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Following ICH guidelines for analytical validation, we report a common C18 column stability indicating isocratic reverse phase high performance liquid chromatography method for the determination of the ion channel modulator Bay K8644. Two main forced degradation products and a minor impurity were also tentatively identified by Mass Spectrometry. The mobile phase consisted of a 50/50 acetonitrile/buffer mixture at a flow rate of 2 mL/min. Mean retention time for Bay K8644 was 3.030 minutes. Excellent linearity (r = 0.9998) was achieved in the range 0.10-1.40 μg/mL at 274 nm wavelength. Analytical limits were 16.56 ± 1.04 ng/mL for detection and 55.21 ± 3.48 ng/mL for quantitation respectively. Accuracy and precision studies showed good results (95-105%). Robustness was assessed by varying ±3%, both temperature and flow rate. Five different stress conditions were applied to assess Bay K8644's stability. Only basic and photolytic treatments yielded degradation products, both correctly resolved in a total runtime of 4 minutes. In conclusion, we developed a fast, simple, sensitive, accurate, precise, reliable and stability indicating method for detecting/quantifying Bay K8644, and tentatively characterized its main impurities/forced degradation products., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A direct and precise isocratic RP-HPLC method for simultaneous determination of silver sulfadiazine (SSD) and sodium hyaluronate (SH) in the presence of methyl (MP) and propyl parabens (PP) was developed and validated. Agilent chromatograph with X-Select C18 column (250 × 4.6 mm2, 5 μm) was used. Chromatographic separation was achieved using a mobile phase consisting of acetonitrile and 0.05 M phosphate buffer (pH 5.0 to which added triethyl amine 0.5 ml/L), at a ratio 35: 65 v/v. Elution was used at flow rate of 1.0 mL/min at ambient temperature with UV detection at 205 nm. The retention times for SH, SSD, MP and PP were 1.49, 3.3, 6.7 and 19.5 min, respectively. The presented chromatographic method was fully validated in accordance with ICH requirements, it was valid over linearity ranges of (0.80-100.00 μg/mL) and (3.20-100.00 μg/mL) for SSD and SH, respectively. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges and the sensitivity of the method, as the limits of detection and quantification for each active ingredient was also determined. The validated method was successfully applied for the quantification of SSD and SH in pharmaceutical cream formulation and the mean recovery % ± SD were 100.93 ± 0.985 and 100.05 ± 0.668 for SSD and SH; respectively, indicating satisfactory accuracy of the method., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This research aimed to develop a new method for simultaneously estimating the presence of azithromycin (AZT) and rifampicin (RIF) in a capsule formulation using reverse-phase high-performance liquid chromatography. The developed method utilized a Gemini column with a 60:40% v/v acetonitrile and potassium dihydrogen phosphate mobile phase, a flow rate of 1 mL/min, and an injection volume of 20 μL. The detection wavelengths of 210 and 254 nm for AZT and RIF, respectively, were used. Validation ensures specificity with a peak purity index > 0.99999 for AZT and >0.99995 for RIF, affirming unambiguous analyte detection. The system suitability test, within acceptable limits, validates method reliability. Linearity calibration curves (R2 = 0.998) cover a 25-150% target concentration range. Accuracy studies employing the standard addition method yield recovery values between 96.6 and 103.9% for both drugs, confirming method accuracy. Precision studies reveal % relative standard deviation values consistently below 2%, highlighting reproducibility. Robustness testing supports method reliability under varying conditions. Application to a pharmaceutical capsule formulation demonstrates the method's practicality, accurately quantifying AZT (98.30%) and RIF (99.37%). This study provides a validated analytical approach for simultaneous quantification in commercial pharmaceutical products containing both drugs, enhancing pharmaceutical quality control for critical antibiotics in complex formulations., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The dry powder inhalation formulation containing vilanterol trifenatate, umeclidinium bromide and fluticasone furoate intended for the therapy of bronchospasm related to chronic obstructive pulmonary disease and bronchial asthma was selected for the development and validation of a novel, selective, accurate, precise, quick and cost-efficient reversed-phase, high-performance liquid chromatography method. Neither an official monograph nor a single method has yet been published for the simultaneous estimation of these three compounds, which makes this method novel. The stationary phase of an ACE-C18-PFP column (250 mm × 4.6 mm, 5 μ) was used with a mobile phase of 25-mM sodium perchlorate buffer (pH 2.5 adjusted with ortho-phosphoric acid) and acetonitrile (40:60% v/v) at a flow rate of 1 mL/min to optimize chromatographic variables. The column temperature was kept at 40°C, and detection was at 224 nm, which was the isosbestic point of these three drugs. Well-resolved good peak symmetry was obtained for all three molecules by isocratic elution in less than 10 min, and the retention times of vilanterol trifenatate, umeclidinium bromide and fluticasone furoate were found to be 3.7, 5.4 and 8.3 min, respectively. The proposed method was validated as per ICH Q2 (R1) guidelines, and the calibration curves were linear in concentration ranges of 5-35 μg/mL for vilanterol trifenatate, 5-80 μg/mL for umeclidinium bromide and 5-150 μg/mL for fluticasone furoate, with mean % recoveries of 99-100%. The limits of detection and quantitation are 0.15 and 0.45 μg/mL for vilanterol trifenatate, 0.58 and 1.77 μg/mL for umeclidinium bromide and 0.32 and 0.96 μg/mL for fluticasone furoate, respectively. Hence, the proposed RP-HPLC technique was successfully used to quantify the inhalation formulation containing all three compounds., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A rapid, simple and highly sensitive stability-indicating reverse-phase high-performance liquid chromatographic technique, coupled with a photodiode array detector, was developed and validated for the estimation of Deferasirox (DFS). The chromatographic separation was achieved using a C-18 (250 × 4.6 mm, 5 μm) stationary phase and a mobile phase composed of 0.1% orthophosphoric acid and acetonitrile at a flow rate of 1 mL/min. The detection was carried out at a wavelength of 245 nm with a constant injection volume of 10 μL throughout the analysis. With an R2 value of 0.9996, the calibration curve was determined to be linear over an appropriate concentration range of 50-500 ng/mL. According to the International Conference on Harmonization (ICH) Q1 (R2) guideline, DFS was evaluated under stress conditions that included hydrolytic (acid, alkali and neutral), oxidative and thermal degradation. The findings demonstrated that significant degradation was observed in acidic degradation conditions, whereas drug substance was found to be stable when exposed to neutral, basic, oxidative and thermal degradation. The developed method was validated as per ICH guidelines. The developed method was employed successfully to estimate the amount of DFS in bulk and pharmaceutical formulation., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Central composite design based RP-HPLC method optimization for the synchronized analysis of Efonidipine Hydrochloride Ethanolate (EFE) and Chlorthalidone (CHL) in tablet. The effective separation was performed using Inertsil ODS C18 column (250 × 4.6 mm, 5 μm), PDA detector with 0.05 M KH2PO4 Buffer (pH 4.5): Acetonitrile (40:60%v/v) mobile phase. Independent variables were investigated include the concentration of KH2PO4 (X1) and flow rate of mobile phase (X2). Based on responses obtained (retention time, resolution and tailing factor), the optimum condition selected was X1 = 40% and X2 = 1 ml/min. Optimized HPLC condition was validated by assessing validation parameters and it meets the acceptance criteria set by ICH. The linear calibration curve was found to be in the quantity range 6.25-18.75 and 20-60 μg/ml Assay of drugs was 100.94 and 100.06% for CHL and EFE. The validated RP-HPLC-PDA method can be used for routine analysis of EFE and CHL in tablet., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Fipronil is a commonly used pesticide in the agricultural and animal health industries for the protection of crops and control of fleas, ticks, and chewing lice. It is difficult to obtain reproducible retention time and relative retention time (RRT) for a common hydrolytic degradation product of fipronil with the current European Pharmacopeia (EP) monograph for assay and estimation of related substances of fipronil. This situation causes misidentification, mislabeling, and/or false out-of-specification results for this hydrolytic degradation product of fipronil in bulk commercial batches during batch release and/or in the stability samples during the shelf life of a released batch., Objective: This study aimed to develop a reversed-phase ultra performance liquid chromatography (UPLC) method for assay and identification of