ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 1, с. 72-77
ОРИГИНАЛЬНЫЕ СТАТЬИ =
УДК 543
ENANTIOSEPARATION OF TERBUTALINE BY ON-LINE CONCENTRATION CAPILLARY ELECTROPHORESIS COUPLING WITH PARTIAL FILLING TECHNIQUE © 2015 Shuya Cui*, Jiaqin Liu**, Xiaoli Hu*1, Juan Li*
*College of Chemistry and Chemical Engineering, Mianyang Normal University Mianyang 621000, P.R.China 1E-mail: huxlcsy@hotmail.com **School of Physics and Chemistry, Xihua University Chengdu 610039, P.R.China Received 22.07.2011; in final form 08.06.2014
A novel, simple and sensitive method for the enantioseparation of terbutaline enantiomers was developed using capillary zone electrophoresis (CZE) in combination with partial filling technique and field-amplified sample concentration. Under optimal conditions, the racemic terbutaline was resolved in less than 21 min by partially filling a fused-silica capillary with 15 mM P-cyclodextrin ф-CD, 1.9 psi, 90 s) and carrying out an electrophoresis with 50 mM phosphate buffer (pH 1.8) at 15 kV separation voltage. The sensitivity was further improved by using field-amplified sample injection (0.2 psi, 2.5 s water plug). Compared with conventional CZE, the resolution and sensitivity of two enantiomers were much improved (the detection limit decreased by 18-25-fold).
Keywords: terbutaline, partial lling technique, on-line concentration, capillary electrophoresis.
DOI: 10.7868/S0044450215010181
Terbutaline is a p2-adrenergic agonist used in the treatment of asthma and lung diseases. Like many other sympathomimetic amines, terbutaline exists as two enantiomers and only the (-)-enantiomer showed the desired pharmacological effects [1, 2]. There are many studies about the separation of terbutaline enanti-omers such as HPLC [3—5], hollow fiber supported liquid membrane technique [6], flow-injection chemiluminescence [7], electrospray high-field asymmetric waveform ion mobility spectrometry coupled to mass spectrometry (ESI—FAIMS—MS) [8], capillary electrophoresis (CE) [9], capillary electrophoresis— ion spray mass spectrometry [10], and capillary electrophoresis—electrospray ionization spectrometry (CE—ESIMS) [11]. However, HPLC give no satisfactory efficiency and requires a number of pretreatment steps. Because CE offers high resolution, rapid analysis and lower running cost, it has gained acceptance as a relatively new chiral separation technique. Ultraviolet (UV) on-column detector is usually used in CE methods, but it provides low concentration sensitivity. Therefore, in order to improve the sensitivity of detection of terbutaline enantiomers, a suitable on-line sample concentration step prior to CE separation is necessary.
The partial filling technique (PFT) in CE is an efficient system where only 50—800 nanolitres of a chiral
selector solution need to be added to each run. PFT is especially applicable when these additives to the background electrolyte (BGE) are expensive or absorb UV light. In this technique, the selector dissolved in the BGE is applied to the capillary as a plug, shorter than the effective length of the capillary, prior to application of the analyte. During the run both ends of the capillary are connected to the BGE. The technique has been successfully applied in a number of studies including enantiomeric separation with a variety of selectors [12]. So far, there has been no report using an on-line sample concentration technique in combination with PFT—CZE to carry out enantioseparation and determination of (±)-terbutaline. Herein, a sensitive method based on PFT—CZE coupling field-amplified sample injection (FASI) was developed for the enantioseparation and determination of terbutaline racemate. The method was validated and used for the determination of (±)-terbutaline in Bricanyl tablets with simple extraction procedures.
EXPERIMENTAL
A P/ACE MDQ capillary electrophoresis system equipped with a UV detector (Beckman-Coulter, Ful-lerton, CA, USA) was used. The system was controlled with 32-Karat Software. The separation was carried
The results of regression analysis on calibration curves and the LOD values
Enantiomer Regression equation y = a + Ьх3 Correlation coefcient Linear range, p.g/mL LODb, ng/mLa
(-)-Terbutaline y =8458.86 + 178469.70x 0.9983 7.88 x 10-3-2.27 0.8
(+)-Terbutaline y =7855.83 + 187710.12x 0.9984 7.88 x 10-3-2.27 1.0
a y and x are the peak areas and the concentrations of the analytes, respectively. b The LOD was defined as the concentration where the signal-to-noise ratio is 3.
out on a 50 cm (40 cm to the detector) x 75 ^m i.d. fused-silica capillary (Yongnian Photoconductive Fiber Factory, Hebei, China). The capillary was treated prior to its first use by flushing with methanol, 1.0 M HCl, 1.0 M NaOH and distilled water for 10 min each. Between two runs, the capillary was rinsed with 1.0 M HCl, 1.0 M NaOH and distilled water for 3 min each. The capillary was maintained at 20°C. The water plug was injected with a pressure of 1.38 kPa (0.2 psi) and the sample was electrokinetically injected. The detection wavelength was set at 214 nm. A PHs-10A acidity meter (Xiaoshan Science Instrumentation Factory, Zhejiang Province, China) was used for the pH measurements.
