Anal. Chem. 2004, 76, 736-741
Derivatization of Chiral Amino Acids in Supercritical Carbon Dioxide Marı´a del Mar Caja Lo´pez, Gracia P. Blanch, and Marta Herraiz*
Instituto de Fermentaciones Industriales CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
A new method is proposed to perform the derivatization of chiral amino acids occurring in complex samples using supercritical carbon dioxide as both the reaction medium and the agent used to extract the obtained derivatives prior to accomplishing the subsequent enantiomeric chromatographic analysis. The derivatization step under supercritical conditions involves the esterification of the carboxyl group and the acylation of the amino group of the amino acids without using a catalyst. A Chirasil-L-Val capillary column enabled the separation of the D- and L-forms of the amino acids as their N(O)-pentafluoropropionyl 1-propyl esters. Relative standard deviation values obtained from the gas chromatographic analysis for the derivatized amino acids ranged from 5 to 15%. There is current interest in developing new methods for the reliable determination of the enantiomeric purity of relevant chiral compounds as this knowledge can elucidate genetically controlled biosynthetic pathways or, to give another example, can allow establishment of microbial transformations giving rise to the occurrence of undesired compounds in a particular sample.1,2 Amino acids are often considered to be the single most important and ubiquitous group of chiral compounds known. In fact, many R-amino acids contain a chiral center and, consequently, can exist as both D- and L-enantiomers. While L-amino acids seem to be more prevalent in foods and beverages, the presence of the D-forms, although certainly less common, has also been already described (e.g., in those samples that have been exposed to either microbiological activity or to extreme conditions of pH, heat, etc., which may eventually cause acid- or base-catalyzed racemization).1 As it has been previously reported that the D-amino acids may alter the biological activity and safety of foods, it is evident that it would be convenient to perform stereochemical analysis of chiral food components in general and, particularly, of chiral amino acids.3-11 * Corresponding author. Telephone: 91-5622900. Fax: 91-564 48 53. E-mail:
[email protected]. (1) Ekborg-Ott, K. H.; Armstrong, D. W. In Chiral separations: applications and technology; Ahuja, S., Ed.; American Chemical Society; Washington, DC, 1997. (2) Marchelli, R.; Dossena, A.; Palla, G. Trends Food Sci. Technol. 1996, 7, 113-119. (3) Bru ¨ ckner, H.; Hausch, M. HRC, High Resolut. Chromatogr. 1989, 12, 680684. (4) Bru ¨ ckner, H.; Hausch, M. Milchwissenschaft 1990, 45, 357-360. (5) Bru ¨ ckner, H.; Lu ¨ pke, M. Chromatographia 1991, 31, 123-128. (6) Bru ¨ ckner, H.; Westhauser, T. Chromatographia 1994, 39, 419-426. (7) Gandolfi, I.; Palla, G.; Delprato, L.; de Nisco, F.; Marchelli, R.; Salvadori, C. J. Food Sci. 1992, 57, 377-379.
