Analytical Letters

ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: http://www.tandfonline.com/loi/lanl20

Determination of Tryptophan in Pharmaceutical

Formulations Using a Sequential Injection - Zone
Fluidic - Chemiluminescence Tubular Reactor

Ramona Georgescu-State & Jacobus (Koos) Frederick van Staden

To cite this article: Ramona Georgescu-State & Jacobus (Koos) Frederick van Staden
(2017): Determination of Tryptophan in Pharmaceutical Formulations Using a Sequential
Injection - Zone Fluidic - Chemiluminescence Tubular Reactor, Analytical Letters, DOI:
10.1080/00032719.2017.1362420

To link to this article: http://dx.doi.org/10.1080/00032719.2017.1362420

Accepted author version posted online: 11

Aug 2017.

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Download by: [Australian Catholic University] Date: 13 August 2017, At: 05:59
 Super Title: Flow Injection Analysis

Determination of tryptophan in pharmaceutical

formulations using a sequential injection - zone fluidic -

chemiluminescence tubular reactor

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Ramona Georgescu-State

Jacobus (Koos) Frederick van Staden*

Process Analytical Technology Laboratory (PATLAB) Bucharest, National Institute of Research

and Development for Electrochemistry and Condensed Matter (INCDEMC), Bucharest,

Romania

Address correspondence to Jacobus (Koos) Frederick van Staden, Process Analytical

Technology Laboratory (PATLAB) Bucharest, National Institute of Research and Development

for Electrochemistry and Condensed Matter (INCDEMC), 202 Splaiul Independentei Str.,

Bucharest 060021, Romania. Tele: + 40 74 169 5743. E-mail: koosvanstaden2012@yahoo.com

ABSTRACT

Tryptophan is an important amino acid for humans with a significant role in cell

metabolism. Depletion of tryptophan in the human body may contribute to diseases and

development of disorders among the human population. It is therefore very important to have a

reliable, stable, sustainable, and cost-effective analytical method for the determination of

tryptophan. Tryptophan was determined using sequential injection - zone fluidics analysis with

1
 luminol-hydrogen peroxide and the Firefly with its unique liquid core waveguide flow-cell design

as chemiluminescence tubular reactor with a high sensitivity photomultiplier tube. This was based

on an intense chemiluminescence formation of tryptophan in luminol-hydrogen peroxide inside

the tubular reactor for measurement. The chemiluminescence intensity was linear with tryptophan

in the range of 1.0 × 106 to 1.0 × 103 mol /L and the limit of detection was 7.5 × 107 mol /L. The

precision for the method was 3.6% (relative standard deviation) for six measurements of 1.0 × 104

mol /L tryptophan. The proposed method has been used to determine tryptophan in pharmaceutical
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formulations. The system is relatively fast for on-line assays. Eighty seconds are required to

complete one cycle providing a throughput of forty-five samples/hour. The proposed sequential

injection analysis - zone fluidics - chemiluminescence system for the assay of tryptophan in certain

specific pharmaceutical capsules is simple, reliable, sustainable, and convenient with relatively

low cost consumption of reagents.

KEYWORDS: chemiluminescence, luminol, hydroge peroxide, photomultiplier tube, sequential

injection analysis, tryptophan, zone fluidics

ARTICLE HISTORY

Received 23 June 2017

Accepted 28 July 2017

Introduction

Tryptophan, IUPAC name (2S )-2-amino-3-(1H-indol-3-yl)-propionic acid, tryptophan is

an essential amino acid for humans with a significant role in cell metabolism as a protein building

block and a precursor for serotonin (a neurotransmitter) (Fernstrom 1983; Schaechter and

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 Wurtman 1990), melatonin (a neurohormone) (Wurtman and Anton-Tay 1969), and niacin (Ikeda

et al. 1965). The depletion of tryptophan in the human body may result in low levels of serotonin

that may cause fluctuation in the mood, sleep, drowsiness, depression, aggression, and in blocking

the analgesic effect of morphine (Kochen and Steinhart 1994). Deficiency of tryptophan has also

been associated with Alzheimer’s Disease. Depletion of tryptophan in the human body may

furthermore contribute to a number of diseases and development disorders in humans. It is however

also a vital element for normal growth in infants and for the nitrogen balance in adults
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(Tashkhourian, Daneshi, and Nami-Ana 2016). Tryptophan cannot be synthesized directly in the

human body (Wu, Tong, and Zhang 2011; Çevikkalp et al. 2016; Tashkhourian, Daneshi, and

Nami-Ana 2016) and can only be supplemented through the intake of food products and

pharmaceutical formulations.

