Sensitive HPLC Method Research Articles

A General Strategy to Rapid and Sensitive Determination of Five High Boiling Point Solvents in Pharmaceuticals by RP-HPLC Combined with Precipitation Method.

The difficulty on determination of high boiling point solvents, always exist due to their low vapor pressure in headspace (HS) vial. What's worse, the commonly used GC diluents, such as N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, N, N-dimethylformamide and N-methylpyrrolidone are often remained in HS instrument after frequent injection into HS, thus would result in terrible positive interference problem. In this work, a fast and sensitive HPLC method in combination with a sample precipitation method, was proposed for the determination of above five high boiling point solvents in drug substances within 14.5min. The final method was validated with regard to limit of detection and limit of quantitation, precision, linearity and accuracy in accordance with ICH Q2 (R1). Finally, the proposed strategy was successfully applied for the analysis of high boiling point solvents used in three active pharmaceutical ingredients. The established generic strategy would provide another choice for the determination of high boiling point solvents in pharmaceuticals.

Sensitive HPLC Method to Support Pre-formulation Studies and to Determine Critical Quality Attributes of Therapeutic Contact Lenses Containing Dorzolamide Hydrochloride

Background: Dorzolamide hydrochloride (DRZ) is a carbonic anhydrase inhibitor used to treat glaucoma and ocular hypertension. Drug-eluting contact lenses, such as Acuvue Theravision ™ with Ketotifen, offer improved drug delivery and reduced side effects compared to eye drops. Drug-loaded nanoparticle-loaded contact lenses can sustain drug release and enhance comfort for extended wear. Objective: High buffer concentration and low pH increase the risk of damage to silica-bonded columns. Therapeutic contact lenses face challenges related to critical lens parameters, including the estimation of drug incorporation and release due to interference of lens matrix leaching. There is currently no analytical method available for estimating DRZ in contact lenses. Method: The HPLC method, which was developed and validated using ICH Q2 (R1) criteria, used a C18 column (250 mm × 4.6 mm, 5 μm) as a stationary phase and methanol:water (70:30 v/v) as the mobile phase. The detecting wavelength was 253 nm. Moreover, to support the efficiency of the developed method, the marketed formulation of DRZ eye drops, drug purity, and loading in contact lenses were analysed. The method was also employed to determine the Critical Quality Attributes (CQAs) of therapeutic contact lenses and drug release and drug leaching during the sterilization process. Result: The developed HPLC method shows Rt for DRZ at 2.881 minutes with good linearity (r2 > 0.998) between 2-32μg/mL, precision (RSD < 2%), accuracy (Recovery > 99.5%), sensitivity, and specificity for quantifying DRZ in marketed formulations and therapeutic contact lenses. The developed method is devoid of any buffer or modifier in the mobile phase, making it safer for the stationary phase. This method mitigates the interference of lens matrix leaching, which induces an overestimation of DRZ. All the result for therapeutic contact lenses was found to be closely aligned with theoretically expected results, confirming the reliability of the developed HPLC method for therapeutic contact lenses. Conclusion: This method is specific, accurate, and precise for quantifying DRZ in commercial formulations and newly developed therapeutic contact lenses. It effectively evaluates the critical quality attributes of these lenses, demonstrating their reliability for assessing their performance and ensuring quality in therapeutic applications.

Scientifc Validation and Chromatographic Method Development for the Estimation of Piperine in Herbal Formulation Babbularishta: A Quality Control Strategy for Critical Quality Attributes

Background: Babbularishta is an Ayurvedic medicine in liquid form used for the treatment of phthisis, skin diseases, urinary disorders,, and breathing problems. It is also beneficial in cases of blood disorders, acne, dermatitis, hemoptysis, lung injury, asthma, persistent cough, whooping cough, etc. Objective: The functional role of this study is to develop a simple, novel, precise, accurate, reliable, and selective RP-HPLC method for the estimation of piperine content in herbal drug formulations (Babbularista). Methods: A traditional approach to method development could fail to meet the desired separation. Consideration of the current regulatory requirement for analytical method development and RP-HPLC method for routine analysis of piperine in the herbal formulation has been optimized using an analytical Quality by Design (QbD) approach. Three laboratory formulations and three marketed preparations of Babularishta were investigated for organoleptic parameters, physicochemical parameters, and validation. The mobile phase composition (methanol and water % as A), pH (B), and flow rate (mL/min as C) were selected as independent variable,. In contrast, retention time (min. as Y2), area (AU as Y1) is selected as dependent variables. Results: The fingerprinting method was developed and optimized by using the AQbD approach. Piperine content was found to be in Piper nigrum (Marica) 3.01±0.01527 % w/w and in Piper longum (Pipali) 2.74±0.01154% w/w in Babbularista. Conclusion: An attempt has been made to develop quality control parameters of Babbularista by developing a suitable and sensitive HPLC methods for the quantitation of piperine in herbal drug formulations (Babbularista). The developed method was found to be simple, accurate, precise,, and economical for the estimation of piperine in herbal formulations.

