Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Ezetimibe in Human Plasma and Pharmaceutical Formulations

An analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed and validated for the determination of ezetimibe in human plasma. Ezetimibe and etoricoxib (internal standard) were extracted from the plasma by liquid-liquid extraction and separated on a C₁₈ analytical column (50 × 3.0 mm I.D.) with acetonitrile:water (85:15, v/v) as mobile phase. Detection was carried out by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The chromatographic separation was obtained within 2.0 min and was linear in the concentration range of 0.25–20ng mL⁻¹ for free ezetimibe and of 1–300ng mL⁻¹ for total ezetimibe. The mean extraction recoveries for free and total ezetimibe from plasma were 96.14 and 64.11%, respectively. Method validation investigated parameters such as linearity, precision, accuracy, specificity and stability, giving results within the acceptable range. The proposed method was successfully applied to the quantitation of ezetimibe and its glucuronide in human plasma to support clinical and pharmacokinetic studies. Moreover, the method was used for the quality control analysis of pharmaceutical dosage forms. Revised: 4 January and 3 February 2006     

Development and Validation of High-Throughput Bioanalytical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Newly Synthesized Antitumor Carbonic Anhydrase Inhibitors in Human Plasma

In the present study, a sensitive and fully validated bioanalytical high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitative determination of three newly synthesized carbonic anhydrases inhibitors (CAIs) with potential antitumor activity in human plasma. The analytes and the internal standard (IS) were extracted using 1.5 mL acetonitrile from only 450 µL aliquots of human plasma to achieve the desired protein precipitation. Chromatographic separations were achieved on Phenomenex Kinetex^® C₁₈ column (100 × 4.6 mm, 2.6 µm) using a binary gradient elution mode with a run time of less than 6 min. The mobile phase consisted of solvent (A): 0.1% formic acid in 50% methanol and solvent B: 0.1% formic acid in acetonitrile (30:70, v/v), pumped at a flow rate of 0.8 mL/min. Detection was employed using triple quadrupole tandem mass spectrometer (API 3500) equipped with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was selected for quantitation through monitoring the precursor-to-parent ion transition at m/z 291.9 → 173.0, m/z 396.9 → 225.1, m/z 388.9 → 217.0, and m/z 146.9 → 91.0 for AW-9a, WES-1, WES-2, and Coumarin (IS), respectively. Linearity was computed using the weighted least-squares linear regression method (1/x²) over a concentration range of 1–1000, 2.5–800, and 5–500 ng/mL for AW-9a, WES-1, and WES-2; respectively. The bioanalytical LC-MS/MS method was fully validated as per U.S. Food and Drug Administration (FDA) guidelines with all respect to linearity, accuracy, precision, carry-over, selectivity, dilution integrity, and stability. The proposed LC-MS/MS method was applied successfully for the determination of all investigated drugs in spiked human plasma with no significant matrix effect, which is a crucial cornerstone in further therapeutic drug monitoring of newly developed therapeutic agents.     

Voltammetric Determination of Rosiglitazone in Pharmaceutical Preparations and Human Plasma

Abstract

The voltammetric behavior of rosiglitazone was studied using direct current (DC_t), differential pulse (DPP), and alternating current (AC_t) polarography. The drug manifests cathodic waves over a pH range of 2–11.2. In Britton‐Robinson buffer (BRb; pH 4), the diffusion current–concentration relationship was found to be rectilinear over a range of 4–24 µg · mL^?1 and 0.1–16 µg · mL^?1 using DC_t and DPP modes, respectively, with minimum limits of detection (LOD) of 0.15 µg · mL^?1 and 0.07 µg · mL^?1 using the DC_t and DDP modes, respectively. The diffusion‐current constant (I _d) was 6.63±0.03 (n=5). The proposed method was successfully applied to the determination of the studied compound both in pure form and in formulations. The mean percentage recoveries in tablets were 100.09±1.18 and 100.85±0.88 (n=5) using DC_t and DPP modes, respectively. Furthermore, the proposed method, adopting the DPP mode, was applied to the determination of rosiglitazone in spiked human plasma and the obtained mean percentage recoveries were 99.14±3.29 (n=4).     

