SPDS Aloe Vera Method Book

2018 SPDS Method Submission Form - ALO-01

Submission Date

2018-02-16 11:34:35

Submitter Name

charles metcalfe

Submitter Email

laptop@calabs.us

Organization

Custom Analytics

Aloe Vera

Method Type

Method Name

Aloe Polysaccharides by O-Acetyl

Method Author(s)

Charles Metcalfe

Method Applicability

Raw Materials and Finished Products

Statement:

Custom Analytics LLC

Standard Operating Procedures

Subject: Aloe Polysaccharides by O-Acetyl Department: Laboratory Original Date: 06/20/2016 Revision #: 1

Procedures #: CA17011

Approval Date:

1. Purpose To determine amount of aloe polysaccharides in raw materials. 2. Reference Aloe Products for Food Raw Material, Chinese National Standard, QB/T 2489 2007 The acetyl groups on polysaccharides are reacted with hydroxylamine to form acetohydroxamic acid. The resulting acetohydroxamic acid is reacted with ferric trichloride to form a ferric ‐ acethydroxamic acid complex and measured 540 nm. 3. Scope This method is intended for raw materials and finished product. Products may contain acetic acid that would react to acetylcholine resulting in false positive results. 4. Materials and Equipment 4.1. UV-VIS spectrophotometer set to 540 nm and 1 cm path length cell

4.2. Ultrasonic bath 4.3. Deionized water 4.4. Hydroxylamine hydrochloride [5470-11-1] 4.5. Acetylcholine chloride [60-31-1] 4.6. Ferric chloride hexahydrate [10025-77-1] fw 270 4.7. Hydrochloric acid, concentrated [7467-01-0] 4.8. Sodium hydroxide [1310-73-2] fw 40 4.9. Sodium acetate trihydrate [6131-90-4] fw 136 5. Preparation of Solutions 5.1. Hydrochloric acid (4 M)

5.1.1.Dilute 33.3 ml of concentrated HCl to 100 ml with DI water. 5.2. Hydrochloric acid (0.1 M) 5.2.1.Dilute 2.5 ml of 4 M HCl to 100 ml with DI water. 5.3. Sodium Hydroxide (3.5 M) 5.3.1.Dissolve 14.0 g in 100 ml of DI water. 5.4. Ferric chloride – hydrochloric acid (0.37 M) 5.4.1.Dissolve 10.0 g in 100 ml of 0.1 M HCl 5.5. Sodium acetate (0.001 M, pH 4.5) 5.5.1.Dissolve 136 mg in 1000 ml of DI water 5.6. Hydroxylamine hydrochloride (2M) 5.6.1.Dissolve 13.9 g in 100 ml of DI water (keep refrigerated for up to one week)

Custom Analytics LLC

Standard Operating Procedures

Subject: Aloe Polysaccharides by O-Acetyl Department: Laboratory Original Date: 06/20/2016 Revision #: 1

Procedures #: CA17011

Approval Date:

6. Preparation of Alkaline Hydroxylamine (prepare on day of use) Mix equal volumes of hydroxylamine hydrochloride and sodium hydroxide. 7. Optimization of pH 7.1. The optimum pH for color development needs to be in the range of 1.0 – 1.4. 7.2. Mix 1.0 ml of DI water, 2.0 ml of alkaline hydroxylamine, 0.7 ml 4 M HCl and 1.0 ml of ferric chloride. 7.3. The pH of this solution should be 1.0 – 1.4. Modify the amount of HCl until the pH is in this range. If the pH is not acidic enough (> pH 3) a brown precipitate will form. 8. Preparation of Calibration Standards (0.2 – 1.0 mg/ml), (prepare on day of use) 8.1. Weigh 50 mg (to the nearest 0.01 mg) of acetylcholine chloride to a 50 ml volumetric flask and dilute to volume with sodium acetate solution. ml Acetylcholine ml Water 0.0 1.0 0.2 0.8 0.4 0.6 0.6 0.4 0.8 0.2 1.0 1.0 8.2. Add 2.0 ml alkaline hydroxylamine and mix. Allow the solutions to sit at room temperature for 4 minutes. 8.3. Add the amount of HCl as determined in 7 . 8.4. Add 1.0 ml of ferric chloride. Gas bubbles will form in the solution which are easily removed by a few minutes in an ultrasonic bath. 8.5. If the pH of the solution is within the 1.0 – 1.4 range, the blank sample will be yellow and the standards and samples will be a reddish orange color.

