C18 Column Research Articles

A liquid chromatography - Tandem mass spectrometry method for determination of ocrelizumab in serum of patients with multiple sclerosis

Ocrelizumab is a second generation recombinant humanized IgG1 monoclonal antibody used for the treatment of multiple sclerosis that selectively target B cells expressing the CD20 antigen. This study aimed to develop and validate a UPLC-MS/MS method for quantification of ocrelizumab in human serum, which can be used in clinical applications for therapeutic drug monitoring.The analysis of ocrelizumab was performed using a bottom-up approach on a liquid chromatography coupled to tandem mass spectrometry detection. The method involved immunoglobulin precipitation with cold methanol followed by peptide digestion with trypsin. The resulting specific peptides were separated on an Acquity UPLC BEH C18 column at 55 °C using gradient elution. The method was validated according to European Medicines Agency (EMEA) guidelines and demonstrated intra- and inter-assay precision with coefficients of variation ranging from 1.6 % to 6.1 % and accuracies between 90.2 % and 107.2 %. Stability testing, including autosampler, long-term and freeze-thaw stability, showed no more than 15 % variation. The method was successfully applied to 169 patient samples, revealing ocrelizumab concentrations ranging from 0.5 to 21.8 mg/L in patients on 6-month dosing regimen and 20.5–65.0 mg/L in 16 patients receiving an initial two-week dose of 300 mg. The newly developed UPLC-MS/MS method met all criteria for accuracy, precision and stability, confirming its suitability for clinical use in monitoring ocrelizumab levels in multiple sclerosis patients.

Quantification of tricyclic glycopeptide in human plasma by UHPLC-MS3 coupled with counter-extraction follow by protein precipitation to enhance sensitivity

An ultra-high performance liquid chromatography tandem mass spectrometry cubed (UHPLC/MS3) assay coupled with protein precipitation and counter-extraction for detection of tricyclic glycopeptide vancomycin in human plasma was established and validated in this study. After protein precipitation and counter-extraction with dichloromethane, chromatographic separation of vancomycin and norvancomycin were performed on a reversed phase column (XBridge Peptide BEH C18 column, 2.1 × 100 mm I.D, 3.5 μm). The transition (parent ions → fragment ions → further fragment ions) at m/z 725.3 → 144.1 → 100.1 was used for quantification of vancomycin. The transition (parent ions → fragment ions) at m/z 718.3 → 144.2 was used for detection of norvancomycin. The linear range of the developed analytical method for quantification of vancomycin was 0.5–100 µg/mL (r = 0.9989). The range of intra- and inter-day precisions of the assay among low, medium and high concentrations is between 1.88 % and 6.33 %. The sensitivity of the analytical method was significantly improved by using MS3 technique as monitoring mode and counter-extraction with dichloromethane followed by protein precipitation as sample processing assay. The developed UHPLC/MS3 assay was successfully applied for clinical therapeutic drug monitoring (TDM) of vancomycin in 45 human plasma samples.

Pharmacokinetic and tissue distribution study of pectolinarigenin in rats using UPLC-MS/MS

Pectolinarigenin (PEC), a natural flavonoid isolated from Cirsium japonicum, exhibits promising therapeutic potential for multiple cancers. In present study, a simple and sensitive UPLC-MS/MS method was established for the quantification of PEC in rat plasma and tissues. The assay procedure involved a one-step protein precipitation with tadalafil as the internal standard, and separation on a Welch Xtimate UHPLC C18 column by gradient elution of acetonitrile/aqueous formic acid (0.1 %, v/v) at a flow rate of 0.2 m L·min−1. The detection was conducted using multiple-reaction monitoring via an electrospray ionization source in positive ionization mode. The established method was proved to be highly sensitive with a good linearity (R2 > 0.99) in respective concentration range (0.1–100 ng·mL−1 in plasma and 1–10000 ng·mL−1 in tissues) and acceptable extraction recovery (≥71.17 %), matrix effect and stability, which was applied to study the pharmacokinetics and tissue distribution of PEC after intravenous (100 μg·kg−1) and oral administration (10, 20 and 40 mg·kg−1). PEC was promptly absorbed (Tmax ≤ 0.222 h) and maintained at a low level with slow elimination (t1/2 z ≥ 14.47 h) in rats after oral administration, resulting in extremely low bioavailability (0.56–0.68 %). However, PEC is widely distributed in rat tissues with high exposure in GI tract, liver and kidney. The bioavailability and tissue affinity were firstly revealed, which will guide directions for further development of PEC as an anti-tumor drug candidate.

