Method Validation Guidelines Research Articles

The use of surrogate matrices in bioanalytical preclinical safety testing using chromatographic methods: a recommendation from the European Bioanalysis forum.

Within the bioanalytical community, the use of blank matrix from preclinical animals for bioanalytical method validation and sample analysis is common practice and required in the context of guidelines for bioanalytical method validation. At the same time, its use has been challenged by the scientific community for decades, since there is ample scientific evidence to allow the use surrogate matrices for this purpose. Nevertheless, legacy and current regulatory thinking continues to be reluctant to allow the use of surrogate matrices in bioanalytical testing except for so-called rare matrices. As part of ongoing discussions in relation to the ICH M10 Guideline, the European Bioanalysis Forum re-challenges the unnecessary use of blank matrices from preclinical animals and believes that, as part of community responsibility and ethical standards and when supported by data, the use of surrogate matrices should become widely accepted. It is in this context that targeted experiments were conducted within the European Bioanalysis Forum to gather additional data and re-open the discussions with all involved and that it should become acceptable to use surrogate matrices wherever possible.

Development and Validation of a New Eco-Friendly HPLC-PDA Bioanalytical Method for Studying Pharmacokinetics of Seliciclib

Background and Objectives: Seliciclib (SEL) is the first selective, orally bioavailable potential drug containing cyclin-dependent kinase inhibitors. Preclinical studies showed antitumor activity in a broad range of human tumor xenografts, neurodegenerative diseases, renal dysfunctions, viral infections, and chronic inflammatory disorders. To support the pharmacokinetics and aid in therapeutic monitoring of SEL following its administration for therapy, an efficient analytical tool capable of quantifying the concentrations of SEL in blood plasma is needed. In the literature, there is no existing method for quantifying SEL in plasma samples. This study introduces the first HPLC method with a photodiode array (PDA) detector for the quantitation of SEL in plasma. Materials and Methods: The chromatographic resolution of SEL and linifanib as an internal standard (IS) was achieved on Zorbax Eclipse Plus C18 HPLC column (150 mm length × 4.6 mm internal diameter, 5 µm particle size), with a mobile phase composed of acetonitrile–ammonium acetate, pH 5 (50:50, v/v) at a flow rate of 1.0 mL min−1. Both SEL and IS were detected by PDA at 230 nm. The method was validated according to the ICH guidelines for bioanalytical method validation. Results: The method exhibited linearity in concentrations ranging from 50 to 1000 ng mL−1, with a limit of quantitation of 66.1 ng mL−1. All remaining validation parameters satisfied the ICH validation criteria. The environmental sustainability of the method was verified using three extensive tools. The proposed HPLC-PDA method was effectively utilized to study the pharmacokinetics of SEL in rats after a single oral administration of 25 mg/kg. Conclusions: The proposed method stands as a valuable tool for studying SELs for pharmacokinetics in humans. It aids in achieving the targeted therapeutic advantages and safety of treatment with SEL by optimizing the SEL dosage and dosing schedule.

Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Dalbavancin in Plasma of Pediatric and Young Adult Patients.

Dalbavancin, an antimicrobial lipoglycopeptide, is authorized in Europe for treating acute bacterial infections of the skin and skin structures in adults and pediatric patients aged 3 months and older. However, off-label dosing regimens have been proposed for various indications beyond acute bacterial infections of the skin and skin structures. This study presents a novel bioanalytical method using liquid chromatography-tandem mass spectrometry to quantify dalbavancin in low-volume plasma samples (50 μL). The method underwent validation in accordance with international guidelines for bioanalytical method validation and was applied to 9 clinical samples obtained from pediatric and young adult patients undergoing dalbavancin therapy. Liquid chromatography-tandem mass spectrometry analyses were conducted at the G. Gaslini Institute in Genoa, Italy, utilizing an Ultimate 3000 ultra high performance liquid chromatography system coupled to a TSQ Quantiva Triple Quadrupole system (Thermo Fisher Scientific, Milan, Italy). The analytical procedure involved the addition of deuterated dalbavancin as internal standard and a rapid extraction from 50 µL of human plasma, followed by chromatographic separation on a Thermo Scientific Accucore Polar Premium column. Accurate quantification of the analyte was achieved through multiple reaction monitoring detection. The assay exhibited linearity within the concentration range of 0.66-400 mcg/mL in plasma, demonstrating accuracy and reproducibility in the absence of matrix effects. Stability testing was conducted on both quality controls and real samples to establish a robust protocol under real-life conditions. This fast and reliable dalbavancin quantitation method could improve current pediatric clinical practice by enabling data collection for future dose recommendations in special patient populations.

