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Chemiluminescent determination of tryptophan in a flow injection system
ANALmcA CHIMICA
ACTA ELSEVIER
Analytica
Chimica Acta 317 (1995) 233-237
Chemiluminescent determination of tryptophan in a flow injection system Abdulrahman A. Alwarthan Chemistry Department, College of Science, King Saud UnnCersify,P.O. Box 2455, Riyadh-11451, Saudi Arahiu Received 25 November
1994; revised 13 July 1995; accepted 19 July 1995
Abstract A method is described for the determination of tryptophan The method is rapid and precise and solutions can be analysed 0.45% at 12.5 pg ml-’ and a limit of detection of 0.1 monosaccharides which may be present along with tryptophan Keywords: Chemiluminescence;
Flow injection; Tryptophan;
in which it reacts with cerium(IV) in 0.15 M sulphuric acid. at a rate of 211 h-i with a relative standard deviation of ca. pg ml -I. The effect of some common amino acids and in tissue or protein hydrolysates has been investigated.
Cerium
1. Introduction Tryptophan [L-2-amino-3-(indol-3-yl)propionic acid] is a vital constituent of proteins and is indispensable in human nutrition for establishing and maintaining a positive nitrogen balance. As an essential amino acid, it is considered exceptional in its diversity of biological functions. In particular, it is the precursor of the neurotransmitter serotonin [l-3]. The formation of tryptophan-derived antinutritional and potentially toxic compounds occurs during processing and storage of food and feedstuffs. Manufacturers of nutritional products are often required to demonstrate an adequate tryptophan content in their products [2,4]. Modifications of existing analytical methods and the development of new methods are important areas of tryptophan research. Although there are several methods available for its determination, they are tedious and the results often uncertain. Many of the spectrophotometric methods involve 0003.2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003.2670(95)00390-8
condensation with an aldehyde such as 4-dimethylaminobenzaldehyde and oxidation of the product. However, it is difficult to control the oxidation step to give a stable colour and reproducible results [5]. Methods have also been proposed which are based on oxidation with nitrous acid and coupling with N-(l-naphthyl)ethylenediamine [6] or nitration [l]; such chemical derivatizations are often tedious and completeness of reaction may be a problem. Although tryptophan is normally determined after alkaline hydrolysis of proteins, improvements to prevent oxidative destruction of its indole group during acid hydrolysis have also been made [7,8]. 2-Hydroxy-5nitrobenzyl bromide is thought to react with all tryptophan residues in intact proteins [9] but low and inconsistent results have also been reported. Spectrophotometric [lo] and fluorimetric [l 1I methods utilizing the ultraviolet WV) absorption and native fluorescence of tryptophan, respectively, are not selective or sensitive. Recently, Chen et al. [12] have
234
Ad. Alwarthan /Analytica
studied the chemiluminescent characteristics of some indole derivatives, including tryptophan. The method developed by Chen et al. for the determination of indole compounds by CL is sensitive and selective, and could possibly constitute a specific method for the determination of tryptophan [12]. There is a constant search for simple, reliable, automated and semiautomated methods for the rapid quantification substances of biochemical and pharmaceutical interest. Analytical procedures using chemiluminescence (CL) combine the advantages of speed and sensitivity and have been used for the determination of drugs [13-161. It is well known that methods based on CL are capable of much better limits of detection than spectrophotometric methods. This is achieved by the almost complete absence of background emission. Flow injection (FI) is an easy and inexpensive way to automate analytical determinations and can be applied in several situations to reduce reagent consumption and increase the repeatability, selectivity and accuracy of the determinations. Since FI allows good reproducibility of both sample and reagent mixing, and these characteristics are essential for precise CL studies. This paper describes a flow-injection method for determination of tryptophan based on its reaction with Ce(IV) in acidic medium in a FI system.
Chimica Acta 317 (1995) 233-237
ml mid
S
Ce (IV) 3.4 5x 10-SM H2SQl
3.4
5xl0-3M
I-J P Fig. 1. FI manifold for the chemiluminescent determination of tryptophan: P, peristaltic pump; S, sample port; L, luminometer; W, waste.
two basic components, the detector housing and the flow-through system, which allow mixing of the reacting solutions just before the detector. Both were assembled from parts in our laboratory and have been described in detail elsewhere [17]. 2.3. Recommended procedure for calibration The FI manifold described in Fig. 1 was used. A loo-p1 portion of tryptophan solution was injected into a stream of cerium(IV) solution which was combined with a stream of 5 X 10m3 M sulphuric acid before the injection port, and the resulting peak height was measured. A calibration graph was prepared by plotting the peak height versus tryptophan concentration over the range 1.6-12.5 ,ug ml-‘.