fipronil including identification and estimation of its related substances in bulk drug substance batches of fipronil and provide consistent retention time of the hydrolytic degradation product., Methods: Fipronil and its related substances were separated by gradient elution on a Halo C18 column (50 mm × 2.1 mm id, 2.0 µm particle size) maintained at 40°C with 0.1% H3PO4 in H2O as mobile phase-A and acetonitrile-methanol (50 + 50, v/v) as mobile phase-B. Fipronil and its related substances were detected and quantified at 280 nm with a quantitation limit of 0.05% of the target (analytical) concentration., Results: The UPLC method was able to separate all analytes of interest by gradient elution with a total run time of 7 min (approximately 40% faster than EP)., Conclusion: In this paper, we report the development and validation of a fast, stability-indicating reversed-phase UPLC method for assay and estimation of related substances of fipronil in stability samples and bulk batches of fipronil., Highlights: The new UPLC method is approximately 40% faster than the current Ph. Eur. monograph for fipronil assay and the new method provides reproducible retention of a common hydrolytic degradation product of fipronil., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In this work, an eco-friendly simple, precise reverse phase high-performance liquid chromatography (HPLC) method has been developed and validated for Favipiravir in bulk and tablet dosage form followed by its force degradation study. The proposed method was validated to obtain official requirements including stability, accuracy, precision, linearity, robustness and selectivity as per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guidelines. The estimation was developed on C (18) column reversed-phase using the mobile phase composition as methanol:water (10:90 v/v). The flow rate was set as 1 ml/min, and the maximum absorption was observed at 323 nm using Shimadzu Photo Diode Array detector. The Favipiravir, drug showed a precise and good linearity at the concentration ranges of 10-50 μg/mL. The Revearse Phase High Perforance Liquid Chromatography assay showed the highest purity ranging from 99.90 to 100.02% for Favipiravir, tablet dosage form, and 100.15% was the mean percentage purity. The percent recovery was found within the acceptance limit of (98.6-100.0%). Intra- and inter-day precision studies of the method were less than the maximum allowable limit percentage of relative standard deviation ≤ 2.0. The Favipiravir retention time was found to be 5.00 min. To examine the stability of the drug, various forced degradation studies were conducted on Favipiravir Active Pharmaceutical Ingredient. The developed method was validated according to the ICH guidelines. A very quick, cost-effective, precise and accurate HPLC method for the determination of Favipiravir has been developed and validated in compliance with ICH guidance Q2., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

An isocratic HPLC method has been developed and validated for estimating paracetamol and diclofenac sodium simultaneously with three skeletal muscle relaxants, namely, methocarbamol, tizanidine hydrochloride and chlorzoxazone in their pure standard mixtures and in different multi-component dosage forms in a single chromatographic run. HPLC separation was achieved on a C18 Inertsil ODS-3V 5 μm column (250 × 4.6 mm) using a mobile phase mixture containing acetonitrile and 25 mM phosphate buffer (pH 7.4 adjusted with NaOH) in the proportion of (39.7:60.3, v/v) pumped at 1.2 mL/min flow rate with UV detection at 220 nm. An experimental design was used by applying Plackett-Burman design for screening the most critical predictors affecting the chromatographic separation and Box-Behnken design for optimizing the selected predictors and creating the response surface between the selected predictors and the interested responses. ICH recommendations were applied for validating the proposed method with regard to linearity, precision, accuracy, selectivity, limits of detection and quantitation, and robustness. There are many applications for the optimized method that can be applied for routine estimation of the cited drugs in laboratories of quality control and pharmaceutical industries to save money and time and to reduce material waste and effort., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A reverse phase high-performance liquid chromatography (HPLC) method has been developed for the quantification of a typical drug Dasatinib (DST) and its related impurities in pharmaceuticals. Kinetex C18 (4.6 × 150 mm, 5 μm) column was used in the chromatographic separations, using buffer (1.36 g of KH2PO4 in 1000 mL of water, pH = 7.8; adjusted with diluted KOH solution) with solvent as acetonitrile and mode of elution as the gradient. The flow rate is 0.9 mL/min, column oven temperature as 45°C and the overall gradient run time as 65 min. The developed method was found to produce symmetric and good separation between the process-related and degradation impurities. Method optimization is achieved with photodiode array at 305 nm over the concentration range of 0.5 mg/mL and degradation studies were carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to demonstrate the stability indicating capability of the method. Two major impurities were found in forced degradation studies in the HPLC analysis, the unknown, acid degradants were enriched and isolated by preparative HPLC, then characterized through high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. The unknown acid degradation impurity was showing Exact Mass of 521.11, molecular formula C22H25Cl2N7O2S and its chemical name as 2-(5-chloro-6-(4-(2-hydroxyethyl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino)-N-(2-chloro-6-methylphenyl) thiazole-5-carboxamide. Another impurity (oxidative degradant) found as known DST N-oxide Impurity-L and its chemical name as 4-(6-((5-((2-chloro-6-methylphenyl) carbamoyl) thiazol-2-yl) amino)-2-methylpyrimidin-4-yl)-1-(2-hydroxyethyl) piperazine 1-oxide. The analytical HPLC method was further validated as per ICH guidelines., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Many analytical methods are reported for simultaneous estimation of pharmaceutical dosages form. However, only a few are reproducible at an industrial scale. The proposed research aims to establish a technology transfer (TT) protocol between two laboratories (Lab-X, originator) with binary and (Lab-Y, receiver) with quaternary high-performance liquid chromatography (HPLC) system. Thus, utilizing reverse-phase HPLC (RP-HPLC), a robust, sensitive and repeatable analytical method has been developed, validated and TT between two laboratories for simultaneous estimation of Andrographolide (AG) and Paclitaxel (PTX). The method has been developed on a Phenomenex Luna C18 column (150 x 4.6, 5) sustained at 40°C and validated under the International Conference on Harmonisation (ICH) Q2 (R1) regulatory guideline and TT USP chapter 1224. The mobile phase consisted of MilliQ (pH = 3) and a combination of acetonitrile and methanol (1:1) in the ratio 50:50 with a flow rate of 0.45 mL/min, linear gradient elution in both labs. The AG and PTX were detected on the PDA detector at 224 and 227 nm wavelength with retention time of 4.5 ± 0.34 and 8.2 ± 0.02 min and limit of detection was found 0.028 ± 0.004 μg/mL, and 0.028 ± 0.0007 μg/mL, whereas limit of quantification as 0.086 ± 0.011 μg/mL and 0.088 ± 0.0014 μg/mL respectively in both labs. Throughout, this approach we have proved that proposed method is repeatable in both labs, and it can be used to quantify AG and PTX in developed pharmaceutical nano-formulations., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Levamisole hydrochloride (LVM) is an anthelmintic drug substance with immunomodulatory and anticancer activities. LVM has also found usage as a cutting agent in street cocaine., Objective: This study was aimed to develop and validate an alternative and improved stability-indicating reversed-phase ultraperformance liquid chromatography (RP-UPLC) method for the determination of LVM and the estimation of its related compounds., Method: The UPLC method for the assay was optimized in terms of organic solvents consumed, pH, column temperature, and flow rate. Determination of LVM and its related compounds was performed using a gradient elution on a Waters ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm i.d., 130 Å). The column temperature was maintained at 35°C. Mobile phase A was composed of aqueous 5 mM ammonium hydroxide, and mobile phase B was composed of acetonitrile. All the analytes were monitored by UV detection at 215 nm with a flow rate of 0.7 mL/min. The total runtime of the method with column equilibration is 4.0 min., Results: The developed method met all the acceptance criteria of the current International Council for Harmonization [ICH Q2 (R1)] guidelines. The method was tested in terms of specificity, linearity (R2 > 0.999), limit of detection (LOD; 0.06 μg/mL), limit of quantitation (LOQ; 0.2 μg/mL), accuracy, precision, and robustness. With a short analysis time (<2.5 min) and reduced consumption of organic solvents, the proposed method is considered a greener alternative to conventional chromatographic methods., Conclusions: An alternative and improved UPLC method was successfully developed and validated in accordance with the ICH guidelines for the determination of LVM and the estimation of its related compounds., Highlights: Due to its high degree of selectivity, speed, and accuracy, the developed method can significantly benefit the end-users with laboratory efficiency and throughput during routine analysis of production batches and stability monitoring of LVM-related drug products., (© The Author(s) 2023. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A simple, robust stability indicating RP-HPLC method was developed for simultaneous quantification of serdexmethyl phenidate and dexmethyl phenidate in a fixed capsule dosage form. This is the first method to be reported for simultaneous estimation and quantification of degradation products produced from forced stressing of the dosage form as per ICH guidelines. The chromatographic separation was attained on Waters X-terra C18 column using a mixture of trifluoro acetic acid and acetonitrile (70:30 v/v) as mobile phase with a flow rate of 1 mL, monitored at 265 nm over a run time of 10 min. Serdexmethyl phenidate and dexmethyl phenidate were eluted with retention times of 2.71 min and 7.33 min, respectively. The method displayed linear responses in the range of 4.2-63 μg/mL (0.9994) for serdexmethyl phenidate and 0.9 to 3.5 μg/mL (0.9998) for dexmethyl phenidate, respectively. The percentage recoveries of the two drugs were found within the acceptable limits. Forced degradation was conducted and showed considerable degradation in various stress conditions. It also confirms the specificity of the method as no interference peaks were observed concerning for to stress products. This method can be routinely used in quality control labs for simultaneous determination of respective drugs in marketed dosage form., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The combination of carvedilol (CAR) and ivabradine (IVA) is used for a greater reduction in heart rate and for achieving better exercise capacity in a patient with chronic heart failure. Numerous reverse-phase high-pressure liquid chromatography (RP-HPLC) and hyphenated techniques have been reported for the simultaneous estimation of CAR and IVA, but the high-performance thin-layer chromatographic (HPTLC) method has not been reported yet. Hence, the robust HPTLC method has been developed by the implementation of an enhanced analytical quality by design approach based on the principles of analytical failure modes critical effect analysis (AFMCEA) and design of experiments (DoE) as per the upcoming ICH Q14 guideline. The AFMCEA was started by the identification of potential analytical failure modes followed by their critical effect analysis by a DoE-based screening design. The high-risk failure modes were optimized by DoE-based response surface methodology. The method operable design ranges and control strategy was framed for optimized chromatography conditions. The HPTLC method was validated as per ICH Q2 (R1) guideline. The HPTLC method was applied for the assay of FDC of CAR and IVA, and results were found in compliance with the labeled claim. The developed method can be used as an alternative to the published RP-HPLC method for quality control of FDC of CAR and IVA in the pharmaceutical industry., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Delmopinol hydrochloride is widely used as an active ingredient (AI) in many human oral hygiene products. To the best of our knowledge (via literature search), there is no stability-indicating liquid chromatography method available in the public domain for assay and estimation of related substances of Delmopinol hydrochloride. A fast stability-indicating Reversed-Phase Ultra High-Performance Liquid Chromatography (RP-UHPLC) method has been developed and validated for identification, assay and estimation of related substances in commercial bulk batches of this AI. The major peak of the AI and its related compounds are adequately separated in 6 min by a gradient elution on a hybrid silica-based C18 column (Waters Acquity UPLC® BEH C18, 50mm × 2.1 mm I.D., 1.7 μm particle size) maintained at 50°C. Mobile phase-A is composed of aqueous 10 mM NH4OH and mobile phase-B is ACN. The AI and its related compounds are detected with UV detection at 220 nm and quantitated by an external Delmopinol hydrochloride reference standard. The quantitation limit of the method is 0.1% of the target analytical concentration. This UHPLC method is fast and green and has been demonstrated to be specific, accurate, linear, precise, sensitive and robust as per ICH guidelines., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The Central Drugs Standard Control Organization approved a bilayered, uncoated, fixed-dosage combination tablet formulation containing pregabalin (75 mg) and etoricoxib (60 mg) in November 2019 to control prolonged back pain with neuropathic components., Objective: This research aims to create an analytical quality by design-assisted reversed phase (RP)-HPLC method for quantifying pregabalin and etoricoxib in tablet formulation., Methods: The best chromatographic conditions were a Phenomenex C8 column (250 × 4.6 mm, 5.6 μm) and a mobile phase of 0.35% orthophosphoric acid -acetonitrile (1 + 1, by volume). A flow rate of 1 mL/min was used with a detection wavelength of 220 nm., Results: The optimized reversed phase HPLC method was successfully validated by the International Conference on Hormonization guideline Q2(R1). Moreover, the percentage assay of pregabalin and etoricoxib tablet formulation was 99.05 and 100.02% w/w, respectively, using the validated RP-HPLC method., Conclusion: The newly developed analytical quality by design-assisted RP-HPLC method has several advantages, including shortened analytical time (2.65 min and 7.45 min pregabalin and etoricoxib, respectively), efficient separation in terms of well-defined peaks, and a simple mobile phase combination., Highlights: The use of analytical quality by design principles and the design of the experiment tool allowed the detection of influential parameters critical for achieving the most favourable chromatographic conditions for accurately quantifying individual drugs using the RP-HPLC method. The compliance of the results with the ICH criteria validated the method. As a result, adopting the analytical quality by design methodology ensured the development of a more robust method that can generate consistent, dependable, and quality data throughout the process while also saving time., (© The Author(s) 2022. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for the identification and assay of Ivermectin, including the identification and estimation of its process-related impurities and degradation products in bulk drug substance of Ivermectin. Analytes were separated on a HALO C18 column (100 mm × 4.6 mm I.D., 2.7 μm particle size) maintained at 40 °C (column temperature) with gradient elution. All analytes of interests were adequately separated within 25 min. All degradation products, process-related impurities and assay were monitored by ultraviolet detection at 254 nm. The new HPLC method described here successfully separated an isomer peak of the active pharmaceutical ingredient (API) from the major API peak. This newly separated isomer peak is around 1.2 to 1.5% (peak area) in typical API samples, and coelutes with the major API peak by all current HPLC methods. Quantitation limit of the HPLC method is 0.1% of target analytical concentration (~1.0 μg/mL). This method has been demonstrated to be accurate, robust, significantly higher degree of selectivity compared to the HPLC methods of Ivermectin drug substance reported in the literature and in the compendial HPLC methods prescribed in the current USA and European Pharmacopeia., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Telmisartan is an antihypertensive drug and several FDC products of telmisartan are available in the market for the treatment of hypertension. The multipurpose reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated as economical and eco-friendly alternative to numerous published chromatography methods for synchronous estimation of FDC of telmisartan to save time, cost and solvent for analysis. The analytical quality risk management was initiated with identification of potential method parameters using cause-effect diagram followed by assessment of their risk by risk priority number ranking and filtering method. The risk of critical method parameters was controlled by their optimization using design of experiments-based full-factorial design. The method operable design ranges was identified for high-risk method parameters, and control strategy was set for mitigation of their risk throughout the life cycle of the developed RP-HPLC method. The RP-HPLC method was validated as per the ICH Q2 (R1) guideline. The RP-HPLC method was applied for simultaneous estimation of seven FDC products of telmisartan, and results were found compliance with labeled claim. The developed RP-HPLC method is fulfilling requirements of numerous RP-HPLC and high performance thin layer chromatography methods for the said estimation. It can be used as a multipurpose RP-HPLC method for quality control of FDC products of telmisartan in the pharmaceutical industry to save solvent, cost and time of analysis., (Published by Oxford University Press 2021.)

