Rand Jenkins

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry.

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid-liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.

Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics

ABSTRACTThis paper represents the consensus views of a cross-section of companies and organizations from the USA and Canada regarding the validation and application of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for bioanalysis of protein biotherapeutics in regulated studies. It was prepared under the auspices of the AAPS Bioanalytical Focus Group’s Protein LC-MS Bioanalysis Subteam and is intended to serve as a guide to drive harmonization of best practices within the bioanalytical community and provide regulators with an overview of current industry thinking on applying LC-MS/MS technology for protein bioanalysis. For simplicity, the scope was limited to the most common current approach in which the protein is indirectly quantified using LC-MS/MS measurement of one or more of its surrogate peptide(s) produced by proteolytic digestion. Within this context, we considered a range of sample preparation approaches from simple in-matrix protein denaturation and digestion to complex procedures involving affinity capture enrichment. Consideration was given to the method validation experiments normally associated with traditional LC-MS/MS and ligand-binding assays. Our collective experience, thus far, is that LC-MS/MS methods for protein bioanalysis require different development and validation considerations than those used for small molecules. The method development and validation plans need to be tailored to the particular assay format being established, taking into account a number of important factors: the intended use of the assay, the test species or study population, the characteristics of the protein biotherapeutic and its similarity to endogenous proteins, potential interferences, as well as the nature, quality, and availability of reference and internal standard materials.

Reference measurement procedures for Alzheimer’s disease cerebrospinal fluid biomarkers: definitions and approaches with focus on amyloid β42

Cerebrospinal fluid (CSF) biomarkers for Alzheimers disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid β42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid β42.

Determination of octreotide and assessment of matrix effects in human plasma using ultra high performance liquid chromatography–tandem mass spectrometry

A selective UHPLC-MS/MS method for determination of the therapeutic peptide octreotide in human plasma was developed and validated. This assay used a UHPLC C(18) column with 1.7 μm particle size for efficient separation and an ion-exchange SPE for selective extraction. Octreotide and its labeled internal standard, [(13)C(6)Phe(3)] octreotide, were extracted from human plasma using a simple Oasis® WCX μElution SPE method and analyzed with a total chromatographic run time of 7.5 min. Matrix effects were studied during method development by direct monitoring of representative phospholipids. On-line removal of phospholipids using column switching and pre-column back-flushing was carried out to trap and remove any residual phospholipid matrix interferences. The UHPLC column provided baseline separation between the analyte and matrix peaks. The chromatographic conditions yielded optimal retention and excellent peak shape for both the analyte and internal standard. The assay was linear in the concentration range of 0.025-25.0 ng/ml, inter- and intra-assay precision and accuracy were within 6.1% and ±1.93%, respectively. Recovery was ∼73%. Post-extraction addition experiments showed that matrix effects were less than 4%. This method for octreotide in human plasma has been validated and utilized to support of clinical pharmacokinetic studies.

CSF Aβ1–42 – an excellent but complicated Alzheimer's biomarker – a route to standardisation

The 42 amino acid form of amyloid β (Aβ1-42) in cerebrospinal fluid (CSF) has been widely accepted as a central biomarker for Alzheimers disease. Several immunoassays for CSF Aβ1-42 are commercially available, but can suffer from between laboratory and batch-to-batch variability as well as lack of standardisation across assays. As a consequence, no general cut-off values have been established for a specific context of use (e.g., clinical diagnostics) and selection of individuals for enrolment in clinical trials (patient stratification) remains challenging. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has initiated a working group for CSF proteins (WG-CSF) to facilitate standardisation of CSF Aβ1-42 measurement results. The efforts of the IFCC WG-CSF include the development of certified reference materials (CRMs) and reference measurement procedures (RMPs) for key biomarkers. Two candidate RMPs for quantification of Aβ1-42 in CSF based on liquid chromatography tandem mass spectrometry have been developed and tested in two ring trials. Furthermore, two commutability studies including native CSF pools, artificial CSF and spiked materials have been completed. On the basis of these studies, human CSF pools containing only endogenous Aβ1-42 at three concentrations were selected as the format for future CRMs that are now being processed.

Round robin test on quantification of amyloid-β 1–42 in cerebrospinal fluid by mass spectrometry

Cerebrospinal fluid (CSF) amyloid‐β 1–42 (Aβ42) is an important biomarker for Alzheimers disease, both in diagnostics and to monitor disease‐modifying therapies. However, there is a great need for standardization of methods used for quantification. To overcome problems associated with immunoassays, liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) has emerged as a critical orthogonal alternative.

Dual universal peptide approach to bioanalysis of human monoclonal antibody protein drug candidates in animal studies

BACKGROUND There is a need for general and reliable LC-MS assays capable of supporting the bioanalysis of a variety of human monoclonal antibody-based therapeutic drug candidates in animal PK/TK studies. RESULTS We present herein improvements in our previously reported universal peptide approach to the bioanalysis of human monoclonal antibody protein drug candidates in animal studies. These improvements include incorporation of a second, light chain-based universal peptide into the assay, thus introducing the concept of a dual universal peptide assay; and incorporation of a universal stable isotope-labeled monoclonal antibody into the assay. CONCLUSION Improvements reported herein to the universal peptide assay will enable more reliable quantification of human monoclonal antibody protein drug candidates in animal studies.

An improved LC-ESI-MS-MS method for simultaneous quantitation of rosiglitazone and N-desmethyl rosiglitazone in human plasma.

A fast, sensitive, and selective method for the simultaneous quantitation of rosiglitazone and N-desmethyl rosiglitazone in human plasma, using rosiglitazone-d(4) and N-desmethyl rosiglitazone-d(4) as the respective internal standards, has been developed and validated. The analytes in human plasma (50 microL sample aliquot) were isolated through supported liquid/liquid extraction (SLE) and separated by isocratic HPLC over a 3-min period. The precursor and product ions were detected by ESI-MS-MS with multiple reaction monitoring (MRM) in a triple quadrupole mass spectrometer. For both rosiglitazone and N-desmethyl rosiglitazone, the lower limit of quantitation (LLOQ) was 1.00 ng/mL, and the quantitation range was 1.00-500 ng/mL (with an average correlation coefficient >0.9990). The intra-assay and inter-assay precision had a maximum %CV of 9.37%, and the accuracy had a maximum %difference from theoretical of 12.7%. This method was applied to a clinical study where 16 healthy volunteers were administered a single dose of 4.0mg rosiglitazone. The pharmacokinetic parameters of rosiglitazone and N-desmethyl rosiglitazone were consistent with the results reported in the literature.

Bioanalytical method validation considerations for LC–MS/MS assays of therapeutic proteins

This paper highlights the recommendations of a group of industry scientists in validating regulated bioanalytical LC-MS/MS methods for protein therapeutics in a 2015 AAPSJ White Paper. This group recommends that most of the same precision and accuracy validation criteria used for ligand-binding assays (LBAs) be applied to LC-MS/MS-based assays where proteins are quantified using the LC-MS/MS signal from a surrogate peptide after proteolytic digestion (PrD-LCMS methods). PrD-LCMS methods are generally more complex than small molecule LC-MS/MS assays and may often include LBA procedures, leading to the recommendation for a combination of chromatographic and LBA validation strategies and appropriate acceptance criteria. Several key aspects of this bioanalytical approach that are discussed in the White Paper are treated here in additional detail. These topics include selectivity/specificity, matrix effect, digestion efficiency, stability and critical reagent considerations.

Conference Report: 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.