Philippine Journal of Science RESEARCH NOTE 149 (4): 1119-1125, December 2020 ISSN 0031 - 7683 Date Received: 08 Jun 2020

Using a Glucometer for Home-based Quantification of Sugar in Baked Goods

Erwin Oliver V. Fundador* and Kriza Faye A. Calumba

Department of Food Science and Chemistry, College of Science and Mathematics University of the Mindanao, Mintal, Davao City 8022 Philippines

Weight reduction can be achieved by reducing the amount of sugar in the diet. However, a lot of bakeries sell products without nutritional labels. This makes it difficult to implement a weight management plan that is based on the reduction of sugar. To solve this problem, a cheap analytical technique suitable for home use has to be developed. Commercial glucometers are -based electrochemical sensors used for levels. These devices are easy to purchase, cheap (as low as USD 7.00 for the unit and USD 0.2 for the strip), portable, and can measure glucose levels in less than 10 seconds. In this paper, the suitability of a glucometer for determining sugar in baked goods was determined by comparing the results obtained with the use of HPLC-RID. The results obtained using both instruments were not significantly different (P < 0.05). Assuming the HPLC-RID values as the standard, the % error values for the measured amount of sugar per serving of egg cookies, graham crackers, and breadsticks were 0.41%, –1.85%, and 6.02% respectively, which were all less than 10%. This study suggests that the glucometer used is a suitable instrument for home-based quantification of sugar in unlabeled baked goods.

Keywords: baked goods, glucometer, HPLC-RID, invertase, sugar

INTRODUCTION sugar is removed (Mitra et al. 2010). Another study has shown that a deliberate reduction of sugar would lead to a Sugar plays an essential role in food products and is one decrease in body weight even without strict control of food of the primary ingredients in baked goods. It is not only (Te Morenga et al. 2013). It appears that the removal of a cheap flavor enhancer (Luo et al. 2019), but it also sugar not only cuts calories but also improves the feeling functions to improve the structure, mouthfeel, and shelf life of fullness. Therefore, the restriction of sugar from the of baked products (Sahin et al. 2019). However, long term diet should be part of a good weight management plan. consumption of sugar may blunt biochemical processes that promote satiation (Mitra et al. 2010) and lead to overeating. In the Philippines and probably in some countries, a lot The excess calories will then lead to weight gain, which of baked goods are sold without disclosing the amount of increases the risk of developing (Al-Goblan et al. sugar in the products. This makes it difficult to implement 2014) and other metabolic diseases. a weight management plan that is based on the reduction of sugar. To solve this problem, an analytical method For a weight management plan to be successful, cutting suitable for home use has to be developed. calories while still feeling full is key. One study suggests that there is an increased sensitivity to fullness when Most colorimetric methods are designed for reducing sugars (Negrulescu et al. 2012) or just glucose (Liu et al. *Corresponding Authors: [email protected] 2020), while sucrose – a non-reducing sugar – is the most

1119 Philippine Journal of Science Fundador and Calumba: Glucometer for Home- Vol. 149 No. 4, December 2020 based Quantification of Sugar in Baked Goods common sugar present in baked goods (van der Sman and Reagents. ACS reagent grade sucrose was used as standard. Renzetti 2019). Invertase is already being used to convert Invertase (I4504) from Saccharomyces cerevisiae was sucrose to glucose and fructose and commercial kits are purchased from Sigma-Aldrich (St. Louis, MO, USA). available for this (Sigma Aldrich 2004). However, aside from being costly, these kits require a 96-well plate reader Instrument Qualification for Determining Sucrose that is not only bulky but also expensive for home use. Linearity. Different sucrose concentrations (1, 2, 3, and 4 Electrochemical sensors for the detection of sucrose have mg/mL) in 100 mM pH 5.5 acetate buffer were prepared. also been published (Cruz et al. 2018; Stredansky et al. One milliliter (1 mL) of each solution was mixed with 2018). In comparison to colorimetric techniques, these 20 µl Invertase (10 mg/ml in the same buffer) and then are relatively easy to miniaturize. However, these sensors incubated at 37 °C for 2 h. The coefficient of determination have not yet been commercialized. Also, the demand for (R2) of sucrose concentration vs. glucometer reading (mM these sensors is probably not that high. It is, thus, unlikely glucose) was then determined. that mass production will be able to lower the price to Day-to-day signal consistency. Glucometer readings for 2 an acceptable point. Glucometers, on the other hand, are mg/mL Invertase-hydrolyzed sucrose were taken at Days already mass-produced, cheaper, and used extensively 1, 4, and 10. The relative standard deviation (RSD) of the (Liyanage et al. 2019). values was calculated. Commercial glucometers are enzyme-based electrochemical sensors used for monitoring blood Determining the Suitability of a Glucometer for glucose levels (Hettiaratchi 2012). These devices are easy Determining Sugar in Baked Goods to purchase, cheap (as low as USD 7.00 for the unit and The accuracy of the glucometer in determining the amount of USD 0.2 for the strip), portable, and can measure glucose sugar in baked goods was assessed by comparing the results levels in less than 10 seconds. For baked goods, detection to those obtained by HPLC-RID. Precision was measured can be achieved by converting sucrose into glucose. If in terms of repeatability and intermediate precision. The adapted for use in these food products, health-conscious repeatability was established by performing measurements in individuals should be able to check the sugar content of triplicate and getting the variation. The intermediate precision unlabeled baked products at home. However, there is no (i.e. different days) was determined by comparing the results published information on the reliability of these devices from two different days and getting the % difference. for sugar determination in food.