A novel stability-indicating isocratic reverse phase high-performance liquid chromatographic method was developed for the quantitative determination of perampanel in the presence of degradation products and its process-related impurities. Good resolution was achieved between the peaks corresponding to process-related impurities and degradation products from the analyte using Shim-pack GIST C18 column with mobile phase, potassium dihydrogen phosphate (pH 2.5; 30 mM)-acetonitrile (45: 55, v/v) at a wavelength of 294 nm. Perampanel and its impurities were well-separated within a run time of 7 min. To show the stability-indicating power of the method, forced degradation studies were performed on bulk samples of perampanel as per ICH prescribed stress condition using acid, base, hydrolytic, oxidative and photolytic degradation conditions. The degradation products were resolved from main peak and its impurities. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also applied for assay and related substances determination of perampanel in bulk and pharmaceutical dosage form., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Reverse phase liquid chromatography (RP-LC) is considered the most extensively used chromatographic technique in drug separation and quantification, yet its routine use shows increasing environmental challenges about energy consumption, solvent used, and waste engendered. From this perception, efforts for greening the developed RP-LC methods are focused on using green solvents and reducing the amount of solvent used and waste produced., Objective: The target of this work is to develop an eco-friendly RP-LC method for the synchronous separation of six widely used drugs in the treatment of cerebrovascular and vestibular disorders, namely neurotropic piracetam, antihistamines dimenhydrinate, cinnarizine, and fluranizine, and vasodilators vincamine and vinpocetine., Method: Separation and quantification are accomplished at ambient temperature by isocratic elution mode using acetonitrile-0.2% triethylamine (85:15 by volume) as a mobile phase at a flow rate of 1.5 mL/min on a Hypersil C18 column. UV detection and quantification are carried out at 225 nm., Results: The method is fully validated per ICH guidelines, where all analytical parameters are within the acceptable range (r > 0.9999), accuracy (≥99.55%), and precision (0.02-0.97%)., Conclusions: The method is applied for the routine analysis of the cited drugs in their single and combined drug products, and it is considered excellent in terms of greenness with respect to Eco-Scale assessment., Highlights: As the routine use of the RP-LC technique shows increasing environmental challenges, efforts for greening are attempted focusing on the use of green solvents and reducing the amount of solvent used and waste produced. A simple eco-friendly RP-LC method for the synchronous separation of six widely used drugs in the treatment of cerebrovascular and vestibular disorders is developed. The method is fully validated per ICH guidelines, where all analytical parameters are within the acceptable range. The method is applied for the routine analysis of the cited drugs in their single and combined drug products., (© The Author(s) 2022. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Chromatography is one of the most popular methods for the separation of compounds in modern pharmaceutical industry and science. Despite the extensive use of the reversed phase chromatography in analytical and preparative applications, the normal phase adsorption chromatography has a special place in purifying post-reaction mixtures or the separation of natural extracts, especially in wet load mode, because of simplicity and high velocity of preparation. Complex mixtures, more difficult to separate, require gradient methods to obtain better results of separations. These methods can be developed by external software, but the automatic methods are often not very accurate and the negative impact of wet load application on separation quality is considerable in them. Therefore, we present the thin-layer chromatography (TLC) gradient optimization strategy for wet load separations to obtain repeatable results of separations for different compounds without worrying about negative impact of wet loading on separation quality. The strategy provides information about an elution model of desired compound, which is used to develop the gradient method. The strategy also allows to standardize the separation length, because gradient methods performed by the TLC gradient optimization strategy have a very similar duration time in column volumes. The method can also be simply scaled because of using the column volume as a base unit in calculations., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Accurate determination of active pharmaceutical ingredients and impurities is essential for ensuring the safety and effectiveness of medications. This study focuses on the validation of a high-performance liquid chromatography (HPLC) method for quantifying atorvastatin and its impurities, addressing a critical aspect of pharmaceutical analysis., Objective: The primary objective is to conduct a comprehensive validation study for the HPLC method, covering specificity assessment, response function establishment, and a detailed analysis of precision, trueness, and tolerance intervals. The emphasis is on demonstrating the method's precision, accuracy, and stability-indicating capabilities across various concentrations and compounds., Methods: The HPLC method is validated through rigorous assessments, including specificity, response function establishment, and analyses of precision, trueness, and tolerance intervals. Induced degradation experiments are conducted to explore atorvastatin's behavior under extreme conditions. Insights into the compound's synthesis and degradation pathways are provided through a proposed mechanism for intramolecular esterification., Results: The results affirm the precision, accuracy, and stability-indicating capabilities of the validated HPLC method. The method effectively differentiates between atorvastatin and its impurities, showcasing its suitability for pharmaceutical quality control., Conclusions: The validated HPLC method emerges as a robust and reliable tool for atorvastatin analysis, contributing significantly to pharmaceutical research and quality control. Its application ensures the safety and efficacy of medications, reinforcing its role in pharmaceutical analysis., Highlights: This study not only validates a crucial HPLC method for atorvastatin analysis but also provides insights into the compound's behavior under extreme conditions and its synthesis and degradation pathways. The validated method serves as a cornerstone in pharmaceutical research and quality control, ensuring medication safety and efficacy., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We investigated the dual retention mechanism in thin-layer chromatography taking place on three stationary phases of different polarity (C-18, plain silica gel and DIOL) and using binary mobile phases composed of acetonitrile as the main component and water, or methanol as a modifier. As the test analytes, we selected a set of 12 compounds of pharmaceutical importance and considerably different chemical structure, i.e. the imidazoline and serotonin receptor ligands, and their related compounds. Retention of each analyte in each investigated chromatographic system was determined in a wide enough range of the mobile phase composition, with volume fraction of the mobile phase modifier ranging from 0.10 to 0.90. Calculation of the exact turning point values as a proof of occurrence of the reversed-phase hydrophilic interaction chromatography (HILIC/RP) retention mechanism was based on the multimodal retention model. The dual retention mode was described with the use of the volume fraction of the mobile phase modifier, the total polarity and the total solubility models. For the DIOL, C-18 and silica gel stationary phase, the dual (HILIC/RP) retention mechanism was confirmed. In the case of the DIOL stationary phase and acetonitrile/methanol mobile phase, the observed retention mechanism was more complicated than the dual HILIC/RP one., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Non-depolarizing neuromuscular blocking agent Rocuronium bromide was quantified in drug substance and drug product using reversed-phase liquid chromatographic method. Forced degradation studies were conducted for Rocuronium bromide in drug substance under acidic (2MHCl), basic (2MNaOH), oxidative (3% H2O2), thermal (135°C) and photolytic (254 nm) stress conditions. An Agilent H12 C18 column was used for separation using diammonium hydrogen phosphate buffer (pH 8; 0.04M)- acetonitrile (50:50; v/v) as mobile phase at flow rate of 1 mL/min. The quantification was done using UV detection at 210 nm. The limit of quantification and detection was 11.1 and 3.66 μg/mL, respectively, and the recovery percentage was 99% in drug substance and drug product. ICH guidelines were adopted for method validation. The proposed LC method monitored the degradation profile for Rocuronium bromide under various stress conditions and provided a specific LC method for its routine analysis. Besides, the MS data were used to identify all Rocuronium bromide degradation products and the possible degradation pathway was designated., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Pranlukast hydrate is an anti-asthmatic drug and used in the treatment of acute asthma. Stability-indicating RP-HPLC method for pranlukast hydrate has been developed and validated. The reverse phase high performance liquid chromatographic method was developed using Shimadzu Column: Kromosil 100 C18 (150 mm × 4.6 mm × 5 μm) and mobile phase Acetonitrile: 0.1% Glacial acetic acid (85: 15% v/v). Eluent was monitored with UV-detector at 262 nm with a flow rate of 0.5 mL/min, temperature maintained at 30°C. Stress testing was carried out in acidic, alkaline, oxidative, photolytic and dry heat degradation conditions. The method was validated as per the International Conference for Harmonization guidelines and includes specificity, accuracy, precision, linearity and limit of quantitation and detection parameters. A relative standard deviation <2% indicates the developed method was precise. The accuracy of the method was represented by recovery studies ranging between 99.41 and 99.72%. In acid, alkaline, oxidative stress conditions, pranlukast hydrate degrades significantly and in photolytic, dry heat, hydrolytic conditions remain stable. This proposed method is suitable for the analysis of pranlukast hydrate in its laboratory mixture., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Chromatographic behavior of different substances in normal and reversed phases is an interesting area in the scientific community., Objective: The work aimed to optimize and validate chromatographic separation methods for simultaneous determination of paracetamol (PAR) and phenylephrine HCl (PHE) in the presence of PAR impurities, namely p-aminophenol, p-nitrophenol, acetanilide, and p-chloroacetanilide with further quantification of these toxic impurities., Methods: TLC method based on normal phase separation was carried out on aluminum sheets of silica gel 60 F254 using ethanol:chloroform:ammonia as a developing system, followed by densitometric measurements. While HPLC is based on reversed phase separation using a C18 column against acetonitrile:phosphate buffer pH 5 as a mobile phase., Results: PAR and PHE were determined by the TLC-densitometric method in concentration ranges of 3-25 and 0.1-3 µg/band, respectively, and determined by the HPLC method over concentration ranges of 5-400 and 2-80 µg/mL, respectively. Both methods were optimized and validated. Furthermore, they were successfully applied for pharmaceutical formulations with precision <2%. Moreover, results of a statistical comparison with the official methods confirm the methods' validity claims., Conclusion: Two eco-friendly chromatographic methods were developed to