Chiral stability-indicating HPLC method for analysis of arotinolol in pharmaceutical formulation and human plasma

2.1 Chemicals and reagent

S-(+)- and R-(−)-arotinolol were gifts from Sumitomo Pharmaceutical Co. (Osaka, Japan), and haloperidol was obtained from Sigma Chemical Co. (St. Louis, MO, USA). HPLC-grade methanol, analytical grade triethylamine, glacial acetic acid and ethyl ether were purchased from BDH Chemicals (Poole, UK). Analytical grade ammonium acetate, hydrochloric acid, sodium hydroxide and hydrogen peroxide were purchased from WINLAB (UK). Deionized water was purified using a cartridge system (Picotech water system, RTP, NC, USA). Human plasma was obtained from King Khalid University hospital (Riyadh, KSA), and was kept frozen until use.

2.2 HPLC system

The HPLC instrument (Jasco, Japan) is equipped with a pump (model PU-980), a UV/vis detector (model UV-975) and injection valve with 20 μl sample loop. Signal acquisition and data handling were performed with LG computer connected to the instrument. The CSP used in this study was the macrolide-type antibiotic vancomycin, known as Chirobiotic V (150 × 4.6 mm i.d.) purchased from Advanced Separation Technologies (Whippany, NJ, USA). The mobile phase was methanol:glacial acetic acid:triethylamine (100:0.02:0.03, v/v/v). The mobile phase was filtered through a Millipore membrane filter (0.2 μm) from Nihon, Millipore (Yonezawa, Japan) and degassed before use. The flow rate was 1 ml/min and the detection wavelength (UV) was set at 315 nm.

2.3 Preparation of stock and standard solution

Stock solutions of individual S-(+)- and R-(−)-arotinolol hydrochloride were prepared in methanol to give a concentration of 80 μg/ml. The internal standard haloperidol was prepared in methanol to give a concentration of 1 mg/ml. Appropriate dilutions of the individual stock solutions of arotinolol were made and used for constructing the calibration curves, for conducting the stability study and for spiking the plasma.

2.4 Degradation studies

All degradation studies were performed at a drug concentration of 200 ng/ml in methanol. Acid hydrolysis was performed in 0.1 and 1 M HCl at room temperature and samples were withdrawn at different time intervals. The study in alkaline condition was carried out in 0.01, 0.1 and 1 M NaOH at room temperature and samples were withdrawn at different time intervals. Oxidative study was carried out at room temperature in 3%, 10%, 20% and 30% hydrogen peroxide and samples were also withdrawn at different time intervals. Photo-degradation study was performed in methanol. The solution was exposed to sunlight for 3 days. Suitable control was kept under the dark. Additionally, thermal degradation was induced by storing the drug at 80 °C for a period of 7 days. Samples were withdrawn at appropriate time and subjected to HPLC analysis.

2.5 Preparation of plasma samples

Human plasma sample (0.5 ml) was placed into individual 1.5 ml Eppendrof tube and accurately measured aliquots of S-(+)- and R-(−)-arotinolol were added. Then 25 μl of the internal standard solution was added to each tube and diluted with water to 1 ml and mixed well to give final concentrations of 80, 150 and 300 ng/ml of each arotinolol enantiomers. The mixture was treated with 100 μl of 3 M ammonium acetate (pH 9) and sonicated for 5 min. The aqueous phase was extracted with 3 × 1 ml ethyl ether. The organic phase was evaporated to dryness. The residue was dissolved in 200 μl methanol and injected into an HPLC system. Blank human plasma samples were processed in the same manner using deionized water instead of arotinolol enantiomers.

2.6 Preparation of tablet solutions

Ten prepared tablets were grounded and powdered. An accurately weighed portion equivalent to 10 mg arotinolol was transferred to 100 ml volumetric flask diluted to the mark with methanol. The solution was sonicated for 15 min, centrifuged at 3000 rpm for 10 min. Accurately measured aliquots of the supernatant were transferred to 5 ml volumetric flasks containing 100 μl of the internal standard and diluted to 5 ml with methanol to give final concentration of 100, 220 and 340 ng/ml of arotinolol.

2.7 Recovery

The absolute recoveries of each enantiomer from plasma were calculated by comparing drug peak area of the spiked analyte samples to unextracted analyte of stock solution that has been injected directly into an HPLC system. The assay absolute recovery for each compound, at each concentration, was computed using the following equation: $$\text{absolute recovery}=(\text{peak area of extract}/\text{mean peak area of direct injection})\times 100\text{.}$$

2.8 Validation of the method

3.1 Optimization of the chromatographic condition

The complex structures of macrocyclic antibiotics are responsible for their chiral selectivity in different modes (Aboul-Enein and Ali, 2002). The possible interactions involved are π–π complexation, hydrogen bonding, inclusion complexation, dipole interactions, steric interactions, anionic and cationic bindings (Aboul-Enein and Ali, 2000). These interactions take place individually or in combination, which can result in the very high chiral recognition capacities for these antibiotics.

