of Germany. Thii oil was analyzed as part of the process for obtaining a certified value for a-tocopherol in the SRM (SRM 1588). Approximately 0.7 g of the oil was diluted with 1 mL of the internal standard tocol solution (14 mg/100 mL) prior to HPLC analysis. The oil also contained certified concentration values of selected chlorinated pesticides and polychlorinated biphenyl (PCB) congeners and information values for additional chlorinated pesticides, polycyclic aromatic hydrocarbons (PAH), polychlorinated dibenzo-p-dioxins, and octachlorodibenzofuran (10). Analysis of Cod Liver Oil (SRM 1588) by Direct-Injection HPLC. The cod liver oil sample was also analyzed for or-tocopherol by using a direct-injectionnormal-phase HPLC procedure. The oil sample was diluted with hexane and approximately 18 mg of the solution was injected onto a semipreparative WBondapak NH2 column utilizing isocratic conditions of 25% methylene chloride in hexane at 2 mL/min with UV absorption detection at 292 nm. Determination of Retinyl Acetate and Ergocalciferol in Coconut Oil (SRM 1563-2). The multidimensional HPLC procedure used to separate retinyl acetate and ergocalciferol from the fortified coconut oil used a 10-lm gel column to eliminate the bulk of the lipid material in the coconut oil matrix prior to separation of the vitamins using normal-phase HPLC on a polar chemically bonded semi-preparative aminocyano column. The oil was diluted with the standard tocol solution and approximately 9-10 mg of the resulting solution was injected onto the gel column. The lipid material was eluted from the gel column and discarded to waste by using 30% methyl tert-butyl ether/methylene chloride (30:70)in hexane. The aminocyano column was switched into the solvent flow, and the vitamins eluting from the gel column were introduced onto the semipreparative aminocyano column. Subsequently, the gel column was switched out of the solvent flow, and a linear 15-min gradient of 30% methyl tert-butyl ether/ methylene chloride (3070) in hexane to 100% methyl tert-butyl ether/methylene chloride (3070) was used to separate the analytes on the aminocyano column. Methyl tert-butyl ether was used in the mobile phase to improve the peak shape and to decrease the retention time of ergocalciferol. The flow rate was 2 mL/min and UV absorption detection at 292 nm was used to monitor the separation. Determination of dl-a-Tocopheryl Acetate in Coconut Oil (SRM 1563-2). Separation of a-tocopheryl acetate from the coconut oil was achieved by using the conditions described above. The lipid material in the oil was eliminated by using the 10-hm gel column, and a fraction (based on retention volume) containing dl-a-tocopheryl acetate and tocol was obtained from the aminocyano column by using the previously described chromatographic conditions. The fraction was evaporated to dryness under a nitrogen stream and reconstituted with the appropriate HPLCgrade solvent for reversed-phase HPLC analysis on a polymeric column with UV absorption detection at 284 nm. An 18-min linear gradient of 50% solvent A to 95% solvent B was used, where solvent A was (60:10:30) methanol/propanol/water and solvent B was (89.5:100.5) methanol/propanol/water.The flow rate was 1.5 mL/min. Determination of a-Tocopherol in Cod Liver Oil (SRM 1588). For the determination of a-tocopherol in the cod liver oil sample (SRM 1588), the GPC procedure was used to eliminate the bulk of the lipid material from the oil matrix and to isolate the a-tocopherol from the other constituents by using 30% tert-butyl methyl ether/methylene chloride in hexane at 2 mL/min with UV absorption detection at 295 nm. The fraction containing a-tocopherol and tocol (approximately 8 mL) was
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Flgure 1. Chromatogram of fortified coconut oil using normal-phase HPLC: (1) dl-a-tocopheryl acetate, (2) retinyl acetate, (3) tocol, and (4)
ergocalciferol.
collected, evaporated to dryness under a nitrogen stream, and reconstituted in an appropriate HPLC-grade solvent for reversed-phase HPLC analysis. The reversed-phase HPLC determination of a-tocopherol was achieved on a polymeric c18 column with an isocratic solvent composition of (30/30)solvent A/solvent B where solvent A was (6010:30) methanol/butanol/ water and solvent B was (89.5/10/0.5) methanol/butanol/water. A flow rate of 1.5 niL/min and UV absorption detection at 295 nm were used. As in the previous analyses, peak identification was based on standard solutions that were run prior to the sample analysis. Analyte concentrations were determined from peak areas of the analyte and internal standard, based on HPLC response factors for the analytes.
