Determination of Methionine and Selenomethionine in Yeast by

Anal. Chem. 2004, 76, 5149-5156

Determination of Methionine and Selenomethionine in Yeast by Species-Specific Isotope Dilution GC/MS Lu Yang,* Zolta´n Mester, and Ralph E. Sturgeon

Institute for National Measurement Standard, National Research Council Canada, Ottawa, Ontario, Canada K1A 0R6

A method for the simultaneous determination of methionine (Met) and selenomethionine (SeMet) in yeast using species-specific isotope dilution (ID) gas chromatography/mass spectrometry (GC/MS) is described. Samples were digested by refluxing for 16 h with 4 M methanesulfonic acid. Analytes were derivatized with methyl chloroformate and extracted into chloroform for GC/MS analysis. In addition to use of commercially available 13Cenriched Met and SeMet spikes for species specific ID analysis, a 74Se-enriched SeMet spike was also available for comparison of results. In selective ion monitoring mode, the intensities of ions at m/z 221, 222, 269, 270, and 263 were used to calculate the 221/222, 269/270, and 269/263 ion ratios for quantification of Met and SeMet. Concentrations of 5959 ( 33 and 3404 ( 12 µg g-1 (one standard deviation, n ) 6) with relative standard deviations of 0.55 and 0.36% for Met and SeMet, respectively, were obtained using 13C-enriched spikes. A concentration of 3417 ( 8 µg g-1 (one standard deviation, n ) 6) was obtained using the 74Se-enriched SeMet spike. The concentration of SeMet measured in the yeast is equivalent to 66.43 ( 0.24% of total Se and 30.31 ( 0.11% of total Met is in the form of SeMet. Method detection limits (three times the standard deviation) of 3.4 and 1.0 µg g-1 were estimated for Met and SeMet, respectively, based on a 0.25-g subsample of yeast with 1 mL of extract used for derivatization. A similar concentration of 5930 ( 29 µg g-1 (one standard deviation, n ) 4) for Met and a lower concentration of 2787 ( 49 µg g-1 (one standard deviation, n ) 4) for SeMet were obtained for this yeast sample using species-specific ID analysis based on GC/MS with 13C-enriched Met and SeMet spikes when a 2-h open microwave digestion approach using 8 M methanesulfonic acid was used. Selenium is an essential metabolic trace element with intake derived mainly from foods. Many benefits of Se have been reported; it protects cells against the effects of free radicals and is essential for normal functioning of the immune system and thyroid gland.1-3 It has also been reported to provide protection against various forms of cancers, including lung, colorectal, and * Corresponding author. Fax: 613-993-2451. E-mail: [email protected]. (1) Levander, O. A. J. Nutr. 1997, 127, 948s-950s. (2) Arthur, J. R. Can. J. Physiol. Pharmacol. 1991, 69, 1648-1652. 10.1021/ac049475p CCC: $27.50 Published 2004 Am. Chem. Soc. Published on Web 07/24/2004

prostate cancers.4-7 Since common foods in some regions in the world have very low selenium content, consumption of Se-enriched yeast supplements has recently become more popular. However, high levels of Se may produce adverse health effects, including a condition called selenosis.8 The nutritional bioavailability and the toxicity of Se are dependent on its chemical forms and concentrations. Selenomethionine (SeMet) and related species in selenized yeast are considered less toxic than inorganic selenium, whereas their bioavailability is higher.9-12 In general, SeMet is the dominant Se species in yeast supplements. As a result, the development of analytical methods for the speciation of Se in yeast has increased dramatically over the past few years. The most frequently used procedures for the extraction of SeMet from yeast are based on enzymatic hydrolysis9-12 with protease, proteinase K, or a mixture of proteolytic enzymes. A different approach, using cyanogen bromide to liberate SeMet from yeast, was recently reported by Wolf et al.13 However, incomplete degradation of proteins with cyanogen bromide has also been reported,14 which could bias results for determination of SeMet. More recently, methanesulfonic acid digestion has been used to efficiently extract SeMet from yeast and nuts,15 with much higher efficiency than enzymatic extraction using proteinase K. Gas chromatography (GC) and high-performance liquid chromatography are currently the most commonly used separation techniques for SeMet speciation in combination with detection by flame photometry,16 atomic emission,17 mass spectrometry,13,17-19 (3) Corvilain, B.; Contempre, B.; Longombe, A. O.; Goyens P.; Gervy-Decoster, C.; Lamy, F.; Vanderpas, J. B.; Dumont, J. E. Am. J. Clin. Nutr. 1993, 57, 244S-248S. (4) Russo, M. W.; Murray, S. C.; Wurzelmann, J. I.; Woosley, J. T.; Sandler, R. S. Nutr. Cancer 1997, 28, 125-129. (5) Knekt, P.; Marniemi, J.; Teppo, L.; Heliovaara, M.; Aromaa, A. Am. J. Epidemiol. 1998, 148, 975-982. (6) Fleet, J. C. Nutr. Rev. 1997, 55, 277-279. (7) Combs, G. F.; Clark, L. C.; Turnbull, B. W. Biomed. Environ. Sci. 1997, 10, 227-234. (8) Koller, L. d.; Exon, J. H. Can. J. Vet. Res. 1986, 50, 297-306. (9) Larsen, E. H.; Sloth, J.; Hansen, M.; Moesgaard, S. J. Anal. At. Spectrom. 2003, 18, 310-316. (10) Huerta, V. D.; Reyes, L. H.; Marchante-Gayo´n, J. M.; Sa´nchez, M. L. F.; Sanz-Medel, A. J. Anal. At. Spectrom. 2003, 18, 1243-1247. (11) Larsen, E. H.; J.; Hansen, M.; Fan, T.; Vahl, M. J. Anal. At. Spectrom. 2001, 16, 1403-1408. (12) B’Hymer, C.; Caruso, J. A. J. Anal. At. Spectrom. 2000, 15, 1531-1539. (13) Wolf, W. R.; Zainal, H.; Yager, B. Anal. Bioanal. Chem. 2001, 370, 286290. (14) Kaiser, R.; Metzka, L. Anal. Biochem. 1999, 266, 1-8. (15) Wrobel, K.; Kannamkumarath, S. S.; Wrobel, K.; Caruso, J. A. Anal. Bioanal. Chem. 2003, 375, 133-138.

