Anal. Chem. 1995, 67, 3319-3323
Enantiomer Discrimination by Continuous Precipitation Evaristo Ballesteros, Mercedes Gallego, Miguel ValcBmel, and Felix Grases* Department of Analytical Chemistry, Faculty of Sciences, E- 14004 C&rdoba, Spain
A continuous turbidimetric method for the discrimination of enantiomers (L-and D-lysine) by the inhibitory action of L-lysine on the crystalkation of glutamic acid is proposed. A multidetection flow system including an open-closed loop and a single detector permits the determinationof kinetic parameters for the crystallization of glutamic acid in the presence of 2-propanol. L-Lysine can thus be determined in the presence of a 20 times higher D-lysine concentration. The proposed method was applied to the determination of L-lysine in pharmaceutical preparations with good results. Some organic substances act as crystallization inhibitors for organic molecules with similar chemical structures (or a slightly different bulk component of the molecular crystal) .l,z The inhibitory effect can be assigned to selective interactions with the foreign molecule at specific points in the crystallizing substance which induce marked changes in the crystallization rate at very low inhibitor concentrations. These processes have found application in analytical chemistry3 (e.g., in the determination of amino acids using batch procedures) .45 The flow injection Q technique is a major alternative to manual methods of Amino acids have been determined by FI using a liquid chromatograph and chemiluminiscence8 or electrochemical detection?Jo Pohlmann et al." reported a method for the continuous determination of L-lysine using L-lysine a-oxidase and spectrophotometric detection; the peroxide produced in the reaction was reacted with phenol and Caminoantipyrine in the presence of peroxidase to obtain a quinone imine dye that was monitored at 500 nm. Various FI systems have been used in conjunction with turbidimetric measurements for the determina* Author to whom correspondence should be addressed. Present address: Department of Chemistry, University of the Balearic Islands, E-07071 Palma de Mallorca, Spain. (1) Addadi, L.; Weinstein, S.; Gati, E.; Weissbuch, I.; Lahav, M. J. Am. Chem. SOC.1982,104, 4610-4617, (2) Addadi. L.; Berkovitch-Yellin. Z.; Weissbuch, I.; Mil, J.; Shimon, L. J. W., Lahav, M.; Leiserowitz, L. Angew. Chem., Int. Ed. Engl. 1985,24, 46f485. (3) Grases, F.;March, J. G. Trends Anal. Chem. 1991,10, 190-195. (4) Grases. F.; Genestar, C. Talanta 1993,40, 1589-1593. (5) Grases, F.; Costa-Bauza, A; Forteza, R ; March, J. G. Anal. Len. 1994,27, 2781-2787. (6) Ruzicka, J.; Hansen, E. H. Flow Injection Analysis, 2nd ed.; Wiley: New York, 1988. (7) Valcarcel. M.; Luque de Castro, M. D. Flow Injection Analysis. Principles and Applications; Ellis Horwood, Chichester, 1987. (8) Hanaoka, N.; Tamaka, H.; Nakamoto, A; Takada, M. Anal. Chem. 1991, 63, 2680-2685. (9) Luo, P.; Zhang, F.; Baldwin, R P. Anal. Chem. 1991,63, 1702-1707. (10) Tsai, H.; Weber, S . G. J. Chromatogr. 1991,542, 343-350. (11) Pohlmann, A; Stamm, W. W.; Kusakabe, H.; Kula. M. R Anal. Chim. Acta 1990,235, 329-335. 0003-2700/95/0367-3319$9.00/0 0 1995 American Chemical Society
