NOTICE: This method: The laboratories have to study the instrumental conditions appropriate for their own instrumentation.

Transcription

1 Determination of Cadmium and Lead in powdered infant formula by Electrothermal Atomization Atomic Absorption Spectrometry with Zeeman Effect Correction NOTICE: This method: - has to be considered only as guideline to help the NRLs to approach the analytical problems of this matrix; - has been developed on the basis of the EU-RL CEFAO experience on GF-AAS technique and it has been applied only on the brand of infant formula used in the 17 th PT; - is not completely validated and the application on different brands and formulations of infant formula is foreseen; - has been developed by EURL CEFAO using the facilities available in its laboratories. The laboratories have to study the instrumental conditions appropriate for their own instrumentation.

2 CONTENTS 1. SCOPE ACRONYMS AND CONVENTIONS METHOD DESCRIPTION REAGENTS SAMPLE TREATMENT- Dry Ashing INSTRUMENTAL ANALYSIS CALCULATION EXPRESSION OF RESULTS...8 Annex 1: Instrumental Settings...9 Page 2 of 10

3 1. SCOPE The method describes the procedure for the determination of cadmium and lead in powdered infant formula by Z-ETA-AAS, after dry ashing digestion. 2. ACRONYMS AND CONVENTIONS Z-ETA-AAS Electrothermal Atomization Atomic Absorption Spectrometry with Zeeman Effect Correction Sp Suprapure grade Up Ultrapure grade c Concentration expressed in units of milligrams/micrograms per litre RSD r Repeatability relative standard deviation EDL Electrodeless Discharge Lamp THGA Transversely Heated Graphite Atomizer Tubes 3. METHOD DESCRIPTION The determination of Cd and Pb is performed by Z-ETA-AAS after dry ashing sample digestion. Instrumental quantification is based on the matrix matched calibration approach. The sample is digested according to the procedure described at point 5. The calibration curve is prepared as specified in section for Cd and in section for Pb, respectively. 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. APPARATUS and EQUIPMENT - Z-ETA-AAS (AAnalyst 800, Perkin Elmer) - Lead: - Tube THGA with end-cap - EDL Lamp - Cadmium: - Tube THGA - EDL Lamp - Analytical balance with 0.1 mg of resolution - Hot plate - Programmable muffle-furnace - Quartz beaker (not less than 50ml) 4. REAGENTS The reagents must have the required 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. 4.1 HNO 3 Sp, not less than 65% mass fraction, d(hno 3 ) ~1,4 g/ml HNO 3 20% v/v. from 4.1 with Up-water. 4.2 Up-water, specific resistance 17 MΩ cm for all samples preparation and dilutions. 4.3 Certified Element Standard Solutions, c = 1000 µg/ml each of Cd and Pb Cd 1 mg/l Page 3 of 10

