*2.1. Materials*

Acetonitrile (gradient HPLC grade) was obtained from Fischer Scientific (Hampton, NH, USA). Ultrapure water was obtained with a Milli-Q water system (Millipore Merck, Darmstadt, Germany). Acetone (≥99.8%), n-hexane (≥95%), HCl (37%) were obtained from Honeywell (Charlotte, NC, USA), NaOH (p.a.), NH3 (25%), acetic acid (glacial), NaH2PO4 × 2H2O (p.a.) and NaHCO3 (p.a.) from Merck (Darmstadt, Germany). Dansyl chloride (≥99%) and amines were obtained from Sigma-Aldrich (St. Louis, MO, USA): 1,7-diaminoheptane (98%), agmatine sulfate (≥97%), phenethylamine (99%), histamine (≥97%), cadaverine (≥96.5%), putrescine (≥98.5%), spermidine (≥98%), spermine (≥97%), tyramine (≥98.5%) and tryptamine (TRP) (≥98%).

Fenugreek (*Trigonella foenum-graecum*), lentil (*Lens esculentum*), alfalfa (*Medicago sativa*), and daikon radish (*Raphanus sativus*) seeds designated for sprouting were supplied by Amarant (Kresnice, Slovenia).

#### *2.2. Seed Sprouting*

The seeds of each species were rinsed, then soaked in tap water (23 ◦C, pH 7.5, 450 μS/cm) at room temperature for 6 h. The soaked seeds were germinated in Schnitzer (Offenburg, Germany) sprouting trays (diameter 18.5 cm, depth 4 cm) with a built-in drainage system. Four trays, each containing different seeds, were organized in a vertical tower to retain moisture. Every 8 h, the tower was dismantled, and the germinated seeds were rinsed in separate trays with tap water (approx. 500 cm<sup>3</sup> per tray) to prevent microbial spoilage. After the water had been drained <sup>o</sup>ff, the tower was reassembled. The germinated seeds were incubated at 23 ± 1.5 ◦C for 4 days. The seeds of all four species were germinated three times (independent experiments).

#### *2.3. Growing Microgreens*

The seeds were soaked, as described in Section 2.2, and spread on a fully hydrated Urbanscape rockwool slab 12 × 12 × 2 cm (Knauf insulation, Škofja Loka, Slovenia) with half-strength Hoagland nutrient solution [57]. The hydrated slabs were placed separately in plastic trays and filed up to a height of 1 cm with half-strength Hoagland nutrient solution. The germinated seeds were incubated for 10 days at 23 ± 1.5 ◦C (relative humidity 60%) under 16 h/8 h (light/dark cycle) photoperiod and a photon flux density of 36 μmol m<sup>−</sup>2s−<sup>1</sup> provided by cool-white fluorescent lamps MASTER TL D 58W/840 (Philips, Amsterdam, The Netherlands). The germinating seeds and later the seedlings were moistened once a day by spraying with distilled water. The loss of solution in the trays was compensated by a daily addition of distilled water. Microgreens of all four species were grown four times (independent experiments).

#### *2.4. Sample Preparation*

#### 2.4.1. Extraction Procedure

All sprouts and microgreens were homogenized fresh, unless otherwise indicated. Approximately 1.5 g (known mass) of sprouts or microgreens (upper two-thirds of the seedling height) were weighed into 50 mL polypropylene centrifuge tubes, filled with 15 mL of 0.4 M HCl containing 10 mg/mL of 1,7-diaminoheptane (IS) and immediately homogenized (30 s homogenization/30 s resting time-repeated 3 times) with the T-25 Ultra-Turrax (Ika-Labortechnik, Staufen, Germany) at 13,500 rpm. The ungerminated seeds were homogenized in the same way as sprouts and microgreens. The homogenized samples were left at room temperature for 5 min and then centrifuged at 4000× *g* for 5 min. Aliquots of the partially cleared homogenates were transferred to 2 mL centrifuge tubes and further centrifuged at 15,000× *g* for another 5 min. The supernatant was transferred to new centrifuge tubes and used for derivatisation, which was performed within 2 h after homogenization. The polyamines extracted in 0.4 M HCl can also be stored at −20 ◦C for one week, as this storage had no influence on the determined polyamine content.

