Utilizing experimental design and desirability function in optimizing RP-HPLC method for simultaneous determination of some skeletal muscle relaxants and analgesics.

An experimental design and response surface methodologies using Plackett-Burman and Box-Behnken designs were applied for selecting and optimizing the most appropriate parameters which significantly affect the separation and quantitative estimation of five skeletal muscle relaxants and four analgesic drugs (baclofen, methocarbamol, dantrolene sodium, orphenadrine citrate, cyclobenzaprine hydrochloride, ketoprofen, etoricoxib, ibuprofen, and mefenamic acid) with a relatively short duration of analysis in a single run. For the separation of the nine drugs, an INERTSIL ODS-V3-5 µm C18 column (250 × 4.6 mm I.D.) was used with the optimum mobile phase conditions (45.15 mM ammonium acetate buffer pH 5.56 adjusted with acetic acid, acetonitrile, and methanol in a ratio of 30.5:29.5:40, v/v/v with a flow rate of 1.5 mL/min) and UV-detection at 220 nm. The optimized method was successfully subjected to the validation steps as described in ICH guidelines for linearity, precision, accuracy, robustness, and sensitivity. The optimized and validated method was effectively applied to determine the content of the studied drugs in their pharmaceutical preparations and to expand its applicability to the counterfeit estimation of etoricoxib in different brands of tablet dosage forms.

Resolution of overlapping spectra using chemometric manipulations of UV-spectrophotometric data for the determination of Atenolol, Losartan, and Hydrochlorothiazide in pharmaceutical dosage form.

Simultaneous determination of atenolol (ATN), losartan potassium (LOS), and hydrochlorothiazide (HCZ) in presence of HCZ impurity B was conducted by chemometric approaches and radial basis function network (RBFN) using UV-spectrophotometry without preliminary separation. Three chemometric models namely, classical least-squares (CLS), principal component regression (PCR), and partial least-squares (PLS) along with RBFN were utilized using the ternary mixtures of the three drugs. The multivariate calibrations were obtained by measuring the zero-order absorbance of the mixtures from 250 to 270 nm at the interval of 0.2 nm. The models were built covering the concentration range of (4.0 to 20.0), (3.8 to 20.2), and (0.9 to 50.1) µg mL-1 for ATN, LOS, and HCZ, respectively. The regression coefficient was calculated between the actual and predicted concentrations of the 3 drugs using CLS, PCR, PLS and RBFN. The accuracy of the developed models was evaluated using the root mean square error of prediction (RMSEP) giving satisfactory results. The proposed methods were simple, accurate, precise and were applied efficiently for the quantitation of the three components in laboratory-prepared mixtures, and in dosage form showing good recovery values. In addition, the obtained results were compared statistically with each other using ANOVA test showing non-significant difference between them.

In Vivo Evaluation of the Pharmacokinetic Interaction between Levothyroxine and Amiodarone in Rat Plasma: Evaluation of Importance of Therapeutic Drug Monitoring during Co-Therapy.

Amiodarone-induced thyrotoxicosis (AIT) is a common condition in patients who are receiving amiodarone for cardiac arrhythmia. This risk is elevated in iodine-deficient regions. Levothyroxine is the standard treatment for patients with hypothyroidism. This investigation is concerned with the evaluation of the possible pharmacokinetic interaction between amiodarone and levothyroxine upon co-therapy in rats and to investigate the cause of thyrotoxicosis. A selective, sensitive and precise RP-HPLC method was developed for the simultaneous determination of levothyroxine and amiodarone in rat plasma. A stationary phase of C18 Xterra RP column and a mobile phase consisting of acetonitrile: acidified water with 0.1% trifluoracetic acid (pH = 4.8) with gradient elution were used. The experiment was conducted at ambient temperature with flow rate of 1.5 mL/min for the chromatographic separation and quantitation of the investigated drugs. Protein precipitation with methanol was applied for the analysis of the two drugs in rat plasma. The method was linear over concentration range of 5-200 µg/mL for both levothyroxine and amiodarone. The European Medicines Agency guideline was applied for the validation of the developed bioanalytical method. The method was successfully applied to in vivo pharmacokinetic study in which levothyroxine and amiodarone were quantified in plasma of rats after receiving an oral dose of levothyroxine and amiodarone. After the calculation of the pharmacokinetic parameters, a statistical analysis was performed to elucidate the existence of significant difference between test and control groups in rats. The combination of levothyroxine and amiodarone significantly decreased levothyroxine bioavailability in rats, making the therapeutic drug monitoring mandatory in patients receiving levothyroxine and amiodarone. In addition, the increased clearance of levothyroxine upon the co-administration with amiodarone may explain the reported hypothyroidism.

