Metformin In Human Plasma Research Articles

A rapid and efficient approach for quantifying metformin in human plasma using Ultra-High performance liquid chromatography with UV-Detection: Relevance in Non-Diabetes clinical studies

Metformin, a first-line drug to treat type-2 diabetes, has gained attention in various non-diabetes studies, such as anti-aging, reduced cancer incidence and mortality, and reduced symptomatic viral infection due to its immunomodulatory properties. This study aims to develop and validate a simple and cost-effective ultra-high-performance liquid chromatography method with UV detection (uHPLC-UV) for quantifying metformin in human plasma amidst operational challenges and restricted accessibility to mass spectrometry. Plasma samples were protein precipitated with acetonitrile, and metformin was separated from endogenous plasma components using a Waters Acquity UPLC BEH Amide column, 1.8 µm, (2.1 x 100 mm) with gradient elution using 0.1 M potassium phosphate adjusted with phosphoric acid to pH 5.8, and acetonitrile as mobile phases and detected at 240 nm. Method validation was performed according to U.S. FDA guidelines. Total run time achieved was 3 min and linearity was demonstrated over the range of 0.1–40 mg/L based on linear regression using 1/x weighting. The lowest quantitation limit of 0.1 mg/L was achieved. Excellent recoveries (90.3–98.4 %) and negligible matrix factors (94.6–108.1 %) were observed in the sample preparation, negating the need for using any internal standard. Finally, the developed assay was utilised to measure metformin plasma levels from healthy volunteers recruited in a randomised, double-blind, placebo-controlled study investigating potential immunomodulatory effects of metformin influence on yellow fever viremia.

Smart nano-actuators for electrochemical sensing of Metformin in human plasma

Smart artificial nano-actuators were developed using electroactive acrylamide polymer nanoparticles by molecular imprinting. These nanoparticles are receptors that mimic the specificity of antibodies and replace enzymes in traditional biosensors allowing highly sensitive and selective monitoring. Electroactive polymer nanoparticles can be synthesized by the including of ferrocene, which confers signaling functions. These actuators operate by a swelling mechanism in response to a specific electrochemical stimulus. Molecular recognition is based on molecular imprinting and binding of the target molecule. The polymer nanoparticles were computationally designed to bind to Metformin and synthesized by controlled free-radical polymerization. Polymer nanoparticles were incorporated on the surface using screen-printed electrodes. As a result, these nano-actuators allow extremely sensitive monitoring of Metformin in human plasma. Metformin is essential for treating various chronic diseases, and monitoring is necessary to reduce collateral drug side effects. Herein, sensing of Metformin was achieved by differential pulse voltammetry (DPV) in a concentration range between 100 and 2000 pM, with a limit of detection of 9 pM and sensitivity at 31.5 nA pM−1 (0.998) in plasma. No cross-reactivity was observed against potential interferences (Sitagliptin and Paracetamol). This technology can be potentially applied for rapid point-of-care testing as an alternative to laboratory-based techniques, reducing the time and cost of the analysis.

Stability indicating RP-UPLC method development and validation for the simultaneous determination of ertugliflozin and metformin in plasma

New stability-indicating RP-UPLC technique was developed for the quantification of ertugliflozin and metformin in human plasma and validated as per the regulatory guidelines. Both the drug components and internal standard were spiked to blank plasma and subjected to liquid-liquid extraction with the mobile phase. The resultant solution was infused into Acquity BEH-C18 (1.7 μ, 100×2.1mm) non-polar column comprising NaH2PO4 buffer (pH-3.5), methanol and acetonitrile in the ratio of 50:10:40% v/v/v as mobile phase. The detector response and flow of the mobile phase were monitored at 240nm and 0.5ml/min, respectively. The linearity plot was made in the concentration range of 0.1-3.0 µg/ml for metformin and 0.05-1.5 µg/ml for ertugliflozin with correlation coefficient value of more than 0.999. The developed method was subjected for bench-top, freeze and thaw, long-term and short-term stability studies and the drug components were stable over the respective conditions. The Lower limit of quantification (LLOQ) for ertugliflozin and metformin were 0.05 and 0.1 µg/ml, respectively. The findings of precision and accuracy were present in between 2.6 to 4.2 %RSD and -2 to 3.99 %RE, respectively. The findings of the stability data were presented below. The %stability of ertugliflozin and metformin were varying from 96% to 104% for ertugliflozin and 96% to 105% for metformin.

