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Determination of penicillin residues in different industrial effluents including wastewater and air samples is important to prevent exposure to residual amounts of penicillin and the development of antibiotic resistance. A green high performance liquid chromatography (HPLC) method coupled with diode array detection has been developed and validated for multiplex determination of nine penicillin antibiotics in the industrial air dust and wastewater environmental samples of penicillin facility in addition to the monitoring of facility surface cleaning. Separation was performed on C18 column with gradient elution of methanol and phosphate buffer (pH 4) at a flow rate of 1.5 mL min-1 and ultra violet (UV) detection at 220 nm. Low limits of detection were achieved (0.1–0.3 μg mL-1) indicating good sensitivity of the proposed. The method was applied for ensuring the efficiency of cleaning validation after worst-case selection. Recovery studies of the studied penicillins from fortified stainless steel and polycarbonate surfaces and swabs were between 91.91 and 100.22% with relative standard deviation 0.11–1.79%. The presence of any of the studied penicillins in wastewater samples from penicillin plant drainage was checked. Also, total air dust concentration (mg m–3) and % of penicillin active material residues in air dust were calculated from the area of the exposed group in suspension, tablet and vial production lines. The proposed method can be recommended for routine analysis of air and wastewater environmental samples for the detection of penicillin antibiotics at low levels as well as monitoring of facility surface cleaning with high accuracy and precision.

Microfluidic capillary electrophoresis (MCE) is the novel technique resulted from the CE mininaturization as planar separation and analysis device. This review presents and discusses various application fields of this advanced technology published in the period 2017 till mid-2019 in eight different sections including clinical, biological, single cell analysis, environmental, pharmaceuticals, food analysis, forensic and ion analysis. The need for miniaturization of CE and the consequence advantages achieved are also discussed including high-throughput, miniaturized detection, effective separation, portability and the need for micro- or even nano-volume of samples. Comprehensive tables for the MCE applications in the different studied fields are provided. Also, figure comparing the number of the published papers applying MCE in the eight discussed fields within the studied period is included. The future investigation should put into consideration the possibility of replacing conventional CE with the MCE after proper validation. Suitable validation parameters with their suitable accepted ranges should be tailored for analysis methods utilizing such unique technique (MCE).

Simple and rapid spectrophotometric methods are described for determination of two mixtures of tamsulosin (TM), as minor component, with either solifenacin (SL) or tolterodine (TL). The proposed methods involve treatment of the absorbance ratio spectra or zero order spectra by derivative or discrete Fourier function. TM and TL mixture could not be resolved by manipulation of their zero order spectra due to the strong overlap between both spectra and only derivative or Fourier function coefficients of ratio spectra could resolve their spectra. TM and SL mixture was fully resolved by the manipulation of both ratio and zero order spectra. The values of the derivative or the Fourier function coefficients of ratio spectra and/or zero order spectra, at either peak or trough points, were correlated to the concentration of each drug in each mixture. Calibration graphs were linear in ranges 2.5-40 and 30-500 μg.mL−1 using derivative ratio and Fourier function ratio, 5-40 and 80–600 μg.mL−1 using direct derivative and 2.5–40 and 30–300 μg.mL−1 using direct Fourier function for TM and SL, respectively. The plots of derivative ratio amplitude and the Fourier function ratio coefficient versus concentration were linear over ranges 2.5–20 and 25–250 μg.mL−1 for TM and TL, respectively. Higher sensitivity as indicated by lower values of detection and quantitation limits were obtained using Fourier convoluted spectra (ratio or zero order) compared to derivative methods. All validation aspects per ICH guidelines are included. The proposed methods were also applied for the studied drugs assay in their tablets and capsules.

Two high-performance liquid chromatography-diode array detection methods have been developed and validated for the simultaneous quantification of genistein (GNS) and all trans retinoic acid (ATRA) as a novel anticancer combination therapy in their co-formulated nanoparticles and in rat plasma. Separation was performed on C18 column (250 × 4.6 mm, 5 μm) using celecoxib as internal standard. A mobile phase containing acetonitrile and water adjusted to pH 3 using 1% trifluoroacetic acid was delivered in gradient elution modes with time programmed UV detection. For extraction of the drugs and the internal standard from rat plasma, liquid- liquid extraction was applied. The proposed methods were validated as per International Conference on Harmonisation (ICH) guidelines (in the range 0.1–10 μg/mL for analysis of GNS and ATRA in nanoparticles) or according to Food and Drug Administration (FDA) guidance on bioanalytical method validation (in the range 0.025–20 μg/mL for analysis of GNS and ATRA in rat plasma). Pharmacokinetic study in six rats was performed following intravenous (IV) administration of a single dose of 0.5 mg/Kg of GNS and ATRA. The drugs’ concentrations were measured up to 24 hours, and different pharmacokinetic parameters were calculated. The obtained parameters were comparable with the reported values for IV administration of each drug alone in rats. This confirms the applicability of the proposed method in monitoring the levels of the two drugs in vivo following their coadministration and indicating that the two drugs could be coadministered as a promising novel combination therapy for the treatment of lung cancer without great alteration in their pharmacokinetic parameters compared with their individual IV administration.