Custom Analytics LLC

Standard Operating Procedures

Subject: Aloe Polysaccharides by O-Acetyl Department: Laboratory Original Date: 06/20/2016 Revision #: 1

Procedures #: CA17011

Approval Date:

9. Preparation of Samples 9.1. Prepare samples using weights and dilutions as shown below. Sample Type mg ml

% Assay Range

1X Liquids

6000 1000 200 200

10 10 10 50 50

0.03 – 0.17

5X, 10X Liquids 20X, 40X Liquids 100X / 200X Powders Purified Aloe (>20 %)

0.2 - 1 1 - 5 5 - 25

50

20 - 100

9.2. Transfer 1.0 ml of sample to a vial and add 2.0 ml alkaline hydroxylamine and mix. Allow the solution to sit at room temperature for 4 minutes. This should be done in duplicate. 9.3. Add the amount of HCl as determined in 7 . 9.4. Add 1.0 ml of ferric chloride. Gas bubbles will form in the solution which are easily removed by a few minutes in an ultrasonic bath. 10. Analysis 10.1. Zero the spectrophotometer with at 540 nm with the 0 ml standard. 10.2. Measure the absorbance of the other standards and generate a calibration curve. An example curve is show below. The regression coefficient must be ≥ 0.98. c 10.3. Measure the sample and determine the mg/ml using the linear regression equation. 10.4. Calculate the concentration of aloe PS using the following equation: % = 100

Custom Analytics LLC

Standard Operating Procedures

Subject: Aloe Polysaccharides by O-Acetyl Department: Laboratory Original Date: 06/20/2016 Revision #: 1

Procedures #: CA17011

Approval Date:

Validation Data

Precision & Reproducibility Day 1 1X Gel Liquid

Day 2 1X Gel Liquid

Reproducibility Days 1 & 2

% PS

% PS

0.0513 0.0507 0.0492 0.0507 0.0507 0.0500

0.0497 0.0496 0.0500 0.0503 0.0495 0.0499

Days 1 & 2

0.0502 0.0006

average

sd

1.2

% rsd

0.0505 average 0.0007 sd 1.4 % rsd

0.0499 average 0.0003 sd 0.6 % rsd

100X Powder

Purified Polysaccharide

% PS

% PS 75.41 77.96 77.26

9.02 9.06 8.81 8.77 8.77 8.88 8.88 0.14 1.60

76.88

average

1.32

SD

1.7

% RSD

average

sd

% rsd

Custom Analytics LLC

Standard Operating Procedures

Subject: Aloe Polysaccharides by O-Acetyl Department: Laboratory Original Date: 06/20/2016 Revision #: 1

Procedures #: CA17011

Approval Date:

Linearity

1.2

y = 0.9879x - 0.0067 R² = 0.9988

1

0.8

0.6

mg/ml

0.4

0.2

0

0

0.2

0.4

0.6

0.8

1

1.2

Abs 540 nm

Custom Analytics LLC

Standard Operating Procedures

Subject: Aloe Polysaccharides by O-Acetyl Department: Laboratory Original Date: 06/20/2016 Revision #: 1

Procedures #: CA17011

Approval Date:

Recovery Tablets 16.26% PS

mg/ml

added

Total

Assayed

ABS 540

g

ml 50 50 50

mg/ml Sample

mg/ml 0.0861 0.0861 0.0861

mg/ml 0.4149 0.4071 0.4214

mg/ml 0.4144 0.3960 0.4169

% Recovery

0.1011 0.0987 0.1031

2.02 1.97 2.06

0.3288 0.3210 0.3353

0.673 0.643 0.677

99.9 97.3 98.9 98.7

Average

Stdev % RSD

1.3 1.3

0.1065 0.1823 0.1878

50 50 50

2.13 3.65 3.76

0.3472 0.5943 0.6122

0.4303 0.4303 0.4303

0.7775 1.0246 1.0425

1.274 1.295 1.318

0.7836 1.0752 1.0943

100.8 104.9 105.0 103.6

Average

Stdev % RSD

2.4 2.3

Limit of Detection - 0.025 mg/ml Limit of Quantitation - 0.1 mg/ml mg/ml ABS 540 nm 0.05145 0.044 0.1029 0.110 0.2058 0.217 0.4116 0.437 0.6174 0.644 0.8232 0.848 1.029 1.029

Custom Analytics LLC

Standard Operating Procedures

Subject: Aloe Polysaccharides by O-Acetyl Department: Laboratory Original Date: 06/20/2016 Revision #: 1

Procedures #: CA17011

Approval Date:

Recovery 1X Gel Liquid 0.0796 % PS

PS

Sample mg/ml

PS

added mg/ml 0.01315 0.01315 0.01315

Total

mg

ml 10 10 10

mg/ml 0.3845 0.3839 0.3850

mg/ml 0.3977 0.3971 0.3982

ABS 540

mg/ml 0.4247 0.4178 0.4079 average

% Recovery

4849.0 4841.3 4855.6

484.9 484.1 485.6

0.437 0.430 0.420

106.8 105.2 102.4 104.8

SD

2.2 2.1

% RSD

PS

Sample

PS

added

Total

ABS 540

mg

ml 10 10 10

mg/ml

mg/ml 0.3658 0.3911 0.4215

mg/ml 0.3288 0.3288 0.3288

mg/ml 0.6946 0.7199 0.7503

mg/ml 0.6670 0.6970 0.7715 average

% Recovery

4613.0 4931.6 5315.6

461.3 493.2 531.6

0.689 0.720 0.797

96.0 96.8

102.8

98.6

SD

3.7 3.8

% RSD

Custom Analytics LLC

Standard Operating Procedures

Subject: Aloe Polysaccharides by O-Acetyl Department: Laboratory Original Date: 06/20/2016 Revision #: 1

Procedures #: CA17011

Approval Date:

Interlab Assays

Sample

CA Labs

Lab 1 10.00

Lab 2

Average

Std. Dev

% RSD

200X 200X 200X 200X 100X 100X 100X 100X

9.78 9.55

9.44

9.65

0.45

4.69

8.93

10.21

10.22 10.33 10.22 10.33

9.68 9.29 9.22 9.12

10.15 10.17

9.97

0.4

4.04

9.84 9.93

9.78

0.5

5.16

10.8

11.43 11.52

11.25 10.66

11.09

0.36

3.22

10.88

1X Gel 1X Gel 1X Gel 1X Gel 1X Leaf 1X Leaf 1X Leaf 1X Leaf

0.050 0.049 0.065 0.065 0.348 0.346 0.095 0.095

0.050 0.046 0.059 0.062 0.287 0.262 0.083 0.092

0.046 0.047 0.065 0.061 0.410 0.414 0.094 0.091

0.048

0.002

3.95

0.063

0.003

4.08

0.345

0.062

18.01

0.092

0.005

4.96

2018 SPDS Method Submission Form ALO-02

Submission Date

2018-01-31 17:22:07

Submitter Name

quanyin Gao

Submitter Email

quanying@herbalife.com

Organization

Herbalife

Method Type

Aloe Vera (Quant or ID

Method Name

Identification and quantitation of Aloe Vera Using 1H NMR

Method Author(s)

Issac Lee and Quanyin Gao

OFFICIAL DOCUMENT

Date Issued: Supersedes:

Page:

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Procedure Title: Identification and quantitation of Aloe Vera Using 1 H NMR

N/A

Effective Date:

TBD

SOP #:

Revision #:

00

Prepared By: Department: Applies To:

Isaac Lee Quality Herbalife QC Laboratories

CONTENTS Procedures Definitions References Forms/Attachments Ownership/Management Sign-off Revision History

SECTION 1.0 2.0 3.0 4.0 5.0 6.0

PURPOSE This SOP establishes the process for identification and quantitation of Aloe Vera in products by proton NMR spectroscopy. SCOPE This procedure applies to dietary supplements and ingredients that contain Aloe vera. 1.0 PROCEDURES 1.1 Safety and precaution 1.1.1 The instrument possesses very strong magnetization. Even though the instrument is shielded, the stray field still exists and it is important to take precaution when getting near to the instrument. 1.1.2 Ferromagnetic objects or person with the pacemaker should not be getting into the stray zone. Figure 1 illustrates the stray zone and cautions that should take when entering the stray field. 1.1.3 Items such as smart phone, ATM cards, watch, and other electronic devices may get damaged by the magnetic field. Avoid bringing such items in the stray zone.

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Procedure Title: Identification and quantitation of Aloe Vera Using 1 H NMR

N/A

Effective Date:

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SOP #:

Revision #:

00

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Isaac Lee Quality Herbalife QC Laboratories

Figure 1. Safety precautions within the stray field 1.1.4

The cryogens (liquid nitrogen and liquid helium) need to be refilled regularly to maintain the magnetizm on the superconducting magnet. Proper personal protective equipments (thermal gloves, safety gloves, lab coat, and any other necessary equipments) are required when handling the cryogens. 1.1.5 The cryogens (liquid nitrogen and liquid helium) need to be refilled regularly to maintain the magnetizm on the superconducting magnet. Proper personal protective equipments (thermal gloves, safety gloves, lab coat, and any other necessary equipments) are required when handling the cryogens.

