AOACSPIFANMethods-2017Awards

ostetler

ournal of

AOAC I

nternational

100, N

3, 2017

liquids, 25 g sample plus 200 g water for powder samples, or

diluted 1:1 by weight for liquid concentrates] in accordance

with

Standard Method Performance Requirement

(SMPR

)

2014.014 (21). The test sample concentrations calculated in

H(f, g)

are expressed per 100 g on an as is basis for all samples,

allowing each laboratory to apply appropriate reconstitution

factors.

A. Principle

Test samples (reconstituted powders, liquid RTFs, and liquid

concentrates) are spiked with an internal standard and saponified

with potassium hydroxide. Samples are then extracted with

MTBE and THF, followed by hexane. The supernatants from

the liquid–liquid extraction are dried under nitrogen and

reconstituted in 2-propanol (isopropyl alcohol; IPA). Separation

is performed by reversed-phase chromatography on a C30

column. All-

trans

-lutein and β-carotene are separated from their

major

cis

isomers, as well as from zeaxanthin, α-carotene, and

lycopene.

B. Apparatus

Note

: Carotenoids are light sensitive, and all work should be

done under protection from UV light.

(a)

Ultra-HPLC (UHPLC) system

.—Consisting of binary

pump, autosampler, thermostatted column compartment, UV-

Vis detector with 60 mm flow cell, and data acquisition software.

(b)

Analytical column

.—C30 carotenoid column, 3 μm,

2.0 × 250 mm (Part No. CT99S03-2502WT; YMC, Kyoto,

Japan).

(c)

Guard column

.—C30 guard column, 3 μm, 2.1 × 10 mm

(Part No. CT99S03-01Q1GC; YMC).

(d)

Guard cartridge holder

.—Part No. XPGCH-Q1 (YMC).

(e)

Spectrophotometer

.—Wavelength range of 200–700 nm,

with 1 cm quartz cells.

(f)

Top-loading balance

.—Capable of weighing to 0.1 g.

(g)

Analytical balance

.—Capable of weighing to 0.01 mg.

(h)

Ultrasonic water bath

.—40 kHz.

(i)

Reciprocating shaker

.—Capable of 200 rpm.

(j)

Evaporator

.—With pure nitrogen supply.

(k)

Laboratory centrifuge

.—With adapters for 50 mL

centrifuge tubes.

(l)

Centrifuge tubes

.—50 mL, polypropylene.

(m)

Syringes

.—1 mL, disposable.

(n)

Syringe filters

.—0.2 μm, PTFE.

(o)

Class A volumetric flasks

.—Various sizes; clear and

amber.

(p)

Scintillation vials

.—12 mL, amber.

(q)

HPLC vials

.—Amber, with 300 μL inserts.

(r)

Class A volumetric pipets

.—Various sizes.

C. Reagents

Note

: Reagent volumes may be scaled up or down provided

good laboratory practices are followed.

(a)

Laboratory water

.—>18 megohm-cm.

(b)

Methanol (MeOH)

.—HPLC grade.

(c)

MTBE

.—HPLC grade.

(d)

n-Hexane

.—HPLC grade.

(e)

Potassium hydroxide (KOH)

.—Pellets, ACS grade.

(f)

Reagent alcohol (ROH)

.—Denatured ethanol, HPLC

grade.

(g)

α-Tocopherol (vitamin E)

.—95%.

(h)

Pyrogallic acid (pyrogallol)

.—ACS grade.

(i)

IPA

.—HPLC grade.

(j)

THF

.—99.9%, stabilized with BHT.

(k)

Ammonium acetate

.—HPLC grade, 98%.

(l)

Potassium hydroxide solution, 50% (w/w).—

Add 50 mL

water to a 250 mL beaker. Weigh 50 g KOH and slowly transfer

to the beaker under constant stirring. When dissolved and

cooled, transfer to a media bottle and store at room temperature

for up to 6 months.

(m)

MTBE/vitamin E.—

Dissolve 2.2 g α-tocopherol in

500 mL MTBE. Store in the refrigerator for up to 1 month.

(n)

Pyrogallol solution (0.2 M pyrogallic acid in ethanol).—

Dissolve 6.3 g pyrogallic acid in 250 mL ROH. Store in the

refrigerator for up to 1 month. Solution should be clear at room

temperature; discard if colored.

(o)

Extraction solution [10 mM vitamin E in MTBE–THF

(1 + 1)].—

Dissolve 2.2 g α-tocopherol in 250 mL MTBE and

250 mL THF. Store in the refrigerator for up to 1 month.

(p)

Sample solvent (10 mM vitamin E in IPA).—

Dissolve

4.4 g α-tocopherol in 1000 mL IPA. Store in the refrigerator for

up to 1 month.

(q)

Mobile phase for LC system

.—(

)

Phase A

.—20 mM

ammonium acetate in methanol–water (98 + 2). Combine

980 mLMeOH, 20 mLwater, and 1.54 g ammonium acetate and

mix to dissolve. Store at room temperature for up to 1 month.

(2)

Phase B

.—MTBE.

D. Standards

(a)

Lutein

.—ChromaDex (Part No. ASB-00012453; Irvine,

CA), or equivalent.

(b)

β-Carotene

.—USP (Part No. 1065480; Rockville, MD),

or equivalent.

(c)

Apocarotenal (β-apo-8′-carotenal)

.—USP Part No.

1040854, or equivalent.

(d)

Lutein containing approximately 10% zeaxanthin

.—USP

Part No. 1370804.

(e)

β-Carotene system suitability reference standard

.—USP

Part No. 1065491.

E. Standards Preparation

Standard solution preparation is summarized in Table

2016.13A

and detailed below.

(a)

Carotenoid stock solutions (20000 μg/100 mL).—

Weigh

(to 0.01 mg) approximately 10 mg each of the lutein,

D(a)

;

β-carotene,

D(b)

; and apocarotenal,

D(c)

reference standards

into separate 50 mL volumetric flasks. Add approximately

40 mL MTBE/vitamin E,

C(m)

, to each, sonicate for 2 to

3 min, and dilute to volume with MTBE/vitamin E. Store stock

solutions at −20°C for up to 6 months and check their purity

each time new standard solutions are made from them.

(b)

Standard measuring solutions for UV-Vis spectroscopy

potency check (200 μg/100 mL).—

Transfer 1.0 mL aliquots

of lutein and β-carotene standard stock solutions,

E(a)

, to