Determination of the extremolyte ectoine in plasma and a pharmacokinetic study in rats by a validated and BAGI-evaluated UPLC-MS/MS method

Ectoine (ECT) has recently gained considerable interest in the healthcare sector due to its promising therapeutic benefits in a variety of human disorders. This research aimed to quantify the ECT plasma level in rats by creating and optimizing a sensitive and validated UPLC-MS/MS method. Prior to analysis, ECT extraction from the plasma samples was conducted via a protein precipitation procedure, using hydroxyectoine as an internal standard (IS). A 1.7 μm UPLC C8 column (100 mm × 2.1 mm) was selected for the chromatographic separation, using a gradient mobile phase consisting of acetonitrile and 0.05% formic acid. The electrospray ionization mass spectrometry (ESI-MS) was used to detect ECT in the positive ion mode. To determine the specific precursor and the product ions of ECT, multiple reaction monitoring (MRM) methods were carried out. The selected ion pair of ECT was 143.1 > 97 and 159.1 > 113.13 for the IS. The ECT’s linearity range in rat plasma was found to be 1-1000 ng/mL, with a recovery rate of 96.48–97.37%. Consistent with FDA guidelines for bio-analytical method validation, the suggested method was validated. The method was efficiently employed to quantify the studied drug in spiked rat plasma with good accuracy and precision with no significant matrix effects. Furthermore, it was effectively used to investigate the pharmacokinetic behavior of ECT in rats after a single oral dose of 30 mg/kg.

B-115 Software Data Processing and Review LC-MS/MS Workflow Improvements for Clinical Research

Abstract Background In the field of quantitative LC-MS/MS, the need for efficient, accurate and user-friendly data review software is paramount, with the data generated in the clinical laboratory meeting local guidelines, as well as rigorous method performance characteristics defined by the FDA Bioanalytical Method Validation and CLSI C62-Ed2 guidelines. The quantitative LC-MS/MS workflow is dependent on interfacing to LMS/LIMS for sample traceability, batch analysis using matrix-matched external calibrators and quality controls for quantification and acceptance, and stable-isotope labeled internal standards to compensate for sample extraction, recovery, matrix effects and analytical variability. The demands on the laboratory to run multiplexed analyte panels and analyze a greater number of samples produces large amounts of analytical data which must still go through batch and quality review before release from the laboratory to the clinicians. The Waters Quan Review Software significantly reduces the burdensome aspects of batch quality control by introducing a series of innovative features. These include improvements in workflow, a modernized interface, and the ability to flag exceptions based on acceptance criteria. Methods Samples for three multiplexed LC-MS/MS panels with previously defined analytical method performance characteristics were acquired using Waters IVD Systems controlled by MassLynx software. The data was processed, quantified, and reviewed using both the existing TargetLynx XS IVD Application Manager and the new Quan Review Software. The three quantitative LC-MS/MS methods were the analysis of four immunosuppressants drugs in whole blood, a twelve steroid panel in plasma and a urine dilute and shoot method for a drugs of abuse panel containing greater than 80 analytes. Each method used matrix matched calibrators and QCs for quantification and batch control with performance characteristics including calibration linearity, carryover, precision and accuracy. Results The analytical performance data generated using the new MS Quan data review software met the same performance criteria as the TargetLynx XS data. For immunosuppressants, the % difference of the mean repeatability and total precision (%CV) for the individual analyte QCs processed using both software was within ±1.3% and ±4.4% respectively. The mean % bias of External Quality Assurance Samples (n=40) for each immunosuppressant processed using both software was within ±7.9%. The modernized, task-oriented accelerated workflows and focused review by exception reduced review time by up to 50%. Conclusions The new Quan Review Software generated results equivalent to the existing TargetLynx XS Application Manager. The software’s enhanced workflow capabilities streamlined the review process in a new user-friendly interface which reduced the training burden enabling scientists to generate high-quality data quickly and easily. The exception focused review functionality allowed tailored rule sets which reduced the time spent reviewing data by up to 50%. Whilst the batch-level review and task-oriented workflow improved batch acceptance and simplified the review of complicated datasets into easy tasks. By integrating these features, Data Review software offers a comprehensive solution that not only improves the efficiency and accuracy of batch quality control but also contributes to the overall reliability and integrity of quantitative LC-MS/MS analysis. For Research Use Only. Not for Use in Diagnostic Procedures.

