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Quantification of Karrikins in Smoke Water Using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry


Quantification of Karrikins in Smoke Water Using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

Jakub Hrdlička et al. Plant Methods.


Background: Karrikins (KARs) are plant growth regulators that promote seed germination and the subsequent growth and development of seedlings of many plant species. In nature they are generated and released by combustion of plant material and promote the restoration of burned ecosystems. Smoke water can be artificially prepared as a saturated extract of all substances in smoke produced by burning plants, and it has various horticultural and agricultural applications.

Results: We have developed, validated and applied the first fast, specific and sensitive method, based on ultra-high performance liquid chromatography-tandem mass spectrometry, for quantifying KARs in smoke water. To assist these efforts and further analyses, standards of the main KARs (which are not commercially available) were synthesized. Due to the complex matrix of smoke waters, two quantification approaches (standard dilution with a structural KAR analogue and standard addition) were compared. The standard addition method allowed absolute quantification of KARs in six of eight smoke water samples of diverse origins and ages.

Conclusions: Our findings reveal differences in both total and relative levels of KARs in smoke water, and indicate that differences in its KAR composition may be linked to variations in its bioactivity.

Keywords: Karrikins; Smoke water; Standard addition method; Standard dilution method; Tandem mass spectrometry (MS/MS); Ultra-high performance liquid chromatography (UHPLC).

Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.


Fig. 1
Fig. 1
Structures of known karrikin compounds, endogenously occurring in smoke water
Fig. 2
Fig. 2
Preparation scheme of karrikins. a TEMPO, NCS, TBAB; b (EtO)2POCH2CO2Et, NaH; c CF3COOH; d ClCOOEt, pyridine; e Et3N, CH2Cl2; f Pd(PPh3)4, THF; g NBS, CHCl3; h CH3B(OH)2, Pd(OAc)2, S-Phos, tBuOK, toluene
Fig. 3
Fig. 3
Stability of karrikin standards in McIlvaine buffer, pH 5.0. Solutions of KAR1 (black squares) and KAR2 (white diamonds) standards (10−5 M) were incubated for 12 days (short-time treatment; a, b) and 12 weeks (long-time treatment; c, d), at two temperatures: + 22 °C (a, c) and + 4 °C (b, d). Values are mean ± SD (n = 3)
Fig. 4
Fig. 4
Full-scan (a) and product ion (b) mass spectra of the studied compounds obtained using the optimised UHPLC-ESI(+)-MS/MS method. MS spectra of KAR1 (top) and KAR2 (bottom) were analysed with a tandem mass spectrometer Xevo TQ-S in positive ion mode. c Separation of KAR standards by the UHPLC-ESI(+)-MS/MS method using an Acquity UPLC BEH C18 2.1 × 50 mm column. Multi-MRM chromatograms of KAR1, KAR2 and KAR-Br (internal standard) samples, containing 1 pmol of each derivative per injection
Fig. 5
Fig. 5
Representative MRM chromatograms of SW5 samples, non-spiked and spiked with a mixture of KAR1 (a) and KAR2 (b) at two concentrations (0.5 and 1 μmol/l). Reproducibility is shown in triplicates (1st injection blue, second injection red, third injection black). In the boxes, representative calibration curves constructed from five SW matrix-based solutions spiked with a mixture of KAR1 and KAR2 at known concentrations (0, 0.25, 0.5, 0.75 and 1.0 μmol/l). The samples were analysed by UHPLC-ESI(+)-MS/MS using the standard addition method

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