Whiteness and greenness assessments of a sensitive HPLC method with fluorimetric detection for dapagliflozin quantitation in human plasma: Application to a healthy human volunteer.

The evident ecological impact of human actions, like air pollution, global warming, and ozone depletion, underscores the need for environmentally friendly approaches across various domains, including analytical chemistry. This study aimed to establish a validated, eco-friendly, and sustainable approach utilizing a fluorescence detector coupled with high-performance liquid chromatography for quantifying the antihyperglycemic agent dapagliflozin (DAPA), in human plasma. This method employed a C18 Microsorb MV (4.5 × 250 mm, 5 μm [particle size]) column at 40°C, with 40:60% v/v isocratic elution of acetonitrile and (0.1%) orthophosphoric acid as the mobile phase at 1 mL/min flow rate. DAPA and the internal standard demonstrated their greatest response by performing excitation at 225 nm (λex) and recording chromatograms at an emission wavelength (λem) equal to 305 nm. The presented approach demonstrated high linearity between 50 and 2000 ng/mL and full adherence to the guidelines of the US Food and Drug Administration regarding the validation of bioanalytical methods. The described technique was effectively used for quantification of DAPA in human plasma samples from a healthy male participant who received a tablet of 10 mg DAPA. Analytical Eco-Scale, Analytical GREEnness metric, and the recently created ChlorTox Scale were utilized for greenness assessment. Additionally, the "Red, Green, and Blue 12" model was used in whiteness evaluation.

A novel QbD HPLC approach for the concurrent analysis of amoxicillin, amprolium, and ethopabate in food samples and dosage forms

A simple, rapid, and sensitive reversed-phase HPLC method coupled with photodiode array detection (PDA) was developed for the simultaneous estimation of three co-administered antibacterial drugs, amprolium, ethopabate, and amoxicillin for the first time. An analytical quality-by-design (QbD) approach was adopted to optimize the developed approach. A two-level full factorial design (25 FFD) was implemented to optimize how variable factors affected chromatographic responses. On a Hypersil BDS C18 column, the chromatographic separation was carried out using isocratic elution at 40.1 °C. The mobile phase consisted of methanol: potassium phosphate buffer containing 5.76 mM heptane sulphonate (9.05 mM; pH 3.32, 44.8:55.2 % v/v) pumped at a flow rate of 0.8 mL/min and an injection volume of 20.0 μL. In less than 7 min, the suggested method was able to separate the ternary mixture with a challenging ratio of 1: 15.6: 1 for amoxicillin, amprolium, and ethopabate, respectively. The calibration curves showed excellent linearity over the concentration ranges of 0.50–30.0, 0.30–30.0, and 0.05–20.0 μg/mL, respectively. Recovery percentages ranged from 90.30 % to 111.13 % with %RSD less than 2 % were obtained upon spiking to chicken tissues, liver, and egg samples, indicating the suitability of the proposed method for determining of the studied drugs in complicated food matrices. Furthermore, the proposed method was successfully applied to determine the studied drugs in their commercial formulations. Full validation was carried out according to ICHQ2 (R1) guidelines. Finally, the method's greenness and ecofriendliness were evaluated using various metrics tools, including the Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE).

