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Journal of Chromatography B 1105 (2019) 203–209

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Copper-dependent hydrolysis of trichloronate by turkey serum studied with T


use of new analytical procedure based on application of chiral
chromatography and UV/Vis spectrophotometry
Damianys Almenares-Lópeza,b, Alina Juantorena-Ugasa,b, Katia Rosales-Espinosaa,b,
Eugenio Vilanovac, Camilo Ríosd, Antonio Monroy-Noyolaa,b,

a
Laboratorio de Neuroprotección, Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
b
División de Ingenierías y Ciencias Agropecuarias, Universidad Popular de la Chontalpa, H. Cárdenas, Tabasco, Mexico
c
Instituto de Bioingeniería, Universidad Miguel Hernández, Elche, Alicante, Spain
d
Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía, M.V.S. Ciudad de México, Mexico

ARTICLE INFO ABSTRACT

Keywords: Trichloronate is a racemic organophosphate, which has been used for the manufacture of insecticides. This
Trichloronate compound induces delayed neuropathy in hen and humans. This study shows the Cu2+-dependent hydrolysis of
Chiral HPLC trichloronate by turkey serum using UV/Vis spectrophotometry and chiral chromatography. The CHIRALCEL OD
UV/Vis spectrophotometry column and mobile phase of heptane allowed a resolution of 1.15 of its two enantiomers, while the liquid-liquid
Hydrolysis
extraction showed a recovery of 95–98%. The optimum linear response was of 50 to 800 μM with a detection and
Serum
quantification limit of 0.6 and 2 μM for (+)-trichloronate, and 0.7 and 2.3 μM for (−)-trichloronate. The levels
Turkey
of Cu2+-dependent hydrolysis (μM remaining concentration) quantified for 60 min at 37 °C and pH 7.4 were
statistically higher (p ˂ 0.05) for (−)-trichloronate (65%) than (+)-trichloronate (32%). This stereoselective
hydrolysis was confirmed by UV/Vis spectrophotometry using 2,4,5‑trichlorophenol as standard, each of the
enantiomers (93–95% purity) collected by HPLC, as well as aminoantipyrine and ferricyanide reagents to yield a
colored product. This method exhibited an optimal linearity (r > 0.99) and a higher Cu2+-dependent hydrolysis
(p < 0.05) to (−)-trichloronate (47%) than its corresponding (+)-form (31%). This results shows the Cu2+-
dependent stereoselective hydrolysis of a racemic OP in its thio form (P = S) by an A-esterase of the turkey
serum through the development of a colorimetric method and optimization of an existing chiral chromato-
graphic method.

1. Introduction these xenobiotics [3]. It is as also needed to propose the safer formulation
of pesticides towards humans and the environment.
Around of 25% of the commercial pesticides are formulations of ra- The A-esterases or PTEs enzymes, such as paraoxonase-1 (PON1) of
cemic mixtures that include organophosphorus compounds (OPs) which the mammal's serum is the main detoxification system of OPs in ver-
feature a chiral center on the phosphorus atom, such as methamidophos, tebrate animals. It is Ca2+-dependent and the most known and studied
fenamiphos, and trichloronate [1]. The chiral OPs insecticides have been A-esterase in the mammals serum which includes that of human serum.
the most commonly employed pesticide worldwide. The optical properties PON1 hydrolyzes non-chiral OP insecticides, such as paraoxon, dia-
of its enantiomers give rise to toxicity differences in biological systems [2]. zoxon, and chlorpyrifos oxon. Meanly, its hydrolyzing activity towards
The systemic stereospecific degradation in tissues and organs is a factor to OPs in their thio form is nonexistent, and very limited about chiral OPs
be considered in the acute neurotoxicity of OPs. It has been suggested that with oxo form [4]. Moreover, when catalytic activity was it observed, it
there is a need to carry out in vitro and in vivo laboratory studies of the was mainly against the less toxic enantiomer as in the case with the
hydrolysis of OPs by A-esterases or phosphotriesterases (PTEs) (EC 3.1.8) nerve agent sarin [5] and with O-hexyl O-(2,5‑dichlorophenyl) phos-
of birds and mammal's tissues of metabolic importance and targets in phoramidate (HDCP), an analog compound of the insecticide metha-
order to elucidate the biochemical mechanisms of the neurotoxicity of midophos [6].


Corresponding author at: Laboratorio de Neuroprotección, Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001 Col.
Chamilpa, C.P. 62209 Cuernavaca Morelos, Mexico.
E-mail address: amonroy@uaem.mx (A. Monroy-Noyola).

https://doi.org/10.1016/j.jchromb.2018.12.026
Received 31 May 2018; Received in revised form 4 December 2018; Accepted 22 December 2018
Available online 24 December 2018
1570-0232/ © 2018 Published by Elsevier B.V.
D. Almenares-López et al. Journal of Chromatography B 1105 (2019) 203–209

Cl Cl
Sampling

QuEChERS
S extrac!on

Cl O P O
Chromatographic isomers
separa!on collec!on

Fig. 1. The chemical structure of trichloronate.


