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Rapid Determination of Total Tryptophan in Yoghurt by Ultra High Performance Liquid Chromatography with Fluorescence Detection


Abstract and Figures

Tryptophan (TRP) is an essential amino acid which cannot be synthesized by humans and animals, but has to be supplied by exogenous sources, notably through the diet. The bulk of dietary TRP flows into the synthesis of body's proteins, but the TRP metabolism also involves several biochemical reactions (i.e., serotonin and kynurenine pathways). Defects in the TRP transport mechanism or catabolism are related to a large number of clinical abnormalities. Therefore, dietary TRP intake is necessary not only for the body's growth but also for most of the body's metabolic functions. Among protein-based foods, milk proteins provide a relatively high amount of TRP. In this paper, a rapid chromatographic method for TRP determination in yoghurt, by ultra high performance liquid Chromatography on a reversed-phase column with fluorescence detection (280 nm Ex; 360 nm Em), is provided. A linear gradient elution of acetonitrile in water allowed TRP analysis in 8.0 min. The limit of detection and limit of quantification of the method were 0.011 ng/µL and 0.029 ng/µL, respectively, using 5-methyl-l-tryptophan as the internal standard. The analytical method was successfully applied to commercial yoghurts from different animal species, and the TRP values ranged between 35.19 and 121.97 mg/100 g (goat and cow Greek type yoghurt, respectively).
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Technical Note
Rapid Determination of Total Tryptophan in Yoghurt
by Ultra High Performance Liquid Chromatography
with Fluorescence Detection
Mena Ritota and Pamela Manzi *
CREA-Centro di Ricerca Alimenti e Nutrizione, Via Ardeatina 546, 00178 Rome, Italy;
Academic Editors: Daniel Cozzolino and Susy Piovesana
Received: 5 October 2020; Accepted: 27 October 2020; Published: 29 October 2020
Tryptophan (TRP) is an essential amino acid which cannot be synthesized by humans
and animals, but has to be supplied by exogenous sources, notably through the diet. The bulk
of dietary TRP flows into the synthesis of body’s proteins, but the TRP metabolism also involves
several biochemical reactions (i.e., serotonin and kynurenine pathways). Defects in the TRP transport
mechanism or catabolism are related to a large number of clinical abnormalities. Therefore, dietary
TRP intake is necessary not only for the body’s growth but also for most of the body’s metabolic
functions. Among protein-based foods, milk proteins provide a relatively high amount of TRP. In this
paper, a rapid chromatographic method for TRP determination in yoghurt, by ultra high performance
liquid Chromatography on a reversed-phase column with fluorescence detection (280 nm Ex; 360 nm Em),
is provided. A linear gradient elution of acetonitrile in water allowed TRP analysis in 8.0 min. The limit
of detection and limit of quantification of the method were 0.011 ng/
L and 0.029 ng/
L, respectively,
using 5-methyl-l-tryptophan as the internal standard. The analytical method was successfully applied
to commercial yoghurts from dierent animal species, and the TRP values ranged between 35.19 and
121.97 mg/100 g (goat and cow Greek type yoghurt, respectively).
Keywords: tryptophan; ultra high performance liquid chromatography; yoghurt
1. Introduction
Tryptophan (TRP) is an essential amino acid needed for normal growth, and is involved in the
synthesis of dierent bioactive compounds, such as nicotinamide (vitamin B6), melatonin, tryptamine,
kynurenine, 3-hydroxykynurenine, and quinolinic and xanthurenic acids [1].
In lower organisms, TRP is formed through the condensation of serine with an indole group by
the action of tryptophan synthase [
]. In humans and animals, though, TRP cannot be synthesized
because they are lacking in tryptophan synthase [
]. Therefore, TRP has to be supplied to the body by
exogenous sources, especially through the diet [
]. Besides participating in the formation of the body’s
proteins, TRP is involved in numerous chemical reactions.
TRP and its metabolites seem to have the potential to contribute to the therapy of autism,
multiple sclerosis, cardiovascular, chronic kidney and inflammatory bowel diseases, cognitive function,
depression, and microbial infections [
]. TRP transport through the cell membranes is competitively
inhibited by the other large neutral amino acids (NAA), such as valine, leucine, isoleucine, phenylalanine
and tyrosine [3].
From these premises, it is clear that TRP intake through the diet, or the intake of TRP-rich proteins,
is necessary not only for the body’s growth, but also to carry out most of the body’s metabolic functions.
However, the wide use of TRP as a dietary supplement for its potential health benefits has raised the
Molecules 2020,25, 5025; doi:10.3390/molecules25215025
Molecules 2020,25, 5025 2 of 11
issue of assessing its safety [
], so much so that an upper limit of safe intake for diet-added tryptophan
of 4.5 g/day has been proposed in young adults [4].
Among food proteins introduced through the diet, cereals, and especially maize, are generally
poor in tryptophan content; as such, TRP may represent the nutritionally limiting amino acid in these
food items [
]. Egg, soy, beans, seafood and poultry proteins, instead, have been described as good
sources of tryptophan [6].
Milk proteins are particularly rich in TRP, which can be released by the proteolytic enzymes either
as a free amino acid or as a part of small peptides with functional activities [
]. Furthermore, it has
been shown that TRP released during
in vitro
gastrointestinal digestion is one of the main factors
responsible for the radical scavenging activity of digested bovine milk [
]. TRP intake through the diet
considerably varies depending on the food protein type, since TRP distribution can significantly dier
among the various fractions [
]; in cow’s milk proteins, for example,
-lactalbumin contains around
6% TRP, whereas bovine serum albumin and
-casein are extremely poor in TRP [
]. Furthermore,
the TRP bioavailability through dietary intake might be reduced during food processing or cooking,
mainly by oxidative degradation or cross-linking among proteins [
], as well as by decarboxylation
reactions [12].
