Anal. Chem. 1995, 67,2004-2009
Electrospray Liquid Chromatography-Mass Spectrometry of Carotenoids Richard B. van Breemen Department of Medicinal Chemistry and Phatmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, W C 781, Chicago, Illinois 60612-7231
The first method for electrosprayliquid chromatographymass spectrometry (LC-MS) of carotenoids is reported, which uses a c30 reversed-phase HPLC column and a gradient solvent system containing methanoVmethyltertbutyl ether/ammonium acetate at a flow rate of 1.0 mW min. The entire HPLC column effluent passes through a photodiode array absorbance detector and then into the electrospray LC-MS interface without solvent splitting. In this way, maximum sensitivity is achieved for both the photodiode array detector, which records the W/vis spectra of each carotenoid, and the mass spectrometer, which measures the molecular ions of each carotenoid. Molecular ions, M + , without evidence of any fiagmentation, were observed in the electrospray mass spectra of both xanthophylls and carotenes. In order to enhance the formation of molecular ions, solution-phase carotenoid oxidation was carried out by means of postcolumn addition of a halogenated solvent to the HPLC effluent. Several different halogenated solvents were evaluated, including chloroform, 2,2,3,3,4,4,4-heptatluoro1-butanol, 2,2,3,3,4,4,4-heptafluorobutyricacid, 1,1,1,3,3,3hexafluor0-2-propano1,and trifiuoroacetic acid. Among 1these halogenated solvents, 2,2,3,3,4,4,4-heptafluorobutanol at a concentration of 0.1% (v/v) was found to produce the best combination of carotenoid molecular ion abundance and reproducibility. The limits of detection for lutein and/?-carotenewere between 1and 2 pmol each, which was 100-fold lower than the detection limit of the photodiode array detector signal.
Astaxanthin
OH
HO 0
@Cryptoxanthin
HO
HO
a-Carotene
D-Carotene
Figure 1. Structures of selected ail-trans carotenoids including the xanthophylls astaxanthin, P-cryptoxanthin, and lutein and the carotenes a-carotene and p-carotene.
High-performance liquid chromatography (HPLC) is regarded as the preferred method for the separation, identification, and
quantitation of carotenoids found in biological tissues.5 Unfortunately, complex mixtures of carotenoids, many of which are closely related structurally, are often present in biological matrices, rendering unequivocal identification by HPLC using retention and fixed wavelength data alone unacceptable. The advent of photodiode array detection, allowing for continuous collection of spectral data during HPLC analysis, has provided a powerful tool for carotenoid r e ~ e a r c h .However, ~ tentative identification of carotenoids using HPLC with photodiode array detection requires retention time measurement, complete chromatographic resolution of absorbing species so that spectrophotometric data for the analyte alone are observed, and comparison of W/vis spectra and retention times with those of authentic standards. Mass spectrometric and tandem mass spectrometric analyses,6 which provide molecular weight and characteristic fragmentation pat-
(1) Spom, M. B.; Clamon, G. H.; Dunlop, N. M.; Newton, D. L.; Smith, J. M.; Saffiotti, U . Nature (London) 1975,253,47-50. (2) Ziegler, R G. Am. J. Clin. Nutr. 1991,53,S251-SZ59. (3) Bendich, A.; Olson, J. A.F A S E B j . 1989,3,1927-1932. (4) Malone, W. F. Am. J. Clin. Nutr. 1991,53,S305-S313.
(5) Taylor, R F.; Farrow, P. E.; Yelle, L. M.; Harris, J. C.; Marenchic. I. G. In Carotenoids: Chemistry and Biology; Krinsky, N . I., et al., Eds.; Plenum Press: New York, 1990; pp 105-123. (6) van Breemen, R B.; Schmitz, H. H.; Schwartz, S. J. 1,Agric. Food Chem. 1995,43.384-389.
The nutritional importance of certain carotenoids as metabolic precursors of vitamin A in man is well known.' Additional functions for carotenoids, including some non-provitamin A carotenoids, have been suggested to include cancer prevention and imm~noenhancers,~ and in vivo antioxidants! Increased interest in carotenoids, coupled with frequently inadequate analytical determination of these compounds when present in many biological tissues, such as serum, liver, and foods, emphasizes the need for new methods which can positively identify individual carotenoids (see carotenoid structures in Figure 1).