Assessment of the in vitro metabolic stability of CEP-37440, a selective FAK/ALK inhibitor, in HLMs using fast UPLC-MS/MS method: in silico metabolic lability and DEREK alerts screening.

CEP-37440 was synthesized and supplied by the research and development division of Teva Branded Pharmaceutical Products (West Chester, PA, United States). CEP-37440 represents a newly developed compound that exhibits selectivity inhibition of Focal Adhesion Kinase and Anaplastic Lymphoma Kinase FAK/ALK receptors, demonstrating novel characteristics as an orally active inhibitor. The simultaneous inhibition of ALK and FAK can effectively address resistance and enhance the therapeutic efficacy against tumors through a synergistic mechanism. The objective of this research was to create an LC-MS/MS method that is precise, efficient, environmentally friendly, and possesses a high level of sensitivity for the quantification of CEP-37440 in human liver microsomes (HLMs). The aforementioned approach was subsequently employed to evaluate the metabolic stability of CEP-37440 in HLMs in an in vitro setting. The validation procedures for the LC-MS/MS analytical method in the HLMs were performed following the bio-analytical method validation guidelines set out by the US-FDA. The AGREE program was utilized to assess the ecological impacts of the current LC-MS/MS methodology. The calibration curve linearity was seen in the range of 1-3000ng/mL. The inter-day accuracy (% RE) exhibited a range of -2.33% to 3.22%, whilst the intra-day accuracy demonstrated a range of -4.33% to 1.39%. The inter-day precision (% RSD) exhibited a range of 0.38% to 3.60%, whilst the intra-day precision demonstrated a range of 0.16% to 6.28%. The determination of the in vitro half-life (t1/2) and moderate intrinsic clearance (Clint) of CEP-37440 yielded values of 23.24min and 34.74mL/min/kg, respectively. The current manuscript is considered the first analytical study for CEP-37440 quantification with the application to metabolic stability assessment. These results suggest that CEP-37440 can be categorized as a pharmaceutical agent with a moderate extraction ratio. Consequently, it is postulated that the administration of CEP-37440 to patients may not lead to the accrual of dosages within the human organs. According to in silico P450 metabolic and DEREK software, minor structural alterations to the ethanolamine moiety or substitution of the group in drug design have the potential to enhance the metabolic stability and safety profile of novel derivatives in comparison to CEP-37440.

Assessing pharmacokinetics and drug-drug interactions of the combination therapy of myelofibrosis with ruxolitinib and lenalidomide by a new eco-friendly HPLC method for their simultaneous determination in plasma.