Sunscreens (SSs) are highly applied all over the world on large areas of human body. Benzophenone chemical group constitute a major part in most SSs. Benzophenones are reported to induce changes in nucleic acids upon UV-irradiation. These alterations may potentially lead to DNA mutation, cell apoptosis, and eventually skin cancer. This work compares the kinetics of the induced DNA damage by some SSs after UV-irradiation. Six commonly used SSs; 4-t-butyl-4-methoxy dibenzoyl methane, 4-methoxycinnamic acid, 2-hydroxy-4-methoxybenzophenone (BZ-3), Dibenzoyl methane, 2,2'-dihydroxy-4-methoxy benzophenone (BZ-8) and p-methylbenzoic acid; are investigated. In this work, terbium chloride bioluminescent genosensor is used for sensitive, simple and inexpensive determination of induced DNA-damage. Results reveal that only BZ-3 and BZ-8 induced DNA-damage upon UV-irradiation that are confirmed by both absorption spectroscopy and viscosity measurements. Moreover, viscosity studies indicated the possible intercalation of the SS into DNA prior to initiation of DNA damage. Furthermore, the potency of BZ-3 and BZ-8 to induce DNA damage upon UVA irradiation was assessed on calf thymus DNA. The low cost of the proposed bioluminescent genosensor allows it to be an automatic simple process for the investigation of any DNA-drug interactions without the need of coupling with other analytical methods.

Two simple validated and highly selective methods for analysis of paracetamol, codeine, guaifenesin and pseudoephedrine or phenylephrine quaternary mixtures were developed. The first method is a high performance liquid chromatography with diode array detection method where separation was successful using Agilent C18 (150 × 4.6 mm) column, gradient elution of phosphate buffer pH 3, methanol and acetonitrile and diode-array detection at 210 nm. The second method is a HPTLC method followed by densitometric measurement of the spots at 257 nm. Separation was carried out on Merck HPTLC aluminum sheets of silica gel using methylene chloride: methanol: glacial acetic acid: ammonia (17.8: 1.68: 0.4: 0.12, v/v) mobile phase. The methods were applied successfully for analysis of both quaternary mixtures in laboratory-prepared tablets and also validated in regards to linearity, precision, accuracy, sensitivity and stability.

Aim: Assessment of pharmacokinetic interaction between linagliptin (LNG) and tadalafil (TDL) in healthy males. Methods: First, a novel LC–MS method was developed; second, a Phase IV, open-label, cross-over study was performed. Volunteers took single 20-mg TDL dose on day 1 followed by wash out period of 2 weeks then multiple oral dosing of 5-mg/day LNG for 13 days. On day 13, volunteers were co-administered 20-mg TDL. Results: LNG and TDL single doses did not affect QTc interval. Smoking did not alter pharmacokinetics/pharmacodynamics of LNG and TDL. Co-administration of LNG with TDL resulted in TDL longer time to reach maximum plasma concentration (Tmax), decreased oral clearance (Cl/F) and oral volume of distribution (Vd/F), increased its maximum plasma concentration (Cmax), area under concentration-time curve (AUC), muscle pain and QTc prolongation. Conclusion: LNG and TDL co-administration warrants monitoring and/or TDL dose adjustment.

A simple offline coupling voltammetry-MALDI/TOF MS procedure is presented for studying electrochemical reactions. It was utilized for the characterization of the electro-reduction products of febuxostat in methanolic acetate buffer (0.1 M, pH 5). The MS analysis reveals that the carboxylic and nitrile groups are the electro-reducible groups at −0.9338 and −1.5503 V with the conversion to aldehydic and amino groups, respectively. The developed voltammetric method was validated and applied successfully for the drug determination in pharmaceutical tablets and real plasma samples within the linearity ranges 0.03–2 and 0.4–5 μg mL−1, respectively.

Aim: To develop a simple HPLC-DAD method for simultaneous determination of febuxostat (FEB) and diclofenac (DIC) in biological samples to assess pharmacokinetic outcomes of their coadministration. Methodology & results: Sample preparation was performed by liquid–liquid extraction. Drugs analysis was done on C18 column using methanol-formic acid pH 2.1 (76:24, v/v) as mobile phase and time-programmed UV detection. Lower limits of quantitation for FEB and DIC were 10 and 20 ng/ml, respectively. Baseline pharmacokinetics were similar to published data on either drug alone. Coadministration led to more than twofold increase in FEB Cmax and AUC together with a reduced hepatic uptake in rats. Conclusion: DIC interfered with initial distribution and terminal clearance of FEB potentially due to reduced FEB hepatic uptake.

The presented work comprehensively discusses omeprazole (OMZ) and its S-active isomer (esomeprazole, ESZ) different methods of analysis indexed in Web of science, Scopus and Pub-med from 2016 till now. Chromatographic methods with different detectors each to fulfill the aim of the analysis were discussed. These chromatographic methods aimed to analyze OMZ and ESZ in biological fluids in presence of other drugs and metabolites for studying drug kinetics or drug-drug interaction and enzyme polymorphism. Moreover, in-vitro chromatographic methods were discussed for analyzing OMZ and ESZ in pharmaceuticals alone or in presence of other drugs. In addition, different chiral chromatographic methods separating the two enantiomers of OMZ (R-OMZ and ESZ) alone or with other chiral drugs or chiral impurities have been thoroughly discussed where no previous reviews have dealt with the chiral separation methods for OMZ and ESZ. Also, environmental analysis of OMZ and ESZ in various environmental samples was found as they are from the most popular drugs used and there is a high incidence that they may be present in wastewater. Moreover, reported spectroscopic and electrochemical methods for OMZ and ESZ analysis were discussed showing the structural features of OMZ/ESZ that lead to their successful analysis using spectroscopy and electrochemistry. Finally, the important figures of merit of all the discussed articles are shown in comprehensive tables and the article comprises 4 sections (chromatographic, electrochemical, spectroscopic and miscellaneous methods) and 7 tables.

