1 h-nmr quantification of major saccharides in açaí raw materials: a comparison of...

Research Article

Received: 30 January 2013, Revised: 16 March 2013, Accepted: 18 March 2013 Published online in Wiley Online Library: 23 May 2013

(wileyonlinelibrary.com) DOI 10.1002/pca.2442

1H-NMR Quantification of Major Saccharidesin Açaí Raw Materials: a Comparison of theInternal Standard Methodology with theAbsolute Intensity qNMR MethodCole Sterling,a Ronald Crouch,b David J. Russellb and Angela I. Calderóna*

ABSTRACT:Introduction – While the use of internal standard methodology for qNMR is a proven and reliable form of quantification,simplified alternative approaches are needed. Agilent’s absolute intensity qNMR utility software is a valuable alternative thathas not yet been subjected to validation in the peer-reviewed literature.Objective – To provide validation of Agilent’s absolute intensity qNMR method with a specific application to natural productquantification by measuring saccharide content in açaí materials.Methods – In order to validate the method, calibration test samples of ibuprofen were prepared in DMSO-d6 at nine differentconcentrations and measured with 1H-NMR. A minimum of 40 spectra were collected for each sample, and the absolute inten-sity utility was used for quantification. The same methodology was then applied to the açaí materials, creating triplicates foreach of the materials and using 3-(trimethylsilyl)-1-propanesulphonic acid sodium salt in water-d2 as both the solvent andinternal standard. 1H-NMR spectra were collected, and the amounts of glucose, sucrose and fructose were determined usingboth the internal standard approach and the absolute intensity qNMR method.Results – Applying the absolute intensity utility to the ibuprofen samples demonstrated a linear response (R2 = 0.99943). Forthe açaí investigations, results obtained from the absolute intensity method were comparable to those obtained from theinternal standard approach, with percentage differences ranging from 0.5–6.2%.Conclusion – This study demonstrates the accuracy, precision and reliability of Agilent’s absolute intensity qNMR method. Inaddition, practical information is provided for assessing the saccharide contents of açaí materials. Copyright © 2013 JohnWiley & Sons, Ltd.

Keywords: NMR; 1H-qNMR; absolute intensity qNMR; açaí; saccharides; raw materials; Euterpe oleracea (Arecaceae)

* Correspondence to: A. I. Calderón, Department of Pharmacal Sciences,Harrison School of Pharmacy, Auburn University, 4306B Walker Building,Auburn, AL 36849 USA. E-mail: [email protected]

a Department of Pharmacal Sciences, Harrison School of Pharmacy, AuburnUniversity, 4306B Walker Building, Auburn, AL, 36849, USA

b Agilent Technologies, Research Products Division, Santa Clara, CA, 95051,USA

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IntroductionQuantitative NMR represents an accurate and precise methodthat can be used as a routine tool for quantification in manyanalytical laboratories (Pauli et al., 2012). Although chemicalreferencing with respect to internal standards is a proven andaccurate qNMR approach, this technique can be tedious andrequires adulteration of the sample (Griffiths and Irving, 1998).Several electronic referencing approaches have been used inan attempt to provide accurate and precise quantitative resultswithout the need for an internal chemical standard, such as ERETIC(electronic referencing to access in vivo concentrations), PIG (pulseinto gradient), ARTSI (amplitude-corrected referencing throughsignal injection), PULCON (pulse length-based concentrationmeasurements) and QUANTAS (quantification by artificial signal).Although these techniques have demonstrated potential as accu-rate and reliable methods of quantification, they are not widelyused in practice for various reasons (Burton et al., 2005; Marroet al., 2008; Mehr et al., 2008; Ziarelli et al., 2008; Farrant et al.,2010). For instance, techniques such as ERETIC (Ziarelli et al.,2008), ARTSI (Mehr et al., 2008) and PULCON (Farrant et al., 2010)require non-standard cabling of the spectrometer and rely onthe injection of an artificial signal.

