Development of Different Separation Techniques for Impurity Profiling of Neostigmine Methylsulfate: A Comparative Study.

Different separation techniques have been employed to resolve mixtures of multicomponent preparations over the last few decades. They could be efficiently applied for impurity profiling of active drug substances. Impurity profiling has become a critical procedure in pharmaceutical companies to comply with numerous regulatory standards. Isolation and characterization of impurities are crucial for obtaining data that proves biological safety and efficacy. In this contribution, different HPLC, capillary electrophoresis (CE) and TLC-densitometric methods were developed for the determination of neostigmine methylsulfate (NEO) along with 3-hydroxyphenyltrimethyl ammonium methylsulfate (3-HPA) and 3-dimethylaminophenol (DAP) as its impurities in the presence of citric acid. The linearity for NEO was attained in the range of 5-120 µg/mL and 10-60 µg/mL for the HPLC and CE methods, respectively. Regarding the impurities, linearity was obtained over the range of 10-30 µg/mL for 3-HPA and 5-30 µg/mL for DAP in the two proposed methods. For the TLC method, NEO and DAP were determined within the range of 1-11 µg/band, whereas 3-HPA was assayed over the range of 2-12 µg/band. The suggested methods can be securely utilized for routine analysis of the cited components in quality control laboratories.

Different Approaches in Manipulating Ratio Spectra for Analyzing Amlodipine Besylate and Irbesartan Combination.

BACKGROUND: Hypertension is a key risk factor for ischemic heart disease and atherosclerosis. Most patients require a combination of antihypertensive medications to accomplish their therapeutic goals. Antihypertensive medicines such as calcium channel blockers and angiotensin receptor blockers are indicated for patients whose high blood pressure cannot be controlled with monotherapy. The combination of amlodipine besylate (AML) with irbesartan (IRB) is an example of this synergistic activity in lowering blood pressure. OBJECTIVE: In this regard, the goal of the research is to develop sensitive spectrophotometric methods for the simultaneous determination of amlodipine besylate and irbesartan. METHODS: Three simple ratio spectra-manipulating spectrophotometric methods namely, ratio difference, mean centering of ratio spectra, and derivative ratio, were developed for the simultaneous assay of the cited mixture. RESULTS: Linear correlations were attained over the concentration range of 1-35 µg/mL and 2-35 µg/mL for amlodipine besylate and irbesartan, respectively. The methods were validated according to the International Conference on Harmonization guidelines with good results. CONCLUSION: The methods developed were successfully applied for the assay of the cited drugs in their marketed formulation. They could be efficiently used for routine analysis of the mentioned drugs in QC laboratories. HIGHLIGHTS: The proposed approaches do not require expensive solvents or complex instruments. They could be used in routine laboratory tests where time and cost are crucial.

Solid Phase Extraction and Simultaneous Chromatographic Quantification of some Non-steroidal Anti-inflammatory Drug Residues; an Application in Pharmaceutical Industrial Wastewater Effluent

Abstract Two sensitive and selective methods were developed for the simultaneous determination of four commonly used non-steroidal anti-inflammatory drugs (NSAIDs), namely; paracetamol (PCM), diclofenac sodium (DCF), ibuprofen (IBP), and indomethacin (IND) in wastewater effluents. The first method used HPLC for the determination of the studied drugs using a mobile phase consisting of phosphate buffer (pH 3.0) and acetonitrile at a flow rate of 1 mL/min. in gradient elution mode and detection at 220 nm. The separation process was performed on BDS Hypersil Cyano column (250 x 4.6 mm, 5 µm). The second method was a TLC-densitometric one which was performed using n-Hexane: ethyl acetate: acetic acid in the ratio (6:3.5:0.5) as a developing system. The proposed chromatographic methods were successfully applied for the selective determination of the four studied drugs in simulated and real pharmaceutical wastewater samples after their solid-phase extraction

Validated Chromatographic Methods for Simultaneous Determination of Calcipotriol Monohydrate and Betamethasone Dipropionate in the Presence of Two Dosage Form Additives.

