Anal. Chem. 2003, 75, 2349-2354
Hydrophilic Interaction Liquid Chromatography Coupled to Electrospray Mass Spectrometry of Small Polar Compounds in Food Analysis Hedwig Schlichtherle-Cerny, Michael Affolter,* and Christoph Cerny
Nestle´ Research Center, Nestec Ltd., Vers-chez-les-Blanc, P.O. Box 44, CH-1000 Lausanne 26, Switzerland
Reversed-phase liquid chromatography (RPLC) is commonly used to analyze nonvolatile components in food. However, polar low-molecular-weight compounds such as hydrophilic amino acids, di- and tripeptides, and organic acids are often not sufficiently retained and represent a challenge for RPLC. Hydrophilic interaction liquid chromatography in combination with electrospray mass spectrometry (HILIC-ESI-MS) on a carbamoyl-derivatized stationary phase was successfully employed to separate free amino acids and small polar peptides in complex food matrixes such as wheat gluten hydrolysate and Parmesan cheese. Glutamyl dipeptides were separated in a sequencespecific order with peptides with N-terminal glutamic acid residues eluting prior to their reverse sequence analogues. ESI-MSn detection in the positive ionization mode provided the necessary information to unambiguously identify isobaric peptides due to their characteristic fragmentation patterns. The technique also proved useful to separate and identify glycoconjugates between amino acids and reducing sugars (Amadori compounds). The investigation of organic acids present in food used a mobile phase comprising ammonium acetate buffer at pH 7 and mass spectrometric detection in the negative ionization mode. Liquid chromatography coupled to mass spectrometry was introduced as early as 1973 by Baldwin and McLafferty.1 With new interface techniques such as atmospheric pressure ionization,2 electrospray ionization (ESI),3 and, more recently, matrix-assisted laser desorption ionization,4 LC-MS has become the method of choice for the analysis of nonvolatile compounds. HPLC on reversed phases such as octadecylsilyl (ODS)derivatized stationary phases is commonly used in the analysis of peptides and protein digests,5 therapeutic drugs such as antibiotics,6 food contaminants,7 and food additives.8 Recently, * Corresponding author. Fax: +41-21-785-8549. E-mail: michael.affolter@ rdls.nestle.com. (1) Baldwin, M. A.; McLafferty, F. W. Org. Mass Spectrom. 1973, 7, 11111112. (2) Horning, E. C.; Carroll, D. I.; Dzidic, I.; Haegele, K. D.; Horning, M. G.; Stillwell, R. N. J. Chromatogr. 1974, 99, 13-21. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (4) Murray, K. K.; Lewis, T. M.; Beeson, M. D.; Russell, D. H. Anal. Chem. 1994, 66, 1601-1609. (5) Herraiz, T. Anal. Chim. Acta 1997, 352, 119-139. 10.1021/ac026313p CCC: $25.00 Published on Web 04/16/2003
© 2003 American Chemical Society
RPLC on a graphitic carbon column was proposed for analysis of the mutagen acrylamide in foodstuffs.9,10 However, polar compounds are very often poorly or not separated due to weak retention on the reversed phase. To overcome this limitation, hydrophilic peptides and amino acids can be derivatized with 9-fluorenylmethyl chloroformate (FMOC),11,12 the resulting FMOC derivatives being more hydrophobic and easier to separate on RP stationary phases. However, this involves an additional step in sample preparation and does not work for other classes of hydrophilic compounds. Also, high-performance ion-exchange chromatography13 (IEC) and ion pair reversed-phase chromatography of small peptides14 have been suggested, but interfacing IEC with MS is challenging due to high salt concentrations. Another alternative in the separation of polar compounds is hydrophilic interaction liquid chromatography (HILIC), which was introduced by Alpert15 and later used by Strege in tandem with MS in drug research.16 A hydrophilic stationary phase is eluted with a more hydrophobic mobile phase. Thus, the retention times increase with the hydrophilicity of the solutes. Suitable stationary phases are amide, cyclodextrin, cyano, and amino-based column materials.15,16 Yoshida used a carbamide phase for HILIC to separate longer chain peptides.17 Small bioactive peptides from a tryptic protein digest were analyzed by HILIC on a polyhydroxyethyl aspartamide-derivatized stationary phase.18 Recently, HILICESI-MS/MS was employed to separate and quantify folates in human plasma19 and to detect hydrophilic metabolites in plant samples.20 (6) Bobbitt, D. R.; Ng, K. W. J. Chromatogr. 1992, 624, 153-170. (7) Malisch, R.; Heusinger, G. In High Performance Liquid Chromatography in Food Control and Research; Mattisek, R., Wittkowski, R., Eds.; Behr: Hamburg, 1993; pp 299-346. (8) Veerabhadrarao, M.; Narayan, M. S.; Kapur, O.; Sastry, C. S. J. Assoc. Off. Anal. Chem. 1987, 70, 578-582. (9) Rosen, J.; Hellenaes, K. E. Analyst 2002, 127, 880-882. (10) Tareke, E.; Rydberg, P.; Karlsson, P.; Eriksson, S.; Toernqvist, M. J. Agric. Food Chem. 2002, 50, 4998-5006. (11) Einarsson, S.; Josefsson, B.; Lagerkvist, S. J. Chromatogr. 1983, 282, 609618. (12) Roturier, J. M.; Le Bars, D.; Gripon, J. C. J. Chromatogr. 1995, 696, 209217. (13) Dizdaroglu, M. CRC Handb. HPLC Sep. Amino Acids Pept. Proteins 1984, 2, 23-43. (14) Petritis, K.; Brussaux, S.; Guenu, S.; Elfakir, C.; Dreux, M. J. Chromatogr. 2002, 957, 173-185. (15) Alpert, A. J. J. Chromatogr. 1990, 499, 177-196. (16) Strege, M. A. Anal. Chem. 1998, 70, 2439-2445. (17) Yoshida, T. Anal. Chem. 1997, 69, 3038-3043. (18) Lane, T. F.; Iruela-Arispe, M.; Johnson, R. S.; Sage, E. H. J. Cell Biol. 1994, 125, 929-943.