The nutritional and safety aspects of tryptophan emphasize the need for reliable analytical
methods for its determination in food. The first rapid method for tryptophan determination dates back
to 1970, when Gaitonde and Dovey [
] proposed a colorimetric method, whereby TRP reacted with
acid ninhydrin giving a yellow product, which was spectrophotometrically revealed at
λ=390 nm.
However, this method needed to be corrected for tyrosine absorption [
]. Later, Inglis [
] developed
a method for amino acid determination by preventing tryptophan degradation, which generally occurs
in the acid conditions of protein hydrolysis (HCl 6 N at 110
C for 22 or 24 h [
]), by protecting
TRP with tryptamine. Yamada et al. [
] improved this method by modifying the proteins with
vapor-phase S-pyridylethylation before the hydrolysis, and then treated the modified proteins with
mercaptoethanesulfonic acid at 176
C. However, all these early methods were applied to pure proteins,
without considering the matrix eect.
When referring to a food product, one of the main concerns is certainly the release of the protein-bound
tryptophan from the matrix. Acid hydrolysis generally carried out for the total determination of most
amino acids cannot be performed for TRP analysis, because it is destroyed during acid hydrolysis [
Therefore, alkaline hydrolysis, as reported by Steven and Jorg as far back as 1989 [
], is currently the
pre-treatment method of choice for tryptophan determination in foods.
TRP detection and quantification can be carried out with dierent analytical techniques. Near
infrared spectroscopy (NIRS) was used to determine TRP, as well as other amino acids in dairy
products, and in particular to evaluate cheese ripening [
]. Even if these spectroscopy methods oer
the advantages of a short time of analysis and a poor sample preparation, they need huge amounts
of samples for the calibration and validation of the models. Ion exchange chromatography (IEC)
with fluorescence detection was also employed to determine TRP in pure proteins and feedstuffs [20],
but IEC needed post-column derivatization with o-phthalaldehyde (OPA), thus resulting in further
time- and chemical-consuming steps. The determination of tryptophan in infant formula by high
performance liquid chromatography (HPLC) with UV detection (
=254 nm) needed a derivatization step,
with phenylisothiocyanate (PITC), in addition to the protein hydrolysis prior to the HPLC analysis [
Derivatization was necessary due to the poor absorbance of TRP in the UV spectral region, but this
could result in a time- and chemicals-consuming procedure. Furthermore, Tsopmo et al. [
], regardless
of the use of the more sensitive tandem mass spectrometry (MS/MS) detector, derivatized TRP with
PITC, in order to evaluate the TRP in human milk, infant plasma and peptide fraction.
Fluorimetric detectors could be employed to increase the selectivity and sensibility of an HPLC
method [
]. Furthermore, TRP exhibits a strong native fluorescence [
], which allows one to avoid
the derivatization generally needed for most amino acids determined by HPLC.
Molecules 2020,25, 5025 3 of 11
Therefore, the aims of this paper were as follows: 1) to develop a robust, rapid and cost-eective
method for tryptophan detection in yoghurt based on ultra high performance liquid chromatography
(UHPLC) by means of a fluorescence detector; 2) to evaluate the levels of tryptophan in commercial
yoghurts from the milk of dierent animal species. The analytical technique of choice in this study
was UHPLC, since this chromatography has the advantages of speed, enhancing resolution and peak
eciency, as well as requiring smaller amounts of solvent compared to the traditional HPLC, so it can
be used for fast and eco-friendly analysis. To the best of our knowledge, this is the first time that this
technique has been applied for the analysis of TRP in yoghurt samples.
2. Results
2.1. Chromatographic Method Validation
The alkaline hydrolysis with NaOH, according to the method of Steven and Jorg [
], was
carried out to extract TRP from the yoghurt samples. Partial disruption of TRP can occur during
hydrolysis: these losses can be corrected based upon the recovery of an internal standard [
and 5-methyl-l-tryptophan (M-TRP) has been revealed as the preferred internal standard for TRP
analysis [
]. Furthermore, the presence of foreign substances in a matrix may cause a bias by increasing
or decreasing the signal attributed to the measurand [
]. Due to the absence of a suitable reference
material to estimate the potential influence of the interferences of the yoghurt matrix on the analysis of
TRP (the so called “matrix eect”), the approach of recovery tests (using spiked samples) was used [
In the recovery value test, the original concentrations of TRP in the yogurt samples were determined
using the calibration curve described below. Each of the yoghurt samples was then spiked, prior to the
extraction, with a known concentration of TRP (at dierent levels, ranging from 0.061 to 0.152
and the total TRP concentrations of the spiked samples were calculated using the same calibration
curve. The recovery values, adjusted for the value of M-TRP, ranged between 97.36% and 100.12%,
with a mean value of 97.27% (Table 1).
Table 1. Accuracy of the method expressed as Recovery (%) in yoghurt samples.
Spiked Levels TRP Addition (µg/mL) Recovery (%)
1 0.061 97.36 ±0.70
2 0.091 98.90 ±1.58
3 0.122 92.79 ±1.09
4 0.152 100.12 ±1.16
The linearity range of the method was evaluated by injection, in triplicate, of the following
TRP standard solutions: 1.105
g/mL: 0.848
g/mL; 0.424
g/mL; 0.553
g/mL 0.1696
g/mL and
g/mL. The linearity range investigated covered the entire measurement range of the samples.
