Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences.

Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.

Eco-friendly monitoring of triclosan as an emerging antimicrobial environmental contaminant utilizing electrochemical sensors modified with CNTs nanocomposite transducer layer.

Environmental appearance of antimicrobials due to frequent use of personal care products as recommended by WHO can cause serious flare-up of antimicrobial resistance. In this work, three eco-friendly microfabricated copper solid-state sensors were developed for measuring triclosan in water. Multi-walled carbon nanotubes were incorporated in sensor 2 and 3 as hydrophobic conductive inner layer. Meanwhile, ß-cyclodextrin was incorporated in sensor 3 as an ionophore for selective binding of TCS in presence of interfering compounds. The obtained linear responses of sensors 1, 2 and 3 were (1 × 10- 8-1 × 10- 3 M), (1 × 10- 9-1 × 10- 3 M) and (1 × 10- 10- 1 × 10- 3 M), respectively. Limit of detection was 9.87 × 10- 9 M, 9.62 × 10- 10 M, and 9.94 × 10- 11 M, respectively. The miniaturized sensors were utilized for monitoring of triclosan in water samples.

Point-of-care diagnostics for rapid determination of prostate cancer biomarker sarcosine: application of disposable potentiometric sensor based on oxide-conductive polymer nanocomposite.

One of the most important reasons for an increased mortality rate of cancer is late diagnosis. Point-of-care (POC) diagnostic sensors can provide rapid and cost-effective diagnosis and monitoring of cancer biomarkers. Portable, disposable, and sensitive sarcosine solid-contact ion-selective potentiometric sensors (SC-ISEs) were fabricated as POC analyzers for the rapid determination of the prostate cancer biomarker sarcosine. Tungsten trioxide nanoparticles (WO3 NPs), polyaniline nanoparticles (PANI NPs), and PANI-WO3 nanocomposite were used as ion-to-electron transducers on screen-printed sensors. WO3 NPs and PANI-WO3 nanocomposite have not been investigated before as ion-to-electron transducer layers in potentiometric SC sensors. The designated sensors were characterized using SEM, XRD, FTIR, UV-VIS spectroscopy, and EIS. The inclusion of WO3 and PANI in SC sensors enhanced the transduction at the interface between the screen-printed SC and the ion-selective membrane, offering lower potential drift, a longer lifetime, shorter response time, and better sensitivity. The proposed sarcosine sensors exhibited Nernstian slopes over linear response ranges 10-3-10-7 M, 10-3-10-8 M, 10-5-10-9 M, and 10-7-10-12 M for control, WO3 NPs, PANI NPs, and PANI-WO3 nanocomposite-based sensors, respectively. From a comparative point of view between the four sensors, PANI-WO3 nanocomposite inclusion offered the lowest potential drift (0.5 mV h-1), the longest lifetime (4 months), and the best LOD (9.95 × 10-13 M). The proposed sensors were successfully applied to determine sarcosine as a potential prostate cancer biomarker in urine without prior sample treatment steps. The WHO ASSURED criteria for point-of-care diagnostics are met by the proposed sensors.

Smart Eco-Friendly Spectrophotometric Methods Resolving Highly Overlapping Spectra: Application to Veterinary Antibiotic Injections.

