Replicates Number for Drug Stability Testing during Bioanalytical Method Validation—An Experimental and Retrospective Approach

Background: The stability of a drug or metabolites in biological matrices is an essential part of bioanalytical method validation, but the justification of its sample size (replicates number) is insufficient. The international guidelines differ in recommended sample size to study stability from no recommendation to at least three quality control samples. Testing of three samples may lead to results biased by a single outlier. We aimed to evaluate the optimal sample size for stability testing based on 90% confidence intervals. Methods: We conducted the experimental, retrospective (264 confidence intervals for the stability of nine drugs during regulatory bioanalytical method validation), and theoretical (mathematical) studies. We generated experimental stability data (40 confidence intervals) for two analytes—tramadol and its major metabolite (O-desmethyl-tramadol)—in two concentrations, two storage conditions, and in five sample sizes (n = 3, 4, 5, 6, or 8). Results: The 90% confidence intervals were wider for low than for high concentrations in 18 out of 20 cases. For n = 5 each stability test passed, and the width of the confidence intervals was below 20%. The results of the retrospective study and the theoretical analysis supported the experimental observations that five or six repetitions ensure that confidence intervals fall within 85–115% acceptance criteria. Conclusions: Five repetitions are optimal for the assessment of analyte stability. We hope to initiate discussion and stimulate further research on the sample size for stability testing.

Tandem Mass Spectrometry (MS/MS) in the Preclinical Pharmacokinetic Study of a Highly Prescribed SNRI Drug for Depression and Its Application to Oral Extended Release Solid Dosage Formulations in Rabbits

Preclinical pharmacokinetic (pK) studies in animal models during the formulation development phase give preliminary evidence and near clear picture of the pK behavior of drug and/or its dosage forms prior to clinical studies on humans and help in tailoring of the dosage form according to the expected and requisite clinical behavior. The present work reports first of its kind preclinical pK study on oral extended release (ER) solid dosage formulations of venlafaxine (VEN) in New Zealand White rabbits. The VEN is a highly prescribed and one of the safest and most effective therapeutic agents used in the treatment different types of depression disorders worldwide. The LC-MS/MS bioanalytical method developed for this purpose demonstrated enough reliability in simultaneously quantitating VEN and its equipotent metabolite O-desmethylvenlafaxine (ODV) in rabbit plasma. The method described uses solid phase extraction for sample preparation followed by an ultra-fast LC-MS/MS analysis. The chromatographic separation was achieved isocratically with a predominantly polar mobile phase by employing RPLC. The triple quadrupole LC/MS/MS system operated in MRM mode used an ESI probe as an ion source in positive polarity. The validation results are within the permissible limits of US FDA recommendations and acceptance criteria for bioanalytical method validation.

Characterization of Alpelisib in Rat Plasma by a Newly Developed UPLC-MS/MS Method: Application to a Drug-Drug Interaction Study

Alpelisib, an oral selective and small-molecule phosphoinositide 3-kinase inhibitor, was lately approved in the United States to treat breast cancer. A sensitive method to quantify alpelisib levels in rat plasma on the basis of ultra-performance liquid chromatography–tandem mass spectrometry technique was established and validated, which was successfully employed to explore the effects of CYP3A4 inhibitors on alpelisib pharmacokinetics in rats. A C18 column named Acquity UPLC BEH C18 was applied to achieve the separation of alpelisib and internal standard duvelisib after protein precipitation with acetonitrile. The mobile phase in this study had two components, namely, acetonitrile and water having 0.1% formic acid, and a program with gradient elution method was used at a flow rate of 0.40 ml/min. Mass spectrometry in a positive multiple reaction monitoring mode was operated. In the scope of 1–5,000 ng/ml, this assay had excellent linearity. Our newly developed assay was verified in all aspects of bioanalytical method validation, involving lower limit of quantification, selectivity, accuracy and precision, calibration curve, extraction recovery, matrix effect, and stability. Then, this assay was used to detect the plasma levels of alpelisib from a drug-drug interaction investigation, where ketoconazole remarkably increased the plasma concentration of alpelisib and changed alpelisib pharmacokinetics more than itraconazole. This study will help better understand the pharmacokinetic properties of alpelisib, and further clinical studies should be done to confirm this result in patients.

Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS

Objectives Bile acids serve as biomarkers for liver function and are indicators for cholestatic and hepatobiliary diseases like hepatitis, cirrhosis, and intrahepatic cholestasis of pregnancy (ICP). Sulfation and renal excretion of bile acids are important elimination steps. The power of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allows specific profiling of primary and secondary bile acids as well as their sulfated counterparts. Methods Twenty-four sulfated and non-sulfated primary and secondary bile acids were quantified in urine with 15 corresponding stable isotope labeled internal standards by using two-dimensional UHPLC-MS/MS. The sample preparation was based on a simple dilution with a methanolic zinc sulfate solution followed by an automated online solid phase extraction clean up. Results The validation results of the method fulfilled the criteria of the European Medicine Agency (EMA) “Guideline on bioanalytical method validation”. To verify fitness for purpose, 40 urine samples were analyzed which showed an average of 86% sulfation, 9.1% taurine-conjugation, 14% non-conjugation, and 77% glycine-conjugation rates. Conclusions Lossless one-pot sample preparation, automated sample purification, and high number of internal standards are major innovations of the presented profiling method, which may allow diagnostic application of BA profiling in the future.

