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.

Repurposing of Omarigliptin as a Neuroprotective Agent Based on Docking with A2A Adenosine and AChE Receptors, Brain GLP-1 Response and Its Brain/Plasma Concentration Ratio after 28 Days Multiple Doses in Rats Using LC-MS/MS.

The authors in the current work suggested the potential repurposing of omarigliptin (OMR) for neurodegenerative diseases based on three new findings that support the preliminary finding of crossing BBB after a single dose study in the literature. The first finding is the positive results of the docking study with the crystal structures of A2A adenosine (A2AAR) and acetylcholine esterase (AChE) receptors. A2AAR is a member of non-dopaminergic GPCR superfamily receptor proteins and has essential role in regulation of glutamate and dopamine release in Parkinson's disease while AChE plays a major role in Alzheimer's disease as the primary enzyme responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine into choline and acetate. Docking showed that OMR perfectly fits into A2AAR binding pocket forming a distinctive hydrogen bond with Threonine 256. Besides other non-polar interactions inside the pocket suggesting the future of the marketed anti-diabetic drug (that cross BBB) as a potential antiparkinsonian agent while OMR showed perfect fit inside AChE receptor binding site smoothly because of its optimum length and the two fluorine atoms that enables quite lean fitting. Moreover, a computational comparative study of OMR docking, other 12 DPP-4 inhibitors and 11 SGLT-2 inhibitors was carried out. Secondly, glucagon-like peptide-1 (GLP-1) concentration in rats' brain tissue was determined by the authors using sandwich GLP-1 ELISA kit bio-analysis to ensure the effect of OMR after the multiple doses' study. Brain GLP-1 concentration was elevated by 1.9-fold following oral multiple doses of OMR (5 mg/kg/day, p.o. for 28 days) as compared to the control group. The third finding is the enhanced BBB crossing of OMR after 28 days of multiple doses that had been studied using LC-MS/MS method with enhanced liquid-liquid extraction. A modified LC-MS/MS method was established for bioassay of OMR in rats' plasma (10-3100 ng/mL) and rats' brain tissue (15-2900 ng/mL) using liquid-liquid extraction. Alogliptin (ALP) was chosen as an internal standard (IS) due to its LogP value of 1.1, which is very close to the LogP of OMR. Extraction of OMR from samples of both rats' plasma and rats' brain tissue was effectively achieved with ethyl acetate as the extracting solvent after adding 1N sodium carbonate to enhance the drug migration, while choosing acetonitrile to be the diluent solvent for the IS to effectively decrease any emulsion between the layers in the stated method of extraction. Validation results were all pleasing including good stability studies with bias of value below 20%. Concentration of OMR in rats' plasma were determined after 2 h of the latest dose from 28 days multiple doses, p.o, 5 mg/kg/day. It was found to be 1295.66 ± 684.63 ng/mL estimated from the bio-analysis regression equation. OMR passed through the BBB following oral administration and exhibited concentration of 543.56 ± 344.15 ng/g in brain tissue, taking in consideration the dilution factor of 10. The brain/plasma concentration ratio of 0.42 (543.56/1295.66) was used to illustrate the penetration power through the BBB after the multiple doses for 28 days. Results showed that OMR passed through the BBB more effectively in the multiple dose study as compared to the previously published single dose study by the authors. Thus, the present study suggests potential repositioning of OMR as antiparkinsonian agent that will be of interest for researchers interested in neurodegenerative diseases.

Development of Advanced Chemometric-Assisted Spectrophotometric Methods for the Determination of Cromolyn Sodium and Its Alkaline Degradation Products.

