Vol.:(0123456789)1 3

Journal of the Iranian Chemical Society (2019) 16:1131–1138 https://doi.org/10.1007/s13738-018-01588-w

ORIGINAL PAPER

Application of a new sample preparation method based on surfactant-assisted dispersive micro solid phase extraction coupled with ultrasonic power for easy and fast simultaneous preconcentration of toluene and xylene biomarkers from human urine samples

Fariborz Omidi1 · Mohammad Behbahani2 · Monireh Khadem3 · Farideh Golbabaei3 · Seyed Jamaleddin Shahtaheri3,4

Received: 30 June 2018 / Accepted: 31 December 2018 / Published online: 5 April 2019 © Iranian Chemical Society 2019

AbstractA fast and easy surfactant-assisted dispersive micro-solid phase extraction method coupled with ultrasonic power was used for the simultaneous preconcentration and determination of low levels of hippuric and methyl hippuric acid in human urine samples. In the first step, magnetic nanoparticles was coated by titanium oxide nanoparticles (Fe3O4/TiO2 NPs), afterward characterized by the scanning electron microscopy, energy-dispersive X-ray spectroscopy, and Fourier-transform infrared spectroscopy. In the next step, anionic surfactants were thus combined with the synthesized magnetic nanoparticles to create a new adsorbent for increasing the simultaneous extraction of hippuric and methyl hippuric acid. After elution of extracted target molecules by the sorbent, the concentration of these molecules were measured by high-performance liquid chromatography (HPLC–UV). Meanwhile, a statistical approach known as Box–Behnken design was applied for optimiz-ing significant parameters. With the optimum parameters anticipated by the experimental design, the limit of quantification (LOQ) acquired was reported to be 3 µg L− 1, and the calibration curve was linear within the concentrations of 3–1000 µg L− 1. Finally, the method was effectively implemented for the determination of low levels of hippuric and methyl hippuric acid in human urine samples.

Keywords Hippuric and methyl hippuric acid · Surfactant-assisted dispersive micro solid phase extraction · Box–Behnken design · Human urine samples · High-performance liquid chromatography

Introduction

Aromatic hydrocarbons such as toluene and xylene are fre-quently utilized in industries for various applications includ-ing industrial paints, glues and adhesives, printing inks and cleaning agents [1]. Exposure to these compounds happens not only in the public community via trace residues in tap water, air pollution, and cosmetics but also in people work-ing in the petrochemical and other related industries [2]. The central nervous system is the major target organ for toluene and xylene. Long-term exposure to toluene cause dizziness, and visual disturbance and also affects the reproductive sys-tem of women. Chronic exposure to xylene may affect some organs such as the digestive system, liver, kidney, and lungs [3, 4]. Due to the biotransformation process, these chemi-cals are oxidized, then conjugated with glycine and changed to hippuric acid (HA) and methyl hippuric acid (MHA) and

Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1373 8-018-01588 -w) contains supplementary material, which is available to authorized users.

* Seyed Jamaleddin Shahtaheri shahtaheri@tums.ac.ir

1 Research Center for Environmental Determinants of Health (RCEDH), Kermanshah University of Medical Sciences, Kermanshah, Iran