Pharmacokinetic Analysis of Gatifloxacin and Dexamethasone in Rabbit Ocular Biofluid Using a Sensitive and Selective LC-MS/MS Method.

Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC-MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56-400 ng mL-1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation.

UPLC-MS/MS analysis of axitinib and pharmacokinetic application in beagle dogs

ObjectiveTo develop a method for determining the concentration of axitinib in beagle dog plasma and utilize this method to investigate the pharmacokinetics of orally administered axitinib in beagle dogs. MethodsPlasma samples were processed using acetonitrile precipitation and analyzed by UPLC-MS/MS with sunitinib as an internal standard (IS). Chromatographic separation was achieved on a Waters Acquisition UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm) with a gradient elution of acetonitrile and 0.1 % formic acid. Mass spectrometry uses an electrospray ion source for positive ion detection in a multiple reaction monitoring mode. The monitored ion transitions for axitinib and sunitinib were m/z 387 → 355.96 and m/z 399.3 → 282.96, respectively. Six beagle dogs were administered 0.33 mg/kg of axitinib orally, and venous blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h post-dose for pharmacokinetic analysis. ResultsThe assay demonstrated a linear range of 0.5–100 ng/mL (r2 = 0.9992), and the lower limit of quantification was up to 0.5 ng/mL. Precision, as assessed by relative standard deviation (RSD), was within 8.64 % for both intraday and interday variability. The relative error (RE) for precision from −2.77 %–1.20 %. The recovery rate of the analytes exceeded 85.28 % and the matrix effect was approximately 100 %. Plasma samples maintained stability under various conditions, including room temperature storage for 12 h, processed on an automatic sampler at 4 °C for 6 h, three freeze-thaw cycles, and long-term storage at −80 °C for 60 days. Pharmacokinetic parameters were determined using DAS 2.0 software, revealing a half-life (T1/2) of 6.05 h and an area under the curve (AUC (0 →∞)) of 97.13 ng h/mL for axitinib. ConclusionsThe UPLC-MS/MS method developed in this study offers high specificity, rapid analysis, high recovery, excellent linearity, and minimal plasma volume requirements, making it well-suited for pharmacokinetic and drug interaction studies in beagles dogs.

The Pharmacokinetic Interaction of Tirabrutinib with Voriconazole, Itraconazole, and Fluconazole in SD Rats by UPLC-MS/MS.

Tirabrutinib is an orally effective, approved, and highly selective second-generation Bruton's tyrosine kinase (BTK) inhibitor for the treatment of recurrent or refractory primary central nervous system lymphoma (PCNSL). This study aimed to develop an ultra-high performance liquid chromatography- tandem mass spectrometry (UPLC-MS/MS) method for the determination of tirabrutinib concentration in rat plasma, where zanubrutinib was used as an internal standard (IS). This method was also applied to study whether tirabrutinib would interact with voriconazole, itraconazole, and fluconazole in rats, providing a reference value for clinical medication guidance. In the current study, the organic solvent protein precipitation method was used to treat plasma samples, which is simple and reproducible. Tirabrutinib (m/z 455.32 → 320.21) and zanubrutinib (m/z 472.13 → 455.04) were separated on a Waters Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm) and detected by multiple reaction monitoring (MRM) in positive ionization mode. The method showed good linearity in the range of 5-3000 ng/mL for tirabrutinib with the lower limit of quantification (LLOQ) of 5 ng/mL. The recovery and matrix effects were 85.7-91.0% and 102.0-113.3%, respectively. The accuracy, precision, stability, and carry-over effect were also acceptable. The method could also be used for determining the pharmacokinetic interaction of tirabrutinib in rats. The results showed AUC0→∞ of tirabrutinib to be increased by 139.3% and 83.9% in the presence of voriconazole and fluconazole, respectively, while itraconazole had little effect. It is necessary to monitor the concentration of tirabrutinib in patients when it is combined with voriconazole and fluconazole to achieve a better therapeutic effect and reduce the risk of adverse reaction. Further research should be conducted in the future.

