Bioanalytical Method Validation Research Articles

Development and Validation of Bioanalytical Method for Estimation of Rivaroxaban using RP-HPLC with Liquid liquid extraction in Human Blood Plasma and its application in Bioequivalence Study

Rivaroxaban is andirect acting oralanticoagulant and factor Xa inhibitor. A simple, selective, precise and rapid RP-HPLC method for estimation of Rivaroxaban (RIVA) in human blood plasma was developed and validated. The sample spike in plasma was extracted using liquid liquid extraction were extracted with the organic solvent ethyl acetate as organic solvent. Apixaban as an internal standard. The compounds were analysed by Agilent HPLC was used with control panel software using UV detector on a Inertsil ODS (250mm x 4.6mm ID;5μ) column with an Flow rate of 1.2mL/min, an isocratic mobile phase consisting of 0.02M Ammonium acetate buffer: Acetonitrile (70:30%v/v). Different sample pre-treatment techniques were evaluated, but Liquid Liquid extraction was found to be satisfactory, with good recovery values of 93.70% for RIVA. The developed method is validated by ICHM10 and USFDA guidelines over the concentration range of 5.00 to 200.00 ng/ml in human blood plasma with R² =0.9993. Within-day precisions and accuracy for RIVA were found in 0.36% to 4.73% and 92.58% to101.82% respectively. The validated RP-HPLC method has been used successfully for both preliminary pharmacokinetic studies and therapeutic drug monitoring

Quantitative Analysis of Cenobamate and Concomitant Anti-Seizure Medications in Human Plasma via Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

Cenobamate (CNB) is a new anti-seizure medication (ASM) recently introduced in clinical practice after approval by the FDA and EMA for the add-on treatment of focal onset seizures in adult patients. Although its mechanism of action has not been fully understood, CNB showed promising clinical efficacy in patients treated with concomitant ASMs. The accessibility of CNB could pave a way for the treatment of refractory or drug-resistant epilepsies, which still affect at least one-third of the patients under pharmacological treatment. In this context, therapeutic drug monitoring (TDM) offers a massive opportunity for better management of epileptic patients, especially those undergoing combined therapy. Here, we describe the first fully validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantification of CNB and concomitant ASMs in human plasma, with samples extracted either manually or by means of a liquid handler. Our method was validated according to the most recent ICH International Guideline M10 for Bioanalytical Method Validation and Study Sample Analysis. The method proved to be selective for CNB and displayed a linear range from 0.8 to 80 mg/L; no matrix effect was found (98.2 ± 4.1%), while intra-day and inter-day accuracy and precision were within the acceptance range. Also, CNB short- and long-term stability in plasma under different conditions was assessed. Leftover human plasma samples were employed as study samples for method validation. Our method proved to be highly sensitive and selective to quantify CNB and concomitant ASMs in human plasma; therefore, this method can be employed for a routinely TDM-based approach to support physicians in the management of an epileptic patient.

Recommendations and feedback from the European Bioanalysis Forum Workshop: 1 year into ICH M10 - keeping our finger on the pulse.

The ICH M10 guideline on bioanalytical method validation and sample analysis is being adopted since 2023. However, and inevitably, some paragraphs or requirements remain ambiguous and are open for different interpretations. In support of a harmonized interpretation by the industry and health authorities, the European Bioanalysis Forum organized a workshop on 14 November 2023 in Barcelona, Spain, to discuss unclear and/or ambiguous paragraphs which were identified by the European Bioanalysis Forum community and delegates of the workshop prior to the workshop. This manuscript reports back from the workshop with recommendations and aims at continuing an open scientific discussion within the industry and with regulators in support of a science-driven guideline for the bioanalytical community and in line with the ICH mission - that is, achieve greater harmonization worldwide to ensure that safe, effective and high-quality medicines are developed and registered in the most resource-efficient manner.

