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A New Approach on the Amino Acid Lysine Quantification by UV-Visible

Spectrophotometry

Article  in  Revista de Chimie -Bucharest- Original Edition- · August 2020

DOI: 10.37358/RC.20.8.8290

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Revista de Chimie
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A New Approach on the Amino Acid Lysine Quantification by

UV-Visible Spectrophotometry
HABIB BOUZID1,2,3, CESAR M. C. FILHO2, JOANA RITA C. MARQUES1,
ARTUR J. M. VALENTE2, LICINIO M. GANDO-FERREIRA1*
1
University of Coimbra, CIEPQPF, Department of Chemical Engineering, Rua Silvio Lima - Pólo II, Pinhal de Marrocos,
3030-790 Coimbra, Portugal
2
University of Coimbra, CQC, Department of Chemistry, Rua Larga, 3004-535Coimbra, Portugal
3
Department of Process Engineering, University of Mostaganem, BP. 227 Route de Bel-Hacel, 27000 Mostaganem, Algeria

Abstract. The methodology of the amino acid lysine (Lys) analysis using ninhydrin (Nin) reagent by
spectrophotometric method was in-depth studied and improved. The optimization of time, Lys to Nin
volume ratio and temperature were explicitly investigated. A specific wavelength of 479 nm was set
according to the peak of the absorbance spectrum. The reaction time between Lys and Nin, in aqueous
solution, occurs during 50 min. The Lys/Nin ratio 1.67 appeared most effective for this reaction.
Moreover, the absorbance increased with temperature until a stable value was achieved nearly at
85°C. It was also found that the reaction is more efficient at initial pH 6 corresponding to formation of
high amount of coloured product. Furthermore, the pH of post-reaction mixture was around 4 for
different concentrations of lysine and initial pH range 3–10. For lysine solution contaminated with
potassium (a representative interfering species) and after variation of the reaction conditions, the
maximum wavelength (479 nm) was not affected. Mastering the use of these parameters leads to a
good usage of this analytical technique which is simple, fast, accurate, less expensive and especially
less harmful towards the environment and human health.

Keywords: amino acid lysine, ninhydrin reagent, UV-Visible spectrophotometric analysis, optimization

1.Introduction
There is a growing demand for amino acids as value-added substances since they have many
potential applications in the food, clinical, pharmaceutical and cosmetic industries, among others [1-7].
Amino acids have also an essential role in regulating neural activities in vivo [7] and have a great
importance in all basic biological processes in the cell [8]. They are also used in the synthesis of
certain biomaterials such as silk fibroin-magnetite for wounds healing [9].
Most L-amino acids are currently produced by fermentation processes leading to desired amino
acids and inorganic species [10,11]. The implementation of simple and reliable techniques of amino
acid analysis is an essential issue in the field of industrial biotechnology applied to production of
added-value chemicals. Leca-Bouvier and Blum [12] reported that the colorimetric procedure is very
successful when it relies on using representative calibration standards. High-performance liquid
chromatography (HPLC) is the most popular method for quantifying amino acids but a derivatizing
reagent, which selectively reacts with the amino group, should be used [12,13]. The reaction of
ninhydrin (2,2-dihydroxy-1,3-indanedione) with a primary amino group to form a coloured compound
has been extensively employed in quantitative and qualitative amino acid analysis. Taking into
consideration the additional steps of the pretreatment of samples for the retention time of lysine, the
analysis time considerably increases and the method becomes times-consuming when compared with
the ninhydrin-based UV-vis spectrophotometric method. In addition, no expensive equipment is
required for the latter methodology when compared to HPLC, which also needs a high qualified
operator [12,13].