2004 Analytical Chemistry, Vol. 67, No. 73,July 7, 7995
0003-2700/95/0367-2004$9.00/0 0 1995 American Chemical Society
terns, may then provide h a 1 confirmation of individual carotenoid identities when used in conjunction with retention and spectral characteristics.' Several ionization methods have been reported for mass spectrometric analysis of carotenoids,including electron impacts and fast atom bombardment (FAB)? Electron impact requires sample vaporization prior to ionization, which is a disadvantage in the analysis of the thermally labile and nonvolatile carotenoids. Recently, a positive ion electrospraymass spectrum of /?-carotene was obtained by Van Berkel and ZhoulO using solution-phase oxidation, which showed a doubly charged molecular ion and [M2+ - H+]+. However, electrospray LC-MS was not demonstrated in their investigation.'O Previous reports from our own laboratory and others exploring the application of LC-MS to carotenoid analysis used a heated moving belt interface with electron impact i~nization,~ a particle beam interface with chemical ionization and a heated ion source,11 or continuous-flow FAB mass spectr~metry.~J~ Among these methods, only continuous-flow FAB does not require the use of gas-phase ionization or a heated ion source, which might lead to carotenoid pyrolysis. However, the flow rate of continuous-flow FAB is l i i t e d to < 10 pL/min, so capillary HPLC columns must be used or solvent splitting of the analytical scale HPLC effluent must be carried out prior to mass spectrometric analysis. In the present investigation, we report development of an electrospray LC-MS method that is compatible with analytical scale HPLC column flow rates of 1.0 mL/min. Furthermore, compatibility of this new electrospray LC-MS method will be demonstrated with the C30reversed-phase HPLC stationary phase recently engineered by Sander et for the separation of carotenoids and carotenoid isomers. EXPERIMENTAL SECTION Mass spectra were obtained using a Hewlett-Packard (Palo Alto, CA) 5989B MS engine quadrupole mass spectrometer equipped with a ChemStation data system and high-flow @e., 1 mL/min) pneumatic nebulizer-assisted electrospray LC-MS interface. The mass spectrometer was interfaced to a HewlettPackard 109OL gradient HPLC system equipped with an autoinjector and a photodiode array W/vis absorbance detector. The quadrupole analyzer was maintained at 120 "C, and unit resolution was used for all measurements. Nitrogen at a pressure of 80 psi was used for nebulization of the HPLC effluent, and nitrogen bath gas at 350 "C and a flow rate of 1.0 Wmin was used for evaporation of solvent from the electrospray. The electrospray needle was maintained at ground potential, while the counterelectrode was at -5100 V. The range m/z 200-610 was scanned over -2 s during LC-MS. HPLC separations were carried out using a reversed-phase column, 4.6 mm x 12.5 cm, containing a C30 silica stationary phase, which was engineered speci6cally for carotenoid separations.13 (7) Schmitz, H. H.; van Breemen, R B.; Schwartz, S. J. Methods Enymol. 1992, 213,322-336. (8)Vetter, W.;Meister, W. Og.Mass Spectrom. 1985,20, 266. (9) Caccamese, S.; Garozzo, D. Og. Mass Spectrom. 1990,25,137-140. (10) Van Berkel, G. J.; Zhou, F. Anal. Chem. 1994,66,3408-3415. (11) Khachik, F.; Beecher, G. R; Goli, M. B.; Lusby, W. R; Smith,J. C.,Jr.Ana1. Chem. 1992,64, 2111-2122. (12) van Breemen, R B.; Schmitz, H. H.; Schwartz, S. J. Anal. Chem. 1993,65, 965-969. (13) Sander, L C.; Sharpless, K E.; Craft, N. E.; Wise, S. A. Anal. Chem. 1994, 66, 1667-1674.