AGREE and ESA for Greenness Assessment of a Novel Validated RP-HPLC Method for Simultaneous Determination of Aspirin, Warfarin and Clopidogrel in Rat Plasma: Application to Pharmacokinetic Study of the Possible Interaction between the Three Drugs.

The green profile of the developed method is assessed and compared with previously reported methods. Percutaneous coronary intervention is a procedure where a catheter is utilized to place a stent in order to facilitate opening of the blood vessels in the heart. Triple antithrombotic therapy includes oral anticoagulation as warfarin and dual antiplatelet therapy (composed of aspirin and clopidogrel bisulfate). The aim of the current study was to evaluate the pharmacokinetic parameters of ASP, WAR and CLP and to investigate the possible interaction between the three drugs upon co-administration in rats. A selective and precise RP-HPLC method was developed for the simultaneous determination of ASP, WAR and CLP in rat plasma. Pharmacokinetic study was conducted in rats that received ASP, WAR and CLP as an application of the developed method. From the statistical evaluation of the pharmacokinetic parameters, it was observed that the co-administration of ASP, WAR and CLP significantly increased the ASP and CLP bioavailability in rats. A significant drug-drug interaction was confirmed in the current study. The elevated Cmax of ASP and CLP upon the co-administration of ASP, WAR and CLP may explain the reported bleeding.

The potential off-target neuroprotective effect of sister gliflozins suggests their repurposing despite not crossing the blood-brain barrier: From bioanalytical assay in rats into theory genesis.

Gliflozins are successfully marketed antidiabetic agents with a reported neuroprotective effect, and this study tests their blood-brain barrier crossing ability. Henceforward, a computational hypothesis interpreting their effects was reasonable after failure to cross into the brain. A chromatographic bioassay for canagliflozin, dapagliflozin, and empagliflozin was developed, validated, and applied to the rat's and rat's plasma and brain. HPLC method robustness was tested over two levels using Design of Experiment on MINITAB. It is the first method for gliflozins' detection in rats' brain tissue. The method was applied on 18 rats and six for each drug. Concentrations in plasma were determined but neither of them was detected in brain at the described chromatographic conditions. A computational study for the three drugs was endorsing two techniques. First, ligand-based target fishing reveals possible targets for gliflozins. They showed an ability to bind with human equilibrative nucleoside transporter 1, a regulator of adenosine extracellularly. Second, a docking study was carried out on this protein receptor. Results showed perfect alignment with a minimum of one hydrogen bond. Dapagliflozin achieved the lowest energy score with two hocking hydrogen bonds. This is proposing gliflozins ability to regulate equilibrative nucleoside transporter 1 receptors in peripheries, elevating the centrally acting neuroprotective adenosine.

A Turn-On-Type Fluorescence Resonance Energy Transfer Eco-friendly Method for Nitazoxanide Quantification in Pharmaceutical Dosage Form and Spiked Plasma: Evaluation of Greenness Profile Using Different Assessment Tools.

A brand-new class of anti-infective drugs that work against bacteria, viruses, and protozoan parasites is nitazoxanide and related thiazolides. Thiazolides have also been shown to cause cell cycle arrest and apoptotic cell death in cancer cells in recent years. In this study, an eco-friendly, spectrofluorimetric technique that is verified, easy, and sensitive has been proposed for quantifying nitazoxanide (NTZ), a broad-spectrum antiparasitic drug. When NTZ is reduced with zinc (Zn) powder in an acidic media, a highly fluorescent product is produced. To get the highest sensitivity, different experimental conditions impacting the response were examined and optimized. Following excitation at 299 nm, scanning of the fluorescent product was done at 440 nm. The intensity of the fluorescence was proportional to the drug concentration in the range of 0.1-0.6 µg/mL. The approach was validated according to International Conference on Harmonization (ICH) guidelines, and the outcome was satisfactory. The detection and quantitation limits were calculated to be 0.013 and 0.038 µg/mL, respectively. The suggested technique was successful in analyzing commercially available NTZ dosage forms. Furthermore, the proposed technique was used to assess NTZ levels in human plasma and it was bio-analytically validated according to European Medicines Agency (EMA) guidelines. The suggested method can be used in quality control laboratories as well as in pharmacokinetic studies. In order to picture the green profile of the developed method, four greenness assessment tools have been applied. National Environmental Methods Index (NEMI), analytical Eco-Scale Assessment (ESA), Green Analytical Procedure Index (GAPI) and Analytical Greenness metric (AGREE) are the relatively most widely used metrics. So, they were utilized to perform a detailed greenness comparison between the proposed method and some of the reported methods for the determination of NTZ. The developed method was found to be an excellent green method with the highest AGREE score.