*email: lferreira@eq.uc.pt

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The motivation behind the choice of lysine (Lys), is related not only with its importance in the
global market of animal feed additives but also for its necessity to children’s physical growth
[5,10,14,15]. The current industrial process to purify Lys solutions is based on ion-exchange
multicolumn systems that require an excessive consumption of water and ammonia, with a consequent
generation of environmentally harmful effluents. Studies on alternative strategies based on clean
technologies for separation/purification of Lys will lead to the fastest and easiest experimental
methodology for analyzing this amino acid during the tests for assessment of the separation efficiency.
Thus, we have improved and optimized an experimental method based on UV-visible spectrophoto-
metric analysis for determination of Lys, by using ninhydrin.
There are a few methods in the literature that address the quantification of amino acids [16],
including Lys [17], in the food products. The production of Lysine is based on a industrial
fermentation process [18-21], which generates a culture broth formed by Lys and low concentration of
contaminant species [21], being the ninhydrin colorimetric method the most used one to evaluated that
reaction. Aiko et al. (1971) [22] found that salts, sugars, urea and ammonia, usually present in the
culture broth, do not interfere with ninhydrin at the maximum absorbance wavelength found (475 nm).
Therefore, the interference of the above mentioned contaminant species will not occur in the present
work since the maximum absorbance wavelength found (479 nm) is similar to that previously reported
[22]. On the other hand, the interference of contaminant species can also be neglected since Lysine
concentration is very high in the culture broth or in the original samples as reported by Bordons (1986)
[18]. It is worth noticing that due to the absence of lysine manufacturing process in Portugal, no real
samples were tested. However, a model solutionof lysine skipped with potassium, acting as a
representative interfering inorganic specie was tested, by following the suggestion of Nagai and Carta
(2004) [2, 21]. It should also be taken into account that the maximum absorbance wavelength depends
on the solvent and added reactants in the reaction mixture [20].
At the best of our knowledge, the optimization of important variables, such as the added volume of
lysine and ninhydrin reagent solutions or the time, temperature and pH of reaction, are not reported in
the literature. For this reason, our main goal was to improve the method’s reaction rate and go deeper
on the optimization of parameters that might affect the quantitative analysis of Lys. Moreover, it is
imperative to reduce the reaction time, around 2-3 h [23,24], and the temperature (ca. 100ºC) [13, 18,
19, 23] of reaction between Lys and ninhydrin reagent, in order to become feasibly for practical terms.
The effect of pH on the color development in ninhydrin method was previously studied [13] for a
variety of amino acid concentrations and in a restricted pH range (5.3–8.05), while in the present study
was specifically analyzed for amino acid lysine and in a large pH range (2–11). Apart from this, the
ratio of Lys to Nin was also investigated and identified as the most important parameter affecting the
reaction. In this paper this method has been improved, by enhancing the reaction rate, and revisited
from a different perspective to standardise the most varied reaction conditions. The optimization of
reaction time, needed volumes of lysine and reagent solutions, reaction temperature, and pH was
successfully achieved, leading to both better accuracy and lower cost, and contributing for a lower
environmental impact as well.

2.Materials and methods

Reagent and solutions
Ninhydrin (2,2-dihydroxyindan-1,3-dione, 99%) reagent was purchased from Sigma-Aldrich
(Bucks, Switzerland), and its solution was daily prepared by dissolving 0.35 g of ninhydrin in 100 mL
of ethanol [24-26]. Lysine (2,6-diaminohexanoic acid, 98%), molar weight of 146.1876 g mol-1and
isoelectric point, pI, of 9.74 [27], was purchased from Sigma-Aldrich (Bucks, Switzerland). To study
the interference of potassium, which is the representative contaminant species of lysine during its
industrial fermentation process [21]. Thus, the Lysine-KCl solution was used in which it represents a
lysine solution with KCl 10% solution. Adjustments of pH in the range 2-11 were carried out by using
hydrochloric acid (1 mol L-1) and sodium hydroxide (1 mol L-1) under magnetic stirring. HCl (37

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wt.%), potassium dihydrogen phosphate (99.5%), potassium chloride (99%), NaOH (pellets,98%) and
ethanol (99.8%) were purchased from Ridel-de-Haën (Seelze, Germany). All solutions were freshly
prepared using ultra-pure grade water, purified with a Milli-Q system from Millipore (Billerica, MA,
USA).

