## 3 Results and Discussion
### Validation of the HPLC Analytical Method Performance  
#### 3.2.1 Sensitivity and Linearity of the Test Method  
Preparation of HNS Simulated Wastewater at Different Concentration Gradients: Accurately weigh 50 mg of HNS drug and dissolve it in 10 mL of DMF under ultrasonic conditions to obtain what will be labeled as Sample 1. A 2 mL aliquot of Sample 1 was diluted to 25 mL with ultrapure water to prepare Sample 2. Similarly, a 2 mL aliquot of DMF organic solvent was diluted to 25 mL with ultrapure water to obtain Sample 3. Under the HPLC conditions described in Section 3.1, test these varying concentrations of HNS standard solutions in triplicate. To minimize interference, samples should be injected in sequence from lowest to highest concentration. Record the peak areas from each injection, calculate the average, and use this average as the vertical axis for plotting. Plot the mass concentrations (mg/L) of each standard on the abscissa to perform linear regression and establish a calibration curve. Further, a 150.00 mg/L HNS standard solution was diluted with ultrapure water until the response value reached three times the instrument's baseline noise. This established a signal-to-noise ratio (S/N) of 3 as the detection limit. When the S/N ratio reaches ten times the baseline noise (S/N = 10), the quantitation limit is established. Stepwise dilution reveals that at an HNS concentration of 0.10 mg/L, the S/N ratio is 3, and at 0.40 mg/L, the S/N ratio is 10. Therefore, twelve concentrations of HNS  
Figure 5: High-performance liquid chromatography chromatograms of DMF and HNS at dual wavelengths, with an injection volume set at 10 \(\upmu\)L, (a) 226 nm (b) 271 nm. \begin{table}
\begin{tabular}{c c} \hline \hline
**Chromotographic Parameter** & **Chromotographic Condition** \\ \hline Chromatographic instrument & Ultimate 3000 UHPLC \\ chromatographic column & ACCUCORE C30, 2.6 \(\upmu\)m, 150 \(\times\) 4.6 mm \\ UV detection channel wavelength & 226 nm and 271 nm \\ Liquid phase mobile phase ratio & MeOH:water = 70:30 \\ Liquid flow rate & 0.5 mL min\({}^{-1}\) \\ Column temperature & 30 \({}^{\circ}\)C \\ Liquid phase injection quantity & 10 \(\upmu\)L \\ Sample test time & 6.5 min \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Conditions for the detection of HNS by HPLC. were prepared: 0.10, 0.50, 2.00, 5.00, 10.00, 15.00, 20.00, 25.00, 30.00, 60.00, 90.00, and 150.00 mg/L. For each concentration, three replicates were prepared, totaling 36 samples to establish the calibration curve. Six samples, labeled sample 4 through sample 9 (0.50, 1.00, 20.00, 30.00, 60.00, 150.00 mg/L), were prepared as detailed in Table 4. The corresponding volumes of sample 1, sample 2, and sample 3 were placed in test tubes and diluted with ultrapure water to a final volume of 50 mL in colorimetric tubes. This process prepared a series of HNS standard solutions at concentrations of 0.50, 1.00, 20.00, 30.00, 60.00, and 150.00 mg/L. To achieve more accurate detection, HNS needs to be extracted from the aqueous solution for testing. As shown by Curve e in Figure 1, ACN demonstrates superior performance in extracting HNS from the aqueous solution, and thus ACN was selected as the extractant. To prepare the HPLC samples, HNS standard solution was mixed with acetonitrile in volume ratios of 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, and 1:3. Each mixture was shaken thoroughly and then filtered through a 0.22 \(\upmu\)m organic nylon 66 filter membrane into a brown chromatographic vial. Investigation revealed that a volume ratio of 1:2 or higher between the HNS standard solution or the wastewater to be tested and acetonitrile significantly enhanced the detection accuracy. For cost-effectiveness, a 1:2 volume ratio of HNS standard solution to acetonitrile was employed for sample preparation. The means of the peak areas obtained from the detection were linearly fitted against the corresponding HNS concentrations to construct a standard calibration curve for HNS detection.