736 Analytical Chemistry, Vol. 76, No. 3, February 1, 2004
The tremendous expansion during the past few years of effective chiral stationary phases suited to gas chromatography, liquid chromatography, and supercritical fluid chromatography (as well as of chiral additives used in electrochromatography) has allowed the development of different separation strategies adequate to achieve finally the enantioselectivity required for specific pairs of enantiomers.12-15 However, experimental conditions typically used during preparation of complex samples for analysis are often inadequate in enantiomeric studies as some of them (e.g., high temperatures) can bring about the racemization of the chiral compound of interest. For that reason, the sample preparation step usually required to achieve the necessary sensitivity to perform the enantiomeric chromatographic separation is a critical aspect of the entire analysis. On the other hand, the potential of supercritical fluid extraction (SFE)16-18 as an alternative to conventional extraction procedures has already been demonstrated with a wide variety of samples.19-22 Actually, in those cases in which the use of mild experimental conditions for sample extraction is specifically demanded (e.g., when analyzing thermally unstable or high reactive compounds), variation of the solvent strength achievable by modifying the pressure and temperature of supercritical fluids is of special interest. In fact, during the past decade, different authors have (8) Gandolfi, I.; Palla, G.; Marchelli, R.; Dossena, A.; Puelli, S.; Salvadori, C. J. Food Sci. 1994, 59, 152-154. (9) Ooghe, W.; Kasteleyn, H.; Temmerman, I.; Sandra, P.HRC&CC, High Resolut. Chromatogr. Chromatogr. Commun. 1984, 7, 284-285. (10) Palla, G.; Marchelli, R.; Dossena, A.; Casnati, G. J. Chromatogr. 1989, 475, 45-53. (11) Zukowski, J.; Pawlowska, M.; Armstrong, D. W. J. Chromatogr. 1992, 623, 33-41. (12) Beesley, T. E.; Scott, R. P. W. Chiral Chromatography. John Wiley & Sons: Chichester, U.K., 1998. (13) Schurig, V. J. Chromatogr., A 1994, 666, 111-129. (14) Schurig, V. J. Chromatogr., A 2001, 906, 275-299. (15) Subramanian, G., Ed. Chiral Separation Techniques: A Practical Approach; Wiley-VCH: Weinheim, 2001. (16) Chester, T. L.; Pinkston, J. D.; Raynie, D. E. Anal. Chem. 1994, 66, 106R130R. (17) Chester, T. L.; Pinkston, J. D.; Raynie, D. E. Anal. Chem. 1996, 68, 487514. (18) Lee, M. L., Markides, K. E., Eds. Analytical Supercritical Fluid Chromatography and Extraction; Chromatography Conferences, Inc.: Provo, UT, 1990. (19) Blanch, G. P.; Iba´n ˜ez, E.; Herraiz, M.; Reglero, G. Anal. Chem. 1994, 66, 888-892. (20) Blanch, G. P.; Reglero, G.; Herraiz, M. J. Agric. Food Chem. 1995, 43, 12511258. (21) King, M. B., Bott, T. R., Eds. Extraction of Natural Products Using NearCritical Solvents; Chapman & Hall: Glasgow, Scotland, 1993. (22) Rizvi, S. S. H., Ed. Supercritical Fluid Processing of Food and Biomaterials; Blackie Academic & Professional: London, England, 1994. 10.1021/ac034638f CCC: $27.50
© 2004 American Chemical Society Published on Web 12/30/2003
exploited the technical advantages of supercritical fluids by developing a number of extraction and separation processes that have been beneficial to many scientific disciplines and have been applied to a wide variety of real-world samples. However, it is now recognized that the potential benefits of supercritical fluids as reaction media have not been fully realized. Concretely, the possibility of increasing reaction rates by accelerating the rate-limiting mass-transfer step represents an interesting opportunity to discover a range of new applications.23,24 In this respect, the use of supercritical carbon dioxide is specially recommended as it is nonflammable, nontoxic, inexpensive, environmentally benign, and readily accessible due to its low critical temperature and pressure (31 °C and 73 atm, respectively). The objective of this work was to perform the derivatization of chiral amino acids using supercritical fluids. To this aim, supercritical carbon dioxide was used as the reaction medium as well as the agent used to extract the amino acid derivatives for subsequent chromatographic analysis. A further objective was the development of a new method suited to the determination of the enantiomeric ratio of chiral amino acids in complex samples. EXPERIMENTAL SECTION Reagents and Chemicals. Acetyl chloride and the antioxidant 2,6-di-tert-butyl-p-cresol (BHT) were