Smart Multivariate Spectrophotometric Determination of Two Co-Administered Autoimmune Drugs; Sulfasalazine and Pentoxifylline; in Bulk and Spiked Human Plasma.

BACKGROUND: Sulfasalazine and pentoxifylline are co-prescribed together to treat psoriasis and pemphigus vulgaris. Sulfasalazine is an anti-inflammatory, immunosuppressant, and antibiotic drug, while pentoxifylline is a vasodilator and immunosuppressant. The spectra of the two drugs and plasma suffer from severe overlap. OBJECTIVE: This work aims to simultaneously determine sulfasalazine and pentoxifylline in their binary mixture and spiked human plasma by the assessment of their UV spectral data. METHODS: Two model updated chemometric methods were established using principal component regression and partial least-squares regression models. The two models were validated in accordance with the U.S. Food and Drug Administration guidelines for bioanalysis and were applied for the determination of both drugs in synthetic mixtures or spiked human plasma. RESULTS: Accuracy and precision were within the accepted limits. In addition, three different assessment methods were used to evaluate the environmental greenness of the proposed models. CONCLUSION: The two updated models are simple, rapid, sensitive, and precise, and could be easily applied in QC laboratories for determination of sulfasalazine and pentoxifylline, without any preliminary separation steps or interference from plasma matrix. HIGHLIGHTS: Two updated chemometric models called principlal component regression and partial least-squares regression were established for determination of sulfasalazine and pentoxifylline in spiked human plasma using UV spectrophotometric data.

Development and validation of synchronous spectrofluorimetric method for the simultaneous determination of duvelisib and moxifloxacin: greenness metric assessment and application to a pharmacokinetic study in rats.

Duvelisib (DUV) is a potent anticancer drug whereas Moxifloxacin (MOX) is an antimicrobial drug with anti-proliferative potency against cancerous cells, which is empirically administered in cancer treatment. DUV and MOX combination is commonly prescribed to combat infections in patients while they are under chemotherapy treatment. This study describes, for the first time, the development of a simple and green synchronous spectrofluorimetric (SSF) method for the simultaneous estimation of DUV and MOX in plasma. DUV and MOX were quantified at 273 and 362 nm, respectively without interference between each other at Δλof 120 nm. The experimental variables influencing fluorescence intensities were thoroughly investigated and the optimum conditions were established. At pH 3.5, the optimum synchronous fluorescence intensity (SFI) was achieved in water solvent by using sodium acetate buffer solution. Calibration curves for DUV and MOX, correlating the SFI with the corresponding drug concentration, were linear in the range of 50-1000 ng mL-1for both drugs, with good correlation coefficients. The method was extremely sensitive, with limits of detection of 24 and 22 ng mL-1, and limits of quantitation of 40 and 45 ngmL-1for DUV and MOX, respectively. The SSF method was validated according to the Food and Drug Administration (FDA) guidelines for validation of analytical procedures, and the validation parameters were acceptable. The proposed SSF method was applied to the pharmacokinetic and bioavailability studies in rats' plasma after single concurrent oral administration of both drugs. The results of the study revealed that caution should be taken with DUV dose when concurrently administered with MOX. The greenness of SSF method was assessed by three different metric tools namely Analytical Eco-scale, Green Analytical Procedure Index, and Analytical Greenness Calculator. The results confirmed that SSF method is an eco-friendly and green analytical approach. In conclusion, the proposed SSF method is a valuable tool for pharmacokinetic/bioavailability studies and therapeutic drug monitoring of simultaneously administered DUV and MOX.

Development of HPLC-UV Method for Simultaneous Determination of Corticosteroid Co-Administered Immune Therapy.

