Accepted Manuscript

A simplified HPLC method for determination of tryptophan in some cereals and

legumes

Senem A. Çevikkalp, Gül B. Löker, Mustafa Yaman, Birdem Amoutzopoulos

PII: S0308-8146(15)00299-X
DOI: http://dx.doi.org/10.1016/j.foodchem.2015.02.108
Reference: FOCH 17207

To appear in: Food Chemistry

Received Date: 11 February 2014

Revised Date: 3 September 2014
Accepted Date: 22 February 2015

Please cite this article as: Çevikkalp, S.A., Löker, G.B., Yaman, M., Amoutzopoulos, B., A simplified HPLC method
for determination of tryptophan in some cereals and legumes, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/
j.foodchem.2015.02.108

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1 A SIMPLIFIED HPLC METHOD FOR DETERMINATION OF TRYPTOPHAN IN SOME CEREALS

2 AND LEGUMES†
† th
3 Presented in part at 10 International Food Data Conference, Granada, Spain, September 2013.

4
1 1 1 1
5 Authors' names: Senem A. Çevikkalp, * Gül B. Löker, Mustafa Yaman, Birdem Amoutzopoulos,

6 *1The Scientific and Technological Research Council of Turkey (TUBITAK) Marmara Research Center,

7 Food Institute, 41470, Kocaeli, Turkey. E-mail: senemakkus@gmail.com

1
8 *Correspondence to: Senem A. Çevikkalp, TUBITAK, Marmara Research Center, Food Institute,

9 41470, Kocaeli, Turkey. Tel: +902626773214 Fax: +902626412309 E-mail: senemakkus@gmail.com

10 ABSTRACT

11 In the present study, a simple analytical method is proposed for determining tryptophan, and method is

12 validated on some cereal and legume samples.

13 In the method alkaline hydrolysis of proteins was used due to the destruction of tryptophan structure

14 during acid hydrolysis. Following alkaline hydrolysis (120 °C for 12 h), hydrolysates are filtered through

15 ashless filter paper and pH values are adjusted with HCl solution. Separation and detection of

16 tryptophan are performed on a reversed-phase column with fluorescence detection within 10 min by

17 using a mobile phase of acetonitrile and acetate buffer of pH 6.3 (1:9, v/v).

18 For determination of tryptophan content, the procedure described in the study offers an alternative

19 analysis method by enabling high speed analysis and the use of simple extraction process to the other

20 available methods.

21

22 Keywords: Tryptophan, HPLC, fluorescence detection, TürKomp, amino acids.

23

24 1 INTRODUCTION

25 Tryptophan (Trp) is an important component of the food composition databases since it is one of

26 essential amino acids in the human nutrition and plays critical role in the metabolism of mammals

27 (Diem et al., 2000). Beside protein biosynthesis, Trp serves as a biochemical precursor of typical

28 physiological activity substances including serotonin, melatonin, tryptamine, niacin and tryptophan

29 degradation products (Molnar-Perl, 1997; Annemieke et. al, 2013; Zhang et. al 2009; Yonekura et. al,

30 2007; Young, S.N., Leyton, M., 2002). The human body cannot synthesize Trp and is dependent on
31 dietary sources of Trp (Solomon & Fryhle, 2000; Caballero, Trugo & Finglas, 2003). Tryptophan is

32 found in foods that naturally contain protein and also in dietetic and fortified food products, and

33 specific pharmaceutical formulations. Therefore, Trp is found in the content of various food groups

34 such as milk and dairy products, meat, nuts, seeds, legumes, cereals, fish and fish products

35 (Keszthelyi et. al, 2009). Food processing and preparation practices including heating process may

36 lead to oxidative degradation and crosslinking between protein molecules and decrease the

37 bioavailability of Trp. The nutritional importance and range of its sources makes Trp as an important

38 component in the content of food composition databases.

39 Many methods have been developed for specifically determining tryptophan in food samples, such as

40 spectroscopy, high-performance liquid chromatography, fluorometric methods, capillary

41 electrophoresis and electro analysis (Nurit et. al, 2009). Fluorometric techniques have probably been

42 the most popular due to its high sensitivity, high accuracy, simple operation mode and low cost (Xi Ba

43 et. al, 2013).

44 The free amino acids are usually analysed without hydrolyzing the sample, but acid hydrolysis (usually

45 100–120 ºC, 6N HCl and 22–24 h) is necessary when the total amino acids in protein are determined

46 and Trp is unstable under these conditions (Caballero, Trugo & Finglas, 2003; The JP Request, 2002).