Validated LC/MS/MS Method for the Determination of Rivastigmine in Human Plasma: Application to a Pharmacokinetic Study in Egyptian Volunteers to Determine the Effect of Gender and Body Mass Index.

The effect of gender and body mass index (BMI) on the pharmacokinetics of rivastigmine was studied in Egyptian human subjects using new bio-analytical validated LC/MS/MS method. In this study, Rivastigmine was estimated in human plasma using Escitalopram as an internal standard (IS). Rivastigmine and Escitalopram were extracted from human plasma samples by liquid-liquid extraction using diethyl ether (DEE)-dichloromethane (DCM) (70:30, v/v). Chromatographic separation was performed on a reversed phase C18 INERTSIL ODS column using 0.05% aqueous formic acid, acetonitrile in the ratio (50:50, v/v) as a mobile phase. Multiple reaction monitoring (MRM) was applied and operated by positive mode electrospray ionization. A significant difference between male and female Cmax (maximum plasma concentration) (P = 0.0205; CL = 95.4) was found using Mann-Whitney U test. Also, a moderate negative correlation was found between BMI and Tmax (time to peak plasma concentration) using spearman rho test. The calculated results confirm the difference of Rivastigmine pharmacokinetics between male and female subjects. Furthermore, it indicates that Rivastigmine dose adjustment may be necessary. The method was applied for the estimation of pharmacokinetic parameters in volunteers (n = 26, 17 male and 9 female) and the effects of gender and BMI were investigated.

Application of Box-Behnken experimental design and response surface methodology for selecting the optimum RP-HPLC conditions for the simultaneous determination of methocarbamol, indomethacin and betamethasone in their pharmaceutical dosage form.

An isocratic RP-HPLC method has been developed for the separation and determination of methocarbamol (MTL), indomethacin (IND), and betamethasone (BET) in combined dosage form using an Inertsil ODS-3v C18 (250 × 4.6 mm, 5 µm) column with UV- detection at 235 nm. Experimental design using Box-Behnken design (BBD) was applied to study the response surface during method optimization and to achieve a good separation with a minimum number of experimental runs. The three independent parameters were pH of buffer, % of acetonitrile and flow rate of the mobile phase while the peak resolution of IND from MTL and the peak resolution of BET from IND (R2) were taken as responses to obtain mathematical models. The composite desirability was employed to optimize a set of responses overall (peak resolutions). The predicted optimum assay conditions include a mobile phase composition of acetonitrile and phosphate buffer (pH 5.95) in a ratio of 79:21, v/v, pumped at a flow rate of 1.4 mL min-1. With this ideal condition, the optimized method was able to achieve baseline separation of the three drugs with good resolution and a total run time of less than 7 min. The linearity of MTL, IND, and BET was determined in the concentration ranges of 5-600 µg mL- 1, 5-300 µg mL- 1, and 5-300 µg mL- 1 and the regression coefficients were 0.9994, 0.9998, and 0.9998, respectively. The average percent recoveries for the accuracy were determined to be 100.41 ± 0.60%, 100.86 ± 0.86%, and 100.99 ± 0.65% for MTL, IND, and BET, respectively. The R.S.D.% of the intra-day precision was found to be less than 1%, while the R.S.D.% of the inter-day precision was found to be less than 2%. The RP-HPLC method was fully validated with regard to linearity, accuracy, precision, specificity, and robustness as per ICH recommendations. The proposed method has various applications in quality control and routine analysis of the investigated drugs in their pharmaceutical dosage forms and laboratory-prepared mixtures with the goal of reducing laboratory waste, analysis time, and effort.

Bioanalytical LC-MS/MS method for simultaneous estimation of atorvastatin, its major active metabolites and ezetimibe.

