Authentic and stable isotope labelled standards (Table 1) were provided from in-house standard library of JAs (cis-OPDA, OPC-8, OPC-6, OPC-4, dn-OPDA, JA, MeJA, 9,10-dhJA, 11-OHJA, 12-OHJA, JA-Val, JA-Ile, JA-Trp, JA-Phe and [2H5]-OPDA, [2H6]-JA, [2H2]-(-)-JA-Ile, [2H6]-(±)-MeJA) , AUXs (IAA, oxIAA, IAA-Asp, IAA-Glu, IAA-glc, oxIAA-glc and [13C6]-IAA, [13C6]-oxIAA, [13C6]-IAA-Asp, [13C6]-IAA-Glu, [13C6]-IAA-glc, [13C6]-oxIAA-glc) , ABAs (ABA, PA, DPA, neoPA, 7´-OHABA and [2H6]-ABA, [2H3]-PA, [2H3]-DPA, [2H3]-neoPA, [2H4]-7´-OHABA)  and salicylates – SA (SA and [2H4]-SA). Methanol, acetonitrile, formic acid, all LC-MS grade, acetic acid of gradient grade and hydrochloric acid were purchased from Sigma-Aldrich (St. Luis, MO, USA). Purified Milli-Q water from a Simplicity 185 System was used for preparation of aqueous solutions.
Plant Material And Growth Conditions
Arabidopsis thaliana - seedlings of ten-day-old Arabidopsis thaliana ecotype ‘Col-0’ grown in vitro in Murashige and Skoog medium Petri dishes in a growth chamber at 20°C, under long-day photoperiod (16 h light, 8 h dark) with a light intensity of 90 µmol photons m− 2 s− 1, were used for optimisation of the extraction and purification protocol, method validation and determination of phytohormones in a series of matrices from different specimens.
Populus tremula x alba - leaves from 90-day-old hybrid poplar seedlings grown under long-day conditions (16 h light at 22°C, 8 h dark at 18°C) as described in  were used in this study for phytohormone quantification.
Brassica rapa - leaves from 4-week-old Brassica rapa (ecotype ‘Pekinensis’) seedlings, cultivated in a growth chamber under long-day conditions at 21°C as described in , were used for phytohormone quantification.
Solanum lycopersicum - leaves from 8-week-old tomato seedlings (ecotype ‘Tornado’) cultivated in soil under environmentally controlled greenhouse conditions during spring 2020 (Olomouc, Czech Republic), were used for phytohormone quantification.
Nicotiana tabacum - leaves from 8-week-old tobacco seedlings, cultivated in a growth chamber under long-day conditions (16 h light at 20°C, 8 h dark at 18°C) as described in , were used for phytohormone quantification.
Triticum aestivum - leaves from 7-day-old seedlings of winter type wheat (ecotype ‘Turandot’) were used in this study for phytohormone quantification. Seedlings were obtained from dormant seeds that were soaked for 5 hours in tap water to germinate, sown into vermiculite, cultivated under long-day conditions at 21°C and irrigated every second day with 0.5 L of Hoagland’s nutrient solution .
Picea abies − 14-day-old spruce whole seedlings, grown in a growth chamber under long-day conditions (16 h light at 22°C, 8 h dark at 18°C) as described in , were used in this study for phytohormone quantification.
Physcomitrella patens − 3-week-old whole gametophores from Physcomitrella patens (Hedw.) Bruch & Schimp (ecotype ‘Gransden 2004’) wild type were used in this study for phytohormone quantification. The plants were grown axenically in 9-cm Petri dishes on Knop medium (100 mg/L Ca(NO3)2·4H2O, 25 mg/L KCl, 25 mg/L KH2PO4, 25 mg/L MgSO4·7H2O and 1.25 mg/L FeSO4·7H2O, pH 5.8). Knop medium was supplemented with 92.05 mg/L ammonium tartrate, 0.5 mg/L nicotinic acid, 0.125 mg/L p-amino benzoic acid, 2.5 mg/L thiamine HCl, trace-element solution (0.614 mg/L H3BO3, 0.389 mg/L MnCl2·4H2O, 0.059 mg/L NiCl2·6H2O, 0.055 mg/L CoCl2·6H2O, 0.055 mg/L CuSO4·5H2O, 0.055 mg/L ZnSO4·7H2O, 0.0386 mg/L Al2(SO4)3·18H2O, 0.028 mg/L KBr, 0.028 mg/L KI, 0.028 mg/L LiCl, 0.028 mg/L SnCl2·2H2O) and 200 mg/L glucose. Medium was solidified with 1.5% (w/v) plant agar. Plants were cultured under standard conditions in a growth chamber at 20 ± 1°C, under a 16/8 h light/dark photoperiod with a light intensity of 90 µmol photons m− 2 s− 1. Plants were subcultured onto fresh medium every 3 weeks.