47 Stability and recovery of Trp is poor in conditions where acid and oxygen are present as well as acid

48 hydrolysis due to the indole group of Trp. NaOH is the most effective alkaline used for protein

49 hydrolysis with or without a carbohydrate or thiodiglycol as an antioxidant. Tryptophan has often being

50 ignored in the assessment of overall amino acid profile of foods. Because, tryptophan exhibits a strong

51 native fluorescence, and fluorometric method has been widely used to determine tryptophan content

52 without derivatization (Delgado-Andrade et. al, 2006; Sánchez-Machado et. al, 2008; Boselli et. al,

53 2003; Vignau et. al, 2004).

54 The aim of this study was to develop a robust and cost-effective method for tryptophan analysis in

55 foods.

56 2 EXPERIMENTAL

57 2.1 Reagents and Materials

58 Standard of L-Tryptophan were from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, ammonium

59 acetate, sodium hydroxide and hydrochloric acid were purchased from Merck (Darmstadt, Germany).

60 All aqueous solutions were prepared with doubly distilled water obtained from Milli-Q System
61 (Millipore, Bedford, MA, USA). During hydrolysis and sample preparation extra-pure nitrogen was

62 used.

63 Samples of wheat, rye, chickpea, red lentil, barley, kidney beans were purchased from local markets in

64 Turkey and grounded to flour in laboratory.

65 2.2 Instrumentation

66 The high performance liquid chromatography (HPLC) system (Shimadzu LC-20AT, Tokyo, Japan)

67 consisting of Model LC-20AT multi-solvent delivery system and Model RF-10AXL fluorescence

68 detector were used for the analysis. For recording and data processing, LC Solution software from

69 Shimadzu (Tokyo, Japan) was used.

70 The analytical column was a Lichrospher® 60 RP-Select B (250mmx4mm), 5µm of pore size

71 purchased from Agilent (Santa Clara, CA, USA).

72 2.3 Standard Tryptophan Preparation

73 Standard stock solution of tryptophan (100 µg/mL) was prepared in acidic water (doubly distilled water

74 adjusted to pH 6.3 with 0.1 N hydrochloric acid) (Yust et al., 2004; Zhang et. al 2009) and freshly

75 prepared in each day of analysis. Individual working standard solutions of five concentration levels

76 were prepared from the stock solution.

77 2.4 Sample Preparation

78 The analytical method was carried out with a little modification of sample preparation procedure and

79 chromatographic conditions reported by Yust et. al (2004) and Zhang et. al (2009). In order to

80 determine the optimal condition, four different hydrolysis times (4, 12, 16, 24 h) were applied for red

81 lentil and wheat samples at 120 °C. The results show that 4 h of hydrolysis time (Yust et al., 2004)

82 resulted in lower amount of tryptophan comparing to the other hydrolysis times. Beside this, no

83 significant difference was observed between 12, 16 and 24 h of hydrolysis time. All results are shown

84 in Figure 1.

85 0.1-1 g of each sample was weighed in 50 mL hydrolysis bottle with Teflon-lined screw-cap and then

86 treated with 20 mL of 5 N sodium hydroxide solutions under nitrogen atmosphere. After hydrolysing in

87 an oven at 120 °C for 12 hours, the hydrolysates were cooled down to room temperature. Following

88 filtration through ashless filter papers, 1 mL of filtrate was diluted with 60 mL of acidic water (pH 6.3)

89 and the final pH level was adjusted to 6.3 by diluted HCl solution (Yust et al., 2004; Zhang et. al 2009).
 90 The final solution was diluted to 100 mL in brown volumetric flask with doubly distilled water and

91 filtered through 0.20 µm filter into HPLC vials.

92 2.5 Chromatographic Conditions

93 In addition to modification of the hydrolysis time, acetate buffer was used to stabilize the pH value of

94 mobile phase which is preferred in long-term studies. The mobile phase was prepared by mixing 900

95 mL of ammonium acetate buffer, pH 6.3 and 100 mL of acetonitrile. The mixture was filtered through a

96 0.22 µm filter.

97 10 µL of sample solutions were injected and flow rate was 1 mL/min. Tryptophan was detected at 280

98 and 340 nm for excitation and emission wavelengths (Zhang et. al 2009; Delgado-Andrade et al.,

99 2006). Running time was 10 min. and retention time for tryptophan was 5.8 ± 0.2 min. The

100 quantification was performed by correlating peak areas of the samples with the concentrations

101 according to the calibration curve.

102 3 RESULTS

103 3.1 Method Validation

104 Linearity was evaluated by analysing five different concentrations of standard solutions in triplicate.

105 The calibration curve was obtained in a concentration range from 0.1 µg/mL to 0.8 µg/mL and found to

106 be linear for tryptophan with coefficient of determination greater than 0.998 as shown in Table 1.