Simultaneous determination of picroside I and picroside II in dietary supplements by high performance liquid chromatography with PDA detector

Picroside I and picroside II are two compounds found in the Picrorhiza Kurroa plant, which have anti-cirrhosis, antioxidant, anti-inflammatory, fever, and asthma effects, etc. They are included in the ingredients of many dietary supplement products that help protect the liver and bile, prevent oxidation, etc. Therefore, determining the content of these two active ingredients in dietary supplements is necessary to evaluate product quality. In this study, highperformance liquid chromatography (HPLC-PDA) was used to simultaneously determine picroside I and picroside II in dietary supplements. The analytes in the samples were extracted with methanol for 60 min using a horizontal shaking technique. The extract was then filtered and determined by the HPLC–PDA method with the following conditions: C18 Column (250 mm x 4.6 mm; 5 µm) and a corresponding pre-column (3.9 mm x 5 mm; 5 µm), the mobile phase consists of a solvent mixture of acetonitrile and water according to the gradient program, detection wavelength 265 nm. The method has good specificity, the calibration curve of picroside I and picroside II has a linear coefficient R2 > 0.9999 and the repeatability and recovery meet the AOAC requirements. The method limits of detection (MDL) for these two substances were 4.3 mg/kg and 3.2 mg/kg, respectively. The method was applied to simultaneously determine picroside I and picroside II in 30 different dietary supplement products. The results detected analytes in 3 samples positive with picroside I and picroside II concentrations ranging from 98.8 to 148 mg/kg.

Simultaneous quantitative analysis of some main components in cinnamon leaves (Cinnamomum cassia) by HPLC-PDA

Cinnamon leaves, a part of the cinnamon tree, are used as raw materials to extract cinnamon essential oil and as a spice in food processing. Trans-cinnamaldehyde is the main ingredients that have biological effects and high concentration in cinnamon, cinnamic acid is often used as a marker in the quantification methods of cinnamon, coumarin is the ingredient that needs to be controlled due to risk of liver toxicity. So that, cinnamaldehyde (CAL), cinnamic acid (CA) and coumarin (CM) have been choosen to determin quality of cinnamon leaves. The analytes in the test sample were extracted with methanol:water ratio 9:1 by ultrasound twice 30 minutes and resting for 30 minutes between extractions. The ultrasonic extract was analyzed using an HPLC-PDA, C18 column (250×4.6 mm, 5 µm) at 25°C, mobile phase system consisting of acetonitrile and 0.05% phosphoric acid is eluted according to the program's gradient, detection wavelength 280 nm. The method was determined according to AOAC 2016 guidelines with appropriate specificity and linearity ranging from 51.90-691.31 µg/mL for coumarin, 1.00-20.10 µg/mL for cinnamic acid and 52.45-609.8 µg/mL for trans-cinnamaldehyde, good repeatability with RSD from 0.46% to 0.61%, accuracy of coumarin, cinnamic acid and cinnamaldehyd were 101.0%, 100.7% and 100.7% respectively, meets AOAC requirements at 3 concentration levels. The study applied quantitative research methods to quantify coumarin, cinnamic acid and transcinnamaldehyde on 17 C. cassia leaves samples collected in provinces in Vietnam. In result, analyte concentrations varied widely between samples, with coumarin ranging from 0.53- 2.47%, cinnamic acid from 0.01-0.08%, and cinnamaldehyde from 0.95-2.56%.

Determination of some NSAIDs content in meat by liquid chromatography with tandem mass spectrometry (LC-MS/MS)

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used group of drugs worldwide for pain relief, anti-inflammation, blood clot prevention, and fever reduction, but they can cause some side effects bad for health if used incorrectly. NSAIDs are on the list of drugs that must be monitored according to Council Directive 96/23/EC. The fact that NSAIDs are also used in animals for anti-inflammation, fever reduction, and pain relief raises concerns that animal products such as meat, eggs, and milk may be contaminated with NSAIDs, affecting human health if consumed like eating right. Therefore, developing a method to analyze NSAIDs in meat products is necessary, contributing to assessing the quality of meat products currently circulating on the market. In this study, 4 common substances in the NSAIDs group were studied and identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) using a Symmetry C18 chromatographic column with an ESI (+) positive electron spray ionization source and multiple reaction monitoring (MRM) mode. The validity of the method was confirmed according to the guidelines of the Association of Official Analytical Chemists (AOAC). The results showed that the method has good specificity, the linearity range from 5.0 to 100 µg/kg, the detection limit of 1.5 µg/kg, the quantification limit of 5.0 µg/kg; precision and accuracy with relative standard deviation (RSD) less than 11% and recoveries ranging from 81.1 to 111%, meeting AOAC requirements. The method was used to analyze the content of NSAIDs in 15 meat samples in Hanoi. The results showed that 7 samples showed tolfenamic acid exceeding the quantitative limit of the method.