Two chromatographic methods were developed, optimized and validated for simultaneous determination of calcipotriol monohydrate (CPM) and betamethasone dipropionate (BMD) in the presence of two dosage form additives named; butylated hydroxytoluene (BHT) and alpha-tocopherol (TOCO). The proposed methods were accurate, sensitive and specific. The first method based on using aluminum thin-layer chromatographic plates precoated with silica gel GF254 as a stationary phase and chloroform-ethyl acetate-toluene (5:5:3, by volume) as a developing system. This was followed by densitometric measurement of the separated bands at 264 nm. Whereas the second method is RP-HPLC where OnyxMonolithic C18® column was used with a gradient profile using methanol, water and acetic acid at flow rate 2.0 mL min-1. Detection was carried out at 264 nm. The methods were validated according to ICH guidelines. The specificity of the developed methods was investigated by analyzing the pharmaceutical dosage form. The validity of the proposed methods was assessed using the standard addition technique. The obtained results were statistically compared with those obtained by the official methods, showing no significant difference with respect to accuracy and precision at P = 0.05.

Development and Validation of Eco-Friendly Liquid Chromatographic and Spectrophotometric Methods for Simultaneous Determination of Coformulated Drugs: Phenylephrine Hydrochloride and Prednisolone Acetate.

Five simple, sensitive, and eco-friendly LC and UV spectrophotometric methods have been developed for the simultaneous determination of phenylephrine hydrochloride (PHE) and prednisolone acetate (PRD) in their combined dosage form. The first method was reversed-phase (RP) LC using methanol-water-heptane-1-sulfonic acid sodium salt (75 + 25 + 0.1, v/v/w) as a mobile phase. Separation was achieved using an XSelect HSS reversed-phase C18 analytical column (250 × 4.6 mm, 5µm). The flow rate was 1.0 mL/min and UV detection was done at 230 nm. Quantification was achieved over the concentration ranges of 5-50 µg/mL for PHE and 2-90 µg/mL for PRD. Four spectrophotometric methods were proposed, namely dual wavelength, first derivative of ratio spectra, ratio difference, and mean-centering of ratio spectra. Linearity was observed in the concentration ranges of 10-120 and 5-35 µg/mL for PHE and PRD, respectively, for the spectrophotometric methods. Green solvents were used in the proposed methods because they play a vital role in the analytical methods' influence on the environment. The suggested methods were validated regarding linearity, accuracy, and precision according to the International Conference on Harmonization guidelines, with satisfactory results. These methods could be used as harmless substitutes for routine analysis of the mentioned drugs, with no interference from excipients.

Simultaneous Determination of Aspirin, Dipyridamole and Two of Their Related Impurities in Capsules by Validated TLC-Densitometric and HPLC Methods.

Aspirin (ASP) and dipyridamole (DIP) are widely used as a combination in pharmaceutical formulations for treatment of strokes. Many of these formulations are containing tartaric acid as an excipient (in DIP pellets formulation for sustained release), which increases the probability of formation of dipyridamole tartaric acid ester impurity (DIP-I). On the other hand, salicylic acid (SAL) is considered to be one of the synthesis impurities and a degradation product of ASP. In this work, two chromatographic methods, namely, TLC-densitometry and HPLC, have been established and validated for simultaneous determination of ASP, DIP, SAL and DIP-I. Good separation was achieved by using silica gel as stationary phase and toluene-methanol-ethyl acetate (2:3:5, by volume) as mobile phase in the case of TLC-densitometry and Zorbax ODS column with mobile phase consisting of phosphate buffer (pH 3.3)-acetonitrile-triethylamine (40:60:0.03, by volume) for HPLC. Influence of different organic solvents in mobile phase composition has been studied to optimize the separation efficiency in TLC densitometry. Moreover, factors affecting the efficiency of HPLC, like pH of the buffer used, organic solvent ratio in the mobile phase and flow rate, have been carefully studied using one variable at a time approach. Finally, the proposed methods were validated as per ICH guidelines.

Chemometrics Tools in Detection and Quantitation of the Main Impurities Present in Aspirin/Dipyridamole Extended-Release Capsules.