purchased from Sigma (St. Louis, MO), 1-propanol was purchased from Scharlau (Barcelona, Spain), pentafluoropropionic anhydride was acquired from Aldrich (Milwaukee, WIS), dichloromethane was from SDS (Peypin, France), and carbon dioxide was obtained from Praxair (Madrid, Spain). Solid racemic amino acid standards (i.e., alanine, threonine, valine, norvaline, isoleucine, leucine, norleucine, proline, serine, methionine, phenylalanine, tyrosine, aspartic acid, and glutamic acid) were provided by Sigma. Amino Acid Derivatization in a “Reacti-vial”. The free amino acids were derivatized following the method previously proposed by Bru¨ckner and Lu¨pke.5 A 3-mg weight of a standard mixture of racemic amino acids was placed in a Reacti-vial (Bu¨chi Glas, Uster, Switzerland) and heated for 1 h at 100 °C after having added a 500-µL volume of 1-propanol/acetyl chloride (8:2, v/v) and ∼1.5 mg of BHT. The reagents were then removed in a stream of nitrogen, and a 200-µL volume of dichloromethane and 100 µL of pentafluoropropionic anhydride were added. The mixture was then heated for 20 min at 100 °C and the reagent excess again removed in a stream of nitrogen, the residue was dissolved in 200 µL of dichloromethane, and 0.2-µL aliquots were sampled into the gas chromatograph as detailed below. Amino Acid Derivatization in Supercritical Carbon Dioxide. The amino acid derivatizations were carried out in the 7-mL thick-walled stainless steel thimble conventionally used as the extraction cell of a Hewlett-Packard 7680A extraction module (Palo Alto, CA).19,20 The thimble containing the sample is placed in the extraction chamber where the components are then extracted. When the extraction chamber is closed, the porous frits contained in the caps at the ends of the vessel produce high-pressure seals and also allow one to hold the sample in place. The unit includes a nozzle/trap assembly, which acts as a controllable variable restrictor and enables the instant depressurization of the super(23) Baiker, A. Chem. Rev. 1999, 2, 453-473. (24) Jessop, P. G.; Ikaruya, T.; Noyori, R. Chem. Rev. 1999, 2, 475-493.
critical fluid as well as the independent control of both the pressure and the supercritical fluid flow rate. This variable restrictor keeps the extraction vessel under pressure, reduces the typical risk of plugging of fix restrictors and, therefore, provides an interface adequate to operate at atmospheric pressure. The extraction module is fully automated and is provided with an internal trap with a solid material. The supercritical fluid with the sample components enters the trap through the nozzle, where it is depressurized. The components are then retained on the trap while the supercritical fluid evaporates and leaves the trap to the vent. Subsequently, the material collected on the trap is dissolved in the appropriate rinsing solvent and moved from the trap through an exit line either to a vial or waste. Esterification and Acylation in Supercritical Carbon Dioxide. Experimental conditions used to perform the esterification and acylation of the chiral amino acids were as follows: supercritical CO2 density, 0.65 g/mL; cell temperature, 50 °C; sample weight in the extraction cell, 3 mg of each amino acid standard. The esterification reaction was performed by adding a 500-µL volume of a solution of 1-propanol/acetyl chloride (8:2, v/v) to the amino acid mixture placed into the cell (i.e., into the vessel which can be considered as a reactor for this specific application) of the SFE unit. After closing the cell, it was maintained 5 min under the conditions detailed above, which imply the operation in the static mode. The vessel was then opened and dried under a nitrogen stream, and subsequently, a 200-µL volume of dichloromethane and 100-µL volume of pentafluoropropionic anhydride were added and the cell was again tightly closed to carry out the acylation reaction. The mixture was then kept for 5 min at the selected temperature and CO2 density; the cell was then reopened and the reagent excess again removed in a stream of nitrogen. Isolation of the Derivatized Amino Acids with Supercritical Carbon Dioxide. The N(O)-pentafluoropropionyl 1-propyl esters of the amino acid enantiomers were isolated from the reactor of the SFE module in which the derivatization had been previously performed under the following experimental conditions: cell temperature, 40 °C; supercritical CO2 density, 0.25 g/mL; supercritical CO2 