Chiral UV-Vis
Trichloronate (O-ethyl O-2,4,5‑trichlorophenyl ethylpho- chromatographic spectrophometric
sphonothioate) is a chiral phosphothionate insecticide (Fig. 1). It has valida!on valida!on
been used in the agriculture, mainly by the control of the fly of the
genus Pylla rosae and its larvae. This insecticide has been commercia-
lized as FENOPHOSPHON, FITOSOL, AGRISIL and PHYTOSOL. An
aquatic study employing C. dubia or D. magna and trichloronate en- Chromatographic
UV-Vis Analysis
antiomers in a range of 0.1–2.0 μg/L at 96 h has shown the stereo- analysis
selective toxicity of this racemic OP, because, the CL50 of (−)-tri-
chloronate proved to be 8–11 times more toxic than (+)-trichloronate
[7]. Others, In vivo toxicity studies have reported that the racemic
mixture of trichloronate causes acute cholinergic toxicity by inhibition
Results
Results
of acetylcholinesterase (AChE) as well as irreversible ataxia, a second Remaining TCP
syndrome known as organophosphate induced delayed polyneuropathy TCP released
isomers
(OPIDP) in hens, rats and sheep. This delayed neuropathy is associated
to the inhibition and aging of the neuropathy target esterase (NTE) [8]. Fig. 2. Flow diagram of the procedure of Cu2+-dependent hydrolysis of tri-
Other, in vitro studies have shown that the oral administration of the chloronate enantiomers in turkey serum samples by chiral HPLC and UV/Vis
racemic mixture of trichloronate in its oxo form exhibits a greater in- spectrophotometric methods.
hibition ability of AChE as compared to NTE. However, in vivo studies
in hens (the usual OPIDP model) showed the neuropathic ability of its and quantification. Also in this study were used the chromatographic
thio-form. This results suggest its enzymatic bioactivation to the oxo conditions; mobile phase, flow velocity, chiral column and detector for
form by induces inhibition and aging of hen brain NTE [9,10]. Jędr- the resolution of trichloronate enantiomers reported by Ellington and
zejowska and coworkers [11]. have reported that chiral trichloronate coworkers [1]. While, similar conditions about the concentration of
induces OPIDP in human, electrophysiologically characterized for da- enantiomers, cofactor metallic, chemical reagents (Tris, sodium dodecyl
mage in the motor and sensory fibers as well as demyelination of distal sulfate, 4-aminoantipyrine and potassium hexacyanoferrate), calibra-
axons. tion curve (trichlorophenol standard), as well as reaction volume, ab-
Other preclinical studies of NTE aging by racemic HDCP have pro- sorbance value and physiological conditions used in the colorimetric
posed the stereospecific induction of OPIDP [12]. For this reason, is quantification of dichlorophenol release in the stereoselective hydro-
important to carry out in vitro and in vivo metabolic studies of tri- lysis of HDCP [17] were employed in the present UV/Vis spectro-
chloronate by A-esterases in avian and mammal tissues [3]. For which it photometric method. The trichloronate insecticide was selected because
is necessary to develop a chiral chromatography method that include it is a chiral compound with delayed neurotoxic properties and turkey
quick, easy, cheap, effective, rugged and safe (QuEChERS) liquid-li- serum was select due to recent report of Cu2+-dependent A-esterase
quido liquid liquid extraction technique as it has been developed by activity in chicken serum [18].
pesticides quantification in biological products [13]. As well as QuE-
ChERS colorimetric method based on the quantification of the phenolic
2. Experimental
product of the OPs hydrolysis developed for dichlorophenol and p-ni-
trophenol in the case of the hydrolysis of HDCP and paraoxon in-
In Fig. 2 is schematized the methodology developed in this study
secticide, respectively [14].
since obtaining blood turkey, trichloronate enantiomers until quantifi-
In this study we development a QuEChERS liquid-liquid extraction
cation of the copper-dependent stereoselective hydrolysis of tri-
technique of trichloronate in combination with chiral high performance
chloronate in turkey serum by chiral chromatography and UV/Vis
liquid chromatography (quiral HPLC) and also we collected tri-
spectrophotometry. Both analytical methods were validated in their
chloronate enantiomers with high purity by development an UV/Vis
recovery, linearity, repeatability, limit of detection and quantification
spectrophotometric method with the purpose to demonstrating the
parameters.
Cu2+-dependent estereoselective hydrolysis of trichloronate ex vivo by
turkey serum. Both analytical methods were evaluated according to the
Note for Guidance on Validation of Analytical Methods: Methodology. 2.1. Chemicals and serum samples
International Conference on Harmonization [15] and guidelines for
data acquisition and data quality evaluation in environmental chem- Trichloronate (O-ethyl O-2,4,5‑trichlorophenyl ethylpho-
istry [16] for their recovery, linearity, repeatability, limit of detection sphonothioate) with a purity of 98% was purchased from Chem Service,

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D. Almenares-López et al. Journal of Chromatography B 1105 (2019) 203–209