Phytochem. Anal. 2013, 24, 631–637 Copyright © 2013 John

In contrast, the qNMR utility in the Agilent VnmrJ softwarepackage directly measures the absolute intensity of an NMRsignal to determine concentration, rather than comparing thatsignal to a reference signal. This method is predicated on thereproducibility and linearity of a modern NMR spectrometer(Mo et al., 2010) and uses a directly sampled, digital receiverarchitecture. Once the response factor of an NMR system hasbeen measured from data acquired on a sample of knownconcentration, that response factor can be used to scale thesignal returned from an unknown sample, thereby yielding theabsolute concentration of the unknown. The advantage of thismethod is that the quantification of all subsequent spectra isan inherent component of the data collection process without

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any additional effort. Moreover, the fully automated softwarecalculation of quantities and errors is a very useful tool. It notonly reduces the manual work but also avoids the risk of errorsdue to entry mistakes and miscalculations.

While the method described here relies on standard software,its validation and application are nowhere to be found in thepeer-reviewed literature. In order to test the utility of thismethod as a routine research tool for future investigations, thisstudy compares the absolute intensity qNMR method againstthe most widely accepted qNMR technique, the internal stan-dard approach, to the measurement of saccharides in açaí rawmaterials.

Euterpe oleracea (Mart.), which is native to Brazil, Colombiaand Suriname (Schauss et al., 2006), is well known for itsnutritious açaí berries. Perhaps the most studied health benefitassociated with açaí is its anti-oxidant and free radical scaveng-ing properties (Schauss et al., 2006; Horiguchi et al., 2011; Udaniet al., 2011). In addition, it has demonstrated anti-inflammatory(Matheus et al., 2006; Jensen et al., 2008) and tumour cellproliferation inhibitory properties (Hogan et al., 2010; Pacheco-Palencia et al., 2010). In terms of consumption, açaí is a populardietary supplement and common ingredient in commercialproducts, which range from tablets and capsules to juices andenergy drinks (Taylor, 2005).

In this study, the validity of the absolute intensity qNMRmethod is tested using ibuprofen, and its application iscompared against the internal standard approach by measuringsaccharide contents in three forms of açaí raw materials – organic,freeze-dried non-organic and processed non-organic açaípowders. The spectra generated from the plant materials clearlyreveal signals of glucose, sucrose and fructose to varying degrees.Peak assignments were validated through close comparison oftheir chemical shifts and coupling patterns against those obtainedfrom the spectrum of the pure sugars. Due to the widespread pop-ularity of açaí in the commercial market, the quantitative methodpresented in this study could provide useful information forassessing the quality of raw materials involved in the manufactur-ing of the numerous products containing açaí. Furthermore, thesethree saccharides were deemed good target compounds forassessing the applicability of Agilent’s qNMR utility on plant mate-rials and other natural products; unlike the anti-oxidantcompounds, these sugars were abundant in the plant materialsand did not require any extensive extraction prior to quantification.

Experimental

Chemicals

All chemicals were purchased from Sigma-Aldrich, Inc St. Louis, MO.These include deuterium oxide (99.9%), dimethyl sulphoxide-d6 (99.8%),a-D-glucose (96% purity), D-(�)-fructose (≥ 99% purity), sucrose (≥ 99.5%purity), sodium azide (≥ 99.5% purity), 3-(trimethylsilyl)-1-propanesulphonicacid sodium salt (DSS; 97% purity) and ibuprofen (≥ 98% purity).

Accuracy and precision of the absolute intensity qNMRmethod using a pure compound

Methodology for qNMR study of ibuprofen. The accuracy andprecision afforded by the absolute intensity qNMR method was investi-gated using ibuprofen as a test compound. A stock solution was preparedby dissolving an analytically weighed sample in dimethyl sulphoxide-d6 toa concentration of 200.00mg/mL (969.5mM). A series of concentrationstandards from 131.94mM to 0.515mMwas then prepared by serial dilution.