Background: Hyperlipidemia is one of the most common chronic diseases encountered globally, and atorvastatin (ATV) is mainly metabolized into two major active metabolites. Methodology: Hence, we aimed to estimate ATV and ezetimibe (EZE) simultaneously in the presence of ATV major and active metabolites using a validated LC-MS/MS method. Conclusion: The proposed method was linear (r2 >0.99), accurate (92.02-109.94%) and precise (CV% ≤14) over the concentration range of 0.50-120 ng/ml, 0.20-48 ng/ml, 0.50-120 ng/ml and 0.20-48 ng/ml for ATV, EZE, 2-hydroxy ATV and 4-hydroxy ATV, respectively. The applied liquid-liquid extraction gave rise to reliable extraction recoveries of 84.91 ± 1.14%, 85.20 ± 1.62%, 85.46 ± 0.41% and 105.46 ± 2.35% for ATV, EZE, 2-hydroxy ATV and 4-hydroxy ATV, respectively.

Study of gender-related pharmacokinetics of ezogabine in Egyptian volunteers by a validated LC-MS/MS bioanalytical method.

Gender-based pharmacokinetics and/or pharmacodynamics differences can result in differences in treatment which can accordingly affect the drug safety and/or efficacy. A new validated bio-analytical LC-MS/MS method was developed for the estimation of ezogabine, a third-generation antiepileptic drug, in human plasma using oxcarbazepine as an internal standard (IS) and to study the gender effect on the pharmacokinetic parameters in Egyptian human subjects. Liquid-liquid extraction of plasma samples was performed with diethyl ether: dichloromethane. The separation was accomplished in an isocratic mode with a mobile phase of a mixture of 5 mM ammonium acetate: methanol: acetonitrile pumped on a reversed phase C18 INERTSIL ODS-3 (5 µm, 150 × 4.6 mm). Multiple reaction monitoring was applied and operated by positive mode electrospray ionization. Male and female Cmax (p = 0.0308; CL = 95) and t1/2 (p = 0.0301; CL = 95) were found to be significantly different using Mann-Whitney U test. These findings highlight the difference of ezogabine pharmacokinetics among populations. Further, gender-based ezogabine dose adjustment may be considered.

Bio-analytical methods for investigating the effect of age, body mass index and gender on the PK/PD ratio of antibiotics.

The effectiveness of antibiotics (ABs) is governed by achieving the adequate pharmacokinetic (PK)/pharmacodynamics (PD) ratio. In this study, fast LC-MS/MS methods were developed and validated for the bioanalysis of cefaclor (CFC), ciprofloxacin (CFC), roxithomycin (RXM) and clindamycin (CLD). Chromatographic separation was performed on a C18 Zorbax-Eclipse Plus (3.5 µm, 100 × 4.6 mm) using isocratic elution. Detection was performed by positive electrospray ionization. The methods were applied for the assessment of PK parameters in volunteers (n = 101, 64 male and 37 female) and the effects of age, body mass index (BMI) and gender were investigated. Good linearity (r2 ≥ 0.99), accuracy (>86%), precision (CV% ≤ 11) and extraction recovery (>83%) were observed for CFC, CFX, RXM and CLD. Application to PK studies revealed that age and BMI affected the Thalf and the AUC of RXM and CLD (p < 0.023). Gender difference affected the critical PK parameters of the four ABs (Thalf (U = 18; P = 0.036) of CFC, the Cmax of CFX (U = 30; P = 0.017), the Thalf (U = 23; P = 0.009) and AUC (U = 26; P = 0.008) of RXM and CLD), respectively. These results highlight the significance of age and BMI variations for RXM and CLD dosing. Furthermore, it indicates that the gender difference may be considered when adjusting the AB dose.

Validated Liquid Chromatographic Method for the Determination of (Canagliflozin, Dapagliflozin or Empagliflozin) and Metformin in the Presence of (1-Cyanoguanidine).

A simple and accurate liquid chromatographic method has been developed and validated for the determination of either canagliflozin, dapagliflozin propandiol monohydrate or empagliflozin and metformin in presence of metformin major degradation product;1-cyanoguanidine. The Liquid Chromatographic (LC) method was based on isocratic elution on Prontosil (Lichrosorb 100-5-NH2) column using a mobile phase consisting of NaH2PO4 buffer (10 mM, pH 2.8):acetonitrile (18.5:81.5, v/v), at a flow rate of 2 mL/min-1. Quantitation was achieved with UV detection at 225 nm. The validation of the method was assessed according to International Conference on Harmonization (ICH) guidelines. Linearity, accuracy and precision were satisfactory over the concentration ranges of 12.5-100, 3.75-30, 0.3075-2.46, and 0.3125-2.5 µg/mL for metformin HCl, canagliflozin, dapagliflozin propandiol monohydrate and empagliflozin, respectively. Limits of detection and quantitation were found to be 0.068, 0.135, 0.077 and 0.069 µg/mL and 0.206, 0.410, 0.233 and 0.210 µg/mL for metformin HCl, canagliflozin, dapagliflozin propandiol monohydrate and empagliflozin, respectively. The developed method is suitable for the quality control and routine analysis of the cited drugs separately or in combinations.