Inc. 2,4,5‑trichlorophenol (TCP) with a purity of 99.5%, copper(II) 2.4. Analytical validation of the chiral chromatography method
sulphate, potassium hexacyanoferrate, 4‑aminoantipyrine, Tris (hy-
droxymethyl) aminomethane and SDS (Sodium dodecylsulfate) were 2.4.1. Equipment and chromatographic conditions
obtained from Sigma Aldrich Co. The chromatography grade heptane Separation of enantiomers were carry out with the chromatography
used was Burdick and Jackson (Morris Township, NJ). Turkey serum system and conditions described above. This method is based on that
was obtained from Facultad de Ciencias Agropecuarias of the described by Ellington and coworkers [1].
Universidad Autonoma del Estado de Morelos (UAEM), following the
Mexican Official Norm NOM-062-ZOO-1999. Technical specifications 2.4.2. Linearity
for the production, care and use of laboratory animals. Standard curves of racemic trichloronate were prepared with 50,
Blood samples (5 mL) from turkey (Meleagris gallopavo) were ob- 100, 200, 400, 600 and 800 μM by triplicate. The colorimetric re-
tained from three animals by venous puncture of their right wing. The sponses were considered linear when adjusted to a correlation coeffi-
tubes employed to collect the samples were free from anticoagulants. cient of a least 0.99.
The blood was centrifuged at 1000g for 10 min to obtain the serum
which was labelled and stored at −70 °C until the time of carrying out 2.4.3. Limits of detection (LOD) and quantitation (LOQ)
the hydrolysis assays. Experiments were carried out in accordance with Both limits were determined according to the ACS committee on
the guidelines of the European Union Council (2010/63/EU), guide- Environmental Improvement. [16]. In both cases, we considered the
lines, following the Spanish regulations for the use of laboratory ani- mean of the blank, plus three times (LOD), or 10 times (LOQ) their
mals; Approval of the accredited Ethical Committee of the Miguel standard deviation (Table 4).
Hernández University, was obtained. (Reference UMH.iB.EVG.01.17).
2.4.4. Reproducibility
Three aliquots of the racemic trichloronate standard (200, 400 y
2.2. Preparation of trichloronate enantiomers
800 μM equivalent to 100, 200 and 400 of each enantiomer) were
analyzed by three analysts in three different days to determine the
For the analysis of enantiomers, 150 μL of a 400 μM standard solu-
inter-assay variation. Again, both retention times and concentration
tion of racemic trichloronate dissolved in dry acetone was injected into
were tested (Table 5).
a high performance liquid chromatography system (HPLC) (Waters
2996) equipped with a photodiode array detector (Waters 2996), auto
2.4.5. Repeatability
sampler (Waters 717) and CHIRALCEL OD column [1]. The retention
Three aliquot mixture of the racemic trichloronate standard (200,
time was 8.49 and 9.92 min for (+)-trichloronate and (−)-tri-
400 y 800 μM equivalent to 100, 200 and 400 of each enantiomer) and
chloronate, respectively. The resolution was 1.15 between both peaks.
a turkey serum sample were analyzed by three analysts in three dif-
For the collection of the enantiomers were adapted a fraction collector
ferent work days to determine the intra-assay variation. Both retention
(Waters Fraction Collector III) to the chromatographic system. 150 μL
times and concentrations were assayed for variation (Table 5).
injections of 400 μM racemic trichloronate dissolved in heptane were
programmed. Once collected, the fractions containing each of the en-
2.4.6. Stability
antiomers were concentrated on the rotary evaporator (BÜCHI R-200)
The same standard mixtures of racemic trichloronate standard
in order to remove the heptane. The new desired concentration for each
above were analyzed by three analysts in three different work days. To
enantiomer was made by adding anhydrous acetone, which were
determine the effect of multiple freezing-thawing cycles in enantiomers
checked by injecting again into the HPLC to determine their purity and
instability, three samples of turkey serum were thawed, analyzed and
true concentration through a calibration curve of standard racemic
frozen for three different days. Others aliquots of the same sample was
trichloronate. The (+)-trichloronate was obtained with a purity of 97%
stored at 4 °C all the time and analyzed in the same way for comparison.
and its corresponding (−)-form was with 93% (Fig. 3). These en-
antiomer solutions were employed for the enzymatic hydrolysis assays
2.4.7. Exactness
by UV/Vis spectrophotometry.
Exactness is verified by means of the recovery test. The relative
recoveries (n = 3) and intra-day precision (n = 3) were obtained
2.3. Stereoselective hydrolysis of trichloronate by chiral chromatography through the addition of the mixture of enantiomers to the serum sam-
ples at three different concentrations (100, 200 and 400 μM for the
The hydrolysis assays of chiral trichloronate by HPLC consisted of trichloronate isomers).
the incubation of turkey serum (10 μL) with an aliquot (0.5 mL) of
400 μM racemic trichloronate (~200 μM of each enantiomer) in the 2.4.8. Precision
presence of 300 μM copper(II) sulphate, at pH = 7,4 and 37 °C for The inter-day precision (n = 9) was obtained in three different
60 min. The reaction was stopped by adding 20 μL of 0.2 M HCl. The concentrations (200, 400 y 800 μM, for the racemate mixture tri-
remaining released trichloronate was removed by liquid-liquid extrac- chloronate, equivalent to 100, 200 and 400 of each enantiomer) with
tion with 2 mL of heptane and shaking for 5 min in an analog Multi-tube three analysts in three different work days (Table 5).
Vortexer (Thomas Scientific). Afterwards, this reaction volume was
centrifuged at 1000g for 15 min. 20 μL of the organic solvent (heptane) 2.5. Hydrolysis of trichloronate by UV/Vis spectrophotometry measuring
were injected into the chiral chromatographic system employing the TCP release
conditions of stationary and mobile phase developed by Ellington and
coworkers [1]. Therefore, was assigned the first peak of the chroma- A new colorimetric method was developed for the measurement of
togram to (+)-trichloronate and the second to (−)-trichloronate. The TCP released (leaving group in the enzymatic reaction) based on the
Cu2+-dependent hydrolysis was expressed as the μM residual con- formation of its complex with 4-aminoantipyrine and potassium ferro-
centrations (not hydrolyzed) of each enantiomer using a calibration cyanide (K3[Fe(CN)6]). The hydrolyzing activity was assayed in 10 μL
curve obtained with 50, 100, 200, 400, 600 and 800 μM of standards of of turkey serum with 320 μM of substrate (trichloronate racemic mix-
racemic trichloronate. The data were checked for their spontaneous ture or (+)-trichloronate or (−)-trichloronate enantiomer) in the pre-
hydrolysis that were in the range of 3–5% for 60 min. In all cases, the sence of 300 μM of copper(II) sulphate for 60 min at pH 7.4 and 37 °C.
yield of the extraction was higher than 95%. The assays were done by The final volume of the reaction was 500 μL. The enzymatic reaction
triplicated. was stopped by adding and mixing 375 μL of sodium dodecyl sulfate/4-