Aspirin (ASP) and dipyridamole (DIP) in combination is widely used in the prevention of secondary events after stroke and transient ischemic attack. Salicylic acid is a well-known impurity of ASP, and the DIP extended-release formulation may contain ester impurities originating from the reaction with tartaric acid. UV spectral data analysis of the active ingredients in the presence of their main impurities is presented using multivariate approaches. Four chemometric-assisted spectrophotometric methods, namely, partial least-squares, concentration residuals augmented classical least-squares (CRACLS), multivariate curve resolution (MCR) alternating least-squares (ALS), and artificial neural networks, were developed and validated. The quantitative analyses of all the proposed calibrations were compared by percentage recoveries, root mean square error of prediction, and standard error of prediction. In addition, r(2) values between the pure and estimated spectral profiles were used to evaluate the qualitative analysis of CRACLS and MCR-ALS. The lowest error was obtained by the CRACLS model, whereas the best correlation was achieved using MCR-ALS. The four multivariate calibration methods could successfully be applied for the extended-release formulation analysis. The application results were also validated by analysis of the stored dosage-form solution, which showed a susceptibility of DIP esterification in the extended-release formulation. Statistical comparison between the proposed and official methods showed no significant difference.

Stability-indicating chromatographic methods for determination of flecainide acetate in the presence of its degradation products; isolation and identification of two of its impurities.

In this work, two stability-indicating chromatographic methods have been developed and validated for determination of flecainide acetate (an antiarrhythmic drug) in the presence of its degradation products (flecainide impurities; B and D). Flecainide acetate was subjected to a stress stability study including acid, alkali, oxidative, photolytic and thermal degradation. The suggested chromatographic methods included the use of thin layer chromatography (TLC-densitometry) and high-performance liquid chromatography (HPLC). The TLC method employed aluminum TLC plates precoated with silica gel G.F254 as the stationary phase and methanol-ethyl acetate-33% ammonia (3:7:0.3, by volume) as the mobile phase. The chromatograms were scanned at 290 nm and visualized in daylight by the aid of iodine vapor. The developed HPLC method used a RP-C18 column with isocratic elution. Separation was achieved using a mobile phase composed of phosphate buffer pH 3.3-acetonitrile-triethylamine (53:47:0.03, by volume) at a flow rate of 1.0 mL/min and UV detection at 292 nm. Factors affecting the efficiency of HPLC method have been studied carefully to reach the optimum conditions for separation. The developed methods were validated according to the International Conference on Harmonization guidelines and were applied for bulk powder and dosage form. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous determination of Fluticasone propionate and Azelastine hydrochloride in the presence of pharmaceutical dosage form additives.

Fluticasone propionate (FLU) and Azelastine hydrochloride (AZE) are co-formulated with phenylethyl alcohol (PEA) and Benzalkonium chloride (BENZ) (as preservatives) in pharmaceutical dosage form for treatment of seasonal allergies. Different spectrophotometric methods were used for the simultaneous determination of cited drugs in the dosage form. Direct spectrophotometric method was used for determining of AZE, while Derivative of double divisor of ratio spectra (DD-RS), Ratio subtraction coupled with ratio difference method (RS-RD) and Mean centering of the ratio spectra (MCR) are used for the determination of FLU. The linearity of the proposed methods was investigated in the range of 5.00-40.00 and 5.00-80.00µg/mL for FLU and AZE, respectively. The specificity of the developed methods was investigated by analyzing laboratory prepared mixtures containing different ratios of cited drugs in addition to PEA and their pharmaceutical dosage form. The validity of the proposed methods was assessed using the standard addition technique. The obtained results were statistically compared with those obtained by official or the reported method for FLU or AZE, respectively showing no significant difference with respect to accuracy and precision at p=0.05.

Stability-indicating spectrophotometric methods for determination of the anticoagulant drug apixaban in the presence of its hydrolytic degradation product.

Apixaban (a novel anticoagulant agent) was subjected to a stress stability study including acid, alkali, oxidative, photolytic, and thermal degradation. The drug was found to be only liable to acidic and alkaline hydrolysis. The degradation product was then isolated and identified by IR and GC-mass spectrometry. Four spectrophotometric methods, namely; first derivative (D(1)), derivative ratio (DR), ratio difference (RD) and mean centering of ratio spectra (MCR), have been suggested for the determination of apixaban in presence of its hydrolytic degradation product. The proposed methods do not require any preliminary separation step. The accuracy, precision and linearity ranges of the proposed methods were determined, and the methods were validated as per ICH guidelines and the specificity was assessed by analyzing synthetic mixtures containing different percentages of the degradation product with the drug. The developed methods were successfully applied for the determination of apixaban in bulk powder and its tablet dosage form.