RESULTS AND DISCUSSION Analyses of Coconut Oil (SRM 1563). The major recommendation of the workshop on Reference Materials for Organic Nutrient Measurements held at the National Bureau of Standards (Gaithersburg, MD) was the development of a SRM for fat-soluble vitamins and cholesterol in a foodlike oil matrix. Coconut oil was selected as the matrix for this SRM because it has a low level of unsaturated lipid material and fatty acids at room temperature and because it is a major component in infant formula (2). Another recommendation of the workshop was to fortify the oil matrix with selected fat-soluble vitamins. These vitamins were added to the coconut oil matrix at levels between 25% and 50% of the United States recommended daily allowance (2). Prior to the adoption of the multidimensional HPLC protocol, the coconut oil sample was diluted with the tocol in hexane and injected directly onto the semipreparative aminocyano column yielding the chromatogram shown in Figure 1. Chemical interferences from the lipid material in the oil were prevalent and there was an indication of column degradation. On the basis of these results, the direct-injection HPLC procedure was not feasible for the quantitation of the vitamins in the SRM. As part of the characterization process for two new SRMs representing foodlike oil matrices, multidimensional HPLC procedures were developed for the determination of selected fat-soluble vitamins. An on-line GPC/normal-phase HPLC procedure was used to eliminate the need for time-consuming and laborious solvent extraction procedures to isolate the vitamins from the oil matrix. The GPC step was implemented to remove the bulk of the lipid material prior to HPLC analysis. The chromatogram obtained from the analysis of the fortified coconut oil using the GPC/normal-phase HPLC procedure is illustrated in Figure 2. Total analysis time was
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988 -Gel
4
Column
Aminocyano Column
1931
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W
(12 mL)
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1
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Volume (mL)
Figure 3. Fractionation of fortified coconut oil SRM 1563-2 using GPChormal-phase HPLC: (1) dl-a-tocopheryl acetate and (2) tocoi.
R e t e n t i o n Time ( B i n )
Figure 2. Chromatogram of fortified coconut oil SRM 1563-2 using GPCInormai-phase HPLC: (1) dl-a-tocopheryi acetate, (2) 2,6ditert-butyl-4-methylphenoi, (3) retinyi acetate, (4) tocol, and (5) ergocalciferol. Table I. Concentrations of Fat-soluble Vitamins in Fortified Coconut Oil (SRM 1563-2) as Determined by Multidimensional GPC/Normal-Phase HPLC
E C
c0ncentration.O UPIP ~~~
mean std dev 5% std dev
dl-a-tocopheryl acetate
retinyl acetate
ergocalciferol
193.1 (158.2)* 6.0 3.1
11.9 (12.6)b 0.3 2.6
11.3 (10.5)* 0.2 1.9
"The concentrations reported represent the mean of 24 HPLC measurements A1 standard deviation of a single measurement. Values in parentheses denote the concentrations of analytes added to the matrix. approximately 45 min (including the GPC step). As shown in Figure 2, separation of the anal* from the oil matrix using the multidimensional HPLC procedure is satisfactory and the recovered concentrations, except for dl-a-tocopheryl acetate, are suitable for quantitation. The concentrations of dl-a-tocopheryl acetate, retinyl acetate, and ergocalciferol in the fortified coconut oil, as detennined by using the GPC/normal-phaee HPLC method, are reported in Table I. These concentrations are expressed as micrograms per gram and represent the mean of 24 HPLC measurements. The uncertainty is A1 standard deviation. Except for the concentration of dl-a-tocopheryl acetate, the measured concentrations of the selected fat-soluble vitamins were within 8% of the gravimetric concentrations shown in parentheses in Table I. The HPLC value for dl-a-tocopheryl acetate was higher than the gravimetric value due to the presence of a coeluting compound. A sample of unfortified coconut oil (SRM1563-1) was analyzed by using the described multidimensional HPLC method to verify the presence of the coeluting compound. On the basis of retention data, it was concluded that dl-a-tocopheryl acetate and another compound coeluted from the column. A fraction containing the coeluting compound was collected and analyzed by mass spectrometry (MS) to confirm that the compound was not native dl-a-tocopheryl acetate.
acetate
0
5
110
115
2Io
215
Retention Time (min)
Flgure 4. Chromatogram of fortified coconut oil SRM 1563-2 using off-line reversed-phase HPLC after GPChormai-phase HPLC fractionatlon. On the basis of the mass spectral scan, this compound was identified as a triglyceride constituent. Because of these findings, the on-line GPC/normal-phase HPLC procedure was modified to include a final off-line reversed-phase HPLC step for quantification of the dl-a-tocopheryl acetate in the oil. The fractionation of the coconut oil sample using the GPC/normal-phase HPLC procedure is shown in Figure 3. The fraction eluting between 3 and 22 mL on the aminocyano column contains dl-a-tocopheryl acetate and the internal standard tocol. The reversed-phase HPLC analysis of this fraction is shown in Figure 4. The GPC, normal-phase HPLC, and reversed-phase HPLC steps were necessary in the multidimensional HPLC procedures to adequately isolate and quantify the analytes. When only the GPC procedure was used prior to reversed-phase HPLC analysis, residual oil droplets (which caused chemical interferences in the coconut oil samples) were often observed in the fraction. The results from the analyses of six fortified coconut oil samples by reversed-phase HPLC after fractionation using the gel and semipreparative aminocyano columns are summarized in Table 11. The measured concentration (158.0pg/g) of dl-a-tocopheryl acetate in the fortified oil is in excellent agreement with the gravimetric value (158.2pg/g).
