Three accurate, sensitive, simple, and precise spectrophotometric methods along with thin-layer chromatography (TLC)–densitometric method were developed, optimized, and validated for the determination of folic acid in the presence of its two impurities (photodegradation products), namely, pteroic acid and para-aminobenzoic acid. Method A is the ratio difference spectrophotometric method (RDSM) which depends on measuring the difference value in the ratio spectrum, where the difference between 291 and 313 nm was used for the determination of folic acid, while the difference between 305 and 319 nm was selected for the estimation of para-aminobenzoic acid; on the other hand, pteroic acid can be determined using the first derivative of ratio spectra spectrophotometric method at 262 nm. Method B is the double-divisor spectrophotometric method (DDSM); this method is based on using the ratio spectrum obtained by the division of the spectrum of ternary mixture by the spectrum of binary mixture containing two of the three mentioned components, and in this method, folic acid, para-aminobenzoic acid, and pteroic acid were measured at 242, 313, and 258 nm, respectively. Method C is the mean-centering of ratio spectra spectrophotometric method (MCR); in this method, folic acid, para-aminobenzoic acid, and pteroic acid can be measured using the mean-centered second ratio spectra amplitudes at 317–318 (peak to peak), 264–265 (peak to peak), and 232 nm, respectively. Lastly, method D is a TLC‒densitometric one that depends on the separation and quantification of the mentioned components on TLC silica gel 60 F254 plates, using methanol‒ iso-propanol‒water‒acetic acid (9:0.5:0.5:0.2, by volume) as the developing system, followed by densitometric measurement of the separated bands at 280 nm. Method validation was carried N.W. Alia, N.S. Abdelwahaba, M.M. Abdelrahmana, and S.I. Tohamy, Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Alshaheed Shehata Ahmed Higazy St., 62514, Beni-Suef, Egypt; and B.A. El-Zeiny, Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr EL-Aini Street, ET 11562, Cairo, Egypt. *E-mail: email@example.com out according to the International Conference on Harmonisation (ICH) guidelines, and the proposed methods were successfully applied to the analysis of folic acid in pharmaceutical formulations, where no interference from additives has been found. The results obtained by the proposed methods were statistically compared with those obtained by the official reversed-phase high-performance liquid chromatography (RP-HPLC) method, in which no significant difference was observed.
Accurate, selective, and sensitive thin-layer chromatography (TLC)—densitometry and reversed-phase high-performance liquid chromatography (RP-HPLC) methods have been developed and validated for the simultaneous determination of vitamin E (VIT E) and vinpocetine (VINP) in the presence of the alkaline-induced degradation product of vinpocetine (DEG). The proposed TLC— densitometric method depends on the separation and quantitation of VIT E, VINP, and VINP alkaline-induced degradation product on TLC silica gel 60 F254 plates, using methanol—chloroform—ethyl acetate—glacial acetic acid—ammonia solution (6:2:2:0.5:0.1, by volume) as the developing system followed by densitometric measurement at 235 nm. The studied components were well resolved from each other with significantly different Rf values of 0.81, 0.62, and 0.41 for VIT E, VINP, and DEG, respectively. On the other hand, the developed RP-HPLC method was based on the separation of the studied components using 0.05 M KH2PO4 (adjusted to pH = 3) and methanol in gradient elution mode on C8 column at a flow rate of 1.5 mL min−1 and ultraviolet (UV) detection at 235 nm. The studied components were well resolved from each other with significantly different Rt values of 10.90, 2.89, and 1.90 min for VIT E, VINP, and DEG, respectively. The developed methods were validated according to the International Conference on Harmonization (ICH) guidelines demonstrating good accuracy and precision. The results were statistically compared with those obtained by the reported method, and no significant difference was found. The developed methods are the first developed stability-indicating assay methods (SIAMs) for the analysis of the studied binary mixture.
The presented study was intended to design two validated, simple, and precise chromatographic methods for the determination of chlorzoxazone (CHZ) and diclofenac potassium (DIC) in the presence of chlorzoxazone nephrotoxic degradation product, 2-amino-4-chlorophenol (ACP) which was reported to be its main impurity. Reversed-phase high-performance liquid chromatography (RP-HPLC) was the first method where chromatographic separation was performed on ZORBAX Eclipse Plus C8 column using methanol—water—phosphoric acid (75:25:0.05, by volume) as the mobile phase at a flow rate of 1 mLmin−1. CHZ, DIC, and ACP retention times were found to be 4.26, 7.94, and 3.17, respectively, using photodiode array detector (DAD) at 230 nm. The calibration curves showed good linear relationships in the concentration ranges of 3–45 μg mL−1 for CHZ, 3–40 μg mL−1 for DIC, and 5–45 μg mL−1 for ACP. The second method was thin-layer chromatography (TLC) at which chromatographic separation was carried out on Merck TLC silica gel 60 F254 aluminum plates followed by measurement of separated bands at 230 nm and using chloroform—ethanol—triethylamine (9:1:0.1, by volume) as the developing system. The studied components were successfully separated with significantly different Rf values (CHZ, Rf = 0.63; DIC, Rf = 0.35; ACP, Rf = 0.42). Linearity was constructed in the range of 1.2–5 μg band−1 for CHZ, 0.5–4 μg band−1 for DIC, and 0.4–4 μg band−1 for ACP. The developed methods were applied to Declophen plus® capsules, and no interference from excipients was observed. The methods were validated as per the United States Pharmacopeia (USP) guidelines, and they were compared favorably with the reported method.
Three simple, sensitive, and validated methods were developed for the quantitative determination of fosinopril sodium (FOS) and hydrochlorothiazide (HCZ) in the presence of an HCZ impurity, chlorothiazide (CZ). The first method (I) was the ratio difference spectrophotometric method (RD), in which a standard spectrum of 8 µg mL−1 HCZ was used as a divisor, and the difference in amplitude values at 204.6 and 231.2 nm and 290 and 302.6 nm was used for the determination of FOS and CZ, respectively. Meanwhile, for the determination of HCZ, a standard spectrum of 6 µg mL−1 CZ was the chosen divisor, and the amplitude difference at 275 nm and 293.6 nm was selected for the calculation of its concentrations. The second method (II) was mean centering of ratio spectra spectro-photometric method (MCR), which depended on the implementation of the mean-centered ratio spectra in two successive steps and the measurement of the amplitudes of the mean-centered second ratio spectra at 243.4 nm for CZ and peak-to-peak amplitudes at 215.6 and 215.8 nm for FOS and at 223.8 and 224 nm for HCZ. On the other hand, the third method (III) was thin-layer chromatography (TLC)-densitometry at which the chromatographic separation of this ternary mixture was performed using pre-activated silica gel 60 F254 TLC plates and a developing system mixture consisting of ethyl acetate-chloroform-methanol-formic acid (60:40:5:0.5, by volume) with ultraviolet (UV) scanning at 215 nm. The developed methods were validated according to the International Conference of Harmonization (ICH) guidelines and were successfully used for the determination of FOS and HCZ in their pharmaceutical formulations. Also, a statistical comparison between the developed methods and the reported HPLC method was attained. Using Student's t-test and F-test, the results confirmed that there was not any significant difference between them regarding accuracy and precision.