Assessing MS-based quantitation strategies for low-level impurities in peptide reference materials: application to angiotensin II.

Identification and quantitation of related impurities is vital in obtaining corrected purity values for peptide certified reference materials. The sensitivity and selectivity of high-resolution mass spectrometry (MS) renders it an indispensable technique in this arena. Typical quantitation efforts involve constructing external calibration curves, although analysis of dilute peptide solutions can be complicated by analyte adsorption to vial walls, instrument tubing, etc. The standard addition method alleviates many concerns associated with this sample loss as the calibrant solutions more closely match the matrix of the samples. Yet, both strategies require acquisition of synthetic impurity peptide standards. Label-free proteomics relies on electrospray ionization (ESI)-MS signals to quantify identical peptides across multiple samples; however, peptides of differing sequence can exhibit widely disparate ESI-MS responses. This study explores the use of peak area ratios to quantitate sequence-related peptide impurities in an angiotensin II candidate certified reference material. Using synthetic standards of five abundant substances, impurity mass fractions calculated via the relative response method are in reasonable agreement with those determined from standard addition experiments, whereas external calibration measurements frequently overestimate impurity amounts. For a synthetic peptide and its related sequence impurities, the relative response method can expedite analysis and lower expenditures, and in some cases improve data quality.

Purity assignment for peptide certified reference materials by combining qNMR and LC-MS/MS amino acid analysis results: application to angiotensin II.

The purity value assignment of metrologically traceable peptide reference standards requires specialized primary methods. