AOACSPIFANMethods-2017Awards

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1400  Jing et al. : J ournal of AOAC I nternational Vol. 98, No. 5, 2015

10.93% (w/w) reconstitution and spiked with a known amount of choline (100 µL of a 593.4 µg/mL solution = 59.3 µg, as choline hydroxide in this case) and carnitine (61 µL of a 77.9 µg/mL solution = 4.75 µg). These spike amounts are at the SMPR required LOQ limit: approximately 60 µg/3.0 g RTF = 20 µg/g = 2.0 mg choline hydroxide/100 g RTF; 4.75 µg/3.0 g RTF = 0.16 mg/100 g RTF. The placebo was analyzed seven times to establish a baseline value, and then seven spiked placebos were analyzed and compared to the baseline to determine spike recovery. A spike recovery of 90–110% would establish the spike level as at, or above, the required LOQ. ( c )  Accuracy (trueness).— SRM 1849a was analyzed with this method, and results were compared to certified values for both choline and carnitine. The 11 SPIFAN matrixes were also spiked with known amounts of choline and carnitine at two different levels, 40 and 80% of the baseline product level, to ensure spike recoveries were in the 90–110% range as required by the SMPR. The spiking was performed on 3 different days, and duplicate spike samples were prepared on each day, at each spiking level. For carnitine, the whole experiment was repeated with acetylcarnitine (Carbosynth LLC, >98% purity, San Diego, CA) instead of L-carnitine, to prove reduction of that species to carnitine with the method’s basic hydrolysis. Finally, accuracy of this method was established by comparison of choline results to independent methods. AOAC 999.14 results were obtained from a commercial laboratory (Covance Laboratories, Madison, WI) over 2 days, in duplicate. AOAC 2012.20 results were obtained from the Thermo/Dionex website (5). ( d )  Precision .—Each of the 11 SPIFAN products was analyzed by the method on 6 days in duplicate, for both choline and carnitine, running the method for both free analyte (no microwave digestion or subsequent basic hydrolysis) and total choline/carnitine. The data were collected by two analysts on two different instruments (Waters Acquity TQD, and Xevo TQS), splitting the days evenly among the analyst/instruments. The repeatability precision and the intermediate precision were calculated from the resulting data using analysis of variance calculations from Microsoft (Redmond, WA) Excel. ( e )  Specificity .—Secondary ion transitions were monitored throughout the validation and assessed afterwards for the specificity information they may provide. Chromatography .—Example chromatograms for the SRM 1849a analysis are shown in Figure 1. Choline and its IS elute first at about 2.5 min followed by the carnitine and its IS at about 4.4 min. Four additional traces are shown in Figure 1 representing the secondary ion transitions that were followed for specificity verification. These data are discussed at the end of this report. Linearity .—The lowest correlation coefficient observed over the course of the study was 0.9992 for either choline or carnitine. Table 3 shows the results of the 3-day linearity study. The average recovery of independent standard solutions run as samples was 99.1–101.2% at various points along the calibration curve, meeting SMPRs. Note that the method should not be used outside this proven region of linearity. Thus, the lowest concentrations checked (3.4 µg/L carnitine and 30 µg/L choline) become the practical LOQ for the method, even if the Validation Results

Table 3. Linearity results a Nominal carnitine, µg/L

Recovery, % (avg. 3 days)

RSD, %

3.4 6.9

101.2

1.8 1.2 1.1 1.5 0.7 1.5 2.0 1.7 2.2 1.4 2.4

99.3

17.2 34.5 51.7 68.9 29.7 59.3 119 237 356 510

100.3 100.0

99.4

100.1

Nominal choline, µg/L

Recovery, % (avg. 3 days)

RSD, %

100.3

99.6

100.3

99.3

100.1

99.4 1.1 a  Calibration curve was 7.5–75 µg/L carnitine and 30–300 µg/L choline.

( d ) For this analysis (for either choline or carnitine), given the mass of IS added to the samples and the concentration of IS in the standards, the DF can be shown to be: DF = 3.125/sample weight (g) (2) ( e ) Thus, the final result (C x ) can be calculated by combining Equations 1 and 2. An SLV protocol was carried out to ensure that the method meets the requirements of the SMPRs for choline (SMPR 2012.013 ) and carnitine (SMPR 2012.010 ). The SMPRs are summarized in Table 1. The full suite of 11 “SPIFAN matrixes” were used in this SLV. These materials (Table 2 ) were made by the manufacturers specifically for the SPIFAN project to be representative of nearly the entire body of infant and adult nutritional products. By SPIFAN convention, the powders were all prepared as reconstitutions (11.1% by weight), and the indicated aliquot sizes were taken. The three liquid/ready- to-feed (RTF) samples were weighed as-is. The SLV probed the usual parameters of linearity, LOQ/LOD, repeatability, intermediate precision, spike recovery (for accuracy), and agreement of results to independent methods or SRMs. ( a )  Linearity .—On each of 3 different days, calibration curves were prepared for choline and carnitine, and then five independently made standard solutions of differing concentrations were injected as samples. The standard solutions covered the range of the calibration curve from 30 to 300 µg/L choline and from 8 to 80 µg/L carnitine, except the carnitine linearity test included a solution at ½ WS1 and the choline linearity test included a solution that was well above WS3 in concentration. The 3-day mean concentration results from these standard solutions were compared to their nominal concentrations, with an expected agreement of better than ±5%. ( b )  LOD/LOQ. —The adult powder was selected as a suitable placebo in lieu of other possible placebos included in the SPIFAN materials kit. The placebo was weighed at 3.00 g of a Validation Protocol