tion of diphenhydraminelz and, more frequently, sulfate.13 Continuous precipitation systems integrated in an FI manifold including an atomic absorption detector have frequently been employed for the preconcentration of metal traces14 and the indirect determination of organic compounds.15 Nos et al. developed an unsegmented flow system for multidetection using a single detector where the sample plug was confined for repeated passage through the detector; the kinetic curve thus obtained afforded calculation of partial reaction orders and rate constants.16 This paper reports an FI system for the discrimination of L-lysine enantiomers by multidetection with a single detector. The use of an open-closed system permits the derivation of the kinetic parameters that iduence the crystallization of L-glutamic acid. The presence of L-lysine in the sample inhibits crystallization of the acid. The proposed method permits the selective determination of L-lysine in the presence of D-lysine and other amino acids with no need for a prior separation. EXPERIMENTAL SECTION Reagents. L-Glutamic acid, L-lysine, and the other amino acids used were purchased from Sigma (St. Louis, MO). The solvents (2-propanol, ethanol, methanol, and acetonitrile), sodium hydroxide, and hydrochloric acid were obtained from Merk (Darmstadt, Germany). Glass beads (Sigma), 425-600 pm in size, were used to construct a single bead string reactor (SBSR). L-Lysine (2 g/L) and L-glutamic acid (4 g/L) solutions were prepared in Milli-Q water and remained stable for at least a week. Apparatus. A Unicam 8625 W/vis spectrophotometer connected to a Radiometer REC-80 Servograph recorder and furnished with a Hellma flow-through cell (10 mm light path, l mm i.d., 18 pL inner volume) was used. The instrument was set at 550 nm for turbidimetric measurements. The continuous flow system used comprised two Gilson Minipuls-2 peristaltic pumps, two Rheodyne 5041 injection valves, and PTFE tubing of 0.5 mm i.d. for coils. Poly(viny1 chloride) and Solvailex pumping tubes were used for aqueous and organic solutions, respectively. Pharmaceutical Preparations. A volume of 1 mL of liquid sample [drinking ampule (Acticinco; Pierre Fabre S.A.E., Barcelona, Spain), oral drops (Crecibaby; Sociedad Espafiola de Especialidades Fhnacc-Terapeuticas S A , Barcelona, Spain), and (12) Martinez-Calatayud.J.; Snchez-Sampedro, A; Navasquillc-%on. S. Analyst 1990,115. 855-858. (13) Filha, M. M. S.; Reis, B. F.; Krug, F. J.; Collins, C. H.; Baccan, N. Talanta 1993,40, 1529-1534. (14) Santelli, R. E.; Gallego. M.; Valcarcel, M. Anal. Chem. 1989,61, 14271430. (15) Eisman, M.: Gallego. M.; Valcarcel, M. Anal. Chem 1992,64, 1509-1512. (16) Nos, A; Luque de Castro, M. D.; Valcarcel, M. Anal. Chem. 1985,57, 1803-1809.
Analytical Chemisfty, Vol. 67, No. 18, September 15, 7995 3319
. r
2-PROPANOL r
D
sv
I
vr
PI
SAMPLE
n
p2
200 cm
Figure 1. Schematic diagram of the setup used for the continuous determination of L-lysine: (A) introduction of sample or blank and 2-propanol and (B)multidetection of signal. P, pump; SBSR, single bead string reactor; IV, injection valve; W, waste; R, reactor; 0,detector; SV, switching valve.