The intake and appropriate dose of tryptophan therefore depends very much on several

factors such as the user’s age, health, pregnancy, breast-feeding, and several other conditions; and

extreme care has to be taken in this regard. Therefore, a simple, rapid, reliable and sustainable

method for the determination of tryptophan with relative low-cost instrumentation is essential and

needed in pharmaceutical preparations and formulations.

A number of analytical methods based on electrochemistry (Liu and Xu 2007; Guo et al.

2010; Khaleghi et al. 2016; Tashkhourian, Daneshi, and Nami-Ana 2016), ion exchange

chromatography (Hugli and Moore 1972; Ravindran and Bryden 2005), high-performance liquid

chromatography (Jagannathan, March, and Venitz 1995; Mattivi, Vrhovšek, and Versini 1999;

Cubero et al. 2010; Çevikkalp et al. 2016; Islam et al. 2016), spectrophotometry (Ren et al. 2007),

and spectrofluorometry (Bagheri et al. 2015) have been developed for the determination of

tryptophan. However, the advantages associated with flow injection analysis (Barnes and van

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 Staden 1992; van Staden 1998; van Staden and Stefan-van Staden 2010; van Staden and Stefan-

van Staden 2012; van Staden 2015a, 2015b) and sequential injection analysis (Taljaard and van

Staden 1998; van Staden 1998; van Staden 2002; van Staden and Stefan 2004; van Staden 2015a,

2015b) as inexpensive flow analysis systems are very attractive and suitable for implementation

as automated process instrumental devices in real industrial processes and environments.

Therefore a number of flow injection analysis (Costin, Francis, and Lewis 2003; Liang and

Song 2005; Qiu et al. 2012) and sequential injection analysis (Gao et al. 2010; Gao and Fan 2014)
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methods based on the direct chemiluminescence reaction of tryptophan with some oxidants have

been reported for the determination of tryptophan. These include oxidants such as MnO 4 and

Mn(III) (Liang and Song 2005), peroxynitrous acid (Qiu et al. 2012), and Ce(IV) (Gao et al. 2010;

Gao and Fan 2014). A major advantage of sequential injection analysis (Taljaard and van Staden

1998; van Staden 1998; van Staden 2002; van Staden and Stefan 2004; van Staden 2015a, 2015b)

is that the samples and reagents with very fast reactions may be time controlled in small stacks of

separated well-defined zones before mixing, reaction, and detection to improve reliable results.

Furthermore, the optimum level of final product maximum in vivo detection level performance

may be obtained with a proper combination of zone mixing and penetration with stopped flow in

a tubular cell that has not been reported by flow injection. This type of detection format by

sequential injection analysis (Taljaard and van Staden 1998; van Staden 2002; van Staden 2015a,

2015b) and zone fluidics (Marshall, Wolcott, and Olson 2003) was previously used (van Staden

and State 2016) for the determination of dopamine.

The use of photomultiplier tubes for photon counting was described in detail by Foord et

al. (1969). Hence, here we used the Firefly with its unique liquid core waveguide flow-cell design

as the chemiluminescence detector (Global FIA 2017) for sequential injection analysis. The liquid

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 core waveguide cell serves as both a reactor and a light carrying conduit and as mixing of the

sample and reagent occur within the liquid core waveguide cell, very fast reactions can be

accommodated. Therefore with the liquid core waveguide, most of the light generated within the

cell is efficiently carried to the ends of the cell where it is transferred by optical fibers to a high

sensitivity photomultiplier tube. Furthermore, the oxidant should be environmental friendly and

hydrogen peroxide as the oxidant decomposes in the luminol reaction to oxygen and water, and

therefore we used hydrogen peroxide alone in this system to see if it is suitable as oxidant for the
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determination of tryptophan. Hence, here we used luminol-hydrogen peroxide in our sequential

injection analysis - zone fluidics - chemiluminescence - Firefly system.