4 Pipette 0,1 ml of Cd stock solution (4.3) and make up to 100 ml with Up-water in a certified decontaminated volumetric flask Pb 1 mg/l Pipette 0,1 ml of Pb stock solution (4.3) and make up to 100 ml with Up-water in a certified decontaminated volumetric flask. Prepare these standard solutions in triplicate, transfer to a plastic decontaminated storage bottle (e.g. Kartell), thoroughly mix. 4.4 Ammonium dihydrogen phosphate: NH 4 H 2 PO 4 (99.99 Sp) Ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ) 100 g/l Example: weigh 10g of NH 4 H 2 PO 4 (4.4) and make up to 100 ml with Up-water in a volumetric flask. 4.5 Magnesium nitrate: Mg(NO 3 ) 2 6H 2 O (99.99 Sp) Magnesium nitrate (Mg(NO 3 ) 2 ) 6g/l Example: weigh 0.6g of Mg(NO 3 ) 2 (4.5) and make up to 100 ml with Up-water in a volumetric flask Magnesium nitrate (Mg(NO 3 ) 2 ) 2g/l Example: weigh 0.2g of Mg(NO 3 ) 2 (4.5) and make up to 100 ml with Up-water in a volumetric flask 4.6 Palladium: Pd (as palladium nitrate (Pd(NO 3 ) 2 )): 10000mg/l in 15% HNO Triton X-100: TX: reagent for analysis Triton X-100 1% (TX1) in water Even if the reagent is liquid, it is easier to prepare the solution by weighting. 5. SAMPLE TREATMENT- Dry Ashing 5.1 Quartz beaker decontamination The use of beakers instead of crucibles is recommended in order to avoid the spattering of the sample. Moreover, to maintain the contamination level under control, it is very important to apply a strict decontamination procedure. Before using the quartz beakers, fill them using HNO 3 20% v/v (4.1.1) and put them on a hot plate at T of about 100 C till evaporation of about 2/3 of the liquid volume, then repeat the procedure two times avoiding the total evaporation of liquid. Rinse the beakers with Up-water (4.2) and let them dry before the use, paying attention to any contamination source. 5.2 Dry Ashing Weigh an amount of about 4g of sample in a quartz beaker and prepare at least one Procedure Blank for each muffle furnace used. Add 2.5 ml of Mg(NO 3 ) 2 g/l (4.5.2) and 5 ml of Up-water (4.2) and dry over a hot plate being careful to properly increase the temperature to avoid the spattering of the sample. After dryness is reached, put the sample in a muffle at an initial T not higher than 200 C; increase the T at a maximum rate of 50 C/h to 400 C. Let the sample stand 12 h. Take the beakers out of the furnace and let them cool; add 2 ml of Up-water (4.2) and 2 ml of HNO 3 Sp (4.1) and put it on the hot plate, take carefully to dryness and repeat the muffle furnace cycle. Repeat the procedure until the ashes are white or slightly coloured. Dissolve the ashing adding 2ml of Up-water (4.2) and 2ml of HNO 3 Sp (4.1) and put it on the hot plate slightly warming till all the ashes are dissolved. Page 4 of 10

5 Take to a final volume of about 10ml into plastic tubes. Treat the Procedure Blank in the same way of the samples. NOTICE: weigh Falcon tubes before transferring the digested solution and weigh them again after taking to the final volume 6. INSTRUMENTAL ANALYSIS 6.1 Determination of Cadmium Cd calibration solution: this solution shall be daily prepared c (Cd) = 0,025 µg/l, into a 1ml cuvette for instrumental analysis (example): pipette 1 ml of Up-water (4.2) take off 25µl add 25µl of Cd (5.4.1) mix the solution by pipetting several times a) please note: New tips were used and they were rinsed twice with Up-water, before the use. The micropipette used for mixing shall not be the same used for dilution, nor one used for sample/standard preparation purposes (e.g:. declassified micropipette and used only for mixing) Matrix modifiers Matrix Modifier Cd: 5g/l NH 4 H 2 PO g/l Mg(NO 3 ) 2 + TX 0.1% Example: pipette 2.5ml of NH 4 H 2 PO g/l (4.4.1), 2.5 ml of Mg(NO 3 ) 2 6g/l (4.5.1) and 0.5ml TX 1% (4.7.1) in a 50ml volumetric flask Calibration Standards In order to save enough solution for analysis, when preparing the calibration curve, it is recommended to mix little aliquots from each of the digested samples (pool of sample). Standards for calibration curve and scheme of preparation: Cd Standard Cd ( ) 0.025µg/l (µl) HNO3 20% v/v (4.1.1) (µl) Up-water (4.2) (µl) Blank Base Std 1 (0.25 µg/l) Std 2 (0.5 µg/l) Std 3 (0.75 µg/l) Std 4 (1.0 µg/l) Std 5 (1.5 µg/l) Page 5 of 10 Pool of sample (µl) Preparation of calibration standards: Blank: a cuvette for instrumental analysis (example): example: pipette 500 µl Up-water (4.2) µl of HNO 3 20% v/v (4.1.1) (µl) mix thoroughly with a dedicated micropipette (see a) Base: in a cuvette for instrumental analysis (example): pipette 500 µl Up-water (4.2) µl of HNO 3 20% v/v (4.1.1) (µl) +250 µl of Pool of sample mix thoroughly by pipetting (see a)