syrup (Trimetabol; J. Uriach & Cia SA, Barcelona, Spain)] was disolved in 50 mL of Milli-Q water. For tablet formulations (Aminoacidos Esenciales; Nutri Sport S A , Barcelona, Spain), 10 tablets were ground in a mortar to a fine mesh, and a portion of -1 g of the resulting powder was accurately weighed. The powder was transferred into a 100 mL vessel, diluted to 50 mL with Milli-Q water, and stirred magnetically for 1h. The solution was filtered and the residue washed with Milli-Q water, and then the filtrate was diluted to volume with water in a 250 mL calibrated flask. An amount of 3 g of protein-vitamin supplement powder (Gevral Proteina; Cyanamid Iberica S.A., Madrid, Spain) was mixed with 50 mL of water in a 100 mL vessel, and the mixture was stirred magnetically for 1h; subsequently, the procedure was continued as for the tablet formulations. For continuous flow analysis, aliquots of 50-250 pL of these solutions (containing about 100 pg of L-lysine) were placed in 10 mL calibrated flasks containing 2.6 g/L L-glutamic acid and diluted to the mark. Procedure. The manifold used for enantiomer discrimination is depicted in Figure 1. In the first step (Figure lA), the pharmaceutical sample or standard solution, containing 0.5-20 mg/L L-lysine and 2.6 g/L L-glutamic acid, was continuously introduced into the flow system at 0.3 mL/min and merged with a stream of 2-propanol circulated at 1.5 mL/min. Mixing of both phases was boosted by the SBSR reactor used (30 cm long, 0.8 mm i.d.). The mixture was continuously circulated through the loop of the injection valve 0. Simultaneously, the open-closed flow loop was filled with carrier (2-propanol) via valve SV. In the second step (Figure lB), the sample-2-propanol mixture was 3320 Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
injected via IV into the 2-propanol carrier at 0.9 mL/min, and SV was switched in order to close the loop. Changes in the reaction mixture were monitored at 550 nm until physical and chemical equilibrium of the system was reached. After the kinetic curve was recorded, SV was switched again, and the open-closed system was flushed with water instead of 2-propanol. RESULTS AND DISCUSSION Signal Proae: Measurement Modes. Figure 2 shows two typical signals obtained by using the manifold of Figure 1 in the presence (sample) and absence (blank) of L-lysine. The initial portion of the signal (0-tJ in Figure 2A corresponds to the homogenization of the closed loop. Signal changes arose from changes in the refractive index from pure 2-propanol to the 5:l 2-propanol-water mixture. Crystallization of L-glutamic acid started at tz. The induction period (t3) was calculated graphically as shown in Figure 2A. In the abscence of L-lysine (Figure 2B), crystallization started before the loop contents were fully homogeneous, so tz was shorter than in Figure 2A For this reason, tl and tz were indistinguishable in the blank signal. There were three ways of drawing analytical information from the signals. The starting crystallization time ( t ~could ) not be used for this purpose because, at low concentrations of L-lysine, signal changes were the result of two processes (homogenization and precipitation) and hence led to poor precision. Also, the slope of the rising portion of the spanning signal crystallization at variable concentrations of L-lysine provided inadequate differences for implementation of the analytical method. The best choice was
h
A)
z
-
40-
Y
2 min
30
n c
.-0
~
20-
L
0
I 0
5
10
15
20
L-lyslne concentration ( mg/L )
Figure 3. Calibration graphs for L-lysine at variable concentrations of L-glutamic acid.
‘3
T I M E (min)
Figure 2. Typical signals for L-glutamic acid crystallization in the presence (sample) and absence (blank) of L-lysine. The sample solution (A) contained 2.6 g/L L-glutamic acid plus 7.0 mg/L L-lysine, while the blank (B) contained 2.6 g/L L-glutamic acid only. fl,
homogenization time; period.
