Mohamed A. Hammad

Validated spectrofluorimetric method for determination of selected aminoglycosides

New, sensitive, and selective spectrofluorimetric method was developed for determination of three aminoglycoside drugs in different dosage forms, namely; neomycin sulfate (NEO), tobramycin (TOB) and kanamycin sulfate (KAN). The method is based on Hantzsch condensation reaction between the primary amino group of aminoglycosides with acetylacetone and formaldehyde in pH 2.7 yielding highly yellow fluorescent derivatives measured emission (471 nm) and excitation (410 nm) wavelengths. The fluorescence intensity was directly proportional to the concentration over the range 10-60, 40-100 and 5-50 ng/mL for NEO, TOB and KAN respectively. The proposed method was applied successfully for determination of these drugs in their pharmaceutical dosage forms.

Development of spectrofluorimetric method for determination of certain aminoglycoside drugs in dosage forms and human plasma through condensation with ninhydrin and phenyl acetaldehyde

A simple and sensitive spectrofluorimetric method has been developed and validated for determination of amikacin sulfate, neomycin sulfate and tobramycin in pure forms, pharmaceutical formulations and human plasma. The method was based on condensation reaction of cited drugs with ninhydrin and phenylacetaldehyde in buffered medium (pH 6) resulting in formation of fluorescent products which exhibit excitation and emission maxima at 395 and 470nm, respectively. The different experimental parameters affecting the development and stability of the reaction products were carefully studied and optimized. The calibration plots were constructed with good correlation coefficients (0.9993 for tobramycin and 0.9996 for both neomycin and amikacin). The proposed method was successfully applied for the analysis of cited drugs in dosage forms with high accuracy (98.33-101.7)±(0.80-1.26)%. The results show an excellent agreement with the reference method, indicating no significant difference in accuracy and precision. Due to its high sensitivity, the proposed method was applied successfully for determination of amikacin in real human plasma.

Enhancement of the sensitivity of valacyclovir and acyclovir for their spectrofluorimetric determination in human plasma

Two rapid, simple and highly sensitive spectrofluorimetric methods have been developed and validated for determination of valacyclovir hydrochloride (VAC) and acyclovir (ACV). The first method is based on measuring the intrinsic fluorescence of VAC or ACV in an aqueous acidic medium (pH 1.3) at 370 nm after excitation at 280 nm. The fluorescence intensity–concentration plots of VAC and ACV were rectilinear over the concentration ranges of 0.4–5.0 and 0.3–4.0 μg ml−1, respectively. The second method was based on the enhancement of the fluorescence intensity using sodium dodecyl sulfate (SDS) as a micellar system in an aqueous acidic medium (pH 1.3). This is the first attempt for enhancement of the sensitivity of these drugs by spectrofluorimetry. The addition of 2% w/v SDS, produced about 2.7 and 2.9 fold enhancements in the relative fluorescence intensity of VAC and ACV, respectively. The linear range for the second method was 0.2–2.5 and 0.1–1.25 μg ml−1, respectively. The proposed methods were successfully applied for determination of VAC and ACV in pharmaceutical preparations without interference from the common excipients. The high sensitivity of the micellar method permits its application for determination of ACV in human plasma with good percentage recovery.

Specific and highly sensitive spectrofluorimetric method for determination of febuxostat in its tablets and real human plasma. Application to stability studies

A simple, rapid, specific and highly sensitive spectrofluorimetric method has been developed for determination of febuxostat (FEB) in its tablets and real human plasma. The proposed method is based on the investigation of the fluorescence spectral behavior of FEB in an aqueous acidic system. The fluorescence intensity was measured at 400 nm after excitation at 335 nm. The fluorescence–concentration plot was rectilinear over the range 0.5–20.0 ng ml−1, with a limit of detection of 38.9 pg ml−1 and limit of quantification of 117.3 pg ml−1. The high sensitivity of the proposed method permits its application for the determination of FEB in real human plasma with good percentage recovery (81.73 ± 2.61). The application of the proposed method was further extended to stability studies of FEB after exposure to different forced degradation conditions, such as acidic, alkaline and oxidative conditions, according to ICH guidelines. A proposal for the degradation pathways was postulated.