6 Standards 1, 3: in a 5ml falcon tube (example): pipette 1000 µl of Up-water (4.2) take off the amount of standard to be added (e.g. for standard 1, take off 20µl) add 500 µl of HNO 3 20% v/v (4.1.1) µl of Pool of sample add the calibration solution and mix thoroughly pipette 1ml of the obtained solution in a cuvette for instrumental analysis Standards 2, 4, 5: in a cuvette for instrumental analysis (example): pipette 500 µl of Up-water (4.2) take off the amount of standard to be added (e.g. for standard 2, take off 20µl) add 250 µl of HNO 3 20% v/v (4.1.1) +250 µl of Pool of sample add the calibration solution and mix thoroughly by pipetting (see a) Sample Dilution Minimum dilution 1:4: into a 1 ml cuvette for instrumental analysis (example): pipette 500µl of Up-water (4.2) add 250µl of HNO 3 20% (4.1.1) + 250µl of sample and mix thoroughly by pipetting (see a) Further dilutions, e.g. 1:10: into a 1 ml cuvette for instrumental analysis (example): pipette 500 µl of Up-water (4.2) add 400 µl of HNO 3 20% v/v (4.1.1) add 100 µl of sample and mix thoroughly by pipetting (see a) Drift Check Drift is checked by analyzing blank and first standard: at the end of an analysis, if the number of samples analyzed is less than 8; after each series of 8 samples; at the end of an analysis, if more than 3 samples have been run after the last check. If the instrumental drift is higher than 7%, repeat the calibration curve and read the previous samples up to the last acceptable check Spectrometer Settings Optimize the instrument in accordance with the recommendation described in the internal procedures. Set ashing and atomization temperature taking into account the instrument used and the ageing of the spare parts (tubes, contacts, furnace). Ashing and atomization temperature shall anyway fall in the following ranges: Ashing temperature: C. Atomization temperature: C. 6.2 Determination of Lead Pb calibration solutions: this solution shall be daily prepared (example): c(pb) = 250 µg/l, into a 1 ml cuvette for instrumental analysis pipette 750 µl of Up-water (5.3) add 250 µl of Pb (5.4.2) and thoroughly mix by pipetting (see a) Page 6 of 10

7 6.2.2 Matrix modifiers : 0.25g/l Pd + 0.2g/l Mg(NO 3 ) 2 Example: pipette 1.25ml of Pd (4.6), 1.7 ml of Mg(NO 3 ) 2 6g/l (4.5.1) in a 50ml volumetric flask Calibration standards In order to save enough solution for analysis, when preparing the calibration curve, it is recommended to mix little aliquots from each of the digested samples (pool of sample). Standards for calibration curve and scheme of preparation: Pb Standard Pb 250 µg/l ( ) (µl) HNO3 20% v/v (4.1.1) (µl) Up water (4.2) (µl) Pool of sample (µl) Blank Base Std 1 (2.5 µg/l) Std 2 (5 µg/l) Std 3 (10 µg/l) Std 4 (15 µg/l) Std 5 (20 µg/l) Preparation of calibration standard Blank: in a cuvette for instrumental analysis (example): pipette 500 µl Up-water (4.2) µl of HNO 3 20% v/v (4.1.1) and thoroughly mix by pipetting (see a) Base: in a cuvette for instrumental analysis (example): pipette 500 µl Up-water (4.2) µl of HNO 3 20% v/v (4.1.1) µl of Pool of sample and thoroughly mix by pipetting (see a) Standards 1: in a 5ml falcon tube (example): pipette 1000 µl Up water (4.2) take off the amount of standard to be added (20µl) add 500 µl of Up-water (4.2) µl of Pool of sample add the calibration solution and mix thoroughly pipette 1ml of the obtained solution in a cuvette for instrumental analysis Standards (2, 3, 4, 5) in a cuvette for instrumental analysis (example) pipette 500 µl of Up-water (4.2) take off the amount of standard to be added (e.g. for standard 3, take off 40µl) add 250 µl of HNO 3 20% v/v (4.1.1) µl of Pool of sample add the calibration solution and thoroughly mix by pipetting (see a) Sample Dilution Minimum dilution 1:4: into a 1 ml cuvette for instrumental analysis (example): pipette 500 µl of Up-water (4.2) add 250 µl of HNO 3 20% v/v (4.1.1) add 250 µl of sample and thoroughly mix by pipetting (see a) Further dilutions, e.g. 1:10: into a 1 ml cuvette for instrumental analysis (example) : pipette 500 µl of Up-water (4.2) Page 7 of 10