In this system the analyte sample was zone stacked and sandwiched between alkaline

luminol and hydrogen peroxide solutions by aspiration within the head and tail zones inside the

holding coil with a selection valve. These zones were reversed and rapidly dispensed directly in

front of the fiber optic cable into the reactor where initial mixing of different zones with zone

penetration started at the entrance of the waveguide tubing. With further continuation of flux of

the penetrated sample/reagents zones through the waveguide tubing, mixing and chemistry occurs

in the waveguide channel. As a result the light generated is delivered through the waveguide flow-

cell and fiber optic cable to the photomultiplier tube for measurement and data processing. The

advantage of this system includes simplicity, reliability, sustainability, and convenience with

relatively low cost consumption of reagents with which the assay of tryptophan can be performed

and data can be acquired and processed.

Experimental

Chemicals

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 All chemicals were of analytical-reagent grade and solutions were prepared using

deionized water obtained from a Direct-Q 3 Water Purification system (Millipore Corporation,

France).

L-Tryptophan was purchased from Sigma Aldrich. The stock standard solution of

tryptophan (1.0 × 102 mol /L) was prepared by dissolving 0.1021 g  of L-tryptophan in 50 mL  of

deionized water. The solution was kept in the refrigerator at 4 ºC. Standard working solutions were
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prepared daily by appropriate dilution of the stock solution with deionized water.

The luminol was obtained from Fluka. The luminol stock solution (1.0 × 102 mol /L) was

prepared by dissolving luminol in a 0.02 mol /L sodium hydroxide solution. The working solutions

of luminol were prepared from the stock solution by serial dilution with deionized water. The pH

of the luminol solutions were 9.1.

The hydrogen peroxide was purchased from Riedel-de-Haën. The hydrogen peroxide stock

solution (1.0 mol /L) was prepared by volumetric dilution of 30% (v/v) H2O2.

Apparatus

We used a Mini-FloPro Sequential Injection Analysis - Zone Fluidics system purchased

from Global FIA. It was constructed from 2 electrically actuated valves (valve A, a 18-port

selection valve and valve B, a 10port selection valve), a milliGAT pump, and a serpentine

plumbing as holding coil. The photon counter was a Global FIA FireFly detector for

chemiluminescence detection. The system is controlled by a portable personal computer using the

Flow Zone Fluidics process development software for device sequence control.

Sample preparation

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 Ten pharmaceutical capsules of Product A purchased from a local pharmacy were used.

Each capsule contains 1.5% (15 mg /mL) tryptophan and other excipients. The weight of powder

in a capsule was approximately 0.35 g. The powder from each capsule was inserted in a 100 mL 

volumetric flask and dissolved in deionized water to the mark to give a stock solution. After stirring

for a few minutes, the solutions were carefully filtered through a 0.2 μm pore-size filter to isolate

the insoluble excipients. The stock solutions were diluted with deionized water to obtain the

working solutions with the concentration of tryptophan within the range of the calibration curve.
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Procedure

The device sequence for one cycle of the sequential injection analysis - zone fluidics

system (van Staden and State 2016) contains 5 steps as follows: an air bubble was first aspirated

from port 18 (step 1) followed by 100 µL of luminol solution at a flow rate of 4 µL/s from port P2

(step 2). Then, in step 3, 100 µL of tryptophan solution (standard or sample) was aspirated at 4

µL/s through port P3, followed in step 4 by 200 µL of hydrogen peroxide solution at 8 µL/s from

the 18 port selection valve. Finally, in step 5, an air bubble from port 18 was aspirated to sandwich

the tryptophan between the luminol and hydrogen peroxide solutions in the holding coil. These

aspirations were all done through the inlet ports of the 18 port valve A and these zones were

therefore all stacked after each other in the holding coil.

The air bubble zone was stacked first followed by the luminol zone, the tryptophan zone,

hydrogen peroxide zone, and completed by another air bubble zone on the other side. With this

configuration, the three solution zones were stacked in a sandwich mode with tryptophan

sandwiched between luminol and hydrogen peroxide solution separating the luminol and hydrogen

peroxide zones preventing any contamination and reaction mixture for an unwanted reaction to

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 occur before detection. Thereafter, outlet Port 10 of the 18 port selection valve A and inlet port 10

of the 10 port selection valve B were selected and the stacking zones were reversed and rapidly

dispensed directly in front of the fiber optic cable into the reactor where initial mixing of different

zones with zone penetration started at the entrance of the waveguide tubing. With further

continuation of the flux mixing with chemical reactions of penetrated sample/reagents zones

occurs during the flow through the waveguide tubing. As a result, the light generated is delivered

through the unique liquid core waveguide flow-cell and fiber optic cable to the photomultiplier
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tube for measurement and data processing. Finally, the reaction solution mixtures from the detector

and carrier stream were dispensed and washed to waste following the same route as before.

Results and Discussion

Chemiluminescence of luminol and hydrogen peroxide with L-tryptophan

The chemiluminescence signal of the luminol-hydrogen peroxide reaction with tryptophan

was established using 104 mol /L tryptophan, 103 mol /L luminol, 0.8 mol /L hydrogen peroxide,

and 0.02 mol /L NaOH. The results in Figure 1 showed a maximum chemiluminescence peak

intensity at 434 nm. This value is similar to the maximum peak intensity of 425 nm for the alkaline

luminol-H2O2 reaction for dopamine in our previous article (van Staden and State 2016), but with

a slight shift in the spectrum value due to tryptophan instead of dopamine. The mechanisms of

both our articles are the same as that given previously (van Staden and State 2016), but with

tryptophan as the analyte in this work.

Effect of hydrogen peroxide concentration

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 The effect of hydrogen peroxide concentration on the chemiluminescence intensity in the

presence of 1.0 × 104 mol /L tryptophan and 1.0 × 103 mol /L luminol was investigated over the

range of 0.2–1.0 mol /L H2O2. As shown in Figure 2, the number of photons counted

(chemiluminescence intensity) increases as the hydrogen peroxide concentration increased from

0.2 to 0.8 mol /L, where after the chemiluminescence intensity dropped sharply up to a

concentration of 1.0 mol /L hydrogen peroxide. Therefore, 0.8 mol /L hydrogen peroxide was used

in further experiments.
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Effect of luminol concentration

The effect of luminol concentration on the chemiluminescence intensity was examined by

using 1.0 × 104 mol /L tryptophan and 0.8 mol /L hydrogen peroxide over the range of 1.0 × 107

to 1.0 × 102 mol /L (0.1 to 10000 µmol/L) luminol. As shown in Figure 3, the number of photons

counted (chemiluminescence intensity) increases until the maximum photons counted (maximum

chemiluminescence intensity) were obtained when the concentration of luminol was 103 mol /L

(1000 µmol/L), where after the chemiluminescence intensity dropped sharply to a concentration

of 102 mol /L (10000 µmol/L) luminol. Therefore, 103 mol /L luminol was chosen throughout

the experiments.

Performance, calibration curve, detection limit, and precision

The performance of the sequential injections analysis - zone fluidics - chemiluminescence

- Firefly system on a series of standard tryptophan solutions at a throughput of forty-five

samples/hour under optimized experimental conditions is given in a graph with overlay peaks in

Figure 4. Under the selected experimental conditions, the chemiluminescence intensity was linear

for tryptophan concentrations within the range of 1.0 × 106 to 1.0 × 103 mol /L (1 to 1000

9
 µmol/L). The linear regression equation was I = 1477.85 - 0.68 × C, where I is chemiluminescence

intensity (photons counts from the Global Flow injection analysis Firefly - liquid core waveguide

flow-cell and photomultiplier tube detector) of the luminol - hydrogen peroxide - tryptophan

product in the tubular reactor detector system and C is the concentration of tryptophan,

respectively, with a correlation coefficient of 0.9989 (n = 6). The limit of detection was calculated

from three-times the standard deviation of the blank signal (3σ) and the limit of quantitation

defined as ten-times standard deviation of the blank (10σ). The limit of detection was 7.5 × 107
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mol /L (153.1 ng/mL), limit of quantification was 1.0 × 106 mol /L (204 ng/mL) and the relative

standard deviation (n = 6) was 3.6% for 1.0 × 104 mol /L tryptophan. These results obtained and

the response on the calibration curve show that the use of hydrogen peroxide as oxidant satisfies

all requirements needed for the determination of tryptophan with the sequential injection analysis

- zone fluidics - chemiluminescence - Firefly system.

Interference study

In order to evaluate the selectivity of the proposed sequential injection analysis - zone

fluidics - chemiluminescence - Firefly system, the effect of the various species and excipients

commonly used in pharmaceutical formulations on the determination of tryptophan was

investigated by analyzing a standard solution containing 1.0 × 105 mol /L tryptophan and different

amounts of each coexisting species. The tolerated limit of each foreign species was considered to

be the highest concentration that yielded a relative error less than 5% for the determination of

tryptophan. The experimental results are listed in Table 1. The results showed that interferences

were observed for 1000-fold excess of D-glucose, L-leucine, L-isoleucine, and D, L-histidine;

more than a 500-fold excess of L-phenylalanine and dopamine; more than a 100-fold excess of L-

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 alanine, D-valine, and L-tyrosine; and more than a 10-fold excess of ascorbic acid, D, L-aspartic

acid, uric acid, and L-cysteine.

Applications

The sequential injection analysis - zone fluidics system with chemiluminescence detection

was applied to the determination of tryptophan in pharmaceutical capsules and the results are listed

in Table 2. As shown in Table 2, the recoveries are between 96.3 and 105.7% (n = 6). It can be
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seen that the results obtained are in good agreement with the specified values for tryptophan, as

well as with satisfactory recoveries and the relative standard deviation was lower than 7% (n = 6).

It was found that the detected values compared also very well with square wave voltammetry

(Khaleghi et al. 2016) and linear sweep voltammetry (Deng, Fei, and Feng 2011) as reference

methods.

Conclusions

The sequential injection analysis - zone fluidics - chemiluminescence - Firefly system

reported here is more advantageous to comparable literature procedures for flow injection analysis

(Costin, Francis, and Lewis 2003; Liang and Song 2005; Qiu et al. 2012) and sequential injection

analysis (Gao et al. 2010; Gao and Fan 2014). In this work, we used the Firefly with its unique

liquid core waveguide flow-cell as both a tubular reactor and a light carrying conduit where the

light generated within the cell is efficiently carried to the ends of the cell and where it is transferred

by optical fibers to a high sensitivity photomultiplier tube. A further advantage here is that a more

reliable milliGAT pump is used to aspirate the tryptophan samples, luminal, and hydrogen

peroxide reagents more efficiently with precise time control into small stack of well-defined zones

kept apart into a holding coil. The same reliable milliGAT pump is used to reverse these zones and

11
 rapidly dispense them directly in front of the fiber optic cable into the reactor where initial mixing

of different zones with zone penetration started at the entrance of the waveguide tubing. With the

flux of penetrated sample/reagents zones through the waveguide tubing mixing, the chemistry

occurs during the flow through the waveguide and the light generated is delivered through the

waveguide and fiber optic cable to the photomultiplier tube for measurement and data processing.

As the mixing of sample and reagent occur within the cell, very fast chemiluminescence reactions

can be accommodated.
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The use of hydrogen peroxide as oxidant was shown to be suitable for sequential injection

analysis chemiluminescence providing a green reagent without toxic by-products. The advantage

of this system includes simplicity, reliability, sustainability, and convenience with relatively low

cost consumption of reagents with which the assay of tryptophan can be performed and data can

be acquired and processed. Eighty seconds were required to complete one cycle providing a

throughput of forty-five samples/hour. The proposed method was successfully applied to the

determination of tryptophan in pharmaceutical formulations with satisfactory results. The

sequential injection analysis - zone fluidics - chemiluminescence -Firefly system performs

continuously and reliably at a sampling rate of 45 samples/hour over a period of more than 6

months with the advantage to be used as a process analyzer for the mentioned industries.

