Measurement of bempedoic acid and its keto metabolite in human plasma and urine using solid phase extraction and electrospray LC-MS/MS.

Bempedoic acid, a new therapeutic for treatment of hypercholesterolemia, inhibits hepatic ATP-citrate lyase in the cholesterol synthesis pathway after its conjugation with coenzyme A. Sensitive and selective methods were required to study the pharmacokinetic behavior of bempedoic acid and its active 8-keto metabolite in clinical studies. A mixed mode anion exchange extraction on 96-well plates was developed to favor high, selective recoveries of these dicarboxylic acids from urine or plasma. Adsorptive losses in urine led to inaccurate measurements unless samples were acidified and diluted with isopropanol prior to any specimen transfers. Tandem mass spectrometry with negative ion electrospray ionization permitted lower limits of measurement of 20 and 10 ng/mL for the drug and metabolite in either matrix. The methods were validated to current regulatory standards and have been the basis for pharmacokinetic measurements in 26 clinical studies involving over 15,000 samples.

Quantitation of free and total N-acetylcysteine amide and its metabolite N-acetylcysteine in human plasma using derivatization and electrospray LC-MS/MS.

Studies of N-acetylcysteine amide (NACA) in nonclinical models have demonstrated various antioxidant, anti-apoptotic, anti-inflammatory and neuroprotective effects, and it is currently being developed as a treatment for retinitis pigmentosa. Sensitive LC-MS/MS methods were developed and validated to quantitate reduced and total NACA and its major metabolite, N-acetylcysteine (NAC), in human plasma to support clinical studies involving NACA. To trap and stabilize reduced NACA and NAC at the time of collection, whole blood was immediately treated with 2-chloro-1-methylpyridinium iodide (CMPI) to convert free thiols to 1-methylpyridinyl thioether derivatives. Plasma was harvested and frozen until samples were assayed using protein precipitation and an LC-MS/MS separation based on hydrophilic-interaction chromatography (HILIC). To process NACA and NAC present as disulfides, an intermediate portion of the extract was further subjected to reduction with tris(2-carboxyethyl) phosphine; the released thiols were then reacted with CMPI, extracted, and analyzed as before, to measure total thiols. The method for NACA and NAC, whether free/reduced or total, covered a range from 50 ng/mL to 50 µg/mL in human plasma and required a single 25 µL plasma sample. Up to 180 samples could be assayed in a single session. The inter-run mean bias and precision (%CV) were within ±5% for the free thiol method and within ±8.5% for the total thiol method. Benchtop, freeze/thaw, and long-term stability were evaluated and acceptable. The NAC/NACA method applied to a clinical study demonstrated incurred sample reproducibility of 95.5% for NAC and 99.1% for NACA.

Measurement of lidocaine and 2,6-dimethylaniline in minipig plasma, skin, and dermal tapes using UHPLC with electrospray MS/MS.

Sensitive LC-MS/MS methods were developed to measure lidocaine and its metabolite 2,6-dimethylaniline (2,6-DMA) with application to transdermal studies. The methods for lidocaine in minipig plasma, tissue biopsies, and dermal tapes utilized mixed mode/SCX solid phase extraction, with lower quantitation limits of 25 pg/mL in plasma, 15 ng/g tissue, and 5 ng/tape. 2,6-DMA was measured in plasma and skin tissue homogenates by ultrafiltration and (for tissue) by further derivatization with 4-methoxybenzoyl chloride to form the corresponding benzamide derivative, which extended the lower limit of quantitation to 200 pg/mL. The methods allowed local measurement of lidocaine in stratum corneum, punch biopsies, and plasma and of 2,6-DMA in plasma and biopsies obtained from minipigs dosed with experimental transdermal formulations. Quantitation limits were approximately 7-fold lower than previously reported for lidocaine and 3-fold lower for 2,6-DMA.

8th GCC: consolidated feedback to US FDA on the 2013 draft FDA guidance on bioanalytical method validation.

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.

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.

2013 White Paper on recent issues in bioanalysis: 'hybrid'--the best of LBA and LCMS.

The 2013 7th Workshop on Recent Issues in Bioanalysis was held in Long Beach, California, USA, where close to 500 professionals from pharmaceutical and biopharmaceutical companies, CROs and regulatory agencies convened to discuss current topics of interest in bioanalysis. These 'hot' topics, which covered both small and large molecules, were the starting point for fruitful exchanges of knowledge, and sharing of ideas among speakers, panelists and attendees. The discussions led to specific recommendations pertinent to bioanalytical science. Such as the previous editions, this 2013 White Paper addresses important bioanalytical issues and provides practical answers to the topics presented, discussed and agreed upon by the global bioanalytical community attending the 7th Workshop on Recent Issues in Bioanalysis.

Implementation of an electronic laboratory notebook to accelerate data review in bioanalysis.

Electronic laboratory notebooks increase opportunities for collaboration and information exchange when compared with paper records. Depending on the degree of implementation, a laboratory- or enterprise-wide system can unify the collection, review and dissemination of data to improve laboratory efficiency and productivity. The advantages of an electronic laboratory notebook for speeding data review in bioanalysis are discussed, through the use of validated templates and organizational constructs to block errors in real-time and reduce manual audit tasks.

Recommendations on bioanalytical method stability implications of co-administered and co-formulated drugs by Global CRO Council for Bioanalysis (GCC).

An open letter written by the Global CRO Council for Bioanalysis (GCC) describing the GCC survey results on stability data from co-administered and co-formulated drugs was sent to multiple regulatory authorities on 14 December 2011. This letter and further discussions at different GCC meetings led to subsequent recommendations on this topic of widespread interest within the bioanalytical community over the past 2 years.

4th Global CRO Council for Bioanalysis: coadministered drugs stability, EMA/US FDA guidelines, 483s and carryover.

The Global CRO Council for Bioanalysis (GCC) was formed in September 2010. Since then, the representatives of the member companies come together periodically to openly discuss bioanalysis and the regulatory challenges unique to the outsourcing industry. The 4th GCC Closed Forum brought together experts from bioanalytical CROs to share and discuss recent issues in regulated bioanalysis, such as the impact of coadministered drugs on stability, some differences between European Medicines Agency and US FDA bioanalytical guidance documents and lessons learned following recent Untitled Letters. Recent 483s and agency findings, as well as issues on method carryover, were also part of the topics discussed.

Going paperless: implementing an electronic laboratory notebook in a bioanalytical laboratory.

AIT Bioscience, a bioanalytical CRO, implemented a highly configurable, Oracle-based electronic laboratory notebook (ELN) from IDBS called E-WorkBook Suite (EWBS). This ELN provides a high degree of connectivity with other databases, including Watson LIMS. Significant planning and training, along with considerable design effort and template validation for dozens of laboratory workflows were required prior to EWBS being viable for either R&D or regulated work. Once implemented, EWBS greatly reduced the need for traditional quality review upon experiment completion. Numerous real-time error checks occur automatically when conducting EWBS experiments, preventing the majority of laboratory errors by pointing them out while there is still time to correct any issues. Auditing and reviewing EWBS data are very efficient, because all data are forever securely (and even remotely) accessible, provided a reviewer has appropriate credentials. Use of EWBS significantly increases both data quality and laboratory efficiency.

Recommendations on: internal standard criteria, stability, incurred sample reanalysis and recent 483s by the Global CRO Council for Bioanalysis.

"The Global CRO Council (GCC) for Bioanalysis was formed in an effort to bring together many CRO leaders to openly discuss bioanalysis and the regulatory challenges unique to the outsourcing industry"