1932
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988
Table 11. Determination of dl-a-Tocopheryl Acetate in Fortified Coconut Oil (SRM 1563-2) by Using Off-Line Reversed-Phase HPLC after GPC/Normal-Phase HPLC Fractionation analysis
concn, wg/g 162.6 151.7 158.3 153.3 159.5 162.3
mean
158.0 f 4.5" (158.2)b
"The uncertainty of the average is fl standard deviation of a single measurement. Parentheses denote the concentration of the analyte added to the matrix. Table 111. Determination of a-Tocopherol in Cod Liver Oil (SRM 1588) by GPC/Off-Line Reversed-Phase HPLC and by Direct Injection Normal-Phase HPLC
sample no. 1 2
3 mean
1
concentration," pglg multidimensional normal-phase HPLCb HPLC" (direct (GPC/ injection) reversed-phase) 109.7 f 1.1 104.4 f 2.4 113.5 f 4.0 109.2 f 5.0
1
8
1
5 10 15 20 Retention time ( m i d Flgure 5. Chromatogram of cod liver oil SRM 1588 using off-line reversed-phase HPLC after GPC fractionation: (1) tocol and (2) a-tocopherol.
111.6 f 4.4 117.8 f 2.7 114.7 f 5
"Three measurements were made for each sample in the normal-phase HPLC analysis; the external standard method was used to quantify a-tocopherol. Values reported represent the mean fl one standard deviation of a single measurement. bFour measurements were made for each sample in the multidimensional HPLC. Quantification of a-tocopherol was achieved by using the internal standard method. Values reported represent the mean fl standard deviation of a single measurement. The concentration of a-tocopherol in the coconut oil was determined to be 4 pg/g by using the multidimensional HPLC procedure. However, the coconut oil SRM was not certified for this constituent. Analysis of Cod Liver Oil (SRM 1588). A multidimensional GPC/reversed-phase HPLC procedure was used to
measure a-tocopherol in a cod liver oil sample (SRM 1588). This procedure was used as a second technique for comparison with results determined in an earlier study by using a method involving direct-injection normal-phase HPLC. The results of these two HPLC procedures were used to provide a certified value for the concentration of a-tocopherol in SRM 1588. A typical chromatogram of the analysis of the cod liver oil fraction obtained by using reversed-phase HPLC after GPC fractionation is shown in Figure 5. The concentration of a-tocopherol in the cod liver oil sample, as determined by using the multidimensional HPLC procedure, is in agreement, within 6%, with the results obtained by using a direct-injection normal-phase HPLC method (see Table 111). In the direct-injection method, the external standard method was used to quantify a-tocopherol in the oil, whereas an internal standard method was used in the
Table IV. Summary of Results for the Determination of Fat-soluble Vitamins in Fortified Coconut Oil and Cod Liver Oil SRMs Using Multidimensional HPLC Coconut Oil
gravimetric retinyl acetate dl-a-tocopheryl acetate ergocalciferol
concentration," pg/g multidimensional HPLC GPC/normal-phase/ GPC/normal-phase reversed-phase
12.6 158.2 10.5
11.9 f 0.3 193.1 f 6.OC 11.3 f 0.2
158.0 f 4.5
certified* 12.2 f 0.8 158.0 f 6.0 10.9 f 0.8
Cod Liver Oil normal-phase HPLC direct injection a-tocopherol
109.2 f 5.0
concentration." ueie multidimensional HPLC GPC/reversed-phase 114.7 f 5.0
certifiedd 112.0 f 5.0
Concentrations are expressed as @g/g. Uncertainties for the certified values are &2 standard deviations. Those associated with the multidimensional HPLC measurements are f l standard deviation of a single measurement. bSee ref 9. 'This value includes the presence of a coeluting compound and was not used in determining the certified value. dValue was derived from the mean of normal-phase and reversed-phase HPLC measurements.