Development and validation of impurity-profiling UPLC method for the determination of sodium cromoglicate and tetryzoline hydrochloride: Application on rabbit aqueous humor.

Sodium cromoglicate (SCG), antihistaminic agent, and tetryzoline hydrochloride (TZH), a sympathomimetic agent, are formulated together as an ophthalmic preparation. An ultra-performance liquid chromatographic method with UV detection (UPLC-UV) was developed and validated for the quantitative determination of SCG and TZH in rabbit aqueous humor. Due to the instability of both SCG and TZH under alkaline conditions, the UPLC method was applied for their determination in the presence of their possible degradation impurities. The separation was performed using C18 column (1.7µm particle size) and isocratic elution system with methanol: 1% o-phosphoric acid (65: 35, v/v).The optimum flow rate was 0.5ml/min and the detection was done at 230nm. The suggested method was validated in compliance with the ICH guidelines and was successfully applied for determination of sodium cromoglicate (SCG) and tetryzoline HCl (TZH) as prepared synthetically in laboratory mixtures, and in the presence of their alkali-induced degradation impurities. The suggested method was effectively applied the determination of spiked rabbit aqueous humor samples as well as commercial pharmaceutical formulation.

Computation of geometric representation of novel spectrophotometric methods used for the analysis of minor components in pharmaceutical preparations.

Novel spectrophotometric methods were applied for the determination of the minor component tetryzoline HCl (TZH) in its ternary mixture with ofloxacin (OFX) and prednisolone acetate (PA) in the ratio of (1:5:7.5), and in its binary mixture with sodium cromoglicate (SCG) in the ratio of (1:80). The novel spectrophotometric methods determined the minor component (TZH) successfully in the two selected mixtures by computing the geometrical relationship of either standard addition or subtraction. The novel spectrophotometric methods are: geometrical amplitude modulation (GAM), geometrical induced amplitude modulation (GIAM), ratio H-point standard addition method (RHPSAM) and compensated area under the curve (CAUC). The proposed methods were successfully applied for the determination of the minor component TZH below its concentration range. The methods were validated as per ICH guidelines where accuracy, repeatability, inter-day precision and robustness were found to be within the acceptable limits. The results obtained from the proposed methods were statistically compared with official ones where no significant difference was observed. No difference was observed between the obtained results when compared to the reported HPLC method, which proved that the developed methods could be alternative to HPLC techniques in quality control laboratories.

Stability-indicating spectrofluorometric method for the determination of some cephalosporin drugs via their degradation products.

A stability-indicating spectrofluorometric method was investigated for the determination of three cephalosporin drugs, namely, cefpodoxime proxetil (CPD), cefixime trihydrate (CFX), and cefepime hydrochloride (CPM), via their acid and alkali degradation products. The three drugs were determined via their acid degradation at 432, 422, and 435 nm using an excitation wavelength of 310, 330, and 307 nm for CPD, CFX, and CPM determination, respectively, and via their alkali degradation at 407, 411, and 405 nm using an excitation wavelength of 310, 305, and 297 nm for CPD, CFX, and CPM determination, respectively. Linearity was achieved in the ranges of 0.35-3.50, 0.4-4.0, and 0.3-3.0 µg/mL for the acid degradation products of CPD, CFX, and CPM, respectively, and in ranges of 0.05-0.5, 0.1-1.0, and 0.08-0.80 µg/mL for the alkali degradation products of CPD, CFX, and CPM, respectively. The method was validated for various parameters according to International Conference on Harmonization guidelines. The method was successfully applied for the determination of these cephalosporin drugs in pharmaceutical dosage forms with good accuracy and precision. The results obtained by the proposed spectrofluorometric method were compared with good agreement to the official HPLC method.

Validated stability-indicating spectrophotometric methods for the determination of cefixime trihydrate in the presence of its acid and alkali degradation products.