The solvent system consisted of a 60 min linear gradient from 8515 to 1090 methanol/methyl tert-butyl ether containing 1.0 mM ammonium acetate at a flow rate of 1 mVmin. Absorbance spectra were recorded from 250 to 600 nm using a photodiode array detector located in-line between the HPLC column and the electrospray LC-MS interface. Based on the method of Van Berkel and Z~OU,~O carotenoids were oxidized in solution by postcolumn addition of methanol/methyl tert-butyl ether solution (5050 v/v) containing a halogenated oxidant (Le., 2% v/v 2,2,3,3,4,4,4heptafluorel-butanol)at a flow rate of 50 pL/min. Optimization experiments were carried out in which different halogenated compounds at various concentrations were evaluated. During some experiments, flow injection of carotenoid solutions was used in combination with selected ion monitoring, in order to measure the abundance of M + ions, such as m/z 536 for /?-carotene or m/z 568 for lutein. The selected ion monitoring dwell time was 100 ms/ion. Carotenoid standards were injected using a Rheodyne (Cotati, CA) Model 7725 injector equipped with a custom-made 1.5 pL fused silica capillary sample loop that was completely filled for each injection. An Applied Biosystems (Foster City, CA) Model 140A dual syringe HPLC pump was used for delivery of methanol (or acetonitrile)/methyl tert-butyl ether (7030 v/v) containing the appropriate halogenated oxidant (typically 0.1% v/v 2,2,3,3,4,4,4heptafluoro-l-butanol) during flow injection measurements at a flow rate of 5 or 10 pL/min. Other carrier solvents investigated included methanol/methyl tert-butyl ether/water (containing 10 mM ammonia or 0.1%v/v glacial acetic acid) (50:46:4 v/v/v) or methanol/ethyl acetatehater (with or without 10 mM sodium acetate) (3069:l v/v/v). HPLC solvents were purchased from Fisher Scientific (Springfield, NJ). The halogenated compounds chloroform, 2,2,3,3,4,4,4 heptafluorel-butanol,2,2,3,3,4,4,4heptanuorobutyric acid, 1,1,1,3,3,2hexafluore2-propanol, and tduoroacetic acid were reagent grade or HPLC grade and were obtained from Aldrich Chemical Co. (Milwaukee, WI). Lutein, a-carotene, and p-carotene standards were purchased from Sigma Chemical Co. (St. Louis, MO) and purified as necessary by using HPLC, and the purity of each standard was confirmed by using HPLC with photodiode array absorbance detection. Standard solutions of lutein, a-carotene, and/or /?-carotene with concentrations up to 25 ng/mL were prepared in methyl tert-butyl ether/methanol (or acetonitrile) (50: 50 v/v). Processed, canned sweet potatoes were extracted and prepared in an analogous manner to carrot and tomato extracts, which was previously des~ribed.~ RESULTS AND DISCUSSION In order to develop an electrospray liquid chromatographymass spectrometry (LC-MS) method, flow injection analyses were carried out to determine suitable electrospray parameters and mobile phase composition. Initially, negative ion electrospray mass spectra were obtained while flow-injecting carotenoids into a solution of methanol, methyl tee-butyl ether, and water. Under these conditions, deprotonated molecules of polar xanthophylls such as astaxanthin at m/z 595 were observed, but no signals were detected for the hydrocarbon carotenes (see carotenoid structures in Figure 1). Addition of ammonia to the carrier solution increased the abundance of deprotonated xanthophyll molecules, but no carotene ions were observed. Positive ion electrospray mass spectrometry of carotenoids was investigated using a mobile phase containing methanol, ethyl acetate, and water. Protonated molecules and traces of sodium Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
2005
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Figure 2. Optimization of trifluoroacetic acid content during solutionphase oxidation of p-carotene. The abundance of the molecular ion of p-carotene (0.25 pglinjection) was monitored at m/z 536 during flow injection positive ion electrospray mass spectrometry. The carrier solvent was acetonitrile/methyl tert-butyl ether (70:30 v/v) at 10 pU min. Each data point represents the average of at least three measurements (& standard deviation).
