Elution Mode Research Articles

Stability indicating RP-HPLC-UV method for quantitative estimation of isomer and other organic impurities of atopic dermatitis drug abrocitinib in drug substance and pharmaceutical dosage form using quality by design (QbD) approach

A modest, reliable, robust, stability-indicating RP-HPLC-UV method was developed and validated following ICH Q2(R2) for the separation and quantification of isomer and other organic impurities of Abrocitinib (Janus kinase inhibitor). Quality by design (QbD) approach was used to achieve the desired specifications. The chromatographic separation was achieved for impurities method of Abrocitinib within 35 min of chromatographic run time on Agilent ZORBAX Eclipse plus C18 150*4.6 mm, 3.5 µm column with gradient elution mode: time (min)/B (%): 0.01/0, 2.5/15, 8.0/20, 15.0/40, 20.0/50, 25.0/50, 25.1/0, 35.0/0 with mobile phase-A (MP-A): 10 mmol/L CH3COONH4 and acetonitrile with the ratio of 900:100 (v/v), mobile phase-B (MP-B): 10 mmol/L CH3COONH4 and acetonitrile with the ratio of 100: 900 (v/v). The detection wavelength, mobile phase flow rate, column temperature, and injection volume were used at 287 nm, 1.0 mL/min, 40 °C, and 20 µl respectively. Utilizing forced degradation and mass balance investigations, the new method’s stability-indicating properties were assessed. The developed method was validated as per ICH and found to be specific, precise, rugged, robust, accurate, linear, and with a limit of quantification values,0.0359 µg/mL for the impurity IMP-A, 0.0192 µg/mL for the impurity IMP-B, 0.0322 µg/mL for the impurity IMP-C and 0.0201 µg/mL for abrocitinib.

Pharmacokinetics and Tissue Distribution of Itampolin A following Intragastric and Intravenous Administration in Rats Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Itampolin A, a natural brominated tyrosine alkaloid isolated from the sponge Iotrochota purpurea, has been shown to have good inhibitory effects in lung cancer cells as a p38α inhibitor. A simple, sensitive, and reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been established, validated, and applied to the study of the pharmacokinetics and tissue distribution of itampolin A following intragastric and intravenous administration. Itampolin A and theophylline (internal standard, IS) were extracted by the simple protein precipitation technique using methanol as the precipitating solvent. Chromatographic separation was achieved by using the optimized mobile phase of a 0.1% formic acid aqueous solution and acetonitrile in the gradient elution mode. Itampolin A and IS were detected and quantified using positive electrospray ionization in the multiple reaction monitoring mode with transitions of m/z 863.9 → 569.1 for itampolin A and m/z 181.1 → 124.1 for IS, respectively. The assay exhibited a linear dynamic range of 1-1600 ng/mL for itampolin A in biological samples and the low limit of quantification was 1 ng/mL. Non-compartmental pharmacokinetic parameters indicated that itampolin A was well-absorbed into the systemic circulation and rapidly eliminated after administration. The apparent distribution volume of itampolin A was much higher after intragastric administration than that after intravenous administration. A tissue distribution study showed that itampolin A could be detected in different tissues and maintained a high concentration in the lung, which provided a material basis for its effective application in lung cancer. The pharmacokinetic process and tissue distribution characteristics of imtapolin A were expounded in this study, which can provide beneficial information for the further research and clinical application of itampolin A.

Liquid chromatography and liquid chromatography coupled with tandem mass spectrometry studies for the identification and characterization of degradation products of lobeglitazone

AbstractThe objective of the present research work was to demonstrate the application of liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) for the separation, identification, and characterization of degradation products (DPs) of the drug lobeglitazone. The drug was subjected to various stress conditions such as oxidation, hydrolysis, thermal, and photolytic degradation as per the guidelines of the International Conference on Harmonization Q1A (R2). The drug was stable under thermal stress conditions, whereas it was susceptible to degradation in the other stress conditions forming four DPs. DPs were separated using a high‐performance LC system equipped with Zorbax SB C18 column (250 × 4.6 mm, 5 µm particles) using isocratic elution mode. The DPs were identified and characterized using MS and MS/MS. The chemical structure and fragmentation pattern were predicted for each degradant from the data obtained by mass studies. The drug and identified DPs were assessed by in‐silico approaches for predicting absorption, distribution, metabolism, and toxicity behavior. The in‐silico tools used for prediction were the pkCSM web server, ToxTree, and OSIRIS property explorer.