Two binary mixtures containing codeine (COD) with either ibuprofen (IBU), mixture 1, or with phenylephrine hydrochloride (PE), mixture 2, were analyzed using three simple eco-friendly spectrofluorimetric methods without the need to a prior separation step. The first method is derivative emission spectrofluorimetry using λex = 236 nm and 275 nm for mixtures 1 and 2, respectively. The second method is constant-wavelength synchronous spectrofluorimetry using ∆λ = 100 nm and 60 nm for mixtures 1 and 2, respectively. The last method is constant-energy synchronous spectrofluorimetry where a wave number interval of −7000 cm−1 was used for the analysis of the two binary mixtures. All measurements were performed in acetate buffer pH 5 and thus no toxic volatile solvents were used increasing method greenness. High sensitivity was attained for the three studied drugs where the lower limits of quantitation of COD, IBU and PE reached 0.064, 0.512 and 0.087 μg/mL, respectively. Analysis of the two binary mixtures in their tablet and liquid dosage forms was performed with good accuracy and precision using the developed methods. The results of the proposed and reported methods were statistically evaluated using one-way ANOVA test and no significant difference among them was obtained. In addition, all aspects of ICH guidelines on analytical method validation were conducted.

Objective and Significance: Methocarbamol (MET) and aspirin (ASP) are widely used as a muscle relaxant combination. The USP reports guaifenesin (GUA) and salicylic acid (SAL) as related substances and hydrolytic products of MET and ASP, respectively. This work aimed at developing and validating a simple and sensitive RP-HPLC method for the determination of both drugs as well as their related substances (at their pharmacopeial limits) in their bulk powders, laboratory prepared mixtures, and MET-ASP combined tablets. Methods and Results: Chromatographic separation was achieved in less than 9 min with the required resolution, peak symmetry, and accuracy on C

This study represents simple inexpensive chromatographic determination of ciprofloxacin (CIP) and tinidazole (TIN) simultaneously in human plasma using HPLC-DAD followed by a pharmacokinetic application. C18 column was used as stationary phase with isocratic elution of a mobile phase composed of acetic acid solution (2%) and acetonitrile (85: 15, v/v) and ornidazole as internal standard (IS) with UV detection at 318 nm. The two drugs and the IS were separated at 6.55, 7.91 and 11.07 min for CIP, TIN and IS, respectively, with good selectivity and sensitivity for their analysis in presence of plasma matrix components and the drugs' metabolites. Sample preparation involved only protein precipitation without any complicated extraction procedures decreasing analysis time. For method validation, FDA regulations for analysis in biological fluids were followed. Pharmacokinetic (PK) study on six healthy volunteers was conducted after single oral dose administration of 500 and 600 mg of CIP and TIN, respectively. Dugs' plasma levels were followed for 12 or 72 h post dosing for CIP and TIN, respectively, and different PK data for the two drugs were calculated and they were comparable to the reported values demonstrating successful future application of the presented method in PK, bioequivalence and bioavailability studies.

For several decades, DNA has been well-known as the cellular target for various cytotoxic anticancer drugs. An active research area between chemistry, medicine and molecular biology is studying mechanism of interaction of drug molecules with DNA. In this study, we introduce a novel inexpensive and simple fluorescent biosensor to directly quantitate the DNA damage induced by epirubicin, an anticancer agent, and investigate the underlying molecular mechanisms of damage. The proposed fluorescent biosensor is established using terbium chloride. In damaged DNA, there are ssDNA regions facing the damaged nucleobases. In the damaged DNA, the terbium biosensor coordinates directly with the unpaired nucleobases accompanied by enhancement of its fluorescence, generating a measureable signal proportionate to the amount of damage. Moreover, dithiothreitol is used in this study to asses any possible DNA damage mechanisms that may occur other than the DNA oxidative damage that is commonly induced by various anthracyclines. In this work, the kinetics of the damage induced in DNA by epirubicin in absence and presence of dithiothreitol were compared using the proposed Tb3+ fluorescence biosensor. The results were confirmed using MALDI-TOF mass spectrometric analysis. It revealed that in absence of dithiothreitol, epirubicin induces oxidative DNA damage due to the interaction with reactive oxygen species causing strand breaks where the mass spectra show a significant reduction of the molecular-ion-peak of DNA, and appearance of peaks of smaller m/z indicating the DNA strand-breaks formation. While, in presence of dithiothreitol, epirubicin covalently binds to DNA to form DNA adducts with m/z values larger than the molecular-ion-peak of DNA target. Furthermore, the potency of epirubicin was examined on calf-thymus natural DNA samples with the proposed fluorescence biosensor. Whilst the interaction mechanism of epirubicin with DNA in presence of dithiothreitol shows slower DNA damage kinetics, but it has more potent damaging effect on DNA than in the case when no dithiothreitol is added. This introduces a novel alternative anticancer therapy that requires further investigation. Moreover, the low cost of the proposed fluorescent biosensor allows it to be an automatic simple process for the investigation of any DNA-drug interactions without the need to couple it with any other analytical method.