2 Faculty of Engineering, Shohadaye Hoveizeh University of Technology, Dasht-e Azadegan, Susangerd, Iran

3 Department of Occupational Health Engineering, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

4 Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran

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finally exerted by urine [5]. Quantitative evaluation of HA and MHA in the urine samples is the best way for assessment of simultaneous exposure to toluene and xylene, which indicate a good correlation between the concentration levels of these compounds in the air and the quantity of metabolites excreted [6]. Taking into consideration all the above-mentioned, deter-mination of the HA and MHA, the biomarkers of toluene and xylene, in urine samples is of great importance. In the litera-ture, several methods such as gas chromatography equipped to flame ionization detector (GC–FID) [7], gas chromatog-raphy–mass spectrometry (GC–MS) [8], spectrophotometry [9], high-performance liquid chromatography–ultraviolet (HPLC–UV) [10], liquid chromatography–tandem mass spec-trometry (LC–MS/MS) [11] have been applied for measure-ment of HA and MHA. Among the above-mentioned meth-ods, HPLC–UV has been regarded as an acceptable procedure for quantification of HA and MHA in urine samples by the National Institute for Occupational Safety and Health (NIOSH 8301) [12]. Despite the high selectivity and sensibility of the advanced analytical instruments, there is an essential need for sample pretreatment of targets prior to the analysis, because of the trace amount of analytes and presence of interfering agents. Moreover, the mentioned methods have disadvantages such as sophisticated sample preparation steps, laboriousness, and excessive solvent consumption. For instance, derivatiza-tion step with some chemicals, which are usually toxic, is required prior to measurement of HA and MHA based on gas chromatography [13]. Considering matrix effect and trace level of HA and MHA in human urine, an efficient sample pretreatment method with a high clean up and pre-concen-tration factor is desirable. Generally, traditional solid phase extraction (SPE) and liquid–liquid extraction (LLE) are the most frequently utilized sample pretreatment methods for determination of HA and MHA in urine specimens [14, 15]. Traditional LLE procedure is tedious and uses a large amount of organic solvents which cause environmental problems due to the generation of hazardous wastes [16]. In conventional SPE, the prepared sorbent should be packed in the SPE col-umn and then the sample is passed through the column which is a laborious and time-consuming process and also chan-neling and blocking of SPE cartridge may occur [17]. To overcome disadvantages of the conventional SPE, dispersive solid-phase extraction (d-SPE) method as a rapid and sim-ple pre-concentration procedure was introduced [18]. In this method, the sorbent nanoparticles are dispersed in the solu-tion which increases the absorption capacity of the adsorbent [18, 19]. Until now, modern sample preparation methods have been developed for trace determination of analytes [20–23]. Heretofore, new sample preparation procedures, based on magnetic nanoparticles (MNPs), have been introduced. These particles are of great interest to analysts. The outstanding dis-persibility and superparamagnetic characteristics of the MNPs help to be easily isolated and re-dispersed in the sample by

just applying an external magnetic field [24]; therefore, the extraction of analytes occur in a shorter period of time [25]. Furthermore, surface modifications of MNPs with functional groups increase the selectivity and efficiency towards targets and avoid nanoparticles aggregation [26]. Coupling of sur-factant with MNPs by the formation of mixed hemimicelles and admicelles provides a sort of mechanisms to link with targets [27]. This procedure has already been outlined for the determination of organic target with well effective results [28]. Nowadays, the pre-concentration method has been widely used for trace detection of target ions in real samples [29, 30].

In this study, first, Fe3O4@TiO2 nanoparticles were syn-thesized and characterized by instrumental techniques. The MNPs were then modified via ionic surfactants and were applied for the simultaneous extraction of HA and MHA from human urine matric. Mentioned metabolites were then captured by the surface of prepared sorbent. After the elu-tion process, the metabolites were analyzed by HPLC–UV. To the best of our knowledge, this study is novel and for the first time present the application of the noted sorbent for the simultaneous extraction of the HA and MHA in human urine samples.

Experimental section

Reagents and chemicals

The detailed information regarding reagents and chemicals is provided in the electronic supplementary materials [Elec-tronic supplementary materials (ESM)].

HPLC–UV analysis

The information about HPLC–UV analysis is provided in the electronic supplementary materials (ESM).

Preparation of  Fe3O4@TiO2 nanoparticles

The detailed information regarding this part is fully provided in the electronic supplementary materials (ESM).

Extraction process

Following multiple steps are described by the extraction pro-cedure of the analytes:

1. An optimized value (26 mg) of the Fe3O4@TiO2 nano-particles and 0.92 mmol L− 1 of Sodium dodecyl sulfate (SDS) were added to 8 mL of HA and MHA (known concentration) aqueous solution for which pH had been regulated at 1.0.

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2. To facilitate mass transfer and adsorption of the analytes onto the adsorbent, the solution was transferred to the ultrasonic bath for 3.2 min.

3. Using a magnetic field, the magnetic-based nanosorbent was rapidly separated from the mixture and deposited on the conical test tube. Then, the obtained sorbent was rinsed with 3 mL of deionized water to elute loosely connected unwanted compounds from the sorbent sur-face.

4. The target molecules were eluted from the sorbent sur-face with 1.0 mL methanol/NaOH (1%; w/v) during 1-min sonication.

5. As the final step, the eluates were evaporated under a soft flow of N2 and dissolved in 50 µL of mobile phase and eventually 20 µL of which was introduced into the HPLC loop.

Figure 1 shows the proposed extraction method of HA and MHA by the optimized dispersive micro solid phase extraction method.