Separation of bufadienolides from Helleborus thibetanus Franch. by a combination approach involving macroporous resin column chromatography and gradient countercurrent chromatography.

In this study, a combination approach involving macroporous resin (MR) column chromatography and gradient countercurrent chromatography (CCC) was employed to enrich and purify bufadienolides from the roots and rhizomes of Helleborus thibetanus Franch. Initially, a D101 MR-packed column chromatography was utilized for fractionation and enrichment of the bufadienolides, which were effectively eluted from the column using a 60% ethanol solution. CCC was subsequently introduced to separate the enriched product using the ethyl acetate/n-butanol/water (EBuWat, 4:1:5, v/v) and EBuWat (5:0:5, v/v) solvent systems in a gradient elution mode. As results, five bufadienolides, including 6.1mg of hellebrigenin-3-O-β-D-glucoside (1), 2.2mg of tigencaoside A (2), 8.3mg of deglucohellebrin (3), 3.5mg of 14 β-hydroxy-3β-[β-D-glucopyranosyl-(1→6)-(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (4), and 3.0mg of 14β-hydroxy-3β-[(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (5), were effectively separated from 300mg of the enriched product. The respective high-performance liquid chromatography purities were as follows: 95.2%, 75.8%, 85.7%, 82.3%, and 92.8%. This study provides valuable insights for the efficient enrichment and separation of bufadienolides from Helleborus thibetanus Franch.

HPLC analysis of synthetic dyes in wastewater

The analysis of synthetic dyes in wastewater is an urgent task of modern analytical chemistry since an increased content of dyes in wastewater can have an adverse effect on the environment. Reverse phase high performance liquid chromatography (RP HPLC) is often used to control the content of dyes in various objects, which makes it possible to reliably determine the content of specified substances. The purpose of this work was to create and evaluate the metrological characteristics for the method of analysis of synthetic dyes in the industrial wastewater with a high content of inorganic salts. The chromatographic system used in this work was a Knauer high-pressure pump. The separation was performed on a Kromasil 100-5-C18 (w) reverse phase column (4.6 × 250 mm) in the gradient elution mode. Acetonitrile and ammonium acetate solution were used as the mobile phase. Synthetic dyes were detected on an Azura diode array detector. The paper presents the results of concentrating 10 synthetic dyes on commercially available solid phase extraction cartridges that were suitable for working with aqueous solutions with a high content of inorganic salts. For each substance, the degree of concentration from distilled and model wastewater was calculated, which varied from 2 to 98%. Among the analysed cartridges, the highest degree of concentration during operation in conditions close to real wastewater was shown by the Diapak P cartridge, at a pH of the concentrated solution equal to 4. The results of this study were used to develop and validate a method for a simultaneous detection of 10 synthetic food dyes (SFD) (E102, E104, E110, E122, E124, E129, E131, E133, E142, E151) in aqueous and organic solutions. The results of the study can be used to provide analytical control of the content of synthetic dyes in the industrial wastewater. The limit of intermediate precision of the method did not exceed 5%, and the expanded uncertainty of the measurements was less than 10%.

