Chromatographia

https://doi.org/10.1007/s10337-020-03896-x

ORIGINAL

Dansyl Chloride as a Derivatizing Agent for the Analysis of Biogenic

Amines by CZE‑UV
Jéssica Oliveira Fernandes Mantoanelli1   · Luís Moreira Gonçalves2   · Elisabete Alves Pereira1 

Received: 29 January 2020 / Revised: 28 March 2020 / Accepted: 9 April 2020

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Biogenic amines (BAs) are important compounds that can be used in the quality control of food and beverages. BA analysis
is a challenging task that can be made easier by applying a derivatizing agent like dansyl chloride (DNS). The optimized
capillary zone electrophoresis (CZE) separation of the DNS-BA derivates (derivates of cadaverine, histamine, putrescine,
tryptamine, and tyramine) was performed using benzylamine as an internal standard, a potential of 18 kV, a temperature of
23 °C, a running buffer consisting of phosphoric acid, 120 mmol L ­ −1, pH 2.5, and an hydrodynamic injection at 25 mBar for
6 s. All calibration curves had r higher than 0.99, and limits of detection (LODs) ranged from 7 to 50 µg L−1. The developed
2

methodology was tested in cheese and yogurt samples. DNS showed to be an alternative derivatization reagent not only
because it produced UV-detectable derivates (214 nm), but also because of its stability, aqueous solubility, high selectivity
and sensitivity, reduced impurities, and simple preparation steps.

Keywords  Dairy products · Derivatization · Food analysis · Food quality · Sample preparation

Introduction by diverse biochemical paths as a consequence of microbial

Biogenic amines (BAs) are low molecular weight (LMW)
nitrogenous substances, commonly classified according to
their chemical structures as aliphatic (e.g., spermine, sper-
midine, putrescine, and cadaverine), aliphatic volatile (e.g.,
methylamine, ethylamine, and isopentylamine), aromatic
(e.g., phenyethylamine, tyramine, and serotonin), and het-
erocyclic (e.g., histamine and tryptamine) [1–4]. BAs are
formed by decarboxylation of amino acids or by amina-
tion and transamination of ketones and aldehydes during
metabolic processes. These compounds play an important
role in essential physiological functions, however, at high
concentrations, they display toxic effects such as nausea,
headache, and blood pressure deregulation, even leading to,
in severe cases, to death [3, 5]. Since BAs are formed in food
* Elisabete Alves Pereira
ealves@ufscar.br
1
Departamento de Física, Química e Matemática,
Universidade Federal de São Carlos (UFSCar), Sorocaba,
SP, Brazil
2
Departamento de Química Fundamental, Instituto de
Química, Universidade de São Paulo (USP), São Paulo, SP,
Brazil
enzymatic activity and deterioration, its determination is
important not only because they are harmful to the health of
consumers, but also because their presence can be used as
food quality indicators. In effect, individual or BAs’ groups
have been used as markers of the degree of freshness and/
or deterioration in foods and beverages; that includes meat,
seafood, dairy products, chocolate, coffee, fruits, vegetables,
soybean and fermented beverages like beer and other alco-
holic beverages [6–10].
There are several analytical methods for the determina-
tion of BAs in food and beverages described in the litera-
ture [6, 11]. Most of these methods are based on separation
techniques like high-performance liquid chromatography
(HPLC) [2, 10, 12–14], gas chromatography (GC) [15–19],
and capillary electrophoresis (CE) [20]. Although CE has
frequently been used as a separation technique for BAs,
few articles have described its use with UV–Vis detection.
Table 1 provides information on some of the methods devel-
oped over the past 10 years for the determination of BAs in
foods using CE. As can be observed, most of the methods
are not spectrophotometric. BAs do not have any chromo-
phoric groups in their structures, and so they cannot be
analyzed directly. Thus, either derivatization is required, to
incorporate a chromophore or a fluorophore moiety in their

13
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Table 1  Literature survey of different analytical methodologies for BAs using CE

CE mode Detection Derivatizing agent BA (in parenthesis LOD in mg L
­ −1) Sample References