The calibration and quantification of TRP in the yogurt samples were obtained by the standard
addition method. The calibration curve (y =502654x) was obtained with a correlation factor
with the error of curve equal to 1751. The limit of detection (LOD) and limit of quantification (LOQ) of
the method were 0.011 ng/µL and 0.029 ng/µL, respectively.
The method’s precision was evaluated through repeatability and reproducibility measurements;
the method resulted in a good precision, and the intra- and inter-day relative standard deviation
(RSD %), on pure standards and on yoghurt samples, are shown in Tables 2and 3, respectively.
Molecules 2020,25, 5025 4 of 11
Table 2. Repeatability and reproducibility performances on pure standards.
std TRP
Intra Day Inter Day
1 Day
2 Day
3 Day
1.105 1.23 1.00 0.99 1.08
0.848 1.68 1.06 1.09 1.28
0.424 1.66 1.33 1.21 1.40
0.553 1.50 2.00 1.44 1.65
0.170 1.66 0.84 1.37 1.29
0.085 1.13 1.11 1.02 1.09
Table 3. Repeatability and reproducibility performances on yoghurt samples.
TRP Level
(mg/100 g)
Intra Day Inter Day
1 Day
2 Day
3 Day
121.97 2.59 2.87 1.79 2.59
62.96 1.11 1.14 1.18 1.71
35.19 1.36 1.55 1.27 1.76
Furthermore, the relative standard deviation in retention times was less than 0.2% and 0.3%,
for intra-day and inter-day, respectively.
The proposed chromatographic method allowed TRP determination in yoghurt samples in
a relatively short time; the total chromatographic running time was 8.0 min, including column
reconditioning, with a TRP retention time equal to 1.197 min and a 5-methyl-l-tryptophan (M-TRP)
retention time equal to 1.564 min (Figure 1).
Figure 1.
Chromatographic profile of a TRP and M-TRP standard solution and yoghurt samples
extracts, according to the chromatographic conditions reported in Section 4.
2.2. Tryptophan Levels in Commercial Yoghurts
The proposed method was applied to the analysis of the TRP contents in various commercial
yoghurts obtained from the milk of dierent animal species. The results, reported in Table 4, showed a
great variability.
Molecules 2020,25, 5025 5 of 11
Table 4. TRP contents (mg/100g) in commercial yoghurts from milk of dierent animal species.
Yoghurt Sample Number Mean SD
Cow yoghurt (whole milk plain) 1. 41.94 0.37
2. 41.28 0.08
3. 41.44 0.20
4. 41.70 0.60
5. 40.78 0.26
6. 42.00 0.13
7. 45.48 0.12
8. 44.36 1.03
9. 48.82 0.38
10. 49.22 0.17
11. 48.91 0.06
12. 49.31 0.14
Ewe yoghurt (whole milk plain) 1. 62.91 1.20
2. 62.96 1.45
Goat yoghurt (whole milk plain) 1. 35.19 1.18
2. 37.61 1.33
Bualo yoghurt (whole milk plain) 1. 53.01 1.23
2. 52.87 0.95
Cow yoghurt (lactose-free whole milk plain) 1. 47.93 0.47
2. 50.36 0.39
Cow yoghurt (Greek-type whole milk plain) 1. 121.97 2.69
2. 120.98 2.24
Among the whole yoghurt samples, there were significant dierences in the TRP levels (P<0.05):
ewe milk yoghurts showed the highest TRP concentration (on average 62.94 mg/100 g), while goat
milk yoghurts had the lowest one (36.40 mg/100 g). Furthermore, whole cow yoghurt samples showed
the greatest sample heterogeneity, with TRP levels ranging between 40.78 and 49.31 mg/100 g.
Among the cow yoghurts, the highest TRP content was observed in the Greek type samples
(on average 121.47 mg/100 g), followed by lactose free and whole cow yoghurts (49.15 mg/100 g and
44.60 mg/100 g, respectively). The highest TRP value reported in the Greek type samples is justified by
their high protein contents (about 9 g/100 g), due to the particular technological process whereby the
liquid whey is removed, obtaining a thick and creamy product.
3. Discussion
3.1. Method Validation
Due to the presence of the indole ring in the tryptophan structure, which is degraded under the
acid conditions generally used in the protein hydrolysis, TRP cannot be analyzed by the standard
method for amino acid analysis [
]. Many attempts have been made over the years, from enzymatic
to modified acid hydrolysis, but all these procedures suer from incomplete TRP recovery [
Therefore, alkaline hydrolysis is the common procedure for TRP analysis in foods. Among the dierent
alkalis used for protein hydrolysis [
], NaOH does not suer for the inconvenience of solubility in
water, unlike Ba(OH)
and LiOH, and it avoids the precipitation/adsorption problems associated with
the use of Ba(OH)
]. Therefore in this study, an alkaline hydrolysis with NaOH 4.2 M, according to
the method of Steven and Jorg [18], was performed.
While many scientific attempts have been made for TRP determinations in biological and
pharmaceutical samples [
], as well as in non-dairy food items [
], very few works have
been reported in the literature about TRP determinations in dairy products, and in particular in yoghurt.