A stereoselective high performance liquid chromatographic method with diode array detection (HPLC-DAD) was introduced for S-naproxen and esomeprazole determination in tablets. The separation was achieved on a Kromasil Cellucoat chiral column using a mobile phase consisting of hexane: isopropanol: trifluoroacetic acid (TFA) (90:9.9:0.1 v/v/v). The proposed system was found to be suitable for the enantioseparation of naproxen and omeprazole biologically active isomers. After optimization of the chromatographic conditions, resolution values of 3.84 and 2.17 could be obtained for naproxen and omeprazole isomers, respectively. The method was fully validated for the determination of S-isomers of each drug in their dosage form. Also, the enentiomeric purity was determined in commercial tablet containing S-naproxen and esomeprazole. The enantiomeric purity was calculated for each drug and the chiral impurities (R-isomers) could be determined at 1% level. The method was validated and good results with respect to linearity, precision, accuracy, selectivity and robustness were obtained. The limits of detection (LOD) and quantification (LOQ) were 2.00, 6.50 and 0.10, 0.35μgmL-1 for S-naproxen and esomeprazole, respectively.

An accurate, precise, rapid, specific and economic high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitative determination of febuxostat (FEB) and diclofenac potassium (DIC). The chromatographic separation was performed on precoated silica gel 60 GF254 plates with chloroform-methanol 7:3 (v/v) as the mobile phase. The developed plates were scanned and quantified at 289 nm. Experimental conditions including band size, mobile phase composition and chamber-saturation time were critically studied, and the optimum conditions were selected. A satisfactory resolution (Rs = 2.67) with RF 0.48 and 0.69 and high sensitivity with limits of detection of 4 and 7 ng/band for FEB and DIC, respectively, were obtained. In addition, derivative ratio and ratio difference spectrophotometric methods were established for the analysis of such a mixture. All methods were validated as per the ICH guidelines. In the HPTLC method, the calibration plots were linear between 0.01-0.55 and 0.02-0.60 µg/band, for FEB and DIC, respectively. For the spectrophotometric methods, the calibration graphs were linear between 2-14 and 4-18 µg/mL for FEB and DIC, respectively. The simplicity and specificity of the proposed methods suggest their application in quality control analysis of FEB and DIC in their raw materials and tablets. A comparison of the proposed methods with the existing methods is presented.

This work aims to develop HPLC-DAD method for linagliptin (LNG) quantitation in the presence of its degradation products in tablets and to study its degradation kinetics. LNG samples were extracted from tablets and diluted. Various ICH degradation conditions were applied to study LNG degradation kinetics. LNG was assayed by a C18 column using a simple isocratic mobile phase (methanol:water containing 0.3% TEA, 40:60, pH 4.5) pumped at 1 mL/min. The drug was detected at 225 nm. The method showed high linearity (r2 > 0.999) with CV% and % error of the mean

A simple stability-indicating high-performance liquid chromatography-diode array detection (HPLC-DAD) method has been developed for the simultaneous determination of triamterene (TRI) and xipamide (XIP) in presence of the degradation products generated in studies of forced decomposition. Drugs were subjected to stress by hydrolysis (acidic, alkaline, and neutral), oxidation, photolysis (254 and 365 nm), and dry and wet heat treatments. Degradation occurs under acidic and alkaline conditions (TRI only), oxidative stress (TRI and XIP), and by photolysis (XIP only), but both drugs were stable under other stress conditions investigated. Separation of the two drugs from all the degradant peaks was achieved within 11 min using C8 column (250 × 4.6 mm, 5 μm) and mobile phase consisting of acetonitrile and 0.05 M phosphate buffer adjusted to pH 4 delivered at a flow rate of 1 mL min−1 using gradient elution system. The drugs were quantified at 220 nm using photodiode array detector, based on peak area. Peak homog...

A binary mixture of Lesinurad (LES) and Allopurinol (ALP) is a newly marketed anti-gout combination where no stability indicating methods have been reported for its analysis. In this work, a simple, green stability indicating capillary zone electrophoretic method coupled with a diode array detector (CZE-DAD) has developed and validated for analyzing this mixture in their combined dosage form. Separation was achieved using fused silica capillary (55 cm × 50 μm id) using 50 mM borate buffer adjusted to pH 10 with 0.5 M NaOH. The linearity concentration ranges were 5–50 and 10–100 μg/mL for LES and ALP, respectively. The stability indicating ability of the proposed method was investigated by analyzing the two drugs after being subjected to several stress conditions as per ICH guidelines. The developed method was able to separate the two intact drugs from their degradation products formed under different stress conditions (acidic, basic, oxidation). In addition, the developed method has been applied to investigate the degradation kinetics of LES and ALP under acidic, basic and oxidative conditions. The degradation of the two drugs was found to follow pseudo-first order kinetics and degradation half-lives were also calculated for both drugs under different stress conditions. The developed CZE method is the first in the literature for determination of both LES and ALP in the presence of their related degradation products with high separation efficiency, minimum solvent consumption, enhanced method greenness and low operation cost. Thus, the proposed method is an efficient simple eco- friendly analytical tool for the routine analysis of LES and ALP in their combined dosage form in presence of their degradation products in quality control laboratories.