Validation of a sensitive bioanalytical method for metronidazole extraction from human plasma

Metronidazole (MTZ) is a broad-spectrum antibiotic with numerous routes of administration, including topical. Topical application of MTZ gel or cream results in very low systemic absorption, resulting in the need for a sensitive extraction method to quantify plasma concentrations. Currently published methods are not suitable for analysis of plasma concentrations after topical application, as undetectable MTZ concentrations commonly occur. We validated a simple extraction method for MTZ recovery from plasma and quantified it using an LC-MS/MS analytical method. Methods: Plasma samples were spiked with MTZ (0.5 – 5 ng/mL) and internal standard (tinidazole, 2 ng/mL). MTZ was extracted by liquid-liquid extraction using ethyl acetate and acetonitrile mixture (4:1) as the extraction solvent. A quadrupole mass spectrometer interfaced with an Acquity H-Class HPLC was used to quantify MTZ concentrations in positive ion mode. A Kinetix C18 analytical column (150 mm × 4.6 mm i.d., 5 µm particle size) was used for separation. The plasma extraction method was validated for various parameters, including % recovery, precision, accuracy, and stability. Results: The extraction method demonstrated high MTZ recovery, ranging from 93.7 – 97.5%. The calibration curve prepared using MTZ samples extracted from plasma (0.5 – 5 ng/mL) had excellent linearity with R2 = 0.999. The extracted samples also showed higher autosampler and freeze-thaw stability over a 72-hr period. The mean intra- and inter-day accuracy and precision of the extraction assay ranged from 97 to 101.6% and 2.7 – 4.8% RSD, respectively. The assay was highly efficient, with a limit of quantification (0.53 ± 0.04 ng/mL) lower than previously published methods (≥5 ng/mL). The extraction method was successfully validated using LC-MS/MS and can be used to extract and detect trace amounts of MTZ in plasma after topical application.

Screening stabilisers for cyanoenone triterpenoid TX101 in rat plasma samples by simultaneous analysis of parent drug and the epoxidation product

AbstractIn the development of bioanalytical methods, stabilizing drug molecules in biological matrices is crucial for ensuring reliable exposure data in pharmacokinetic and toxicokinetic sample analyses. This study focuses on the evaluation of stabilizing effects on the synthetic triterpenoid TX101, a cyanoenone triterpenoid Nrf2 activator with known instability in plasma samples. The molecule's unsaturated double bond is susceptible to oxidation, either nonenzymatically via oxygen or enzymatically through cytochrome P450 enzyme‐catalyzed epoxidation. The research explores the impact of antioxidants (L‐ascorbic acid, sodium metabisulfite, sodium sulfite) and P450 enzyme inhibitors (sodium diethyldithiocarbamate, memantine hydrochloride, 1‐aminobenzotriazole) on TX101 stability in rat plasma samples. Results reveal that adding 2.5 mg/mL sodium sulfite or sodium metabisulfite effectively inhibits the nonenzymatic oxidation of TX101 to TX101‐epoxide, while L‐ascorbic acid shows minimal stabilizing effect. Among P450 enzyme inhibitors, sodium diethyldithiocarbamate and memantine hydrochloride exhibit modest stabilizing effects, likely attributed to their antioxidant activity. The developed High‐formance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method, incorporating Supported Liquid Extraction for sample cleanup, allows simultaneous monitoring of TX101 and TX101‐epoxide. Application of this method in a rat dose‐range finding study confirms successful inhibition of TX101‐epoxide formation in samples treated with sodium sulfite or sodium metabisulfite. Overall, the study emphasizes the importance of stabilizers in preventing nonenzymatic oxidation reactions during sample storage, providing valuable insights for bioanalytical method development and validation.

Development and validation of pharmacokinetics assays for a novel HER2-targeting antibody-drug conjugate (SHR-A1201): Application to its dose-escalation pharmacokinetic study

Approximately 25% of breast cancer patients with HER2 overexpression tend to have a high risk of disease progression and death. Various HER2-targeting therapies have been approved for treatment. Recently, a novel antibody-drug conjugate, SHR-A1201, is being researched and developed. For the pharmacokinetic study of SHR-A1201, suitable bioanalytical methods are needed for quantifying unconjugated cytotoxin, cytotoxin-conjugated antibodies and total antibodies. In this research, bioanalytical methods involving a highly sensitive LC-MS/MS assay for unconjugated cytotoxic payload DM1 in human plasma, ELISA strategies for DM1-conjugated trastuzumab and total trastuzumab in human serum were developed, validated and successfully applied to a phase I dose-escalation pharmacokinetic study of SHR-A1201. The pharmacokinetic properties and exposure-to-dose proportionality was evaluated for SHR-A1201. According to the bioanalytical method validation guidance, the bioanalytical methods were fully validated and the validation results met the acceptance criteria. The nonspecific binding of DM1 and dimer was avoided for the LC-MS/MS assay. In the dose-escalation pharmacokinetic study of SHR-A1201, a potential dose-proportional pharmacokinetics was observed over the dose from 1.2 mg/kg to 4.8 mg/kg. The validated bioanalytical strategies are robust and reproducible and these bioanalytical methods will contribute to better understanding of the pharmacokinetic properties of SHR-A1201.

