SPDS Aloe Vera Method Book

Date Issued: Supersedes:

Page:

2 of 7

Procedure Title: Determination of Organic Acid Contents in Aloe vera Fresh Leaf and Concentrated Juice Powder by HPLC Method

N/A

Effective Date:

QCL

SOP #:

Revision #:

Prepared By: Yuehong Zhang, Zhichao Bao Department: R&D Applies To: Herbalife International

- Flow rate: 0.4 mL/min - Column temperature: 20 o C - Sample temperature: ambient - Wavelength: 210 nm - Injection volume: 10 μL - Run time: 45 minutes

1.0

PROCEDURES 1.1

Mobile Phase Preparation 1.1.1 Mobile Phase A: Add 1.176 mL of Phosphoric acid (85%-90%) into a 500 mL volumetric flask and dilute to volume with water. Mix well. 1.1.2 Mobile Phase B: Acetonitrile. 1.2 Standard Preparation 1.2.1 Standard Solutions 1.2.1.1 Isocitric Acid Standard Stock Solution: Weigh ~40 mg of DL-Isocitric acid trisodium salt hydrate standard into the 10 mL volumetric flask. Dissolve and dilute to volume with ultrapure water. Store the Stock Standard Solution in refrigerator (2 - 8 ℃ ). 1.2.1.2 Isocitric acid Lactone Standard Stock Solution: Weigh ~25 mg of isocitric acid lactone standards into the 10 mL volumetric flask. Dissolve and dilute to volume with ultrapure water. Store the Stock Standard Solution in refrigerator (2 - 8 ℃ ). 1.2.1.3 Mixed Organic Acid Standard Stock Solution: Pipette ~0.5 mL of formic acid,

~1.25 mL of lactic acid and ~1.25 mL of acetic acid into the same 50 mL volumetric flask, dissolve and dilute to volume with ultrapure water (Solution 1). Weigh ~250 mg of oxalic acid dehydrate and ~10 mg of fumaric acid into the same 100 mL volumetric flask, dissolve and dilute to volume with ultrapure water (Solution 2). Weigh ~50 mg of malic acid, ~36 mg of isocitric acid trisodium salt hydrate, ~25 mg of isocitric acid lactone, ~25 mg of citric acid into the same 10 mL volumetric flask. Pipette 1mL of ‘Solution 1’ and 1mL of ‘Solution 2’ into the volumetric flask, dissolve and dilute to volume with ultrapure water. The mixed organic acid standard stock solution was prepared at the concentrations of fumaric acid (~0.01 mg/mL), oxalic acid (~0.2 mg/mL), tartaric and formic acid (~1 mg/mL each), malic acid (~5 mg/mL), isocitric, lactic, acetic, citric and succinic acid (~2.5 mg/mL each), and isocitric acid lactone (~2.5 mg/mL). Store the Stock Standard Solution in refrigerator (2-8 o C). 1.2.1.4.1 ~2500 μg/mL Standard solution: Pipette 5 mL of the Isocitric Acid Standard Stock Solution into a 5 mL volumetric flask. 1.2.1.4.2 ~500 μg/mL Standard solution: Pipette 1 mL of the Isocitric Acid Standard Stock Solution into a 5 mL volumetric flask and dilute to volume with ultrapure water. Mix well. 1.2.1.4.3 ~100 μg/mL Standard solution: Pipette 1 mL of the Isocitric Acid Standard Stock Solution into a 25 mL volumetric flask and dilute to volume with ultrapure water. Mix well.

1.2.1.4 Isocitric Acid Working Standard Solution:

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