Acknowledgments

The authors would like to acknowledge the financial support received from project

Program Ideas by PN-II-ID-PCE-2011-3-0538/2012-2014, financed by contract 100/27.10.2011.

References

12
 Bagheri, H., M. Ahmadi, T. Madrakian, and A. Afkhami. 2015. Preconcentration and
spectrofluorometric determination of L-tryptophan in the presence of D-tryptophan using a chiral
magnetic nanoselector. Sens. Actuators B: Chem. 221:681–87. doi:10.1016/j.snb.2015.07.013.
Barnes, D. E., and J. F. van Staden. 1992. Flow-injection analysis in the evaluation of supported
liquid membranes. Anal. Chim. Acta 261 (1–2):441–51. doi:10.1016/0003-2670(92)80225-v.
Çevikkalp, S. A., G. B. Löker, M. Yaman, and B. Amoutzopoulos. 2016. A simplified HPLC
method for determination of tryptophan in some cereals and legumes. Food Chem. 193:26–29.
doi:10.1016/j.foodchem.2015.02.108.
Costin, J. W., P. S. Francis, and S. W. Lewis. 2003. Selective determination of amino acids using
flow injection analysis coupled with chemiluminescence detection. Anal. Chim. Acta 480:67–77.
doi:10.1016/s0003-2670(02)01645-8.
Downloaded by [Australian Catholic University] at 05:59 13 August 2017

Cubero, J., F. Toribio, M. Garrido, M. T. Hernández, J. Maynar, C. Barriga, and A. B.

Rodríguez. 2010. Assays of the amino acid tryptophan in cherries by HPLC-fluorescence. Food
Anal. Methods 3:36–39. doi:10.1007/s12161-009-9084-1.
Deng, P., J. Fei, and Y. Feng. 2011. Sensitive voltammetric determination of tryptophan using an
acetylene black paste electrode modified with a Schiff’s base derivative of chitosan. Analyst
136:5211–17. doi:10.1039/c1an15351j.
Fernstrom, J. D. 1983. Role of precursor availability in control of monoamine biosynthesis in
brain. Physiol. Rev. 63 (2):484–546.
Foord, R., R. Jones, C. J. Oliver, and E. R. Pike. 1969. The use of photomultiplier tubes for
photon counting. Appl. Opt. 8 (10):1975–89. doi:10.1364/ao.8.001975.
Gao, C. Y., N. Chu, and S. Fan. 2010. Chemiluminescence behavior and application of Ce(IV)-
Tween20-tryptophan system. Anal. Lett. 43:2142–51. doi:10.1080/00032711003698770.
Gao, C., and S. Fan. 2014. Determination of tyrosine and tryptophan by sequential injection
analysis and chemiluminescence detection. Anal. Lett. 47:178–89.
doi:10.1080/00032719.2013.764532.
Global FIA. 2017. www.GlobalFia.com.
Guo, Y., S. Guo, Y. Fang, and S. Dong. 2010. Gold nanoparticle/carbon nanotube hybrids as an
enhanced material for sensitive amperometric determination of tryptophan. Electrochim. Acta
55:3927–31. doi:10.1016/j.electacta.2010.02.024.
Hugli, T. E., and S. Moore. 1972. Determination of the tryptophan content of proteins by ion
exchange chromatography of alkaline hydrolysates. J. Biol. Chem. 247:2828–34.
Ikeda, M., H. Tsuji, S. Nakamura, A. Ichiyama, Y. Nishizuka, and O. Hayaishi. 1965. Studies on
the biosynthesis of nicotinamide adenine dinucleotide. II. A role of picolinic carboxylase in the
biosynthesis of nicotinamide adenine dinucleotide from tryptophan in mammals. J. Biol. Chem.
240 (3):1395–401.

