Anal. Chem. 2000, 72, 4999-5003
A Liquid Chromatography-Mass Spectrometry Method for the Quantification of Bioavailability and Bioconversion of β-Carotene to Retinol in Humans Yan Wang,† Xiaoying Xu,† Machteld van Lieshout,‡ Clive E. West,‡ Johan Lugtenburg,§ Michiel A. Verhoeven,§ Alain F. L. Creemers,§ Muhilal,| and Richard B. van Breemen*,†
Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612, Division of Human Nutrition & Epidemiology, Wageningen University, The Netherlands, Leiden Institute of Chemistry, Leiden University, The Netherlands, and Nutrition Research and Development Centre, JI Dr Sumeru 63, Bogor 16112, Indonesia
A method based on high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (APCI LC-MS) was developed for the quantification of the bioavailability of retinyl palmitate and β-carotene and the bioconversion of β-carotene to retinol in humans. Following oral administration of [8,9,10,11,12,13,14,15,19,20-13C10]-retinyl palmitate and [12,13,14,15,20,12′,13′,14′,15′,20′-13C10]-β-carotene at physiological doses to children between 8 and 11 years of age, blood samples were drawn and serum was prepared. Retinol and β-carotene were extracted from 0.2and 1.0-mL serum samples, respectively, and analyzed using reversed-phase HPLC with a C30 column interfaced to an APCI mass spectrometer. Unlike other LC-MS assays for carotenoids, no additional purification steps were necessary, nor was any derivatization of retinol or β-carotene required. APCI LC-MS showed a linear detector response for β-carotene over 4 orders of magnitude. Using selected ion monitoring to record the elution profile of protonated circulating β-carotene at m/z 537 and [13C10]-β-carotene at m/z 547, the limit of detection was determined to be 0.5 pmol injected on-column. To assess the ratio of labeled to unlabeled retinol, selected ion monitoring was carried out at m/z 269, 274, and 279. These abundant fragment ions corresponded to the loss of water from the protonated molecule of circulating retinol, [13C5]-retinol (metabolically formed from orally administered [13C10]-β-carotene), and [13C10]-retinol (formed by hydrolysis of [13C10]-retinyl palmitate). The ratios of labeled to unlabeled retinol and the ratio of labeled to unlabeled β-carotene were calculated. Combined with standard HPLC measurement of β-carotene and retinol concentration and a mathematical model, these results showed that this simple LC-MS method can be used to quantify β-carotene bioavailability and its bioconversion to retinol at physiologically relevant doses. Carotenoids are the primary source of vitamin A in the human diet in developing countries. Lack of vitamin A increases risk of * Corresponding author: (telephone) (312) 996-9353 ; (fax) (312) 996-7107; (e-mail)
[email protected]). † University of Illinois at Chicago. ‡ Wageningen University. § Leiden University. | Nutrition Research and Development Centre. 10.1021/ac000454e CCC: $19.00 Published on Web 08/24/2000
© 2000 American Chemical Society
morbidity and mortality.1 There has been concern that the bioavailability of β-carotene and retinol and the bioconversion of β-carotene to retinol are less than previously thought.2 In nutrition, the term bioavailability means the fraction of an ingested nutrient that is available for utilization in normal physiologic functions and for storage.3 In nutrition, bioconversion means the fraction of a bioavailable nutrient (here: absorbed provitamin A carotenoids) that is converted to the active form of a nutrient (here: retinol). Among the more than 600 carotenoids, ∼50 have provitamin A activity in humans, and among them, β-carotene is the most nutritionally active.4 Besides its provitamin A activity, β-carotene is also a singlet oxygen scavenger,5 and epidemiological studies indicate that consumption of fruits and vegetables rich in β-carotene is associated with reduced risks of heart disease6 and cancer.7 Despite its nutritional significance, β-carotene bioavailability and bioconversion to retinol remain poorly characterized in humans because of the lack of suitable bioanalytical methods. Unlike bioavailability (as defined in pharmaceutical research) and pharmacokinetic measurements of typical drugs, studies with nutrients such as β-carotene and retinol are complicated by high circulating concentrations (0.1-1.0 µM) of these nutrients in blood and tissues. Isotopic labeling may be used to distinguish an administered dose from circulating material. However, labeling with stable isotopes is preferred to labeling with radioactive isotopes, especially in studies involving children, because of safety issues. Therefore, recent studies on the bioavailability and bioconversion of β-carotene have used labeling with stable isotopes. Current approaches to the measurement of the bioavailability of β-carotene and retinol involve either administration of high doses of β-carotene (12-30 mg/d)8,9 or deuterated compounds (1) Beaton, G. H.; Martorell, R.; Aronson, K. J. Effectiveness of vitamin A supplementation in the control of young child morbidity and mortality in developing countries. United Nations. 