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and inductively coupled plasma mass spectrometry (ICPMS).9-12,20-24 In particular, ICPMS has been used as a sensitive and selective detector for speciation analysis for the past decade. In addition to its high sensitivity, large dynamic range, and multielement capability, isotope dilution (ID) can also be implemented. Isotope dilution mass spectrometry (ID-MS) has been widely employed for trace element analysis in a variety of sample matrixes as it serves to improve both accuracy and precision.25 Applications of ID-MS to species-specific determinations have been limited due to a lack of commercially available species specific spikes.26 If ID can be performed, a number of advantages accrue, including the following: enhanced precision and accuracy in results as the species-specific spike serves as an ideal internal standard; matrix effects are accounted for since quantitation is based on ratio measurements; nonquantitative analyte recovery during subsequent sample manipulation does not impact on the final results; species alteration during sample workup can be assessed;27 and an alternative and comparative quantitation strategy is provided. Despite the advantages offered by species-specific isotope dilution methodology, very few applications of species-specific ID for the determination of SeMet have been published.9,13 Gas chromatography/mass spectrometry (GC/MS) has been used as a powerful technique for characterizing organic molecules, including SeMet in various sample matrixes, due to its inherent capability for structure identification. Moreover, the cost of GC/ MS instrumentation is much lower than that of GC/ICPMS. Although ICPMS has been successfully used for speciation analysis, determination of low concentrations of Se is problematic due to isobaric interferences from argon dimers on most Se isotopes, including the most abundant isotope at m/z 80 and its high ionization potential (9.75 eV), resulting in a relatively poor detection limit. Despite the advantages offered by GC/MS, isotope dilution calibration has rarely been applied due to the complex molecular fragmentation spectrum generated and the need to calculate relative isotopic abundance of molecular ions. Only recently, Wolf et al.13 reported an ID GC/MS method using a synthesized 74Se-enriched SeMet for its quantitation in yeast based on the SeCN+ fragment ion. Despite the dramatically increased interest in such determinations, the accuracy of the data cannot be directly verified due to (16) Kataoka, H.; Miyanaga, Y.; Makita, M. J. Chromatogr., A 1994, 659, 481485. (17) Haberhauer-Troyer, C.; AÄ lvarez-Llamas, G.; Zitting, E.; Rodrı´guez-Gonza´lez, P.; Rosenberg, E.; Sanz-Medel, A. J. Chromatogr., A 2003, 1045, 1-10. (18) Isciogˇlu, B.; Henden, E. Anal. Chim. Acta 2004, 505, 101-106. (19) Myung, S.; Kim, M.; Min, H.; Yoo, E.; Kim, K. J. Chromatogr., B 1999, 727, 1-8. (20) Chassaigne, H.; Che´ry, C. C.; Bordin, G.; Rodriguez, A. R. J. Chromatogr., A 2002, 976, 409-422. (21) Casiot, C.; Szpunar, J.; Łobin´ski, R.; Potin-Gautier, M. J. Anal. At. Spectrom. 1999, 14, 645-650. (22) Vonderheide, A. P.; Montes-Bayon, M.; Caruso, J. A. Analyst 2002, 127, 49-53. (23) Devos, C.; Sandra, K.; Sandra, P. J. Pharm. Biomed. Anal. 2002, 27, 507514. (24) Pela´ez, M. V.; Bayo´n, M. M.; Garcı´a Alonso, J. I.; Sanz-Medel, A. J. Anal. At. Spectrom. 2000, 15, 1217-1222. (25) De Bie`vre, P. Isotope dilution mass spectrometry. In Trace Element Analysis in Biological Specimens; Herber, R. F. M., Stoeppler, M., Eds.; Elsevier: Amsterdam, 1994. (26) Kingston, H. M. S.; Huo, D.; Lu, Y.; Chalk, S. Spectrochim. Acta, B 1998, 53, 299-309. (27) Hintelmann, H.; Falter, R.; Ilgen, G.; Evans, R. D. Anal. Bioanal. Chem. 1997, 358, 363-370.

