Different Eco-Friendly Spectrophotometric Approaches Including Direct and Manipulations of Zero and Ratio Spectra for Simultaneous Determination of Novel Nasal Spray Combination Used in Seasonal Allergic Rhinitis.

BACKGROUND: The presentation of rhinitis has drawn increasing attention in recent years due to the possibility of overlap or confusion between allergic rhinitis symptoms and those of COVID-19. Azelastine hydrochloride (AZH) and mometasone furoate (MOF) are two of the most efficient combinations for enhancing the symptoms of seasonal allergic rhinitis. OBJECTIVE: This work concerns applying and validating different accurate and simple spectrophotometric approaches for simultaneous quantification of the binary mixture of AZH and MOF in raw material, laboratory-prepared mixtures, and pharmaceutical preparation. Moreover, assessment of the environmental impact of the applied approaches on the environment was also a key goal of this study. METHODS: AZH was determined using the direct spectrophotometric (D0) method, while four reliable spectrophotometric approaches namely, induced dual wavelength (IDW), ratio subtraction (RS), ratio difference (RD), and ratio derivative (1DD) were used for MOF determination. RESULTS: The methods were validated in line with the International Conference of Harmonization standards. In the AZH range of (5-56 µg/mL) and MOF range of (2-20 µg/mL), the linearity of the proposed approaches was investigated with high accuracy findings. There were no significant differences between the obtained results and those of the reported method when compared statistically. Furthermore, the applied spectrophotometric methods were deemed to be eco-friendly according to Green Analytical Procedure Index (GAPI) and Analytical Greenness Calculator (AGREE) assessment metrics. CONCLUSIONS: The applied spectrophotometric methods are simpler, more eco-friendly, and take a shorter time to precisely estimate many measurements compared to the only reported chromatographic analysis. HIGHLIGHTS: Neither publications of novel spectrophotometric methods nor reported green ones have been available for simultaneous determination of the binary mixture of AZH and MOF, so this work has a great significance and novelty in the area of pharmaceutical analysis.

Development and validation of a simultaneous analytical method for non-steroidal therapeutic compounds in cosmetics using liquid chromatography-tandem mass spectrometry.

Atopic dermatitis is a typical chronic inflammatory skin disease that affects all age groups and requires basic skin care for treatment. Anti-inflammatory and antiallergy steroids are the most frequently used treatments but they are limited due to their side effects caused by a weakening of the immune system. Many consumers focus on performance as a criterion for selecting cosmetics. However, steroids have been illegally used to improve the performance of cosmetics, and consumers have been adversely affected by the corresponding side effects. In this paper, we propose a simple and rapid method using liquid chromatography-tandem mass spectrometry to simultaneously analyze ten non-permitted atopic therapeutic compounds in cosmetic products: chlorpheniramine maleate, ketotifen fumarate, doxepin hydrochloride, azelastine hydrochloride, bufexamac, clotrimazole, tranilast, fusidic acid, tacrolimus, and pimecrolimus. Additionally, the major characteristic fragment ions for tacrolimus, pimecrolimus, and clotrimazole were identified by time-of-flight mass spectrometry. The specificity, linearity, limit of detection, limit of quantification, recovery, precision, accuracy, and stability of the proposed method were validated. The limit of detection and quantification were in the ranges of 5.05-203.30 pg/mL and 15.15-609.90 pg/mL, respectively. The proposed analysis method could help improve the safety management of cosmetics.

In vitro metabolism of olaparib in liver microsomes by liquid chromatography/electrospray ionization high-resolution mass spectrometry.

RATIONALES: Olaparib is a Poly (ADP-ribose) Polymerase (PARP) inhibitor which has been developed as an anti-cancer agent. The purpose of this study was to characterize the metabolites of olaparib from liver microsomes and to reveal the interspecies differences between animals and humans. METHODS: Olaparib (20 µM) was incubated with different species of liver microsomes at 37°C for 1 h in the presence of NADPH. The incubation samples were analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) operated in positive ion mode. The metabolites were characterized by accurate masses, MS2 spectra and retention times. RESULTS: A total of 12 metabolites were detected and the structures of the metabolites were characterized based on their accurate masses, fragment ions and retention times. Four metabolites, i.e., M1, M10, M11 and M12, were unambiguously identified by using reference standards. The metabolic pathways of olaparib included hydroxylation, bis-hydroxylation, hydrolysis, dealkylation, dehydrogenation, and alcohol oxidation. CONCLUSIONS: Compared with animal species, no human-specific metabolite was found in HLM. Dog also had a closer metabolic profile to humans. This study will be helpful for a better understanding of the species difference in pharmacokinetics/pharmacodynamics.

