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Anal. Chem. 1996, 68, 322-326
Sequential Determination of D- and L-Glutamic Acid by Continuous Fractional Crystallization Evaristo Ballesteros, Mercedes Gallego, and Miguel Valca´rcel*
Department of Analytical Chemistry, Faculty of Sciences, University of Co´ rdoba, E-14004 Co´ rdoba, Spain
A method involving turbidimetric multidetection of the signal produced in the continuous crystallization of D- and L-histidine in the presence of an organic solvent is proposed. It uses the inhibitory effect of D- and L-glutamic acid on the crystallization of D- and L-histidine, respectively. Calibration graphs are linear down to 40 mg/L Dand L-glutamic acid, with relative standard deviations between 2.6 and 2.9%. The proposed method was used for the determination of L-glutamic acid in pharmaceutical preparations and the determination of D- and L-glutamic acid in a racemate of DL-glutamic acid. L-Amino acid enantiomers are the more commonly occurring in living systems; however, D-amino acid forms have been found in some microorganisms such as bacteria. Amino acids such as D-glutamic acid can be used as chiral labels for the specific detection of such microorganisms. However, when an amino acid is synthetized in the laboratory, a racemic mixture is generally obtained that is optically inactive.1 Different analytical techniques have been used for the separation of chiral amino acids. High-performance liquid chromatography (HPLC) is the most frequently used for the resolution of enantiomeric compounds.2-6 Alternative techniques have been proposed for the industrial resolution of racemic mixtures by fractional crystallization of diastereoisomer conglomerates.7 When crystals of an organic compound are grown in the presence of a growth inhibitor, a change in crystal morphology is observed. A stereochemical correlation exists between the crystal structure, its modified morphology, and the molecular structure of the inhibitor.8 The last can be adsorbed on surface sites only in such a way that the different counterpart emerges from the crystal surface; once adsorbed, the admixture inhibits the regular crystallization process. These substances are known as “tailor-made” inhibitors. The potential applications of these crystallization inhibition processes in analytical chemistry have been pointed out elsewhere.9 Recently, several methods for the kinetic resolution of amino acid enantiomers based on the interaction between a
(1) Lehninger, A. L. Biochemistry; Worth Publishers, Inc.: New York, 1985. (2) Dyremark, A.; Ericsson, M. Chromatographia 1990, 29, 51-53. (3) Merino-Merino, I.; Blasco-Gonza´lez, E.; Sanz-Medel, A. Anal. Chim. Acta 1990, 234, 127-131. (4) Galaverna, G.; Corradini, R.; de Munari, E.; Dossena, A.; Marchelli, R. J. Chromatogr. 1993, 657, 43-54. (5) Brueckner, H.; Haasmann, S.; Langer, M.; Westhauser, T.; Wittener, R.; Godel, H. J. Chromatogr. 1994, 666, 259-273. (6) Vogt, C.; Georgi, A.; Werner, G. Chromatographia 1995, 40, 287-295. (7) Collet, A.; Jackes, J.; Wilen, S. H. Racemates, Enantiomers and Resolution; Wiley: New York, 1981. (8) Addadi, L.; Berkovitch-Yellin, Z.; Weissbuch, I.; van Mil, J.; Shimon, L. J. W.; Lahav, M.; Leiserowitz, L. Angew. Chem., Int. Ed. Engl. 1985, 24, 466485. (9) Grases, F.; March, J. G. Trends Anal. Chem. 1991, 10, 190-195.
