Incurred Sample Stability Research Articles

Pharmacokinetic profile and incurred sample stability of hydroxychloroquine in Volumetric Absorptive Microsampling (VAMS) using high-performance liquid chromatography-photodiode array

Background: Hydroxychloroquine (HCQ) is a quinoline compound derived from chloroquine used to treat SLE. An in vitro stability evaluation was conducted to develop and validate the hydroxychloroquine analysis method. However, an assessment of the incurred sample stability is needed to ensure drug effectiveness, as in vitro evaluation doesn't reflect drug metabolism processes in the body. Objective: This research aims to obtain a pharmacokinetic profile of hydroxychloroquine using Volumetric Absorptive Microsampling (VAMS) as a safe sampling technique to use during the COVID-19 pandemic and evaluate the incurred sample stability of hydroxychloroquine samples. Methods: The chromatographic conditions used were column C18 (Waters, XBridge; 250 × 4.6 mm; 5μm); mobile phase acetonitrile-1% diethylamine (65:35); flow rate of 0.8 mL/min; column temperature 45°C; PDA detector analysis wavelength of 332 nm; and chloroquine as internal standard. Results: The pharmacokinetic profile of hydroxychloroquine in VAMS samples gave results that the Cmax ranged from 322.61-505.32 ng/mL with an average of 425.33 ± 65.90 ng/mL; tmax was 4 hours; mean t1/2 was 23.32 ± 9.65 hours; mean AUC0-72 was 5103.63 ± 1419.66 ng.h/mL; mean AUC0-∞ was 5763.97 ± 2155.26 ng.h/mL, and the AUC ratio was above 80%. Conclusion: The incurred sample stability of hydroxychloroquine in VAMS met the 2011 EMEA Bioanalytical Guideline requirements up to day 30.

Clinical validation and assessment of feasibility of volumetric absorptive microsampling (VAMS) for monitoring of nilotinib, cabozantinib, dabrafenib, trametinib, and ruxolitinib

Volumetric absorptive microsampling (VAMS) has emerged as a minimally invasive alternative to conventional sampling. However, the applicability of VAMS must be investigated clinically. Therefore, the feasibility of at-home sampling was investigated for the kinase inhibitors nilotinib, cabozantinib, dabrafenib, trametinib and ruxolitinib and evaluated regarding the acceptance of at-home microsampling, sample quality of at-home VAMS and incurred sample stability. In addition, clinical validation including three different approaches for serum level predictions was performed. For this purpose, VAMS and reference serum samples were collected simultaneously. Conversion of VAMS to serum concentration was based either on a linear regression model, a hematocrit-dependent formula, or using a correction factor. During the study period 591 VAMS were collected from a total of 59 patients. The percentage of patients who agreed to perform VAMS at home ranged from 50.0 % to 84.6 % depending on the compound. 93.1 % of at-home VAMS were collected correctly. Regarding the drug stability in dried capillary blood, no stability issues were detected between on-site and at-home VAMS. Linear regression showed a strong correlation between VAMS and reference serum concentrations for nilotinib, cabozantinib, dabrafenib and ruxolitinib (r 0.9427 – 0.9674) and a moderate correlation for trametinib (r 0.5811). For clinical validation, the acceptance criteria were met for all three approaches for three of the five kinase inhibitors. Predictive performance was not improved by using individual hematocrit instead of population hematocrit and was largely independent of conversion model. In conclusion, VAMS at-home has been shown to be feasible for use in routine clinical care and serum values could be predicted based on the measured VAMS concentration for nilotinib, cabozantinib, and dabrafenib.

Pharmacokinetic Study and Incurred Sample Stability of Esomeprazole in Dried Blood Spot Sample Using High Performance Liquid Chromatography-Photodiode Array