13
MEKC UV/Vis Benzoyl chloride Cad (0.29), His (0.43), Put (0.44), Spd (0.21), Spm (0.29), Try Fish [38]
(0.27) and Tyr (0.73)
MEKC UV/Vis Benzoyl chloride Cad (10), His (10), Phe (10), Put (10), Spd (10), Spm (10) and Fish [39]
Try (10)
MEKC UV/Vis 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate Cad (0.26), His (4.4), Put (0.44), Spd (3.6), Spm (5.1), Try Wine, chive, sausage [59]
(0.16) and Tyr (0.34)
MEKC LIF o-Phthaldehyde Cad (6.3), His (5.1), Phe (5.1), Put (3.6), Spd (8.4), Spm (5.1), Fish and wine [40]
Tyr (5.8) and Try (6.8)
MEKC LIF Fluorescein isothiocyanate Cad (0.13 × 10–3), His (0.14 × 10–3), Put (0.11 × 10–3), Spd Wine and pomegranate molasses [60]
(0.18 × 10–3), Try (0.20 × 10–3) and Tyr (0.17 × 10–3)
MEKC LIF Fluorescein isothiocyanate Cad (64 × 10–6), His (0.11 × 10–3), Put (0.14 × 10–3), Spd Fish [61]
(0.15 × 10–3), Try (66 × 10–6) and Tyr (58 × 10–6)
CZE LIF Fluorescein isothiocyanate Agm (0.0500), But (0.053), Cad (0.13), Dib (0.033), Dim Beer [62]
(0.0067), Eth (0.0050), Hex (0.20), His (0.017), Put (0.11)
and Tyr (0.023)
CZE LIF 3-(2-Furoyl)quinoline-2-carboxaldehyde Cad (0.25 × 10–3), His (56 × 10–6), Phe (1.2 × 10–3), Put Tobacco leaf [63]
(0.44 × 10–3), Spd (0.36 × 10–3), Spm (0.51 × 10–3), Try
(0.16 × 10–3) and Tyr (0.14 × 10–3)
CEC LIF 4-Fluoro-7-nitro-2,1,3-benzoxadiazole His (0.2 × 10–3), Spd (0.1 × 10–3), Spm (0.2 × 10–3), Tyr Soy sauce and chicken [64]
(0.4 × 10–3) and Try (1 × 10–3)
MCE LIF Fluorescein isothiocyanate Cad (0.13 × 10–3), His (0.40 × 10–3), Put (0.16 × 10–3) and Try Fish [65]
(0.29 × 10–3)
MEKC LIF 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde Cad (0.14 × 10–3), His (0.17 × 10–3), Phe (48 × 10–6), Put Soy sauce, fish and wine [66]
(1.322.10–4), Spd (0.20 × 10–3), Spm (2.4 × 10–3) and Tyr
(0.16 × 10–3)
CZE MS Isobutyl chloroformate Cad (0.015), Eth (0.015), Die (0.015), His (0.015), Phe Wine and fruit wine [22]
(0.015), Put (0.015), Spd (0.015), Spm (0.015), Tet (0.015),
Try (0.015) and Tyr (0.015)
CZE MS _ Cad (0.002), His (0.001), Phe (0.001), Ser (0.001); Spd Beer and wine [23]
(0.002), Put (0.002); Try (0.002); Tyr (0.002) and Uro:
(0.002)
CZE MS _ Cad (0.015), Eth (0.013), His (0.027), Isa (0.005), Isp (0.024), Wine [24]
Pen (0.007), Phe (0.007) and Tyr (0.018)
CZE C4D _ Cad (0.045), His (0.054), Put (0.041), Spd (0.066) and Tyr Cheese and yoghurt [7]
(0.098)
4
CZE CD _ Cad (0.055), His (0.046), Phe (0.098), Put (0.045), Spd Hard liquor and water [25]
(0.044), Spm (0.057), Try (0.15) and Tyr (0.11)
CZE C4D _ His (0.35), Phe (0.33) and Tyr (0.37) Wine [26]
Microchip Amperometry _ Eth (1.4), Trp (6.8) and Try (4.3) Beer [27]
J. O. F. Mantoanelli et al.
 Table 1  (continued)
CE mode Detection Derivatizing agent BA (in parenthesis LOD in mg L
­ −1) Sample References

CZE Electrochemi- _ His (8.4 × 10–3), Phe (96 × 10–3), Put (0.92 × 10–3), Spd Oysters [28]
lumines- (0.60 × 10–3) and Tyr (12 × 10–3)
cence
MEKC Indirect UV _ His, Phe, Trp and Try Wine [67]
MEKC UV/Vis – Dop (0.023), Epi (0.022), Hyd (0.15), Hyr (0.068), Nor Wine and beer [1]
(0.019), Trp (0.098), Tys (0.024), Try (0.098) and Tyr
(0.037)
CZE Indirect UV _ Cad (0.5), Phe (0.6), Put (0.4), Spd (0.6), Spm (0.1), Tma Fish and meat [32]
(0.6), Try (0.5) and Tyr (0.5)
CZE Indirect UV _ Cad (0.20), Car (0.54), Dop (0.061), Epi (0.073), His (0.41), Beer [33]
Hyd (0.16), Nor (0.068), Oct (0.092), Phe (0.24), Put (0.15),
Spd (0.29), Try (0.096) and Tyr (0.082)
CZE Indirect UV _ Cad (0.18), Eta (0.08); His (0.093), Iso (0.08), Isp (0.11), Met Wine [34]
(0.1), Phe (0.06), Pro (0.11), Put (0.092) and Tyr (0.05)
CZE Indirect UV _ Dim (0.45), Tma (0.59) and Tmo (0.75) Fish [35]
CZE Indirect UV _ Die (0.40), Dim (0.23), Met (0.65), Pro (0.47), Tea (0.32) and Fish [36]
Tma (0.49)

