Beruflich Dokumente
Kultur Dokumente
Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb
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
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
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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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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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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
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
+).
3. Results
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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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