TRACE LEVEL DETERMINATION OF 4-MBC METABOLITES IN URINE SAMPLES USING THE AGILENT 6495 LC/MS/MS

Importance of the topic

Environmental monitoring of UV-filter metabolites in human urine is critical to assess exposure and potential health risks. 4-Methylbenzylidene camphor (4-MBC) is a common sunscreen agent with endocrine-disrupting concerns. Sensitive and reliable methods to quantify its major metabolites at trace levels support regulatory compliance, toxicological studies and biomonitoring efforts.

Objectives and Study Overview

This study aimed to develop and validate a robust LC/MS/MS method on the Agilent 6495 platform for routine trace-level determination of two primary 4-MBC metabolites (MBC-CX and MBC-OH) in pre-treated urine samples. Key goals included achieving excellent linearity, precision and matrix tolerance over the 0.2–50 µg/L calibration range using matrix-stripping chromatography.

Methodology and Instrumentation

Sample pretreatment involved standard urine cleanup procedures followed by injection (10 µL) into a two-column HPLC system with back-flush configuration. Matrix-stripping chromatography removed early-eluting polar interferents to prolong source cleanliness. Chromatographic separation utilized:

• Analytical column: Poroshell 120 Phenyl-Hexyl (2.1×100 mm, 2.7 µm)
• Stripping column: ZORBAX RRHD Eclipse Plus C18 (2.1×30 mm, 1.8 µm)
• Flow rate: 0.4 mL/min, total run time 9.6 min

Instrumentation details comprised:

• Agilent 1290 Infinity LC for gradient separation
• Agilent 1260 Infinity Binary Pump for sample loading and stripping
• Agilent Quick-Change 6-port, 2-position valves for column switching
• Agilent 6495 Triple Quadrupole MS with Jet Stream ion source

Internal standards were deuterated analogues of both metabolites.

Main Results and Discussion

The matrix-stripping approach effectively diverted matrix residues to waste, reducing source contamination. Calibration curves for MBC-CX and MBC-OH demonstrated linear responses (R² > 0.995) across 0.2–50 µg/L. Precision studies (six replicates) yielded RSD values below 12% at 0.5 µg/L and below 7% at higher levels. Analysis of six real urine samples identified metabolite concentrations up to 1.0 µg/L, confirming method suitability for low-level monitoring.

Benefits and Practical Applications

• High sensitivity and reproducibility enable reliable trace-level quantification.
• Matrix stripping extends maintenance intervals by protecting the ion source.
• Short run times (9.6 min) support high sample throughput.
• Method adaptability allows integration into routine environmental and clinical laboratories.

Future Trends and Opportunities

Emerging directions include automation of sample preparation, coupling with high-resolution MS for non-targeted screening, and expansion to other UV-filter metabolites. Integration with biobanking studies can enhance exposure assessment in vulnerable populations. Miniaturized systems and green chromatography approaches may further reduce solvent use and operational costs.

Conclusion

The presented Agilent 6495 LC/MS/MS method offers a sensitive, precise and efficient solution for trace-level determination of 4-MBC metabolites in urine. Matrix-stripping chromatography ensures instrument robustness, making the approach well suited for routine environmental and biomonitoring applications.

Reference

[1] Scientific Committee on Consumer Products (SCCP). Opinion on 4-Methylbenzylidene camphor (4-MBC). SCCP/1184/08, June 2008.