The present study aimed to develop and validate a simple, rapid and stability-indicating high-performance liquid chromatographic (HPLC) method for the simultaneous quantification of nystatin (Nys), neomycin sulfate (NS), triamcinolone acetonide (TAC) and gramicidin D (Gr) in bulk drug samples and transferosomal gel formulations. Chromatographic separation was achieved on an Agilent Eclipse C18 (250 × 4.6 mm, 5 μm) using a mobile consisting of methanol and 0.1% orthophosphoric acid (30:70, v/v). The flow rate was maintained at 1.0 mL/min, and detection was carried out at 260 nm using a Photo Diode Analyzer (PDA) detector. Under the optimized chromatographic conditions, all four analytes were eluted with symmetrical peaks, satisfactory resolution and acceptable theoretical plate counts within a run time of 12 min. The method exhibited excellent linearity over concentration ranges of 25-150 μg/mL for TAC, 62.5-375 μg/mL for Nys, 250-1,500 μg/mL for NS and 6.25-37.5 μg/mL for Gr, with correlation coefficients greater than 0.999 for all analytes. The developed method was validated according to International Council for Harmonization guidelines and demonstrated acceptable accuracy, with percentage recoveries ranging from 98 to 102% and precision, with percentage relative standard deviation values below 2%. Forced degradation studies were performed under acidic, alkaline, oxidative, reductive, thermal, photolytic, peroxide and hydrolytic conditions. The analytes exhibited degradation ranging from ~11 to 12.1%, confirming the stability-indicating capability of the method. The proposed method is simple, accurate, precise, robust and suitable for the routine quantification of Nys, NS, TAC and Gr in bulk drug substances, transferosomal gel formulations and marketed pharmaceutical products.
Erectile dysfunction (ED) is defined as the persistent inability to achieve or maintain an erection sufficient for satisfactory performance for a duration of >3 months. However, chewable tadalafil formulations for treating ED and corresponding dissolution analysis methods for their quantification are lacking. Therefore, this study aims to investigate Quality by Design considerations for developing a reversed-phase high-performance liquid chromatography method. The test method was designed to perform a quantitative dissolution analysis of tadalafil chewable tablets to enhance medication compliance. This method required an Agilent C8 column (4.6 × 5.0 cm, 5 μm) as the stationary phase and a mobile phase comprising a 1:1 methanol-water mixture. The column temperature and detection wavelength were maintained at 40°C and 225 nm, respectively. The development and validation of the dissolution method resulted in a relative standard deviation of 0.03% for system suitability, assessed in a test solution containing a solubilizer. The test method demonstrated specificity by clearly distinguishing the active pharmaceutical ingredient from excipients. Tadalafil accuracy was confirmed with an average recovery rate of 100.22%, and the precision showed a relative standard deviation of 0.35%. Excellent linearity (R2 = 1.000) was observed over the concentration range of 1.5 μg/mL to 9.0 μg/mL. Additionally, the detection and quantification limits for tadalafil were 0.003 μg/mL and 0.008 μg/mL, respectively. The developed method facilitated rapid quantification of tadalafil in chewable tablets, completing each run within 5 min and reducing analysis time by ~60% compared to conventional methods. Additionally, reagent consumption and operational costs were reduced by ~45%, improving overall efficiency. These findings validate the suitability of the method for reliable industrial quality control of tadalafil chewable tablets and new formulations.
The primary objective of this study was to develop and validate a liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometric (LC-APCI-MS/MS) method for the determination of trace levels of N-nitroso vonoprazan (NVNP) in vonoprazan active pharmaceutical ingredient (API) and pharmaceutical products. Chromatographic separation was achieved on an Acquity® Premier HSS T3 column under isocratic conditions using 0.1% formic acid in water and acetonitrile as the mobile phase. Detection was performed in positive ion mode using multiple reaction monitoring. The method was validated according to ICH Q2(R2) guidelines for selectivity, linearity, accuracy, precision, robustness, matrix effect, limit of detection (LOD), and limit of quantification (LOQ). The method was linear over the concentration range of 0.24-50.0 ng/mL (R2 = 0.9971), with recoveries of 85.6%-99.2% and relative standard deviation values below 6.1%. The validated LOD and LOQ were 0.10 and 0.24 ng/mL, respectively; the latter corresponds to 0.24 μg/g (240 ppb) in the pharmaceutical product. Matrix effect evaluation showed 91.6% response (8.4% ion suppression), indicating minimal matrix interference. The validated LC-APCI-MS/MS method was successfully applied to both API and finished products and is suitable for routine quality control, stability studies, nitrosamine risk assessment, and regulatory monitoring of NVNP.