Bioanalytical Method Using Ultra-High-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry (UHPL-CHRMS) for the Detection of Metformin in Human Plasma.

Metformin is the first-line medicine for the treatment of type 2 diabetes. Drug interactions between metformin and other drugs, food, or beverages cannot only cause changes in the pharmacokinetic profiles but also affect the efficacy of metformin. The purpose of this study was to develop a rapid and reliable bioanalytical method for the detection of plasma metformin concentration in humans. To remove interfering substances in plasma, acidified acetonitrile (acetonitrile containing 0.1% formic acid) was added to samples. Ultra-high-performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry (HRMS) was used to analyze metformin and its internal standard (metformin-d6). Analyte separation was performed on a BEH HILIC analytical column (100 × 2.1 mm, 1.7 μm) using a gradient elution of 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The total chromatographic run time was 2 min. The developed method was validated for its linearity, accuracy and precision, selectivity (signal of interfering substance; analyte, lower limit of quantification (LLOQ) ≤ 20%; IS, IS ≤ 5%), sensitivity (LLOQ, 5 ng/mL; S/N ratio ≥ 10), stability (low quality control (LQC, 15 ng/mL), 2.95–14.19%; high quality control (HQC, 1600 ng/mL), −9.49–15.10%), dilution integrity (diluted QC (4000 ng/mL); 10-folds diluted QC (400 ng/mL); 5-folds diluted QC (800 ng/mL); accuracy, 81.30–91.98%; precision, ≤4.47%), carry-over (signal of double blank; analyte, LLOQ ≤20%; IS, IS ≤5%), and matrix effect (LQC, 10.109%; HQC, 12.271%) under various conditions. The constructed calibration curves were shown linear in the concentration range of 5–2000 ng/mL, with within- and between-run precision values of <8.19% and accuracy in the range of 91.13–105.25%. The plasma metformin concentration of 16 healthy subjects was successfully measured by applying the validated bioanalytical method.

Development of LC-MS/MS method for simultaneous determination of Canagliflozin and Metformin in human plasma and its pharmacokinetic application in Indian population under fast and fed conditions

A novel, selective and sensitive method is developed for simultaneous estimation of canagliflozin and metformin and successfully applied to fast and fed pharmacokinetic studies in healthy Indian volunteers. The current study reports the development, optimization, and validation of liquid chromatography-mass spectrometry (LC-MS/MS) method for simultaneous quantification of canagliflozin and metformin in human plasma using deuterated canagliflozin D4 and metformin D6 as an internal standard (IS). The solid-phase extraction technique was employed where strata X polymeric reverse phase (30 mg-1 cc) SPE cartridges were used for the extraction of analytes and IS from plasma. The ACE 5 C18 column (50 × 4.6 mm, 5µ) was used to chromatograph the prepared samples. The mobile phase consisted of methanol and 5 mM ammonium trifluoroacetate in water, pH 5 (50:50, v/v) at a flow rate of 0.8 mL/min.Detection was performed by positive ion Turbo ion spray in Multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 461.9 → m/z 191.1 and m/z 461.9 → m/z 267.2, for quantification of canagliflozin. The response of canagliflozin fragments m/z 461.9 → m/z 191.1 and m/z 461.9 → m/z 267.2 was combined. Also, for metformin transitions were monitored at m/z 130.0 → m/z 71.1. Full validation of the method was performed according to the United States Food and Drugs Administration (USFDA) guidelines. Linearity was in the range of 24.95–2806.55 ng/mL for canagliflozin and 24.99–3400.72 ng/mL for metformin. The mean extraction recovery of canagliflozin, canagliflozin D4, metformin, and metformin D6 was 77.240, 84.663, 66.747, and 67.449, respectively across four QC levels. This rapid method with the run time of 2.80 min allows the analysis of more than 400 plasma samples per day.