1.2

Standards and Reagents 1.2.1

Nicotinamide Analytical Standard, Supelco, or equivalent D-Glucose (Dextrose), USP Reference Standard, or equivalent

1.2.2 1.2.3 1.2.4

Malic Acid, USP Reference Standard, or equivalent

DSS (Sodium 2,2-dimethyl-2-silapentane-5-sulfonate), Cambridge Isotope Laboratories, Inc., or equivalent

1.2.5

Deuterium Oxide, Acros organics, 99.8% atom % D or equivalent

1.3

Equipment and materials

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Procedure Title: Identification and quantitation of Aloe Vera Using 1 H NMR

N/A

Effective Date:

TBD

SOP #:

Revision #:

00

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Isaac Lee Quality Herbalife QC Laboratories

1.3.1 Not less than 300 MHz NMR spectrometer (e.g. Bruker, Ascend 400 with Topspin software, or equivalent) 1.3.2 Lyophilizer (e.g. Free Zone Plus 4.5L Cascade Dry System or equivalent) 1.3.3 Vortex mixer 1.3.4 Sonicator 1.3.5 5 mm NMR tube (e.g. Norell, part# 507-HP-7, or equivalent) 1.3.6 Test tubes 1.3.7 Glass bottle 1.3.8 Glass vial with Teflon lined cap 1.4 NMR Conditions ( The following parameters can be modified for optimization ) 1.4.1 Acquisition parameters 1.4.1.1 Relaxation delay (d1) 2-6 sec 1.4.1.2 Number of scans (NS) Not less than 16 depending on the concentration 1.4.1.3 Sweep width (SW) 15 ppm 1.4.1.4 Pulse angle 30 o to 90 o depending on the sample (it is recommended to use water suppression pulse sequence with 90 o angle on the sample containing water) 1.4.1.5 Temperature 20 o C – 25 o C 1.4.1.6 Date points: Not less than (NLT) 64K 1.4.1.7 Dummy scans: 4

1.5

Blank preparation 1.5.1

Dissolve 3mg of DSS with 0.7mL of D 2

O in a test tube. Transfer the solution to a NMR

tube.

1.6

Reference Standard Preparation 1.6.1

Weigh 5 mg of Dextrose and Malic acid reference standards in the separate 12x75 mm test tubes and add 3 mg of DSS. Add 0.7 mL of deuterium oxide and vortex to dissolve. Transfer the solution to a NMR tube. 1.6.2 Filter each dilution through a 0.45 μm filter prior to NMR analysis, if required. 1.7 Nicotinamide solution 1.7.1 Weigh 357.14mg of Nicotinamide reference standard and 214.29mg of DSS in a glass bottle, and add 50mL of deuterium oxide. Mix the solution until it observes the clear dissolution.

1.8

System suitability solution 1.8.1

Transfer 0.7mL of Nicotinamide solution to a NMR tube.

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Procedure Title: Identification and quantitation of Aloe Vera Using 1 H NMR

N/A

Effective Date:

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SOP #:

Revision #:

00

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Isaac Lee Quality Herbalife QC Laboratories

1.9

Sample Preparation (When only requires identification of aloe vera) 1.9.1 Powder Aloe Samples

1.9.1.1 For raw materials, weigh 7-14 mg of sample depending on concentration of material in a 12x75 mm test tube and add 0.7 ml of Nicotinamide solution. Vortex the solution and transfer it to a NMR tube. 1.9.1.2 For finished product, weigh about 20mg of sample in a 12 x 75 mm test tube, and add 0.7mL of Nicotinamide solution. Vortex the solution, and transfer it to a NMR tube. 1.9.1.3 Filter each dilution through a 0.45 μm filter prior to NMR analysis, if required. Liquid Aloe Samples 1.9.2.1 For raw materials, transfer about 0.7 mL of sample in a12x75 mm test tube, and add 0.7 mL of Nicotinamide solution. Vortex the solution and transfer 0.7mL of prepared sample to a NMR tube. 1.9.2.2 For finished product, transfer about 3.5 mL of sample in a 16x125 mm test tube, and add 0.7 mL of Nicotinamide Solution. Vortex and transfer 0.7 mL of prepared sample to a NMR tube. 1.9.2.3 Specifically for highly diluted aloe product, lyophilize 20-30 ml of the sample using a lyophilizer. Weigh about 40 mg of freeze dried sample in a12x75 mm tube, and add 0.7mL of Nicotinamide Solution. Vortex and transfer to a NMR tube. 1.9.2.4 Filter each dilution through a 0.45 μm filter prior to NMR analysis, if required. 1.10.1.1 For raw materials, weigh 7-14 mg of sample, depending on the materials concentration, 5mg of Nicotinamide, and 3mg of DSS in a glass vial. Add 0.7mL of D 2 O, and vortex the solution until the complete dissolution and transfer to a NMR tube. 1.10.1.2 For finished product, weigh 20mg of sample, 5mg of Nicotinamide, and 3mg of DSS in a glass vial. Add 0.7mL of D 2 O, and vortex the solution until the complete dissolution and transfer to a NMR tube. 1.10.1.3 Sonicate the solution if the complete dissolution is not reached using the vortex mixer alone. 1.10.1.4 If complete dissolution is not reached after the sonication, check the ingredients if the product contains any non-water soluble ingredients. Lower the sample amount if all ingredients should be dissolved in water. 1.10.2.1 For raw materials, weigh 0.7 mL of sample, 5mg of Nicotinamide, and 3mg of DSS in a glass vial. Add 0.3mL of D 2 O, and vortex the solution. Transfer 0.7mL of prepared sample to a NMR tube. 1.10.2.2 For finished product, lyophilize 20-30 ml of the sample using a lyophilizer. Weigh about 40 mg of freeze dried sample, 5mg of Nicotinamide, and 3mg of