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a lack of reference materials certified for SeMet content. The National Research Council Canada has undertaken a project to address the need for a yeast Certified Reference Material (CRM) for validation of measurements of Met, SeMet, and total Se. As certification requires good agreement between at least two independent analytical methods, development of independent methodologies possessing the highest possible accuracy and precision is required. The objective of this study was to evaluate the application of isotope dilution to GC/MS for the simultaneous determination of Met and SeMet using commercially available high-purity 13C-enriched species-specific spikes. A 74Se-enriched SeMet spike synthesized by the Food Composition Laboratory of the U.S. Department of Agriculture (Beltsville, MD) was also used for the determination of SeMet by GC/MS for comparison purposes. To the best of our knowledge, this is the first report of the application of species-specific isotope dilution calibration for the simultaneous determination of Met and SeMet in yeast samples using GC/MS with 13C-enriched spikes. EXPERIMENTAL SECTION Instrumentation. The sector field ICPMS (SF-ICPMS) instrument used for measurement of total Se content was a ThermoFinnigan Element2 (Bremen, Germany) equipped with a Scotttype double-pass glass spray chamber and a PFA self-aspirating nebulizer (Elemental Scientific, Omaha, NE). A plug-in quartz torch with a sapphire injector and a Ag guard electrode were used. A Hewlett-Packard HP 6890 GC (Agilent Technologies Canada Inc., Mississauga, ON, Canada) fitted with a DB-5MS column (IsoMass Scientific Inc., Calgary AB, Canada) was used for the separation of the Met and SeMet in preparations of yeast extract. Detection was achieved with an HP model 5973 mass-selective detector (MS). A CEM (Matthews, NC) MDS-2100 microwave digester equipped with Teflon vessels was used for closed vessel highpressure decomposition of yeast for total Se determination. A Microdigest model 401 (2.45 GHz, maximum power 300 W) open vessel microwave digester (Prolabo, Paris, France) equipped with a TX32 programmer was used to investigate microwaveassisted extraction of SeMet and Met from the yeast. Reagents and Solutions. Nitric acid was purified in-house prior to use by subboiling distillation of reagent grade feedstock in a quartz still. Environmental grade ammonium hydroxide was purchased from Anachemia Science (Montreal, PQ, Canada). OmniSolv methanol (glass-distilled) and cholorform were purchased from EM Science (Gibbstown, NJ). High-purity deionized water (DIW) was obtained from a NanoPure mixed-bed ionexchange system fed with reverse osmosis domestic feedwater (Barnstead/Thermolyne Corp.). Certified grade chloroform was sourced from Fisher Scientific (Ottawa, Canada). Methanesulfonic acid (98% purity) and methyl chlorofomate (99% purity) were obtained from Sigma Aldrich Canada (Oakville, ON, Canada). Enriched isotope 82Se (elemental), purchased from the Oak Ridge National Laboratory (Oak Ridge, TN), was used for the determination of total Se. A stock solution of 82Se ( ∼90.5 µg mL-1) was prepared by dissolution of this material in a few milliliters of HNO3 and diluted with DIW. The concentration of this 82Se spike was verified by reverse spike isotope dilution using naturalabundance Se standards prepared from high-purity elemental Se.

Natural-abundance high-purity Met, SeMet, 13C-enriched Met, and 13C-enriched SeMet compounds were purchased from Sigma Aldrich Canada. Individual stock solutions of 1000-2500 µg mL-1 were gravimetrically prepared in 1% HCl solution and kept refrigerated until used. A 74Se-enriched SeMet (74SeMet) compound was obtained from W. Wolf (Food Composition Laboratory, USDA, Beltsville, MD) and used to prepare a stock solution of ∼450 µg mL-1 in 1% HCl. The concentration of 74SeMet spike was verified by reverse spike isotope dilution using the natural-abundance SeMet standards. Safety Considerations. Methyl chloroformate is a highly toxic and flammable substance. Material Safety Data Sheets must be consulted and essential safety precautions employed for all manipulations. Sample Preparation for Determination of Total Se in Yeast by ICPMS. Four 0.25-g subsamples of yeast were accurately weighted into individual precleaned Teflon digestion vessels. A suitable amount of the enriched inorganic 82Se spike was then added to each vessel. Three process blanks (spiked with 10% of the amount of enriched isotope solution used for the samples) were processed along with the samples. After 5 mL of nitric acid and 0.2 mL of H2O2 were added, the vessels were capped and digested in a CEM MDS-2100 microwave oven. The heating conditions were as follows: 10 min at a pressure of 20 psi and 40% power, 10 min at 40 psi and 50% power, 10 min at 80 psi and 50% power, 20 min at 100 psi and 60% power, and 30 min at 120 psi and 70% power. After cooling, 0.25-mL volumes of the digested solutions were transferred to precleaned polyethylene screwcapped bottles and diluted to 25 mL with 1% HNO3. No dry weight correction was applied to the final results. Calibration of the 90.5 µg mL-1 82Se-enriched spike was achieved by reverse spike isotope dilution. This measurement was performed at the same time as the total Se measurements in the yeast digests. Six replicate samples were prepared by accurately pipetting 0.1003-mL volumes of 90.5 µg mL-1 82Se spike solution into precleaned polyethylene screw-capped bottles. Aliquots of 0.0700 mL of the first 1000 µg mL-1 natural-abundance Se stock solution were added to the first three bottles; this was repeated for the other three bottles using the second 1000 µg mL-1 Se stock solution. The contents of each bottle were then diluted with 15 mL of 1% HNO3 for SF-ICPMS analysis. The digested yeast samples and the six reverse spike ID calibration samples were analyzed by SF-ICPMS on the same day. Mass bias correction was implemented based on the theoretical natural-abundance ratio of an isotope pair divided by the mean value of the isotope pair measured in a nature-abundance Se standard. The Element2 SF-ICPMS was optimized daily following recommendations by the manufacturer. Detector dead time was determined according to the procedure described by Nelms et al.28 using three different concentrations of U. The SF-ICPMS operating conditions used are summarized in Table 1. Open Vessel Microwave Extraction with Methanesulfonic Acid. Various concentrations of methanesulfonic acid and heating time were investigated. In brief, 0.25 g of yeast along with 20 mL of methanesulfonic acid in the concentration range of 4-12 M was heated in a Prolabo microwave digester at full power (300 (28) Nelms, S. M.; Que´tel, C. R.; Prohaska, T.; Vogl, J.; Taylor, P. D. P. J. Anal. At. Spectrom. 2001, 16, 333-338.