Indirect synchronous fluorescence spectroscopy and direct high-performance thin-layer chromatographic methods for enantioseperation of zopiclone and determination of chiral-switching eszopiclone: Evaluation of thermodynamic quantities of chromatographic separation.

Economic and enantioselective synchronous fluorescence spectroscopy and high-performance thin-layer chromatography methods have been developed and validated as per ICH guidelines for the separation of zopiclone enantiomers using L-(+)-tartaric acid as a chiral selector, followed by determination of the chiral-switching eszopiclone. Synchronous fluorescence spectroscopy was successfully applied for chiral recognition of R & S enantiomers of zopiclone at ∆λ = 110 nm based on creating of diastereomeric complexes with 0.06M tartaric acid in an aqueous medium containing 0.2M disodium hydrogen orthophosphate. Synchronous fluorescence intensities of eszopiclone were recorded at 296 nm in concentration range 0.2- to 4-µg/mL eszopiclone. High-performance thin-layer chromatography method depends on resolution of zopiclone enantiomers on achiral HPTLC silica-gel plates using acetonitrile:methanol:water (8:2:0.25, v/v/v) containing L-(+)-tartaric acid as a chiral mobile-phase additive followed by densitometric measurements at 304 nm in concentration range of 1 to 10 µg/band of eszopiclone. The effect of chiral-selector concentration, pH, and temperature on the resolution have been studied and optimized for the proposed methods. The cited procedures were successfully applied to determine eszopiclone in commercial tablets of pure and racemic forms. Enantiomeric excess was evaluated using optical purity test and integrated peak area to describe the enantiomeric ratio. Thermodynamics of chromatographic separation, enthalpy, and entropy were evaluated using the Van't Hoff equation. The proposed methods were found to be selective for identification and determination of the eutomer in drug substances and products.

Development and Validation of Spectrophotometric Methods for the Determination of Canagliflozin or Gliclazide and Metformin in the Presence of Metformin Impurity (1-Cyanoguanidine).

Background: Quantitative multicomponent analysis is considered an analytical goal to save time and cost in analysis. Objective: This work aimed to provide sensitive and selective [successive ratio subtraction coupled with constant multiplication (SRS-CM) and chemometric] methods for the determination of coformulated antidiabetic drugs, namely canagliflozine and metformin or gliclazide and metformin in presence of metformin degradation product, 1-cyanoguanidine. Methods: SRS-CM method was developed for the determination of canagliflozin and metformin at 292 and 237 nm, respectively, using 14 µg/mL canagliflozin as a divisor in the first step to cancel the spectrum of canagliflozin. Then, 18 µg/mL metformin was used as a divisor in the second step to cancel the spectrum of metformin. Finally, we obtained the spectrum of cyanoguanidine. Chemometric method was applied for the determination of the gliclazide and metformin mixture in a 225-235 nm range. Sample enrichment technique was used to increase the concentration of canagliflozin or gliclazide to allow its simultaneous determination with metformin without prior separation. Results: Validation parameters according to the International Conference on Harmonization guidelines were satisfactory over the concentration ranges of 5 to 16 µg/mL for canagliflozin and metformin as well as 2.5 to 12.5 and 6 to 24 µg/mL for gliclazide and metformin, respectively. Conclusions: The method provides sufficient selectivity and accuracy to be applied for routine analysis and quality control in laboratories for the cited drugs. Highlights: This work shows two examples to how to select a suitable UV spectrophotometric method to overcome the spectral overlap.

Optimized bio-analytical methods development and comparative pharmacokinetic studies of four antidepressants in Egyptian population based on gender difference.