f2,
starting crystallization time;
f3,
induction
thus the induction period, which was calculated in the presence and absence of L-lysine. Chemical Variables. The effects of chemical variables were studied by using a continuous flow system similar to that depicted in Figure 1. Two aqueous solutions containing 3 g/L L-glutamic acid (blank) or 3 g/L L-glutamic acid plus 10 mg/L L-lysine (sample) were employed. Supersaturated solutions of L-glutamic acid were prepared by altering the solvent composition. Thus, addition of an organic solvent to aqueous solutions of this amino acid allowed us to prepare unstable supersaturated solutions. For this purpose, various organic solvents [methanol, ethanol, 7:3 (v/v) ethanolacetonitrile, and 2-propanol1 were continuously mixed with an aqueous solution of L-glutamic acid to induce crystallization of the amino acid. Crystallization in methanol and ethanol was rather slow (about 40 and 15 min, respectively). On the other hand, crystallization in the 7:3 ethanol-acetonitrile mixture or 2-propanol took only 5 min. Simultaneous experiments with L-glutamic acid solutions containing L-lysine (sample) revealed that L-glutamicacid crystallization proceeded similarly in mixtures with the organic solvents tested and in the absence of L-lysine; methanol and ethanol were discarded owing to the long time required for the crystallization of L-glutamic acid. 2-Propanol was thus the optimal choice because Solvaflex pumping tubes are more resistant to it than they are to the ethanol-acetonitrile mixture. 2-Propanol was also selected as the carrier for the open-closed system as it furnished a suitable medium for crystallizing L-glutamic acid. The L-glutamic acid concentration was optimized by running several calibration graphs for L-lysine at variable concentrations
of L-glutamic acid between 2 and 3 g/L. As can be seen in Figure 3, the length of the induction period increased with decreasing L-glutamic acid concentration; at low L-glutamic concentrations, the crystallization time for the amino acid increased more markedly in the presence of L-lysine than in its absence, which resulted in greater sensitivity. A compromise between sensitivity and sample throughput was made by choosing an L-glutamic acid concentration of 2.6 g/L for further experiments. The sensitivity of the method increased with use of a lower L-glutamic acid concentration. However, this was unnecessary because the L-lysine concentrations in the pharmaceutical preparations studied were at the gram-per-liter level, so the proposed FI system was also appropriate for sample dilution (the sample was diluted &fold in 2-propanol before injection into the closed loop). In aqueous solutions, amino acids are present as cations, zwitterions, or anions depending on the pH. We therefore studied the effect of pH between 2 and 5 (adjusted with 0.1 mol/L HCl or 0.1 mol/L NaOH) on the crystallization of L-glutamic acid. The optimal pH was estimated to be from 3 to 4; outside this range, crystal growth of L-glutamic acid was significantly delayed (the pK1 and pK2 values for L-glutamic acid are 2.19 and 4.25, respectively); pH 3.4, obtained by dilution of the blank and sample with water, was finally selected. Optimization of the Flow Discrimination System. The flow variables influencing the performance of the proposed continuous flow system were optimized by introducing an aqueous solution containing 2.6 g/L L-glutamic acid (blank) or 2.6 g/L L-glutamic acid plus 10 mg/L L-lysine. The sample was merged with a 2-propanol stream and the mixture passed through an SBSR for hom~genization.’~ The sample flow rate was kept at 0.3 mL/min, while that of 2-propanol was varied between 0.3 and 2.0 mWmin. Increasing the 2-propanol flow rate had two opposing effects, viz., diluting the sample (which delayed crystallization of L-glutamic acid) and increasing the proportion of 2-propanol (which boosted crystallization of the amino acid). Because the induction period increased with increasing flow rate, the diluting effect prevailed over the concentrating effect. Below 1mL/min, throughput and reproducibility were both lower. A 2-propanol flow rate of 1.5 mL/ min was thus selected as a compromise between adequate (17) Reijn, J. M.; Poppe, H.;van der Linden, W. E. Anal. Chem. 1984,56,943948.
Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
3321
25
-
-Ee
I\
A
20
v
0
8n e IOC
I
.-08
15
n
C
.=0
\
10
0
z o .{l5
-
70
without
3
-
L-lysine
0
C
5
0
120
170
220
270
320
25
75
125
Injected volume ( pL )
175
225
275
coil length (cm)
Figure 4. Effect of the injected volume on the crystal growth of L-glutamic acid. Sample, 10 mg/L L-lysine and 2.6 g/L L-glutamic acid; blank 2.6 g/L L-glutamic acid.