Table 1. SF-ICPMS Operating Conditions rf power plasma Ar gas flow rate auxiliary Ar gas flow rate Ar carrier gas flow rate sampler cone (nickel) skimmer cone (nickel) dead time resolution data acquisition

1200 W 15.0 l min-1 1.05 l min-1 1.20 l min-1 1.1 mm 0.8 mm 18 ns 300 and 10000 E-scan, 5 runs 50 passes, 5 (low resolution) and 100% (high resolution) mass window, 0.0050 s sample time.

W) for times ranging from 5 min to 12 h. Following the microwave extraction, 0.250 mL of 2193.4 µg mL-1 13C-enriched SeMet and 1.00 mL of 1093.1 µg mL-1 13C-enriched Met were added to the digested mixture for quantitation of Met and SeMet extracted. The derivatization procedure followed that reported by Haberhauer-Troyer et al.17 with use of a 1-mL volume of extract. After addition of 0.48 mL of ammonium hydroxide and 0.75 mL of methanol/pyridine (3:1 v/v) to a 10-mL glass vial containing 1 mL of extract, 0.250 mL of methyl chloroformate was slowly added. The vial was shaken manually for 1 min with venting. One milliliter of chloroform was then added, and the vial was shaken manually for 1 min. The chloroform layer was then transferred to a 1-mL glass vial for subsequent GC/MS analysis. Once the extraction conditions were optimized, final quantitation of Met and SeMet in the yeast was achieved by isotope dilution. Three sample blanks and six replicate subsamples of yeast were prepared. In brief, 0.25 g of yeast spiked with 0.250 mL of 13C-enriched SeMet and 1.00 mL of 13C-enriched Met along with 20 mL of 8 M methanesulfonic acid were heated in the Prolabo microwave digester for 2 h. A 1-mL volume of extract was used for derivatization, and derivatized analytes were extracted into chloroform for GC/MS analysis, as described above. Methanesulfonic Acid Reflux for Sample Preparation. The yeast extraction procedure used in this study followed that described by Wrobel et al.15 with small modification. Three sample blanks and six subsamples of yeast were prepared at the same time. In brief, 0.25 g of yeast was spiked with 0.250 mL of 2193.4 µg mL-1 13C-enriched SeMet and 1.00 mL of 1093.1 µg mL-1 13Cenriched Met. After addition of 16.75 mL of DIW and 6 mL of methanesulfonic acid (resulting in a concentration of 4 M for methanesulfonic acid and 24 mL volume in total), the contents were refluxed on a hot plate for 16 h. Subsequent derivatization of the analytes was as described above. For comparison, a separate set of samples was prepared using the 74Se-enriched SeMet for quantitation of SeMet. RESULTS AND DISCUSSION Results for Total Se in Yeast. Isotope dilution and reverse isotope dilution were used for accurate quantitation of total Se content in the yeast sample. The following equation was used for this purpose:

C ) Cz

vy vz Ay - ByRn BxzR′n - Axz - Cb mx v′y BxzRn - Axz Ay - ByR′n

(1)

where C is the blank-corrected total Se concentration (µg g-1) in Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

