PANIC 2014 - Edwards Presentation - Aloe - ED Herbal - Acacia Gum - 2-3-14

Process NMR Associates

NMR Based Authentication of Nutraceuticals, Herbal Supplements, and Food Additives: Economic- and Efficacy-Driven Adulteration of Aloe Vera, Herbal

Erectile Dysfunction Supplements, and Acacia Gum

Presented By

John Edwards, Ph.D.

Process NMR Associates, LLCDanbury, Connecticut

February 3, 20142nd PANIC, Charlotte, NC

Process NMR Associates

Acquisition Time 3-8 SecondsRelaxation (Recycle) Delay 2-6 Seconds

Frequency, MHz 300-500 MHzNucleus 1H

Number of Pulse Accumulations* 16-256Original FID Points 16384-84000Zero-filled Points 32768-262144Pulse sequence Single pulse

Solvent D2OSweep width, ppm 16

Temperature Ambient (25 ºC)Line Broadening 0.35 Hz

Steady State Pulses 8Pre-Acquisition Delay 60 seconds

NMR Systems and Experimental Conditions

Solids -200

Liquids-300

60 MHz

Process NMR Associates

Advantages of Using NMR in Complex Mixture Analysis

• Minimal Sample Preparation• Reproducible (site to site, country to country)• Linear and Quantitative Spectral Response• Targeted AND Non-Targeted• Qualitative• Quantitative• Compound Specific• Rapid – 10 Minutes• Affordable

1H NMR Method for qNMR Analysis of Aloe Vera Leaf Juice is Included in the American Herbal Pharmacopoeia Monograph on Aloe Vera Leaf, Leaf Juice & Inner Leaf Juice

# Chemical Characterization of the Immunomodulating Polysaccharide of Aloe Vera L., W.J.Goux, et al,

http://www.iasc.org/AloeStructure080604.pdf

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Process NMR Associates

Compound IASC Certification requirement

Acemannan ≥ 5% dry weight

Glucose Present

Aloin

10 ppm or less in 0.5% aloe vera solids solution, analysed by HPLC or other fit for purpose methodology approved by IASC

Isocitrate ≤ 5% dry weight

Maltodextrin

Must be listed on label and analysis must meet label claims. If undeclared, is considered an adulterant.

Solids≥ 0.46% in single-strength juice (for example, a 10x concentrate should have ≥ 4.6%)

Ash ≤ 40%

Aloe Vera Inner Leaf Juice Constituents and Additives that need to be Analyzed and Reported for IASC Certification

Included in the American Herbal Pharmacopoeia Monograph on Aloe Vera Leaf, Leaf Juice & Inner Leaf Juice

Process NMR Associates

Substance Signal Type and N Parameter Chemical shift, ppm

Acetylated Polysaccharides Broad Group of CH3 Singlets (N=3) 2.0-2.3

Isocitric acid CH, Doublet (N=1) 4.25

Malic acid CH, 4 peak multiplet (N=1) 4.45

-Glucose CH Doublet (N=1) 4.6

-Glucose CH Doublet (N=1) 5.2

Isocitric lactone CH Doublet (N=1) 5.05

Characteristic chemical shift values, peak multiplicity, protonated carbon type and N values used for detection and quantitation of the major natural components of aloe vera leaf juice

Process NMR Associates

Freeze-Dried Inner Leaf Juices with (a) and without (b) preservatives

Process NMR Associates

Commercial freeze-dried 200x aloe vera leaf juice powder

Compound Type of compound Signal type Chemical shift, ppm

Propylene glycol Additive CH3, doublet (N=3) 1.1Ethanol Degradation product or additive CH3, triplet (N=3) 1.15Lactic acid Degradation product CH3, doublet (N=3) 1.33Potassium sorbate Preservative CH3, doublet (N=3) 1.82

Acetic acid Degradation product CH3, singlet (N=3) 1.96Pyruvic acid Degradation product CH3, singlet (N=3) 2.35Citric acid Naturally present or added as pH

regulator or preservative2 x CH2, Multiplet (N=4) 2.5-3.0

Succinic acid Degradation product 2 x CH2, singlet (N=4) 2.6Glycerol Additive CH2 and CH, multiplet 3.5Glycine Additive CH2, singlet (N=2) 3.51Sucrose Additive CH, doublet (N=1) 5.4Fumaric acid Degradation product 2 x CH, singlet (N=2) 6.5Sodium benzoate Preservative 2 x CH, doublet (N=2) 7.95

Formic acid Degradation product CH, singlet (N=1) 8.2-8.3

Chemical shift values, peak and chemistry descriptions, molar conversion factors that can be used for detection and quantitation of aloe vera leaf juice preservatives, additives, and degradation products

Process NMR Associates

Freeze Dried Aloe Vera Leaf Juice – Acidification Required for Isocitrate Quantification.