Ruxolitinib (RUX), a Janus kinase 2 (JAK2) inhibitor, and lenalidomide (LEN), an immunomodulatory agent, have recently been proposed as a combined treatment for myelofibrosis (MF). This combination has demonstrated improved efficacy, safety, and tolerability compared to monotherapy. To further refine these findings, an efficient analytical tool is needed to simultaneously determine RUX and LEN concentrations in blood plasma. This tool would enable the study of their pharmacokinetics, drug-drug interactions, and therapeutic monitoring during MF therapy. Unfortunately, such a method has not been existed in the literature. This study presents the first HPLC method with UV detection for the simultaneous quantitation of RUX and LEN in plasma. The method was validated according to the ICH guidelines for bioanalytical method validation. It exhibited linearity in the concentration ranges of 10 to 3150 ng mL- 1 for RUX and 80 to 5200 ng mL- 1 for LEN. The limits of quantitation were determined to be 25 and 90 ng mL- 1 for RUX and LEN, respectively. All other validation parameters were satisfactory. The HPLC-UV method was successfully employed to study the pharmacokinetics and drug-drug interactions of RUX and LEN in rats following oral administration of single doses. The results demonstrated that the pharmacokinetics of both drugs were changed substantially by their coadministration. LEN exhibited synergistic effects on the maximum plasma concentration (Cmax) and total bioavailability of RUX, meanwhile it exhibited diminishing effect on the values of volume of distribution (Vd) and clearance (CL). Additionally, RUX decreased the Cmax and total bioavailability of LEN, meanwhile it increased its Vd and CL. These data suggest that the use of RUX, as a combination with LEN, is a better therapeutic approach for MF, compared with RUX as a monotherapy. The effects of LEN on the pharmacokinetics of RUX should be considered and can be useful in determining the appropriate RUX dosage and dosing regimen to achieve the desired therapeutic effect when used as a combination therapy with LEN. The method's environmental friendliness was confirmed through three comprehensive tools. This method represents a valuable tool for determining the appropriate dosage and dosing regimen of RUX in combination therapy with LEN to achieve the desired therapeutic effect. Furthermore, it can aid in predicting drug distribution in different patients and assessing the drug accumulation or insufficient drug levels in specific body compartments.

Development and Validation of a Simple HPLC–UV‐Based Bioanalytical Method for Estimation of Acalabrutinib in Rat Plasma and Its Application in Evaluation of Drug‐Loaded Nanocrystal Formulation

ABSTRACTA robust and reproducible HPLC–UV‐based bioanalytical method for acalabrutinib (ACP) was developed and validated in rat plasma using prednisone as internal standard. The samples were prepared by single‐step protein precipitation method using methanol with 1% v/v formic acid. A polar C18 stationary phase was used with a combination of 10 mM ammonium acetate (pH 3.5) and methanol (60:40 ratio), flow rate 1.1 mL/min, and 50 µL injection volume. ACP and internal standard were retained at ∼5.96 and 8.29 min, respectively. The method was validated according to the United States Food and Drug Administration and ICH M10 bioanalytical method validation guidelines. It was linear in the calibration range (0.08–5 µg/mL) with as 0.9989; accurate (% bias ≤ 0.50 ± 0.67), and precise (%RSD ≤ 0.986) for any quality control standard. Further, the method was applied in estimating ACP in rat plasma samples collected during in vivo pharmacokinetic study of the bulk drug as well as a nanocrystal (NC) suspension formulation. ACP‐NCs were formulated by bottom‐up solvent–antisolvent precipitation with mean particle size 266.32 ± 29.90 nm, 0.25 ± 0.05 PDI, with 92.7% ± 2.3% yield. The NC formulation improved both in vitro dissolution and in vivo absorption, supporting a further development of ACP‐loaded nanodrug delivery systems.

Determination of amphetamines in hair samples using microextraction by packed sorbent and gas chromatography-mass spectrometry.

Several protocols for the analysis of amphetamine-type stimulants (ATS) in hair have been developed over the years, with microextraction by packed sorbent (MEPS) being used for drugs like opiates, cocaine and ketamine. However, concerning ATS determination in hair samples, this approach has only been applied so far to amphetamine (AMP) and methamphetamine (MAMP). This study aimed at developing and validating a MEPS-based procedure for the determination in hair of not only AMP and MAMP but also of 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 1-(1,3-benzodioxol-5-yl)propan-2-yl (ethyl)amine (MDE) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-aminobutane (MBDB) as well. Hair, 50 mg, was incubated with 1M sodium hydroxide (NaOH) at 45°C overnight, neutralization with 10M hydrochloric acid (HCl) and centrifugation followed. The design of experiments approach was used for MEPS optimization, with the final optimized conditions including conditioning (250 μL methanol and deionized water), loading (18 × 100 μL) and elution (7× 100 μL 2% NH4OH in acetonitrile). The eluted extract was evaporated to dryness and underwent microwave-assisted derivatization with N-methyl-bis(trifluoroacetamide) (MBTFA), and it was afterwards injected onto the gas chromatography-mass spectrometer (GC-MS). The obtained recoveries ranged between 8% and 14% for AMP, 14% and 20% for MAMP, 10% and 15% for MDA, 18% and 28% for MDMA, 25% and 43% for MDE and 34% and 52% for MBDB, and the method was linear from 0.2 to 5.0ng/mg. Precision and accuracy were in accordance with international method validation guidelines. This novel method involving MEPS coupled to GC-MS offers a swift, eco-friendly and cost-effective alternative to traditional procedures for detecting these AMPs in hair samples.

