2.1. Chemicals and reagents

Terbutaline sulfate and the internal standard, salbutamol, were obtained from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade n-hexane, 1,2-dichloroethane, ethyl acetate, acetonitrile, and methanol were obtained from the Duksan Pure Chemicals Co. (Ansan, Kyeonggi, Korea). Deionized water was prepared in the laboratory. All other chemicals were of analytical reagent grade.

2.2. Equipment

The HPLC equipment (Fig. 2) consisted of the following components: two LC-9A pumps, two RF-10AXL fluorescence detectors with excitation/emission wavelengths of 276/306 nm, and a FCV-2AH sixport switching valve driven and time controlled by a SCL-6B controller (Shimadzu, Kyoto, Japan). The samples were applied using a Rheodyne model 7725i sample loop injector with an effective volume of 200 μL.

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Fig.2.

Schematic diagram of the column-switching apparatus in the HPLC system. P-1: valve position 1 (bold line), P-2: valve position 2 (dotted line), MP 1: mobile phase 1, MP 2: mobile phase 2, I: injector, C1: C18 column, C2: silica column, D1: UV detectors, D2: fluorescence detectors, Trap: silica guard column, V: switching valve.

For the LC columns, a Phenomenex Luna C18 (2) column (250×4.6 mm I.D., 5 μm, Torrance, CA, USA) and a Phenomenex Luna Silica (2) column (250×4.6 mm I.D., 5 μm, SCAS, Osaka, Japan) were used as the C18 column and silica column, respectively. A Phenomenex silica guard column (30×4.6 mm I.D., 5 μm, Torrance, CA, USA) was chosen as the trap column. The acquisition of chromatograms and integration were obtained using a CR4A integrator (Shimadzu, Kyoto, Japan). The vacuum manifold chamber and Sep-Pak Plus Silica, Sep-Pak C18, and OASIS HLB solid phase extraction cartridges were obtained from the Waters Corporation, USA.

2.3. Sample preparation

Ten microliters of internal standard solution (salbutamol, 0.25 μg/mL, in water) were spiked into 1 mL of plasma. The mixture was vortex-mixed for 10 s and applied to a Sep-Pak silica cartridge pre-conditioned with methanol (6 mL) and water (6 mL). The cartridge was washed with acetonitrile (2 mL). The analytes were eluted with 7 mL of methanol. The eluate was concentrated to dryness, and the residue was reconstituted in mobile phase 1 (500 μL) for an assay sample.

2.4. Chromatographic procedures

The column-switching system was able to introduce the objective compounds into the silica column and remove the large biological materials from the HPLC system. A schematic diagram of the column-switching system is shown in Fig. 2.

Two hundred microliters of plasma sample solution were injected into the C18 column, and terbutaline and the internal standard were separated from plasma interferences with a mobile phase consisting of 5 mM ammonium phosphate buffer (pH 4)-methanol (90:10, v/v) at a flow rate of 1.0 mL/min. After 9.5 min, the fraction containing the compounds of interest, terbutaline, and the internal standard was sent into the trap column for 2.5 min. The compounds were then eluted from the trap column and transferred to the silica column with the mobile phase consisting of 20 mM ammonium phosphate buffer (pH 4)-methanol (20:80, v/v) at a flow rate of 1.0 mL/min. Ratios of terbutaline to the internal standard versus concentrations of terbutaline were used for quantitative computations.

2.5. Validation studies

The method was validated according to the currently accepted US Food and Drug Administration (FDA) bioanalytical method validation guidance (FDA, 2001 and 2003). Spiked plasma samples were prepared by adding known amounts of terbutaline and the internal standard (salbutamol) to drug-free plasma at seven concentration levels (0.4, 0.5, 1.0, 2.0, 5.0, 10, and 20 ng/mL) and used for evaluation of linearity, accuracy, and precision. The precision and accuracy of the method were assessed through the analysis of spiked plasma samples (n = 5) at three concentrations (0.5, 2.0, and 10 ng/mL). Inter-day assays were evaluated via the analysis of samples for five different days.

2.6. Application of the method

The analytical method was applied to a pharmacokinetic study. The study was conducted on eight healthy male subjects administered two tablets of Beta-2® containing 2 mg of terbutaline sulfate from CG Pharm Ltd. (Republic of Korea) after they had been informed of the purpose protocol and risk of the study. All subjects gave written informed consent and the local ethics committee approved the protocol. The study was conducted strictly in accordance with the current Good Clinical Practices (GCP), International Conference on Harmonization (ICH), and FDA guidelines. The protocol for the study was approved by the Institutional Review Board (IRB) of the Research Institute of Pharmaceutical Science, Kangwon National University, which comprised a panel of medical practitioners. The subject were not allowed to consume any other medicine or alcohol for 2 weeks prior to or during the study. The health of the participants was judged via their clinical history, physical examination, and laboratory testing, that is, hemoglobin, hematocrit, white blood cell (WBC) and red blood cell (RBC) counts, platelets, differential counting of WBC BUN, creatinine, total protein, albumin, SGOT, SGPT, total bilirubin, cholesterol, glucose fasting, alkaline phosphatase-specific gravity, color, pH, sugar, albumin, bilirubin, ability to communicate efficiently with the researcher, and willingness to adhere to the protocol requirements. Plasma samples were obtained at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 9, 12, 15, and 24 h after intake of the drug and were stored at -70 ℃ until analysis.