Multi-Steps Fragmentation-Ion Trap Mass Spectrometry Coupled to Liquid Chromatography Diode Array System for Investigation of Olaparib Related Substances.

A high-performance liquid chromatography-diode array-mass spectrometric (LC-DAD-MS) method was developed and validated to investigate the related substances of olaparib (OLA) in bulk form. OLA was exposed to acid⁻base hydrolysis, boiling, oxidation with hydrogen peroxide, and UV light followed by LC-DAD-MS analysis. OLA and OLA-related substances were simultaneously and quantitatively monitored by DAD at 278 nm and triple quadrupole mass spectrometry (QQQ-MS). The investigated compounds were auto-scanned by an ion trap MS which applied positive and negative modes separately. The fragmentation pathway was confirmed by applying multi-steps fragmentation to identify the resulted cleaved ions and their parent ion. OLA was found to be sensitive to the alkaline hydrolysis and less sensitive to UV light. Two major hydrolytic degradation products, including the protonated molar ions m/z 299 and m/z 367, were identified. Three potential impurities were also characterized. The LC-MS limit of detection (LOD) and limit of quantification (LOQ) were 0.01 and 0.05 ng/µL, respectively. The quantitative results obtained by LC-DAD was comparable with that of LC-QQQ-MS. The proposed method shows good intra-day and inter-day precision with relative standard deviation (RSD) <2%.

Study of aldehyde oxidase with phthalazine as substrate using both off-line and on-line capillary electrophoresis.

An optimized and economical capillary electrophoretic method for both off-line and on-line study of the enzyme aldehyde oxidase and its substrate phthalazine was developed. The separation of the substrate phthalazine and its metabolite 1-phthalazinone was achieved using micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulphate in the background electrolyte (BGE). The BGE consists of 25 mM sodium phosphate buffer containing 50 mM sodium dodecyl sulphate at pH 7.4. A bare-fused-silica capillary with a capillary length of 40 cm, 50 µm ID and effective length of 30 cm was used to develop the capillary electrophoresis method. Improved separation conditions were elaborated and the separation method was validated based on the ICH and EMA guidelines. The limit of detection for phthalazine and 1-phthalazinone was 8 µM and 3 µM, respectively. The limit of quantification was 25 µM for phthalazine and 10 µM for 1-phthalazinone. The linearity of the detector response was checked for 1-phthalazinone at nine different concentrations in the range 10-500 µM and the determination coefficient was 0.9994. Accuracy was tested by comparing the corrected peak area of 1-phthalazinone reference solution at 20 µM and 50 µM with the corrected peak area of 20 µM and 50 µM 1-phthalazinone in the presence of human liver cytosol (HLC). Accuracy values of +5.3% and -2.5% were obtained at 20 µM and 50 µM, respectively. The on-line enzymatic reaction was successful with the application of the method of transverse diffusion of laminar flow profiles (TDLFP), which enables the mixing as well as separation of the enzyme and substrate inside the nanoliter-scale capillary. TDLFP is examined to be precise when performing 5 consecutive injections, with a relative standard deviation of 7.16% which is within the limitation of EMA standards. This miniaturized and low-cost incubation and separation method could be further introduced into industry and extended to other substrates.

Validated stability indicating assay method of olaparib: LC-ESI-Q-TOF-MS/MS and NMR studies for characterization of its new hydrolytic and oxidative forced degradation products.

Olaparib (OLA) is a poly ADP ribose polymerase (PARP) enzyme inhibitor used to treat prostate, ovarian and breast cancer. The drug substance OLA was subjected to forced degradation as per ICH prescribed guidelines. It was degraded in hydrolytic (acidic and basic) and oxidative stress conditions and yielded four degradation products (DPs) while it remained stable in neutral hydrolytic, dry heat and photolytic stress conditions. A stability indicating assay method was developed to separate OLA and its DPs using InertSustain C18 column (250 × 4.6 mm, 5 µm) with a gradient mobile phase of 10 mM ammonium acetate (pH 4.5) and acetonitrile (ACN) at a flow rate of 1 mL min-1. The characterization of DPs was carried out by using liquid chromatography-electrospray ionization-quadrupole-time of flight tandem mass spectrometry (LC-ESI-Q-TOF-MS/MS). Major degradation products (DP-1 and DP-2) were isolated by using preparative HPLC and structures were further confirmed by using NMR spectroscopy. All the obtained DPs were new and not reported previously. The developed chromatographic method was validated as per ICH Q2 (R1) guideline and USP general chapter on method validation.