Research Progress in the Separation of Chemical Components from Essential Oils by High-Speed Countercurrent Chromatography

Essential oils (EOs) are vital secondary metabolites in plants. They have garnered substantial attention owing to their distinct flavors and desirable attributes, including potent antioxidant, antibacterial, and antitumor properties. Nevertheless, the active constituents of EOs exhibit intricate chemical structures, and conventional separation techniques are inadequate for purifying the individual chemical components from EOs. High-speed countercurrent chromatography, based on the principles of a hydrodynamic equilibrium system, has emerged as a liquid–liquid chromatographic separation method renowned for its ability to handle substantial single injection volumes and the absence of irreversible adsorption. Consequently, in recent years, this technique has been widely employed in the isolation and refinement of natural products. In this review, a comprehensive analysis is conducted, contrasting the merits and demerits of high-speed countercurrent chromatography with conventional separation methods. The solvent systems, elution modes, commonly employed detectors, and practical applications are reviewed in the context of high-speed countercurrent chromatography for essential oil separation and purification. Furthermore, this review offers a glimpse into the potential prospects of applying this technique, with the intention of serving as a valuable reference for the use of high-speed countercurrent chromatography in the purification of EOs.

Isolation, identification and quantification of flavonoids from the flowers of <i>Staphylea pinnata</i> L.

Introduction. Search for new plant species containing biologically active substances (hereinafter – BAS) is one of the leading tasks of pharmacognosy as a science. The search for flavonoid glycosides in plant raw materials is especially relevant, since they have anti-inflammatory, antioxidant, immunostimulating, as well as weak fungicidal and bacteriostatic action. Staphylea pinnata L. is an endemic plant of the Caucasus, cultivated not only in Georgia, but also in the Russian Federation in the Northern and Northwestern Caucasus. In foreign literature there are studies of antioxidant, antiproliferative and cytotoxic activity of leaf extracts of several species of S. pinnata L., as well as inhibitory activity of COX-1, COX-2 and LTB4 formation. Meanwhile, no serious Russian-language scientific studies on either the chemical composition or pharmacological action of generative organs of S. pinnata were found in the literature. This work is part of a comprehensive phytochemical study of S. pinnata. The aim of the work is to study the qualitative and quantitative composition of flavonoids in the studied object.Aim. To isolate, identify and quantify flavonoids in flowers and buds of Staphylea pinnata L.Materials and methods. Alcohol-water extracts from dried generative organs of the studied plant were used as analyzed solutions. Solutions were analyzed on a spectrophotometer SF-2000 (LLC "OKB Spectr", Russia) after sample preparation with aluminum chloride and on an HPLC Nexera-i LC-2040 (Shimadzu Corporation, Japan) equipped with a column and sample thermostat, degasser and autosampler using an individually selected elution gradient of the mobile phase (0.1 % orthophosphoric acid/acetonitrile solution). The primary data were processed using LabSolutions Single LC software (Shimadzu Corporation, Japan). Compounds from the flavonoid group were identified by retention times. Detection was carried out using a UV detector with an absorption wavelength of 365 ± 2 nm.Result and discussion. Alcohol-water extracts from flowers and buds of S. pinnata L. were obtained. Quantitative evaluation by spectrophotometry for flavonoid content was carried out. A gradient elution mode for HPLC was selected for simultaneous determination of 7 flavonoid glycosides. These chromatographic conditions allowed the identification and quantification of astragaline, cynaroside, cosmosiin, narcissin and rutin in flowers and buds of Staphylea pinnata L. Flavonoid glycosides: raponticin and kaempferol were not detected.Conclusion. Flavonoid glycosides were isolated from the generative organs of S. pinnata L., a technique for quantitative determination of flavonoid glycosides in alcohol-water extracts was developed, astragalin, cynaroside, cosmosiin, narcissin and rutin were detected and quantified.

Chemo- and enantioselective analysis of high hydrophilic organophosphate pesticides by liquid chromatography under different elution modes