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A calibration standard was also prepared from the stock solution at a con-centration of 15.14mM. All samples were transferred to 5mm NMR tubes,with final volumes of 0.65mL in each tube.

NMR measurements for ibuprofen samples. The ibuprofen quantifi-cation experiments were performed on an Agilent 500MHz Direct DriveNMR spectrometer operating at a proton frequency of 499.86MHz and ata regulated temperature of 25 �C. The system was equipped with a 5mmOneNMR Probe and a 7600AS sample changer. Shimming and lockingwere performed automatically for each sample, and all data were col-lected non-spinning. The probe was initially tuned to the calibrationstandard and left in that configuration for all subsequent measurements.

A minimum of 40 1H-NMR spectra were collected on each sample,with a sample change occurring between the collection of each dataset. For each measurement, eight transients were collected with an ac-quisition time of 3.0 s, a relaxation delay of 5.0 s, and a pulse width of12.1 ms (90� tip angle). T1 values for the methine, methyl, and isopropylmethyl resonances were determined to be 1.3, 0.54 and 0.75 s, respec-tively, indicating a sufficiently long relaxation delay. A 0.3 Hz line broad-ening was used for apodisation and the data sets were zero-filled to 256kpoints prior to processing. The scaled integral values for the isopropylmethyl resonance (~0.82 ppm), the methyl resonance (~1.33 ppm) andthe isopropyl methine resonance (~1.77 ppm) were adjusted for thenumber of nuclei present (i.e., 6, 3 and 1, respectively) and then averagedto determine the experimental concentration for each data set. All pro-cessing operations were performed automatically.

The calibration spectrum was acquired on the calibration standard usingthe same parameters and conditions as above, except only a single transientwas collected. The methyl doublet resonance at 1.33 ppm was used as thereference signal for calibration, and the calibration was accomplished usingthe qNMR utility provided in the system’s standard software package.

Comparison of the absolute intensity method to the internalstandard approach using raw plant materials

Methodology for qNMR study of saccharides in açaí plantmaterials. Quantification of sugars in E. oleracea was carried out usingboth the absolute intensity qNMR method and an internal standard(DSS). A single sugar solution consisting of glucose, sucrose and fructoseserved as the external standard for all analyses. The use of a singlestandard solution, rather than one for each sugar, was chosen in orderto account for any possible interactions between these sugars, whichmight be present in the plant materials (Weberskirch et al., 2011). Inaccordance with similar qNMR experiments involving saccharide contentin vegetables (Weberskirch et al., 2011), a single peak was chosen torepresent each sugar for quantification: glucose, 3.25 ppm (H2b proton,triplet); sucrose, 5.42 ppm (H1 proton, doublet); and fructose, 4.12 ppm(H3 and H4 protons, doublet). These peaks are labelled in Fig. 1.

Plant materials. Three different forms of açaí (E. oleracea Mart.) rawmaterials were used in the study: processed non-organic (ADSR-1, Na-ture’s Sunshine Products, Spanish Fork, UT, Lot No. Q166545); organic(ADSR-2, Live Superfoods, Bend, OR, Lot No. LSF077); and freeze-driednon-organic (ADSR-3, FutureCeuticals, Momence, IL, Lot No. N424).

Preparation of plant materials. Processed non-organic açaí powder(ADSR-1) was prepared from the fruits of E. oleracea from Brazil atNature’s Sunshine Products, Spanish Fork, UT. Processing includeddefrosting the fruits and adding chlorinated potable water; this mixturewas prepared in a homogenisation tank. The contents were then filtered,stored in an equilibration tank, pasteurised at 90–100�C, and dried in aspray drier. This processed açaí powder also contains 25% maltodextrinfrom non-GMO corn starch. Organic açaí powder (ADSR-2) was preparedfrom Brazilian organic açaí fruits. The fruits were freeze-dried within 24 hof being picked and stored without preservatives in air-tight bags at LiveSuperfoods until use. Freeze-dried, non-organic açaí powder (ADSR-3)was prepared from fresh, non-organic whole açaí fruits from Brazil. Thesefruits were freeze-dried and powdered at FutureCeuticals. All materialswere stored at 4�C prior to being prepared for NMR.

