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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.

Single-Laboratory Validation (SLV)

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,

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

Table 1. Method performance requirements:

trans

-vitamin K

1

a

Analytical range

1–100

b

LOQ

≤1

b

RSD

r

, %

1–10

b

≤8

>10

b

≤5

Recovery, %

90 to 110 of mean spiked recovery

over the range of the assay

RSD

R

, %

1–10

b

≤15

>10

b

≤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

Sample type

No. of replicates

(duplicates on multiple days)

Mean,

µg/100 g RTF SD

r

RSD

r

, % SD

IP

RSD

IP

, %

Child formula powder, placebo

6

2.03

0.040

2.0

0.045

2.2

Infant elemental powder, placebo

6

2.03

0.020

1.0

0.025

1.2

Adult nutritional RTF high protein, placebo

6

3.47

0.021

0.6

0.040

1.2

Adult nutritional RTF high fat, placebo

6

3.07

0.034

1.1

0.034

1.1

Infant formula RTF milk based, placebo

6

2.16

0.038

1.8

0.038

1.8

SRM 1849a

12

1.11

a

0.022

2.0

0.025

2.3

Adult nutritional powder milk protein based

12

3.26

0.097

3.0

0.142

4.4

Infant formula powder partially hydrolyzed milk based

12

7.69

0.123

1.6

0.170

2.2

Infant formula powder partially hydrolyzed soy based

12

8.99

0.141

1.6

0.226

2.5

Adult nutritional powder low fat

12

2.92

0.102

3.5

0.102

3.5

Child formula powder

12

2.66

0.049

1.8

0.050

1.9

Infant elemental powder

12

7.57

0.129

1.7

0.451

6.0

Infant formula powder milk based

12

6.09

0.105

1.7

0.131

2.2

Infant formula powder soy based

12

6.26

0.105

1.7

0.211

3.4

Infant formula RTF milk based

12

9.01

0.168

1.9

0.189

2.1

Adult nutritional RTF high protein

16

9.10

0.283

3.1

0.299

3.3

Adult nutritional RTF high fat

12

10.7

0.120

1.1

0.179

1.7

a

 Results reported as mg/kg powder.

92