Isotope Dilution Liquid Chromatography - Mass Spectrometry Methods

Anal. Chem. 2011, 83, 92–98

Isotope Dilution Liquid Chromatography - Mass Spectrometry Methods for Fat- and Water-Soluble Vitamins in Nutritional Formulations Karen W. Phinney,* Catherine A. Rimmer, Jeanice Brown Thomas, Lane C. Sander, Katherine E. Sharpless, and Stephen A. Wise Analytical Chemistry Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899, United States Vitamins are essential to human health, and dietary supplements containing vitamins are widely used by individuals hoping to ensure they have adequate intake of these important nutrients. Measurement of vitamins in nutritional formulations is necessary to monitor regulatory compliance and in studies examining the nutrient intake of specific populations. Liquid chromatographic methods, primarily with UV absorbance detection, are well established for both fat- and water-soluble measurements, but they do have limitations for certain analytes and may suffer from a lack of specificity in complex matrices. Liquid chromatography-mass spectrometry (LC-MS) provides both sensitivity and specificity for the determination of vitamins in these matrices, and simultaneous analysis of multiple vitamins in a single analysis is often possible. In this work, LC-MS methods were developed for both fat- and water-soluble vitamins and applied to the measurement of these analytes in two NIST Standard Reference Materials. When possible, stable isotope labeled internal standards were employed for quantification. Vitamins have numerous biochemical functions and play an important role in essential processes in the human body, including protein metabolism, maintenance of blood glucose levels, and regulation of cell growth and differentiation. Vitamins can be divided into two categories: the water-soluble vitamins (WSVs) including the B vitamins and vitamin C, and the fat-soluble vitamins (FSVs), including vitamins A, E, D, and K. Recommended intakes of vitamins are established by the Food and Nutrition Board of the Institute of Medicine and are reviewed on a regular basis. Intake of more than the recommended allowance of WSVs is rarely associated with adverse health effects, and any unneeded amounts are excreted from the body.1 However, excess intake of FSVs can be harmful because these nutrients are stored in the liver and adipose tissues and eliminated more slowly than the WSVs.2 Vitamin deficiencies tend to be rare in developed countries and can result from insufficient intake or from medical conditions * To whom correspondence should be addressed. (1) Rasmussen, S. E.; Andersen, N. L.; Dragsted, L. O.; Larsen, J. C. Eur. J. Nutr. 2006, 45, 123–135. (2) Penniston, K. L.; Tanumihardjo, S. A. Am. J. Clin. Nutr. 2006, 83, 191– 201.

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that prevent effective absorption or use of the vitamin. Individuals affected by cystic fibrosis are at risk of vitamin K deficiency because of malabsorption of the vitamin or because chronic use of antibiotics kills beneficial intestinal flora that produce vitamin K.3 Vitamin B6 deficiencies are more common in individuals affected by alcoholism4 and have been associated with the use of anticonvulsant medications to treat epilepsy.5 Elderly or institutionalized individuals with poor diets are more likely to suffer from vitamin deficiencies, and there is evidence that requirements for certain vitamins increase with age.6 Dietary supplements such as multivitamins and other nutritional products can meet micronutrient needs that are not met through the diet. Despite a lack of clear evidence of benefit, more than half of all American adults also utilize supplements in the hope of improved health and quality of life.7 Measurement of vitamins in foods and nutritional supplements is important for quality assurance purposes, regulatory compliance, and for establishing nutrient intakes of various populations.8-11 Several different types of methodology have been employed to measure the fat- and water-soluble vitamins in such matrices. Microbiological methods are among the most widely used methods for the B vitamins, particularly in food matrices, and there are several AOAC Official Methods of Analysis for B vitamins in food extracts and infant formulas.12 These techniques utilize specific strains of microorganisms that require the vitamin of interest in order to grow, and the methods are relatively simple and inexpensive to implement. Drawbacks to this approach include (3) Rashid, M.; Durie, P.; Andrew, M.; Kalnins, D.; Shin, J.; Corey, M.; Tullis, E.; Pencharz, P. B. Am. J. Clin. Nutr. 1999, 70, 378–382. (4) Glo´ria, L.; Cravo, M.; Camilo, M. E.; Resende, M.; Cardoso, J. N.; Oliveira, A. G.; Leitaõ, C. N.; Mira, F. C. Am. J. Gastroenterol. 1997, 92, 485–489. (5) Schwaninger, M.; Ringleb, P.; Winter, R.; Kohl, B.; Fiehn, W.; Rieser, P. A.; Walter-Sack, I. Epilepsia 1999, 40, 345–350. (6) Russell, R. M.; Suter, P. M. Am. J. Clin. Nutr. 1993, 58, 4–14. (7) Ann. Intern. Med. 2006,145, 364-371. (8) Perales, S.; Alegrıá, A.; Barbera´, R.; Farre´, R. Food Sci. Technol. Int. 2005, 11, 451–462. (9) Dwyer, J. T.; Holden, J.; Andrews, K.; Roseland, J.; Zhao, C.; Schweitzer, A.; Perry, C. R.; Harnley, J.; Wolf, W. R.; Picciano, M. F.; Fisher, K. D.; Saldanha, L. G.; Yetley, E. A.; Betz, J. M.; Coates, P. M.; Milner, J. A.; Whitted, J.; Burt, V.; Radimer, K.; Wilger, J.; Sharpless, K. E.; Hardy, C. J. Anal. Bioanal. Chem. 2007, 389, 37–46. (10) Haytowitz, D. B.; Pehrsson, P. R.; Holden, J. M. J. Food Compos. Anal. 2008, 21, S94-S102. (11) Sichert-Hellert, W.; Wenz, G.; Kersting, M. J. Nutr. 2006, 136, 1329–1333. (12) Official Methods of Analysis, 18th ed.; AOAC International: Gaithersburg, MD, 2006. 10.1021/ac101950r Not subject to U.S. Copyright. Publ. 2011 Am. Chem. Soc. Published on Web 11/30/2010