Development and validation of an UPLC-MS/MS method for the determination of irinotecan (CPT-11), SN-38 and SN-38 glucuronide in human plasma and peritoneal tumor tissue from patients with peritoneal carcinomatosis

Irinotecan (CPT-11), an antineoplastic drug, is used for the treatment of colorectal and pancreatic cancer due to its topoisomerase I inhibitory activity. CPT-11 is a prodrug which is converted to its active metabolite SN-38 by carboxylesterases. SN-38 is further metabolized to its inactive metabolite SN-38 glucuronide. When evaluating the pharmacokinetic properties of CPT-11 and its metabolites, it is important to accurately assess the concentrations in both plasma as well as tumor tissues. Therefore, the aim of the current study was to develop and validate a robust and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method to quantify the concentration of CPT-11 and its metabolites (SN-38 and SN-38 glucuronide) in human plasma and peritoneal tumor tissue. The sample preparation of plasma and tumor tissue consisted of protein precipitation and enzymatic digestion/liquid–liquid extraction, respectively. Chromatographic separation was achieved with an Acquity UPLC BEH C18 column combined with a VanGuard pre-column. The mobile phases consisted of water +0.1 % formic acid (mobile phase A) and acetonitrile +0.1 % formic acid (mobile phase B). Mass analysis was performed using a Xevo TQS tandem mass spectrometer in the positive electrospray ionization mode. Method validation was successfully performed by assessing linearity, precision and accuracy, lower limit of quantification, carry over, selectivity, matrix effect and stability according to the following guidelines: “Committee for Medicinal Products for Human use, Guideline on Bioanalytical Method Validation”. A cross-validation of the developed method was performed in a pilot pharmacokinetic study, demonstrating the usefulness of the current method to quantify CPT-11 and its metabolites in the different matrices.

Rapid and simple quantification of belimumab in human plasma using ultra-high performance liquid chromatography with tandem mass spectrometry

ObjectiveBelimumab is a monoclonal antibody against the B-lymphocyte stimulating factor and is approved for the treatment of patients with systemic lupus erythematosus (SLE) not responding adequately to existing therapies. In this study, we established and validated an assay for quantifying belimumab in human plasma. MethodsFrom the peptides generated by trypsin digestion of belimumab, in silico analysis was used to search for unique peptides to determine the surrogate peptides. Samples were trypsin digested, pretreated with solid phase extraction, and analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) to quantify the surrogate peptide in the samples. The assay was validated according to the Food and Drug Administration (FDA) bioanalytical method validation guidance. We used the established assay to quantify plasma belimumab concentrations in two SLE patients treated with belimumab. ResultsAmong the unique peptides identified by the in silico analysis, the peptide with the best peak shape when measured by UHPLC-MS/MS was selected as the surrogate peptide. The validation results of this assay met the acceptable criteria recommended by the FDA guidance. The lower limit of quantification (LLOQ) for belimumab was 2 µg/mL. Recovery rates and matrix effects when corrected for internal standards were 91.5–114.3 % and 96.9–108.4 %, respectively. Plasma concentrations of belimumab were measured in 12 samples from two belimumab-treated SLE patients. All concentrations were within the calibration range. ConclusionsWe have established and validated a method for measuring plasma belimumab concentrations using UHPLC/MS-MS. By measuring plasma belimumab concentrations in more patients, this method is expected to contribute to appropriate use of belimumab.