Instrumentation
Absorbance spectra were recorded on UV-Visible UVWin5 Software V5.0.5 spectrophotometer
T60 (UK), in the wavelength range 400 nm to800 nm and a medium speed scan of 2 nm s-1.
The chromatography method for the separation and quantification of L-lysine was performed using
a VWR-Hitachi LaChrom Elite HPLC system (Hitachi, Japan), equipped with a degasser, auto
sampler, column oven, and DAD. For better separation of L-Lysine, an analytical column (0.25 m ×
4.6 mm, 5 µm film) Purospher® Star RP-18 endcapped (Merck-Millipore, Germany) was used.
EZChrom Elite software (Agilent, USA) was used for data acquisition and processing.

Experimental procedure
At the first stage, 1 mL of ninhydrin solution was added to 5 mL of sample in a series of caped test
tubes [25,26] which were placed at 80 °C in the water-bath with a gentle stirring fixed at 70 rpm. This
temperature was chosen in the interval between 80–100°C according to the previous studies with
ninhydrin [13,18,19] and 2-hydroxynaphthaldehyde reagents [28]. After cooling the reaction tubes at
room temperature, the solution absorbance corresponding to 1 h of reaction was measured by using a
UV-Visible spectrophotometer. The maximum absorbance wavelength for of lysine and Lysine-KCl
solutions, at different concentrations (0.1-0.9 mmol L-1), was recorded at ca. 479 nm (Figure 1). It is
also worth noticing that the maximum absorbance wavelength does not depend on different
experimental parameters as, for example, reaction time, ratio of lysine to ninhydrin, reaction
temperature and pH (Figure S1). The optimization of experimental conditions was carried out by using
a full-factorial design methodology. Hence, potassium, as a representative interfering species [2,21],
did not interfere with ninhydrin at 479 nm, in agreement with the work reported by Aiko et al. [22] and
Bordons [18].

Figure 1. UV/vis absorbance spectra of a) aqueous lysine solutions and

b) Lysine-KCl solution after reaction with ninhydrin
(0.35 g Nin/100 mL ethanol) for 1 hour, at 80ºC. Lysine concentrations:
1) 0.1 mmol L-1, 2) 0.2 mmol L-1, 3) 0.4 mmol L-1,
4) 0.6 mmol L-1, 5) 0.8 mmol L-1, 6) 0.9 mmol L-1

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The analytical methodology for quantification of lysine by HPLC was based on the method
reported elsewhere [29]. In summary the mobile phase containing 10 mmol L-1 of potassium
dihydrogen phosphate and the pH was adjusted at 7.5 by using trimethylamine. The detection was
carried at 195 nm. The flow rate of the mobile phase was set to 0.5 mL min-1, while the temperature of
column maintained at 25 ºC. The injection volume was 20 µLand each test required 5 min. All samples
solutions, standards and mobile phase were filtered through membrane filter of 0.45 µm.
All the lysine solutions were prepared by placing appropriate amounts of L-lysine in a 100 mL
volumetric flask containing ultrapure water. Lysine-KCl solutions were prepared using a stock solution
of 40 mg KCl/200 mL of ultrapure water (Table S1 in the supplementary material).

Method validation
Linearity
The standard calibration curves were obtained using aqueous solutions of lysine and Lysine-KCl
solution in the concentration range 0.1-0.9 mmol L-1. Five replicate of each concentration were
analyzed using the optimized parameters of ninhydrin method with UV-visible spectrophotometry. The
linear regression equation and the corresponding determination coefficient (R2) were computed, as
reported by ICH (International Conference on Harmonisation) [30], from a plot of absorbance versus
lysine concentration and Lysine-KCl solution concentration.

Precision
The relative standard deviation (RSD) of the determinations was computed as follows [31-34]:

SD
( RSD %) = ( )  100 (1)
Abs m
where SD is the standard deviation of the five/ten replicate, respectively, measured at two different
days [day 1 (n=5), day 2 (n=10)] [29,32], and Absm is the mean value of absorbance for the five/ten
replicate of each concentration varying from 0.1 to 0.9 mmol L-1.