The moisture content of sprouts and microgreens was determined by oven drying the samples at 105 ◦C to constant weight (≈6 h).

#### 2.4.2. Freezing and Thawing

Liquid nitrogen was poured over the sprouts to induce immediate freezing. Frozen sprouts were immediately transferred into polypropylene bags and stored at −20 ◦C. One week storage at −20 ◦C of frozen sprouts did not result in lower polyamine content if they were immediately transferred to 0.4 M HCl containing 10 mg/<sup>L</sup> IS and homogenized as explained in Section 2.4.1. To assess the influence of thawing on the polyamine content, frozen sprouts were evenly spread on a glass Petri dish and homogenized in 0.4 M HCl containing 10 mg/<sup>L</sup> of IS after 5, 20, 60, and 180 min of thawing at room temperature.

#### 2.4.3. Fenugreek Sprouts as a Source of Amine Oxidases

Fresh fenugreek sprouts (5 g) were homogenized (30 s homogenization/30 s resting time-repeated twice) with the T-25 Ultra-Turrax (Ika-Labortechnik, Staufen, Germany) at 13,500 rpm in 25 mL of MQ water. 4 mL of fresh homogenates were transferred into 80 mL glass beakers containing a mixture of 100 mM bu ffer with suitable pH (6 mL) and 10 mL of a mixture of biogenic amines. Bu ffers (100 mM) with pH 4 and 5 were prepared previously from acetic acid with the addition of NaOH. Bu ffers (100 mM) with pH 6, 7, and 8 were prepared from sodium dihydrogen phosphate with the addition of NaOH. The mixture of polyamines (100 mg/<sup>L</sup> of CAD, HIS, PHE, PUT, TRP, and TYR) was previously adjusted to pH 7 by the addition of HCl solution. The concentration of individual polyamines in the reaction mixtures was 50 mg/L, 30 mM for bu ffer, and 33 g/<sup>L</sup> for fenugreek sprouts (assuming that the densities of all solutions/sprouts are approximately 1 g/mL). The reaction mixtures were incubated at 25 ◦C on a magnetic stirrer at a stirring speed of 250 min−1.

At specified time intervals (2, 5, 12, 25, 60, and 120 min), 750 μL of the reaction mixtures were transferred into 2 mL centrifuge tubes containing 750 μL of IS (20 mg/L) in 0.8 M HCl, thoroughly mixed to stop the reaction, centrifuged and proceed as described in Section 2.4.1.

As the pKa values of all amino groups (except the imidazole group of HIS) are above 9, the neutral solution of polyamines had no significant influence on the pH value of the reaction mixtures. The pH value of the reaction mixtures was also checked at the end of the incubation period (120 min), and we found that it did not di ffer from the initial pH value by more than for ±0.1. Bu ffers at the concentration used in the reaction mixture did not seem to a ffect the derivatization yield of the biogenic amines.

#### *2.5. Preparation of Standard Solutions and Derivatization*

#### 2.5.1. Internal Standard

1,7-diaminoheptane (IS) was used as an internal standard in amine standard solutions and at various levels of sample preparation to control all steps of sample manipulation from homogenization, derivatization, and injection into HPLC. Stock IS solution with a concentration of 1.0 g/<sup>L</sup> was prepared by weighing 10 mg of IS and dissolving it in 10 mL of 0.4 M HCl or 0.8 M HCl.

#### 2.5.2. Amine Standards and Calibration Solutions

Standard solutions of individual amine (AGM sulfate, TRP, PEA, PUT, CAD, HIS, TYR, SPD, and SPM) were prepared with a concentration of 1.0 g/L. Then, 10 mg of solid amines (AGM sulphate, TRP, HIS, TYR, and SPM) were dissolved in 10 mL of 0.4 M HCl solution containing 10 mg/<sup>L</sup> of IS. Afterwards, 10 μL of liquid amines (CAD, PUT, SPD, PEA) were pipetted and dissolved in di fferent amounts of 0.4 M HCl with IS, according to their density (ρ(CAD) 0.873 g/mL, ρ(PUT) 0.877 g/mL, ρ(SPD) 0.925 g/mL, ρ(PEA) 0.962 g/mL). Mixed calibration standard solutions containing all 9 amine compounds were prepared in the concentration range of 0.3–45.0 mg/L, with a 0.4 M HCl solution containing 10 mg/<sup>L</sup> of IS.