2. Experimental 2.1. Reagents All chemicals were of analytical-reagent grade and the solutions prepared with doubly distilled water. m-Tryptophan (Fluka, Buchs, Switzerland, 5 X 10v3 M stock solution) was prepared by dissolving 0.1021 g of tryptophan in 100 ml of distilled water. Working solutions were prepared daily by suitable dilution. An aqueous 5 X 10e3 M cerium(IV) sulphate (BDH, Poole, UK) was prepared by dissolving 1.01 g of the chemical in 500 ml of 0.15 M sulphuric acid with further dilutions as required. 2.2. Instrumentation The chemiluminescent measurements were made with a continuous-flow CL analyser which features
3. Results and discussion Tryptophan reacts with cerium(IV) in acidic solutions to give CL. The manifold schematized in Fig. 1 was used to investigate the effect of chemical variables on the peak height. The studies were performed by altering each variable in turn while keeping the others constants (univariate method). The composition of the Ce(IV> stream had a strong influence on the peak height. Four different acids (HCl, HClO,, HNO, and H,SO,) were tested in order to ascertain which was the most suitable. Nitric acid could not be used owing to the inhibitory effect of nitrate on the CL of the Ce(III) produced (see below) [17]. The emission intensity obtained in presence of perchloric or hydrochloric acid was
AA. Alwarthan /Analytica
235
Chimica Acta 317 (1995) 233-237
ml min-’ produced a maximum peak height CL at ca.7.5 ml min-’ (3.75 ml min-’ for each reagent). A total flow rate of 6.8 ml min-’ was chosen because higher rates led to both high pressures in connectors and greater consumption of reagents, with little gain in sensitivity. The variation of the CL emission with the injected sample volume in the lo-500 ~1 range was studied. The results, also given in Fig. 3, show maximum intensity at 100 ~1. For larger sample volumes (> 200 ~1) mixing of the sample with the reagents was insufficient, and broadening of the peaks appeared. Thus a 100 ~1 sample loop was chosen. The selected values for the FI variables, therefore, are flow rate, 6.8 ml mini’ and injection volume, 100 ~1. 3.2. Emitting species
[Ce (IV)]
/M
Fig. 2. Influence of Ce(IV) concentration on peak height. Injected tryptophan solution (250 ~0, 25 Fg ml- ’
slightly less than that obtained in the presence of sulphuric acid. Therefore, sulphuric acid was chosen as a diluent for Ce(IV). The effect of sulphuric acid concentration in the range 1 X 10e3-0.2 M was also studied and 0.15 M was chosen because it gave the greatest peak height. The effect of the concentration of the Ce(IV) stream on the CL intensity is shown in Fig. 2. The greatest CL response was obtained at 5 X 10-j M, which was selected for further studies. 3.1. Optimization
Cerium(III), which is formed by the reduction of cerium (IV), is a well known fluorescent ion 1181 and therefore is a possible CL emitter. However, other reducing agents such as arsenite, titanium(II0, hexacyanoferrate(II1, folic acid, ascorbic acid and tartaric acid were injected instead of tryptophan, but did not
of the FI variables
Because the oxidation of tryptophan by Ce(IV) under the recommended conditions is very fast, a simple manifold (Fig. 1) using a 5 X lop3 M cerium(IV) sulphate solution in 0.15 M sulphuric acid as carrier was found to be the most suitable. Optimization of the different variables influencing the FI system was carried out using the univariate method. Fig. 3 shows the effect of the flow rate and sample volume. The effect of flow rate is important. Too low or too high flow rates result in the CL occurring before or after the flow cell, respectively. Variation of the total flow rate over the range 3.1-9
201
I
loo
I
J
200
Sample
300
500
volume (@I
I
I
1
1
3
5
7
9
Flow-rate
400
(ml mine’ )
Fig. 3. Effect of (0) sample volume, (a) flow-rate on peak height. The arrows mark the values of the parameters selected.
