Bo-Hao
Chen
a and
Shiuh-Jen
Jiang
*ab
aDepartment of Chemistry, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
bDepartment of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. E-mail: sjjiang@faculty.nsysu.edu.tw; Fax: +886-7-5253908
First published on 30th October 2020
In this study a procedure based on liquid chromatography (LC) and dynamic reaction cell inductively coupled plasma mass spectrometry (DRC ICP-MS), coupled with ultrasonic nebulization, for chromium speciation in rice was reported. Chromium (Cr) containing species, including Cr(III) and Cr(VI), were well separated by reversed phase HPLC with a C18 column as the stationary phase and a solution containing 0.5 mmol L−1 tetrabutyl ammonium phosphate (TBAP), 0.1 mmol L−1 EDTA and 2% (v/v) methanol (pH 6.9) as the mobile phase using isocratic elution. The chromatographic separation was complete in 4.5 min. Ammonia gas was employed as the reactive gas in the DRC to minimize spectral interference due to 40Ar12C+ and 35Cl16OH+ and 40Ar13C+ and 35Cl18O+ ions on the signal of 52Cr+ and 53Cr+, respectively. The detection limits were in the range 0.011–0.012 ng Cr mL−1. To verify the accuracy, the developed procedure was applied on a certified reference material (NIST SRM 1573a Tomato Leaves) wherein the sum of the concentrations of individual species agreed with the total certified concentration of Cr. Species were extracted using microwave heating with 1% (v/v) HF and 2 mmol L−1 EDTA. After ensuring the accuracy, this procedure has been applied to determine various Cr compounds present in rice and rice cereal. Supernatants were injected directly into the LC-ICP-MS system after dilution. The spike recovery was in the range of 95–105% and precision between sample replicates was better than 5% for all determinations. The LC-ICP-MS results showed satisfactory agreement with the total Cr concentrations obtained by ICP-MS analysis. Both Cr(III) and Cr(VI) were present in the samples analyzed. An unknown Cr compound was found in all the rice samples analyzed.
ICP-MS is an elemental analysis methodology capable of detecting most of the elements in the periodic table at milligram to nanogram levels per liter. It is used in the analysis of samples for environmental monitoring, geochemical analysis, characterization of metallurgical compounds and pharmaceuticals, and also clinical research. Cr speciation using liquid chromatography (LC)4–11 and capillary electrophoresis (CE)12,13 coupled with ICP-MS has been reported for a variety of samples. Coupling of LC with ICP-MS is attractive due to the ease of operation, simplicity of interfacing between LC and ICP-MS, availability of isotope ratio information and specificity of the signal intensity of the determined element.14 The determination of Cr by quadrupole ICP-MS is typically compromised by isobaric interference from 40Ar12C+ and 35Cl16OH+ on 52Cr+; 40Ar12CH+ and 37Cl16O+ on 53Cr+. These interferents are reported to be alleviated by employing the dynamic reaction cell (DRC) method with O2 and/or NH3 as the reaction gas.2,15–17
Ultrasonic nebulizers (USNs) with desolvation are employed in ICP based procedures to improve sensitivity and also avoid a high solvent loading. An ultrasonic nebulizer improves the nebulization efficiency compared to a pneumatic nebulizer. The desolvation system of an USN removes most of the water and solvents used for LC separation that might have negative effects on the ICP-MS process. The aims of the present work are to develop a rapid and accurate procedure for the speciation analysis of Cr(III) and Cr(VI) in rice using liquid chromatography DRC-ICP-MS. Herein, optimization of the operating conditions for the LC-USN-DRC-ICP-MS system and its analytical figures of merit when used for the speciation analysis of Cr in rice and rice cereal are presented. An effective microwave-assisted extraction procedure for the extraction of Cr compounds from rice samples has also been presented.