Limits of quantification (LOQ) and of detection (LOD)

The LOQ and LOD were calculated based on the standard deviation of the response at low
concentrations (SD) of the calibration curve and respective slope (S) using the following equations
[30,32,35]:
SD
LOQ = 10  ( ) (2)
S
SD
LOD = 3.3  ( ) (3)
S
Accuracy
The accuracy of the proposed method was evaluated by determining the recovery values for five
replicate of each concentration, as reported by AOAC International [36], varying from 0.1 to 0.9 mmol
L-1. The average percent recovery was calculated through the equation below [30,32,33,36,37]:
C
Recovery (%) = ( obs )  100 (4)
C spi
where Cobs and Cspi are the experimental and spiked concentrations, respectively.

3.Results and discussions

In this section, optimization studies aiming the optimal values of key variables that affect the
reaction between lysine and ninhydrin solutions will be discussed. The general reaction is shown in
Figure 2.

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Figure 2. Chemical reaction of ninhydrin with amino acid Lysine

in positively charged form (Lys+) to yield Ruhemann's Purple
(adapted from Wigfield et al. (1980) [38]and Lehninger et al. (1993) [27])

Effect of reaction time

Figure 3 shows the dependence on the maximum absorbance of Lys/Nin solutions on the reaction
time (from 5 to 70 min).It can be seen that the absorbance increases by increasing the reaction time,
reaching a maximum absorbance at approximately 50 min. From the analysis of Figure 3, we can
concluded that the rate constant is constant up to 50 min The establishment of the plateau revealed the
existence of a limiting concentration for Ruhemann's Purple formation and can be explained by the
cancellation of the reaction rate. These results indicate that the optimal reaction time is of 50 minutes
and show that the required time for analyzing lysine with this methodology is lower than that the one
needed to obtain the retention time of lysine peak by HPLC reported in previous works [39,40]. On the
other hand, this reaction time (50 min) is feasible for practical terms when taking into consideration all
additional steps of the experimental previously reported procedure [13,18,24]. In addition, a reduced
consumption of ninhydrin quantity, corresponding to about one sixth [13] and one third [18], and
reagents [13,18], was noted.

Figure 3. Dependence of the maximum absorbance at 479 nm on the lys-ninhydrin

reaction time; a) [Lys]= 0.5 mmol L-1and (b) [Lys]= 1 mmol L-1, at 80°C and [Lys/Nin]= 5
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Effect of volume of Lysine and reagent solutions

The effect of volume of Lys, at two different concentrations (0.5 and 1 mmol L-1), and ninhydrin
(Nin) reagent solutions, at a fixed time (50 min), have been studied. By changing the volume of
ninhydrin solution and kept the volume of Lys as equal to 2.5 mL, we will be able to modify the
volume Lys/Nin ratio from 0.33 to 10. The obtained results are shown in Figures 4 and 5. These
figures show a similar trend for both Lys concentrations; i.e., the absorbance increases gradually until
reaching a maximum at 0.592 and 1.184, for Lysine concentrations of 0.5 and 1 mmol L-1 respectively.
It can be concluded that the optimal volumes of Lys and ninhydrin reagent solutions are 2.5 and 1.5
mL, respectively, corresponding to a volume ratio Lys/Nin of 1.67. At higher volume ratios, the
reaction rate is not efficient and lead to low absorbance values. On the other hand, the excess of
ninhydrin may lead to side reactions [20,41].

Figure 4.Variation of absorbance with volume of 0.5 mmol L-1 lysine (a) and
Lys/Nin ratio (b) at 80°C for 50 min

Figure 5.Variation of absorbance with volume of 1 mmol L-1lysine

(a) and Lys/Nin ratio (b) at 80°C for 50 min
Effect of reaction temperature
The reaction temperature was optimized by using 0.5 and 1 mmol L-1 Lysine solutions. The
dependence of A on the T shows an interesting S-shape (Figure 6), reaching a plateau at T around 80-
85ºC. An insight on such behavior can arise from the kinetic mechanism analysis. At temperatures
below 60-70ºC the activation energy barrier to reach the transition state is not reached and,
consequently, the absorbance due to Ruhemann’s purple complex is low [42,43]. By increasing the T,
a pre-steady-state kinetic [44] is reached and, consequently, the reaction rate increases leading to the
formation Ruhemann’s purple complex. At 80-85 ºC a decrease in the Ruhemann’s purple complex
formation is observed; this can be justified by a slowdown in the reaction rate followed by the addition
of small activation energy thus slight increase in Gibbs free energy (ΔG) and in Ruhemann’s purple
complex productions. One hypothesis explaining the specific behavior is based on the fact that the
enthalpy variation of the reaction to form the Ruhemann’s purple complex is endothermic. This
conclusion arises from the dependence of the reaction product concentration as a function of
temperature. However, the decrease in the algebraic value of the Gibbs energy variation is only

