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The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies’ Input. Part 1 (Small molecules bioanalysis using LCMS) was published in the Bioanalysis issue 6(22) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the Bioanalysis issue 6(24).

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for Large molecules bioanalysis using LBA and Immunogenicity. Part 1 (Small molecules bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input) were published in the Bioanalysis issues 6(22) and 6(23), respectively.

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies’ input) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the upcoming issues of Bioanalysis.

Background: In the past several years, the impact of changing counter ions while keeping the same anticoagulant in bioanalytical LC–MS/MS methods has become a highly discussed topic. In order to confirm that there is no impact from counter ions, matrix effect and stability evaluations were performed on bicalutamide LC–MS/MS bioanalytical methods. Results: Independently from the anticoagulant counter ion used, the matrix effect evaluation met acceptance criteria, even when using conditions expected to increase matrix effect, such as protein precipitation with an analog internal standard. Freeze–thaw along with storage stabilities, namely short- and long-term, demonstrated less than 8% deviation regardless of the counter ion used. Conclusion: Differences in the anticoagulant counter ion used has no impact on the bicalutamide bioanalytical LC–MS/MS method.

Quantification of analytes by Dried Blood Spots (DBS) on different paper cards has been extensively reported in the past several years. However, some factors limit the robustness of the precision and accuracy of DBS such as: hematocrit level, blood viscosity, analyte nature, spotting technique and spotting conditions. As such, the paper material used for DBS must meet strict quality control criteria to produce reliable quantification of drugs: uniformity, no chemical leaching and no chromatographic effect. To overcome these variables, especially the hematocrit impact, a modification of the traditional DBS, named Pre-Cut Dried Blood Spot (PCDBS), is presented. In contrast to the classical DBS technique, the new PCDBS procedure demonstrates no variation in response, within � 3%, independently of the hematocrit level or of the type of card used. The impact of the hematocrit level on the analyte recovery is discussed for both DBS and PCDBS approaches. Moreover, for quantification of naproxen by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), the PCDBS technique was demonstrated to be as precise (%CV ≤3.1%) and accurate (%nominal between 95.4 and 104.4%) as the classical DBS procedure. Copyright © 2011 John Wiley & Sons, Ltd.

Background: Dried blood spots (DBS) sampling is a well-known technology for qualitative determination such as DNA analysis and screening of newborn metabolic disorders. The scientific community has recently expressed interest in applying the DBS technique for quantitative determination of drugs in biological fluid. Results: Two new bioanalytical assays were developed and validated for the determination of naproxen in human plasma and in DBS samples using liquid chromatography coupled with tandem MS. Furthermore, plasma and DBS clinical samples were collected from four subjects enrolled as part of a bioequivalence study. Concentration data for plasma and DBS samples were determined and pharmacokinetic (PK) profiles in plasma and in DBS samples were compared. Conclusions: A strong correlation between PK data obtained by the DBS and conventional plasma method was observed, which makes DBS a valuable technique for further naproxen bioavailability and PK investigations and studies.

Internal standards (IS) with similar physicochemical properties to the analyte provide multiple advantages in liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalytical methods such as: reduction of the analysis run time, improvement in the intra-injection reproducibility, impact reduction of matrix and ionization effects. However, it is important to evaluate the purity of the IS prior to their use. Indeed, a minor impurity in the IS may lead to an important issue during bioanalytical method development. Stable labelled internal standards are usually appropriate IS for bioanalysis. The use of oxycodone-D3, ursodiol-D5 and atovaquone-D4 as internal standards in three different bioanalytical methods was evaluated. During oxycodone, oxymorphone and noroxycodone simultaneous quantification method development, oxymorphone was identified as a contaminant in oxycodone-D3. Since the limit of quantification for oxymorphone was very low (10 pg/mL), the presence of an even low percentage of oxymorphone in oxycodone-D3 leads to the change of the stable labelled IS for an analogue, ethylmorphine. 23-Nordeoxycholic acid was preferred to ursodiol-D5 as internal standard for the ursodiol, tauroursodiol and glycoursodiol simultaneous quantification method. Indeed, more than 7% of ursodiol was identified in the ursodiol-D5 which could not be bypassed by decreasing the IS concentration without compromising the linearity. An atovaquone-D4 reference standard revealed the non-negligible presence of atovaquone-D5 to atovaquone-D8 that has a large impact on the method validation. Therefore, atovaquone-D4 was sent for recertification since its isotopic purity was found to be much less than the isotopic purity mentioned on its certificate of analysis. Consequently, during bioanalytical method development, the purity of the IS should be scrutinized.