1.9.2

1.10

Sample Preparation (When requires quantitation of aloe vera) 1.10.1 Powder Aloe Samples

1.10.2 Liquid Aloe Samples

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00

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DSS in a glass vial. Add 0.7mL of D 2

O and vortex the solution until the

complete dissolution. Transfer the solution to a NMR tube. 1.10.2.3 Sonicate the solution if the complete dissolution is not reached using the vortex mixer alone. 1.10.2.4 If complete dissolution is not reached after the sonication, check the ingredients if the product contains any non-water soluble ingredient. Lower the sample amount if all ingredients should be dissolved in water.

1.11

1 H NMR spectrums

DSS

Acetylated Glucomannan

Malic Acid

Glucose

Nicotinamide

Figure 1, Typical 1 H NMR Spectrum of aloe vera drink (finished produt)

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DSS

Acetylated Glucomannan

Nicotinamide

Malic Acid

Glucose

Figure 2, Typical 1 H NMR Spectrum of Aloe Vera 5X

DSS

Nicotinamide

Acetylated Glucomannan

Malic Acid

Glucose

Figure 3, Typical 1 H NMR Spectrum of Purified Aloe Vera 100X

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1.12

Analysis 1.12.1 Follow the sample sequence to analyze standards and samples.

Blank (Deuterium Oxide)

1 Run 5 Run 1 Run 1 Run 1 Run 1 Run 1 Run 1 Run 1 Run 1 Run

Reference Standard (Nicotinamide Solution)

Reference Standard (Glucose) Reference Standard (Malic Acid)

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6

Reference Standard (Nicotinamide Solution) 1 Run Run a reference standard (Nicotinamide Solution) after every 6 sample runs and at the end of each batch run. 1.13 System Suitability 1.13.1 The chemical shift (ppm) for Nicotinamide reference standard peaks must be within 0.2 ppm in five (5) replicate runs. 1.14 1 H NMR spectrums & Acceptance Criteria: 1.14.1 Acquire a free induction decay (FID) and Fourier transformed spectra are recorded. The

DSS methyl signal should be set to 0.0 ppm. All spectra are phased manually or automatically, and baseline correction is applied before peak identification.

1.14.2 Identification of aloe vera:

Using the NMR spectrum of Standard solution, identify NMR peaks corresponding to Nicotinamide (~8.9, 8.7, 8.2, and 7.6 ppm), glucose (~5.2ppm), Malic acid (~4.5ppm), and acetyl groups of Acetylated glucomannan (between 2.0 – 2.3ppm). Acceptance criteria: The characteristic marker compounds of Aloe Vera are Glucose, Malic Acid and Acetylated glucomannan. The presence of these three components indicates the presence of Aloe Vera. Note: presence of the four (4) common adulterants (Acacia gum, Locust bean gum, Carrageenan, and Maltodextrin) can be identified as follows.

For Use Only By Herbalife International. This document contains confidential and proprietary information belonging to Herbalife International - It must not be reproduced or disclosed to others without prior written approval Acacia gum As shown in Figure 4, no Glucose (α- at 5.2 ppm and β- at 4.6 ppm) and Malic acid (4.3 ppm) peaks were observed in Acacia gum while there is a multiplet featuring similar chemical shift but slightly different peak shape profile compared with Acetylated glucomannan (2.0-2.3 ppm). Our

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Procedure Title: Identification and quantitation of Aloe Vera Using 1 H NMR

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Isaac Lee Quality Herbalife QC Laboratories

method requires the presence of all the peaks for glucose, malic acid and Acetylated glucomannan while Acacia gum NMR spectrum is missing glucose and malic acid peaks. In addition, the spectrum of Acacia gum contains distinguishable peaks around at 1.26ppm region, so any aloe vera sample diluted with the acacia gum is detectable (see figure 5). This ID method can screen out this adulterant for aloe vera ID and provide reliable conclusion for identification.

Figure 4. 1 H NMR spectrum of Acacia gum

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Procedure Title: Identification and quantitation of Aloe Vera Using 1 H NMR

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Isaac Lee Quality Herbalife QC Laboratories

Figure 5. Comparison of 1 H NMR spectra of Acacia gum and Aloe vera samples Locust bean gum 1 H NMR spectrum of Locust bean gum showed no Glucose (α- at 5.2 ppm and β- at 4.6 ppm), Malic acid (4.3 ppm), and Acetylated glucomannan (2.0-2.3 ppm) peaks (Figure 6). Therefore, this material will be confirmed as not an aloe vera material. In addition, the spectrum of Locust bean gum contains distinguishable peaks around at 1.06ppm region, so any aloe vera sample diluted with the locust bean gum is detectable (see figure 7). Therefore, this proton NMR ID method gives correct and reliable conclusion.

