Inorganic arsenic determination in fresh mussels using water bath extraction and anion exchange chromatography-inductively coupled plasma mass spectrometry DISCLAIMER: This method: - has to be considered only as guideline to help the NRLs to approach the analytical problems of inorganic arsenic determination in fresh mussels; - has been developed by the EURL-CEFAO using the facilities available in its laboratories; The laboratories have to study the instrumental conditions appropriate for their own instrumentation. Page 1 of 8
CONTENTS 1. SCOPE... 3 2. ACRONYMS AND CONVENTIONS... 3 3. METHOD DESCRIPTION... 3 4. EQUIPMENT... 3 5. REAGENTS AND SAMPLES... 4 6. PROCEDURES... 4 7. INSTRUMENTAL ANALYSIS... 5 8. VALIDATION PARAMETERS... 7 9. FINAL CONSIDERATIONS... 7 Page 2 of 8
1. SCOPE The method describes a general guideline for speciation of inorganic arsenic in fresh mussels. 2. ACRONYMS AND CONVENTIONS ICP-MS Inductively Coupled Plasma Mass Spectrometry HPLC High Performance Liquid Chromatography Inorganic Arsenic i-as Ammonium dihydrogen phosphate NH 4 H 2 PO 4 Ammonium nitrate NH 4 NO 3 Methanol CH 3 OH Ammonia NH 4 OH Disodium hydrogen arsenate heptahydrate Na 2 HAsO 4 *7H 2 O sp Suprapure grade up Ultrapure grade c concentration expressed in units of milligrams/micrograms per litre LoD Limit of detection LoQ Limit of quantification RSDr Relative standard deviation under repeatability conditions RSD R Relative standard deviation under within-laboratory reproducibility conditions 3. METHOD DESCRIPTION The determination of i-as is performed by anion exchange chromatography-inductively coupled plasma mass spectrometry after water bath extraction. The proposed extraction procedure leads to the complete oxidation of As(III) into As(V), which results as a well separated peak in the anion exchange HPLC-ICP-MS chromatogram. The i-as is quantified as As(V). The method was validated using samples of Mediterranean mussels containing roughly 3.8 mg/kg of total As. Instrumental quantification is based on at least three point external calibration curve. WARNING: the application of the method includes the use of hazardous materials, operations and equipment. The user is responsible for the appropriate use of safety tools. 4. EQUIPMENT The following apparatus and equipment have been used for the EURL-CEFAO method development: - ICP-MS (ELAN DRC II, Perkin Elmer): Interface Pt skimmer and sampler cones Meinhard quartz concentric nebulizer used in conjunction with a PC3 Peltier-cooled quartz cyclonic spray chamber Page 3 of 8
- HPLC system (Perkin Elmer Series 200) - Shaking water bath (VWR International) - 0.22 µm PVDF syringe filters - ph meter - Centrifuge - Hamilton PRP-X100 Column - Polypropylene falcon tubes - Plastic Pasteur pipettes - Polypropylene syringe without needle - Stericup filter units (or similar) - Analytical scale (0.1 mg precision) 5. REAGENTS AND SAMPLES The reagents must have adequate purity. Sp or up grade guarantees that the concentration of the analyte in reagents and water is negligible compared with the level of concentration to be determined. - Nitric acid up (70% v/v) - Hydrogen peroxide sp (30% v/v) - NH 4 H 2 PO 4 (99.99% Suprapur) - NH 4 NO 3 (pro analysis) - CH 3 OH (HPLC grade) - Up-water, specific resistance 17 MΩ cm for all samples preparation and dilutions - Na 2 HAsO 4 *7H 2 O - Certified Element Standard Solutions, c = 1000 µg/ml of As, and Rh - ERM-CE278K - Fresh mussels from the Mediterranean sea (Mytilus spp.) 6. PROCEDURES 6.1 Sample extraction Weigh circa 1.0 g of fresh mussels or 0.3 g of freeze-dried samples in the case of, e.g., lyophilised reference materials in 50 ml polypropylene test tube (e.g. Falcon type). Add 10 ml of a 1% (v/v) HNO 3 and 1% (v/v) H 2 O 2 solution and let stand overnight at room temperature (pre-digestion step). Afterwards, place the tubes in a water bath at 95 C for 1 hour under gentle shaking. Let the samples cool to room temperature and centrifuge the tubes at 10000 rpm for at least 10 minutes (centrifuge temperature set at 4 C). Collect the clear supernatant by means of a 3 ml plastic Pasteur pipette in 15 ml polypropylene test tubes after filtration through 0.22 µm filters. If the filtration is particularly difficult, the centrifugation time can be increased and the filtration through 0.22 µm filters can be preceded by a further step performed using 0.45 µm filters. If the extracts are not analysed straight after filtration, aliquot them and store the resulting aliquots at a temperature of -20 C or lower till analysis. Page 4 of 8