crystallizing substrate and a stereospecific tailor-made growth inhibitor were reported.10,11 Flow injection (FI) methods have also been used for the determination of glutamine using an amperometric biosensor,12 and several amino acids using a spectrophometer and multivariate calibration.13 L-Lysine was determined by using a continuous turbidimetric method.14 In this work, we developed a turbidimetric method for the indirect determination of D- and L-glutamic acid by the inhibitory effect of these substances on crystal growth of D- and L-histidine, respectively. This continuous method permits the sequential determination of D- and L-glutamic acid in a multidetection flow system, including an open-closed loop and a single spectrophotometer. EXPERIMENTAL SECTION Apparatus. Turbidimetric measurements were made on a Unicam 8625 UV/vis spectrophotometer (wavelength, 550 nm) connected to a Radiometer REC-80 Servograph recorder and furnished with an Hellma flow-through cell (10 mm light path, 1 mm i.d., 18 µL). The flow manifold consisted of two Gilson Minipuls-2 peristaltic pumps, a Rheodyne 5041 injection valve, and two Rheodyne 5301 switching valves. Poly(vinyl chloride) and Solvaflex pumping tubes were used for aqueous and organic solutions, respectively; all coils were constructed from PTFE tubing of 0.5 mm i.d. Reagents. D- and L-glutamic acid, racemic DL-glutamic acid, D- and L-histidine, and the other amino acids used were purchased from Sigma (St. Louis, MO). HPLC-grade solvents (2-propanol, ethanol, and methanol), sodium hydroxide, and hydrochloric acid were obtained from Merck (Darmstadt, Germany). Standard solutions containing 1 g/L D- or L-glutamic acid were prepared in Milli-Q water. Solutions containing 4 g/L of D- or L-histidine were used as substrates. All solutions were stable for at least 1 week. Sample Preparation. For tablet formulations (Amiopia Medical, S.A., Co´rdoba, Spain; Glutaneurina-B6 fuerte, Zyma Farmace´utica, S.A., Barcelona, Spain), 10 tablets were placed in a mortar and ground to a fine mesh. For capsule formulations (Tebetane Compuesto, Laboratorios Elfar-Drag, S.A., Madrid, Spain), the contents of 10 capsules were mixed. The weighed amount of tablets or capsules (0.2-0.3 g) was mixed with 75 mL of Milli-Q water and shaken electromagnetically for 90 min. The solution was filtered, and the aqueous solution was diluted to volume with (10) Grases, F.; Genestar, C. Talanta 1993, 40, 1589-1593. (11) Grases, F.; Costa-Bauza´, A.; Forteza, R.; March, J. G. Anal. Lett. 1994, 27, 2781-2787. (12) Huang, Y. L.; Khoo, S. B.; Yap, M. G. S. Anal. Lett. 1995, 28, 593-603. (13) Saurina, J.; Herna´ndez-Cassou, S. Analyst 1995, 120, 305-312. (14) Ballesteros, E.; Gallego, M.; Valca´rcel, M.; Grases, F. Anal. Chem. 1995, 67, 3319-3323.
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Figure 1. Flow injection manifold for the sequential determination of D- and L-glutamic acid. (A) Introduction of the sample and reagents into the system. (B) Closed system and signal multidetection. (C) Typical signal for histidine crystallization. SV, switching valve; P, pump; R1, R2, and R3, coils of 50, 75, and 75 cm (0.5 mm i.d.), respectively; IV, injection valve (volume, 100 µL); D, spectrophotometer; ta, homogenization time; tb, starting crystallization time; tc, induction period.
water in a 250 mL calibrated flask. For continuous flow analyses, aliquots of 400-500 µL of these final solutions were placed in 10 mL calibrated flasks and diluted to the mark (pH 5-6). Procedure. The manifold used for the sequential determination of D- and L-glutamic acid is depicted in Figure 1. In the first step for the determination of D-glutamic acid (Figure 1A), a sample containing 1-40 mg/L D- and L-glutamic acid at pH 3-10 was continuously introduced into the system at 0.5 mL/min and merged with a substrate solution containing 4 g/L D-histidine (flow rate, 0.4 mL/min). The mixture was then merged with a stream of 2-propanol (flow rate, 2.0 mL/min). The final solution was homogenized in coil R2 (75 cm) and then circulated through the injection valve (IV). Simultaneously, a 2-propanol stream (carrier) was introduced into the open system by means of the second pump (P2) at 1.2 mL/min. In the second step (Figure 1B), the loop contents (100 µL) of IV were injected into the carrier, and SV2 was switched in order to close the loop. Changes in crystal growth of D-histidine were monitored at 550 nm until physicochemical equilibrium in the closed system was attained. Milli-Q water was used as blank. For the determination of L-glutamic acid, SV1 was switched and the same sample was merged with a solution containing 4 g/L L-histidine. The procedure then continued as described above for the determination of D-glutamic acid. After each determination, the open-closed system was flushed with Milli-Q water for 1 min by switching SV2. The signal profile obtained in the crystallization of histidine can be seen in Figure 1C. Of the three choices for deriving
