Quantification of polyamines by high performance liquid chromatography (HPLC)

The cultures of Staphylococcus spp. in DSMZ 92 medium were sampled in the growing and stationary phases. They were centrifuged (18,700×g, 1 min), and the cells and culture supernatant were collected. For measurement of polyamine concentration in the cells, the collected cells were washed twice with PBS (18,700×g, 5 min). They were then resuspended in 300 µL of 5% (v/v) trichloroacetic acid and incubated in boiling water for 15 min. After centrifugation (18,700×g, 5 min), the supernatant was filtered through Cosmonice filter W (Merck, Darmstadt, Germany), and the polyamines in the cells were analyzed by HPLC. Cell debris was dissolved in 300 µL of 0.1 N NaOH. The protein concentration in the NaOH solution was measured by Bradford method using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercule, CA, USA) and standardized by the protein concentration in the cells.

For the measurement of the polyamine concentration in the culture supernatant, 20 µL of 100% TCA was added to 200 µL of culture supernatant to treat the protein in the culture supernatant. Centrifugation (18,700×g, 5 min) was performed, and the supernatant was filtered with Cosmonice filter W. Polyamines in the filtered supernatants were analyzed by HPLC. An HPLC system (Chromaster, Hitachi Ltd., Tokyo, Japan) equipped with a cation-exchange column (#2619PH, 4.6×50 mm; Hitachi) maintained at 67°C was used to measure polyamine concentrations. Polyamines were eluted by mobile phase A (45.2 mM tri-sodium citrate, 63.3 mM sodium chloride, and 60.9 mM citric acid) and mobile phase B (200 mM tri-sodium citrate, 2 M sodium chloride, 5% ethanol, and 5% 1-propanol). The concentration of mobile phase B was linearly increased from 50 to 85% during minutes 0–6, maintained at 85% during minutes 6–12, increased to 100% during minutes 12–18, maintained at 100% during minutes 18–45, and then returned to 50% for minutes 45–60. Eluted polyamines were derivatized with o-phthalaldehyde using the post-column method and detected using a fluorescence detector (λ_ex 340 nm, λ_em 435 nm). For the derivatization of polyamines, reaction solution 1 (0.4 N NaOH) and reaction solution 2 (234 mM boric acid, 0.05% Brij-35, 5.96 mM o-phthalaldehyde, 0.2% 2-mercaptoethanol) were mixed with the eluate at 67°C. The concentration of each polyamine was calculated based on a standard curve created using standards of known concentrations. The standards used and their retention times were as follows: agmatine, 34.8 min; cadaverine, 20.6 min; carboxyspermidine, 8.4 min; putrescine, 15.2 min; spermidine, 26.0 min; and spermine, 39.1 min.