A Simple LC-MS/MS Method for the Quantification of PDA-66 in Human Plasma

The treatment of cancer is one of the most important pharmacotherapeutic challenges. To this end, chemotherapy has for some time been complemented by targeted therapies against specific structures. PDA-66, a structural analogue of the inhibitor of serine–threonine kinase glycogen synthase kinase 3β SB216763, has shown preclinical antitumour effects in various cell lines, with the key pathways of its anticancer activity being cell cycle modulation, DNA replication and p53 signalling. For the monitoring of anticancer drug treatment in the context of therapeutic drug monitoring, the determination of plasma concentrations is essential, for which an LC-MS/MS method is particularly suitable. In the present study, a sensitive LC-MS/MS method for the quantification of the potential anticancer drug PDA-66 in human plasma with a lower limit of quantification of 2.5 nM is presented. The method was successfully validated and tested for the determination of PDA-66 in mouse plasma and sera.     

Development and Validation of a Liquid Chromatographic Method for Determination of Efavirenz in Human Plasma

A simple, rapid, and sensitive high-performance liquid chromatographic method for estimation of efavirenz in human plasma has been developed and validated. Chromatography was performed with C₁₈ analytical column and 50:50 acetonitrile–phosphate buffer (pH 3.5) as mobile phase. Compounds were monitored by UV detection at 247 nm. The retention time for efavirenz was 6.45 min and that for the internal standard, nelfinavir, was 2.042 min. Response was a linear over the concentration range of 0.1 μg–10 μg mL⁻¹ in human plasma. The method was simple, specific, precise and accurate and was useful for bioequivalence and pharmacokinetic studies of efavirenz.     

Analytical Performance and Greenness Evaluation of Five Multi-Level Design Models Utilized for Impurity Profiling of Favipiravir,a Promising COVID-19 Antiviral Drug

In 2018, the discovery of carcinogenic nitrosamine process related impurities (PRIs) in a group of widely used drugs led to the recall and complete withdrawal of several medications that were consumed for a long time, unaware of the presence of these genotoxic PRIs. Since then, PRIs that arise during the manufacturing process of the active pharmaceutical ingredients (APIs), together with their degradation impurities, have gained the attention of analytical chemistry researchers. In 2020, favipiravir (FVR) was found to have an effective antiviral activity against the SARS-COVID-19 virus. Therefore, it was included in the COVID-19 treatment protocols and was consequently globally manufactured at large-scales during the pandemic. There is information indigence about FVR impurity profiling, and until now, no method has been reported for the simultaneous determination of FVR together with its PRIs. In this study, five advanced multi-level design models were developed and validated for the simultaneous determination of FVR and two PRIs, namely; (6-chloro-3-hydroxypyrazine-2-carboxamide) and (3,6-dichloro-pyrazine-2-carbonitrile). The five developed models were classical least square (CLS), principal component regression (PCR), partial least squares (PLS), genetic algorithm-partial least squares (GA-PLS), and artificial neural networks (ANN). Five concentration levels of each compound, chosen according to the linearity range of the target analytes, were used to construct a five-level, three-factor chemometric design, giving rise to twenty-five mixtures. The models resolved the strong spectral overlap in the UV-spectra of the FVR and its PRIs. The PCR and PLS models exhibited the best performances, while PLS proved the highest sensitivity relative to the other models.     