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D. Almenares-López et al. Journal of Chromatography B 1105 (2019) 203–209

1
(+)-Trichloronate 2
a (–)-Trichloronate

Fig. 3. Separation and obtaining of trichloronate enantiomers by chromatography. One hundred fifty microliters of 400 μM of racemic trichloronate (a) were injected
in to the high performance liquid chromatography system equipped with a chiral column and a fraction collector. Obtaining enantiomer (+)-trichloronate (peak 1)
(b) and (−)-trichloronate (peak 2) (c) with a purity of 97% and 93%, respectively.

aminoantipyrine (2%/1.23 mM). Then, 375 μL of potassium hex- 2.7. Quality control
acyanoferrate III (0.4%) was added to produce the color complex with
TCP-antipyrine by the reduction of the complex AAP-TCP [19]. It was The performance of the chiral chromatography system, including
quantified at 510 nm in a UV/Vis spectrophotometer (Perkín Elmer, the racemic trichloronate extraction from turkey serum, and the mea-
Lambda 25) [14,17]. For the quantification of the hydrolysis, a cali- surement of the micromolar concentrations of (+)-trichloronate and
bration curve was employed using concentrations of 20, 40, 60, 80 y (−)-trichloronate were monitored by using control charts [20]. The
100 μM of the TCP standard. The absorbance values were checked for quality control was performed using internal standards of 0 (blank) and
the spontaneous hydrolysis of the reactants. Finally, the hydrolysis 400 μM of racemic tricloronate (~200 μM for each isomer) with 99%
values were expressed in μM concentration of TCP formed for 60 min. purity in 1 mL of TRIS (10 mM) containing 10 μL of turkey serum pull
This spontaneous hydrolysis was in the range of 3–5%. without copper sulphate. Those internal standards were analyzed by
triplicate at the the start of each batch of samples. The average ± SD of
2.6. Precautions residual concentration (μM) analyzed from the internal standard for
15 days allowed the elaboration of quality control charts during the
Due to trichloronate being a toxic organophosphorus compound period in which the study was conducted.
(inhibitor of cholinesterases and inductor of delayed neuropathy in
humans) throughout this study, this compound was handled wearing 2.8. Statistical analysis
double gloves (nitrile), laboratory coat, safety glasses and mouth pro-
tection. The trichloronate solutions were prepared inside a fume hood The remaining trichloronate enantiomers values of the experimental
in the laboratory. The materials employed in the preparation of the group (turkey serum plus copper) versus control group (turkey serum
solutions, test tubes and residues of the assayed reactions were treated without copper) were analyzed by the one-way ANOVA statistical test.
with basic solution (2 M sodium hydroxide) and rinsed with distilled For comparison of the hydrolysis of (+)-trichloronate versus (−)-tri-
water. chloronate of the experimental group a t-Student test was applied.

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D. Almenares-López et al. Journal of Chromatography B 1105 (2019) 203–209

Table 1
The resolution (Rs) and separation factor (a) of trichloronate on the CHIRALCEL OD column. Heptane mobile phase, UV–Vis detector, 235 nm, with a flow velocity of
1 mL/min, injection volume 20 μL of 400 μM of racemic trichloronate and analysis time of 15 min. Each sample was analyzed in triplicate and injected twice into the
chromatography system.
Retention time (min) Peak width (min) Capacity factor c

T0 T+ T− w+ w− k′+ k′− α Rs

3.354 7.258 8.080 0.75 0.65 1.164 1.41 1.2 1.15

T0: retention time of solvent; “+” and “_” indicate the (+) and (_) enantiomers; k0þ = (T+ _ T0)/T0; k0 _ = (T_ _ T0)/T0; a = k0 _ = k0 þ; Rs = 2(k0 _ k0þ)/(w_ + w
+).

p < 0.05 was used as a value of statistical significance. These analyzes


were carried out with the SPSS v.19 software and the graphics were
designed with the Sigma Plot v.12 software.