8 add 400 µl of HNO 3 20% v/v (4.1.1) add 100 µl of sample and thoroughly mix by pipetting (see a) Drift Check Drift is checked by analyzing blank and the first standard: at the end of an analysis, if the number of samples analyzed is less than 8; after each series of 8 samples; at the end of an analysis, if more than 3 samples have been run after the last check. If the instrumental drift is higher than 10%, repeat the calibration curve and read the previous samples up to the last acceptable check Spectrometer Settings Optimize the instrument in accordance with the recommendation described in internal procedures. Set ashing and atomization temperature taking into account the instrument used and the ageing of the spare parts (tubes, contacts, furnace). Ashing and atomization temperature shall anyway fall in the following ranges: Ashing temperature: C. Atomization temperature: C. 7. CALCULATION Calculate the content, w, of the chemical element as mass fraction mg/kg as follows: w = D a V m 1000 where: a = is the concentration of the element in the test solution in μg/l V = final volume of the solution in ml m = weight of the sample in g Please note: the concentration of procedure blank is not considered in the final calculation. Its value in the procedural digested blank was for Cd <0.1 µg/l and for Pb <1 µg/l. Calculate V as follows: V= (Pf-Pi)/d where: Pi = weight of the empty falcon tubes Pf = weight of the falcon tubes containing the digested solution d = weight of 1 ml of digested solution (please note, if you perform this procedure after digestion, allow samples to reach room temperature) 8. EXPRESSION OF RESULTS Express the final result, always considering the average of three independent sample preparations. Page 8 of 10

9 PARAMETERS OF THE EURL CEFAO METHOD LoD and LoQ were determined using 21 aliquots of a sample of commercial infant formula. Repeatability was evaluated using a powdered infant formula spiked with Cd and Pb and then freeze-dried: 12 aliquots were used. The recovery was performed on milk sample of the 14th PT 2nd Round, quantified using the calibration curve prepared in the pool of samples of the infant formula. The parameters obtained during the method development are summarized as follows: Parameter Cd Pb LoD (=3*SD) µg/kg LoQ (=10*SD) µg/kg 2 12 RSDr % Recovery % on 14 th PT, 2 nd round FINAL CONSIDERATIONS This method has been developed on the basis of the EU-RL CEFAO experience on GF-AAS technique. As for Pb, the use of the Tube THGA with end-caps allows the achievement of a suitable sensitivity. The external calibration approach is possible for Cd, but it is necessary to investigate the effect of acidity/matrix. Page 9 of 10

10 Annex 1: Instrumental Settings The annexes are the instrumental report in paper form Annex 1a: Cd (4 pages) Annex 1b: Pb (4 pages) Page 10 of 10