BACKGROUND: Over the last few years, mathematical manipulation has proved to be a very powerful means of successfully resolving severely overlapped spectra for various multicomponent mixtures. OBJECTIVE: Smart and environmentally friendly spectrophotometric determination approaches were used for two binary mixtures of fixed dose veterinary injections containing flunixin meglumine (FLU) combined with either florfenicol (FLR), or oxytetracycline HCl (OXY). METHODS: Regarding the first mixture, both FLU and FLR were determined by three successive resolution techniques, which were; constant multiplication coupled with spectrum subtraction (CM-SS), derivative ratio (DD1), and ratio difference (RD), and two progressive resolution techniques which were absorbance subtraction (AS) and amplitude modulation (AM). Also, graphical representation of concentration of the two drugs through concentration value (CNV) method was also applied. Concerning the second mixture, both FLU and OXY showed severely overlapped spectra and a comparative study was conducted for the determination of each drug by constant center (CC), ratio subtraction via amplitude difference coupled with spectrum subtraction (RS/AD-SS), constant value via amplitude difference (CV-AD), and advanced concentration value (ACV) methods. RESULTS: Calibration graphs of the first mixture were linear over the range 5-40 µg/mL for FLU, and 3-40 µg/mL for FLR. The proposed methods overcame the problem of the overlapped spectra and the presence of a minor component in the mixture. Regarding the second mixture, calibration graphs were linear over the range 2.5-24 µg/mL for FLU and 4-28 µg/mL for OXY. CONCLUSION: The proposed methods were successfully validated as per International Council for Harmonization (ICH) guidelines. The obtained results were statistically compared with the official or reported methods, showing no significant difference concerning accuracy and precision. The methods were evaluated for greenness by three different assessment tools: NEMI, analytical ecoscale, and GAPI. HIGHLIGHTS: The methods were successfully applied for the simultaneous determination of the two combinations in synthetic mixtures and their marketed antibiotic veterinary injections: Megluflor® and Floxon®.

Simultaneous quantitative determination of insulin aspart and insulin degludec in human plasma using simple shoot LC method.

Combining short-acting and long-acting insulin analogs was a real challenge that was overcome by NovoNordisk through the co-formulation of insulin aspart and insulin degludec in single-dosage form. The proposed study provides a simple, short, and reliable HPLC method with diode array detection that is developed and validated for the simultaneous determination of insulin aspart and insulin degludec in human plasma. The proposed method achieved good separation between the two analytes utilizing a C8 column at 35°C in a very short run time (6 min), with a simple, low-cost, and reliable extraction method through precipitation of plasma protein. Gradient elution was applied using a mobile phase consisting of 0.1 M sodium sulfate (pH 3.4) and acetonitrile. The method was validated according to EMA Guideline on Bioanalytical Validation. The proposed method had a linear range from 3.0 to 300 µg/mL for insulin aspart and from 3.5 to 300 µg/mL for insulin degludec. The intra- and inter-day precision of insulin aspart were 0.36-3.33% and 1.59-8.84%, respectively, and accuracy was between 10.06 and 3.09% The intra- and inter-day precision of insulin degludec were 0.29-1.93% and 0.89-5.14%, respectively, and accuracy was between -5.29 and 3.91%.

A comprehensive stability assessment of insulin degludec using New customized validated RP-HPLC and SEC-HPLC methods in an orthogonal testing protocol.

Stress testing of biopharmaceuticals plays an important role in preparation of their stability profiles through investigation of possible degradation pathways and identification of degradation products, so in this study Insulin Degludec which is a new generation ultra-long-acting basal insulin is subjected to stress conditions as different temperatures, different pH, oxidation, mechanical agitation, and repeated freeze and thaw cycles to generate possible degradation products and aggregation that are investigated by two new validated RP-HPLC and SEC-HPLC methods in addition to dynamic light scattering (DLS) and native polyacrylamide gel electrophoresis (Nu-PAGE). SEC-HPLC was used to investigate formation of aggregates whose results were correlated with those obtained from DLS and Nu-PAGE, while RP-HPLC was used to investigate any possible chemical modifications. The Proposed RP-method had limit of detection (LOD) and limit of quantitation (LOQ) of 0.012 mg/mL and 0.045 mg/mL respectively and accuracy of 99.22 ± 1.07 %, while the SEC methods had limit of detection (LOD) and limit of quantitation (LOQ) of 0.012 mg/mL and 0.031 mg/mL, respectively. The degradation pattern due to high temperature effect and oxidation is investigated by LC- tandem mass spectrometry. Results showed that Insulin degludec is highly stable under low temperature, mechanical agitation and repeated freeze and thaw stress conditions but elevated temperature and high acidic condition lead to formation of aggregates and also chemical modifications including deamidation, isomerization and oxidation. Such different chemical degradation pathways are due to presence of variable reactive moieties in Insulin degludec structure. Insulin degludec is highly vulnerable to oxidation at the sulfur containing cysteine residue in B chain in position B7 forming trioxidation derivative when exposed to hydrogen peroxide. Formation of A21-Asp and A18-Asp deamidated variants as well as B3-Asp and B3-isoAsp deamidated variants are prominent degradation pathways at neutral pH but at elevated temperature.