Development of simultaneous determination of empagliflozin and metformin in human plasma using liquid chromatography-mass spectrometry and application to pharmacokinetics.

A rapid and sensitive liquid chromatography-mass spectrometry method was developed, optimized, and validated for simultaneous quantification of empagliflozin and metformin in human plasma using empagliflozin D4and metformin D6 as an internal standard. Analytes and internal standard were extracted from plasma by optimized solid-phase extraction technique using Strata X polymeric reverse phase (30 mg-1cc) solid-phase extraction cartridges. The prepared samples were chromatographed on Orosil C18 column (150 × 4.6 mm, 3 µ). Separation was done by pumping isocratic mobile phase consisting of methanol and 10 mM ammonium trifluoroacetate (90:10, v/v) in positive ion mode at a flow rate of 0.8 mL/min. The API 3200 liquid chromatography-mass spectrometry system having turbo ion spray as an ion source coupled with Shimadzu Prominence ultrafast liquid chromatography system was operated under the selected reaction monitoring mode. Turbo ion spray ionization was used for mass transition of m/z 468.070/355.100 and m/z 130.072/71.200 for empagliflozin and metformin, respectively. A method was successfully validated for concentration range of 10.09-5013.46 ng/mL for both the analytes and according to the United States Food and Drugs Administration guidelines. The linearity was found to be in the range of 10.09-403.46 ng/mL for empagliflozin and 25.44-5013.46 ng/mL for metformin. The limit of quantification was found to be 10.09 ng/mL for empagliflozin and 25.44 ng/mL for metformin. Intra- and inter-day/between batch precision determination for empagliflozin and metformin, expressed as coefficient of variation were within the acceptance limits and ranged below 13.16%. A short run time of 3.3 min allows analysis of more than 400 plasma samples per day. The developed method was successfully applied to fasting pharmacokinetic study in healthy human volunteers. Results of incurred sample re-analysis were within the acceptance range of ±20% of original value, for 97.2% of samples reanalyzed for empagliflozin and 100% of samples reanalyzed for metformin.

Simultaneous Determination of Canagliflozin and Metformin in Human Plasma by LC–MS/MS Assay and its Application to a Human Pharmacokinetic Study

Simultaneous Determination of Canagliflozin and Metformin in Human Plasma by LC–MS/MS Assay and its Application to a Human Pharmacokinetic Study

A validated LC-MS/MS bioanalytical method for the simultaneous determination of dapagliflozin or saxagliptin with metformin in human plasma

An LC-MS/MS method was developed and validated for the simultaneous quantitation of dapagliflozin or saxagliptin with metformin in human plasma. Separation and detection of dapagliflozin (DAP), saxagliptin (SAX), metformin (MET) and their internal standards was achieved on Zorbax C18 column (50 × 4.6 mm, 5 μm) using acetonitrile: 0.1% formic acid (45:55, v/v) as a mobile phase applying electrospray ionization (ESI) source in positive ion mode. Sequential liquid-liquid extraction and precipitation techniques were used for the extraction due to the difference in the physicochemical properties of drugs. The MS/MS response was linear over the concentration ranges from 5 to 500 ng/ml, 2–50 ng/ml and 10–4000 ng/ml for DAP, SAX and MET, respectively. Precision, accuracy, selectivity, recovery, and stability were within the acceptable limits as FDA guidelines. The method is suitable for further application of pharmacokinetic studies.

