Published on February 17, 2014
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, LLC Danbury, Connecticut February 3, 2014 2nd PANIC, Charlotte, NC
Solids -200 Process NMR Associates NMR Systems and Experimental Conditions Acquisition Time Relaxation (Recycle) Delay Frequency, MHz Nucleus Number of Pulse Accumulations* Original FID Points Zero-filled Points Pulse sequence Solvent Sweep width, ppm Temperature Line Broadening Steady State Pulses Pre-Acquisition Delay 3-8 Seconds 2-6 Seconds 300-500 MHz 1H 16-256 16384-84000 32768-262144 Single pulse D2 O 16 Ambient (25 ºC) 0.35 Hz 8 60 seconds 60 MHz Liquids-300
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
x y w w d c m z a m b
Process NMR Associates Aloe Vera Inner Leaf Juice Constituents and Additives that need to be Analyzed and Reported for IASC Certification 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 Ash ≥ 0.46% in single-strength juice (for example, a 10x concentrate should have ≥ 4.6%) ≤ 40% Included in the American Herbal Pharmacopoeia Monograph on Aloe Vera Leaf, Leaf Juice & Inner Leaf Juice
Process NMR Associates 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 Substance Signal Type and N Parameter Chemical shift, ppm Broad Group of CH3 Singlets (N=3) 2.0-2.3 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 Acetylated Polysaccharides Isocitric acid
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
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 Compound Type of compound Signal type Propylene glycol Ethanol Lactic acid Potassium sorbate Additive Degradation product or additive Degradation product Preservative CH3, doublet (N=3) CH3, triplet (N=3) CH3, doublet (N=3) CH3, doublet (N=3) Chemical shift, ppm 1.1 1.15 1.33 1.82 Acetic acid Pyruvic acid Citric acid CH3, singlet (N=3) CH3, singlet (N=3) 2 x CH2, Multiplet (N=4) 1.96 2.35 2.5-3.0 Succinic acid Glycerol Glycine Sucrose Fumaric acid Sodium benzoate Degradation product Degradation product Naturally present or added as pH regulator or preservative Degradation product Additive Additive Additive Degradation product Preservative 2 x CH2, singlet (N=4) CH2 and CH, multiplet CH2, singlet (N=2) CH, doublet (N=1) 2 x CH, singlet (N=2) 2 x CH, doublet (N=2) 2.6 3.5 3.51 5.4 6.5 7.95 Formic acid Degradation product CH, singlet (N=1) 8.2-8.3
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 Acetylated Polysaccharide Quantitation 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/mol Now 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. C AP ( WNic * I AcMann * N Nic * MWAcMann WNic * I AcMann * N Nic * MWMann * (0.22 / 0.78) WNic * I AcMann * N Nic * MWGlu * (0.16 / 0.84) 1 )* *100% I Nic * N AcMann * MWNic I Nic * N AcMann * MWNic I Nic * N AcMann * MWNic Wsample WNic * I AcMann N Nic 1 * * ( MWAcMann MWMann * 0.28 MWGlu * 0.19) * *100% I Nic N AcMann * MWNic Wsample WNic * I AcMann 4 * * (204.2 (162.2 * 0.47)) *100% I Nic *Wsample 3 *122.1 WNic * I AcMann * 3.06 *100% I Nic *Wsample AcMann = acetylmannosyl, Mann=Mannosyl, Glu=glucosyl N = number of protons in the group - molar conversion factor - NNic=4, NAP=3 CAP = 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 standard MWAcMann = Molecular Weight of acetylated polysaccharides (204.2 g/mol) MWNic = molecular weight of nicotinamide standard (122.1 g/mol) MWMann=MWGlu=162.2
Isocitrate 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) * 6.29 CICA = 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 standard MWICA = 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
Aloe Gel 10x Component Dry Matter Composition Wt% Product Composition Wt% 11.77 Acemannan Wt% 0.742 13.33 Glucose Wt% 0.840 27.71 Malic Acid Wt% 1.747 0.11 Lactic Acid 0.007 8.68 Citric Acid 0.547 0.00 Pyruvic Acid 0.000 0.79 Sorbate 0.122 0.92 Benzoate 0.153 0.00 Acetoin 0.000 0.00 2,3-butandiol 0.000 Not Observed Whole Leaf Marker (WLM) Not Observed Isocitrate (WLM-1) 0.00 0.000 Isocitrate Lactone (WLM-2) 0.00 0.000 0.18 Acetic Acid 0.011 0.00 Succinic Acid 0.000 0.00 Formic Acid 0.000 0.00 Fumaric Acid 0.000 0.06 Ethanol 0.028 0.00 Glycine 0.000 0.00 Propylene Glycol 0.000 0.00 Sucrose 0.000 Dry Matter Weight (%) 6.31
Question: Why is this competitors product so good? Ingredients : Oyster, Barberry, Dextrose, Snow Lotus, Bombyx Mori L, Ginger Root, Salfron Crocus. Sample preparation: Extract with 20:80 D2O:CD3CN Centrifuge or Settle Supernatant analyzed. Answer: Also contained ~10 wt% Sulfoaildenafil A Viagra Analog
Typical Herbal Extracts
60 MHz 300 MHz
Adulteration of Acacia senegal – aka Gum Arabic Emulsifier 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)
PANIC 2014 - Edwards Presentation - Aloe ... PANIC 2014 - Edwards Presentation - Aloe - ED Herbal - Acacia Gum - 2-3-14 Aug 29, 2014 Technology john ...
NMR Based Authentication of Nutraceuticals, Herbal ... Supplements, and Acacia Gum Presented By John Edwards, ... 3, 2014 2nd PANIC, ...
Share ENC 2014 - MNova Users Meeting Presentation Aloe Vera, Beer, PDE5i Adulterants in Dietary SupplementsAcacia Gum ... Aloe - ED Herbal - Acacia Gum - 2 ...