analytical information from the signals (viz., starting crystallization time, induction period, and crystallization rate), the induction period (tc) was found to result in the best precision. RESULTS AND DISCUSSION Influence of Chemical Variables. First, the inhibitory effects of L-glutamic acid on the crystallization of L-lysine or L-histidine were comparatively studied in order to select the best substrate for its determination. For this purpose, a sample containing 10 mg/L L-glutamic acid was introduced into the flow system (Figure 1) and merged with a substrate solution containing 4 g/L L-lysine or L-histidine. L-Glutamic acid exhibited a similar inhibitory effect on the crystallization of both amino acids; however, when L-histidine was used as the substrate, the effect of L-glutamic acid was more selective. This was confirmed by introducing a sample containing 10 mg/L L-glutamic acid plus 50 mg/L D-glutamic acid into the system and using both substrates individually. The induction period was similar (8 min) for the sample (10 mg/L L-glutamic acid) with and whithout D-glutamic acid if L-histidine was used; if L-lysine was used as the substrate, the induction period for the sample containing D-glutamic acid (15 min) increased relative to the sample without D-glutamic acid (10 min). The same results were obtained if the analyte was D-glutamic acid and the substrate D-lysine or D-histidine. From the above results, the effect of D- or L-glutamic acid as inhibitor for the crystallization of D- or L-histidine, respectively, was chosen. A supersaturated solution of the substrate (D- or L-histidine) was obtained by changing the solvent composition. Thus, various
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Figure 2. Calibration graphs for L- and D-glutamic acid (solid and dashed lines, repectively) at variable concentrations of L- and D-histidine, respectively. The induction period is the difference of the induction periods between sample and blank solutions.
organic solvents (methanol, ethanol, and 2-propanol) were continuously mixed with the aqueous solution of D- or L-histidine to induce crystallization of the substrate. Crystallization of D- and L-histidine (4 g/L) was more effective in the presence of 2-propanol (induction period, 6 min) than in ethanol or methanol (18 and 35 min, respectively). Experiments in the presence of the inhibitor (D- or L-glutamic acid) were carried out in parallel with D- or L-histidine as substrate, respectively; the induction period was too long with ethanol and methanol, however. Thus, 2-propanol was selected as the solvent for inducing crystal growth of D- and L-histidine and as the carrier for the open-closed system (Figure 1), as it resulted in the optimum induction period for increasing sample throughput. To select the best concentration of D- and L-histidine, several calibration graphs for D- and L-glutamic acids were constructed at three different concentrations of D- or L-histidine (3.6, 4, and 4.4 g/L). As can be seen in Figure 2, the induction period increased with decreasing D- or L-histidine concentration. The crystallization of D-histidine was more favorable than that of L-histidine because its induction period was shorter; as a result, the slope of the calibration graph and the sensitivity for the L-glutamic acid were higher. A concentration of 4 g/L D- or L-histidine was thus selected as a compromise between sensitivity and sample throughput for further experiments. The effect of the sample (or blank) pH on histidine crystallization was studied in the range 3-10 (adjusted with 0.01 mol/L NaOH or 0.01 mol/L HCl), where the induction period was found not to be affected. A blank of water or a sample pH of 5-6 was thus selected, which was directly obtained when the solutions of glutamic acids were prepared in water. Selection of Flow Variables. The flow variables studied were flow rates, coil lengths, and injected volume. For this purpose, a blank of water or sample containing 10 mg/L D- or L-glutamic acid was merged with the substrate solution containing 4 g/L D- or L-histidine, respectively. The effects of the flow rates of the aqueous solutions (sample and substrate) and organic solvent (2-propanol) were studied by changing one at a time while keeping all others constant. First, the flow rate of the sample (10 mg/L glutamic acid) was studied between 0.2 and 0.7 mL/min, while keeping those of the substrate
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Figure 3. Influence of the sample flow rate on the crystallization of L- and D-histidine. Samples: a,b, L- and D-glutamic acid, respectively. Blank: c,d, Milli-Q water. The substrates were L-histidine for a and c and D-histidine for b and d.