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

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

Investigation of the spectrofluorimetric behavior of azelastine and nepafenac: Determination in ophthalmic dosage forms.

The first spectrofluorimetric report investigating the fluorimetric behavior of the antihistaminic drug, azelastine (AZEL), and the non-steroidal anti-inflammatory drug, nepafenac (NEP), either in bulk or in their dosage forms, eye drops and ophthalmic suspension. After a full investigation of the factors that may influence their spectrofluorimetric behavior: pH, different organized media and organic solvents, the optimum factors were set in order to enable the analysis of each drug with maximum sensitivity. The AZEL spectrofluorimetric analysis was set at 286/364 (λex/λem) in distilled water while for NEP, the analysis was set at 228/303 (λex/λem) in methanol. The linearity range for AZEL was from 0.1 to 1.5 µg/mL while that of NEP was from 0.2 to 1.5 µg/mL. The linearity yielded good regression parameters with low LOD (0.022 and 0.032 µg/mL for AZEL and NEP, respectively) and LOQ (0.073 and 1.08 µg/mL for AZEL and NEP, respectively) when compared with those obtained from many previous spectroscopic and chromatographic reports in literature. The method was ICH validated and was applied to the analysis of AZEL and NEP with good selectivity regarding the inactive ingredients.

Validated spectroscopic methods for determination of anti-histaminic drug azelastine in pure form: Analytical application for quality control of its pharmaceutical preparations.

Two simple, sensitive, rapid, validated and cost effective spectroscopic methods were established for quantification of antihistaminic drug azelastine (AZL) in bulk powder as well as in pharmaceutical dosage forms. In the first method (A) the absorbance difference between acidic and basic solutions was measured at 228nm, whereas in the second investigated method (B) the binary complex formed between AZL and Eosin Y in acetate buffer solution (pH3) was measured at 550nm. Different criteria that have critical influence on the intensity of absorption were deeply studied and optimized so as to achieve the highest absorption. The proposed methods obeyed Beer's low in the concentration range of (2.0-20.0µg·mL-1) and (0.5-15.0µg·mL-1) with % recovery±S.D. of (99.84±0.87), (100.02±0.78) for methods (A) and (B), respectively. Furthermore, the proposed methods were easily applied for quality control of pharmaceutical preparations without any conflict with its co-formulated additives, and the analytical results were compatible with those obtained by the comparison one with no significant difference as insured by student's t-test and the variance ratio F-test. Validation of the proposed methods was performed according the ICH guidelines in terms of linearity, limit of quantification, limit of detection, accuracy, precision and specificity, where the analytical results were persuasive.

Chemical characterisation of aerosols emitted by electronic cigarettes using thermal desorption-gas chromatography-time of flight mass spectrometry.

A non-targeted analytical method, using thermal desorption-gas chromatography-time of flight mass spectrometry, to detect, identify and semi-quantify volatile and semi-volatile organic constituents of e-cigarette aerosols is presented. A heart-cutting process with a Deans Switch has been applied to avoid saturation of the mass analyser by high-abundance bulk components. Between 30 and 90 compounds were detected in four aerosol samples generated by e-cigarettes, depending on the added flavourings. The method analyses in a single 80mL, 3-second puff, GC-amenable compounds with volatilities ranging between those of propane (C3) and octacosane (C28) and abundance greater than approximately 5ng. Method sensitivity is suitable for the application of thresholds of toxicological concern for product assessment at an exposure threshold of 1.5µg per day. The method is compatible with the high-throughput screening of GC-amenable compounds in e-cigarette aerosols.

Validated sensitive spectrofluorimetric method for determination of antihistaminic drug azelastine HCl in pure form and in pharmaceutical dosage forms: application to stability study.

A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of azelastine HCl (AZL) in either its pure state or pharmaceutical dosage form. The proposed method was based on measuring the native fluorescence of the studied drug in 0.2 M H2 SO4 at λem  = 364 nm after excitation at λex  = 275 nm. Different experimental parameters were studied and optimized carefully to obtain the highest fluorescence intensity. The proposed method showed a linear dependence of the fluorescence intensity on drug concentration over a concentration range of 10-250 ng/mL, with a limit of detection of 1.52 ng/mL and limit of quantitation of 4.61 ng/mL. Moreover, the method was successfully applied to pharmaceutical preparations, with percent recovery values (± SD) of 99.96 (± 0.4) and 100.1 (± 0.52) for nasal spray and eye drops, respectively. The results were in good agreement with those obtained by the comparison method, as revealed by Student's t-test and the variance ratio F-test. The method was extended to study the stability of AZL under stress conditions, where the drug was exposed to neutral, acidic, alkaline, oxidative and photolytic degradation according to International Conference on Harmonization (ICH) guidelines. Copyright © 2016 John Wiley & Sons, Ltd.