Background: Phospholipids from biological samples are a source of matrix effect in liquid chromatography mass spectrometry. While the behavior of phospholipids has been documented under reversed-phase chromatography, there is a lack of information concerning the selectivity of hydrophilic interaction chromatography (HILIC) towards phospholipids. Results: Human plasma extracts were used to evaluate retention times and matrix effects associated with phospholipids under HILIC conditions. It was observed that phosphatidylcholine and lysophosphatidylcholine phospholipid retention times vary greatly between columns operated in different HILIC conditions. Therefore, matrix effects associated with phospholipids could present a quantitation problem if not evaluated thoroughly during method development. Conclusions: Analytical chemists should carefully choose the right combination of sample preparation and chromatographic conditions when working under HILIC conditions to avoid variable results.

Background: Matrix effects are one of the major drawbacks of ESI–MS/MS. It is majorly caused by lipids in plasma, which can be overcome by using different extraction techniques. Results: In this investigation, a major matrix effect was observed in samples containing a co-administered drug. Unknown compounds appeared over time in the human plasma samples spiked with co-administered drug creating major ion suppression. The changes in matrix integrity were associated with the organic solvent content in the plasma samples. Conclusion: The amount and type of organic solvent added to human plasma along with the storage conditions must be carefully determined during method development in order not to impact quantitation.

Background: The challenge of quantifying two compounds in a single assay with drastic dynamic ranges is to obtain linearity without source or detector saturation at the mass spectrometer. Results: In positive-ionization mode, the nonlinear relationships for Desmethyl Mebeverine Acid (DMAC) were demonstrated using three common strategies to overcome this issue: using offset voltage parameters, less-sensitive product ion or 13C mass SRM transitions. On the contrary, nonlinear relationships for DMAC were overcome if negative-ionization mode was used. Due to Mebeverine analytical LLOQ, dilution was not suitable for a single assay of Mebeverine and DMAC. However, polarity switching in negative mode for DMAC was successfully found to compensate for the nonlinearity at the mass spectrometer while preserving Mebeverine linear regression model in positive mode. Conclusion: The polarity switching strategy has demonstrated the advantage of improving linearity for analytes having different ionization polarities and three orders of magnitude difference in concentration.

Quantification of analytes by Dried Blood Spots (DBS) on different paper cards has been extensively reported in the past several years. However, some factors limit the robustness of the precision and accuracy of DBS such as: hematocrit level, blood viscosity, analyte nature, spotting technique and spotting conditions. As such, the paper material used for DBS must meet strict quality control criteria to produce reliable quantification of drugs: uniformity, no chemical leaching and no chromatographic effect. To overcome these variables, especially the hematocrit impact, a modification of the traditional DBS, named Pre-Cut Dried Blood Spot (PCDBS), is presented. In contrast to the classical DBS technique, the new PCDBS procedure demonstrates no variation in response, within ±3%, independently of the hematocrit level or of the type of card used. The impact of the hematocrit level on the analyte recovery is discussed for both DBS and PCDBS approaches. Moreover, for quantification of naproxen by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), the PCDBS technique was demonstrated to be as precise (%CV ≤3.1%) and accurate (%nominal between 95.4 and 104.4%) as the classical DBS procedure.

Background: Anticoagulants are used to prevent coagulation in blood samples. The plasma pH may change with a different counter ion and anticoagulant; thus, it is essential to determine effects on drug stability and the matrix effect during the bioanalytical method development. Results: Cross-validation of multiple compounds between different counter ions was performed and no impact from the counter ion nature was demonstrated. Moreover, plasma stabilities and matrix effects for both fluconazole and granisetron were investigated thoroughly in numerous counter ions/anticoagulants (K3ethylenediaminetetraacetic acid [K3EDTA], K2EDTA, NaEDTA, NaHeparin and LiHeparin). Sirolimus, a large cyclic molecule, was also tested in different whole-blood EDTA counter ions. Results showed percentage deviation less than 8.5% and percentage cross-validation less than 8.4%. Conclusion: None of the compounds tested had an impact on the matrix stabilities and matrix effect.

Due to the presence of endogenous components in biofluids, ionization suppression or enhancement may occur for bioanalytical assays using LC–MS or LC–MS/MS technologies. The matrix effect may affect the precision and accuracy of a bioanalytical method and, therefore, compromise the quality of the results. Protein precipitation sample preparation along with LC–MS/MS is a high-throughput method most commonly used in bioanalysis and is largely affected by the matrix effect. In order to eliminate the matrix effect during the method development, some considerations may be used: cleaner sample preparations, more sensitive instruments, which allow less material to be injected, different chromatographic separations and much more must be investigated. More than giving tools to adequately assess the matrix effect during the method development, this review gives scientists numerous ways to eliminate or reduce the matrix effect based on novel sample-preparation techniques, new chromatographic optimization methods and new technologies.