In this paper, the suitability of a cheap glucometer for Sample and Standard Preparation quantifying sugar in baked goods was determined. This Three different baked products (egg cookies, graham was done through instrument qualification (i.e. linearity crackers, and breadsticks) were purchased from a and day-to-day consistency) and partial validation (i.e. supermarket in Davao City, Philippines. The baked goods accuracy and precision) for use in baked goods. Accuracy were oven-dried at 130 °C for 1 h and pulverized using a was assessed by comparing the measured sugar values mortar and pestle. An amount of sample was then placed using a glucometer to those using HPLC-RID (high- in a 100-mL volumetric flask, diluted to volume with performance liquid chromatography – refractive index 100 mM pH 5.5 acetate buffer, and sonicated for 2 min. detector). The amount of sample (X) used was determined using Equation 1. The resulting solution served as the sample preparation. Standard preparations of 1, 2, 3, and 4 mg/ mL of sucrose in the same buffer were also prepared. MATERIALS AND METHODS (1) Equipment Glucometer. A blood glucose meter with dimensions where: of 100 x 58 x 23 mm (GA-3, Sannuo Yizhun, ) X = amount of sample dissolved (mg) was used. The apparatus came with 100 test strips and Y = serving size (g) additional test strips were purchased separately. Z = sugar per serving (g)

HPLC-RID. A Shimadzu LC-20AD™ HPLC equipped Determination of Sugar Concentration Using a with a Shimadzu 10AS VP column oven and a Glucometer Shimadzu RID-10A™ was used. The column used was a One milliliter (1 mL) of each of the samples and SUPELCOGEL 610H. standard solutions was mixed with 20 µl Invertase (10 mg/mL in the same buffer) and then incubated at 37 °C

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for 2 h. Glucometer signals (mM glucose) for both the generated against Invertase-hydrolyzed sucrose. Good sample and the standard were obtained. The sugar in calibration curves are achieved if the signals are linear the sample preparation was determined using multiple and stable. Therefore, the suitability of glucometers for point calibration. The measured sugar per serving (A) the determination of sugar in baked goods is determined was calculated using Equation 2. The experiment was by the linearity and consistency of the readings. conducted in triplicate.

(2) Linearity A linear relationship between the sucrose concentration and glucometer response was observed (Figure 1). The where: obtained R2 value of 0.994 was considered good enough A = measured sugar per serving (g) for the intended purpose. Glucometers are designed to B = measured concentration of the sample preparation detect glucose concentrations of up to 6 mg/mL (Togashi (mg/mL) et al. 2016). If all of the glucose came from sucrose, this Z = serving size (g) should translate to 11.4 mg/mL of sucrose. However, in X = amount of sample dissolved (mg) this study, the linearity of the glucometer was tested only for sucrose solution concentrations of 1–4 mg/mL. Except Determination of Sugar Concentration Using for bland tasting products, the sugar in most baked goods HPLC-RID (e.g. cookies and cakes) does not differ by more than The HPLC system as described above was used. Water four times. Therefore, with the validated linear range, a was used as the mobile phase, the flow rate was 1 mL/ standardized sample preparation protocol can be created min, and the temperature was 40 °C. The same standard even if the amount of sugar in the sample is not known. and sample preparations were used with the addition of For example, if 1000 mg of sample is dissolved in 100 a 0.2-micron filtration step. Sugar concentration in the mL water and the concentration of sugar in the sample is sample preparation was determined using a single-point approximately 10–40%, then the concentration of sugar calibration with 2 mg/mL sucrose as standard. Sugar per in the sample preparation should range from 1–4 mg/mL. serving was calculated using Equation 2. The experiment was conducted in triplicate. As seen in the succeeding discussions, the method can accurately detect sugars in sample preparations containing approximately 2 mg/mL of sucrose (Table 2). It was also Statistical Analysis All experiments were conducted in triplicate. Data were shown that the method is linear from 1–4 mg/mL. This statistically analyzed using the Student’s t-test at 0.05 implies that the calibration curve applies to all data points level of significance. within that range and not only to 2 mg/mL. Therefore, the method should be accurate enough for all sample preparations that fall within 1–4 mg/mL.