ICP-MS instrument | PerkinElmer SCIEX ELAN 6100 DRC II |
ICP parameters | |
RF power | 1300 W |
Plasma gas flow rate | 15 L min−1 |
Auxiliary gas flow rate | 1.1 L min−1 |
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Mass spectrometer settings | |
Scan mode | Peak hopping |
Dwell time | 50 ms |
Sweeps/reading | 21 |
Reading/replicate | 200 |
Replicates | 1 |
Isotopes monitored | 50Cr, 52Cr, and 53Cr |
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HPLC system | |
Pump | Hitachi Model CM-5110 LC pump |
Injector | Rheodyne model 7725i |
Stationary phase | PerkinElmer C18 reversed phase column, 5 μm diameter particles, 4.6 mm i.d. × 150 mm length |
Mobile phase | 0.5 mmol L−1 TBAP, 0.1 mmol L−1 EDTA, 2% (v/v) methanol, pH 6.9 |
Flow rate | 1.0 mL min−1 |
Sample loop | 100 μL |
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Ultrasonic nebulizer conditions | |
Desolvation temperature | 120 °C |
Condenser temperature | −4 °C |
Carrier gas flow rate | 1.1 L min−1 |
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DRC parameters | |
Reaction gas | NH3 |
Reaction gas flow rate | 0.6 mL min−1 |
Rpq | 0.6 |
Rpa | 0 |
Axial field voltage | 50 V |
The HPLC system is equipped with a HPLC pump (Hitachi, Model CM-5110), an injector (Rheodyne 7725i) and a reversed phase HPLC column (PerkinElmer, Univ L-RP18, 5 μm diam. particles, 4.6 mm i.d. × 150 mm length). The samples were loaded with a syringe into a 100 μL sample loop. All separations were performed at room temperature under isocratic conditions. The LC conditions were selected to maximize the resolution while lowering the retention times (Table 1). Polytetrafluoroethylene (PTFE) tubing was used to connect the column outlet to the USN of the ICP-MS system.
A CEM MARS 6 (Matthews, NC, USA) microwave digester equipped with temperature and pressure sensors was used for the extraction and digestion of Cr species from rice samples. Samples of SRM 1573a tomato leaves and rice (0.3 g each) were weighed into 15 mL polyethylene centrifuge tubes and then the extraction solution (5 mL) containing 0.5 mmol L−1 TBAP, 2% (v/v) methanol, 2 mmol L−1 EDTA, and 1% (v/v) HF was added. The tubes were inserted into a 250 mL beaker (Pyrex glass) containing water (120 mL) and exposed to microwave heating. The microwave system was programmed to maintain the temperature at 90 °C for 50 min; the ramp time was set at 10 min. After microwave heating, the solutions were cooled and centrifuged (3743g, 10 min) directly (MIKRO 22R, Hettich, Germany). The supernatants of different rice samples and SRM 1573a Tomato Leaves were diluted 0 to 12-fold (12 fold for the SRM, 2 fold for Rice 1 and 2, and no further dilution for Rice 3 and Rice cereal) with the mobile phase, filtered through a PVDF filter (Pall; porosity: 0.22 μm) and then injected for LC-USN-DRC-ICP-MS analysis. The concentrations of Cr species were determined through external calibration based on peak areas.
The spike recoveries of the individual compounds were determined by spiking the samples with suitable amounts of Cr(III) and Cr(VI), and then extracting them with the extraction solution. The Cr(III) and Cr(VI) standards were spiked at a level of 1 μg g−1 each in the SRM 1573a tomato leaves; 0.217 and 0.15 μg g−1 in Rice 1; 0.083 and 0.067 μg g−1 in Rice 2; 0.020 and 0.0067 μg g−1 in Rice 3; and 0.083 and 0.020 μg g−1 in Rice cereal. The supernatant solutions were filtered through a PVDF filter before LC separation. The concentration of Cr species was determined by an external calibration procedure based on the peak area. Recoveries were then calculated against the theoretical concentrations.
The extraction efficiency of the Cr compounds was verified through comparison of the total Cr concentrations in the extracts with the certified values and/or the values obtained using the total dissolution procedure. For total dissolution, the samples were digested as follows. To the sample (ca. 0.2 g) taken in a PTFE vessel HNO3 (3 mL) was added. The closed vessel was heated at 150 °C for 20 min, 180 °C for 20 min, and 200 °C for 20 min. The power of the microwave was set at 900 W. The digest was diluted to 5 mL. After suitable dilution the total concentration of Cr in the sample was analyzed through pneumatic nebulization DRC ICP-MS (internal standard: Rh, 1 ng mL−1).
The effect of the concentration of EDTA in the mobile phase on the chromatogram was studied at 0.1, 0.15 and 0.2 mmol L−1 at pH 6.9. As shown in Fig. S1 (ESI†), the retention time of Cr(VI) decreased with the EDTA concentration, which could be due to the increase in the ionic strength of the mobile phase. For better separation of Cr(VI) and Cr(III)–EDTA, 0.1 mmol L−1 EDTA was selected. The influence of the concentration of ion pairing reagent TBAP at 0.25, 0.5 and 0.75 mmol L−1, on the chromatogram is shown in Fig. S2 (ESI†). The retention time of Cr(VI) and Cr(III)–EDTA increased with the increase in the TBAP concentration. A TBAP concentration of 0.5 mmol L−1 was considered optimum. The concentration of methanol in the mobile phase also influences the chromatogram and hence the effect of the methanol concentration in the mobile phase on the chromatogram, at 1, 2, and 3% (v/v), was studied. The retention times of Cr(VI) and Cr(III)–EDTA decreased slightly with the methanol concentration. Furthermore better S/B could also be obtained when 2% methanol was added. In the following experiments, 2% (v/v) methanol was added to the mobile phase. The LC operating conditions used in this work are listed in Table 1.
