Analysis of L-dopa in human serum.

Among the drugs acting on the dopaminergic system, L-dopa is the most important one for treatment of patients with Parkinson’s disease (2). The monitoring of L-dopa concentrations in plasma has clarified and solved several pharmacokinetic problems, such as recognizing pharmacokinetic interactions and producing new types of preparations such as controlled release drugs (2). Sagar and Smyth (3) recently published an advanced column-switching method for the analysis of L-dopa and referenced 18 methods from 1962– 1986, but not more modern and simpler methods. In studies of the pharmacokinetics of the drug (1), we used an HPLC method combined with a simple clean-up procedure employing the properties of boronate gel to capture chemical compounds that, similar to Ldopa, contain vicinal hydroxyls. After washing the sample adsorbed to the gel, L-dopa was eluted in a weakly acidic solution and subjected to HPLC. However, the boronate affinity gel PBA-30 (Amicon, Danvers, MA, USA) we used disappeared from the market, and in search for a substitute, we found a similar preparation, Prosep®-PBA (Millipore, Consett, Durham, UK), which was suitable for our purposes. We also investigated the boronate binding of αmethyldopa as a promising internal standard candidate. We used L-dopa from Merck (Darmstadt, Germany) and α-methyldopa from Sigma (St. Louis, MO, USA). We prepared 1 mL boronate gel in Econocolumns (0.7 × 4.0 cm; Bio-Rad Laboratories, Hercules, CA, USA) by equilibrating the gel with 25 mL 100 mM phosphate buffer, pH 8.3, containing 2.7 mM EDTA and 5.3 mM sodium metabisulphite. The columns could be re-equilibrated and reused several times. Stock solutions of L-dopa and DL-αmethyldopa (both 10 mM) were prepared in an aqueous solution of 10 mM hydrochloric acid and 0.2 mM EDTA. These solutions were freshly prepared on the day of analysis. We prepared a working standard solution by diluting Ldopa and α-methyldopa to 2.0 μM with mobile phase (0.1 mM formic acid, 0.2 mM EDTA, pH 3.1) and made solutions with varying concentrations of the calibrator and internal standard for experimental studies. The α-methyldopa was diluted to 100 μM with the hydrochloric acid-EDTA solution and added to the samples as an internal standard. For the analysis of L-dopa, we added 1.3 mL serum sample to analytical tubes. Then 26 μL 100 μM α-methyldopa were added, the samples were thoroughly mixed, and 35 μM EDTA were added and mixed. For protein precipitation, 200 μL 4 M HClO4 were added, samples centrifuged at 3000× g at 4°C for 10 min, 1.1-mL aliquots of the supernatants were added to new tubes containing 1.3 mL 1 M K2HPO4, and the samples were thoroughly mixed. The samples were centrifuged at 3000× g at 4°C for 10 min, and 2.2 mL of the supernatants were transferred to the equilibrated Prosep-PBA boronate columns. The column was washed with 1.5 mL 100 mM phosphate buffer, pH 8.3, containing 2.7 mM EDTA and 5.3 mM sodium metabisulphite. For elution of L-dopa and α-methyldopa, 0.75 mL 100 mM phosphate buffer, pH 2.3, and 0.2 mM EDTA were added. This fraction was discarded. Then, 1 mL of the same acid buffer was added, and the fraction was collected in plastic vials for analysis by HPLC. The sample injection volume was 20 μL. The equipment used was a model 590 HPLC pump from Waters (Milford, MA, USA), an automatic sample injector model 231 XL from Gilson (Middleton, WI, USA), and an electrochemical detector model 460 from Waters. A model 740 data module (Waters) was used for integration and calculations. The analytical column was a Supelcosil C18 column (250 × 4.6 mm i.d., particle size 5 μm) from Supelco (Bellefonte, PA, USA). The temperature was maintained at 30°C by a Temperature Control Module thermostat and column heater (Waters). The mobile phase was a solution of 0.1 mM formic acid and 0.2 mM EDTA with pH adjusted to 3.1 with sodium hydroxide. The solution was filtered through a 0.45-μm cellulose acetate filter (Sartorius, Göttingen, Germany). Venous blood samples were obtained from Parkinson’s patients on treatment with L-dopa. Blood samples were collected in 10-mL vacuum tubes without additives (Becton Dickinson). After coagulation, the samples were centrifuged at 3000× g at 4°C for 10 min within 4 h of collection. Serum was then stored at -70°C until analysis. The L-dopa and α-methyldopa peaks were well separated on the chromatogram with retention times of 7.8 and 15.4 min, respectively. The detector responses were linear over the range of 0–10 μM for both substances. When the serum samples were analyzed, an unknown peak appeared at 9.3 min. This peak was also well separated from L-dopa. We investigated the optimal equilibration pH for the Prosep-PBA column before loading the sample. The sample was prepared from serum spiked with both L-dopa and α-methyldopa. Very Benchmarks