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Green analytical procedures are gaining popularity in the pharmaceutical research area as a way to reduce environmental impact and improve analyst health safety. The current work presents a green and sensitive electrochemical carbon paste electrode that has been chemically modified with zirconium dioxide and multi-walled carbon nanotubes for estimation of pyridoxine HCl (PYR) and doxylamine succinate (DOX) using the square wave voltammetric technique. Under optimum conditions, the linearity ranges were 20.00–2000.00 ng mL−1 and 2.00–20.00 µg mL−1 for both drugs in the 1st linear segment and 2nd linear segment, respectively. Stability testing assesses how the quality of a drug substance changes over time, depending on environmental and laboratory factors. DOX was found to undergo oxidative degradation when refluxed for 7 h using 30% H2O2 and the degraded product (DOX DEG) (toxic metabolite) was successfully characterized utilizing LC–MS. The developed electrode showed selectivity for the determination of binary mixture in pure form, pharmaceutical form, and in the presence of DOX DEG and common interfering molecules with good recovery. The proposed method was found to be eco-friendlier than the reported method in terms of the use of hazardous chemicals and solvents, energy consumption, and waste generation. Graphical Abstract

Background: PARTIAL Least Squares (PLS) and Principal Component Regression (PCR) are two well-known chemometric methods based on dimension reduction techniques. They can be very practical analyzing a large data set of multiple correlated predictor variables. Objective:In the presented work, the resolving power of spectrophotometric assisted mathematical techniques was implemented for the simultaneous determination of two active ingredients; ephedrine hydrochloride (EPH) and naphazoline nitrate (NAPH), in a matrix of excipients. Methods:To build the PLS and PCR models, a calibration set was prepared where the two drugs, in combination with the interfering parabens, were modeled by multilevel multifactor design. The proposed models successfully predicted the concentrations of both drugs in validation samples with low Root Mean Squared Error of Prediction (RMSEP). Results:The results revealed the ability of the mentioned multivariate calibration models to analyze EPH and NAPH in the presence of the interfering parabens with high selectivity in the concentration ranges of 4.00-20.00 μg mL-1 and 1.00-9.00 μg mL-1, respectively. Conclusion: A commercially available nasal spray was successfully analyzed using the developed methods without interfering with other dosage form additives.

Green analytical methods have gained a growing interest in the field of pharmaceutical research to reduce impacts on the environment and enhance analysts’ health safety. Chloramphenicol (CHL), dexamethasone sodium phosphate (DSP) and tetrahydrozoline HCl (THZ) form an ophthalmic ternary mixture that is co-formulated for conjunctivitis treatment. In the present work, for time saving and higher sensitivity, two green thin-layer chromatography (TLC) methods were developed for the determination of this ophthalmic ternary mixture in the absence or presence of p-nitroacetophenone (PNA), a synthetic precursor of chloramphenicol. In both proposed methods, silica gel 60 F254 plates were used as the stationary phase. The mobile phase used for method (A) was ethanol‒water‒ammonia (7.0:2.5:0.5, V/V), while, for method (B), acetonitrile‒water‒ammonia (10.0:3.0:0.5, V/V) was used as the mobile phase. TLC separation was followed by quantitative determination of the aforementioned drugs at wavelengths 242.0 nm and 220.0 nm. Both methods were validated in compliance with the International Conference on Harmonisation (ICH) guidelines, where both methods were found to be reliable, reproducible, and selective. Statistical comparison of the developed methods was done with a reported high-performance liquid chromatography (HPLC) method where no significant difference was found. Analytical eco-scaling depends on penalty point which was calculated to be 92, 88 and 87 for methods A, B and the reported HPLC, respectively, suggesting that the proposed methods are eco-friendlier with penalty point scoring very high on the scale than the reported one.

Background The combination of pyridoxine hydrochloride (PYR) and doxylamine succinate (DOX) as an antiemetic binary mixture is used to treat nausea and vomiting during pregnancy. Objective Two validated, accurate, and selective chemometric models were developed to assay binary mixture in the presence of DOX oxidative degradation product (DOX DEG) that could be characterized using LC-MS. Methods Partial least squares (PLS) regression and principal component regression (PCR) were selected for the determination of our binary mixture in presence of degradation. To exhibit a training set of 25 mixtures that had various percentages of tested substances in five level 3 variables, an experimental design was chosen. A set of 18 synthetic mixtures in the concentration range 10.0–50.0 μg/mL, 12.00–20.0 μg/mL, and 6.0–30.0 μg/mL for PYR, DOX, and DOX DEG, respectively, were used in the construction of the calibration models. Then set of seven synthetic mixtures with different concentrations were used in the construction of the validation models. Results In validation samples with low root mean square error of prediction (RMSEP), the suggested models successfully predicted the concentrations of our drugs. The models developed were evaluated by RMSEP calculation, and the values obtained were 0.341, 0.196, and 0.388 for PYR, DOX, and DOX DEG, respectively, using PLS. While using PCR, RMSEP calculation and the values obtained were 0.400, 0.256, and 0.375 for PYR, DOX, and DOX DEG, respectively. The developed models were validated according to ICH strategies. Conclusions The corresponding methods are suitable to determine PYR and DOX in pure form, pharmaceutical dosage form, and in the presence of DOX DEG product. Highlights The study of drug breakdown pathways is very important nowadays, so even in the presence of degradation and extreme spectral overlapping, the suggested PLS and PCR spectrophotometric approaches were able to identify PYR and DOX.