Scutellariae barbatae Herba, the dried whole plant of S.barbatae D. Don, has been documented in the Chinese Pharmacopoeia since 1985. The chemical constituents of S. barbatae Herba are complex. And comprehensive analysis of the chemical components in S. barbatae Herba is still scarcely studied. In this study, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used in both negative and positive ion modes to comprehensively analyze chemical constituents of S. barbatae Herba. Moreover, the fragmentation pathways of compounds were explained in brief. According to tandem mass spectrometry fragmentation data and previous reports, a total of 39 compounds were identified, including 19 flavonoids, 4 neo-clerodane diterpenoids, 1 neo-clerodane diterpenoid alkaloid, 3 triterpenes, 5 fatty acids, 1 lignin, 1 monoterpene phenol, and 4 other constituents. More fragments of information are formed in the compounds, and the fragmentation pathways were explained simply. The developed analytical method is simple, reliable, and effective, providing comprehensive information on the metabolite profile of S. barbatae Herba, which may enhance its quality control and further utilization.
The simultaneous determination of Abemaciclib and Olaparib in in-houseprepared tablets was accomplished by the development and validation of a novel and rapid and sensitive reverse-phase ultra-performance liquid chromatography (RP-UPLC) technique. Using a Waters X-Bridge C18 (4.6 × 100 mm, 3.5 μm) column and a mobile phase made up of 0.1% orthophosphoric acid in water, acetonitrile and methanol (70:20:10% v/v/v) in an isocratic elution mode at a flow rate of 1 mL/min, the chromatographic separation was accomplished. High sensitivity and selectivity were made possible by performing the detection at 219 nm. Following ICH Q2(R1) criteria, the technique was validated and showed high linearity (R2 > 0.999) for both drugs over the concentration range of 10-60 μg/mL. Within acceptable boundaries (<2% Relative Standard deviation (RSD)), both intra-day and inter-day precision and accuracy (recovery percentage) were proven. Forced degradation studies confirmed the method's stability-indicating capability, Stability studies indicated significant degradation under acid, alkali, peroxide and thermal degradation conditions. The degradation products were characterized by LC-MS technique, and possible degradation pathways were proposed. Additionally, in vitro pharmacokinetic evaluation, including dissolution and drug release kinetics, was performed to assess formulation performance. The developed method proved simple, precise and robust, making it suitable for routine quality control, stability study and pharmacokinetic studies of Abemaciclib and Olaparib co-formulated tablets.
A new cooled side arm tube headspace single drop microextraction (CSAT-HS-SDME) method was successfully applied to the extraction of Carum copticum volatile components. The aim of this research is to design and fabricate a new device for simultaneous cooling of the drop and evaporated sample. The innovated technique was evaluated by the extraction and Gas chromatography-mass spectrometer (GC-MS) determination of p-cymene, γ-terpinene and thymol in C. copticum samples. Optimization of the effective extraction parameters was carried out by the simplex method. Under the optimized conditions (i.e. cold and heat temperature, -10°C and 70°C; water addition, 20 μL and extraction time, 20 min), the relative standard deviations of peak areas obtained from five replications were between 3.62% and 4.69%. Compared to other similar equipment for cooling the extraction phase and simultaneously heating the sample, this device has advantages such as simplicity, very low cost, ease of use, portability and no need for additional equipment such as a liquid carbon dioxide cylinder or heater-stirrer. The proposed method was successfully applied to the extraction and identification of volatile components of some C. copticum samples.