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

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

D5 receptor agonist 027075 promotes cognitive function recovery and neurogenesis in a Aß1-42-induced mouse model.

In this study, a high throughput screening system was set up to identify D5 receptor agonists-027075. Then, a series of behavior tests were used to evaluate the beneficial effects of 027075 in Aß1-42-induced mice model including morris water maze, passive avoidance, active avoidance, open field and step-down test. The neuroprotective effect of 027075 was assessed by a high content screening in vitro. In behavior tests, the cognitive function impairment caused by Aß1-42 was significantly ameliorated by 027075 in a dose-dependent manner. 027075 (8 mg/kg) significantly prolonged the time spent in the target quadrant when compared to the model group in morris water maze test. The latency was significantly increased and the number of errors was decreased in both passives avoidance task and step down test when compared to the model group. In active avoidance and open field test, latency, stimulation time, number of errors were significantly reduced, while number of avoidance and line crossing and central distance were increased by 027075 (8 mg/kg). All the results above was significantly reversed by 027075-H + SCH39166 (5 mg/kg) when compared to 027075-H (8 mg/kg). The neuroprotective effect of 027075 was demonstrated by promotion of cell differentiation and extension of neurite length. But the effects were abrogated by the specific D1/D5 antagonist, SCH39166. These results indicate that D5 receptor might be used as an ideal target for the treatment of AD and its agonists might become a new AD drugs in the future.

A High-Temperature, High-Throughput Method for Monitoring Residual Formaldehyde in Vaccine Formulations.

Formaldehyde has long been used in the chemical inactivation of viral material during vaccine production. Viral inactivation is required so that the vaccine does not infect the patient. Formaldehyde is diluted during the vaccine manufacturing process, but residual quantities of formaldehyde are still present in some current vaccines. Although formaldehyde is considered safe for use in vaccines by the Food and Drug Administration, excessive exposure to this chemical may lead to cancer or other health-related issues. An assay was developed that is capable of detecting levels of residual formaldehyde in influenza vaccine samples. The assay employs incubation of dosage formulation suspensions with hydralazine hydrochloride under mildly acidic conditions and elevated temperatures, where formaldehyde is derivatized to yield fluorescent s-triazolo-[3,4-a]-phthalazine. The assay has been traditionally run by high-performance liquid chromatography, where runtimes of 15 minutes per sample can be expected. Our laboratory has developed a plate-based version that drastically improved the throughput to a runtime of 96 samples per minute. The assay was characterized and validated with respect to reaction temperature, evaporation, stability, and selectivity to monitor residual formaldehyde in various influenza vaccine samples, including in-process samples. Heat transfer and evaporation will be especially considered in this work. Since the assay is plate based, it is automation friendly. The new assay format has attained detection limits of 0.01 µg/mL residual formaldehyde, which is easily able to detect and quantify formaldehyde at levels used in many current vaccine formulations (<5 µg/0.5-mL dose).

Alternative two-step matrix application method for imaging mass spectrometry to avoid tissue shrinkage and improve ionization efficiency.

Mass spectrometry (MS) was used to measure the concentrations of drug and biological compounds in plasma and tissues. Matrix-assisted laser desorption/ionization (MALDI) imaging MS (IMS) has recently been applied to the analysis of localized drugs on biological tissue surfaces. In MALDI-IMS, matrix application process is crucial for successful results. However, it is difficult to obtain homogeneous matrix crystals on the tissue surface due to endogenous salts and tissue surface heterogeneity. Consequently, the non-uniform crystals degrade the quality of the spectrum and likely cause surface imaging artifacts. Furthermore, the direct application of matrix solution can cause tissue shrinkage due to the organic solvents. Here, we report an alternative two-step matrix application protocol which combines the vacuum deposition of matrix crystals and the spraying of matrix solution to produce a homogeneous matrix layer on the tissue surface. Our proposed technique can also prevent cracking or shrinking of the tissue samples and improve the ionization efficiency of the distributed exogenous material.

Identification and determination of ketotifen hydrogen fumarate, azelastine hydrochloride, dimetindene maleate and promethazine hydrochloride by densitometric method.

