Metabolome Analysis of Hydrophilic Metabolites in Saliva Using LCMS™-8060NX Triple Quadrupole Mass Spectrometer

Importance of the Topic

Saliva contains diverse metabolites produced by oral bacteria, including short-chain fatty acids and hydrophilic compounds. Tracking these molecules is critical for understanding oral health, disease pathways in periodontitis, and the broader impact of the oral microbiome on human physiology.

Objectives and Study Overview

This study applied comprehensive metabolome analysis to evaluate how storage temperature affects the profile of hydrophilic metabolites in human saliva. Using a triple quadrupole LC-MS/MS platform, the work aimed to quantify short-chain fatty acids, organic acids, amino acids, nucleosides, and related compounds after saliva was stored at –80 °C, 4 °C, 25 °C, and 40 °C for three days.

Methodology

Sample Preparation:

• Saliva from a healthy adult male was centrifuged and aliquoted for storage at four temperatures.
• For short-chain and organic acids, samples were derivatized with 3-nitrophenylhydrazine, pyridine, carbodiimide, and 2-ethylbutyric acid (internal standard), then diluted 1:5 with methanolic formic acid.
• For general hydrophilic metabolites, saliva was diluted 1:5 with water containing 2-morpholinoethanesulfonic acid (1 µmol/L) as internal standard.

Chromatographic and Mass Spectrometric Conditions:

• Reversed-phase column with gradient elution using 0.1 % formic acid in water and acetonitrile.
• Flow rates of 0.35 mL/min for organic acids and 0.25 mL/min for hydrophilic metabolites; injection volumes of 3 µL.
• Electrospray ionization in positive/negative MRM mode; multiple gas flows and temperatures optimized for sensitivity.

Used Instrumentation

• Shimadzu Nexera X3 high-performance liquid chromatograph
• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer with IonFocus unit
• Peakintelligence AI-driven peak integration software within LabSolutions Insight
• SIMCA 16 for multivariate statistical analysis

Main Results and Discussion

IonFocus Unit:
The use of focus electrodes allowed the ion source to be positioned further from the inlet, reducing matrix effects without sacrificing ion transfer efficiency. Sensitivity improved by an average of 1.4-fold.

Peak Integration:
Peakintelligence applied AI-trained algorithms to separate overlapping signals, reducing manual correction and improving quantitation accuracy.

PCA Findings:
– Short-chain fatty acids and organic acids: samples stored at 40 °C clustered separately (high succinic and glutamic acids), and those at 25 °C showed elevated pyruvic acid, histidine, and tyrosine. Storage at 4 °C and –80 °C yielded similar profiles.
– Hydrophilic metabolites: 50 compounds including amino acids, nucleosides, and organic acids displayed temperature-dependent variation, with pronounced changes at elevated storage temperatures.

Temperature Profiles:
Peak area ratios for arginine, glutamic acid, histidine, lactic acid, pyruvic acid, and succinic acid increased or decreased significantly at 25 °C and 40 °C, indicating ongoing bacterial metabolism during storage.

Benefits and Practical Applications

This LC-MS/MS method offers high sensitivity, robustness against complex matrices, and reliable quantitation of over 100 metabolites in saliva. It supports oral microbiome research, clinical diagnostics, and quality control in pharmaceutical or nutraceutical development.

Future Trends and Potential Applications

Emerging approaches may include integration of ion mobility spectrometry, real-time monitoring of oral metabolites, and expansion to other biofluids. Advanced AI algorithms will further automate data processing and enhance detection of low-abundance compounds.

Conclusion

The presented metabolome analysis workflow enables comprehensive and sensitive profiling of saliva-derived hydrophilic metabolites. Storage temperature critically influences measured levels due to bacterial activity, underscoring the need for consistent sample handling in clinical and research settings.