Result and discussion

Characterization of the prepared magnetic based sorbent

Investigation of the morphology of Fe3O4 nanoparticles and Fe3O4 @TiO2 nanocomposite was conducted by

scanning electron microscopy. As Fig. 1 indicate, the size of synthesized Fe3O4 nanoparticles was about 25 nm. In Fig. 1, Fe3O4@TiO2 core–shell nanocomposites are also spherical and have a diameter of approximately 45 nm. To more morphological characterization of the sorbent TEM (EM 208S Philips, Netherlands) was applied and the TEM image is shown in Fig. 2. To further examine the structure of the sorbent, N2 adsorption–desorption isotherms were employed. The surface area and mean pore size of the sorbent determined by BET (BELSORP MINI II) method was about 117.25 m2 g− 1 and 11.9 nm, respectively. The

Fig. 1 The SEM micrograph for Fe3O4 and Fe3O4@TiO2 nanoparticles

Fig. 2 The TEM image for the synthesized nano-sorbent

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N2 adsorption/desorption isotherm of the synthesized magnetic nano-sorbent is presented in Fig. 3.

Figure  4 provides the FT-IR spectra pattern of Fe3O4 and Fe3O4@TiO2 nanoparticles in the range of 3000–400 cm− 1. According to Fig. 4, there was observed a broad peak in the range of 700–500 cm− 1, which is assigned to the Metal-O functional group vibration. The detected peak around 560 cm− 1 is ascribed to the oscilla-tion of the Fe–O functional group. The absorption peak of Ti–O approximately appeared in 510 cm− 1. As can be seen in the figure, the observed peak in the mentioned region is rather broader for Fe3O4@TiO2, and the observation is related to the overlapping of Ti–O and Fe–O peaks. In the FT-IR of Fe3O4@TiO2, the emerged peaks at 1120 and 1460 cm− 1 could also be attributed to the existence of stretching oscillations of Ti–O and Fe–O–Ti bonds. The H–O–H bending vibration bands at 1628 cm− 1 describing that multiple surfaces of OH groups exist in the prepared sorbent due to TiO2 coating.

Energy dispersive X-ray (EDX) spectrum was also utilized for elemental analysis of synthesized magnet-ized nanosorbent. EDX of Fe3O4@TiO2 [Fig. 1S (ESM)] revealed the existence of Fe, O, and Ti, which con-firmed the existence of TiO2 on the surface of magnetic nanoparticles.

Optimization of the sorption stage

Box–Behnken design (BBD) was employed for investigation and optimization of four effective variables including sam-ple’s pH, SDS concentration, amount of sorbent and time

of sonication on the mean extraction recovery of HA and MHA by the surfactant-assisted dispersive micro-solid phase extraction procedure. Because the number of main param-eters on the extraction of the studied molecules using the nanosorbent was minimum, BBD method was used without a screening step. Rotatable or “nearly” rotatable is a substi-tute option to fit quadratic models requiring 3 grade for each variable. The BBD is an independent quadratic design which does not comprise an embedded factorial or fractional facto-rial design [19, 31]. One variable at a time was applied for selecting of factors range levels and the selected ranges are shown in Table 1S (ESM). BBD is suitable to find quadratic response surface. The presented model for BBD is as follow:

where Y is the response parameter (area), bo is an inter-cept, bi, bii and bij are constant regression coefficients of the model, and Xi, Xj (i = 1,4; j = 1,4 and i ≠ j) present the coded level of an independent parameter. The number of runs (N) to perform the study was N = 2 k (k−1) + C0, where k is the number of variables and C0 is the center point numbers. In the present work, k and Co were fixed at 4 and 3, respec-tively, which indicated that 27 tests need to be conducted [19, 29]. The acquired data expressed an accurate fitting with the second order polynomial equations. Obtained R-square (98.1%) and adjusted R-square (96%) were suitable. The given findings explain that theoretical and experimental data have an appropriate fitting.

Provided Pareto chart [Fig. 2S (ESM)] demonstrate the impact of parameters on the simultaneous mean extraction recovery of HA and MHA using applied magnetic-based sorbent. The factors draw out the line, associate to the sta-tistically meaningful parameters at the 95% confidence level. Reduction or increment of simultaneous extraction of HA and MHA using the proposed extraction method are shown with the negative or positive sing, respectively [19, 29]. Optimization of variables using BBD revealed that four elected variables for the optimization had a remarkable impact; it is also worth noting that the influence of SDS con-centration, sonication time and sorbent amount was positive while the impacts of pH on the retention of the analytes by the applied sorbent under sonication condition was nega-tive. Mean extraction recovery of target analytes (HA and MHA) based on magnetic-based sorbent was determined as the response. By elevating pH value, the extraction of target molecules was decreased. The effective extraction of target molecules was carried out in the lower pH and the obser-vation can be attributed to the cation-pi, hydrophobic and hydrogen bonding interaction of neutral target molecules with the surfactant magnetic based sorbent. According to the above-mentioned interpretation and obtained data from BBD, pH 1.0 was used for the next experiments. As the