Stereospecific separation method of (±) betaxolol, (±) carvedilol, and (±) sotalol using High Performance Thin Layer Chromatography (HPTLC) and β-cyclodextrin as chiral selector has been developed and validated. The Box-Behnken surface response design was selected for optimizing the operating variables based on 15 trials design. The optimized method involves separation on Fluka HPTLC silica gel plates 60 F254 (20 × 10 cm) using acetonitrile-methanol-acetic acid-water (3.4:3.6:0.18:1 v/v) as a mobile phase containing 0.57 mM β-cyclodextrin. Densitometric measurements were made at 220 nm for betaxolol and sotalol or at 245 nm for carvedilol. Maximum separation of the enantiomers of the three drugs was obtained by optimizing concentration of chiral selector, the mobile phase composition including acetonitrile amount in the organic part of the mobile phase and the volume of acetic acid added. The proposed method enables estimation of (-) and (+) enantiomers of betaxolol in drug substance and in various pharmaceuticals. The detection limit of betaxolol was 0.15 and 0.13 μg band-1 for (-) and (+) enantiomers, respectively. The detection limits were found to be 0.2 and 0.3 μg band-1 for carvedilol and sotalol, respectively, as racemate. In addition, the proposed method was applied in checking the enantiomeric purity of (-) BET in the presence of (+) BET at 1% level where the inactive (+) enantiomer was quantified with good accuracy and precision at 1% level in the active (-) enantiomer.

The first spectrofluorimetric report investigating the fluorimetric behavior of the antihistaminic drug, azelastine (AZEL), and the non-steroidal anti-inflammatory drug, nepafenac (NEP), either in bulk or in their dosage forms, eye drops and ophthalmic suspension. After a full investigation of the factors that may influence their spectrofluorimetric behavior: pH, different organized media and organic solvents, the optimum factors were set in order to enable the analysis of each drug with maximum sensitivity. The AZEL spectrofluorimetric analysis was set at 286/364 (λex/λem) in distilled water while for NEP, the analysis was set at 228/303 (λex/λem) in methanol. The linearity range for AZEL was from 0.1 to 1.5 μg/mL while that of NEP was from 0.2 to 1.5 μg/mL. The linearity yielded good regression parameters with low LOD (0.022 and 0.032 μg/mL for AZEL and NEP, respectively) and LOQ (0.073 and 1.08 μg/mL for AZEL and NEP, respectively) when compared with those obtained from many previous spectroscopic and chromatographic reports in literature. The method was ICH validated and was applied to the analysis of AZEL and NEP with good selectivity regarding the inactive ingredients.

High performance liquid chromatographic (HPLC) method with a pre-column derivatization based on Hantzsch condensation reaction was applied for simultaneous determination of alendronate sodium (ALN) and its main related impurity, 4-Aminobutanoic acid (ABA) at its pharmacopeial limit. The separation of colored condensation products of ALN and ABA were achieved on Agilent Zobrax Eclipse SB-C18 analytical column (250 × 4.6 mm, 5 μm) using a mobile phase composed of acetonitrile–0.1 M acetate buffer, pH 5.0 (15:85, v/v). The flow rate was 1 mL min−1. The detection was carried out at 340 nm using photo-diode array detector. Peak areas were used for the linear regression line in the range of 10–500 and 0.2–40 μg mL−1 for ALN and ABA, respectively. Different conditions for the optimization of the derivatization reactions as well as for the HPLC measurement were studied. The proposed method was validated for linearity, precision, accuracy, specificity and robustness. This method was used to check the purity of ALN in the presence of ABA (related impurity) at the pharmacopeial limit (0.5%). For comparison purpose, another method was proposed which involves synchronous fluorescence measurement after ALN reaction with fluorescamine. In this method, the third derivative synchronous spectra were estimated as peak to peak measurement from 339 to 370 nm for ALN determination with LOD and LOQ of 24 and 73 ng mL−1, respectively, showing very high sensitivity. Both methods have been applied for determination of the alendronate sodium (ALN) in bulk and pharmaceutical preparations without interference of additives in tablets or oral solution. Keywords: Pre-column derivatization, Derivative synchronous fluorometry, Alendronate sodium, 4-Aminobutanoic acid, Pharmaceutical preparations

Methotrexate (MTX) is widely used to treat rheumatoid arthritis (RA), mostly along with non-steroidal anti-inflammatory drugs (NSAIDs), the most common of which is aspirin or acetyl salicylic acid (ASA). Since NSAIDs impair MTX clearance and increase its toxicity, it was necessary to develop a simple and reliable method for the monitoring of MTX levels in urine samples, when coadministered with ASA. The method was based on the spectrofluorimetric measurement of the acid-induced hydrolysis product of MTX, 4-amino-4-deoxy-10-methylpteroic acid (AMP), along with the strongly fluorescent salicylic acid (SA), a product of acid-induced hydrolysis of aspirin and its metabolites in urine. The overlapping emission spectra were resolved using the derivative method (D method). In addition, the corresponding derivative emission spectra were convoluted using discrete Fourier functions, 8-points sin x i polynomials, (D/FF method) for better elimination of interferences. Validation of the developed methods was carried out according to the ICH guidelines. Moreover, the data obtained using derivative and convoluted derivative spectra were treated using the non-parametric Theil's method (NP), compared with the least-squares parametric regression method (LSP). The results treated with Theil's method were more accurate and precise compared with LSP since the former is less affected by the outliers. This work offers the potential of both derivative and convolution using discrete Fourier functions in addition to the effectiveness of using the NP regression analysis of data. The high sensitivity obtained by the proposed methods was promising for measuring low concentration levels of the two drugs in urine samples. These methods were efficiently used to measure the drugs in human urine samples following their co-administration.