Ramucirumab (RAMU) is a recently US Food and Drug Administration-approved monoclonal antibody that is included in various anticancer protocols. It has a structural complexity and high degradation risk that have a significant effect on its safety and effectiveness. The major aim of this work was to assess the degradation pattern of RAMU based on physicochemical characterization. Mechanical agitation, repeated freeze-thaw cycles, pH and temperature were the selected stress conditions to which RAMU samples were subjected. The SE-HPLC method was applied and validated to monitor the RAMU monomer along with its aggregates and/or fragments. The purity of the separated peaks together with system suitability parameters were determined through the calculation of percentage purity and percentage drop in RAMU concentration. The results were interpreted by correlating them with those of dynamic light scattering and reducing and non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Samples incubated at pH 2.0-10.0 and 37°C for up to 4 weeks were analysed, recording detection of reversed phase (RP) aggregates and low molecular weight peptide fragments. Similarly, samples under short-term storage conditions of 4 weeks at different temperatures (-20, 2-8, 25, 37 and 50°C) showed low molecular weight peptide fragments but to a lesser extent. These results highlight the alarming effect on RAMU multidose vial efficacy and safety.

Biosimilar products present a growing opportunity to improve the global healthcare systems. The amount of accepted variability during the comparative assessments of biosimilar products introduces a significant challenge for both the biosimilar developers and the regulatory authorities. The aim of this study was to explore unsupervised machine learning tools as a mathematical aid for the interpretation and visualization of such comparability under control and stress conditions using data extracted from high throughput analytical techniques. For this purpose, a head-to-head analysis of the physicochemical characteristics of three Trastuzumab (TTZ) approved biosimilars and the originator product (Herceptin®) was performed. The studied quality attributes included the primary structure and identity by peptide mapping (PM) with reversed-phase chromatography-UV detection, size and charge profiles by stability-indicating size exclusion and cation exchange chromatography. Stress conditions involved pH and thermal stress. Principal component analysis (PCA) and two of the widely used cluster analysis tools, namely, K-means and Density-based Spatial Clustering of Applications with Noise (DBSCAN), were explored for clustering and feature representation of varied analytical datasets. It has been shown that the clustering patterns delineated by the used algorithms changed based on the included chromatographic profiles. The applied data analysis tools were found effective in revealing patterns of similarity and variability between i) intact and stressed as well as ii) originator and biosimilar samples.

Functional foods provide nutritional and health benefits, yet they could be contaminated with residues like pesticides and polychlorobiphenyls. These residues affect the safety, quality, and consequently the commercial value of functional foods. Therefore, the validity and efficiency of residue determination methods constitute a major analytical concern. Reduction of matrix effect (ME) has always been the golden key for guaranteed sensitivity, selectivity, and high throughput analysis. This study aims for accurate determination and streamlined quantification of 200 pesticide residues in 16 matrices. Hence, QuEChERS protocol coupled to GC-MS/MS was then employed and separations were obtained in 25 min. Dilution of the final extracts of fresh and herbal samples was carried out to achieve an acceptable balance between sensitivity and peak characteristics. Dilution factors of 1x and 5x were selected for fresh and herbal samples, respectively. Principal component analysis (PCA) was then independently applied on the digitally exported total ion chromatograms (TICs) of the studied matrices and the calculated ME%. PCA score/loading plots of TICs demonstrated the key matrix constituents that influenced the obtained trends. Similarly, three main clusters were obtained after PCA of ME% indicating a dependent relationship between matrix type and the obtained effects. Out of the obtained three clusters, an appropriate representative matrix-matched calibration (R-MMC) was selected for ME compensation. Based on the EU validation guidelines, the proposed protocol was validated at 2 and 10 μg Kg−1 with acceptable method performance. Four proficiency testing (PT) and commercial samples were successfully analyzed. The proposed protocol would help laboratories to increase sample processing capacity and to ensure the safety of functional food products. This work should serve in setting standards that warranty the quality/safety of functional foods by national regulatory authorities.