Sensitive Derivative Synchronous and Micellar Enhanced Ecofriendly Spectrofluorimetric Methods for the Determination of Atenolol, Diclofenac, and Triclosan in Drinking Tap Water.

BACKGROUND: Nowadays, emergence of unexpected contaminants in drinking water is a challenging environmental problem facing humanity. OBJECTIVE: Two eco-friendly spectrofluorimetric methods were proposed for the determination of three unexpected contaminants in drinking tap water. METHODS: The first method is first derivative synchronous spectrofluorimetric method which was developed for simultaneous determination of atenolol (ATN) and diclofenac (DCF) without prior separation at Δλ = 70 nm and at Δλ = 80 nm for ATN and DCF, respectively. The second method was based on using sodium dodecyl sulfate (SDS) as fluorescent enhancer of triclosan (TCS) native fluorescence. TCS exhibits enhanced fluorescence at λ emission = 600 nm upon excitation at λ excitation = 299.4 nm. Solid phase extraction was carried out in both methods. RESULTS: Linear calibration curves were obtained in concentration range of (4-3000 ng/mL) for ATN and (4-2000 ng/mL) for DCF, by measuring first derivative signal of fluorescence at 300 nm and 375.2 nm, respectively. TCS exhibits linear range (0.1-1 ng/mL) at 600 nm. Mean percentage recoveries were 101.04 ± 0.571, 99.66 ± 1.443, and 99.73 ± 0.566 for ATN, DCF, and TCS, respectively. CONCLUSIONS: Validation of both methods were performed according to the International Conference on Harmonization guidelines. Results obtained were statistically compared with published methods and no significant differences were found. The proposed methods' greenness is evaluated using analytical Eco-scale and Green Analytical Procedure Index. A greenness comparison with previously published methods has been performed. HIGHLIGHTS: Both methods were found to be eco-friendly and were successfully applied for the determination of the emerging contaminants in drinking tap water.

In situ monitoring of triclosan in environmental water with subnanomolar detection limits using eco-friendly electrochemical sensors modified with cyclodextrins.

The environmental emergence of unexpected contaminants has gained the attention of the scientific community. A broad spectrum antimicrobial compound named triclosan (TCS) was detected in the environment as an emerging contaminant. Owing to its inherent toxicity, we have proposed eco-friendly potentiometric liquid state sensors to be used for monitoring and quantifying TCS in environmental water samples. The proposed sensors have been optimized by modifying the inner filling solution using hydrophilic 2-hydroxypropyl ß-cyclodextrin as a complexing agent to be capable of minimizing the trans-membrane ion flux and hence improving the selective and sensitive determination of TCS in environmental matrices with low LOD values. The obtained linear response of the optimized sensor was (1 × 10-9 to 1 × 10-5 M) compared to the control sensor (1 × 10-8 to 1 × 10-4 M). The obtained limit of detection (LOD) value was found to be 9.86 × 10-10 M compared to 9.78 × 10-9 M of the control sensor. The modification of the inner filling solution of the sensor with 2-hydroxypropyl ß-cyclodextrin improves not only its sensitivity but also its response time to be only 5 seconds. The electrical performance of the proposed sensor was evaluated following IUPAC recommendations. Both the pH and temperature effects were studied and optimized. Two different greenness assessment tools, Analytical Eco-scale and Green Procedure Index, were adopted upon the evaluation of the proposed sensors' greenness.

Analytical comparability study of anti-CD20 monoclonal antibodies rituximab and obinutuzumab using a stability-indicating orthogonal testing protocol: Effect of structural optimization and glycoengineering.