Bioanalytical method development and validation for the simultaneous estimation of Olanzapine and Samidorphan in rabbit plasma by using HPLC–MS/MS and application to pharmacokinetic study

BackgroundSamidorphan is an opioid antagonist while Olanzapine is an effective medication for schizophrenia and bipolar disorder. A unique and accurate MS/HPLC approach due to simultaneous measurement of Olanzapine and Samidorphan is, therefore, more urgently required. Simultaneous quantification of Olanzapine and Samidorphan in rabbit plasma using HPLC-MS. Using a buffer composed of 1 mL of formic acid in 1 L of water and a mixture of two components, buffer and acetonitrile in a ratio of 50:50 and a flow rate of 1 mL/min at room temperature, we separated compounds on an Inertsil ODS column (250 × 4.6 mm, 5 m).ResultsAnalysis was performed within 8 min over a satisfactory linear concentration range of 2–40 ng/mL for Olanzapine (r2 = 0.99901 0.024) and 2–40 ng/mL for Samidorphan (r2 = 0.99927 0.012). The matrix effect recoveries of Olanzapine and Samidorphan at various QC concentration levels were 104.5, 100.51% and 110.36, 99.25%, respectively. The precision and recovery study outcomes fall within the acceptable range. An electrospray ionization source was used to analysis of Olanzapine and Samidorphan at m/z 313.40 → 192.54, m/z 371.45 → 220.61 for Olanzapine and Samidorphan, m/z 316.40 → 237.58, m/z 374.41 → 223.61 for D3 Olanzapine and D3 Samidorphan that were ion pairs of mass analysis.ConclusionsLiquid–liquid extraction was used to remove Olanzapine (0.17 mg/kg) and its reference standard (D3-Olanzapine) from rabbit plasma. Both the active compound Samidorphan (0.17 mg/kg) and its reference, D3-samidorphan, were isolated from rabbit plasma. We conducted stability studies to ensure that the medications would remain stable in accordance with USFDA regulations.

Salt-assisted liquid–liquid microextraction for determination of haloperidol in human plasma by LC-MS/MS

Haloperidol is an antipsychotic used in the treatment of schizophrenia. Compared to other antipsychotics, it is widely used in developing countries due to its affordable price. Haloperidol has a narrow therapeutic range and variable pharmacokinetics; therefore, therapeutic drug monitoring (TDM) is recommended. For this reason, in this study, an easily applicable, fast, selective, accurate, reliable, and economical LC-MS/MS method was developed for the determination of haloperidol in human plasma for use in TDM and also method was validated according to European Medicines Agency (EMA) Bioanalytical method validation guidelines. In the developed method, analyte and internal standard were extracted from plasma by salt-assisted liquid-liquid microextraction (SALLME) technique and after that injected to the LC system. The limit of quantification of haloperidol was determined as 1 ng/ml. The calibration curve was validated between 1-15 ng/ml, with correlation coefficients >0.99. In addition, the developed method was used to determine drug concentration levels in the plasma of real patients.

Spectrometric Bioanalytical Method Development and Validation of Tolvaptan in Spiked Human plasma Followed by Forced degradation Studies

Tolvaptan in bulk and formulation in spiking human plasma were estimated using a brand-new, straightforward, speedy, precise, and accurate bioanalytical method that was developed and validated using UV-visible spectrophotometer. The acetonitrile-based solvent chosen for the tolvaptan UV study was scanned over the UV spectrum from 200 to 400nm using a solution containing 10g/ml. At 267nm, Tolvaptan exhibits its greatest absorption. The accuracy investigations were conducted at three distinct levels, i.e., 80%, 100%, and 120%, and recovery was found to be in the range of 99.4%. The tolvaptan showed linearity over the range of 5-160g/mL with correlation coefficientnt (r2) of 0.999. The thresholds for detection and quantification were 0.471 and 1.435 g/ml, respectively. In accordance with ICH requirements, all the parameters were validated.

A QbD Assisted Bioanalytical Method Development and Validation for Simultaneous Estimation of Montelukast and Bilastine by LC-MS Technique

This study describes how to use internal standards (IS) under the Analytical Quality by Design (AQbD) methodology to measure montelukast and bilastine in artificial rabbit plasma. The protein precipitation method was used for sample preparation and extraction. The chromatographic analysis was performed on the processed materials. The measurement range of the calibration was between 2 to 40 ng/mL for Bilastine and 1 to 20 ng/mL for montelukast. Optimizing mobile phase composition, flow rate, and pH were used to determine peak area and retention time. A significant reduction in method variability was observed as a result of the method of experimental design during optimization, and this enhanced the stability of the technique. A validation study demonstrated linearity, accuracy, precision, selectivity, sensitivity, and stability for estimating bilastine and montelukast in rabbit plasma using the developed method. The chemical composition of the drug was not altered during tests on its stability in human plasma. Overall, the investigations showed that the established approach is straightforward, reliable, and affordable for use in regular pharmacokinetic and bioequivalence research.