adducts were observed for xanthophylls but not carotenes. Acidification of the solution by addition of acetic acid slightly increased the abundance of protonated molecules, but sodium adducts were still observed. When sodium acetate was added to the mobile phase instead of acetic acid, no protonated molecules were observed; instead, abundant [M Nal+ ions were detected for xanthophylls such as astaxanthin ( F i r e 1). Because carotenes lack any heteroatoms such as oxygen to which protons or sodium cations might attach, no ions were detected for these hydrocarbons during electrospray under these conditions. In order to ionize carotenes such as /?carotene during electrospray LC-MS, trifluoroacetic acid was added postcolumn to the mobile phase in order to carry out solution-phase oxidation as described by Van Berkel and Zhou.'O Although Van Berkel and Zhou observed doubly charged molecular ions and [M - HI+ species during solution-phase oxidation of /?-carotene, only molecular ion radicals, M*+, were observed for the carotenoids investigated during the present investigation. The primary differences between these two studies were the composition of the mobile phase and the use of a quadrupole instead of an ion trap mass spectrometer. Previously,'o methylene chloride was used as the carrier solvent for flow injection electrospray mass spectrometry, which might have been at least partly responsible for oxidation of p-carotene to a doubly charged molecular ion. Methanol and methyl tert-butyl ether were used instead of methylene chloride in the present LC-MS study for optimum carotenoid chromatography using the C ~ silica O HPLC column developed by Sander et al.13 Because molecular ions, M + , were preferred over doubly charged species or [M - HI+ ions, no further attempts were made to reproduce the results of Van Berkel and Z~OU.~O A range of trifluoroacetic acid concentrations, including 0.01%, 0.02%,0.05%,0.1%,0.25%,and 0.5%,were evaluated for the solutionphase oxidation of /?-carotene,and the most effective composition was found to contain 0.1%trifluoroacetic acid (v/v) (see Figure 2). The highest level of trifluoroacetic acid investigated, 0.5%,was as ineffective as no trifluoroacetic acid at all, which suggested that the electrospray was not forming efficiently in the presence
+
2006 Analytical Chemistry, Vol. 67,No. 13, July 1, 7995
of higher concentrations of trifluoroacetic acid,I4p-carotene ions were being quenched in the gas phase by trifluoroacetic acid, or /?-carotene ions could not evaporate from charged droplets containing high concentrations of acid. As the conductivity of the mobile phase increases with increasing concentrations of trifluoroacetic acid, the 'Taylor cone" from which charged droplets are emitted can become unstable so that sensitivity decreases.I4 This source of interference with the formation of a stable electrospray might account for the loss of sensitivity at 0.25% and 0.5% trifluoroacetic acid compared to lower concentrations of acid. Because molecular ions of /?carotene were observed in the absence of solution-phase oxidants such as trifluoroacetic acid, the electrospray process itself was shown to contribute to the oxidation and ionization of p-carotene. Electrospray ionization of neutral carotenoids probably occurs by electrophoretic charging and field ionization at the metal-liquid interface of the electrospray capillary, as described by Blades et al.I5 In this manner, the electrospray interface may be viewed as an electrolytic cell. The formation of carotenoid molecular ions by electrochemical oxidation is also similar to oxidation of metallocenes reported by Xu et a1.,16 who concluded that formation of metallocene ions occurred either in solution or at the needle-solution interface. Like in the electrospray mass spectra of carotenoids reported here, Xu et al. observed primarily molecular ions, M + , and no doubly charged ions. Although Xu et al. found that addition of trifluoroacetic acid had no effect upon formation of metallocene ions,16 carotenoid ionization was enhanced by the addition of trifluoroacetic acid, as described by Van Berkel et aL1O>I7 In order to determine what properties of trifluoroacetic acid were important for the formation of molecular ions of carotenoids during electrospray and to possibly identify a superior halogenated oxidant for this application, several other halogenated solvents were investigated as solution-phase oxidants, including chloroform, 2,2,3,3,4,4,4heptafluorobutytic acid, 2,2,3,3,4,4,4heptafluoro1-butanol,and 1,1,1,3,3,3-hexafluoro-2-propanol. A comparison of the abundances of /?