107 Limit of detection (LOD) and limit of quantification (LOQ) for tryptophan were calculated from the

108 calibration line at lowest concentration using the following equations: LOD= 3xSD, LOQ=10xSD,

109 where SD is standard deviation. LOD& LOQ values have been shown in the Table 1. In practice, LOQ

110 value was found to be suitable compared to signal-to-noise ratio (10:1).

111 FAPAS® Test Material 2584 amino acids in infant formula, wheat and red lentil samples were used for

112 precision and accuracy assessment in the present study. The precision study of the analytical method

113 included repeatability and reproducibility tests. The repeatability was tested by analysing the same

114 sample in ten replicate within the same day and the reproducibility was determined by analysing the

115 same sample in sixteen replicate in three independent days. The relative standard deviation (RSD)

116 values of repeatability are less than 3.7% and RSD values of reproducibility are less than 6% as

117 shown in Table 1.

®
118 The accuracy of the method was evaluated by analysing FAPAS Test Material and the measured

119 mean value (17.45 ± 2.62 mg/g protein) is close to the assigned value (17.2 mg/g protein),
120 demonstrating the validity of the assays. The recoveries for the red lentil and wheat samples were

121 determined by spiking standard solution at concentration of 0.2 µg/mL and 0.6 µg/mL. Recoveries

122 were found to be in the range of 95.2-99.2% for tryptophan as shown in Table 1.

123 Blank samples of ultrapure water and reagents were also prepared using the same procedures as for

124 the food samples. In the determination of tryptophan no obvious interferences was observed from the

125 blank chromatograms. The results indicate the method was accurate for tryptophan determination in
®
126 FAPAS Test material, cereals and legumes.

127 The uncertainty budget was estimated based on the general principles of EURACHEM/CITAC Guide

128 (2012). Using a coverage factor (k) of 2 and at the 95% confidence level, the relative expanded

129 uncertainty was estimated to be 11% for wheat, 11% for red lentil and 15% for FAPAS® Test Material,

130 respectively.

131 In the present study, the quality procedures of analytical method were based on ISO/IEC 17025

132 requirements. A Standard Reference Material (SRM 1849) Infant/Adult Nutritional Formula was

133 purchased from National Institute of Standards & Technology (Gaithersburg, MD, USA) and was

134 proceed in a similar way to the unknown samples.

135 3.2 Application to Sample

136 The analytical method was successfully applied to determine the tryptophan content of 6 cereal and

137 legume samples, wheat, rye, chickpea, red lentil, barley and kidney bean results are shown in Table 2.

138 Figure 2 shows typical chromatograms for standard solution and samples.

139

140 4 CONCLUSION

141 A quantitative method has been developed for the determination of Trp in cereals and legumes by

142 using HPLC with fluorescence detection. The proposed method would considerably reduce the time

143 required for sample preparation procedures and increase the analytical throughput in addition is

144 precise and accurate confirming its utility for routine analysis.

145 This modified method for the determination of tryptophan in cereals and legumes is used during

146 formation of Turkish Food Composition Database (TürKomp) (Löker et. al, 2011). With its precise and

147 accurate confirming use in cereals and legumes, this analytical method can be proposed for

148 determination of Trp in the future food composition studies.

149
150

151 Acknowledgement:

152 This work has been funded by TÜBİTAK KAMAG TARAL-1007 Research Programme, 107G208.

153

154 References:

155 Annemieke T. Van der Goot and Ellen A.A. Nollen, (2013), Tryptophan metabolism: entering the field

156 of aging and age-related pathologies, Trends in Molecular Medicine, 19,336-344.

157 Boselli, E., Caboni, MF, Sabatini AG, Marcazzan, GL and Lercker G., (2003). Determination and

158 changes of free amino acids in royal jelly during storage, Apidiologie, 34, 129-137.

159 Caballero, B., Trugo, L. C., Finglas P.M., (Eds), (2003). Encyclopedia of Food Sciences and Nutrition.

160 (2nd Edition), Amino acids (pp. 188-200). Oxford: Academic Press.

161 Delgado-Andrade, C., Rufian-Henares, J., Jimenez-Perez, S., Morales, F. J., (2006). Tryptophan

162 determination in milk-based ingredients and dried sport supplements by liquid chromatography with

163 fluorescence detection, Food chemistry, 98, 580-585.

164 Diem, S., Bergmann, J., Herderich, M., (2000). Tryptophan-N-glucoside in fruits and fruit juices, J.

165 Agric. Food Chem. 48, 4913-4917.

166 Keszthelyi, D., Troost, F. J., Masclee, M., (2009). Understanding the role of tryptophan and serotonin

167 metabolism in gastrointestinal function, Neurogastroenterol Motil., 21, 1239-1249.

168 Löker G.B., Ozkoc S.O., Amoutzopoulos B., Yaman M., Akkus S., Sanli F., Kucuk F. (2011).

169 Establishing a food composition database for Turkey based on European Standarts, Nutrition Bulletin,

170 36, 254-258.

