The Development of Spectrophotometric and Validated Stability- Indicating RP-HPLC Methods for Simultaneous Determination of Ephedrine HCL, Naphazoline HCL, Antazoline HCL, and Chlorobutanol in Pharmaceutical Pomade Form.

BACKGROUND: Allergic rhinitis, acute nasal congestion and sinusitis are one of the most common health problems and have a major effect on the quality of life. Several medications are used to improve the symptoms of such diseases in humans. Pharmaceutical pomade form containing Ephedrine (EPD) HCl, Naphazoline (NPZ) HCl, Antazoline (ANT) HCl, and Chlorobutanol (CLO) is one of them. OBJECTIVE: For these reasons, this study includes the development of spectrophotometric and chromatographic methods for the determination of EPD HCl, NPZ HCl, ANT HCl, and CLO active agents in the pharmaceutical pomade. METHOD: In the spectrophotometric method, third-order derivative of the amplitudes at 218 nm n=5 and the first-order derivative of the amplitudes 254 nm n=13 was selected for the determination of EPD, ANT, respectively while NPZ was determined by the second derivative at 234 nm and n=21. Colorimetric detection was applied for assay analysis of CLO at 540 nm. Furthermore, a reverse phase high performance liquid chromatographic (RP- HPLC) method has been developed and optimized by using Agilent Zorbax Eclipse XDB C18 (75 mm x 3.0 mm, 3.5µm) column. The column temperature was 40°C, binary gradient elution was used and the mobile phase consisted of 15 mM phosphate buffer in distilled water (pH 3.0) and methanol, and the flow rate was 0.6 mL min-1 and the UV detector was detected at 210 nm. The linear operating range was obtained as 11.97-70, 0.59-3, 2.79-30, and 2.92-200 µg mL-1 for EPD HCl, NPZ HCl, ANT HCl, and CLO respectively. RESULTS: The LOD values were found to be 3.95, 0.19, 0.92 and 0.96 µg mL-1 for EPD HCl, NPZ HCl, ANT HCl, and CLO in the spectrophotometric method, respectively. The linear ranges in the RP-HPLC method were 8.2-24.36 µg mL-1, 0.083 - 0.75 µg/mL, 2.01-7.5 µg mL-1 and 2.89-24.4 µg mL-1 for EPD HCl, NPZ HCl, ANT HCl, and CLO, respectively. The LOD values in the validation studies were 2.7, 0.025, 0.66 and 0.86 µg mL-1 for EPD HCl, NPZ HCl, ANT HCl, and CLO in RP-HPLC method respectively. CONCLUSION: The results of the spectrophotometric and chromatographic methods were compared and no differences were found between the two methods.

Determination of parabens in human milk and other food samples by capillary electrophoresis after dispersive liquid-liquid microextraction with back-extraction.

Dispersive liquid-liquid microextraction (DLLME) with back-extraction was used prior to capillary electrophoresis (CE) for the extraction of four parabens. Optimum extraction conditions were: 200 µL chloroform (extraction solvent), 1.0 mL acetonitrile (disperser solvent) and 1 min extraction time. Back-extraction of parabens from chloroform into a 50mM sodium hydroxide solution within 10s facilitated their direct injection into CE. The analytes were separated at 12°C and 25 kV with a background electrolyte of 25 mM borate buffer containing 5.0% (v/v) acetonitrile. Enrichment factors were in the range of 4.3-10.7 and limits of detection ranged from 0.1 to 0.2 µg mL(-1). Calibration graphs showed good linearity with coefficients of determination (R(2)) higher than 0.9957 and relative standard deviations (%RSDs) lower than 3.5%. DLLME-CE was demonstrated to be a simple and rapid method for the determination of parabens in human milk and food with relative recoveries in the range of 86.7-103.3%.

Dispersive liquid-liquid microextraction combined with field-amplified sample stacking in capillary electrophoresis for the determination of non-steroidal anti-inflammatory drugs in milk and dairy products.

Dispersive liquid-liquid microextraction (DLLME) was coupled with field-amplified sample stacking in capillary electrophoresis (FASS) for the determination of five non-steroidal anti-inflammatory drugs (NSAIDs) in bovine milk and dairy products. After extraction, the enriched analytes were back-extracted into a basic aqueous solution for injection into CE. Under optimum conditions, enrichment factors were in the range 46-229. Limits of detection of the analytes ranged from 3.0 to 13.1 µg kg(-1) for all matrices analysed. Calibration graphs showed good linearity with coefficients of determination (R(2))≥ 0.9915 and relative standard deviations (RSD%) of the analyses in the range of 0.6-6.2% (n=5). Recoveries of all NSAIDs from bottled milk, raw milk, yogurt and white cheese samples were in the ranges of 86.6-109.3%, 84.3-100.5%, 77.4-107.3%, and 90.9-101.6%, respectively. DLLME-FASS-CE was demonstrated to be a rapid and convenient method for the determination of NSAIDs in milk and dairy products.