Development of a SPE-LC-MS Method for the Quantitation of Palbociclib and Abemaciclib in Human Plasma

Palbociclib and abemaciclib are two cyclin-dependent kinases 4 and 6 used for breast cancer treatment. Levels of these medicines present a significant interindividual variability, so monitoring those concentrations might be necessary in therapy. Most of the methods presented so far in the literature use simple protein precipitation of plasma proteins as sample preparation method followed by direct injection of the supernatant into the LC instrument, preceded or not by a simple filtration step. Within that approach, the probability of injecting proteins in the chromatographic system is increased. With the purpose of obtaining a cleaner extract of the drugs, we developed and validated a simple and accurate LC-MS method for determining palbociclib and abemaciclib in human plasma. Solid phase extraction (SPE) using Oasis PRiME HLB^® cartridges was used for plasma sample preparation. The method provided clean extracts with a recovery extraction higher than 85% for both compounds. Separation was achieved by high-performance liquid chromatography (HPLC), using a C18 (4.6 × 50 mm) column, with a gradient elution of ammonium acetate/acetic acid-acetonitrile as the mobile phase. Detection was performed by mass spectrometry (MS) in single ion recording (SIR) mode. Intra-day and inter-day precision data for both analytes were 3.8–7.2% and 3.6–7.4%, respectively. Calibration curves were both linear between 2 and 400 ng/mL with a correlation coefficient higher than 0.998. The LC-MS method can be used to quantify the drugs in human plasma in routine analysis. The method proved to be useful in determining real plasma levels in patients involved in cancer therapy. Drug concentrations were determined in a 10 min run-time, including re-equilibration of the column.     

Two Green Micellar HPLC and Mathematically Assisted UV Spectroscopic Methods for the Simultaneous Determination of Molnupiravir and Favipiravir as a Novel Combined COVID-19 Antiviral Regimen

Following the spread of the COVID-19 pandemic crisis, a race was initiated to find a successful regimen for postinfections. Among those trials, a recent study declared the efficacy of an antiviral combination of favipiravir (FAV) and molnupiravir (MLP). The combined regimen helped in a successful 60% eradication of the SARS-CoV-2 virus from the lungs of studied hamster models. Moreover, it prevented viral transmission to cohosted sentinels. Because both medications are orally bioavailable, the coformulation of FAV and MLP can be predicted. The developed study is aimed at developing new green and simple methods for the simultaneous determination of FAV and MLP and then at their application in the study of their dissolution behavior if coformulated together. A green micellar HPLC method was validated using an RP-C18 core-shell column (5 μm, 150 × 4.6 mm) and an isocratic mixed micellar mobile phase composed of 0.1 M SDS, 0.01 M Brij-35, and 0.02 M monobasic potassium phosphate mixture and adjusted to pH 3.1 at 1.0 mL min⁻¹ flow rate. The analytes were detected at 230 nm. The run time was less than five minutes under the optimized chromatographic conditions. Four other multivariate chemometric model methods were developed and validated, namely, classical least square (CLS), principal component regression (PCR), partial least squares (PLS-1), and genetic algorithm–partial least squares (GA–PLS-1). The developed models succeeded in resolving the great similarity and overlapping in the FAV and MLP UV spectra unlike the traditional univariate methods. All methods were organic solvent-free, did not require extraction or derivatization steps, and were applied for the construction of the simultaneous dissolution profile for FAV tablets and MLP capsules. The methods revealed that the amount of the simultaneously released cited drugs increases up until reaching a plateau after 15 and 20 min for FAV and MLP, respectively. The greenness was assessed on GAPI and found to be in harmony with green analytical chemistry concepts.     

Development and Validation of a Reliable UHPLC-MS/MS Method for Simultaneous Quantification of Macrocyclic Lactones in Bovine Plasma