3.1. Chromatography

The compositions of the mobile phase were investigated to determine the optimal chromatographic conditions. In the column switching technique, similar mobile phases have to be used for the two HPLC system because the solvent employed for the C18 column system could flow into the silica column system. Terbutaline and salbutamol (I.S.) were separated by HPLC on the silica column using 20 mM ammonium phosphate buffer (pH 4)-methanol (20:80, v/v) as a mobile phase. A similar mobile phase was investigated for HPLC on the C18 column. We selected 5 mM ammonium phosphate buffer (pH 4)-methanol (90:10, v/v) as the optimum mobile phase for C18 column. Under these conditions, terbutaline and the internal standard were eluted as a single peak, with a peak width of approximately 1.2 min; a total fraction containing them was transferred to the trap column via a six-port switching valve. The trapping capacity of the silica pre-column was examined by altering the transfer time. shows the relationship between peak areas of the analytes and trapping windows. The peak areas of terbutaline and salbutamol reached the highest levels at the transfer window of 1.5 min, and neither their peak areas nor the peak area ratio of terbutaline to the internal standard changed when a transfer time of 2.5 min was used. The peak areas decreased with a transfer time ≥ 3.0 min. These results indicate that the transfer of a peak containing terbutaline and internal standard was complete within 2.0 min and the trapping capacity was not less than 2.5 min. Thus, the 2.5 min window for the switch was selected and satisfactory results were obtained in validation studies.

The use of 20 mM ammonium phosphate buffer (pH 4)-methanol (20:80, v/v) as a mobile phase for silica column resulted in the baseline separation of terbutaline, the internal standard, and the interference peak. Under silica column conditions, the retention times of terbutaline and the internal standard were 24.5 and 27.0 min, respectively. Fig. 3 shows chromatograms obtained from the analysis of plasma samples at 1 h after oral administration of 4 mg of terbutaline sulfate. No interferences with terbutaline and the internal standard from blank plasma were detected.

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Fig.3.

Representative chromatograms of plasma samples at 1 h after oral administration of 4 mg terbutaline sulfate on the column switching system. (A) C18 column chromatogram, (B) silica column chromatogram after column switching. Peak a: terbutaline, peak b: internal standard (salbutamol). C18 column system: column: Phenomenex Luna C18(2) column (250 × 4.6 mm I.D., 5 μm), mobile phase: 5 mM ammonium phosphate buffer (pH 4)-methanol (90:10, v/v), detection: UV 276 nm, flow rate 1.0 mL/min. Silica column system: column: Phenomenex Luna Silica (2) column (250 × 4.6 mm I.D., 5 μm), mobile phase: 20 mM ammonium phosphate buffer (pH 4)-methanol (20:80, v/v), detection: fluorescence detector (excitation wavelength 276 nm, emission wavelength 306 nm, flow rate 1.0 mL/min).

3.2. Linearity and limit of detection

The calibration curves were obtained by analyzing spiked plasma samples. Calibration curves showed good linearity in the concentration range of 0.4~20 ng/mL in plasma. The equation of the calibration line obtained was as follows: Y = 0.1834 X + 0.0244. The correlation coefficient was 0.9999 (Fig. 4). The limit of detection was 0.1 ng/mL and the limit of quantitation was 0.4 ng/mL, which was sensitive enough for the pharmacokinetic study of terbutaline.

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Fig.4.

Calibration curve of terbutaline.

3.3. Accuracy and precision

The accuracy and precision of the method were determined through a replicated analysis of blank human plasma spiked with terbutaline at three concentrations (0.5, 2.0, and 10 ng/mL). Five replicates of each concentration were analyzed on five separate days. The results obtained are shown in Table 1. The intra-day precision for each concentration was C.V. 2.4-7.8% and the inter-day precision was C.V. 5.2~6.3%. The accuracy determined for each concentration ranged from 99.1% to 102.4% (Table 1).

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Table1.

Precision and accuracy for determining terbutaline in human plasma (n = 5)

3.4. Recovery

The absolute recoveries were evaluated for terbutaline by comparing peak areas of extracted samples to the unextracted pure authentic standard solutions’ peak areas at three concentrations (1.0, 7.5, and 15 ng/mL). The results ranged from 93.1% (C.V. 5.7%) to 99.9% (C.V. 9.9%).

3.5. Application

This analytical method was applied to analyze plasma samples obtained after the administration of a single dose of two 2 mg tablets of terbutaline sulfate to eight healthy male volunteers for pharmacokinetic study. Plasma sample were obtained from 0 to 24 h after intake of the drug and were stored at -70 ℃ until analysis. The mean plasma concentration-time profile of the eight volunteers is represented in Fig. 5.

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Fig.5.

Representative data showing the mean plasma concentration-time profile of eight healthy volunteers after the administration of an oral single dose of two 2 mg terbutaline sulfate tablets. The error bars represent±standard deviation (SD).

The pharmacokinetics of terbutaline were demonstrated in the extent (AUC0-t and AUC0-inf) and rate (Cmax and Tmax) of absorption. The mean Cmax of terbutaline was 4.61±1.20 ng/mL. The mean Tmax for terbutaline was 3.13±1.87 h. The mean AUC0-t and AUC0-inf value of terbutaline was 28.16±5.75. The mean t1/2 value of terbutaline was 3.99±0.98.