A simple validated high-performance thin-layer chromatographic (HPTLC) method was developed for the determination of two binary mixtures containing omeprazole with ketoprofen (mixture 1) and esomeprazole with naproxen (mixture 2). The separation was carried out on Merck HPTLC aluminum plates precoated with silica gel 60 F254 (20 × 10 cm) with 250-μm thickness using chloroform-ethyl acetate (3:7, v/v) as the mobile phase. HPTLC separation of the four drugs in the two binary mixtures, followed by densitometric measurement, was carried out in the absorbance mode at 302 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r > 0.999 in the concentration ranges of 0.03–0.3 μg band−1 for omeprazole (OME) and esomeprazole (ESO), 0.2–2 μg band−1 for ketoprofen (KETO), and 0.5–6 μg band−1 for naproxen (NAP). The proposed method was validated and successfully applied for the analysis of pharmaceutical formulations and synthetic mixtures containing the two combination...

Two simple, sensitive and specific high-performance thin-layer chromatographic (HPTLC) methods were developed for the determination of febuxostat (FEB) individually, and simultaneously with diclofenac (DIC) in human plasma. Method A presents the first HPTLC-ultraviolet attempt for FEB determination in human plasma. FEB was separated from endogenous plasma components (at hRF = 70) with ethyl acetate-methanol-water (9:2:1, v/v) mixture as mobile phase and quantified by densitometry at its λmax (315 nm). Method B is considered the first attempt for the simultaneous determination of FEB and DIC in human plasma. A mixture of petroleum ether-chloroform-ethyl acetate-formic acid (7.5:1:2.5:0.25, v/v) was used as the mobile phase. The two drugs were separated at hRF of 39 and 60 for FEB and DIC, respectively. FEB and DIC were quantified by densitometry at their isoabsorptive point (289 nm). FEB calibration plots were linear between 0.1 and 7 μg mL−1 in both methods A and B. In method B, DIC showed linear response in the range of 0.08–8 μg mL−1. Sample preparation was performed by liquid-liquid extraction using diethyl ether. Both methods did not record any interference from plasma matrix, the studied drugs' metabolites or their decomposition products. They were successfully applied for the determination of the studied drugs in healthy male volunteers after oral administration of FEB or FEB/DIC dosage forms. FEB plasma concentration increased significantly when given with DIC. The proposed methods provided very simple, rapid and cheap approaches that might be attractive for the future pharmacokinetic and bioavailability studies of FEB and/or DIC.

Two novel combination therapies for the treatment of benign prostatic hyperplasia were analyzed using simple and enhanced spectrofluorimetric methods based on derivative and derivative ratio techniques. The two combinations contained tamsulosin hydrochloride (TAM) as a minor component with tolterodine tartrate (TOL) or solifenacin succinate (SOL). The fluorescence of the three drugs under study was measured in methanolic water solution. For the TAM and SOL mixture, successful resolution between both drugs was achieved by derivative manipulation of both ratio and zero-order emission spectra with good linearity in the ranges of 0.75-3.50 and 2.5-15.0 μg ml-1 for TAM and SOL, respectively. Extensive emission spectral overlap was observed for the TAM and TOL mixture. Therefore, only derivative application of the ratio emission spectra resolved such overlap and quantitated TAM and TOL simultaneously in the ranges 0.75-3.50 and 2.5-20.0 μg ml-1 for TAM and TOL, respectively. Optimization of various experimental parameters that affected the fluorescence intensity of the three drugs was performed. Successful application of all proposed methods was achieved for analysis of the two drugs in each combination therapy in their laboratory-prepared mixtures and dosage forms with good accuracy and precision.

A new combination of ibuprofen (NSAID) and famotidine (H2 receptor antagonist) was recently approved by the FDA. It was formulated to relief pain while decreasing the risk of ulceration, which is a common problem for patients receiving NSAID. A rapid and simple derivative emission spectrofluorimetric method is proposed for the simultaneous analysis of this combination in their pharmaceutical preparation. The method is based upon measurement of the native fluorescence intensity of the two drugs at λex = 233 nm in acetonitrile. The emission data were differentiated using the first (D1) derivative technique. The plots of derivative fluorescence intensity versus concentration were rectilinear over a range of 2-35 and 0.4-8 µg/mL for both ibuprofen (IBU) and famotidine (FAM), respectively. The method was sensitive as the limits of detection were 0.51 and 0.12 µg/mL and limits of quantitation were 1.70 and 0.39 µg/mL, for IBU and FAM respectively. The proposed derivative emission spectrofluorimetric method was successfully applied for the determination of the two drugs in their synthetic mixtures and tablets with good accuracy and precision. The proposed method was validated as per ICH guidelines.

