Clinical Chemistry 49: 2056-2066, 2003.
First published November 13, 2003; 10.1373/clinchem.2003.022681
(Clinical Chemistry. 2003;49:2056-2066.)
© 2003 American Association for Clinical Chemistry, Inc.
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Automation and Analytical Techniques |
Simultaneous Determination of Tocotrienols, Tocopherols, Retinol, and Major Carotenoids in Human Plasma
Bee-Lan Lee1,
Ai-Li New1 and
Choon-Nam Onga,1
1 Department of Community, Occupational and Family Medicine, Faculty of Medicine, National University of Singapore, Singapore 117597.
aAuthor for correspondence. Fax 65-6779-1489; e-mail cofongcn{at}nus.edu.sg.
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Abstract
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Background: Epidemiologic evidence suggests that the concentrations of antioxidant vitamins in human plasma may play an important role in numerous chronic diseases, such as cancer and cardiovascular disease. However, methods for simultaneous measurement of these antioxidants are scarce. We developed and validated a new HPLC method for simultaneous determination of these vitamers in human plasma that uses a novel column-switching approach.
Methods: The new method uses liquid–liquid extraction and isocratic separation with two monomeric C18 columns maintained at 35 and 4 °C coupled with ultraviolet–visible and fluorometric detection. This method could separate 14 vitamers and 3 internal standards within 27 min. No additional modifier was required; the mobile phase was acetonitrile–methanol (65:35 by volume), and the flow rate was 1 mL/min.
Results: For photodiode array detection, the detection limits (signal-to-noise ratio >3) were 0.02 mg/L for ß-carotene, lutein, zeaxanthin, and canthaxanthin; 0.01 mg/L for all-trans-retinol, ß-cryptoxanthin, 
-carotene, and lycopene; and 0.1 mg/L for all tocopherols and tocotrienols. The detection limit was at least 25-fold lower (0.004 mg/L) when fluorometry was used for measurement of 
-, 
-, and -tocotrienol and -tocopherol compared with ultraviolet detection. The recovery and imprecision of the assay were generally >90% and <10%, respectively.
Conclusions: This new method separates a wide range of fat-soluble antioxidant vitamins in human plasma, including six carotenoids, three isoforms of tocotrienols and tocopherols (-, -, and -), and all-trans-retinol. The overall findings suggest that our method is faster, more sensitive, and more comprehensive than existing methods.
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Introduction
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Vitamin A (all-trans-retinol) and vitamin E analogs, i.e., the tocopherols and tocotrienols, are lipid-soluble vitamins essential for human health. Both groups have free-radical-scavenging properties that allow them to function as physiologic antioxidants (1)(2)(3). Although - and -tocopherol are considered to be the more biologically active forms of vitamin E, recent evidence suggests that the tocotrienols may be more potent than the tocopherols in preventing both cardiovascular disease and cancer and are thus worthy of further scientific investigation (4)(5). Another group of lipophilic vitamin, the carotenoids, found in abundance in vegetables and fruits, also have antioxidant properties similar to those of vitamins A and E. Moreover, higher serum concentrations of carotenoids have been associated with a reduced risk of cancer incidence as well as cardiovascular disease in humans (6)(7).
Existing reversed-phase HPLC methods using a C18 stationary phase coupled with spectrophotometric and fluorometric detection are well suited for determining both vitamin A and E and some major carotenoids (8)(9)(10)(11)(12)(13)(14)(15). However, most of these methods are designed for the separate analysis of either the tocopherols or carotenoids in biological samples. Furthermore, most methods are unable to provide clear separation of lutein and zeaxanthin. Although this problem has been overcome by adopting a C30 reversed-phase column methodology, this methodology requires increased analysis time and suffers from poor sensitivity (16)(17)(18)(19). To circumvent these problems, photodiode array (PDA)1
detection coupled with mass spectrometric (MS) or nuclear magnetic resonance technologies has been suggested. Both techniques provide information such as peak purity (PDA) and unequivocal structural information (MS and nuclear magnetic resonance), but the systems are expensive and require a great deal of expertise in both method development and data interpretation, making them less suitable for many clinical laboratories. In addition, both techniques are also hampered by the fact that the nonpolar carotenoids and tocopherols cannot be easily ionized by standard electrospray ionization and often require derivatization with halogen-containing eluents, ferrocene-based derivatives, or silver ions to make them more amenable to the ionization process (20)(21)(22). To date, atmospheric pressure chemical ionization has afforded the best liquid chromatography–MS approach for the identification of carotenoids. Nevertheless, it is not suitable for the simultaneous determination of tocopherols (23). Likewise, the use of nuclear magnetic resonance for tocopherol isomer analysis is limited by its low sensitivity (24). No single method exists that can simultaneously analyze tocotrienols, tocopherols, retinol, and carotenoids in biological samples.