determine PAR and PHE in their binary mixtures, pharmaceutical formulations, and in the presence of PAR-related impurities with further quantification of these toxic impurities., Highlights: These simple chromatographic methods are the first methods developed for simultaneous determination of PAR and PHE in the presence of PAR-related impurities., (© AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Present study reports design of experiment (DoE) based development and validation of a simple, rapid and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for estimation of efavirenz (EFZ), a non-nucleotide reverse transcriptase inhibitor (NNRTs), used in the treatment of acquired immunodeficiency syndrome (AIDS). Plackett-Burman design was explored to screen the critical method variables (CMVs) for the RP-HPLC method. A response surface Box-Behnken design was employed to optimize the screened CMVs which affect the analytical responses (ARs) of RP-HPLC method. Using the optimized CMVs the HPLC method was developed and validated according to International Conference on Harmonization (ICH) guidelines. EFZ in marketed formulation was estimated using the validated method. Acetonitrile proportion, pH of the phosphate buffer and mobile phase flow rate were the CMVs and retention time and number of theoretical plates were the ARs for the study. The optimized chromatographic parameters were acetonitrile proportion in mobile phase: 51.17%v/v, pH of phosphate buffer: 4.04 and flow rate: 1.25 mL/min. Use of these optimized parameters resulted in retention time of 11.031 min and 9,498.787 number of theoretical plates as ARs of the HPLC method. The method was further validated in harmony with current ICH guidelines Q2 (R1). The method was capable of the successful estimation of EFZ in marketed formulation. The study depicts successful development and validation of a simple RP-HPLC method of EFZ using DoE approach., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A stability-indicating reverse phase high-performance liquid chromatography method was developed and validated for simultaneous quantification of apremilast (APL) and betamethasone dipropionate (BD) in bulk as well as drug loaded microsponges. Various mobile phase systems were screened to check the system suitability followed by force degradation analysis to determine APL and BD stability under varying stress conditions. A central composite design model was used to optimize the column temperature and flow rate using Design Expert® (9.0.1). One factor at a time approach with five independent factors were used to validate the robustness of the method. Finally, APL and BD were precisely and accurately quantified from drug loaded microsponges using the validated method. A favorable separation of APL and BD was obtained on a Phenomenex® Luna C18 column using a mixture of 50 mM phosphate buffer containing 0.1% triethylamine (pH 6.1) and acetonitrile (60:40%v/v) as mobile phase. Both the drugs were found to be stable when exposed to stressors such as heat-, light-, alkali-, acid- and peroxide-induced degradation. The calibration curves were found to be linear with appreciable limit of detection and limit of quantification. Recovery and percentage relative standard deviation of peak areas for APL and BD were found to be < 2.0% and 99-100% in bulk drug solution and <2.0% and 99-103% in microsponge formulation, respectively. Statistical analysis using analysis of variance indicated that the model was significant (P < 0.001). Hence, the developed method can be effectively used to quantify APL and BD, both in bulk as well as microsponge formulations., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Numerous RP-HPLC and HPTLC methods have been reported for estimation of fixed-dose combination (FDC) products of aspirin with anti-hypertensive and anti-lipidemic drugs. Each FDC of aspirin needs separate and dedicated chromatographic conditions for analyses. No chromatographic method has been reported for simultaneous estimation of FDC products of aspirin using a single chromatography condition., Objective: A multipurpose HPTLC method was developed for simultaneous estimation of some FDC products of aspirin using an enhanced analytical quality-by-design approach based on a design of experiment (DoE) and risk-based define, measure, analyse, improve and control (DMAIC) principle to save solvent, cost, and time of analysis., Method: The risk-based DMAIC process was carried out with identification of potential method risk parameters and their assessment using risk priority number (RPN) ranking and filtering. The DoE-based DMAIC process was carried out by the implementation of fractional factorial and full factorial design., Results: The mobile phase composition and volume of modifier were found to be critical method risk parameters for resolution of all peaks. The developed method was found to be validated, and assay results of all FDC products of aspirin were found to be in good agreement with their respective labelled claim., Conclusions: The developed method is found to be solvent, cost, and time saving and also fulfilled the analytical requirements of many reported chromatography methods. Hence, the developed method is a multipurpose chromatography for analysis of FDC products of aspirin., Highlights: DoE and risk-based DMAIC principle to development of the multipurpose-chromatography method. The developed method was applied for the estimation of eight different FDC products of aspirin., (© AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Doramectin is a broad-spectrum antiparasitic drug used in veterinary medicine. It belongs to the family of avermectins, which possess a macrocyclic lactone structure, and is widely used for the treatment of parasites., Objective: This study aimed to develop a stability-indicating reversed-phase (RP) HPLC method for the assay and identification of doramectin including identification and estimation of its related substances in commercial batches of doramectin drug substance., Method: Methanol was used to dissolve and prepare doramectin samples. Doramectin and its related substances were separated on a HALO C8 (100 mm × 4.6 mm i.d., 2.7 µm particle size) column maintained at 40 °C using an isocratic HPLC method with a mobile phase composed of acetonitrile-water (70 + 30, v/v). Analytes were detected with UV detection at 245 nm and quantitated against a single point external reference standard of doramectin. The LOQ of the method is 0.1% of the target concentration as described in the method., Results: The HPLC method can separate all analytes of interest by an isocratic elution within 10 min. The method was validated according to the guidelines described in the International Conference on Harmonization Q2(R1)., Conclusions: The HPLC method for assay of doramectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability-indicating., Highlights: This is the first known paper to report an HPLC method for assay of doramectin and estimation of its related substances in commercial batches of doramectin drug substance., (© AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Dronedarone HCl (DRN) is an anti-arrhythmic drug indicated for atrial fibrillation. DRN has a low solubility of 2 µg/mL and 4% bioavailability, thus it is formulated as a co-amorphous system to enhance its solubility by using quercetin (QCT) as a co-former. A sensitive, accurate, and economic method for the simultaneous quantification of DRN and QCT in formulation is not found in the literature., Objective: To develop a Reverse Phase -HPLC method for the simultaneous estimation of DRN and QCT in a DRN-QCT co-amorphous system., Method: The co-amorphous system was prepared using a solvent evaporation technique with DRN and QCT in a 1:1 molar ratio. The separation was achieved on a Purospher® STAR C18 (250 mm × 4.6 mm × 5 μm id (internal diameter)) column with the mobile phase comprising of acetonitrile and a 25 mM phosphate buffer pH 3.6 (60:40%, v/v)., Results: DRN and QCT were retained on the column for 6.7 and 3.5 min, respectively. For both molecules, the method was developed with a wide linearity range of 0.2-500 µg/mL. The LOD for DRN was found to be 0.0013 µg/mL and for QCT it was found to be 0.0026 µg/mL. The LOQ for DRN was found to be 0.0041 µg/mL, and for QCT it was 0.0078 µg/mL., Conclusions: The method was validated as per International Conference on Harmonization (ICH) guidelines for linearity, precision, accuracy, and robustness. The method was used in simultaneous quantification of DRN and QCT in co-amorphous samples., Highlights: The method developed was used for the analysis of content uniformity and solubility samples of co-amorphous system, where the method was able to successfully quantify DRN and QCT. Low detection and quantification limits contribute to the sensitivity of the method and wide linearity range assures the robust and precise quantification of molecules., (© AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Apixaban (APX) is a novel anti-coagulant drug approved by USFDA. According to referred literature, numerous chromatographic methods such as RP-HPLC and high-performance thin-layer chromatography have been published for the stability study of APX. But these chromatographic methods have been developed using toxic organic solvents that are hazardous to the environment and unsafe for analysts. Hence, green and sustainable analytical chemistry-driven chromatographic method has been developed for the stability study of APX using safe organic solvents for the safety of analysts and the protection of the environment. APX was subjected to forced degradation for the development of a stability-indicating assay method. The method development was carried out by the implementation of chemometric and DoE approaches for minimizing solvent wastage. Principal component analysis was applied for the identification of critical method risk variables (MRVs) and method performance attributes. DoE-based response surface modelling was applied for the optimisation of critical MRVs. The greenness profile scales of published and developed chromatographic methods have been assessed by NEMI and AGREE methods for the estimation of APX. The developed method was found to be more eco-friendly and robust than the published chromatographic methods for the estimation of APX., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Infant formulas, and pediatric and adult nutritional products, are being fortified with bovine lactoferrin (bLF) due to its beneficial impacts on immune development and gut health. Lactoferrin supplementation into these products requires an analytical method to accurately quantify the concentrations of bLF to meet global regulatory and quality standards., Objective: To develop and validate a lactoferrin method capable of meeting the AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR®) 2020.005., Methods: Powder formula samples are extracted using warm dibasic phosphate buffer, pH 8, then centrifuged at 4°C to remove insoluble proteins, fat, and other solids. The soluble fraction is further purified on a HiTrap heparin solid-phase extraction (SPE) column to isolate bLF from interferences. Samples are filtered, then analyzed by LC-UV using a protein BEH C4 analytical column and quantitated using an external calibrant., Results: The LOQ (2 mg/100 g), repeatability (RSD: 2.0-4.8%), recovery (92.1-97.7%), and analytical range (4-193 mg/100 g) all meet the method requirements as stated in SMPR 2020.005 for lactoferrin., Conclusion: The reported single-laboratory validation (SLV) results demonstrate the ability of this lactoferrin method to meet or exceed the method performance requirements to measure soluble, intact, non-denatured bLF in infant and adult nutritional powder formulas., Highlights: The use of a heparin affinity column to isolate lactoferrin from bovine milk products combined with a selective analytical chromatographic column provides suitable analyte specificity without requiring proprietary equipment or reagents., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL.)