Conditions for determination of: ketotifen hydrogen fumarate, azelastine hydrochloride, dimetindene maleate and promethazine hydrochloride by densitometric method in substances and pharmaceuticals were provided. Maximum wavelenghts were: 228 nm for ketotifen hydrogen fumarate, 295 nm for azelastine hydrochloride, 265 nm for dimetindene maleate and 255 nm for promethazine hydrochloride. The limits of quantification were in the ranges of 0.2-5 microg/spot. The statistical data showed adequate accuracy and precision of developed methods.

LC-MS/MS bioanalytical method development for AMG 900: resolution of an isobaric interference in rodent in vivo studies.

AMG 900 is an orally available small molecule that is a highly potent and selective pan-aurora kinase inhibitor currently in development for the treatment of advanced human cancers. A co-eluting, isobaric interference was discovered in preliminary LC-MS/MS analyses of rodent in vivo pharmacokinetic samples during preclinical evaluation of AMG 900. The interference was identified as a major circulating N-oxide metabolite which partially converted to an [M+H-O](+) ion under the conditions of atmospheric pressure chemical ionization. A selective liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of AMG 900 and its N-oxide metabolite in plasma was developed and successfully applied for the bioanalysis of discovery stage preclinical rodent pharmacokinetic studies.

Effective on-line extraction of drugs from plasma using a restricted-access media column in column-switching HPLC equipped with a dilution system: application to the simultaneous determination of ER-118585 and its metabolites in canine plasma.

The present paper describes the on-line extraction of drugs in plasma using a restricted-access media (RAM) column in a column-switching high performance liquid chromatography (HPLC) apparatus that was equipped with an on-line dilution system. The use of a six- to eightfold on-line dilution ratio for plasma samples resulted in almost 100% recovery of both acidic and basic drugs from plasma. It was found that the relationship between the on-line dilution times and drug recovery efficiencies from plasma was explained in terms of the binding constant between the drug and albumin. The applicability of column-switching HPLC with an on-line dilution system and the effectiveness of the extraction procedure were confirmed by a simultaneous determination of the basic compound, ER-118585, and its metabolites in canine plasma.

Comparison of the properties of polymeric and C8 based materials for solid phase extraction.

The extraction properties of two polymeric solid phase extraction materials, styryldivinyl benzene (SDB) and 'Oasis' have been compared with those of a base deactivated C8 bonded silica gel using a range of acidic and basic test analytes. In the case of the two polymer phases good extraction of all the test compounds from aqueous buffer was obtained over the pH range 2-10. On the C8 material, efficient extraction of the most polar acidic analyte, anisic acid, was only obtained between pH 2 and 6. The use of methanol water mixtures, or methanol water mixtures modified with either trifluoroacetic acid (TFA) or triethylamine (TEA) as eluents was investigated for the recovery of the analytes following extraction. The use of TFA or TEA as ionic modifiers strongly influenced the efficiency of the elution step. The effect of a plasma matrix on extraction efficiency was also investigated, with the result depending upon the analyte. An approach to assessing the performance of the three phases has been developed based on the percentages of methanol in the eluent resulting in the recovery of 50% of the analyte, and in determining the difference between eluents giving recoveries of 10 and 90%.

Investigation of the stereoselective in vitro metabolism of the chiral antiasthmatic/antiallergic drug flezelastine by high-performance liquid chromatography and capillary zone electrophoresis.

An achiral HPLC method using a silica gel column as well as two independent chiral analytical methods by HPLC and capillary zone electrophoresis (CZE) were developed in order to investigate the in vitro metabolism of the racemic antiasthmatic/antiallergic drug flezelastine. The chiral HPLC analysis was performed on a Chiralpak AD column, which also allowed the simultaneous separation of the N-dephenethyl metabolite. The chiral separation by CZE was achieved by the addition of beta-cyclodextrin to the run buffer. The stereoselectivity of the in vitro biotransformation of flezelastine was investigated using liver homogenates of different species. Depending on the species, diverse stereoselective aspects were demonstrated. The determination of the enantiomeric ratios of flezelastine and of N-dephenethylflezelastine after incubations of racemic flezelastine with liver microsomes revealed that porcine liver microsomes showed the greatest enantioselectivity of the biotransformation. (-)-Flezelastine was preferentially metabolized. After incubations with bovine liver microsomes the enantiomer of N-dephenethylflezelastine formed from (+)-flezelastine dominated. Incubations of the pure enantiomers of flezelastine with induced rat liver microsomes resulted in the stereoselective formation of a hitherto unknown metabolite, which was only detected in samples of (+)-flezelastine. Initial structure elucidation of the compound indicated that the new metabolite was most likely an aromatically hydroxylated derivative of the N-dephenethylflezelastine.