In the past years, Esomeprazole (EMP) was analyzed in human plasma samples, which still has stability issues; thus, the new biosampling technique known as Dried Blood Spot (DBS) might solve the issue. This research aims to evaluate the incurred sample stability of esomeprazole in dried blood spot using high performance liquid chromatography-photodiode array with lansoprazole as an internal standard. The analytical separation was performed on a C-18 column (Waters, Sunfire™ 5 μm; 250 × 4.6 mm) at 40 °C. The mobile phase used was acetonitrile–phosphate buffer pH 7.6 (40:60% v/v) with a flow rate of 1.00 mL/min; and was detected at 300 nm. The analyte was extracted from dried blood spot by methanol. Incurred sample stability was evaluated from 6 healthy subjects on day 0, 7, 14, and 28, respectively. This method was linear in the range concentration of 70–1400 ng/mL with r > 0.98. Pharmacokinetic study shows that the average of AUC0–t of EMP in the DBS sample was 1765.41 ngh/mL. The highest percent difference value of esomeprazole’s incurred samples stability on day 7, 14, and 28 from 6 healthy subjects were 9.81%. This result fulfilled the acceptance criteria, which is the percent difference should not be greater than 20%, and 67% of total samples have to fulfill the criteria. The incurred sample stability result showed that esomeprazole was stable in the DBS sample at least until 28 days with the highest value of percent difference is 9.81%.

New approaches for biomarker stability determination in regulated bioanalysis: trending, bridging and incurred samples.

Aim: Determining the stability of biomarkers continues to present challenges. Disease states, complex matrices and differences between recombinant and endogenous analytes require new approaches to maintain stability and measure it. In this report, we determine stability for two assays using trending and statistical analysis. Methodology & results: Monitoring trends helps identify out of specification measurements and determine whether concerns are due to the stability of the analyte. We also describe challenges presented when measuring arginase activity in human sputum, a complex matrix, for respiratory diseases. We controlled preanalytical protease activity and collection heterogeneity and monitored incurred sample stability to improve stability of arginine. Conclusion: These new approaches to achieving and determining biomarker stability may provide solutions for increasingly complex biomarker measurements.

PHARMACOKINETIC PROFILE AND INCURRED ESOMEPRAZOLE SAMPLE STABILITY IN PLASMA USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY - PHOTODIODE ARRAY

Objective: Esomeprazole (ESO) is one of the proton-pump inhibitors and is used to treat gastroesophageal reflux. It is sensitive to low pH, heat,moisture, and oxidation, which often means that ESO in clinical samples is degraded at the time of storage, affecting analysis results. This study aimedto analyze the in vivo stability of ESO in subjects’ plasma samples by testing the incurred sample stability (ISS) of ESO in plasma following 7, 14, and28 days of storage at two concentrations close to Cmax and one concentration in the elimination phase.Methods: Samples were analyzed using high-performance liquid chromatography with a C18 column with detection at 300 nm using a photodiodearray detector. Lansoprazole was used as an internal standard.Results: The ESO pharmacokinetics profile in the plasma samples yielded the values of Cmax 704.57–1425.85 ng/mL; tmax is 2.25 h; and AUC0-t is2444 ng.h/mL. ISS testing of plasma samples values were 6.50%, 5.73%, and 4.57% on first Cmax concentration; 3.55%, 4.84%, and 3.68% on 2nd Cmaxconcentration; and 4.04%, 4.80%, and 4.98% on elimination phase concentration.Conclusion: ISS testing results of plasma samples from six healthy subjects who were administered doses of 40 mg of ESO stored for 28 days showedthat it fulfilled the acceptance criteria (<20%) of the 2011 EMEA Bioanalytical Guidelines with a %diff value in all incurred samples of 6.5%.

Incurred sample stability of ASP3258 in the presence of its acyl glucuronide

Incurred sample stability of ASP3258 in the presence of its acyl glucuronide

Development and validation of LC-MS/MS assay for the determination of Butoconazole in human plasma: Evaluation of systemic absorption following topical application in healthy volunteers

Butoconazole is an imidazole antifungal that is more effective than miconazole and clotrimazole for treatment of vaginal candidiasis. A highly sensitive tandem mass spectrometric assay was developed and validated to evaluate systemic absorption of Butoconazole following intravaginal administration. Chromatographic separation was achieved using Waters Xterra C18 column (3µm, 3.0×50.0mm). Liquid-liquid extraction using tert-butyl methyl ether was used for preparation of plasma samples. The mobile phase was solvent A: 0.1% formic acid in water and solvent B: acetonitrile: methanol (30:70, v/v), using gradient elution mode at 0.5mL/min. Detection at positive electrospray ionization in the MRM mode was then employed. Analysis was carried out within 5.5min over a linear concentration range of 0.10–30.00ng/mL. Validation was carried out according to US FDA guidelines for bioanalytical method validation. Matrix effect, recovery efficiency and process efficiency have been investigated for the analyte and internal standard in neat solvent, post-extraction matrix and plasma. The mean percentage recoveries were higher than 80%, the accuracy was 93.51–106.85% and the RSD was below 10% throughout the studied concentration range. Results indicated sufficient stability of the target analyte in plasma at the employed experimental conditions. Results of incurred sample re-analysis and incurred sample stability revealed less than 5% variability. The applicability of the assay for monitoring of the systemic absorption of Butoconazole following intra vaginal application to healthy volunteers was demonstrated. Results confirmed that Butoconazole was detected shortly after intra vaginal administration with Cmax and tmax of 30ng/mL and 6h, respectively.