Advanced and sensitive spectrophotometric and chemometric analytical methods were successfully established for the stability-indicating assay of cromolyn sodium (CS) and its alkaline degradation products (Deg1 and Deg2). Spectrophotometric mean centering ratio spectra method (MCR) and chemometric methods, including principal component regression (PCR) and partial least square (PLS-2) methods, were applied. Peak amplitudes after MCR at 367.8 nm, 373.8 nm and 310.6 nm were used within linear concentration ranges of 2-40 µg mL-1, 5-40 µg mL-1 and 10-100 µg mL-1 for CS, Deg1 and Deg2, respectively. For PCR and PLS-2 models, a calibration set of eighteen mixtures and a validation set of seven mixtures were built for the simultaneous determination of CS, Deg1 and Deg2 in the ranges of 5-13 µg mL-1, 8-16 µg mL-1, and 10-30 µg mL-1, respectively. The authors emphasize the importance of a stability-indicating strategy for the investigation of pharmaceutical products.

Structural re-positioning, in silico molecular modelling, oxidative degradation, and biological screening of linagliptin as adenosine 3 receptor (ADORA3) modulators targeting hepatocellular carcinoma.

Chemical entities with structural diversity were introduced as candidates targeting adenosine receptor with different clinical activities, containing 3,7-dihydro-1H-purine-2,6-dione, especially adenosine 3 receptors (ADORA3). Our initial approach started with pharmacophore screening of ADORA3 modulators; to choose linagliptin (LIN), approved anti-diabetic drug as Dipeptidyl peptidase-4 inhibitors, to be studied for its modulating effect towards ADORA3. This was followed by generation, purification, analytical method development, and structural elucidation of oxidative degraded product (DEG). Both of LIN and DEG showed inhibitory profile against hepatocellular carcinoma cell lines with induction of apoptosis at G2/M phase with increase in caspase-3 levels, accompanied by a downregulation in gene and protein expression levels of ADORA3 with a subsequent increase in cAMP. Quantitative in vitro assessment of LIN binding affinity against ADORA3 was also performed to exhibit inhibitory profile at Ki of 37.7 nM. In silico molecular modelling showing binding affinity of LIN and DEG towards ADORA3 was conducted.

Factorial design optimization of micelle enhanced synchronous spectrofluorimetric assay of Omarigliptin: Applied to content uniformity testing and in vitro drug release.

A micelle enhanced spectrofluorimetric method was developed for determination of Omarigliptin (OMG) based on its native fluorescence behavior. The interaction of OMG with surfactants and macromolecules was studied. In aqueous solution, the relative fluorescence intensity (RFI) of OMG was enhanced by 24% in the presence of Tween 80 at pH 3.5. The optimal conditions for the micelle enhanced fluorescence were attained by Minitab® program using Plackett-Burman factorial design. Pareto chart, contour plots and surface plots were used to exclude the insignificant variables and optimize the significant factors. The spectrofluorimeter was operated under synchronous mode using ∆λ = 30 nm and recording the RFI of the intense narrow band at 267 nm for OMG in 0.5% w/v Tween 80 + 0.2 M acetate buffer (pH 3.5) system using water as diluent. Using synchronous scan mode offered many advantages including considerable reduction of spectral overlap and enhanced linearity of the calibrators. Validation parameters were satisfied over the concentration range 0.1-2 µg/ml. The developed method was the first analytical procedure for OMG assay in Marizev® tablets. Moreover, content uniformity testing and in vitro drug release of tablets were performed.

A stability-indicating HPLC assay of ten different vitamins in a food supplement: Appraisal of the method's greenness, whiteness, and blueness.