the necessity of maintaining cultures of the microorganisms and the relatively long analysis time. In addition, certain preservatives, lipids, or other ingredients in the formulation can have either a stimulatory or inhibitory effect on the bacteria being used.13 Liquid chromatographic (LC) methods, primarily utilizing UV absorbance detection, have been developed as alternatives to microbiological methods for WSVs. Most LC methods for WSVs are performed in the reversed-phase mode with aqueous-organic mobile phases.14,15 Ion-pairing reagents have been used in some cases to facilitate the determination of several WSVs in a single chromatographic run.16 Some of the potential advantages of LC methods for the B vitamins include enhanced specificity and the ability to quantify different forms of the vitamin (known as vitamers) or multiple vitamins in a single analysis.17 Fluorescence detection has also been employed in LC methods for the WSVs; the analyte may have intrinsic fluorescence (such as riboflavin) or be converted to a form that fluoresces.17,18 A comprehensive overview of LC methods for WSVs has been given by Blake.19 Although there is a significant body of literature related to the analysis of WSVs in food and nutritional supplements, often these methods have only been suited to analytes with UV absorbance, or only applicable to formulations or matrices with higher concentration of the analytes such as B-complex vitamins.20 Liquid chromatographic methods are commonly employed for fat-soluble vitamins.8,21 Both reversed-phase and normal-phase approaches have been reported in conjunction with UV absorbance or fluorescence detection.13 There are AOAC-approved methods for vitamins A, D, E, and K in food and nutritional products, but many of these were developed in the 1980s and have become outdated as new formulation strategies for these products have been adopted.22 A few methods have also purported to achieve the simultaneous determination of FSVs and WSVs,23,24 but such approaches are generally not widely applicable because fat- and water-soluble vitamins often require different extraction conditions. Liquid chromatographic methods incorporating single quadrupole or triple quadrupole mass spectrometric detection (LC-MS and LC-MS/MS) are gaining popularity for the determination of both fat- and water-soluble vitamins in various matrices.25-28 Chen et al. described an LC-MS method with electrospray (13) Eitenmiller, R. R.; Ye, L.; Landen, W. O. Vitamin Analysis for the Health and Food Sciences;, 2nd ed.; CRC Press: Boca Raton, FL, 2008. (14) Chatzimichalakis, P. F.; Samanidou, V. F.; Verpoorte, R.; Papadoyannis, I. N. J. Sep. Sci. 2004, 27, 1181–1188. (15) Heudi, O.; Kilinç, T.; Fontannaz, P. J. Chromatogr., A 2005, 1070, 49–56. (16) Albala´-Hurtado, S.; Veciana-Nogue´s, M. T.; Izquierdo-Pulido, M.; Marine´Font, A. J. Chromatogr., A 1997, 778, 247–253. (17) Vin ãs, P.; Balsalobre, N.; Lo´pez-Erroz, C.; Hernańdez-Co´rdoba, M. J. Agric. Food Chem. 2004, 52, 1789–1794. (18) Vin ãs, P.; Balsalobre, N.; Lo´pez-Erroz, C.; Hernańdez-Co´rdoba, M. Chromatographia 2004, 59, 381–386. (19) Blake, C. J. Anal. Bioanal. Chem. 2007, 389, 63–76. (20) Markopoulou, C. K.; Kagkadis, K. A.; Koundourellis, J. E. J. Pharm. Biomed. Anal. 2002, 30, 1403–1410. (21) Sundaresan, P. R. J. AOAC Int. 2002, 85, 1127–1135. (22) Blake, C. J. J. AOAC Int. 2007, 90, 897–910. (23) Klejdus, B.; Petrlova´, J.; Potesˇil, D.; Adam, V.; Mikelova´, R.; Vacek, J.; Kizek, R.; Kubań, V. Anal. Chim. Acta 2009, 520, 57–67. (24) Vazquez, R.; Hoang, M.-D. L.; Martin, J.; Yahia, Y. A.; Graffard, H.; Guyon, F.; Do, B. Eur. J. Health Pharm. Sci. 2009, 15, 28–35. (25) Leporati, A.; Catellani, D.; Suman, M.; Andreoli, R.; Manini, P.; Niessen, W. M. A. Anal. Chim. Acta 2005, 531, 87–95. (26) Chen, P.; Wolf, W. R. Anal. Bioanal. Chem. 2007, 387, 2441–2448. (27) Nelson, B. C.; Sharpless, K. E.; Sander, L. C. J. Chromatogr., A 2006, 1135, 203–211.