Prednisolone (PDS) has recently been utilized to treat a variety of medical disorders, including autoimmune illnesses and cancer. It is also used to treat coronavirus disease 2019 infection-related respiratory problems. Because it may induce health problems including gastrointestinal lesions and ulceration, it has to be used alongside other drugs like esomeprazole (ESM), which acts as a proton pump antagonist to reduce the probability of ulceration. As a result, the goal of this research is to create an environmentally safe and sensitive high-performance liquid chromatography (HPLC) approach for determining PDS and ESM in their binary combination and spiked human plasma. C8 column (100 × 4.6 mm, 5 µm) and gradient mobile phase elution were used to separate the studied drugs with ultraviolet recognition at 290 nm. Caffeine was utilized as an internal standard to adjust the sample variance. Plasma, caffeine, ESM and PDS all had tR values of 1.4, 3.5, 6.3 and 7.3, respectively. The suggested method's greenness features were evaluated using three greenness evaluation tools: green analytical procedure index, analytical greenness metric approach and analytical eco-scale, and the findings were approved and satisfied. Validation parameters were evaluated in accordance with US-FDA recommendations in order to meet the global desires for biological analysis technique, acceptable limits were obtained.

Chromatographic analysis of ciprofloxacin and metronidazole in real human plasma: green analytical chemistry perspective.

Background: Ciprofloxacin and metronidazole are beneficial for treating mixed aerobic/anaerobic infections. Methods: Following the oral administration of ciprofloxacin and metronidazole in healthy volunteers, TLC and HPLC methods were described for their analysis in plasma samples. In the first method, a stationary phase of silica gel TLC F254 plates was used using acetone/water/triethylamine/glacial acetic acid (8:2:0.25:0.1 v/v). The second approach used a C18 column and methanol/aqueous 0.05% triethylamine (25:75 v/v), with a flow rate of 1 ml/min and detection at 325 nm. Four green metrics were used to evaluate the approaches' environmental impact. Conclusion: The study provided the sensitivity required for determination of the two drugs in the collected samples. The findings showed that results were within permitted ranges with minimal environmental impact.

A validated ecofriendly chromatographic method for determination of myasthenia gravis combined medications in spiked human plasma.

Recently, the main interest of analytical chemistry researchers has been the development of green analytical methods to minimize harmful effects on the environment and natural life. Therefore, an RP-HPLC method was developed and assessed regarding its greenness criteria using three greenness assessment tools: an analytical eco-scale, an analytical greenness metric approach and a green analytical procedure index. This method aims to separate and quantitatively determine three co-administered drugs, namely pyridostigmine bromide (PYR), 6-mercaptopurine (MRC) and prednisolone (PRD), in their tertiary mixture and spiked human plasma. These drugs are co-administered to manage myasthenia gravis autoimmune disease. The separation was done using a C18 column and a gradient elution of a mixture of 0.1% H3 PO4 aqueous solution (pH 2.3) and methanol. The flow rate was adjusted to 1 ml/min and detection was done at 254 (for PYR and PRD) and at 330 nm (for MRC). The lower limits of quantitation were 15, 2, and 5 µg/ml for PYR, MER and PRD, respectively. Linear correlations were obtained and found to be near 1. In addition, the proposed method was validated according to the US Food and Drug Administration's instructions, and the results proved its success to determine the three studied drugs in their tertiary mixture and spiked human plasma.

Tailoring Two Chemometric Models for Determination of Three Neuromuscular Combined Medications and Application to Spiked Human Plasma.

BACKGROUND: Prednisolone, 6-mercaptopurine, and pyridostigmine bromide are co-administered together to treat a neuromuscular autoimmune disease called myasthenia gravis. Prednisolone and 6-mercaptopurine are immunosuppressant drugs. 6-Mercaptopurine is the active form of the pro-drug azathioprine. Pyridostigmine bromide is a cholinesterase enzyme inhibitor. Curently, green characteristics are taken into account by analysts when they develop new methods. OBJECTIVE: The spectra of the three compounds and plasma are highly overlapped, so this study aims to remove this overlap and determine the three components quantitatively in raw powders and spiked human plasma using green methods. METHODS: Two multivariate updated chemometric models called principle component analysis and partial least-squares were developed. Three greenness assessment tools: the eco-scale, the analytical greenness metric approach, and the green analytical method index, were used to evaluate the greenness behavior of the generated models. RESULTS: The two models were verified in accordance with Food and Drug Administration requirements, and the results were within acceptable limits. In addition they are relatively green in accordance with the abovementioned greenness evaluation tools. CONCLUSION: The developed models succeeded in determining the proposed drugs in their tertiary combinations and spiked human plasma with satisfactory precision, accuracy, and good greenness behavior. HIGHLIGHTS: Two ecologically evaluated, multivariate spectrophotometric methods were developed for the determination of pyridostigmine bromide, 6-mercaptopurine, and prednisolone in spiked human plasma.