Due to their susceptibility to degradation, vitamin levels in food formulations may differ from those found in the finished product. Vitamin levels can be impacted by processing and storage. In this work, the ingredients of Strong B50 ® film-coated tablets were estimated simultaneously using simple efficient stability indicating HPLC method. Strong B50 ® film-coated tablets contain thiamine (VB1), riboflavin (VB2), calcium pantothenate (VB5), pyridoxine (VB6), vitamin C (VC), folic acid (FA), biotin (BT), inositol (IS), niacin (NC), para-aminobenzoic acid (PB), cyanocobalamine (B12), choline bitartarate, and iron gluconate. Hypersil BDS C18 column was used for achieving reasonable separation. Mobile phases (A) and (B) were utilized, the mobile phase (A) consisted of 0.015 M Hexane sulfonic acid sodium salt + 0.1 % Triethylamine and orthophosphoric acid was used to adjust the pH to (2.9) while (B) system consisted of acetonitrile. Validation of the method was assessed using International Conference of Harmonization (ICH) parameters, where linearity, accuracy, selectivity, and robustness of the method were investigated. Correlations were above 0.99, accuracy results ranged from 97.6 to 102.8 % and limits for detection and quantitation (LOD and LOQ) values were determined for each vitamin in µg/mL except for FA and BT in ng/mL. LOD values were between 0.006 and 15.08 µg/mL while LOQ values ranged from 0.031 to 49.77 µg/mL. Stability studies were conducted under stressed conditions and degradation percentages were computed. Where, VB5, VB6, FA and PB, VC, and NC were the most degradable vitamins. Whiteness evaluation using the modern RGB 12 algorithm compared our method and the old reported one by Sasaki et al., 2020. The comparison favored our newly developed method in terms of analytical performance, practical applicability and greenness. Besides, AGREE and GAPI soft wares were used to assess the greenness of the method. It was clear that the results of colored pictograms confirm low hazardous impact and that the new method is greener with AGREE score of 0.66. Furthermore, the functionality and applicability of the novel HPLC approach were concluded via the Blue Applicability Grade Index (BAGI) tool with a final score of 82.5.

Omarigliptin attenuates rotenone-induced Parkinson's disease in rats: Possible role of oxidative stress, endoplasmic reticulum stress and immune modulation.

The current study aimed to explore the potential neuroprotective effect of omarigliptin (OG), an antidiabetic drug that crosses the blood-brain barrier (BBB), in a Parkinson's disease (PD) rotenone-based rat-model. Results showed that OG attenuated motor impairment, histological aberrations, α-synuclein accumulation, and rescued the dopaminergic neurons in rotenone-administered rats. Furthermore, OG halted rotenone-induced oxidative stress; as shown by reduced lipid peroxidation, decline in the oxidative stress sensor (nuclear factor erythroid 2-related factor 2) and its downstream heme oxygenase-1. In addition, OG abrogated neuroinflammation and apoptosis in rotenone-treated rats. Moreover, OG ameliorated endoplasmic reticulum (ER) stress in rotenone-administered rats; as evidenced by reduced levels of ER resident proteins such as glucose-regulated protein 78, C/EBP homologous protein and apoptotic caspase-12. In conclusion, this study implies repurposing of OG, as a novel neuroprotective agent due to its antioxidant properties, its effects on ER stress in addition to its anti-inflammatory and anti-apoptotic activities.

Investigation of Pharmacokinetic Parameters of Trelagliptin in Egyptian Volunteers Using Sensitive LC-MS/MS: A Comparative Study with a Japanese Population.

Trelagliptin (TLN) is a novel once-weekly antidiabetic drug that enhanced the patient compliance in type 2 diabetes. TLN analysis and bioanalysis literature review showed many methods for TLN assay either in dosage form or as biological fluids (pharmacokinetic parameters), but all those methods did not consider the full details dealing with biological assay of TLN. Studies that included information about pharmacokinetic parameters did not mention the used analytical procedures for those determinations and parameters. Although some LC-MS/MS and UPLC-UV methods were reported for TLN bioassay in rats' plasma, they used direct precipitation techniques, and the current described procedure showed lower LLOQ than all the reported methods in spite of that working on human plasma is more complicated than on rats' plasma. In this study, LC-MS/MS bioanalysis of TLN in human plasma (4-1000 nM) was employed successfully with LLOQ of 4 nM which is lower than all reported methods in rats' plasma followed by a preliminary pharmacokinetic study. Alogliptin was used as internal standard (IS) because of its structure similarity to TLN. Pharmacokinetic parameters of TLN were investigated in Egyptian volunteers, and they had been compared to Japanese. Liquid-liquid extraction showed more sensitive results than direct precipitation. The proposed method was successfully applied to a pharmacokinetic study conducted on Egyptian volunteers. No dose modification is required upon comparing the pharmacokinetic parameters of the current study and previous studies on non-Egyptian volunteers.

