Erin E. Chambers

Performance of plasma free metanephrines measured by liquid chromatography-tandem mass spectrometry in the diagnosis of pheochromocytoma

BACKGROUNDnPlasma free metanephrines have proved a highly sensitive biochemical test for the diagnosis of pheochromocytoma. We have developed and validated a simple, LC-MS/MS method to determine plasma metanephrines and compared the diagnostic efficacy of the method with an enzyme immunoassay procedure in 151 patients, 38 with histologically confirmed pheochromocytoma.nnnMETHODSnOff-line solid phase extraction in a 96-well plate format was used to isolate metanephrines from 100-microL of plasma, followed by rapid separation with hydrophilic interaction chromatography. Mass spectrometry detection was performed in multiple-reaction monitoring mode using a tandem quadrupole mass spectrometer with positive electrospray ionization.nnnRESULTSnDetection limits were <0.1nmol/l with method linearity up to 23.0nmol/L for normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT). Method comparison with an automated LC-MS/MS yielded Deming regression slopes of r=0.94 for NMN, r=0.98 for MN and r=0.94 for 3-MT. Method comparison with enzyme immunoassay revealed regression slope of r=1.28 (NMN) and 1.25 (MN) with values approximately 25% lower than LC-MS/MS. Plasma metanephrines by LC-MS/MS identified all 38 patients with phaeochromocytoma compared with 36 cases by immunoassay.nnnCONCLUSIONSnPlasma metanephrines measured by LC-MS/MS are a reliable and sensitive test for the biochemical detection of pheochromocytoma.

Quantitation of amyloid beta peptides Aβ1–38, Aβ1–40, and Aβ1–42 in human cerebrospinal fluid by ultra-performance liquid chromatography–tandem mass spectrometry

Critical events in Alzheimers disease (AD) involve an imbalance between the production and clearance of amyloid beta (Aβ) peptides from the brain. Current methods for Aβ quantitation rely heavily on immuno-based techniques. However, these assays require highly specific antibodies and reagents that are time-consuming and expensive to develop. Immuno-based assays are also characterized by poor dynamic ranges, cross-reactivity, matrix interferences, and dilution linearity problems. In particular, noncommercial immunoassays are especially subject to high intra- and interassay variability because they are not subject to more stringent manufacturing controls. Combinations of these factors make immunoassays more labor-intensive and often challenging to validate in support of clinical studies. Here we describe a mixed-mode solid-phase extraction method and an ultra-performance liquid chromatography tandem mass spectrometry (SPE UPLC-MS/MS) assay for the simultaneous quantitation of Aβ(1-38), Aβ(1-40), and Aβ(1-42) from human cerebrospinal fluid (CSF). Negative ion versus positive ion species were compared using their corresponding multiple reaction monitoring (MRM) transitions, and negative ions were approximately 1.6-fold greater in intensity but lacked selectivity in matrix. The positive ion MRM assay was more than sufficient to quantify endogenous Aβ peptides. Aβ standards were prepared in artificial CSF containing 5% rat plasma, and quality control samples were prepared in three pooled CSF sources. Extraction efficiency was greater than 80% for all three peptides, and the coefficient of variation during analysis was less than 15% for all species. Mean basal levels of Aβ species from three CSF pools were 1.64, 2.17, and 1.26 ng/ml for Aβ(1-38); 3.24, 3.63, and 2.55 ng/ml for Aβ(1-40); and 0.50, 0.63, and 0.46 ng/ml for Aβ(1-42).

Improved sensitivity of the nano ultra-high performance liquid chromatography-tandem mass spectrometric analysis of low-concentrated neuropeptides by reducing aspecific adsorption and optimizing the injection solvent

Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolar concentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides to Eppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is presented to reduce adsorption of these peptides during standard preparation. Within this respect, all procedural steps from dissolution of the lyophilized powder until the injection of the sample onto the system are investigated. Two peptides of the neuromedin family, i.e. neuromedin B and neuromedin N, and a neuromedin N-related neuropeptide, neurotensin, are evaluated. The first part of this study outlines a number of parameters which are known to affect peptide solubility. The main focus of the second part involves the optimization of the sample composition in the UHPLC vial by using design of experiments. Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix components. They are found important for injection of the peptides into the system, but crucially need to be excluded from the dilution solvent. Furthermore, the type of surface material, temperature and the pipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results of the central composite design reveals that the highest peptide responses are obtained with the injection solvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can be identified as the main contributor to the gain in method sensitivity. Since the reduction of peptide adsorption and the optimization of the injection solvent resulted in a clear and quantifiable signal of the three peptides, optimization of both issues should be considered in the early stage of method development, in particular when the analysis of low-concentration peptide solutions is envisaged.

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.

Quantitative Analysis of Therapeutic and Endogenous Peptides using LC/MS/MS Methods

Historically, new drug entities arose from combinatorial chemistry of small molecules. Today, a rapidly increasing number of new drugs, called biopharmaceuticals, are being developed which are based on biological molecules such as peptides, proteins, and oligonucleotides.

Bioanalysis Zone: overcoming matrix effects Q&A

Experts Erin Chambers (Waters Corporation), John Kagel (University of North Carolina) and David Bell (Sigma-Aldrich) took part in a recent live panel discussion on overcoming matrix effects as part of the Bioanalysis Zone spotlight on the topic. Here they answer questions from our readers, which were submitted during the discussion and as part of our survey on the topic.