Xylitol, a sugar substitute frequently used in sugar-free gum, is generally considered harmless to humans but it can be extremely toxic to dogs. Dog-owning customers are becoming increasingly aware of the risks associated with xylitol-containing chewing gums. However, there remains some uncertainty if these chewing gums are still dangerous to dogs after they have been partially consumed. In this work, a reliable low-cost analytical method has been developed to quantify the xylitol in sugar-free gum samples. Xylitol was extracted from gum samples using water as a solvent. Extractions were analyzed by GC-MS with direct aqueous injection (DAI). This method was successfully applied to over 120 samples including fresh gum and 5 min, 15 min, and 30 min chewed gum samples.
Xylitol can be found naturally in low concentrations in fruits, vegetables, mushrooms, and sugar cane. It is a type of artificial sweetener known as a sugar alcohol [
Xylitol has a varied safety margin in mammals. The LD50 of xylitol in mice is 20 g of xylitol per kilogram [
The amount of xylitol and number of fresh gum sticks that can cause hypoglycemia in dogs [
Size of the dog | Amount of xylitol to cause hypoglycemia in dog (g) (dose: 0.1 g of xylitol per kg of dog) | Required number of gum sticks | ||
---|---|---|---|---|
Ice breakers (1.5 g of xylitol/gum stick) | Stride (0.2 g of xylitol/gum stick) | Trident (0.2 g of xylitol/gum stick) | ||
2 kg Chihuahua | 0.2 | 1 | 1 | 1 |
4 kg Yorkie | 0.4 | 1 | 2 | 2 |
6 kg Jack Russell Terrier | 0.6 | 1 | 3 | 3 |
12 kg Border Collie | 1.2 | 1 | 6 | 6 |
25 kg Golden Retriever | 2.5 | 2 | 12 | 12 |
Xylitol’s presence in chewing gum and other consumer products makes it readily available to dogs with detrimental effects often requiring veterinary care. This study was inspired by many such incidences. Today, many dog-owning customers are aware of the risks associated with xylitol-containing chewing gums. However, there remains some uncertainty as to whether these chewing gums are still dangerous to dogs after they have been partially consumed. The primary goal of this study was to provide conclusive evidence of the toxicity of xylitol-containing chewing gums after partial consumption. Herein, a reliable low-cost analytical method has been developed to quantify the xylitol in sugar-free gum samples.
Numerous methods are commonly employed for the analysis of polyols [
A direct aqueous injection GC-MS method utilizing an Agilent 7890A GC/5975C MS has been developed in this study. Direct aqueous injection (DAI) is key to this analysis because polyols are more soluble in water than any other common solvent [
Glycerol (CAS-56-81-5, assay 99.5%, MW 92.09 g mol−1), xylitol (CAS-87-99-0, assay 99%, MW 152.15 g mol−1), DL-threitol (CAS-7493-90-5, assay 97%, MW 122.12 g mol−1), and sorbitol (CAS-50-70-4, assay 99%, MW 182.17 g mol−1) were purchased from Sigma-Aldrich. Xylitol-containing Trident sugar-free gum was purchased from Walmart (regular 0.17–0.20 mg of xylitol/piece). DI water was used to prepare all samples and standard stock solutions. Similar weight gum pieces were chosen for analysis.
An Agilent 7890A/5975C gas chromatography-mass spectrometry (GC-MS) system was used with a water resistant 60 m × 320
A flow diagram of the method is shown in Figure
A flow diagram of xylitol analysis method.
Sample collection: 4 gum packs, each containing 18 gum sticks, were randomly selected from a commercial package containing 14 packs. A total of 12 fresh gum sticks were collected from the gum packs including the 1st, 9th, and 18th gum sticks of each pack to determine xylitol content.
A fresh gum stick was carefully cut into about 6–7 small pieces. Gum pieces were ground for 5 min with 10 mL of DI water. This extraction step was repeated 9 times requiring a total of 90 mL DI water and 45 min to extract xylitol completely from a single gum piece (Figure
Amount of xylitol extracted after each extraction.
Sample collection: 12 volunteers, between 20 and 40 years old, participated in this study. Participants were asked to wash their mouth with water before they chewed a gum stick. Participants chewed their gum pieces with similar starting masses (1.724 g ± 0.008) for 5 min before placing the chewed gum into a sterilized container. Three gum samples from each participant were collected within a 2-day interval (a total of 36 gum samples). The xylitol content of each 5 min chewed gum piece was determined as follows.