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the yeast; Cz is the concentration of primary assay Se standard (µg mL-1); vy is the volume (mL) of spike used to prepare the blend solution of sample and spike; mx is the mass (g) of yeast sample used; vz is the volume (mL) of primary assay Se standard; v′y is the volume (mL) of spike used to prepare the blend solution of spike and primary assay Se standard solution; Ay is the abundance of the reference isotope in the spike; By is the abundance of the spike isotope in the spike; Axz is the abundance of the reference isotope in the sample or primary assay standard; Bxz is the abundance of the spike isotope in the sample or primary assay standard; Rn is the measured reference/spike isotope ratio (mass bias corrected) in the blend solution of sample and spike; R′n is the measured reference/spike isotope ratio (mass bias corrected) in the blend solution of spike and inorganic Se standard; Cb is the analyte concentration in the blank (µg g-1) normalized to a sample weight of 0.25 g. In addition to its high mass resolution and high sensitivity, the Element2 SF-ICPMS provides unique flat-topped peaks in lowresolution mode to provide for more accurate and precise isotope ratio measurements.29-37 Thus, low resolution (300) was initially tested for isotope pairs of 77Se/82Se and 78Se/82Se. Mass biascorrected ratios of 0.8712 ( 0.0036 and 2.715 ( 0.015 (one standard deviation, n ) 3) obtained in an unspiked digest yeast were not significantly different from the expected naturalabundance ratios of 0.8745 and 2.723 for 77Se/82Se and 78Se/82Se, respectively. This confirmed that no significant spectroscopic interference on any of the three isotopes arises from the sample matrix, permitting accurate results to be obtained using either isotope pair. Total Se concentrations of 2072 ( 21 and 2064 ( 21 µg g-1 (one standard deviation, n ) 4) were obtained in yeast using 77Se/82Se and 78Se/82Se ratios, respectively. A subsequent, comparative analysis of these samples was performed using high resolution (10 000) to separate any spectroscopic interferences arising from Ar dimers or argon chlorides on the selected Se isotopes. Concentrations of 2064 ( 51 and 2052 ( 78 µg g-1 (one standard deviation, n ) 4) were obtained in the yeast using the 77Se/82Se and 78Se/82Se pairs, respectively, in agreement with values generated at low resolution. As expected, enhanced precisions of 1.02 and 1.04% RSD in the measured Se concentrations derived from the 77Se/82Se and 78Se/82Se, respectively, were obtained with low resolution compared to 2.46 and 3.81% RSD obtained using high-resolution mode. Optimization of GC/MS Parameters. The effects of injector temperature, transfer line temperature, He column flow rate, and initial column temperature on the separation and sensitivities of Met and SeMet were investigated uisng injections of 1 µL of a 50 µg mL-1 Met and SeMet standard solution in chloroform. Injector temperatures in the range of 180-280 °C in the splitless mode were examined. Intensities for both Met and SeMet peaks increased rapidly as temperature increased from 180 to 220 °C. Only slight increase in peak heights was observed as temperature increased from 220 to 280 °C. An initial injector temperature of 280 °C was used for all subsequent studies. (29) Latkoczy, C.; Prohaska, T.; Stingeder, G.; Teschler-Nicola, M. J. Anal. At. Spectrom. 1998, 13, 561-566. (30) Vanhaecke, F.; Moens, L.; Dams, R. Anal. Chem. 1996, 68, 567-569. (31) Que´tel, C. R.; Prohaska, T.; Hamester, M.; Kerl, W.; Taylor, P. D. P. J. Anal. At. Spectrom. 2000, 15, 353-358. (32) Rehka¨mper, M.; Mezger, K. J. Anal. At. Spectrom. 2000, 15, 1451-1460. (33) Galy, A.; Pomie`s, C.; Day, J. A.; Polrovsky, O. S.; Schott, J. J. Anal. At. Spectrom. 2003, 18, 115-119. (34) White, W. M.; Albare`de, F.; Te´louk, P. Chem. Geol. 2000, 167, 257-270. (35) Anbar, A. D.; Knab, K. A.; Barling, J. Anal. Chem. 2001, 73, 1425-1431. (36) Hintelmann, H.; Lu, S. Analyst 2003, 128, 635-639.

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Transfer line temperatures in the range of 260-300 °C were investigated with no significant impact on SeMet peak height. The intensity of the Met peak remained constant as temperature increased from 260 to 280 °C but decreased slightly as temperature increased from 280 to 300 °C. A transfer line temperature of 260 °C was thus selected for both analytes. The retention times for Met and SeMet decreased as the He column flow rate increased from 0.8 to 2.4 mL min-1. High and constant responses for both Met and SeMet were obtained using He column flow rates in the range of 1.2-1.6 mL min-1. Sensitivities decreased at both lower and higher flow rates. A 1.5 mL min-1 was chosen for all subsequent studies to ensure the highest sensitivities for both analytes as well as adequate separation with the shortest retention times possible. As expected, retention times of Met and SeMet were significantly influenced by the initial column temperature and 120 °C was selected in an effort to achieve shortest retention time as well as permitting an appropriate delay in the solvent elution peak. Isotope Dilution Calibration Using GC/MS. As shown in Figure 1, good separation and peak profiles for Met and SeMet were obtained under optimized conditions and the entire chromatographic run was accomplished in 6 min. All species arising from various combinations of isotopes of the reference and spike ions must be included in calculations to derive the true abundance of the reference and spike ions needed for the final quantitation. A software program (Isotope Pattern Calculator v 3.0) developed by Yan38 was used to calculate relative abundance of the derivatized Met molecular ion (C8H15O4NS+) and the derivatized SeMet molecular ion (C8H15O4NSe+) for different C or Se isotopes. Both molecular ions were selected for quantitation as a result of better signal-to-noise ratio in these masses as well as the attached 13Cenriched methyl group. The CG/MS operating conditions are presented in Table 2. The calculated relative abundances based on the IUPAC-recommended isotopic abundance of C, H, N, S, and Se and enriched C or Se are presented in Table 3. The measured isotopic patterns are shown in Figure 1b and c. Ions at m/z 221 and 222 were selected as reference and spike ions for ID analysis using a 13C-enriched Met to generate the final concentration of Met in the yeast. Similarly, ion pairs of m/z 269/ 270 and 269/263 were selected for ID analysis using a 13C-enriched SeMet spike and a 74Se-enriched SeMet spike, respectively. All five ions were monitored under selective ion monitoring (SIM) mode. Peak areas were used to calculate reference ion and spike ion ratios, from which the analyte concentrations were calculated. With information on calculated relative abundances, calculation of the Met or SeMet concentrations in the yeast using ID GC/ MS can be undertaken using eq 2,

vy Ay - ByRn AWx Cx ) Cy mx BxRn - Ax AWy

(2)

where Cx is the analyte concentration (µg g-1); Ay is the abundance of reference ion in the spike; By is the abundance of spike ion in the spike; Ax is the abundance of reference ion in the sample; Bx is the abundance of spike ion in the sample; Rn is the measured reference/spike ion ratio (mass bias corrected) in the blend solution of sample and spike; AWx is atomic weight of analyte in the sample and AWy is atomic weight of analyte in the spike. As clearly expressed by this equation, only the reference/spike ion (37) Yang, L.; Mester, Z.; Sturgeon, R. E. J. Anal. At. Spectrom. 2003, 18, 13651370. (38) Yan, J. http://www.geocities.com/junhuayan/pattern.htm, 2001.