Study on determination of cynarin in dietary supplement containing artichoke by high performance liquid chromatography

There has been no announcement on the standardization and testing of cynarin in dietary supplements containing artichokes in Vietnam. The high-performance liquid chromatography (HPLC) method was developed to determine cynarin in dietary supplements containing artichokes. The sample was extracted twice with 80% methanol solvent and ultrasonicated at 50°C for 20 minutes. Centrifuge at 6000 rpm for 5 minutes, filter, and inject into HPLC system with XBridge C18 column (250 mm x 4,6 mm, 5 µm) and corresponding pre-column, mobile phase consisting of two solvents phosphoric acid 0,1% and acetonitrile eluted according to gradient program, flow rate 1mL/min, detection wavelength 322nm. The calibration curves for cynarin had R2 = 1, and the validation parameters met AOAC standards. The method detection limits and the limit of quantification were 3.00 mg/kg and 10.0 mg/kg, respectively. The validated method was applied to determine cynarin in 15 dietary supplement samples. The content of cynarin varied depending on the manufacturer

Simultaneous determination of acid carnosic and carnosol in rosemary-containing foods by high-performance liquid chromatography with photodiode array detection (HPLC–PDA)

The research was conducted to develop and validate a simultaneous quantification method for acid carnosic (CA) and carnosol (CAR) in rosemary-containing foods using highperformance liquid chromatography coupled with photodiode array detection (HPLC–PDA), with a simple and fast sample processing procedure. Analytical samples were extracted using a methanol–phosphoric acid mixture (99.5:0.5, v/v) at room temperature for 20 minutes. The chromatographic conditions were as follows: Sunfire C18 column (4.6 mm x 250 mm; 5 µm), mobile phase consisting of 0.1% phosphoric acid – acetonitrile (40:60, v/v), flow rate of 1 mL/min, injection volume of 10 µL, detection at 230 nm. The method was validated according to the Association of Official Analytical Chemists (AOAC) criteria. The results showed that the method had good specificity, and the standard curves of acid carnosic and carnosol were linear in the concentration range of 2.5 – 200 µg/mL. The limits of detection (LOD) for acid carnosic and carnosol were 0.3 µg/mL and 0.25 µg/mL, respectively. The limits of quantification (LOQ) for acid carnosic and carnosol were 1 µg/mL and 0.75 µg/mL, respectively. The recovery rates were in the range of 98.8 – 100.6% (acid carnosic) and 97.5 – 102.2% (carnosol). Repeatability and internal reproducibility met the requirements of AOAC. The method was applied to evaluate the acid carnosic and carnosol levels in 20 food samples collected from the market, such as rosemary leaf powder, dried rosemary leaves, rosemary herbal tea,...

A sustainable RP-HPLC method for concurrent estimation of capecitabine and celecoxib in liposomal formulation: Greenness and whiteness appraisal.

Liposomes have been reported for combination therapy due to their ability to carry both hydrophilic and lipophilic drugs together. The current investigation aims to develop a novel, eco-friendly, and sustainable reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of capecitabine and celecoxib co-encapsulated in liposomes. The method reported herein uses a C18 column (4.6 × 250 mm2, 5 μm) and a mobile phase consisting of water, and acetonitrile/methanol in a ratio of 60:40, containing 0.1% formic acid in both the phases. The flow rate is maintained at 1 mL/min, with an injection volume of 10 μL in the gradient mode. Detection is set at λmax = 240 nm for capecitabine and 252 nm for celecoxib. The developed liposomes are mono-disperse with a surface potential of -6.93 mV. The average size of the liposomes is 142 nm. The percentage entrapment efficiency for capecitabine is 52.39 ± 0.94%, and for celecoxib, it is 77.13 ± 0.74%. The Analytical Greenness Metric of 0.61 and Analytical Eco-Scale Score of 75 signify the greenness of the developed method. Also, the Red-Green-Blue model shows excellent whiteness, with a score of 83.2. Thus, the developed method offers a reliable, accurate, precise, buffer-free, and environment-friendly RP-HPLC approach for the simultaneous analysis of capecitabine and celecoxib co-encapsulated in liposomes.