and 2-propanol constant at 0.35 and 2.0 mL/min, respectively. As can be seen in Figure 3, the induction period for histidine crystallization increased with increasing sample flow rate through increased dilution of the substrate and increased concentration of D- or L-glutamic acid in the final mixture. The effect of the blank (Milli-Q water) was less strong, since only the substrate was diluted. The influence of flow rate of the substrate (D- or L-histidine) was the opposite to that of the sample; thus, the induction period decreased with increasing substrate flow rate through increased final concentration of histidine in the manifold. Increasing the flow rate of organic phase (2-propanol) had an effect similar to that of increasing the organic solvent volume in the final mixture and the sample and substrate dilution; thus, the induction period increased with an increase in the 2-propanol flow rate. Flow rates of 0.5, 0.4, and 2.0 mL/min were thus selected for the sample, substrate, and 2-propanol solvent, respectively, as compromises between sensitivity and sample throughput. The flow rate of the carrier (2-propanol) in the closed system was studied over the range 0.5-1.5 mL/min. Above 0.75 mL/min, the signal remained constant for both the blank and the sample because complete homogenization of the system was achieved; at lower flow rates, the induction period for D- and L-histidine crystallization decreased slightly, owing to increased dispersion of the sample/substrate mixture in the closed loop. Therefore, a flow rate of 1.2 mL/min for 2-propanol (the carrier) was chosen to unload the sample loop contents into the closed system. The influence of the coil length for the sample/substrate mixture (R1 in Figure 1) was investigated between 25 and 150 cm (0.5 mm i.d.). The induction period remained constant throughout the interval studied. A 50-cm coil length was selected. The effect of the length of the second coil (R2) was also studied between 25 and 150 cm. The length of R2 did not affect the induction period above 50 cm at an inner diameter of 0.5 mm. A homogenization coil of 75 cm was finally selected. The injected volume of valve IV, located between the pump 2 and the detector, had a significant effect on the D- and L-histidine crystallization. The induction period decreased with increasing injected volume between 50 and 200 µL. Also, the reproducibility decreased as a result of an increasing homogenization time of the sample plug. An injected volume of 100 µL was chosen, taking into account both effects. Finally, the influence of the length of the coil located
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Table 1. Tolerated Levels of Various Amino Acids and Other Chiral Organic Acids in the Determination of L- or D-Glutamic Acid (10 mg/L) L-glutamic
acid determination
compound L-ascorbic, L-lactic L-
malic, L-tartaric acids acid L-leucine, L-methionine, L-isoleucine, L-phenylalanine, L-alanine, L-tyrosine, L-valine, L-penicillamine L-serine, L-asparagine L-glutamine, L-aspartic acid, L-threonine, L-cysteine L-lysine L-ornithine, L-arginine D-glutamic
D-glutamic
max tolerated concn (mg/L)
max tolerated concn (mg/L)
compound D-lactic, D-malic, D-tartaric
acids acid D-alanine, D-phenylalanine, D-valine, D-penicillamine D-aspartic acid D-lysine D-ornithine, D-arginine
>200 >200 >100
inside the closed system (R3 in Figure 1B) on the substrate crystallization was studied between 25 and 250 cm. The crystallization induction period increased with increasing the length of R3 because the sample and blank were more extensively diluted as a result of the overall volume of the closed system increasing with increasing coil length. A length of 75 cm was chosen for further experiments. The temperature is an environmental variable that can affect D- and L-histidine crystallization. Therefore, its effect on the substrate crystallization was studied over the range 15-50 °C by inmersing coil R3 in a water bath. The crystallization induction period in the absence (blank) and presence of D- or L-glutamic acid (sample) remained constant throughout the interval studied, so the closed system was maintained at room temperature. Figures of Merit of the Proposed Method. By using the optimum values of the variables and the manifold depicted in Figure 1, a calibration graph for the determination of D- and L-glutamic acid was constructed. For this purpose, solutions containing variable concentrations of D- or L-glutamic acid (between 1 and 40 mg/L) were introduced into the system and merged with a substrate stream containing 4 g/L D- or L-histidine, respectively. The equations of the graphs were obtained from nine points in each instance: Y ) 0.09 + 0.20[D-glutamic acid], and Y ) -0.11 + 0.26[L-glutamic acid], where Y is the difference between the induction periods for the sample and blank (in min), and concentrations are expressed in mg/L. The correlation coefficients were 0.995 and 0.998 for D- and L-glutamic acid, respectively. The detection limits (0.7 and 0.6 mg/L for D- and L-glutamic acid, respectively) were calculated as 3 times the standard deviation of the induction period for 11 injections of the blank (Milli-Q water). The precision for each enantiomer was established by analyzing 11 samples containing 10 mg/L of each; it turned out to be 2.9 and 2.6% (as relative standard deviations) for D- and L-glutamic acid, respectively. Study of Interferences. To evaluate the selectivity of the proposed method, the effects of amino acids and chiral organic acids exhibiting behaviour similar to that of D- or L-glutamic acid were studied in detail in order to establish their tolerated levels. The tolerated limits for the compounds studied in the determination of 10 mg/L of D- or L-glutamic acid are given in Table 1. A study of potential interferences with the determination of Lglutamic acid more detailed than that for D-glutamic acid was carried out because pharmaceutical preparations contain the L-form predominantly. The most significant finding in this respect was that the proposed method for the determination of L- and D-glutamic acid is highy selective; in fact, either enantiomer is tolerated at concentrations at least 20 times higher than that of
acid determination
>200 >200 >100
L-glutamic
75 35 5 2.5
15 4 2
Table 2. Determination of L-Glutamic Acid in Pharmaceutical Preparations trade name Amiopia (tablets) Glutaneurina-B6 (tablets) Tebetane (capsules) a
nominal content founda (mg/tablet (mg/tablet or capsule) or capsule) 200 400 265
200.1 ( 7.2 383.1 ( 13.2 270.3 ( 6.7
Mean ( standard deviation (n ) 5).