SPECTROPHOTOMETRIC ESTIMATION OF METFORMIN IN HUMAN PLASMA

To develop a simple, selective and sensitive spectrophotometric method and validated for the determination of metformin in human plasma. The method was applied for the determination of metformin from pharmaceutical preparations and plasma samples and after spiking with metformin. The results were checked by standard addition method. A number of pharmaceutical additives and plasma matrix did not affect the determination of metformin. The proposed method was found to be accurate and precise for routine estimation of metformin hydrochloride in human plasma.&#x0D; Keywords: Metformin, spectrophotometric, standard addition, plasma, pharmaceutical preparation

A Validated Ultra Performance Liquid Chromatography Method for Quantification of Metformin in Human Plasma

Metformin is oral hypoglycemic or blood sugar-lowering drug which is used for the first- line drug in the treatment of diabetes mellitus type 2. This study presented the validated Ultra Performance Liquid Chromatography UltraViolet (UPLC-UV) method for the determination of metformin in human plasma. Metformin levels were measured using UPLC with a UV detector and liquid-liquid extraction method. Separation was carried out on an Acquity UPLC HSS T3 100mm × 2.1mm i.d. column (1.8μm particle size) using gradient elution of acetonitrile and phosphate buffer 0.02 M (0.6 mL/min) as mobile phase at 30°C. The analyte was monitored at 236 nm. No endogenous substances were found to interfere with the peaks of drug and internal standard. The value of percent deviation and the coefficient variation obtained respectively less than the percentage set in the FDA guidelines. The linearity factor values were more than 0.997 and LOD was 0.01µg/mL. UPLC with UV detector is able to analyze metformin in a short time with good precision and accuracy which is useful for bioequivalence and bioavailability studies.

SIMULTANEOUS DETERMINATION AND QUANTITATION OF METFORMIN AND TENELIGLIPTIN IN HUMAN PLASMA BY LC-ESI-MS/MS WITH AN APPLICATION TO PHARMACOKINETIC STUDIES

‘Gliptins’ is the nickname given to a group of anti-diabetic medicines-DPP-4 inhibitors, such as sitagliptin, vildagliptin, sexagliptin, linagliptin, alogliptin and teneligliptin, which are used with metformin for combination therapy of type 2 diabetes mellitus. Advantages of this combined therapy are low risks of hypoglycemia and weight gain compared with other classes of anti-diabetic drugs. In the present study efforts, were made to develop and validate a bioanalytical method for simultaneous estimation of metformin and teneligliptin in human plasma by LC-MS/MS with an application to quantification of plasma samples obtained from the comparative pharmacokinetic studies on healthy human volunteers. The developed method for simultaneous determination and quantification of teneligliptin and metformin in human plasma was also validated as per the US-FDA guidelines. The validation parameters were found to be within the specified regulatory limits, hence the method acceptable. The present method had a short run time (3.5min) and easy extraction process. Therefore, developed method was found to be simple, specific, highly selective, sensitive and reproducible. This was applied for the analysis of volunteer plasma samples obtained from comparative pharmacokinetic study. On the basis of comparison of AUC0-t, the relative bioavailability of test preparation was found to be 97.64% and 91.37% for teneligliptin and metformin, with respect to that of the reference preparation.

Validation of an HPLC-UV Method for the determination of metformin hydrochloride in spiked-Human plasma for the application of therapeutic drug monitoring

Widely used as the mainstay therapy of type-2 diabetes patients, the plasma level of metformin should remain within therapeutic range (0.75–5.00 μg/mL) to gain optimal effectiveness without lactic acidosis. An HPLC-UV method has been developed and validated for the determination of metformin in spiked-human plasma according to the FDA Guidance of Bioanalysis. Optimum determination was achieved by using a mixture of 55% buffer pH 5.2 (6 mM potassium dihydrogen phosphate-6 mM sodium dodecyl sulphate) and 45% acetonitrile with Sunfire® C-18 column, 150x4.6mmx5μm from Waters, and SM7 injector with UV detector at 233 nm of wavelength. The analyte was extracted from plasma using 10% trichloroacetic acid. The assay was linear over the therapeutic steady-state concentration range of 0.50–7.00 μg/mL for metformin HCl with correlation coefficient 0.9999. The limit of detection was 0.14 μg/mL. The obtained results from accuracy, precision, and selectivity tests complied well with the generally accepted criteria for bioanalytical assay. The validated method could be used for determining metformin in human plasma and monitoring the metformin therapy in a clinical setting for dose adjustment, particularly for the elderly and patients with renal impairment as well as for measuring metformin accumulation.