Background: Estimation of the drug and development of the method is a critical aspect of formulation development and a critical factor for analytical scientists. Gefitinib is a poorly soluble anticancer drug., Objective: The present research focuses on the topic of the development of innovative quality by design methods for the estimation of gefitinib (GF) from bulk, pharmaceutical tablet formulation, and complex nanoformulations., Methods: To simplify the estimation of poorly soluble drugs such as GF, response surface methodology (RSM) was adopted with effective leverages to obtain precise computation design space using the Box-Behnken design (BBD) model. The major three mixed-effect independent factors (percentage of buffer, pH of buffer, and flow rate) were screened with three prominent dependent responses (viz., theoretical plate, retention time, and tailing factor) selected for optimal analysis. Furthermore, co-processed steps were employed for the estimation of the analyte from the complex formulation., Results: The RP-HPLC method uses the quality by design (QbD) approach can effectively estimate the analyte concentration of less than 4.5 min. The developed method was economically robust and sensitive and shows a relative standard deviation (RSD, %) of less than 2% for all the selected validation parameters. The estimated design space suggests the highest desirability (R2-0.998) at 60% of buffer in the mobile phase, pH 4.25, and flow rate of 0.7 mL/min., Conclusions: The QbD approach was used to design and develop the method by understanding the interaction between dependent and independent variables to get the optimum values. The developed method was validated successfully and can be useful for formulation scientists to estimate drug concentration and drug release profiles from complex nanoformulations., Highlights: The analytical approach was designed and quantified using a quality-by-design approach to make the RP-HPLC method more robust and efficient for the estimation of analytes from complex nanoformulations. The method is also useful to eliminate the interfering molecule during estimation by employing co-processing steps. The developed method saves time and cost of solvent and employs QbD as a requirement of recent regulatory concern., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The application of Quantitative Structure-Property Relationship (QSPR) modeling to the prediction of reversed-phase liquid chromatography retention behavior of synthetic cannabinoids (SC), and its use in aiding the untargeted identification of unknown SC are described in this paper. 1D, 2D molecular descriptors and fingerprints of 105 SC were calculated with PaDEL-Descriptor, selected with Boruta algorithm in R environment, and used to build-up a multiple linear regression model able to predict retention times, relative to JWH-018 N-pentanoic acid-d5 as internal standard, under the following conditions: Agilent ZORBAX Eclipse Plus C18 (100 mm × 2.1 mm I.D., 1.8 μm) column with Phenomenex SecurityGuard Ultra cartridge (C18, 10 mm × 2.1 mm I.D., < 2 μm) kept at 50°C; gradient elution with 5-mM ammonium formate buffer (pH 4 with formic acid) and acetonitrile with 0.01% formic acid, flow rate 0.5 mL/min. The model was validated by repeated k-fold cross-validation using two-thirds of the compounds as training set and one-third as test set (Q2 0.8593; root mean squared error, 0.087, ca. 0.56 min; mean absolute error, 0.060) and by predicting relative Retention Times (rRT) of 5 SC left completely out of the modeling study. Application of the model in routine work showed its capacity to discriminate isomers, to identify unexpected SC in combination with mass spectral information, and to reduce the length of the list of candidate isomers to ca. one-third, thus reducing significantly the time required for predicting high-resolution product ion spectra to be compared to the unknown using a computational Mass Spectrometry (MS) search/identification approach., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Rufinamide is a triazole derivative that is structurally dissimilar to other marketed antiepileptic drugs, has been assumed a marketing authorization, by the European Union and FDA, for use as a complementary therapy for seizures associated with Lennox-Gastaut syndrome., Objective: This work is concerned with development of two methods for determination of rufinamide (RUF) in presence of 1-[(2,6-difluorophenyl)methyl]-1H-1,2,3-triazole-4 carboxylic acid as its alkaline degradation product in dosage form., Methods: The first method was capable of determing RUF in the presence of its alkaline degradation product and in dosage form. Kromasil C8 column and mobile phase consisting of acetonitrile-water (50:50, v/v) were used and UV detection at 210 nm. In the second method, first derivative ratio spectrophotometry, RUF was determined by measuring peak amplitude at 269.5 nm over 5-30 μg/mL., Results: The linearity range of RUF was 10-90 μg/mL for HPLC method covering its therapeutic range with r2 = 0.9999. Forced degradation under alkaline conditions was carried out, the degradation product was isolated and its structure was confirmed. Both methods were validated in accordance to ICH guidelines. Statistical analysis revealed no significant difference between obtained results and reported ones., Conclusion: The present study is useful for therapeutic drug monitoring and routine analysis of RUF in quality control laboratories., Highlights: Kinetics of the alkaline degradation of RUF was studied by following the concentration of the remaining drug until complete degradation was achieved., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A rapid, reliable and precise integrated solid-phase extraction (SPE) and reversed-phase liquid chromatography method was developed and validated to determine bupivacaine in human serum using single protein-coated analytical columns. The protein-coated columns were packed with four different sorbents: TSK-ODS, LiChrosorb RP-8, LiChrosorb RP-2 and μ-Bondapak CN-bonded silica. The method involved direct injection of serum sample onto the columns for trapping of the analyte, clean-up from weakly retained serum endogenous components, as well as the final separation. The protein-coated columns operated in two different chromatographic modes. Serum proteins were extracted and cleaned up by SPE, whereas the final separation of bupivacaine was based on reversed-phase chromatography. The protein-coated TSK-ODS column resulted in more accurate peak integration and more reproducible results. A linear relationship between the concentrations of drug and peak areas was confirmed in the range of 100-2000 ng/mL. Detection and quantification limits were 24.85 and 85.36 ng/mL, respectively. The average recovery for bupivacaine ranged from 96.48% to 98.81%. The present methodology was successfully applied, with a high degree of confidence, to analyze clinical samples obtained from patient receiving 0.5% bupivacaine therapy., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Ponceau 4R (E124) and carmoisine (CMS; E122) are frequently utilized azo synthetic dyes in the food industry owing to their aesthetically pleasing coloration and broad consumer acceptability. It is imperative to prioritize environmentally favorable technologies for quantifying these dyes, as excessive consumption of these poses significant health risks., Objective: The primary objective of this research was to establish a reversed-phase (RP)-HPLC method that could simultaneously detect Ponceau 4R and CMS, implementing green analytical chemistry (GAC) and analytical quality by design (AQbD), using an ultrasound-assisted extraction (UAE) technique in commercial food samples., Methods: An Agilent Eclipse Plus column (C18, 250 × 4.6 mm id, 5 µm) was utilized for effective separation with a mobile phase of ethanol-acetate buffer pH 5 (60:40, v/v), flow rate of 1 mL/min, and detection wavelength of 515 nm. Critical variables selected for method optimization were ethanol percentage and flow rate, determined using central