AA. Alwarthan /Analytica
236
12.5
Chimica Acta 317 (1995) 233-237 Table 1 Determination of tryptophan in pure samples by the spectrophotometric (Sp) and CL methods Claimed (pgml-‘) 1.6 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
Cow. found ( pg ml-’ ) a
Recovery (o/o)
SP
CU f S.D.) b
Sp
CL
1.58 2.0 2.48 3.05 3.5 3.95 4.47 4.97 5.35 6.0 6.6 6.90 7.44 8.1
1.62 f 0.33 2.01 f 0.10 2.5 +_0.25 3.0 + 0.40 3.5 rto.45 3.97 +_0.38 4.46 f 0.26 4.96 f 0.22 5.45 *0.31 6.01 i-o.15 6.5 + 0.42 6.95kO.16 7.48 + 0.29 7.98kO.39
98.8 100.0 99.2 101.7 100.0 98.8 99.3 99.4 97.3 100.0 101.5 98.6 99.2 101.3
101.3 100.5 100.0 100.0 100.0 99.3 99.1 99.2 99.1 100.2 100.0 99.3 99.7 99.8
a Average of 4 injections. b Standard deviation.
Time Fig. 4. Typical recording for the determination of tryptophan. numbers on the peaks are fig ml-’ of tryptophan injected.
The
This method was compared with the spectrophotometric nitrous acid method [l] by measuring the tryptophan content of pure solutions covering a wide range of concentrations (Table 1). The results obtained by the two methods are in good agreement. 3.4. Interferences
generate CL with cerium(IV). This suggests that the simple formation and decay of excited cerium(II1) is unlikely to be the cause of the emission, and an oxidation product of tryptophan is a more likely emitter. 3.3. Determination
of tryptophan
Once chemical and instrumental variables were optimized to achieve maximum CL emission, a series of standard solutions containing 1.6, 3.1, 6.25 and 9.25 and 12.5 pg ml-’ tryptophan was pumped, each as five replicates (Fig. 4) to test the linearity of the calibration graph. A plot of the emission intensity versus concentration of tryptophan in the sample injected was linear over the range 1.6-12.5 pg ml-‘. The statistical study performed on 13 replicate samples at 12.5 pug ml-’ yielded a relative standard deviation level of 0.45%. The detection limit (2 X noise) was 0.1 pg ml-’ (100 ~1, sample).
An interference study aimed at the determination of tryptophan was performed. Samples containing a fixed concentration of tryptophan (10 pg ml-‘) and various concentrations of foreign substances were injected into the FI system. The results are shown in Table 2. Cysteine, one of the essential amino acids, interferes severely in tryptophan determination, which should be removed by chromatography before the analysis. No interference was observed from other amino acids such as alanine, glutamine or histidine. Methionine interfered at high concentration (2 50 pg ml-l), but the effect is reduced by dilution. Arabinose, fructose, galactose, glucose, glucosamine and nicotinamide showed no interference and gave recoveries in the range 96-102%. Ascorbic acid gave a decreased response. It was found to react with Ce(IV). The interference can only be decreased by large dilutions but such dilutions would also de-
AA. Alwarthan/Analytica Table 2 Recovery of 10 &g ml-’ tryptophan from solutions various excipients at different concentrations Claimed
containing
the method can be automated an automatic sampling unit.
0.5 Kg ml-’
Acknowledgements
231
fully by the addition of
Recovery (%) 50 pg ml-’
D-C- )-Arabinose D-C-)-Fructose p( + )-Galactose ~-(+)-Glucose D( + )-Glucosamine L-( + )-Alanine L-c - )-Glutamine L-Histidine L-Methionine L-Cysteine L-( + )-Ascorbic acid Nicotinic acid Nicotinamide 2-Mercaptoethanol Casein
Chimica Acta 317 (1995) 233-237
99.6 101.9 91.1 95.9 91.1 101.5 98.9 101.5 88.7 15.0 28.2 92.1 98.1 93.9 90.2
10 I.Lg ml-’
5 pg ml-’
This work was supported partially by a Research Fund Grant-1994-from The Royal Society of Chemistry (London).
