## 2 Materials and Methods
### Chlorination Reaction
#### 2.2.3 Chlorination Procedure and Product Isolation  
Amoxicillin (1 g, 2.74 mmol), dissolved in milliQ water (2 L), was treated for 5 min with 5% hypochlorite (molar ratio AMO/NaOCl 1:2; concentration spectroscopically determined at a \(\uplambda\)max of 292 nm, \(\varepsilon\) = 350 dm\({}^{3}\)/mol cm) at room temperature [38]. The pH of the solution increased immediately from the initial pH of 8.0 to 10.5, and the pH remained at this value during the reaction. After 5 min, the solution was quenched using an excess of thiosulphate, with respect to NaOCl, and dried by lyophilization, and the residue was dissolved in a saturated Na\({}_{2}\)CO\({}_{3}\) solution and extracted with ethyl acetate (EA). The EA fraction (351 mg) was separated with the silica gel column chromatography (CC) using a gradient of methylene chloride/methanol (100:0 to 10:90, \(v/v\)) to yield 15 fractions. The EA5 fraction (25 mg), eluted with methylene chloride/methanol (90:10, \(v/v\)), was analyzed via HPLC using a Supelcosil LC-18 column, 25 cm \(\times\) 4.6 mm ID, and 5 \(\upmu\)particles. The solvent system was a gradient of acetonitrile/tetrahydrofuran/water (A, 30:10:60, \(v/v/v\)) and acetonitrile/water (B, 60:40, \(v/v\)), starting with 0% B for 1 min and installing a gradient to obtain 100% B over 20 min, at a solvent flow rate of 1.5 mL/min. The column effluent was monitored at 360 nm. Identification of DP6 and DP4 was achieved by comparison with standard compounds. The fraction EA7 (33 mg), eluted with methylene chloride/methanol (75:25, \(v/v\)), was analyzed via HPLC using a Supelcosil LC-8 column, 15 cm \(\times\) 4.6 mm I.D., and 5 \(\upmu\)m particles. The solvent system used was a gradient of acetic acid/methanol (A, 1:99, \(v/v\)) and acetic acid/water (B, 1:99, \(v/v\)), starting with 65% B for 1 min and installing a gradient to obtain 100% A over 25 min and returning to 65% B for 5 min at a solvent flow rate of 1.5 mL/min. The column effluent was monitored at 280 nm. Identification of DP7 and DP8 was achieved by comparison with standard compounds. The fraction EA8 (29 mg), eluted with methylene chloride/methanol (70:30, \(v/v\)), was dried, dissolved in an appropriate volume of methylene chloride (100 \(\upmu\)L), and analyzed using a gas chromatograph with a flame ionization detector (Shimadzu 2010 series, Milano, Italy). The gas chromatograph was equipped with an EquityTM-5 capillary column (30 m \(\times\) 0.25 mm I.D. \(\times\) 0.25 \(\upmu\)m film thickness). The following parameters were set during the experiments: detector temperature, 340 \({}^{\circ}\)C, carrier gas, helium (25 cm/s), injected samples, and 1.0 \(\upmu\)L, introduced into the injector using an AOC-20i auto sampler (Shimadzu, Milano, Italy) and heated to 225 \({}^{\circ}\)C with a split ratio of 100:1. The initial temperature was 40 \({}^{\circ}\)C with a 2 min hold, followed by a 8 \({}^{\circ}\)C/min ramp to 300 \({}^{\circ}\)C, with a 2 min hold. Identification of DP9 and DP10 was achieved by comparison with standard compounds. Figure 1: Time-conversion plot for the reaction of amoxicillin (AMO) with one equivalent NaOCl at buffered pH = 3.0 (**a**) and pH = 7 (**b**). \(\bullet\): AMO consumption by reaction with NaOCl (green); \(\Box\): Disappearance of AMO in the absence of NaOCl (red). Figure 2: (**a**) Time-conversion plot for the reaction of AMO with one equivalent NaOCl at pH = 9.0. \(\blacksquare\): NaOCl consumption in the presence of AMO (green); \(\bullet\): AMO consumption by reaction with NaOCl (blue); \(\Box\): Disappearance of NaOCl in the absence of AMO (red); \(\Box\): Disappearance of AMO in the absence of NaOCl (black); (**b**) AMO disappearance by NaOCl at pH basic no-buffered after a 5 min reaction. Figure 3: Chemical structures of amoxicillin and its degradation byproducts. etate/methanol (70:30, \(v/v\)), was analyzed via HPLC using a Discovery RP-Amide C16 column, 15 cm \(\times\) 4.6 mm I.D., and 5.0 \(\upmu\)m particles.