## 3 Results and Discussion
### Method Validation
#### 3.2.7 Quantification of Metformin Uptake by HEI-OC1  
Good viability of HEI-OC1 to different metformin concentrations was noticed with respect to control non-treated cells (Figure 5). Two-way ANOVA results revealed that metformin concentrations are significant variables in the experimental uptake results, while the experimental day outcomes are not. Bonferroni post hoc tests also revealed that increasing metformin concentration resulted in significant uptake of HEI-OC1 cells of the supplemented biguanide up to 5 mM concentration, after which, 10 mM metformin did not improve the cellular uptake by HEI-OC1 cells (Figure 5A,B). Exposure of HEI-OC1 cells to 1 mM metformin resulted in 19.34 ug/mL \(\pm\) 2.23 uptake, which was increased to 51.67 ug/mL \(\pm\) 7.96 upon exposure to 2.5 mM metformin (Figure 5A). When HEI-OC1 cells were exposed to 5 mM metformin, the cellular uptake of the biguanide significantly increased to 373.32 ug/mL \(\pm\) 24.03, after which, the cellular uptake of HEI-OC1 to metformin slightly increased to 377.82 ug/mL \(\pm\) 5.06 upon exposure to 10 mM concentration (Figure 5A). Figure 3: HPLC method linearity and sensitivity. (**A**) represents calibration curves (upper limit), (**B**) represents calibration curves (lower limit), and (**C**) represents limit of detection (LOD) and limit of quantification (LOQ) for the three analytes. These results were also presented in Figure 5B, which shows that exposure of HEI-OC1 to 1 mM resulted in 11.68% \(\pm\) 1.34 uptake of metformin with respect to original metformin supplemented. The uptake was slightly increased to 12.48% \(\pm\) 1.93 with exposure to 2.5 mM metformin. Cellular uptake of metformin by HEI-OC1 was significantly increased to 45.08% \(\pm\) 2.90 when metformin concentration increased to 5 mM. On the contrary, when HEI-OC1 cells were exposed to 10 mM metformin, their cellular uptake was only 22.81% \(\pm\) 0.31 with respect to the initial metformin concentration added (Figure 5B). Although 10 mM metformin did not harmfully affect the cell viability of HEI-OC1 cells, metformin cellular uptake was not significantly improved. These data suggest saturation of the cellular uptake of HEI-OC1 to the biguanide above 5 mM concentration. Figure 4: Recovery results for metformin, glipizide and gliclazide at three different concentrations (1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, 2 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 5 \(\mathrm{\SIUnitSymbolMicro g}\)/mL). (**A**) represents chromatograms of recovered analytes from marketed pharmaceutical products and (**B**) represents recovery % of these analytes from marketed tablets at three different concentration levels (1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, 2 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 5 \(\mathrm{\SIUnitSymbolMicro g}\)/mL). (*) in this image stands for the peaks of metformin, glipizide and gliclazide recovered from their marketed pharmaceutical products at different concentration levels and appearing at the same retention time for every analyte. The otoprotective effect of metformin was previously investigated on HEI-OC1 at several concentrations (1 mM) [48], (2 mM) [47] and (5 mM) [60]. In this paper, we tried 10 mM metformin concentration to investigate the cellular uptake of metformin by HEI-OC1 cell lines in addition to the previously reported concentrations. Cellular metformin uptake was reported to be mediated through different types of cellular transporters such as the organic cation transporters (OCTs), carnitine/organic cation transporter (OCTN1), thiamine transporter (THTR-2), serotonin reuptake transporter (SERT), plasma membrane monoamine transporter (PMAT) and multidrug and toxin extrusion transporters (MATEs) [4]. [PERSON] et al. investigated the transport/uptake of radiolabelled metformin on HEK-293 cell lines (a transformed type of embryonic human kidney cell line) [61]. They reported concentration-dependent uptake of the biguanide mediated through OCT2 that is saturated by increasing metformin concentration [61].