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Figure 1. 1H-NMR spectra of açaí plant materials: (a) ADSR-1, (b) ADSR-2 and (c) ADSR-3. The labelled peaks were used for saccharide quantification in theplantmaterials. Chemical structure for each of the sugars is shown, and hydrogen atoms used for quantification are those attached to the indicated carbons.

Quantification of Sugars in Açaí using Absolute Intensity 1H-QNMR

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Sample preparation of plant materials for NMR. In an attempt toprevent any bacteria from contaminating these solutions andmetabolisingthe target sugars, 0.2 mg of sodium azide was added directly to the


deuterium oxide solvent. An internal standard solution was then preparedby dissolving 31 mg of DSS in 15 mL of deuterium oxide. This DSS solutionserved as the solvent for all solutions. The sugar standard solution, which

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served as the external standard for all qNMR measurements, was preparedbyweighing each of the pure compounds and dissolving them into a singlesolution with 1.0 mL of DSS/deuterium oxide. This produced concentra-tions in the 10–100 mM range for each sugar, since similar concentrationswere expected in the plant solutions. The resulting solutions were thentransferred to a 5 mm NMR tube and studied with 1H-NMR. Next, 20 mgof each of the three açaí raw materials was weighed and dissolved in1.0 mL of DSS/deuterium oxide. The resulting solutions were sonicatedfor 10 min, vortexed briefly, and sonicated again for 20 min. This wasfollowed by 5 min of centrifugation at 13000 rpm at room temperature.The solutions were then filtered using Pasteur pipettes filled with glasswool and transferred to 5 mm NMR tubes. Triplicates were prepared foreach of the plant materials, yielding a total of nine samples.

NMR measurements for plant samples. 1H-NMR spectra were run ona Varian MR400 spectrometer equipped with a PFG AutoX DB probe op-erating at a proton frequency of 399.76 MHz and a temperature of 25�C.Shimming was performed automatically, and both the sample spinningand steady state transients were turned off. For each sample, 32 scanswere performed with an acquisition time of 1.8 s, a relaxation delay of25 s, pulse width of 11.30 ms (90� tip angle), and a receiver gain of 24.A 0.5 Hz line broadening was used for apodisation and the data setswere zero filled to 256k points prior to processing.

Results and discussion

Accuracy and precision of the absolute intensity qNMRmethod

The results obtained from the absolute intensity qNMR methodapplied to the ibuprofen samples demonstrated a linear correla-tion coefficient of 0.99943. The precision and accuracy of themethod is shown for each concentration sample in Table 1.

The data obtained in the ibuprofen study clearly demonstratethe validation of the absolute intensity qNMR method. The accu-racy and precision are comparable to, if not better than, those

Table 1. The average concentration measured for each ibuprofenfor each experimental average. A minimum of 40 replicates were

1 2 3

Analytical concentration (mM) 0.515 1.03 2.06qNMR average concentration (mM) 0.531 1.049 2.098Standard deviation 0.006 0.008 0.012Accuracy (% error) 3.02 1.87 1.86% standard deviation 1.07 0.79 0.59

Table 2. Average weight percentages of selected saccharides in thADSR-3). All values shown are expressed as percentages� standahave been compared against those obtained from the internal sta

ADSR-1

Saccharide

Glucose Sucrose Fructos

Absolute intensity average weight % << 34.0� 0.8 <<Internal standard average weight % << 33.5� 1.0 <<Percentage difference – 1.6 –


reported for the internal standard method (Maniara et al., 1998).In contrast to any other widely used quantitative NMR technique,the absolute intensity utility remains viable over a very large con-centration range. For example, the data in this study representchanges of nearly three orders of magnitude in concentration.