Glycoengineering and biosimilarity are the key factors for growing, promising and progressive approaches in monoclonal antibodies development. In this study, the physicochemical stability of anti-CD20 rituximab (RTX); originator and biosimilar was compared to its glycoengineered humanized version; obinutuzumab (OBZ). An orthogonal stability-indicating protocol using a set of validated bioanalytical techniques; size exclusion high performance liquid chromatography (SE-HPLC), reversed phase liquid chromatography (RP-HPLC), quantitative gel electrophoresis by TapeStation, receptor binding assay and dynamic light scattering (DLS) was used to investigate the effect of different stress factors on the pattern and kinetics of degradation. SE-HPLC results supported with spectral purity showed similar degradation extent with a different pattern of degradation between RTX and OBZ. A lower tendency to form degraded fragments and a relatively higher favorability for degradation through aggregate formation has been revealed in case of OBZ. Results were in agreement with those of DLS and receptor binding assay which showed specificity to the intact antibodies in the presence of their degradation products. Furthermore, results were additionally confirmed through denaturing quantitative gel electrophoresis which suggested reducible covalent bonds as the mechanism for aggregates formation. RP-HPLC results showed two oxidized forms via excessive oxidation of RTX and OBZ with nearly the same degradation percent. Comparability data of RTX and OBZ using the applied methodologies showed that although glycoengineering; carried out to enhance the therapeutic and biological activity of OBZ altered the pattern of degradation but did not significantly affect the overall stability. Results showed also consistent stability profile between the biosimilar and its originator RTX products.

HPLC Method for Simultaneous Determination of Bisphenol-A-Diglycidyl Ether and Some of Its Reaction Products in Canned Foods Using Photodiode Array Detector.

Gradient reversed-phase high-performance liquid chromatography with photodiode array detection was used for separation, detection and quantification of bisphenol-A-diglycidyl ether (BADGE) and some of its reaction products, namely, BADGE·HCl·H2O, BADGE·H2O and BADGE·2HCl in pure form and in canned foods, where canned beans and tuna were used as representatives of aqueous and oil-in-water food matrices, respectively. The proposed method had a linear range of 0.01-0.5 µg g-1 for BADGE·HCl·H2O, BADGE·H2O, BADGE·2HCl and 0.02-0.7 µg g-1 for BADGE in aqueous food matrices. In oil-in-water matrices, the method was proven to be sensitive over a linear range of 0.01-0.5 µg g-1 for BADGE·HCl·H2O, BADGE·H2O and 0.02-0.7 µg g-1 for BADGE·2HCl and BADGE. The limits of detection and quantification ranged from 0.24 to 1.22 ng g-1 and 0.73 to 14.07 ng g-1, respectively. Excellent intraday and interday precision (n = 9) were obtained with RSD% of 0.84-2.19% and 1.88-2.52%, respectively. Accuracy was measured at five concentration levels and the recoveries ranged from 96.31% to 98.76% with an acceptable variation of ±0.9-2.87. Results suggest that the proposed method could be applied for the routine analysis of the studied compounds in their laboratory-prepared mixtures and in various types of canned foods following the limits and regulations of the European Union.

Validated chemometrics-assisted spectrophotometric methods for simultaneous determination of bisphenol-A-diglycidyl ether and some of its reaction products in canned foods in the Egyptian market.

Two multivariate calibration methods, namely principal component regression (PCR) and partial least squares (PLS-2) have been developed, validated and compared for the simultaneous determination of bisphenol-A-diglycidyl ether (BADGE) and some of its reaction products, including BADGE·HCl·H2O, BADGE·H2O and BADGE·2HCl. Chemometrics allowed rapid, accurate and precise simultaneous quantification of the analytes of interest which was not possible by other spectrophotometric methods due to their severe spectral overlap. PCR and PLS-2 techniques successfully quantified BADGE, BADGE·HCl·H2O, BADGE·H2O and BADGE·2HCl in the ranges of 1.4-3.4, 1-5, 1-4.2 and 1-7 µg mL-1, respectively. The constructed models were validated according to the International Conference on Harmonization guidelines and successfully applied for the determination of these compounds in pure form, laboratory prepared mixtures and in various types of canned foods following the limits and regulations of the European Union (EU) where satisfactory recovery results were obtained.

Liquid chromatographic and spectrofluorimetric assays of empagliflozin: Applied to degradation kinetic study and content uniformity testing.