The limit of detection (LOD) of this method (0.011 ng/
L) is comparable to that reported
by Yılmaz and Gökmen (0.0165 ng/
L in yoghurt) [
], who even used a more sensitive detector
(tandem mass spectrometry) to determine TRP and its derivatives in foods. Furthermore, our LOD
Molecules 2020,25, 5025 6 of 11
is lower compared to that obtained by Liu and Xu for TRP analysis in milk [
], who employed a
selective electrochemical sensor; the method proposed, in fact, showed a limit of detection of 6
corresponding to 1.226 ng/
L. Only the methods proposed by Wang et al. [
], who developed a
modified electrochemical sensor to determine TRP in milk, and by Baytak and Aslanoglu, who had to
resort to a nanosensor for TRP determination in cow’s milk [
], showed lower LOD values (0.0035 ng/
and 0.00135 ng/
L, respectively). Even though the electrochemical sensors generally allow a simple and
rapid determination of the analytes in food matrices without any sample pretreatment, TRP analysis
requires a modification procedure of the electrodes, since under the traditional working electrode
conditions TRP oxidation suers from high overpotential and sluggish kinetics [
]. These electrode
modifications may result in chemical reagent consumption. Furthermore, electrochemical sensors are
still little used in the laboratories for routine analysis.
The proposed chromatographic method allowed TRP determination in yoghurt samples in a
relatively short time (total chromatographic running time =8.0 min). This was possible thanks to the
high speed of the UHPLC technique. Furthermore, Delgado-Andrade et al. [
] proposed a liquid
chromatography-based method with fluorescence detection for TRP analysis in milk-based ingredients,
but the total chromatographic time, even if not specified by the authors in the text, was at least 10 min.
The running chromatographic time of this proposed method was even shorter than that proposed by
La Cour et al. [43]
with UHPLC–single quadrupole mass spectrometry, who reported a total analysis
time of 11.5 min for TRP analysis in plant materials and dog foods, even if the author stated a possibility
of shortening the running time.
The results reported in this study show that the proposed chromatographic method can be used
for the routine analysis of TRP in foods; it is low in terms of time and chemical consumption, it ensures
a low sensitivity, and the analytical equipment is easy to access and much less expensive than the more
sensitive mass spectrometry.
3.2. Nutritional Evaluation of Eryptophan Levels in Commercial Yoghurts
Regarding the TRP levels in the yoghurt samples, the results obtained in this study are in agreement
with those previously reported by Gambelli et al. [
] in commercial yoghurt samples, through a similar
analytical method, but using an HPLC. The TRP levels reported in this study are also of the same order
of magnitude as those reported by Posati and Orr [
] on similar commercial samples. Furthermore,
az et al. [
] evaluated the total TRP content in commercial yoghurts, but they reported
slightly higher levels (374 mg/kg) by using a completely dierent method based on heavy atom-induced
room temperature phosphorescence.
On the contrary, our results are very high compared to those reported by Bertazzo et al. [
] on
similar food items (TRP =0.7
g/mL in yoghurt samples); the main dierence is due to the fact that the
authors [
] did not hydrolyze the commercial yoghurt samples, but carried out the HPLC analysis
on the supernatants directly after centrifugation, so their results referred to the “free” TRP content of
yoghurts. Furthermore, Yılmaz and Gökmen [48] reported similar results for free TRP in commercial
yoghurts, with values ranging between 3.2 and 13.4 mg/kg dry weight. The dierences in the free amino
acid contents observed by the authors [
] could be due to the dierent microorganisms involved
in the production of the yoghurts, since free amino acid content has been shown to be influenced by
the interactions between the microorganisms involved in the yoghurt fermentation [
], and by the
dierent strains of the microorganisms employed [49].
Dierences in the total TRP levels of yoghurt samples from the milk of dierent animal species
are essentially due to the dierent protein contents [
]. However, Yılmaz and Gökmen [
] observed
the presence of TRP, together with its derivatives, in the kynurenine pathway in commercial yoghurts,
suggesting a fermentation eect on the level of TRP and its derivatives. Similar findings were reported
also by Bertazzo et al. [
], who observed TRP and its derivatives in both the serotonin and kynurenine
pathways in milk and fermented dairy products. The authors also reported an increase in the free
Molecules 2020,25, 5025 7 of 11
TRP levels with increasing fermentation, due to the proteolytic action of the added cultures, thus
corroborating the hypothesis of Yılmaz and Gökmen [37].
The highest TRP content of yoghurt from ewe milk could be due to the higher general TRP content
in ewe milk compared to milk from other animal species [
]. Similar values were reported for TRP in
cow and bualo milk [
], while in some cases slightly lower levels of TRP were reported in goat milk
compared to other animal species [
]. Furthermore, the nutrient composition of yoghurts has been
shown to be highly dependent on the technological process [45].
The typical recommended daily intake for tryptophan has been set by FAO/WHO at 4 mg/kg of
body weight per day for adults [52], that is to say, 280 mg/day for a 70 kg adult [3].
According to the data reported in this study, three daily recommended servings of cow yoghurt
(125 grams for a serving [
]) supply almost 60.6% of the recommended TRP daily intake for adults,
while 84.3%, 48.8% and 70.9% of the recommended TRP daily intake is supplied by three servings of
ewe, goat and bualo yoghurt, respectively. It is worth noting that only two Greek yoghurt servings
are enough for achieving the entire TRP recommended daily intake (108.5%), thanks to its high
protein content.
TRP levels in yoghurts are generally of the same order of magnitude in milk [
], so a yoghurt
serving (with the exception of Greek yoghurt, due to its typical production process) provides almost the
same TRP intake of a milk serving. However, yoghurt’s shelf life is longer than milk’s, and yoghurt can also
be consumed by lactose-intolerant people. Furthermore, according to the study of
Bertazzo et al. [47]
the TRP content in yoghurt does not decrease during the storage, so the TRP intake is guaranteed until
the expiration date of the yoghurt. For all these reasons, yoghurt can be considered a good source of
TRP, and its consumption should be encouraged, not only for ensuring the recommended daily intake
of TRP, but also for its overall nutritive value, above all the probiotic properties and the calcium intake.