-carotene molecular ions at two different concentrations of each solution-phase oxidant is shown in Figure 3. Acidification of the solution with 0.1%acetic acid resulted in no molecular ion formation, so the acidity of trifluoroacetic acid was probably not beneficial for oxidation and ionization of /?-carotene. A comparison of heptafluorobutyric acid and h e p tafluorobutanol (Figure 3) also shows that the presence of the carboxylic acid group in the oxidant does not contribute to and probably suppresses the ionization of ,&carotene. Among the oxidants evaluated, hexafluoropropanol and heptafluorobutanol facilitated production of the most abundant molecular ions of /?-carotene. Because heptafluorobutanol at either 0.1%or 0.5%produced abundant molecular ions of ,+carotene with high reproducibility (see standard deviations of the measurements in Figure 3), the optimum concentration of this oxidant was determined, and the results are shown in Figure 4. A plateau of maximum molecular ion formation of p-carotene was reached from 0.02% to 0.25% heptafluorobutanol (Figure 4). All subsequent electrospray analyses of carotenoids were carried out using heptafluorobutanol at (14) Eshraghi, J.; Chowdhury, S. K. Anal. Chem. 1993,65,3528-3533. (15) Blades, A. T.; Ikonomou, M. G.; Kebarle, P. Anal. Chem. 1991,63,21092114. (16) Xu. X.; Nolan, S. P.; Cole, R B. Anal. Chem. 1994,66, 119-125. (17) Van Berkel. G. J.; McLuckey, S. A; Glish, G. L. Anal. Chem. 1992,64, 1586-1593.
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Figure 3. Comparison of halogenated solvents as oxidants during positive ion electrospray mass spectrometry of ,%carotene. Selected ion monitoring was used to measure the abundance of the molecular ion of ,&carotene (0.25 pg) introduced by flow injection into a solution of acetonitrile/methyl teff-butyl ether (70:30 v/v). Each value represents the average of three measurements (& standard deviation).
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Figure 4. Optimization of heptafluorobutanol content during solution-phase oxidation of p-carotene. Using selected ion monitoring, the abundance of the molecular ion of p-carotene (0.25 pg/injection) was monitored at m/z 536 during flow injection positive ion electrospray mass spectrometry. The carrier solvent was acetonitrile/methyl teff-butyl ether (70:30 v/v) at 10 pUmin. Each data point represents the average of at least three measurements (& standard deviation).
0.1%(the middle of the plateau region in Figure 4) as the solutionphase oxidant. The libnit of detection of positive ion electrospray mass spectrometry for carotenoid analysis was determined using the xanthophyll, lutein, and the carotene, B-carotene, as standards (Figure 5). Because the electrospray mass spectra of lutein and Bcarotene (and all other carotenoids used in this study) contained no fragment ions, selected ion monitoring was carried out of the molecular ions at m/z 568 and 536, respectively. Serial dilutions of each carotenoid were prepared and flow-injected. The limits of detection of lutein and p-carotene were determined to be between 1 and 2 pmol (Figure 5). These limits of detection are comparable to those obtained of continuous-flow FAB, which were reported to be -9.0 and 28 pmol for lutein and a-carotene, respectively.12 Note that the continuous-flowFAB detection limits were determined using an HPLC column while scanning the mass spectrometer, whereas the electrospray detection limit was measured using flow injection with selected ion monitoring. Although acetonitrile and methyl ted-butyl ether were used as cosolvents during the limit of detection measurements with the expectation that protic solvents might interfere with ionization and should be avoided, substitution of methanol for acetonitrile
produced similar limits of detection. This result is consistent with those of Xu et al.,16 who found that protic solvents did not interfere with metallocene ionization during electrospray. In contrast, Van Berkel et a l l 7 reported that protic and/or nucleophilic solvents such as methanol would quench molecular ions and should be avoided. Because gradients of methanol to methyl tert-butyl ether produced carotenoid separations that were superior to those obtained with gradients using acetonitrile, methanol was used in all LC-MS analyses using the C ~ reversed-phase O column. 