A spectrofluorimetric method was used for the estimation of closely related fluorescent reaction products, Fluoxetine and Olanzapine, in their mixture after derivatization of both drugs using 4-chloro-7- nitrobenzo - 2 -oxa-1,3 - diazole (NBD-Cl) in borate buffered medium (pH 9.5) to form highly fluorescent products. The method based on the use of first and second derivative ratio of the emission data along with their convolution using 8-points sin x i or cos x i polynomials (discrete Fourier functions). The proposed method facilitates their simultaneous determination despite the presence of a minor component (Olanzapine) and strong overlapped spectra of the two NBD-Cl fluorescent products of fluoxetine and olanzapine. The accurate and precise estimation of the minor component was achieved after the convolution of the derivative ratio curves. Moreover, the obtained data were subjected to non-parametric linear regression analysis (Theil’s method). The work combines the advantages of convolution of derivative ratio curves using discrete Fourier functions together with the reliability and efficacy of the non-parametric analysis of data.

A sensitive high-performance thin-layer chromatographic (HPTLC) method was developed for the estimation of metformin in human urine. The drug was extracted from urine using ion-pair solid-phase extraction (IPSPE). Separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using a mixture of chloroform-methanol-ammonia 27% w/v (5:5:0.2, v/v) as a mobile phase. This system was found to give dense and compact spots for metformin (retardation factor, RF, 0.25) following densitometric measurements of the spots at 237 nm. The proposed method was applied to generate urinary excretion data for metformin after administration of two metformin formulations (500 mg extended release, XR, Formulation R, and microbeads controlled release, Formulation T) to six healthy human volunteers in a two-treatment, crossover design. Various pharmacokinetic parameters like peak excretion rate ((dAU/dt)max), cumulative amount (Ae0–24) of metformin excreted, elimination half-life (t1/2), and terminal elimination ...

HPLC-DAD method based on solid-phase extraction was developed for residual determination of metronidazole (MET) and its metabolite hydroxymetronidazole (MET-OH) in two strains of hen eggs (Alexandria egg line, AE and Fayoumi egg line, PP). The LC separation was performed on a RP C-18 analytical column (150 mm × 4.6 mm, 5 µm) with an isocratic elution system of 0.0 5 M phosphate buffer of pH 2.5–acetonitrile (95:5) as the mobile phase. The procedure was evaluated and fully validated. The calibration graphs for MET and MET-OH were rectilinear in the range of 0.3–16.0 µg g−1 for MET and 0.2–20.0 µg g−1 for MET-OH. No residues could be detected before the 3rd day or after 11th day after the drug administration. The drug and metabolite concentration levels in eggs for the two strains of hens were different owing to their change in genetic potential. PP has less ability to keep MET and MET-OH residues than AE.

Two stability-indicating chromatographic methods are described for simultaneous determination of amiloride hydrochloride (AMI), atenolol (ATE), and chlorthalidone (CHL) in combined dosage forms. The first method was based on HPTLC separation of the three drugs followed by densitometric measurements of their bands at 274 nm. The separation was carried out on Merck HPTLC silica gel 60F254 aluminum sheets using chloroform–methanol–ammonia 27%, w/w (9 + 2 + 0.3, v/v/v) mobile phase. Analysis data was used for the linear regression graph in the range of 0.1–0.5, 0.8–5.0, and 0.3–1.5 μg/band for AMI, ATE, and CHL, respectively. The second method was based on an RP-HPLC separation of the cited drugs performed on an RP stainless steel C18 analytical column (250 × 4.6 mm id) with a gradient elution system of methanol and 0.05 M aqueous phosphate buffer adjusted to pH 4 as the mobile phase, at the flow rate of 1.0 mL/min. Quantitation was achieved with photodiode array detection at 275 nm for AMI and 225 nm for ATE and CHL. The calibration graphs for each drug were rectilinear in the range of 2–50, 25–150, and 2–100 μg/mL for AMI, ATE, and CHL, respectively. The proposed chromatographic methods were successfully applied for determination of the investigated drugs in pharmaceutical preparations. Both methods were validated in compliance with International Conference on Harmonization guidelines in terms of linearity, accuracy, precision, robustness, LOD, and LOQ.

A validated and selective high-performance thin-layer chromatography (HPTLC) method was developed for the analysis of mixures of tamsulosin hydrochloride (TAM) with either tolterodine tartrate (TOL) or solifenacin succinate (SOL) in bulk drug and in combined dosage forms. The proposed method is based on HPTLC separation of the three drugs followed by densitometric measurements of their spots at 224 nm. Separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using ethyl acetate-methanol-ammonia (6:4:0.05, v/v) as mobile phase. The linear regression analysis data were used for the regression line in the range of 0.1-0.7, 0.4-4 and 1-6 μg band-1 for TAM, TOL and SOL, respectively. The proposed method was validated and successfully applied for the analysis of their pharmaceutical formulations and laboratory-prepared mixtures containing the two bicomponent combinations. The method was validated and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability.

