# Nutritional Quality and Safety Characteristics of Imported Biscuits Markered in Basrah, Iraq
## 3 Results and Discussion
### HMF and AA Content of Biscuits Samples  
The collected biscuits were also examined for their content of HMF and AA. The HPLC resolution for HMF was good (Figure 1a) and peaks were very clear, with the highest level of HMF found in the BSC2-U sample (Figure 1b). Figure 2a shows the HPLC chromatogram for the AA standard and Figure 2b shows the sample with the highest level of AA, i.e., BSC2-U. Again, the HPLC resolution was good and the peaks were very neat. Table 3 shows the content of HMF, and AA ascertained in the examined biscuit samples. Extremely high variability in HMF content was observed among samples within the same category or country, from ND to 62.08 mg/kg, which made not significant the influence of the \"Biscuit type\" and \"Country\" variables and made not significant also the effect of the \"Biscuit type\"Country\" interaction. The observed variation might be due to the type of sweetener used in the biscuit recipe. Invert sugar syrup was utilized as a sweetener in certain samples, as opposed to sucrose, which was used in other biscuits. [PERSON] et al. [47] demonstrated that HMF rises more rapidly from glucose and fructose than it does from sucrose, which may account for the observed differences. [PERSON] et al. [48] also observed that the generation of HMF  
Figure 1: (**a**) HPLC chromatogram of HMF standard; (**b**) HPLC chromatogram of HMF content of the biscuit sample (BSC2-U) which showed the highest level of HMF. is temperature-dependent (200 \({}^{\circ}\)C, 250 \({}^{\circ}\)C, and 300 \({}^{\circ}\)C) and sugar-dependent (fructose, glucose, and sucrose). At a higher baking temperature, the amount of HMF was greater: biscuits prepared at temperatures \(>\)300 \({}^{\circ}\)C contained between 10 and 100 times the amount of HMF (167.4 and 1100.1 mg/kg) as those baked at 200 \({}^{\circ}\)C (9.9 to 39.6 mg/kg) [48]. \begin{table}
\begin{tabular}{c c c} \hline
**Biscuit Type** & **HMF (mg/kg)** & **AA (\(\upmu\)/kg)** \\ \hline Cookies & & \\ BSCI-S & ND & ND \\ BSC2-S & \(1.85\pm 0.05\) & ND \\ BSC3-S & ND & ND \\ BSC1-I & \(3.31\pm 0.15\) & ND \\ BSC2-I & \(5.64\pm 0.12\) & ND \\ BSC3-I & \(32.62\pm 1.50\) & \(639.9\pm 4.1\) \\ BSC1-U & ND & ND \\ BSC2-U & \(62.08\pm 3.11\) & \(1421.7\pm 5.7\) \\ BSC3-U & \(9.27\pm 1.03\) & ND \\ BSC1-T & \(19.03\pm 1.14\) & \(397.9\pm 3.1\) \\ BSC2-T & ND & ND \\ BSC3-T & ND & ND \\ Crackers & & \\ BSCr1-S & \(0.60\pm 0.08\) & ND \\ BSCr2-S & \(6.30\pm 0.74\) & ND \\ BSCr3-S & \(13.55\pm 0.87\) & \(243.4\pm 2.9\) \\ BSCr1-I & \(60.66\pm 2.81\) & \(1061.9\pm 6.1\) \\ BSCr2-I & \(16.95\pm 1.04\) & \(254.3\pm 2.7\) \\ BSCr3-I & \(56.52\pm 2.86\) & \(802.4\pm 4.3\) \\ BSCr1-U & \(8.77\pm 1.01\) & ND \\ BSCr2-U & \(19.55\pm 1.24\) & \(404.2\pm 3.0\) \\ BSCr3-U & \(13.65\pm 1.04\) & \(312.9\pm 3.2\) \\ BSCr1-T & \(9.73\pm 0.75\) & \(327.2\pm 3.8\) \\ BSCr2-T & \(10.66\pm 1.11\) & \(323.2\pm 3.1\) \\ BSCr3-T & \(54.80\pm 3.11\) & \(742.7\pm 4.2\) \\ Digestives & & \\ BSD1-S & \(5.45\pm 0.06\) & ND \\ BSD2-S & ND & ND \\ BSD3-S & \(7.53\pm 0.62\) & \(226.2\pm 3.1\) \\ \hline \end{tabular}
\end{table} 
Table 3: Content (\(\upmu\)/kg) of 5-hydroxymethyl-2-furural (HMF) and acrylamide (AA) ascertained in biscuits marketed in Basrah, Iraq. Figure 2: (a) HPLC chromatogram of acrylamide standard. (b) HPLC chromatogram of acrylamide of a biscuit sample (BSC2-U). Additionally, the ingredients have an effect on HMF formation: sucrose-containing bisciucuits had the lowest HMF content (9.9 mg/kg) at 200 \({}^{\circ}\)C than bisciucuits whose formulation included fructose and glucose (39.6-34.2 mg/kg), owing to sucrose's thermal breakdown [48]. Another factor able to dramatically increase the HMF content is the use of ammonium bicarbonate [49], which was present as a leavening agent in the formulation of many of the examined samples (Supplementary Table S1). However, [PERSON] et al. [50] stated that the variations in HMF are more related to the type of thermal treatment used in the manufacturing process than to any specific ingredient.