Quantification of Whole Leaf Markers in Aloe Vera Raw Materials and Products by 1H NMR Spectroscopy
Process NMR Associates has developed an extension of its 1H NMR analysis of Aloe Vera raw materials and products analysis. Powder aloe vera raw materials (100x/200x) can be analyzed directly and juice samples must be freeze dried for this analysis. The whole leaf markers utilized in this test to indicate the presence of whole leaf material are iso-citrate and iso-citrate lactone which are formed in the green leaf part of the aloe plant as part of the citric acid cycle. It is possible to assign and quantify the following components of an aloe vera juice or powder:
* aloe vera components: acemannan, glucose, malic acid
* whole leaf markers: isocitrate, iso-citrate lactone, citric acid
* degradation products: lactic acid, acetic acid, fumaric acid, pyruvic acid, formic acid, succinic acid, and ethanol
* adulterants: maltodextrin
* preservatives: sorbate, benzoate
* additives: sucrose, fructose, flavorants (contain ethanol and propylene glycol)
For the components above it is possible to obtain a wt% of that component in a juice sample – the freeze drying process is then used to determine the wt% solids in the juice and the dry weight concentrations of the organic components in the solids.
Currently testing of aloe vera raw materials revolve around acemannan and whole leaf marker content and the presence of glucose. Process NMR Associates method can perform this analysis but also tell you the “quality” of the aloe vera raw material – the absolute concentrations of degradation products can tell you a lot about the exposure of the raw material to:
- excessive heat (hydrolyzation of acemannan acetyl groups to acetic acid (vinegar) or formic acid)
- lactobacillus bacteria (found on the skin of the aloe vera plant – these bacteria “eat” malic acid and produce lactic acid)
- enzymatic processes (enzymes found in the aloe vera plant itself can cause degradation to succinic acid or fumaric acid
The figure below shows the NMR assignment of a whole leaf juice that has been freeze dried. From the molar ratios of the various 1H NMR peaks it is possible to obtain a wt% value for all components by comparison with a nicotinamide internal standard (spectral range not shown) and the molecular weight of the component molecule or monomer unit.
1H NMR spectroscopy observes signals from all protons in the sample simultaneously. Aloe vera components, preservatives, and degradation products yield peaks at specific chemical shifts which can be integrated and quantified. Observations are made on the following peaks:
Nicotinamide – Internal NMR Standard: 1) 8.85 ppm, 2) 8.2 ppm (coincides with formic acid), 3) 7.55 ppm, 4) 8.65 ppm
Glucose – C1 proton for alpha conformation at 5.2 ppm (doublet), C1 proton for beta conformation at 4.6 ppm (doublet)
Malic Acid – CH at 4.35 ppm (multiplet), CH2 at 2.4-2.8 ppm (multiplet)
Acemannan – CH3 resonances of acemannan acetylation – fingerprint distribution of methyl resonances from 2.0-2.2 ppm
Lactic Acid – CH3 Peak at 1.33 ppm (doublet)
Acetic Acid – CH3 peak at 1.92 ppm (singlet)
Succinic Acid – 2 x CH2 peak at 2.5 ppm (singlet)
Formic Acid – Aldehyde Resonance at 8.2 ppm (singlet)
Ethanol – CH3 peak at 1.18 ppm (triplet)
Pyruvic Acid – CH3 peak at 2.35 ppm (singlet)
Citric Acid – 2 x CH2 resonances at 2.4 to 3.0 ppm (multiplet)
Iso-Citrate (Whole Leaf Marker) – CH at 4.25 ppm (doublet)
Iso-Citrate Lactone (Whole Leaf Marker) – CH at 5.05 ppm (doublet)
Benzoate – ortho-protons (2H) give peaks at 7.8 ppm.
Sorbate – CH3 peak is observed at 1.77 ppm (doublet) olefin protons observed at 5.7, 6.15, &7 ppm.
Fumaric Acid – CH peak at 6.55 ppm (singlet)
Sucrose – C1 proton observed at 5.4 ppm (doublet)
Maltodextrin – qNMR protons observed at 5.4 ppm
Maltodextrin is readily observed and quantified by 1H NMR – in the figure below is an example of a 100x and a 200x aloe vera gel powder – the 100x sample contains 50 wt% maltodextrin – the peaks at 5.4 and in the 3.5-4.0 ppm region are used to quantify the presence of maltodextrin.
Process NMR Associates is currently the only company providing a detailed breakdown of the components of aloe vera including quantitation of the whole leaf markers. Dry weight limits of acemannan, whole leaf markers, and maltodextrin are required for aloe vera raw material certification and quality control.
What is interesting in the food world is that most fruits and beverages and dairy products are a complex mixture of these same chemistry components. 1H NMR has a unique advantage in the analysis of these types of materials as the analysis requires very little sample preparation and the data can be used to unequivocally identify the presence of single component chemistry and quantify it’s presence. Process NMR Associates are available to develop NMR based methodologies on any of these types of food systems and would welcome discussion of this with potential customers. Customers are not charged for initial consultation and project scoping. These NMR methods are universally applicable on NMR systems from 200-900 MHz. If you are interested please contact John Edwards (Tel: 203 744-5905)