Ibolya Molnftr-Perl(Editor)

Quantitation of Amino Acids and Amines by Chromatography

Journal of ChromatographyLibrary,Vol. 70 9 2005 ElsevierB.V. All rights reserved

1.2.5. HPLC of Amino Acids as Dansyl and Dabsyl Derivatives Toyohide Takeuchi
Contents

Summary 1. Introduction 1.1. Dansyl derivatives 1.2. Dabsyl derivatives 2. Derivatization conditions 2.1. Dansyl derivatization 2.2. Dabsyl derivatization 3. Chromatographic conditions for dansyl and dabsyl amino acids 3.1. Dansyl amino acids 3.1.1. Detection 3.1.2. Separation 3.2. Dabsyl amino acids 3.2.1. Detection 3.2.2. Separation 4. Applications 4.1. Dansyl amino acids Amino acids / enantiomeric separation / on-column fluorimetric detection 4.2. Dabsyl amino acids

Summary
Pre-column derivatization of amino acids with 5-dimethylaminonaphthalene-l-sulfonyl chloride (dansyl chloride) or 4-dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride) is described. These pre-column derivatization methods enable a sensitive HPLC analysis of amino acids. Dansyl derivatization has originally been applied to the sequential analysis of peptides and proteins. It finds another use in biochemistry for the fluorogenic labeling of proteins and enzymes. Dansyl chloride is the most widely used for the derivatization of amino acids. Dansyl chloride readily reacts with primary and secondary amino groups of amino acids. The re-

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Toyohide Takeuchi

action medium is usually an aqueous-organic mixture (e.g., 1:1 acetone-water) adjusted to a pH of 9.5-10. Dansylation reaction is usually carried out at elevated temperatures. Various dansylation reaction conditions are reported, involving 60 ~ for 60 min or at 38 ~ for 90120 min. Dansyl amino acids absorb light in the UV region. For example, absorption maxima are observed at 214, 246 and 325 nm for dansyl glycine, and the absorption at 214 nm is the strongest. Dansyl group is also fluorescent and dansyl amino acids can therefore be detected by a fluorimetric detector. The excitation and emission wavelengths for dansyl glycine are 324 and 559 nm, respectively. Dansyl amino acids are mostly separated in the reversed-phase mode on a C8 or C 18 column with a linear gradient. Dabsyl chloride has a number of advantages over other derivatization methods, including a simple derivatization procedure, very good stability, good reproducibility and a good limit of detection for the method, complete HPLC separation of all the amino acids, and specific detection at a wavelength in the visible region. Dabsyl chloride also reacts with primary and secondary amino groups of amino acids as does dansyl chloride. Dabsylation reagent solution can be prepared by dissolving dabsyl chloride in acetone or in acetonitrile, followed by mixing with buffer (e.g., carbonate, pH 8.5-9.5). Derivatization of amino acid with dabsyl chloride is also carded out at elevated temperatures as for dansylation. For example, samples were incubated at 70 ~ for 15-30 min. There have been a number of literatures dealing with application of dansylation to the determination of amino acids contained in various sample sources, involving biological fluids, tissues, foods, peptide or protein hydrolyzates, etc. Dabsylation also covers nearly the same application areas as for dansylation. Enantiomeric separation of dansyl and dabsyl amino acids can be carded out by ligand-exchange chromatography. The use of native, and derivatized 13- and ~,-cyclodextrin stationary phases as well as the use of 13-cyclodextrin mobile phase additive are other options for the chiral separation of these derivatized amino acids.

1. Introduction

1.1. Dansyl derivatives

Pre-colunm derivatization with 5-dimethylaminonaphthalene-l-sulfonyl chloride (dansyl chloride) is known to enable a sensitive HPLC analysis of amino acids, as do derivatization with o-phthalaldehyde, phenylisothiocyanate, fluorescamine or 2,4-dinitrofluorobenzene. Dansyl derivatization has originally been developed by Gray and co-workers [1-3] and applied to the sequential analysis of peptides and proteins. It finds another use in biochemistry for the fluorogenic labeling of proteins and enzymes.

