OLIGOSACCHARIDE FINGERPRINTING TO VERIFY AUTHENTICITY

Using GC to Detect Food Adulteration

Determining the authenticity of food has become a challenging problem. Detecting the adulteration of foods rich in carbohydrates, such as fruit juices, honey, and maple syrup is particularly difficult, because a variety of commercial sweeteners exist which exactly match the major carbohydrate profiles of these foods. The undeclared -- that is, not labelled -- addition of a commercial sweetener to a food is considered adulteration.

The driving force behind food adulteration is economics. In the case of apple juice, a worldwide shortage of apple concentrate in 1995 caused the market price to fluctuate between $7 and $14 per gallon. By contrast, the wholesale price of inexpensive sweeteners is stable and ranges from $0.70 to $1.40 per lb/solids. Similar economic situations are encountered for other fruit juices, honey, and maple syrup. Estimates on the extent of marketplace adulteration of foods rich in carbohydrate range from a low of 5% to as high as 25%.

Adding Less Expensive Carbohydrates

Carbohydrates account for more than 98% of the total soluble solids present in honey, maple syrup, and most fruit juices. The major carbohydrates naturally present in these foods are glucose, fructose, and sucrose. For example, the monosaccharides fructose and glucose are the major carbohydrate constituents of apple juice, and the fructose/glucose ratio is generally over 2.0. A facile yet sophisticated method of adulterating apple juice (and any food rich in carbohydrates) would involve the addition of less expensive carbohydrate syrups that resemble the major carbohydrate composition of apple juice.

Three commercially available sweeteners that approximate the carbohydrate composition of apple juice are beet/cane invert sugar (IS; 1:1 ratio of fructose to glucose), high-fructose corn syrup (HFCS, specifically HFCS 55; 55% fructose and 40% glucose), and high-fructose inulin syrup (HIS; fructose-to-glucose ratios of 8:1 to 3:1).

Analysis of pure apple juice and apple juice with added 10% high-fructose corn syrup.

Gas chromatogram of pure apple juice and pure apple juice adulterated with 10% beet invert sugar.

Analysis of pure apple juice and pure apple juice with 10% high-fructose inulin syrup added.

Detection is Difficult

To illustrate how difficult it is to detect an adulterated food, two authentic apple juice samples were intentionally adulterated with 5, 10, and 15% high-fructose corn syrup. These samples were then subjected to a wide range of chemical analyses; the results are shown in the table. Comparison of these intentionally adulterated samples with the authentic range for pure apple juices clearly indicates how difficult it is to identify an adulterated juice.

The Clue: Presence of FPOs

Recently,1-4 our research group developed a capillary gas chromatographic method for detecting the addition of inexpensive sweeteners to fruit juices (apple, orange, and grapefruit), honey, and maple syrup. This method is based on the presence of fingerprint oligosaccharides (FPOs) in inexpensive sweeteners which are either not present or are present at very low concentrations in the aforementioned pure foods.

We used an HP 6890 gas chromatograph with flame ionization detection for this analysis. The GC system was operated in the constant-flow mode, which offered exceptional retention time reproducibility of the FPOs. Hydrogen was employed as the carrier gas which, compared to helium, provided improved resolution of the FPOs and reduced analysis times. The added safety features of the HP 6890 (automated system shutdown when a hydrogen leak is detected) reduces the concerns associated with the use of hydrogen as a carrier gas.

Preparing the Sample

Sample preparation is facile. An appropriate dilution (typically 100 µL of a 5.5 Brix solution) of the food sample is transferred to a GC autosampler vial (12 x 32 mm), followed by freeze-drying of the sample. The carbohydrates in the sample are derivatized using 0.5 mL of Tri-Sil Z®, the resulting solution is heated at 70°C for 30 minutes, then analyzed with the HP 6890 employing an HP- 5 capillary column (0.32 mm x 30 m; 0.25 µm film thickness).

Detection in a Single Run

The efficacy of this method is clearly demonstrated in the capillary gas chromatograms of apple juice samples intentionally adulterated with IS , HFCS, and HIS (see figures). The fingerprint oligosaccharide marker peaks used for the detection of each of these commercial sweeteners occur at approximately 39.1 and 39.8 (IS); 41.2 and 44.6 (HFCS); and 20.0 and 30.1 (HIS) minutes. All three commercial sweeteners can be detected in a single chromatographic run, and the limits of detection for each of these materials is approximately 5%.

Chemical Composition Data for Selected Pure and Intentionally Adulterated Apple Juice Samples
Sample	So (%)	G (%)	F (%)	S (%)	F/G	CGA (ppm)	M (g/100mL)	C (g/100mL)	K (ppm)	Ca (ppm)	Mg (ppm)	ð13C
PAJ -1	0.42	1.75	6.66	1.70	3.81	97	420	22	1000	78	60	-25.6
PAJ -1 + 5% HFCS	0.40	1.70	6.62	1.66	3.89	95	390	21	959	71	57	-24.9
PAJ -1 + 10% HFCS	0.36	1.66	6.90	1.49	4.16	88	351	20	858	70	52	-23.9
PAJ -1 + 15% HFCS	0.30	1.46	7.14	1.44	4.89	80	360	20	888	65	55	-23.1
PAJ -2	0.39	2.00	6.16	1.89	3.08	47	440	20	1006	83	49	-25.9
PAJ -2 + 5% HFCS	0.33	1.93	6.37	1.80	3.30	46	444	19	1011	82	44	-25.2
PAJ -2 + 10% HFCS	0.34	1.80	6.50	1.68	3.61	44	400	19	929	80	44	-24.5
PAJ -2 + 15% HFCS	0.28	1.67	6.63	1.60	3.97	44	379	20	897	75	39	-23.7
Literature Values	0.16- 1.20	0.89- 3.99	3.00- 10.5	0.88- 5.62		>20	200- 900	<30	685- 1510	30- 120	40- 70	<-20.2
All samples were at 11.5 °Brix C=citric acid; Ca=calcium; CGA=chlorogenic acid; F=fructose; G=glucose; HFCS=high fructose corn syrup; M=malic acid; K=potassium; Mg=magnesium; PAJ=pure apple juice; S=sucrose; So=sorbitol

References

1. Low, N. H. J. AOAC Int. 79, 724-737 (1996).
2. Low, N. H. and Hammond, D. A. Fruit Processing 4, 135-139 (1996).
3. Low, N. H. Fruit Processing 11, 362-367 (1995).
4. Low, N. H., Brause, A., and Wilhelmsen, E. J. AOAC Int. 77, 965-975 (1994).

Tri-Sil Z is a registered trademark of Pierce Chemical Co.