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Procedure Title: Identification and quantitation of Aloe Vera Using 1 H NMR

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Effective Date:

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SOP #:

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00

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Isaac Lee Quality Herbalife QC Laboratories

Figure 6. 1 H NMR spectrum of Locust bean gum

Figure 7. Comparison of 1 H NMR spectra of Locust bean gum and Aloe vera samples

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Procedure Title: Identification and quantitation of Aloe Vera Using 1 H NMR

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Effective Date:

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00

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Isaac Lee Quality Herbalife QC Laboratories

Lambda-Carrageenan, Kappa-Carrageenan, and Iota-Carrageenan For Carrageenan, the 1 H NMR spectra of Lambda-Carrageenan, Kappa-Carrageenan, and Iota- Carrageenan were collected to demonstrate the specificity (Figure 8 to Figure 10). These three (3) types of carrageenans are most widely used in food industry. As seen from the NMR spectra result, no Glucose, Malic acid, or Aloverose diagnostic peaks were observed in both Kappa- Carrageenan and Iota-Carrageenan products. The spectrum of Lambda-Carrageenan contains Glucose peak, but the other two chemical marker are missing

Figure 8. 1 H NMR spectrum of Lambda-Carrageenan

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Figure 9. 1 H NMR spectrum of Kappa-Carrageenan

Figure 10. 1 H NMR spectrum of Iota-Carrageenan

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A sample diluted with lambda carrageenan can be detected by looking at this region.

Maltodextrin

Test data for Maltodextrin sample indicated no Glucose, Malic acid, and Aloverose peaks (Figure 11) thus confirming the material is not aloe vera. In addition, the spectrum of Maltodextrin contains distinguishable peak at 5.4ppm region. The aloe vera sample contains doublet at 5.4ppm region, which belongs to sucrose. Maltodextrin peak is not a doublet and it is broader than the sucrose peak. So, any aloe vera sample diluted with maltodextrin is detectable (see figure 13).

Figure 11. 1 H NMR spectrum of Maltodextrin

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Figure 12. Comparison of 1 H NMR spectra of Maltodextrin and Aloe vera samples

1.14.3 Quantitation of aloeverose: Identify NMR peaks corresponding to acetyl groups of aloeverose in the Sample solution between 2.0 – 2.3ppm. Integrate peaks in this area. Calculate the percentage of Acetylated glucomannan in the sample solution using the following equation:

Result (Acetylated glucomannan %) = 3.06 x ( W N x I A

)/( W A

x I N

) x (Purity, %)

= weight of added Nicotinamide internal standard (mg)

W N

= integration area of acetylation methyls (multiple peaks in 2.0-2.3 ppm region) in aloeverose = sum of integration areas of the 4 aromatic CH peaks of the Nicotinamide standard = conversion factor of molecular weight and proton numbers for acetylated polysaccharide vs. Nicotinamide standard = weight of Aloe juice in Sample solution (mg)

I A

I N

W A

3.06

Purity, %

= Purity of Nicotinamide Standard

2.0

For Use Only By Herbalife International. This document contains confidential and proprietary information belonging to Herbalife International - It must not be reproduced or disclosed to others without prior written approval DEFINITIONS Acetylated glucomannan: Acetylated polysaccharides derived from aloe vera. It is also called Aloeverose or Acemannan.

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3.0

REFERENCES 3.0 Journal of AOAC International Vol. 93, No. 3, 2010 – Quantitative 1 H-NMR Spectrometry Method for Quality Control of Aloe Vera Products 3.1 AOAC SMPR 2017.010, Standard Method Performance Requirements (SMPRs) for Identification of Aloe Vera in Dietary Supplements and Dietary Ingredients 3.2 QCL331 Operation, Performance Verification and Maintenance of Bruker NMR Magnet System 3.3 QAH100 Material Review 3.4 QCL100 Report Investigating and Documenting OOS Results for Physical and Chemical Testing 3.5 QCL327 Operation of Free Zone Plus 4.5L Cascade Dry System

4.0

FORMS/ATTACHEMENTS N/A

5.0

OWNERSHIP/MANAGEMENT SIGN-OFF Approvals Refer to ECO57859

6.0

REVISION HISTORY Revision Date

Revised By

Summary of Items Revised

January 2018

Isaac Lee

New SOP

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2018 SPDS Method Submission Form ALO-03

