In this section, we will not describe the ba-sic principles of high performance liquid chromatography, which is a standard analyti-cal technique routinely used in the laboratory.
Rather, we will detail the experimental condi-tions and procedures, including drug extrac-tion from the tapes, employed during these studies.
Since the FTIR method permitted only a relative quantification of the drug content in SC, it was necessary to validate the procedure by another analytical method which allowed absolute quantification of terbinafine (TBF).
This was achieved by chromatographically quantifying the absolute amount of TBF con-tained in the tape-removed layers of SC.
The absorbed TBF was first extracted from the sample tapes containing the removed SC.
For each sample set, the tapes were placed in a polypropylene tube (the first 10 individu-ally, the last 10 combined in pairs, thus pro-viding up to 15 samples) containing 7ml of a 80:20 mixture of acetonitrile and 0.72 mM aqueous triethylamine at pH 2.5 as buffer. All the tapes were extracted, including the first one, which was not discarded. After 16 h ex-traction (horizontal back-and-forth shaking, 400 min-1), the solution was filtered with a polyamide (nylon) syringe filter (Nalgene® 0.45 µm, Nalge, Rochester, NY) and the
fil-trate was transferred to 1 ml HPLC glass vials for quantification.
The recovery of TBF by this procedure was validated by spiking tape stripped sam-ples of untreated SC with 100 µl of a 10 mg/ml solution of TBF (corresponding to 1 mg TBF per tape) and submitting them to the protocol described above. The extraction permitted a 96.6% recovery of TBF (n=5).
Chromatographic measurements were per-formed with a Waters system (Waters-Millipore, Milford, MA): a pump 600, a tun-able UV detector 486, an autosampler 717 Plus and a manager integrator Millennium version 2.0. A reversed phase C18 column of 120 mm lenght, 4.6 mm internal diameter, filled with 5 µm diameter silica particles (Par-tisphere RP-18, Whatman, Clifton, NJ) was used. All solvents (HPLC grade) and buffer components were obtained from Sigma-Aldrich, Steinheim, Germany.
The mobile phase was isocratic, and con-tained a mixture of 50% acetonitrile, 14.3%
tetrahydrofuran, and 35.7% tetramethylam-monium hydroxide pentahydrate 1.59 mM at pH 7.8 as a buffer. The flow rate was set at 2 ml/min under in-line vacuum degassing, and the analyte was detected at 280 nm. The vol-ume of the injected filtrate was 20 µl. Under these conditions and at ambient temperature, the retention time of TBF was about 6 min.
Comparison of the chromatograms of
ex-tracted blank tapes and tapes+SC did not re-veal any interfering peaks with TBF.
The drug concentrations were determined using the AUC method from TBF standard plots generated with the neat compound (kindly supplied by Novartis Pharma, Basel, Switzerland) in acetonitrile (80%) and aque-ous triethylamine 0.72 mM at pH 2.5 in water (20%). Five concentrations (0.5, 25, 50, 75, 100 µg/ml) were prepared, and 20 ml of each was injected. The calibration plots originating from these samples were linear in the range considered (R2 = 0.9998). The detection limit was 10 ng on column. The drug content in each tape strip was normalized with respect to the volume of SC removed, thus expressing a concentration.
The following figures show some repre-sentative chromatograms of terbinafine ex-tracted from the tape-strips. Figure 14 depicts a calibration for the study presented in chapter 7. Figures 15, 16, and 17 show the effect of application time of a solution of terbinafine at 25% saturation in isopropyl myristate and ethanol 50:50 applied to the ventral forearm of a subject during 0.5, 2, and 4 hours. Each chromatogram of a series corresponds to the extracted drug from a single tape-strip.
Figure 14: Chromatograms of a single calibration from 5 solutions of terbinafine in acetonitrile and triethyl-amine 80:20.
Figure 15: Chromatograms of the extracted terbinafine from the serial tape-strips, after 0.5 hours application on the ventral forearm of subject F.
Figure 16: Chromatograms of the extracted terbinafine from the serial tape-strips, after 2 hours application on the ventral forearm of subject F.
Figure 17: Chromatograms of the extracted terbinafine from the serial tape-strips, after 4 hours application on the ventral forearm of subject F.
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