The main objective of this investigation was to develop a robust and cost-effective HPLC method for the rapid and simultaneous determination and quantification of vitamins A and E and various diet-derived carotenoids in human plasma. The sensitivity, accuracy, and reliability of the proposed method were validated with both fresh biological samples collected from healthy individuals taking or not taking vitamin supplements as well as stored plasma samples randomly selected from an ongoing epidemiologic study (25)(26).
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Materials and Methods
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materials
Reagents and chemicals.
All-trans-retinol; -, -, and, -tocopherol; tocopherol acetate, ß- and -carotene; and lycopene were purchased from Sigma Chemical Co. and were of the highest purity. Echinenone was from the DHI Water and Environment Agency. Retinol acetate was from Supelco. Lutein, zeaxanthin, canthaxanthin, and ß-cryptoxanthin were kind gifts from BASF-AG (Luwigshafen, Germany), and -, -, and -tocotrienol were kind gifts from Malaysian Palm Oil Research Institute (Selangor, Malaysia). Methanol, n-butanol, ethyl acetate, acetone, and acetonitrile were gradient grade; absolute ethanol, tetrahydrofuran, n-hexane, butylated hydroxytoluene (BHT), and disodium sulfate were analytical grade and were obtained from Merck. An ethanol–BHT solution containing 0.2 g/L BHT was used for the calibration mixtures and sample preparation.
Sample collections.
For method validation, we randomly selected 43 stored plasma samples collected in 1995 for a cardiovascular cohort study (25)(26) and 11 fresh plasma samples from the staff of a local medical school. Also obtained were two blood samples from a volunteer who had ingested one serving of LifePak® multivitamin plus mineral supplement (Pharmanex) and another individual who had ingested two TocovidTM capsules containing 100 mg of mixed tocotrienols (Hovid). All samples were collected with informed consent. The procedures for blood collection have been described elsewhere (25)(26)(27). All plasma samples collected were protected from light and kept frozen at -70 °C until analysis.
procedures
Preparation and storage of calibrators.
Stock solutions of each calibrator were prepared individually with appropriate solvents. All-trans-retinol was dissolved in ethanol; retinol acetate [internal standard 1 (IS1)] was initially dissolved with 1 volume of acetone before the addition of 9 volumes of acetonitrile. Stock solutions of the -, -, and -isoforms of tocopherol and tocotrienol and tocopherol acetate (IS2) were all prepared in acetonitrile. Stock solutions of carotenoids were prepared in tetrahydrofuran, and echinenone dissolved in ethanol was used as the IS (IS3 and IS4; measured at 450 and 475 nm, respectively). For calibration a mixture of the calibrators containing -, -, and -tocotrienol; - and -tocopherol (each at 100 mg/L); -tocopherol (500 mg/L); all-trans-retinol (50 mg/L); ß-cryptoxanthin, ß-carotene, lutein, zeaxanthin, and lycopene (each at 25 mg/L); and -carotene and canthaxanthin (each at 12.5 mg/L) was prepared in ethanol–BHT. We distributed 200-µL aliquots of this mixture into crimp-capped amber vials and stored them at -70 °C. Each day, a vial of this calibration mixture was thawed to room temperature before mixing with 800 µL of ethanol–BHT solution. After ultrasonification for 20 min, the calibration mixture was further diluted with ethanol–BHT to the concentrations needed for the various calibrators, as listed in Table 1
. The final concentrations of ISs added to 100 µL of calibrator were 0.25 mg/L for IS1, 25 mg/L for IS2, and 0.2 mg/L for IS3 (IS4).