Analytical Chemistry, Vol. 75, No. 10, May 15, 2003 2349
HILIC-ESI-MS in the analysis of complex food samples has not yet been reported to the best of our knowledge. The objective of the present study was to analyze small, highly hydrophilic compounds such as polar di- and tripeptides and glycoconjugates by HILIC. The method should allow sequence-specific separation and identification of isobaric peptides and should prove useful for the analysis of food samples such as Parmesan cheese and protein hydrolysates used as seasonings. EXPERIMENTAL SECTION Chemicals. Peptides were purchased from Bachem (Bubendorf, Switzerland) and Sigma (St. Louis, MO). Acetic acid, ammonium acetate, ammonium hydroxide solution, ethanol, L-glutamic acid, L-lactic acid, L-leucine, DL-malic acid, L-proline, L-pyroglutamic acid, L-serine, and succinic acid were purchased from Fluka (Buchs, Switzerland); L-glutamine was from Sigma. Formic acid, L-threonine, L-tartaric acid, acetonitrile (ACN), and methanol (both HPLC gradient grade) were obtained from Merck (Darmstadt, Germany). Water used in the experiments was deionized and purified by a Milli-Q system (Millipore Inc., Milford, MA). The reference solution for HILIC-ESI-MS contained amino acids (0.5 mg/mL each) and peptides (0.25 mg/mL each, 0.05 mg/mL for WGY) in aqueous ammonium acetate buffer (2.6 mmol/L, pH 5.5) in 60% ACN. The organic acid test solution contained lactic acid, malic acid, pyroglutamic acid, succinic acid, and tartaric acid at a concentration of 0.7 mg/mL in aqueous ammonium acetate buffer (2.6 mmol/L, pH 7.0) in 60% ACN. Amadori Compounds. Samples containing the Amadori compounds N-(1-deoxyfructos-1-yl)glutamic acid (Fru-Glu), N-(1deoxyfructos-1-yl)glutamine (Fru-Gln), and N-(1-deoxyfructos-1yl)lysine (Fru-Lys), respectively, were prepared from equimolar mixtures of glucose and the respective amino acid. Each mixture was dissolved in water at a concentration of 30% and, after adjusting the pH to 5.5, heated at 95 °C for 30 min. The reacted sample was concentrated, dried under vacuum (1 kPa) at 65 °C, and used without purification. In the same way, the Amadori compound N-(1-deoxyfructos-1-yl)glutamyl glutamate [Fru-(GluGlu)] was prepared from glucose and glutamyl glutamic acid, and the Amadori compounds N-[glucosyl-(1f4)-1-deoxyfructos-1-yl]glutamic acid (Maltu-Glu), N-[glucosyl-(1f4)-1-deoxyfructos-1-yl]glutamine (Maltu-Gln), and N-[glucosyl-(1f4)-1-deoxyfructos-1yl]lysine (Maltu-Lys) from maltose and the respective amino acids. Wheat Gluten Hydrolysate. Partially acid-deamidated wheat gluten was obtained by treating wheat gluten (Roquette, Lestrem, France) with hydrochloric acid (0.1 mol/L) at pH 1.0 and 65 °C for 24 h. After neutralization with sodium hydroxide, the enzymatic hydrolysis was carried out in aqueous suspension at pH 6.0 and 55 °C during 16 h using Flavourzyme L 1000 (Novozymes, Bagsvaerd, Denmark). Fractionation of the acid-deamidated wheat gluten hydrolysate was performed by gel permeation chromatography on Sephadex G10 (Amersham Pharmacia Biotech, Uppsala, Sweden) and subfractionation by RPLC on a ODS packed column with hydrophilic end-capping.21 The RPLC subfraction eluting in the void volume was freeze-dried and dissolved by adding (19) Garbis, S. D.; Melse-Boonstra, A.; West, C. E.; van Breemen, R. B. Anal. Chem. 2001, 73, 5358-5364. (20) Tolstikov, V. V.; Fiehn, O. Anal. Biochem. 2002, 301, 298-307. (21) Schlichtherle-Cerny, H.; Amado, R. J. Agric. Food Chem. 2002, 50, 15151522.