Stability assessment in ligand-binding assays: a critical parameter for data integrity.

Stability assessment in ligand-binding assays: a critical parameter for data integrity.

Effects of blood processing and sample storage on the stability of biotherapeutics and anti-drug antibodies.

Pre-analytical factors such as sample processing, handling or storage could affect the stability of biotherapeutics and anti-drug antibodies in clinical samples, potentially impacting the pharmacokinetic and immunogenicity assessments. We used sarilumab, a fully human IgG1 monoclonal antibody, and evaluated the stability of sarilumab (both functional and bound forms) and anti-sarilumab antibodies in blood samples during serum collection and the impact of various processing conditions on the analyte stability in serum for long-term storage. We also assessed the incurred sample stability of these analytes in samples from clinical studies. Assessment of analyte stability can provide relevant information about sample stability under different pre-analytical conditions and improve the confidence in the validity of bioanalytical data generated.

7th GCC Insights: incurred samples use; fit-for-purpose validation, solution stability, electronic laboratory notebook and hyperlipidemic matrix testing.

The Global CRO Council for Bioanalysis (GCC), a global independent consortium bringing together many senior level CRO representatives, was created in 2010 in order to openly discuss and share opinions on scientific and regulatory issues specific to the bioanalytical field [1]. Since its formation, the GCC members have met on a regular basis to discuss various topics and challenges faced by bioanalytical CRO companies. Several conference reports of past GCC meetings were published to share the discussions held during these events [2–4]. In addition, the GCC also published several influential White Papers on topics of widespread interest in bioanalysis [5–9]. These White Papers provide unified GCC recommendations that are helpful to the global bioanalytical community. The 7th GCC Closed Forum for Bioanalysis took place in Long Beach, CA, USA on 8 April 2013, one day before the start of the 7th Workshop on Recent Issues in Bioanalysis (WRIB). In attendance were 46 senior-level participants from six countries, representing 37 bioanalytical CRO companies/sites. This event represented a good opportunity for bioanalytical experts from CROs to share and discuss these issues of concern for the outsourcing industry. The 7th GCC Closed Forum was chaired by Mario Rocci, who started the meeting by communicating the official admonition statement, as has been done in all previous editions [1]. Prior to initiating discussions, participants introduced themselves. As in previous GCC meetings, multiple topics of 7th GCC Insights: incurred samples use; fitfor-purpose validation, solution stability, electronic laboratory notebook and hyperlipidemic matrix testing

Recommendations on Incurred Sample Stability (ISS) by GCC

The topic of incurred sample stability (ISS) has generated considerable discussion within the bioanalytical community in recent years. The subject was an integral part of the seventh annual Workshop on Recent Issues in Bioanalysis (WRIB) held in Long Beach, CA, USA, in April 2013, and at the Global CRO Council for Bioanalysis (GCC) meeting preceding it. Discussion at both events focused on the use of incurred samples for ISS purposes in light of results from a recent GCC survey completed by member companies. This paper reports the consensus resulting from these discussions and serves as a useful reference for depicting ISS issues and concerns, summarizing the GCC survey results and providing helpful recommendations on ISS in the context of bioanalytical method development and application.