Terbutaline sulphate was purchased from Shanghai Jingchun industry Co. Ltd. (Aladdin-reagent, Shanghai, China). Standard stock solution of terbutaline sulphate at concentration of 0.1 mg/mL was prepared in distilled water. Solutions of lower concentrations were prepared by diluting the stock solution with distilled water. Bricanyl tablets (AstraZeneca biopharma-ceutical company, Wuxi, China) were purchased from local pharmacy in Mianyang, China. The running buffers were prepared from 0.1 M Na2HPO4 and 0.1 M NaH2PO4 stock solutions. The pH of the buffers was adjusted with 1.0 M NaOH or 1.0 M HCl. P-Cyclodex-trin was purchased from Chengdu Kelong reagent company (Chengdu, China). Other chemicals were of analytical grade. All solutions for CE were filtered through a 0.45 ^m cellulose acetate filter (Shanghai Xinya Purification Apparatus Factory, Shanghai, China).
The powdered Bricanyl tablets (0.1856 g) were extracted with 25 mL distilled water for 30 min in an ultrasonic bath. The extraction was repeated three times and the extracts were combined together. The sample solutions were prepared by diluting the extracts six times with distilled water. The solution was passed through a 0.45 ^m cellulose acetate filter before being injected directly into the capillary electrophoresis system.
The running buffer used in the CE was 50 mM phosphate buffer (pH 1.8) exchanged before every run to obtain reproducible results. Separation buffer was prepared by dissolving P-CD directly in the running buffer. Fresh capillaries were pretreated by flushing with 1 M NaOH for 3 min, water for 2 min and the
running buffer for 3 min. This procedure was also applied at the beginning of each run. Before introducing a sample, the capillary was first ushed with the running buffer for 2 min and then partially lled with the separation solution at 13.1 kPa (1.9 psi) for 90 s. The sample solution was injected at 8 kV for 5 s. Both ends of the capillary were dipped into the running buffer, and a constant voltage of 15 kV was applied for the separation. In our studies, sample solution, standard solution and running buffer were all ltered through a syringe cellulose acetate filtered (0.45 ^m) prior to use.
RESULTS AND DISCUSSION
The procedures of the PFT—FASI—CZE technique are shown as follows: (1) the capillary is rinsed with the running buffer; (2) separation buffer is introduced into the capillary partially and the rest including the detection window is still lled with running buffer; (3) water or organic solvent plug is injected into the capillary with pressure; (4) sample solution is electro-kinetically introduced into the capillary; (5) the injection end is dipped into the running buffer followed by the application of separation voltage [13]. When the analytes pass through the CD-containing separation buffer, the enantiomers run in different speeds because they form the inclusion complexes with CD. Because the complexes have different mobility, the enanti-omers are separated from each other and clearly observed in the detection cell.
Cyclodextrins have been commonly used in enan-tioseparation. The effect of P-CD concentration on the (—)-terbutaline enantiomer (1) and (+)-terbutaline enantiomer (2) migration behaviors was tested (Fig. 1), it was found that the migration time increased and the resolution was improved when P-CD concentration increased, but the peaks ofterbutaline became broader with cyclodextrin concentration above 20 mM. Considering analysis time and resolution, 15 mM was selected as the optimum concentration.
The pH of the running buffer can affect the elec-troosmotic flow as well as the charge of the analytes, and further affect the migration time and the resolution of the analytes. The pKa value is 8.8 for the first phenolic hydroxyl group, 10.1 for the amine hydrogen
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15.5 15.0 14.5 14.0 13.5 13.0 12.5 12.0 11.5
1.05
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10 15 20 25 30 Cyclodextrin concentration, mM
10
15 20 25
Cyclodextrin concentration, mM
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30
Fig. 1. Effect of P-CD concentration on the chiral separation of terbutaline. Running buffer 50 mM phosphate buffer (pH 1.8); separation buffer 10—30 mM P-CD in running buffer; separation zone 1.9 psi, 90 s; separation voltage 20 kV; electrokinetic injection (10 kV, 5 s); concentration of two enantiomers (1, 2) is 2.27 ^g/mL; detection wavelength 214 nm; 1 — (-)-terbutaline enantiomer, 2 — (+)-terbutaline enantiomer.
and 11.2 for the second phenolic hydroxyl in terbutaline [1]. The influence of pH was carried out using a 50 mM phosphate buffer in PFT—CZE. In the pH range of1.5 to 3.0, two enantiomers were positively charged. The resolution of enantiomers did not improve with the pH increasing from 1.8 to 3.0, and when the pH value was higher than 1.8, the peaks of
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