Y = b◦+

∑

biXi +

∑

biiX2

i+

∑

bijXiXj

Fig. 3 The BET evaluation of Fe3O4@TiO2 nanoparticles

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Pareto chart indicates, the influence of SDS concentra-tion, sonication time and sorbent amount on the simultane-ous extraction of HA and MHA was significant and their related effect on the mean extraction recovery was positive. It should be noted that the influence of SDS on simultaneous extraction recovery of HA and MHA was significant and positive. The acquired optimized conditions for sonication time, magnetic-based sorbent amount and SDS concentra-tion from BBD were 3.2 min, 26 mg, and 0.92 mmol L− 1, respectively. Contour plots and three-dimensional surface were utilized for the investigation of the interaction between independent variables. The resulted plots were exhibited as a function of two parameters while other parameters remained at center level [19, 29]. Figure 5, demonstrate the contour plots and three-dimensional surface for SDS concentration

versus pH of the solution. Other contour plots and the three-dimensional surface was presented in electronic supplemen-tary materials (Fig. 3S).

Fast extraction time is necessary for simultaneous extrac-tion of HA and MHA by the surfactant-assisted dispersive micro-solid phase extraction with the aid of ultrasonic waves. High and quick mass transfer of target molecules for the magnetic based sorbent was reached with the aid of ultra-sonic waves. Ultrasound-assisted extraction help to increase the diffusion coefficient and also facilitated the retention of the analytes by the magnetic based sorbent; the ultrasonic power facilitated the dispersity and contact of the sorbent with the target molecules.

Fig. 4 The FT-IR of Fe3O4 and Fe3O4@TiO2 nanoparticles

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The resulted optimum conditions for simultaneous extrac-tion of HA and MHA using the proposed sorbent based on BBD strategy were as follow:

solution’s pH, 1.0; sonication time, 3.2 min; magnetic based sorbent amount, 26  mg and SDS concentration, 0.92 mmol L− 1.

Matrix effects

The detailed information for this part is provided in the elec-tronic supplementary materials (ESM).

Method validation

Analytical figures of merit

The calibration curve was plotted for HA and MHA in the concentration ranges of 3–1000 µg L− 1. Calibration curve exhibited good linearity with the correlation coefficient greater than 0.999, which demonstrate the degree of linearity between concentration level and signal. Each concentration level was injected three times. The limit of quantification (LOQ) was determined according to the following term: the lowest level of analyte that the accuracy drop across 85% and 115% and have the precision level of ≤ 10% under five successive replication. Given to the mentioned explanation, the LOQ value for the introduced procedure was 3 µg L− 1. The signal-to-noise ratio of 3 was considered as the limits of detections (LOD) which were 0.8 µg L− 1 and 1.0 µg L− 1 for HA and MHA, respectively. Regarding the obtained data, the present method shows high sensibility and vast dynamic linear range and also good method repeatability.

Applicability of the introduced method for the real samples analysis

To examine the applicability and accuracy of the optimized method in water samples, three concentration levels of target molecules in water samples were considered. The effective parameters on the extraction of analytes were set in the opti-mum values as mentioned earlier.

The samples were then spiked with 3, 50 and 500 µg L− 1 of each target molecules to investigate the power of the developed method.

Moreover, the necessary experiments to estimate relative recovery (RR %) were carried out at the above-mentioned levels (the number of replicates were 3). As Table 1 indi-cates, the obtained relative recoveries were in the range of 99.2 and 102.4%.

The relative standard deviation (RSD%) values for three identical experiment within a day (repeatability) and in 3 successive days (reproducibility) were fell inside the span of 3.1–6.5% which highlight the good precision of the method (Table 1).

In general, the obtained data show excellent recoveries with proper precision suggesting the proposed extraction procedure is greatly effective for simultaneous determina-tion of HA and MHA in real samples.

Analysis of target molecules in human urine samples revealed that strongly sophisticated matrices had a nearly slight effect on the extraction and determination of HA and MHA. This fact is also approved by Fig. 6 indicating the HPLC–UV chromatograms for human urine sample (urine sample 2) analysis by the proposed sample prepara-tion method. It should be noted that for human urine sam-ples analysis, standard addition method was applied. The obtained data for two separate human urine sample analysis is presented in Table 2.