ionization (ESI) for 10 WSVs in multivitamin tablets and utilized hippuric acid as the internal standard.29 However, the mobile phase in this method incorporated an ion-pairing reagent in order to achieve separation of the analytes of interest. The use of such additives in LC-MS is less than ideal because they can suppress ionization of the analytes of interest or result in other matrix effects. Publications describing mass spectrometric detection for the FSVs in nutritional products have been limited. One reason for this may be that the FSVs are considered poor candidates for ESI, and the number of successful applications of LC-MS and LC-MS/MS to FSVs did not begin to increase until the introduction of atmospheric pressure chemical ionization (APCI).30,31 Most of the reported applications of LC-MS or LC-MS/MS for quantification of fat- and water-soluble vitamins have utilized external standard approaches to calibration or have employed internal standards that are not structurally related to the analytes being determined.26,29,32 Such methodologies may fail to account for analyte losses during sample preparation, something that can be significant for FSVs such as vitamin D and K that are present at relatively low concentrations and may require multiple steps for isolation prior to their analysis.22 Vitamin D2 has been used as an internal standard for quantification of vitamin D3,30,33 but this approach is only valid if the form of vitamin D present is known in advance. While the use of nonlabeled standards can provide some improvement, the use of stable isotope labeled internal standards is anticipated to be superior in accounting fully for sample losses during preparation as well as any potential matrix effects on ionization when mass spectrometry is used.34-36 In this work, LC-MS methods were developed for both fatand water-soluble vitamins and applied to the measurement of these vitamins in two Standard Reference Materials (SRMs) recently developed by NIST, SRM 3280 Multivitamin/Multielement Tablets and SRM 1849 Infant/Adult Nutritional Formula. Stable isotope labeled internal standards were used when possible for quantification of the analytes of interest. The results of this work demonstrate the advantages of the methodology in terms of specificity and sensitivity as well as their applicability to a range of sample matrices. This is the first report of the application of isotope dilution approaches to LC-MS quantification of both fatand water-soluble vitamins in a variety of nutritional formulations. EXPERIMENTAL SECTION Standard Reference Materials. SRM 3280 Multivitamin/ Multielement Tablets was prepared by a manufacturer of multivitamin tablets. The tablets were prepared in a noncommercial production run but in a similar manner to commercial multivitamin (28) Huang, M.; Winters, D.; Crowley, R.; Sullivan, D. J. AOAC Int. 2009, 92, 1728–1738. (29) Chen, Z.; Chen, B.; Yao, S. Anal. Chim. Acta 2006, 569, 169–175. (30) Heudi, O.; Trisconi, M. J.; Blake, C. J. J. Chromatogr., A 2004, 1022, 115– 123. (31) Kalman, A.; Mujahid, C.; Mottier, P.; Heudi, O. Rapid Commun. Mass Spectrom. 2003, 17, 723–727. (32) Gentili, A.; Caretti, F.; D’Ascenzo, G.; Marchese, S.; Perret, D.; Di Corcia, D.; Rocca, L. M. Rapid Commun. Mass Spectrom. 2008, 22, 2029–2043. (33) Byrdwell, W. C. J. Agric. Food Chem. 2009, 57, 2135–2146. (34) Tan, A.; Hussain, S.; Musuku, A.; Masse´, R. J. Chromatogr., B 2009, 877, 3201–3209. (35) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Anal. Chem. 2003, 75, 3019–3030. (36) Huang, M.; LaLuzerne, P.; Winters, D.; Sullivan, D. J. AOAC Int. 2009, 92, 1327–1335.