References 95.6 43.5 75.8 100.8
66.1 86.3
94.8 99.6
96.8 100.0
crease the tryptophan concentration. Hence, the method cannot be used for the determination of tryptophan in biological samples without pretreatment. Nicotinic acid, nicotinamide, 2-mercaptoethano1 and casein interfered slightly when they were present at high concentration (2 50 pg ml-‘), but the effect can easily be decreased by dilution.
4. Conclusions The proposed CL-F1 method has been shown to be applicable to the determination of tryptophan. The CL measurement was readily carried out in a flow injection system with good precision and high sample throughput. The method has a lower detection limit than spectrophotometric methods due to the absence of background signals, The reagents and instrumentation for this analysis are inexpensive and
[l] K.K. Verma, A. Jain and .I. Gasparic, Talanta, 35 (1988) 35. [2] T. Simat, K. Meyer and H. Steinhart, J. Chromatogr., 661 0994) 93. [3] A.C. Moffat, J.V. Jackson, MS. Moss and B. Widdop, Clarke’s Isolation and Identification of Drugs, 2nd edn., The Pharmaceutical Press, London, 1986, p. 1056. [4] S.E. Garcia and J.H. Baxter, J. AOAC Int., 75 (1992) 1113. [5] P. Lindroth and K. Mopper, Anal. Chem., 51 (1979) 1667. [6] A.K. Goswami, Analyst, 99 (1974) 657. [7] H. Matsubura and R.M. Sasaki, Biochem. Biophys. Res. Commun., 35 (1969) 175. [8] T.Y. Liu and Y.H. Chang, J. Biol. Chem., 246 (197 I) 2842. [9] R.H. Horton and D.E. Koshland, J. Am. C’hem. Sot., 87 (1965)
1126.
[lo] W.L. Bencze and K. Schmid, Anal. Chem., 29 (1057) 1193. [ll] D.E. Duggan and S. Udenfriend, J. Biol. Chem.. 223 (1956) 313. [12] G.-N. Chen, X.-Q. Xu, J.-P. Duan, M.-M. Hc and F. Zhang, Analyst, 120 (1995) 1699. [13] P. Vinas, I. Lopez Garcia and J.A. Martinez Gil. J. Pharm. Biomed. Anal., 11 (1993) 15. 1141 S. Luterotti and D. Maysinger, J. Pharm. Biomed. Anal., 12 (1994) 1083. 1153 A.A. Alwarthan, S.A. Al-Tamrah and A.A. Akel. Anal. Chim. Acta, 292 (1994) 201. [16] A.A. Alwarthan, S.A. Al-Tamrah and A.A. Al-Akcl. Anal. Sci., 10 (1994) 449. [17] A.A. Alwarthan and A. Townshend, Anal. Chim. Acta. 185 (1986) 329. [18] P. Cukor and R.P. Weberling, Anal. Chim. Acta, 41 (1968) 404.
ACTA ELSEVIER
Analytica
Chimica Acta 317 (1995) 233-237
Chemiluminescent determination of tryptophan in a flow injection system Abdulrahman A. Alwarthan Chemistry Department, College of Science, King Saud UnnCersify,P.O. Box 2455, Riyadh-11451, Saudi Arahiu Received 25 November
1994; revised 13 July 1995; accepted 19 July 1995
Abstract A method is described for the determination of tryptophan The method is rapid and precise and solutions can be analysed 0.45% at 12.5 pg ml-’ and a limit of detection of 0.1 monosaccharides which may be present along with tryptophan Keywords: Chemiluminescence;
Flow injection; Tryptophan;
in which it reacts with cerium(IV) in 0.15 M sulphuric acid. at a rate of 211 h-i with a relative standard deviation of ca. pg ml -I. The effect of some common amino acids and in tissue or protein hydrolysates has been investigated.