11 Method Name: Cd_inf_form_add Define Element Method Description : Milk dry ashing + infant formula Spectrometer Element : Cd Wavelength (nm) : Slit Width (nm) : 0.7L Signal Type : AA - BG Measurement : Peak Area Settings Read Parameters Time (sec) : 3.0 Delay Time (sec) : 0.0 BOC Time (sec) : 2 Replicates... Same for All Samples : 3 Lamp Current Use value entered in Lamp Setup window Furnace Program Step Temp( C) Ramp Time Hold Time Internal Flow Gas Type Normal Normal Normal Normal Normal Read Step : 4 Injection Temperature( C) : 20 No extra furnace cleanout. Furnace Autosampler Sample Volume : 20 ul Diluent Volume : 0 ul Diluent Location : 1 Matrix Modifiers #1 Volume : 5 ul Location : 40 Add to calibration blank and standards. Add to reagent blank and samples. #2 Volume : 0 ul Location : 1 Add to calibration blank and standards. Add to reagent blank and samples. Autosampler and Furnace Sequence Step Actions and Parameters A Pipet diluent + modifier1 + spike + sample/std B Run furnace steps 1 to end Pipet Speed... Take up : 100% Dispensing : 100% Page 1 12/06/ :56:20

12 Method Name: Cd_inf_form_add Calibration Equation and Units Equation : Method of Additions Calibrate Maximum Decimal Places : 3 Maximum Significant Figures : 4 Analyst-prepared additions. Calibration Units : ug/l Sample Units : ug/l Calibration Standard Concentrations ID Concentration A/S Loc Stock(uL) Diluent(uL) Calib Blank Bianco 1 20 Addition 1 st 0.25 ppb Addition 2 st 0.5 ppb Addition 3 st 0.75 ppb Addition 4 st 1.00 ppb Addition 5 st 1.50 ppb Initial Calibration Options When opening this method manually: Clear calibration curve(s) and construct a new calib curve. When using this method in a multimethod sequence: Start by constructing new calibration curves. Calibration Check Correlation coefficient checking is not enabled in this method. Recalibration Periodic recalibration is not enabled in this method. Analyze standards at end of analysis is not enabled in this method. Precision Checks Precision checking is not enabled in this method. Beyond Calibration Range Beyond calibration range checking is not enabled in this method. Matrix Recovery Matrix recovery checking is not enabled in this method. Automatic Recovery Page 2 12/06/ :56:20

13 Method Name: Cd_inf_form_add Automatic recovery checking is not enabled in this method. QC Sample Definition A/S Count as Subtract QC Sample ID Loc Sample Reagent Blank QC Sample Concentrations and Limits Schedule for QC Analyses After After QC Sample ID Init Cal Recal Periodic Freq At End Periodic Timing of Analyses Frequency... Same for all QC's : 1 Count : Samples Failure Actions for After-Calibration QC's Times to When All Additional QC Sample ID Retry QC Tries Fail Message Failure Actions for Periodic QC's Times to When All Additional QC Sample ID Retry QC Tries Fail Message Failure Actions for At-End QC's Times to When All Additional QC Sample ID Retry QC Tries Fail Message Maximum Retries After QC Failure After a group of standards or unknowns has been reanalyzed 1 times, then Continue Options Include in Results Display and Printed Log: Headers: * Analytical Header * Method Header (Short) * Sample Header (Short) Start each sample on a new page Sample Data Items: Summary Items: * Replicate Data Analysis List * Means and Statistics * Matrix Test Reports Transient Peak Plots (First) * Calibration Summary * Calibration Curves Save with Results: * Transient Peak Profiles Page 3 12/06/ :56:20

14 Method Name: Cd_inf_form_add Remarks: Mod: NH4H3PO4 5g/l + Mg(NO3)2 0.3 g/l + Triton X 0.1% Standards prepared in HNO3 20% diluted 1:2 with up water. Samples diluted at least 1:4 (500 ul H2O ul HNO3 20% ul sample) Page 4 12/06/ :56:20

