Comparing Diode Array Detectors, with MS, and MS/MS for the Analysis of Plant Hormones in Plant Extracts

Significance of the Topic

Plant hormones such as abscisic acid, melatonin, N-acetylserotonin, serotonin and tryptamine play central roles in growth regulation, stress response and secondary metabolism. Quantifying these analytes at endogenous levels (pg/g to µg/g) in complex plant matrices poses significant challenges due to low concentrations and coeluting interferences.

Aims and Overview of the Study

• Compare three detection platforms—diode array (DAD), single-quadrupole MS (SIM) and triple-quadrupole MS/MS (MRM)—for plant hormone analysis.
• Evaluate selectivity, sensitivity and limits of quantitation (LOQs) for five tryptophan-derived and isoprenoid hormones spiked into hempseed oil.

Methodology and Instrumentation

• Chromatography: Agilent 1290 Infinity II UHPLC with Zorbax SB-AQ 150×2.1 mm, 1.8 µm column; gradient 75→5→5→75 % A over 6 min at 0.25 mL/min; oven 45 °C; injection 2 µL.
• Detection:
• DAD at 254 nm (ABA, melatonin) and 280 nm (others).
• Single-quadrupole MS (Agilent iQ MSD) in SIM mode.
• Triple-quadrupole MS/MS (Agilent 6470) in dynamic MRM with ESI + and Agilent Jet Stream.
• MS settings: drying gas 9 L/min at 190 °C; sheath gas 12 L/min at 390 °C; capillary 4500 V; fragmentor and collision energies optimized per analyte.
• Sample preparation: 1 g hempseed oil extracted with 2 mL acetonitrile; shake 5 min; centrifuge 3500 rpm 6 min; inject supernatant.

Main Results and Discussion

• DAD detection suffered severe matrix interferences in hemp oil, yielding LOQs of 410 000–720 000 ppb and false positives (e.g., tryptamine coelution).
• SIM improved selectivity and lowered LOQs to 0.41–0.70 ppb but remained vulnerable to unmonitored interferences.
• MRM achieved instrument LODs down to 4 fg on column, linear dynamic ranges 0.002–50 ng/mL (R2≥0.997), and sample LOQs in the low-ppb to sub-ppb range.
• Matrix effects ranged from suppression to enhancement (50–150 % recovery); all quantitation met criteria: S/N≥10, ion ratio within ±30 %, r2≥0.99.

Benefits and Practical Applications

• Triple-quadrupole MRM offers robust sensitivity and selectivity for trace quantification of plant hormones in demanding matrices.
• Simplified acetonitrile extraction and rapid UHPLC separation enable high-throughput workflows for quality control and research.
• DAD and SIM remain useful for high-abundance targets or initial screening.

Future Trends and Potential Applications

• Adoption of high-resolution MS to further reduce false positives and broaden analyte coverage.
• Automated sample handling and data processing to accelerate time-to-result.
• Expansion to additional phytoactives such as cannabinoids using multi-reaction monitoring.
• Development of field-deployable or miniaturized MS platforms for on-site plant metabolite profiling.

Conclusion

Comparative evaluation demonstrates that while HPLC-DAD and SIM can detect abundant plant hormones, triple-quadrupole MRM provides the sensitivity, selectivity and quantitative performance required for reliable trace analysis in complex plant matrices such as hempseed oil.

Reference

• Sue D’Antonio, Hui Zhao, Anthony Macherone. Comparing Diode Array Detectors with MS and MS/MS for the Analysis of Plant Hormones in Plant Extracts. ASMS 2021 Poster FP143, Agilent Technologies.