Acephate and malathion are prevalent chiral organophosphate insecticides, giving rise to more toxic chiral metabolites such as methamidophos and malaoxon, respectively. Enantioselective separation of chiral pesticides is crucial due to the differing pharmacological, environmental, and toxicological profiles of enantiomers. This study systematically evaluates an enantio‑ and chemoselective performance of eight polysaccharide-based chiral columns (Chiralcel OB-H, Chiralcel OD-H, Chiralcel OJ-R, Chiralpak AD-H, Chiralpak AS-H, Chiralpak IG-3, Chiralpak IK-3, and Lux-Amylose-2) and ten mobile phases across multimodal elution (normal-phase, polar organic, and reversed-phase conditions). Within the 80 combinations per analyte, the chiral screening highlights specific enantioselectivity trends concerning hydrophilic and hydrophobic compounds when utilizing amylose- or cellulose-based polymers under different conditions. Notably, a set of columns including IK-3, OJ-R, AS-H, and AD-H demonstrated superior coverage, achieving at least a 62 % success rate for enantioseparation. For the resolution between the pairs acephate/methamidophos and malathion/malaoxon, where no further optimizations were undertaken, the success rate for enantio‑ and chemoselectivity was 10 % and 30 %, respectively.

Analysis of varietal advantages of leaf form <i>Apium graveolens</i> L. according to chlorogenic acid content

Introduction. Special studies on the benefits of agricultural crops – current research regarding the prospects for the development of their products based on functional and specialized nutrition. Substances with a phenolic profile carry out various types of pharmacological activity aimed at treating metabolic and other socially significant phenomena, which allows them to be positioned as generally accepted main ingredients. The leafy botanical form of celery is a rich source of phenolic compounds.Aim. Development and validation of a method for the quantitative analysis of chlorogenic acid to assess the varietal advantages of Apium graveolens L. raw materials using HPLC.Materials and methods. For the analysis, we used leaf celery grass of different varieties grown at two sites (the village of Lembolovo and SNT "Ruchey"). Sample preparation of raw materials was carried out by extraction in an ultrasonic bath. Chromatography conditions: column Luna 5 μm C 18(100) Å LC Column 250 × 4.6 mm, thermostat temperature 40 °C, detection wavelength 327 nm. PF A 0.05 % TFA, PF B acetonitrile. Elution mode: 0 min 12 % B, 20 min 30 % PF B, 23 min 12 % PF B.Results and discussion. According to the data obtained, the accumulation of chlorogenic acid in celery raw materials depends on both the plant variety and the cultivation conditions. However, the difference in the quantitative content of the target component correlates between varieties grown on different sites. The highest content of chlorogenic acid was noted in the variety Nezhny (SNT "Ruchey") and amounted to 0.464 ± 0.012. The variety Summer Boom can be distinguished as the best variety.Conclusion. The results of the study can be used in the development and quality control of functional and specialized nutrition based on the leafy botanical form of odorous celery due to the high content of phenolic substances.

Development and Validation of a Rapid and Sensitive LC-APCI-MS/MS Method for the determination of three N-nitrosamine impurities in Varenicline tartrate drug substance and drug products

Nitrosamines impurities may be present in low levels in a variety of products that people are exposed to every day. After thorough investigations, regulatory agencies have outlined that the formation of nitrosamines is possible in the presence of secondary, tertiary, or quaternary amines and nitrite salts under acidic reaction conditions. For safety purpose, all API manufacturers must be developed methods to monitor the low levels of each nitrosamine impurity before the release the individual batches. In this article, we have developed novel, rapid and sensitive liquid chromatography Atmospiric pressure chemical ionization Mass spectroscopy (LC-APCI-MS/MS) method for the determination of N-Nitroso Dimethylamine (NDMA), N-Nitroso Diethylamine (NDEA) and N-Nitroso varenicline impurities in the Varenicline Tartrate drug substance and Drug products. Chromatographic separation is achieved using Inert sustain AQ C18 150 × 4.6mm, 3.0μ column with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B at the 0.6 mL/min flow rate by using gradient mode of elution upto the 25 minutes total run time. The three impurities are successfully ionized and quantified in positive mode of atmospheric pressure chemical ionization (APCI) using multiple reaction monitoring (MRM). This method was validated according to ICH Q2 (R1) guidelines. The method provided excellent S/N ratios with a high linearity range of 0.66-19.88 ppm with respect to Varenicline Tartrate sample concentrations with a regression coefficient >0.995. The recovery for the method was established with a protocol of three-step sample preparation and was satisfactory within 85-115%. The determined LOQ (0.66 ppm) and LOD (0.22 ppm) values are very low with respect to sample concentration which shows the sensitivity Performance of the method. The method can be routinely applied for the detection of N-nitrosamine impurities (NDMA, NDEA and N-Nitroso varenicline) in Varenicline Tartrate drug substance and Drug products.