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tablets. Each tablet weighs approximately 1.5 g. The directcompression tablets were dispensed into plastic bottles, each containing 30 tablets. The tablets contain both fat- and watersoluble vitamins as well as minerals and excipients that are representative of those found in multivitamin formulations.37 SRM 1849 Infant/Adult Nutritional Formula is a milk-based powder formulation. The SRM was prepared by a producer of infant formula and adult nutritional products. It was developed as a hybrid material with constituents that are representative of both types of formulations in order to broaden the potential applicability of the SRM. The powered material was packaged in single-use pouches, and each pouch contains 10 g of the formulation.38 Water-Soluble Vitamins. Materials. Niacinamide, thiamine hydrochloride, riboflavin, calcium pantothenate, and pyridoxine hydrochloride were obtained from the U.S. Pharmacopeia (Rockville, MD). Information provided by the vendor indicated that all compounds had a purity of 99% or better. Purity of the standards for thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, and niacinamide was also assessed by LC-UV. Nicotinamide-[2H4] was obtained from IsoSciences LLC (King of Prussia, PA). Thiamine chloride-[13C3], calcium pantothenate monohydrate[13C3,15N], and pyridoxine hydrochloride-[13C4] were obtained from Cambridge Isotope Laboratories (Andover, MA). All solvents used were HPLC grade. Calibration Solutions. Two stock solutions of the unlabeled vitamin standards were prepared in 30 mL 1% acetic acid (volume fraction) in water. Individual solutions for each of the four labeled internal standards (B1, B3, B5, B6) were also prepared. Four calibration solutions were then prepared by combining the stock solutions with the solutions of labeled internal standards. The calibrants were prepared so that the concentrations of the standard and internal standard were approximately equal for each of the analytes. Sample PreparationsSRM 3280. Six bottles of SRM 3280, each containing 30 tablets, were selected for analysis. All sample preparation was performed under reduced lighting to minimize potential analyte degradation. Fifteen tablets from each bottle were ground for 10 min with a Retsch RM-100 (Newtown, PA) automated mortar grinder, and the ground material was transferred to an amber bottle. Two subsamples were analyzed from each bottle. For analysis, 0.25 g (exact mass known) of SRM 3280 and 1.5 mL of each internal standard solution (exact mass known) were added to a 50 mL polypropylene centrifuge tube. The extraction solvent (1% acetic acid in water, 24 mL) was added to the tube (total liquid volume ≈30 mL), the tube was tightly capped, and the contents were vortexed for 30 s to disperse the solid material in the extraction liquid. The tubes were placed in an ultrasonic bath and sonicated (no heat) for 30 min. The tubes were then centrifuged at 3000 rpm (314 rad/s) for 15 min. A 5 mL portion of the (37) Sander, L. C.; Sharpless, K. E.; Wise, S. A.; Long, S. E.; Mackey, E. A.; Marlow, A. F.; Nelson, B. C.; Phinney, K. W.; Porter, B. J.; Rimmer, C. A.; Sieber, J. R.; Spatz, R. O.; Thomas, J. B.; Turk, G. C.; Wood, L. J.; Yu, L. L.; Zeisler, R.; Yen, J. H.; Duewer, D. L.; Atkinson, R.; Chen, P.; Goldschmidt, R.; Greene, E.; Harnly, J.; Wolf, W. R. ; Ho, I.-P.; Betz, J. M. Anal. Chem. (accepted). (38) Sharpless, K. E.; Lindstrom, R. M.; Nelson, B. C.; Phinney, K. W.; Rimmer, C. A.; Sander, L. C.; Schantz, M. M.; Spatz, R. O.; Thomas, J. B.; Turk, G. C.; Wise, S. A.; Wood, L. J.; Yen, J. H. J. AOAC Int. 2010, 93 12621274.

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yellow-orange supernatant was withdrawn with a disposable 10 mL syringe. The liquid was filtered through a 0.45 µm nylon filter into autosampler vials for analysis. Sample PreparationsSRM 1849. Six packets of candidate SRM 1849 were selected for analysis and allowed to reach room temperature. The packets were shaken briefly by hand to distribute the contents. All sample preparation was performed under reduced lighting to minimize potential analyte degradation. Two subsamples were analyzed from each packet. For analysis, 2.0 g (exact mass known) of SRM 1849 and 0.15 mL of each internal standard solution (exact mass known) were added to a 50 mL polypropylene centrifuge tube. The extraction solvent (1% acetic acid in water, 29 mL) was added to the tube (total liquid volume ≈ 30 mL), the tube was tightly capped, and the contents were vortexed for 30 s to disperse the solid material in the extraction liquid. The tubes were placed in an ultrasonic bath and sonicated (no heat) for 30 min. A small aliquot of acetonitrile (50 µL) was added to each tube, and the tubes were placed in a freezer (-20 °C) overnight to aid coagulation of the lipid layer at the top of the solution. The tubes were then centrifuged at 3000 rpm (314 rad/s) for 15 min. A 5 mL portion of the yellow-orange supernatant was withdrawn with a disposable 10 mL syringe and filtered through a 0.45 µm RC filter into autosampler vials for analysis. LC-MS Analysis. Analyses were performed on an Agilent 1100 LC/MSD with an electrospray ionization source in positive ionization mode. The relevant instrumental parameters include the nebulizer pressure (0.34 MPa, 50 psig), nebulizer gas temperature (350 °C), drying gas flow (13.0 L/min), capillary voltage (4000 V), and fragmentor voltage (110 V). Liquid chromatographic separation of the WSVs was performed using a Cadenza CD-C18 stationary phase (4.6 × 250 mm, 3 µm particles) from Silvertone Sciences (Philadelphia, PA). A gradient elution method was used. Mobile phase A was 20 mM ammonium formate (pH 4.0) and mobile phase B was methanol. The gradient program was as follows: 0-6 min, isocratic at 100% A (volume fraction); 6-20 min, linear gradient from 0% B to 50% B; 20-30 min, isocratic at 50% B; 30-35 min, return to 100% A; 35-45 min, equilibrate at 100% A. The flow rate was 0.8 mL/min, and the column was thermostatted at 22 °C. The injection volume was 2 µL. Fat-Soluble Vitamins. Materials. Retinyl acetate, retinyl palmitate, dl-R-tocopherol acetate, and phylloquinone (vitamin K1) were obtained from Sigma-Aldrich (St. Louis, MO). Ergocalciferol (vitamin D2) was obtained from Chromadex (Irvine, CA) and cholecalciferol (vitamin D3) was from the U.S. Pharmacopeia. Ergocalciferol-[2H3] and cholecalciferol-[2H3] were obtained from IsoSciences LLC. Phytonadione-[2H4], retinyl acetate-[2H6], and retinyl palmitate-[2H4] were from Cambridge Isotope Laboratories. Calibration Solutions. Five independently weighed stock solutions of the four FSVs of interest were prepared in ethanol. Individual solutions of the labeled internal standards were also prepared in ethanol at appropriate concentrations. Calibration solutions were prepared by combining the standard solutions and labeled internal standard solutions to obtain approximately equal amounts of the labeled and unlabeled forms of the analytes. Sample PreparationsSRM 3280. Fifteen tablets from a representative bottle were ground for 10 min in an automated mortar grinder. Approximately 0.6 g of the sample was then accurately