With the growing number of biosimilars, healthcare providers opt to switch costly originator products with biosimilars. However, extensive data are required to support biosimilarity and interchangeability. In this study, factors affecting physical and chemical stability of infliximab were assessed under various stress conditions to reveal possible degradation products. A set of orthogonal stability- and purity-indicating assays involving size exclusion, reversed phase and ion exchange chromatography in addition to quantitative TapeStation gel electrophoresis, dynamic light scattering, and functional receptor binding assay was developed and used for validation. An objective, statistically-guided interpretation of peptide mapping data using principal component analysis (PM-PCA) was performed to establish a “biosimilarity fingerprint”. SE-HPLC and dynamic light scattering were used to evaluate the formation of aggregates and/or small molecular weight fragments under non-denaturing conditions. Results were correlated with those obtained under denaturing conditions using TapeStation gel electrophoresis. Results indicated that aggregates were formed via non-reducible covalent bonds. RP-HPLC showed no oxidized forms, yet the infliximab biosimilar was relatively more hydrophilic than its innovator counterpart. Ion exchange chromatography revealed slight differences in abundance of the charge variants that were not revealed using other techniques. PM-PCA and receptor binding assay showed differences, yet they were statistically non-significant (P 0.05). Results confirmed similarity in the primary structure, higher order structure and receptor-binding pattern. In-depth investigations of the critical quality attributes of infliximab along with setting up the similarity and range were required. These helped with assessment of the impact of such slight differences on the biosimilarity and interchangeability of the studied product. The developed PM-PCA along with the orthogonal assays helped locate variability that could potentially hinder both biosimilarity and interchangeability of other complex biopharmaceuticals.

Spectrophotometric data analysis using multivariate approaches has many useful applications. One of these applications is the analysis of active ingredients in presence of impurities. Four chemometric-assisted spectrophotometric methods, namely, principal component regression (PCR), partial least-squares (PLS), artificial neural networks (ANN) and multivariate curve resolution-alternating least squares (MCR-ALS) were proposed and validated. The developed chemometric methods were compared to resolve the severely overlapped spectrum of Paracetamol (PAR) and Phenylephrine HCl (PHE) along with PAR impurities namely, P-Aminophenol (PAP), P-Nitrophenol (PNP), Acetanilide (ACT) and P-Chloroacetanilide (CAC). The four multivariate calibration methods succeeded in simultaneous determination of PAR and PHE with further quantification of PAR impurities. So, the proposed methods could be used with no need of any separation step and successfully applied for pharmaceutical formulation analysis. Furthermore, statistical comparison between the results obtained by the proposed chemometric methods and the official ones showed no significant differences.

The environmental impact of analytical procedures is a major concern nowadays. As a result, scientists are now working to reduce the adverse environmental impact of chemicals and solvents. Stability testing is an important stage in the pharmaceutical industry because it determines how a drug product's quality changes over time as a result of environmental and laboratory conditions. This study demonstrates the use of green reversed phase (RP-HPLC) to determine pyridoxine HCl (PYR) and doxylamine succinate (DOX) in the presence of DOX oxidative degradation product (DOX DEG). DOX DEG was identified by using (LC-MS). The chromatographic separation was achieved using a mobile phase consisting of ethanol and 0.01M phosphate buffer pH=5.0 (10: 90, v/v) with isocratic elution, pumped at 1.0 mL/min on an Xterra C18 column (100 mm x 4.6mm x 5m). DAD was used for detection at 254.0 nm, and the retention times for PYR, DOX, and DOX DEG were 1.41 min, 5.89 min, and 7.36 min, respectively. This procedure was valid over linearity ranges of 10.00-120.00 g/mL and 5.00-100.00 g/mL, with mean percentage recoveries100.09± 0.949 and 100.29±0.519 for PYR and DOX, respectively. The presented chromatographic method was fully validated in accordance with ICH requirements and was statistically compared with those of the reported method and there was no significant difference between them. The proposed approach was shown to be more eco -friendly in terms of the use of hazardous chemicals and solvents, energy consumption, and waste generation than the reported one.

Peptide mapping (PM) is a vital technique in biopharmaceutical industry. The fingerprint obtained helps to qualitatively confirm host stability as well as verify primary structure, purity and integrity of the target protein. Yet, in-solution digestion followed by tandem mass spectrometry is not suitable as a routine quality control test. It is time consuming and requires sophisticated, expensive instruments and highly skilled operators. In an attempt to enhance the fuctionality of PM and extract multi-dimentional data about various critical quality attributes and comparability of biosimilars, coupling of PM generated using immobilized trypsin followed by HPLC-UV to principal component analysis (PCA) is proposed. Recombinant human growth hormone (rhGH); was selected as a model biopharmaceutical since it is available in the market from different manufacturers and its PM is a well-established pharmacopoeial test. Samples of different rhGH biosimilars as well as degraded samples: deamidated and oxidized were subjected to trypsin digestion followed by RP-HPLC-UV analysis. PCA of the entire chromatograms of test and reference samples was then conducted. Comparison of the scores of samples and investigation of the loadings plots clearly indicated the applicability of PM-PCA for: i) identity testing, ii) biosimilarity assessment and iii) stability evaluation. Hotelling's T2 and Q statistics were employed at 95% confidence level to measure the variation and to test the conformance of each sample to the PCA model, respectively. Coupling of PM to PCA provided a novel tool to identify peptide fragments responsible for variation between the test and reference samples as well as evaluation of the extent and relative significance of this variability. Transformation of conventional PM that is largely based on subjective visual comparison into an objective statiscally-guided analysis framework should provide a simple and economic tool to help both manufacturers and regulatory authorities in quality and biosimilarity assessment of biopharmaceuticals.

