Improved Method for the Direct Determination of Available Carbohydrates in Low Carbohydrate
Products Using High-Performance Anion Exchange Chromatography
David Ellingson1, Brian Potts1, Phillip Anderson1, Greg Burkhardt1, Wayne Ellefson1, Darryl Sullivan1, Wesley Jacobs2 and Robert Ragan2
1Covance Laboratories Inc., Madison, WI; 2Abbott Laboratories, Columbus, OH Corresponding author: david.ellingson@covance.com
Introduction
As the popularity of low carbohydrate products continues to increase in the food and beverage
industry, the need for an official method for direct determination of available “net” carbohydrates
has become a priority. Available carbohydrates are defined as carbohydrates that are
hydrolyzed to monosaccharides and absorbed in the small intestine to enter the pathways of
carbohydrate metabolism.1 The traditional method by difference2 is nonspecific and inaccurate
in low carbohydrate products. A method published in 2005 for direct determination using
HPAEC-PAD was a dramatic improvement upon the method by difference.3 However, a
modification was needed to increase the accuracy, specificity, and detection levels for analysis
in low carbohydrate products. This new method utilizes a more extensive enzymatic digestion
to hydrolyze all common sugars, starches, and starch derivatives down to the monosaccharides
glucose, fructose, and galactose. This modification results in greater specificity by having to
quantify only the three monosaccharides. It also allows all available (or potentially available)
carbohydrates present in the sample to be captured through pooling of these three
monosaccharides, which increases the accuracy of determining the levels in the product and
improves the detection limits. Faster run times are now possible with having to quantitate only
three compounds. Another modification is the use of high purity enzymes. This decreases the
background monosaccharide contribution seen from the enzymes themselves, resulting in very
small corrections. It also reduces the possibility of contaminating activity that could potentially
release monosaccharides from unavailable carbohydrates. The new method was validated for a
very low carbohydrate soy infant formula (RCF®). Method validation consisted of ten days of
analyzing various levels of spiked samples. The linearity, sensitivity, precision, and accuracy
were captured from the ten days of analyses. Specificity was determined through the treatment
of samples with enzymes that either oxidized or phosphorylated the three analytes. This
allowed removal of the analytes from their respective retention times to reveal any possible
interferences and identify the peaks as glucose, fructose, and galactose.
Materials and Methods
Spike Preparation
■ Fructose, galactose, glucose, lactose, maltose, sucrose, raffinose, and stachyose were
spiked into RCF® at four concentration levels. Corn starch and maltodextrin (DE 18) were
also spiked separately into RCF® at four concentration levels.
Table 1. Actual Concentrationsa(µg/g) in Mixed Sugar, Maltodextrin, and
Corn Starch Spiked Samples
Method
Extraction and Enzyme Step 1
■ 2 g RCF® sample weighed into 25-mL volumetric flask (two blanks per run were also
prepared to correct for enzyme contribution)
■ MES-TRIS buffer pH 8.2 and 0.1 mL α-amylase (10,000 U/mL) added to flask
■ Incubated at 80˚C in a shaking water bath for 30 minutes
■ Cooled and flask brought to volume with MES-TRIS buffer
Enzyme Step 2
■ 1 mL aliquot added to screw top test tube
■ 1 mL of acetic acid (0.8% v/v) added to adjust pH to 4.6
■ 0.2 mL each of amyloglucosidase (1,500 U/mL), α-galactosidase (200 U/mL), and β-
galactosidase (200 U/mL) added to test tube
■ Stir bar added, capped, and incubated in 50˚C water bath with gentle stirring for 30 minutes
Enzyme Step 3
■ Uncapped test tube and added 0.2 mL of invertase (1,500 U/mL)
■ Capped and placed back in 50˚C water bath with gentle stirring for 30 minutes
■ Test tubes removed and placed in steam bath 100˚C for 5 minutes to quench enzymes and
then cooled.
■ Appropriate dilutions with water taken and then filtered into injection vials for HPAEC-PAD
analysis.