171 Molnar-Perl, I. (1997). Tryptophan analysis in peptids and proteins, mainly by liquid chromatography,

172 Journal of Chromatography A, 763, 1-10.

173 Nurit, E., Tiessen, A., Pixley, K. V., Palacios-Rojas, N. (2009). Reliable and inexpensive colorimetric

174 method for determining protein-bound tryptophan in maize kernels, J. Agric. Food Chem. , 57, 7233-

175 7238.

176 Sánchez-Machado DI, Chavira-Willys B, López-Cervantes J. (2008), High-performance liquid

177 chromatography with fluorescence detection for quantitation of tryptophan and tyrosine in a shrimp

178 waste protein concentrate, J Chromatogr B Analyt Technol Biomed Life Sci, 863, 88-93.
179 Solomon, G., Fryhle, C. (2000). Organic Chemistry, (7th Edition), New York: John Willey&Sons,

180 (Chapter 14).

181 The Japanese Pharmacopoeia Request, (Revised July 2002). Stage 5-Proposal Global Document,

182 Amino acid analysis.

183 Vignau, J., Jacquemont, MC, Lefort, A., Imbenotte, M and Lhermitte, M. (2004). Simultaneous

184 determination of tryptophan and kynurenine in serum by HPLC with UV and fluorescence detection,

185 Biomedical Chromatography, 18, 872-874.

186 Xi Ba, Liqiang L., Yaping, D., Xiao, L. (2013). Determination of l-tryptophan in the presence of ascorbic

187 acid and dopamine using poly(sulfosalicylic acid) modified glassy carbon electrode, Sensors and

188 Actuators B: Chemical, 187, 27–32.

189 Yonekura, R., Itoh, Y., Asayama, M., Shibata, K. (2007). The effects of dietary tryptophan metabolism

190 on the vigor and confusion factor of mood, International Congress Series, 1304, 180-183.

191 Young, S.N., Leyton, M. (2002). The role of serotonin in human mood and social interaction

192 Insight from altered tryptophan levels, Pharmacology, Biochemistry and Behavior, 71, 857–865.

193 Yust , M. M., Pedroche, J., Giron-Calle, J., Vioque, J., Millan, F., Alaiz, M. (2004). Determination of

194 tryptophan by high-performance liquid chromatography of alkaline hydrolysates with

195 spectrophotometric detection, Food Chemistry, 85, 2, 317-320.

196 Zhang, J. Z., Xue, X. F., Zhou, J. H., Chen ,F., Wu, L. M., Li, Y., Zhao, J. (2009). Determination of

197 tryptophan in bee pollen and royal jelly by high-performance liquid chromatography with fluorescence

198 detection, Biomedical Chromatography, 23, 9, 994–998.

199

200 Figure 2 Chromatograms for tryptophan under chromatographic conditions of the analytical method
201 (a) Lentil, red; (b) Wheat, durum; (c) Chickpea; (d) Standard of tryptophan

202

203

204

205

206

207

208

209

210

211

212

213 Figure 1 Effect of different hydrolysis time

214
215 Table 1 Results for validation parameters

Fapas Test Material Wheat, Durum Lentil, Red

Linear regression equation y=395544x+3174,1 y=395544x+3174,1 y=395544x+3174,1
r2 0.9994 0.9994 0.9994
Range of linearity (µg/mL) 0.1-0.8 0.1-0.8 0.1-0.8
Repeatability (RSD, n=10) 3.3% 1.5% 0.9%
Reproducibility (RSD of intra-day studies, n=16) 4.4% 1.5% 0.9%
LOD (µg/mL) 0.002 0.002 0.002
LOQ (µg/mL) 0.006 0.006 0.006
a b a b a b
Accuracy (a; recovery, b; RSD, n=10) 98.84% , 3.3% 95.2-99.2% , 1.5% 96.0-96.8% , 0.9%
2
x; amount (µg/mL), y; peak area, r ; correlation coefficient
216
217

218 Table 2 Tryptophan content of some cereals and legumes*

Tryptophan
Samples
(mg/100g)

Wheat, durum 169 ± 2,56

Rye 125 ± 0,11

Barley 165 ± 0,18

Chickpea 220 ± 0,14

Lentil, red 139 ± 1,23

Kidney beans 240 ± 0,27

* Values are mean ±SD (n=2)

219

220

221
222 Highlights

223 • A simple modified analytical method for determination of tryptophan is proposed.

224 • We evaluate the performance of the analytical method.
225 • The time required for sample preparation procedures are reduced.
226 • The analytical throughput in addition is precise and accurate confirming its utility for routine
227 food analysis is increased.

228