The background at Cr major masses m/z 52 and m/z 53 was too high to report the better detection limit when ICP-MS was operated in standard mode. Quadrupole ICP-MS instruments equipped with a reaction cell and/or collision cell are known to reduce polyatomic ion interference in the presence of a suitable reaction gas.19,20 Both NH3 and O2 reaction gases are known to lower the background at Cr masses when used as reaction gases in the DRC.2,15,16 In this study, NH3 was selected due to marginally superior estimated detection limits (EDLs). The optimization of DRC conditions has been carried out by continuous introduction of 3 ng mL−1 Cr solution using conventional pneumatic nebulization. The mobile phase containing 300 ng mL−1 Cl− was treated as the blank for this experiment. Fig. S4 (ESI†) shows the effect of the NH3 gas flow rate on the signal of 52Cr+ and 53Cr+, and the blank signals at m/z 52 and 53, respectively. The results indicated that background due to 40Ar12C+, 35Cl16OH+ and 40Ar12CH+, and 37Cl16O+ has been suppressed effectively when NH3 was used as the reaction gas. As shown in Fig. S4,† the blank signal at m/z 52 was suppressed significantly when the NH3 gas flow rate was greater than 0.5 mL min−1. The estimated detection limit (EDL) was determined based on the concentration necessary to yield a net signal equal to three-times the square root of the background. Better detection of 52Cr+ and 53Cr+ could be obtained when the NH3 flow rate was 0.6 mL min−1. Although it did not have a significant effect on the EDL when the rejection parameter q (Rpq) was varied between 0.25 and 0.6, a Rpq value of 0.6 was selected based on the lower background for 52Cr+ and 53Cr+. The rejection parameter a (Rpa) value was set at 0 in this study and the axial field voltage (AFV) was optimized to be 50 V. The DRC conditions used in this work are listed in Table 1.
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Fig. 1 Typical mass-selective chromatogram for Cr(VI) and Cr(III)–EDTA: (a) with standard mode, each Cr species was present at 50 ng Cr mL−1; and (b) with DRC mode, each Cr species was present at 5 ng Cr mL−1. No reaction gas was used in the standard mode. Other LC conditions are given in Table 1. |
Under the optimized conditions, the following parameters have been computed. The relative standard deviation of the peak areas was less than 1.9% and the repeatability of retention time was better than 3% for five consecutive injections at 5 ng mL−1 (as the element) each of the two Cr species. Calibration curves (five points) based on the peak height and peak area, were linear with the correlation coefficient (r2) better than 0.9996 for each species in the range studied (0.1–10 ng mL−1). The detection limit was estimated from the peak height versus concentration plot and based on the concentration (as the element) necessary to yield a net signal equal to three times the standard deviation of the baseline fluctuation of the chromatogram. The LC-ICP-MS detection limit obtained in this study was 0.011 and 0.012 ng mL−1 for Cr(III) and Cr(VI), respectively. These results are compared with those of other similar techniques in Table 2. Overall, the detection limits obtained in this work are better than previous results with similar techniques.5,8,16–18,21–26 The lower limit of detection of Cr could be due to the use of the USN as the sample introduction device in this work. A slightly better detection limit could be obtained for 53Cr+ compared to 52Cr+. Hence, in this study, 53Cr+ was used for quantification work.