RESULTS AND DISCUSSION

Instrument Qualification for Determining Sucrose According to the US FDA, glucometers are allowed to deviate by 15% from the true value in 95% of the cases (Binder 2018). Moreover, a recent study reported that even the popular of glucometers could have biases of up to 18.7% (Choukem et al. 2019). In this study, a 2-mg/mL sample of hydrolyzed sucrose consistently gave readings of around 6.5 mM glucose (Table 1). At that concentration, the theoretical amount of glucose released after sucrose hydrolysis should be around 5.85 mM. To Figure 1. Linearity of glucometer reading to sucrose concentration. correct for possible biases, calibration curves need to be

Table 1. Day-to-day consistency of glucometer response (mM Day-to-day Signal Consistency glucose) with 2 mg/mL sucrose standard. Similar to most popular brands, the home glucometer Day 1 Day 4 Day 10 % RSD used is not designed to be recalibrated. It is assumed that 6.53 ± 0.06 6.76 ± 0.11 6.60 ± 0.06 1.66% the glucose readings are consistent for the duration of the warranty. While most popular brands have warranties of

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up to 10 years (ACCU-CHECK 2020; CONTOUR NEXT HPLC-based protocols are considered to be highly specific 2020), no warranty was provided for the glucometer due to the separation step done before detection. Thus, used in the study. Also, one study has shown poor we considered the HPLC-RID measured value as the reproducibility for some home glucometers (Salacinski reference value. et al. 2014). Thus, the consistency of the readings of the glucometer used in this study had to be confirmed. With To ensure the optimal accuracy of the HPLC-RID that, the glucometer readings at 1, 4, and 10 days had an method, we chose to do a single-point calibration instead RSD of 1.66% (Table 1). The low variability suggests that of multiple-point calibration. Calibration curves are new calibration curves are not needed for at least 10 days. not perfectly linear, which is also why all USP HPLC protocols (2008) used to check active ingredients in drug manufacturing are based on single-point calibration (US Determining the Suitability of a Glucometer for Pharmacopeia 2008). Determining Sugar in Baked Goods Total sugars on the label of food products include those However, this assumption is true only if the concentration that are naturally present as well as any added sugars in the sample preparation matches or is at least close to (US FDA 2020). In the case of baked goods, the most the concentration in the standard preparation. In this common type of sugar is sucrose (van der Sman and study, we tried to prepare the standard solution such that Renzetti 2019). In some cases, high-fructose syrup can the concentration is close to the standard preparation, also be used (Zargaraan et al. 2016). However, the sugars which is 2 mg/mL. This is done by modifying the sample present in high-fructose syrup are mostly glucose and preparation protocol based on the amount of sugar per fructose at almost equimolar amounts (Tou et al. 2011). serving, as seen in Equation 1. The composition of high-fructose syrup is almost the same Moreover, single-point calibration is just used for the as invertase-hydrolyzed sucrose. Therefore, the protocol HPLC-RID to ensure that the values taken are as accurate used in this study is also applicable to baked goods that as possible. The glucometer method developed in this use high-fructose syrup. article used multiple-point calibration. Multiple-point The obtained values for the amount of sugar per serving calibration is needed for our method because it will of the food products used in this study were compared to eventually be used for baked goods without labeled claims. those reported in the labeled claims, and results showed The accuracy and precision of the glucometer method are only minimal differences (Table 2). It is worth noting reported in Tables 2 and 3. In this study, % error, RSD, that differences between the actual sugar content and and % difference values of less than 10% were considered the labeled claim are expected due to variations in the acceptable. For most people, determining the sugar manufacturing processes. Thus, the accuracy of the content of unlabeled baked goods at an accuracy of more glucometer was not based on the labeled claim, but on or less 10% should be acceptable for weight management the results obtained using HPLC-RID. purposes. Nutrition labels of some snack foods do not even The accuracy of the method is dependent not only on the appear to be that accurate (Jumpertz et al. 2013). suitability of the glucometer but also on the ability of the sample preparation to quantitatively extract the sugar. Accuracy While the sample extraction protocol needs validation, the Correct quantification of food composition is necessary suitability of the glucometer was proven by comparing its for diet purposes (Silveira et al. 2009). While several results to that of an HPLC-RID protocol whose sample analytical techniques have been used for sugars, HPLC extraction was the same as that of the glucometer method methods are the most specific. HPLC-based protocols are performed. In this way, the difference would be due to considered to be highly specific because of the separation the instrumental measurement itself (n.b., which includes step that occurs before detection (Hadjikinova et al. 2017). the calibration technique) and not the sample extraction. For this reason, the results obtained using HPLC-RID were