Five simple, accurate, precise, and economical spectrophotometric methods have been developed for the determination of cefixime trihydrate (CFX) in the presence of its acid and alkali degradation products without prior separation. In the first method, second derivative (2D) and first derivative (1D) spectrophotometry was applied to the absorption spectra of CFX and its acid (2D) or alkali (1D) degradation products by measuring the amplitude at 289 and 308 nm, respectively. The second method was a first derivative (1DD) ratio spectrophotometric method where the peak amplitudes were measured at 311 nm in presence of the acid degradation product, and 273 and 306 nm in presence of its alkali degradation product. The third method was ratio subtraction spectrophotometry where the drug is determined at 286 nm in laboratory-prepared mixtures of CFX and its acid or alkali degradation product. The fourth method was based on dual wavelength analysis; two wavelengths were selected at which the absorbances of one component were the same, so wavelengths 209 and 252 nm were used to determine CFX in presence of its acid degradation product and 310 and 321 nm in presence of its alkali degradation product. The fifth method was bivariate spectrophotometric calibration based on four linear regression equations obtained at the wavelengths 231 and 290 nm, and 231 and 285 nm for the binary mixture of CFX with either its acid or alkali degradation product, respectively. The developed methods were successfully applied to the analysis of CFX in laboratory-prepared mixtures and pharmaceutical formulations with good recoveries, and their validation was carried out following the International Conference on Harmonization guidelines. The results obtained were statistically compared with each other and showed no significant difference with respect to accuracy and precision.

A comparative study of different aspects of manipulating ratio spectra applied for ternary mixtures: derivative spectrophotometry versus wavelet transform.

This work represents a comparative study of different aspects of manipulating ratio spectra, which are: double divisor ratio spectra derivative (DR-DD), area under curve of derivative ratio (DR-AUC) and its novel approach, namely area under the curve correction method (AUCCM) applied for overlapped spectra; successive derivative of ratio spectra (SDR) and continuous wavelet transform (CWT) methods. The proposed methods represent different aspects of manipulating ratio spectra of the ternary mixture of Ofloxacin (OFX), Prednisolone acetate (PA) and Tetryzoline HCl (TZH) combined in eye drops in the presence of benzalkonium chloride as a preservative. The proposed methods were checked using laboratory-prepared mixtures and were successfully applied for the analysis of pharmaceutical formulation containing the cited drugs. The proposed methods were validated according to the ICH guidelines. A comparative study was conducted between those methods regarding simplicity, limitation and sensitivity. The obtained results were statistically compared with those obtained from the reported HPLC method, showing no significant difference with respect to accuracy and precision.

Column performance study of different variants of liquid chromatographic technique: an application on pharmaceutical ternary mixtures containing tetryzoline.

High-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) and rapid resolution liquid chromatographic (RRLC) methods have been developed and validated for the separation and quantitation of both or either of two ternary mixtures present in ophthalmic solutions. The first mixture contains chloramphenicol, dexamethasone sodium phosphate and tetryzoline HCl (TZH); while the second one contains ofloxacin, prednisolone acetate and TZH. Both preparations contain benzalkonium chloride as a preservative. The columns used were a HPLC column (C18 5 µm particle size), a RRLC column (C18 2.6 µm particle size) and a UPLC column (C18 1.7 µm particle size). A comparative study was conducted to illustrate the effect of the change in column particle size and dimensions on the other chromatographic conditions, backpressure and the separation of both ternary mixtures. The methods were validated as per ICH guidelines where accuracy, repeatability, interday precision and robustness were found to be within the acceptable limits. The RRLC column provided shorter run time and better resolution than HPLC, while the UPLC column gave the shortest run time for all columns. The RRLC column resulted in minimum backpressure, so it could be used with any HPLC instrument, which makes the method more practical and economic. The results obtained from the proposed methods were statistically compared with official ones where no significant difference was observed.

Novel two wavelength spectrophotometric methods for simultaneous determination of binary mixtures with severely overlapping spectra.