Simultaneous quantification of mirabegron and vibegron in human plasma by HPLC-MS/MS and its application in the clinical determination in patients with tumors associated with overactive bladder

Mirabegron and vibegron, both newly identified beta-3 adrenergic agonists, have significantly improved the quality of life for patients suffering from overactive bladder. In order to comprehensively assess the plasma exposure levels of these agents, the development of a rapid and highly sensitive bioanalytical method becomes imperative. The primary objective of this study was to establish a robust high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the concurrent quantification of mirabegron and vibegron in human plasma. The analytes were extracted from a 100 μL plasma sample through protein precipitation, employing 300 μL of methanol. Subsequently, samples underwent separation and quantification using a Waters XBridge C18 column (2.1 × 100 mm, 3.5 µm), with a mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The mass analysis was conducted using positive electrospray ionization (ESI+) operated in a multiple reaction monitoring (MRM) mode. The proposed method was meticulously validated in accordance with the guidelines set forth by the U.S. Food and Drug Administration (FDA) for bioanalytical method validation. The regression equations demonstrated exceptional linearity for both mirabegron (r² ≥ 0.994) and vibegron (r² ≥ 0.996) across the concentration range of 0.5 – 200 ng/mL. Furthermore, the assay exhibited accuracy (inter-day relative error ≤ 6.90%) and precision (inter-day coefficient of variation ≤ 8.88%). The average recoveries of the analytes were found to range from 81.94% to 102.02%, with mean matrix effects falling within the range of 89.77% to 110.58%. As a result, this method was deemed highly suitable for the precise determination of the concentrations of both mirabegron and vibegron in the context of therapeutic drug monitoring and bioequivalence studies.

Design of experiment-oriented development and validation of novel bioanalytical reverse phase high performance liquid chromatography method of palbociclib in rat plasma and tissues, and its application in pharmacokinetic studies.

The current research involved the development and validation of an easy, cost-effective, and sensitive bioanalytical reverse-phase high-performance liquid chromatography method for the assessment of palbociclib (PAL) in rat plasma and kidney, liver, spleen and heart. A response surface methodology-based Box-Behnken design was used to optimize critical chromatographic conditions such as buffer pH, organic phase concentration and flow rate to attain good sensitivity, tailing factor and retention time. The conditions were: pH of buffer, 4.5; organic phase concentration, 40%; Shimpac column with 1.0ml/min flow rate. The responses were: tailing factor, 1.29 ± 0.03, sensitivity, 366,593 ± 8,592; and retention time, 4.5 ± 0.05 min. The samples were extracted by a protein precipitation method, and absolute recoveries were in the range of 88.99-95.08%. The linearity of the developed method was validated over the range 100-2,000 ng/ml (r2 ≥ 0.994) in all tested matrices. The developed bioanalytical method showed greater accuracy (0.98 and 4.01%) and precision (<4.88%). The method was optimized for the sensitive analysis of the PAL in rat plasma, and the kidney, liver, spleen and heart were effectively applied to pharmacokinetic studies.

Bioanalytical Method Development and Validation of Doxorubicin and Lapatinib in Rat Plasma Using UHPLC-HESI-LTQ-MS