Anal. Chem. 1988, 60, 1933-1936
1933
technique. The certified value for the a-tocopherol in the cod liver oil SRM is also provided in Table IV. The multidimensional HPLC procedures provide rapid analysis time, minimal sample handling, and analyte specificity. The accuracy of the multidimensional HPLC method is M % , and the precision is within 6%. The availability of SRMs 1563 and 1588 will provide the analyst with materials for use in the validation and comparison of analytical methods for the determination of fat-soluble vitamins in lipidic foodlike matrices.
c1
E N
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ACKNOWLEDGMENT The authors thank Hoffman-La Roche, Basel, Switzerland, for the gift of tocol and Karlheinz Ballschmiter, University of Ulm, Federal Republic of Germany, for the cod liver oil sample. We also thank Gary D. Byrd and Richard G. Christensen for their technical assistance in establishing the purity of the reference compounds used in this study and Robert C. Paule for statistical analysis of the analytical data. Registry No. Retinyl acetate, 127-47-9;vitamin D2,50-14-6; dl-a-tocopheryl acetate, 52225-20-4; a-tocopherol, 59-02-9.
m
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5
10
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1
15
LITERATURE CITED
20
Retention time (min)
F W e 6. Chromatogram of cod liver oil SRM 1588 using normal-phase HPLC.
GPC/reversed-phase HPLC analysis. A chromatogram from the analysis of the cod liver oil using direct-injection normal-phase HPLC is shown in Figure 6. CONCLUSIONS Table IV provides both gravimetric values and the concentrations of retinyl acetate, dl-a-tocopheryl acetate, and ergocalciferol in SRM 1563-2 as determined by the multidimensional HPLC procedures. The measured values are in agreement, in all cases, with the gravimetric amounts of each vitamin added to the coconut oil. The certified concentrations for SRM 1563-2 (fortified coconut oil) were derived from a weighted combination of the HPLC results and the gravimetric data. The uncertainty associated with these values is f 2 standard deviations of the mean values determined by each
(1) Method of Vitamin Assay, 3rd ed.; Interscience: New Ywk, 1966. (2) Margolls. S. A. Reference MaferMs for Organic Nutrienf Measurem n f ; Special Publication 635; National Bureau of Standards: Galthersburg, MD, 1982. (3) Landen, W. 0. J . Assoc. Off. Anal. Chem. 1980, 6 3 . 131-135. (4) Landen, W. 0.; Eienmuiier, R. R. J . Assoc. Off. Anal. Chem. 1979, 6 2 , 283-289. (5) Williams, R. C.; Schmidt, J. A.; Henry, R. A. J . Chromafogr. Sci. 1972, 70, 494-501. ( 6 ) Ueda, F.; Makino, T.; Kazama, A. Vitamins 1969, 39, 176-160. (7) Ueda. F.; Makino, T.; Kazama, A. Vitamins 1969, 40, 84-90. (8) Holasov6, M.; BlattnB, J. J . Chmmafogr. 1976, 723, 225-230. (9) Cerflflcafe of Analysis , SRM 1563, Chokferol and Fat-Soluble Vita mlns in Coconut oil; National Bureau of Standards: Gaithersburg, MD. 1987. (10) Cerfiffcafeof Analysis, SRM 7588, Organics in Cod Liver Oil; National Bureau of Standards: Gaiihersburg, MD, 1988.
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RECEIVED for review February 5,1988. Accepted May 2,1988. Certain commercial products are identified to specify adequately the experimental procedure. Such identification does not imply endorsement or recommendation by the National Bureau of Standards, nor does it imply that the materials identified are necessarily the best available for the purpose.
Detection of Neutral Products of Chemical Ionization Reactions Charles Allgood and Burnaby Munson* Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 The neutral products of chemical Ionization (CI) reactions have been detected In an unmodified commercial chemical ionization mass spectrometer source. The neutral products are ionized in subsequent ion/moiecule reactions with reagent gas ions. The reiatlve abundances of these ions show a characteristic increase with increasing electron current. The neutral product of benzene has been detected at m / z 79, CsH7+, in the CH, C I spectrum of bibenzyi and 4-methylbenzophenone. Chlorobenzene, the dominant neutral product from C I reactions, was Identified in the CH, C I spectrum of l,l-bls(p-chlorophenyl)-2,2,2-trlchloroethane ( p ,p’-DDT). I n addition, thls method has been used to determine that protonated benzil, CsHsCOCOCeHs, fragments to form C,H,CO+ and neutral benzaldehyde, C,H,CHO, rather than C,H, and
co. 0003-2700/88/0360-1933$01.50/0
Table I. Relative Ion Intensities for Selected Ions in the CHd CI Mass Spectrum of Bibenzyl as a Function of Total Source Pressurea pressureb
183c/105d
7ge/105
0.425 0.500 0.550 0.600 0.650
0.0195 0.0190 0.0190 0.0189 0.0180
0.0290
0.0370 0.0480 0.0500 0.0505
Electron current = 0.65 mA. Source pressure in Torr. mlz 183 = (M + H)+. d m l z 105 = C,H,C,H,+. ‘ m l z 79 = C,H,+. The neutral products of ion/molecule reactions have been detected in radiation chemistry experiments (I, 2 ) , in ultralow-pressure reactors (3), in ion cyclotron resonance (ICR) 0 1988 American Chemical Society