13
 Islam, J., H. Shirakawa, T. K. Nguyen, H. Aso, and M. Komai. 2016. Simultaneous analysis of
serotonin, tryptophan and tryptamine levels in common fresh fruits and vegetables in Japan using
fluorescence HPLC. Food Biosci. 13:56–59. doi:10.1016/j.fbio.2015.12.006.
Jagannathan, V., C. March, and J. Venitz. 1995. Determination of unbound L-tryptophan in
human plasma using high-performance liquid chromatography with fluorescence detection.
Biomed. Chromatogr. 9:305–08. doi:10.1002/bmc.1130090626.
Khaleghi, F., A. E. Irai, V. K. Gupta, S. Agarwal, M. Bijad, and M. Abbasghorbani. 2016.
Highly sensitive nanostructure voltammetric sensor employing Pt/CNTs and 1-butyl-3-
methylimidazolium hexafluoro phosphate for determination of tryptophan in food and
pharmaceutical samples. J. Mol. Liq. 223:431–35. doi:10.1016/j.molliq.2016.08.058.
Kochen, W., and H. Steinhart. 1994. L-Tryptophan: Current prospects in medicine and drug
Downloaded by [Australian Catholic University] at 05:59 13 August 2017

safety. Berlin: de-Gruyter.

Liang, Y. D., and J. F. Song. 2005. Flow-injection chemiluminescence determination of
tryptophan through its peroxidation and epoxidation by peroxynitrous acid. J. Pharm. Biomed.
Anal. 38:100–06. doi:10.1016/j.jpba.2004.12.010.
Liu, Y., and L. Xu. 2007. Electrochemical sensor for tryptophan determination based on copper-
cobalt hexacyanoferrate film modified graphite electrode. Sensors 7:2446–57.
Marshall, G. D., D. Wolcott, and D. Olson. 2003. Zone fluidics in flow analysis: Potentialities
and applications. Anal. Chim. Acta 499:29–40. doi:10.1016/j.aca.2003.09.003.
Mattivi, F., U. Vrhovšek, and G. Versini. 1999. Determination of indole-3-acetic acid,
tryptophan and other indoles in must and wine by high-performance liquid chromatography with
fluorescence detection. J. Chromatogr. A 855:227–35. doi:10.1016/s0021-9673(99)00696-2.
Qiu, H., C. Luo, M. Sun, F. Lu, L. Fan, and X. Li. 2012. Determination of L-tryptophan based on
graphene oxide–magnetite-molecularly imprinted polymers and chemiluminescence. Talanta
98:226–30. doi:10.1016/j.talanta.2012.06.078.
Ravindran, G., and W. L. Bryden. 2005. Tryptophan determination in proteins and feedstuffs by
ion exchange chromatography. Food Chem. 89:309–14. doi:10.1016/j.foodchem.2004.05.035.
Ren, J., M. Zhao, J. Wang, C. Cui, and B. Yang. 2007. Spectrophotometric method for
determination of tryptophan in protein hydrolysates. Food Technol. Biotechnol. 45:360–66.
Schaechter, J. D., and R. J. Wurtman. 1990. Serotonin release varies with brain tryptophan
levels. Brain Res. 532 (1–2):203–10. doi:10.1016/0006-8993(90)91761-5.
Taljaard, R. E., and J. F. van Staden. 1998. Application of sequential injection analysis as
process analysers. Lab. Rob. Autom. 10 (6):325–337. doi:10.1002/(sici)1098-
2728(1998)10:6<325::aid-lra3>3.0.co;2-l.
Tashkhourian, J., M. Daneshi, and S. F. Nami-Ana. 2016. Simultaneous determination of
tyrosine and tryptophan by mesoporous silica nanoparticles modified carbon paste electrode
using H-point standard addition method. Anal. Chim. Acta 902:89–96.
doi:10.1016/j.aca.2015.10.037.

14
 van Staden, J. F. 1998. Flow and sequential injection systems in process control and monitoring.
Achievements and challenges. Curr. Trends Anal. Chem. 1:89–118.
van Staden, J. F. 2002. Solving the problems of sequential injection systems as process
analyzers. Anal. Chim. Acta 467 (1–2):61–73. doi:10.1016/s0003-2670(02)00205-2.
van Staden, J. F. 2015a. Analytical continuous flow systems. Where two worlds collide! From
gravimetry and test tubes to flow systems to FIA to SIA to PAT and from Orsat to control room
to PAT to TAP. Rev. Ro. Chim. 60 (5–6):403–14.
van Staden, J. F. 2015b. Application of phthalocyanines in flow- and sequential-injection
analysis and microfluidics systems. Talanta 139:75–88. doi:10.1016/j.talanta.2015.02.026.
van Staden, J. K. F., and R. State. 2016. Determination of dopamine using the alkaline luminol-
hydrogen peroxide system for sequential injection-zone fluidics analysis. Anal. Lett. 49
Downloaded by [Australian Catholic University] at 05:59 13 August 2017