Stichococcus bacillaris - lyophilised green algae isolated from a pond in a region of the town Nové Hrady (Czech Republic) in summer 2013 were used in this study for phytohormone quantification.
When harvested, all plant tissues were immediately frozen in liquid nitrogen and stored at -80°C.
Plant tissue was homogenised and powdered with a mortar and pestle under liquid nitrogen and reweighed into samples of approximately 10 mg FW or 1 mg of DW (Stichococcus bacillaris). The samples were extracted in cold 1 mol/L formic acid in 10% aqueous methanol so that 1 mL of the extraction solvent contained 10 mg FW or 1 mg DW. Internal standards were added to the samples to give 5 pmols of [13C6]-IAA, [13C6]-oxIAA, [13C6]-IAA-Asp, [13C6]-IAA-Glu, [13C6]-IAA-glc, [13C6]-oxIAA-glc, [2H5]-OPDA, [2H2]-(-)-JA-Ile, [2H6]-(±)-MeJA and 10 pmols of [2H6]-JA, [2H6]-ABA, [2H3]-PA, [2H3]-DPA, [2H3]-neoPA, [2H4]-7´-OHABA, [2H4]-SA per 1 mL. To each of the samples, 4 ceria-stabilised zirconium oxide 2 mm beads (Retsch GmbH, Haan, Germany) were added, the samples were placed on an MM 400 mixer mill (Retsch GmbH, Haan, Germany) (29 Hz, 10 min, precooled holders), and centrifuged (25 200 g, 20 min, 8°C), and 100 µL (1 mg of FW, 0.1 mg DW) or 200 µL (2 mg of FW, 0.2 mg of DW) of supernatant was loaded on a stagetip (Fig. 2A). The samples were handled at 8°C in a CoolRack® in a CoolBox™ (Biocision, Larkspur, CA, USA) in the course of the sample preparation steps.
In-tip Microspe High-throughput Purification
For in-tip microSPE columns, the stagetips  were self-assembled using ordinary pipette tips (2–200 µL, Eppendorf) and 3 layers of SDB-XC and C18 sorbent (3M™ Empore™, St. Paul, MN, USA) similarly to the method published for auxin metabolite profiling  (Fig. 2B).
A 96-place tip-holder was projected in 123D Design (AutoDesk Inc., San Rafael, CA, USA) and printed by DeltiX (Trilab, Czech Republic) using polylactic acid filament. This holder was designed to be compatible with regular 96-well plates and a centrifuge rotor for 96-well plates (M-20, 75003624, Heraeus Megafuge 16K centrifuge, Thermo Fisher Scientific, Waltham, MA, USA) (Fig. 2B). The design and dimensions of the device are provided in Additional file 1: Fig. S4. Depending on number of samples, the stagetips were accommodated in 3D-printed 96-place tip-holders adaptable to process different number of large sets of samples, up to 192 samples in one run.
The purification protocol was adopted from  with modifications (Fig. 2A). The stagetips, located in a centrifuge, were conditioned with 50 µL of acetone (320 g, 10 min, 8°C), 50 µL of methanol (320 g, 10 min, 8°C), and 50 µL of water (627 g, 15 min, 8°C). 100 or 200 µL of sample extract (1742 g, 20 min; 8°C) was applied, washed with 50 µL of 0.1% aqueous acetic acid in water (819 g, 20 min, 8°C) and eluted with 80% aqueous methanol (1280 g, 20 min, 8°C) into a 96-well plate suitable for direct injection onto an LC-MS/MS system. Apart from the sample loads, all liquids were applied with an eight-channel pipette (Fig. 2B). Finally, the eluents in the 96-well plate were lyophilized (Labconco, Kansas city, MO, USA) and the plate stored at -20°C until required for analysis. When evaporation techniques tested TurboVap LV system (Caliper Life Sciences, Hopkinton, MA, USA) for evaporation under gentle stream of nitrogen or Acid-Resistant CentriVaps benchtop concentrator (Labconco, Kansas city, MO, USA) for evaporation in vacuo were used.