Process NMR Associates

Commercial 200x inner leaf juice powder

Commercial 100x inner leaf juice powder containing 50% maltodextrin

Process NMR Associates

Aloe Vera Leaf Juice Freeze Dried and Mixed 80% with Maltodextrin

Process NMR Associates

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sampleNic

AcMannNic

sampleNic

AcMannNic

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GluMannAcMann

NicAcMann

Nic

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sampleNicAcMannNic

GluNicAcMannNic

NicAcMannNic

MannNicAcMannNic

NicAcMannNic

AcMannNicAcMannNicAP

WI

IW

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WMWMWMW

MWN

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IW

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MWNIW

MWNI

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MWNIWC

AcMann = acetylmannosyl, Mann=Mannosyl, Glu=glucosylN = number of protons in the group - molar conversion factor - NNic=4, NAP=3CAP = content of acetylated polysaccharides (AP) in the sample, wt%WNic = weight of added internal standard (mg), WSample = weight of sample (mg)IAcMann = integration area of acetylation methyls (multiple peaks in 2.0-2.3 ppm region)INic = sum of integration areas of the 4 aromatic CH peaks of the nicotinamide standardMWAcMann = Molecular Weight of acetylated polysaccharides (204.2 g/mol)MWNic = molecular weight of nicotinamide standard (122.1 g/mol)MWMann=MWGlu=162.2

Demonstrated that the acetylation of the mannose monomer units is at 78% (Ref. 2, Manna et al., 1993).

Mannose represents 84% of the polysaccharide backbone with the remainder being composed of glucose, galactose, and a few other saccharides (Ref 3., Chow et al., 2005).

The acetylation content and the presence of 16% other saccharides must be taken into account so as not to underestimate the AP content.

The molecular weight of AP is calculated under the assumption that 1 water molecule is removed upon condensation of acetyl with mannosyl monomer, and each mannosyl unit shares in the loss of a single water molecule upon condensation form the predominantly mannose based polysaccharide.Thus, MWmannosyl = Mwmannose – (MWwater)/2 = 180.2 - 18 = 162.2 g/mol

MwAcMann = Mwacetyl group + MWmannosyl - MWwater = 60 + 162.2 - 18 = 204.2 g/molNow taking into account the 0.78/1 acetyl/mannosyl residue ratio as well as the presence of ~16% non-mannosysl saccharides in the AP we can calculate the concentration of AP (CAP) by the following equation.

Acetylated Polysaccharide Quantitation

CICA (wt%) = 100% * (WNic * IICA * NNic * MWICA) / (INic * NICA * MWNic * WSample) = 100% * (WNic * IICA * 4 * 192.1) / (INic * 1 * 122.1 * WSample) = 100% * (WNic*IICA/INic*WSample) * 6.29CICA = content of isocitrate in the sample, weight%

WNic = weight of added nicotinamide internal standard (mg), WSample = weight of sample (mg)IICA = integration area of CH proton peak of isocitrate (doublet at 4.45 ppm (dissolved in D2O and acidified with DCl)INic = sum of integration areas of the 4 aromatic CH peaks of the nicotinamide standardMWICA = Molecular Weight of Isocitrate (192.1 g/mol), MWNic = molecular weight of nicotinamide standard (122.1 g/mol) N = number of protons in the group - molar conversion factor, NNic=4, NICA=1

IsocitrateQuantitation

Aloe Gel 10x

Component Dry Matter Composition Wt% Product Composition Wt%

Acemannan Wt% 11.77 0.742

Glucose Wt% 13.33 0.840

Malic Acid Wt% 27.71 1.747

Lactic Acid 0.11 0.007

Citric Acid 8.68 0.547

Pyruvic Acid 0.00 0.000

Sorbate 0.79 0.122

Benzoate 0.92 0.153

Acetoin 0.00 0.000

2,3-butandiol 0.00 0.000

Whole Leaf Marker (WLM) Not Observed Not Observed

Isocitrate (WLM-1) 0.00 0.000

Isocitrate Lactone (WLM-2) 0.00 0.000

Acetic Acid 0.18 0.011

Succinic Acid 0.00 0.000

Formic Acid 0.00 0.000

Fumaric Acid 0.00 0.000

Ethanol 0.06 0.028

Glycine 0.00 0.000

Propylene Glycol 0.00 0.000

Sucrose 0.00 0.000

Dry Matter Weight (%) 6.31

Ingredients : Oyster, Barberry, Dextrose, Snow Lotus, Bombyx Mori L, Ginger Root, Salfron Crocus.

Sample preparation:Extract with 20:80 D2O:CD3CNCentrifuge or SettleSupernatant analyzed.

Answer:Also contained ~10 wt% SulfoaildenafilA Viagra Analog

Question:Why is this competitors product so good?

Typical Herbal Extracts

60 MHz

300 MHz

Adulteration of Acacia senegal – aka Gum ArabicEmulsifier in Many Food, Beverage, Pharmaceutical and Cosmetic Products

Acknowledgments

Aspect Imaging and Cosa Xentaur – 60 MHz NMR system

Tal Cohen – Modcon – 60 MHz NMR System Improvements

Aloe Work - Adam, Daniel and Paul at PNA

Control ED Herbal Project - Unknown customer (who didn’t pay for analysis and thereby gave me permission to publish)

Chris Berliner – ISC (Provided Gums)