RESULTS AND DISCUSSION

preparation procedures and detection strategies.39 LC methods are well established in our laboratory for vitamin measurements, and the incorporation of mass spectrometric detection represents a next step in optimizing method specificity. Therefore, as part of the certification of SRM 3280 and SRM 1849, NIST developed LC-MS and LC-MS/MS methods for the WSVs and FSVs in these formulations. This report focuses on the LC-MS measurement of five WSVs (B1, B2, B3, B5, and B6) and four FSVs (A, E, D, K) in these SRMs. The manufacturers of the two SRMs provided a listing of the ingredients, including the specific forms of the vitamins added. Full descriptions of the SRM preparation and value assignment process for SRM 1849 and SRM 3280 are given in related publications.37,38 Method Development for Water-Soluble Vitamins. Our existing methodology for analysis of WSVs was based upon reversed-phase separation on a C18 stationary phase and an aqueous:organic mobile phase that incorporated a phosphate buffer. In order to develop a second, independent method for these analytes, we pursued development of a chromatographic separation of the WSVs that utilized an MS-compatible mobile phase. Successful separation of the target analytes was achieved on a C18 stationary phase with a volatile ammonium formate buffer as the aqueous component of the mobile phase. By adjusting the pH of the buffer and incorporating a gradient program, baseline resolution of the WSVs was achieved, with good retention of the analytes of interest. Figure 1 shows a comparison of the existing WSV methodology (Figure 1a) to the newly developed chromatographic separation (Figure 1b). To facilitate comparison, UV detection was used for both approaches. As shown in the figure, both C18 columns provide baseline resolution of the WSVs of interest, but the elution order differs between the two columns. The newly developed separation shown in Figure 1b also incorporates a smaller particle size chromatographic column (3 µm) that reflects recent advancements in LC column technology, while the previous method (Figure 1a) uses a 5 µm column. Although nicotinic acid, an alternate form of vitamin B3, was not listed as an ingredient in either SRM 3280 or SRM 1849, we investigated its chromatographic retention to ensure that it would not interfere with any of the analytes of interest if it were present. We also made certain that other WSV species, such as folic acid and vitamin B12, would not coelute with any of the analytes of interest. Vitamin B5 is not evident in the newly developed separation in Figure 1 because it lacks a useful UV chromophore; nevertheless, it was well separated from the other analytes, as will be described in subsequent sections. Direct infusion coupled with ESI was used to optimize the MS parameters and to identify the appropriate masses for quantification. The specific ions that were used for quantification of the WSVs are listed in Table 1. In general, these ions represent [M+H]+ species. For thiamine, the ion monitored represents the loss of associated chloride, and calcium pantothenate was determined as pantothenic acid. Selected ion monitoring was employed to maximize selectivity and sensitivity for application to the nutritional formulations.

Value assignment of Standard Reference Materials at NIST typically incorporates the use of two independent analytical methods. In ideal situations, these methods utilize different sample

(39) Sharpless, K. E.; Thomas, J. B.; Christopher, S. J.; Greenberg, R. R.; Sander, L. C.; Schantz, M. M.; Welch, M. J.; Wise, S. A. Anal. Bioanal. Chem. 2007, 389, 171–178.