composite design (CCD). In order to adhere to the 12 principles of green chemistry, hazardous solvents were substituted with ethanol, which is distinguished by its ease of use, effectiveness, and ecological sustainability. The greenness assessment was conducted utilizing the green analytical procedure index (GAPI), analytical eco-scale (AES), and analytical greenness metrics (AGREE)., Results: The respective retention times for Ponceau 4R and CMS were 2.276 and 3.450 min. The recovery rate of Ponceau 4R and CMS fluctuated between 70% and 102% and 80% and 102%, respectively, across various marketed food samples. The procedure passed validation in accordance with the International Conference on Harmonization Q14 guidelines., Conclusion: The devised method demonstrates that the validation parameters like linearity, precision, sensitivity, and reproducibility are within the specified limits of ICH guidelines. The greenness assesment tools GAPI, AES, and AGREE produced the most favorable results., Highlights: In future, environmentally sustainable, solvent-based, robust AQbD methodologies for assessing varieties of food colorants may be adopted and improved commercially., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Analytical quality by design (AQbD) affords a systematic scaffolding to triumph a continuously validated, robust assay as well as life cycle management. The resuscitative repurposed drug favipiravir, an oral drug approved for reemerging pandemic influenza in Japan in 2014, is used for the treatment of life-threatening pathogens such as Ebola, Lassa virus, and currently COVID-19. Favipiravir is gaining a great deal of medical importance due to its pharmaceutical applications., Objective: To develop and validate a risk-based stability-indicating RP-HPLC method employing an AQbD approach using Central Composite Design (Design Expert Software 13.0) for the estimation of favipiravir., Method: The Quality Target Product Profile optimized were flow rate and mobile phase composition, thus assessing the critical analytical attributes (retention time, tailing factor, and number of theoretical plates) as the constraints of method robustness. The proposed technique was optimized with a C18 (150 × 4.6 mm, 5 µm) column and 0.1% orthophosphoric acid buffer-acetonitrile (50:50, v/v) as the mobile phase at a flow rate of 1 mL/min using diode-array detector (230 nm) eluted favipiravir at 2.3 min., Results: The optimized method validated as per ICH guideline Q2 (R1) was found to be eco-friendly, simple, precise (RSD 0.0051-1.2%), accurate (99.86-100.22%), linear (25-150 µg/mL), rugged (RSD 0.70%), and robust (RSD 0.6-1.6%) with a limit of detection and limit of quantitation of 1.140 µg/mL and 4.424 µg/mL, respectively., Conclusion: Forced degradation studies (acidic, alkaline, thermal, photolytic, and oxidative conditions) revealed the suitability of the AQbD method for the analysis of favipiravir in tablet formulation.The developed and validated AQbD method is less time consuming and can be used in the industry for routine quality control/analysis of bulk drug and marketed Favipiravir products., Highlights: A robust Design of Experiment enhanced stability-indicating analytical method was developed and validated for the estimation of favipiravir. Furthermore, the contemporary method would aid in extending the analysis of favipiravir in other formulations., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

For the purpose of identifying enantiomeric impurities in the drug substance and pharmaceutical dosage forms of the novel anti-TB medication pretomanid, an RP-high-performance liquid chromatography method was devised. To ensure the robustness of the optimized approach, analytical quality by design was used. Studies on factor screening and risk evaluation helped pinpoint the critical method parameters (CMPs); resolution (R1), analyte retention time (R2) and tailing factor (R3) are those terms. Pareto charts, half-normal plots, 3D surface plots, 2D contour plots and 3D cube plots were used to study the effects of solo and interactive CMPs on critical analytical attributes. Analysis of variance (ANOVA) was used to verify the technique parameters' confirmation of significance (P = 0.05). With a Chiral Cel OJ-3R (150 × 4.6 mm, 3 µm) and a mobile phase consisting of 20 mM of ammonium trifluoroacetate, pH = 2.5, and acetonitrile in a gradient mode, chromatographic separation was accomplished. At 30°C and 330 nm, the column's temperature and wavelength, respectively, were recorded. The procedure is stability-indicating and is LC-MS compatible. According to the International Conference on Harmonization tripartite guidelines, the method demonstrated appropriate specificity, sensitivity, linearity, accuracy, precision and robustness. The LOD and LOQ were, respectively, 0.09 and 0.3 μg/mL. With a correlation coefficient of >0.990, it was discovered that the established method for enantiomeric impurity was linear over the concentration range of 0.3-2.25μg/mL. The approach exhibits adequate accuracy (%recovery = 85-115%), robustness (%RSD = 5.0) and precision (%RSD = 5.0). The method was also shown to be stability-indicating and was shown to provide effective separation in the presence of degradation products through the use of forced degradation tests., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The green profile of the developed method is assessed and compared with previously reported methods. Percutaneous coronary intervention is a procedure where a catheter is utilized to place a stent in order to facilitate opening of the blood vessels in the heart. Triple antithrombotic therapy includes oral anticoagulation as warfarin and dual antiplatelet therapy (composed of aspirin and clopidogrel bisulfate). The aim of the current study was to evaluate the pharmacokinetic parameters of ASP, WAR and CLP and to investigate the possible interaction between the three drugs upon co-administration in rats. A selective and precise RP-HPLC method was developed for the simultaneous determination of ASP, WAR and CLP in rat plasma. Pharmacokinetic study was conducted in rats that received ASP, WAR and CLP as an application of the developed method. From the statistical evaluation of the pharmacokinetic parameters, it was observed that the co-administration of ASP, WAR and CLP significantly increased the ASP and CLP bioavailability in rats. A significant drug-drug interaction was confirmed in the current study. The elevated Cmax of ASP and CLP upon the co-administration of ASP, WAR and CLP may explain the reported bleeding., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Recently, analytical separation techniques have the potential toward green approaches to reduce the environmental impact. This study focuses on the development of an analytical method for the determination of paracetamol and chlorzoxazone in their pharmaceutical combination. The separation was achieved using a home-made capillary column (0.10 mm i.d. × 200 mm length) filled with porous cross-linked hexyl polymethacrylate as monolithic stationary phase. The method proved to be simple, fast, sensitive, efficient, cost-effective and green approach due to the combination of the amazing properties of a monolithic material and a miniaturized liquid chromatography, which would be considered as a step toward reducing the analytical costs and the environmental impact of chromatographic applications. Both components were detected using a 3-nL nano-UV cell fixed at 270 nm wavelength. The optimized mobile phase was composed of 1% aqueous formic acid solution and acetonitrile at 40:60 ratio, 1.0 μL/min flow rate, 4.0 nL injection volume and 50°C column temperature. Under the optimized conditions, paracetamol and chlorzoxazone have been separated in about 6.5 min with chromatographic resolution of 2.37. The prepared column and the analysis method was fully validated and compared with other reported works. All findings allow to conclude that the prepared column and proposed method are applicable for quality control and routine analysis of the two drugs.

