Bioanalytical Method Validation Research Articles

Bioanalytical method development and validation for quantification of amivantamab in rat plasma by LC-MS/MS

BackgroundAmivantamab is a monoclonal bispecific anti-EGFR-MET antibody used to treat non-small cell lung cancer. There were no published methods using a liquid chromatographic—tandem mass spectrometric approach to develop and validate a feasible, novel, and thoroughly validated method for quantifying amivantamab in rat plasma.ResultsThe liquid–liquid extraction method was used to extract the analyte from rat plasma. The analyte was separated using acetonitrile–ammonium formate buffer (40:60) as a mobile phase on waters, alliance e-2695 model high-pressure liquid chromatographic system having Agilent eclipse C18, 150 mm × 4.6 mm, 3.5 µm column. The overall runtime was 6 min at a 1.0 ml/min flow rate. The method showed significant sensitivity and acceptable linearity over the 5.00–100.00 ng/ml concentration range. Accuracy was proved by mean percent recovery ranging from 98.03 to 99.99%. The intraday precision coefficient of variation (%) ranged between 0.31 and 5.43. Also, the findings such as Cmax, tmax, AUC0− t, AUC0− ∞, and half-life values of amivantamab showed that the technique was helpful for pharmacokinetic studies.ConclusionsAll the validated parameters were found to be within the acceptable range. The validated method was found to be simple, accurate, precise, and reproducible and hence can be used for the routine analysis of amivantamab, such as in-process quality control by liquid chromatographic—tandem mass spectrometry.

Development and validation of bioanalytical methods to support clinical study of disitamab vedotin.

Disitamab vedotin (RC48), a humanized anti-HER2 antibody conjugated with monomethyl auristatin E (MMAE), is the first antibody-drug conjugate in China with an approved biological license application. A bioanalytical method was established for three analytes (total antibody, conjugate antibody and free payload) to help characterize their pharmacokinetic behavior in clinical settings. The bioanalytical methods were validated according to M10 guidance. Electrochemiluminescence assaymethods were used for thequantitative measurement of total antibody and conjugated antibody in human serum. A LC-MS/MSmethod was used to quantify the concentration of MMAE in human serum. The method had high specificity and sensitivity with a quantitative range of 19.531-1250.000ng/ml (total antibody), 39.063-5000.000ng/ml (conjugated antibody)and 0.04-10.0ng/ml (MMAE), respectively.

Miniaturized on-spot protein denaturation/microwave-assisted extraction for spectrofluorimetric determination of favipiravir in dried plasma: Application to real human sample and inclusive incurred sample reanalysis study

The development of high-throughput sample preparation methods is a key step for upgrading the throughput of classical analytical techniques. In this work, a miniaturized on-spot protein denaturation coupled with microwave-assisted extraction sample treatment method was developed for extraction and subsequent spectrofluorimetric determination of favipiravir (FAV) in human plasma spots. Dried plasma spotting was performed using only 10 µL of plasma on filter paper discs followed by on-spot protein denaturation using 10 µL methanol. Maximum extraction efficiency was achieved using water: methanol (1:1) as extraction solvent with 50 % microwave power for 60 sec. The fluorescence intensity of extracted FAV was measured at 432 nm after excitation at 361 nm. According to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH -M10) guidelines for bioanalytical method validation, spiked plasma samples showed very good linearity between 100 and 1000 ng/mL. The accuracy and precision of the developed method were indicated by mean recovery (%) within 100 ± 15 % for all tested concentrations levels. The obtained results were further compared with a reported protein precipitation method using both F-test and t-test revealing no significant difference between either method. Moreover, the method was successfully applied for FAV determination in real human samples for an incurred sample reanalysis study revealing no metabolite back conversion. The developed method showed superior green performance using analytical Eco-scale, Green Analytical Procedure Index (GAPI), and Analytical Greenness Metric Approach (AGREE) tools compared to a classical protein precipitation method. According to Blue Applicability Grade Index (BAGI), and whiteness tools, the developed method outperformed all reported methods for FAV determination in plasma in terms of practicality and analytical functionality. Compared to reported sample treatment methods, this method exhibited excellent analytical performance, increased throughput, hundred-fold reduction of sample volume (10 μL), significant reduction of organic solvent consumption (260 μL methanol) and produced negligible wastes. Thus, it can be adopted for routine work in pharmacokinetics and clinical trial studies.

Molnupiravir Bioanalytical Method Development and Validation in Rat Plasma by LC-MS/MS Detection and Application to a Pharmacokinetic

Molnupiravir Bioanalytical Method Development and Validation in Rat Plasma by LC-MS/MS Detection and Application to a Pharmacokinetic

Bioanalytical Method Development and Validation of UV Spectrophotometric Method for Estimation of Sitagliptin in Urine sample

Bioanalytical Method Development and Validation of UV Spectrophotometric Method for Estimation of Sitagliptin in Urine sample

A RP-HPLC approach to Bioanalytical Method Development and Validation: A Review

A RP-HPLC approach to Bioanalytical Method Development and Validation: A Review

Edman Degradation Reveals Unequivocal Analysis of the Disulfide Connectivity in Peptides and Proteins.

Disulfide bridges in peptides and proteins play an essential role in maintaining their conformation, structural integrity, and consequently function. Despite ongoing efforts, it is still not possible to detect disulfide bonds and the connectivity of multiply bridged peptides directly through a simple and sufficiently validated protein sequencing or peptide mapping method. Partial or complete reduction and chemical cysteine modification are required as initial steps, followed by the application of a proper detection method. Edman degradation (ED) has been used for primary sequence determination but is largely neglected since the establishment of mass spectrometry (MS)-based protein sequencing. Here, we evaluated and thoroughly characterized the phenyl thiohydantoin (PTH) cysteine derivatives PTH-S-methyl cysteine and PTH-S-carbamidomethyl cysteine as bioanalytical standards for cysteine detection and quantification as well as for the elucidation of the disulfide connectivity in peptides by ED. Validation of the established derivatives was performed according to the guidelines of the International Committee of Harmonization on bioanalytical method validation, and their analytical properties were confirmed as reference standards. A series of model peptides was sequenced to test the usability of the PTH-Cys-derivatives as standards, whereas the native disulfide-bonded peptides CCAP-vil, μ-conotoxin KIIIA, and human insulin were used as case studies to determine their disulfide bond connectivity completely independent of MS analysis.

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.

In 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.

Determination and validation of tiaprofenic acid in human plasma: A detailed LC-MS/MS-based analysis following ICH M10 guidelines and the accuracy profile approach

The validation of bioanalytical methods holds critical importance for regulatory agencies and organizations dedicated to ensuring the safety, efficacy, and quality of pharmaceuticals. In this context, the recent release of the ICH M10 guideline in May 2022 represents a significant milestone in standardizing bioanalytical method validation globally. However, this guideline lacks explicit experimental protocols for implementation. In this study, we address the practical implementation of the newly released ICH M10 guideline by providing a detailed validation protocol for a bioanalytical method. Our method specifically targets tiaprofenic acid, a widely used nonsteroidal anti-inflammatory drug. Tiaprofenic acid is a critical component in bioequivalence studies, underscoring the necessity for precise and accurate quantification within complex biological matrices. The integration of the accuracy profile approach, a statistical tool, enhances the significance of this work. This approach aids in assessing the accuracy and precision of bioanalytical methods, establishing confidence intervals around measured concentrations, and quantifying the level of accuracy and precision expected when using the validated method.

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.