Polyamine hydrochlorides and derivatizing agent-tosyl chloride, were purchased from Fluka (Buchs, Switzerland). All salts, acids and bases used for preparation of the buffers and adjusting the pH of the donor and acceptor phases were of analytical grade and were obtained from POCH (Gliwice, Poland). Acetonitrile of HPLC grade was obtained from Merck (Darmstadt, Germany). Water was purified with Milli-Q-RO4 system (Millipore, Bedford, MA, USA).
Liquid membrane solvent - dihexyl ether, and membrane carrier - di-(2-ethylhexyl)phosphoric acid (D2EHPA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Fluoropore FG flat circular sheet membranes of 9 cm diameter (porosity 0.7, pore size-0.2 μm, thickness115 μm) were purchased from Milipore. Q3/2 Accurel PP. Hollow fiber membrane in a shape of capillary with 0,6 mm inner diameter and 200 μm wall thickness (0,2 μm average pore size) was obtained from Membrana (Wuppertal, Germany).
HPLC system used for determination of polyamines consisted of Ultimate 3000 series modules (Dionex, Sunnyvale, CA, USA): vacuum degasser, gradient pump, autosampler with 50 μL loop working in the partial injection mode (20 μL of sample was injected) and diode array detector. Analytes were separated on Gemini C-18 column (Phenomenex, Torrance, CA, USA) with 5 μm average diameter of particles and 4,6 mm × 250 mm dimensions. Chromatographic data were collected and analyzed using Chromeleon 6.08 software (Dionex).
Tissue collection
Tissue samples were collected from four patients who were operated at The First Department and Clinic of General, Gastroenterological and Endocrinological Surgery of Wrocław Medical University. All patients were euthyroid and had a normal level of thyroid-stimulating hormone (TSH). They were not treated with any drugs before the surgery. Thyroid specimens (the tumor tissues and the healthy tissues from the second, healthy thyroid lobe that is routinely resected in such situations) were collected intraoperatively. Each sample was snap frozen in liquid nitrogen and stored at −80 °C. Histological assessment and classification of these tumors as carcinoma papillare were conducted according to the criteria of the WHO [17].
Preparation of donor phases
The 5 mM stock solution of polyamines was prepared using degassed water and polyamine hydrochlorides. The solution was degassed with a stream of nitrogen and divided into portions that were next frozen (to prevent oxidation of amines). The portions were stored in a freezer up to 3 weeks and were thawed before usage.
Fresh, morning urine of healthy person was adjusted to desired pH using 1 M HCl or solid KOH under a control of pH-meter (Beckman, Fullerton, USA) and supplemented with polyamine after pH adjustment.
100 mg of porcine kidney or human thyroid tissue was homogenized with 0.5 mL 10% trichloroacetic acid and 0.5 mL water in 1 mL Wheaton glass homogenization vessel (Milville, NJ, USA). Then the sample was centrifuged for 15 min (10 000 rpm). 1 mL of supernatant was taken and its pH was adjusted to 7 using 5 M KOH under the control of pH meter with very thin electrode ERH-13–6 (Hydrometr, Gliwice, Poland). Then the electrode was rinsed with water to gather any remaining analytes. The neutralized sample was subjected to SLM extraction. Depending on experiment, some of the samples were supplemented with polyamines before tissue homogenization and some after pH adjustment.
Supported liquid membrane extraction
All extractions were conducted in at least three repetitions. Error of the results is represented by standard deviation or by coefficient of variation (CV).
The flat sheet supported liquid membrane was prepared by immersing Fluoropore FG for several seconds in organic solvent consisting of 20% v/v D2EHPA in dihexyl ether. After impregnation membrane was rinsed with water and placed in an extraction unit described earlier [16]. The extraction module consisted of four cylindrical blocks of 12 cm diameter and 1 cm thickness. Supported liquid membrane was place between two inner blocks made from PTFE that one surface had grooved channel (0.25 mm deep, 1.5 mm wide and 2.5 m long) arranged as Archimedes spiral. Each channel had volume of 0.94 mL. The PTFE blocks were clamped by two aluminum blocks to make the construction more rigid and stable and were tightened with six screws. Sample (10 mL of studied solution) was pumped through the channels of the module using peristaltic pump Minipuls 3 (Gilson Medical Electronics, Villiersle-Bel, France). Diluted hydrochloric acid was used as acceptor phase.
Q3/2 Accurel PP hollow fiber was cut into 51 mm long pieces. One side of each fiber was melted by soldering tool and closed using forceps. To remove any impurities the prepared fibers were immersed in acetone, and dried. A lumen of the fiber was filled with acceptor phase (13 μl) using 25 μl HPLC syringe. The filled fiber (still attached to the syringe needle) was immersed in membrane organic liquid for 3–5 s in order to impregnate the pores of polypropylene support and thus to make SLM. Then, the fiber was rinsed with water to remove the excess of the liquid membrane phase. Finally, the fiber was taken out from syringe needle and put onto soft wire (tin-lead-silver alloy, 0.7 mm diameter). Membrane could be used immediately for extraction or could be stored for 2–3 h in the beaker filled with water. During extraction the whole membrane was immersed in the sample placed in 2.0 mL donor phase (0.1–1.0 M HCl) in Eppendorf test tube. Extraction vessels were shaken using orbital shaker TTS2 at 600 rpm (IKA Werke, Staufen, Germany). After extraction, the fiber was taken out from the sample, dried with soft paper tissue and emptied with the use of an HPLC syringe filled with the air. The acceptor phase was pushed out into PCR tube (0.2 mL) and analyzed by means of HPLC.
Derivatization and HPLC analysis
Derivatization was performed according to the published procedure with some modifications. Thus, to the sample (5 μL) placed in 100-μL glass vessel equal volume of saturated solution of disodium carbonate, four volumes of potassium borate buffer (0.5 M, pH 11) and four volumes of derivtizing reagent (5 mg/mL of tosyl chloride in acetonitrile) were added. The samples were shortly vortex-mixed and left for 10 min. Afterward, one volume of 1 M hydrochloric acid was added, samples were stirred again and subjected to HPLC analysis. Water and acetonitrile were used as mobile phase while the elution program was maintained in dependence to sample matrix. The basic binary elution program was set up starting from 62% of acetonitrile up to 85% in 15 min. The flow rate of mobile phase was 1 mL/min and the detection wavelength was set on 229 nm.
Calculations
The extraction parameters that were used in this paper are enrichment factor and extraction efficiency. The enrichment factor (Ee) was calculated from Eq. 1:
$$ {E}_e=\frac{C_A}{C_D^0} $$
(1)
where CA is the concentration of analyte in the acceptor phase after SLM extraction and C is the initial concentration of analyte in the donor phase (sample).
The extraction efficiency (E) was calculated from Eq. 2:
$$ E={E}_e\times \frac{V_A}{V_D}\times 100=\frac{C_A{V}_A}{C_D^0{V}_D}\times 100\kern1em \left[\%\right] $$
(2)
where VA is volume of the acceptor phase and VD is volume of the donor phase.