The combination of pyridoxine HCl (PYR) and doxylamine succinate (DOX) was proved to be effective and safe acting as the first line of pregnancy medication for vomiting and nausea under a trade name; Vomibreak® delayed release tablets. This combination has been available in the Egyptian market since 2016. Dissolution study is a meaningful tool that represents a predictor of output because the rate controlling steps in any drug's absorption is the rate of discharging from its medicinal formulation. Generally, the dissolution test of all delayed release tablets is operated at two stages: first the acid stage then the buffer stage. In our work, the acid stage was performed in 0.1 N hydrochloric acid (0.1 M HCl) and the buffer one was in 0.2 M sodium phosphate buffer (0.2 M Na-PB), pH = 6.8, according to FDA guidelines. In present work, for the first time, this binary mixture was quantitatively determined by applying four spectrophotometric methods. PYR was directly determined by zero order spectra method (D0) at 291.0 nm in the range 2.0–26.0 μg/mL in the acid stage and at 325.0 nm in the range 5.0–35.0 μg/mL in the buffer stage, where DOX show no interference in both cases. However, DOX was determined by three methods, namely, Dual wavelength (DW), Ratio difference (RD) and Derivative ratio (DD1). DD1 was the chosen method for determination of DOX in the two-phase dissolution study of Vomibreak® tablets at 249.0 nm in the range 2.0–44.0 μg/mL and 273.0 nm in the range 5.0–100.0 μg/mL in acid and buffer phases, respectively. All of the suggested methods were tested in compliance with ICH guidelines, where all methods were found to be reliable, reproducible, and selective. A statistical comparison was computed between two analytical techniques of critical importance in the development of two media dissolution profile: proposed UV- spectrophotometric and reported HPLC methods where no significant difference was found. Difference (ƒ1) and similarity (ƒ2) factors were calculated for PYR and DOX and shown that ƒ1 was 1.490 and 1.654 and ƒ2 was 94.431 and 92.396 for PYR and DOX, respectively.

Two chromatographic methods were developed and validated for the simultaneous determination of Sodium Cromoglycate (SCG) and Oxymetazoline Hydrochloride (OXMT). SCG and OXMT are administered in combination for effective treatment of nasal congestion and allergy. The first chromatographic method was based on usingaluminum TLC plates pre-coated with silica gel GF254 as the stationary phase and chloroform: methanol: toluene: triethylamine (5: 2: 4:1, by volume) as the mobile phase followed by densitometric measurement of the separated bands at 235 nm. The second method is a high performance liquid chromatographic method for separation and determination of SCG and OXMT using reversed phase C18 column with isocratic elution. The mobile phase composed of acetonitrile: methanol (2: 1, v/v) at flow rate of 1.0 mL/ min. Quantitation was achieved with UV detection at 220 nm. The validity of the proposed methods was assessed using the standard addition technique. The obtained results were statistically compared with those obtained by the official methods, showing no significant difference with respect to accuracy and precision at p = 0.05.

Sodium cromoglycate (SCG) and oxymetazoline hydrochloride (OXMT) are administered in combination for effective treatment of nasal congestion and allergy. In this work, SCG was determined using direct spectrophotometry by measuring its zero order absorption spectra at its λ max 320.6 nm where OXMT showed zero absorbance. On the other hand, four simple, sensitive and precise spectrophotometric methods were developed and validated for the determination of OXMT in the presence of SCG in their laboratory prepared mixtures and pharmaceutical formulation, without preliminary separation; Method A: first derivative spectrophotometric method [ 1 D], Method B: first derivative of ratio spectra method [ 1 DD], Method C: ratio difference spectrophotometric method [RDSM] and Method D: ratio subtraction method [RSM]. Ratio manipulating methods (Method B, C and D) were done using divisor of 10.00 µg/mL SCG. Linear correlation was obtained in range 4-22 µg/mL for OXMT by methods A, B and D and 6-22 µg/mL for method C. All methods were validated in compliance with the International Conference on Harmonization (ICH) guidelines and satisfactory results were obtained. No significant difference was noted between the developed methods and the official one with respect to accuracy and precision.