13. 1993 (ACC/SCN state of the art series: nutrition policy discussion paper). (2) de Pee, S.; West, C. E.; Muhilal; Karyadi, D.; Hautvast, J. G. A. J. Lancet 1995, 346, 75-81. (3) Jackson, M. J. Eur. J. Clin. Nutr. 1997, 51 (Suppl, 1), S1-S2. (4) Demming-Adams, B.; Gilmore, A. M.; Adams, W. W. FASEB J. 1996, 10, 403-412. (5) Burton, G. W.; Ingold, K. U. Science 1984, 224, 569-573. (6) Kritchevsky, S. B. J. Nutr. 1999, 129, 5-8. (7) Smith, T. A. Br. J. Biomed. Sci. 1998, 55, 268-275. (8) Brown, E. D.; Micozzi, M. S.; Craft, N. E.; Bieri, J. G.; Beecher, G.; Edwards, B. K.; Rose, A.; Taylor, P. R.; Smith, J. C. Am. J. Clin. Nutr. 1989, 49, 1258-1265.
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such as [10,10′,19,19,19,19′,19′,19′-2H8]-β-carotene.10,11 In the first approach, the large dose facilitated the measurement of the administered compound in the serum, but total plasma β-carotene increased 110-2600 times at the same time. This raises the question of whether absorption and bioconversion would be the same at physiological doses that do not perturb total β-carotene levels. In the second approach, the HPLC retention times of β-carotene and [2H8]-β-carotene were different. Since deuteration of β-carotene changes its HPLC retention time, there is concern that other physicochemical properties might be altered. 13CLabeling and GC/MS with an isotope ratio mass spectrometer have also been used to study β-carotene kinetics in humans.12 In that study, sample preparation included semipreparative HPLC followed by saponification, hexane extraction, liquid-liquid partition, reversed-phase HPLC, and finally hydrogenation catalyzed by platinum oxide. The analytical step required gas chromatography and then combustion of the purified sample followed by measurement using a specialized isotope ratio mass spectrometer. The elaborate sample preparation and specialized equipment required for the approach limit its accessibility and applicability to large-scale human intervention studies. Based on our liquid chromatography-mass spectrometry (LC-MS) methods for the analysis of retinol and carotenoids in human serum,13,14 a quantitative method was developed to quantify the bioavailability of β-carotene and its bioconversion to retinol in humans. In our approach, 13C was used instead of deuterium to minimize any physicochemical differences between labeled and unlabeled compounds. Furthermore, extrinsically specifically labeled β-carotene and retinyl palmitate (See Figure 1) were administered to human volunteers at physiological doses, so that circulating levels of these compounds would not be perturbed. Here, we report our LC-MS method and preliminary data from a human intervention study investigating β-carotene bioavailability and its bioconversion to retinol in children in Indonesia. EXPERIMENTAL SECTION Chemicals. Unlabeled all-trans-retinol and all-trans-β-carotene were purchased from Sigma Chemical (St. Louis, MO), and [13C10]β-carotene and [13C10]-retinyl palmitate were synthesized on on the basis of published methods.15 The structures of these compounds and the sites of 13C-labeling are shown in Figure 1. HPLC grade or better solvents were purchased from Fisher Scientific (Fair Lawn, NY). Sample Preparation. Although the study design and complete results will be reported separately, examples of our study of β-carotene and retinol bioavailability and bioconversion of β-carotene to retinol in Indonesian school children will be used (9) Micozzi, M. S.; Brown, E. D.; Edwards, B. K.; Bieri, J. G.; Taylor, P. R.; Khachik, F.; Beecher, G. R.; Smith, J. C. Am. J. Clin. Nutr. 1992, 55, 11201125. (10) Dueker, S. R.; Jones, A. D.; Smith, G. M.; Clifford, A. J. Anal. Chem. 1994, 66, 4177-4185. (11) Novotny, J. A.; Dueker, S. R.; Zech, L. A.; Clifford, A. J. J. Lipid Res. 1995, 36, 1825-1838. (12) Parker, R. S.; Swanson, J. E.; Marmor, B.; Goodman, K. J.; Spielman, A. B.; Brenna, J. T.; Viereck, S. M.; Canfield, W. K. Ann. N. Y. Acad. Sci. 1993, 691, 86-95. (13) van Breemen, R. B.; Nikolic, D.; Xu, X.; Xiong, Y.; van Lieshout, M.; West, C. E.; Schilling, A. B. J. Chromatogr., A 1998, 794, 245-251. (14) van Breemen, R. B.; Huang, C. R.; Tan, Y.; Sander, L. C.; Schilling, A. B. J. Mass Spectrom. 1996, 31, 975-981. (15) Lugtenburg, J. Eur. J. Clin. Nutr. 1996, 50 (Suppl. 3), S17-S20.