3. Results

3.1. Hydrolysis of trichloronate by turkey serum

In order to characterize the hydrolysis of the racemic mixture of


trichloronate, a separation method of the enantiomers of trichloronate
by HPLC was developed. The analytical results were consistent with the
ones reported by Ellington et al. [1] as far as the retention time and
resolution are concerned. The (+)-trichloronate enantiomer eluted first
with a retention time of 8.49 min; the second peak was assigned to
(−)-trichloronate at 9.92 min and the resolution (Rs) was 1.15
(Table 1). The present chiral chromatographic method has been vali-
dated by the hydrolysis of trichloronate in turkey serum. The analytical
parameters did not show interferences due to the biological matrix in
Fig. 4. Cu2+-dependent stereoselective hydrolysis of trichloronate in turkey
the quantification of the residual concentrations of each of the two serum by chromatography chiral. Each bar (A) shows the average and its
enantiomers. The calibration curves (50–800 μM) showed an optimal standard deviation of three experiment. Ten microliters of turkey serum were
linear behavior for each of the enantiomers (r > 0.99), as well as ac- incubated with 400 μM of racemic trichloronate (~200 μM for each isomer; (+)
ceptable values of precision (2–7%), intermediate precision and re- and (−)(B) in the absence of copper (1) or 300 μM copper (2), for 60 min at
producibility (5–10%). The detection and quantification limits were 0.6 pH 7.4 and 37 °C. *1 vs 2, p ˂ 0.05 (one-way ANOVA). **(−)-trichloronate vs
and 2 μM for (+)-trichloronate, and 0.7 and 2.3 μM for (−)-tri- (+)-trichloronate, p ˂ 0.05 (t-Student).
chloronate. On the other hand, the percent recovery in the liquid-liquid
extraction was around 98% using HPLC grade heptane. The hydrolysis Table 2
was determined in an indirect manner through the residual con- Validation parameters for the identification and quantification of TCP
centration (μM) of each enantiomer (not hydrolyzed) using the values by UV/Vis spectrophotometry.
of the chromatographic area (Table 4).
Detection limit 17.00 μM
Limit of quantification 29.29 μM
3.2. Stereoselective hydrolysis of turkey serum analyzed by chiral Accuracy (coefficient of variation) 4–9%
chromatography Accuracy (relative error%) 2.9%
Recovery (%) 98%
Linearity (r2) > 0.99
Our results show a Cu2+-dependent stereoselective hydrolysis, be-
cause the (+)-trichloronate enantiomer showed a residual concentra-
tion of [136 μM]. In hydrolysis term, this enantiomer was hydrolyzed parameters of analytical validation (Table 2). The hydrolysis values
by 32% [64 μM], while the (−)-trichloronate was hydrolyzed by 65% obtained for each of the enantiomers through the quantification of TCP
[70 μM residual], using 10 μL of turkey serum at 60 min of reaction time show a stereoselective hydrolysis similar to that obtained by chiral
(Fig. 4). These hydrolysis values are significantly different (p < 0.05, chromatography. The (−)-trichloronate was significantly (p < 0.05)
one-way ANOVA) when the comparison was made between groups hydrolyzed in higher proportion (47%) than the (+)-trichloronate
(control group vs experimental group). The comparison of the hydro- which was hydrolyzed by 31% (Table 3). The racemic mixture showed
lysis between both enantiomers of trichloronate in the experimental an intermediate hydrolysis value of 35%.
group (turkey serum + copper) is also statistically different (p < 0.05,
t-Student).
4. Discussion
3.3. Stereoselective hydrolysis analyzed by UV–Vis spectrophotometry with
isolated enantiomers Our results show a stereoselective Cu2+-dependent hydrolysis of
(−)-trichloronate by turkey serum employing chiral HPLC analysis of
In order to verify the stereoselective hydrolysis of racemic tri- the enantiomers and the subsequent confirmation by colorimetric
chloronate observed by analysis with chiral HPLC, assays were con- analysis of the phenol product testing with pure isolated enantiomers.
ducted with the purified enantiomers, for which pure enantiomers were In particular, the chromatographic resolution parameters (1.15) and the
isolated by preparative HPLC (Fig. 3). The 320 μM solution of (+)-tri- coefficient of selectivity (1.2) were similar to those reported by
chloronate or 320 μM of (−)-trichloronate in dry acetone were pre- Ellington [1], for the separation of the enantiomers of trichloronate (1.3
pared. The present colorimetric method complied with the optimum and 1.1 respectively). Because, in this study were used the same

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D. Almenares-López et al. Journal of Chromatography B 1105 (2019) 203–209