4. Materials and Methods
4.1. Samples Preparation
In total, 22 commercial brands of yoghurt from dierent species were purchased in the local markets:
-12 dierent brands of whole cow yoghurt;
-2 dierent brands of whole ewe yoghurt;
-2 dierent brands of whole goat yoghurt;
-2 dierent brands of whole bualo yoghurt;
-2 dierent brands of whole cow yoghurt, lactose-free;
-2 dierent brands of whole cow Greek yoghurt.
All samples were stored at 4 C, as indicated on the label, prior to testing.
Tryptophan (TRP) was extracted by alkaline hydrolysis according to the method of Steven and
Jorg [
]. In brief, 8 mL of NaOH 4.2 M was added to 0.5 g of the yoghurt samples. An appropriate
amount of the internal standard (5-methyl-l-tryptophan) was added, then the oxygen was removed to
avoid the oxidative degradation of TRP. The hydrolysis was carried out at 110
C for 20 h. Afterwards,
the samples were cooled in an ice bath and neutralized with HCl, added with EtOH and filled to the
mark with phosphate buer 0.2 M. The samples were filtered (0.2 µm) prior to the UHPLC analysis.
All the 22 commercial brands of yoghurt were analyzed in triplicate.
4.2. Chemicals
l-tryptophan (TRP) and 5-methyl-triptophan (M-TRP) were purchased from Sigma (Sigma-Aldrich
Co., St. Louis, MO, USA).
The hydrochloric acid, sodium hydroxide and acetonitrile of HPLC grade were from Merck
(Darmstadt, Germany). All the other chemicals used were of analytical purity. All solvents were
filtered through 0.2 µm membrane filters (Phenomenex Inc., Torrance, CA, USA).
Molecules 2020,25, 5025 8 of 11
4.3. UHPLC Equipment and Conditions
A Nexera UHPLC system (Shimadzu Corporation, Kyoto, Japan), equipped with two LC-30AD
pumps, an RF-20A fluorimetric detector and an SIL 30-AC autosampler, was employed for the
chromatographic analyses. A Shim-Pak ODS II column (2.2
m; 75 mm
2 mm i.d., Shimadzu
Corporation, Kyoto, Japan), maintained at 25
C during the analysis, was used for the separation.
The selected elution system, for a total of running time equal to 8.0 min, is reported in Table 5. The flow
rate was set at 0.4 mL/min, while the injection volume was set at 1
L. Tryptophan and 5-methyl-
tryptophan were detected at 280 and 360 nm for excitation and emission wavelengths.
Table 5. Gradient elution system.
Time (Min) CH3CN % H2O % mL/min
0.0 10 90 0.4
3.0 80 20 0.4
4.0 10 90 0.6
7.9 10 90 0.6
8.0 10 90 0.4
4.4. Method Validation
Standard stock solution of TRP (21.2 mg in 25 mL of deionized water) was prepared. Individual
working standard solutions were prepared at six dierent levels by dilution in deionized water
of the standard stock solution (0.085
g/mL 0.170
g/mL; 0.424
g/mL; 0.553
g/mL; 0.,848
1.105 µg/mL).
Quantities of 100
L of the internal standard 5-methyl-l-tryptophan (856
g/mL in NaOH 0.05 M)
were added before hydrolyzing all the samples.
The method’s performance parameters were evaluated according to the EURACHEM Guide 2014 [
In more detail, the method precision was evaluated through the measurements of repeatability (intra-day
precision) and reproducibility (inter-day precision) upon direct injection of TRP standard solutions
at six levels, respectively, on three replicates and on three non-consecutive days. The repeatability
and reproducibility on yogurt samples at three levels, respectively, on three replicates and on three
non-consecutive days, were considered as well to evaluate the variation due to the entire analytical
procedure. Precision was expressed as relative standard deviation (RSD %).
The limit of detection (LOD) and limit of quantification (LOQ) for TRP were calculated according
to the following equations: LOD =X
, LOQ =X
, where X
was the blank mean
value (n=10) and SD
the blank standard deviation [
]. Procedural blanks [
], where deionized
water was used in place of the yoghurt matrix, were used for determining LOD and LOQ.
The recovery was calculated by means of spiked samples, and expressed as relative spike recovery,
according to the following equation: R (%) =[(x
– x)/x
100. Here, x
is the mean value of the
spiked sample, x is the mean value of the non-spiked sample and x
is the added concentration [
4.5. Statistics
All the analyses were performed in triplicate. Data were reported as mean value with standard
deviation (SD). Mean values were subjected to one-way analysis of variance (ANOVA), coupled with the
Tukey’s post hoc test. Statistical analysis was performed using the PAST Software (2.17c version) [
Author Contributions:
Conceptualization and methodology, P.M.; writing—original draft preparation, M.R.;
writing—review and editing, M.R. and P.M. All authors have read and agreed to the published version of
the manuscript.
This research was funded by the Italian Ministry “Ministero delle Politiche Agricole, Alimentari e
Forestali (MiPAAF)” within the Project: QUALIFU “QualitàAlimentare e Funzionale”, D.M. 24292/7303/14.
Conflicts of Interest: The authors declare no conflict of interest.