Simultaneous quantification of two commonly used anticoagulant drugs and three hypolipidemic drugs in human plasma using gradient high-performance liquid chromatography

The combination of an anticoagulant and a hypolipidemic agent is commonly used in patients at risk for stroke because it controls thrombosis and lowers cholesterol levels. However, the use of anticoagulants carries a high risk of life-threatening bleeding. To address the need for personalized clinical monitoring and risk management, we developed a rapid and sensitive HPLC method with a streamlined sample preparation process. This method enables the simultaneous determination of concentrations for two commonly used anticoagulant drugs (apixaban and rivaroxaban) and three hypolipidemic drugs (rosuvastatin, atorvastatin calcium, and simvastatin). The chromatographic conditions and sample preparation methods were optimized. The drugs were well separated by the gradient elution of phosphoric acid (0.01%, v/v) and acetonitrile at a flow rate of 1.0 mL/min within 40 min on an Athena C18-WP column (4.6 × 150 mm, 5-μm particle size). The proposed method was linear (r 2 ≥ 0.994) within the concentration ranges of 0.22–20 μg·mL−1 for the five analytes, including the expected range of therapeutic concentrations. This method enables simultaneous monitoring of the levels of the mentioned drugs in clinical practice. Furthermore, the chromatographic conditions were simple and feasible, making them highly suitable for emergency clinical medication monitoring in basic medical institutions.

Determination of 3-acetyl-11-keto-β-boswellic acid in analytical and biological samples using streamlined and robust RP-HPLC method.

AKBA (3-acetyl-11-keto-β-boswellic acid) is a phytoconstituent derived from Boswellia serrata extract and utilized in the management of rheumatoid arthritis. Drug delivery approaches showed interest in delivering AKBA with advanced nanotechnology. There is a need for a simple, sensitive, and robust HPLC method that can determine AKBA in complex nanoformulation and in vitro and ex vivo samples. In the proposed work, the RP-HPLC method was developed using a mobile phase comprising a mixture of acetonitrile : milli Q (90 : 10) at detection λmax 250 nm. The method exhibited a linearity of 250 to 20 000 ng mL-1 with a high correlation coefficient of 1. The limit of detection and limit of quantification for the analytes were found to be 41.32 ng mL-1 and 125.21 ng mL-1, respectively. In the accuracy study, the % recovery of AKBA was found to be 98% to 102%, and the precision study showed less than 2% relative standard deviation. The developed method was found to be robust under chromatographic conditions with changes in pH and mobile phase mixture ratio. The method was also explored for forced degradation study, and the results showed the successful separation of degradation products from the AKBA. Further, the RP-HPLC method was applied for the quantification of AKBA in topical nanoformulations and different matrices, such as skin matrices and adhesive tapes. The method was able to measure entrapment efficiency (93.13 ± 1.94%), drug loading (25.83 ± 0.54%), drug assay in a gel matrix (96.99 ± 3.89%), drug amount in stratum corneum (7.90 ± 0.62 μg cm-2), and drug amount in viable skin layers (33.94 ± 0.21 μg cm-2) with high-speed reproducibility. The developed method can be utilized for the routine analysis of AKBA in conventional and complex formulations in academia and industry.

Two Steps Non-Enzymatic Synthesis of Molnupiravir, which is Free from Mutagenic Impurity and Analytical Method Development for Estimation of Genotoxic Impurity (Hydroxylamine Hydrochloride Content) by using RP-HPLC Technique

Genotoxic impurity free Molnupiravir was synthesised by a novel and an elegant non-enzymatic method, which is simple operationally and ecologically. The genotoxic raw material, Hydroxylamine, was used in the initial stages of synthesis to get the mutagenic impurity free Molnupiravir. A new and sensitive HPLC method was developed and validated for the determination of hydroxylamine in Molnupiravir drug substance according to ICH guidelines. The HPLC method was developed and optimized on Zorbax SB C18 150×4.6mm, 3.5µm column with oven temperature maintained at 40°C. 1.0 mL of Orthophosphoric acid in 1000 mL water was selected as mobile phase in isocratic reverse phase mode. Chromatographic parameters are flow rate: 0.8 ml/min, wavelength detection: 252 nm, injection volume: 10µl and run time: 20 min. Based on the validation data; the method was found to be specific, sensitive, accurate and precise. This method can be used as a good quality control tool for quantification of hydroxylamine at the low level. The experimental data was deliberated in detail in this research paper.