Cerium
1. Introduction Tryptophan [L-2-amino-3-(indol-3-yl)propionic acid] is a vital constituent of proteins and is indispensable in human nutrition for establishing and maintaining a positive nitrogen balance. As an essential amino acid, it is considered exceptional in its diversity of biological functions. In particular, it is the precursor of the neurotransmitter serotonin [l-3]. The formation of tryptophan-derived antinutritional and potentially toxic compounds occurs during processing and storage of food and feedstuffs. Manufacturers of nutritional products are often required to demonstrate an adequate tryptophan content in their products [2,4]. Modifications of existing analytical methods and the development of new methods are important areas of tryptophan research. Although there are several methods available for its determination, they are tedious and the results often uncertain. Many of the spectrophotometric methods involve 0003.2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003.2670(95)00390-8
condensation with an aldehyde such as 4-dimethylaminobenzaldehyde and oxidation of the product. However, it is difficult to control the oxidation step to give a stable colour and reproducible results [5]. Methods have also been proposed which are based on oxidation with nitrous acid and coupling with N-(l-naphthyl)ethylenediamine [6] or nitration [l]; such chemical derivatizations are often tedious and completeness of reaction may be a problem. Although tryptophan is normally determined after alkaline hydrolysis of proteins, improvements to prevent oxidative destruction of its indole group during acid hydrolysis have also been made [7,8]. 2-Hydroxy-5nitrobenzyl bromide is thought to react with all tryptophan residues in intact proteins [9] but low and inconsistent results have also been reported. Spectrophotometric [lo] and fluorimetric [l 1I methods utilizing the ultraviolet WV) absorption and native fluorescence of tryptophan, respectively, are not selective or sensitive. Recently, Chen et al. [12] have
234
Ad. Alwarthan /Analytica
studied the chemiluminescent characteristics of some indole derivatives, including tryptophan. The method developed by Chen et al. for the determination of indole compounds by CL is sensitive and selective, and could possibly constitute a specific method for the determination of tryptophan [12]. There is a constant search for simple, reliable, automated and semiautomated methods for the rapid quantification substances of biochemical and pharmaceutical interest. Analytical procedures using chemiluminescence (CL) combine the advantages of speed and sensitivity and have been used for the determination of drugs [13-161. It is well known that methods based on CL are capable of much better limits of detection than spectrophotometric methods. This is achieved by the almost complete absence of background emission. Flow injection (FI) is an easy and inexpensive way to automate analytical determinations and can be applied in several situations to reduce reagent consumption and increase the repeatability, selectivity and accuracy of the determinations. Since FI allows good reproducibility of both sample and reagent mixing, and these characteristics are essential for precise CL studies. This paper describes a flow-injection method for determination of tryptophan based on its reaction with Ce(IV) in acidic medium in a FI system.
Chimica Acta 317 (1995) 233-237
ml mid
S
Ce (IV) 3.4 5x 10-SM H2SQl
3.4
5xl0-3M
I-J P Fig. 1. FI manifold for the chemiluminescent determination of tryptophan: P, peristaltic pump; S, sample port; L, luminometer; W, waste.
two basic components, the detector housing and the flow-through system, which allow mixing of the reacting solutions just before the detector. Both were assembled from parts in our laboratory and have been described in detail elsewhere [17]. 2.3. Recommended procedure for calibration The FI manifold described in Fig. 1 was used. A loo-p1 portion of tryptophan solution was injected into a stream of cerium(IV) solution which was combined with a stream of 5 X 10m3 M sulphuric acid before the injection port, and the resulting peak height was measured. A calibration graph was prepared by plotting the peak height versus tryptophan concentration over the range 1.6-12.5 ,ug ml-‘.