weighed into a 50 mL polyethylene centrifuge tube. A 10 mL aliquot of 1% (mass concentration) EDTA solution was added to the sample and the centrifuge tube was immersed in a 45 °C water bath for 1 h to dissolve the gel matrix which encapsulates some of the fat soluble vitamins. Three internal standard solutions were then added to the sample by mass with approximately 0.9 g retinyl acetate-d6 solution, 0.7 g vitamin K1-d4 solution, and approximately 0.8 g vitamin D2-d3 solution. The samples were placed in an ultrasonic bath for 10 min. The aqueous solution was then extracted with ≈20-30 mL of hexane overnight through agitation on a modified roto-vap setup. The samples were centrifuged at 2500 rpm (209 rad/s) for 20 min, the hexane was removed from the aqueous solution, and the samples were immersed in an ultrasonic bath for 30 min. Another 20 mL aliquot was added to the vial and extracted on the modified roto-vap setup for 1 h, then centrifuged and the hexane removed. This extraction was repeated five times. Sample PreparationsSRM 1849. Before opening the infant/ adult nutritional formula was gently mixed. A 1.5-2.5 g sample of infant formula was accurately weighed into a 50 mL polyethylene centrifuge tube. Separate aliquots of each of the isotopically labeled internal standard solutions in ethanol were gravimetrically delivered to the sample in the centrifuge tube via syringe. A 40-50 mL portion of ethyl acetate was added to the sample and mixed by hand. The sample was placed in an ultrasonic bath for 30 min, then allowed to rotate/mix overnight (approximately 16 h). The sample was centrifuged and the ethyl acetate was removed for later analysis. A second aliquot of ethyl acetate was added to the sample, the tube placed in an ultrasonic bath for 30 min followed by a 30 min period of rotating/mixing, then centrifuged and the ethyl acetate pooled with the previous aliquot. This procedure was repeated three more times for a total of five extractions. The pooled ethyl acetate solution was reduced to approximately 10 mL under nitrogen gas. The final samples were centrifuged and injected without further cleanup. All of the above steps were performed in a room with subdued lighting and when possible the samples were fully shielded from light. LC-MS Analysis. Analyses were performed on an Agilent 1100 LC/MSD with an atmospheric pressure chemical ionization (APCI) source in positive ionization mode. The relevant instrumental parameters include the capillary voltage (3500 V), fragmentor voltage (100 V), corona current (4 µA) nebulizer gas temperature (350 °C), vaporizer temperature (350 °C), drying gas flow (6 L/min), and nebulizer pressure (0.24 MPa, 35 psig). Liquid chromatographic separation of the FSVs was performed on an ACE C18 stationary phase (4.6 × 250 mm, 5 µm particle size) from Advanced Chromatography Technologies. The separation was achieved under isocratic conditions using a mobile phase comprised of 40% methanol (volume fraction) and 60% acetonitrile with 5 mmol/L ammonium acetate. The column was thermostatted at 25 °C and the flow rate was 1.0 mL/min. The injection volume was 5 µL for vitamins A and E and 20 µL for vitamins D and K.


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Figure 1. Comparison of two reversed phase separations of WSVs in SRM 3280 Multivitamin/Multielement Tablets. The separation in (a) was performed on a YMC C18 Pro column with an acetonitrile:phosphate buffer mobile phase. The separation in (b) was performed on a Cadenza CD-C18 stationary phase with a methanol:ammonium formate buffer mobile phase. Table 1. Ions Used for Quantification of Water- And Fat-Soluble Vitamins analyte

unlabeled (m/z)

labeled (m/z)

vitamin B1 vitamin B2 vitamin B3 vitamin B5 vitamin B6 vitamin D2 vitamin D3 Vitamin K1 vitamin A (palmitate) vitamin A (acetate) vitamin E (acetate)

265 377 123 220 170 398 385 452 269 328 473

268 -a 127 224 174 401 388 456 273 334 -b

a Riboflavin was quantified using the labeled vitamin B6 as the internal standard. b Vitamin E (tocopherol) acetate was quantified using the labeled vitamin A (retinyl) acetate.

The sample preparation procedure for the WSVs in SRM 3280 was relatively simple and consisted primarily of an acidified water extraction with sonication and centrifugation. Four time periods (15 min, 30 min, 45 min, and 60 min) were evaluated to determine the optimal extraction time. No significant differences in analyte recovery were observed for sonication periods less than 45 min. Longer sonication was associated with degradation of some of the analytes and therefore 30 min was selected as the extraction time. The adult/infant nutrition formula (SRM 1849) poses different measurement challenges than SRM 3280. The level of most vitamins is approximately 2 orders of magnitude lower in this SRM (mg/kg) when compared to SRM 3280 (mg/g). In addition, SRM 1849 contains substantial amounts of safflower, soy, and coconut oils that give this matrix a high lipid content (≈31% fat). Therefore 96


a modified sample preparation procedure was needed to isolate the WSV analytes from this matrix. A small amount of acetonitrile was added to help coagulate the protein/lipid layer that formed at the top of the extraction liquid, and the extracts were placed in the freezer overnight to help congeal this material. A portion of the liquid from under this layer could then be removed and filtered for analysis. Method Development for FSVs. The FSVs were chromatographically resolved under isocratic conditions on a C18 phase with a methanol-acetonitrile mobile phase. These conditions were selected because they provided good selectivity and were readily compatible with APCI-MS. Ammonium acetate was added to the mobile phase to enhance ionization and also to improve the recovery of carotenoids that were measured in related work. Direct infusion coupled with APCI was used to identify the ions to be used for quantification and to develop a selected ion monitoring program. The specific ions that were used for quantification are shown in Table 1. In general, these ions represent [M+H]+ species. Figure 2 provides an illustration of the chromatographic retention for each analyte of interest as well as the masses selected for quantification of each FSV. Both vitamins D2 and D3 were considered during method development because they were known to be present in either SRM 3280 (vitamin D2) or SRM 1849 (vitamin D3). Conversion of vitamin D to previtamin D can occur during sample processing, and this conversion can occur for both vitamin D2 and D3.22 In our approach, the vitamin D and previtamin D species were resolved chromatographically and integration was performed by summing the two peaks because the two species arise from the same compound and exist in equilibrium.

