Quick and Routine Research Quantification of Melatonin in Plasma with the Agilent Ultivo LC/TQ

Importance of the topic

Melatonin is a key hormone for regulation of circadian rhythm and sleep. Low level quantification in human plasma supports research in pharmacokinetics and endogenous production. Sensitive methods are required to measure sub ppt concentrations reliably in complex biological matrices.

Goals and overview

The study demonstrates a rapid and sensitive method for quantification of melatonin in plasma using an Agilent 1260 Infinity II Prime liquid chromatograph coupled with Ultivo triple quadrupole mass spectrometer with electrospray ionization. The target limit of quantification is 10 pg per mL using a simple protein precipitation extraction.

Methodology

The procedure uses protein precipitation of plasma samples with acetonitrile followed by centrifugation and vacuum drying. Extracts are reconstituted in methanol and subjected to LC MS analysis. A stacked injection approach with a 3 microL injection volume is applied. Calibration standards of deuterated melatonin are prepared in plasma extract over a range from 10 pg per mL to 100 ng per mL.

Used instrumentation

• Agilent 1260 Infinity II Prime flexible pump
• Agilent 1260 Infinity II multisampler with cooler
• Agilent 1260 Infinity II multicolumn thermostat
• Agilent Ultivo triple quadrupole LC MS system
• Agilent electrospray ionization source

Main results and discussion

The optimized method achieves a limit of detection of 5 pg per mL in extract corresponding to 10 pg per mL in plasma and a lower limit of quantification of 10 pg per mL. The calibration curve is linear over four orders of magnitude with correlation coefficient of 0.998. Method precision is better than 10 percent relative standard deviation and average accuracy ranges from 95 to 105 percent. Extraction recovery of deuterated melatonin is 95 percent with one standard deviation of 6 percent. Measurement of endogenous melatonin in pooled plasma yields an average concentration of 40.8 pg per mL with 7.3 percent RSD.

Benefits and practical applications

• Fast sample preparation by simple protein precipitation
• High sensitivity suitable for low ppt level detection
• Robust linear response across a wide concentration range
• Fit for routine research use in clinical and pharmacokinetic studies

Future trends and possibilities

• Integration with high throughput automation
• Expansion to multiplex hormone panels
• Further miniaturization and increased sensitivity
• Applications in chronobiology and personalized dosing strategies

Conclusion

The Agilent Ultivo LC TQ with ESI source provides an economical fit for purpose solution for trace level quantification of melatonin in plasma. The method offers high sensitivity precision and reproducibility with minimal sample handling and is well suited for routine research applications.

Reference

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