The Origin-Adjusted Approach for Reliable Quantification of Endogenous Analytes in Mass Spectrometric Bioanalysis.

The reliably accurate and precise quantification of biomarkers is a priceless objective in the drug development and diagnostic arenas. To employ a technique that brings such reliability and furthermore involves a simpler, faster, and inexpensive regime would only underline the potential importance of the concept and technique. To the existing established approaches for biomarker quantification in bioanalytical LC-MS, surrogate matrix (SUR-M) and surrogate analyte (SUR-A), in this Letter we present an approach that fulfills the aforementioned advantages. The concept builds on the historic method of standard addition (SA), in which one source of biological matrix is spiked with analyte to form a calibration curve. With the SA curve back-calculated, the heart of this procedure is the subsequent adjustment of the intercept to zero, the origin, and using only the slope of the curve for interpolation giving calculated sample concentrations. In SA, the concentration axis intercept indicates the endogenous analyte concentration, and our zeroing of this is equivalent to removing the endogenous level. This key shift of the calculated line to the origin unveils our novel origin-adjusted (OA) approach. It enables use akin to a regular xenobiotic method, with no need to ultimately account for the endogenous analyte level in the control matrix used for calibrants. We present a comparison of OA against the control approach of SUR-M in a representative application for kynurenine and tryptophan in human plasma by LC-MS. A numerical performance analysis performed is demonstrative of equivalence between the two approaches for both analytes.

Novel hydrophilic-phase extraction, HILIC and high-resolution MS quantification of an RNA oligonucleotide in plasma.

Aim: In the theme of quantitative LC-MS bioanalysis of oligonucleotides free of ion-pairing, a 22-mer RNA oligonucleotide took center stage. The focus was on a unique polar-based retention scheme to produce a high-recovery extraction presenting a high-performance alternative extraction means, also there was the opportunity to involve hydrophilic-interaction liquid chromatography and contemporary high-resolution MS as the end point. Results: Original LC-MS methodology was developed for the oligonucleotide and the performance was robust for both nominal and accurate mass detection, the latter affording 10× improvement in sensitivity and 4000-fold linear dynamic range, 500 pM to 2000 nM. Conclusion: A novel means of solid-phase extraction is exhibited within a robust pair-free methodology, reaching pM sensitivity with the demonstrably beneficial accurate mass platform.

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

The 13th GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5th, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.

12th GCC Closed Forum: critical reagents; oligonucleotides; CoA; method transfer; HRMS; flow cytometry; regulatory findings; stability and immunogenicity.

The 12th GCC Closed Forum was held in Philadelphia, PA, USA, on 9 April 2018. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: critical reagents; oligonucleotides; certificates of analysis; method transfer; high resolution mass spectrometry; flow cytometry; recent regulatory findings and case studies involving stability and nonclinical immunogenicity. Conclusions and consensus from discussions of these topics are included in this article.

An oligonucleotide bioanalytical LC-SRM methodology entirely liberated from ion-pairing.

Aim: Reliable quantitative LC-MS methodology has been established and validated for an oligonucleotide in plasma in a fresh and unique fashion, free of ion-pairing reagents and the various associated deleterious effects from primary solution preparation through sample preparation and extraction to the LC-MS analytical end point, offering a highly selective mixed-mode solid-phase extraction with hydrophilic-interaction liquid chromatography as the chromatographic element prior to SRM detection. Results: Inter- and intra-assay accuracy and precision ranged from 97.9 to 111% and 2.75 to 9.66%, respectively. Recoveries of 50% were attained, and there was no significant matrix effect manifestation. Conclusion: The method demonstrated rugged performance and reliability under the optimized conditions, indicating a possible exciting new avenue, free of ion-pairing, for general application in oligonucleotide quantitative LC-MS.

Recommendations for classification of commercial LBA kits for biomarkers in drug development from the GCC for bioanalysis.

Over the last decade, the use of biomarker data has become integral to drug development. Biomarkers are not only utilized for internal decision-making by sponsors; they are increasingly utilized to make critical decisions for drug safety and efficacy. As the regulatory agencies are routinely making decisions based on biomarker data, there has been significant scrutiny on the validation of biomarker methods. Contract research organizations regularly use commercially available immunoassay kits to validate biomarker methods. However, adaptation of such kits in a regulated environment presents significant challenges and was one of the key topics discussed during the 12th Global Contract Research Organization Council for Bioanalysis (GCC) meeting. This White Paper reports the GCC members' opinion on the challenges facing the industry and the GCC recommendations on the classification of commercial kits that can be a win-win for commercial kit vendors and end users.

A novel sequential electrostatic-HILIC SPE within a quantitative method for bivalirudin in human plasma by LC-SRM.