flow, 4 mL/min; extraction time, 3 min; trap temperature, 30 °C; restrictor temperature, 45 °C. Analytes were trapped by letting the solute-containing supercritical fluid decompress onto a solid sorbent material (Hypersil octadecylsilica, 30-40 µm) placed in the internal trap of the SFE module. Subsequently the trap was rinsed with 1 mL of dichloromethane at a rate of 2 mL/ min and the obtained extract was sampled into the gas chromatograph as detailed below. Gas Chromatographic Analysis. All analyses were performed using a model HP 6890 (Hewlett-Packard) gas chromatograph. The 50 m × 0.25 mm i.d. fused-silica column of Permabond L-Chirasil-Val (Macherey Nagel, Du ¨ ren, Germany) was programmed from 45 °C at 2 °C/min to 160 °C and held for 30 min at the final temperature. Helium was used as the carrier gas, 1.5 mL/min being the flow rate. When the GC analysis of the amino acid derivatives obtained in both the Reacti-vial and the supercritical medium was performed, the sample was injected into the gas chromatograph using a splitless injector kept at 250 °C. Mass Spectrometric Analysis. The gas chromatograph was fitted with a mass selective detector (HP 5973) with hyperbolic quadrupole, 250 °C being the source temperature. Identification Analytical Chemistry, Vol. 76, No. 3, February 1, 2004
737
Figure 1. Elution profile of N(O)-pentafluoropropionyl 1-propyl esters of a standard mixture of racemic amino acids derivatized in supercritical carbon dioxide using acetyl chloride as a catalyst. Capillary column, 50 m × 0.25 mm i.d. of Chirasil-L-Val; temperature program, 45 °C at 2°/min to 160 °C (30 min). See Experimental Section for further details.
of the enantiomers of the investigated amino acids in the real sample (a sport nutritional supplement commercially available as powder capsules) was established by matching the typical fragment ions (and their relative intensities) of the 70-eV mass spectra with those recorded for the compounds resulting from the derivatization of the corresponding amino acids available as standards. Safety Considerations. Special care must be taken in the experimentation with supercritical carbon dioxide to ensure that the fittings used are capable of withstanding the required pressures. RESULTS AND DISCUSSION Study on the Amino Acid Derivatization in Supercritical Carbon Dioxide. The new method of amino acid derivatization proposed in this work involves the use of supercritical carbon dioxide as reaction medium to carry out both the esterification of the carboxyl group and the acylation of the amino group of the amino acids. Supercritical carbon dioxide was also used as extractant agent to isolate the obtained derivatized amino acid enantiomers as their N(O)-pentafluoropropionyl 1-propyl esters to perform the subsequent gas chromatographic analysis. Experimental conditions to perform the esterification and acylation in supercritical carbon dioxide were established by studying the effect of the carbon dioxide fluid density and temperature on the solubility parameters of all the investigated amino acid derivatives. In this respect, it was considered that experimentation at low densities could eventually hinder the solubilization of the compounds of interest. It was clear that if the supercritical CO2 density became greater due to the increase of the pressure, the consequent enhancement of the solvating power promoted the solubilization, but as a counterpart, the solute vapor pressure could then be not high enough to allow the solubilization of some specific derivatives. As in the present study, the aim was the solubilization in a single run of 14 amino acid derivatives of different size, shape, hydrophobicity, and reactivity; it was considered mandatory to establish the required balance 738 Analytical Chemistry, Vol. 76, No. 3, February 1, 2004
