Ceftiofur is a broad-spectrum cephalosporin antibiotic extensively used in veterinary medicine. Accurate determination of ceftiofur by HPLC is essential for quality control of pharmaceutical products. According to the pharmacopoeial method, a client reported that when using a Supersil ODS2 column, the retention time of ceftiofur decreased progressively with consecutive injections, compromising method robustness and reliability. This study addresses this issue by evaluating the SinoPak BEH T-C18 column as a stable alternative. The results demonstrate that the SinoPak BEH T-C18 column maintains consistent retention times over multiple injections, effectively overcoming the drift problem.
Standards: Ceftiofur (CAS 80370-57-6)
Reagents: deionized water (18.2 MΩ·cm), methanol (HPLC grade), tetrabutylammonium hydroxide (10% in water), ammonium acetate (99%), tetrahydrofuran (THF, HPLC grade), acetic acid (AR). Other solvents and chemicals as required for sample pretreatment (e.g., extraction solvents, filtration membranes)
0.05 mol/L ammonium acetate solution: Dissolve 0.385 g of ammonium acetate in 100 mL of water.
Mobile phase: Weigh 3.95 g of ammonium acetate, add 67 mL of 10% tetrabutylammonium hydroxide solution, and dilute to 700 mL with water. Adjust the pH to 6.3 with acetic acid. Mix with methanol and THF in the ratio ammonium acetate solution / methanol / THF = 700 / 200 / 110 (v/v/v).
Accurately weigh about 50 mg of ceftiofur sample into a 50 mL volumetric flask. Add 0.5 mL of DMF and shake to dissolve. Dilute to volume with 0.05 mol/L ammonium acetate solution. Pipette 1 mL of this solution into a 10 mL volumetric flask and dilute to volume with 0.05 mol/L ammonium acetate solution. Mix well and filter through a 0.45 μm membrane before injection.
HPLC System: EClassical 3200 equipped with quaternary pump, UV-vis detector, autosampler, column oven, solvent bottle tray, Kromstation CDS.
Pretreatment equipment: analytical balance (0.0001 g sensitivity), ultrasonic cleaner, centrifuge, solvent filtration apparatus, membrane filters, pipettes, etc.
Column: SinoPak BEH T-C18 (5 μm, 4.6 × 150 mm
Mobile Phase: Ammonium acetate solution/methanol/THF = 700/200/110 (v/v/v, prepared as above).
Flow Rate: 1.0 mL/min
Detection: UV at 254 nm
Injection Vol.: 10 μL
Column Temp.: 30°C
Retention Time Drift with Supersil ODS2 Column
The client's Supersil ODS2 column was tested under the pharmacopoeial conditions. The ceftiofur sample was injected 13 times consecutively. As shown in Table 1, the retention time decreased from 13.307 min in the first injection to 12.772 min in the thirteenth injection, a significant shift of 0.535 min. This progressive drift compromises peak identification and quantitative accuracy.
Table 1. Ceftiofur peak parameters – Supersil ODS2 column (1st vs. 13th injection)
| Injection No. | Retention Time (min) | Peak Height (mAU) | Peak Area (mAU·s) | Asymmetry | Plate Number (N) |
|---|---|---|---|---|---|
| 1st | 13.307 | 179.779 | 4350.04 | 0.990 | 7165 |
| 13th | 12.772 | 201.123 | 4372.56 | 0.997 | 7102 |
Stable Performance with SinoPak BEH T-C18 Column
The same ceftiofur sample was analyzed using the SinoPak BEH T-C18 column under identical conditions. Thirteen consecutive injections were performed. As summarized in Table 2, the retention time remained essentially unchanged (9.428 min for the first injection vs. 9.345 min for the thirteenth), with a negligible shift of only 0.083 min. Peak area, height, asymmetry, and plate number also showed excellent consistency, confirming the column's robustness and stability.
Table 1. Ceftiofur peak parameters – SinoPak BEH T-C18 column (1st vs. 13th injection)
| Injection No. | Retention Time (min) | Peak Height (mAU) | Peak Area (mAU·s) | Asymmetry | Plate Number (N) |
|---|---|---|---|---|---|
| 1st | 9.428 | 249.600 | 4065.07 | 1.278 | 7984 |
| 13th | 9.345 | 252.367 | 4066.79 | 1.286 | 7952 |
The results clearly demonstrate that the Supersil ODS2 column exhibits significant retention time drift when used for ceftiofur analysis under pharmacopoeial conditions, which can lead to unreliable quantification and peak misidentification. In contrast, the SinoPak BEH T-C18 column provides exceptional retention time stability over multiple injections, with a drift of less than 0.1 min after 13 consecutive runs. This stability is attributed to the advanced BEH particle technology and optimized bonding chemistry of the SinoPak BEH T-C18 column, which minimizes column degradation and ensures consistent chromatographic performance. Therefore, the SinoPak BEH T-C18 column is a superior choice for routine quality control of ceftiofur.
The SinoPak BEH T-C18 column effectively overcomes the retention time drift issue observed with conventional C18 columns for ceftiofur analysis. It delivers stable retention times, consistent peak shape, and reliable performance over multiple injections, meeting the requirements of pharmacopoeial methods. The client is recommended to adopt this column for their ceftiofur testing to ensure accurate and reproducible results.
