Optimization of a methodology for speciation of arsenic, selenium and mercury in blood samples based on the deep eutectic solvent

Graphical abstract

Sodium selenite and sodium selenate (both purchased from Merck, Darmstadt, Germany) were used for preparation of stock standard solutions of selenite and selenate with a concentration of 1000 mg l À1 . Stock standard solutions of As(III) and As(V) with a concentration of 1000 mg l À1 were obtained by dissolving appropriate amounts of As 2 O 3 and Na 2 HAsO 4 (Merck, Darmstadt, Germany). An Hg (II) standard stock solution (1000 mg l À1 in 1% nitric acid, 250 ml) was purchased from Fluka, Buchs, Switzerland. The CH 3 Hg + 1000 mg l À1 stock solution was prepared by dissolving appropriate amount of its chloride (Merck, Darmstadt, Germany) in the smallest possible volume of methanol and diluting to volume with deionized water. The stock standard solutions were diluted to prepared a required standard solutions with 0.1 M HCl. The analytical grade diethyldithiophosphoric acid (DDTP) as chelating agent was obtained from Merk (Darmstadt, Germany). Iridium solution with concentration of 1000 mg l À1 in hydrogen chloride (Sigma-Aldrich, St. Louis, Missouri, USA) was used as the chemical modifier. Decanoic acid and choline chloride were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

Instrumentation
An Analytik Jena AG Model nov AA 400 (Jena, Germany) atomic absorption spectrometer equipped with a deuterium background correction system, a transversely heated graphite tube atomizer and a MPE60 auto-sampler were employed throughout measurements. Pyrolytic graphite coated tubes with PIN-graphite platform (Analytik Jena, Part No.:407-A81.026, Jena, Germany) were applied for the optimizations with aqueous standards. A hollow cathode lamp was used as the radiation source. Argon 99.999% was used as purge and protective gas. Integrated absorbance (peak area) was used exclusively for signal evaluation. The sample injection volume was 20 ml in all experiments. The instrumental parameters and temperature program for the graphite atomizer are listed in Table 1. The pH values were measured by Metrohm pH meter Model 692 (Herisau, Switzerland). A Multi-Wave 3000 microwave-assisted UV system (MUV, Anton Paar, Graz, Austria) was used for converting R-Hg to Hg 2+ .

Sampling and sample preparation
Blood samples were collected from five children (3 girls and 2 boys) who were patient under treatment, kindly provided by the Clinic of Mohammad Kermanshahi Hospital (Kernanshah, Iran). The age was in the range 2-10 years. To preparation of sample, 1.0 ml of blood sample was placed in a 10-ml glass tube and 600 ml acetonitrile and 900 ml of 15% (w/v) ZnSO 4 was added. The glass tube was vortexed for 5 min, maintained at 5 C for 10 min followed by centrifugation at 5000 rpm for 3 min. Then, the supernatant was collected in another tube and this solution was diluted to 10.0 ml using ultrapure water. The resulting solution was then subjected to the LPME-SDES procedure.

LPME-SDES procedure
An aliquot of 10.0 ml of ultra-pure water or pretreated/diluted blood sample spiked or not with target analytes was placed in a 10-ml test tube and 60 ml of DES (extraction solvent) containing 15.0 mL DDTP (chelating agent) was rapidly injected into the sample solution with a 100-mL syringe (Gastight, Hamilton, Reno, NV, USA). The mixture was then shaken using a vortex agitator for 5 min to ensure full contact of the DES and target ions inside the sample solution. The mixture was then centrifuged for 4 min at 5000 rpm in order to separate the mixture into phases. After centrifugation, the fine droplets of DES float at the top of the test tube. The test tube was then transferred into an ice bath and the DES was solidified after few minutes. Then obtained solidified DES was transferred into a conical vial where it was melted immediately. To decrease viscosity and simply injected into the GFAAS, a 20 ml acidic ethanol added into the vial. Finally, for quantitation of target ions, 30.0 ml of the extract using an auto-sampler was injected into the GFAAS and was subjected to the temperature program, shown in Table 1.