Sample preparation.
In an amber microcentrifuge tube, a 100-µL aliquot of plasma was deproteinized with 100 µL of ethanol–BHT containing the same amounts of ISs as were added to the calibrators. For the enriched sample, 100 µL of pooled plasma was deproteinized with 100 µL of different concentrations of calibrators as indicated in Table 2
. Approximately 20 mg of disodium sulfate and 100 µL of butanol–ethyl acetate (1:1 by volume) were added to the deproteinized sample and vortex-mixed for 5 s. Samples were then extracted with 500 µL of n-hexane for 5 min in a VortempTM vortex-mixing incubator (UniEquip; shaking speed set at 1350 rpm and temperature at 25 °C). After centrifugation (15 000g for 2 min), 500 µL of supernatant was transferred into an amber microcentrifuge tube. Samples were dried under a stream of nitrogen for 20 min in Supelco VisidryTM and VisprepTM devices before being reconstituted in 60 µL of ethanol–BHT solution. Samples were then placed in the autosampler compartment (temperature set at 20 °C). The injection volume was 20 µL.
For reproducibility and recovery studies, 6 sets of enriched samples were prepared to give the concentrations listed in Table 2
. Analyses were carried out within 24 h for within-day imprecision, and between-day variation was determined by repeating the same assay once a week for 6 consecutive weeks.
HPLC apparatus and configuration.
The HPLC system was set up in the configuration shown in Fig. 1
. The Waters Alliance 2695 Separations Module was connected with a guard cartridge (Jour Guard C18) and two analytical columns (as described below), a Model 996 PDA detector, and a Model 2475 fluorescence detector. A Waters NovaPak C18 column [150 x 3.9 mm (i.d.); 4 µm bead size] was used as column 1 and maintained at 35 °C in a thermostated column compartment, and a Whatman Partisphere-5 C18 replaceable cartridge [110 x 4.7 mm (i.d.); 5-µm bead size] was used as column 2 and chilled to 4 °C in a Gilson thermostated cuvette (Model 832). The chromatographic separation was performed by isocratic elution with a mixture of acetonitrile and methanol (65:35 by volume) at a flow rate of 1 mL/min. A two-position, six-port SynergiTM fluid processor (Model AVO-6082; Phenomenex) was used to perform column-switching with an analytical cycle initiated by the autosampler. The method was initiated with the fluid processor set at position 1 for 6 min; it then switched to position 2 from 6 to 20 min and back to position 1 from 20 to 27 min. Compounds identified by PDA detection were monitored at 292 nm (tocopherols, tocotrienols, and IS2), 326 nm (retinol and IS1), 450 nm (ß-carotene, -carotene, lutein, zeaxanthin, ß-cryptoxanthin, and IS3), and 475 nm (lycopene, canthaxanthin, and IS4). The concentrations of -, -, and -tocotrienol and -tocopherol were also quantified by fluorescence detection using an excitation wavelength of 296 nm and emission wavelength of 330 nm (gain of 10). Data acquisition and peak purity tests were performed with Waters EmpowerTM software.
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Figure 1. Schematic representation of the column-switching system, showing the configuration during sample injection.