6.2 Inorganic arsenic determination 6.2.1 Preparation of the mobile phase (10mM NH 4 H 2 PO 4, 10mM NH 4 NO 3 2% (v/v) CH 3 OH) Prepare a solution of 2% (v/v) CH 3 OH (at least 1 L of solution). Dissolve circa 1.15 g of NH 4 H 2 PO 4 and 0.8 g of NH 4 NO 3 in 1 L of the solution 2% (v/v) CH 3 OH. Adjust to ph 5.5 adding a diluted solution of ammonia (1:10, v/v) or a solution of diluted HNO 3 while the solution is maintained under stirring. Before use this solution has to be filtered (e.g. by using a stericup filter unit connected to a vacuum pump). 6.2.2 Preparation of stock standard solutions All the solutions should be prepared by weight. As (V) 1000 mg/kg (sol A): prepare a 1000 mg/kg aqueous standard solution of arsenate (expressed as As) starting from Na 2 HAsO 4 *7H 2 O (e.g. weigh 0.208 g Na 2 HAsO 4 *7H 2 O in 50 g up water). The solution has to be stored at - 20 C. As (V) 1.0 mg/kg (sol B): dilute sol A up to 1 mg/kg with up water. This solution has to be properly stored in a freezer or a refrigerator. In any case the actual concentration of As (V) in this solution has to be ascertained by ICP-MS, whereas the chromatographic purity has to be assessed by HPLC-ICP-MS. As 1.0 mg/kg (sol C): prepare an aqueous solution preferably by weight, e.g. weigh 0.05 g of the certified As standard solution at 1000 mg/kg and dilute up to 50 g with acidified (HNO 3; 1% v/v) up water. Rh 1.0 mg/kg (sol D): prepare an aqueous solution preferably by weight, e.g. weigh 0.05 g of the certified Rh standard solution at 1000 mg/kg and dilute up to 50 g with acidified (HNO 3; 1% v/v) up water. 7. INSTRUMENTAL ANALYSIS 7.1 Working standard solutions These solutions must be daily prepared preferably by weight. As (V) 0.1 mg/kg (sol E): prepare an aqueous solution preferably by weight (e.g. weigh 1.0 g of sol B and dilute up to 10 g with up water). This solution is used to check the actual concentration of the sol B so as to correct the title of the starting 1000 mg/kg As (V) solution. It is also used to prepare the calibration curve to quantify the concentration of As (V) in the samples. Rh 0.1 mg/kg (sol IS): prepare an aqueous solution preferably by weight (e.g. weigh 1.0 g of sol D and dilute up to 10 g with up water) As 0.1 mg/kg (sol F): prepare an aqueous solution preferably by weight (e.g. weigh 1.0 g of sol C and dilute up to 10 g with up water). 7.2 Check of the actual concentration of As (V) 1.0 mg/kg Prepare a three point external calibration curve by using sol IS and sol F (e.g. the following calibration points can be used: 50 ppb; 100 ppb; 200 ppb with IS at 5 ppb) and quantify the concentration of sol E on this curve. On the basis of the result correct the concentration of sol B. This right concentration value has to be used for the subsequent analyses and calculations. Page 5 of 8
7.3 Speciation of i-as The analysis is performed by HPLC-ICP-MS using a Hamilton PRPX-100 column and the elution is carried out in isocratic mode (total time for each sample: 30 minutes). The samples have to be diluted two-fold (w/w) before the analysis. The i-as is identified as As(V) by comparing the retention time of the chromatographic peak of the samples with that of the peak obtained by spiking the sample extracts with As(V) standard solution. The quantification is based on the measurement of peak areas and is performed using an external calibration curve made of at least three points. The calibration curve has to be reacquired after each series of 6-10 samples. ArCl interference on m/z 75 is assessed by monitoring m/z equal to 77 and 82. The operating conditions of the HPLC-ICP-MS system are summarized in Table 1. A typical chromatogram of an extract of the samples used for this validation is shown in Figure 1. Table 1. Operating conditions HPLC-ICPMS HPLC Column Hamilton PRPX-100 (250 x 4.6 mm, 5µm particle size) Temperature 22 C Injection volume 50 µl Mobile phase 10 mm NH 4 H 2 PO 4 +NH 4 NO 3 2% CH 3 OH (ph=5.5) Flow rate 1.0 ml/min Elution mode Isocratic Run time 30 minutes As(V) retention time 10 minutes ICP-MS Plasma flow 15 L/min Auxiliary gas flow 1.0 L/min Nebulizer gas flow Daily optimized RF Power 1500 W Figure 1. Typical chromatogram obtained for an extract of the Mediterranean mussels used for validation 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 AsV Mussels 1% HNO 3 + 1% H 2 O 2 water bath extraction 0 0 5 10 15 20 25 30 Page 6 of 8
