2.2 M ETHODS
2.2.11 High-Performance Liquid Chromatography (HPLC)
High-Performance Liquid Chromatography (HPLC) is a chromatographic technique used to separate single components of a complex mixture with the purpose of identifying, quantifying and purifying the individual components of the mixture. HPLC uses high pressure to force eluent through a chromatographic column density packed with micron-size particles. The high number of small size particle used leads to more precise separations. The principle of HPLC is that the solvent competes with the solute for the available adsorption sites on the stationary phase. Elution with a single solvent or a constant solvent mixture is called isocratic elution. If one solvent does not discriminate adequately between the components of a mixture or if the solvent does not provide sufficiently rapid elution of all components, the gradient elution can be used. In this case with increasing acetonitrile fraction in a water-acetonitrile mixture, solutes which were initially strongly retained by stationary phase (silica column) are eluted.
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2.2.11.1 Quantification of intracellular tramadol and O-desmethyl-tramadol by HPLC
Stock solutions for Tramadol and O-Desmethyl-Tramadol:
1) Tramadol (C16H25NO2, MW 263.4 g/mol)
10 mM stock solution: 10 mg of tramadol (Sigma) was solved in 4.012 ml ddH2O and thereof 1 ml aliquots were prepared and stored at -20oC.
1 mg/ml (3.7mM): 379 µl of 10mM tramadol was mixed with 621 µl ddH2O and stored at 4oC.
4 ng/µl: 1 mg/ml solution was diluted (1:10) with ddH2O to get 100 ng/µl. Then 40µl of the 100ng/µl solution was diluted with 960 µl ddH2O and stored at 4oC.
0.4 ng/µl: 4 ng/µl solution was diluted (1:10) with ddH2O to prepare 1 ml solution
0.04 ng/µl: 0.4 ng/µl solution was diluted (1:10) with ddH2O to prepare 1 ml solution
1 ng/µl: 1 mg/ml solution was diluted (1:10) with ddH2O to get 100 ng/µl. Then 10µl of the 100ng/µl solution was diluted with 990 µl ddH2O and stored at 4oC.
Standard test 100ng Tramadol with 25ng O-desmethyl-tramadol as internal standard:
100µl of 4ng/µl tramadol stock solution was mixed with 100µl of 1ng/µl O-desmethyl-tramadol stock solution (see below) and 50µl from this solution was injected by HPLC apparatus.
Standard test 10ng Tramadol with 25ng O-desmethyl-tramadol as internal standard:
100µl of 0.4ng/µl tramadol stock solution was mixed with 100µl of 1ng/µl O-desmethyl-tramadol stock solution (see below) and 50µl from this solution was injected by HPLC apparatus.
Standard test 1ng Tramadol with 25ng O-desmethyl-tramadol as internal standard:
100µl of 0.04ng/µl tramadol stock solution was mixed with 100µl of 1ng/µl
O-83 desmethyl-tramadol stock solution (see below) and 50µl from this solution was injected by HPLC apparatus.
2) O-Desmethyl-Tramadol (C15H23NO2, MW 249.349 g/mol)
5 mM stock solution: 5 mg of O-desmethyl-tramadol (Sigma) was solved in 4.012 ml ddH2O. 1 ml aliquots were prepared and stored at -20oC.
1 mg/ml (4 mM): 802 µl of 5mM O-desmethyl-tramadol was mixed with 198 µl ddH2O and stored at 4oC.
4 ng/µl: 1 mg/ml solution was diluted (1:10) with ddH2O to get 100 ng/µl. Then 40µl of the 100ng/µl solution was diluted with 960 µl ddH2O and stored at 4oC.
0.4 ng/µl: 4 ng/µl solution was diluted (1:10) with ddH2O to prepare 1 ml solution and then was stored at 4oC.
0.04 ng/µl: 0.4 ng/µl solution was diluted (1:10) with ddH2O to prepare 1 ml solution and stored at 4oC.
1 ng/µl: 1 mg/ml solution was diluted (1:10) with ddH2O to get 100 ng/µl. Then 10µl of the 100ng/µl solution was diluted with 990 µl ddH2O to get 1 ng/µl and stored at 4oC.
Standard test 100ng O-desmethyl-tramadol with 25ng tramadol as internal standard:
100µl of 4ng/µl O-desmethyl-tramadol stock solution was mixed with 100µl of 1ng/µl tramadol stock solution and 50µl from this solution was injected by HPLC apparatus.
Standard test 10ng O-desmethyl-tramadol with 25ng tramadol as internal standard:
100µl of 0.4ng/µl O-desmethyl-tramadol stock solution was mixed with 100µl of 1ng/µl tramadol stock solution and 50µl from this solution was injected by HPLC apparatus.
Standard test 1ng O-desmethyl-tramadol with 25ng tramadol as internal standard:
84 100µl of 0.04ng/µl O-desmethyl-tramadol stock solution was mixed with 100µl of 1ng/µl tramadol stock solution and 50µl from this solution was injected by HPLC appa-ratus.