5000 Analytical Chemistry, Vol. 72, No. 20, October 15, 2000
Figure 1. Structures of all-trans-[12,13,14,15,20,12′,13′,14′,15′,20′-13C10]-β-carotene and all-trans-[8,9,10,11,12,13,14,15,19,2013C ]-retinyl palmitate and their conversion to all-trans-[13C ]- and 10 5 [13C10]-retinol in the body. (* denotes position of 13C-label.)
to illustrate the practical application of the LC-MS method. Children (8-11 years old) were administered physiological doses of [13C10]-retinyl palmitate and [13C10]-β-carotene (80 µg each) twice a day as supplements to a low-carotenoid and low-retinol diet. Blood samples were drawn at various times up to 10 weeks after starting the experiment. Serum was prepared by centrifugation of whole blood (750g for 10 min at room temperature) and stored on dry ice or in a -80 °C freezer until analysis. Retinol and β-carotene were extracted from serum without saponification. Each serum sample (1.0 mL for β-carotene and 0.2 mL for retinol analysis) was mixed with 1 mL of 30% NaCl (aq) and 1 mL of 70% EtOH and then extracted three times with 3-mL portions of hexane. The hexane extracts were combined and evaporated to dryness under vacuum. The extraction procedure was carried out under subdued light. The residue was redissolved in 200 µL of methanol/methyl tert-butyl ether (1:1; v/v) for LC-MS analysis. LC-MS Analysis of β-Carotene. LC-MS was carried out using a Hewlett-Packard (Palo Alto, CA) G1946A LCMSD quadrupole mass spectrometer equipped with a series 1100 HPLC system consisting of a binary pump, automatic solvent degasser, autosampler, a YMC (Wilmington, NC) C30 column (3 µm; 250 × 4.6 mm), and a C30 guard cartridge. The solvent system consisted of a gradient from 15 to 30% methyl tert-butyl ether in 12 min, followed by 30% methyl tert-butyl ether for 13 min. The cosolvent was methanol containing 1 mM ammonium acetate, and the flow rate was 1 mL/min. The column was then flushed with 100% methyl tert-butyl ether for 5 min and then equilibrated with 15% methyl tert-butyl ether for 10 min before the next injection. For
ether/acetic acid (50:50:0.5; v/v/v). The C30 column (3 µm; 100 × 2.1 mm) was flushed out with 100% solvent B for 5 min to remove strongly retained compounds and reequilibrated at 30% B for 10 min before the next injection. For each analysis, 20 µL of the serum extract (200 µL total volume) was injected onto the HPLC column. The positive ion APCI conditions for retinol analysis included a nitrogen nebulizer pressure of 35 psi (2.4 bar), a vaporizer temperature of 150 °C, a nitrogen drying gas temperature of 275 °C at 5 L/min, a capillary voltage of 2200 V, a corona current of 2.0 µA, and a fragmentor voltage of 50 V. Because retinol fragments during positive ion APCI form a base peak of m/z 269,13 SIM was used to record the signals at m/z 269 for unlabeled retinol and the corresponding ions of m/z 274 and 279 for [13C5]-retinol and [13C10]-retinol, respectively. [13C5]-Retinol was expected to be formed in vivo by metabolic conversion of [13C10]β-carotene (Figure 1). The ratios of [13C5]-retinol and of [13C10]retinol to unlabeled (circulating) retinol were calculated. The concentrations of retinol and β-carotene in serum were determined at the same time using the HPLC method of Craft et al.16
Figure 2. Standard curves for the analysis of all-trans-β-carotene using (A) positive ion APCI mass spectrometry and (B) positive ion electrospray mass spectrometry. The base peak of the mass spectrum at m/z 537 [M + H]+ was recorded using SIM. Note that the electrospray curve shows a nonlinear response, while APCI gave a linear response over the entire range of concentrations investigated.