Simultaneous spectrophotometric determination of fluphenazine HCl and nortriptyline HCl in presence of their potential impurities perphenazine and dibenzosuberone in bulk and pharmaceutical formulation.

Fluphenazine HCl (FLU) is an anxiolytic, while Nortriptyline HCl (NOR) is an anti-depressant. They are co-formulated together to treat depression and schizophrenia. Perphenazine (PER) and dibenzosuberone (DBZ) are the pharmacopeial impurities of FLU and NOR, respectively. Four spectrophotometric and multivariate chemometric methods were developed to determine the two drugs together or in presence of their two impurities in their bulk and pharmaceutical formulation. Method (A) is the triple divisor-ratio derivative (TDR) method, where the zero order spectrum of each component was divided by a mixture of the other 3 components, then the peak amplitudes of the first derivative spectra of FLU, NOR and DBZ were measured at 265, 245.4 and 283.2 nm, respectively. Method (B) is the double divisor-ratio difference-dual wavelength (DD-RD-DW) method, in which each component spectrum mixture was divided by a binary mixture of 2 of the interfering components. In the resulting ratio spectra, the amplitude difference is calculated between 2 wavelengths at which the third interfering component has zero difference. Methods (C and D) are the principle component analysis (PCA) and partial least squares (PLS) models. Methods (A and B) failed to quantify PER (FLU impurity), while (C and D) succeeded to quantify all components. The four methods have been applied for the prediction of the FLU and NOR in their pharmaceutical formulation with good accuracy and precision. The proposed methods have been validated according to the ICH guidelines and the results were within the acceptable limits.

US FDA-validated TLC method with four greenness assessment evaluations for simultaneous determination of prednisolone and esomeprazole in spiked human plasma.

Recently, prednisolone has been used in treating many medical conditions, such as autoimmune diseases and cancer. It is also prescribed to mitigate the respiratory complications caused by COVID-19 infection. It can cause some health complications, such as GIT ulcers, so it should be co-administered with proton-pump inhibitors, such as esomeprazole, to prevent the risk of ulcers. This work aims to develop an ecofriendly and sensitive TLC method for simultaneous determination of esomeprazole and prednisolone in their binary mixtures and spiked human plasma. Separation was performed using a mixture of ethyl acetate, methanol, and ammonia (9.5:0.5:0.1, v/v/v) as an eluting system with UV scanning at 245 nm. Dapoxetine was used as an internal standard to correct the variation during sampling. The resulting Rf values for plasma, esomeprazole, prednisolone, and dapoxetine were 0.03, 0.51, 0.72 and 0.85, respectively. Four greenness assessment tools-national environmental method index, eco-scale assessments, analytical greenness metric approach (AGREE), and green analytical procedure index (GAPI)-were used to evaluate the greenness characteristics of the proposed method to the environment, and the results were acceptable and satisfactory. Validation parameters were checked according to the US FDA guidelines to achieve the international requirements for bioanalytical method validation, and the results were within the accepted ranges.

Multivariate Model Update Chemometric Methods for Determination of Prednisolone and Esomeprazole in Spiked Human Plasma: A Comparative Study.