Development and validation of TLC–densitometric method for simultaneous determination of two binary antihypertensive mixtures containing felodipine in fixed dose combinations

A new densitometric thin-layer chromatographic method has been developed for simultaneous determination of two binary mixtures containing felodipine in combination with either metoprolol (mixture I) or ramipril (mixture II). The two mixtures were quantitatively separated on 60 F254 silica gel plates using toluene-ethyl acetate-methanol-ammonia as mobile phase with UV detection at 233 and 229 nm for mixtures I and II, respectively. The studied drugs were satisfactorily resolved with retention factor (Rf ) values of 0.34 ± 0.03 and 0.65 ± 0.03 for metoprolol and felodipine, respectively, in mixture I and 0.35 ± 0.03 and 0.74 ± 0.03 for ramipril and felodipine, respectively, in mixture II. Linearity ranges were 2000-7000 and 200-700 ng/band for metoprolol and felodipine, respectively, in mixture I and 1500-4000 ng/band for both ramipril and felodipine in mixture II. Correlation coefficient (r) values were 0.9968 for both metoprolol and felodipine in mixture I and 0.9993 for ramipril and 0.9989 for felodipine in mixture II. The method has been validated according to International Conference on Harmonization guidelines and has been successfully applied for determination of the studied drugs in their dosage forms without interference from commonly encountered excipients.

Spectrofluorimetric and micelle-enhanced spectrofluorimetric methods for determination of Felodipine and Nimodipine in pharmaceutical preparations and human plasma.

Rapid, simple and sensitive two spectrofluorimetric methods have been developed for determination of Felodipine (FLD) and Nimodipine (NDP). The first method is based on measuring the native fluorescence of either Felodipine or Nimodipine at 426 nm after excitation at 385 nm. The fluorescence intensity-concentration plots of Felodipine and Nimodipine were rectilinear over the concentration ranges (0.2-3.0) and (0.5-4.0) μg ml(-1), respectively. The second method is based on measuring the fluorescence intensity of the studied drugs in micellar media (0.3% Tween-80) at λex=385 nm and λem=423 nm. In the presence of 0.3% Tween-80, about 1.6-fold and 2.1-fold enhancement can be achieved in the relative fluorescence intensity (RFI) of Felodipine and Nimodipine, respectively. The fluorescence intensity-concentration plots of Felodipine and Nimodipine with Tween-80 were rectilinear over the concentration ranges (0.05-4.0) and (0.1-4.0) μg ml(-1), respectively with determination coefficients (r(2)) of 0.9981 and 0.9990, and limit of quantitation of 0.05 and 0.027μg ml(-1) for FLD and NDP, respectively. The proposed methods were validated according to ICH guidelines and have been successfully applied to the analysis of these drugs in their commercial tablets with high accuracy (97.6-98.8±0.50-1.42%, n=5). The high sensitivity of micellar method permits its application for determination of the cited drugs in spiked human plasma with % recovery (91.9-106.6±0.66-1.7%, n=6).

Highly sensitive spectrofluorimetric method for determination of doxazosin through derivatization with fluorescamine; Application to content uniformity testing.

A highly sensitive, simple and selective spectrofluorimetric method has been developed and validated for determination of doxazosin mesylate in pure form, pharmaceutical formulations and human plasma. The method is based on the reaction between doxazosin mesylate and fluorescamine in Teorell buffer solution (pH 3) to give highly fluorescent derivative that can be measured at 489 nm using excitation wavelength of 385 nm. Different experimental parameters affecting the reaction were carefully studied and optimized. The calibration plot was constructed over the concentration range of 16-400 ng mL(-1) with quantitation limit of 14.3 ng mL(-1). The developed procedure was validated according to ICH guidelines and the results were satisfactory. The proposed method has been successfully applied to the analysis of the cited drug in its pharmaceutical preparations as well as for content uniformity testing. The results showed excellent agreement with the reported method with respect to precision and accuracy. In addition, the drug concentration was determined in the spiked human plasma by the suggested method with % recovery in the range of 96.2-98.3% (SD; 0.76-0.93, n=5).