between CO2 density and solute vapor pressure changes to try to achieve the solubilization of all the derivatizated amino acids. Specifically, moderate temperature and pressure conditions were observed to be most effective for amino acid derivatization in supercritical CO2 as well as for their subsequent extraction with supercritical CO2. For that reason, the experiment was performed, under the conditions of temperature and pressure given in the Experimental Section (i.e., T ) 50 °C and F ) 0.65 g/mL). Study on the Amino Acid Derivatization in a Reacti-vial. To evaluate the applicability of the new method proposed in this work, the amino acid derivatization procedure previously described by Bru¨ckner and Lu¨pke5 was used as a reference; it involves the use of a Reacti-vial under the conditions given in the Experimental Section. Study on the Performance of the Derivatization Reaction Using Supercritical Carbon Dioxide as Reaction Medium. Figure 1 shows the gas chromatogram obtained when analyzing the N(O)-pentafluoropropionyl 1-propyl esters of the 14 racemic amino acids investigated using supercritical CO2 as the reaction medium. As can be seen, serine derivatization was not achieved, probably due to its lack of solubility in supercritical carbon dioxide under the conditions used. In this respect, the conditional step might be the isolation of the derivative rather than the esterification and acylation reactions since the supercritical carbon dioxide used as the reaction medium is maintained (i.e., static mode) in the vessel acting as a reactor while derivatization is performed. However, upon completion of the derivatization reaction, the supercritical carbon dioxide continuously flows (i.e., is operated in the dynamic mode) through the N(O)-pentafluoropropionyl 1-propyl esters formed, which are finally released through the restrictor to the internal trap of the SFE module to be isolated. In any case, it should be considered that there are other possible causes to explain the absence of the peak for the serine derivative in Figure 1, namely, failure of the derivative to form in the reactor or loss of the serine derivative at any step of the
Table 1. MS Fragments and Relative Intensities Identified in the Derivatives Obtained in Supercritical Carbon Dioxide from Amino Acids Having Aliphatic Side Chains (Alanine, Valine, Norvaline, Isoleucine, Leucine, Norleucine) and Cyclized Side Chain (Proline) alanine
molecular ion (M)•+ M-(C4H7O2) C2F5 C4H3NOF5 C5H2NO2F5/ C6H6NOF5 C5H5NOF5 C5H9 C8H10NO3F5
valine
(%)
m/z 277
norvaline
(%)
m/z
m/z
305
190 119
(100) (10)
(%)
305
isoleucine m/z
leucine
(%)
m/z
319
norleucine
(%)
(%)
m/z
319
319
proline (%)
m/z 303
218 119 176 203
(100) (5) (4) (28)
218 119 176 203
(100) (9) (43) (12)
232 119 176 203
(100) (9) (13) (60)
232 119 176 203
(100) (11) (11) (20)
232 119 176 203
(100) (6) (25) (9)
190
(1)
263
(4)
190 69 263
(7) (6) (10)
190 69 263
(3) (37) (26)
190 69 263
(76) (48) (23)
190 69 263
(7) (25) (7)
216 119 176
(100) (14) (1)
69
(8)
Table 2. MS Fragments and Relative Intensities Identified in the Derivatives Obtained in Supercritical Carbon Dioxide from Amino Acids Having in the Side Chain a Hydroxyl Group (Threonine, Tyrosine), an Aromatic Ring (Phenylalanine), a Sulfur (Methionine), and a Carboxylic Acid (Aspartic Acid, Glutamic Acid) threonine m/z molecular ion (M)•+ M-(C4H7O2) C2F5 C2H5S C5H2NO2F5/ C6H6NOF5 C5H4NO2F5 C5H5NOF5 C6H5NOF5 C6H5NO3F5 C6H6NOF5 C7H5NO2F5/ C11H20NO4 C7H7 C8H10NO3F5 C9H8O2 C10H18NO4/ C6H3NO2F5 C12H8NO2F5 C12 H9NO3F5 C12H14O2 C15H15NO3F5
(%)
307 119
203
263
tyrosine m/z
(%)
369 (39)
218 119
(0.8) (19)
phenylalanine m/z
(%)
353
(1)
266 119
(33) (27)
methionine
aspartic acid
m/z
(%)
m/z
337
(37)
363
119 61 203
(15) (50) (100)
(%)
glutamic acid (%)
m/z 377
276 119
(100) (8)
190
(52)
234
(62)
290 119
(72) (6)
202
(100)
230
(91)
(100) 91
(98)
148
(100)
(7)
263
(88) 216
293 310
(25) (100)
352
(58)
190
analysis (e.g., collection in the trapping material or rinse with dichloromethane). On the other hand, it should be pointed out that although the enantioselectivity or efficiency of the column used was not adequate to separate the enantiomeric forms of proline and aspartic acid as well as the L-threonine and D-valine, it is evident the acceptable enantiomeric resolutions were generally observed. It is interesting to emphasize that the mass spectra obtained from each amino acid derivatized separately enabled the performance of the derivatization reaction under supercritical conditions to be demonstrated. The typical fragment ions (and their relative intensities) of the spectra recorded for each amino acid derivative are given in Tables 1 and 2, which also show the most representative radical losses undergone by each compound and, consequently, the pathways followed in the fragmentation of the different structures considered. From data in Table 2, it seems clear that the carboxyl groups in the side chain of aspartic acid and glutamic acid are also esterified under the proposed conditions. Specifically, the deriva-
(35)
(61)