It is worth noting that the error in the measurements made forthis study tends to increase near the ends of the concentrationrange. This is not an inherent limit of the technique but rather a re-flection of the study design. A fixed number of transients and a fixedpulse width were used for all spectra. At the lower concentrations,the signal-to-noise ratio obtained was limited, thereby reducingthe accuracy of the integration. This condition would be remediedby averaging more transients (Holzgrabe, 2010; Pauli et al., 2012).Similarly, at the high end of the concentration range there are sev-eral factors that could contribute to compression of the recordedsignal. As these samples are very concentrated and yield very highsignal-to-noise with even a single transient, the use of a smaller ex-citation pulse would produce more robust results (Mo et al., 2010).Additionally, in order to obtain more precise results for the absoluteintensity method, the calibration should be remeasured for eachnew batch of samples – something that is likely due to variationsin external conditions (e.g. temperature from day to day).

Comparison of the absolute intensity method to the internalstandard approach

The 1H-NMR spectra for each of the three açaí materials, as wellas the peaks used for saccharide quantification, are shown inFig. 1. While the glucose content in ADSR-1 was above the de-tection threshold, its amount was too low for quantificationand thus was not included in the study. Quantitative resultsobtained from the internal standard and the absolute intensitymethods are compared in Table 2.

sample, with the standard deviation and accuracy determinedcollected for each value

Sample number

4 5 6 7 8 9

4.12 8.24 16.49 32.98 65.97 131.944.171 8.269 16.571 33.014 67.813 129.1410.024 0.048 0.103 0.293 0.492 1.0911.23 0.35 0.49 0.10 2.79 �2.120.57 0.58 0.62 0.89 0.73 0.84

ree different forms of açaí plant materials (ADSR-1, ADSR-2, andrd deviation. Results obtained using absolute intensity qNMRndard (DSS) using percent difference

Açaí material

ADSR-2 ADSR-3

Saccharide Saccharide

e Glucose Sucrose Fructose Glucose Sucrose Fructose

7.5� 0.4 70.4� 7.3 9.6� 0.2 2.6� 0.5 << 1.4� 0.17.5� 0.3 71.4� 7.5 9.3� 0.4 2.7� 0.6 << 1.5� 0.20.5 1.5 2.9 3.7 – 6.2


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The results of the açaí investigations strongly reinforce the ac-curacy and precision of the absolute intensity qNMR approachby comparing this method to the internal standard approach.The use of an internal standard is widely accepted as the primaryapproach to quantitative NMR (Pauli et al., 2005; Shao et al.,2007; Holzgrabe, 2010), and compares a peak integral of the tar-get compound against that of an internal standard, or referencecompound, which is physically added to the analyte. In contrast,the absolute intensity method relies on a one-time calibration ofthe instrument against an externally-generated referencesample. Thus, the two methods used in this study are entirelyindependent.

Methodology of absolute intensity qNMR

The absolute intensity calibration is accomplished by collectingan NMR spectrum on a sample of known concentration. Theintegral area for a resonance representing a known number ofprotons is defined and selected for calibration. The softwarethen captures the tip angle, gain value and number of transientsused to collect the reference spectrum. These values, along withthe intensity of the reference signal, are recorded in the probefile. As an NMR spectrometer using a directly sampled digitalreceiver architecture has a very linear response, an integral valueextracted from any subsequent spectrum can be adjusted fordifferences in the acquisition parameters (i.e., tip angle and sin(θ) relationship; receiver gain and logarithmic relationship;number of transients and 2n relationship), then scaled by thecalibrated response factor measured for the reference sample.Once adjusted for the number of nuclei associated with the res-onance of interest, this yields a direct measurement of absoluteconcentration of the unknown sample.