Table 3 the hydrolysis of this racemic OP by measuring the leaving group TCP.
Copper-dependent hydrolysis of trichloronate enantiomers by Others toxicological studies have reported the usefulness of this col-
UV/Vis spectrophotometry. The μM values of TCP represent the orimetric method of quantification of phenols using 4-aminoantipyrine
triplicate of the hydrolysis of 10 μL of turkey serum incubated and potassium ferricyanide for the evaluation hydrolysis of chiral
with 320 μM of racemic trichloronate or of each of its en-
phosphoramidates, which are neuropathic because of their interactions
antiomers in presence of 300 μM of Cu2+, at pH 7.4, 37 °C and
with esterases [12,24]. The present colorimetric method may be uti-
60 min. Asterisk indicates statistical difference between the en-
lized for the study of the interaction with A and B esterases in biological
antiomers (p < 0.05, t-Student test).
tissues of other compounds whose leaving group is TCP. The chromo-
Chemical compound [μM] TCP phore TCP-AAP‑potassium ferricyanide of the present method is sup-
Racemic trichloronate 120 ± 5 (35%)
ported by the high affinity of the AAP towards the OH group of the
(+)-trichloronate 100 ± 4 (31%)⁎ phenol, and to the subsequent formation of a colored compound made
(−)-trichloronate 148 ± 5 (47%)⁎ up by aminoantipyrine and potassium ferricyanide in alkaline condi-
tions (pH 8). This reaction had been suggested by Emerson et al. [19]
for substituents such as carboxyl, halogen, methoxy and sulphonic acid
Table 4 at the para position in phenols. The results obtained in the Cu2+-de-
Parameters of validation of chiral chromatography method. pendent hydrolysis of trichloronate identified in this work, are relevant
Enantiomer (analyte) Linear equation r LOD (μM) LOQ (μM) in the field of the delayed neurotoxicity of OPIDP due to trichloronate
which is a chiral OP insecticide neuropathic in hens [9].
(+)-trichloronate Y = 5060× + 187,949 0.995 0.6 2.0
The stereoselective metabolism of (−)-trichloronate is also relevant
(−)-trichloronate Y = 5120× + 188,712 0.993 0.7 2.3
due to the fact that this enantiomer is 8–11 times more toxic as compared
to (+)-trichloronate [7] in toxicity studies with Ceriodaphnia dubia y
Table 5 Daphnia magna. This Cu2+-dependent stereoselective hydrolysis of OPs in
Liquid-liquid extraction of racemic trichloronate. turkey serum is the first report of a hydrolysis of an OP racemic insecticide
in its thio form by an A-esterase Cu2+-dependent from the serum of a
Enantiomers [μM] Recovery (%) Precision (RSD %)
vertebrate. These results suggest the convenient of the identification and
(n = 3)
Intra-day Inter-day production of the responsible protein in turkey serum in order to carry out
(n = 3) (n = 9) stereoselective hydrolysis studies of OPs in the biomedical field of ex-
position to insecticides and nerve agents [25,26], as well as in the en-
(+)-trichloronate 400 98 ± 1 1.54 5.4
vironmental remediation of OPs. On the other hand, our results of the
200 94 ± 1 0.31 3.4
100 100 ± 1 0.85 0.85 Cu2+-dependent stereoselective hydrolysis of trichloronate incubating
(−)-trichloronate 400 100 ± 2 1.35 5.1 400 μM for 60 min with turkey serum coincide with those reported for
200 100 ± 1 0.30 5.7 HDCP for 30 min in chicken serum in the presence of 100–300 μM de
100 95 ± 2 1.33 2.37 Cu2+. In that case, the R-(+)-HDCP is hydrolyzed [18], which is the en-
antiomer with the greater inhibiting power and aging capacity of NTE. On
n = 3. “ ± ” indicate the standard error of the mean.
the contrary, PON1 of human serum [6] and other PTEs of bacterial origin
[27], hydrolyze the less toxic isomer of chiral OPs. Therefore, is of special
conditions of chiral column (Chiralcel OD), mobile phase (Heptane interest the reported hydrolyzing stereoselectivity of the enantiomer
grade HPLC), chromatographic flow (1 m/min) and detector wave- (−)-trichloronate which is more toxic.
length (235 nm). However, our resolution parameter obtained between This study reinforces the findings reported on the Cu2+-dependent
both isomers of trichloronate was lower than that obtained by Liu and A-esterase activity in chicken serum known as “antagonistic stereo-
coworkers [7], which was 4.03 using Chiralcel OJ chiral column and n- selectivity” [18] associated to albumin protein [28], thus suggesting
hexane/n-heptane/EtOH (90/5/5, v/v/v) as mobile phase. In this that birds could present a greater protection against this chiral OPs
study, the same stereochemical configuration for the enantiomers has because of their hydrolyzing stereoselectivity of the enantiomers of
been assigned; (+)-trichloronate is the isomer that elutes first in the higher toxicity in phosphorothioates and phosphoramidates such as
chromatographic run. On the other hand, The development of the trichloronate and HDCP respectively. This study also reinforces that
present chiral chromatographic method shows a QuEChERS extraction reported by Landis and Shough [29], who described the presence of non
technique, because the trichloronate racemic extraction from the vo- Ca2+-dependent A-esterases or PTEs in avian kidney and liver, since
lume reaction was done with heptane grade HPLC, without the use of those tissues in the presence of manganese hydrolyze the nerve agent
salts (NaCl or standard MgSO4) nor clean-up by dispersive solid phase diisopropylfluorophosphate (DFP). In this study is reported the hydro-
extraction (SPE) using primary and secondary amine bonded silica lysis of trichloronate by turkey serum in the presence of Cu2+ sug-
(PSA) as has been used by determination of pesticides including orga- gesting that the birds present A-esterase activities metal-dependent
nophosphorus insecticides in environmental and biological matrices different to those reported in mammals, such as PON1 which is a Ca2+-
[21,22]. This QuEChERS liquid-liquid extraction strategy has been used dependent A-esterase as well as other typical metal-dependent PTEs of
by our research group in the chiral HPLC method by determination of bacterials.
HDCP hydrolysis for avian and mammal tissues [14] including human In conclusion, this study reports for the first time the development
serum [6] and chicken serum [18]. The present chiral HPLC method of a method of chiral chromatography complemented with other of UV/
complied with a quality control procedure and with the parameters of Vis spectrophotometry for the ex vivo study of the stereoselective hy-
linearity, repeatability and accuracy by means of the recovery test drolysis of the insecticide trichloronate by animal serum. Both analy-
(International Conference on Harmonization, 1995) [15]. Some its tical methods have the advantage of presenting an initial QuEChERS
parameters as linearity, recovery and detection limit were similar to the technique, since the chiral HPLC method includes a direct liquid-liquid
HPLC methods reported in the ex vivo hydrolysis of others chiral OPs as extraction of the enantiomers, while for the colorimetric it is not re-
HDCP in hen plasma [23] and fenamiphos in human serum [4] and liver quired. However, the chromatographic has the disadvantage of using
microsomes [22]. organic solvent (2 mL), while the colorimetric requires quantities of
With respect to the colorimetric method with 4-aminoantipyrine enantiomers with high purity (95%). Their validation evidenced the ex
and potassium ferricyanide, for the quantification of the hydrolysis of vivo stereoselective Cu2+-dependent hydrolysis of trichloronate (a
trichloronate it is worth noting that it is the first study that quantifies chiral OP insecticide in its thio form) by the turkey serum.