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justified by a decrease in the entropy variation; i.e. ΔS<ΔH / T [45] which, in the limit, prevents the
formation of the Ruhemann's purple complex. Above 85°C there was no further production of
Ruhemann’s purple complex once all the activated molecules of the reactants have already been
forwarded to the transition state by forming a limiting concentration of the Ruhemann’s purple
complex.
The results obtained of the adopted methodology suggest that the optimum conditions are 50 min
of reaction time (Figure 3), 1.67 as Lys/Nin ratio (Figures 4 and 5) and 85 ºC of reaction temperature
(Figure 6). It should be noted that the optimal reaction temperature is lower than those reported
elsewhere [13,18,19].

Figure 6. Variation of absorbance with reaction temperature for lysine concentrations

of 0.5 mmol L-1 (a) and 1 mmol L-1 (b) with Lys/Nin ratio of 1.67 for 50 min

Effect of initial pH
It is well known that the amount of colored product formed in the reaction between the amino acid
and ninhydrin reagent is dependent on the pH at which the reaction is carried out. However, some
chemical issues behind the reaction of the protonated or unprotonated form of the amino acid in
solution with ninhydrin are still veiled. From the analysis of Figure 7, the effectiveness of the reaction
increases by increasing the pH from 2 to 6. So, it seems that the reaction is more efficient at initial pH
6 when the dominant form of the amino acid is positively charged (Lys+) according to the different
forms of Lysine shown by Garrett and Grisham (2010) [46]. This optimum pH (6) found for lysine is
near that one (5.5) reported by Friedman (2004) for all amino acids. It can be interestingly observed
that pH 6 is the mean of pKa of the carboxylic acid group (–COOH), which is of 2.18, and pI (9.74) of
Lysine. It can be concluded that when the pH is higher than pKa(–COOH) and lower than pI the lysine
is in its most dissociated state notably at pH 6 and, consequently, the chemical reaction (Figure 2)
occurs with a significant rate. On the other hand, a zwitterionic form (Lys±) predominates at pI value
when the overall charge of the lysine molecule is cancelled causing a minimum solubility (and a lower
reaction rate) of Lysine in solution.

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Figure 7. Effect of pH on the reaction of ninhydrin with lysine

1 mmol L-1 for t=50 min and at T=85 oC and Lys/Nin ratio=1.67

The pH of post-reaction mixture (final pH) reveals further understanding of the reactional
mechanism between lysine and ninhydrin. It was observed (Figure 8a) that the final pH was around 4
for different concentrations of lysine aqueous solutions (initial pH 9.7). Consequently, the pH of post-
reaction mixture is independent of the concentration of lysine. Figure 8b shows the relationship
between initial and final pH values with Lysine 1 mmol L-1. In fact, it has not been seen any color of
samples at initial pH lower than 2, which can be explained by the absence of the decarboxylation of
lysine caused by the existence of the form Lys2+ in solution. When the initial pH of lysine solutions are
below 4, the final pH exhibited an upward trend towards the values around 4. For initial pH above 4,
the final pH had a downward trend stabilizing at approximately 4. This can be explained by the
amphoteric character of lysine. In addition, the decrease in pH to about 4 for initial pH higher than 4,
can probably be explained by the formation of a weak acid, namely by the carbonation of the solution,
occurred due to lysine decarboxylation during the heating process, as stressed by Bordons (1986) [18].
However, there was no impact on the ninhydrin reagent since dissolved carbon dioxide helps to
provide more H+ which improves the reaction rate. For initial pH values lower than 4, there were
sufficiently protons H+ thus the increasing of H+ concentration shifted the equilibrium towards the
direction to reduce the protons H+, this can be achieved by controlling in the production of CO2. It can
be concluded that CO2 acts as a pH regulator in the reaction mixture which is justified by the shape of
the curve of Figure 8b.