Enhanced Detection of Lamotrigine Through HPLC Methodology in Bioanalysis

Background: Lamotrigine, an anticonvulsant drug, requires sensitive detection methods in biological and pharmaceutical matrices. Current methods struggle with interference and sensitivity, necessitating the development of improved analytical techniques. Objective: This study aims to develop and validate a highly sensitive HPLC method enhanced by salicylaldehyde derivatization for detecting lamotrigine in pharmaceutical and biological samples, and to differentiate it from Piracetam when co-administered. Methods: A reverse-phase HPLC method was employed, utilizing salicylaldehyde as a derivatizing agent to induce a bathochromic shift in lamotrigine from 315 nm to 415 nm. Optimization of the derivatization reaction included adjustments of pH, reagent concentration, temperature, and time. The standard addition method was applied to evaluate the recovery rates from spiked samples. Results: The optimized method demonstrated a linear calibration range of 1-5 µg/ml with a determination coefficient (R²) of 0.998. Recovery of lamotrigine from pharmaceutical preparations averaged 96.5%, while recovery from deprotonated serum and urine samples of healthy volunteers was 96% with a relative standard deviation (RSD) of 6%. Conclusion: The developed method offers a robust and highly sensitive approach to detect and distinguish lamotrigine in the presence of Piracetam, proving effective for both pharmaceutical and biological sample analysis.

Development of sensitive stability indicating HPLC method for quantification of process related impurities of Fluphenazine; LC–MS/MS elucidation of their degradation products and degradation pathway

This study focused on the development of a simple and sensitive HPLC method for the resolution and quantification of process related impurities of fluphenazine and further assessment of the forced degradation behavior of fluphenazine. Chromatographic separation was achieved on phenomenex™ Gemini C18 column (250×4.6mm, 5.0μm) at room temperature using acetonitrile, methanol and TFA in 65:30:05 (v/v) at pH 4.2 as the mobile phase was pumped isocratically at a 0.9 mL/min flow rate. The detection wavelength was selected as 255 nm. In the proposed conditions, the retention time of the analytes was noticed to be 6.23 min for fluphenazine and 4.57, 5.30, 9.01 and 8.29 min for impurity 1-4 respectively. The method produces sensitive detection limits of 0.003 μg/mL, 0.013 μg/mL, 0.018 μg/mL and 0.022 μg/mL for impurity 1 to 4 respectively with calibration range of 75-450 μg/mL for fluphenazine and 0.075 - 0.45 μg/mL for impurities. The other validation parameters were noticed to be within the acceptable levels for fluphenazine and its impurities. The drug was exposed to different stress conditions (acid, base, peroxide, thermal and UV light) according to the ICH Q1A (R2) guidelines. The DPs formed during the stress study were identified and characterized by LCMS/MS in the ESI positive mode.

Therapeutic drug monitoring of vancomycin in surgical patients using a validated HPLC method.

Vancomycin is being used for the treatment of a variety of infections caused by methicillin resistant Staphylococcus aureus and methicillin susceptible Staphylococcus aureus. Therapeutic drug monitoring (TDM) is highly recommended for ensuring the safe and effective therapy with vancomycin. A reliable and cost-effective bioanalytical method is required for TDM as well as pharmacokinetic studies of vancomycin. A selective, sensitive, and cost effective HPLC method was developed and validated for quantification of vancomycin concentrations in human plasma. The mobile phase was a mixture of buffer (50 mM ammonium dihydrogen phosphate, pH 2.4) and acetonitrile 88 : 12 v/v. The separation was carried on C18 column (125 × 4.6 mm, particle size 5 µm) with isocratic flow rate of 0.370 mL/min at room temperature with UV detection at 215 nm. The method was validated for sensitivity, accuracy, and precision as well as stability of vancomycin in human plasma by following European Medicine Agency (EMA) guideline. Therapeutic drug monitoring of vancomycin was performed by quantifying the trough concentrations of vancomycin in 65 human plasma samples after administration of therapeutically relevant dose. The developed method was sensitive enough to quantify vancomycin concentrations as low as 0.25 mg/L in human plasma. Moreover, the method was proved accurate and precise in terms of quantifying the unknown concentration of vancomycin. The evaluation of short-term, long-term, and freeze-thaw stability proved the stability of vancomycin in human plasma. The TDM of vancomycin by using this method showed that 39 (60%) samples were within the target trough concentration range (TTCR), i.e. 10 - 20 mg/L, while 23 samples (35.4%) were below the TTCR, and 3 samples (4.6%) were above this range. The developed method is sensitive and cost effective for quantification of vancomycin in human plasma. The results of sample analysis shows that the developed method can be used reliably for TDM of vancomycin.