Stability-indicating high-performance liquid chromatography (HPLC) and spectrofluorimetric methods were developed for determination of empagliflozin (EGF). EGF was subjected to oxidation, wet heat, photo-degradation, acid hydrolysis and alkali hydrolysis. The alkaline degradation pathway was subjected to a kinetics study as the major product obtained after stress conditions. Arrhenius plots were constructed and the activation energies of the degradation process were calculated. HPLC was used for the kinetic study as it enabled simultaneous determination of EGF and the degradation product while the spectrofluorimetric assay was applied to content uniformity testing due to its higher sensitivity and lower limit of detection (LOD). Isocratic chromatographic elution was attained for HPLC on a Intersil® C18 column (150 mm × 4 mm, 5 µm), using a mobile phase of acetonitrile-potassium dihydrogen phosphate buffer pH 4, (50:50, v/v) at a flow rate of 1 ml/min with ultraviolet (UV) detection at 225 nm. The relative fluorescence intensity was recorded by spectrofluorimeter applying synchronous mode using ∆λ = 70 nm at 297.6 nm. Linearity ranges were found to be 5-50 µg/ml and 50-1000 ng/ml for HPLC and spectrofluorimetric methods, respectively.

Mimicking new receptors based on molecular imprinting and their application to potentiometric assessment of 2,4-dichlorophenol as a food taint.

Innovative host-tailored polymers were prepared, characterized and used as recognition elements in potentiometric transducers for the selective quantification of 2,4-dichlorophenol (DCP).The polymer beads were synthesized using DCP as a template molecule, acrylamide (AM),methacrylic acid (MAA) and ethyl methacrylate (EMA) as functional monomers and divinylbenzene (DVB) and ethylene glycol dimethacrylate (EGDMA) as cross-linkers. The sensors were fabricated by the inclusion of MIPs in plasticized polyvinyl chloride (PVC) matrix. Response characteristics of the proposed sensors revealed anionic slopes of -59.2, -49.7 and -80.6 mV/decade with detection limits of 5.6 × 10-5,5.9 × 10-5 and 13.2 × 10-5 mol/L for MIP/AM/DVB, MIP/MAA/DVB and MIP/EMA/EGDMA membrane based sensors, respectively. Good selectivity was observed over common inorganic/organic anions. Validation of the assay method according to IUPAC recommendations was justified ensuring the synthesis of good reliable novel sensors for DCP determination. The method was successfully applied for routine analysis of food taint in fish and fish farms water samples.

Sensitive Synchronous Spectrofluorimetric Study of Certain Sunscreens Using Fluorescence Enhancers in Cosmeceutical Formulations.

Synchronous spectrofluorimetric methods could be successfully adopted for simultaneous determination of Octinoxate (OMC), Avobenzone (AVO), Octyltriazone (OT), and Phenyl benzimidazole sulfonic acid (PBSA) in moisturizing sunscreen lotion, utilizing ß-CD as fluorescence enhancer, and determination of Avobenzone (AVO), Homosalate, Tinosorb M and Phenyl benzimidazole sulfonic acid (PBSA) in presence of Octocrylene (OCR) in whitening sunscreen cream, using micellar medium of Sodium Dodecyl Sulfate (SDS) to enhance fluorescence intensity. For first product, zero order synchronous spectrofluorimetric method was used for determination of OMC and AVO, and derivative synchronous spectrofluorimetric technique was utilized for OT and PBSA in quaternary mixture. Linear calibration curves were obtained in a concentration range of 0.5-8 µg mL- 1 for OMC and AVO, and in range of 0.05-3 µg mL- 1 for OT and 0.001-5 µg mL- 1 for PBSA, by measuring the fluorescence at 370, 405, 333.2 and 340.6 nm, respectively. For second product, first derivative synchronous fluorescence method was used for each UV-filter. A linear calibration curves were obtained in a concentration range of 0.5-8 µg mL- 1 for AVO, in range of 0.1-8 µg mL- 1 for Homosalate, 2-10 µg mL- 1 for Tinosorb M and 0.001-5 µg mL- 1 for PBSA, by measuring the fluorescence at 409.8, 373, 307.2 and 316.8 nm, respectively. The detection limits are well below the maximum admissible concentration. The proposed methods were validated according to ICH guidelines and successfully applied to determine sunscreens in pure form and in Cosmeceutical formulations. All the results obtained were compared with those of published methods, where no significant difference was observed.