Method validation
The analytical characteristics of the method, i.e., precision, detection limits and linearity, were investigated under the chosen experimental conditions. The results are listed in Table 2. The percent relative standard deviations (RSDs %) were between 2.2 and 4.1. The limit of detection, defined as C L = 3S B /m (where C L , S B , and m are the limit of detection, standard deviation of the blank and slope of the calibration graph, respectively), were obtained between 0.015 and 0.10 mg l À1 for different metal ions. Linear ranges (LRs) of 0.15-40 mg l À1 for As, 0.05-5.0 mg l À1 for se and 0.30-60 mg l À1 for Hg were obtained. The correlation coefficient of the calibration curves were in the range of 0.993-0.999. The enhancement factor, obtained from the slope ratio of calibration graph after and before extraction, were in the range of 98-106.  Table 3 Determination of As(III) and As(V) in blood samples, and relative recovery of spiked arsenic in these samples a .

Determination of As in blood samples
After applying the proposed methodology, total inorganic arsenic (iAs) was then measured after the reduction of As(V) to As(III) with sodium thiosulfate and potassium iodide. As(V) was calculated by the difference between the total As and As(III). As(III) and As(V) in all blood samples were detected at different concentration levels and they were confirmed by spiking As(III) and As(V) into the all samples. The concentration of As(III) and As(V) in the blood samples are shown in Table 3. Blood samples were spiked with As(III) and As(V) standards to assess matrix effects. The relative recoveries of As(III) and As(V) from blood samples at spiking different levels are listed in Table 3. In addition, the accuracy of the proposed method was evaluated by analyzing a standard reference material (SRM) NIST-2669 Inorganic Arsenic Species in Frozen Human Urine from National Institute of Standards and Technology (NIST), with certified As(III) and As(V) content of 1.47 AE 0.10 mg l -1 and 2.41 AE 0.30 mg l -1 , respectively. No significant difference was found between the result obtained by employing the proposed method and the certified value ( Table 3).

Determination of Se in blood samples
Determination of total inorganic selenium is based on the conversion of Se(VI) species into Se(IV), which are then analyzed by GFAAS. Gentle boiling in 5 mol l À1 HCl medium for one hour and adjusting pH to 3 were used as the appropriate conditions for rapid reduction of Se(VI) to Se(IV). Finally, the concentration of Se(VI) was calculated by subtracting the Se(IV) concentration from the total inorganic selenium concentration. Results in Table 4 show that all samples contain selenite and selenate with different concentrations. To validate the method and evaluate the effects of the matrix, all samples were spiked with standard selenite and selenate solutions. Relative recovery of selenite and selenate in spiked blood samples at various concentration levels is shown in Table 4, ranging from 91 to 105%. Moreover, the accuracy of the LPME-SDES was appraised by analyzing a standard reference material Table 4 Determination of Se(IV) and Se(VI) in blood samples, and relative recovery of spiked Se in these samples.

Samples
Analyte (SRM) NIST-1598A inorganic constituent in frozen animal (Bovine and Porcine) serum, with certified Se content of 134.4 AE 5.8 mg l -1 . No remarkable difference was observed between the result gained by applying the presented method and the certified value ( Table 4).

Determination of Hg in blood samples
Determination of total mercury (t-Hg) is based on the conversion of organic mercury(R-Hg) species into Hg 2+ , which are then analyzed by GFAAS. Ultraviolet (UV) light and microwave were used as the appropriate sources for rapid conversion of R-Hg to Hg 2+ . In this work, we used MUV 3000, a closed vessel microwave assisted-UV system, equipped with both the thermal and radiant (UV) energies. Total Hg was obtained when the aqueous samples containing both Hg 2+ and R-Hg was put on vessel in special conditions (180 C, UV) for 15 min. All R-Hg species were converted to Hg 2+ . Finally, the concentration of R-Hg was calculated by subtracting the Hg 2+ concentration from the t-Hg concentration. Mercury in all blood samples were detected at different concentration levels (Table 5). Blood samples were spiked with Hg 2+ standard solution to assess matrix effects. The results of relative recovery and the concentrations obtained in spiked studies of the blood samples are also included in Table 5. In addition, the accuracy of the proposed methodology was evaluated by analyzing a standard reference material (SRM) NIST-955C Toxic Metals in Frozen Caprine Blood, with certified contents of 9.0 AE 1.3 and 17.8 AE 1.6 mg l -1 inorganic mercury and total mercury, respectively. No significant Table 5 Determination of Hg 2+ , R-Hg and t-Hg in blood samples and relative recovery of spiked mercury in these samples. difference was found between the results obtained by employing the proposed method and the certified value. The determined value of 8.6 AE 1.4 mg l -1 for inorganic mercury and 18.2 AE 1.5 mg l -1 for total mercury are in satisfactory agreement with the certified values.