Robust U-HPLC Method Development of Desonide and its Application to In Vitro Release Testing (IVRT) of Topical Cream Products

Background: The study focuses on establishing In Vitro Release Testing (IVRT) parameters for Desonide cream, following the guidelines of the Topical Classification System (TCS), to assess the bioequivalence between the Reference Listed Drug (RLD) and test. Aim: This study aimed to develop a reliable IVRT method using Franz diffusion cells. An environmentally friendly U-HPLC method was created to analyze Desonide in the samples. Objectives: To evaluate the drug release in Desonide products in accordance with SUPAC guidance, quantify the drug concentration using an analytical method, as per bioanalytical method validation guidelines, and ensure that the results meet the acceptance criteria. Linearity was established from 0.50 μg/mL to 40 μg/mL with acceptable regression values. Precision was confirmed three times, with an average % RSD of below 15% for 3 sets of 6QC level sample preparations. Stability tests demonstrated Desonide stability in receptor fluid (LLOQ and ULOQ) for 72 hours at 2-8°C and 25°C. Autosampler stability at LQC and HQC levels was proven at 25°C for 72 hours. Additionally, the stock solution remained stable at both 25°C and 2-8°C for 72 hours. Methods: The study involved evaluating the dosing regimen, release medium, and membrane while optimizing the U-HPLC method based on three variables including column temperature, mobile phase composition, and flow rate. After experimentation, it was determined that Nylon membrane and 0.9% NaCl: Methanol release media (70:30 v/v) with 1000 mg dose were used to maximize the release profile of desonide. Results: The created explanatory strategy is precise, delicate, and exact for measuring Desonide, with satisfactory Limits of Location LOD and Lower Limits of Measurement LLOQ measured at 0.15 and 0.50 ng /mL, respectively. The Regression coefficient r2 was identified to be 0.9996. The degree of Desonide measurement lessening was considered palatable, basically since the recuperation was underneath 30.00, additionally due to the favourable linear relationship watched within the Desonide discharge rates amid the IVRT study. Conclusion: All three generic products analyzed were found to be equivalent to the RLD, meeting for "sameness" outlined in the FDA's SUPAC-SS guidance. A novel U-HPLC method was developed for Desonide, covering the range from 0.5 to 40 μg/ml, with intra and inter-day variability below 2% RSD. Additional characterizations were established, and the stability of Desonide was successfully determined.

Ultra-fast UPLC–MS/MS approach for estimating X-376 in human liver microsomes: Evaluation of metabolic stability via in silico software and in vitro analysis

X-376 is a novel anaplastic lymphoma kinase (ALK) inhibitor that is capable of penetrating the blood brain barrier. This makes it suitable for use in patients with ALK-positive non-small cell lung cancer (NSCLC) who have metastases in the central nervous system. This study developed a highly sensitive, fast, eco-friendly, and reliable UPLC-MS/MS approach to quantify X-376 in human liver microsomes (HLMs). This approach was used to evaluate X-376's metabolic stability in HLMs in vitro. The UPLC-MS/MS analytical technique validation followed US-FDA bio-analytical method validation guidelines. StarDrop software, containing P450 metabolic and DEREK modules, was utilized to scan X-376's chemical structure for metabolic lability and hazardous warnings. X-376 and Encorafenib (ENF as internal standard) were resoluted on the Eclipse Plus C18 column utilizing an isocratic mobile phase method. The X-376 calibration curve was linear from 1 to 3000 ng/mL. The precision and accuracy of this study's UPLC-MS/MS approach were tested for intra- and inter-day measurements. Inter-day accuracy was −1.32% to 9.36% while intra-day accuracy was −1.5% to 10.00%. The intrinsic clearance (Clint) and in vitro half-life (t1/2) of X-376 were 59.77 mL/min/kg and 13.56 min. The high extraction ratio of X-376 supports the 50 mg twice-daily dose for ALK-positive NSCLC and CNS metastases patients. In silico software suggests that simple structural changes to the piperazine ring or group substitution in drug design may improve metabolic stability and safety compared to X-376.