each analysis, 60 µL of the serum extract (200 µL total volume) was injected onto the HPLC column. Positive ion atmospheric pressure chemical ionization (APCI) and electrospray were compared, and APCI was selected for all subsequent studies because of the necessity for a wide dynamic range (see Figure 2 and Results and Discussion). The optimum positive ion APCI conditions for β-carotene analysis included a nitrogen nebulizer pressure of 45 psi (3.1 bar), a vaporizer temperature of 325 °C, a nitrogen drying gas temperature of 200 °C at 11 L/min, a capillary voltage of 2800 V, a corona current of 4.0 µA, and a fragmentor voltage of 70 V. Selected ion monitoring (SIM) was used to record the abundances of the protonated molecules of unlabeled and [13C10]-β-carotene at m/z 537 and 547, respectively. The ratio of labeled to unlabeled (circulating) β-carotene was calculated. The serum concentration of β-carotene was measured using HPLC with UV/visible absorbance detection according to published procedures.16 LC-MS Analysis of Retinol. Retinol was analyzed using LCMS as described above with the following modifications. Compared to the carotenoid analysis, a more polar solvent system was used for the HPLC analysis of retinol. The solvent system consisted of gradient elution at 0.2 mL/min from 30 to 46% solvent B in 8 min, 46 to 50% B in 4 min, and then isocratic elution at 50% B for 8 min. Solvent A consisted of methanol/water/acetic acid (50:50:0.5; v/v/v), and solvent B was methanol/methyl tert-butyl (16) Craft, N. E.; Wise, S. A.; Soares, J. H. J. Chromatogr. 1992, 589, 171-176.
RESULTS AND DISCUSSION Since it is preferable to administer doses of labeled compounds that do not perturb the steady state, an LC-MS method is required that can measure low concentrations of these labeled compounds in serum. Furthermore, these measurements must be carried out in the presence of higher concentrations of circulating compounds coeluting from the HPLC. Therefore, an LC-MS method was required with a wide dynamic range for the simultaneous determination of trace amounts of labeled retinol and β-carotene in the presence of large quantities of the corresponding unlabeled compounds. Over the range of β-carotene concentrations, 0.4968-99.36 pmol/µL (0.25-1987 pmol oncolumn), positive ion APCI produced a linear response with R2 ) 0.9984 (Figure 2A). In contrast, positive ion electrospray did not produce a linear response for β-carotene over the same range of concentrations (Figure 2B). Similar results were observed for retinol.13 Therefore, APCI was used throughout this investigation instead of electrospray, because of its wider dynamic range and greater linearity of detector response. The positive ion APCI mass spectrum of β-carotene (Figure 3) showed a base peak at m/z 537 corresponding to the protonated molecule. The abundance of all the other ions was less than 25%. Therefore, SIM of the abundant protonated molecule of β-carotene was used in all subsequent measurements. A low limit of detection of 0.25 pmol on-column was obtained (defined as a signal-to-noise ratio of 3:1). The lower limit of quantification was determined to be 560 fmol on-column with a coefficient of variation of 4.82% (n ) 6). Comparable limits of detection were obtained for retinol and retinyl palmitate by monitoring the fragment ion at m/z 269 (13). Because all human serum samples contained circulating β-carotene, no blank serum was available for limit of detection determinations. Therefore, standard solutions were prepared in methanol/methyl tert-butyl ether (1:1, v/v) instead of serum. Our previous report validated the use of mobile phase instead of serum for the preparation of retinol standards for similar studies.13 Examples of LC-MS analyses of β-carotene in serum samples obtained at day 0 and day 21 are shown in Figure 4. At day 0 (Figure 4A), only unlabeled all-trans-β-carotene was observed in the serum sample, and there was no compound with m/z 547 (that Analytical Chemistry, Vol. 72, No. 20, October 15, 2000
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Figure 3. Positive ion APCI mass spectrum of β-carotene. The protonated molecule was detected as the base peak at m/z 537.