AIM: This work set out to realize an idea for a novel means of extracting the peptide therapeutic bivalirudin from human plasma in what would be a uniquely selective means of SPE, a mixed-mode protocol involving electrostatic interactions followed by HILIC. RESULTS: Inter and intra-assay relative error ranged from 3.52 to 8.23%, and 2.37 to 6.90%, respectively. Inter and intra-assay precision ranged from 2.64 to 7.12%, and 0.855 to 2.90%, respectively. Recoveries of 80% were attained, and there was no hint of discernible manifestation of matrix effects. CONCLUSION: The method was shown to perform excellently in the assessment tantamount to method validation. The essence of the extraction method presents a new option for highly selective extraction of peptides from biological matrices.

LC-MS/MS quantification of parathyroid hormone fragment 1-34 in human plasma.

BACKGROUND: It was desired to use contemporary knowledge and technology to develop a highly selective and reliable LC-MS/MS method for teriparatide in human plasma to begin to supplant existing ELISA methodologies. RESULTS: The method was developed using SPE of intact teriparatide and the internal standard rat analogue parathyroid hormone fragment 1-34 from human plasma, and UPLC-MS/MS analysis. Inter- and intra-batch accuracy and precision ranged from 97.5 to 109%, and 1.78 to 12.4%, respectively. Mean analyte extraction recoveries of 80% were attained. CONCLUSION: All relevant acceptance criteria were met in a procedure akin to method validation without the stability aspects, and with a basis of excellent signal stability all performance aspects of the method were unassailable.

Silica hydride-based chromatography of LC-MS response-altering compounds native to human plasma.

BACKGROUND: An investigation was carried out into the chromatographic behavior, on a silica hydride-based phase and a comparator silica-based phase, of an important group of lipids endogenous to human plasma, which are associated with matrix effect and in the context of quantitative peptide analysis. RESULTS: The propensity for aqueous normal phase (ANP) retention on the silica hydride-based phase was strong and extensive in comparison with the silica-based comparator, and the lipophilic interferences in question were readily eluted using the ANP mode, a contrast to over-retention issues with accompanying implications for method ruggedness typically found with silica-based phases. CONCLUSION: The silica hydride-based phase, with ANP operation, offered selectivity conducive to rapid lipophilic interferent elimination and the bimodal retention involved in suitable gradient elution was appropriate for general peptide analytical application.

Implication of free cholesterol in LC-MS response enhancement.

BACKGROUND: In the context of matrix effects in bioanalytical LC-MS/MS, a speculative link between free cholesterol, the recoveries of the compound from three common extraction procedures, and response enhancement was qualitatively investigated. RESULTS: Injections on-column of cholesterol both in solution and extracts, in conjunction with post-column infusion of three representative drugs, reveal a direct role in pronounced response enhancement for two out of three analytes, under one set of LC-MS/MS conditions, for the majority of typical plasma extraction procedures, where electrospray-based gaseous ion generation is used. CONCLUSION: Cholesterol has been shown to have a strong association with LC-MS/MS response enhancement and ideally should be monitored during method development, reinforcing the reasoning behind minimizing SPE elution volumes and avoiding less selective means of sample preparation.

Evaluation of acetone as organic modifier in SPE for bioanalytical quantitative LC-MS/MS.

BACKGROUND: To assess the suitability of acetone as an alternative to acetonitrile in SPE under otherwise commonly used conditions, with a focus on selectivity with regard to the most abundant phospholipid class. Two representative analytes were included, leuprolide and tramadol, a peptide and a small molecule, respectively. RESULTS: The use of acetone resulted in analogous elution profiles of all monitored compounds in the majority of conditions. The only significant difference was on silica-based C18, where acetone effected markedly enhanced elution of lysophosphatidylcholines than did acetonitrile. Unmodified silica was shown to operate in per aqueous LC mode in highly aqueous conditions. CONCLUSION: Acetone was established as a selectively similar and slightly more eluotropic alternative to acetonitrile in bioanalytical SPE.

Stable-labeled analogues and reliable quantification of nonprotein biomarkers by LC-MS/MS.

BACKGROUND: The aim was to develop, and establish as suitable to begin assessment by full validation, a quantitative LC-MS/MS method for asparagine in human plasma. Therein, to utilize a stable-labeled analogue of asparagine to act as surrogate analyte, producing complete calibration curves and corresponding QC samples and another m/z distinct stable-labeled analogue to act as internal standard. RESULTS: From two candidates, the surrogate analyte was selected through statistical comparisons of concentration-response data and the resultant method employed protein precipitation and LC on an unmodified silica column with multiple reaction monitoring detection mode. The calibration range was 50-10,000 ng/ml. CONCLUSION: This method was successfully proven to meet the accuracy and precision acceptance criteria of current bioanalytical method validation guidelines.