2. Experimental 2.1. Reagents All chemicals were of analytical-reagent grade and the solutions prepared with doubly distilled water. m-Tryptophan (Fluka, Buchs, Switzerland, 5 X 10v3 M stock solution) was prepared by dissolving 0.1021 g of tryptophan in 100 ml of distilled water. Working solutions were prepared daily by suitable dilution. An aqueous 5 X 10e3 M cerium(IV) sulphate (BDH, Poole, UK) was prepared by dissolving 1.01 g of the chemical in 500 ml of 0.15 M sulphuric acid with further dilutions as required. 2.2. Instrumentation The chemiluminescent measurements were made with a continuous-flow CL analyser which features
3. Results and discussion Tryptophan reacts with cerium(IV) in acidic solutions to give CL. The manifold schematized in Fig. 1 was used to investigate the effect of chemical variables on the peak height. The studies were performed by altering each variable in turn while keeping the others constants (univariate method). The composition of the Ce(IV> stream had a strong influence on the peak height. Four different acids (HCl, HClO,, HNO, and H,SO,) were tested in order to ascertain which was the most suitable. Nitric acid could not be used owing to the inhibitory effect of nitrate on the CL of the Ce(III) produced (see below) [17]. The emission intensity obtained in presence of perchloric or hydrochloric acid was
AA. Alwarthan /Analytica
235
Chimica Acta 317 (1995) 233-237
ml min-’ produced a maximum peak height CL at ca.7.5 ml min-’ (3.75 ml min-’ for each reagent). A total flow rate of 6.8 ml min-’ was chosen because higher rates led to both high pressures in connectors and greater consumption of reagents, with little gain in sensitivity. The variation of the CL emission with the injected sample volume in the lo-500 ~1 range was studied. The results, also given in Fig. 3, show maximum intensity at 100 ~1. For larger sample volumes (> 200 ~1) mixing of the sample with the reagents was insufficient, and broadening of the peaks appeared. Thus a 100 ~1 sample loop was chosen. The selected values for the FI variables, therefore, are flow rate, 6.8 ml mini’ and injection volume, 100 ~1. 3.2. Emitting species
[Ce (IV)]
/M
Fig. 2. Influence of Ce(IV) concentration on peak height. Injected tryptophan solution (250 ~0, 25 Fg ml- ’
slightly less than that obtained in the presence of sulphuric acid. Therefore, sulphuric acid was chosen as a diluent for Ce(IV). The effect of sulphuric acid concentration in the range 1 X 10e3-0.2 M was also studied and 0.15 M was chosen because it gave the greatest peak height. The effect of the concentration of the Ce(IV) stream on the CL intensity is shown in Fig. 2. The greatest CL response was obtained at 5 X 10-j M, which was selected for further studies. 3.1. Optimization
Cerium(III), which is formed by the reduction of cerium (IV), is a well known fluorescent ion 1181 and therefore is a possible CL emitter. However, other reducing agents such as arsenite, titanium(II0, hexacyanoferrate(II1, folic acid, ascorbic acid and tartaric acid were injected instead of tryptophan, but did not
of the FI variables
Because the oxidation of tryptophan by Ce(IV) under the recommended conditions is very fast, a simple manifold (Fig. 1) using a 5 X lop3 M cerium(IV) sulphate solution in 0.15 M sulphuric acid as carrier was found to be the most suitable. Optimization of the different variables influencing the FI system was carried out using the univariate method. Fig. 3 shows the effect of the flow rate and sample volume. The effect of flow rate is important. Too low or too high flow rates result in the CL occurring before or after the flow cell, respectively. Variation of the total flow rate over the range 3.1-9
201
I
loo
I
J
200
Sample
300
500
volume (@I
I
I
1
1
3
5
7
9
Flow-rate
400
(ml mine’ )
Fig. 3. Effect of (0) sample volume, (a) flow-rate on peak height. The arrows mark the values of the parameters selected.
AA. Alwarthan /Analytica
236
12.5
Chimica Acta 317 (1995) 233-237 Table 1 Determination of tryptophan in pure samples by the spectrophotometric (Sp) and CL methods Claimed (pgml-‘) 1.6 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
Cow. found ( pg ml-’ ) a
Recovery (o/o)
SP
CU f S.D.) b
Sp
CL
1.58 2.0 2.48 3.05 3.5 3.95 4.47 4.97 5.35 6.0 6.6 6.90 7.44 8.1
1.62 f 0.33 2.01 f 0.10 2.5 +_0.25 3.0 + 0.40 3.5 rto.45 3.97 +_0.38 4.46 f 0.26 4.96 f 0.22 5.45 *0.31 6.01 i-o.15 6.5 + 0.42 6.95kO.16 7.48 + 0.29 7.98kO.39
98.8 100.0 99.2 101.7 100.0 98.8 99.3 99.4 97.3 100.0 101.5 98.6 99.2 101.3
101.3 100.5 100.0 100.0 100.0 99.3 99.1 99.2 99.1 100.2 100.0 99.3 99.7 99.8
a Average of 4 injections. b Standard deviation.
Time Fig. 4. Typical recording for the determination of tryptophan. numbers on the peaks are fig ml-’ of tryptophan injected.