Table 2. Summary of Results for Water-Soluble Vitamins in SRM 3280 (mg/g) and SRM 1849 (mg/kg)a NIST LC/MS

NIST LC/UV

certified valueb

SRM 3280 thiamine hydrochloride (B1) riboflavin (B2) niacinamide (B3) pantothenic acid (B5) pyridoxine hydrochloride (B6)

1.17 (1.6) 1.47 (8.2) 13.9 (1.5) 8.1 (1.6) 1.86 (2.1)

1.06 (2.9)

1.06 ± 0.12 1.32 ± 0.17 14.10 ± 0.23 7.30 ± 0.96 1.81 ± 0.17

SRM 1849 thiamine chloride (B1) riboflavin (B2) niacinamide (B3) pantothenic acid (B5) pyridoxine hydrochloride (B6)

15.2 (4.6) 16.6 (5.4) 98.8 (2.8) 66.0 (3.0) 14.0 (5.0)

15.2 (1.3) 18.1 (3.9) 98.4 (4.3)

14.3 (1.7) s 1.75 (1.1)

13.3 (3.0)c

15.8 ± 1.3 17.4 ± 1 97.5 ± 2.3 64.8 ± 2.2 14.2 ± 1.5

a Relative standard deviations (% RSD) for the measurements are given in parentheses. b A detailed description of the certification process for these two SRMs is given in refs 37 and 38. c Vitamin B6 was determined using LC with fluorescence detection.

For the FSVs in SRM 3280, it was necessary to address the issue of encapsulation of the FSVs as part of the sample preparation. This mechanism is used to stabilize the vitamins and reduce the likelihood of degradation through oxidation.22 An aqueous EDTA solution was used to release the FSVs from the gelatin encapsulation.40 Examination of the extracts from sequential extractions of SRM 3280 and SRM 1849 also revealed that a single extraction was not sufficient to isolate the majority of the analytes. Therefore the extraction process was repeated, the extracts combined, and the solution concentrated under nitrogen prior to analysis. Quantification of FSVs and WSVs. The use of LC-MS and LC-MS/MS for quantification requires careful consideration of factors that may not affect the accuracy of measurements by LC-UV. Components in the sample matrix may either enhance or suppress ionization of the species of interest.34 These matrix effects can be minimized through selection of appropriate sample preparation procedures and chromatographic resolution of the analytes from potential interferences.35 The use of isotopically labeled internal standards has been suggested as the preferred approach to minimizing the impact of matrix effects on quantification by LC-MS and LC-MS/MS. In our work, an isotope dilution approach was used for quantitation of both water- and fat-soluble vitamins. Calibration solutions containing labeled and unlabeled forms of the analyte were prepared to achieve approximately 1:1 ratios of the two forms. By utilizing calibration standards that bracket the concentration of the analyte, the effect of any nonlinear detection response should be minimized. Labeled internal standards were also added to the sample matrices at the beginning of sample processing to achieve approximately a 1:1 ratio between

the unlabeled and labeled form of the analyte. Because these internal standards are likely to behave in the same manner as the unlabeled analytes during sample preparation and analysis, they are anticipated to compensate for any sample losses and/or matrix effects that may occur.31 Table 2 compares the results of LC-MS measurements for the five B vitamins with those by LC-UV at NIST for the two SRMs. These values have been corrected for the purity of the standards used. The certified values for the WSVs determined in this work are also provided in Table 2. The certified values were obtained by combining NIST measurements with data from collaborating laboratories, and this process is described in detail elsewhere.37,38 As shown in the table, the LC-MS and LC-UV methods from NIST yield comparable results for the selected B vitamins. Riboflavin was not measured by LC-UV in SRM 3280, and therefore no comparison can be made. The results obtained for SRM 3280 by our LC-MS method are also in good agreement with results from related work using LC-MS/MS.41 The measurement of pantothenic acid (vitamin B5) illustrates the advantages of LC-MS methodology for determination of multiple WSVs in a single analysis. Pantothenic acid lacks a useful chromophore and could not be determined in either nutritional formulation by LC-UV because of insufficient sensitivity. Through the use of a labeled internal standard, this vitamin was easily quantified with excellent precision (CV < 2%) in SRM 3280 and in SRM 1849 (CV ) 3%). In the work reported here, this vitamin can be quantified simultaneously with four other vitamins, and confirmation of identity is made through monitoring of specific ions. Figure 3 gives an example of the extracted ion chromatograms obtained during simultaneous determination of five WSVs in SRM 1849. As noted earlier, the concentrations of the WSVs in SRM 1849 are significantly lower than in SRM 3280. We believe this is the first application of an isotope dilution LC-MS approach to simultaneous measurement of these five vitamins in a matrix like SRM 1849. Table 3 summarizes the LC-MS results for the FSVs in the two SRMs and provides a comparison to LC-UV measurements

(40) Thomas, J. B.; Sharpless, K. E.; Yen, J. H.; Rimmer, C. A. J. AOAC Int. (accepted).