The genus Tabebuia is widely used in traditional medicine of tropical and subtropical countries. We recently reported the antitumor activity toward several human cell lines of the ethyl acetate extract of the leaves of Tabebuia hypoleuca, a species endemic to Cuba, together with the high content of triterpenic acids. This work describes the development and the validation of a reversed-phase high-performance liquid chromatography with diode array detection method for the simultaneous quantitation of the contents of oleanolic acid (OA) and ursolic acid (UA). The method was carried out on a C18 column, using methanol and water as mobile phase (ratio 95:5 v/v), at the flow rate of 0.4 mL/min, recorded at λ = 210 nm. The retention times were 16.371 ± 0.045 and 16.943 ± 0.052 min for OA and UA, respectively, with a resolution between peaks of 1.29. The average contents of OA and UA within three sets of the ethyl acetate extract, considered as an active pharmaceutical ingredient (API) were 102.93 and 244.28 mg/g, respectively. The linearity of the response was found to be 10-700 μg/mL for OA and its equation was y = 0.2946x - 2.0432 (r2 = 0.9924). The linearity range for UA was 5-1000 μg/mL and its equation was y = 0.3284x - 17.508 (r2 = 0.9865). The limit of detection and limit of quantification of these constituents were 6.50 and 19.81 μg/mL for OA, 0.75 and 1.98 μg/mL for UA. OA and UA may be used as markers for the quality assessment of formulations containing this API. The developed method is accurate, specific, precise, reproducible and may be applied as a simple and rapid quality-control procedure for medicinal plant extracts with similar triterpene patterns.
In recent years, the government of India has banned single-use plastic, as it is more customer-friendly but poses major disadvantages, such as leaching. As impurities can be present in any source, from the raw material to the end product, and so on, these are scientifically termed extractables and leachables. To identify these types of impurities from the pharmaceutical formulation, predominantly parenteral and ophthalmic or any other products available in plastic packaging, a rapid, selective, and simultaneous method for four different analytes has been developed with the help of gas chromatography-mass spectrometry and validated completely according to International Council for Harmonization Q2(R1) guidelines. Selected extractables and leachables, namely cyclohexanone, 2,4,7,9-tetramethyl-5-decyne-4,7-diol, bis(2-ethylhexyl) sulfosuccinate sodium salt, and benzophenone, were detected by mass spectrometry and identified using the NIST17 library of the instrument. The developed method was within the range.
Atrazine (ATZ) is a persistent herbicide found in aquatic environments worldwide, and the evaluation and monitoring of potential contamination by this pesticide are of utmost importance. A properly validated and well-established analytical method is important to detect ATZ in the environment. In this work, a dispersive liquid-liquid microsyringe extraction method was developed for the pre-concentration of ATZ in lake water samples for subsequent determination in a gas chromatograph with an electron capture detection (GC-ECD) system. The method showed excellent linearity in the range of 2 μg L-1 to 30 μg L-1, with correlation coefficient (R) values ≥0.995. In addition, the method is selective, and the sample matrix does not interfere with the determinations (≤20%). The limits of detection and limits of quantification were 0.6 μg L-1 and 2 μg L-1, respectively. The method is accurate, with recoveries ranging from 100% to 114%, and precise (13%). The extraction method associated with GC-ECD is reliable, sensitive, simple, fast, reproducible, environmentally friendly and economical. The method was applied to water samples from the Municipal Lake of the city of Cascavel (PR, Brazil) and no ATZ was detected in any of the samples.
There are nine different varieties of Angelica sylvestris plants found in Asia, America, and Europe, and all of them, especially their roots, have medicinal properties. It is important to determine the surface properties of medicinal A. sylvestris that can be easily found in nature. In this study, it was aimed to investigate the surface characteristics of the root, stem, and flower parts of the A. sylvestris. For this purpose, gas chromatography columns were prepared by separating the root, stem, and flower parts of the plant, and the surface properties were determined by the inverse gas chromatography (IGC) method. The IGC study was performed at infinite dilution with polar and non-polar solvents. Surface characteristics of different parts of the plant were determined through analysis of retention diagrams. From IGC experiments, the changes in the acidic and basic character of the parts of the plant were determined and compared with each other. As a result of this comparison, it was found that all KD/KA values were less than 1 and changed as flower > root > stem.
