AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

Fluor-03 (Feb. 2019) FOR ERP USE ONLY DO NOT DISTRIBUTE

H. IC analysis (a) IC and dialysis parameters.— See paragraph G for instrument operating conditions and dialysis setup, respectively. (b) Instrument startup .—Prepare the mobile phases A and B, UPW, MeOH rinsing solution, and the suppressor regenerant (see paragraph D for preparation). Turn on the IC system and equilibrate for at least 30 min. Verify that the detector is stable before starting the analysis. (c) Standard and sample analysis. —Subsequent system equilibration run at least three UPW blank samples to equilibrate the system with the gradient conditions. Afterwards start with the calibration series. Inject one standard at each concentration level ( Table 2 ). Run a blank sample (UPW) afterwards and a set of four check standards in duplicate (see Table 3 ). The check standards should be repeated every 12 h and at the end of each sample series (analytical batch). Each nested sample run takes approximately 65 and involves the dialysis and transfer time, the signal recording of 10 min, followed by the column cleaning step using the eluent gradient ( Table 5 ) and the sample path cleaning (including autosampler needle and dialysis cell) with MeOH (50%) and UPW. By UPW blanks carry-over can be checked. (d) System shutdown. —After all samples and standards of the analytical batch have been analyzed, inject several UPW blanks for a final system rinse. Despite the automatic cleaning, run a 20% MeOH mixture for the final instrument cleaning and store the analytical columns in the manufacturer recommended standard mobile phase. I. Quality Control (a) Quality control. —Validated check standards ( Table 3 ) are used to check the calibration as described above. Inject at least two replicates of the check standards at the beginning and the end of each sequence (low, high, and mid-range of calibration) and run if necessary additional check standards every 12 h. Spike recoveries are evaluated according to the AOAC recommended guidelines [2-4] (see method validation). The dialysis set-up can be controlled by running check standards with transfer time variation (see G.). J. Calculation of sample concentration Before calculating the fluoride concentrations in samples compare the fluoride standard peaks with the fluoride sample peaks and confirm their retention time. The fluoride retention time should be around 7.5±0.5 min depending on the individual analytical column. Furthermore, the peak height of the samples should be within the range of the peak heights determined for the calibration standards. Else, the samples need to be diluted or run with a higher sample weight amount for a less intense dilution. (a) Calibration. —The standards for the calibration curve are injected at the beginning of each sample series (analytical batch). The nine standards cover a concentration range of 5 to 1500 μg/kg, spanning an analytical range based on 2.5 g sample weight in a total volume of 50 g of 10 to 3000 µg/ 100 g final product ( Table 2 ). To achieve a higher accuracy, the calibration is split into a high level calibration (anions high) and a low level calibration (anions low) (see Table 2 ). The quantitative evaluation is performed via peak-height on base of a horizontal baseline (see Figure 4 and Figure 5 for a typical standard chromatogram and the overlay of all standards). Prepare the standard curve using an unweighted quadratic function (intercept = 0 for the low calibration level) on the concentrations versus the determined standard peak heights (see Figure 6 ). The obtained calibration curve should have acceptable correlation coefficient of r 2 ≥ 0.9999. Calibration curve residuals (relative error) must be ≤2% for both calibration levels [2] .

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