Impurity assessment, development and validation of an RP-HPLC method for the determination of eleven potential impurities of eltrombopag precursor

Eltrombopag is an oral non-peptide thrombopoietin receptor (TPO-R) agonist indicated for the treatment of thrombocytopenia in patients with persistent or chronic immune thrombocytopenia (idiopathic thrombocytopenic purpura, ITP) or chronic hepatitis C infection and the treatment of severe aplastic anemia. The purpose of this research was to assess the possible impurities that may carry over to eltrombopag from its precursor Eltro-1 (3′-amino-2′-hydroxy-[1,1′-biphenyl]-3-carboxylic acid) and to develop a specific analytical method for the determination of these impurities. Eltro-1 samples synthesized by two different synthesis routes were investigated during the evaluation and method development studies. Besides the expected process-related impurities (Eltro-1A – Eltro‐1J), e.g., starting materials, intermediates, and/or compounds formed from their further reactions, an unknown impurity detected above 0.10% was identified by LC-MS, synthesized and fully characterized by NMR, MS and FTIR (Eltro‐1K). Accordingly, an HPLC-RP method for the determination of eleven impurities (Eltro-1A – Eltro‐1K) in Eltro-1 was developed and validated according to ICH Q2. The control limits for impurities in Eltro-1 were set at ≤ 0.15% for Eltro-1A – Eltro‐1J and ≤ 1.0% for Eltro‐1K based on fate, spike-purge and carryover studies and in accordance with the ICH M7 classification for impurities in drug substance. Eltro-1 and eleven impurities at the specification limit were separated from each other and the diluent peaks with sufficient resolution without interference. Separation was performed on a Waters XBridge C18 column (150 × 4.6 mm, 3.5 μm) at 40 °C with a 10 µL injection volume at a detection wavelength of 220 nm and 15 °C sample temperature. The gradient elution is performed at a flow rate of 1.0 mL/min for 40 min with mobile phase A (0.1% orthophosphoric acid in water) and B (acetonitrile) according to the following program: Time (min) / Acetonitrile (%): 0/0, 35/70, 36/0, 40/0. Test and standard solutions were prepared at a concentration of 1.0 mg/mL and 1.0 µg/mL, respectively, using a mixture of mobile phase A and acetonitrile (75/25) as diluent. This is the first specific, selective, sensitive, linear, precise, accurate, and robust HPLC method for the determination of Eltro-1A – Eltro‐1K in Eltro-1, which showed no significant degradation under thermal stress, photostability (UV and VIS), and standard accelerated and long-term stability conditions.

A specific chiral HPLC method for lifitegrast and determination of enantiomeric impurity in drug substance, ophthalmic product and stressed samples

Lifitegrast is a lymphocyte function-associated antigen-1 (LFA-1) antagonist used to treat the indications and symptoms associated with dry eye disease (DED), one of the most common ocular surface diseases. Lifitegrast has a chiral center, and the S-enantiomer (S-Lif) is responsible for the therapeutic effects, while the R-enantiomer (R-Lif) lacks efficacy in the treatment of DED. Lifitegrast ophthalmic solution containing 5% lifitegrast was approved by the United States Food and Drug Administration (FDA) in July 2016 for the treatment of DED in patients 17 years of age and older. The objective of this study was to develop a chiral HPLC method for the determination of the enantiomeric impurity of lifitegrast in the drug substance and in the ophthalmic product. In addition, we aimed to investigate the effect of stress and stability conditions on the enantiomeric purity of lifitegrast in both drug substance and ophthalmic solution. During the method development studies, four known lifitegrast impurities (Lif. Imp. A-D) and stressed lifitegrast samples were injected to ensure the specificity of the developed method. The enantiomers of lifitegrast are well separated with a resolution of higher than 4.0. They are also well separated from the peaks of the diluent, impurities, and the placebo used to prepare the ophthalmic solution without interference in 20 min. Chiral separation was achieved using a Chiralpak AD-H column (250 × 4.6 mm, 5.0 μm) at 40 °C with a mobile phase consisting of a mixture of n-hexane, 2-propanol, and formic acid (500:500:2, v/v/v) at a flow rate of 1.0 mL/min and a detection wavelength of 260 nm. Methanol was used as the diluent, and the drug substance solution was found to be stable for 48 h at 15 °C. The optimized chiral HPLC method for lifitegrast was validated according to ICH Q2, and the calibration curves showed excellent linearity for R-Lif (0.0369 – 1.816 µg/mL). This is the first stability-indicating, specific / selective, sensitive, linear, precise, accurate, and robust chiral HPLC method for the determination of R-Lif in S-Lif. The amount of enantiomeric impurity R-Lif in S-Lif increased under all stress and photostability test conditions without exceeding the acceptable impurity limit, with the most significant increase observed at elevated temperatures (105 °C) for both the drug substance in powder form and the ophthalmic drug solution.