Agm agmatine, But butylamine, C4D capacitively coupled contactless conductivity detector, Cad cadaverine, Car carnosine, CE capillary electrophoresis, CZE capillary zone electrophoresis,
Dib dibutylamine, Die diethylamine, Dim dimethylamine, Dip dipropylamine, Dop dopamine, Epi epinephrine, Eth Ethylonamine, Hex hexanediamine, Hep heptylamine, His histamine, Hyd
hydroxytryptamine, Hyr hydroxytryptophan, Isa isoamylamine, Iso isopentylamine, Isp isopropylamine, LIF laser-induced fluorescence, MECK micellar electrokinetic chromatography, MCE
Microchip electrophoresis, Met methylamine, MS mass spectrometry, Nor norepinephrine, Oct octapimine, Pen pentylamine, Phe phenylethylamine, Pro propylamine, Put putrescine, Ser serine,
Dansyl Chloride as a Derivatizing Agent for the Analysis of Biogenic Amines by CZE‑UV

Spd spermidine, Spm spermine, Tea triethylamine, Tet triethanolamine, Tma trimethylamine, Tmo trimethylamine-n-oxide, Trp tryptophan, Try tryptamine, Tys tyrosine, Tyr tyramine, Uro uro-
canic acid

13

structures [21], or analysts should apply other detection tech-
niques like mass spectrometry (MS) [22–24], capacitively
coupled contactless conductivity detection (­ C4D) [7, 25,
26], or electroanalytical detectors [27, 28]. Indirect detec-
tion making use of a background electrolyte containing an
adsorbing co-ion (light-absorbing compound called probe)
with absorbance in the UV–Vis region is also possible [29,
30]. When a non-absorbing amine passes through the detec-
tor, it causes a decrease in absorbance, and a negative peak is
registered in the baseline [31]. Copper sulfate and imidazole
are some of the probes most used for indirect CE determi-
nation [32–36]. Although indirect detection is a tool that
can solve the issue of the absence of analytes containing
chromophore groups, this type of analysis implies spend-
ing time with the clean-up process since food and beverage
samples are complex matrices containing different interfer-
ing species.
Most derivatizing reactions for amines can be divided
into four types [21, 37]: acylation (e.g., benzoyl chloride [38,
39]), carbamate formation, Schiff base formation (e.g., with
o-phthaldehyde [40]), and sylation). Nevertheless, a different
type of reaction is herein suggested, using dansyl chloride
(DNS). DNS is an important reagent in numerous biochemi-
cal methods to analyze several amine containing molecules
like amino acids [41]. The sulfonyl group (–S(=O)2Cl) in
the DNS ‘swaps’ the chloride for the amine (Fig. 1), thus
each different amine produces a different derivate. DNS is
a non-toxic derivatizing agent for BAs, and the reaction can
occur in mild conditions [42, 43]. Furthermore, according to
recent literature [2], it may be advantageous, when compared
to other derivatizing agents, in terms of “higher sensitivity,
accuracy, precision and stability of the derivatives”.
In this work, several BAs (namely: putrescine, cadaver-
ine, tyramine, histamine, and tryptamine) were determined
in cheese and yogurt samples using DNS. To the best of the
authors’ knowledge, it is the first time that DNS has been
applied as a derivatizing reagent for BAs’ determination in
Fig. 1  Derivatization reaction
between DNS and a BAs
13
J. O. F. Mantoanelli et al.
an analytical procedure using capillary zone electrophoresis
with UV detection (CZE-UV).
Experimental
Chemicals and Samples
All reagents used were of analytical grade and were used
without further purification. Ultrapure water (resistivity
not less than 18.2 Ω cm at 298 K) from a Direct-Q 3UV
water purification system (Millipore) was used in all experi-
ments. Acetonitrile (ACN), ethanol (EtOH) and methanol
(MeOH), hydrochloric acid (HCl) and phosphoric acid were
purchased from Merck; sodium hydroxide, sodium tetrabo-
rate (STB), triethylamine (TEA), benzylamine, 1,7-diamino-
heptane, cadaverine, histamine, tryptamine, trichloroacetic
acid (TCA) and DNS were obtained from Sigma-Aldrich;
putrescine was purchased from ChemCruz; and tyramine
from Alfa Aesar.
Stock solutions of each amine (1000 mg L ­ −1) were pre-
pared in an aqueous solution of 0.1 mol ­L−1 of HCl and
stored, up to 1 month, protected from the light at 4 °C.
Working solutions were prepared daily by appropriate
dilution from the stock solutions with aqueous solution of