Two spectrophotometric methods are presented for the simultaneous determination of ezetimibe/simvastatin and ezetimibe/atorvastatin binary mixtures in combined pharmaceutical dosage forms without prior separation. The first is the derivative ratio method where the amplitudes of the first derivative of the ratio spectra (1DD) at 299.5 and 242.5 nm were found to be linear with ezetimibe and simvastatin concentrations in the ranges 0.5–20 µgml−1 and 1–40 µgml−1, respectively, whereas the amplitudes of the first derivative of the ratio spectra (1DD) at 289.5 and 288 nm were selected to determine ezetimibe and atorvastatin in the concentration ranges 5–50 µgml−1 and 1–40 µgml−1, respectively. The second is the H-point standard additions method; absorbances at the two pairs of wavelengths, 228 and 242 nm or 238 and 248 nm, were monitored while adding standard solutions of ezetimibe or simvastatin, respectively. For the analysis of ezetimibe/atorvastatin mixture, absorbance values at 226 and 248 nm or 212 and 272 nm were monitored while adding standard solutions of ezetimibe or atorvastatin, respectively. Moreover, differential spectrophotometry was applied for the determination of ezetimibe in the two mixtures without any interference from the co-existing drug. This was performed by measurement of the difference absorptivities (ΔA) of ezetimibe in 0.07 M 30% methanolic NaOH relative to that of an equimolar solution in 0.07 M 30% methanolic HCl at 246 nm. The described methods are simple, rapid, precise and accurate for the determination of these combinations in synthetic mixtures and dosage forms. Copyright © 2010 John Wiley & Sons, Ltd.

Differential pulse voltammetric method was developed for determination of Silymarin (SMR)/Vitamin E acetate (VEA) mixture in pharmaceuticals. SMR and VE gave well-resolved diffusion-controlled anodic peaks at +756 and +444 mV, respectively (versus Ag/AgCl) in Britton–Robinson buffer at pH 2.8. The solution conditions and instrumental parameters were optimized for their quantitative determination. The linear response was obtained in the range 0.1–4.0 mg L−1 with a detection limit of 0.03 mg L−1 for SMR and 0.05–4.0 mg L−1 with a detection limit of 0.01 mg L−1for VEA.

A new rapid and highly sensitive stability-indicating spectrofluorimetric method was developed for the determination of two stereoisomers anticancer drugs, doxorubicin (DOX) and epirubicin (EPI) in pure form and in pharmaceutical preparations. The fluorescence spectral behavior of DOX and EPI in a sodium dodecyl sulfate (SDS) micellar system was investigated. It was found that the fluorescence intensity of DOX and EPI in an aqueous solution of phosphate buffer pH4.0 and in the presence of SDS was greatly (about two fold) enhanced and the mechanism of fluorescence enhancement effect of SDS on DOX was also investigated. The fluorescence intensity of DOX or EPI was measured at 553nm after excitation at 497nm. The plots of fluorescence intensity versus concentration were rectilinear over a range of 0.03-2μg/mL for both DOX and EPI with good correlation coefficient (r>0.999). High sensitivity to DOX and EPI was attained using the proposed method with limits of detection of 10 and 9ng/mL and limits of quantitation of 29 and 28ng/mL, for DOX and EPI, respectively. The method was successfully applied for the determination of DOX and EPI in biological fluids and in their commercial pharmaceutical preparations and the results were concordant with those obtained using a previously reported method. The application of the proposed method was extended to stability studies of DOX following different forced degradation conditions (acidic, alkaline, oxidative and photolytic) according to ICH guidelines. Moreover, the kinetics of the alkaline and oxidative degradation of DOX was investigated and the apparent first-order rate constants and half-life times were calculated.

A single, simple, selective and validated high performance thin layer chromatographic (HPTLC) method was developed for the determination of either linagliptin (LGP), saxagliptin (SGP) or vildagliptin (VGP) in their binary mixtures with metformin (MET) in pharmaceutical preparations using environmentally preferable green mobile phase system. Separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using methanol-0.5% w/v aqueous ammonium sulfate (8 : 2, v/v) as mobile phase. Densitometric measurement of the spots was performed at 225 nm for LGP/MET mixture and at 208 nm for both SGP/MET and VGP/MET mixtures. The linear regression analysis data were used for the regression line in the range of 0.05-0.5 µg/band for LGP and SGP and 0.2-2 and 5-40 µg/band for VGP and MET, respectively. The method was validated and showed good performances in terms of linearity, limits of detection and quantitation, precision, accuracy, selectivity and specificity. The calculated percentage relative error values and percentage relative standard deviation for intra- and interday precision studies did not exceed 2%. The developed method was satisfactorily applied for the analysis of pharmaceutical preparations and proved to be specific and accurate for the quality control of the cited drugs in their dosage forms.

An efficient chromatographic method for the simultaneous determination of triamterene (TRI) and xipamide (XIP) in urine samples, based on high performance liquid chromatography with photodiode array detector (HPLC-DAD) has been developed. The HPLC separation was performed on a RP stainless-steel C-18 analytical column (250 mm × 4.6 mm, 5 μm) with a gradient elution system of 0.05 M phosphate buffer adjusted to pH 4.0 and methanol as the mobile phase. The method was used to determine the urinary excretion profile and to calculate different urinary pharmacokinetic parameters following oral dose of their combination compared with single oral doses of each drug and hence comparing their bioavailability. Quantitation was performed using chlorthalidone as internal standard. The calibration graphs of each drug were rectilinear in the range of 0.2-40 μg/mL urine for TRI and 0.2-15 μg/mL urine for XIP. An HPLC-DAD method was also successfully developed for the simultaneous determination of the investigated drugs in pharmaceutical preparations. The methods were validated in terms of linearity, accuracy, precision, selectivity, limits of detection and quantitation and other aspects of analytical validation.