The object of the analytical work is to develop an analytical multivariate optimization for the determination of Favipiravir (FAV), a SARS-CoV-2 molecule, by the reverse-phase liquid chromatographic method using the analytical quality by design approach. FAV is used as an antiviral drug. Box-Behnken design is utilized for the optimization of the experiment and to identify the critical method parameters like the volume of acetonitrile, temperature and flow rate. Further, these factors are used to design the suitable mathematical models and illustrate their effect on various responses. This newly developed method utilized C18 column (5μm, 100 × 4.6 mm) and a temperature of 40°C with a flow rate of 0.5 mL/min. The mobile phase is composed of acetonitrile and ammonium acetate buffer (pH 4), in the ratio of 20:80v/v and the wavelength of HPLC UV-Detector was fixed to 323nm. This method is validated according to International Council for Harmonization Q2 (R1) guidelines. The System suitability is performed and the retention time of Favipiravir is 3.4min. The linearity range is obtained at 0.062 - 4 μg/mL with a correlation coefficient (r2 = 0.9979). The recovery is found to be in the range of 98.84-100%. Thus, the intended method is found to be simple and robust., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

An HPLC procedure on a polar reversed-phase column with mobile phases containing ionic liquid (IL) was developed for the analysis of selected alkaloids from different chemical groups. We aimed to obtain optimal conditions for the separation of alkaloids because widely used silica-based stationary phases exhibit a silanol effect, rendering analysis of basic analytes extremely difficult. Retention, separation selectivity, peak symmetry, and system efficiency were examined in various eluent systems containing different concentrations of IL and acetonitrile. The obtained results revealed substantial influence from the concentrations of IL, the organic modifier, and temperature on the retention behavior of the investigated alkaloids. The most selective and efficient chromatographic systems were applied for the analysis of several alkaloids in a plant extract.

A rapid and sensitive HPLC method was developed and validated for selective determination of the human growth hormone (hGH) somatropin in the presence of its deamidated and oxidized degradation products. Reversed-phase chromatography with an acetonitrile and ammonium bicarbonate mobile phase in gradient elution mode was used. A short run time of 15 min allowed rapid and cost-effective analysis, with an average retention time of 7.4 min for native hGH, 6.2 min for its deamidated form, and 4.3 and 6 min for its oxidized variants. The method was validated for selectivity, linearity, and intra- and interday variations according to International Conference on Harmonization guidelines. The proposed method was successfully applied for rapid evaluation of the quantity of hGH during downstream processing, formulation, and storage.

A rapid and sensitive reversed-phase high-performance liquid chromatography (HPLC) method using novel salting-out assisted liquid-liquid extraction technique has been developed for the quantitative determination of febuxostat (FEB), used for the treatment of gout, in rat plasma. The method was validated according to US FDA guideline. Separation was achieved using a Phenomenex Luna-C 18 (250 × 4.60 mm, 5 µm) column and mobile phase composed of potassium dihydrogen orthophosphate buffer 25 mM, adjusted to pH 6.8 with triethylamine:methanol in a ratio of 35:65 (v/v) showing retention time 5.56 and 8.86 min for FEB and internal standard, respectively. The optimal salting-out parameters; 1 mL of acetonitrile and 200 µL of 2 M ammonium acetate salt showed extraction recovery >90% for FEB from plasma. This extraction procedure afforded clear samples resulting in convenient and cost-saving procedure and showed good linear relationship (r > 0.9997) between peak area ratio and concentration from 0.3 to 20 µg/mL. The results of pharmacokinetic study showed that absorption profile of spherical agglomerate of FEB compared to marketed formulation was higher indicating greater systemic absorption. In conclusion, the developed SALLE-HPLC method with simple ultraviolet detection offered a number of advantages including good quantitative ability, wide linear range, high recovery, short analysis time as well as low cost., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A new reverse phase-liquid chromatography (RP-HPLC) method has been developed for simultaneous determination of entacapone and its pharmacopoeia impurities, in-house impurities and degradation impurities (total 17 analytes). Chromatographic separation was achieved on a C18 column (size: 250 × 4.6 mm; 5 µm particle size) at a flow rate of 1.0 mL/min with 210 nm detection. The mobile phase (MP) consists of 1.361 g of potassium di-hydrogen phosphate and 1.742 g of di-potassium phosphate in 1.0 L water, pH adjusted to 2.5 with ortho phosphoric acid (MP-A) and acetonitrile (MP-B) through gradient elution. The product was subjected to stress conditions such as acid, base, peroxide, thermal and photolytic degradation. Two new impurities above 2% level were observed and isolated through preparative HPLC and well characterized. However, no interference observed due to degradation impurities and entacapone and its EP impurities, in-house impurities. As part of the method validation, specificity, limit of detection, limit of quantitation (LOQ), linearity, accuracy, precision, robustness and ruggedness were determined. LOQ values were achieved between 0.01 and 0.04%. Good linearity (r(2) > 0.99) was obtained ranging from LOQ to 150%. Recovery was verified for all impurities at concentrations ranging from LOQ to 150%. Hence, a newly developed RP-HPLC method was capable for well separation of all analytes with acceptable resolution and tailing factor., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A simple and fast high-performance liquid chromatography method with UV detection for determination of vitamin D3 in stability studies as well as in solutions, nutritional supplements and pharmaceuticals was developed. Successful separation of vitamin D3 from its degradation products was achieved on a Gemini C18 100 × 3.0 mm column using a mixture of acetonitrile and water (99:1, v/v) as а mobile phase. The method was successfully validated according to the ICH guidelines. The described reversed-phase HPLC method is favorable compared with other published HPLC-UV methods because of its stability-indicating nature, short run time (3.3 min) and wide analytical range with outstanding linearity, accuracy and precision. The method was further applied for quantification of vitamin D3 in selected liquid and solid nutritional supplements and prescription medicines, confirming its suitability for routine analysis. Degradation products, formed under stress conditions (hydrolysis, oxidation, photolysis and thermal degradation), were additionally elucidated by suitable equipment (LC-DAD-MS) to confirm the stability-indicating nature of the developed method., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Two stability-indicating reversed-phase liquid chromatographic methods were developed and validated for the determination of fluorometholone (FLU) in its mixtures with sodium cromoglycate (SCG) and tetrahydrozoline hydrochloride (THZ). The first HPLC method (Method 1) was based on isocratic elution of FLU and SCG along with their alkaline degradation products on a reversed phase C18 column (250 × 4.6 mm id)-ACE Generix 5, using a mobile phase consisting of methanol-water (70 : 30, v/v), pH adjusted to 2.5 using orthophosphoric acid at a flow rate of 1.2 mL min(-1) Quantitation was achieved with UV detection at 240 nm. The second HPLC method (Method 2) was based on isocratic elution of FLU, its alkaline degradation product and THZ on a reversed phase C8 column (250 × 4.6 mm)-ACE Generix 5, using a mobile phase consisting of acetonitrile-50 mM potassium dihydrogen orthophosphate (40 : 60, v/v) at a flow rate of 2 mL min(-1) Quantitation was achieved by applying dual-wavelength detection, where FLU and its alkaline degradation product were detected at 240 nm and THZ was detected at 215 nm at ambient temperatures. Linearity, accuracy and precision were found to be acceptable over the concentration range of 5-50 and 10-500 μg mL(-1) for FLU and SCG (Method 1) and over the concentration range of 5-80 and 5-60 μg mL(-1) for FLU and THZ (Method 2), respectively. Besides, the FLU alkaline degradation product was verified using IR, NMR and LC-MS spectroscopy. The two proposed methods could be successfully applied for the routine analysis of the studied drugs either in their pure bulk powders or in their pharmaceutical preparations without any preliminary separation step., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Treosulfan (l-threitol-1,4-di-methanesulfonate) is a prodrug of a bifunctional alkylating agent that is being used increasingly in pediatric bone marrow transplantation regimens. The activation pathway is a complex reaction, which consists of two consecutive reactions leading to epoxybutane derivatives which are responsible for DNA alkylation. A simple, sensitive high performance liquid chromatography method for the determination of the sum of treosulfan and its epoxy metabolites by UV detection after derivatization with sodium diethyldithiocarbamate in human plasma was developed and validated. Plasma samples containing treosulfan and epoxy metabolites were converted into thiocarbamate derivative with 10% sodium diethyldithiocarbamate. Dinitrobiphenyl was used as an internal standard. The analysis was carried out using a reversed phase C18 column with a mobile phase consisting of methanol-water (65:35, v/v) at a flow rate of 1 mL/min. The eluent was monitored at 254 nm. The standard calibration curve was established between 2.5 and 50 µg/mL, with a correlation coefficient of 0.9987. Intra- and interday precision and accuracy of the method was <8% and met the analytical criteria. Pharmacokinetic parameters were determined in six children who received intravenous treosulfan (dose range 12-24 g/m(2)) in combination with fludarabine prior to blood or marrow transplantation., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)