The gum stick was ground using a mortar and pestle for 5 min with 10 mL of DI water. This extraction step was repeated
Sample collection: the same 12 volunteers who participated in the 5 min chewed gum sampling also participated in this study. A similar procedure was used as above. Participants washed their mouth with water before chewing gum pieces with almost identical masses (1.724 g ± 0.008) for 15 min. The chewed gum pieces were collected into sterilized containers before extraction. Three replicates were performed by each participant (total 36 gum samples).
A similar extraction method was used as above with the 5 min samples. However, with these 15 min samples, the 10 mL extraction step was repeated
Sample collection: the same 12 volunteers who participated for both the 5 and 15 min chewed samples also participated in this study. An identical method was used with the exception being 30 min of chewing before extraction. Xylitol was then extracted and analyzed using an identical method as above for the 15 min samples.
Water is generally considered as a poor solvent in GC analysis due to problems related to backflash and chemical reactivity. Common GC solvents such as hexane, ethyl acetate, acetone, and dichloromethane have vapor-to-liquid volume ratios in the range of 100–300, [
Laminar cup splitter design.
Analysis of xylitol (boiling point 216°C) in a complex mixture requires both high temperature injection and temperature programming to ensure chromatographic separation. The GC oven was programmed with an initial temperature at the boiling point of xylitol to facilitate high temperature analysis, while reducing column bleed and improving limits of detection (Figure
GC oven program.
The effect of injection temperature was studied to facilitate complete vaporization of xylitol in the GC inlet. Raising the injection temperature from 200°C to 280°C increased xylitol peak area by a factor of 5. However, only a 5% increase was observed when the inlet temperature was increased from 280°C to 300°C (Figure
Effect of injection temperature on xylitol peak area. Note: error bars indicate standard deviation (
Choosing a correct internal standard (IS) can improve a method’s accuracy and precision. Method development for GC-MS often utilizes an internal standard to account for routine variation of the instrument response and injection volumes. An internal standard should be chemically similar to the analyte but it should not be naturally present in any of the samples to be analyzed. A Trident spearmint flavor gum piece contains three polyols in large quantities: xylitol, glycerol, and sorbitol. Mannitol is also present but at low concentrations compared to the xylitol and sorbitol content of the gum piece (Figure
Total ion chromatogram (TIC) of Trident gum extract.
Various chemicals were tested as an internal standard for this study including ethylene glycol, 3,5-dimethoxyphenol, 2-methoxyphenol, terpineol, 2-nonanol, and DL-threitol. Both 2-methoxyphenol and DL-threitol were found to be suitable for analysis in the terms of retention times, since they did not overlap with any peaks of the Trident gum extract. However, DL-threitol was picked as the better internal standard because it has the same functional groups as xylitol. More importantly, glycerol, threitol, xylitol, and sorbitol are members of a series of sugar alcohols where the difference between any two compounds in a sequence varies by one carbon atom, two hydrogen atoms, and one oxygen atom, as CH–OH unit (Figure
Total ion chromatogram (TIC) of Trident gum extract with internal standard.
Recovery tests were performed to evaluate extraction efficiency. Gum base left after the 10th extraction was spiked with a known amount of solid xylitol (180.0 mg). The xylitol was thoroughly ground into the gum base using a mortar and pestle before extraction and DAI GC-MS analysis using the fresh gum method described above. This procedure was repeated for five gum-base samples (GB-1 through GB-5). Recovery values (Table
Precision and recovery of fresh gum analysis method (
Gum base no. | Spiked (mg) | Measured (mg) | Recovery (%) | RSD (%) |
---|---|---|---|---|
GB-1 | 178.2 | 177.2 | 99.4 | 0.31 |
GB-2 | 180.3 | 178.4 | 98.9 | 0.18 |
GB-3 | 179.4 | 177.2 | 98.8 | 0.17 |
GB-4 | 180.6 | 176.6 | 97.8 | 0.24 |
GB-5 | 178.4 | 169.9 | 95.1 | 0.72 |
Limit of detection (LOD) and limit of quantification (LOQ) were determined. An internal standard calibration graph was plotted using the ratio of peak areas of the internal standard (DL-threitol) versus the concentration of xylitol. Good linearity was observed, with a square of the correlation coefficient (
Analysis of variance with Tukey’s honestly significant difference test (
Effect of chewing rate on xylitol release was studied. Four volunteers were randomly selected for this experiment. Each participant was given three gum sticks with similar masses (1.724 g ± 0.008). Chewing rates were selected after considering general chewing habits. We observed that a person starts chewing a gum piece at a rate between 30 and 120 chews per minute for the first two minutes due to the sugar taste. Chewing rates then decrease with time due to decreased sugar content of the gum. Three chewing rates 30, 60, and 120 chews per minute were selected. Participants were asked to chew gum pieces at the selected rates for 2 minutes. The method, which was used for the fresh gum analysis, was used to determine the xylitol content of chewed gum samples in this experiment (Figure
Amount of xylitol remaining in a gum piece chewed by four participants at three different rates (numbers sharing similar lower case letters indicate no significant difference).