A quality by design assisted RP-HPLC method utilizing central composite design for the assay of eliglustat and its organic impurities in drug

This study aimed to develop a stability-indicating RP-HPLC assay method for eliglustat and its organic impurities using a central composite design approach. The chromatographic separation was achieved with a 10 mM phosphate buffer at pH 4.0 on a Thermo Accucore C18 column (150 x 4.6 mm, 2.6 μm) at a wavelength of 210 nm. Gradient elution was performed using a mobile phase-A consisting of buffer and acetonitrile in a 90:10 v/v ratio and a mobile phase-B composed of water and acetonitrile in a 35:65 v/v ratio, at a flow rate of 1.1 mL/min at 45°C for a total run time of 55 minutes. Based on preliminary experiments, central composite design was utilized to evaluate the effects of independent variables such as flow rate, acetonitrile ratio and column oven temperature on the response factors. Eliglustat and its impurities (EGS-5A, EGS-Diastereomers, and EGS-N-Oxide) had retention time of 3.5 minutes, 21.5 minutes, 23.2 minutes and 25.9 minutes, respectively. The limits of detection and quantitation for eliglustat and its impurities were established in relation to the test concentration, achieving a desirability of 1. The developed method was validated as per regulatory guidelines and successfully applied in bulk drugs and formulation.

Rapid determination of 54 dye components in hair dyes by liquid chromatography coupled to quadrupole orbitrap high-resolution mass spectrometry.

Chemical hair dye components can have allergenic, reproductive, and carcinogenic risks. Detecting restricted and prohibited ingredients in these products is challenging due to product diversity, isomer separation, instability, and wide polarity range. A method was developed using HPLC-high-resolution mass spectrometry for the qualitative and quantitative analysis of 54 hair dye components in various products. Samples were extracted with a 70% methanol solution, ultrasonicated in an ice bath, centrifuged, filtered, diluted with 25% methanol solution and 25% methanol solution containing 0.05% D-isoascorbic acid. Separation was achieved using an ACE Excel 3 C18 column (2.1 mm × 150 mm, 3 μm) with analysis conducted via quadrupole Orbitrap mass spectrometry. It showed good linear correlations, with detection limits of 0.1-23.5 ng mL-1, and quantitation limits of 0.2-78.1 ng mL-1. Average recovery ranged from 60.0% to 118.4%, with repeatability from 4.0% to 14.9%. Stability was confirmed within 48 hours. When applied to 20 batches of commercially available hair dyes, 24 hair dye components were found within permissible levels. The method is crucial for quality control of hair dyes, covering 10 common prohibited and 44 permissible hair dye components outlined in the Safety and Technical Standards for Cosmetics (2015 Edition). Compared to the standard methods, it can separate isomers in a single mobile phase system within 15 minutes in positive ion mode while maintaining sensitivity for phenol, hydroquinone, and other components in negative ion mode. Moreover, the pre-treatment strategy significantly improved stability and accuracy, enabling precise analysis of the 54 hair dye components.

Quantification of Letrozole, Palbociclib, Ribociclib, Abemaciclib, and Metabolites in Volumetric Dried Blood Spots: Development and Validation of an LC-MS/MS Method for Therapeutic Drug Monitoring