Optimization of an ammonia assay based on transmembrane pH-gradient polymersomes

Reliable ammonia quantification assays are essential for monitoring ammonemia in patients with liver diseases. In this study, we describe the development process of a microplate-based assay for accurate, precise, and robust ammonia quantification in biological fluids, following regulatory guidelines on bioanalytical method validation. The assay is based on transmembrane pH-gradient polymersomes that encapsulate a pH-sensitive ratiometric fluorophore, the fluorescence signal of which correlates with the ammonia concentration in the sample. Using a four-parameter logistic regression, the assay had a large quantification range (30–800 μM ammonia). As for selectivity, the presence of amino acids or pyruvate (up to clinically relevant concentrations) showed no assay interference. In samples with low bilirubin levels, polymersomes containing the fluorophore pyranine provided accurate ammonia quantification. In samples with high bilirubin concentrations, billirubin’s optical interference was alleviated when replacing pyranine with a close to near-infrared hemicyanine fluorophore. Finally, the assay could correctly retrieve the ammonia concentration in ammonia-spiked human plasma samples, which was confirmed by comparing our measurements with the data obtained using a commercially available point-of-care device for ammonia.

Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples

Highly selective multifunctional magnetic nanoparticles containing a thermoresponsive polymer shell were developed and used in the sample pretreatment of urine for the assessment of lysozymuria in leukemia patients. Crosslinked poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) was grown onto silica-coated magnetic nanoparticles by reversible addition fragmentation chain transfer (RAFT) polymerization. The lysozyme binding property of the nanoparticles was investigated as a function of time, protein concentration, pH, ionic strength and temperature and their selectivity was assessed against other proteins. High-abundant proteins, like human serum albumin and γ-globulins did not interfere with the binding of lysozyme even at elevated concentrations characteristic of proteinuria. A sample cleanup procedure for urine samples has been developed utilizing the thermocontrollable protein binding ability of the nanoparticles. Method validation was carried out according to current bioanalytical method validation guidelines. The method was highly selective, and the calibration was linear in the 25 to 1000 µg/mL concentration range, relevant in the diagnosis of monocytic and myelomonocytic leukemia. Intra- and inter-day precision values ranged from 2.24 to 8.20% and 1.08 to 5.04%, respectively. Intra-day accuracies were between 89.9 and 117.6%, while inter-day accuracies were in the 88.8 to 111.0% range. The average recovery was 94.1 ± 8.1%. Analysis of unknown urine samples in comparison with a well-established reference method revealed very good correlation between the results, indicating that the new nanoparticle-based method has high potential in the diagnosis of lysozymuria.

Simultaneous determination of duloxetine and 4-hydroxy duloxetine glucuronide in human plasma and back-conversion study

Aim: To develop an LC-MS/MS method for simultaneous determination of duloxetine and its metabolite, 4-hydroxy duloxetine glucuronide (4HDG) in human plasma and to investigate the potential back-conversion of 4HDG to duloxetine using stability study. Materials & methods: The LC-MS/MS method was validated according to the EMA and USFDA Bioanalytical Method Validation Guidelines and applied to pilot bioequivalence study. Results & conclusion: The method validation results were within the acceptance limits. The stability study and incurred sample reanalysis results ruled out the occurrence of back-conversion. The study highlighted the conduct of back-conversion test and the advantages of LC-MS/MS method in terms of sensitivity, specificity and low consumption of organic solvents.

Applying context of use to quantitative polymerase chain reaction method validation and analysis: a recommendation from the European Bioanalysis Forum

Polymerase chain reaction (PCR) is widely used in various fields of laboratory testing, ranging from forensic, molecular biology, medical and diagnostic applications to a wide array of basic research purposes. COVID-19 infection testing has brought the three-letter PCR abbreviation into the vocabulary of billions of people, making it likely the most well-known laboratory test worldwide. With new modalities and translational medicine gaining importance in pharmaceutical research and development, PCR or more specifically, quantitative PCR (qPCR) is now becoming a standard tool in the (regulated) bioanalytical laboratory, driving the bioanalytical community to define best practices for method development, characterization and validation. In absence of specific guidance from health authorities, qPCR may be vulnerable to scope creep from pharmacokinetics (PK) assay validation as defined in bioanalytical method validation guidance/guidelines. In this manuscript, the European Bioanalysis Forum builds a rationale for applying context of use principles when defining requirements for qPCR assay performance and validation criteria.

A Sensitive LC–MS/MS Method for the Quantification of 3-Hydroxybenzo[a]pyrene in Urine-Exposure Assessment in Smokers and Users of Potentially Reduced-Risk Products

Benzo[a]pyrene (BaP), a human carcinogen, is formed during the incomplete combustion of organic matter such as tobacco. A suitable biomarker of exposure is the monohydroxylated metabolite 3-hydroxybenzo[a]pyrene (3-OH-BaP). We developed a sensitive LC–MS/MS (liquid chromatography coupled with tandem mass spectrometry) method for the quantification of urinary 3-OH-BaP. The method was validated according to the US Food and Drug Administration (FDA) guideline for bioanalytical method validation and showed excellent results in terms of accuracy, precision, and sensitivity (lower limit of quantification (LLOQ): 50 pg/L). The method was applied to urine samples derived from a controlled clinical study to compare exposure from cigarette smoking to the use of potentially reduced-risk products. Urinary 3-OH-BaP concentrations were significantly higher in smokers of conventional cigarettes (149 pg/24 h) compared to users of potentially reduced-risk products as well as non-users (99% < LLOQ in these groups). In conclusion, 3-OH-BaP is a suitable biomarker to assess the exposure to BaP in non-occupationally exposed populations and to distinguish not only cigarette smokers from non-smokers but also from users of potentially reduced-risk products.