Makoto Niwa

Measurement of the novel decapeptide cetrorelix in human plasma and urine by liquid chromatography–electrospray ionization mass spectrometry

A sensitive LC-MS quantitation method of cetrorelix, a novel gonadotropin releasing hormone (GnRH) antagonist, was developed. Plasma and urine samples to which brominated cetrorelix was added as an internal standard (I.S.) were purified by solid-phase extraction with C8 cartridges. The chromatographic separation was achieved on a C18 reversed-phase column using acetonitrile-water-trifluoroacetic acid (35:65:0.1, v/v/v) as mobile phase. The mass spectrometric analysis was performed by electrospray ionization mode with negative ion detection, and the adduct ions of cetrorelix and I.S. with trifluoroacetic acid were monitored in extremely high mass region of m/z 1543 and 1700, respectively. The lower limit of quantitation was 1.00 ng per 1 ml of plasma and 2.09 ng per 2 ml of urine, and the present method was applied to the analysis of pharmacokinetics of cetrorelix in human during phase 1 clinical trial.

Chemical derivatization in LC-MS bioanalysis: current & future challenges.

Chemical derivatization is an important tool in bioanalysis. Because of the extensive amount of background information available for classic and established derivatization techniques, we suggest using these techniques initially to solve bioanalytical issues. In this paper, we will present current technical challenges that require modified derivatization and related analytical techniques. In real laboratory circumstances, sometimes classic derivatization techniques lead to liquid chromatographic problems induced by excess hydrophobicity and chemical complexity of derivatives. With regard to derivatization techniques, another concern is procedural complexity. We will present some recent technique modifications that can be used to overcome these difficul ties. In addition, we will present some unexplored fields related to derivatization. We will describe the combined use of derivatization with microflow LC–MS and introduce the idea of new chemical tagging techniques.

Sensitive measurement of vinorelbine in dog plasma by liquid chromatography–electrospray ionization tandem mass spectrometry utilizing transitions from double‐charged precursor ions

A sensitive high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for measuring vinorelbine was developed. A 100 µL aliquot of plasma was spiked with deuterium-labeled internal standard and subjected to solid-phase extraction using an Oasis HLB μ-elution plate. Two microliters of the extracted samples was directly injected into LC/MS/MS. Chromatographic separation was achieved on a Capcell Pak C18 UG column (2 × 75 mm) with a gradient elution of methanol (mobile phase B) against 0.05% formic acid in aqueous 10 mm ammonium formate (mobile phase A). The LC flow rate was set to 0.28 mL/min and the gradient (solvent B concentration) was processed from 40 to 90%. In mass spectrometric detection, observation of the reaction from a double-charged precursor ion [M + 2H](2+) (m/z 390) to product ion m/z 122 provided very high sensitivity. The method was validated with a lower limit of detection of 0.2 ng/mL with 0.1 mL of plasma, and the method was used to determine the plasma pharmacokinetics of vinorelbine in dogs.

Tiered approach to metabolite quantification: regional practices reviewed by Japan Bioanalysis Forum discussion group