Dispersive liquid-liquid microextraction based on solidification of floating organic drop combined with field-amplified sample injection in capillary electrophoresis for the determination of beta(2)-agonists in bovine urine.

Dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) was for the first time combined with field-amplified sample injection (FASI) in CE to determine four ß(2)-agonists (cimbuterol, clenbuterol, mabuterol, and mapenterol) in bovine urine. Optimum BGE consisted of 20 mM borate buffer and 0.1 mM SDS. Using salting-out extraction, ß(2)-agonists were extracted into ACN that was then used as the disperser solvent in DLLME-SFO. Optimum DLLME-SFO conditions were: 1.0 mL ACN, 50 µL 1-undecanol (extraction solvent), total extraction time 1.5 min, no salt addition. Back extraction into an aqueous solution (pH 2.0) facilitated direct injection of ß(2)-agonists into CE. Compared to conventional CZE, DLLME-SFO-FASI-CE achieved sensitivity enhancement factors of 41-1046 resulting in LODs in the range of 1.80-37.0 µg L(-1). Linear dynamic ranges of 0.15-10.0 mg L(-1) for cimbuterol and 15-1000 µg L(-1) for the other analytes were obtained with coefficients of determination (R(2)) ≥ 0.9901 and RSD% ≤5.5 (n = 5). Finally, the applicability of the proposed method was successfully confirmed by determination of the four ß(2)-agonists in spiked bovine urine samples and accuracy higher than 96.0% was obtained.

Ultrasound-assisted emulsification microextraction for the determination of ephedrines in human urine by capillary electrophoresis with direct injection. Comparison with dispersive liquid-liquid microextraction.

Ultrasound-assisted emulsification microextraction and dispersive liquid-liquid microextraction were compared for extraction of ephedrine, norephedrine, and pseudoephedrine from human urine samples prior to their determination by capillary electrophoresis. Formation of a microemulsion of the organic extract with an aqueous solution (at pH 3.2) containing 10% methanol facilitated the direct injection of the final extract into the capillary. Influential parameters affecting extraction efficiency were systematically studied and optimized. In order to enhance the sensitivity further, field-amplified sample injection was applied. Under optimum extraction and stacking conditions, enrichment factors of up to 140 and 1750 as compared to conventional capillary zone electrophoresis were obtained resulting in limits of detection of 12-33 µg/L and 1.0-2.8 µg/L with dispersive liquid-liquid microextraction and ultrasound-assisted emulsification microextraction when combined with field-amplified sample injection. Calibration graphs showed good linearity for urine samples by both methods with coefficients of determination higher than 0.9973 and percent relative standard deviations of the analyses in the range of 3.4-8.2% for (n = 5). The results showed that the use of ultrasound to assist microextraction provided higher extraction efficiencies than disperser solvents, regarding the hydrophilic nature of the investigated analytes.

Quantitative determination of ketoprofen in gels and ampules by using flow-injection UV spectrophotometry and HPLC.

A flow-injection UV spectrophotometric method for the determination of ketoprofen in gels and ampules was developed. Quantitative determination of ketoprofen was realized by using distilled water as a carrier for gels and citrate buffer, pH 6.5, for ampules at 261 nm. No spectrophotometric interferences from additives of gels, carboxypolymethylene and triethanolamine, were observed. There were also no spectrophotometric interferences resulting from additives of ampules named as benzyl alcohol and arginine. The detection limits were 0.436 and 0.303 microg/ml for gels and ampules, respectively. Throughout the study, the flow rate, loop volume and the number of injection per hour were 13.8 mlmin(-1), 193 mcirol and 85, respectively. Analytical signal of the ketoprofen was linear in the concentration range of 7.5-75 microg/ml. Quantitative results of ketoprofen in gels, 25.25+/-0.27 (mean+/-S.D.), and in ampules, 99.42+/-0.44 were in good agreement with the labeled quantities (25 mg/1g gel, 100 mg/2 ml ampule). The recoveries were in the range of 98.65-100.63 and 99.1-101.5% for gels and ampules, respectively. Results obtained were in accordance with those obtained by HPLC. It was seen that the proposed method was fast, accurate, precise and suitable for automation as an analytical method.