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.

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.

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.