Novel RP-HPLC method for estimation of a newly developed combination of tizanidine and etoricoxib in rat plasma: Eight criteria for greens evaluation.

A novel environmentally friendly reversed-phase high-performance liquid chromatography (RP-HPLC) method has been effectively validated for simultaneously measuring a prospective conjunction of tizanidine (TIZ) and etoricoxib (ETC), the combined medicine, in rat plasma. The technique employs diclofenac potassium as the internal standard, guaranteeing dependable and precise outcomes. This study aimed to assess the impact of the suggested combination therapy on treating inflammation resulting from rheumatoid arthritis (RA) in a rat model. The procedure was performed using an Agilent series 1200 model HPLC apparatus. The chromatographic conditions consist of isocratic elution mode, C18 column with dimensions of 150 mm × 4.6 mm × 5 µm, flow rate of 1.5 mL/min, wavelength of 230 nm, temperature of 50°C, and injection volume of 10 µL. The elution was performed using a mobile phase consisting of a phosphate buffer with a pH of 3.5 and acetonitrile in a ratio of 80:20 v/v. Calibration curves were conducted for TIZ and ETC within the 1-50 µg/mL range, demonstrating linear trends with R2 values over 0.999. The effectiveness and eco-friendliness of the proposed method were evaluated using eight separate environmentally conscious metrics. The addition of TIZ and ETC to arthritic rodents amplified these effects significantly. Furthermore, TIZ and ETC significantly reduced serum levels in arthritic rodents, and safety investigations revealed normal complete blood count, liver, and renal functions. TIZ and ETC appear to have antiarthritic, anti-inflammatory, and safe combinations, making them viable future treatment options for RA that are also safe and efficacious. Following validation by United States Food and Drug Administration(US-FDA) rules, all goods met the criteria.

Adapting Fabric Phase Sorptive Extraction as an Innovative Multitool for Sample Transfer and Extraction in Pharmacokinetic Analysis Followed by LC-MS Determination of Levofloxacin in Plasma Samples.

Fabric phase sorptive extraction (FPSE) is a simple microextraction technique that allows analytes to be rescued from matrix components while using a small volume of samples to analyze complex biological systems. This study used FPSE as a microextraction tool and a sample storage and transfer device. Levofloxacin as a model molecule was applied intravenously (IV) to New Zealand male rabbits. The samples were simultaneously extracted by using FPSE and protein precipitation methods. The final solutions were analyzed using LC-MS equipped with an ACE C18 LC Column (150 mm × 4.6 mm, 5 μm) at 25 °C employed in isocratic elution mode using solution A (0.1% formic acid in water)/solution B (0.1% formic acid in acetonitrile) (80:20, v/v). The total analysis time was less than 15 min. The developed method was validated using the ICH M10 bioanalytical method validation and study sample analysis guidelines. The results obtained using FPSE were statistically identical to those obtained using protein precipitation. The plasma samples applied onto FPSE (10 μL onto 1.0 cm × 1.0 cm Biofluid Sampler) were stored in three different temperatures [refrigerator (2-8 °C), at ambient temperature (20 ± 5 °C), and in the stability cabinet (40 °C, 75% humidity)] and three different storage conditions (Eppendorf tubes, plastic containers, and straw paper envelopes). Levofloxacin in plasma samples adsorbed by FPSE biofluid sampler remained stable at 2-8 °C in Eppendorf tubes for at least 1 week. This study showed that FPSE could be used as a sample storage and transfer device for pharmacokinetic applications that need to work with small sample volumes and discard aggressive cold chains to store and transfer the plasma samples.

