6. AOACSPIFANMethods-2018Awards

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Bidlack et al.: J ournal of AOAC I nternational V ol. 98, N o. 5, 2015  1383

RP chromatography, and it has been shown to under-recover vitamin K in some more complex infant, pediatric, and adult nutritional matrixes (5, 6). This new NP HPLC method with postcolumn reduction and fluorescence detection allows for the quantitative determination of trans vitamin K 1 in infant, pediatric, and adult nutritionals. Vitamin K 1 is extracted from products with iso-octane after precipitation of proteins and release of lipids with methanol. Prepared samples are injected onto a silica HPLC column where cis and trans vitamin K 1 are separated with an iso-octane– isopropanol mobile phase. The column eluent is mixed with a dilute ethanolic solution of zinc chloride, sodium acetate, and acetic acid, and vitamin K 1 is reduced to a fluorescent derivative in a zinc reactor column. The resulting hydroquinone is then detected by fluorescence at an excitation wavelength of 245 nm and an emission wavelength of 440 nm. Experimental To verify the applicability of this method, an SLV with all 12 SPIFAN infant, pediatric, and adult matrixes was completed. To establish method precision, all fortified and unfortified matrixes were prepared and analyzed in duplicate on 6 days. National Institute of Standards and Technology Standard Reference Material (SRM) 1849a (Infant/Adult Nutritional Formula) was reconstituted by dissolving the entire contents of the sachet (10 g) in 90 mL water. All other powders were reconstituted by dissolving 25 g powder in 200 mL laboratory water. New reconstitutions were prepared each day. Method accuracy was established by spiking aliquots of each SPIFAN matrix with vitamin K 1 at approximately 50 or 100% of the previously determined trans vitamin K 1 level. Approximately 40–250 µL vitamin K 1 , dissolved in ethanol, Single-Laboratory Validation (SLV)

Table 1. Method performance requirements: trans -vitamin K 1 a Analytical range 1–100 b

≤1 b

LOQ RSD r

1–10 b

, %

≤8 ≤5

>10 b

Recovery, %

90 to 110 of mean spiked recovery over the range of the assay

1–10 b

, %

≤15

RSD R

>10 b

was added to 25 g aliquots of each SPIFAN matrix, and all sample and spike weights were recorded. On 2 days each sample matrix was spiked at 100% of the previously determined trans vitamin K 1 level, and on the third day each matrix was spiked at 50%. On each day spike blanks were also prepared by adding 75–250 µL vitamin K 1 , dissolved in ethanol, to 25 mL iso-octane. Spiked samples and blanks were thoroughly mixed and stored refrigerated for at least 24 h to allow vitamin K 1 incorporation into the sample matrix. After at least 24 h, spiked and unspiked samples were prepared and analyzed in duplicate as described in the method. Spike blanks were diluted to appropriate concentrations with iso-octane and analyzed along with the spiked and unspiked sample preparations. Method linearity was evaluated by injecting five or six standards with trans vitamin K 1 concentrations ranging from approximately 2 to 90 µg/L before and after every set of samples analyzed during validation. Calibration curves were ≤10 a  Concentrations apply to (a) RTF liquids “as is”, (b) reconstituted powders (25 g into 200 g of water), and (c) liquid concentrates diluted 1:1 by weight. b  µg/100 g reconstituted final product.

Table 2.  Trans vitamin K 1

SLV data—precision

No. of replicates (duplicates on multiple days)

Mean, µg/100 g RTF SD r

Sample type

RSD r

, % SD IP

RSD IP

, %

Child formula powder, placebo Infant elemental powder, placebo

6 6 6 6 6

2.03 2.03 3.47 3.07 2.16 1.11 a 3.26 7.69 8.99 2.92 2.66 7.57 6.09 6.26 9.01 9.10 10.7

0.040 0.020 0.021 0.034 0.038 0.022 0.097 0.123 0.141 0.102 0.049 0.129 0.105 0.105 0.168 0.283 0.120

2.0 1.0 0.6 1.1 1.8 2.0 3.0 1.6 1.6 3.5 1.8 1.7 1.7 1.7 1.9 3.1 1.1

0.045 0.025 0.040 0.034 0.038 0.025 0.142 0.170 0.226 0.102 0.050 0.451 0.131 0.211 0.189 0.299 0.179

2.2 1.2 1.2 1.1 1.8 2.3 4.4 2.2 2.5 3.5 1.9 6.0 2.2 3.4 2.1 3.3 1.7

Adult nutritional RTF high protein, placebo Adult nutritional RTF high fat, placebo Infant formula RTF milk based, placebo

SRM 1849a

12 12 12 12 12 12 12 12 12 12 16 12

Adult nutritional powder milk protein based

Infant formula powder partially hydrolyzed milk based Infant formula powder partially hydrolyzed soy based

Adult nutritional powder low fat

Child formula powder Infant elemental powder

Infant formula powder milk based Infant formula powder soy based Infant formula RTF milk based Adult nutritional RTF high protein Adult nutritional RTF high fat a  Results reported as mg/kg powder.