Reagents Manufacturer
Methanol Merck, Germany
Acetonitrile Merck, Germany
Buffer A (pH 5.7) Volume
NaH2PO4*H2O (50 mM) 6.9 g
Na2HPO4 *2H2O (2.5 mM) 0.45 g
H2O 1L
Buffer B Volume
Acetonitrile 50% (v/v)
Buffer A 50% (v/v)
Buffer A and B were degassed with vacuum water jet pomp before running
The concentrations of tramadol and O-desmethyl-tramadol were analyzed by HPLC using a LaChrom system (Merck Hitachi, Darmstadt, Germany) consisting of an inter-face (D-7000,Merck Hitachi), a pump (L-7100, Merck Hitachi), an automatic sampler (L-7200, Merck Hitachi), a Fluorescence detector (L-7485, Merck Hitachi) and a degasser (L-7614,Merck Hitachi). Separation was carried out on a LiChrospher 100 RP-18e (5 μm, 4 × 150 mm) column with a LiChrospher100CN guard column (5 μm, both from Merck). The peaks were detected by fluorescence monitoring with excitation and emission wavelengths of 230 nm and 296 nm, respectively. Gradient elution was performed with an initial mobile phase of 77% (v) buffer A and 23% (v) buffer B and then the concentration of acetonitrile was increased to reach to 5% (v) buffer A and 95% (v) buffer B during 13 min and after that declined to the initial concentration (77%
(v) buffer A and 23% (v) buffer B) at a flow-rate of 1.5 ml/min at room temperature.
The peaks of tramadol and O-desmethyl-tramadol were detected with retention times of 3.8 min and 10.8 min, respectively, and were quantified using peak area with external
85 standardization. The amount of intracellular tramadol and O-desmethyl-tramadol indicated as area under curve (AUC) was normalized to the total amount of protein measured by BCA assay (section 2.2.3.1).
2.2.11.2 Quantification of intracellular debrisoquine and 4-hydroxy debrisoquine by HPLC
The concentrations of debrisoquine were analysed by HPLC using a LaChrom system (Merck Hitachi, Darmstadt, Germany) consisting of an interface (D-7000,Merck Hita-chi), a pump (L-7485, Merck HitaHita-chi), an automatic sampler (L-7200, Merck HitaHita-chi), a fluorescence detector (L-7400, Merck Hitachi) and a degasser (L-7614,Merck Hitachi).
Separation was carried out on a LiChrospher 100 CN (5 μm, 4 × 150 mm) column with a LiChrospher 4-4,100CN guard column (5 μm, both from Merck). The peaks were detected by fluorescence monitoring with excitation and emission wavelengths of 210 nm and 290 nm, respectively according to the previous report (Cerqueira et al., 2000).
Isocratic elution was performed with a mobile phase of 5% (v) acetonitrile and 95% (v) 0.05 mol/l sodium acetate buffer (pH 5.0) at a flow-rate of 0.7 ml/min at room temperature. Venlafaxine was used as internal standard, and the peaks of debrisoquine and venlafaxine were detected with retention times of 9 min and 16 min, respectively, and were quantified using peak area with external standardization. The amount of intracellular debrisoquine indicated as AUC was normalized to the total amount of protein measured by BCA assay (See section 2.2.3.1).
Individual stock solutions of debrisoquine and venlafaxine as internal standard (IS) were prepared at concentrations of 100 µg ml-1 in water. The procedure for preparation of standard solutions was similar to those described for Tramadol and O-desmethyl tra-madol (see section 2.2.11.1). To do this, aliquots of the stock solutions of debrisoquine were diluted with mobile phase to prepare a series of standard solutions containing 4, 0.4 and 0.04 µg ml-1 of the drug. The initial stock solution of the internal standard (venlafaxine) was diluted with mobile phase to yield a 1 µg ml-1 and then aliquots of the standard solutions and internal solution were mixed together in same volume and then 50 µl of these final solution was injected in column.
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2.2.11.3 Quantification of intracellular tropisetron and ondansetron by HPLC
Tropisetron and ondansetron quantifications were performed using a modification of a previously described HPLC method (BAUER et al. 2002). Briefly, tropisetron and ondansetron were quantified using a LaChrom HPLC system (Merck Hitachi, Darm-stadt, Germany) consisting of an interface (D-7000, Merck Hitachi), a pump (L-7100, Merck Hitachi), an automatic sampler (L-7200, Merck Hitachi), an ultraviolet detector (L-7400, Merck Hitachi) and a degasser (L-7614, Merck Hitachi). The compounds were separated at room temperature on a LiChrospher 100 reverse phase-18e (5 mm, 4_150mm) column with a LiChrospher 100CN guard column (5 mm, both from Merck) and quantified by ultraviolet detection at 284nm for tropisetron and 305nm for ondansetron. The mobile phase consisted of 20% acetonitrile and 80% 0.05M sodium acetate buffer (pH 5.0) and was delivered at a flow rate of 1.5 ml min–1. Tropisetron and ondansetron were detected as peaks with retention time of 5.7 and 4.3 min, respectively, and quantified using peak area with external standardization. The amount of intracellular tropisetron and ondansetron indicated as AUC was normalized to the total amount of protein measured by BCA assay (See section 2.2.3.1).
The procedure for preparation of standard solutions was like those described for tra-madol and O-desmethyl tratra-madol (see section 2.2.11.1).