Specificity The change from MOPS (3-(N-Morpholino) propanesulfonic acid) to a MES-TRIS buffer for Table 5. Summary of Total Table 6. Day-to-Day, Within-Day, and Overall
controlling pH in the enzymatic digestion steps allowed a simple pH adjustment after the initial Carbohydrate Recoveries for Relative Standard Deviations
■ A spiking solution consisting of sucrose, lactose, maltose, raffinose, and stachyose were Spiked Samplesa
digestion with α-amlyase to make it suitable for conditions required for the combination of
spiked into RCF®.
enzymes in the second digestion.4,5 The initial incubation at 80˚C instead of 100˚C also helps
■ Solution treated per the method, with the exception of the omission of extraction and enzyme reduce the possibility of capturing resistant starches.6
step 1, acetate buffer (pH 4.6) used instead of MES-TRIS, and a higher concentration of
amyloglucosidase (3000 U/mL). The calibration data over the ten days of analyses resulted in coefficients of determination (r2)
of at least 0.999 for all three analytes, except for day 2 galactose at 0.996. The average
■ Aliquots after method treated with:
calibration error was not significantly different from zero for any of the analytes for all five
- Hexokinase, MgCl2, TRIS/HCl buffer pH 8, ATP (phosphorylate fructose and glucose) Or, levels, which shows a lack of significant bias associated with the calibration model.
- Galactose dehydrogenase w/mutarotase, TRIS/HCl buffer pH 8.6, NAD+ (oxidize
Specificity experiments showed that all the enzymes used in the enzymatic digestion were able
galactose)
to fully hydrolyze the sugars present to galactose, glucose, and fructose (Figure 2). All
■ Injected onto HPAEC-PAD with gradient used to separate all sugars in the spiking solution
galactose was successfully removed from this extract through treatment with galactose
to confirm complete digestion of the sugars in the spiking solution, and also for removal of
dehydrogenase, which converts galactose to galactonic acid. Similarly, glucose and fructose
the three monosaccharides from their relative retention times to show presence of
were completely removed from this extract through treatment with hexokinase, which converts
interferences.
Table 2. Detector glucose and fructose to glucose-6-phosphate and fructose-6-phosphate, respectively.
Together, these treatments showed an absence of any probable interference peaks in the
Waveform Settings
Instrumentation product matrix.
■ Dionex ICS-3000 with ED cell
■ Dionex ED Au conventional working electrode
■ Dionex Ag/AgCl reference electrode
Blank data showed that only low levels of glucose and galactose were present. This data was
■ Dionex CarboPac PA1 analytical (4 mm x 250 mm)
used to generate a limit of detection (LOD) of 0.0034 g/100g (LOD = Avg. Total Blank + (3 x
■ Dionex CarboPac PA1 guard (4mm x 50 mm)
Total Blank SD)) and a limit of quantitation (LOQ) of 0.0074 g/100g (LOQ = Avg. Total Blank
+ (10 x Total Blank SD)). The range that was chosen for standards equated to a range of
Instrument Conditions
analysis very near the LOD. The LOQ is well below the label claim of 0.069 g/100g and this
■ Flow rate = 1.2 mL/min Table 3. Method Gradient
supports the adequacy of the method for monitoring carbohydrate content at relevant levels.