Figure 8. a) Effect of lysine concentration at natural pH (initial pH 9.7 for all lysine concentrations)
on the final pH and b) relationship between initial and final pH values for 1 mmol L-1 lysine solutions

Optimization of lysine analysis using an experimental design

In order to find optimal conditions for lysine analysis, 18 experiments based on a full-factorial
design with 4 factors at two levels (Table 1), including two replicates at central point, were performed.
The lysine concentration was set at 1 mmol L-1. Table 2 shows the results of the response variable

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(absorbance) for different experimental conditions established by the full-factorial design. Considering
that the main goal is to maximize the absorbance, it is noticeable from Table 2 that the region of the
optimum response is obtained for high contact time and temperature, and intermediate values for the
lysine/ninhydrin. These results are consistent with ones obtained previously where the effect of each
variable was evaluated in separate.

Table 1. Experimental range and levels of the factors for lysine analysis
Factor Low level (-1) Central point (0) High level(+1)
Time (min) 20 40 60
Lys/Nin ratio 1 3 5
pH 2 6 10
Temperature (oC) 75 80 85

Table 2.Results from experimental design for the lysine analysis

Run Time (min) Lys/Nin ratio pH T (oC) Abs
1 20 1 2 75 0.280
2 60 1 2 75 0.778
3 20 5 2 75 0.159
4 60 5 2 75 0.411
5 20 1 10 75 0.312
6 60 1 10 75 1.109
7 20 5 10 75 0.415
8 60 5 10 75 0.477
9 20 1 2 85 0.734
10 60 1 2 85 1.213
11 20 5 2 85 0.369
12 60 5 2 85 1.383
13 20 1 10 85 0.574
14 60 1 10 85 1.315
15 20 5 10 85 0.257
16 60 5 10 85 0.462
17 40 3 6 80 0.902
18 40 3 6 80 0.964

The Pareto chart showed in Figure 9 allows evaluating the importance of each factor as well as
their interactions on the response variable for a 95% confidence interval (p  0.05). It can be concluded
that the variable most relevant for achieving the highest values for the absorbance is the time.
However, the Lys/Nin ratio and temperature may be also considered statistically significant whereas
the solution pH has little effect on the response variable.

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Figure 9. Pareto chart obtained from experimental design

The optimal values of the studied parameters enabled to perform the calibration curves of the
absorbance vs lysine concentration (Figure 10a) and Lysine-KCl solution concentration (Figure 10b)
under optimal conditions. Thus, it will be possible to analyze lysine solutions in a frequent way with
ease, swiftness, and accuracy while avoiding polluting the environment.
It is worth mentioning that the calibration curves illustrated in Figures 10a and 10b were performed
with lysine solutions at natural pH since it has little effect on the response variation compared to other
optimized parameters. Further studies involving the adsorption of Lysine by a cationic resin will be
carried out under these conditions.

Figure 10. Calibration curves for a) lysine and b) Lysine-KCl solution under
optimal conditions (at 85°C, natural pH (9.7), t = 50 min and Lys/Nin ratio = 1.67)

Method validation
Calibration curves and linearity
As shown, the determination coefficients (R2) obtained from the linear regression analysis of data
corresponding to absorbance vs lysine concentration, Figure 10a, and absorbance vs Lysine-KCl
solution concentration, Figure 10b, are higher than 0.99 (0.996 and 0.998 respectively), which
goodness of the fitting [36,47].
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Precision
The RSD, as repeatability (for the 1st day), values computed from the calibration curve, Absorbance
vs Lysine concentration, ranged from 4.4 to 19.6 % in which the amino acid concentration was
changed from 0.1 mmol L-1 to 0.9 mmol L-1 (Table 3). These values meet the USEPA (United States
Environmental Protection Agency) [48] quality control criteria. In the case of the calibration using
Lysine-KCl solution, the RSD values, for the same day, varied between 2.78 and 9.07% for
concentrations in the range from 0.1-0.9 mmol L-1. These values meet the IUPAC (International Union
of Pure and Applied Chemistry) [49] and USEPA [48] quality control criteria, RSD% lower than 20%
shows the high-level precision of the method [32,33, 49,50,51]. For the 2nd day, the RSD, as
intermediate precision, ranged from 4.6 to 17.8 % for lysine varying from 0.1 mmol L-1 to 0.9 mmol L-
1
. These values are within the USEPA quality control criteria. For Lysine-KCl solution, the RSD (2nd
day) varied from 4.0 to 14.4 % for the same concentration range. These values are in compliance with
the USEPA quality control criteria [48].