Clomiphene citrate (CLO) and co-enzyme Q10 (Q10) are "ovulatory stimulants" used to treat female infertility and CLO-resistant Polycystic Ovary Syndrome (PCOS). The study aimed to create a unique and well-validated stability-indicating RP-HPLC method for quantifying CLO and Q10 in bulk and tablet formulation, supported by UV analysis. The λ max values of CLO and Q10 were determined at 293 nm and 273 nm, respectively. The chromatographic separation was achieved on a Waters XBridge phenyl 5 μm column (4.6 × 250 mm) using an acetonitrile: tetrahydrofuran (80:20 v/v) with 0.5% triethylamine as a mobile phase at a flow rate of 0.5 mL/min with photodiode array detector at 279 nm (isobestic point). Resveratrol (RES) served as an internal standard (ISTD), with retention durations of 6.504, 9.319 and 5.831 minutes for CLO, Q10 and RES, respectively. Calibration curves were linear (R2 = 0.9987 for CLO and 0.9993 for Q10) and %RSD values for all validation parameters were below 2%, confirming precision and accurate. Forced degradation studies through various stress conditions, assessed the method's stability-indicating nature by exhibiting clear separation of degradation products from the analytes and degradation levels within ICH limits (≤ 20%). The validated method was found to be reliable, specific, and applicable for routine analysis in quality control.
The co-administration of ibuprofen (IBU) and phenyramidol HCl (PHE) is frequently preferred for the management of musculoskeletal disorders to enhance therapeutic efficacy. This study aimed to develop and validate a rapid, sensitive and cost-effective high-performance liquid chromatography-photodiode array (HPLC-PDA) method for the simultaneous determination of IBU and PHE in human plasma and pharmaceutical preparations, and to demonstrate its applicability in pharmacokinetic studies. Chromatographic separation was achieved on a C18 column using a mobile phase consisting of phosphate buffer (pH 6.0 ± 0.05) and methanol (30:70, v/v) at a flow rate of 1.0 mL·min -1. The retention times of IBU and PHE were 3.7 and 2.7 min, respectively, with a total analysis time of 6 min. The method showed excellent linearity within the range of 0.05-40.0 μg·mL -1 for both drugs, with LLOQ values of 0.05 μg·mL-1 for IBU and 0.0025 μg·mL-1 for PHE. The plasma samples were collected at four time points (0.0, 1.0, 4.0 and 8.0 h) after oral administration of IBU and PHE. Cmax, Tmax and AUC0-8 were calculated, with Cmax of 16.14 μg·mL-1 for IBU and 3.71 μg·mL-1 for PHE, while Tmax was 1.0 h for both. Despite limitations in the number of sampling time points, the method proved highly applicable for determining pharmacokinetic profiles and for routine quality control analyses. Its short analysis time, high sensitivity and low sample volume requirement make it a powerful tool for clinical research and therapeutic monitoring. This is the first validated HPLC-PDA method applied to the simultaneous pharmacokinetic evaluation of IBU and PHE in human plasma.
The present research focuses on developing a high-performance thin-layer chromatographic (HPTLC) method using a design of experiment approach for simultaneously estimating Tadalafil and Mirabegron. Chromatographic separation was achieved on aluminum plates precoated with silica gel 60 F254 using an optimized mobile phase of MeOH: ACN: n-Butanol: TEA (6:2:2:0.1%v/v/v/v). A fractional factorial design was applied for the robustness study and the independent variables selected were chamber saturation time (A), wavelength (B), mobile phase composition (C) and solvent front (D). It was statistically revealed that the volume of MeOH affected the Rf of both drugs resulting in stricter control of MeOH volume compared to the other three variables. Rf for Tadalafil and Mirabegron was 0.75 and 0.45 at 242 nm. Calibration plots were linear for Tadalafil (100-300 ng/band) and Mirabegron (500-1500 ng/band), with recovery rates of 98.15%-100.34% and 98.46%-102.14%, respectively. The method was validated per ICH Q2(R1) guidelines, demonstrating linearity, precision, accuracy and selectivity, with %RSD values below 2% for both drugs. This HPTLC method is simple, accurate, reproducible and suitable for routine quality control of pharmaceutical formulations.