Abstract Cancer is considered a silent killer. The complexity of cancer makes it earn that title. So far there are only a few approaches to treat cancer. Among them, chemotherapy is considered the best approach. Many chemotherapeutical compounds are commercially available in the market. Among them, doxorubicin (DOX) and lapatinib (LAP) are considered blockbuster molecules. However, DOX suffers from poor bioavailability and exhibits cardiotoxicity. Interestingly, a fixed dose combination of DOX and LAP significantly decreases the cardiotoxic effect of DOX. To enhance the oral bioavailability of DOX and to avail the synergistic effect of LAP, many formulations have been made. To quantify both compounds in any formulation or biological matrix, an Liquid chromatography-Mass Spectrometry (LC-MS) method is required. In this present study, a simple and rapid Ultra High-Performance Liquid Chromatography - Heated Electron Spray Ionization - Mass Spectrometry (UHPLC-HESI-MS) bioanalytical method was developed. The developed method was validated as per the regulatory guidelines. The validated bioanalytical method had a lower limit of quantification of 0.75 ng. A simple protein precipitation technique was optimized to extract the compounds from the rat plasma. All the validation parameters were found to be within the limits as per the regulatory guidelines. A novel and rapid analytical method was successfully developed and validated. This developed method can be used to quantify the DOX and LAP in any formulation and biological matrix.

Smartphone based colorimetric point-of-care sensor for abused drugs: case of baclofen determination in urine

As a GABA-β receptor agonist, the central muscle relaxant Baclofen (BAC) has a potential of abuse. Unfortunately, the sense of wellbeing and pleasure is obtained at very high BAC doses. This is associated with many life-threating or even fatal cases due to neurological and respiratory failures. Moreover, having narrow therapeutic index makes BAC a high-risk drug. This is potentiated in case of long-treatment regimen or off-label use in smoking and alcohol cessation protocols. Until now, there is no rapid diagnostic test available for BAC screening. Therefore; It is quite difficult to routinely monitor cases on BAC regimen. On the other hand, smartphone-based colorimetric point of care testing (POCT) is displacing conventional analytical approaches in the detection and assay of abused drugs as well as therapeutic drug monitoring. It offers on-site, rapid, easy, affordable and interpretable analysis. Incorporating smartphone as a portable device facilitates its application, especially in remote areas and low-income countries. For the first time, the current work presents a smartphone-based colorimetric POCT for BAC analysis in urine without interference from urine matrix. It depends on BAC reaction with naphthoquinone sulfonate (NQS) in highly alkaline aqueous medium. The developed color was captured in a customized photo box using smartphone camera. Then, intensity of the blue channel was measured by a software application “Color Analyzer”. All parameters were optimized with respect to the colorimetric reaction, photographing and smartphone-based analysis. All parameters were successfully investigated according to FDA guidelines for bioanalytical method validation. Also, all POCT criteria were considered as per WHO requirements. This method could determine BAC, linearly, from 0.02 to 0.21 mmol L−1 in urine. Moreover, LLOQ was lower than the expected BAC therapeutic concentrations in urine. The proposed method proved high reliability and suitability to analyze BAC in urine. This strongly recommends its routine application in screening BAC abusers and BAC therapeutic monitoring.Graphical

Development and validation of a LC–MS/MS method for the quantification of phenolic compounds in human saliva after intake of a procyanidin-rich pine bark extract

Plant-derived phenolic compounds are regularly ingested as food compounds or as food supplements. Concentrations of individual compounds and metabolites are typically measured in serum or urine samples. This, however, allows no conclusion on the distribution into organs and tissues. An easily accessible biofluid is saliva. At this point, it was not clear yet, whether polyphenols circulating in the blood would be secreted or diffuse into saliva. The purpose of the present study was to develop and validate a method using liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for analysis of phenolic compounds in human saliva. Method validation for the quantification of taxifolin, ferulic acid, caffeic acid, gallic acid, para-coumaric acid, and protocatechuic acid and the gut microbial catechin metabolite δ-(3,4-dihydroxyphenyl)-γ-valerolactone (M1) in human saliva was performed according to current guidelines for bioanalytical method validation. The lower limit of quantification ranged from 0.82 ng/ml for M1 to 8.20 ng/ml for protocatechuic acid. The method was successfully applied to an authentic saliva sample of a volunteer after swallowing of procyanidin-rich pine bark extract capsules (dietary supplement Pycnogenol®). All polyphenols except ferulic acid were quantified at concentrations ranging from 1.20 ng/ml (M1) to 10.34 ng/ml (gallic acid). Notably, in contrast to serum samples, all phenolic compounds were present without sulfate or glucuronic acid conjugation in saliva, suggesting an enzymatic deconjugation, e.g., by a β-glucuronidase activity, during compound transfer from serum to saliva. Since M1 is only produced in the gut, its presence in saliva ruled out the possibility of sample contamination by phenolic compounds residing in the oral cavity after food intake. To the best of our knowledge, this is the first time that the gut microbiota-derived metabolite M1 has been detected in saliva. To further investigate the role of phenolic compounds in saliva, the described analytical method can be applied in clinical studies investigating the biodistribution of polyphenols and their metabolites.