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D. Almenares-López et al. Journal of Chromatography B 1105 (2019) 203–209

Acknowledgments (2018) E2582, , https://doi.org/10.3390/molecules23102582.


[14] A. Monroy-Noyola, M.A. Sogorb, E. Vilanova, Stereospecific hydrolysis of a phos-
phoramidate as a model to understand the role of biotransformation in the neuro-
This project has been supported by CONACYT 257092. Almenares- toxicity of chiral organophosphorus compounds, Toxicol. Lett. 170 (2007) 157–164,
López had received financial support of PRODEP UPC-PTC-032 for the https://doi.org/10.1016/j.toxlet.2007.03.002.
elaboration of this manuscript. The assistance of M.S. Juan Antonio [15] International Conference on Harmonisation, Note for Guidance on Validation of
Analytical Methods: Methodology, International Conference on Harmonisation,
Regla in the translation of the present manuscript is greatly appre- November 29 1995.
ciated. [16] Daniel MacDougall, Warren B. Crummett, et al., Guidelines for data acquisition and
data quality evaluation in environmental chemistry, Anal. Chem. 52 (1980)
2242–2249.
References [17] A. Monroy-Noyola, M.A. Sogorb, E. Vilanova, Enzyme concentration as an im-
portant factor in the in vitro testing of the stereospecificity of the enzymatic hy-
[1] J.J. Ellington, J.J. Evans, K.B. Prickett, W.L. Champion Jr., High performance liquid drolysis of organophosphorus compounds, Toxicol. in Vitro 13 (1999) 689–692.
chromatographic separation of the enantiómeros of organophosphorus pesticides on [18] A. Monroy-Noyola, M.A. Sogorb, N. Díaz-Alejo, E. Vilanova, Copper activation of
polysaccharide chiral stationary phases, J. Chromatogr. A 928 (2001) 145–154, organophosporus compounds detoxication by chicken serum, Food Chem. Toxicol.
https://doi.org/10.1016/S0021-9673 (01)01138-4. 106 (2017) 417–423, https://doi.org/10.1016/j.fct.2017.05.055.
[2] M.G. Nillos, J. Gan, D. Schlenk, Chirality of organophosphorus pesticides: analysis [19] E. Emerson, The condensation of aminoantipyrine. II. A new color test for phenolic
and toxicity, J. Chromatogr. B 878 (2010) 1277–1284, https://doi.org/10.1016/j. compounds, J. Org. Chem. 8 (1943) 417–428.
jchromb.2009.11.022. [20] P. Konieczka, J. Namieśnik, Estimating uncertainty in analytical procedures based
[3] J.M. Battershill, P.M. Edwards, M.K. Johnson, Toxicological assessment of isomeric on chromatographic techniques, J. Chromatogr. A 1217 (2010) 882–891, https://
pesticides: a strategy for testing of chiral organophosphorus (OP) compounds for doi.org/10.1016/j.chroma.2009.03.078 (Epub 2009 Apr 1).
delayed polyneuropathy in a regulatory setting, Food Chem. Toxicol. 42 (2004) [21] J.F. Garcia-Reyes, C. Ferrer, J.M. Gómez-Ramos, A. Molina-Diaz, A.R. Fernández-
1279–1285, https://doi.org/10.1016/j.fct.2004.03.004. Alba, Comprehensive screening of target, non-target and unknown pesticides in
[4] D. Almenares-López, M.F. Martínez-Salazar, M.L. Ortiz-Hernández, R. Vazquez- food by LC-TOF-MS, TrAC Trends Anal. Chem. 26 (2007) 828–841, https://doi.org/
Duhalt, A. Monroy-Noyola, Fenamiphos is recalcitrant to the hydrolysis by allo- 10.1016/j.trac.2007.06.006.
forms PON1 Q192R of human serum, Toxicol. in Vitro 27 (2013) 681–685, https:// [22] N.C.P. de Albuquerque, J.V. de Matos, A.R.M. de Oliveira, In-line coupling of an
doi.org/10.1016/j.tiv.2012.11.014. achiral-chiral column to investigate the enantioselective in vitro metabolism of the
[5] M. Valiyaveettil, Y.A. Alamneh, P.B. Doctor, Crossroads in the evaluation of para- pesticide Fenamiphos by human liver microsomes, J. Chromatogr. A 1467 (2016)