Revolutionizing biomarker analysis one tip at a time—Coupling antibody to Volumetric Absorptive Microsamplers for sensitive LC-MS analysis

The constant pursuit of efficient and cost-effective methods for analyzing human samples drives ongoing innovation in biomedical research. Introduced in 2014, Volumetric Absorptive Microsampling (VAMS) technology ensures fixed blood volume absorption and enhanced sample uniformity. Our study aims to enhance VAMS functionality by integrating sample preparation onto the device primarily used for sample collection. This involves the adsorption of antibodies onto VAMS tips, enabling instant sample clean-up at the time of sample collection. Using human chorionic gonadotropin (hCG) as a model analyte, we evaluated the qualitative and quantitative performance of Affinity-VAMS. First, we showed that adsorbing and covalently binding monoclonal antibodies to VAMS tips results in capturing of the target analyte in comparison to unmodified VAMS. Due to the ease of preparation, we moved forward using antibody adsorption to VAMS tip and optimized the procedure. Optimization of the procedure involved fine-tuning the antibody coupling step, washing process, and determining the optimal amount of antibody required, leading to a streamlined process with significant time savings up to two days. Recovery experiments demonstrate successful capture of the target analyte by the Affinity-VAMS, while matrix effects and stability assessments indicate no negative effects from serum matrix or storage conditions. Finally, quantitative analysis shows promising performance of the Affinity-VAMS in detecting different concentrations of the target analyte in the concentration range between 7.5 - 25 ng·mL−1. The calculated correlation factor was R2 of 0.9988 and limit of detection 2.5 ng·mL−1. The precision lays within the ICH guidelines for bioanalytical method validation. While this report is focused on the proof of concept of the Affinity-VAMS, our findings demonstrate the potential for the usage of modified VAMS in remote sampling and integrated sample processing, enhancing biomarker analysis in clinical and research settings.

Comparison of Analytical Method validation guidelines used for release, stability in Biosimilar Manufacturing process

Comparison of Analytical Method validation guidelines used for release, stability in Biosimilar Manufacturing process

A New and Rapid LC-MS/MS Method for the Determination of Cysteamine Plasma Levels in Cystinosis Patients.

Cystinosis is a rare lysosomal storage disorder caused by autosomal recessive mutations in the CTNS gene that encodes for the cystine transporter cystinosin, which is expressed on the lysosomal membrane mediating the efflux of cystine. Cysteamine bitartrate is a cystine-depleting aminothiol agent approved for the treatment of cystinosis in children and adults. In this study, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of cysteamine levels in plasma samples. This LC-MS/MS method was validated according to the European Medicines Agency (EMA)'s guidelines for bioanalytical method validation. An ultra-performance liquid chromatograph (UPLC) coupled with a 6470 mass spectrometry system was used for cysteamine determination. Our validated method was applied to plasma samples from n = 8 cystinosis patients (median, interquartile range (IQR) = 20.5, 8.5-26.0 years). The samples were collected before cysteamine oral administration (pre-dose) and 1 h after (post-dose). Our bioanalytical method fulfilled the regulatory guidelines for method validation. The cysteamine plasma levels in pre-dose samples were 2.57 and 1.50-3.31 μM (median and IQR, respectively), whereas the post-dose samples reported a cysteamine median concentration of 28.00 μM (IQR: 17.60-36.61). Our method allows the rapid determination of cysteamine plasma levels. This method was successfully used in cystinosis patients and, therefore, could be a useful tool for the evaluation of therapy adherence and for future pharmacokinetic (PK) studies involving a higher number of subjects.