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... As a matter of fact, devising a simple approach to make Trp determination easier is important. Several techniques for determining Trp have been developed, including liquid chromatography with fluorescence detection [4], chemiluminescence [5], gas chromatography-mass spectrometry [6], high-performance liquid chromatography [7], and infrared spectroscopy [8]. These techniques can determine trace quantities of Trp because of their great sensitivity. ...
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A tryptophan (Trp) sensor was investigated based on electrochemical impedance spectroscopy (EIS) of a molecularly imprinted polymer on a lysozyme amyloid fibril (MIP-AF). The MIP-AF was composed of aniline as a monomer chemically polymerized in the presence of a Trp template molecule onto the AF surface. After extracting the template molecule, the MIP-AF had cavities with a high affinity for the Trp molecules. The obtained MIP-AF demonstrated rapid Trp adsorption and substantial binding capacity (50 µM mg−1). Trp determination was studied using non-Faradaic EIS by drop drying the MIP-AF on the working electrode of a screen-printed electrode. The MIP-AF provided a large linear range (10 pM–80 µM), a low detection limit (8 pM), and high selectivity for Trp determination. Furthermore, the proposed method also indicates that the MIP-AF can be used to determine Trp in real samples such as milk and cancer cell media.
... Having this in mind, the importance of determining this amino acid in biological samples, as well as in food samples can be accomplished. Although a number of classical methods [5][6][7] are available to quantify this target analyte, electrochemical techniques have become important in this research field. Voltammetry is an electrochemical and electroanalytical technique based on measuring current as a function of applied potential. ...
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One of the goals of this research was to develop an electrochemical sensor that had the ability to determine the target analyte and was both cheap and non-toxic. Another goal was to influence the reduction of electronic waste. In accordance with these, a graphite rod from zinc-carbon batteries was used to prepare an electrochemical sensor for the determination of L-tryptophan in Britton–Robinson buffer solution. Two electrochemical methods were used in the experimental research, differential pulse voltammetry and cyclic voltammetry. The effect of different parameters, including the pH value of supporting solution, scan rate, as well as the concentration of L-tryptophan on the current response, was studied. The pH value of Britton–Robinson buffer influenced the intensity of L-tryptophan oxidation peak, as well as the peak potential. The intensity of the current response was the highest at pH 4.0, while the peak potential value became lower as the pH increased, indicating that protons also participated in the redox reaction. Based on the obtained data, electrochemical oxidation of L-tryptophan at the graphite electrode was irreversible, two electron/two proton reaction. In addition, it was observed that the oxidation peak increased as the scan rate increased. According to the obtained electrochemical data, it was suggested that the oxidation of L-tryptophan was mixed controlled by adsorption and diffusion. The linear correlation between oxidation peak and L-tryptophan concentration was investigated in the range 5.0–150.0 µM and the obtained values of limit of detection and limit of quantification were 1.73 µM and 5.78 µM, respectively. Also, the prepared electrochemical sensor was successful in determination of target analyte in milk and apple juice samples.
... Reference Chromatography [18]] Spectroscopy [19][20][21][22] Colorimetry [23] High-performance liquid chromatography (HPLC) [19,21,24,25] Capillary electrophoresis [26] Chemiluminescence [27] Atomic force spectroscopy [ Figure 1. The pathway for the synthesis of TRP (adapted from [3]). ...
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This study describes the development of a new sensor with applicability in the determination and quantification of yjr essential amino acid (AA) L-tryptophan (L-TRP) from pharmaceutical products. The proposed sensor is based on a carbon screen-printed electrode (SPCE) modified with the conductor polymer polypyrrole (PPy) doped with potassium hexacyanoferrate (II) (FeCN). For the modification of the SPCE with the PPy doped with FeCN, the chronoamperometry (CA) method was used. For the study of the electrochemical behavior and the sensitive properties of the sensor when detecting L-TRP, the cyclic voltammetry (CV) method was used. This developed electrode has shown a high sensibility, a low detection limit (LOD) of up to 1.05 × 10−7 M, a quantification limit (LOQ) equal to 3.51 × 10−7 M and a wide linearity range between 3.3 × 10−7 M and 1.06 × 10−5 M. The analytical performances of the device were studied for the detection of AA L-TRP from pharmaceutical products, obtaining excellent results. The validation of the electroanalytical method was performed by using the standard method with good results.
... Neither the addition of salt nor the drying method significantly affected the content of the majority of essential amino acids (EAA) (threonine, valine, methionine, phenylalanine, histidine, and lysine) and non-EAA (serine, glycine, alanine, cystine, and proline). Tryptophane was not reported as it is generally destroyed during the acid hydrolysis step of AA analysis (Ritota & Manzi, 2020). However, the concentration of two EAA (isoleucine and leucine) was significantly higher in SdSa jameed compared with FdUs jameed. ...