The
This method was compared with the spectrophotometric nitrous acid method [l] by measuring the tryptophan content of pure solutions covering a wide range of concentrations (Table 1). The results obtained by the two methods are in good agreement. 3.4. Interferences
generate CL with cerium(IV). This suggests that the simple formation and decay of excited cerium(II1) is unlikely to be the cause of the emission, and an oxidation product of tryptophan is a more likely emitter. 3.3. Determination
of tryptophan
Once chemical and instrumental variables were optimized to achieve maximum CL emission, a series of standard solutions containing 1.6, 3.1, 6.25 and 9.25 and 12.5 pg ml-’ tryptophan was pumped, each as five replicates (Fig. 4) to test the linearity of the calibration graph. A plot of the emission intensity versus concentration of tryptophan in the sample injected was linear over the range 1.6-12.5 pg ml-‘. The statistical study performed on 13 replicate samples at 12.5 pug ml-’ yielded a relative standard deviation level of 0.45%. The detection limit (2 X noise) was 0.1 pg ml-’ (100 ~1, sample).
An interference study aimed at the determination of tryptophan was performed. Samples containing a fixed concentration of tryptophan (10 pg ml-‘) and various concentrations of foreign substances were injected into the FI system. The results are shown in Table 2. Cysteine, one of the essential amino acids, interferes severely in tryptophan determination, which should be removed by chromatography before the analysis. No interference was observed from other amino acids such as alanine, glutamine or histidine. Methionine interfered at high concentration (2 50 pg ml-l), but the effect is reduced by dilution. Arabinose, fructose, galactose, glucose, glucosamine and nicotinamide showed no interference and gave recoveries in the range 96-102%. Ascorbic acid gave a decreased response. It was found to react with Ce(IV). The interference can only be decreased by large dilutions but such dilutions would also de-
AA. Alwarthan/Analytica Table 2 Recovery of 10 &g ml-’ tryptophan from solutions various excipients at different concentrations Claimed
containing
the method can be automated an automatic sampling unit.
0.5 Kg ml-’
Acknowledgements
231
fully by the addition of
Recovery (%) 50 pg ml-’
D-C- )-Arabinose D-C-)-Fructose p( + )-Galactose ~-(+)-Glucose D( + )-Glucosamine L-( + )-Alanine L-c - )-Glutamine L-Histidine L-Methionine L-Cysteine L-( + )-Ascorbic acid Nicotinic acid Nicotinamide 2-Mercaptoethanol Casein
Chimica Acta 317 (1995) 233-237
99.6 101.9 91.1 95.9 91.1 101.5 98.9 101.5 88.7 15.0 28.2 92.1 98.1 93.9 90.2
10 I.Lg ml-’
5 pg ml-’
This work was supported partially by a Research Fund Grant-1994-from The Royal Society of Chemistry (London).
References 95.6 43.5 75.8 100.8
66.1 86.3
94.8 99.6
96.8 100.0
crease the tryptophan concentration. Hence, the method cannot be used for the determination of tryptophan in biological samples without pretreatment. Nicotinic acid, nicotinamide, 2-mercaptoethano1 and casein interfered slightly when they were present at high concentration (2 50 pg ml-‘), but the effect can easily be decreased by dilution.
4. Conclusions The proposed CL-F1 method has been shown to be applicable to the determination of tryptophan. The CL measurement was readily carried out in a flow injection system with good precision and high sample throughput. The method has a lower detection limit than spectrophotometric methods due to the absence of background signals, The reagents and instrumentation for this analysis are inexpensive and
[l] K.K. Verma, A. Jain and .I. Gasparic, Talanta, 35 (1988) 35. [2] T. Simat, K. Meyer and H. Steinhart, J. Chromatogr., 661 0994) 93. [3] A.C. Moffat, J.V. Jackson, MS. Moss and B. Widdop, Clarke’s Isolation and Identification of Drugs, 2nd edn., The Pharmaceutical Press, London, 1986, p. 1056. [4] S.E. Garcia and J.H. Baxter, J. AOAC Int., 75 (1992) 1113. [5] P. Lindroth and K. Mopper, Anal. Chem., 51 (1979) 1667. [6] A.K. Goswami, Analyst, 99 (1974) 657. [7] H. Matsubura and R.M. Sasaki, Biochem. Biophys. Res. Commun., 35 (1969) 175. [8] T.Y. Liu and Y.H. Chang, J. Biol. Chem., 246 (197 I) 2842. [9] R.H. Horton and D.E. Koshland, J. Am. C’hem. Sot., 87 (1965)
1126.
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