SPDS Aloe Vera Method Book

Date Issued: Supersedes:

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Procedure Title: Determination of Absolute Molecular Weight and Content of Polysaccharides in Aloe vera Fresh Leaf and Concentrated Juice Powder by SEC-MALS-RI

N/A

Effective Date:

QCL

SOP #:

Revision #:

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

1.0

PROCEDURES 1.1

100mM NaCl Mobile Phase Preparation 1.1.1

Mobile Phase: Weigh 5.844mg of sodium chloride (NaCl) into a 1-L volumetric flask and dilute to volume with water. Mix well and filter the mobile phase through 0.22μm Nitrocellulose membrane.

1.2

100mM NaCl Extraction Solution Preparation 1.2.1

Extraction Solution: Weigh 5.844 g of NaCl into a 1-L volumetric flask and dilute to volume with water. Mix well.

1.3

Standard Preparation 1.3.1

Standard Solutions 1.3.1.1 Bovine serum albumin standard solution (5mg/mL): Pipette 1mL into a glass HPLC vial Sample Preparation 1.4.1 Fresh Leaf 1.4.1.1 Preparation of fresh leaf samples: for whole leaf sample, cleaned the aloe leaf by water and cut the leaf tip, butt, barbs and edges with a knife. Grind the entire aloe leaves in a high speed blender until the blend was flowing smoothly and no large chunks remained. Squeeze the juice out of the leaf residue and lyophilize the juice to concentrated whole leaf juice powder. For inner leaf, filet the cleaned leaf with knife to separate the rind from the inner leaf and lyophilize inner leaf to aloe powder. 1.4.1.2 Weigh ~100mg of the powder raw material sample into a 50 mL volumetric flask, dissolve and dilute to volume with 100 mM Sodium chloride solution. Vortex the sample for 30 seconds, and allowed to swell for 20-24 hours at 4 o C. Centrifuge the sample liquid with 4000-5000 rpm for 10 min. Pipette 1-mL supernatant into a glass HPLC vial. Concentrated Juice Powder 1.4.2.1 Weigh ~250 mg of concentrated juice powder into a 50mL volumetric flask, dissolve and dilute to volume with 100mM NaCl solution, vortex the sample for 30 seconds, and allowed to swell for 20-24 hours at 4 o C. Centrifuge the sample liquid with 4000-5000 rpm for 10 min. Pipette 1-mL supernatant into a glass HPLC vial. 1.4.2 Run a bovine serum albumin (BSA) standard solution to check the system suitability. The difference of molecular weight (M w ) of BSA between the theoretical and detected values should be less than ±5%; otherwise the system needs to be normalized with BSA standard solution. Calculations Properly integrate the RI chromatogram showing the peaks of interest. The weight-average molecular weight (M w ), number-average molecular weight (M n ), z-average molecular weight (M z ) and polydispersity (PD = M w /M n ) of polysaccharides are determined by Wyatt ASTRA 6.1.5.22 software. A refractive index increment (dn/dc) is 0.14 mL/g and use Zimm extrapolation method. System Suitability 1.5.1

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