HPLC of Amino Acids as Dansyl and Dabsyl Derivatives

231

Dansyl chloride is the most widely used for the derivatization of amino acids. Dansyl chloride readily reacts with primary and secondary amino groups of amino acids. It can also react with phenols, thiols and imidazoles less rapidly, and very slowly with alcohols. Therefore, some amino acids can form a few different dansylated derivatives depending on the derivatization conditions. For example, tyrosine forms N-dansylated, O-dansylated and didansylated derivatives [4]. Dansyl amino acids have usually been separated in the reversed-phase mode, followed by fluorimetric or UV spectrophotometric detection. They have mostly been separated by gradient elution, but isocratic separations have also been reported.

1.2. Dabsyl derivatives

After introduction of 4-dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride) as a precolumn derivatization agent for amino acids [5], numerous applications have been published, and most of them related to the analysis of amino acids in biological fluids or protein hydrolyzates by HPLC [6, 7]. Dabsyl chloride has a number of advantages over other derivatization methods, including a simple derivatization procedure, very good stability, good reproducibility and a good limit of detection for the method, complete HPLC separation of all the amino acids, and specific detection at a wavelength in the visible region [5-8]. Dabsyl chloride also reacts with primary and secondary amino groups of amino acids as does dansyl chloride, and it forms mono-dabsyl derivatives as well as bis-dabsyl derivatives with amino acids such as lysine, tyrosine and histidine [9].

2. Derivatization conditions

2.1. Dansyl derivatization

The reaction medium is usually an aqueous-organic mixture (e.g., 1:1 acetone-water) adjusted to a pH of 9.5-10. Figure 1 shows the reaction of a primary or secondary amino acid (HNR1R2) with dansyl chloride. Dansylation reaction is usually carried out at elevated temperatures. Various dansylation reaction conditions are reported, involving 60 ~ for 60 rain [10] or at 38 ~ for 90-120 rain [11]. The dansylation reaction provides high yield derivatization while the yield appears to be independent of the ratio of dansyl chloride to amino acid over 1000-fold range [12]. In addition, dansyl chloride stock solution is kept in a freezer to avoid degradation.

232 H 3 C ~ N ~ CH3

Toyohide Takeuchi H 3 C ~ N I CH3

-tSO2

RI~NIR2 I H
SO2

I C!
Figure 1 Dansyl derivatization of amino acids

I R,,,,N ~R2

2.2. Dabsyl derivatization Dabsylation reagent solution can be prepared by dissolving dabsyl chloride in acetone or in acetonitrile, followed by mixing with buffer (e.g., carbonate, pH 8.5-9.5). Derivatization of amino acid with dabsyl chloride is also carried out at elevated temperatures as for dansylation. For example, samples were incubated at 70 ~ for 15-30 min [ 13, 14]. It should be noted that dabsyl chloride stock solution is also kept in a freezer.

HaC~ N / CH3

H3C~ N I CH3

Rn~ N/ R2
N~ N -!I "~ N~N

I Ci

SO2

I RI,,,,N~R2

SO2

Figure 2 Dabsyl derivatization of amino acids

HPLC of Amino Acids as Dansyl and Dabsyl Derivatives

233

3. Chromatographic conditions for dansyl and dabsyi amino acids

3.1. Dansyl amino acids 3.1.1. Detection

Dansyl amino acids absorb light in the UV region. Figure 3 shows UV absorption spectra of 0.01 and 0.1 mM dansyl glycine aqueous solution. It can be seen that absorption maxima are observed at 214, 246 and 325 nm, and the absorption at 214 nm is the strongest. The molar extinction coefficients of dansyl glycine at 214, 246 and 325 nm are 3.5x104, 1.3x104 and 0.39x 104, respectively (see Table 1). The dansyl group is fluorescent and dansyl amino acids can therefore be detected by a fluorimetric detector [15]. The excitation and emission wavelengths for dansyl glycine are 324 and 559 nm, respectively [16]. The excitation wavelength is close to the wavelength of a He/Cd laser, which leads to highly sensitive detection of dansyl amino acid [17]. Fluorimetric detection with a packed flow cell was applied to dansyl amino acids in LC using conventional size columns [18, 19] and microcolumns [20]. Both peak area and peak height of analytes increased with increasing retention owing to focusing and environmental effects of the stationary phase [18-20]. Chromatograms obtained in the presence of the stationary phase are demonstrated in Figure 4 [18].