0.1 mol ­L−1 of HCl. The 0.75 g ­L−1 DNS solution was pre-
pared daily in ACN. The stock solution of phosphoric acid
(200 mmol ­L−1) was prepared in ultrapure water and stored
under refrigeration for 1 month. All electrolyte solutions
were prepared daily.
All food samples were purchased in a local supermarket.
CZE Separations
CZE separations were performed using a CE system, model
7100A (Agilent technologies), with a diode array detector
(DAD) set to 214 nm and temperature control. The fused
Dansyl Chloride as a Derivatizing Agent for the Analysis of Biogenic Amines by CZE‑UV
silica capillary (Polymicro Technologies) had 48.5 cm total
length, 40 cm of effective length, 75 µm of internal diameter
and 375 µm of outside diameter.
The fused capillaries were daily conditioned with sodium
hydroxide, 1 mol ­L−1, for 30 min, followed by 30 min of
water and then 30 min of the electrolyte; between each anal-
ysis there was a 3 min conditioning step with the electrolyte.
Sample Preparation Procedures
2.5 g of previously homogenised sample were placed in
8 mL of an aqueous solution of HCl, 1 mol L ­ −1. The mix-
ture was stirred for 30 min and subsequently centrifuged for
15 min at 605 RCF. The supernatant was diluted with water,
completing 10 mL of volume and filtered with syringe filter
with 0.45 μm porosity [7].
Amine derivatization was performed by mixing 2 mL of
the filtered solution, 2 mL of the DNS solution and 0.8 mL
Fig. 2  a Initial electropherogram of a mixture of BAs (100 mg L ­ −1 of
each amine) derivatized with DNS, electrolyte consisted of 40 mmol
­L−1 phosphate buffer, pH 2.7, potential was 10  kV, temperature of
25  °C, injection at 50 mBar for 3  s. b Electropherogram of a mix-
ture of BAs (25 mg ­L−1 of each amine) derivatized with DNS, elec-
trolyte consisted of 40 mmol L ­ −1 of phosphoric acid, pH 2.5 adjusted
with TEA. c Apparent mobility of the different derivates by addition
of modifiers to the electrolyte (40 mmol L ­ −1 of phosphoric acid, pH
2.5 adjusted with TEA), namely methanol, 10% (v/v), acetonitrile,
10% (v/v), and β-cyclodextrin, 15  mmol L ­ −1. d Apparent mobility
of the different derivates by changing the percentage of acetonitrile,
experiments performed with electrolyte consisting of 40 mmol ­L−1 of
phosphoric acid, pH 2.5 adjusted with TEA. e Electropherograms of
a mixture of BAs (25 mg ­L−1 of each amine) derivatized with DNS,
60 mmol ­L−1 of phosphoric acid, acetonitrile 10% (v/v) with different
pH values: 2.0 and 2.5 (pH adjusted with TEA), potential was 20 kV,
of sodium hydroxide, 6 mol ­L−1. Reaction occurred pro-
tected from the light. The organic phase (the one on top)
was used for further analysis. The derivatization reaction
conditions were optimized in this work.
It was possible to add an enrichment step (also called as
‘pre-concentration’) by evaporation by means of nitrogen
flux at 40 °C. The residue was dissolved in 250 µL of ACN.
Results and Discussion
Method Development
Initially, the electrolyte consisted of phosphate buffer pH 2.7
[44], being the separation performed with constant poten-
tial of 10 kV at 25 °C (Fig. 2a). Since asymmetrical peaks
were observed, along with a slow separation, other elec-
trolytes based on the literature were evaluated for further
temperature of 29 °C, injection at 50 mBar for 3 s. f Effect of derivat-
ization conditions (sodium hydroxide, carbonate and borate). Electro-
lyte: 60 mmol ­L−1 of phosphoric acid, acetonitrile 10% (v/v) pH 2.5
adjusted with TEA, potential was 20 kV, temperature of 29 °C, injec-
tion at 50 mBar for 3 s. g Testing of different phosphoric acid concen-
trations in the electrolyte, *means impossible to differentiate between
peaks. In both a–c 25  mg L ­ −1 of each amine, potential was 20  kV,
temperature of 29  °C, injection at 50 mBar for 3  s. h Final optimi-
zation of the acetonitrile percentage. Electropherogram of a mixture
of BAs (25 mg L ­ −1 of each amine) derivatized with DNS, electrolyte
consisted of 120 mmol L ­ −1 of phosphoric acid, pH 2.5 adjusted with
TEA, potential was 20 kV, temperature of 29 °C, injection at 25 mBar
for 6 s. For a, b, f, g and h (1) DNS-putrescine, (2) DNS-cadaverine,
(3) DNS-histamine 1, (4) DNS-tyramine, (5) DNS-histamine 2, and
(6) DNS-tryptamine
13