15 Method Name: Pb_milk_infant_form Define Element Method Description : Milk dry ashing Spectrometer Element : Pb Wavelength (nm) : Slit Width (nm) : 0.7L Signal Type : AA - BG Measurement : Peak Area Settings Read Parameters Time (sec) : 3.0 Delay Time (sec) : 0.0 BOC Time (sec) : 2 Replicates... Same for All Samples : 3 Lamp Current Use value entered in Lamp Setup window Furnace Program Step Temp( C) Ramp Time Hold Time Internal Flow Gas Type Normal Normal Normal Normal Normal Normal Read Step : 5 Injection Temperature( C) : 20 No extra furnace cleanout. Furnace Autosampler Sample Volume : 20 ul Diluent Volume : 0 ul Diluent Location : 1 Matrix Modifiers #1 Volume : 5 ul Location : 40 Add to calibration blank and standards. Add to reagent blank and samples. #2 Volume : 0 ul Location : 1 Add to calibration blank and standards. Add to reagent blank and samples. Autosampler and Furnace Sequence Step Actions and Parameters A Pipet diluent + modifier1 + spike + sample/std B Run furnace steps 1 to end Pipet Speed... Take up : 100% Dispensing : 100% Page 1 12/06/ :57:41

16 Method Name: Pb_milk_infant_form Calibration Equation and Units Equation : Method of Additions Calibrate Maximum Decimal Places : 3 Maximum Significant Figures : 4 Analyst-prepared additions. Calibration Units : ug/l Sample Units : ug/l Calibration Standard Concentrations ID Concentration A/S Loc Stock(uL) Diluent(uL) Calib Blank Bianco 1 20 Addition 1 st 2.5 ppb Addition 2 st 5 ppb Addition 3 st 10 ppb Addition 4 st 15 ppb Addition 5 st 20 ppb Initial Calibration Options When opening this method manually: Clear calibration curve(s) and construct a new calib curve. When using this method in a multimethod sequence: Start by constructing new calibration curves. Calibration Check Correlation coefficient checking is not enabled in this method. Recalibration Periodic recalibration is not enabled in this method. Analyze standards at end of analysis is not enabled in this method. Precision Checks Precision checking is not enabled in this method. Beyond Calibration Range Beyond calibration range checking is not enabled in this method. Matrix Recovery Matrix recovery checking is not enabled in this method. Automatic Recovery Page 2 12/06/ :57:41

17 Method Name: Pb_milk_infant_form Automatic recovery checking is not enabled in this method. QC Sample Definition A/S Count as Subtract QC Sample ID Loc Sample Reagent Blank QC Sample Concentrations and Limits Schedule for QC Analyses After After QC Sample ID Init Cal Recal Periodic Freq At End Periodic Timing of Analyses Frequency... Same for all QC's : 1 Count : Samples Failure Actions for After-Calibration QC's Times to When All Additional QC Sample ID Retry QC Tries Fail Message Failure Actions for Periodic QC's Times to When All Additional QC Sample ID Retry QC Tries Fail Message Failure Actions for At-End QC's Times to When All Additional QC Sample ID Retry QC Tries Fail Message Maximum Retries After QC Failure After a group of standards or unknowns has been reanalyzed 1 times, then Continue Options Include in Results Display and Printed Log: Headers: * Analytical Header * Method Header (Short) * Sample Header (Short) Start each sample on a new page Sample Data Items: Summary Items: * Replicate Data Analysis List * Means and Statistics * Matrix Test Reports Transient Peak Plots (Last) * Calibration Summary Calibration Curves Save with Results: Page 3 12/06/ :57:41

18 Method Name: Pb_milk_infant_form * Transient Peak Profiles Remarks: Mod.: Pd(NO3) g/l + Mg(NO3)2 0.2 g/l THGA Tube with end caps. Standard prepared in HNO3 20% diluted 1:2 with up water. Samples diluted at least 1:4 (500 ul H2O ul HNO3 20% + 250ul sample). Page 4 12/06/ :57:41