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Results
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chromatographic separation and detection sensitivity
We used the Waters PDA detection system and its Empower software to examine the peak resolution and purity for every chromatographic run until the best chromatographic separation was achieved. The chromatograms of a calibration mixture measured at 292, 326, 450, and 475 nm are shown in Fig. 2A
. The analytes of interest were divided into two groups according to their polarity. Polar vitamers such as all-trans-retinol; IS1; -, -, and -tocotrienol; lutein; zeaxanthin; and canthaxanthin eluted from column 1 and were retained in column 2 from 0 to 6 min. From 6 to 20 min, the less- or nonpolar compounds, such as -tocopherol, -tocopherol, -tocopherol, ß-cryptoxanthin, lycopene, -carotene, ß-carotene, IS2, and IS3 (IS4) were separated and detected from column 1. Those polar compounds retained earlier in column 2 were then separated and detected from 20 to 27 min. The retention times of all analytes were highly reproducible with CV <0.5%. The detection limits of PDA are shown in Fig. 2B
. With the present procedure, concentrations as low as 0.1 mg/L for all tocopherols and tocotrienols; 0.02 mg/L for ß-carotene, lutein, zeaxanthin, and canthaxanthin; and 0.01 mg/L of all-trans-retinol, ß-cryptoxanthin, -carotene, and lycopene could be detected in 100 µL of plasma. Chromatograms of plasma samples collected from healthy individuals taking or not taking dietary supplements, as detected by PDA, are shown in Fig. 3
. The peaks of interest were identified by comparison with known pure compounds based on retention times and peak purity analysis. Panels A and B in Fig. 3
represent the plasma antioxidant vitamers in two healthy individuals not taking or taking vitamin supplements, respectively. Significantly higher plasma concentrations of all-trans-retinol, -tocopherol, lycopene, -carotene, and ß-carotene were observed for the individual taking a dietary supplement. Fig. 3C
demonstrates the significant increase in tocotrienols in another volunteer who ingested 100 mg of tocotrienols. The same calibration mixtures and samples shown in Figs. 2A
and 3A
were further analyzed with fluorometric detection, and the chromatograms are shown in Fig. 4
, panels A and C, respectively. As illustrated in Fig. 4B
, the detection limits for fluorometric detection were at least 25-fold (0.004 mg/L for all compounds of interest at signal-to-noise ratios >3) lower than for spectrophotometric detection, as shown in Fig. 2B
. The detection limits for various analytes in actual plasma samples are summarized in Table 3
.
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Figure 2. Typical chromatograms of aqueous calibrators as detected with a PDA.
(A), calibration mixture containing 5 mg/L -tocopherol; 1 mg/L each of -tocopherol, -tocopherol, -tocotrienol, -tocotrienol, and -tocotrienol; 0.5 mg/L all-trans-retinol; 0.25 mg/L each of ß-cryptoxanthin, ß-carotene, lutein, zeaxanthin, and lycopene; 0.125 mg/L each of -carotene and canthaxanthin; 0.25 mg/L IS1; 2.5 mg/L IS2; and 0.2 mg/L IS3. (B) calibration mixture with concentrations 10-fold lower than those in A. mAU, milliabsorbance units.
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Figure 3. Chromatograms of human plasma samples analyzed with use of PDA detection.
(A), individual not taking a vitamin supplement. (B), volunteer who had ingested one serving of a dietary supplement. Note the obviously higher concentrations of all-trans-retinol, -tocopherol, -carotene, ß-carotene, and lycopene. (C), volunteer after ingestion of 100 mg of mixed-tocotrienol supplement. Measured concentrations were 0.15 mg/L -tocotrienol, 1.04 mg/L -tocotrienol, and 1.54 mg/L -tocotrienol. Measurements were at 292 nm. mAU, milliabsorbance units.
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calibration, reproducibility, and recovery
To calibrate the current analytical procedure, we used the IS additions method. Working solutions of the three ISs stored at -20 °C were stable for at least 1 month, with CV <10%; the details for all compounds examined are summarized in Table 1
. The day-to-day variations (n = 6) in slope and linearity were generally <10% and <0.4%, respectively. The within-day precision, between-day variations, and recovery rates were further examined with use of plasma samples enriched with different concentrations of the analytes of interest. The results of the within-day and between-day CV (n = 6) assays are shown in Table 2
, with CV generally <10% and <15%, respectively. The recovery rates based on direct peak height comparisons of ISs and enriched samples were generally similar, 
130%. These >100% recovery rates were likely attributable to the dilution and concentration procedures involved in sample preparation. The recovery rates corrected with the use of ISs are summarized in Table 2
. For canthaxanthin; -, -, and -tocotrienol; and -tocopherol, which are usually present in lower concentrations in human plasma, the recovery rates were 90%, whereas the rest of the compounds showed recoveries >90%.
