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
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GluMannAcMann
NicAcMann
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sampleNicAcMannNic
GluNicAcMannNic
NicAcMannNic
MannNicAcMannNic
NicAcMannNic
AcMannNicAcMannNicAP
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MWN
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WMWNI
MWNIW
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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)
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