tized aspartic acid undergoes loss of the propoxycarbonyl radical forming a fragment at m/z 276 as the base peak. The fragmentation of this ion via elimination of propene gives m/z 234, while the loss of the second propoxycarbonyl radical and the additional occurrence of a hydrogen transposition produces the ion at m/z 190. Similarly, the ion at m/z 230 observed in the mass spectrum obtained from the glutamic acid derivative represents the losses of a propoxycarbonyl radical and propanol, which suggests again the esterification of the two carboxyl groups. The additional elimination of 28 mass units (carbonyl radical) yields the base peak (m/z 202). With respect to the threonine derivative, mass spectrum data did not demonstrate the esterification of the hydroxyl group in the side chain as the base peak (m/z 203) may result from elimination of either a free hydroxyl group or an esterified hydroxyl group and the additional loss of a propoxycarbonyl radical. Fragmentation of the tyrosine derivative follows a similar pathway that includes the loss of either a free hydroxyl group or an esterified hydroxyl group (m/z 352) and the subsequent Analytical Chemistry, Vol. 76, No. 3, February 1, 2004
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Figure 2. Gas chromatogram of N(O)-pentafluoropropionyl 1-propyl esters of a standard mixture of nonpolar racemic amino acids derivatized in either supercritical carbon dioxide without using catalyst (a) or a Reacti-vial without using catalyst (b). I.S., internal standard (n-C15). Capillary column and chromatographic conditions as in Figure 1.
elimination of a propoxyl radical (m/z 293). Interestingly, it must be stressed that the loss of 59 mass units (i.e., a propoxyl radical) from the molecular ion (m/z 369) corresponding to the tyrosine derivative with the unesterified hydroxyl group in the side chain generates the base peak (m/z 310) observed in the spectrum. A difficult aspect concerning the applicability of methods involving the use of acetyl chloride as catalyst refers not only to its corrosive action but also to its violent reaction with water, which evidently make it impossible to use such procedures for analyzing a wide variety of real-world samples. For that reason we decided to evaluate whether the enhancement of mass transfer and the catalytic effect resulting from the unusual properties of supercritical carbon dioxide could be sufficient to avoid the use of acetyl chloride during the derivatization reaction. The experimental work performed showed the possibility of achieving derivatization of the amino acids having a nonpolar side chain (e.g., DL-alanine, DL-valine, DL-norvaline, DL-leucine DL-norleucine, DL-isoleucine, DLmethionine, and DL-phenylalanine) while such derivatization was not possible for the amino acids having either polar groups in the side chain or a cyclized side chain. Figure 2 gives the 740 Analytical Chemistry, Vol. 76, No. 3, February 1, 2004
chromatograms obtained from the N(O)-pentafluoropropionyl 1-propyl esters of the mentioned amino acids when performing the derivatization reaction either under supercritical conditions (Figure 2a) or using a Reacti-vial (Figure 2b). For the sake of comparison, in both cases, acetyl chloride was not added during the corresponding reactions. As can be seen, similar results were obtained by applying the two evaluated procedures, the reaction yield being higher with the Reacti-vial. However, the analysis time required to perform the overall procedure (i.e., derivatization, isolation of the derivatives, and GC analysis) is significantly different (1 h and 45 min when supercritical carbon dioxide is used as the reaction medium vs 3 h and 45 min when the Reactivial is used). Moreover, as the use of supercritical carbon dioxide as proposed in this work allows one to take advantage of its properties as an extraction agent, it is clear that the sample preparation step can be considered as a part of the overall procedure. However, sample preparation is not only an additional time-consuming step but also a possible source of error when the Reacti-vial is used for the analysis of the enantiomeric composition of amino acids in those real-world samples, which demand the
Figure 3. Gas chromatogram of N(O)-pentafluoropropionyl 1-propyl esters of free amino acids of a sport nutritional supplement derivatized in supercritical carbon dioxide without using catalyst. I.S., internal standard. Capillary column and chromatographic conditions as in Figure 1.
