High-Speed and High Sensitivity Analysis of Active Ingredients in Mouthwash

Importance of Topic

High‐performance liquid chromatography (HPLC) remains a cornerstone in quality control of oral care products. Rapid and sensitive determination of active agents such as cetylpyridinium chloride (CPC), dipotassium glycyrrhizinate (GK2), and isopropyl methylphenol (IPMP) is vital for ensuring efficacy, safety, and regulatory compliance of mouthwashes. Minimizing peak tailing, reducing analysis time, and improving detection limits directly support high‐throughput industrial workflows and reliable batch release.

Objectives and Study Overview

This work aimed to develop a four‐minute HPLC method combining high speed and high sensitivity for simultaneous quantification of CPC, GK2, and IPMP. The study evaluated the use of a Shim‐pack Arata™ C18 column with a phosphate buffer mobile phase to suppress adsorption of basic analytes without ion‐pairing additives. Both mixed standards and commercial mouthwash samples were analyzed to assess method performance.

Methodology and Instrumentation

The analyses employed a Shimadzu Nexera™ XR system with photodiode array (PDA) detection. Key parameters included:

• Column: Shim‐pack Arata C18 (75 × 3.0 mm, 2.2 µm) at 45 °C
• Mobile Phase: A) 20 mmol/L sodium phosphate buffer (pH 2.6), B) Methanol
• Gradient: 60 % B at 0 min→80 % B at 4.00–4.50 min→60 % B at 4.51–7.00 min
• Flow Rate: 0.8 mL/min; Injection Volume: 5 µL
• Detection Wavelengths: 250 nm for GK2, 258 nm for CPC, 280 nm for IPMP

Sample preparation for mouthwash involved 100‐fold dilution with 50 % methanol and 0.2 µm filtration.

Main Results and Discussion

Mixed standards (5 mg/L each) were baseline‐resolved within 4 minutes with no peak tailing. Repeatability (six replicates) demonstrated RSDs of retention times ≤0.12 % and peak areas ≤1.01 %. Calibration over 0.1–10 mg/L yielded r²≥0.999. Limits of quantitation were 0.079 mg/L (GK2), 0.076 mg/L (CPC), and 0.053 mg/L (IPMP). Analysis of commercial mouthwash indicated concentrations of 1.65 mg/L GK2, 4.25 mg/L CPC, and 2.92 mg/L IPMP with RSDs below 0.3 %.

Benefits and Practical Applications

By eliminating ion‐pair reagents and leveraging a low‐absorption phosphate buffer, this method:

• Delivers high throughput for routine quality control
• Ensures accurate quantitation of basic and hydrophobic actives
• Offers flexibility to monitor multiple wavelengths in one run
• Reduces solvent complexity and maintenance concerns

Future Trends and Opportunities

Further enhancements may include coupling with mass spectrometric detection for structural confirmation, exploration of sub‐2 µm or core–shell phases for even faster separations, and integration into automated sampling platforms. Expanding the method to related personal care matrices and adapting greener mobile phases align with industry sustainability goals.

Conclusion

A robust, four‐minute HPLC‐PDA method was established for simultaneous determination of CPC, GK2, and IPMP in mouthwash. The combination of a Shim‐pack Arata C18 column and phosphate buffer mobile phase suppressed peak tailing, achieved excellent repeatability, and provided low detection limits without ion‐pairing agents. This approach supports rapid, reliable quality control in oral care manufacturing.

References

1. U.S. Pharmacopeia 41–NF 36, Official Monograph “Cetylpyridinium Chloride,” 2017.
2. Application News No. 01-00116A, High‐Speed and Simultaneous Analysis of Active Ingredients CPC and GK2 in Mouthwash.