(41) Chen, P.; Ozcan, M.; Wolf, W. R. Anal. Bioanal. Chem. 2007, 389, 343– 347.

Figure 2. Extracted ion chromatograms for fat-soluble vitamin standards. Additional experimental details are provided in the text.


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Figure 3. Extracted ion chromatograms from the analysis of WSVs in SRM 1849 Infant/Adult Nutritional Formula. Experimental details are provided in the text. Table 3. Summary of Results for Fat-Soluble Vitamins in SRM 3280 (µg/g, unless Otherwise Noted) and SRM 1849 (mg/kg)a NIST LC/MS SRM 3280 ergocalciferol (D2) phylloquinone (K1) R-tocopherol (E)c,d retinole SRM 1849 cholecalciferol (D3) phylloquinone (K1) retinolf

8.78 (3.3) 24.1 (4.5) 18.7 (12.0) 0.93 (6.6) 0.251 (14.2) 2.08 (9.3) 14.8 (7.4)

NIST LC/UV

certified valueb

21.9 (1.9) 0.8 (1.5)

9.13 ± 0.71 22.8 ± 2.2 21.4 ± 3.5 0.78 ± 0.19

16.3 (2.6)

0.251 ± 0.027 2.20 ± 0.18 16.4 ± 1.3

a

Relative standard deviations (% RSD) are given in parentheses. A detailed description of the certification process for these two SRMs is given in refs 37 and 38. c Results are in expressed in mg/g. d R-Tocopherol was added to SRM 3280 as tocopheryl acetate. The results are expressed in R-tocopherol equivalents. e The value for retinol in SRM 3280 is a reference value, not a certified value. Retinol was added to SRM 3280 as retinyl acetate. The results are expressed in retinol equivalents. f Retinol was added to SRM 1849 as retinyl palmitate. The certified value is expressed as retinol equivalents. b

made at NIST. The certified and reference values for the FSVs in SRM 3280 and SRM 1849 are also provided. As in the case of the WSVs, the certified and reference values were obtained by combining the NIST measurements with data from collaborating laboratories. Vitamin D was present in SRM 3280 as ergocalciferol (vitamin D2) and in SRM 1849 as cholecalciferol (vitamin D3). Vitamin D could not be determined in either SRM by LC-UV because of insufficient sensitivity. Both forms of vitamin D

98


could be quantified in these matrices through the use of the appropriate labeled internal standard, once again demonstrating the advantages that can be realized through LC-MS approaches. In general, better measurement precision was observed for the FSVs in SRM 3280 than in SRM 1849. As noted previously, the analyte concentrations were significantly lower in SRM 1849 than in SRM 3280, and the high lipid content could reasonably be anticipated to pose challenges for quantification of fat-soluble species. Stable isotope labeled internal standards are generally considered to represent the ideal approach to quantification. However, appropriate labeled internal standards may not be available or may be cost-prohibitive. At the time this work was performed, no labeled internal standard was available for riboflavin. Therefore, the labeled pyridoxine was used for quantification of this analyte. This analyte seemed the best choice in terms of structural similarity and analyte concentration to riboflavin. Examination of the results for riboflavin in SRM 3280 provides evidence of the improvement in precision that can be achieved through the use of a labeled internal standard. Coefficients of variation (CV) were on the order of 2% for the WSVs when labeled internal standards were used, whereas the CV for riboflavin was ≈ 8%. A similar situation was observed for the FSVs in SRM 3280. Quantification of retinyl acetate, vitamin D2, and vitamin K1 using labeled internal standards yielded CVs in the range of 3-7%, whereas tocopherol acetate, which was quantified using labeled retinyl acetate, was associated with greater variability (CV ) 12%). A number of factors may contribute to variability of mass spectrometry-based quantification in complex matrices, including inconsistencies in the ionization process. Stable isotope labeled internal standards are theoretically better suited to account for these differences,31,35 and our results support this conclusion. CONCLUSIONS The results presented here illustrate the versatility of LC-MS methods for the determination of both fat- and water-soluble vitamins in nutritional formulations. Although the applicability of these methods was studied for only two matrices, we anticipate that they will be applicable to other sample types as well. In addition, this work has demonstrated the value of stable isotope labeled internal standards when quantifying vitamins in complex matrices. ACKNOWLEDGMENT Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

Received for review July 23, 2010. Accepted November 2, 2010. AC101950R

Isotope Dilution Liquid Chromatography - Mass Spectrometry Methods

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