optimization [8, 45, 46]. Optimization experiments were car-
ried out using as phosphoric acid, 40 mmol ­L−1, and TEA to
adjust the pH at 2.5; separation was performed with constant
potential of 20 kV at 29 °C (Fig. 2b).
To optimize the separation, the addition of modifiers was
evaluated. The addition of organic solvents often improves
resolution between peaks because it reduces the EOF and
changes retention and selectivity factors [31, 47]. Another
fact to be considered is that solutes featuring hydrophobic
characteristics are not properly separated because of low sol-
ubility in aqueous solutions. Cyclodextrins have a structure
with a hydrophobic cavity and a hydrophilic outer surface,
causing solutes that have a certain hydrophobicity to parti-
tion between cyclodextrin and the aqueous phase-improving
separation [31]. It was tested the addition, to the running
electrolyte, of methanol, 10% (v/v), acetonitrile, 10% (v/v),
and β-cyclodextrin, 15 mmol L ­ −1. Although the addition of
these modifiers to the running electrolyte did not provide the
separation of critical peaks, i.e., cadaverine/histamine 1 and
tyramine/histamine 2 (Fig. 2c), acetonitrile provided peaks
with a more symmetrical shape and shorter time of analysis.
Subsequently, the percentage (v/v) of acetonitrile was opti-
mized from none to 15%; a separation between the critical
peaks at 5 and 10% acetonitrile was not observed, however
the addition of 15% provided coelution between cadaverine/
histamine 1/tyramine and histamine 2/tryptamine. Since the
time of analysis was shorter with 10% ACN, the percentage
was selected for the following experiments (Fig. 2d). The
electrolyte’s pH was studied in the interval from 2.0 to 4.5
with constant run electrolyte and acetonitrile concentration
(60 mmol ­L−1 and 10%, respectively). Values above 3.0 were
not very positive for both peak separation and peak height,
however pH 2.0 resulted in poor peak separation may be due
to a lower electroosmotic flow (EOF), thus a pH value of
2.5 was considered optimum despite insufficient resolution
among critical peaks (Fig. 2e)
Experimental efforts were also dedicated to the derivati-
zation reaction. Derivatization is a simple and ingenious
technique that chemists can apply when the analytes are of
difficult direct analysis [48–52]. Different procedures were
evaluated aiming to obtain the best analytical signal: sodium
tetraborate, 6 mmol ­L−1, at pH 9.3 and 12 [53]; carbon-
ate buffer, 150 mmol ­L−1, pH 10 [54]; phosphate buffer,
42 mmol ­L−1, pH 12 [55]. However, none of the evaluated
procedures gave origin to better results than those observed
for the derivatization reaction with DNS in sodium hydrox-
ide, 6 mol ­L−1, with 5 min of reaction time at room tempera-
ture (Fig. 2f). DNS concentration was optimized between
0.75 and 3.5 g L ­ −1. In general, results did not improve a
lot with a higher concentration, hence 0.75 g ­L−1 was the
chosen concentration.
Injection mode and injection time can be used to
improve the magnitude of the analytical signal. Initially,
13
J. O. F. Mantoanelli et al.
the hydrodynamic injection mode (50 mBar for 3 s) was
used. One of the problems that can occur when using short
injection times is the variation in the amount of sample that
is transferred into the capillary column. One way to mini-
mize this problem is to use longer injection times with lower
pressures, therefore the applied pressure was reduced to 25
mBar. By maintaining a constant pressure of 25 mBar and
varying the injection time (6, 8 and 10 s), it was possible to
observe, as expected, an increase in the magnitude of the
analytical signal as a function of the increase in the injec-
tion time. However, the amount of sample inserted into the
column also increases which resulted in loss of resolution
between peaks. The best compromise between resolution
and signal magnitude was obtained using 25 mBar for 6 s.
Hydrodynamic injection is the most commonly used mode
in CE, however, electrokinetic injection may be used due to
the sample matrix type or for sensitivity reasons. In the elec-
trokinetic injection, the amount of analyte transferred into
the capillary column is a result of the electrophoretic mobil-
ity of the analyte, the sample’s conductivity, the running
electrolyte’s conductivity, and the EOF. Thus, in this type
of injection, there is a discrimination between the analytes
with different mobilities. Effectively, more mobile analytes
easily enter the capillary may be ‘pre-concentrated’ when
compared to less mobile analytes [47]. The use of the elec-
trokinetic injection mode did not present satisfactory results,
as the analyte’s signal magnitude decreased much in relation
to the hydrodynamic injection. In addition, a decrease in the
baseline during the analyte migration was observed, a behav-
iour that makes identification and quantification difficult,
especially with very low concentrations. After the evaluation
of the type and period of injection, the hydrodynamic mode
using 25 mBar for 6 s was selected for the following steps.
In these tests, samples were present in acetonitrile and the
electrolyte consisted of phosphoric acid, 60 mmol ­L−1, with