8. VALIDATION PARAMETERS The following validation parameters have been assessed: LoD, LoQ, repeatability, within-laboratory reproducibility, accuracy. LoD and LoQ were determined using 20 samples of fresh mussels from Mediterranean sea with a low content of i-as. In particular, the standard deviation was multiplied by 3 and 10 so as to calculate the LoD and LoQ, respectively. Repeatability and within-laboratory reproducibility were assessed by analyzing samples of Mediterranean mussels containing higher amounts of i-as abd 3.8 mg/kg of total arsenic. As far as the accuracy is concerned, it was assessed using a Reference Material (ERM- CE278K) and recovery rates of spiked As(III). The satisfactory recovery of As(III) as As(V) confirmed the complete oxidation of As(III) into As(V). The reference value of i-as in ERM- CE278K (0.086 mg/kg ± 0.008 mg/kg) was the value assigned in a dedicated collaborative trial (IMEP-41). The parameters obtained during the method validation are summarized in table 2. Table 2. Parameters of the EURL-CEFAO validation Parameter Parameter value LoD (mg/kg) 0.0012 LoQ (mg/kg) 0.0041 a) RSDr (%) 5.0 b) RSD R (%) 9.2 Recovery (%): c) As(III) 108 ERM-CE278K 94 a) Mean concentration value of i-as = 0.097 mg/kg b) Mean concentration value of i-as = 0.090 mg/kg c) Spiked concentration As(III) = 0.050 mg/kg A provisional value of expanded uncertainty equal to 19% (k=2) was estimated using a formula that combined the within-laboratory reproducibility and the uncertainty of the bias. Please, note that this value was calculated using the assigned value and the relevant uncertainty of the ERM-CE278K as derived from the IMEP-41 trial. The value was assessed by considering the analysis in triplicate. 9. FINAL CONSIDERATIONS During method development particular attention was paid to the use of a shaking water bath as extraction tool since it allows to obtain a greater sample throughput compared with microwave systems. Each extract sample was analyzed with three different anion-exchange HPLC-ICP-MS methods based on two different columns (Hamilton PRP-X100 and ICSep ION-120). As for the quantification of the i-as, both external and standard addition calibration approaches were applied in order to evaluate the occurrence of matrix effects. Due Page 7 of 8
to the good preliminary results obtained the method consisting in the extraction with a HNO 3 /H 2 O 2 mixture and elution with ammonium phosphate and ammonium nitrate in 2% methanol (ph=5.5) on Hamilton PRPX-100 column was considered for validation. In particular, both microwave ovens and shaking water baths were evaluated as extractive apparatus and no difference resulted between the two procedures. Extraction with water bath is less expensive (use of polypropylene disposable tubes and water bath compared with expensive TFM vessels and microwave ovens) and less time consuming, allowing the operator to extract a greater number of samples than with microwaves (roughly 60 vs 10). Furthermore, the difference in repeatability and accuracy between samples extracted with microwave oven and shaking water bath was found to be negligible, leading to the selection of the latter procedure as the one to be implemented. The elution through the Hamilton PRPX-100 was performed in isocratic mode and the i-as was determined as a single species, i.e. As(V), using both external and standard addition calibration for quantification. The comparison of the results showed no differences between the two approaches so the external calibration was selected as quantification method being easier to perform. Both the undiluted and two-fold diluted samples were tested: also in this case no significant differences were found; the two-fold dilution was selected as it implied both a minor wear and tear of the column and a better resolution of the chromatographic peaks. Page 8 of 8