Bio-analytical Method for the Pre-clinical Estimation of Donepezil Hydrochloride in Rabbit Plasma by RP-HPLC

A simple, reliable and sensitive RP-HPLC bioanalytical method with PDA as detector was developed for the estimation of donepezil HCl in the rabbit plasma and validated. The biological samples were extracted and analysed using loratadine as internal standard. The sample analysed with Inertsil ODS 150mm x 4.6mm, 5.0µm particle size column using 70:30 v/v of 0.1% trifluoroacetic acid and acetonitrile mobile phase at flow rate of 1mL/min. All the parameters validated were within the limits proposed in bioanalytical method validation guidelines. The linearity was observed in the range of 5-200ng/mL with LOD of 0.9ng/mL. The method was observed to have short analysis time of 6 min and also found to be successful in studying the pharmacokinetic parameters of donepezil HCl.

A simple and green capillary electrophoresis-mass spectrometry method for therapeutic drug monitoring of colistin in clinical plasma samples

Colistin and other polymyxin antibiotics have become increasingly used in clinical settings as a result of treating multidrug-resistant infections in critically ill patients. The highly variable pharmacokinetics of colistin in these patients is accompanied by a high risk of toxicity or underdosing. An effective tool that allows rational optimization of the drug dosage regimen is therapeutic drug monitoring. Therefore, there is a need to dispose with appropriate, sensitive, and accurate analytical methods. Here, a simple, specific, and accurate on-line capillary electrophoresis – tandem mass spectrometry method was developed and applied for the first time to determine colistin in human plasma. Protein precipitation using acidified acetonitrile was the solitary procedure used to achieve sample pretreatment. A bare fused silica capillary was employed for the separation process, and the background electrolyte used was 50 mM formic acid (pH 2.54). The FDA's bioanalytical method validation guidelines were followed in the validation of the proposed method. For colistin A and colistin B, favorable performance and validation parameters were obtained (such as linearity, limit of detection, lower limit of quantitation, intra-day and inter-day precision, accuracy, and stability).The validated method was then effectively used to analyze real clinical samples taken from patients who were in critical condition. Our newly developed method is comparable with previously published liquid chromatography approaches and has the potential to be applied in the therapeutic monitoring of colistin in routine clinical laboratories. Moreover, according to the greenness assessment, the developed capillary electrophoresis – mass spectrometry method represents a very interesting green and sustainable tool in the field of bioanalysis.

Simple HPLC-UV Method for Therapeutic Drug Monitoring of 12 Antiepileptic Drugs and Their Main Metabolites in Human Plasma

The objective of the present report was to develop and validate a simple, selective, and reproducible high-performance liquid chromatography method with UV detection suitable for routine therapeutic drug monitoring of the most commonly used antiepileptic drugs and some of their metabolites. Simple precipitation of plasma proteins with acetonitrile was used for sample preparation. 10,11-dihydrocarbamazepine was used as an internal standard. Chromatographic separation of the analytes was achieved by gradient elution on a Phenyl–Hexyl column at 40 °C, using methanol and potassium phosphate buffer (25 mM; pH 5.1) as a mobile phase. The method was validated according to the FDA guidelines for bioanalytical method validation. It showed to be selective, accurate, precise, and linear over the concentration ranges of 1–50 mg/L for phenobarbital, phenytoin, levetiracetam, rufinamide, zonisamide, and lacosamide; 0.5–50 mg/L for lamotrigine, primidone, carbamazepine and 10-monohydroxycarbazepine; 0.2–10 mg/L for carbamazepine metabolites: 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine and carbamazepine-10,11-epoxide; 0.1–10 mg/L for oxcarbazepine; 2–100 mg/L for felbamate and 3–150 mg/L for ethosuximide. The suitability of the validated method for routine therapeutic drug monitoring was confirmed by quantification of the analytes in plasma samples from patients with epilepsy on combination antiepileptic therapy.