BACKGROUND: Prednisolone (PRD) is an immunosuppressant and anti-inflammatory drug, although it may cause peptic ulcers as a side effect. Esomeprazole (ESO) is used for the treatment of peptic ulcers, therefore the two drugs are co-administered in cases of organ transplantation and autoimmune diseases. OBJECTIVE: This work aims to determine the two drugs simultaneously, in bulk, and in spiked human plasma by eliminating the overlap of their spectra and the interference of the plasma matrix. METHODS: Two simple and effective updated chemometric models-principal component analysis (PCA) and partial least squares (PLS)-were established using UV spectrophotometric data. RESULTS: The two updated models have been validated according to the U.S. Food and Drug Administration guidelines with acceptable results. The results were statistically compared with those of the reported methods, where no significant difference was found, indicating the validity of the developed methods. The two updated models have been successfully applied for prediction of the proposed drugs with good accuracy and precision. CONCLUSION: The two updated models are simple, rapid, sensitive, and precise and could be easily applied in quality control laboratories for determination of PRD and ESO, without any preliminary separation steps or interference from plasma matrixes. HIGHLIGHTS: Two model updated chemometric models, PCA and PLS, were established for determination of PRD and ESO in spiked human plasma using UV spectrophotometric data.

Quantitative Determination of Anti-Migraine Quaternary Mixture in Presence of p-Aminophenol and 4-Chloroacetanilide.

A validated RP-HPLC method was developed for the estimation of paracetamol, caffeine, metoclopramide and ergotamine simultaneously in bulk and pharmaceutical formulation. The method was extended for the determination of two paracetamol genotoxic and nephrotoxic impurities and degradation products namely p-aminophenol and 4-chloroacetanilide. Separation was done on octadecyl column (15 cm × 2.1 mm, 5 µm). Gradient elution was performed using mobile phase consisting of acetonitrile and water (pH 3) in ratio of (20: 80, v/v) for the first 3 min, (50: 50, v/v) for the next 4.5 min, then (20: 80, v/v) for the final 2.5 min. The flow rate was 0.7 mL/min throughout the run which took 10 min. UV detection was done at 230 nm. The proposed method agreed with ICH guidelines of method validation. The suggested method was applied for the estimation of the proposed drugs in their dosage form. Statistical comparison was done between the results obtained from the suggested method and those obtained from reported HPLC method. The proposed method is more beneficial than the published one because of having higher sensitivity and selectivity as it allows the detection of the proposed drugs in lower concentrations in the presence of paracetamol toxic impurities, p-aminophenol and 4-chloroacetanilide.

Development and Validation of Four Spectrophotometric Methods for Assay of Rebamipide and its Impurity: Application to Tablet Dosage Form.

BACKGROUND: Rebamipide (REB) is quinolinone derivative compound, which is used for the treatment of stomach ulcers. OBJECTIVE: The development of four spectrophotometric methods for quantification of REB and its impurity and degradation product: the debenzoylated isomer of REB (DER). METHODS: Method A is ratio difference spectrophotometry where 254 and 291 nm were selected for REB and 320 and 355 nm were selected for DER, allowing spectral discrimination for both. Method B is derivative ratio spectrophotometry, where the peak amplitudes of the first derivative of ratio spectra at 261 and 350.2 nm for REB and DER, respectively, are determined. Method C is a second derivative approach, which allows quantification of both REB and DER at 337 and 340 nm, respectively. Method D is mean centering of ratio spectra, where electronic absorption spectra of REB and DER were recorded and divided by a suitable divisor from DER and REB, respectively, and then the mean center is represented by the ratio spectrum so obtained. RESULTS: The proposed methods are simple, selective, and sensitive in the quantification of REB and DER. These methods were validated according to International Conference on Harmonization guidelines. Statistical analyses performed on the findings from the suggested methods and those obtained from reported methods revealed high accuracy and good precision. CONCLUSION: The developed and validated methods are useful for quality control assay in routine analyses. HIGHLIGHTS: First derivative, second derivative, derivative ratio, and mean centering methods for quantification of REM and DER. These methods are useful for analysis of REB in pharmaceutical dosage form.

Lipophilicity study of different cephalosporins: Computational prediction of minimum inhibitory concentration using salting-out chromatography.