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Discussion
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analysis of vitamin a and carotenoids
Recently, the measurement of vitamin A and carotenoids has attracted a great deal of attention because of the potential roles these vitamers have in cancer prevention (8)(9)(10)(11)(12)(13). The six most common diet-derived carotenoids are lutein, zeaxanthin, lycopene, ß-cryptoxanthin, -carotene, and ß-carotene. However, critical issues for assays for these antioxidants in human plasma include simultaneous selectivity, sensitivity, and reliability. It is well known that lutein and zeaxanthin are difficult to separate on monomeric C18 columns, and high resolution can be achieved only by use of polymeric C18 or C30 columns with gradient elution (19)(28)(29). Unfortunately, the retention of - and ß-carotene tends to increase with polymeric C18 or C30 with a column temperature >35 °C (17). Furthermore, lycopene is usually retained on the column and eluted after ß-carotene, thus decreasing sensitivity. Therefore, methyl-tert-butyl ether and/or acetone have been used in combination with methanol to reduce the column retention of lycopene (23)(28). Moreover, the authors of several reports have suggested that measurement of retinol, carotenoids, and tocopherol vitamers could be enhanced by use of a gradient system incorporating a third or fourth organic modifier, such as tetrahydrofuran, dichloromethane, or hexane, or by use of two separate chromatographic conditions (11)(12)(13).
To improve the selectivity, ammonium acetate, acetic acid, and triethylamine have been used in the mobile phase (9)(14), but it was recently reported that temperature influences the selectivity to a greater extent than the composition of the mobile phase (30). We therefore carried out a systematic investigation to examine the effects of the reagents used and temperature conditioning. We observed that carotenoids, especially lycopene and ß-carotene, degraded when column temperature exceeded 45 °C. The ideal column temperature for separating these nonpolar compounds was 30–40 °C, and was 4 °C for lutein and zeaxanthin. We applied a column-switching technique and coupled-C18 chromatography with two separate column temperatures to complement the extreme chromatographic behaviors of the polar and nonpolar analytes. In our system, the best results were achieved when we used isocratic elution with acetonitrile–methanol (65:35 by volume), a flow rate of 1 mL/min, and a NovaPak column maintained at 35 °C coupled with a Partisphere column maintained at <4 °C. As demonstrated, all 14 compounds and the 3 ISs could be measured accurately within 27 min without the need of added organic or inorganic modifiers (Figs. 2
and 3
). When we switched from column 1 to column 2 with the second column thermostated at 4 °C, the backpressure increased from 470 to 960 psi. However, this change in psi had no adverse effects on the HPLC system used, as tested over a 3-month period with >1000 samples. The detection limits achieved with PDA were at least twofold lower than those reported for gradient elution with MS detection (21)(23).
tocopherol and tocotrienol analysis
In recent years, there has been growing interest in tocopherols and tocotrienols because of their important biological and nutritional functions (31)(32)(33). Although a great deal of information has been gained from animal studies, reports on tocotrienol measurements in human samples are scarce (34)(35). This could be attributable to the relatively short metabolic half-life in humans (4–5 h) and the lack of a sensitive method for their accurate measurement. Thus the development of a suitable method for simultaneous measurements of all of the lipophilic antioxidants, including the tocotrienols, became a challenge. To overcome autooxidation of lipid-soluble vitamins and carotenoids during sample treatment, BHT (9)(10)(11)(12) or ascorbic acid (8) has been added routinely as an antioxidative preservative. However, we found that BHT was more suitable than ascorbic acid when tocopherols were determined by ultraviolet detection at 292 nm. This was because ascorbic acid caused background interference, whereas BHT was well separated from the tocopherols and eluted as a single peak at 3.5 min (Fig. 3
).