A semi-automated LC–MS/MS method for the determination of LCI699, a steroid 11β-hydroxylase inhibitor, in human plasma

A novel liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of LCI699 was developed and validated with dynamic ranges of 0.0500-50.0 ng/mL and 1.00-1,000 ng/mL using 0.0500 mL and 0.100mL, respectively, of human plasma. LCI699 and the internal standard, [M+6]LCI699, were extracted from fortified human plasma via protein precipitation. After transfer or dilution of the supernatant followed by solvent evaporation and/or reconstitution, the extract was injected onto the LC-MS/MS system. Optimal chromatographic separation was achieved on an ACE C18 (50 mm × 4.6mm, 3 μm) column with 30% aqueous methanol (containing 0.5% acetic acid and 0.05% TFA) as the mobile phase run in isocratic at a flow rate of 1.0 mL/min. The total analysis cycle time is approximately 3.5 min per injection. The addition of an ion-pair reagent, TFA (0.05%, v/v), to the mobile phases significantly improved the chromatographic retention and resolution of the analyte on silica based reversed-phase column. Although addition of TFA to the mobile phase suppresses the ESI signals of the analyte due to its ion-pairing characteristics in the gas phase of MS source, this negative impact was effectively alleviated by adding 0.5% acetic acid to the mobile phase. The current method was validated for sensitivity, selectivity, linearity, reproducibility, stability and recovery. For the low curve range (0.0500-50.0 ng/mL), the accuracy and precision for the LLOQs (0.0500 ng/mL) were -13.0 to 2.0% bias and 3.4-19.2% CV, respectively. For other QC samples (0.100, 6.00, 20.0 and 40.0 ng/mL), the precision ranged from 1.2 to 9.0% and from 3.8 to 8.8% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from -11.3 to 8.0% and -7.2 to 1.6% bias, respectively, in the intra-day and inter-day batches. For the high curve range (1.00-1,000 ng/mL), the accuracy and precision for the LLOQs (1.00 ng/mL) were 1.0-15.0% bias and 7.4-9.2% CV, respectively. For the other QC samples (3.00, 20.0, 200 and 750 ng/mL), the precision ranged from 0.8 to 7.0% and from 1.9 to 5.2% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from -2.5 to 4.0% and 0.7-1.0% bias, respectively, in the intra-day and inter-day batches. Additional assessments of incurred sample stability (ISS) and incurred sample reanalysis (ISR) were conducted to demonstrate the ruggedness and robustness of the assay method. The absence of adverse matrix effect and carryover was also demonstrated. The validated method was successfully used to support rapid turnaround human pharmacokinetic studies.

Conference Report: ‘Less Is More‘: Defining Modern Bioanalysis

The 4th Open Symposium of the European Bioanalytical Forum entitled 'Less is More' was held on 16-18 November 2011 at the Hesperia Tower Hotel, Barcelona, Spain. More than 50 interesting presentations were delivered covering areas with interest for the small- and large-molecule community - biomarker validation; regulations, including an update on new and emerging guidelines and on Global harmonization; technology updates; incurred sample stability; microdosing; dried blood spots and microsampling; challenges of 'free' and 'total' macromolecule quantification; stability issues in ligand binding assays or anomalous results. In excess of 450 delegates from more than 170 institutes and companies (industry, regulators and academia) from all global regions participated in the open and stimulating discussions. This manuscript provides an overview of the highlights discussed at the meeting.

Bioanalysis in clinical development of tasquinimod using liquid chromatography/tandem mass spectrometry

Tasquinimod (ABR-215050) is an oral drug in clinical development for treatment of patients with castrate resistant prostate cancer. This paper describes a method for the determination of tasquinimod in human plasma. The method is based on liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) using stable isotope labeled tasquinimod as internal standard (IS). The plasma samples were processed by protein precipitation using acidic acetonitrile containing the IS. The precipitated samples were centrifuged and the supernatant was injected directly into the LC–MS/MS system. Chromatographic separation was performed on a reversed phase column using fast gradient elution, with a total run cycle time of 4 min. The method was validated with respect to accuracy, precision, dynamic range, lower limit of quantification, selectivity and robustness. Furthermore, the stability of tasquinimod in spiked plasma, in processed extracts and in incurred samples was thoroughly studied. The method was validated in the range of 1.0–2400 nmol/L, defining the lower and upper limits of quantification. The repeatability, reproducibility and overall bias were 1.5–7.1%, 3.5–7.4%, and 1.3–4.7%, respectively, in the range of 1–2000 nmol/L. Excellent selectivity was demonstrated in the validation, as well as in study samples from both healthy volunteers and cancer patients. Robustness was demonstrated by the calculated carry-over as low as 0.06%, and by an incurred sample reproducibility (ISR) experiment where 97% of the reanalyzed samples fulfilled the acceptance criteria of 20% deviation from initial analysis result. Also, tasquinimod was found to be stable in all investigated matrices, including in incurred samples. In an incurred sample stability (ISS) investigation, tasquinimod was demonstrated to be stable for 24 months, and 97% of the reanalyzed samples were within 20% from the initial analysis result. In conclusion, the method was demonstrated to be accurate, precise, robust and reliable for the determination of tasquinimod. The method was successfully used in several clinical studies for the support of pharmacokinetic and pharmacodynamic evaluations.