Fig. 5 Contour plot and three-dimensional surface for assessment of interaction between independent factors (SDS and pH). The resulted plots were exhibited as a function of two parameters while other parameters were remained at center level

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For further validation of obtained data by the proposed method, these data were compared by the obtained data by GC–MS (gas chromatography equipped to mass spec-trometry) analysis. As can be seen, there is a satisfactory agreement between the results achieved by the proposed method and GC/MS analysis.

Comparison of the developed method with other methods

The effectiveness of the developed method for the simulta-neous extraction and determination of HA and MHA was compared with other recently published works. Table 3 summarizes the comparative results. Regarding the limit of detection and precision, the current method is a sensitive and precise procedure for simultaneous trace detection of HA and MHA [7, 8, 14, 15, 32, 33].

Conclusions

In this work, a surfactant-assisted dispersive micro solid phase extraction coupled with ultrasonic power has been successfully applied for simultaneous preconcentration of HA and MHA from human urine samples followed by HPLC–UV quantification. The reported method consider-ably enhanced the separation process and extraction effi-ciency in compared with traditional SPE based approaches. Moreover, the present research integrates the benefits of modified magnetic nanoparticles and mixed hemimicelles, which have been demonstrated to give various advantages including excellent extraction efficiency and simple elution step of the targets. The suitable interaction of analytes (cat-ion-pi, hydrophobic and hydrogen bonding interaction) by the magnetic based sorbent modified with anionic sodium dodecyl sulfate surfactant was used for sample clean-up and preconcentration of HA and MHA from human urine sam-ples. The use of magnetic nanoparticles as sorbent make this method easy and time-saving. The applied surfactant was used to assist the sorbent for extraction of target mol-ecules according to the interaction of target by surfactant. It should be noted that, without the use of surfactant the extraction of target molecules by the sorbent is not effec-tive, because the surface of Fe3O4@TiO2 is polar and it is not suitable for extraction of non-polar compounds. There-fore, we can claim that the method is effective and fast for

Table 1 The simultaneous determination of HA and MHA in tap water samples

Sample Cadded Cfound Repeatability (intra-day preci-sion)

Reproducibility (inter-day precision)

Relative recovery (%)

Tap water HA 0.0 N.D.a – – –MHA 0.0 N.D – – –HA 3.0 µg L− 1 2.98 6.2 6.5 99.3MHA 3.0 µg L− 1 3.1 5.9 6.2 101.3HA 50.0 µg L− 1 51.2 5.1 5.9 102.4MHA 50.0 µg L− 1 50.1 4.8 5.3 100.2HA 500.0 µg L− 1 495.9 3.1 4.3 99.2MHA 500.0 µg L− 1 503.5 3.9 4.5 100.7

Fig. 6 The obtained chromatogram for trace detection of HA and MHA in urine sample (2) by the developed analytical method

Table 2 The simultaneous determination of HA and MHA in human urine samples

Sample number Target HA and MHA concentration (µg L− 1)

Proposed method GC/MS analysis

Urine 1 HA 254.2 (± 0.2) 253.9 (± 0.1)MHA N.D N.D

Urine 2 HA 460.2 (± 0.2) 460.5 (± 0.1)MHA 54.2 (± 0.2) 54.0 (± 0.1)

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extraction of other molecules and target ions in future works. The limit of quantification for the method was 3 µg L− 1 and limits of detections for the presented method were 1 µg L− 1 and 0.8 µg L− 1 for HA and MHA, respectively. Finally, it is strongly expected that the optimized method could be applied as a common procedure for simultaneous measure-ment of HA and MHA in water and human urine samples.

Acknowledgements This work has been funded and supported by Tehran University of Medical Sciences grant (Project No. 35971). The authors would like to acknowledge the University and also the laboratory personnel of occupational health engineering department for their kind help.

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Table 3 Comparison of the developed sample preparation method with recently published articles

Determination method Sample prepara-tion technique

Metabolites LODa RSD % Ref.

HPLC–UV LLE HA and MHAs 5000–6000 5.3–6.7 [32]GC–FID µ-SPE HA 16.5 1.6 [7]GC–MS LLE HA and MHAs 17–20 1–4.2 [8]HPLC–UV LPME MHAs 2–3 3.8–7.3 [15]HPLC–UV MINPs HA 0.15 < 6.1 [14]HPLC–UV – HA 700 3.2 [33]HPLC–UV SA-d-SPE HA and MHA 0.8 < 7.0 This work