the other (analyte). Both determinations are interfered by diaminocarboxylic acids (lysine, ornithine, and arginine), which perturb at concentrations below that of the analyte in some cases. All other amino acids tested were tolerated at higher concentrations. In addition, other chiral organic acids commonly used in pharmaceutical preparations, viz., tartaric, ascorbic, lactic, and malic acids, exhibited no interference at the maximum concentrations tested. Applications. The proposed method was validated by applying it to the determination of L- and D-glutamic acid in pharmaceutical preparations. Samples were prepared as described in the Experimental Section. The results obtained in three determinations of glutamic acid and their standard deviations are listed in Table 2. As expected, only L-glutamic acid was detected, since pharmaceutical preparations of glutamic acid contains only the active form (L) (as is the case with most amino acids except phenylalanine, histidine, methionine, and tryptophan, which are also active in their D forms).15 Because no real samples containing D-glutamic acid were available, variable amounts of this enantiomer were added to the above pharmaceutical samples before dissolution. Three standard additions were done on each preparation following dilution (final concentrations of D-glutamic acid in the diluted samples of 10, 20, and 30 mg/L). The recoveries obtained from three individual additions of equal amounts of D-glutamic acid were close to 100% (94.2 -104.0%) in all instances. Finally, in order to assess the potential of the proposed method for the resolution of both enantiomers, D- and L-glutamic acid were determined in a DL-glutamic acid racemate (Sigma). For this purpose, solutions containing variable concentrations of racemic DL-glutamic acid between 20 and 80 mg/L were analyzed. The results obtained are listed in Table 3. As can be seen, the mean contents of D- and L-glutamic acids were close to 50% of the overall concentration of the DL-glutamic acid racemate analyzed. (15) Del Pozo, A. Farmacia Gale´ nica Especial; Romargraf, S.A.: Barcelona, 1978.
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Table 3. Determination of D- and L-Glutamic Acid in a DL-Glutamic Acid Racemate sample
DL-glutamic acid (mg/L)
D-glutamic acid (mg/L)a
L-glutamic acid (mg/L)a
1 2 3 4 5
20 30 40 60 80
10.5 ( 0.3 14.8 ( 0.4 19.4 ( 0.5 29.8 ( 0.6 40.7 ( 0.8
9.4 ( 0.2 14.9 ( 0.3 20.8 ( 0.6 31.1 ( 0.7 38.9 ( 0.9
a
Mean ( standard deviation (n ) 5).
CONCLUSIONS The D- and L-enantiomers of glutamic acid can be resolved with a nonchromatographic technique, thereby avoiding their typical shortcomings (e.g., the high price of chiral columns and mobile phases and the need to work under strictly controlled conditions). Like its manual counterparts,8-11 the proposed method is based
on Pasteur fractional crystallization; however, it is implemented in a continuous configuration, which facilitates automatation, minimizes manipulations, and ensures good precision. This continuous method can be used for the determination of D- and L-glutamic acid in the same sample, simply by changing the substrate (D- or L-histidine, respectively), as well as for detecting potential adulteration in D-glutamic acid pharmaceutical preparations. ACKNOWLEDGMENT The Spanish Direccio´n General de Investigacio´n Cientı´fica y Te´cnica (DGICyT) is gratefully acknowledged for financial support (Grant No. PB94-0450). Received for review July 5, 1995. Accepted October 27, 1995.X AC9506591 X
Abstract published in Advance ACS Abstracts, December 1, 1995.