2350
Analytical Chemistry, Vol. 75, No. 10, May 15, 2003
successively aqueous ammonium acetate buffer (6.5 mmol/L, pH 5.5), formic acid, and ACN at a ratio of 45:5:50 (v/v/v) to a final concentration of 150 mg/mL for HILIC-ESI-MS analysis. Extract of Parmesan Cheese. Parmesan cheese from a local market (150 g) was ground and freeze-dried and the fat removed by Soxhlet extraction with pentane (750 mL) during 3.5 h. The ground defatted cheese was extracted with water (500 mL), and the water extract was ultrafiltered at 4 °C (molecular weight cutoff Mr > 3000). The ultrafiltrate was freeze-dried and stored at -20 °C until further use. Gel Permeation Chromatography (GPC). Lyophilized cheese extract (1.44 g corresponding to 10 g of Parmesan cheese) was dissolved in water (10 mL), applied onto the Sephadex G10 column (2.6 × 64 cm), and eluted with a solution of 15% ethanol in water at a flow of 1 mL/min. The effluent was monitored for its UV absorbance at 280 nm and for its electrical conductivity and separated into four fractions, which were freeze-dried and stored at -20 °C until use. RPLC. The mobile phase consisted of aqueous ammonium acetate buffer (10 mmol/L) adjusted to pH 6.0 with acetic acid (solvent A) and aqueous ammonium acetate (10 mmol/L, pH 6.0) in 60% methanol (solvent B). Analysis was performed at 50 °C at a flow rate of 0.8 mL/min using an Agilent 1100 system equipped with a quaternary pump model G1311A including a model G1311A on-line degasser, an autosampler model G1313A, a column oven G1316A, and a model G1314A photodiode array detector (Agilent, Palo Alto, CA) and a Grom-Sil 120 ODS 4HE column (4.0 mm i.d. × 1.0 cm guard column and 4.0 mm i.d. × 25.0 cm analytical column, 3 µm, 120 Å) from Grom (Herrenberg, Germany). The gradient was as follows: 0% B from 0 to 7 min, 0-70% B from 7 to 30 min, 70-100% B from 30 to 35 min, and 100% B from 35 to 43 min.21 HILIC-ESI-MS. HILIC-ESI-MS was performed on a TSKGel Amide 80 column (2.0 mm i.d. × 1.0 cm guard column and 1.5 mm i.d. × 25.0 cm, 5 µm, 80 Å) from Tosoh BioSep (Tokyo, Japan) coupled to a Finnigan LCQ ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an ESI source. The LC system consisted of a quaternary Rheos 2000 pump with a CPC-LC on-line degasser (Flux Instruments, Basel, Switzerland) and an HTS PAL autosampler (CTC Analytics, Zwingen, Switzerland). The capillary temperature was set to 200 °C and the spray voltage to 4.0 kV. Nitrogen was used as sheath and auxiliary gas. The scan range was m/z 50-1200. Data acquisition and processing were carried out using the Xcalibur software version 1.2 (Thermo Finnigan). The mass scale of the instrument was calibrated using a solution containing caffeine, the tetrapeptide MRFA, and Ultramark 1621 (Thermo Finnigan) according to the standard procedure described in the instrument manual. A solution of the dipeptide GE (1 mg/mL, MW 204.18) in aqueous ammonium acetate (2.6 mmol/L, pH 5.5) in 60% ACN was used for tuning of the mass scale in the positive ionization mode and a solution of succinic acid (0.2 mg/mL, MW 118.09) in aqueous ammonium acetate (2.6 mmol/L, pH 7.0) in 60% ACN for tuning in the negative ionization mode. MS/MS and MS3 spectra were obtained at a relative collision energy of 35% in the data-dependent scan mode with the signal intensity threshold set to 2.5 × 105 for positive ESI analysis and to 1.0 × 105 for negative ESI. The flow was 100 µL/min. The effluent of the HPLC column was split, and a flow
Table 1. Retention Times of Free Amino Acids and Small Polar Peptides Chromatographed by RPLC and HILIC retention time (min)
retention time (min)
compda
in RPLC
in HILIC
compda
in RPLC
in HILIC
L WGY P T Q S E ET
5.1 27.9