A fast, sensitive, greener, and stability-indicating HPLC method for the standardization and quantitative determination of chlorhexidine acetate in commercial products

A fast, sensitive, greener, and stability-indicating HPLC method for the standardization and quantitative determination of chlorhexidine acetate in commercial products

Developing a highly validated and sensitive HPLC method for simultaneous estimation of cefotaxime and paracetamol in pure and pharmaceutical preparations

An isocratic HPLC technique was exploited and validated for the quick simultaneous separation and measurement of cefotaxime and paracetamol in vials dosage forms, with a total analysis time of 3 minutes. The process of separation was carried out on a Thermo Scientific® Venusil XBPC18 (L) (5µm, 4.6x250 mm) using a mobile phase of ACN: distilled water (70:30, v/v) at the ambient temperature. The flow rate used in the experiment was 1 mL/min, and the highest level of absorption was determined by high-performance liquid chromatography with photodiode array detection (HPLC-PDA) employing a PDA detector set at a wavelength of 255 nm. The established retention times for cefotaxime and paracetamol were 1.79 and 2.97 minutes, respectively, suggesting reduced analysis duration. The observed limits of detection for ceftaxime and paracetamol were 4.2×10-5 and 1.2×10-5 µg/mL, respectively, indicating a significant level of sensitivity in the approach. The approach was subsequently verified in accordance with the requirements set out by the Food and Drug Administration (FDA) for the quantification of medicines in vial dosage form.

A Fast HPLC/UV Method for Determination of Ketoprofen in Cellular Media.

A simple, sensitive and quick HPLC method was developed for the determination of ketoprofen in cell culture media (EMEM, DMEM, RPMI). Separation was performed using a gradient on the C18 column with a mobile phase of acetonitrile and miliQ water acidified by 0.1 % (v/v) formic acid. The method was validated for parameters including linearity, accuracy, precision, limit of quantitation and limit of detection, as well as robustness. The response was found linear over the range of 3-100 μg/mL as demonstrated by the acquired value of correlation coefficient R2=0.9997. The described method is applicable for determination of various pharmacokinetic aspects of ketoprofen in vitro.

Development of stability-indicating HPLC method for quantification of pharmacopeia impurities of Zuclopenthixol and characterization of its stress degradation products by LCMS/MS

BackgroundThe present study focused to develop a simple and sensitive HPLC method for resolution and estimation process-related impurities of zuclopenthixol and further assessment of forced degradation behavior of zuclopenthixol.ResultsThe chromatographic separation of drug substance, process-related impurities and its degradation products (DPs) was achieved on KNAUER C18 (250 mm × 4.6 mm, 5µ id) column at that was maintained at 35 °C temperature using 0.1 M sodium acetate buffer at pH 4.3 and methanol in 20:80 (v/v) as mobile phase A, 0.1% formic acid and acetonitrile in 75:25 (v/v) as mobile phase B. Equal volume of mobile phase A and B was pumped in isocratic elution at 0.8 mL/min. Detection wavelength was selected as 257 nm. In the proposed conditions, zuclopenthixol is identified at 6.91 and 1.91 min and 2.89 min, respectively, for impurity B and A min with acceptable system suitability and specificity. The method produces LOD at 0.009 for impurities with calibration range of 30–180 µg/mL for zuclopenthixol and 0.03–0.18 µg/mL for impurities. The other validation parameters were notices to be with in the acceptable levels for zuclopenthixol and its impurities. The drug was exposed to different stressed conditions (acid, base, peroxide, thermal and UV light) according to ICH Q1A (R2) guidelines. The DPs formed during the stress study were identified and characterized by LCMS/MS in ESI positive mode.ConclusionThe analysis involved a comparison of collision-induced dissociation mass spectrometry data between the degradation products and zuclopenthixol. As a result, potential structures for six degradation compounds were suggested. The results from additional validation studies were similarly pleasing and demonstrated their suitability for the routine analysis of zuclopenthixol and its associated impurities in both bulk drug and pharmaceutical dosage forms. Additionally, these findings can be extended to assess the mechanism of stress degradation in zuclopenthixol.