Figure 1. (a) Total ion chromatogram (m/z 50-300) of a mixed natural-abundance standard solution obtained by GC/MS in scan mode; 40 ng each of Met and SeMet injected in 1 µL of chloroform; (b) spectrum of the derivatized Met peak; (c) spectrum of the derivatized SeMet peak.

Table 2. GC/MS Operating Conditions column injection system injector temp oven temp program carrier gas; flow rate transfer line temp MS SIM parameters MS quad temp MS source temp

DB-5MS 30 m × 0.25 mm i.d. × 0.25 µm df split/splitless injector- splitless mode 280 °C 120-260 °C at 20 °C/min (hold 2 min) helium; 1.5 mL min-1 260 °C HP model 5973 mass selective detector measured ions: m/z 221, 222, 269, 270, 263. dwell times: 25 ms for each m/z 150 °C 250 °C

ratios in the spiked samples need to be measured to derive the final analyte concentrations. The mass bias correction factor can be calculated from the expected-to-measured ratio using a naturalabundance Met and SeMet standard solution.

Results for Microwave-Assisted Extraction Using Methanesulfonic Acid. As noted earlier, Wrobel et al.15 reported much higher extraction efficiency of SeMet from yeast and nuts using a methanesulfonic acid digestion (reflux for 8 h) compared to enzymatic extraction with proteinase K, but the procedure was quite time-consuming. Microwave-assisted extraction generally offers a fast and efficient means of species extraction,39-47 and for (39) Donard, O. F. X.; Lale`re, B.; Martin, F.; Łobin´ski, R. Anal. Chem. 1995, 67, 4250-4254. (40) Szpunar, J.; Schmitt, V. O.; Łbin´ski, R.; Monod, J. L. J. Anal. At. Spectrom. 1996, 11, 193-200. (41) Pereiro, R.; Schmitt, V. O.; Szpunar, J.; Donard, O. F. X.; Łbin´ski, R. Anal. Chem. 1996, 68, 4135-4140. (42) Łbin´ski, R.; Pereiro, I. R.; Chassaigne, H.; Wasik, A.; Szpunar, J. J. Anal. At. Spectrom. 1998, 13, 859-868. (43) Rodriguez, I.; Mounicou, S.; Łbin´ski, R.; Sidelnikov, V.; Patrushev, Y.; Yamanaka, M. Anal. Chem. 1999, 71, 4534-4543. (44) Szpunar, J.; McSheehy, S.; Polec´, K.; Vacchina, V.; Mounicou, S.; Rodriguez, I.; Łbin´ski, R. Spectrochim. Acta, B 2000, 55, 777-793. (45) Yang, L.; Lam, J. W. H. J. Anal. At. Spectrom. 2001, 16, 724-731.

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Table 3. Isotopic Compositions of Derivatized Met (C8H15O4NS+) and SeMet (C8H15O4NSe+) Ions natural abundance C8H15O4NS+ m/z

relative abundance

220 221 222 223 224 225 226

0.00000 0.85793 0.08706 0.04980 0.00445 0.00071 0.00005

natural abundance C8H15O4NSe+

enriched + 7H15O4NS

13CC

m/z

relative abundance

220 221 222 223 224 225 226 227

0.00000 0.00867 0.85932 0.07833 0.04904 0.00392 0.00067 0.00005

enriched + 7H15O4NSe

enriched C8H15O4N74Se+

13CC

m/z

relative abundance

relative abundance

relative abundance

262 263 264 265 266 267 268 269 270 271 272 273 274 275

0.00000 0.00804 0.00075 0.08478 0.07688 0.22229 0.02099 0.45102 0.04210 0.08434 0.00776 0.00098 0.00007 0.00000

0.00000 0.00007 0.00806 0.00144 0.08561 0.07811 0.22202 0.02272 0.45193 0.03805 0.08414 0.00687 0.00091 0.00006

0.00000 0.70229 0.06564 0.04963 0.02306 0.04723 0.00446 0.07149 0.00667 0.02671 0.00248 0.00032 0.00002 0.00000

this purpose, an open vessel microwave-assisted extraction was investigated. A systematic study of methanesulfonic acid concentration, microwave heating time, and power was undertaken to optimize the extraction of both Met and SeMet species from the yeast sample. The effect of methanesulfonic acid concentration in the range of 4-12 M on recovery of both Met and SeMet from the yeast sample was investigated first. A maximum microwave power (300 W) and 60-min heating time were used. Concentrations of Met and SeMet in the extracts were quantified by isotope dilution following addition of 13C-enriched Met and SeMet spikes. The results, which were normalized to the highest concentration measured, are presented in Figure 2. It is evident that the greatest extraction efficiency for SeMet occurred when using 8 M methanesulfonic acid and extraction efficiency decreased at both higher and lower acid concentrations. High and constant extraction efficiency for Met was observed in the range of 8-10 M methanesulfonic acid. An 8 M solution was chosen for subsequent study as highest extraction efficiencies were obtained for both Met and SeMet from yeast under this condition. In a preliminary study, lower concentrations of Met and SeMet in yeast were obtained when less than maximum microwave power was used with 1-h extraction. Maximum power was thus employed for all subsequent studies, and heating times from 5 min to 12 h were tested using 8 M methanesulfonic acid. Results were treated in a manner similar to those described above and are presented in Figure 3. It is evident that no significant difference in the extraction efficiencies for both anlytes was observed for heating (46) Ruiz Encinar, J.; Gonzalez, P. R.; Garcı´a Alonso, J. I.; Sanz-Medel, A. Anal. Chem. 2002, 74, 270-281. (47) Yang, L.; Mester, Z.; Sturgeon, R. E. Anal. Chem. 2002, 74, 2968-2976.