Enhanced LC-MS/MS analysis of alogliptin and pioglitazone in human plasma: Applied to a preliminary pharmacokinetic study.

A new fast LC-MS/MS method was developed for determination of alogliptin and pioglitazone in human plasma. Linearity ranges of 10-400ngmL-1 for alogliptin and 25-2000ngmL-1 for pioglitazone, were found to be suitable for their bioanalysis covering the Cmin and Cmax values of the drugs. Direct precipitation technique was used for simultaneous extraction of the drugs successfully from human plasma samples. Chromatographic separation was achieved on a BEH C18 column (50mm×2.1mm, 1.7µm) with 0.1% aqueous formic acid: acetonitrile (40:60, v/v) at a flow rate of 0.3mLmin-1. The validated method was applied to a preliminary pharmacokinetic study on human volunteers. Monitoring the transition pairs of m/z 340.18 to 116.08 for alogliptin and m/z 356.99 to 133.92 for pioglitazone, using triple quadrupole mass spectrometer with multiple reaction monitoring, was achieved in the positive mode. The validated method is accurate and suitable for further clinical applications and possible bioequivalence studies.

Comparative Study Between Multivariate and Univariate Analysis of Two Antidiabetic Combinations.

New multivariate and univariate methods were developed for the analysis of two novel gliptin combinations by manipulating the zero-order and ratio spectra of empagliflozin and linagliptin in combination, with application on Glyxambi® tablets, and of alogliptin and pioglitazone in combination, with application on Oseni® tablets. Linearity ranges for chemometric approaches using principal component regression and partial least-squares were found to be 2-10, 2.5-12.5, 5-15, and 5-25 µg/mL for empagliflozin, linagliptin, alogliptin, and pioglitazone, respectively, whereas the respective linearity ranges for the spectrophotometric approaches were found to be 5-15, 2-12, 5-15, and 5-15 µg/mL. The proposed spectrophotometric methods included ratio subtraction coupled with extended ratio subtraction, spectrum subtraction coupled with constant multiplication, and mean centering. Acceptable LOD and LOQ values were obtained by all methods. Statistical analysis showed no significant difference between multivariate and univariate methods in comparison with the reference methods. The optimized methods provide fast and economic determination of the recently approved antidiabetic combinations without the complex instrumentation or time-consuming mobile phase preparations that were used in the chromatographic techniques reported in the literature.

Investigation of different spectrophotometric and chemometric methods for determination of entacapone, levodopa and carbidopa in ternary mixture.

New, simple, accurate and sensitive UV spectrophotometric and chemometric methods have been developed and validated for determination of Entacapone (ENT), Levodopa (LD) and Carbidopa (CD) in ternary mixture. Method A is a derivative ratio spectra zero-crossing spectrophotometric method which allows the determination of ENT in the presence of both LD and CD by measuring the peak amplitude at 249.9nm in the range of 1-20µgmL-1. Method B is a double divisor-first derivative of ratio spectra method, used for determination of ENT, LD and CD at 245, 239 and 293nm, respectively. Method C is a mean centering of ratio spectra which allows their determination at 241, 241.6 and 257.1nm, respectively. Methods B and C could successfully determine the studied drugs in concentration ranges of 1-20µgmL-1 for ENT and 10-90µgmL-1 for both LD and CD. Methods D and E are principal component regression and partial least-squares, respectively, used for the simultaneous determination of the studied drugs by using seventeen mixtures as calibration set and eight mixtures as validation set. The developed methods have the advantage of simultaneous determination of the cited components without any pre-treatment. All the results were statistically compared with the reported methods, where no significant difference was observed. The developed methods were satisfactorily applied to the analysis of the investigated drugs in their pure form and in pharmaceutical dosage forms.

Simultaneous Determination of Octinoxate, Oxybenzone, and Octocrylene in a Sunscreen Formulation Using Validated Spectrophotometric and Chemometric Methods.