Therapeutic drug monitoring (TDM) may be beneficial for cyclin-dependent kinase 4/6 inhibitors (CDK4/6is), such as palbociclib, ribociclib, and abemaciclib, due to established exposure–toxicity relationships and the potential for monitoring treatment adherence. Developing a method for quantifying CDK4/6is, abemaciclib metabolites (M2, M20), and letrozole in dried blood spots (DBS) could be useful to enhance the feasibility of TDM. Thus, an optimized LC-MS/MS method was developed using the HemaXis DB10 device for volumetric (10 µL) DBS collection. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column. Detection was performed with a triple quadrupole mass spectrometer, utilizing ESI source switching between negative and positive ionization modes and multiple reaction monitoring acquisition. Analytical validation followed FDA, EMA, and IATDMCT guidelines, demonstrating high selectivity, adequate sensitivity (LLOQ S/N ≥ 30), and linearity (r ≥ 0.997). Accuracy and precision met acceptance criteria (between-run: accuracy 95–106%, CV ≤ 10.6%). Haematocrit independence was confirmed (22–55%),with high recovery rates (81–93%) and minimal matrix effects (ME 0.9–1.1%). The stability of analytes under home-sampling conditions was also verified. Clinical validation supports DBS-based TDM as feasible, with conversion models developed for estimating plasma concentrations (the reference for TDM target values) of letrozole, abemaciclib, and its metabolites. Preliminary data for palbociclib and ribociclib are also presented.

Buffer-free high pH mobile phase LC-MS/MS for determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 and 20 drugs and metabolites in whole blood

BackgroundAcidic mobile phases are commonly used in reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalysis. However, increased sensitivity, improved peak symmetry, and increased retention, especially for basic hydrophilic drugs have been observed using basic mobile phases. In our previous acidic mobile phase LC-MS/MS method we needed two injections (0.4 and 2.0 μL) of each sample for this task, which is inefficient. The aim of this study was to investigate if basic mobile phase LC-MS/MS could be used to determine phosphatidylethanol 16:0/18:1 and 20 other drugs and metabolites with satisfactory sensitivity in one single run. MethodsWhole blood was prepared by 96-well supported-liquid extraction using heptane/ethyl acetate/2-propanol (16:64:20, v:v:v). Chromatographic separation was achieved on an Acquity BEH C18 column (50 × 2.1 mm I.D.), using a mobile phase with 0.025 % ammonia, pH 10.7 (Solvent A) and methanol (Solvent B). All compounds had isotope-labelled internal standards. ResultsThe method was fully validated. Recovery was between 63 and 91 % for 20 compounds and 10 % for benzoylecgonine. Matrix effects were low, except for ion enhancement of buprenorphine and ion suppression for THC. However, internal standards compensated for these effects. Inter-assay precision and accuracy were < ± 20 % for all compounds at five tested concentrations, except for methamphetamine at the highest concentration. ConclusionAn LC-MS/MS method for simultaneous determination of PEth 16:0/18:1 and 20 drugs and metabolites in whole blood were for the first time developed and validated. Retention of PEth 16:0/18:1 was, in contrast to the other 20 compounds, largely affected by mobile phase buffer concentration. The buffer free basic mobile phase ensured that phosphatidylethanol 16:0/18:1 eluted before most of the unwanted phospholipids.

Method Development and Validation of Metformin and Gliclazide in Tablet dosage form by Micellar Liquid Chromatography

Micellar Liquid Chromatography (MLC) has emerged as a promising technique for the analysis of Pharmaceutical compounds due to its versatility, efficiency, and cost-effectiveness. In this experimental research paper, we explore the application of MLC in the simultaneous analysis of two commonly prescribed antidiabetic drugs, Metformin and gliclazide. A combination of these medicines is widely used to treat type 2 diabetes mellitus, so accurate quantification of them is essential for efficient therapeutic management. To separate the drug formulation, reversed phase liquid chromatography (RP-HPLC) was employed using surfactant solutions above Critical Miceller concentration (CMC). The mobile phase included 0.10M SDS and 10% Propanol-1, with a pH of 3.50 maintained by o-Phosphoric Acid and analysis was done using Deoxyprobe C18 column (4.6 x 150 mm, 5 µm). The separation was carried out at room temperature with a flow rate of 1.0 ml/min, and detection was performed at 228 nm. Gliclazide has a retention time of 4.46 minutes, compared to 5.57 minutes for metformin. The percentage composition of the sample under analysis ranged from 90 to 110 percent in accordance with schedule V of the Drug and Cosmetic Act. The suggested practice met ICH requirements. The method was also low-cost, easy to use, secure, and ecologically benign. It can be used to conduct standard quantitative analyses on pills that include metformin and gliclazide.