(17):2783–92. doi:10.1080/00032719.2016.1157691.
van Staden, J. F., and R. I. Stefan. 2004. Chemical speciation by sequential injection analysis. An
overview. Talanta 64 (5):1109–13.
van Staden, J. F., and R. I. Stefan-van Staden. 2010. Application of porphyrins in flow-injection
analysis. A review. Talanta 80 (5):1598–605. doi:10.1016/j.talanta.2009.10.016.
van Staden, J. F., and R. I. Stefan-van Staden. 2012. Flow-injection analysis systems with
different detection devices and other related techniques for the in vivo and in vitro determination
of dopamine as neurotransmitter. A review. Talanta 102:34–43.
doi:10.1016/j.talanta.2012.05.017.
Wu, F., B. Tong, and Q. Zhang. 2011. Application of a new iridium complex as a
chemiluminescence reagent for the determination of tryptophan. Anal. Sci. 27:529–33.
Wurtman, R. J., and F. Anton-Tay. 1969. The mammalian pineal as a neuroendocrine transducer.
Recent Prog. Horm. Res. 25:493–522. doi:http://dx.doi.org/10.1016/b978-0-12-571125-8.50014-
4.

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 Table 1. Tolerable concentration ratios of some interfering species to 1.0 × 105 mol /L
tryptophan.

Substance Tolerable concentration ratio

D-Glucose 1000

L-Leucine 1000

L-Isoleucine 1000

D,L-Histidine 1000
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L-Phenylalanine 500

Dopamine 500

L-Alanine 100

D-Valine 100

L-Tyrosine 100

Ascorbic acid (Vitamin C) 10

D,L-Aspartic acid 10

Uric acid 10

L-Cysteine 10

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 Table 2. Determination of tryptophan in pharmaceutical capsules (n = 6).

Sample Labeled of Found concentration of Recovery Relative

concentration of tryptophan (mg/mL) (%) standard

tryptophan (mg/mL) deviation

(%)

Product A1 15 15.9 105.7 2.0

Product A2 15 15.2 101.5 4.8

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Product A3 15 14.5 96.8 2.9

Product A4 15 14.4 96.3 3.6

Product A5 15 14.5 96.9 4.5

Product A6 15 14.6 97.1 7.1

Product A7 15 15.4 102.8 6.3

Product A8 15 14.7 98.1 6.3

Product A9 15 15.0 100.2 6.5

Product A10 15 15.6 104.1 3.4

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 Figure 1. Chemiluminescence spectrum of luminol with hydrogen peroxide in the presence of
104 mol /L tryptophan. Conditions: 103 mol /L luminol, 0.8 mol /L H2O2 and 0.02 mol /L
NaOH.
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 Figure 2. Effect of hydrogen peroxide concentration on the number of photons counted
(chemiluminescence intensity). Conditions: 104 mol /L tryptophan; 103 mol /L luminol and
0.02 mol /L NaOH.
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 Figure 3. Effect of luminol concentration on the number of photons counted
(chemiluminescence intensity); insert - 0.1 and 100 µmol/L. Conditions: 104 mol /L tryptophan;
0.8 mol /L H2O2 and 0.02 mol /L NaOH.
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 Figure 4. (A) Chemiluminescence as a function of L-tryptophan concentration: (a) 106 mol /L,
(b) 105 mol /L, (c) 104 mol /L, (d) 4 × 104 mol /L, (e) 6 × 104 mol /L and (f) 103 mol /L. (B)
Calibration plot: I = 1477.85  0.68 × C; R2 = 1.00. Conditions: luminol 1.0 × 103 mol /L; H2O2
0.8 mol /L and NaOH 0.02 mol /L.
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