Simultaneous quantification of ramipril, glimepiride and metformin in human plasma by ultra-performance liquid chromatography – tandem mass spectrometry

Rapid high Performance liquid Chromatography- Tandem mass Spectrometry Method For Quantitation of Milnacepran in Human PlasmaUmesh Thorat

Ion-pair HPLC Method for the Quantification of Metformin in Human Plasma and Its Application to a Pharmacokinetic Study

Ion-pair HPLC Method for the Quantification of Metformin in Human Plasma and Its Application to a Pharmacokinetic Study

Development, validation of liquid chromatography-tandem mass spectrometry method for simultaneous determination of rosuvastatin and metformin in human plasma and its application to a pharmacokinetic study.

A new, simple and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of rosuvastatin (ROS) and metformin (MET) in human plasma was developed. The assay procedure involved simple protein precipitation with acetonitrile. Following precipitation, fraction of supernatant was decanted and evaporated under gentle stream of nitrogen at 40°C. The residue was reconstituted in mobile phase and injected. The chromatographic separation was achieved with Thermo Hypurity C18 column (50 mm × 4.6 mm, 5 μ) using a mobile phase composition containing 0.1% v/v formic acid in water and acetonitrile (30:70, v/v) at a flow rate of 0.4 mL/min. The total run time was 3.5 min. The method showed good linearity in the range 0.5–200 ng/mL for ROS and 2–2000 ng/mL for MET with correlation coefficient (r) >0.9994 for both the analytes. The intra and inter-day precision values for ROS and MET met the acceptance criteria as per regulatory guidelines. The battery of stability studies viz., bench-top, freeze-thaw and long term stability were performed. The developed method was applied to a pharmacokinetic study.

Quantitation of Metformin in Human Plasma and Urine by Hydrophilic Interaction Liquid Chromatography and Application to a Pharmacokinetic Study

We describe an analytical method for the quantification of the widely used antihyperglycemic agent, metformin, in human plasma and urine. The separation was performed using isocratic hydrophilic interaction liquid chromatography on a Luna hydrophilic interaction liquid chromatography column (125 × 4.6 mm, 3 μm). The sample preparation was accomplished by solid-phase extraction. Validation of the method was performed in the range 10-2000 ng/mL for plasma and 5-30 mcg/mL for urine. The methods were linear within the investigated range (r(2) > 0.988). Within-day repeatability ranged from 3.1% to 7.5% in plasma and 1.6% to 6.2% in urine. Between-day reproducibility ranged from 2.9% to 5.3% in plasma and 0.6% to 1.8% in urine. The inaccuracy expressed as bias ranged from -3.1% to 1.9% in plasma and from -7.2% to 0.7% in urine. The lower limit of quantification for metformin in plasma was 5 ng/mL and in urine was 40 ng/mL. The method was therefore considered to be precise, accurate, reproducible, and sensitive enough to be appropriate for pharmacokinetic studies of metformin. The applicability of the method for human pharmacokinetic studies was demonstrated by dosing a healthy male volunteer with 500-mg metformin hydrochloride as a single oral dose; plasma and urine concentrations were measured for 24 hours.

Simple HPLC-UV method for the quantification of metformin in human plasma with one step protein precipitation

This study presents the optimization of a simple HPLC-UV method for the determination of metformin in human plasma. Ion pair separation followed by UV detection was performed on deproteinized human plasma samples. The separation was carried out on a Discovery Reversed Phase C-18 column (250×4.6mm, 5μm) with UV detection at 233nm. The mobile phase contained 34% acetonitrile and 66% aqueous phase. Aqueous phase contained 10mM KH2PO4 and 10mM sodium lauryl sulfate. Aqueous phase pH was adjusted to 5.2. The mobile phase was run isocratically. The flow rate of the mobile phase was maintained at 1.3ml/min. The linearity of the calibration curve was obtained in the concentration range of 0.125–2.5μg/ml and coefficient of determination (R2) was found to be 0.9951. The lowest limit of quantification and detection was 125 and 62ng/ml respectively. No endogenous substances were found to interfere with the peaks of drug and internal standard. The intra-day and inter-day coefficient of variations was 6.97% or less for all the selected concentrations. The relative errors at all the studied concentrations were 5.60% or less. This method is time efficient and samples are easy to prepare with minimum dilution. So, it can be applied for monitoring metformin in human plasma.