Table 3. Method precision of the proposed method

Concentration Repeatability (RSD%) Intermediate precision (RSD%)
(mmol L-1) Lysine Lysine-KCl Lysine Lysine-KCl
0.1 19.6 04.34 17.82 06.7
0.2 08.53 09.08 08.48 14.42
0.4 07.86 03.25 08.14 06.73
0.6 05.04 02.79 04.57 07.15
0.8 07.27 05.11 10.02 05.23
0.9 04.41 04.53 06.72 04.01

Limits of detection (LOD) and limits of quantification (LOQ)

The LOD and LOQ values found from data of absorbance vs lys concentration are, respectively,
0.056 mmol L-1 (0.008 mg mL-1) and 0.169 mmol L-1 (0.025mg mL-1). For the calibration data with
Lysine-KCl solution, LOD and LOQ of 0.011 mmol L-1 (0.0016 mg mL-1) and 0.033 mmol L-1 (0.0049
mg. mL-1), respectively, were calculated. The LOD of 0.0016 mg mL-1 is almost 4 times lower than
that previously reported (0.006 mg mL-1) [52]. This suggests the high measurement sensitivity of the
proposed method.

Accuracy
The average recovery value obtained is of 98.53 (±0.08)% (n=5) for lysine 0.1, 0.2, 0.4, 06, 0.8 and
0.9 mmol L-1, which is within the ICH, USEPA and Commission Decision 2002/657/EC [53] quality
control criteria [30-32,48,54,55], where the limits for recovery values are in the range of 80 and 120 %
(Table 4). For Lysine-KCl, the average recovery value is of 102.95 (±0.2) % (n=5) for the same above
mentioned concentrations of lysine. This result show that the method exhibits high accuracy.

Table 4. Method recovery of the proposed method

Concentration Recovery (%)
(mmol L-1) Lysine Lysine-KCl
0.1 85.58 76.83
0.2 96.67 96.12
0.4 109.18 105.35
0.6 102.65 99.64
0.8 99.05 139.14
0.9 98.07 100.64

HPLC–DAD method for quantification of Lysine

Figure 11 shows a representative chromatogram of L-lysine in ultrapure water, at 195 nm. It can be
observed that the peak of the analyzed compound is well defined and with a time below 6 min.

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Figure 11. Chromatogram of lysine at 3 mmol L-1

in water, detected at 195 nm

Figure 12 shows a calibration curve plotting the peak areas of lysine versus its corresponding
concentration (mmol L-1).

Figure 12. Calibration curve of lysine under HPLC optimized conditions

A linear correlation was found between the peak areas and the concentration of Lysine in the
investigated concentration range. Table 5 summarizes the regression analysis data and the computed
values for LOD and LOQ. The linearity of the method can be confirmed since a R2 higher than 0.99
was obtained.

Table 5. Analytical and fitting parameters for the developed method for l-lysine
LOD* LOQ*
Elution time Linearity range
Compound n Regression equation R2 (mmol (mmol L-
(min) (mmol L-1)
L-1 ) 1)

Lysine 4.89 1–9 7 y = 1.15E6x - 1.13106 0.9944 0.57 1.72

*limits of detection (LOD) and of quantification (LOQ)

Lysine quantification in KCl aqueous solutions under HPLC optimized conditions

Potassium chloride was used in order to test the influence of different environment in the
quantification of lysine. The accuracy of the method was evaluated by determining the recovery values
for three replicates using HPLC optimized conditions. Mixed solutions containing potassium chloride
solution and L-lysine, at five concentration levels (1 to 8.6 mmol L-1), were prepared. The recovery
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values experimentally obtained (Table 6) range from 81.6 (±0. 2) to 114.4 (±0.2) %, which are within
the ICH and USEPA quality control criteria (recovery limit between 80 and 120 %) [30,48].