Development and validation of an analytical methodology based on solvent microextraction and UHPLC-MS/MS for determining bisphenols in honeys from different botanical origins

A new analytical methodology was proposed to determine fourteen bisphenols in honeys from different botanical origins using ultra-high performance liquid chromatography-tandem mass spectrometry. A fast, efficient, environmentally-friendly and simple sample treatment (recoveries between 81% and 116%; matrix effect <20% for all studied compounds except for bisphenol E, F and S) was proposed, which involved a solvent microextraction with acetone and a small volume/amount of 1-hexanol. Chromatographic analysis (< 15 min) was performed in a Kinetex EVO C18 column under gradient elution mode. The method was validated in terms of selectivity, limits of detection (0.2–1.5 μg/kg) and quantification (0.5–4.7 μg/kg), linearity, matrix effect, trueness, and precision (relative standard deviation <17%). Finally, thirty honey samples were analyzed, revealing the presence of residues of nine bisphenols in some of them. However, quantification was possible only in two cases for bisphenol A, with a concentration of approximately 13 μg/kg.

Determining flavonoids in the skin of Citrus reticulata

n this work we studied the flavonoid composition of tangerine skins using 10 kinds of tangerines from various producers. The tangerine skins were peeled from fruit purchased from the market in Belgorod and dried at room temperature outside direct sunlight. The compositions of “green” ethanol-based extraction solutions and the “weighed portion of plant material: volume of extraction solution” ratios were chosen for extraction to ensure a high yield of flavonoids. For separation, reverse-phase HPLC on the Kromasil 100-5C18 stationary phase was used in environmentally friendly mobile phase compositions that contained ethanol as an organic modifier. In gradient elution mode all mobile phases contained 0.2 vol. % orthophosphoric acid. To assign extract components, we used samples of a number of substances and parameters of electronic absorption spectra in comparison with published data. It was found that the chromatographic profiles of the extracts of the studied samples were noticeably different. A number of the least lipophilic compounds, derivatives of cinnamic acids, were found in all extracts. After that, we discovered two flavanone glycosides, the main of which was hesperidin (hesperitin-7-rutinoside) with a content of is 26.0-39.9 mg per 1 g of dried raw material, and naringin (naringenin-7-neohesperidiside) in significantly smaller quantities, from 0 to 1.15 mg per 1 g. But of greatest interest were polymethoxylated flavones, among which the main component was nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone) with a content from 0.03 to almost 11 mg/g in dried peel and tangeretin (5,6,7,8,4′-pentamethoxyflavone), found in slightly smaller quantities, 0.1-5.4 mg/g. It was established that nobiletin and tangeretin were mostly accumulated in the skins of small tangerines. On the whole, tangerine skin is a great source of highly biologically active flavonoids, so its processing is an important task for the food industry.

A novel LC-QTOF-MS/MS method for trace level identification and quantification of potential genotoxic impurity, 5-Nitro-2-(propylthio)pyrimidine-4,6-diol in ticagrelor drug substance

An important objective of the present work is to develop and validate a novel LC-QTOF-MS/MS method to identify and quantify 5-Nitro-2-(propylthio) pyrimidine-4,6-diol genotoxic impurity at trace level (∼8.33 ppm) in ticagrelor drug substance, is an antiplatelet medicine used in the treatment of heart attacks and strokes. The potential genotoxic impurities must be controlled below acceptance limit during process of ticagrelor synthesis and release. In reversed-phase mode, the ACQUITY UPLC CSH C18 (2.1 mm x 100 mm, 1.7 μm) with ambient column temperature was used. 0.1% formic acid in Milli-Q water and methanol in the ratio of 30:70 v/v used as mobile phase A and mobile phase B with isocratic elution mode and the run time was 8.0 min. The flow rate was maintained at 0.25 mL/min, autosampler temperature was 5°C, Injection volume was 20 μL. The Retention time of 5-Nitro-2-(propylthio)pyrimidine-4,6-diol was found at 1.345 minutes. Limit of detection (LOD) and limit of quantification (LOQ) were found at 0.56 ppm and 1.67 ppm, respectively. The 5-Nitro-2-(propylthio)pyrimidine-4,6-diol was linear (R2=0.9986) over the concentration range 1.666 to 12.495 ppm. The method was accurate and precise, it was demonstrated by calculating the %mean recovery values ranging from 98.0-106.9%.