Repositioning of dipeptidyl peptidase-4 inhibitors and glucagon like peptide-1 agonists as potential neuroprotective agents.

Repositioning of dipeptidyl peptidase-4 inhibitors and glucagon like peptide-1 receptor agonists is a breakthrough in the field of neural regeneration research increasing glucagon like peptide-1 bioavailability, hence its neuroprotective activities. In this article, the authors suggest not only crossing blood-brain barrier and neurodegenerative disease as off target for dipeptidyl peptidase-4 inhibitors and glucagon like peptide-1 receptor agonists, but also for ophthalmic preparations for diabetic retinopathy, which may be the latest breakthrough in the field if prepared and used in an appropriate nano-formulation to target the retinal nerves. The relation of neurodegenerative diseases' different mechanisms to the dipeptidyl peptidase-4 inhibitors and glucagon like peptide-1 receptor agonists should be further examined in preclinical and clinical settings. The repositioning of already marketed antidiabetic drugs for neurodegenerative diseases should save the high cost of the time-consuming normal drug development process. Drug repositioning is a hot topic as an alternative to molecular target based drug discovery or therapeutic switching. It is a relatively inexpensive pathway due to availability of previous pharmacological and safety data. The glucagon like peptide-1 produced in brain has been linked to enhanced learning and memory functions as a physiologic regulator in central nervous system by restoring insulin signaling. Intranasal administration of all marketed gliptins (or glucagon like peptide-1 receptor agonists) may show enhanced blood-brain barrier crossing and increased glucagon like peptide-1 levels in the brain after direct crossing of the drug for the olfactory region, targeting the cerebrospinal fluid. Further blood-brain barrier crossing tests may extend dipeptidyl peptidase-4 inhibitors' effects beyond the anti-hyperglycemic control to intranasal spray, intranasal powder, or drops targeting the blood-brain barrier and neurodegenerative diseases with the most suitable formula. Moreover, novel nano-formulation is encouraged either to obtain favorable pharmacokinetic parameters or to achieve promising blood-brain barrier penetration directly through the olfactory region. Many surfactants should be investigated either as a solubilizing agent for hydrophobic drugs or as penetration enhancers. Different formulae based on in vitro and in vivo characterizations, working on sister gliptins (or glucagon like peptide-1 receptor agonists), different routes of administration, pharmacokinetic studies, dose response relationship studies, monitoring of plasma/brain concentration ratio after single and multiple dose, and neurodegenerative disease animal models are required to prove the new method of use (utility) for dipeptidyl peptidase-4 inhibitors as potential neuroprotective agents. Furthermore, investigations of glucagon like peptide-1 receptor agonists' neuroprotective effects on animal models will be considered carefully because they crossed the blood-brain barrier in previous studies, enabling their direct action on the central nervous system. Combination therapy of dipeptidyl peptidase-4 inhibitors or glucagon like peptide-1 receptor agonists with already marketed drugs for neurodegenerative disease should be considered, especially regarding the novel intranasal route of administration.

Different Spectrophotometric Methods for Simultaneous Determination of Trelagliptin and Its Acid Degradation Product.

