Separation and Quantitation of Polyamines in Plant Tissue by High Performance Liquid Chromatography of Their Dansyl Derivatives

Abstract

High performance liquid chromatography in combination with fluores-cence spectrophotometry can be used to separate and quantitate polya-mines (putrescine, cadaverine, spermidine, spermine), prepared as their dansyl derivatives, from plant tissue. The procedure gives sensitive and consistent results for polyamine determinations in plant tissue. In a standard mixture, the minimal detection level was less than 1 picomole of polyamines. Several methods have been employed to analyze polyamines in biological tissues. Chromatographic procedures including TLC, ion exchange, and HPLC have been used to separate colored or fluorescent derivatives of polyamines (2, 6). The most sensitive method for detecting polyamines is through their dansyl derivatives (8). Dansylated polyamines are highly fluorescent and detectable in small quantities. However, dansyl chloride (1-dimethylamino-naphthalene-5-sulfonyl chloride) is nonspecific since it reacts with amino groups of many compounds and with phenols and some alcohols (8). Generally, dansylated amines are separated by TLC. The advantage of this method is it allows the simultaneous separation of multiple samples, but they are difficult to quantify directly, so sensitivity is less. Several workers have reported successful separation of dansyl amines from animal tissues and fluids by HPLC (1, 5, 7). HPLC provides several advantages for the separation of dansyl amines including easy column regeneration for repeated separations of large numbers of samples over a short period of time, and the detection of low quantities ofpolyamines (previous reports have been in the order of 20 pmol). In this paper, a reverse phase HPLC method is described for the separation of dansylated polyamines prepared from plant extracts. MATERIALS AND METHODS Plant Material. G2 pea plants (Pistm sativum) were grown in clay pots containing a soilless mixture of vermiculite, peat moss, limestone, and fertilizer. After 3 weeks in the greenhouse, seedlings were transferred to growth chambers with full light (200 ,uE/m2.*s at pot level) provided by a combination of fluorescent and incandescent lamps. Plants received an 18-h photoperiod and 19°C day and 17°C night temperature conditions. Chemicals. Putrescine dihydrochloride, spermidine trihy-'Current address: MSU/DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824. drochloride, spermine tetrahydrochloride, and dansyl chloride were purchased from Sigma. Methanol and acetone (HPLC grade) were purchased from J. T. Baker (Phillisburg, NJ). Tolu-ene used was distilled in glass from Burdick and Jackson (Mu-skegeon, MI). Extraction of Tissues. Apical bud tissue was usually homoge-nized in chilled mortars with pestles in 0.2 N HCI04 (100 mg tissue/ml acid). Smaller quantities of tissue were ground in 5-ml centrifuge tubes with ground glass conical bases using matching pestles (Kontes, Vineland, NJ). Hexanediamine at 1 gmol/g fresh weight of tissue was added to the extracts as an internal standard (7). The homogenates were centrifuged at 4°C in a clinical centrifuge. The supematants were analyzed for polyamines. Samples were usually derivatized immediately or stored for no more than 2 weeks at-200C. Dansylation. The polyamines were derivatized according to the methods of Flores and Galston (3, and personal communication). Fifty-to 100-Ml aliquots of the supernatant were added to 200 u1 of saturated sodium carbonate and 400 ul of dansyl chloride in acetone (7.5 mg/ml) in a 5-ml tapered reaction vial. The mixture was incubated in a thermal reaction block at 60°C for 1 h in the dark. One hundred Ml of proline was added to the mixture to remove the excess dansyl chloride. After 0.5 h, the polyamines were extracted with 500 ,d of toluene with vigorous vortexing for 30 s. The mixture separated into two phases, aqueous and organic. The lower, aqueous phase was removed with a l-ml syringe and discarded. The organic phase, containing the polyamines, was completely dried under nitrogen. The pol-yamine residue was dissolved in 1 ml of methanol, ultrafiltered through nylon membranes (0.2 Mm pore; Rainin, Woburn, MA), and assayed immediately or stored (no more than 1 week) at-20°C. Aliquots of samples were diluted 5-to 20-fold before injection into the HPLC. HPLC Analysis. HPLC was performed on a system consisting of two solvent metering pumps (Altex, model 1lOA) programmed with a microprocessor controller (Altex, model 420). Samples were injected into a fixed 20-,ul loop for loading onto a reverse phase C18 column. The columns used in these investigations included ODS2 hypersil (5-Mm particle diameter, 5 x 250 mm; Shandon, Cheshire, England) and ODS ultrasphere (5-Mm particle diameter, 4.6 x 250 mm; Altex, Berkeley, CA). Samples were eluted from the column with a programmed water:methanol (v/v) solvent gradient, changing from 60% to 95% in 23 min at a flow rate of I ml/min. Elution was completed by 27 min. The column was washed with 100% methanol for 5 min and reequil-ibrated at 60% methanol for 5 min before the next sample was injected. Detection and Quantification. Eluates from the column were 2Abbreviation: ODS, octadecylsilane. 89