10% v/v of acetonitrile, pH adjusted to 2.50 with TEA.
There are other alternatives that may improve sensi-
bility like the online ‘preconcentration’ called stacking.
A band of water is introduced into the capillary prior to
the sample introduction, which should be prepared in a
medium of low electrical conductivity. When voltage is
applied, a region of high electric field is formed in the
water band, larger than in the rest of the solution. The
analytes migrating in the sample band are accelerated
and migrate faster when they meet the water band, and
are decelerated when they leave this band and find the
electrolyte band again, thus concentrating in a thin band
between these two regions, possibly resulting in enrich-
ment. When electric fields are equated, the analytes are
separated within the column [56]. The use of this strategy
increased the magnitude of the analytical signal, but unfor-
tunately there was no stability in the baseline, which would
challenge a suitable analysis.
Dansyl Chloride as a Derivatizing Agent for the Analysis of Biogenic Amines by CZE‑UV
Since any improvement on resolution of peaks critical
was not observed in the range from 40 to 70 mmol L ­ −1,
the phosphoric acid concentration in the electrolyte was
evaluated from 80 to 150 mmol ­L−1. As can be observed
in Fig. 2g, higher concentrations improve the separation
however that is accompanied by a greater Joule effect, thus
120 mmol ­L−1 of phosphoric acid (with pH 2.5, adjusted
with TEA) was the compromise used in the subsequent
experiments. The acetonitrile concentration thus needed to
be adjusted again. The effect of acetonitrile on the separation
was evaluated in range of 5–15% (Fig. 2h). With increasing
acetonitrile concentration, it was observed a loss in resolu-
tion. Therefore 5% acetonitrile was chosen as the optimum
concentration.
Temperature’s influence on the BAs’ separation was eval-
uated from 21 to 29 °C. Reduction in temperature promotes a
reduction in the current values and an increase in the running
electrolyte viscosity. These characteristics can result in a
reduction of the EOF and, consequently, improve the resolu-
tion between peaks, on the other hand, it could be observed
an increase in the analysis time. Since no significant varia-
tions in resolution were observed between the critical peaks
in the evaluated range, the temperature of 23 °C was selected
because it presented a suitable compromise between resolu-
tion and time of analysis, as well as avoiding the formation
of bubbles throughout the capillary due to the Joule effect.
To avoid the peaks’ coelution that may occur as a function of
slight variations during the analysis procedure (electrolyte’s
pH and concentration), the voltage was reduced to 18 kV
(100 µA at 23 °C).
Different substances were evaluated as possible internal
standard (IS): imidazole, glycine, 1,7-diaminoheptane and
benzylamine (all being derivatized in a similar manner). The
Fig. 3  a Electropherogram of a mixture of BAs derivatized with
­ −1 of each
DNS. b Study of adducts stability. In both a and b 8 mg L
amine, potential was 18  kV, temperature of 23  °C, injection at 25
imidazole peak showed up after migration of all amines,
significantly increasing the total analysis time, moreover the
peak was also rather asymmetric. The glycine peak was of
low magnitude even at high concentrations, and coelution
with the putrescine peak could also be observed. 1,7-diami-
noheptane presented a symmetrical peak but with very simi-
lar migration time as tyramine. Benzylamine was detected
before the amines of interest and appeared to be the most
suitable compound to be used as an IS (Fig. 3a).
It was also important to evaluate the stability of the
formed adducts. After the derivatization process, the stand-
ard mixture containing the DNS-BAs adducts was ana-
lysed after different reaction times: 5, 30, 60, 120, 180 and
240 min (Fig. 3b). Tryptamine and cadaverine showed a
maximum peak area at 5 min, with a relatively stable behav-
iour up to 180 min. Putrescine, tyramine and benzylamine
(the IS) were also stable up to 180 min. Histamine presented
the largest variation, the histamine 1 peak area increased as
the histamine 2 peak area decreased almost proportionally,
showing some stability only until 180 min. These tendencies
suggest that one species of histamine is converted to another
one over time. This had already been observed throughout
this work and could be evidenced more clearly in this stabil-
ity study.
Sampling preparation is often the bottleneck of many
analytical methodologies [57] and is usually worth spend-
ing some time and effort on this particular aspect. Two
extraction methodologies were adapted: using TCA [7]
and using HCl [58]. To evaluate which parameter influ-
enced the extraction process the most (either the acid
nature or stirring/time), two procedures were tested for
each case: vortex stirring for 1 min with, and 30 min stir-
ring using a magnetic stirrer. Tests were performed using
mBar for 6 s, electrolyte consisted of phosphoric acid, 120 mmol ­L−1,
and acetonitrile, 5% (v/v), pH 2.50 adjusted with TEA
13