New spectrophotometric and chemometric methods were carried out for the simultaneous assay of trelagliptin (TRG) and its acid degradation product (TAD) and applied successfully as a stability indicating assay to recently approved Zafatek® tablets. TAD was monitored using TLC to ensure complete degradation. Furthermore, HPLC was used to confirm dealing with one major acid degradation product. The proposed methods were developed by manipulating zero-order, first-derivative, and ratio spectra of TRG and TAD using simultaneous equation, first-derivative, and mean-centering methods, respectively. Using Spectra Manager II and Minitab v.14 software, the absorbance at 274 nm-260.4 nm, amplitudes at 260.4 nm-274.0 nm, and mean-centered values at 287.6 nm-257.2 nm were measured against methanol as a blank for TRG and TAD, respectively. Linearity and the other validation parameters were acceptable at concentration ranges of 5-50 µg/mL and 2.5-25 µg/mL for TRG and TAD, respectively. Using one-way analysis of variance (ANOVA), the optimized methods were compared and proved to be accurate for the simultaneous assay of TRG and TAD.

Repositioning of Omarigliptin as a once-weekly intranasal Anti-parkinsonian Agent.

Drug repositioning is a revolution breakthrough of drug discovery that presents outstanding privilege with already safer agents by scanning the existing candidates as therapeutic switching or repurposing for marketed drugs. Sitagliptin, vildagliptin, saxagliptin & linagliptin showed antioxidant and neurorestorative effects in previous studies linked to DPP-4 inhibition. Literature showed that gliptins did not cross the blood brain barrier (BBB) while omarigliptin was the first gliptin that crossed it successfully in the present work. LC-MS/MS determination of once-weekly anti-diabetic DPP-4 inhibitors; omarigliptin & trelagliptin in plasma and brain tissue was employed after 2 h of oral administration to rats. The brain/plasma concentration ratio was used to deduce the penetration power through the BBB. Results showed that only omarigliptin crossed the BBB due to its low molecular weight & lipophilic properties suggesting its repositioning as antiparkinsonian agent. The results of BBB crossing will be of interest for researchers interested in Parkinson's disease. A novel intranasal formulation was developed using sodium lauryl sulphate surfactant to solubilize the lipophilic omarigliptin with penetration enhancing & antimicrobial properties. Intranasal administration showed enhanced brain/plasma ratio by 3.3 folds compared to the oral group accompanied with 2.6 folds increase in brain glucagon-like peptide-1 concentration compared to the control group.

Green Pharmaceutical Analysis of Drugs Coformulated with Highly Different Concentrations Using Spiking and Manipulation of Their Ratio Spectra.

Introducing green analysis to pharmaceutical products is considered a significant approach to preserving the environment. This method can be an environmentally friendly alternative to the existing methods, accompanied by a validated automated procedure for the analysis of a drug with the lowest possible number of samples. Different simple spectrophotometric methods were developed for the simultaneous determination of empagliflozin (EG) and metformin (MT) by manipulating their ratio spectra in their application on a recently approved pharmaceutical combination, Synjardy tablets. A spiking technique was used to increase the concentration of EG in samples prepared from the tablets to allow for the simultaneous determination of EG with MT without prior separation. Validation parameters according to International Conference on Harmonization guidelines were acceptable over a concentration range of 2-12 µg/mL for both drugs using derivative ratio and ratio subtraction coupled with extended ratio subtraction. The optimized methods were compared using one-way analysis of variance and proved to be suitable as ecofriendly approaches for industrial QC laboratories.

DOE Optimization of Nano-based Carrier of Pregabalin as Hydrogel: New Therapeutic &Chemometric Approaches for Controlled Drug Delivery Systems.

Niosomes entrapping pregabalin (PG) were prepared using span 60 and cholesterol in different molar ratios by hydration method, the remaining PG from the hydrating solution was separated from vesicles by freeze centrifugation. Optimization of nano-based carrier of pregabalin (PG) was achieved. Quality by Design strategy was successfully employed to obtain PG-loaded niosomes with the desired properties. The optimal particle size, drug release and entrapment efficiency were attained by Minitab® program using design of experiment (DOE) that predicted the best parameters by investigating the combined effect of different factors simultaneously. Pareto chart was used in the screening step to exclude the insignificant variables while response surface methodology (RSM) was used in the optimization step to study the significant factors. Best formula was selected to prepare topical hydrogels loaded with niosomal PG using HPMC and Carbopol 934. It was verified, by means of mechanical and rheological tests, that addition of the vesicles to the gel matrix affected significantly gel network. In vitro release and ex vivo permeation experiments were carried out. Delivery of PG molecules followed a Higuchi, non Fickian diffusion. The present work will be of interest for pharmaceutical industry as a controlled transdermal alternative to the conventional oral route.