Figure 4. LC-MS SIM chromatograms of human serum extracts showing elution of all-trans-β-carotene and all-trans-[13C10]-β-carotene at m/z 537 and 547. (A) Serum sample drawn immediately before administration of all-trans-[13C10]-β-carotene; (B) serum sample obtained on day 21 of dietary administration of all-trans-[13C10]-βcarotene.
is the m/z of labeled β-carotene) eluting at the same retention time. At day 21 (Figure 4B), both labeled and unlabeled β-carotene were observed coeluting at 22.8 min. There was little difference in the retention time between labeled and unlabeled β-carotene, which confirmed that the physical properties of all-trans-[13C10]β-carotene resemble those of unlabeled β-carotene. This is unlike the results with [2H8]-β-carotene and unlabeled β-carotene reported 5002 Analytical Chemistry, Vol. 72, No. 20, October 15, 2000
Figure 5. LC-MS analysis of retinol in a hexane extract of human serum recorded using SIM of m/z 269 (circulating all-trans-retinol), 274 (all-trans-[13C5]-retinol), and 279 (all-trans-[13C10]-retinol). (A) Serum sample obtained immediately before administration of labeled compounds; (B) serum sample obtained on day 21 of dietary administration of labeled compounds. Note the appearance of 13Clabeled retinol in serum after administration.
by Dueker et al.,10 where there was a distinct difference in retention time of the two compounds. Another advantage of 13Clabeling over deuterium labeling is the elimination of the possibility of isotope scrambling during bioconversion of β-carotene to retinol. Structures of [13C10]-β-carotene and [13C10]-retinyl palmitate and their conversion to retinol in the body are shown in Figure 1. Whether cleavage of β-carotene to retinol is centric or eccentric,
bioconversion of [13C10]-β-carotene would produce [13C5]-retinol. [13C10]-Retinyl palmitate was administered instead of [13C10]-retinol because nearly all retinol in food is present as esters, which are more lipid soluble and stable. Nevertheless, circulating retinol bound to albumin is unesterified. Examples of LC-MS analyses of retinol from human serum are shown in Figure 5. During LCMS, the elution profiles of circulating retinol and labeled retinol were recorded using SIM of m/z 269, 274, and 279. The ion of m/z 269 corresponded to the loss of water from the protonated molecule of retinol ([MH - H2O]+), the ion of m/z 274 corresponded to the [MH - H2O]+ ion of [13C5]-retinol formed in the body from bioconversion of [13C10]-β-carotene, and the ion of m/z 279 corresponded to the [MH - H2O]+ ion of [13C10]-retinol formed in the body from hydrolysis of [13C10]-retinyl palmitate. At day 0 (Figure 5A), only unlabeled retinol was observed at m/z 269 with a retention time of 17.7 min. The absence of coeluting signals at m/z 274 and 279 indicates that there is no interference for the LC-MS analysis of labeled retinol. After 21 days of dietary administration of labeled β-carotene and retinyl palmitate, [13C5]and [13C10]-retinol were detected coeluting with unlabeled retinol during LC-MS analysis of a serum extract (Figure 5B), these results are typical of the data obtained on other days and for other subjects. CONCLUSION An APCI LC-MS method has been developed and applied to the measurement of β-carotene and retinol in human serum. Utilizing administered 13C-labeled compounds, this method is
suitable for the investigation of β-carotene bioavailability and its bioconversion to retinol. By combining simple sample preparation with straightforward LC-MS analysis, the procedure is convenient and sufficiently sensitive and specific for measuring isotopic enrichment of β-carotene and retinol in human serum. The limit of detection and quantification for retinol and β-carotene (