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Quantification of saccharides in açaí materials

Results from the açaí investigations clearly demonstrate theapplication of the absolute intensity qNMR method for qualitycontrol of dietary supplements. However, a few limitations ofthis part of the study should be addressed. The glucose peakat 3.25 ppm in ADSR-2 and the fructose peak at 4.12 ppm inADSR-3 demonstrated crowding and signs of signal overlap, asevident by asymmetrical baselines and disruption of peak integ-rity (see Fig. 1). Although the source of these signals was notidentified due to the complex nature of the materials studied,their chemical shifts suggest an amino acid or carbohydratenot included in this study. These particular integrations probablysuffer from larger errors in terms of the accuracy. However, verysimilar quantitative results were found in all triplicates and bothmethods of study, indicating the precision of their application. Inaddition, the characteristic splitting pattern of the anomericproton in sucrose (5.42 ppm) was not resolved in any of theplant materials. This was probably caused by reduced fieldhomogeneity due to the complex matrix of these samples anddid not affect peak area or quantification.

The most notable difficulty in quantifying sugars is theirisomerisation. When reducing sugars are dissolved in solution,various isomeric forms will be present. Although isomericequilibria have demonstrated stability under controlled condi-tions, environmental factors such as temperature and pH canhave an effect on the outcome of these ratios (Goux, 1985).Additionally, ample time must be given for reducing sugars toreach equilibrium – something that varies depending on the


particular sugar being studied. For example, D-glucose may takebetween 3 and 4 h to equilibrate, whereas D-arabinose may doso in less than 2 h (Pazourek, 2010). In order to ensure full equil-ibration of these sugars, all solutions were left overnight andstudied approximately 20 h later using 1H-NMR.For both of the reducing sugars used in these experiments

(glucose and fructose), specific isomers were selected for quan-titative purposes: the b-glucopyranose and b-fructofuranoseforms. In this instance, 64.5% of glucose was found in theb-glucopyranose form, and 26.2% of fructose was found inthe b-fructofuranose form. These isomeric ratios, which wereestablished by prior studies involving solutions of the puresugars and the internal standard DSS, are consistent withthe longstanding values (Goux, 1985; Pazourek, 2010). Whilevarious metabolites within the plant solutions could haveplayed a minor role in affecting the degree of isomerisation,further testing demonstrated very similar pH values between allsolutions, indicating lesser significance of these metabolites’ con-tributions to isomeric differences. As the same conditions used inthese preliminary experiments were also used in those involvingthe plant materials, the previously mentioned isomeric ratios wereused to study the plant materials. Consequently, each of the inter-nal standard measurements for glucose and fructose was dividedby 0.645 and 0.262, respectively. Since sucrose is not a reducingsugar and no such isomerisation occurred, no additional calcula-tions were necessary.While the anomeric proton peak (H1a, 5.24 ppm) helped to

identify glucose in the plant materials, this signal was not usedfor quantification due to associated water interference(Weberskirch et al., 2011). Prior studies involving solutions ofthe pure sugars and the internal standard DSS confirmed thisproblem; when using the anomeric peak for measurements,the a-glucose anomer erroneously seemed to contribute any-where between 39% and 45% of the total glucose weight, ratherthan the anticipated and more reasonable 35.5%. Although pre-saturation techniques would have minimised this water interfer-ence, such experiments can affect the intensities of signals thatneighbour the suppressed water peak (Holzgrabe, 2010) and thusmay have introduced errors into the glucose measurements.Instead, the H2b signal (3.25 ppm) was used for quantification. Thissignal has been used as a viable alternative for glucose quantifica-tion in similar experiments (Weberskirch et al., 2011).

