A Rapid, Simple and Sensitive Assay for Quantitative Determination of Melatonin in Human Plasma Using Shimadzu LCMS-8045

Significance of the Topic

Melatonin is a key regulator of the sleep–wake cycle and is widely used to treat circadian rhythm disorders. Its endogenous and exogenous concentrations can be very low and highly variable, so sensitive analytical assays are essential for accurate pharmacokinetic and clinical studies. Regulatory guidelines demand low limits of quantification, minimal sample volumes, and robust methods to support low-dose formulation evaluation.

Objectives and Study Overview

This study aimed to develop and validate a rapid, simple, and highly sensitive LC–MS/MS assay using Shimadzu LCMS-8045 for quantifying melatonin in human plasma. The target was to achieve a lower limit of quantification (LLOQ) of 5 pg/mL, short analysis time, and minimal plasma volume to facilitate high-throughput and regulatory-compliant bioanalysis.

Instrumental Setup

• UHPLC system: Nexera X2 equipped with Shim-pack GIST C18 column (75 × 3 mm, 2 µm).
• Mass spectrometer: LCMS-8045 with heated ESI interface and UF-Qarray ion guide.
• Mobile phase: 50:50 (v/v) methanol and 0.1% formic acid in water, isocratic flow at 0.4 mL/min.
• Column temperature: 50 °C; injection volume: 20 µL; total run time per sample: 3.5 min.
• MRM transitions: m/z 232.95 → 173.90 for melatonin; m/z 237.10 → 178.20 for melatonin-D4 internal standard.

Methodology

Sample preparation involved adding 50 µL of melatonin-D4 (5 ng/mL) to 200 µL plasma, followed by liquid–liquid extraction using 2.5 mL ethyl acetate. After vortexing and centrifugation, the supernatant was evaporated under nitrogen at 35 °C and reconstituted in 300 µL of aqueous solution. Chromatographic separation and MRM detection were optimized for sensitivity and selectivity.

Main Results and Discussion

The method was validated according to US guidelines: linear range 5–10,000 pg/mL with r > 0.99; LLOQ at 5 pg/mL showed signal-to-noise >20. Intra- and inter-day precision (%RSD) were ≤15% (≤20% at LLOQ), and accuracy ranged from 80% to 115%. Mean analyte recovery was ~79% with minimal variability, and matrix effects were negligible (normalized factors 1.05–1.12). Selectivity testing in multiple plasma lots confirmed no interfering peaks, and no carryover was detected in blank injections.

Benefits and Practical Applications

• The method requires only 200 µL plasma, preserving sample volume and extending instrument life.
• Short run time (3.5 min) and simple extraction protocol enable high-throughput analysis.
• Validated assay is readily transferable to customer laboratories for clinical pharmacokinetics and regulatory studies.

Future Trends and Opportunities

Advancements may include automation of sample preparation workflows, adoption of microflow or nanoflow LC–MS for further sensitivity gains, integration with real-time data analysis platforms, and expansion to comprehensive metabolite profiling using high-resolution mass spectrometry. These developments could streamline clinical assessments and personalized medicine applications.

Conclusion

The developed LC–MS/MS assay on Shimadzu LCMS-8045 provides a robust, rapid, and ultra-sensitive solution for quantifying melatonin in human plasma. Its low LLOQ, minimal sample requirement, and high throughput address critical needs in bioanalytical and clinical research settings.

References

1. https://go.drugbank.com/drugs/DB01065
2. Gooneratne NS et al. Melatonin pharmacokinetics following two different oral surge-sustained release doses in older adults. J Pineal Res. 2012;52(4):437-445. doi:10.1111/j.1600-079X.2011.00958.x
3. Zhao H et al. Rapid and sensitive analysis of melatonin by LC–MS/MS and its application to pharmacokinetic study in dogs. Asian J Pharm Sci. 2016;11(2):273-280. doi:10.1016/j.ajps.2015.08.004