oxonase 1 for protection against nerve agent and organophosphate toxicity, Toxicol. 326–334, https://doi.org/10.1016/j.chroma.2016.08.039.
Lett. 210 (2012) 87–94, https://doi.org/10.1016/j.toxlet.2012.01.013. [23] N. Diaz-Alejo, E. Vilanova, Chiral high-performance chromatography and gas
[6] A. Monroy-Noyola, B. Trujillo, P. Yescas, F. Martínez-Salazar, S. García-Jiménez, chromatography of the isomers of O-hexyl, O-2,5-dichlorophenyl phosphoramidate,
C. Ríos, et al., Stereospecific hydrolysis of a phosphoramidate used as an OPIDP J. Chromatogr. 622 (1993) 179–186, https://doi.org/10.1016/0378-4347 (93)
model by human sera with PON1 192 alloforms, Arch. Toxicol. 89 (2015) 80264-5.
1801–1809, https://doi.org/10.1007/s00204-014-1327-2. [24] N. Diaz-Alejo, P.C. Pellin, J.L. Vicedo, E. Vilanova, Hen liver and plasma can me-
[7] W. Liu, L. Kunde, G. Jianying, Separation and aquatic toxicity of enantiomers of the tabolize hexyl-DCP phosphoramidate at a rate comparable to that of rat,
organophosphorus insecticide trichloronate, Chirality 18 (2006) 713–716, https:// Neurotoxicol. Teratol. 12 (1990) 615–617, https://doi.org/10.1016/0892-
doi.org/10.1016/j.chemosphere.2010.08.036. 0362(90)90072-K.
[8] A.H. El-Sebae, S.A. Soliman, N.S. Ahmed, A. Curley, Biochemical interaction of six [25] O. Khersonsky, D. Tawfik, Structure-reactivity of serum paraoxonasa PON 1 suggest
op delayed neurotoxicants with several neurotargets, J. Environ. Sci. Health B 16 that its natine activity is lactonase, Biochemistry 44 (2005) 6371–6382, https://doi.
(1981) 465–474, https://doi.org/10.1080/10934528109374998. org/10.1021/bi047440d.
[9] M.K. Johnson, The target for initiation of delayed nerotoxicity by organopho- [26] G. Amitai, L. Gaidukov, R. Adani, S. Yishay, G. Yacov, M. Kushnir, et al., Enhanced
sphorus esters: biochemical studies and toxicological application, Rev. Biochem. stereoselective hydrolysis of toxic organophosphates by directly evolved variants of
Toxicol. 4 (1982) 141–212. mammalian serum paraoxonase, FEBS J. 273 (2006) 1906–1919, https://doi.org/
[10] M.K. Johnson, P. Glynn, Neuropathy target esterase, in: R. Kreiger (Ed.), Handbook 10.1111/j.1742-4658.2006.05198.x.
of Pesticide Toxicology (Volume 2: Agents), Academic Press, 2001, pp. 953–965. [27] S.B. Hong, F.M. Raushel, Stereochemical preferences for chiral substrates by the
[11] H. Jedrzejowska, K. Rowińska-Marcińska, B. Hoppe, Neuropathy due to phytosol bacterial phosphotriesterase, Chem. Biol. Interact. 119-120 (1999) 225–234,
(Agritox). Report of a case, Article in, Acta Neuropathol. 49 (2) (1980) 163–168, https://doi.org/10.1016/S0009-2797(99)00031-9.
https://doi.org/10.1007/BF00690757. [28] A. Monroy-Noyola, M.A. Sogorb, E. Vilanova, Albumin, the responsible protein of
[12] E. Vilanova, M.K. Johnson, J.L. Vicedo, Interaction of some unsustituted phos- the Cu2+-dependent hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate
phoramidate analogs of methamidophos (O,S dimethyl phosphorothioamidate) (HDCP) by chicken serum “antagonistic stereoselectivity”, Food Chem. Toxicol. 120
with acetylcholinesterase and neuropathy target esterase of hen brain, Pestic. (2018) 523–527, https://doi.org/10.1016/j.fct.2018.07.047.
Biochem. Physiol. 28 (1987) 224–238, https://doi.org/10.1016/0048-3575(87) [29] W.G. Landis, N.J. Shough, Discovery initial characterization and comparison of the
90021-6. organophosphate acid hydrolyzing activities of the bob white quail, stilt and mal-
[13] Ż. Bargańska, P. Konieczka, J. Namieśnik, Comparison of two methods for the de- lard, Comp. Biochem. Physiol. C 102 (1992) 527–535.
termination of selected pesticides in honey and honeybee samples, Molecules 23

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