The chromatographic and lipophilicity characters of seven cephalosporins of different four classes (cephradine, cefaclor, cefprozil, cefixime, cefotaxime, ceftazidime and cefepime) were examined by salting out thin-layer chromatography (SOTLC). SOTLC using ammonium sulfate salt was employed to predict the lipophilicity of the proposed drugs via their retention behavior. The calculated RM0 values showed liner relationship with the molar concentration of ammonium sulfate in mobile phase in the range of 0.5-2.5 mol/L. Additionally, quantitative structure retention relationship (QSRR) was generated to figure out the relationship between the calculated chromatographic parameters (RM0 and C0) and log P of the studied cephalosporins. Good correlations were found between the chromatographically obtained retention parameters (RM0 and C0) and some molecular descriptors of the examined drugs. Furthermore, an efficient QSAR model was carried out using the calculated chromatographic parameters (RM0 and C0) and log P of the studied cephalosporins to predict minimum inhibitory concentration (MIC) and blood brain barrier (BBB) penetration of the examined drugs. The study was extended to separate and quantify the selected antibiotics in their pure forms and pharmaceutical formulations. Normal phase thin layer chromatographic (NP-TLC) method using a usable developing system of acetone: methanol: water: ammonium hydroxide: glacial acetic acid (90: 10: 18: 3: 2, by volume) was successfully applied to resolve the studied cephalosporins. Linearity was achieved in the range of 0.2-3 µg/mL for most of the studied antibiotics. The developed SOTLC method can be considered as a good start alternative to reversed phase thin layer chromatography (RP-TLC) for prediction of the lipophilic properties of examined cephalosporins. Moreover, the proposed NP-TLC densitometric method can be easily applied for quality control analysis of the chosen drugs and other structurally related components.

Separation and Determination of Diflunisal and its Impurity by Two Chromatographic Methods: TLC-Densitometry and HPLC.

BACKGROUND: Diflunisal (DIF) has analgesic and anti-inflammatory activity. It is a pharmacopeial drug found in the British Pharmacopoeia (BP), and its major pharmacopeial impurity is biphenyl-4-ol (BPL). OBJECTIVE: DIF has not previously been determined together with BPL. The presence of BPL could significantly affect the dose of DIF in its dosage forms; hence it is crucial to determine DIF and BPL in each other's presence. METHODS: TLC is the first proposed method, where DIF and BPL were separated on silica gel TLC F254 plates. The eluent was toluene-acetone-acetic acid solution (3.5:6.5:1, v/v). Reversed-phase (RP) HPLC is the second suggested method, where a mixture of DIF and BPL was separated on a C18 (5 µm ps, 250 mm and 4.6 id) column using phosphate buffer pH 4 (0.05 M)-acetonitrile (40:60, v/v). Detection was carried out at 254 nm in both methods. RESULTS: For the TLC method, concentration ranges of 0.5-3 and 0.3-1.7 µg/band were used, with mean percentage recoveries of 100.22% (SD 0.893) and 100.52% (SD 0.952) for DIF and BPL, respectively. The RP-HPLC method was carried out over a concentration range of 5-30 and 2-9 µg/mL, with mean percentage recoveries of 100.10% (SD 1.259) and 98.88% (SD 0.822) for DIF and BPL, respectively. CONCLUSION: The TLC and RP-HPLC methods were successfully applied for the determination of DIF and BPL, quantitatively, whether in bulk powder or in pharmaceutical formulations. HIGHLIGHTS: Two chromatographic methods were developed and validated according to International Council on Harmonization guidelines for the assay of DIF and its pharmacopeial impurity.

Different chromatographic methods for determination of alogliptin benzoate, metformin hydrochloride, and metformin impurity in bulk and pharmaceutical dosage form.

Two simple, sensitive, and reproducible methods were developed for the determination of alogliptin and metformin hydrochloride in presence of metformin impurity "melamin" in pure form and in pharmaceutical formulation. Method (A) was a thin layer chromatographic method in which separation was achieved using ethyl acetate-methanol-formic acid (6:3.8:0.2, by volume) as a developing system followed by densitometric scanning at 230 nm. Method (B) was a high-performance liquid chromatography method; separation was achieved on C18 column, the mobile phase consisted of a mixture of sodium lauryl sulfate buffer 0.1% w/v, pH 3: methanol in the ratio 70:30, v/v and measurement was done at 220 nm. System suitability testing parameters were calculated to ascertain the quality performance of the developed chromatographic methods. The proposed methods have been validated regarding accuracy, precision, and selectivity, moreover they have been successfully applied to Westirizide tablets containing both alogliptin and metformin hydrochloride, results indicate that there was no interference from additives. No significance difference was found when these methods were compared to the reported one.