Optimization of Stability-Indicating HPLC Method for Analyzing Process Related Impurities of Penfluridol and Structural Elucidation of Stress Degradation Products by LCMS/MSucidation of Stress Degradation Products by LCMS/MS

This study focused on the development of a simple and sensitive HPLC method for resolution and quantification of process-related impurities of penfluridol and further assessment of forced degradation behavior of penfluridol. The chromatographic separation was achieved on XTerra™ C18 (250×4.6 mm, 5.0μm) column and UV detection at 245nm. The mobile phase comprises of methanol and tetrahydrofuran in 55:45 (v/v) as solvent A and acetonitrile and tetrahydrofuran in 80:20 (v/v) as solvent B. The 60:40 (v/v) composition of solvent A and B were pumped isocratically at 1.0mL/min. In the proposed conditions, the retention time identified as 5.29 min for penfluridol, 4.51 min, 9.95 min and 7.64 min respectively for impurity 1, 2 and 3 with acceptable system suitability. The method produces sensitive detection limit of 0.008μg/mL for impurity 1, 2 and 0.004 μg/mL for impurity 3 with calibration range of 25-150 μg/mL for penfluridol and 0.025-0.150 μg/mL for impurities. The drug was exposed to different stressed conditions (acid, base, peroxide, thermal and UV light) according to ICH Q1A (R2) guidelines. The Degradation Products (DPs) formed during the stress study was characterized by LCMS/MS in ESI positive mode and the possible structures of five DPs with possible degradation pathways were proposed. The outcomes of other validation studies were likewise satisfactory and proven adequate for regular analysis of penfluridol and its process-related impurities in bulk drug and pharmaceutical dosage forms and can also applicable for evaluation of stress degradation mechanism of penfluridol.

Determination of vitamin K in infant formulas by liquid chromatography-online electrochemical reduction based on porous Pt/Ti electrode-fluorescence detection.

A highly sensitive and selective HPLC method for the determination of vitamin K vitamers including phylloquinone (PK) and menaquinones (MK-4) in infant formulas is described. The K vitamers were quantified with a fluorescence detector after online post-column electrochemical reduction occurring in a laboratory-made electrochemical reactor (ECR) equipped with platinum plated porous titanium (Pt/Ti) electrodes. The morphology of the electrode showed that the grain size of Pt was homogeneous and well plated on the porous Ti substrate, resulting in largely improved electrochemical reduction efficiency due to the large specific surface area. In addition, the operation parameters such as mobile phase/supporting electrolyte and working potential were optimized. The detection limits of PK and MK-4 were 0.81 and 0.78ngg-1. Infant formula varying in stages were detected, showing PK ranged from 26.4 to 71.2µg/100g, while MK-4 was not detected.

Development and Application of a Validated HPLC Method for the Determination of Alpelisib and its Process-Related Impurities in Pure Drug and Pharmaceutical Formulations

Impurity analysis plays a significant role in the manufacture of a safe pharmaceutical product that ensures the safety of consumers. Keeping this in consideration, the present study was intended to develop a simple and sensitive HPLC method for the resolution and quantification of 4 process-related impurities namely impurities 1, 2, 3 and 4 in alpelisib pure drug and formulations. The method consumes greener solvents as a mobile phase that resolves the analytes on ProntoSIL ODS-C18 (250×4.6 mm; 5 μ id) column at room temperature as stationary phase, ethanol and 0.1% aqueous acetic acid in 65:35 (V/V) at pH 4.5 as mobile phase at 0.8 mL/min flow rate, UV detection at 246 nm. The method can detect the analytes at retention times of 7.05, 4.52, 6.09, 2.88 and 8.14 min respectively for alpelisib, impurity 1, 2, 3 and 4. The analysis was completed with a run time of 15 min that consume less solvent and the same analysis time. The linearity of the proposed method was perceived in the range of 12.5 to 100 μg/mL for alpelisib and 0.0125 to 0.10 μg/mL for impurities. The method can effectively resolve the unknown stress degradation products generated during the stress exposure of alpelisib along with its known impurities in the study. The outcomes of other validation studies were likewise satisfactory and proven adequate for regular analysis of alpelisib and its process-related impurities in bulk drug and pharmaceutical tablet doses.