6. AOACSPIFANMethods-2018Awards

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the standard’s nominal concentration). The inclusion of a PB (run as a sample; its measured concentration must be <1/2 of the lowest calibration standard), a duplicate sample (relative difference within 10% for Cr, 7% for Se, and 5% for all other elements), and known reference materials serving as control samples (recovery check within control or certified limits) are mandatory for good method performance. If any of these QC checks fails, results should be considered invalid. (c)  The order of analysis should be calibration standards, followed by rinse, blank check (PB run as a sample), check standard, control sample, sample, sample duplicate (up to 10 samples), and finally a repeated check standard. G. Calculations Sample concentrations in ng/g are automatically calculated by the software using a nonweighted least-squares linear regression calibration analysis to produce a best-fit line: = + a blank Y x Note that for the Agilent software used in this work, the sample blank is identical to the Cal Blk and is essentially zero because high purity reagents are used. The analyte concentration in the sample is then calculated: = − × x y blank a DF where x = analyte concentration (ng/g); y = analyte to ISTD intensity ratio, which is the measured count of each analyte’s standard solution data point in the calibration curve divided by the counts of the ISTD at the same level; similarly, the blank = analyte to ISTD intensity ratio, which is the measured count of the blank standard solution data point in the calibration curve divided by the counts of the ISTD at the same level as the blank standard solution; a = slope of the calibration curve (mL/ng); and DF = volume of the sample solution (mL) divided by sample weight (g). H. Method Validation This method has undergone a thorough single-laboratory validation (SLV) using AOAC guidelines to probe its linearity, LOQ, specificity, precision, accuracy, and ruggedness/ robustness. Accuracy has also been affirmed by comparison to ICP-atomic emission spectrometry (AES) results generated in the authors’ own laboratory. In addition, reproducibility was estimated during a limited multilaboratory testing (MLT) study employing six laboratories and four different ICP/MS instruments. Both the SLV and MLT results are summarized in a concurrent publication (6). Specificity The specificity of the method was determined using a single element standard at 50 mg/L for each analyte and checking for apparent signal from the other analytes. None of the standards produced a response above the PLOQ for any of the other 11 analytes (data not shown), demonstrating that each response is Results and Discussion

specific for that analyte. The ISTDs were not tested since they are used at a low concentration of 50 μg/L.

Linearity

Linearity was demonstrated by analyzing various independent standards (made from the same stock) as samples against the normal calibration curve. Linearity standards at nine concentrations of each analyte spanning the range from 50% of the lowest calibration standard to 50% above the highest calibration standard were analyzed twice on each of 3 days using freshly made standards each day. The means of all six analyses are reported in Table 2. At the lowest level, 50% of the lowest calibration standard, all analytes demonstrated acceptable agreement (95–105%, with rounding) with the nominal value. Therefore, 50% of the lowest calibration standard concentration is set as the PLOQ. Overall, the recoveries varied from 91 to 107%, and RSDs varied from 0.3 to 9.3%. The recoveries were nearly all within a desired 95–105% range, though there are no specific criteria in the SMPR for linearity. The only elements that presented any linearity issues were P and Fe, which were routinely under-recovered (P) or over-recovered (Fe) by about 5–6% across the calibration curve. Possibly, the linearity could be improved by adjusting some factors for the analysis of these elements, as they both have relatively low mass with significant background interferences that must be handled by the CRC. In practice, no accuracy issues were observed except for some apparent bias in P results relative to SRM 1849a ( see below). Typical correlation coefficients were 0.9995 or better for all analytes. The PLOQ values from the linearity experiment were converted from a solution concentration (mg/L) to a weight basis (mg/100 g for a typical dilution of 1.0 g RTF to 50 mL) and compared to the SMPR ( see Table 3). The PLOQs meet the SMPR for all elements except Fe, Cu, and Mn. In these cases, the test portion size could be increased to 2–3 g RTF to improve the PLOQ 2–3-fold lower. The lowest concentrations of Mn, Cu, and Fe found in the SPIFAN matrixes were 150 ng/g (0.015 mg/100 g), 580 ng/g (0.058 mg/100 g), and 14000 ng/g (1.4 mg/100 g), respectively, all in the SPIFAN control milk. SMPR for LOQ for Mn, Cu, and Fe are 0.001, 0.001, and 0.01 mg/100 g, respectively, at least 10-fold lower than observed values. LOQ SPIFANmatrixes were tested on 8 days (including two analysts and two instruments) in duplicate, and the results are summarized in Table 4. The SMPRs require RSD r to be ≤5% in all 11 matrixes. All analytes in all matrixes meet this criterion for the within-day duplicates (data not shown), typically in the 1–2% range. This requirement is built into the method due to the criterion that duplicate results must agree to within 5%. When considering intermediate reproducibility precision (among days/analysts/ instruments, but in a single laboratory), of the 12 elements and 11 matrixes, there are 11 instances of RSD iR >5%. Ten of these are for the ultratrace elements, Mo and Cr, and there is one instance for Ca in Adult RTF with high fat. The Adult RTF with high fat matrix has since been shown to be unstable and perhaps Precision