Figure 5. Influence of the coil length (R) on the crystallization of L-glutamic acid. The sample and blank concentrations were the same as in Figure 4.
reproducibility, sensitivity, and throughput. The influence of the length of the SBSR reactor on the homogeneity of the sample2-propanol mixture was studied over the range 10-50 cm. Above 25 cm, homogenization was complete because the induction period obtained in several determinations of the same sample was quite reproducible. An SBSR reactor of 30 cm was thus selected. The injection valve (TV) could, in principle, be placed at three different positions in the open-closed system (Figure 1): before the SV valve for introduction of 2-propanol; between the switching valve (SV) and pump 2; and between pump 2 and the detector. The best results were obtained by placing the IV between pump 2 and the detector (see Figure l), because the flow rate of 2-propanol was more uniform after the peristaltic pump and unloading of the loop contents and the subsequent operations were more reproducible. The effect of the flow rate of carrier (2-propanol) in the open-closed system was studied over the range 0.7-1.1 mL/ min. At low flow rates, the induction period in the crystal growth of L-glutamic acid was somewhat shorter because of the increased sample dispersion in the system. Above 0.85 mL/min, the signal remained virtually constant in the presence and absence of L-lysine because the dispersion was insignificant. A carrier flow rate of 0.9 mL/min was thus chosen. The effect of the injected volume of the sample-2-propanol mixture on L-glutamic acid crystallization at a constant flow rate of carrier (0.9 mL/min) was studied between 70 and 325 ,uL. As can be seen in Figure 4, the induction period decreased with increasing injected volume through an increase in the amount of L-glutamic injected. The difference in crystal growth of L-glutamic acid in the presence and absence of L-lysine was higher at low injected volumes, which resulted in increased sensitivity. An injected volume of 100 ,uL was thus chosen as a compromise between adequate sensitivity and throughput. The influence of the length of the coil located before the detector (R in Figure 1) on L-glutamic acid crystallization was studied between 25 and 300 cm. As can be seen in Figure 5, increasing coil lengths increased the volume of the closed system and hence dilution, which delayed crystallization of L-glutamicacid; the effect was more marked in the presence of L-lysine than in its absence. A coil length of 200 cm was thus chosen for further experiments. The effect of temperature on the crystallization process was studied over the range 10-40 "C by immersing coil R in a water bath. The induction period for L-glutamic acid crystallization
Table 1. Tolerated Limits for Amlno Acids in the Determinationof L-Lysine (5 mg/L)
3322 Analytical Chemisfry, Vol. 67,No. 78,September 75, 7995
amino acids L-L~u,L-Gln, L-AQ L-Phe, L-Thr, L-Ser, L-Ile, L-Met, L-Val, L-Asn, L-CYS,M a , L-Tyr D-LYS L-His L-Om
maximum tolerated
amount (mg/L) '100
100 15 4
remained constant above 15 "C in the presence and absence of L-lysine, so the open-closed system was maintained at 25 "C. Determination of L-Lysine. Under the selected chemical and flow conditions, the manifold depicted in Figure 1 was used to run a calibration gi-aph for the determination of L-lysine. The graph was constructed by introducing solutions containing 2.6 g/L L-glutamic acid and variable concentrations of L-lysine between 0.5 and 20 mg/L in Milli-Q water. The equation for the standard curve was Y= 5.05 1.OlX (Y,induction period, in min; X,L-lysine concentration, in mg/L) , the intercept representing the induction period in the crystallization of the L-glutamic acid solution used as blank. The correlation coefficient (n = 8) was 0.998. The detection limit, calculated as 3 times the standard deviation of the induction period obtained in 10 determinations of the same blank (with 2.6 g/L L-glutamic acid), was 0.3 mg/L. The precision, calculated from 11samples containing 10 mg/L L-lysine,was 2.5% as relative standard deviation. Interferences. The effect of other amino acids on the crystallization of L-glutamic acid was studied. The amino acids selected for this purpose were those commonly accompanying L-lysine in pharmaceutical products. The tolerated limits for the amino acids in the determination of 5 mg/L of L-lysine are listed in Table 1. None was found to interfere, except for diaminocarboxylic acids similar to L-lysine (L-histidine and L-ornithine),which were tolerated at concentrations only 3 and 0.8 times the L-lysine concentration. The tolerated concentration of D-lysine was 20fold that of L-lysine. Synthetic samples containing 5 mg/L L-lysine and variable D-lysine concentrations (25, 50, 75, and 100 mg/L) were used for the determination of L-lysine in the presence of D-lySine. The recovery obtained was almost 100%in all cases. Analysis of Pharmaceutical Products. The proposed method was applied to the determination of L-lysine in commercially
+
Table 2. Determination of L-Lysine in Pharmaceutical Products
sampleu
found6 @/L)
drinking ampule oral drops syrup tablets powder supplement
39.1 i 1.1 98.8 i 3.0 51.6 i 1.8 11.8 f 0.3c 3.2 f 0.lc
Table 3. Recovery of L-Lysine Added to Pharmaceutical Products
nominal contents stated by the manufacturer (g/L) 40.0 100.0 50.0 11.03c 3.33'
sample drinking ampule
oral drops
Manufacturer samples are described under Pharmaceutical Preparations. Mean f standard deviation (n = 5). As % w/w.