Results show that chewing rate had no significant effect on the remaining xylitol content in the 2 min chewed gums (
Trident spearmint flavor gum pieces were selected for this study. In order to determine the range of xylitol content in a gum stick, 4 gum packs were chosen randomly, each with 18 gum sticks. The 1st, 9th, and 18th sticks of each pack were selected for analysis. The amount of xylitol in the fresh gum pieces were determined using the fresh gum analysis method established above. Each sample was analyzed by DAI GC-MS three times, and the results are summarized in Table
Determination of the xylitol content of Trident spearmint flavor gum (regular care).
Gum piece ID | Xylitol content (mg) | SD ( |
Gum piece ID | Xylitol content (mg) | SD ( |
---|---|---|---|---|---|
P1-ST1 | 180.8 | 0.8 | P3-ST1 | 187.8 | 0.5 |
P1-ST9 | 179.1 | 0.2 | P3-ST9 | 179.8 | 0.6 |
P1-ST18 | 179.7 | 0.3 | P3-ST18 | 171.3 | 0.2 |
P2-ST1 | 193.0 | 0.3 | P4-ST1 | 170.7 | 0.4 |
P2-ST9 | 187.8 | 0.6 | P4-ST9 | 172.4 | 0.4 |
P2-ST18 | 174.0 | 0.4 | P4-ST18 | 181.1 | 0.2 |
A total of 36 samples (3 each for the 12 participants) were analyzed to determine the amount of xylitol in 5 min chewed gum samples, and the results are summarized in Figure
Determination of xylitol content in 5 min chewed gum samples.
A total of 36 (3 from each participant) of the 15 min chewed gum samples were also analyzed (Figure
Determination of xylitol content in 15 min chewed gum samples.
There was no xylitol above the LOD in any of the 36 samples collected after a 30 min chewing period. Approximately 99.4% of xylitol is removed from the gum within the first 15 min, and another 15 min of chewing time reduced the xylitol content below the method limits to detect.
Table
Amount of gum sticks required to supply toxic dose to make a dog sick.
Size of the dog | Amount of xylitol to cause hypoglycemia (g) | Number of gum pieces required to supply toxic dose (dose: 0.1 g of xylitol per kg of dog) | ||
---|---|---|---|---|
Fresh (xylitol = 179 mg) | 5 min chewed (xylitol = 7.8 mg) | 15 min chewed (xylitol = 1.1 mg) | ||
2 kg Chihuahua | 0.2 | 1 | 26 | 182 |
4 kg Yorkie | 0.4 | 2 | 51 | 364 |
6 kg Jack Russell Terrier | 0.6 | 3 | 77 | 545 |
12 kg Border Collie | 1.2 | 7 | 154 | 1091 |
25 kg Golden Retriever | 2.5 | 14 | 320 | 2273 |
A method for the analysis of xylitol in sugar-free gum was successfully developed using GC-MS with direct aqueous injection (DAI). Additional cleanup steps and sample derivatization were not required for the analysis, resulting in short analysis times. Spiked recoveries of the sample ranged from 95 to 99% while RSD ranged from 0.17 to 0.72% (
The data used to support the findings of this study are included within the article.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This study was funded by the American College of Veterinary Emergency and Critical Care (ACVECC). The authors would like to thank graduate students and undergraduates of Department of Chemistry, MSU, for providing analytical samples.