Although the tocotrienols coeluted with several unknown compounds with absorbances at 450 nm (Fig. 3
), such interference did not hamper the specificity of quantification. As shown in Fig. 3C
, the concentrations of the tocotrienols in a volunteer taking a mixed-tocotrienol supplement were significantly higher than those in an individual not taking a supplement (Fig. 3, A and B
). The reliability of the tocotrienol determination was further confirmed by fluorometric detection, which was reproducible and gave a recovery >90% (Table 2
). Fluorometric detection was estimated to be 25- to 100-fold more sensitive (Fig. 4B
) than ultraviolet detection (Fig. 2B
) when pure compounds were used for the determinations. As shown in Fig. 4C
, tocotrienols could be detected by fluorometric detection but not ultraviolet detection (Fig. 3A
), suggesting that fluorometric detection is preferable for measurements of the trace amounts of -, -, and -tocotrienol and -tocopherol. When combined with column switching, our approach is also more sensitive than other methods: its detection limit is four times lower than the latest reported detection limits for fluorometry (15) and coulometry (31).
method validation
The proposed method was evaluated with use of 11 fresh samples collected from healthy volunteers not taking dietary supplements and 43 stored samples collected from 1993 to 1995 for a cohort study. The main findings are shown in Table 3
. The values for all-trans-retinol and -tocopherol in the stored samples were comparable with our previous data (25). The results also show that in addition to no significant differences (P >0.05) for these two compounds, the Spearman correlations were very close (r >0.87, data not shown). These findings suggest that all-trans-retinol and -tocopherol are reasonably stable if stored in the dark at -70 °C. Although the observed values of these two vitamins were higher in the stored samples than in samples obtained fresh from the 11 volunteers (Table 3
), they were well within the ranges reported elsewhere (30)(36)(37). In contrast, the measured amounts of - and ß-carotene in the stored samples were lower than in the fresh samples, suggesting that these two carotenes are less stable than the other plasma carotenoids after prolonged storage periods. Nevertheless, the values of most major lipophilic antioxidants obtained from either stored or fresh samples were in close agreement with values reported in different countries (1)(30)(36)(37).
efficiency of sample preparation
Analytical methods for epidemiologic studies must be fast and simple, but have a high degree of precision with consistent reproducibility. In the literature, several procedures for sample preparation have been recommended; however, most are tedious and time-consuming (12)(28) and are therefore not suitable for use with large numbers of specimens. We found that several items are critical for sample preparation. The first is that, because all analytes are bound to plasma lipoproteins, they must be deproteinized with ethanol–BHT before organic extraction. In addition, it is important to avoid deproteinization with chilled ethanol, as suggested previously (26), because this caused a 50% loss of lycopene and a 20% loss of ß-carotene when samples were stored at 10 °C, although other compounds of interest showed no such effects. The second item is that dehydration by use of disodium sulfate is essential because it ensures the stability of the highly lipophilic analytes. The third is that addition of ethyl acetate–butanol (1:1 by volume) enhances separation efficiency for plasma carotenoids. The fourth item is that residue reconstitution in a smaller volume of ethanol–BHT improves the detectability of trace carotenoids.
To date we have analyzed >1100 samples with the proposed method without encountering any major problems. The method is faster and more reliable than other existing methods: 24 plasma samples can be prepared in <2 h, and a total of 48 samples can be analyzed in 1 day.
In summary, this report shows that most of the lipid-soluble antioxidants can be simultaneously quantified by use of the proposed column-switching technique with monomeric C18 chromatography and isocratic elution. The proposed sample preparation procedure avoids numerous time-consuming steps and is highly reproducible. This HPLC method has been shown to be robust, rapid, sensitive, and reliable.
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Acknowledgments
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We thank Drs. P. Rose and Manav for critical comments on the manuscript and Drs. C. Kopsel and H. Ernst (BASF-AG, Luwigschafen, Germany) for the carotenoid calibrators. This study was carried out with the support of the National University of Singapore (186-000-050-112).
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Footnotes
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1 Nonstandard abbreviations: PDA photodiode array; MS, mass spectrometry; BHT, butylated hydroxytoluene; and IS, internal standard. 
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Abstract
Introduction