The chemical structures of S-(+) – (I) and R-(−) – (II) arotinolol and haloperidol (III) are shown in Fig. 1.

The polar organic mode (POM) which has been described to obtain enantioselective separation with macrocyclic antibiotic-based CSPs (Fried et al., 1998) is a good choice for enantioseparation of arotinolol. Easy-to-prepare mobile phases composed of methanol with small amounts of acetic acid (HAc) and triethylamine (TEA) were used. The baseline separation of arotinolol was achieved on the vancomycin CSP column with a polar organic mobile phase consisting of methanol:glacial acetic acid:triethylamine (100:0.02:0.03, v/v/v) (Table 2) and Fig. 2.

Close

Figure 2

Chromatogram of freshly prepared arotinolol sample using Chirobiotic V column (mobile phase: methanol:glacial acetic acid:triethylamine (100:0.020:0.030, v/v/v), flow rate: 1 ml/min, UV detection: 315 nm).

Export to PPT

When using pure methanol as mobile phase, arotinolol did not eluted. A small addition of HAc and TEA was sufficient to elute the enantiomers in an acceptable time. An increase in the acid and base concentrations decreases the enantioresolution values.

3.2 Degradation studies

HPLC studies of samples obtained on stress testing of arotinolol under different conditions suggested the following degradation behaviour.

3.2.1

3.2.1 Acid degradation study

The drug was stable in 0.1 M HCl at room temperature for a period of 7 days. In 1 M HCl, 20.90% of arotinolol was hydrolyzed in the same period (Fig 3).

Close

Figure 3

Chromatogram of arotinolol in 1 M HCl after a period of 7 days using Chirobiotic V column (mobile phase: methanol:glacial acetic acid:triethylamine (100:0.020:0.030, v/v/v), flow rate: 1 ml/min, UV detection: 315 nm).

Export to PPT

3.3 Applications to spiked human plasma

The extraction procedure used in this study afforded percentage recovery ranges of 96–104% for linearity in the range of 50–400 ng/ml for both enantiomers, Table 3. The addition of ammonium acetate was necessary to obtain such recoveries. Chromatograms of a blank human plasma sample (A) and an extracted sample from plasma (B) are in Fig. 4. It is clear that there were no interferences from endogenous plasma contents indicating the high selectivity of the proposed method.

Close

Figure 4

Chromatograms of (A) blank human plasma and (B) spiked with (I) S-(+)-arotinolol, (II) R-(−)-arotinolol, and (III) haloperidol (IS) using Chirobiotic V column (mobile phase: methanol:glacial acetic acid:triethylamine (100:0.020:0.030, v/v/v), flow rate: 1 ml/min, UV detection: 315 nm).

Export to PPT

3.4 Precision and accuracy

A summary of the accuracy and precision results in spiked human plasma is given in Table 4. The acceptance criteria (within-run and between-run %RSD of less than 15% and accuracy between 85% and 115%) were met in all cases. The within-run precision and accuracy (n = 3) expressed as %RSD and %error were 1.9–5.5% and 2.1–3.1%, respectively, for S-(+)-arotinolol and 1.9–7.1% and 2.2–3.4%, respectively, for R-(−)-arotinolol.

The between-run precision and accuracy (n = 9) expressed as %RSD and %error were 4.1–5.7% and 0.5–1.9% for S-(+)-arotinolol, respectively, and 3.8–4.8% and 2.1–3.8% for R-(−)-arotinolol, respectively.

3.5 Limit of detection and limit of quantitation

The LOD and the LOQ in plasma sample as defined in Section 2 were 20 and 50 ng/ml for each enantiomer, respectively (Table 3).

3.6 Application to pharmaceutical formulation

The validity of the method developed here was applied to various concentrations taken from the pharmaceutical formulations for determining their content of arotinolol enantiomers. The values of the overall drug percentage recoveries and the %RSD value of S- and R-arotinolol enantiomers are presented in Table 5, indicating that these values are acceptable and the method is accurate and precise (Fig 5).

Close

Figure 5

Chromatogram of (I) S-(+)-arotinolol (II) R-(−)-arotinolol recovered from tablets spiked with (III) haloperidol (IS) using Chirobiotic V column (mobile phase: methanol:glacial acetic acid:triethylamine (100:0.020:0.030, v/v/v), flow rate: 1 ml/min, UV detection: 315 nm).

Export to PPT

3.7 Selectivity

The analytical figures of merit for this method are shown in Table 1. Arotinolol enantiomers were well separated under the HPLC conditions applied. The drug was completely resolved from its degradation products. No interference was observed in drug free human plasma samples.

Excipients commonly coformulated with the studied drug such as magnesium stearate, cellulose, starch, calcium hydrogen phosphate, colloidal silicon dioxide and coloring agents, also did not interfere with the determination of arotinolol enantiomers, indicating the high selectivity of the method.