This study aimed to assess the biological properties of peptide fractions isolated from dried fermented dairy products (jameed) as influenced by processing. Peptide fractions were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) from salted (Sa) and unsalted (Us) cow milk jameed after drying the fermented curd by sun drying (Sd) or freeze-drying (Fd) and were characterized for their antioxidant capacity and inhibitory activity toward angiotensin I-converting enzyme (ACE) and α-amylase. Sd samples showed more numerous peptide peaks in RP-HPLC chromatograms than Fd samples, regardless of the salt content. High antioxidant activity was evidenced in several peptide fractions from FdUs jameed (including fractions 1, 2, 4, 7, 8, 9, and 10), SdUs jameed (1, 2, 5, 7, and 9), and FdSa jameed (2, 5, 6, and 9). By contrast, peptide fractions from SdSa (1, 2, 3, 5, 8, and 9), SdUs (4, 5, and 10), and FdUs (5, 6, and 8) jameed displayed the highest ACE inhibitory activity. Similarly, the highest inhibition of α-amylase was obtained with fractions from SdSa (1, 2, 3, 4, 5, 6, 8, and 9), SdUs (2 and 6), and FdUs (1, 7 and 9) jameed. A significant negative correlation was evidenced between antioxidant activity and anti-α-amylase activity of peptide fractions from SdSa jameed. These findings demonstrate that cow milk jameed is a source of bioactive peptides with antioxidant, anti-ACE, and anti-α-amylase properties in vitro, which can be tailored by adjusting the salt content and the drying conditions. Practical Application: This study shows that cow milk jameed, a staple fermented food in several Mediterranean countries, can serve as a useful source of multifunctional bioactive peptides with potential antioxidant, hypotensive, and hypoglycemic effects, which may help prevent and manage chronic health conditions such as hypertension, type 2 diabetes, and the metabolic syndrome. The bioactivities of certain peptide fractions were enhanced by lowering the salt content of jameed or by the drying method. The relatively simple RP-HPLC method described in this study can be used to isolate the peptide fractions of interest for further characterization and use as functional ingredients.
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Tryptophan is a key component in many biological processes and an essential amino acid in food and feed materials. Analysis of the tryptophan content in proteins or protein-containing matrices has always been a challenge. We show here that the preparation of samples prior to tryptophan analysis can be significantly simplified, and the time consumption reduced, by using ascorbic acid as antioxidant to eliminate the problem of tryptophan degradation during alkaline hydrolysis. Combined with separation by HPLC and detection by Single Quadrupole Mass Spectrometry, this allows the analytical run time to be reduced to 10 min. The alkaline hydrolysate obtained in the method presented here may be combined with the oxidized hydrolysate obtained when sulfur-containing amino acids are to be measured, thus essentially providing two analyses for the time of one.
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Tryptophan is an essential plant-derived amino acid that is needed for the in vivo biosynthesis of proteins. After consumption, it is metabolically transformed to bioactive metabolites, including serotonin, melatonin, kynurenine, and the vitamin niacin (nicotinamide). This brief integrated overview surveys and interprets our current knowledge of the reported multiple analytical methods for free and protein-bound tryptophan in pure proteins, protein-containing foods, and in human fluids and tissues, the nutritional significance of l-tryptophan and its isomer d-tryptophan in fortified infant foods and corn tortillas as well the possible function of tryptophan in the diagnosis and mitigation of multiple human diseases. Analytical methods include the use of acid ninhydrin, near-infrared reflectance spectroscopy, colorimetry, basic hydrolysis; acid hydrolysis of S-pyridylethylated proteins, and high-performance liquid and gas chromatography-mass spectrometry. Also covered are the nutritional values of tryptophan-fortified infant formulas and corn-based tortillas, safety of tryptophan for human consumption and the analysis of maize (corn), rice, and soybean plants that have been successfully genetically engineered to produce increasing tryptophan. Dietary tryptophan and its metabolites seem to have the potential to contribute to the therapy of autism, cardiovascular disease, cognitive function, chronic kidney disease, depression, inflammatory bowel disease, multiple sclerosis, sleep, social function, and microbial infections. Tryptophan can also facilitate the diagnosis of certain conditions such as human cataracts, colon neoplasms, renal cell carcinoma, and the prognosis of diabetic nephropathy. The described findings are not only of fundamental scientific interest but also have practical implications for agriculture, food processing, food safety, nutrition, and animal and human health. The collated information and suggested research need will hopefully facilitate and guide further studies needed to optimize the use of free and protein-bound tryptophan and metabolites to help improve animal and human nutrition and health.
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Melamine is a nitrogen-rich compound (about 66%) whose fraudulent addition to foods aims to increase the apparent protein content. In 2008, melamine adulteration incidents occurred in China caused several deaths in humans from kidney failure and other health problems. This issue prompted private as well as governmental laboratories to develop several analytical methods in order to determine melamine in foods. This review aims to provide an overview of the analytical techniques currently available in the literature for melamine detection and measurement in milk and dairy products, including a specific section related to sample preparation. Recent studies concerning conventional (both screening and confirmatory) methods are reported, and technical and critical issues are discussed for each technique (liquid and gas chromatography, mass spectrometry, liquid chromatography coupled to mass spectrometry, gas chromatography coupled to mass spectrometry, and capillary electrophoresis). These techniques, very sensitive and specific but also time-consuming, may require tedious sample preparation, costly instrumentation, and skilled personnel. Therefore, new innovative detection methods are also provided, focusing attention on immunoassay, sensor technology, vibrational spectroscopy coupled to chemometrics, hyperspectral imaging, micellar liquid chromatography, magnetic resonance spectroscopy, and chemiluminescence.
In this contribution we investigate the thermal decomposition of four pentapeptides containing a tryptophan moiety. Pentapeptides were heated at 220°C and the resulting reaction mixtures investigated by HPLC coupled to high resolution mass spectrometry and tandem mass spectrometry. A total of 95 thermal decomposition products could be observed and resolved by chromatography. In detail we report on the structure assignment of two types of reaction products common to investigated peptides and introduce two decomposition mechanisms. Pentapeptides react with oxygen to produce hydroxyl-tryptophan derivatives. In addition we observe the C-terminal decarboxylation of two peptides to form N-acyl tryptamine derivatives.