The pharmacokinetics (PK) and pharmacodynamics of many oral antidepressants (OADs) vary substantially among different genders and ethnicities. Likewise is their therapeutic effectiveness, time to response and the incidence of adverse drug events. The aim of this study is to compare the PK of four OADs (desvenlafaxine; DSV, venlafaxine; VLX, escitalopram; ESP, and agomelatine; AGT) among Egyptian males and females. In this study, LC-MS/MS methods were developed and validated for determining the four OADs in human plasma. Samples were prepared by liquid-liquid extraction. Chromatographic separation was performed on reversed-phase C18 columns followed by positive-ion electrospray ionization and MS/MS detection. The assays were applied for the assessment of PK parameters in human volunteers (n = 95). The developed methods were linear, accurate, and precise for the determination of DSV, VLX, ESP and AGT with extraction recovery of 90 ±â€¯2.0, 98 ±â€¯1.0, 90 ±â€¯1.3 and 87 ±â€¯4.3%, respectively. OADs levels were successfully measured in subjects' plasma and PK parameters were calculated. A prevalent inter-individual variation in PK of the studied OAD was observed. The PK profile of DSV, VLX or ESP did not vary significantly between male and female subjects (p = 0.07-0.98; confidence level (CL) = 95) while the PK of AGT exhibited a significant gender-based variation in both the Cmax and the AUC∞ (p = 0.047 and 0.0015; CL = 95). Our results highlight the significance of therapeutic drug monitoring of OADs. Further, it indicates the dose adjustment based on gender difference may not be relevant for DSV, VLX and ESP while it may be considered for AGT.

Rapid bioanalytical LC-MS/MS method for the simultaneous determination of sofosbuvir and velpatasvir in human plasma-application to a pharmacokinetic study in Egyptian volunteers.

A novel, rapid and validated LC-MS/MS bioanalytical method has been developed for the extraction and determination of sofosbuvir and velpatasvir simultaneously in human plasma using ledipasvir as an internal standard (IS). The simple and reproducible protein precipitation technique with acetonitrile was successfully used for the deproteinization and extraction of the analytes from human plasma matrix. The developed method achieved consistent recoveries over different concentrations with average extraction recoveries of 81.72% and 80.46% for sofosbuvir and velpatasvir, respectively. The chromatographic separation was performed within only 2.80 min as a run time by an isocratic elution through C18 Zorbox eclipse plus (100 × 4.6 mm, 5 µm). Optimum mobile phase consisted of 0.1% formic acid in water: acetonitrile: methanol (30:60:10, v/v/v) pumped at a flow rate of 0.55 mL min-1 and injection volume was 10 µL. LC-MS/MS detection was done by multiple reaction monitoring transitions operating at positive ionization mode for both analytes and IS. Bioanalytical method validation as per EMA guidelines was carried out where the proposed method revealed linearity over the concentration range of 5-5000 and 10-1500 ng mL-1 for sofosbuvir and velpatasvir, respectively. After validation, the method was applied to the analysis of the two drugs after a single oral administration of Epclusa 400/100 mg tablets to Egyptian healthy volunteers.

Simultaneous HPLC Determination of Betamethasone Esters-Containing Mixtures: Analysis of Their Topical Preparations.

Topical pharmaceutical preparations containing betamethasone esters are widely prescribed for treatment of severe inflammatory skin conditions. Some betamethasone esters-containing preparations are formulated with either an antibacterial or an antifungal agent or a vitamin D3 derivative. A fast reversed-phase high-performance liquid chromatography method has been developed for the simultaneous determination of three betamethasone esters-containing binary mixtures along with the excipients of their dosage forms using clobetasone butyrate as internal standard. The first mixture was betamethasone valerate and fusidic acid (Mixture I) with chlorocresol as preservative. The second mixture was betamethasone dipropionate (BTD) and clotrimazole (Mixture II) with benzyl alcohol as preservative. The third mixture was BTD and calcipotriol monohydrate (Mixture III). Optimized chromatographic separation was achieved on a Discovery® C18 (4.6 × 250 mm, 5 µm) column, using water: acetonitrile (35:65, v/v) as mobile phase at flow rate of 1 mL/min with UV detection at 230 nm. The method was validated according to ICH guidelines. The regression coefficients were > 0.999 for all drugs. The method was successfully applied for the determination of the studied drugs in bulk, synthetic mixtures and dosage forms. The developed method is accurate, sensitive, selective and precise and can be used for routine analysis in quality control laboratories.

LC-MS/MS bioassay of four proton pump inhibitors.