'Ihe LC-MS analysis of a mixture of acarotene and B-carotene standards (20 ng each) is shown in Figure 6. Both the electrospray mass spectrometer and the photodiode array absorbance detector detected the &trans isomers of a-and m o t e n e ( F i i e 6 panels A and B). However, only the mass spectrometer detected the cis isomers eluting between 8-10 and 13-14 min. Compared to the absorbance chromatogram at 450 nm, the signal-to-noise ratio was considerably higher in the computer reconstructed mass chromatogram of the molecular ions at m/z 536. The mass spectrum of acarotene, which was recorded at an elution time of 10.8 min, contained an abundant molecular ion at m/z 536 and no fragment ions (see mass spectrum in Figure 6C). Next, an extract of heat processed, canned sweet potatoes was analyzed by using electrospray LC-MS. When -20 ng of ,%carotene was injected onto the C ~ reversed-phase O HPLC column, a mixture of cis and trans isomers of ,&carotene was chromatographically resolved and detected by positive ion electrospray mass spectrometry (see the computer reconstructed mass chromatogram in Figure 7 4 . However, only the more abundant alltrans isomers were detected during the same analysis using the on-line photodiode array absorbance detector (Figure 7D). When lWfold more sample was injected onto the HPLC column, the absorbance chromatogram resembled the reconstructed mass Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
2007
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d Z Figure 6. Positive ion electrospray LC-MS analysis of a mixture of a-carotene and p-carotene (20 ng each) using a Cm reversedphase HPLC column with postcolumn addition of heptafluorobutanol as oxidant. (A) Computer-reconstructed mass chromatogram of the Absorbance chromatogram at 450 nm molecular ions at m/z 536. (6) recorded on-line using a photodiode array detector. (C) Electrospray mass spectrum of a-carotene recorded at a retention time of 10.8 min.
chromatogram with respect to signal-to-noise and detection of the cis isomers (Figure 7E). Therefore, the sensitivity of electrospray mass spectrometry for the detection of carotenoids was found to be -1Wfold higher than that of the photodiode array detector used during this investigation. Although p-carotene was the most abundant carotenoid in the sweet potato extract, molecular ions corresponding to other more polar carotenoids were detected at lower abundance, including ions of m/z 568 and 552 which probably corresponded to isomers of lutein and Bcryptoxanthin (see Figure 7, parts B and C). Because no Corresponding absorbance spectra for these compounds could be obtained at this concentration using the photodiode array detector, the assignment of these structures is based only on HPLC retention times and molecular weight. 2008 Analytical Chemistry, Vol. 67, No. 73, July 7, 7995
Figure 7. Positive ion electrospray LC-MS analysis of an extract of heat-processed, canned sweet potatoes using a C ~ reversedO phase HPLC column with postcolumn addition of heptafluorobutanol. (A) Computer-reconstructed mass chromatogram of the p-carotene molecular ion at m/z 536 following injection of -20 ng of extract. (6) Computer-reconstructed mass chromatogram of the ion at m/z 568 correspondingto lutein. (C) Computer-reconstructedmass chromatogram of m/z 552 showing isomers of p-cryptoxanthin. (D) HPLC photodiode array detector absorbance chromatogram at 450 nm recorded on-line during the analysis shown in (A). (E)HPLC absorbance chromatogram for the analysis of 2 pg of sweet potato extract.
CONCLUSIONS Although negative ion electrospray mass spectrometry may be used to detect polar xanthophylls, positive ion electrospray LCMS was shown to be useful for the analysis of both xanthophylls and carotenes. Molecular ions, M + , were observed for all carotenoids investigated, and the mechanisms of ionization included solution-phase oxidation using heptafluorobutanol and electrophoreticcharging and field ionization at the metal-liquid interface. This electrospray LC-MS method is compatible with solvents used for the C30 reversed-phase HPLC column and can be used on-line with photodiode array absorbance detection for additional characterization of carotenoids eluting from the HPLC column. Because no solvent splitting of the 1 mL/min HPLC
effluent is necessary, postcolumn band broadening is minimized.
from Lane Sander of the National Institute of Science and Technology.
ACKNOWLEDGMENT Alexander Schilling and Steven Fischer provided fruitful discussionsand technical assistancewith mass swctrometrv. Use of the electrospray mass spectrometer was generously provided by the Hewlett-Packard Co. The C ~ HPLC O column was a gift
Received for review December 12, 1994. Accepted April ,9,1995.B AC9411987 @Abstractpublished in Advance ACS Abstracts, June 1, 1995.
Analytical Chemistty, Vol. 67, No. 13, July 7, 1995
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