This work presents the application of different spectrophotometric techniques based on two wavelengths for the determination of severely overlapped spectral components in a binary mixture without prior separation. Four novel spectrophotometric methods were developed namely: induced dual wavelength method (IDW), dual wavelength resolution technique (DWRT), advanced amplitude modulation method (AAM) and induced amplitude modulation method (IAM). The results of the novel methods were compared to that of three well-established methods which were: dual wavelength method (DW), Vierordt's method (VD) and bivariate method (BV). The developed methods were applied for the analysis of the binary mixture of hydrocortisone acetate (HCA) and fusidic acid (FSA) formulated as topical cream accompanied by the determination of methyl paraben and propyl paraben present as preservatives. The specificity of the novel methods was investigated by analyzing laboratory prepared mixtures and the combined dosage form. The methods were validated as per ICH guidelines where accuracy, repeatability, inter-day precision and robustness were found to be within the acceptable limits. The results obtained from the proposed methods were statistically compared with official ones where no significant difference was observed. No difference was observed between the obtained results when compared to the reported HPLC method, which proved that the developed methods could be alternative to HPLC techniques in quality control laboratories.

A comparative study of progressive versus successive spectrophotometric resolution techniques applied for pharmaceutical ternary mixtures.

This work represents a comparative study of a novel progressive spectrophotometric resolution technique namely, amplitude center method (ACM), versus the well-established successive spectrophotometric resolution techniques namely; successive derivative subtraction (SDS); successive derivative of ratio spectra (SDR) and mean centering of ratio spectra (MCR). All the proposed spectrophotometric techniques consist of several consecutive steps utilizing ratio and/or derivative spectra. The novel amplitude center method (ACM) can be used for the determination of ternary mixtures using single divisor where the concentrations of the components are determined through progressive manipulation performed on the same ratio spectrum. Those methods were applied for the analysis of the ternary mixture of chloramphenicol (CHL), dexamethasone sodium phosphate (DXM) and tetryzoline hydrochloride (TZH) in eye drops in the presence of benzalkonium chloride as a preservative. The proposed methods were checked using laboratory-prepared mixtures and were successfully applied for the analysis of pharmaceutical formulation containing the cited drugs. The proposed methods were validated according to the ICH guidelines. A comparative study was conducted between those methods regarding simplicity, limitation and sensitivity. The obtained results were statistically compared with those obtained from the official BP methods, showing no significant difference with respect to accuracy and precision.

A comparative study of novel spectrophotometric methods based on isosbestic points; application on a pharmaceutical ternary mixture.

This work represents the application of the isosbestic points present in different absorption spectra. Three novel spectrophotometric methods were developed, the first method is the absorption subtraction method (AS) utilizing the isosbestic point in zero-order absorption spectra; the second method is the amplitude modulation method (AM) utilizing the isosbestic point in ratio spectra; and third method is the amplitude summation method (A-Sum) utilizing the isosbestic point in derivative spectra. The three methods were applied for the analysis of the ternary mixture of chloramphenicol (CHL), dexamethasone sodium phosphate (DXM) and tetryzoline hydrochloride (TZH) in eye drops in the presence of benzalkonium chloride as a preservative. The components at the isosbestic point were determined using the corresponding unified regression equation at this point with no need for a complementary method. The obtained results were statistically compared to each other and to that of the developed PLS model. The specificity of the developed methods was investigated by analyzing laboratory prepared mixtures and the combined dosage form. The methods were validated as per ICH guidelines where accuracy, repeatability, inter-day precision and robustness were found to be within the acceptable limits. The results obtained from the proposed methods were statistically compared with official ones where no significant difference was observed.

Stability-indicating chromatographic methods for the determination of sertindole.

In this work, two chromatographic methods have been developed and validated for the determination of sertindole (an antipsychotic agent) in the presence of its oxidative degradation product. Sertindole was subjected to stress stability studies, including acid, alkali, oxidative, photolytic and thermal degradation. The chromatographic methods included the use of thin-layer chromatography (TLC-densitometry) and high-performance liquid chromatography (HPLC). The TLC method employed aluminum TLC plates precoated with silica gel G.F254 as the stationary phase and methanol-ethyl acetate-33% ammonia (1:9:0.1, by volume) as the mobile phase, and the chromatograms were scanned at 227 nm. The developed HPLC method used a reversed-phase C18 column with isocratic elution. The mobile phase was composed of phosphate buffer pH 3.0-acetonitrile-triethylamine (45:55:0.03, by volume) and run at a flow rate of 1.0 mL/min. Quantitation was achieved with ultraviolet detection at 256 nm. The linearity ranges were found to be 2-14 µg/band and 5-200 µg/mL for TLC and HPLC, respectively. The developed methods were validated according to the International Conference on Harmonization guidelines and were applied for bulk powder and dosage forms.