#### 2.5.3. Derivatization Procedure with Dansyl Chloride (DNS–Cl)

The solution of DNS–Cl with a concentration of 10 g/<sup>L</sup> was prepared in acetone. The derivatization was performed in a 1.5 mL centrifuge tube, as previously described [58]. Then, 250 μL of the calibration solution or sample was pipetted, and then 50 μL of 2 M NaOH, 75 μL of the saturated solution of NaHCO3, and 500 μL of DNS–Cl solution were added, each addition followed by vortexing. The derivatization was carried out in a heating block at 40 ◦C for 60 min. After incubation, 25 μL of a 25% aqueous NH3 solution was added to the solution and left at room temperature for 30 min. Afterwards, 350 μL of acetone was added, the solution was mixed again, and filtered through a 0.45-μm nylon filter before HPLC analysis.

#### *2.6. HPLC Analyses*

The HPLC determinations were performed with UV–vis and fluorescence detectors. All chromatograms were recorded using both detectors (Figure 1). Due to better sensitivity and selectivity of the dansylated amines obtained by a fluorescence detector, the signals for the latter were used for peak-area integration and further evaluation. The only exception was histamine, where spectrophotometric signals were employed because the fluorescence yield of its dansylated derivative was low. Across all samples, the ratio of peak areas in the chromatograms was constant by the respective detectors for the individual amine derivative, which corroborated the supposition that the integrated peak actually reflected the content of the amine analyzed. All peak areas were normalized to those of IS. The matrix showed only a small influence on the derivatization yield of IS. The median derivatization yield of IS in the complex matrix was 86% (upper quartile 91% and lower quartile 80%).

**Figure 1.** Chromatograms of the standard solution (11 mg/L) of the dansylated biogenic amines agmatine (AGM), tryptamine (TRP), phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), 1,7-diaminoheptane (IS), tyramine (TYR), spermidine (SPD), and spermine (SPM). (**a**) fluorescence detector (350/520 nm) and (**b**) UV–vis (254 nm).

Instrumentation: Agilent HPLC system 1100 (Palo Alto, CA, USA), equipped with a degasser, a quaternary pump, an autosampler, a UV–vis and a fluorescent detector was used. The wavelength of the UV–vis detector was 254 nm, the excitation wavelength of the fluorescence detector was 350 nm, and the emission wavelength 520 nm. A Kinetex XB-C18 (5 μm, 100 Å, 150 × 4.6 mm) column with a

guard column of the same particle size was used (Phenomenex, Torrence, CA, USA). The flow rate of the mobile phase was 0.7 mL/min. The separation was performed with a gradient of two eluents. Eluent A was MQ water and eluent B was acetonitrile. The initial composition of the mobile phase was 40% B, which changed linearly from 0 to 25 min to 80% B. At 25 to 30 min, a second linear gradient was used to change the mobile phase from 80% B to 100%, where it remained constant until 35 min. Then the composition changed linearly within 5 min to the initial 40% B. The column was then equilibrated for 2 min.

#### *2.7. Statistical Analysis*

A non-parametric Mann–Whitney test [59,60] based on the data ranking was used for the statistical analysis. The differences in the content of a particular polyamine in ungerminated seeds, sprouts and microgreens were significant at the *p* < 0.05 level.

#### **3. Results and Discussion**

#### *3.1. Polyamine Content in Sprouts and Microgreens of Lentil, Fenugreek, Alfalfa, and Daikon Radish*

Polyamine contents were determined in different stages of plant growth for four different species—lentil, fenugreek, alfalfa, and daikon radish. Sprouting and microgreen formation resulted in a large transformation of polyamines in all species analyzed. In general, a large accumulation of polyamines was observed. In each species, the content of at least one of the polyamines increased by two orders of magnitude compared to that in ungerminated seeds. The changes in polyamine content that occurred during growth from seed to sprout, and, finally, to microgreen, were specific to each species, so the results are presented separately. A comparison of the polyamine content in the different species and growth stages was carried out in terms of dry weight (DW) values.