A fast, accurate and reliable ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantification of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) in bovine plasma. A priority for sample preparation was the eradication of possible infectious diseases to avoid travel restrictions. The sample preparation was based on protein precipitation using 1% formic acid in acetonitrile, followed by Ostro^® 96-well plate pass-through sample clean-up. The simple and straightforward procedure, along with the short analysis time, makes the current method unique and suitable for a large set of sample analyses per day for PK studies. Chromatographic separation was performed using an Acquity UPLC HSS-T3 column, with 0.01% acetic acid in water and methanol, on an Acquity H-Class ultra-high performance liquid chromatograph (UHPLC) system. The MS/MS instrument was a Xevo TQ-S^® mass spectrometer, operating in the positive electrospray ionization mode and two multiple reaction monitoring (MRM) transitions were monitored per component. The MRM transitions of m/z 897.50 > 753.4 for IVER, m/z 921.70 > 777.40 for DORA and m/z 640.40 > 123.10 for MOXI were used for quantification. The method validation was performed using matrix-matched calibration curves in a concentration range of 1 to 500 ng/mL. Calibration curves fitted a quadratic regression model with 1/x2 weighting (r ≥ 0.998 and GoF ≤ 4.85%). Limits of quantification (LOQ) values of 1 ng/mL were obtained for all the analytes, while the limits of detection (LOD) were 0.02 ng/mL for IVER, 0.03 ng/mL for DORA, and 0.58 ng/mL for MOXI. The results of within-day (RSD < 6.50%) and between-day (RSD < 8.10%) precision and accuracies fell within acceptance ranges. No carry-over and no peak were detected in the UHPLC-MS/MS chromatogram of blank samples showing good specificity of the method. The applicability of the developed method was proved by an analysis of the field PK samples.     

Development and Validation of a Method of Liquid Chromatography Coupled with Tandem Mass Spectrometry for Quantification of ST-246 (Tecovirimat) in Human Plasma

The aim of this work was to develop and validate a sensitive and robust method of liquid chromatography coupled with tandem mass spectrometry to quantitate ST-246 (tecovirimat) in plasma using an internal standard (2-hydroxy-N-{3,5-dioxo-4-azatetracyclo [5.3.2.02.6.08.10]dodec-11-en-4-yl}-5-methylbenzamide). The method was validated in negative multiple reaction monitoring mode following recommendations of the European Medicines Agency for the validation of bioanalytical methods. The calibration curve for the analyte was linear in the 10–2500 ng/mL range with determination coefficient R² > 0.99. Intra- and inter-day accuracy and precision for three concentrations of quality control were <15%. Testing of long-term stability of ST-246 (tecovirimat) in plasma showed no degradation at −20 °C for at least 3 months. The method was applied to a clinical assay of a new antipoxvirus compound, NIOCH-14. Thus, the proposed method is suitable for therapeutic drug monitoring of ST-246 (tecovirimat) itself and of NIOCH-14 as its metabolic precursor.     

Development and Validation an LC Method for the Determination of Bendroflumethiazide in Human Plasma and its Pharmacokinetics

A simple, rapid, sensitive high performance liquid chromatography method with fluorescent detection was developed and validated for the determination of bendroflumethiazide in human plasma. Extraction from the plasma was by liquid-liquid extraction using ethyl acetate. Mosapride citrate was used as the internal standard. The chromatographic separation was performed on reverse phase LiChrosphere C18 column with mobile phase comprising of acetonitrile and phosphate buffer (38:62 v/v). The assay precision ranged from 0.9–12.5 and accuracy between 96.8–108.8%, revealing that the method has good reproducibility over the concentration range of 0.98–100.16 ng mL⁻¹. The validated method has been applied to analyze the bendroflumethiazide concentrations for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Rapid Liquid Chromatographic – Tandem Mass Spectrometric Method for the Quantification of Pentoxifylline in Human Plasma

A simple, rapid, sensitive and selective liquid chromatography / tandem mass spectrometry method was developed and validated for the quantification of pentoxifylline, a haemorheological agent. The analyte and internal standard, tamsulosin were extracted by liquid-liquid extraction with ethyl acetate and were separated using an isocratic mobile phase on a reverse phase C₁₈ column. The analytes were analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]⁺ ions, m/z 279/138 for pentoxifylline and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 2–1000 ng mL⁻¹ for pentoxifylline in human plasma. The lower limit of quantification was 2 ng mL⁻¹ with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 1.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. Revised: 4 and 20 October 2005     

