Experimental design optimization of simultaneous enantiomeric separation of atenolol and chlorthalidone binary mixture by high-performance liquid chromatography using polysaccharide-based stationary phases.

In this work, enantiomeric separation of a drug combination of two chiral drugs, namely, atenolol and chlorthalidone, is described. Prior investigation of the effect of different variables on the resolution of the enantiomers' peaks and the total run time represented by the retention time of the last eluted peak was conducted using face-centered composite design. Twenty-two experiments were carried out by varying the chiral stationary phase type as a categorical factor and mobile phase composition including the percentage of ethanol and percentage of diethylamine as continuous factors. According to the optimization process, a mobile phase consisting of hexane:ethanol:DEA:TFA (60:40:0.2:0.1%, v/v/v/v) pumped at flow rate 1 ml min-1 onto Lux-Cellulose 2 stationary phase was applied for the chiral separation and quantification of the drug combination at 230 nm. Application of the developed method to the pharmaceutical formulation of this combination was successfully performed, and satisfactory percentage of recoveries was obtained. The method was also fully validated following International Conference on Harmonization (ICH) guidelines. This method could be of high value and relevance for application in quality control laboratories.

Structure-retention relationship for enantioseparation of selected fluoroquinolones.

Fluoroquinolones are popular class of antibiotics with distinct chemical functionality. Most of them are ampholytes with one chiral center. Stereogeneic center is located either in the side ring of Gatifloxacin (GFLX) or in the quinolone core of Ofloxacin (OFLX). These two amphoteric fluoroquinolones have terminal amino groups in common. The unusual Nadifloxacin (NFLX) is an acidic fluoroquinolone with a core chiral center. Owing to chirality and functionality differences among GFLX, OFLX, and NFLX, we mapped these enantiomers onto structure-retention relationship. Amount of acetic acid modifier was studied in screened mobile phase and cellulose tris(3-chloro-4-methyl phenyl carbamate) (Lux cellulose-2) stationary phase. Experimental design of acetic acid% along with column temperature have been applied. Resolution and enantioselectivity have been related to structural features of the studied enantiomers. High amount of acid (0.4%) was optimum for the separation of either side chirality with a proximate amino group (GFLX) or core chirality without basic functionality (NFLX), while low amount (0.2%) is optimum for core chiral center with distal amino group (OFLX). Temperature has no significant effect on resolution and retention of enantiomers except for OFLX. Enantio-retention explains possible chiral selective and nonselective interactions. The proposed methods have been validated for pharmaceutical analyses.

Enantioseparation of Substituted 1, 3-Diazaspiro [4.5]Decan-4-Ones: HPLC Comparative Study on Different Polysaccharide Type Chiral Stationary Phases.

Enantioseparation of substituted 1,3-diazaspiro[4.5]decan-4-ones (1-14) was achieved using different polysaccharide type chiral stationary phases (CSPs), namely, Chiralcel OJ, Chiralcel OD and Lux-Amylose-2 using different mobile phases which were either n-hexane/2-propanol or n-hexane/ethanol mixtures of various ratios (v/v) at flow rate 1 mL min-1. UV detection was carried out at 254 nm and temperature of 20°C. The retention behavior and selectivity of these CSPs were examined in isocratic normal phase high-performance liquid chromatography mode. The results revealed that the amylose CSP (Lux-Amylose-2) could separate almost all the compounds under investigation in contrast to cellulose CSPs (Chiracel OJ and Chiracel OD) which resolved fewer compounds.

Riluzole: validation of stability-indicating HPLC, D1 and DD1 spectrophotometric assays.

A stability-indicating reversed-phase high-performance liquid chromatographic assay procedure has been developed and validated for riluzole in the presence of alkaline and oxidative degradation products. The liquid chromatographic separation was achieved and compared isocratically on C18 Zorbax ODS and Poroshell 120 EC-C18 columns by using a mobile phase containing methanol-water, pH = 3.10 (70:30, v/v), at a flow rate of 1 mL/min and ultraviolet detection at 264 nm. The method was linear over the concentration ranges of 20-200 µg/mL (r = 0.9997) and 10-200 µg/mL (r = 0.9995). The limit of detection and quantitation for the two columns were 2 and 6 µg/mL and 1 and 3 µg/mL, respectively. Moreover, spectrophotometric methods were applied for the determination of riluzole in the presence of its oxidative degradation products by using first derivative spectrophotometry at 252.5 and 275.0 nm. The method was linear over the concentration range of 1-20 µg/mL (r = 0.9995 and 0.9996) at the studied wavelengths, with limits of detection and quantitation of 0.1 and 0.3 µg/mL. In addition, the first derivative ratio spectrophotometry (DD1) method was applied for the determination of riluzole in the presence of its alkaline degradation product at 252.0, 278.5 and 306.3 nm by using 100 µg/mL of alkaline degraded riluzole as a divisor; riluzole was additionally determined in the presence of its hydrogen peroxide oxidative degradation products at 252.5, 275.0 and 305.0 nm by using 200 µg/mL of oxidative degraded riluzole as a divisor. The DD1 method was linear over the concentration range of 1-20 µg/mL (r = 0.9996, 0.9995 and 0.9996 for the alkaline degradation product at the three studied wavelengths, respectively; and r = 0.9995, 0.9996 and 0.9995 for the oxidative degradation product at the three studied wavelengths, respectively), with limits of detection and quantitation of 0.1 and 0.3 µg/mL for both alkaline and oxidative degradation products. The two studied chromatographic and spectrophotometric methods were comparable and display the required accuracy, selectivity, sensitivity and precision to assay riluzole in bulk and pharmaceutical dosage forms. Degradation products resulting from the stress studies did not interfere with the detection of riluzole, which indicates that these are stability-indicating assays.

A validated HPLC method for separation and determination of promethazine enantiomers in pharmaceutical formulations.

A simple, rapid, and validated method for separation and determination of promethazine enantiomers was developed. Promethazine was separated and quantitated on a Vancomycin Chirobiotic V column (250 x 4.6 mm), using a mixture of methanol, acetic acid, and triethylamine (100:0.1:0.1%, by volume) as a mobile phase at 20 degrees C and at a flow rate of 1 mL/min. The UV-detector was set to 254 nm. Acetyl salicylic acid (Aspirin) was used as an internal standard. The applied HPLC method allowed separation and quantification of promethazine enantiomers with good linearity (r > .999) in the studied range. The relative standard deviations (RSD) were 0.29 and 0.36 for the promethazine enantiomers with accuracy of 100.06 and 100.08. The limit of detection and limit of quantification of promethazine enantiomers were found to be 0.04 and 0.07 microg/mL, respectively. The method was validated through the parameters of linearity, accuracy, precision, and robustness. The HPLC method was applied for the quantitative determination of promethazine in pharmaceutical formulations.