Validated ecofriendly chromatographic method for quantitative determination of anti-migraine quaternary mixture.

A novel ecofriendly, cost and time saving high-performance thin-layer chromatographic method was developed and validated for simultaneous determination of metoclopramide, ergotamine, caffeine, and paracetamol in bulk and pharmaceutical formulation. The separation was carried out on silica gel plates, using ethyl acetate:ethanol:ammonia (9:1:0.1, v/v/v) as a developing system. Ultraviolet detection was carried out at 272 nm. The resulting retention times were 0.15, 0.36, 0.49, and 0.74 min for metoclopramide, ergotamine, caffeine, and paracetamol, respectively. The greenness profile assessment was achieved to the proposed method to evaluate its greenness characters to the environment with acceptable results. Validation parameters were checked according to International Conference of Harmonization guidelines to achieve the international requirements for quality control analysis of the proposed drugs.

Baclofen impurities: Facile synthesis and novel environmentally benign chromatographic method for their simultaneous determination in baclofen.

An efficient, economic and high yielding method was described for the synthesis of baclofen (BAC) pharmacopoeial impurities (impurity A and impurity B) which can be used for gram-scale synthesis. Furthermore, a novel ecofriendly thin-layer chromatographic TLC-densitometric method was established and validated for the determination of BAC and its synthesized impurities. The developed TLC-densitometric method is based on the chromatographic separation using TLC plates (60 F254 ) using a green mobile phase of ethyl acetate-methanol-ammonia solution, 33% (8:2:0.1, by volume) with UV scanning at 220 nm. The proposed method was validated with respect to International Conference on Harmonization guidelines. The validated method was successfully applied for determination of BAC in pure form and in its commercial dosage form. Additionally, the greenness profile of the developed method was evaluated and compared with those of the reported chromatographic methods. The developed method was found to be superior to the published methods, being environmentally benign.

New ecological method for determination of different ß-lactams: application to real human plasma samples.

Recently, the use of antibiotics has become widespread all over the world resulting in bacterial resistance to these antibiotics, which requires alternative medications or higher doses of antibiotics. Implementation of an easy analytical method that can analyze a wide range of ß-lactam antibiotics in a single run is important to reduce the time of therapeutic drug monitoring (TDM) in hospitals and minimize the spreading of bacterial resistance. A novel environmentally harmless HPTLC method was developed and validated following FDA recommendations for analysis of four ß-lactams; cefaclor, cefotaxime, cefepime, and meropenem, in human plasma. A solvent mixture of ethylacetate : methanol : deionized water : formic acid (60 : 30 : 15 : 1, by volume) was the used developing system, detection was carried out at 270 nm, and valacyclovir was used as an internal standard. A lower limit of quantitation (LLOQ) was found to be 0.1 µg per band for all the analyzed drugs. Validation parameters were calculated and found to fulfil the international requirements for bio-analytical method validation. Additionally, each of the studied antibiotics was given to a group of healthy volunteers from which blood samples were collected at t max of each, methanol was used for precipitation of plasma protein, and the developed method was used for calculation of the concentrations in the separated plasma samples. The developed method, being a green one, and time and money saving, can be used for TDM of these drugs in clinical studies as well as for quality control analysis in pharmaceutical companies. The proposed method is the first developed HPTLC method for the simultaneous bio-analysis of the selected ß-lactams.

Efficient UPLC and CE methods for the simultaneous determination of azelastine hydrochloride and its genotoxic impurity.