0.5 0.4l
O

Dansyl glycine

= 0.3
<

0.2
0.1

300 Wavelength / nm

400

Figure3 UV spectra of dansyl glycine

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Toyohide Takeuchi
Table 1. Molar extinction coefficients of dansyl and dabsyl glycine.
Amino acid Wavelength 6

nm
325

M-1 cm-~
0.39x104 1.3x104 3.5x104 1.9x104 0.65x104

Dan-Gly

246 214

Dab-Gly

473

277

When dansyl amino acids eluted from the column are combined at a mixing tee with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)32+] and Ru(bpy)33+ is electrochemically oxidized to Ru(bpy)33+ at the electrode, dansyl amino acids and Ru(bpy)aa+react to emit light. This chemiluminescence detection achieves the detection limit of 2 pmol injected. [21 ]. Dansyl amino acids were separated by reversed-phase gradient HPLC and detected by optical activity and UV absorbance. This method allowed the determination of enantiomeric ratios of dansyl amino acids without physical separation of the isomers [22].

i
I

2o

'

~o

'

~o

TIME (mln)

Figure 4 Fluorimetric detection of dansyl amino acids in the presence of the stationary phase. Column, L-column ODS, 150x4.6 mm I.D. Mobile phase, aqueous acetonitrile (22%, v/v) in 40 mM ammonium acetate (pH 7.20). Flow rate, 1.0 mL/min. Detection, 335 nm for excitation and 522 nm for emission. Samples, 21 laL solution containing 0.21 pmol each dansyl amino acids. (Reproduced from Ref. 18 with the permission from the publisher.)

HPLC of Amino Acids as Dansyl and Dabsyl Derivatives 3.1.2. Separation

235

Amino acids from protein hydrolyzates can be separated in the reversed-phase mode on a C8 or C18 column with a linear gradient. The mobile phase is composed of a mixture of buffer and an organic modifier such as methanol or acetonitrile. The buffer can be selected from Tris-HC1 (pH 7.55) [23], ammonium acetate (pH 7.20) [18], sodium acetate (pH 7.5) [24], phosphate (pH 7.4) [25], etc. Enantiomeric separation of dansyl amino acids can be carried out by ligand-exchange chromatography with (S)- and (R)-phenylalaninamide-modified silica gel as the stationary phase and copper (II) as complexing metal ion [26]. Human serum albumin [27] as well as bovine serum albumin stationary phases [28] allowed the resolution of dansyl amino acids. Enantiomeric separation of dansyl amino acids were also performed on native, and derivatized 13- and ~-cyclodextrin stationary phases [29, 30] as well as on a chiral 13-CD-based polymer [31]. Norvancomycin [32], vancomycin [33] and teicoplanin [34] are another stationary phases for the enantiomeric separation of dansyl amino acids. Enantiomeric separation of dansyl amino acids by reversed-phase micro HPLC with a [3-cyclodextrin mobile phase additive was reported, in which twelve pairs of dansyl amino acids were separated in a single chromatographic run [35].

3.2. Dabsyl amino acids 3.2.1. Detection

Dabsyl amino acids absorb light in the UV region as well as in the visible region. This feature leads to an improvement of the selectivity when dabsyl amino acids are detected at longer wavelengths. Figure 5 shows a UV/visible absorption spectrum of dabsyl glycine. It can be seen that the absorption maxima are observed at 277 and 473 nm, and the latter absorption is much larger than the former. The molar extinction coefficients of dabsyl glycine at 277 and 473 nm are 0.65x 10 4 and 1.9x10 4, respectively (see Table 1). Dabsyl amino acids are normally determined by using visible absorption detector. A linear relation between peak area and concentration was observed from 1.25 to 1,250 pmol and the detection limit was between 0.12 and 0.52 pmol [13].

236

Toyohide Takeuchi

Dabsyl glycine 0.8 0.6

O

-~ < 0.4 0.2

20~

"

'

300

400 500 Wavelength / nm

600

Figure 5. UV-visible spectra of dabsyl glycine.

Detection limits of 0.75 fmol of dabsyl amino acids were achieved by using a 0.25 mm I.D. column and a laser-induced crossed-beam thermal lens detector [36]. Absorbance detection of dabsyl amino acids by laser wave mixing was demonstrated in microbore LC, where an argon ion laser was operated at 488 nm [37]. Tandem mass (MS/MS) detector was used for the determination of dabsyl amino acids, in which common amino acid derivatives had good linear relationship between the peak area and the concentration between 10-250 ~tM [38].