LC-MS/MS Determination of Empagliflozin and Metformin.

A new LC-MS/MS method was developed for determination of empagliflozin and metformin. Bridged Ethylene Hybrid C18 column (50 mm × 2.1 mm, 1.7 µm), isocratic elution based on 0.1% aqueous formic acid:acetonitrile (75:25, v/v) as a mobile phase, column temperature at 55°C and flow rate at 0.2 mL min-1 were used. The mass spectrometer was operated under multiple reaction monitoring mode using electrospray ionization by monitoring the transition pairs (precursor to product ion) of m/z 451.04-71.07 for empagliflozin and m/z 130.11-71.14 for metformin in the positive mode. The validation parameters were acceptable over concentration ranges of 5-1,000 ng mL-1 and 50-25,000 ng mL-1 for empagliflozin and metformin, respectively. The optimized method was validated according to International Conference on Harmonization guidelines. Regression parameters, limit of detection, limit of quantification, accuracy, precision and pharmaceutical formulation analysis were investigated. The developed method was proved to be accurate for the quality control of recently approved Synjardy® tablets.

Suitability of various chromatographic and spectroscopic techniques for analysis and kinetic degradation study of trelagliptin.

Multifaceted comparative analytical methods for trelagliptin (TRL) were investigated, applied to ZAFATEK tablets and HPLC-UV was selected for a degradation kinetic study. UPLC-MS/MS (Method I), UPLC-UV (Method II), HPLC-UV (Method III), UHPLC-UV (Method IV) and direct UV (Method V) methods were developed. Methods (I-V) showed satisfactory results using TRL concentration ranges of 50-800 ng/mL, 2.5-80 µg/mL, 5-100 µg/mL, 5-100 µg/mL and 5-50 µg/mL, respectively. Multiple Reaction Monitoring (MRM) of the transition pairs of m/z 358.176 to 134.127 for TRL and m/z 340.18 to 116.08 for alogliptin (IS) were employed utilizing positive mode Electrospray Ionization (ESI). The degradation kinetic study (Method VI) was carried out using 1 N HCl based on three different temperatures (70 °C, 80 °C and 90 °C). Through the optimized method-3, a good chromatographic separation of TRL from its major degradation product was achieved. Arrhenius plot was used in the kinetic study and the apparent 1st order degradation rate constant (K), t1/2, t90, and the activation energies were calculated for each temperature and at 25 °C. The optimized UPLC-MS/MS method is suitable for further TRL assay either in biological fluids or in the presence of impurities.

Comparative study between different simple methods manipulating ratio spectra for the analysis of alogliptin and metformin co-formulated with highly different concentrations.

Different simple spectrophotometric methods were developed for simultaneous determination of alogliptin and metformin manipulating their ratio spectra with successful application on recently approved combination, Kazano® tablets. Spiking was implemented to detect alogliptin in spite of its low contribution in the pharmaceutical formulation as low quantity in comparison to metformin. Linearity was acceptable over the concentration range of 2.5-25.0µg/mL and 2.5-15.0µg/mL for alogliptin and metformin, respectively using derivative ratio, ratio subtraction coupled with extended ratio subtraction and spectrum subtraction coupled with constant multiplication. The optimized methods were compared using one-way analysis of variance (ANOVA) and proved to be accurate for assay of the investigated drugs in their pharmaceutical dosage form.

Pharmacokinetic Evaluation of Empagliflozin in Healthy Egyptian Volunteers Using LC-MS/MS and Comparison with Other Ethnic Populations.

