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nternational
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99, N
o
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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