5154 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

Figure 2. Effect of methanesulfonic acid concentration (20 mL) on the extraction of Met and SeMet from 0.25-g subsample of yeast: 2, SeMet; b, Met.

Figure 3. Effect of microwave heating time on the extraction of Met and SeMet from a 0.25-g subsample of yeast using 8 M methanesulfonic acid (20 mL): 2, SeMet; b, Met.

times between 1.5 and 6 h. Extraction efficiency for SeMet decreased significantly at 12 h of microwave heating while only a slight decrease was evident for Met. A minimum 2-h microwave heating was chosen for the final study to ensure quantitative extraction of both Met and SeMet from yeast. Prior to the final quantitation of Met and SeMet in the yeast, ion ratios at m/z 221/222 and 269/270 were measured in an unspiked yeast digest to verify the absence of interferences on these ions. Measured ratios of 9.856 ( 0.027 and 10.702 ( 0.032 (mean and one standard deviation) at m/z 221/222 and 269/270, respectively, were not significantly different from the expected

Figure 4. (a) Total ion chromatogram (m/z 50-300) of yeast spiked with 13C-enriched Met and SeMet obtained by GC/MS in scan mode; (b) derivatized Met ion isotope pattern; (c) derivatized SeMet ion isotope pattern.

natural abundance ratios of 9.854 (85.793%/8.706%) and 10.713 (45.10%/4.21%), confirming the absence of any significant spectroscopic interference on selected ions arising from the sample matrix. A total ion chromatogram and selected ion isotopic pattern of a spiked (13C-enriched Met and SeMet) yeast extract are presented in Figure 4. The increased abundance at m/z 222 and 270 in Figure 4b and c reflects the contribution from added 13Cenriched spikes. Equation 2 was used for the final quantitation of both analytes and concentrations of 5930 ( 29 and 2787 ( 49 µg g-1 (one standard deviation, n ) 4) for Met and SeMet, respectively, were obtained using ID GC/MS in this yeast sample. Results for Methanesulfonic Acid Reflux Digestion. There is no CRM available with certified values for Met and SeMet that can be used to verify the accuracy of the above developed method using microwave extraction with ID GC/MS. As a consequence, a methanesulfonic acid reflux digestion reported by Wrobel et al.15 was applied to the determination of Met and SeMet in the yeast. For convenience, a 16-h reflux (overnight) with 4 M methanesulfonic acid was used. The absence of degradation of either Met or SeMet during this prolonged digestion was experimentally confirmed. Three replicate samples of 1 mL each of 2000 µg mL-1 Met and SeMet standard solutions in 16 mL of 4 M methanesulfonic acid were refluxed for 16 h with an additional three replicate samples prepared in the same way being used as control samples without reflux. Concentrations of Met and SeMet in these extracts were quantified by ID analysis following addition of appropriate amounts of 13C-enriched spikes. Concentrations of 2119 ( 5 and 2083 ( 17 µg mL-1 (one standard deviation, n ) 3) for Met and SeMet, respectively, were obtained in the refluxed samples, in agreement with values of 2116 ( 12 and 2087 ( 49

Table 4. Measured Met and SeMet Concentrations in Yeast (µg g-1) by ID GC/MS Using 13C-Enriched Spikes for Sample Weight Study sample wta (g)

Met

SeMet

methanesulfonic acid, 4 M (mL)

0.125 0.250 0.500 1.000 1.000

5955 ( 46 5917 ( 60 5856 ( 49 5836 ( 55 5911 ( 29

3403 ( 19 3407 ( 15 3344 ( 5 3306 ( 14 3398 ( 9

16 16 16 16 64

a

All n ) 3.

µg mL-1 (one standard deviation, n ) 3) obtained in control samples. These results confirmed that there is no degradation of either analyte incurred during the digestion process. A series of experiments was also conducted to study the effect of subsample mass processed with a fixed volume of 4 M methanesulfonic acid on the recovery of species. Sample weights of 0.125, 0.25, 0.50, and 1.0 g of yeast were used. Concentrations of Met and SeMet in these samples measured by ID GC/MS are summarized in Table 4. It is interesting to note that no significant difference in the measured concentrations is evident for either species when the subsample weight used is less than 0.25 g when using 16 mL of 4 M methanesulfonic acid. Both concentrations decreased slightly with sample masses, suggesting that both Met and SeMet, as polar compounds, can partition between the solid phase (yeast matrix) and the liquid phase (4 M methanesulfonic acid). Thus, for a fixed volume of liquid phase, as the mass of Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