Significance of the investigation of saccharides in açaímaterials

This study describes a simple and reliable method for determin-ing the major saccharide content in açaí materials using 1H-NMR,as well as practical information, which could be used in similarstudies involving dietary supplements and other natural prod-ucts. Especially with respect to flavour, sugars play an importantrole in the quality and consistency of commercial food and bev-erage products (Tardieu et al., 2009). Given the vast number ofjuices, energy drinks and edible products containing açaí, thesugar contents of different E. oleracea materials could representa significant consideration for ingredient selection andmanufacturing of such products.Previous LC–MS studies have demonstrated lower quantities of

these sugars in other freeze-dried açaí materials (Schauss et al.,2006). However, such differences are to be expected; it is wellunderstood that metabolite content is strongly affected by plantvariety, harvest site, growing conditions and developmentalstage (Li et al., 2004; Tardieu et al., 2009; Ali et al., 2011; Zhao


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et al., 2011). Since fruit ripening typically involves the conversionof starch to sugars (Dudley, 2004), increased sugar content oftenserves as an indication of fruit maturity and ‘picking stage’(Kader et al., 1977). For example, in sapota fruits, the amountsof glucose and fructose have been found to increase until the‘eating-ripe stage’, while sucrose content continues to increaseuntil maturity and subsequently decline (Chaughule et al., 2002).

Although the primary purpose of the quantitative resultspresented in the current study is to serve as a comparison forthe two 1H-NMR methods of quantification, strong variations insugar content between the forms of açaí studied here shouldnot be ignored – particularly the greater quantities found inthe organic material (ADSR-2) in comparison with the non-organic materials (ADSR-1, ADSR-3). At least one study on açaísuggests that reducing sugars may play a role in anthocyanindegradation and thus affect anti-oxidant stability (Pacheco-Palencia et al., 2007). While sugar content appears to be an im-portant consideration, it is impossible to identify the preciseorigin of the variations observed in the current study; as has al-ready been discussed, there may be a multitude of contributingfactors. Although no evidence exists to necessarily attributethese differences to the organic or non-organic nature of thesematerials, this could serve as the basis for subsequent investiga-tions on açaí materials.

Potential value of the absolute intensity qNMR method

Numerous studies have validated the use of an internal stan-dard to quantify components in natural product mixtures using1H-NMR (Pauli et al., 2005; Lubbe et al., 2009; Dai et al., 2010).Instead, the present study describes the validation and applicationof a new qNMR-based method, which compares extremely wellwith the internal standard approach in terms of accuracy andprecision. The absolute intensity qNMR method is advantageousin certain situations, including the validation of newly developedinternal standards, as well as quantitative studies involving a largenumber of samples. Whereas a reference compound must bephysically added to each sample when an internal standardmethodology is employed, the qNMR utility described hererequires the preparation of only a single reference solution, whichcan be used for any samples over an extended period of time.

In addition to reducing the cost of materials, the use ofthis simpler method can save time and reduce the risk oferror by minimising sample preparation. Furthermore, it isbeneficial to have alternative 1H-qNMR methods, since aninternal standard may not always be found to meet thenecessary requirements for each experiment, such as mutualsolubility and avoidance of signal overlap (Holzgrabe, 2010).In such cases, the absolute intensity method would be anextremely valuable and precise tool for quantification. Even whenthe use of an internal standard is the desired methodology forquantification, the use of multiple qNMR techniques provides agreater certainty for quantitative results. As shown in our investi-gation of açaí materials, this may be particularly suitable foranalysing solutions of natural products as well as other complexmixtures in solutions.

Acknowledgements

The authors extend their thanks to Dr Lorraine Wolf and theAuburn University Undergraduate Research Fellowship Commit-tee for their general support and financial assistance, Dr MikeMeadows of the Department of Chemistry, AU, for all of his


technical advice, and Dr Forrest Smith of the Department ofPharmacal Sciences, AU, for kindly providing us access to theVarian 400 MHz spectrometer. In addition, the authors would liketo thank Dr William J. Keller from Nature’s Sunshine Products andDr William Hurst from the Hershey Company for generouslyproviding the plant materials used in the study.

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