2015 Tiered approach; familiarization is essential A tiered approach [1–3] is an idea of step-bystep characterization of analytical methods in coordination with the stage of drug development and the importance of the analytical results obtained. It is thought that this approach contributes to the optimization of the framework of bioanalytical laboratories by adding flexibility to the analytical methods [4]. Nevertheless, it appears that bioanalysts are not ready to use this tiered approach in their day-to-day work because bioanalytical method validation guidances or guidelines, which are in force in the USA, Europe, Brazil and Japan, do not provide a framework for this approach (although the Japanese guidelines do outline the concept of a tiered approach). In addition, bioanalytical experts in industry or regulatory authorities have not provided detailed scientific requirements for different assay types or stages of drug development [4]. The tiered approach is often accompanied with the idea of case-by-case consideration. This makes it hard for those who are not familiar with this concept to understand the whole picture. The common question is how to implement the tiered approach practically (timing and characterization details of the analytical methods). This question seems to show that detailed models of works for the tiered approach are necessary in drug discovery and development stages. Though one of the ultimate goals for the tiered approach is to add flexibility to the bioanalytical works, we considered that it is useful to be familiarized with the approach at first through the typical workflow model. Complete understanding would contribute to more flexible usage of the methods. This activity may help remove certain hurdles toward general acceptance of the tiered approach in laboratory works [4]. Accordingly, a voluntary discussion group (named DG2013–03) of Japan Bioanalysis Forum (JBF), consisting of eight Japanese researchers from pharmaceutical companies and a contract research organization, tried to get the birds-eye-view of the tiered approach in accordance with drug discovery and development stages with emphasis on metabolite research [5]. This activity was intended to provide detailed scientific requirements in assay type or stage of drug development as described above. As an outcome of JBF DG2013–03, this report outlines tiered characterization of the analytical methods in metabolite quantification in relation to actual regional practices in Japan.

Survey on the tiered approach for Japanese bioanalysts operated by Japan bioanalysis forum DG2014-09.

Background The tiered approach is a strategy to limitedly characterize an analytical method initially and expand the range of characterization gradually to a full validation as the drug development process proceeds [1–3]. This approach has been discussed in European Bioanalysis Forum (EBF) [1,3] and Global Bioanalysis Consortium (GBC) [4,5] and is considered so beneficial that it enables the flexible use of analytical methods in dayto-day laboratory works [6,7]. In principle, it complies with the Japanese bioanalytical method validation (BMV) guideline [2], but the description in the guideline is not sufficient to put this approach into practice. In Japan, the importance of building a bioanalytical community was recognized in recent years, and the Japan Bioanalysis Forum (JBF) was established in 2011 [8]. As one of its activities, voluntary discussion groups (DGs) were organized in 2013. Under this circumstance, ‘tiered approach to the metabolite quantification’ was adopted as one of the DG topics in 2013 (named DG2013-03). In DG2013-03, analytical tiers raised by GBC were put into a typical workflow of drug discovery and development with consideration of regional practices as reported previously [9,10]. As a subsequent activity, a further survey on the tiered approach for Japanese bioanalysts was performed in a follow-up DG (DG2014-09) in the fiscal year 2014 [11]. Survey on the tiered approach A survey and discussion on following items were performed to facilitate the use of tiered approach in metabolite quantification. Initially, general opinions on tiered approach were collected:

Giving consideration to scientific validation in Japanese drug application by Japan Bioanalysis Forum discussion group

Background Scientific validation (SV) is a concept proposed by the European Bioanalysis Forum (EBF) and it utilizes both scientific discretion and validation in characterizing analytical methods. SV could improve drug-development efficiency as a component of a tiered approach by streamlining the process of analytical method establishment and validation. The EBF has summarized and published SV as a White Paper [1]. In the White Paper, a total of five categories are listed as assay-appropriate SV constituting three categories (metabolites in plasma for assessment of ICH-M3(R2) criteria, unchanged form in urine and tissue homogenates) and stage-appropriate SV constituting two categories (unchanged form in plasma at early stage clinical studies and preclinical studies). There are many discussions about b ioanalytical v alidation parameters in each category. The Japan Bioanalysis Forum (JBF) was established in 2011 [2]. As one of its activities, voluntary discussion groups (DGs) have been organized since 2013. Each DG selects and discusses a bioanalytics theme, and presents it at the JBF symposium. In 2015, DG2015–16 selected SV as a main activity theme. First, the DG members shared and discussed the contents of the white paper in detail applicable to the Japanese New Drug Application (NDA). Then the results of discussions were summarized and presented in the form of a poster at the 2016 JBF symposium [3]. Based on the results of the poster presentation, DG201620, which is a new DG for the 2016 fiscal year, performed a survey to collect opinions from the Japanese bioanalytical community (JBF DG s upporters and their colleagues). In this article, we describe our discussion and survey results regarding SV points a pplicable to Japanese NDA submission.