3.2. 2. Separation
Dabsyl amino acids can also be separated in the reversed-phase mode with a gradient consisting of buffer and an organic modifier. Acetonitrile, methanol and ethanol can be employed as the organic modifier, whereas sodium phosphate (pH 6.55) [13] and sodium acetate (pH 4.0) was used as the buffer [39]. It is reported that triethylamine was a very effective additive to optimize separation efficiency [ 13]. Enantiomeric separation of dabsyl amino acids can be carried out by ligand-exchange chromatography with (S)- and (R)-phenylalaninamide-modified silica gel as the stationary phase and copper (II) as complexing metal ion [26]. Addition of L-benzoylproline-copper (II) chelate in the mobile phase allowed enantiomeric separation of dabsyl amino acids using a reversed-phase column (C18) [40]. Native and derivatized 13-cyclodextrin-bonded stationary

HPLC of Amino Acids as Dansyl and Dabsyl Derivatives

237

phased could also be used for the chiral separation of dabsyl amino acids. Dabsyl amino acid enantiomers were resolved best on a (R)- and (S)-naphthyl-ethylcarbamate-13-cyclodextrinbonded phase [41].

4. Application
4.1. Dansyl amino acids

There have been a number of literatures dealing with application of dansylation to the determination of amino acids contained in various sample sources, involving plasma [15, 42], monkey saliva [24], rat cortical tissue [43], bacteria broths [12], forest tree tissues and cell cultures [10], soy sauce [11 ], peptide or protein hydrolyzates [25, 44-52], and NH2-terminal analysis of lysozyme [9].

4.2. Dabsyl amino acids

Amino acids contained in various sources have also been derivatized with dabsyl chloride for the determination. Amino acids from protein hydrolyzates [13], plasma [13], urine [39, 53], various tissues [53], food [13, 54] are typical applications. Figure 6 demonstrates reversed-phase HPLC separation of dabsyl derivatives from human plasma using gradient elution [13], where amino acids and amines are separated in 70 min. The repeatability was reported to be between 0.2 and 3.3%. Selective determination of secondary amino acids such as proline and hydroxylproline are reported [55, 56]. The method involves an HPLC method with two derivatizations, the first with o-phthalaldehyde in order to eliminate interferences due to some primary amino acids eluting with retention times similar to those of L-proline and trans-4-hydroxy-L-proline and the second with dabsyl chloride, was developed and evaluated. The method was applied to the analysis of rat tail collagen, and the contents of hydroxyproline and proline were 1.55 + 0.04 and 2.03 +0.04 nmol/gg, respectively [56].

238

Toyohide Takeuchi

E
C

o z

In

(r. 0 ,<

(fJ m

H 0 v 0 T

A ol
E CS T

ri
I
. . . . .

L
.

t-t'/
0

,~ '~

,
20

30

40

50

I~rl'EOrt'X~N "riME [magi

Figure 6 Reversed-phase HPLC separation of dabsyl derivatives from human plasma. Column, Spherisorb ODS-2, 150x4.6 mm I.D. Mobile phase A, 9 mM sodium dihydrogenphosphate, 4% dimethylformamide and 0.16% triethylamine (pH adjusted to 6.55 with phosphoric acid). Mobile phase B, 80% (v/v) aqueous acetonitrile. Gradient, 8% B (0min) to 100% (66 min), almost linear, see Reference 13 for details. Flow rate, 1 mL/min. Wavelength of detection, 436 nm. (Reproduced from Reference 13 with the permission from the publisher.)

Experimental
5.1. Derivatization of amino acids with dansyl chloride
Standard free amino acids were dissolved in a carbonated buffer (pH 9.7) and mixed with acetonitrile-acetone solution of dansyl chloride, followed by heating at 38~ for 90 min for dansylation. The amino acids could be derivatized with addition of dansyl chloride about 5 fold excess of total amino acids. In addition, some dansyl amino acids are commercially available. Derivatization of amino acids in sake with dansyl chloride can be carried out by the following procedures [ 11 ]. The pH of sake was adjusted to 9.7 with 0.2 M sodium bicarbonate and sodium hydroxide. The prepared solution (1 mL) was then placed in a 5-mL vial and mixed with 0.13 mL of 1% (w/v) dansyl chloride dissolved in acetonitrile-acetone (90:10). The vial was placed in an oven and heated at 38~ for 90 min for dansylation.

