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Experimental central composite design-based dispersive liquid-liquid microextraction for HPLC-DAD determination of diazinon in human urine samples: method development and validation

Reza Mohammadzaheri
Reza Mohammadzaheri
, 
Mehdi Ansari Dogaheh
Mehdi Ansari Dogaheh
, 
Maryam Kazemipour
Maryam Kazemipour
 and 
Kambiz Soltaninejad
Kambiz Soltaninejad
   | Apr 09, 2020
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Article Category: Original article
Published Online: Apr 09, 2020
Page range: 48 - 55
Received: May 01, 2019
Accepted: Feb 01, 2020
DOI: https://doi.org/10.2478/aiht-2020-71-3292
Keywords
© 2020 Reza Mohammadzaheri, Mehdi Ansari Dogaheh, Maryam Kazemipour, Kambiz Soltaninejad, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1

Probability plot of the effects
Probability plot of the effects

Figure 2

Surface plots showing the effects of variables with the highest impact on the recovery of the method (A) The effect of the volume of toluene and methanol; (B) the effect of the volume of methanol and the sodium lauryl sulphate (SLS) concentration; (C) the effect of methanol volume and the sodium chloride (NaCl) concentration
Surface plots showing the effects of variables with the highest impact on the recovery of the method (A) The effect of the volume of toluene and methanol; (B) the effect of the volume of methanol and the sodium lauryl sulphate (SLS) concentration; (C) the effect of methanol volume and the sodium chloride (NaCl) concentration

Figure 3

Specificity of the proposed method for the analysis of diazinon in urine sample Chromatogram A: blank urine; Chromatogram B: urine spiked with: 1 – tramadol, 2 – azinphos-ethyl, 3 – diazinon, 4 – pirimiphosmethyl, and 5 – chlorpyrifos
Specificity of the proposed method for the analysis of diazinon in urine sample Chromatogram A: blank urine; Chromatogram B: urine spiked with: 1 – tramadol, 2 – azinphos-ethyl, 3 – diazinon, 4 – pirimiphosmethyl, and 5 – chlorpyrifos

Variables and their levels for experimental design

Symbol Level 3 Level 2 Level 1 Factor
A methanol acetonitril type of disperser solvent
B 10 0 sonication duration (minute)
C dichloromethane chloroform toluene type of extraction solvent
D 600 300 100 volume of extraction solvent (μL)
E 1000 500 0 volume of disperser solvent (μL)
F 5 3 1 surfactant concentration (% w/v)
G 5 3 1 salt concentration (% w/v)
H 10 7 4 pH

Experimental ranges and levels of independent variables for the central composite design

α- 1- 0 1+ α+
Methanol μL A 200 400 600 800 1000
NaCl % B 0 1 2 3 4
SLS % C 0 1 2 3 4
Toluene μL D 225 300 375 450 525

Experimental conditions according to the central composite design and observed response values

Experiment No. Methanol volume (μL) NaCl conc. (%w/v) SLS conc. (%w/v) Toluene volume (μL) Actual recovery Predicted recovery
1 800 3.00 3.00 300.00 43.70 40.72
2 800 3.00 1.00 300.00 20.10 21.55
3 800 1.00 3.00 450.00 82.00 80.49
4 400 3.00 1.00 450.00 69.27 68.99
5 800 1.00 1.00 450.00 43.52 43.50
6 400 1.00 3.00 300.00 46.3 43.06
7 400 3.00 3.00 450.00 63.00 61.23
8 400 1.00 1.00 300.00 31.80 32.99
9 200 2.00 2.00 375.00 63.00 64.14
10 1000 2.00 2.00 375.00 53.50 54.13
11 600 0.00 2.00 375.00 67.50 67.14
12 600 4.00 2.00 375.00 29.00 28.64
13 600 2.00 0.00 375.00 18.02 15.94
14 600 2.00 4.00 375.00 41.33 45.18
15 600 2.00 2.00 225.00 42.00 42.88
16 600 2.00 2.00 525.00 73.60 74.48
17 600 2.00 2.00 375.00 48.00 47.89
18 600 2.00 2.00 375.00 46.00 47.89
19 600 2.00 2.00 375.00 49.00 47.89
20 600 2.00 2.00 375.00 50.00 47.89
21 600 2.00 2.00 375.00 44.00 47.89

Analysis of variance for the proposed model

Source Sum of Squares df Mean square F value p-value

all p-values are statistically significant

 Prob.> F
Model 6195.89 14 442.56 41.69 0.0053
A 2223.78 1 2223.77 206.49 0.0007
B 72.231 1 72.31 6.81 0.0797
C 726.08 2 363.04 34.2 0.0086
D 862 2 431 40.6 0.0067
E 229.05 2 114.52 10.79 0.0426
F 1029.91 2 514.95 48.51 0.0052
G 915.54 2 457.77 43.12 0.0062
H 137.22 2 68.61 6.46 0.0817
Residual 31.85 3 10.62
Correction Total 6227.774 17

Analysis of variance for central composite design

Source Sum of squares df Mean square F value p-value

all p-values are statistically significant

 Prob. > F
Model 840.2 11 76.37 63.10 <0.0001
A 16.28 1 16.28 13.45 0.0052
B 112.89 1 112.89 93.28 <0.0001
C 142.35 1 142.35 117.63 <0.0001
D 70.52 1 70.52 58.27 <0.0001
AB 27.88 1 27.88 23.04 0.0010
AC 53.21 1 53.21 43.97 <0.0001
AD 44.91 1 44.91 37.11 0.0002
BC 19.36 1 19.36 16.00 0.0031
A^2 31.00 1 31.00 25.62 0.0007
C^2 84.41 1 84.41 69.75 <0.0001
D^2 26.72 1 26.72 22.08 0.0011
Residual 10.89 9 1.21
Lack of Fit 7.37 5 1.47 1.67 0.3196
Pure Error 3.53 4 0.88
Cor Total 850.91 20

Comparison of the proposed DLLME-HPLC-DAD with other analytical methods for determination of diazinon in biological samples

Method Matrix LOD (μg/mL) Correlation coefficient (R2) Recovery (%) Ref. No.
SPE-GC-MS whole blood 0.15 0.9981 78–87 15
SPE-HPLC-DAD plasma 0.15 0.998 77.7–86.3 17
LLE-HPLC-DAD whole blood, serum, urine 0.78 0.9996 97.4–99.01 (for blood and serum)101.1–101.4 (for urine) 16
mini-QuEChERS-LC-MS-MS whole blood, gastric content 0.1 0.95 80–100 18
MEPS-GC-MS-MS whole blood 0.5 0.99 61–77 26
DBS-GC-MS-MS whole blood 0.05 0.998 4.56–5.11 27
DLLME-HPLC-DAD urine 0.15 0.993 75.0–95.6 this study

Method precision and accuracy (intra-day: n=5; inter-day: n=5 series per day, 3 days).

Diazinon concentration (μg/mL) Intra-day (n=5)
 Inter-day (n=5)
Mean±SD CV (%) Recovery±SD (%) Mean±SD CV (%) Recovery±SD (%)
0.5 0.46±0.04 7.4 92.1±1.0 0.48±0.03 6.9 95.6±1.0
1 0.76±0.02 3.3 76.0±2.0 0.75±0.01 1.2 75.0±1.1
3 2.33±0.09 4.0 77.4±0.9 2.31±0.11 4.9 77.1±1.4
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