Formation of a Global Contract Research Organization Council for Bioanalysis

Background Over the last year, the bioanalytical community strongly expressed their need for international harmonization of bioanalytical guidances through numerous international meetings and publications, and this need was acknowledged by several regulatory agencies [1–5]. Following the 4th Calibration and Validation Group (CVG) Workshop on Regulated Bioana lysis hosted in Montreal (April 2010), a unanimous consensus was reached for the global bioanalytical community to identify non-prescriptive, s ciencebased language that could form the basis of a proposed guidance document on bioana lysis [6]. Ideally, such guidance language would describe the rationale behind each bioanalytical requirement and would be presented for consideration by agencies and industry worldwide. A recent concrete action taken towards such global harmonization of bioanalytical guidances was the creation of the Global Bioana lysis Consortium (GBC). Drawing from representatives of scientific associations with involvement in regulated bioana lysis across the globe, the objective of the GBC is to merge existing or emerging bioanalytical guidances and create a unified document that can be presented to the regulatory authorities in various countries and regions. The development of the GBC is currently in process, with the intention to present plans and updates at upcoming bioanalytical meetings and to seek input from the scientists conducting bioana lysis [7]. At the 4th CVG Bioana lysis Workshop in Montreal, Canada, several Contract Research Organizations (CROs) highlighted the importance of having a strong and cohesive CRO contribution to the global harmonization process. Having CROs, academic laboratories and pharma ceutical industries appropriately represented was viewed as critical to identifying optimum language. Consequently, the proposal to build a Global CRO Council (GCC) arose, designed to be a distinct group consisting exclusively of bioanalytical CRO members conducting r egulated bioana lysis business. To initiate creation of the GCC, a special closed forum was held in Montreal on 14 September 2010, where 41 executive representatives from 32 bioanalytical CROs were present to discuss scientific harmonization issues and the institution of the GCC. This first CRO Formation of a Global Contract Research Organization Council for Bioana lysis

Determination of ticagrelor and two metabolites in plasma samples by liquid chromatography and mass spectrometry

Rapid and sensitive analytical methods using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for the determination of ticagrelor, the first reversible oral platelet P2Y 12 receptor inhibitor, and its metabolites AR-C124910XX and AR-C133913XX in human plasma. Ticagrelor and its metabolites were extracted using protein precipitation with acetonitrile. Chromatographic separations were performed on reversed phase columns and detection using atmospheric pressure chemical ionization (APCI). Ticagrelor and AR-C124910XX were analyzed in the same assay, with the internal standard, d7-ZD6140, on a C18 column using negative ionization; AR-C133913XX analyzed separately on a phenyl column using positive ionization. Full validation of the methods was performed including selectivity, lower limit of quantification, accuracy, precision stability and incurred sample reproducibility and incurred sample stability. Total analytical run time was short (2 min). Calibration curves were established in the range 5–5000 ng/mL for ticagrelor, 2.5–2500 ng/mL for AR-C124910XX and 2–1000 ng/mL for AR-C133913XX. Lower limits of quantification for ticagrelor, AR-C124910XX and AR-C133913XX were determined to be 5, 2.5 and 2.0 ng/mL, respectively from 100 μL of human plasma. For ticagrelor, AR-C124910XX and AR-C133913XX, mean intra-batch accuracy was 91.9–109.0%, 86.8–109.2% and 100.5–112.0%, respectively; intra-batch precision was 4.0–8.4%, 5.2–16.9% and 3.9–12.3%, respectively. The methods were also applied to quantification of ticagrelor, AR-C124910XX and AR-C133913XX in rabbit, rat, mouse and marmoset, using 25 μL of animal plasma. A modified methodology was developed to quantify ticagrelor and AR-C124910XX in plasma from dog and cynomolgus monkey. Human incurred samples were found to generate consistent reproducibility and stability results. This method was successfully applied to determine plasma concentrations following administration of ticagrelor in human volunteers and patients, and animal safety evaluation studies. This validated methods has the advantages of being straightforward, robust and allows a fast throughput of samples.