A practical approach to supported liquid extraction and measurement of 18 steroids in plasma and serum by targeted liquid chromatography tandem mass spectrometry

Chromatography combined with mass spectrometry is a gold standard technique for steroid measurement, however the type of sample preparation, the dynamic range and reliability of the calibration curve, the chromatographic separation and mass spectrometry settings ultimately determine the success of the method. The steroid biosynthetic pathway is conserved in higher mammals and literature demonstrates that the concentration ranges of different steroid groups are relatively comparable across species. We sought to develop a robust and reliable multi steroid targeted analysis method for blood that would have wide application across higher mammals. The method was developed following bioanalytical method validation guidelines to standards typically applied to human clinical studies, including isotopically labelled internal standards where at all possible. Here we describe the practical approach to a 96-well supported liquid extraction (SLE) method of extraction from plasma (200 µL) using an Extrahera liquid handling robot (Biotage, Sweden), including quality control samples, followed by a comprehensive separation and targeted LC-MS/MS analysis of 18 steroids in plasma (pregnenolone, progesterone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycortisol, 21-deoxycortisol, cortisol, cortisone, androstenedione, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, estrone, 17β-estradiol and estriol).•SLE in a 96-well format of up to 74 biological plasma samples, enriched with multiple isotopically labelled internal standards, a 12-point aqueous calibration curve, and 6 serum quality controls, designed to monitor long-term performance of the method•Chromatographic separation of multiple steroids along the gradient, with ammonium fluoride mobile phase additive to improve sensitivity, followed by electrospray ionisation and constant polarity switching•Aqueous calibration standards that cover physiologically relevant ranges - high nanomolar glucocorticoids, low nanomolar androgens and picomolar ranges for estrogens and steroid intermediates.

An Isolated Perfused Rat Liver Model: Simultaneous LC-MS Quantification of Pitavastatin, Coproporphyrin I, and Coproporphyrin III Levels in the Rat Liver and Bile.

The isolated perfused rat liver (IPRL) model provides a mechanistic understanding of the organic-anion-transporting polypeptide (OATP/Oatp)-mediated pharmacokinetics in the preclinical evaluation, which often requires the use of control substrates (i.e., pitavastatin) and monitoring endogenous biomarkers (coproporphyrin I and III). This study aimed to develop and validate an LC-MS method allowing the simultaneous quantification of pitavastatin, coproporphyrin I (CPI), and coproporphyrin III (CPIII) in rat liver perfusion matrices (perfusate, liver homogenate, bile). The analysis was performed on a C18 column at 60 °C with 20 μL of sample injection. The mobile phases consisted of water with 0.1% formic acid and acetonitrile with 0.1% formic acid with a gradient flow of 0.5 mL/min. The assay was validated according to the ICH M10 Bioanalytical Method Validation Guideline (2022) for selectivity, calibration curve and range, matrix effect, carryover, accuracy, precision, and reinjection reproducibility. The method allowing the simultaneous quantification of pitavastatin, CPI, and CPIII was selective without having carryover and matrix effects. The linear calibration curves were obtained within various calibration ranges for three analytes in different matrices. Accuracy and precision values fulfilled the required limits. After 60 min perfusion with pitavastatin (1 μM), the cumulative amounts of pitavastatin in the liver and bile were 5.770 ± 1.504 and 0.852 ± 0.430 nmol/g liver, respectively. CPIII was a more dominant marker than CPI in both liver (0.028 ± 0.017 vs 0.013 ± 0.008 nmol/g liver) and bile (0.016 ± 0.011 vs 0.009 ± 0.007 nmol/g liver). The novel and validated bioanalytical method can be applied in further IPRL preparations investigating Oatp-mediated pharmacokinetics and DDIs.

Adapting Fabric Phase Sorptive Extraction as an Innovative Multitool for Sample Transfer and Extraction in Pharmacokinetic Analysis Followed by LC-MS Determination of Levofloxacin in Plasma Samples.

