Impurity profiling of the synthetic peptide LL-37 using high-performance liquid chromatography with combined UV and single quadrupole mass spectrometric detection

Importance of the Topic

The synthetic antimicrobial peptide LL-37 has broad medical applications but requires stringent quality control to ensure safety and efficacy. Impurity profiling is essential to detect low-level process- and product-related by-products that can affect biological activity and regulatory approval. Combining rapid chromatographic separation with complementary detection techniques enhances confidence in impurity identification.

Objectives and Study Overview

This study demonstrates a fast UHPLC-based impurity profiling method for LL-37 using dual UV and single quadrupole mass spectrometric detection. The main goals are to quantify impurity levels via UV absorbance at 214 nm and to confirm peptide identity and detect co-eluting species by full-scan MS up to m/z 2000. Two known LL-37 fragments (RKS and SKE) are spiked into the API to simulate product-related impurities.

Methodology

An Acclaim RSLC 120 C18 column (50 × 2.1 mm, 2.2 µm) at 50 °C is used with a binary gradient of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B). The gradient ramps from 20% to 50% B over 2 min, holds for 0.1 min, then returns to initial conditions for total run time of 5.5 min. Flow rate is 0.5 mL/min; injection volume is 1 µL; UV is monitored at 214 nm (10 Hz). Full-scan MS is performed in positive mode over m/z 500–2000 with 0.2 s dwell time.

Used Instrumentation

• Thermo Scientific Vanquish Flex Binary UHPLC
• Vanquish Binary Pump F
• Vanquish Split Sampler FT
• Vanquish Column Compartment H
• Vanquish Variable Wavelength Detector F (2.5 µL flow cell, 7 mm path)
• Thermo Scientific ISQ EM single quadrupole mass spectrometer

Main Results and Discussion

UV detection of pure LL-37 yields a single major peak at 1.7 min with no visible impurities, but MS confirms absence of co-eluting species by showing clean full-scan spectra. Spiked fragments are baseline resolved within 2 min (RT 0.9 min for SKE, 1.2 min for RKS). The ISQ EM’s extended range detects charge states from +2 to +8; deconvoluted average masses deviate ≤0.5 Da from theoretical values. Comparison of relative peak areas from UV, total ion chromatogram (TIC), and extracted ion chromatogram (XIC) traces reveals variations due to differing UV responses and ionization efficiencies, highlighting challenges in accurate quantitation without individual calibration or isotopically labeled standards.

Benefits and Practical Applications

• Combines UV quantitation with MS-based confirmation for enhanced impurity profiling.
• Rapid gradient method completes analysis in under 6 min, increasing laboratory throughput.
• Extended MS range enables detection of low-charged peptide species and full charge state characterization.
• Applicable to QA/QC of peptide APIs, screening for known and unknown impurities.

Future Trends and Applications

Advancements in high-resolution MS and MS/MS fragmentation will improve structural elucidation of unknown peptide impurities. Integration of automated data analysis and AI-driven interpretation can streamline impurity identification. Coupling with multidimensional chromatography may further enhance separation of complex peptide mixtures. These developments will expand the method’s utility to larger peptides and protein therapeutics.

Conclusion

The described UHPLC-UV/MS approach offers a robust, high-throughput solution for impurity profiling of synthetic peptides such as LL-37. The synergy of rapid chromatographic separation, UV quantitation, and extended-range single quadrupole MS delivers reliable detection, identification, and quantitation of low-level impurities within minutes.

References

• 1. Wu LC, Chen F, Lee SL, Raw A, Yu LX. Building parity between brand and generic peptide products: Regulatory and scientific considerations for quality of synthetic peptides. International Journal of Pharmaceutics. 2017;518:320–334.
• 2. Chen Y, Yang S, Ho EA. Development of an analytical method for the rapid quantitation of peptides used in microbicide formulations. Chromatographia. 2014;77(23-24):1713–1720.
• 3. ICH Guidelines Q3A-Q3D: Impurities in New Drug Substances and Products.