previous isolation of the compounds of interest. The relative standard deviation (RSD) values estimated from the relative peak areas (n ) 3) resulting from the overall procedure (including derivatization, isolation of the derivatives, and GC analysis) ranged from 5 to 15%. Figure 3 shows the gas chromatogram of the N(O)-pentafluoropropionyl 1-propyl esters of some nonpolar chain-free amino acids analyzed in a sport nutritional supplement derivatized in supercritical carbon dioxide without using acetyl chloride. Identified amino acids included the branched-chain amino acids L-valine, L-isoleucine, and L-leucine, especially recommended for consumption during prolonged exercise.25,26 The enantiomeric excesses (ee) obtained were 90 (L-valine and L-isoleucine), 94 (L-leucine), 97 (L-methionine), and 100% (Lphenylalanine). The ee value repeatability estimated by measuring the RSD (from three replicates) was equal to or lower than 2%. Finally, the detection limits (estimated from a signal equal to 5 times the baseline noise) were 2,1 (L-valine), 1.7 (L-isoleucine), 2.5 (L-leucine), 0.2 (L-methionine), and 0.6 µg (L-phenylalanine). It should be mentioned that mass spectra of humps observed in Figures 2 and 3 showed that they were produced by the pentafluoropropionic anhydride used to perform the acylation reaction. Most likely the presence of this reagent in the chromatogram was due to incomplete removal under the nitrogen stream of its excess once the derivatization procedure was finished. However, the cause of the hump in Figure 1 could not (25) Boniglia, C.; Carratu´, B.; Sanzini, E. J. Food Sci. 2002, 67, 1352-1355. (26) Blomstrand, E.; Hassmen, P.; Ekblom, B.; Newsholm, E. A. Eur. J. Appl. Physiol. 1991, 63, 83-88.
be clearly identified on the basis of the corresponding mass spectrum, although the corrosive effect of the acetyl chloride used in this case as calalyst (while chromatograms in Figures 2 and 3 were obtained without it) suggests the possibility of the presence of degradation products resulting from chemical reactions occurring with a given material of the SFE module. In any case, it is worth emphasizing that clean chromatograms were obtained when blanks were performed under the selected experimental conditions. Summarizing, data obtained suggest the potential of supercritical carbon dioxide as an alternative reaction medium to develop new environmentally friendly methods suitable to carry out the in situ derivatization and subsequent extraction of amino acids. Concerning the reliable determination of enantiomeric ratios characteristic of chiral amino acids, the use of supercritical carbon dioxide as reaction medium in derivatization reactions seems to be a valuable alternative as it not only allows the use of mild conditions suited to prevent racemization but it also meets legal regulations regarding the use of contaminant solvents. ACKNOWLEDGMENT Financial support for this work by CICYT (Comisio´n Interministerial de Ciencia y Tecnologı´a) (Project ALI99-1188) is gratefully acknowledged.
Received for review June 11, 2003. Accepted November 13, 2003. AC034638F
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