Development and Validation of a Chiral Liquid Chromatographic Assay for Enantiomeric Separation and Quantification of Verapamil in Rat Plasma: Stereoselective Pharmacokinetic Application

A novel, fast and sensitive enantioselective HPLC assay with a new core–shell isopropyl carbamate cyclofructan 6 (superficially porous particle, SPP) chiral column (LarihcShell-P, LSP) was developed and validated for the enantiomeric separation and quantification of verapamil (VER) in rat plasma. The polar organic mobile phase composed of acetonitrile/methanol/trifluoroacetic acid/triethylamine (98:2:0.05: 0.025, v/v/v/v) and a flow rate of 0.5 mL/min was applied. Fluorescence detection set at excitation/emission wavelengths 280/313 nm was used and the whole analysis process was within 3.5 min, which is 10-fold lower than the previous reported HPLC methods in the literature. Propranolol was selected as the internal standard. The S-(−)- and R-(+)-VER enantiomers with the IS were extracted from rat plasma by utilizing Waters Oasis HLB C18 solid phase extraction cartridges without interference from endogenous compounds. The developed assay was validated following the US-FDA guidelines over the concentration range of 1–450 ng/mL (r² ≥ 0.997) for each enantiomer (plasma) and the lower limit of quantification was 1 ng/mL for both isomers. The intra- and inter-day precisions were not more than 11.6% and the recoveries of S-(−)- and R-(+)-VER at all quality control levels ranged from 92.3% to 98.2%. The developed approach was successfully applied to the stereoselective pharmacokinetic study of VER enantiomers after oral administration of 10 mg/kg racemic VER to Wistar rats. It was found that S-(−)-VER established higher C_max and area under the concentration-time curve (AUC) values than the R-(+)-enantiomer. The newly developed approach is the first chiral HPLC for the enantiomeric separation and quantification of verapamil utilizing a core–shell isopropyl carbamate cyclofructan 6 chiral column in rat plasma within 3.5 min after solid phase extraction (SPE).     

Validation of an LC–MS/MS Method for the Quantification of the CK2 Inhibitor Silmitasertib (CX-4945) in Human Plasma

Silmitasertib (CX-4945) is currently being investigated in clinical trials against various types of cancer. The U.S. Food and Drug Administration (FDA) has already granted orphan drug designation to the compound for the treatment of advanced cholangiocarcinoma, medulloblastoma, and biliary tract cancer. Silmitasertib inhibits the serine/threonine protein kinase CK2, which exerts a proliferation-promoting and anti-apoptotic effect on cancer cells. In view of current and future applications, the measurement of silmitasertib levels in plasma is expected to play an important role in the evaluation of therapeutic and toxic concentrations in cancer patients. In the present work, we therefore present an LC–MS/MS method for the quantification of silmitasertib in human plasma. Using a simple liquid–liquid extraction with ethyl acetate and a mixture of n-hexane and ethyl acetate, this method can be performed in any laboratory with mass spectrometry. The validation was carried out according to the FDA guideline.     

Fast and Sensitive HPLC-ESI-MS/MS Method for Etoricoxib Quantification in Human Plasma and Application to Bioequivalence Study

Etoricoxib is a non-steroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation. The objective of the current study was to develop a sensitive, fast and high-throughput HPLC-ESI-MS/MS method to measure etoricoxib levels in human plasma using a one-step methanol protein precipitation technique. A tandem mass spectrometer equipped with an electrospray ionization (ESI) source operated in a positive mode and multiple reaction monitoring (MRM) were used for data collection. The quantitative MRM transition ions were m/z 359.15 > 279.10 and m/z 363.10 > 282.10 for etoricoxib and IS. The linear range was from 10.00 to 4000.39 ng/mL and the validation parameters were within the acceptance limits of the European Medicine Agency (EMA) and Food and Drug Analysis (FDA) guidelines. The present method was sensitive (10.00 ng/mL with S/N > 40), simple, selective (K prime > 2), and fast (short run time of 2 min), with negligible matrix effect and consistent recovery, suitable for high throughput analysis. The method was used to quantitate etoricoxib plasma concentrations in a bioequivalence study of two 120 mg etoricoxib formulations. Incurred sample reanalysis results further supported that the method was robust and reproducible.     