Submission Date

2018-01-25 19:04:49

Submitter Name

Kan He

Submitter Email

kanh@herbalife.com

Organization

Herbalife International of America

Method Type

Aloe Vera (Quant or ID)

Method Name

Determination of Polysaccharides Molecular Weight in Aloe vera Fresh Leaf and Concentrated Juice Powder by SEC-RI

Method Author(s)

Yuehong Zhang, Zhichao Bao, Zhaoyang Xie, Kan He

Method Applicability Statement:

The method can be used to determine the relative molecular weight of polysaccharides in aloe vera using commercial available polysaccharides, pullulans, to establish a standard curve on SEC. The standard curve can be adjusted by using aloe polysaccharides with known molecular weights determined by MLAS measurement to obtain more accurate results. The method is suitable for polysaccharide molecular weight analysis by SEC, but without detectors for absolute MW determination, such light scattering detection.

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

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CONTENTS Procedures Definitions References Forms/Chromatograms Ownership/Management Sign-off Revision History

SECTION 1.0 2.0 3.0 4.0 5.0 6.0

PURPOSE The purpose of the test method is to determine the polysaccharides molecular weight in Aloe vera fresh leaf and concentrated juice powder by Size Exclusion Chromatography Coupling with Refractive Index Detector (SEC-RI).

REAGENTS 

1 : 0.342, 2 :1.32, 3 : 6.2, 4 : 10.0, 5 : 21.7, 6 : 48.8,

Pullulan Standards and Molecular Weight (MW) (M w

7 :113, 8 : 200, 9 : 366, and 10 : 805 kDa), Sigma-Aldrich, Product No.96351 or equivalent  Sodium chloride, Sinopharm Reagents, Catalog# 10019318 or equivalent  Methanol, HPLC grade  Ultrapure Water or equivalent EQUIPMENT  Analytical Balance  Nitrocellulose Membrane, 0.22μm GSWP or equivalent  Disposable Syringe Filters, 25mm, 0.45 μm Hydrophilic PTFE membrane filter or equivalent  Disposable Syringes (1mL)  Disposable Plastic Pipette  Vortex Mixer  SCIENTZ-12N Lyophilizer  1-L Volumetric Flask  50-mL Volumetric Flask  5-mL Volumetric Flask  1.5-mL Glass HPLC Vials HPLC  HPLC System: Waters 2695 Alliance Separations Module consisting of pump and auto sampler and Empower 3 GPC/SEC Software or equivalent  Waters 2414 Refractive Index Detector or equivalent  Size Exclusion Chromatographic (SEC) Column: Phenomenex PolySep™-SEC GFC-P Linear, LC Column 300 x 7.8 mm, Product No. CHO-9232 or equivalent  Guard Column: Phenomenex PolySep™-SEC GFC-P Guard, LC Guard Column 35 x 7.8 mm, Product No. CHO-9225 or equivalent  HPLC chromatographic conditions: - Mobile phase: 100mM Sodium chloride solution - Flow rate: 0.7 mL/min

- Column temperature: 35 o C - Sample temperature: 20 o C - Refractive index detector temperature: 35 o C - Injection volume: 100 μL - Run time: 30 minutes

1.0

PROCEDURES

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1.1

100mM NaCl Mobile Phase Preparation 1.1.1

Mobile Phase A: Weigh 5.844mg of sodium chloride (NaCl) into a 1-L volumetric flask and dilute to volume with water. Mix well and filled by the filter kit with 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 Pullulan Standards Solution: Weigh ~5 mg each of standards into each 5 mL volumetric flask. Add 10 µL of methanol (MeOH). Dissolve and dilute to volume with 100mM NaCl solution. Vortex for 30 seconds and allowed to swell for 20-24 hours at 4 o C. Filter standard solution through a 0.45μm Hydrophilic PTFE filter into a glass HPLC vial. Aloe Standard Solutions 1.3.2.1 Weigh ~5 mg aloe standard into a 5 mL volumetric flask. Add 10 µL of methanol (MeOH). Dissolve and dilute to volume with 100mM NaCl solution. Vortex for 30 seconds and allowed to swell for 20-24 hours at 4 o C. Filter standard solution through a 0.45μm Hydrophilic PTFE filter into a glass HPLC vial. Fresh Aloe 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 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 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 inner leaf juice powder. 1.4.1.2 Weigh ~100 mg of the leaf juice powder into a 50 mL volumetric flask. Add 100 µL of MeOH. 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. Aloe Concentrated Juice Powder 1.4.2.1 Weigh ~250 mg of the concentrated juice powder into a 50 mL volumetric flask. Add 100 µL of MeOH. 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 at 4000-5000 rpm for 10 min. Pipette 1- mL supernatant into a glass HPLC vial.

