6. AOACSPIFANMethods-2018Awards

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140 C ampos G iménez & M artin : J ournal of AOAC I nternational V ol . 100, N o . 1, 2017

(a)  Ascorbic acid stock solution (500 μg/mL) .—In a 25 mL amber glass volumetric flask, weigh 12.5 mg ascorbic acid. Dissolve and dilute to volume with TCEP solution. This solution can be kept for 3 months if stored at 4°C away from light. (b)  Ascorbic acid intermediate standard solution (50 μg/mL) .— In a 10 mL amber glass volumetric flask, pipet 1 mL stock solution. Dilute to volume with TCEP solution. This solution can be used for 1 month if stored at 4°C away from light. (c)  Ascorbic acid calibration standard solutions (0.5, 1.0, 2.0, 3.0, 5.0, 7.5, and 10.0 μg/mL) .—In 10 mL amber glass volumetric flasks, pipet 0.1, 0.2, 0.4, 0.6, 1.0, 1.5, and 2.0 mL intermediate standard solution. Dilute to volume with mobile phase to prepare the respective concentrations given above. (a)  Reconstitution of powder samples .—( 1 ) Weigh 25 g powder in a 250 mL brown glass beaker and add 10 mg TCEP. ( 2 ) Add 200 g warm water (40°C). Mix well until dissolution is complete. Proceed to (b) as soon as possible as vitamin C can degrade rapidly. Do not let the reconstituted samples stand for >30 min. (b)  Extraction. —( 1 ) Weigh 2 g liquid or reconstituted sample in a 10 mL amber glass volumetric flask. ( 2 ) Add 4 mL TCEP solution and 2 mL TCA (15%). ( 3 ) Dilute to volume with water. ( 4 ) Filter the solution through a folded paper filter. ( 5 ) Transfer 1 mL filtrate to a 10 mL amber glass volumetric flask containing 1 mL acetate solution (pH 5.4) and dilute to volume with mobile phase. ( 6 ) Filter ~2 mL through a 0.22 or 0.45 μm membrane into an HPLC vial. ( 7 ) Proceed to chromatographic analysis using either UHPLC conditions in G(a) or HPLC conditions in G(b) . F. Sample Preparation

using decylamine as an ion-pairing agent in a sodium acetate buffer solution (pH 5.4) containing TCEP.

B. Apparatus

(a)  Balances. —With readability of 0.1 mg and 0.01 g. (b)  pH meter. —Metrohm 691 (Herisau, Switzerland), or equivalent. (c)  Folded paper filters .—Grade 597 ½ . (d)  Syringe filters .—0.22 or 0.45 μm pore size. (e)  Chromatographic system. —HPLC or UHPLC system equipped with a quaternary or binary pump, a sample injector, a UV-Vis detector (or optionally, a photodiode array detector), a degassing system, and data software. (f)  UHPLC column. —Waters Acquity UPLC ethylene bridged hybrid C 18 column (1.75 μm, 2.1×100 mm; or equivalent). (g)  HPLC column. —LiChrospher RP-18 column (5 μm, 250×4.6 mm; or equivalent).

C. Reagents and Standards

(a)  Acetonitrile. —HPLC

grade,

Merck

(Geneva,

Switzerland); or equivalent. (b)  Ascorbic acid. —>99%, Fluka (Buchs, Switzerland), or equivalent. (c)  Decylamine. —Fluka, or equivalent. (d)  Phosphoric acid. —85%, Merck; or equivalent. (e)  Ultrapure water. —Resistivity >18 MΩ/cm. (f)  Sodium acetate trihydrate .— Merck, or equivalent.

(g)  TCA. —Merck, or equivalent. (h)  TCEP. —Fluka, or equivalent. (i)  Isoascorbic acid. —Fluka, or equivalent. (j)  Orotic acid. —Sigma, or equivalent.

D. Preparation of Solutions

(a)  Sodium acetate solution (500 mmol/L, pH 5.4). —In a 500 mL volumetric flask, weigh 34.0 g sodium acetate trihydrate and add ~400 mL water for dissolution. Adjust the pH to 5.4 with phosphoric acid (85%) and dilute to volume with water. (b)  TCA (15%). —In a 500 mL volumetric flask, weigh 75.0 g TCA, dissolve the compound, and dilute to volume with water. (c)  TCEP (250 μg/mL). —In a 500 mL volumetric flask, weigh 125 mg TCEP, dissolve the compound, and dilute to volume with water. (d)  Mobile phase for UHPLC. —In a 250 mL flask, mix 0.4 g decylamine, 2.5 mL acetonitrile, 25 mL sodium acetate solution 500 mmol/L (pH 5.4), and 205 mL water (do not dilute to volume). Adjust the pH to 5.4 with phosphoric acid (85%). Add 10 mg TCEP. (e)  Mobile phase for HPLC. —In a 1000 mL flask, mix 1.6 g decylamine, 80 mL acetonitrile, 100 mL sodium acetate solution 500 mmol/L (pH 5.4), and 820 mL water (do not dilute to volume). Adjust the pH to 5.4 with phosphoric acid (85%). Add 50 mg TCEP.

G. Analysis

(a)  UHPLC conditions .—( 1 ) Injection volume.—5 μL. ( 2 )  Autosampler temperature. —10°C.

( 3 )  Column temperature. —25°C. ( 4 )  Flow rate. —0.35 mL/min. ( 5 )  Run time. —4.0 min.

( 6 )  Mobile phase for UHPLC. — See D(d ): 0.4 decylamin, 2.5 mL acetonitrile, 25 mL sodium acetate 500 mmol/L (pH 5.4), 205 mL water, and 10 mg TCEP (pH 5.4). ( 7 )  Detection wavelength. —265 nm. Note : At the end of each analytical series, rinse the column with acetonitrile–water (1+1, v/v) for 10 min at 0.4 mL/min. (b)  HPLC conditions. —( 1 ) Injection volume.—25 μL. ( 2 )  Autosampler temperature. —10°C. ( 3 )  Column temperature. —25°C. ( 4 )  Flow rate. —1.0 mL/min. ( 5 )  Run time. —20 min. ( 6 )  Mobile phase for HPLC. — See D(e ): 1.6 g decylamine, 80 mL acetonitrile, 100 mL sodium acetate 500 mmol/L (pH 5.4), 820 mL water, and 50 mg TCEP (pH 5.4). ( 7 )  Detection wavelength. —265 nm. Note : At the end of each analytical series, rinse the column with acetonitrile–water (1+1, v/v) for 60 min at 1.0 mL/min.

E. Preparation of Standards

Note : Vitamin C is sensitive to light and oxygen. Conduct operations under subdued light conditions, or use amber glassware. Keep all solutions away from direct light.