*
syrup
available pharmaceutical products by using the manifold depicted in Figure 1. We analyzed five pharmaceutical products in various forms: drinking ampules, oral drops, syrup, tablets, and nutritional supplement powder. Variable volumes of the previously prepared samples (see Experimental Section) were placed in 10 mL calibrated flasks containing 2.6 g/L L-glutamic acid and diluted to the mark. Table 2 lists the results obtained in five individual determinations of L-lysine and their standard deviations. All d u e s were consistent with the nominal contents. Recoveries were calculated by adding variable amounts of L-lysine to each pharmaceutical product prior to preparation in such a way that the final L-lysine concentration was 2.5 or 5.0 mg/ L. The recoveries thus obtained (Table 3) ranged from 96.6 to 102.7%. CONCLUSIONS This is the first reported example of the use of adsorption during crystalline growth for analytical purposes in the FI technique. The main advantages of this technique over manual methods, viz., ease of automation and handling, are clearly reflected in the proposed application, which is also more sensitive than its manual counterpart4 and similarly selective. This can be ascribed to the fact that the crystallization rate of a substance is controlled by a combination of structurally related factors and
tablets
powder supplement
contenP mg/L
added (mg/L)
found" (mg/L)
recovery
4.9 f 0.1 4.9 i.0.1 9.8 i 0.3 9.8 3~0.3 4.8 f 0.1 4.8 f 0.1 9.4 f 0.3 9.4 f 0.3 5.1 i.0.1 5.1 f 0.1 10.3 f 0.4 10.3 f 0.4 5.5 i 0.2 5.5 k 0.2 11.0 f 0.3 11.0 f 0.3 4.8 f 0.1 4.8 i 0.1 9.7 i 0.2 9.7 f 0.2
2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0
7.3 f 0.2 10.0 f 0.3 12.0 f 0.4 14.8 f 0.4 7.4 f 0.2 9.8 f 0.2 11.5 f 0.3 14.5 f 0.4 7.5 f 0.2 10.2 f 0.3 12.6 f 0.3 15.7 f 0.4 7.9 f 0.2 10.6 f 0.3 13.2 f 0.4 16.3 f 0.4 7.5 0.2 9.8 f 0.3 12.4 f 0.3 14.3 f 0.4
98.6 101.0 97.6 100.0 101.4 100.0 96.6 100.7 98.7 101.0 98.4 102.6 98.8 100.9 97.8 101.9 102.7 100.0 101.6 97.3
(%)
Mean f standard deviation (n = 3).
other, external factors, such as the solvent, temperature, supersaturation, and stimng rate. In order to avoid exceedingly long substrate crystallization times in the proposed FI method, we used 2-propanol rather than the ethanol employed as solvent in the manual method. ACKNOWLEDGMENT The Spanish CICyT is acknowledged for financial support awarded for the realization of this work (Grant PB94-0450). Received for review March
13, 1995. Accepted June 21,
1995.B AC950254G Abstract published in Advance ACS Abstracts, August 1, 1995.
Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
3323