Innovative thin-layer chromatographic method combined with fluorescence detection for specific determination of Febuxostat: Application in biological fluids

A newly developed thin-layer chromatographic (TLC) method coupled with fluorescence detection for specific determination of Febuxostat (FEB) was designed. The proposed method adopts exposure of FEB on a developed TLC plate to hydrochloric acid vapors, resulting in a large enhancement of its weak fluorescence, permitting its specific and sensitive determination in real human plasma and urine after excitation at 345nm on 60 F254 silica gel plates using toluene-ethyl acetate-methanol-glacial acetic acid; (30:10:5:0.1,v/v/v/v) as mobile phase. The retention factor (Rf) value for FEB was 0.33 ± 0.03 with a correlation coefficient of 0.9974 in the concentration range of 2.5-50ng/band. Upon using polynomial regression, the correlation coefficient was greatly improved (0.9999), with detection and quantification limits of 0.55 and 1.67 (ng/band), respectively. The proposed method was validated according to the International Conference of Harmonization and was successfully used for specific and selective determination of FEB in its commercial dosage form without excipient interference. Moreover, the proposed method was extended to efficient determination of the studied drug in real human plasma and urine samples in the presence of its metabolites without any interference, allowing clinical application of the proposed method for direct FEB determination in biological fluids as well as in pharmacokinetics studies and for quality control of the pharmaceutical dosage form without sample pretreatment or exhausting extraction steps.

Simultaneous Determination of Tizanidine, Nimesulide, Aceclofenac and Paracetamol in Tablets and Biological Fluids Using Micellar Liquid Chromatography

A simple, sensitive and rapid micellar liquid chromatographic method was developed and validated for simultaneous determination of four drugs, namely, paracetamol (PAR), tizanidine (TZD), aceclofenac (ACF) and nimesulide (NMD). Good chromatographic separation was achieved using Cyano column and micellar mobile phase consisting of 120 mM sodium dodecyl sulfate, 25 mM phosphate buffer and 10% (V/V) butanol. The pH was adjusted to three using phosphoric acid. The total retention time was below 10 min. The analysis was performed at a flow rate of 1 mL/min and a column temperature of 40°C with direct UV detection at 230 nm. Diclofenac sodium was used as the internal standard. The proposed method was validated according to the ICH guidelines and was successfully applied to the analysis of these drugs in their tablet dosage forms with high accuracy. Limits of detection were found to be 0.03, 0.07, 0.033 and 0.11 μg/mL for PAR, ACF, TZD and NMD, respectively. The high sensitivity of developed method permitted its application to the in-vitro determination of the cited drugs in spiked human plasma and urine samples, and the obtained results were satisfactory. However, PAR could not be determined in spiked human urine because its peak overlapped with that of the urine peak.

Utility of 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole for development of a highly sensitive stability indicating spectrofluorimetric method for determination of salmeterol xinafoate; application to human plasma

A new, simple and rapid spectrofluorimetric method was developed and validated for determination of salmeterol xinafoate in dosage form and spiked plasma. The method is considered as the first attempt for the spectrofluorimetric determination of the investigated drug. The proposed method is based on nucleophilic substitution reaction between salmeterol xinafoate and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) at pH (9) to form a highly fluorescent product that can be measured at 543 nm after excitation at 473 nm. The fluorescence–concentration plot was rectilinear over the concentration range (100–1500 ng mL−1) with a detection limit of 19 ng mL−1. The suggested method was validated according to ICH guidelines and successfully applied for determination of salmeterol xinafoate in its pharmaceutical dosage form and spiked human plasma. The developed method was further extended for a stability study of salmeterol xinafoate under different stress circumstances including alkali, acid, oxidative and photodegradation conditions and the method was proved to be able to determine the intact drug in the presence of its degradation products.