The present study considered the pharmacokinetic evaluation of empagliflozin after administration to Egyptian volunteers, and the results were compared with other ethnic populations. The FDA recognizes that standard methods of defining racial subgroups are necessary to compare results across pharmacokinetic studies and to assess potential subgroup differences. The design of the study was as an open labeled, randomized, one treatment, one period, single dose pharmacokinetic study. The main pharmacokinetic parameters estimated were Cmax, Tmax, t1/2, elimination rate constant, AUC0-t and AUC0-inf. The insignificant difference in pharmacokinetic parameters between Egyptians and white German subjects suggests that no dose adjustment should be considered with administration of 25 mg empagliflozin to Egyptian population. A new LC-MS/MS method was developed and validated, allowing sensitive estimation of empagliflozin (25-600 ng mL-1) in human plasma using dapagliflozin as an internal standard (IS). The method was applied successfully on the underlying pharmacokinetic study with enhanced sample preparation that involved liquid-liquid extraction. Multiple Reaction Monitoring (MRM) of the transition pairs of m/z 449.01 to 371.21 for empagliflozin and m/z 407.00 to 328.81 for dapagliflozin (IS) was employed utilizing negative mode Electro Spray Ionization (ESI). The validated LC-MS/MS method is suitable for further toxicodynamic and bioequivalence studies.

Development and validation of simple spectrophotometric and chemometric methods for simultaneous determination of empagliflozin and metformin: Applied to recently approved pharmaceutical formulation.

New univariate spectrophotometric method and multivariate chemometric approach were developed and compared for simultaneous determination of empagliflozin and metformin manipulating their zero order absorption spectra with application on their pharmaceutical preparation. Sample enrichment technique was used to increase concentration of empagliflozin after extraction from tablets to allow its simultaneous determination with metformin without prior separation. Validation parameters according to ICH guidelines were satisfactory over the concentration range of 2-12µgmL(-1) for both drugs using simultaneous equation with LOD values equal to 0.20µgmL(-1) and 0.19µgmL(-1), LOQ values equal to 0.59µgmL(-1) and 0.58µgmL(-1) for empagliflozin and metformin, respectively. While the optimum results for the chemometric approach using partial least squares method (PLS-2) were obtained using concentration range of 2-10µgmL(-1). The optimized validated methods are suitable for quality control laboratories enable fast and economic determination of the recently approved pharmaceutical combination Synjardy® tablets.

Enhanced Chromatographic Determination of Nicotine in Human Plasma: Applied to Human Volunteers.

Development of enhanced UPLC-UV method for determination of nicotine in human plasma was achieved on a Symmetry(®) C18 column (100 mm × 2.1 mm, 2.2 µm) applying isocratic elution based on Methanol: Acetonitrile: Phosphate Buffer (pH: 2.7) with the ratio (20:30:50, v/v/v) as a mobile phase. The ultraviolet detector was operated at 260 nm. The mobile phase was pumped through the column at a flow rate of 0.2 mL min(-1). The column temperature was adjusted to 50ºC and the injection volume was 2 µL. Quinine was selected as an internal standard (IS) due to its structure similarity to nicotine having basic pyridine ring to optimize the liquid liquid extraction procedure using diethyl ether coupled with vacuum evaporation at 40°C. Validation parameters for nicotine were found to be acceptable over the concentration range of 2.5-50 ng ml(-1). The application of the proposed method on four healthy human volunteers was approved by the ethical committee. The study was carried out under fasting conditions and the concerned subjects were informed about the objectives and possible risks involved in the study. The proposed method proved to be simple and fast which is a major advantage to analyze large number of samples per day using the accelerated vacuum evaporation technique. The method showed satisfactory data for all the parameters tested within the limits for bioanalytical assays. The lower limit of quantification (LLOQ) permits the application of the method for further pharmacological and clinical studies.