Fabric phase sorptive extraction (FPSE) is a simple microextraction technique that allows analytes to be rescued from matrix components while using a small volume of samples to analyze complex biological systems. This study used FPSE as a microextraction tool and a sample storage and transfer device. Levofloxacin as a model molecule was applied intravenously (IV) to New Zealand male rabbits. The samples were simultaneously extracted by using FPSE and protein precipitation methods. The final solutions were analyzed using LC-MS equipped with an ACE C18 LC Column (150 mm × 4.6 mm, 5 μm) at 25 °C employed in isocratic elution mode using solution A (0.1% formic acid in water)/solution B (0.1% formic acid in acetonitrile) (80:20, v/v). The total analysis time was less than 15 min. The developed method was validated using the ICH M10 bioanalytical method validation and study sample analysis guidelines. The results obtained using FPSE were statistically identical to those obtained using protein precipitation. The plasma samples applied onto FPSE (10 μL onto 1.0 cm × 1.0 cm Biofluid Sampler) were stored in three different temperatures [refrigerator (2-8 °C), at ambient temperature (20 ± 5 °C), and in the stability cabinet (40 °C, 75% humidity)] and three different storage conditions (Eppendorf tubes, plastic containers, and straw paper envelopes). Levofloxacin in plasma samples adsorbed by FPSE biofluid sampler remained stable at 2-8 °C in Eppendorf tubes for at least 1 week. This study showed that FPSE could be used as a sample storage and transfer device for pharmacokinetic applications that need to work with small sample volumes and discard aggressive cold chains to store and transfer the plasma samples.

RP-HPLC Estimation of Clobetasol Propionate and Salicylic Acid using Quality by Design Approach

ABSTRACT: The RP-HPLC method for CLOP and SA estimation from bulk and pharmaceutical dosage from has been developed and validated. For analytical methods to be robust, current ICH guidelines, Q8 to Q11 were suggested use of analytical quality by design (AQbD) includes adoption of current systematic approaches. The proposed method was optimized and developed using Taguchi orthogonal design. The RP-HPLC method parameters were optimized by box- Behnken design. The stationary phase used C18 Princeton column (150mm × 4.6mm × 5µm) with acetonitrile: 0.05M phosphate buffer (pH 2.5, adjustment with by ortho - phosphoric acid) as mobile phase at ratio of 60:40v/v, 1.0 ml/min of flow rate along with UV-Visible wavelength of detection 240 nm. The linearity over concentration 5-15 µg/ml for CLOP and 600-1500 µg/ml for SA (r2 =0.9969 for CLOP and 0.9943 for SA) was found. The retention time for SA was 2.2 min. and CLOP 7.0 minute. The % recovery was found to be 98.0.3 SA and 97.84 or CLOP. As per ICH analytical method validation guidelines [Q2 (R1)], the RP-HPLC method was validated.

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.

Simultaneous Determination of Ibrutinib, Dihydroxydiol Ibrutinib, and Zanubrutinib in Human Plasma by Liquid Chromatography-Mass Spectrometry/Mass Spectrometry.

Ibrutinib and zanubrutinib are Bruton tyrosine kinase inhibitors used to treat mantle cell lymphoma, chronic lymphocytic leukemia, and small lymphocytic lymphoma. Dihydroxydiol ibrutinib (DHI) is an active metabolite of the drug. A liquid chromatography-tandem mass spectrometry method was developed to detect ibrutinib, DHI, and zanubrutinib in human plasma. The method involved a protein precipitation step, followed by chromatographic separation using a gradient of 10 mM ammonium acetate (containing 0.1% formic acid)-acetonitrile. Ibrutinib-d5 was used as an internal standard. Analytes were separated within 6.5 minutes. The optimized multiple reaction monitoring transitions of m/z 441.1 → 304.2, 475.2 → 304.2, 472.2 → 455.2, and 446.2 → 309.2 were selected to inspect ibrutinib, DHI, zanubrutinib, and the internal standards in positive ion mode. The validated curve ranges included 0.200-800, 0.500-500, and 1.00-1000 ng/mL for ibrutinib, DHI, and zanubrutinib, respectively. The precisions of the lower limit of quantification of samples were below 15.5%, the precisions of the other level samples were below 11.4%, and the accuracies were between -8.6% and 8.4%. The matrix effect and extraction recovery of all compounds ranged between 97.6%-109.0% and 93.9%-105.2%, respectively. The selectivity, accuracy, precision, matrix effect, and extraction recovery results were acceptable according to international method validation guidelines. A simple and rapid method was developed and validated in this study. This method was used to analyze plasma concentrations of ibrutinib and zanubrutinib in patients with mantle cell lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, or diffuse large B-cell lymphoma. The selected patients were aged between 44 and 74 years.