Table 2. Glucometer vs. HPLC-RID results for the amount of sugar per serving of different baked goods. Declared sugar Measured sugar per serving using Measured sugar per serving Product % error per serving (g) glucometer (g) (day 1) using HPLC-RID (g) Egg cookies 14 12.04 ± 0.12a 11.99 ± 0.03a 0.41% Graham crackers 5 4.77 ± 0.11a 4.86 ± 0.04a –1.85% Breadsticks 5 4.22 ± 0.20a 4.48 ± 0.05a 6.02% aMeans with the same letter within the same product are not significantly different (P < 0.05

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considered to be highly reliable not only for sucrose but one study (Hadjikinova et al. 2017). For the intermediate for other sugars as well. The accuracy of the glucometer precision, the values calculated on both days for Graham in determining the amount of sugar in baked goods was crackers and breadsticks were not significantly different assessed by running these two instruments in parallel. (Table 3). There was a statistically significant difference Using HPLC-RID, sucrose – as well as minute amounts of in the sugar values for the egg cookies on both days; glucose and fructose – were detected (Figure 2). As seen however, it was only 4.65%, which is still lower than 10%. in the figure, there were two small peaks of comparable size in addition to the large sucrose peaks. The two extra peaks are most likely the hydrolysis products of sucrose (i.e. glucose, fructose) that were produced during the CONCLUSION AND baking process, indicating that the sugar used in baking RECOMMENDATIONS is practically sucrose. Therefore, measuring sucrose based on the detection of glucose after hydrolysis (i.e. The glucometer gave a linear response and the signal enzymatic) is an appropriate way to determine the total appeared to be stable for up to 10 days, allowing continued sugar in baked goods. use of the device during such a period without calibration. The results from the glucometer were comparable to those obtained from HPLC-RID, signifying good accuracy. The instrument also had good repeatability and intermediate precision in general. Assuming that a 10% error is acceptable for weight management purposes, the glucometer used in this study is a suitable instrument for quantifying sugar in unlabeled baked goods at home. This will help consumers adhere to a weight management plan based on the reduction of sugar. To fully validate the method, we intend to prepare the baked goods ourselves. By doing it this way, the results of the method can then be compared to the amount of sugar that “we” placed in the sample.

Figure 2. HPLC-RID chromatograms for various baked goods.

ACKNOWLEDGMENTS The results obtained using both instruments were not significantly different (P < 0.05). The % error values The authors would like to acknowledge the financial for the measured amount of sugar per serving of the egg support given by the University of the Philippines cookies, Graham crackers, and breadsticks were 0.41%, Mindanao In-house Research Grant. –1.85%, and 6.02% respectively, which were all less than 10% (Table 2).

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Table 3. Repeatability and intermediate precision of glucometer measurements. Day 1 Day 4 Product Mean ± SD Mean ± SD RSD, % RSD, % (mM glucose) (mM glucose) Egg cookies 12.04 ± 0.12b 1.00% 12.61 ± 0.2a 1.67% Graham crackers 4.77 ± 0.11a 2.31% 4.90 ± 0.16a 3.27% Breadsticks 4.22 ± 0.20a 4.74% 4.34 ± 0.04a 0.92% aMeans with the same letter within the same product are not significantly different (P < 0.05).

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