1.3.2

1.4

Sample Preparation 1.4.1

1.4.2

1.5

System Suitability

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1.5.1 Chromatograph a series of pullulan standards. Retention time deviation of the standards (M w 805, 48.8, and 6.2 kDa) should be no more than 0.1 minute, and the Relative Standard Deviation (RSD) for calibration standard should be no more than 2.0%. 1.5.2 Obtain standard calibration curve by plotting the logarithm of pullulan molecular weights (Log M) in narrow mode versus retention time (T) of corresponding standards. Combine aloe standards with known M w , M p , M n , and M z into the above calibration curve in broad mode and use a third-order polynomial fit curve.

1.5.3 1.5.4 1.5.5

Run aloe polysaccharide standards.

Run aloe samples

Run a reference check after every six sample injections and at the end of the run.

1.6

Calculations Integrate the chromatograms showing the peaks of interest. Calculate the molecular weight and molecular weight distribution by Empower 3.0 GPC/SEC software according to: Log M = aT 3 + bT 2 + T + c Where: M: molecular weight; T: retention time (min.); a, b, and c: constants determined by software.

2.0

REFERENCES N/A

3.0

FORMS / CHROMATOGRAMS 3.1

Chromatogram 1: Chromatogram of pullulan standards

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3.2

Chromatogram 2: Chromatogram of Aloe sample.

4.0

OWNERSHIP/MANAGEMENT SIGN-OFF Approvals Refer to

5.0

REVISION HISTORY Revision Date

Revised By

Summary of Items Revised New Test Method

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2018 SPDS Method Submission Form ALO-04

Submission Date

2018-01-25 18:43:14

Submitter Name

Kan He

Submitter Email

kanh@herbalife.com

Organization

Herbalife Internation of America

Method Type

Aloe Vera (Quant or ID)

Method Name

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

Method Author(s)

Yuehong Zhang, Zhichao Bao, Zhaoyang Xie, Kan He

Method Applicability Statement:

The method can be used to determine the contents of organic acids in aloe vera. It can be used for identification of aloe products, quantitate individual organic acid, or used for differentiate aloe whole leaf and inner gel products by organic acid profiles. Can also be used as a alternative method for aloe organic acid analysis when nmr signals overlapped if using nmr method.

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CONTENTS Procedures Definitions References Forms/Chromatograms Ownership/Management Sign-off Revision History

SECTION 1.0 2.0 3.0 4.0 5.0 6.0

PURPOSE The purpose of this test method is to determine the amounts of organic acids in Aloe vera fresh leaf and concentrated juice powder by HPLC to establish the organic acid profile for Aloe vera identification.

REAGENTS 

DL-Malic acid, analytical standard, Sigma-Aldrich, Catalog# 94916 or equivalent  Acetic acid, analytical standard, Sigma-Aldrich, Catalog# 71251 or equivalent  Citric acid, analytical standard, Supelco, Catalog# 46933 or equivalent  DL-Isocitric acid trisodium salt hydrate, Sigma, Catalog# I1252 or equivalent  Isocitric acid lactone , Aldrich, Catalog# I16005 or equivalent  Oxalic acid dihydrate, Sinopharm Reagents, Catalog# 10014818 or equivalent  Tartaric acid, Sinopharm Reagents, Catalog# 10022018 or equivalent  Formic acid, Sinopharm Reagents, Catalog# 80065518 or equivalent  Lactic acid, Sinopharm Reagents, Catalog# 30108518 or equivalent  Succinic acid, Sinopharm Reagents, Catalog# 30171716 or equivalent  Fumaric acid, Aladdin, Catalog# F110741 or equivalent  Acetonitrile, HPLC grade  Phosphoric acid(85%-90%), HPLC grade  Ultrapure water or equivalent

EQUIPMENT 

Analytical Balance

 Disposable syringe filters, 25mm, 0.45 μm Hydrophilic PTFE membrane filter or equivalent  Disposable syringes (1mL)  Disposable plastic pipette  Vortex mixer  Sonicator  SCIENTZ-12N Lyophilizer  5-, 10-, 25-, 50-, 100, 250- and 500-mL Volumetric flasks  1.5-mL Glass HPLC vials HPLC  HPLC System: Waters 2695 Alliance Separations Module consisting of pump and autosampler or equivalent  Waters PDA detector or any variable wavelength UV detector or equivalent  HPLC Column: Phenomenex Luna C-18, 5 µm, 250 × 4.6 mm Part# 00G-4252-E0 and Thermo AcclaimTM OA, 5µm, 250 × 4.6 mm, product No. 062902 connected in series or equivalent  Guard Column: Phenomenex C-18, 4 x 3.0mm, Cat.# AJO-4287 or equivalent  HPLC chromatographic conditions: - Mobile Phase: 98% A = 0.2% Phosphoric Acid in Water 2% B = 100% Acetic Acid in Acetonitrile - Isocratic Condition

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

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- 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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