Table of Contents Table of Contents
Previous Page  106 / 258 Next Page
Information
Show Menu
Previous Page 106 / 258 Next Page
Page Background

2

S

alvati

et al

.:

J

ournal of

AOAC I

nternational

V

ol

.

99, N

o

.

3, 2016

containing compounds tend to tail considerably with conventional

chromatographic equipment because of stainless steel plumbing

and column frits (8). Further, the phosphorylated forms of these

vitamins tend to be a small fraction of the free-form thus putting

pressure on accuracy of quantitation. Instead of trying to overcome

the challenges of direct analysis of the phosphorylated forms,

this method includes an overnight enzymatic digestion with acid

phosphatase to hydrolyze the phosphate yielding the free vitamin

forms (6). Papain and α-amylase are also included in the enzymatic

cocktail to digest protein and complex carbohydrate, respectively,

thus aiding reduction in sample extract complexity (9).

Method performance was demonstrated in SPIFAN II

matrixes. SPIFAN II matrixes are a range of infant formula and

adult nutritionals prepared by a number of the infant formula

manufacturers to enable single- and multilaboratory validation

of methods. SPIFAN II matrixes include partially hydrolyzed,

milk-based infant formula powder; partially hydrolyzed, soy-based

infant formula powder; milk-based toddler formula powder; two

milk-based infant formula powders; a soy-based infant

formula powder; child formula powder; elemental (extensively

hydrolyzed) infant formula powder; infant formula powder with

fructooligosaccharides and galactooligosaccharides; milk-based,

ready-to-feed (RTF) infant formula; low fat, adult nutritional

powder; high protein, RTF adult nutritional; and high fat, RTF

adult nutritional. The matrixes also include the National Institute

of Standards and Technology (NIST) Standard Reference

Material (SRM) for infant formula, SRM 1849a; and five,

unfortified, placebo matrixes matching a select subset of those

previously mentioned: partially hydrolyzed, milk-based infant

formula powder; partially hydrolyzed, soy-based infant formula

powder; milk-based, RTF infant formula; high protein, RTF adult

nutritional; and high fat, RTF adult nutritional. In these matrixes,

precision averaged 3.9%, and over-spike recovery was generally

95–105%. Both free and phosphorylated forms were over-spiked

as part of the recovery experiment. The method was demonstrated

to meet all SPIFAN SMPR.

AOAC Official Method 2015.14

Simultaneous Determination of Total Vitamins B

1

,

B

2

, and B

6

in Infant Formula and Related Nutritionals

Enzymatic Digestion and LC-MS/MS

First Action 2015

[Applicable for simultaneous quantitation of total B

1

, B

2

,

and B

6

in infant formula]

A. Principle

This method facilitates simultaneous quantitation of three

water-soluble vitamins in infant formula and related nutritional

products, including all SPIFAN SMPR relevant forms of vitamins

B

1

, B

2

, and B

6

. Samples are prepared by enzymatic digestion

with papain and α-amylase to hydrolyze protein and complex

carbohydrate and acid phosphatase to free phosphorylated vitamin

forms. Stable-isotope labeled internal standards are incorporated

into the sample preparation to correct for variability in both the

sample preparation and instrument response. A series of six

mixed working standard (MWS) solutions spanning two orders

of magnitude in vitamin concentration are used to generate

calibration curves based on the peak response ratio of the analyte

to its stable-isotope labeled internal standard.

Prepared samples and working standard solutions are

injected onto an ultra-high pressure liquid chromatograph

(UPLC) interfaced to a triple-quadrupole mass spectrometer

for analysis. The mass spectrometer is configured to monitor

parent–daughter (precursor–fragment) ion pairs for each analyte

and internal standard. This reaction forms the basis for method

selectivity. Analytes are quantified by least-squares regression

using the response ratio of the analyte to its internal standard.

B. Apparatus and Materials

(a)

Control sample.

—NIST SRM 1849a, or current lot.

Store at 4°C. National Institute of Standards and Technology,

Gaithersburg, MD.

(b)

Waters Acquity BEH C18 column.

—2.1 × 100, 1.7 μm.

Part No. 186002352. Waters Corp., Milford, MA

(c)

UPLC system.

—Waters Acquity Classic, or equivalent.

Waters

(d)

Tandem quadrupole mass spectrometer with ESI

probe.

—Waters Xevo TQ-S, or equivalent. Waters

(e)

Analytical balances

(

3

)

.

—(

1

) One capable of accurately

weighing 5.00 mg (for standards), six-place balance.

(

2

) An analytical five-place balance for samples.

(

3

) A top loading two-place balance capable of weighing to

several hundred grams.

(f)

Water purifier.

—Millipore Milli-Q Water Purification

System, or equivalent. EMD Millipore, Billerica, MA.

(g)

Reciprocal shaking bath capable of maintaining 37°C.

(h)

Bottle-top dispenser.

—Capable of dispensing volumes of

approximately 24 mL.

(i)

pH meter.

—Capable of measuring a pH of 4.0–5.0.

(j)

 Karl Fischer for moisture determination in the thiamin

hydrochloride United States Pharmacopeia (USP) standard.

Metrohm AG, Riverview, FL.

(k)

Oven.

—For drying riboflavin USP standard at 105°C.

(l)

Vacuum chamber.

—For drying pyridoxine hydrochloride

USP standard.

(m)

Vortex mixer.

(n)

Multiposition magnetic stir plate.

(o)

Teflon coated, magnetic stir bars.

(p)

Hot plate with magnetic stirring.

(q)

Room light shields.

—A.L.P. Protect-A-Lamp, UV cutoff

at 460 nm, or equivalent. A.L.P Lighting Components, Inc.

Chicago, IL.

(r)

Graduated cylinders.

—Various sizes, including 100,

500, 1000, and 2000 mL.

(s)

Beakers.

—Various sizes, including 100, 200, 400, 600,

1000, and 2000 mL.

(t)

Volumetric flasks.

—Various sizes, including 10, 25, 50,

100, 250, and 2000 mL.

(u)

Mobile phase bottles.

—Glass, various sizes, including

250, 500, 1000, and 2000 mL.

(v)

Disposable, plastic Pasteur pipets.

(w)

Amber bottles.

—Volume capacity of 50 and 100 mL (for

stock standard storage).

(x)

Weighing vessels.

—Various, including disposable

weighing boats and glass weighing funnels.

(y)

Positive displacement pipets.

—Gilson Microman: 10,

100, 250, and 1000 μL; Part Nos. F148501, F148504, F148505,

and F148506. Gilson, Inc. Middleton, WI.

(Z)

Positive displacement pipet tips.

—Gilson Capillary

Piston: 10, 100, 250, and 1000 μL; Part Nos. F148312, F148314,

F148014, and F148560. Gilson, Inc.

(aa)

Plastic syringes.

—3 mL.

106