Catch Them Sleeping: Quick and Routine Quantification of Melatonin in Plasma with Ultivo LC/TQ

Significance of Topic

Melatonin regulates circadian rhythms and is widely prescribed for sleep disorders. Precise measurement of its low circulating levels in human plasma is essential for pharmacokinetic research and understanding endogenous production patterns.

Objectives and Study Overview

This work presents a quick and routine LC-MS/MS assay using the Agilent Ultivo triple quadrupole system, aimed at achieving the sensitivity and specificity required to quantify melatonin in plasma.

Methodology and Instrumentation

Samples are prepared by acetonitrile-based protein precipitation to extract melatonin and a deuterated internal standard (MEL-D4). Chromatographic separation is performed on an Agilent InfinityLab Poroshell 120 EC-C18 column (2.1 × 50 mm, 1.9 µm) at 35 °C, with a gradient elution using 5 mM ammonium formate/0.1 % formic acid in water and methanol. Mass spectrometric detection employs an Agilent Ultivo Triple Quadrupole with electrospray ionization in positive mode, monitoring four MRM transitions with optimized fragmentor and collision energies.

Used Instrumentation

• Agilent 1260 Infinity II Prime LC System
• Agilent InfinityLab Poroshell 120 EC-C18 column
• Agilent Ultivo Triple Quadrupole LC/MS with ESI source

Main Results and Discussion

The assay demonstrates a limit of detection below 10 pg/mL and a validated linear range of 10 pg/mL to 100 ng/mL (R² = 0.998). Intra- and interday precision (RSD < 10 %) and accuracy (95–105 %) are excellent across four orders of magnitude. Average recovery of MEL-D4 from plasma is 95 ± 6 %. Endogenous melatonin levels in human plasma are accurately quantified by correcting for sample preparation losses using the internal standard.

Benefits and Practical Applications

• High sensitivity suitable for low-ppt range quantification
• Simple sample preparation streamlines workflow
• Robust performance supports clinical and research studies

Future Trends and Application Opportunities

Ongoing improvements in triple quadrupole technology, ion source design, and software are expected to enhance throughput and sensitivity. Coupling with automated sample introduction and miniaturized systems may further accelerate pharmacokinetic and biomarker studies.

Conclusion

The developed method on the Agilent Ultivo LC/TQ system provides rapid, reliable, and highly sensitive quantification of melatonin in plasma, fulfilling the demands of circadian rhythm research and therapeutic monitoring.