HPLC of Amino Acids as Dansyl and Dabsyl Derivatives 5.2. Chromatography

239

The liquid chromatograph was assembled from two PU-980 LC pumps (Jasco, Tokyo, Japan) with an HG-980-30 mixer, a Model 7000 six-port valve (Rheodyne, Cotati, Ca, USA) with a laboratory-made loop (ca. 20 gL) as injector, a 150x4.6 mm I.D. An L-column ODS separation column (5 gm; Chemical Inspection & Testing Institute, Tokyo, Japan), an FP-920 fluorimetric detector (335 nm for excitation and 522 nm for emission; Jasco), and a Chromatopac C-R4AX data processor (Shimadzu, Kyoto, Japan) [19]. The flow rate of the pump was 1 mL/min. The solutions delivered by the two pumps were 40 mM ammonium acetate and 50% (v/v) acetonitrile in 40 mM ammonium acetate. The composition of acetonitrile was linearly programmed in the high-pressure gradient mode. The acetonitrile concentration was changed from 18% at a slope of 0.35%/min for 21 dansyl amino acids [19]. It should be noted that the dansyl amino acids can also be sensitively detected by a UV detector at 214 nm.

References

[1] W. R. Gray, B. S. Hartley, Biochem. J.89 (1963) 379-380. [2] W. R. Gray, J. F. Smith, Anal. Biochem. 33 (1970) 36-42. [3] B. S. Hartley, Biochem. J. 119 (1970) 805-822. [4] P. L. Felgner, J. E. Wilson, Anal. Biochem. 80 (1977) 601-611. [5] J. K. Lin, J. Y. Chang, Anal. Chem. 47 (1975) 1634-1638. [6] J.-Y. Chang, R. Knecht, D. Braun, Methods Enzymol. 91 (1983) 41-48. [7] H. J. Schneider, Chromatographia, 28 (1989) 45-48. [8] E. H. J. M. Jansen, R. H. Van den Berg, R. Both-Miedema, L. Doom, J. Chromatogr. 553 (1991) 123-133. [9] N. Kaneda, M. Sato, K.Yagi, Anal. Biochem. 127 (1982) 49-54. [10] R. Minocha, S. Long, J. Chromatogr. A 1035 (2004) 63-73. [11] T. Takeuchi, M. Yamazaki, D. Ishii, J. Chromatogr. 295 (1984) 333-339. [12] Y. Tapuhi, D. E. Schmidt, W. Lindner, B. L. Karger, Anal. Biochem. 115 (1981) 123129. [13] I. Krause, A. Bockhardt, H. Neckermann, T. Henle, H. Klostermeyer, J. Chromatogr. A 715 (1995) 67-79. [14] M. A. Castillo, R. C. Castells, J. Chromatogr. A 921 (2001) 121-133.

240 [ 15] J. T. Tyopponen, J. Chromatogr. 413 (1987) 25-31. [ 16] Y. Kitamaki, T. Takeuchi, unpublished data.