Development of an High Performance Liquid Chromatography Method for Simultaneous Determination of Hesperidin and Sodium Benzoate in Kien Ty Syrup

The Kien ty syrup is formulated from six medicinal materials, including Pericarpium Citri Reticulatae, Fructus Aurantii immaturus, Radix Codonopsis, Rhizoma Atractylodis macrocephalae, Fructus Crataegi, Fructus Hordei germinatus and sodium benzoate as a preservative. Hesperidin, a bioflavonoid found in Pericarpium Citri Reticulatae, Fructus Aurantii immaturus, is used to assess the syrup's quality in terms of assay. In this study, an HPLC method for simultaneous determination of hesperidin and sodium benzoate in Kien ty syrup was developed. The method was performed on an Agilent C18 column (250 × 4.6 mm; 5 μm) with the mobile phase being a mixture of 0.05 M phosphate buffer solution pH 5.5: acetonitrile (70:30, v/v), running in a isocratic elution mode. The flow rate was set at 1.0 mL/min, and the injection volume was 10 μL. The analytes were detected at 225 nm. The method was validated and met the requirements according to ICH, AOAC guidelines. Applying the developed quantification method, the analysis of three sirup lots revealed that the content of hesperidin and sodium benzoate ranged from 0.480-0.504 mg/mL and 2.869-2.306 mg/mL, respectively. The study results indicate that this quantification method can be applied for simultaneous determination of hesperidin and sodium benzoate in Kien ty syrup and similar products.&#x0D;

RP HPLC Enabled Novel Analytical Method for the Development and Quantification of Cefuroxime Axetil in its Bulk Drug and Pharmaceutical Formulations

AbstractCurrent investigations aim to develop and validate an accurate, simple, specific, and precise RP‐HPLC method for the detection of cefuroxime axetil, in bulk and tablet dosage forms. Despite extensive literature reviews, no method could be found for estimating the variables mentioned above. The chromatographic separation is achieved on Sunfire C18 column (250 mm × 4.6 mm, 5 µm) of HPLC (Agilent Tech 1100), with 5 µm mobile phase composed of methanol, orthophosphoric acid (0.05%) adjusted to a pH of 3.0 in water, 40:60% v/v triethylamine using isocratic mode of elution, at wavelength 278.0 nm using UV Spectrophotometer, temperature 25 °C, detection is done. The developed method is done as per ICH guidelines. A linear calibration curve is found to be linear in concentrations between 1 and 5 µg mL−1 (r2 = 0.9995). The percentage recoveries of cefuroxime axetil are found to be in the range of 95.68–96.98%. The percentage of relative standard deviations in intraday trials is found to be 0.61, 0.66, and 0.88, respectively, and in interday studies are 0.06, 0.16, and 0.29, respectively, demonstrating good accuracy and little variance. The proposed method is highly sensitive, robust, precise, accurate, and the use of green solvents makes this method safe and efficacious.

Stability Indicating Assay of Empaglifozin and Metformin

The newly developed stability‐indicating RP-HPLC method is simple, robust and validated on the basis of ICH guidelines for the simultaneous determination of Empaglifozin and Metformin hydrochloride in the tablet. Retention times under the optimized condition were 2.104 and 4.0822min for Metformin and Empaglifozin respectively. This research article indicates best separation of Empaglifozin and Metformin from their degradation products. Separation was achieved on a Sunniest ECO C18, 250mm x 4.6mm, 5µm analytical column at wavelength of 225nm, using a mobile buffer (pH-1.8): acetonitrile (50:50) in an isocratic elution mode at a flow rate of 1.2ml/min, Injection volume: 10µl and run time 6mins. The %RSDs for the precision studies was less than 1.5% for both drugs. The %RSD was less than 1.5% in all the parameters of robustness. The forced degradation studies were carried by using 0.1N HCl, 0.1 N NaOH and % 3 H2O2 and there is only % 7.2 degradation in an acidic medium for emplaglifozin and 11.63% for Meformin where as there was nothing degradation in basic and oxidative method. The analyte peaks were clearly separated from the degradant peaks in forced degradation studies. So this method is a fast, sensitive, robust and efficient high performance liquid chromatographic method for the concurrent determination of Metformin and Empaglifozin in their combination, and thus this method can be effectively employed for routine quality control works.