This study aimed to develop an analytical method for the determination of tryptophan and its derivatives in kynurenine pathway using tandem mass spectrometry in various fermented food products (bread, beer, red wine, white cheese, yoghurt, kefir and cocoa powder). The method entails an aqueous extraction and reversed phase chromatographic separation using pentafluorophenyl (PFP) column. It allowed quantitation of low ppb levels of tryptophan and its derivatives in different fermented food matrices. It was found that beer samples were found to contain kynurenine within the range of 28.7±0.7 μg/L and 86.3±0.5 μg/L. Moreover, dairy products (yoghurt, white cheese and kefir) contained kynurenine ranging from 30.3 to 763.8 μg/kg d.w. Though bread samples analyzed did not contain kynurenic acid, beer and red wine samples as yeast-fermented foods were found to contain kynurenic acid. Among foods analyzed, cacao powder had the highest amounts of kynurenic acid (4486.2±165.6 μg/kg d.w), which is a neuroprotective compound.
Tryptophan is one of the eight essential amino acids and plays an important role in many biological processes. For its interaction with human health, environment and relevant commercial interest in biotechnology-based production, rapid and specific quantification method for this molecule accessible to common laboratories is badly needed. We herein reported a simple colorimetric method for free tryptophan quantification with 96-well-plate-level throughput. Our protocol firstly converted tryptophan to indole enzymatically by purified tryptophanases and then used reactivity of indole with hydroxylamine to form pink product with absorption peak at 530 nm, enabling the quantification of tryptophan with simple spectrometry in just two hours. We presented that this method exhibited a linear detection range from 100 μM to 600 μM (R²= 0.9969) with no detection towards other naturally occurring tryptophan analogs or tryptophan residues in proteins. It was very robust in complicated biological samples, as demonstrated by quantifying the titer of 36 mutated tryptophan-producing strains with Pearson correlation coefficient of 0.93 in contrast to that measured by high performance liquid chromatography (HPLC). Our method should be potent for routine free tryptophan quantification in a high-throughput manner, facilitating studies in medicine, microbiology, food chemistry, metabolic engineering, etc.
This study aimed to investigate the formation of tyramine during yoghurt fermentation with the focus on interaction between Streptococcus thermophilus RSKK 04082, Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 and Lactobacillus plantarum RSKK 02030. These microorganisms were used in the yoghurt fermentation as single strains or mixed cultures containing double or triple strains. The interactions between microorganisms have been also revealed by determining total free amino acids and the pH of the medium together with the microbial count of the strains. It was observed that L. delbrueckii subsp. bulgaricus DSM 20081 did not produce tyramine while S. thermophilus RSKK 04082 and L. plantarum RSKK 02030 could produce tyramine depending on the fermentation conditions. Synergistic interactions between S. thermophilus RSKK 04082 and L. delbrueckii subsp. bulgaricus DSM 20081 and, between L. delbrueckii subsp. bulgaricus DSM 20081 and L. plantarum RSKK 02030 were found in terms of tyramine production. It was observed in this study that L. delbrueckii subsp. bulgaricus DSM 20081 had indirect effect on accumulation of tyramine in the yoghurts.
On the basis of research presented during the 9th Amino Acid Assessment Workshop, a No Observed Adverse Effect Level (NOAEL) for diet-added arginine (added mostly in the form of dietary supplements) of 30 g/d and an upper limit of safe intake (ULSI) for diet-added tryptophan (added mostly in the form of dietary supplements) of 4.5 g/d have been proposed. Both recommendations apply to healthy young adults. The total dietary leucine ULSI proposed for elderly individuals is 500 mg · kg⁻¹ · d⁻¹. All 3 recommendations are relevant only to high-quality amino acid-containing products with specifications corresponding to those listed in the US Pharmacopeia. Because the above amino acids are extensively utilized as dietary supplements for various real or perceived benefits, such as vasodilation, spermatogenesis, sleep, mood regulation, or muscle recovery, the above safety recommendations will have an important impact on regulatory and nutritional practices.
Background: Tryptophan is an indispensable amino acid and is a precursor of the neurotransmitter serotonin. Tryptophan metabolites, such as serotonin and melatonin, are thought to participate in the regulation of mood and sleep and tryptophan is used to treat insomnia, sleep apnea, and depression. Objective: This study examined the intake of tryptophan and its associations with biochemical, behavioral, sleep, and health and safety outcomes in adults in a secondary analysis of a large, publicly available database of the US population. Methods: Data from the NHANES 2001-2012 (n = 29,687) were used to determine daily intakes of tryptophan and its associations with biochemical markers of health- and safety-related outcomes, self-reported depression, and sleep-related variables. Data were adjusted for demographic factors and protein intake. Linear trends were computed across deciles of intake for each outcome variable, and P-trends were determined. Results: The usual tryptophan intake by US adults was 826 mg/d, severalfold higher than the Estimated Average Requirement for adults of 4 mg/(kg ⋅ d) (∼280 mg/d for a 70-kg adult). Most health- and safety-related biochemical markers of liver function, kidney function, and carbohydrate metabolism were not significantly (P-trend > 0.05) associated with deciles of tryptophan intake and were well within normal ranges, even for individuals in the 99th percentile of intake. Usual intake deciles of tryptophan were inversely associated with self-reported depression measured by the Patient Health Questionnaire raw score (0-27; P-trend < 0.01) and calculated level (1 = no depression, 5 = severe depression; P-trend < 0.01) and were positively associated with self-reported sleep duration (P-trend = 0.02). Conclusions: Tryptophan intake was not related to most markers of liver function, kidney function or carbohydrate metabolism. Levels of tryptophan intake in the US population appear to be safe as shown by the absence of abnormal laboratory findings. Tryptophan intake was inversely associated with self-reported level of depression and positively associated with sleep duration.