Reaction/collision gas | Separation time | Cr(III) | Cr(VI) | Real sample | Reference |
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a Limit of quantification. | |||||
NH3 | 4.5 min | 0.011 | 0.012 | Rice and cereal | This work |
NH3 | 4 min | 0.1 | 0.1 | Drinking and tap water | 20 |
NH3 | 7 min | — | 0.11a(mg kg−1) | Cosmetic products | 4 |
NH3 | 3 min | 0.05 | 0.05 | Oil | 17 |
He | 7 min | 0.05 | 0.08 | Water | 22 |
NH3 | 3 min | 0.094 | 0.10 | Water | 15 |
NH3 | 5 min | 0.05 | 0.05 | Soil and leaves | 7 |
CH4 | 3 min | 0.5 | 0.6 | Air | 23 |
He | 7 min | 0.01 | 0.07 | Dairy and cereal products | 24 |
H2 | 12 min | 1 | 1 | Rice | 21 |
He | 8 min | 0.08a (μg g−1) | 0.08a (μg g−1) | Dietary supplements | 25 |
Sample and compound | Concentration found/ng g−1 | Spike recovery/% |
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a Values are means of three measurements ± standard deviation. b Column recovery; the value in parentheses is the percentage of the sum of the species to the extracted concentration. c Extraction efficiency; the value in parentheses is the percentage of the extracted concentration to the digested concentration. d NIST certified values. | ||
NIST SRM 1573a | ||
Cr(III) | 950 ± 40 | 102 ± 3 |
Cr(IV) | 1030 ± 40 | 96 ± 2 |
Sum of Cr species | 1980 ± 60b (102%) | |
Total Cr (extract) | 1950 ± 70c (100%) | |
Total Cr (digestion) | 1950 ± 70 | |
Certifiedd | 1990 ± 60 | |
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Rice 1 | ||
Unknown | 52.7 ± 2.1 | |
Cr(III) | 212 ± 6.7 | 105 ± 4 |
Cr(IV) | 135 ± 4.4 | 95 ± 2 |
Sum of Cr species | 400 ± 8.3b (103%) | |
Total Cr (extract) | 388 ± 11c (98%) | |
Total Cr (digestion) | 396 ± 13 | |
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Rice 2 | ||
Unknown | 14.3 ± 0.7 | |
Cr(III) | 73.3 ± 2.7 | 105 ± 3 |
Cr(IV) | 56.4 ± 2.2 | 96 ± 3 |
Sum of Cr species | 144 ± 3.5b (101%) | |
Total Cr (extract) | 143 ± 4.1c (97%) | |
Total Cr (digestion) | 148 ± 4.5 | |
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Rice 3 | ||
Unknown | 11.1 ± 0.5 | |
Cr(III) | 19.9 ± 0.8 | 101 ± 2 |
Cr(IV) | 6.37 ± 0.3 | 99 ± 1 |
Sum of Cr species | 37.4 ± 1.0b (98%) | |
Total Cr (extract) | 38.3 ± 1.3c (98%) | |
Total Cr (digestion) | 39.2 ± 1.1 | |
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Rice cereal | ||
Unknown | 16.9 ± 0.7 | |
Cr(III) | 79.3 ± 2.9 | 104 ± 4 |
Cr(IV) | 21.4 ± 0.8 | 96 ± 3 |
Sum of Cr species | 118 ± 3.1b (98%) | |
Total Cr (extract) | 120 ± 3.5c (99%) | |
Total Cr (digestion) | 121 ± 3.9 |
As the results for the SRM are satisfactory, this procedure was applied for the speciation analysis of Cr in three polished rice samples and a rice cereal sample. The typical mass selective chromatograms (52Cr+ and 53Cr+) obtained for Rice 1 and Rice 3 are shown in Fig. 4 and Fig. 5, respectively. As shown, both Cr(III) and Cr(VI) are present in these samples. There was an unidentified compound near a retention time of 100 s in all rice and rice cereal samples analyzed. To ensure that the unidentified peak is due to Cr, the ratio of the peak area of m/z 52 to m/z 53 for all the chromatographic peaks was computed. The ratio was in the range 8.32–8.52 and there was no significant difference between the peak area ratios of these peaks according to the Student t-test for a confidence level of 95%. It proves that all the chromatographic peaks shown in the chromatogram do correspond to Cr. The concentration of the compound whose standard was not available was estimated against the sensitivity of the nearest peak. The results of rice and rice cereal are also listed in Table 3. As shown, the spike recovery was in the range of 95–105% for Cr species in rice and rice cereal. A slightly lower recovery of Cr(VI) was observed possibly due to the reduction of Cr(VI) to Cr(III) during extraction. With a lower concentration of HF as the extractant, the reduction could be minimized; however the extraction efficiency of Cr might be degraded. As shown in Table 3, the HPLC-USN-DRC-ICP-MS results were compared with the total concentrations of Cr in the extracts and also in completely digested samples. The results are in satisfactory agreement with the total concentrations. Moreover the Cr(VI) results obtained by the current procedure were in good agreement with the results reported in a previous paper.11 With the current procedure, not only the amount of Cr(VI) was determined, but the concentrations of Cr(III) and an unknown Cr containing compound were also determined. Hence, the current procedure offers complete speciation of Cr in rice.
The precision between sample replicates, of different experiments including sample pretreatment, separation and determination (n = 3), was better than 5% for all determinations. This experiment indicated that the Cr compounds in rice and rice cereal could be readily quantified by the proposed HPLC-USN-DRC-ICP-MS procedure.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ja00356e |
This journal is © The Royal Society of Chemistry 2021 |