Extractionless HPLC Method for the Determination of Naproxen in Human Plasma and Urine

Abstract

A simple and rapid (extractionless) high-performance liquid chromatographic method with ultraviolet detection, at 278 nm, is described for the determination of naproxen in human plasma and urine. Niflumic acid is used as internal standard. The chromatographic system consists of a reversed-phase C18-Spherisorb column with acetonitrile/0.1 M sodium acetate (35:65 v/v, pH 6.14) as the mobile phase. The retention time is 3.0 min for naproxen and 3.8 min for niflumic acid. The total run time is 5 min and the typical assay time is 10 min. The method is sufficiently sensitive for biopharmaceutical studies, after the oral administration of a single sustained release dose.     

Improved High Pressure Liquid Chromatographic Method for Phenylpropanolamine in Human Plasma

Abstract

A high-pressure liquid chromatographic analysis of phenylpropanolamine in plasma following extraction, back extraction and pre-column derivitization with O-phthalaldehyde is presented. The method is improved by the use of phenylethanolamine as internal standard. Using fluorescence detection, the method is sufficiently sensitive to quantitate 5 ng/ml in 0.5 ml plasma with a standard error of estimate of 2.7 ng/ml when calibrated over the O to 240 ng/ml range. Analysis of over 2000 clinical samples have shown the method to be highly specific and reliable.     

Development and validation of an RP‐HPLC method for the quantitation of odanacatib in rat and human plasma and its application to a pharmacokinetic study

A simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of odanacatib in rat and human plasma. The bioanalytical procedure involves extraction of odanacatib and itraconazole (internal standard, IS) from a 200 μL plasma aliquot with simple liquid–liquid extraction process. Chromatographic separation was achieved on a Symmetry Shield RP₁₈ using an isocratic mobile phase at a flow rate of 0.7 mL/min. The UV detection wave length was 268 nm. Odanacatib and IS eluted at 5.5 and 8.6 min, respectively with a total run time of 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 50.9–2037 ng/mL (r² = 0.994). The intra‐ and inter‐day precisions were in the range of 2.06–5.11 and 5.84–13.1%, respectively, in rat plasma and 2.38–7.90 and 6.39–10.2%, respectively, in human plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitation of Toremifene and its Major Metabolites in Human Plasma by High-Performance Liquid Chromatography Following Fluorescent Activation

Abstract

A highly sensitive reverse-phase high-performance liquid chromatography assay utilizing fluorescence activation has been developed for the quantitative analysis of the anti-estrogenic compound toremifene and its major metabolites, 4-hydroxy-toremifene and N-desmethyl-toremifene. Plasma samples containing various quantities of toremifene and its metabolites were spiked with an internal standard (nafoxidine), extracted with 2% n-butanol in hexane, and irradiated with high intensity ultraviolet light (254 nm). Aliquots of the extracted plasma components were then injected onto a C-18 reversed phase column and eluted isocratically with a mobile phase of water and triethylamine in methanol. Fluorescence of toremifene, its metabolites, and internal standard was measured at an excitation wavelength of 266 nm. -The sensitivity of this assay was 8.0, 15.0 and 5.0 ng/mL for toremifene, N-desmethyl-toremifene and 4-hydroxy-toremifene, respectively. Linearity was achieved for the concentration range of 25 to 400 ng/mL for all the compounds, with correlation coefficients of greater than 0.994. The assay presented is highly specific, very sensitive and demonstrates reproducible linearity throughout a wide range of clinically relevant plasma toremifene concentrations.