A new validated bio-analytical LC-MS/MS method was developed for the simultaneous extraction and determination of four proton pump inhibitors: esomeprazole, lansoprazole, pantoprazole and rabeprazole in human plasma using escitalopram as an internal standard. The proteins in plasma samples were precipitated using acetonitrile for the extraction of analytes which is a simple economic method. The separation was accomplished using a mobile phase composed of 10 mM ammonium formate: acetonitrile: methanol (20:40:40% v/v) at a flow rate of 0.8 mL/min in isocratic mode on a reversed phase C18 INERTSIL ODS-3 (5 µm, 150 × 4.6 mm) and column temperature of 40 °C. Positive mode electrospray ionization source was used prior to multiple reaction monitoring (MRM) detection using parent and daughter ions: m/z 346.2 → 198.1 for esomeprazole, m/z 370.1 → 252 for lansoprazole, m/z 384.2 → 200.2 for pantoprazole, m/z 360.1 → 242.1 for rabeprazole and m/z 325.2 → 109 for escitalopram. The calibration curves were constructed, and the method was linear in the range of 20-5000 ng/mL applying weighted (1/X2) linear regression coefficient for all drugs. The method was fully validated following US-FDA and EMA guidelines.

A Versatile Liquid Chromatographic Method for the Simultaneous Determination of Metformin, Sitagliptin, Simvastatin, and Ezetimibe in Different Dosage Forms.

A new LC method is introduced with the concept of its versatile application to widely used drugs from different pharmacological classes. Metformin hydrochloride (MTF), sitagliptin phosphate (SIT), simvastatin (SIM) and ezetimibe (EZB) were simultaneously determined with a simple reversed-phase LC method in which a SIT-SIM binary mixture, present in a dosage form brand, was considered central for its development. Chromatographic separation was achieved with a mobile phase of acetonitrile and 0.02 M potassium dihydrogen phosphate (pH 5.2) (77 + 23, v/v) flowing through a C18 column (BDS Hypersil, 250 × 4.6 mm, 5 µm) at 1.2 mL/min at ambient temperature. UV detection was programmed to be carried out at 210 nm for EZB, SIT, and MTF, whereas SIM was detected at 240 nm. The method was validated according to International Conference on Harmonization guidelines. Linearity, accuracy, and precision were satisfactory over concentration ranges 4-40 µg/mL for EZB and SIM, 0.5-50 µg/mL for SIT, and 5-500 µg/mL for MTF. Coefficients of determination were >0.99 for the four drugs. LOQs found were 0.01 µg/mL for EZB, 0.02 µg/mL for SIT, 0.2 µg/mL for MTF, and 0.02 µg/mL for SIM. The developed method is simple, rapid, accurate, precise, and suitable for the routine QC analysis of the cited drugs in pharmaceutical products by conventional HPLC systems.

Simultaneous spectrophotometric determination of glimepiride and pioglitazone in binary mixture and combined dosage form using chemometric-assisted techniques.

In the present work, pioglitazone and glimepiride, 2 widely used antidiabetics, were simultaneously determined by a chemometric-assisted UV-spectrophotometric method which was applied to a binary synthetic mixture and a pharmaceutical preparation containing both drugs. Three chemometric techniques - Concentration residual augmented classical least-squares (CRACLS), principal component regression (PCR), and partial least-squares (PLS) were implemented by using the synthetic mixtures containing the two drugs in acetonitrile. The absorbance data matrix corresponding to the concentration data matrix was obtained by the measurements of absorbencies in the range between 215 and 235nm in the intervals with Δλ=0.4nm in their zero-order spectra. Then, calibration or regression was obtained by using the absorbance data matrix and concentration data matrix for the prediction of the unknown concentrations of pioglitazone and glimepiride in their mixtures. The described techniques have been validated by analyzing synthetic mixtures containing the two drugs showing good mean recovery values lying between 98 and 100%. In addition, accuracy and precision of the three methods have been assured by recovery values lying between 98 and 102% and R.S.D. % ˂0.6 for intra-day precision and ˂1.2 for inter-day precision. The proposed chemometric techniques were successfully applied to a pharmaceutical preparation containing a combination of pioglitazone and glimepiride in the ratio of 30: 4, showing good recovery values. Finally, statistical analysis was carried out to add a value to the verification of the proposed methods. It was carried out by an intrinsic comparison between the 3 chemometric techniques and by comparing values of present methods with those obtained by implementing reference pharmacopeial methods for each of pioglitazone and glimepiride.