Toyohide Takeuchi

[17] B. G. Belen'kii, V. A. Mostovnikov, S. V. Nechaev, A. F. Lobazov, I. V. Nazimov, Zhumal Prikladnoi Spektroskopii 44 (1986) 511-517. [ 18] T. Takeuchi, T. Miwa, Chromatographia 40 (1995) 545-549. [19] T. Takeuchi, T. Miwa, Chromatographia 41 (1995) 148-152. [20] T. Takeuchi, T. Miwa, Anal. Chim. Acta 311 (1995) 231-236. [21] W.-Y. Lee, T. A. Nieman, J. Chromatogr. A 659 (1994) 111-118. [22] B. H. Reitsma, E. S. Yeung, Anal. Chem. 59 (1987) 1059-1061. [23] N. Kaneda, M. Sato, K.Yagi, Anal. Biochem. 127 (1982) 49-54. [24] R. A. Miller, N. E. Bussell, J. R. Wynkoop, II, R. Bongiovanni, T. M. Boehm, Chromatogr. Sei. Series 18 (1981) 235-246. [25] A. R. Martins, A. P. Padovan, J. Liq. Chromatogr. Relat. Techno. 19 (1996) 467-476. [26] B. Galli, F. Gasparrini, D. Misiti, C. Villani, J. Chromatogr. A 666 (1994) 77-89. [27] E. Peyrin, Y. C. Guillaume, N. Morin, C. Guinehard, J. Chromatogr. A 808 (1998) 113120. [28] Y. Abe, S. Fukui, Y. Koshiji, M. Kobayashi, T. Shoji, S. Sugata, H. Nishizawa, H. Suzuki, K. Iwata, Biochim. Biophys. Acta 1433 (1999) 188-197. [29] T. Araki, Y. Kashiwamoto, S. Tsunoi, M. Tanaka, J. Chromatogr. A 845 (1999) 455-462. [30] T.-Y. Kim, H.-J. Kim, J. Chromatogr. A 933 (2001) 99-106. [31] B. Mayr, F. Sinner, M. R. Buehmeiser, J. Chromatogr. A 907 (2001) 47-56. [32] G.-S. Ding, Y. Liu, R.-Z. Cong, J.-D. Wang, Talanta 62 (2004) 997-1003. [33] I. Slama, E. Jourdan, A. Villet, C. Grosset, A. Ravel, E. Peyrin, Chromatographia 58 (2003) 399-404. [34] E. Peyrin, C. Ravelet, E. Nicolle, A. Villet, C. Grosset, A. Ravel, J. Alary, J. Chromatogr. A 923 (2001) 37-43. [35] T. Takeuchi, H. Asai, D. Ishii, J. Chromatogr. 357 (1986) 409-415. [36] T. G. Nolan, N. J. Dovichi, Anal. Chem. 59 (1987) 2803-2805. [37] Z. Wu, W. G. Tong, J. Chromatogr. A 805 (1998) 63-69. [38] Y.-H. Chen, L.-L. Shih, S.-E. Liou, C.-C. Chert, Food Sci. Technol. Res. 9 (2003) 276282. [39] J.-K. Lin, C.-H. Wang, Clin. Chem. 26 (1980) 579-583. [40] S. H. Lee, T. S. Oh, Y. C. Lee, Bull. Kor. Chem. Soc. 11 (1990) 411-414. [41 ] S. H. Lee 1, A. Berthod, D. W. Armstrong, J. Chromatogr. 603 (1992) 83-93.

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[42] F. J. Mfirquex, A. R. Quesada, F. Sfinchez-Jim6nez, I. Nffiez De Castro, J Chromatogr. B 380 (1986) 275-283. [43] M. H. Joseph, J. Halliday, Anal. Biochem. 64 (1975) 389-402. [44] G. J. Schmidt, D. C. Olson, W. Slavin, J. Liq. Chromatogr. 2 (1979) 1031-1045. [45] S. Weiner, A. Tishbee, J. Chromatogr. 213 (1981) 501-506. [46] L. N. Mackey, T. A. Beck, J. Chromatogr. 240 (1982) 455-461. [47] L. F. Congote, J. Chromatogr. 253 (1982) 276-282. [48] S. Weinstein, S. Weiner, J. Chromatogr. 303 (1984) 244-250. [49] R. Badoud, G. Pratz, Chromatographia 19 (1984) 155-164. [50] A. Negro, S. Garbisa, L. Gotte, M. Spina, Anal. Biochem. 160 (1987) 39-46. [51] T. Takeuchi, T. Niwa, D. Ishii, J. Chromatogr. 407 (1987) 141-150. [52] A. M. Rizzi, P. Briza, M. Breitenbach, J. Chromatogr. 582 (1992) 35-40. [53] T. Abe, Y. Kurozumi, W.-B. Yao, T. Ubuka, J. Chromatogr. 712 (1998) 43-49. [54] O. Pinho, I. M. P. L. V. O. Ferreira, E. Mendes, M. O. Bruno, F. Margarida, Food Chem. 75 (2001) 287-291. [55] M. Ikeda, K. Sorimachi, K. Akimoto, Y. Yasumura, J. Chromatogr. 621 (1993) 133-138. [56] V. Bianchi, L. Mazza, J. Chromatogr. B 665 (1995) 295-302.