Migraine is a complex neurological condition that often involves a severe headache characterized by moderate to severe throbbing. Lasmiditan a novel ditan approved by USFDA in 2011 to treat migraine. This contemporary research elaborates the systematic development and validation of Reverse Phase-High Performance Liquid Chromatography bio analytical method for estimating Lasmiditan in human plasma employing analytical quality by design. Bio analysis employing protein precipitation was performed to extract Lasmiditan from human plasma using Dolutegravir as the internal standard. Design of Experiments was incorporated employing central composite design, and the methodology was assessed for critical analytical attributes by varying the critical quality attributes using Design Expert Software Version 13.0. The chromatographic conditions optimized were symmetry C18 (150 × 4.6 mm, 5 μ) column, mobile phase 0.1% orthophosphoric acid: acetonitrile (70:30 (v/v) with a flow rate of 1.0 mL/min at detection wavelength of 260 nm and column temperature of 30°C. Validation studies indicated linearity between 20 and 800 ng/mL (r2 = 0.999), whereas accuracy and precision portrayed a 98.79% recovery. Succinctly, current work exemplifies the significant merits of analytical quality by design approach toward the holistic process in bioanalytical method validation. Furthermore, the method was evaluated using the Whiteness Assessment Tool (Blue Applicability Grade Index), yielding a high score of 82.5, indicating superior performance in terms of analytical efficiency, environmental friendliness and operator safety. Thus, the developed method can be applied for estimation of preclinical and clinical samples of Lasmiditan with improved accuracy and precision.
Quantitative determination of eight characteristic aroma components, namely, benzyl alcohol, phenylethyl alcohol, eugenol, geraniol, nerol, linalool, citronellol, and methyl eugenol, by reversed-phase high-performance liquid chromatography with hydroxypropyl-β-cyclodextrin as mobile-phase additive was investigated. The proposed method was validated and demonstrated with good linearity of 0.994 to 1.000 with a recovery in the range of 94.97-109.38% and exhibited favorable accuracy, intra and inter-day precision based on relative standard deviation values. Additionally, the inclusion complex between eight characteristic aroma components and hydroxypropyl-β-cyclodextrin was investigated, the complex formation was found at a 1:1 molal ratio for eight characteristic aroma components and hydroxypropyl-β-cyclodextrin, and the apparent formation constant between citronellol and hydroxypropyl-β-cyclodextrin was determined to be 71.86 M-1. In those components, ∆H of linalool and citronellol was found to be +877.03 kJ mol-1 and +1433.67 kJ mol-1, and ∆S were positive, which indicated that transfer from mobile phase to the stationary phase of the two analytes was entropically driven. Furthermore, this method was successfully applied to determination of characteristic aroma components in Rosa damascena Mill. hydrosol and R. rugosa cv. "Plena" hydrosol, in which it was found that phenylethyl alcohol, citronellol, and methyl eugenol were principal components.
The main aim of this study was to develop and validate a new reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of tofacitinib citrate (TFC) and berberine chloride (BBC) in pharmaceutical injection formulations and blood plasma. An isocratic RP-HPLC method was used with a mobile phase consisting of phosphate buffer saline (pH 5.5) and acetonitrile at a ratio of 70:30, at flow rate of 1 mL/min at 25°C. Both drugs were detected at 306 nm. The proposed method demonstrated excellent linearity over a concentration range of 5-30 μg/mL for both the TFC and BBC, with correlation coefficients (R2) exceeding 0.999. System suitability parameters, including peak area, tailing factor, theoretical plates and resolution, were within acceptable limits, with relative standard deviations <2%. The recovery studies at 50, 100 and 150% showed mean recovery rates of 99.4-100.7% for TFC and 99.9-100.4% for BBC. The method exhibited high sensitivity, with low detection and quantification limits. The robustness was confirmed under slight variations in flow rate and mobile phase composition. The developed RP-HPLC method is accurate, reliable and suitable for simultaneous quantification of TFC and BBC in pharmaceutical and biological matrices, offering potential for routine quality control and pharmacokinetic studies.