■ Temperature = 30˚C
■ Injection volume = 25 µL
■ Mobile Phase A,B: Purified water
■ Mobile Phase C: 50 mM NaOH
Conclusions
■ Mobile Phase D: 600 mM NaOH Although the validation work reported here was limited to the low carbohydrate soy formula, it
■ Mobile Phase A,B: Purified water is expected that the method could be used for direct determination of available carbohydrates
■ Mobile Phase C: 250 mM NaOH in a variety of low carbohydrate food and beverage products, depending on the specific
■ Mobile Phase D: 600 mM NaOH
Table 4. Specificity Gradient3 carbohydrate composition. The main source of carbohydrates in the soy infant formula is the
soy protein ingredient, which has low levels of sucrose, raffinose, and stachyose. In order to
Calculations monitor and maintain the low carbohydrate levels required in this product, it was a priority to
err on the conservative side. The inclusion of α-galactosidase in the second digestion step
■ Quantitation and identification were
accomplished using Empower ensured that raffinose and stachyose would be fully captured and reported, in spite of their
chromatography software. limited digestibility. For other applications, this may not be desirable. Simply eliminating the α-
galactosidase from the enzyme mixture would limit recovery of monosaccharides from raffinose
■ All statistical computations/
and stachyose to the small amount liberated by invertase. An additional consideration is that
manipulations were done using either
the enzymatic conditions utilized in this method would be expected to capture most, if not all
Minitab® 15 (v 15.1.0.0; Minitab®
short chain fructooligosaccharides (scFOS) and galactooligosaccharides (GOS) that might be
Inc., State College, PA) or SAS® (v9.1: SAS Institute Inc, Cary, NC).
present. In cases where these materials comprise a significant portion of the total
carbohydrate, that might not be desirable.
Figure 2. Specificity testing involving RCF® spiked with mixed sugars (A). The extract
Results was subjected to treatment with galactose dehydrogenase (B) or hexokinase (C).
Digesting carbohydrates down to their monosaccharide components simplifies the Future Work
chromatography, which is shown for a starch spiked sample (Figure 1). Separation of six
The accuracy of the method was determined through duplicate analyses of spiked samples Covance is currently validating a method for direct determination of available carbohydrates by
different analytes was required within one gradient to quantitate available carbohydrates in the
HPAEC-PAD, which is designed to cover a wide range of food and beverage products from
over ten days. Spiked samples were comprised of four levels of common sugars (MS1-4),
previous method.3 This presented challenges in complex matrices to adequately resolve all six,
three levels of maltodextrin (MD1-3), and three levels of corn starch (CS1-3) (Table 1). Spike low to high concentrations that captures the improvements made in the method validated and
and also resulted in longer run times. The run time of this improved method is much faster,
recoveries were calculated from the average of the duplicates for each day (Table 5). Average described here. The main differences of this method from the low carbohydrate method
since the monosaccharides are the first to elute. Greater flexibility is now possible in resolving
recoveries by spike sample ranged from 94.1% to 106% (N = 10) with an overall average of described is the ability to capture only available carbohydrates in the presence of non-
peaks in complex food and beverage matrices.
99.4% (N = 100) across all levels and types. digestible oligosaccharides (raffinose, stachyose, scFOS, etc.), which the enzymes utilized in
the low carbohydrate method will hydrolyze to monosaccharides. It’s also designed to cover a
The precision of the method was estimated from the ten days of data collected for unspiked, large concentration range, so a majority of products can be analyzed. The method will replace
as well as spiked samples. Within-day, day-to-day, and overall RSDs for each sample are given the Lilla et al. 2005 method for routine net carbohydrate analysis upon validation.
in Table 6. Data from all of the samples was also pooled (after normalizing the results for each
to their respective means) in order to obtain overall estimates of the precision metrics. The
pooled estimates (also listed in Table 6) of precision were 3.4%, 3.6%, and 4.9% for within-
day, day-to-day, and overall RSDs, respectively. The pooled estimates are the best overall
Acknowledgements
indicator of method precision. The authors wish to thank Janis Dugle for her assistance with the statistical analyses.
References
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2. Official Methods of Analysis (2000) 17th Ed. AOAC INTERNATIONAL, Gaithersburg, MD, Method 986.25.
3. Lilla, Z., Sullivan, D., Ellefson, W., Welton, K., & Crowley, R. (2005) J. AOAC Int. 88, 714-719.
4. Official Methods of Analysis (2000) 17th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, Method 991.43.
5. McCleary, B.V. (2007) Anal. Bioanal. Chem. 389, 291-308.
6. Berry, C.S. (1986) J. Cereal Sci. 4, 301-314.
Figure 1. Chromatogram of a starch spiked RCF® sample after digestion according to the method.