Accurate, reliable, and sensitive spectrophotometric and chemometric methods were developed for simultaneous determination of octinoxate (OMC), oxybenzone (OXY), and octocrylene (OCR) in a sunscreen formulation without prior separation steps, including derivative ratio spectra zero crossing (DRSZ), double divisor ratio spectra derivative (DDRD), mean centering ratio spectra (MCR), and partial least squares (PLS-2). With the DRSZ technique, the UV filters could be determined in the ranges of 0.5-13.0, 0.3-9.0, and 0.5-9.0 µg/mL at 265.2, 246.6, and 261.8 nm, respectively. By utilizing the DDRD technique, UV filters could be determined in the above ranges at 237.8, 241.0, and 254.2 nm, respectively. With the MCR technique, the UV filters could be determined in the above ranges at 381.7, 383.2, and 355.6 nm, respectively. The PLS-2 technique successfully quantified the examined UV filters in the ranges of 0.5-9.3, 0.3-7.1, and 0.5-6.9 µg/mL, respectively. All the methods were validated according to the International Conference on Harmonization guidelines and successfully applied to determine the UV filters in pure form, laboratory-prepared mixtures, and a sunscreen formulation. The obtained results were statistically compared with reference and reported methods of analysis for OXY, OMC, and OCR, and there were no significant differences with respect to accuracy and precision of the adopted techniques.

New methods for amlodipine and valsartan native spectrofluorimetric determination, with factors optimization study.

Four native fluorescence methods were suggested for simultaneous determination of amlodipine (AML) and valsartan (VAL). These methods were based on excitation of both drugs at λ(ex) 300 nm, in one step, to give maximum emission at λ(em) 378 and 496 nm for AML and VAL, respectively. The first method, single λ(ex) method, was used without any additions. The sensitivity of this method was further increased by the addition of hydroxy propylmethyl cellulose (HPMC) surfactant, ß-cyclodextrin, or ferric oxide magnetite nanoparticles, in the other three methods. Different types of surfactants, and different concentration levels of both ß-cyclodextrin and ferric oxide nanoparticles, were scanned to determine the optimum conditions for enhancing the sensitivity. Some factors affecting the fluorescence intensity of both cited drugs, like the type and volume of the added solvent (to be used as a sensing agent), and pH of measurement were studied and optimized. The proposed methods could be used in determination of AML and VAL in bulk powder, their laboratory prepared mixtures and pharmaceutical formulations. The obtained results were statistically compared to each other and to that of some reported methods. The specificity of the developed methods was investigated, and the methods were validated according to ICH guidelines.

Application of new spectrofluorometric techniques for determination of atorvastatin and ezetimibe in combined tablet dosage form.

Two accurate, reliable, and highly sensitive spectrofluorometric methods were developed for simultaneous determination of the binary mixture of Atorvastatin and Ezetimibe without prior separation steps. The first method is based on double scan synchronous fluorescence spectrometry. Each of Atorvastatin and Ezetimibe can be determined independent of the other when scanned at Δλ=100 nm and 40 nm, respectively. The relative fluorescence intensity-concentration plots at two wavelengths, 272 (Δλ=100 nm) and 266 nm (Δλ=40 nm) were rectilinear over the range of 0.4-8 µg/mL (for Atorvastatin) and 0.6-8 µg/mL (for Ezetimibe), respectively. The second method is based on the technique of simultaneous equations (Vierodt's method), in which two equations are solved simultaneously after using a single excitation wavelength of 273 nm and λEm1=380 nm of Atorvastatin and λEm2=301 nm of Ezetimibe. Under the optimum conditions, linear relationships were found between the relative fluorescence intensity and the concentrations of the investigated drugs in the range of 0.4-8 µg/mL (for Atorvastatin) 0.6-8 µg/mL (for Ezetimibe). The different experimental parameters affecting the fluorescence intensities of the two drugs were carefully studied and optimized. The proposed methods were successfully applied for the determination of the investigated drugs in pure form, dosage form and in synthetic mixtures with good recovery and the results obtained were favorably compared to those obtained with a reference method.