The increasing demand for safe, effective and sustainable alternatives to traditional cough suppressants has brought polyherbal formulations in respiratory ailment management. This report outlines the development and greenness evaluation of a high-performance liquid chromatography method for the simultaneous quantification of glycyrrhizic acid, piperine and ursolic acid from crude plant extracts and commercial polyherbal cough syrups. Chromatographic separation was performed using Agilent Eclipse C18 column (250 × 4.6 mm, 5 μm) under gradient elution using 0.1% formic acid in water and acetonitrile as mobile phases. The optimal absorbance wavelengths during quantification were 254 nm for glycyrrhizic acid, 342 nm for piperine, and 210 nm for ursolic acid at retention times of 21.3, 24.5 and 31.0 min, respectively. Results indicated a method that was sensitive, reproducible and specific to the three analytes quantified as well as validated according to ICH Q2(R1). Evaluating the greenness of the method (using the Analytical GREEnness metric, Green Analytical Procedure Index, and Eco-Scale Assessment confirmed that this method could be considered "green". Upon completion of method validation, the method was used to quantify each marker compound from commercially available products in an effort to better understand production methodologies and directed our quality control and standardization efforts. The results of this systematic investigation point to the advantage of applying green analytical techniques for the assessments of polyherbal medicines, potentially establishing them as credible, environmentally friendly therapeutic options for respiratory ailments.
"Trikatu churna," very famous herbal formulation which is widely used and prescribed for various disease conditions like hypothyroidism, hyperlipidaemia, atherosclerosis and others. It is having well proven anti-inflammatory, hepatoprotective and anthelmintic activities. The preliminary issues in quality of trikatu churna arise due to numerous factors like replacement, omission and adulteration in key ingredients of trikatu churna with spurious alike ingredients. A novel, simple, swift, precise, robust, non-destructive and non-targeted method based on High Performance Thin layer chromatography (HPTLC) coupled with multicomponent analysis was developed to appraise the quality of the samples of trikatu churna. Ten market samples and one standardized in-house sample was used for quality evaluation. Three in house samples were also prepared by deliberately omitting one key ingredient in each. Various chemometrics methods like Principal Component Analysis, and Cluster Analysis based on Hierarchy for further analysis to evaluate the differences in market samples of trikatu churna. Thus, developed method can be used as an effectual and applied approach for quality assessment of trikatu churna.
Per- and polyfluoroalkyl substances (PFAS) are an emerging group of environmental contaminants widely used in consumer products. Public unease regarding the levels of these compounds in the environment has called for methods to keep up with emerging PFAS and regulations. There is a need for analytical methods that are efficient and robust that can be readily used and easily accessed in commercial laboratories. This work discusses the development of an efficient, sensitive and reliable liquid chromatography tandem mass spectrometry method for the analysis of 40 PFAS in aqueous matrices (10 min run time). Initial calibration demonstrated excellent linearity for all PFAS analytes with r 2 values greater than 0.997. Limit of detections (0.2-100 ng/L) and limit of quantifications (1-350 ng/L) show the ability for the method to reach necessary levels to stay up to date with federal and state regulations. Two solid phase extraction techniques were compared based on recovery accuracy and precision. Recoveries ranged from 92.0% to 105.0% and 67.0% to 109.0% for the automated and manual setups, respectively. Evidence was found to support improvements in sample recovery using an automated setup. The validated method was used to analyze samples from six natural water sources in the Baltimore-Metro area, where seven of the forty PFAS compounds were found in at least one sample.
In this study, a straightforward, precise, environmentally friendly, and strong high-performance liquid chromatography technique was established for the examination of Tasimelteon in pharmaceutical formulations. The chemometric factor screening investigation facilitated the identification of crucial technique parameters, which were subsequently improved using the Box-Behnken design to enhance comprehension and enhance the performance of the method. The separation was achieved with using the Supelco Ascentis® Express C8 column (100 × 4.6 mm, id, 2.7 μm) with a mobile phase consisting of methanol:phosphate buffer (0.030 M, pH 2.5) at a ratio of 51:49 (v/v). The flow rate was chosen as 1.2 mL/min. The optimized method was fully validated according to International Council on Harmonization (Q2)R1. The method demonstrated linearity within the concentration range of 10.0 to 75.0 μg/mL, with a coefficient of determination (R2) of 0.999 and a significance level (P) of less than 0.05. Linearity investigations were conducted using the peak normalization approach. The internal standard that was deemed most suitable was agomelatine, at a concentration of 33.0 μg/mL. In addition, the environmental sustainability of the method was proved using different analytical greenness metrics as National Environmental Methods Index, analytical eco-scale, Analytical Greenness Metric and Green Analytical Procedure Index for evaluating its greenness. The applicability of the method was demonstrated in the analysis of Hetlioz® pseudo formulation.
