The Application Notebook
USP assay methods are widely used for quality control and stability testing.
USP assay methods are widely used for quality control and stability testing. Older methods can often be improved to take advantage of newer instrument design and column technologies. Improvements can include increases in sample throughput, method reproducibility, and solvent savings. This application note illustrates how run time and solvent consumption can be reduced when compared against the USP assay method for chlorhexidine gluconate.
The improved method is demonstrated on two different columns: YMC-Triart C18 (hybrid particle) and YMC-Meteoric Core C18 (core-shell particle).
Figure 1: YMC-Triart C18, Part number = TA12S03-1002WT.
Detection: 239 nm
Column: 250 × 4.6 mm, base-deactivated 5 µm packing L1
Column Temp: 40 °C
Flow rate: 1.5 mL/min
Inj. Volume: 50 µL
Total Runtime: 21 min
Mobile Phase A: 0.1M NaH2PO4, 0.5% Triethylamine, pH=3.0
Mobile Phase B: Acetonitrile
Gradient: 0–9 min 0% B, 9–10 min 0–55% B, 10–15 min hold 55% B, 15–16 min 55–0% B, 16–21min hold 0% B
Resolution: NLT 3.0 between chlorhexidine and p-chloroaniline
Detection: 239 nm
Columns: Test done on two different columns: YMC-Triart C18, 3 µm, 2.0 × 100 mm YMC-Meteoric Core C18, 2.7 µm, 2.1 × 100 mm
Column Temp: 40 °C
Flow rate: 0.3 mL/min
Inj. Volume: 4 µL
Total Runtime: 8.8 min
Mobile Phase A: 0.1M NaH2PO4, 0.5% Triethylamine, pH=3.0
Mobile Phase B: Acetonitrile
Gradient: 0–3.6 min 0% B, 3.6–4 min 0–55% B, 4–6 min hold 55% B, 6–6.4 min 55–0% B, 6.4–8.5 min hold 0% B
Resolution: NLT 3.0 between chlorhexidine and p-chloroaniline (same as original method)
Method Transfer Considerations: Scale-down for YMC L1 Packing Materials
Separation with YMC-Triart C18
Highlights:
Separation with YMC-Meteoric Core C18
Highlights:
The method was repeated on both YMC-Triart and YMC-Meteoric Core for 25 consecutive injections each. Both types exhibit excellent reproducibility for peak shape and retention time. Space here does not allow illustration, but the details are available from YMC America.
Figure 2: YMC Meteoric Core, Part number = CAS08SQ7-10Q1PT.
YMC-Triart C18 and YMC Meteoric Core C18 columns exhibit the selectivity and reproducibility necessary to deliver meaningful improvements to the USP method. The smaller particle sizes, shorter column length, and smaller column inner diameter allow for a much faster method (1/3 the runtime) that uses 12× less solvent. The method also meets the original method's resolution requirements of NLT 3.0 without issue.
YMC America, Inc.
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Website: www.ymcamerica.com