Development and validation of highly selective and robust method for simultaneous estimation of pioglitazone, hydroxypioglitazone and metformin in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A simple, selective and robust reverse phase high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous quantitation of pioglitazone (PIO), pioglitazone metabolite M-IV - hydroxypioglitazone (OH-PIO) and metformin (MET) in human plasma using deuterated internal standards (IS) is developed and fully validated as per industrial practices. After acetonitrile-induced protein precipitation of the plasma samples; PIO, OH-PIO, MET and IS were chromatographed on reverse phase column and analyzed in the multiple reaction monitoring in positive ion mode. The ion transitions were monitored at m/z 357.2→134.2 for PIO, 373.0→150.1 for OH-PIO, 130.2→71.0 for MET, 361.1→134.2 for PIO-IS and 136.1→77.1 for MET-IS. The total chromatographic run time was 4.0min. A linear response function (r>0.998) was established for the range of concentrations 15-2500ng/mL, 10-1500ng/mL and 25-3000ng/mL for PIO, OH-PIO and MET respectively in human plasma. The intra and inter-day precision and accuracy values have met the set acceptance criteria. The method is simple, selective, robust economic and has been applied successfully to more than 2000 plasma samples as part of pharmacokinetic study in humans.

The development and validation of a gas chromatography-mass spectrometry method for the determination of metformin in human plasma

A new, simple, specific gas chromatography-mass spectrometry method (GC-MS) was developed for the determination of metformin in human plasma. A number of derivatization approaches (silylation, acylation and methylation) were tested to derivatize metformin prior to GC-MS analysis. Derivatization of metformin was achieved by using N-methyl bis(trifluoroacetamide) (MBTFA). Several parameters such as reaction temperature and time were optimized, which affected the yield of the derivatization reaction. A trifluoroacetyl derivative of metformin was identified and quantified in selected ion monitoring mode (mass-to-charge ratio (m/z): 303). The method was fully validated using parameters such as specificity, carryover, linearity, limits of detection and quantification, precision, accuracy, stability, recovery, robustness and ruggedness. Linearity was demonstrated over the concentration range of 100–3000 ng mL−1 with a coefficient of determination (R2) above 0.996. The limits of quantification and detection were found to be 100 and 40 ng mL−1, respectively. Intra- and inter-day accuracy and precision were within the acceptable limits (<15% for concentration points in the calibration range; <20% for the limit of quantitation). The developed method was successfully applied for the identification and quantification of metformin in the plasma of diabetic patients.

Bioanalytical method development and validation using incurred samples: Quantitative estimation of metformin in human K3EDTA plasma by LC–MS/MS

Bioanalytical methods should be reproducible and consistent when applying to clinical sample analysis. Incurred sample reanalyses (confirmatory reanalyses) are performed to support clinical data, and regulatory agencies evaluate the same before approval of bioequivalent products/drugs. A confirmatory reanalysis was demonstrated for metformin after administration of 2/500 mg repaglinide + metformin fixed dose formulation under fasted and fed conditions. The liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of metformin in human plasma using metformin-d6as an internal standard has been developed and validated. The ions transitions recorded in multiple reaction monitoring (MRM) were m/z 130.1→60.0 and 136.2→60.0 for metformin and metformin-d6, respectively. The compounds were isolated by solid phase extraction and separated on a C12 reverse phase (Synergi MAX-RP 80A) column, using isocratic mobile phase flow at a flow rate of 0.8 mL/min. No matrix effect was observed within t...