All analyses were carried out on an Agilent 6490 Triple Quadrupole LC/MS system coupled to a 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA). The data were processed in the MassHunter Quantitative software package version B.09.00 (Agilent Technologies, Santa Clara, CA, USA).
Purified and freeze-dried extracts were each dissolved in 40 µL of 20% aqueous acetonitrile and injected (10 µL) onto a Kinetex Evo C18 reverse phase column 2.1 x 150 mm, particle size 2.6 µm (Phenomenex, Torrance, CA, USA) protected by an inlet filter, using 10 mmol/L formic acid in water and methanol as mobile phases at a flow rate of 0.3 mL/min and a temperature of 60°C. Gradient elution started at 2 min, going from 20–90% of methanol at 13.5 min, continuing within 0.5 min to 100% methanol. After 1 min of 100% methanol, the methanol content was decreased to 20% over the next 0.5 min and the column was equilibrated with 20% methanol for 3.5 min before the next injection.
The MS system was operated in dynamic multiple reaction monitoring mode in ESI positive and negative ionisation mode simultaneously (Table 1). The MS settings, multiple reaction monitoring transitions, and collision energies were optimised to previous standards. The nozzle voltage was set to 0 V, the capillary voltage to 2800/3000 V positive/negative mode, and the drying gas was at 130°C with a flow rate of 14 L/min. The sheath gas was heated to 400°C and its flow rate was set to 12 L/min.
The method was validated in terms of accuracy and precision at five concentration levels using 2 mg FW of 10-day-old Arabidopsis thaliana seedlings spiked with authentic (0.1, 0.5, 1.0, 5.0 and 10.0 pmol) and internal standards (1 pmol for all AUXs, JAs detected in ESI+, 2 pmol for JAs, ABAs, SA detected in ESI-) (Table 1) with four replicates. The accuracy was calculated using the determined levels of the analytes (pmols) with the endogenous levels subtracted divided by the nominal level of the spike (pmol) and expressed as a percentage of the nominal level. Precision was calculated as the relative standard deviation of determined levels (%). The PE, RE and ME were assessed as in  using 2 mg FW of plant matrix spiked before or after extraction and the purification procedure at one concentration level (4 pmol) with four replicates. Briefly, to evaluate PE, the mean peak area of the IS spiked before sample preparation was divided by the mean peak area of the neat solution of IS without any extraction and purification, expressed as a percentage. RE was calculated as the percentage of the mean peak rate of authentic standards spiked before and after the extraction and purification process. ME was calculated by dividing the mean peak area of authentic standards spiked after sample preparation by the peak area of the neat solution with the same amount of analyte. All validation samples were processed by the optimised extraction and purification protocol (Fig. 2).
To evaluate the influence of the method of drying the samples after purification, 50 µL aliquots of solutions of all authentic standards (at 0.5 pmol) in 80% aqueous methanol were processed using evaporation under a stream of nitrogen, in vacuo drying and lyophilisation, with four replicates of each. The dried samples were redissolved in 50 µL 20% aqueous acetonitrile mimicking the conditions immediately before LC-MS/MS analysis. The recovery was calculated by dividing the peak area of a dried standard by that of the original authentic standard solution, expressed as a percentage.
Calibration And Quantification
The contents of all samples were quantified using logarithmically transformed calibration curves constructed by plotting the responses of calibration standards (analyte peak area divided by IS area multiplied by IS concentration) against their known concentrations. The calibrations spanned from six to three orders of magnitude (Table 1).
Amount Of Plant Matrix Required For Phytohormone Profiling
The weight of sample necessary for quantification of the analytes was evaluated using 1–2 – 4–6 – 8 mg FW or 0.1–0.2–0.4–0.6–0.8 mg DW of plant matrix. Either 1 or 2 mg FW (0.1 or 0.2 mg of DW) plant matrix were loaded on a stagetip, and eluates of 2 mg (0.2 mg of DW) were combined to obtain samples of 4 to 8 mg FW (0.4 to 0.8 mg DW). Each sample amount was analysed in either triplicate, or, for 1 mg FW (0.1 mg DW), in duplicate. The mean concentration of each analyte was calculated from all samples in which the analyte was quantified, e.g. an analyte quantified at 1 mg (n = 2) was also quantified at 2 mg (n = 3), 4 mg (n = 3), 6 mg (n = 3) and 8 mg (n = 3), giving a total of 14 samples (n = 14).