A novel stability-indicating UPLC and CE method was established and validated for the determination of azelastine hydrochloride (AZL) and its genotoxic impurity, benzohydrazide, in the presence of benzalkonium chloride. The developed UPLC method was based on chromatographic separation using a C18 column as a stationary phase and acetonitrile-(0.1% w/v) aqueous sodium lauryl sulfate (55:45, v/v, pH 5 with phosphoric acid) as a mobile phase with a flow rate of 1.2 mL/min and UV detection at 215 nm. The chromatographic run time was ~2 min. The developed CE method depended on using a stationary phase of Standard Bare Fused Silica Capillaries (75 µm i.d. × 59 cm and 50 cm detection length) and the applied voltage was 30 kV using 40 mm phosphate buffer (pH 2 with aqueous H3 PO4 ); the detection wavelength was 225 nm. The analysis time was about 6 min. The suggested methods were successfully applied for the analysis of AZL in a pharmaceutical preparation. The validity of the developed methods was assessed by applying the standard addition technique and no interference from excipients was observed. The results obtained by the proposed methods were statistically analyzed and compared with the manufacturer's method and no significant difference was found between the compared methods.

Determination of flutamide and two major metabolites using HPLC-DAD and HPTLC methods.

Flutamide is a potential antineoplastic drug classified as an anti-androgen. It is a therapy for men with advanced prostate cancer, administered orally after which it undergoes extensively first pass metabolism in the liver with the production of several metabolites. These metabolites are predominantly excreted in urine. One of the important metabolites in plasma is 4-nitro-3-(trifluoromethyl)phenylamine (Flu-1), while the main metabolite in urine is 2-amino-5-nitro-4-(trifluoromethyl)phenol (Flu-3). In this work the two metabolites, Flu-1 and Flu-3, have been synthesized, and then structural confirmation has been carried out by HNMR analysis. Efforts were exerted to develop chromatographic methods for resolving Flutamide and its metabolites with the use of acceptable solvents without affecting the efficiency of the methods. The drug along with its metabolites were quantitatively analyzed in pure form, human urine, and plasma samples using two chromatographic methods, HPTLC and HPLC-DAD methods. FDA guidelines for bio-analytical method validation were followed and USP recommendations were used for analytical method validation. Interference from excipients has been tested by application of the methods to pharmaceutical tablets. No significant difference was found between the proposed methods and the official one when they were statistically compared at p value of 0.05%.

TLC-Densitometric and RP-HPLC Methods for Simultaneous Determination of Dexamethasone and Chlorpheniramine Maleate in the Presence of Methylparaben and Propylparaben.

Validated simple, sensitive, and highly selective methods are applied for the quantitative determination of dexamethasone and chlorpheniramine maleate in the presence of their reported preservatives (methylparaben and propylparaben), whether in pure forms or in pharmaceutical formulation. TLC is the first method, in which dexamethasone, chlorpheniramine maleate, methylparaben, and propylparaben are separated on silica gel TLC F254 plates using hexane-acetone-ammonia (5.5 + 4.5 + 0.5, v/v/v) as the developing phase. Separated bands are scanned at 254 nm over a concentration range of 0.1-1.7 and 0.4-2.8 µg/band, with mean ± SD recoveries of 99.12 ± 0.964 and 100.14 ± 0.962%, for dexamethasone and chlorpheniramine maleate, respectively. Reversed-phase HPLC is the second method, in which a mixture of dexamethasone and chlorpheniramine maleate, methylparaben, and propylparaben is separated on a reversed-phase silica C18 (5 µm particle size, 250 mm, 4.6 mm id) column using 0.1 M ammonium acetate buffer-acetonitrile (60 + 40, v/v, pH 3) as the mobile phase. The drugs were detected at 220 nm over a concentration range of 5-50 µg/mL, 2-90 µg/mL, 4-100 µg/mL, and 7-50 µg/mL, with mean ± SD recoveries of 100.85 ± 0.905, 99.67 ± 1.281, 100.20 ± 0.906, and 99.81 ± 0.954%, for dexamethasone, chlorpheniramine maleate, methylparaben paraben, and propylparaben, respectively. The advantages of the suggested methods over previously reported methods are the ability to detect lower concentrations of the main drugs and to show better resolution of interfering preservatives; hence, these methods could be more reliable for routine QC analyses.