Table 6. Method recovery in potassium chloride matrices

Theoretical value of lysine Experimental value of lysine
Recoverya (%) ±RSD
(mmol L-1) (mmol L-1)
1.02 1.25 (±0.04) 81.6 ± 0.2
2.3 2.01 (0.06) 114.4 ± 0.2
4.6 4.25 (±0.13) 108.2 ± 0.3
6.29 5.93 (±0.18) 106.1 ± 0.3
8.6 8.62 (0.26) 99.77 ± 0.01
a Mean computed from the calculated concentration in mmol L-1 (n=3)

As aforementioned, the LOD and LOQ values for the ninhydrin spectrophotometric method are:
0.056 mmol L-1 and 0.169 mmol L-1, respectively. Using HPLC analysis, a LOD of 0.57 mmol L-1 and
LOQ of 1.72 mmol L-1 were determined. Thus, it can be concluded that the spectrophotometric
procedure for lysine analyses is more sensitive than HPLC technique. In addition, it should be noted
the former method is more accurate, for Lys and Lys-KCl respectively, (Recovery (%) = 98.53 (±0.08)
% and 102.95 (±0.2) %) than the HPLC method (Recovery (%) = 81.6 (±0. 2) - 114.4 (±0.2)).

4. Conclusions
This study reports an optimized methodology, by improving the reaction rate, for quantification of
an amino acid lysine with ninhydrin reagent, by using UV-Visible spectrophotometry. Such
development leads to a more efficient, less time-consuming and less expensive methodology when
compared with those already existing. Besides, the proposed methodology has further advantages, such
as a reduced reaction time (50 min) and reagent consumption, an accurate ratio of Lysine solution
volume to ninhydrin reagent solution volume (1.67) and reduced reaction temperature (85°C).
The initial pH effect on the reaction between lysine and ninhydrin was also investigated. Although
the reaction was somewhat efficient at pH6, it has been found that pH has little effect on the response
variation compared to other optimized parameters. Therefore, the natural (pH 9.7) lysine solution was
used to further reduce the determination procedure time for lysine amino acid.
The linearity, precision, limit of detection, limit of quantification and accuracy showed the
usefulness of the proposed method. The validation of the proposed method was done by comparing
with the HPLC technique which highlights the merit of the present development method. In addition, it
was found that potassium, as a representative interfering species, did not interfere with ninhydrin in
various reaction conditions.
It can be concluded that the application of this method is not harmful towards the environment.
Such results can help to maintain the best conditions for an accurate analysis of Lysine solutions by
UV-Visible spectrophotometric technique.

Acknowledgements: The authors acknowledge the support of Al-IdrisiII Erasmus Mundus (European)
program for HB post-doc grant. CF and AV acknowledge FCT - Fundação para a Ciência e a
Tecnologia for the financial support through the project UID/QUI/00313/2019.

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Manuscript received: 20.04.2020

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SUPLEMENTARY MATERIAL

Figure S1.Absorbance spectrums of product peak from 400

to 800 nm (λmax = 479nm) of Lysine 1 mmol L-1 contaminated
with 10% KCl and reacted with ninhydrin in the water-bath
at different physicochemical conditions: (1) time: 20 min, ratio
1, pH 9.7, T=75 ºC; (2) time: 20 min, ratio 1, pH 2, T=75 ºC;
(3) time: 20 min, ratio 3, pH 6, T=80 ºC; (4) time: 40 min, ratio 3,
pH 6, T=80 ºC; (5) time: 60 min, ratio 3, pH 6, T=80 ºC

Table S1. Composition of lysine solutions

Lysine-KCl solutions KCl stock solution (mL)
Lysine 1 mmol L-1-KCl 3.8
Lysine 2 mmol L-1-KCl 7.5
Lysine 4 mmol L-1-KCl 14.9
Lysine 6 mmol L-1-KCl 22.4
Lysine 8 mmol L-1-KCl 29.8

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