A Liquid Chromatography Coupled to Tandem Mass Spectrometry Method for Simultaneous Estimation of Saxagliptin and Avanafil: Application to Pharmacokinetic Drug‐Drug Interaction in Type 2 Diabetic Rat Model

ABSTRACTErectile dysfunction is a common complication of type 2 diabetes. The combination of saxagliptin and avanafil can effectively manage both conditions in individuals with type 2 diabetes. However, evaluating the potential pharmacokinetic interactions for co‐administration of saxagliptin and avanafil is crucial. Here, we developed a sensitive bioanalytical method for simultaneously quantifying saxagliptin and avanafil in rat plasma using liquid chromatography hyphenated to triple quadrupole mass spectrometry. The chromatographic separation of saxagliptin, avanafil, and irbesartan (internal standard) was achieved on an Aquity BEH C18 column (100 × 2.1 mm, 1.7 µm), using 0.1% formic acid in water and acetonitrile as mobile phase with the gradient elution at a flow rate of 0.2 mL/min. Mass detection was carried out using selective reaction monitoring mode, with the product ions for saxagliptin, avanafil, and irbesartan being 180.2, 375.1, and 207.1 m/z, respectively. The developed liquid chromatography coupled to tandem mass spectrometry method was validated as per the US Food and Drug Administration guidelines in rat plasma. The validation findings confirmed the method's robustness and suitability for accurate quantification of saxagliptin and avanafil in rat plasma with the limit of quantification of 1 ng/mL for both the drugs and were linear in the range of 1–2000 ng/mL. The study found no significant pharmacokinetic interaction between saxagliptin and avanafil, suggesting they can be safely co‐administered to patients with type 2 diabetes.

Novel Green Chromatographic Approaches for Estimation of a Triple Common Cold Pharmaceutical Combination.

Nowadays, there is a strong interest in the scientific community in developing innovative methodologies within a green analytical chemistry framework. Herein, we introduce the first chromatographic approaches for the concurrent estimation of paracetamol (PAR), carbinoxamine (CRX), and pseudoephedrine (PSE) intended to relieve COVID-19 and common cold symptoms. The first method was thin layer chromatography (TLC) densitometry, which depends on the separation of the studied medications on TLC silica gel plates using ethyl acetate: methanol: ammonia (7.0: 3.0: 0.2, by volume) as the developing system, and were scanned at 208.0nm. The data were linear in the ranges of 1-25μg/band for PAR, 1-25μg/band for PSE and 0.1-5μg/band for CRX. The second method was reversed-phase high-performance liquid chromatography separation on a Kromasil C18 column using a mixture of 0.01M phosphate buffer containing 0.1% triethylamine (pH3.5) adjusted with orthophosphoric acid and ethanol at a flow rate of 1.0 mL/min in a gradient program. The separated peaks were detected at 215.0 nm over a concentration range of 10-250μg/mL for PAR, 5-35μg/mL for PSE, and 0.5-25μg/mL for CRX. Both approaches were validated according to International Conference on Harmonization (ICH) guidelines. Finally, the impact of these methods on the environment was evaluated by many tools.

Stability Indicating LC–ESI-MS/MS Method Development and Validation for the Quantitation of Lurbinectedin in Biological Matrices.

The anticancer drug lurbinectedin was measured in plasma samples using a newly designed and validated LC-ESI-MS/MS method. The drug’s examination was conducted using ledipasvir as the internal standard. Following the liquid liquid extraction of 200 μL of plasma, the samples were separated using a ZorbaxSB (25 0mm x4.6 mm, 5 µm)C18 column. The mobile phase comprised of 0.1 %V/V formic acid and acetonitrile in the fraction of 10:90, and an infusion flow rate was 0.80mL/min. The analytes were eluted in 3.5 minutes total. Using a concentration range of 1.50 to 5000ng/mL of lurbinectedin and a correlation coefficient value of 0.9994, the method was validated in compliance with FDA standard standards. Assay accuracy ranged from 95.31% to 104.06% of the nominal values, and both intra- and inter-day precision results were within 4.32%. For the analyte at the LQC level, the matrix factor varies from 94.25% to 104.85% with a %CV of 4.61, whereas at the HQC level, it ranges from 94.62% to 103.88% with a %CV of 4.02. The results of the stability tests show that the approach is quite stable. Regular analysis in bioavailability, quality control, and bioequivalence investigations of biological matrices may be effectively performed using the established approach.