a yogurt sample fortified with amine concentration of
2.5 mg ­L−1. It was observed that the extraction procedure
using HCl, 0.1 mol ­L−1, for 30 min with stirring provided
better results in relation to the magnitude of the analyti-
cal signal (except for tryptamine) (Fig. 4), thus, it was the
procedure selected.
Fig. 4  Comparison of the effectiveness of the extraction procedure:
TCA, stirring: extraction using TCA with 30 min stirring with a mag-
netic bar; TCA, vortex: extraction using TCA with 1  min of vortex;
HCl, stirring: extraction using HCl acid with 30  min stirring with a
magnetic bar; HCl, vortex: extraction using HCl with 1 min of vortex
Fig. 5  a Electropherograms showing selectivity in a real sample
(potential of 18  kV). I—Control: non-spiked yogurt sample with
strawberry extract; II—DNS-BA standards (4  mg L ­ −1) in water;
III—yogurt sample spiked with DNS-BA standards (4  mg L ­ −1). b
Electropherograms of real samples spiked with DNS-BAs standards
13
J. O. F. Mantoanelli et al.
Method Validation
Selectivity was tested using a real sample (yogurt with
strawberry extract), the sample was spiked with 4 mg L ­ −1
of each tested BA (Fig. 5a). It was possible to observe that
the sample analysis had no interfering peaks, thus separa-
tion and identification would not be harmed in real sam-
ples. However, the peak height significantly decreased in the
spiked sample showing that a matrix effect, for quantitative
goals, was occurring.
All method performance parameters were obtained using
the IS benzylamine. The analytical curves, without ‘pre-
concentration’, were constructed with a concentration range
from 1.0 up to 10.0 mg L ­ −1 (n = 5), being all standard solu-
tions analyzed in triplicate. All correlation coefficients (r)
were above 0.990 (Table 2). The limits of detection (LOD)
and quantification (LOQ) were, with pre-concentration
(evaporation according to what is mentioned in the experi-
mental section), in a range from 7 to 50 µg ­L−1 and from
0.03 to 0.2 mg ­L−1, respectively. LOD and LOQ, with pre-
concentration, were based on the signal-to-noise (S/N) ratio
of 3 and 10, respectively.
Precision was evaluated at two levels: repeatability and
intermediate precision (Table 3). Repeatability was per-
formed by comparing the obtained peak areas with three
different concentrations (2.5, 5 and 10 mg ­L−1), performed
in triplicate for each amine in each in the same day. Interme-
diate precision was determined with the same concentrations
in three different days. Recovery tests were carried out with
three different concentrations (2.5, 5 and 10 mg L ­ −1). The
recovery percentages were calculated by comparing the peak
(potential of 20  kV). I—BA-DNS standards (0.5  mg L ­ −1) in water;
II—whole yogurt sample; III—fresh cheese sample. In both a and
b: #non-identified peak; electrolyte: phosphoric acid, 120 mmol ­L−1,
with 5% v/v of acetonitrile, pH adjusted to 2.50 with TEA, tempera-
ture of 23 °C, injection at 25 mBar for 6 s
Dansyl Chloride as a Derivatizing Agent for the Analysis of Biogenic Amines by CZE‑UV