Assessment of pheniramine in alternative biological matrices by liquid chromatography tandem mass spectrometry.

Pheniramine is an over-the-counter antihistamine drug. Its accessibility and low cost made it more popular among drug abusers in Pakistan. In this study, pheniramine was quantified in both conventional and alternative specimens of twenty chronic drug abusers, aged 16-50years, who were positive for pheniramine in comprehensive toxicological screening for drugs by gas chromatography with mass spectral detection in positive electron impact mode. Pheniramine was extracted from biological specimens using solid phase extraction and liquid chromatography tandem mass spectrometry was employed for quantification. Chromatographic separation was carried out on a Poroshell120EC-18 (2.1mm × 50mm × 2.7µm) column using water-acetonitrile in formic acid (0.1%) mobile phase in gradient elution mode with 500μL/min flow rate. Positive electrospray ionization mode and multi-reaction monitoring with ion transitionsm/z241.3 → 195.8 and 167.1 for pheniramine andm/zm/z247.6 → 173.1 for pheniramine-d6 were employed. The quantification method showed good linear ranges of 2-1000ng/mL in blood, urine, and oral fluid; 2-1000ng/mg in hair and 5-1000ng/mg in nail with ≥ 0.985% coefficient of linearity. The retention time of pheniramine was 3.0 ± 0.1min. The detection and lower quantification limits were 1ng/mL and 2ng/mL for blood, urine, oral fluid and hair whereas 2.5ng/mg and 5ng/mg for nail, respectively. Mean extraction recovery and ionization suppression ranged 86.3-95.1% and -4.6 to -14.4% in the studied matrices. Intra-day and inter-day precision were 4.1-9.3% and 2.8-11.2%, respectively. Pheniramine levels in specimens of drug abusers were 23-480ng/mL in blood, 72-735ng/mL in urine, 25-379ng/mL in oral fluid, 10-170ng/mg in hair and 8-86ng/mg in nail specimens. Alternative specimens are of utmost significance in clinical and medico-legal cases. In this study, authors compared matrix-matched calibration curves to blood calibration curve and obtained results within ± 10%; thereby justifying the use of blood calibration curve for urine, oral fluid, hair, and nail specimens.

A newly developed UPLC-MS/MS method for simultaneous quantitative analysis of ajuforrestin A, ajuforrestin B, ajugamacrin and 8-O-acetylharpagide derived from Ajuga plants in mice blood and the in vivo pharmacokinetics

Objective To develop a sensitive and fast detection method via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to assess the concentration of ajuforrestin A, ajuforrestin B, ajugamacrin and 8-O-Acetylharpagide primarily derived from Ajuga plants in mice blood and their pharmacokinetics. Methods Single protein precipitation with high-proportioned acetonitrile is chosen for sample clean-up. The UPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm) column with a mobile phase in gradient elution mode at the flow rate of 0.4 mL/min was used for sample separation. Acetonitrile was selected as the organic phase solution and water containing 0.1% formic acid was chosen as the aqueous solution. A tandem mass spectrometer containing an electrospray ionization (ESI) source in the positive ionization mode was used to detect four compounds via multiple reaction monitoring (MRM). Results The calibration curves (5–1000 ng/mL) of four compounds were linear with correlation coefficients > 0.997. The matrix effects, accuracy, precision, and recovery were all within permissible scope. Conclusions In this approach, the corresponding pharmacokinetic parameters were successfully clarified in mouse for the first time, which provided a theoretical basis for the improvement of the standard of Ajuga plants and the safety of clinical medication. Furthermore, this method may provide the UPLC-MS/MS evidence for the differentiation of the main close relative varieties of genus Ajuga according to these plants contain different mixtures of the four marker compounds.