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Table 5. Results for Met and SeMet in Yeast Met (µg g-1)

method used

SeMet (µg g-1)

SeMet as % of total Se

reflux with 13C spikes (n ) 6) 5955 ( 33 3404 ( 12 reflux with 74C SeMet spike (n ) 6) nd 3417 ( 8 microwave with 13C spikes (n ) 4) 5930 ( 29 2787 ( 49

66.4 ( 0.2 66.7 ( 0.2 54.4 ( 1.0

yeast (active site) increases, the percentage of Met and SeMet partitioned into the liquid phase decreases. This was subsequently confirmed by the fact that higher concentrations for both Met and SeMet were obtained when a 1-g sample was processed with an excess amount of 4 M methanesulfonic acid (64 mL). Therefore, for the final quantitation of Met and SeMet in the yeast, 24 mL of 4 M methanesulfonic acid and 16-h reflux time were used to process a 0.25-g subsample. Six replicate samples of yeast were prepared for the final quantitation of Met and SeMet concentrations using ID GC/MS based on accurately prepared 13C-enriched spike solutions. Results are summarized in Table 5; concentrations of 5959 ( 33 and 3404 ( 12 µg g-1 (one standard deviation, n ) 6) with relative standard deviations of 0.55 and 0.36% for Met and SetMet, respectively, were obtained. For comparison purposes, a 74Se-enriched SeMet spike was also used for quantitation of SeMet in this yeast. Since purity data on the 74Se-enriched SeMet spike were not available, a reverse ID analysis using two independently prepared natural-abundance SeMet standards was applied to accurately quantify the concentration of the spike, thereby ensuring accuracy of the final results. The m/z 269/263 ion pair and eq 1 were used for calculation of the SeMet concentration in the yeast. A concentration of 3417 ( 8 µg g-1 (one standard deviation, n ) 6) was obtained, in agreement with that generated using the 13C-enriched spike. It is of interest to calculate relative percentages of Met and SeMet present in this yeast. Based on results obtained using acid reflux and ID using 13C-enriched spikes presented in Table 5, this yeast contains 39.91 ( 0.22 and 17.36 ( 0.06 µM g-1 (one standard deviation, n ) 6) of Met and SeMet, respectively. The relative percentages of Met and SeMet were calculated using eqs 3 and 4.

Met % )

CMet(µM‚g-1) CMet(µM‚g-1) + CSeMet(µM‚g-1)

SeMet % )

%

CSeMet(µM‚g-1) CMet(µM‚g-1) + CSeMet(µM‚g-1)

%

(3)

(4)

Values of 69.69 ( 0.39 and 30.31 ( 0.11% were obtained for Met and SeMet, respectively. The detection limit for the ID GC/MS technique was evaluated using three spiked blank samples. Values of 3.4 and 1.0 µg g-1 were estimated for Met and SeMet, respectively, based on three

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Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

times the standard deviation of measured concentrations normalized to a 0.25-g subsample with derivatization of a 1-mL volume of methanesulfonic acid extract. It is noteworthy that these detection limits could be improved 24-fold if all 24 mL of extract were used. It is evident from Table 5 that a more efficient release of SeMet occurs from this yeast using acid reflux digestion (16 h using 4 M methanesulfonic acid) as compared to microwave digestion (2 h using 8 M methanesulfonic acid), whereas similar extraction efficiencies were obtained for Met with both digestion methods. This suggests that extended heating with a mild acid concentration may provide the best means of efficiently releasing SeMet from protein without degrading the species. No further test was conducted on the 4 M methanesulfonic acid using much longer microwave extraction times, despite the low extraction efficiency of SeMet obtained even with 2-h microwave heating using 20 mL of 8 M methanesulfonic acid. Clearly, a 4 M methanesulfonic acid reflux digestion in combination with ID GC/MS is the preferred choice for accurate measurement of Met and SeMet in this yeast. Additional studies on the use of other digestion procedures, such as enzymatic hydrolysis9-12 and cyanogen bromide digestion,13 will be conducted in the future for quantitation of Met and SeMet in this yeast. CONCLUSIONS A rapid and precise method has been developed for the simultaneous determination of Met and SetMet in yeast using ID GC/MS. This may provide an important tool for metabolic studies to follow the incorporation rate of Se into the proteome. The concentration of SeMet measured in the yeast using a 13C-enriched SeMet spike is in good agreement with that obtained using a 74Seenriched SeMet spike. Overall, the use of 4 M methanesulfonic acid reflux digestion in combination with ID GC/MS provides superior results for determination of Met and SeMet in this yeast compared to a microwave extraction procedure. Good precision of less than 0.6% RSD for both analytes was obtained using the present method, clearly demonstrating the superior capability of ID. SeMet measured is equivalent to 66.43 ( 0.24% of the total selenium in this yeast sample whereas 30.31 ( 0.11% of total Met is in the form of SeMet. The proposed method has sufficiently low detection limits (3.4 and 1.0 µg g-1 for Met and SeMet, respectively) to make it well suited to the certification of Met and SeMet in any proposed CRM yeast matrial arising from this study. ACKNOWLEDGMENT The authors thank Dr. Wayne Wolf of the Food Composition Laboratory (BHNRC, ARS, USDA, Beltsville, MD) for providing 74Se-enriched SeMet. The authors are grateful to Institute Rosells Lallemand for supporting this research and providing the yeast sample used in this study. Received for review April 6, 2004. Accepted June 17, 2004. AC049475P