Table 2  Analytical parameters
BA Without pre-concentration With pre-concentration
2 −1 −1
Calibration curve r LOD/mg ­L LOQ/mg ­L LOD/mg ­L−1 LOQ/mg ­L−1

Putrescine y = (0.47 ± 0.02) x + (0.02 ± 0.11) 0.996 0.07 0.2 0.01 0.04

Cadaverine y = (0.81 ± 0.02) x + (0.1 ± 0.1) 0.997 0.04 0.2 0.007 0.03
Histamine 1 y = (0.12 ± 0.01) x − (0.03 ± 0.04) 0.989 0.4 2 0.05 0.2
Tyramine y = (0.236 ± 0.008) x + (0.03 ± 0.05) 0.997 0.2 0.5 0.04 0.1
Histamine 2 y = (0.164 ± 0.004) x + (0.02 ± 0.02) 0.998 0.2 0.6 0.03 0.1
Tryptamine y = (1.13 ± 0.05) x + (0.4 ± 0.3) 0.994 0.05 0.2 0.008 0.03

Table 3  Repeatability (n  = 3), intermediate precision (n = 9) and The method was also applied for quantitative determina-
recovery (n = 3) using three different amine concentrations (2.5, 5 and tion in five samples of dairy products: two different fresh
10 mg ­L−1). Recovery experiments were performed in yogurt samples
cheese (A and B) and three different milk drinks with fruit
BA Concentra- Repeat- Intermediary Recovery/% pulp (A, B and C). All samples were subjected to the ‘pre-
tion/mg abil- precision/% concentration’ process as mentioned in the experimental sec-
­L−1 ity/%
tion. The fresh cheese sample A and the dairy drink samples
Putrescine 2.5 1.7 5.5 96.2 ± 1.6 A and B presented none of the amines investigated in this
5.0 1.0 8.8 101.3 ± 1.0 work. The peaks of putrescine in the sample of fresh cheese
10.0 2.2 6.0 100.6 ± 2.2 B and cadaverine in the sample of milk drink C above the
Cadaverine 2.5 1.6 8.0 87.1 ± 1.4 LOD but below the LOQ were identified. In the cheese sam-
5.0 1.1 9.6 96.2 ± 1.1 ple B, it was possible to identify and quantify cadaverine,
10.0 2.3 5.5 100.8 ± 2.3 with a concentration of 3.3 ± 0.1 mg per kg. The presence
Tyramine 2.5 1.1 8.2 105.5 ± 1.2 of cadaverine in this sample may indicate that there was
5.0 2.7 8.8 110.7 ± 3.0 previous contamination or even very rapid degradation of
10.0 2.7 8.2 95.4 ± 2.6 the product, considering the date of production and suitable
Tryptamine 2.5 1.1 6.5 89.9 ± 1.0 conservation. The presence of cadaverine is predominant
5.0 2.9 8.0 95.9 ± 2.8 among different types of cheese [6], although the concentra-
10.0 2.9 6.8 98.4 ± 2.9 tion found was low compared to other analyzes performed on
Total histamine 2.5 3.6 15 87.4 ± 3.1 the same type of product (364 ± 2 mg per kg of cadaverine
5.0 9.9 14 113 ± 11 in fresh cheese) [7].
10.0 8.4 15 104.0 ± 8.7 This methodology has the potential to be applied not only
to similar samples but also, with minor adaptations in the
sample preparation procedure, it might be used for other
areas of the spiked and non-spiked yogurt samples with the types of matrices, such as meat or fish products.
peak areas of standard solutions at the same concentrations.
The proposed methodology was the first time that deri-
vatization with DNS was associated with CE aiming the Conclusions
analysis of BAs. It showed several advantages like the
simplicity of the derivatization procedure (5 min at room CE-UV with derivatization using DNS proved to be suitable
temperature) and rapid analysis (less than 11 min). In addi- for the determination of BAs in dairy food samples. The
tion, CE uses reduced samples, reagents and solvent vol- main advantages of the method were short time of analysis
umes when compared with HPLC and GC. As a proof of and cost-effectiveness. After optimization of the analytical
concept, the method was applied to evaluate the presence and instrumental parameters, the use of a pH 2.5 running
of BAs as indicatives of product degradation. The samples electrolyte allowed the DNS-BAs adducts to acquire charge
(whole yogurt and fresh cheese) were stored for 2 months and the analysis to be performed using the CZE mode. In
under refrigeration and analyzed (Fig. 5b). In the cheese overall, the evaluated validation parameters were satisfac-
sample, it was possible to identify, by spiking and compar- tory. The use of stirring for 30 min in HCl showed the best
ing migration times, the peaks of putrescine, cadaverine, performance for BAs’ extraction. The method proved to be
and tyramine, probably resulting from the storage period, effective for degradation tests and quantitative determina-
showing product degradation. tions in dairy products.

13

Acknowledgements  Authors wish to acknowledge Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES: 001), MSc
fellowship granted to JOFM by Programa de Pós-Graduação em Bio-
tecnologia e Monitoramento, UFSCAr (Campus Sorocaba), Con-
selho Nacional de Desenvolvimento Científico e Tecnológico (CNPq
132680/2019-0), and Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP 2019/03582-7 and 2018/14425-7) for financial
support.
Compliance with Ethical Standards  high-performance liquid chromatography. Food Anal Methods
Conflict of Interest  All authors declare no conflicts of interest.
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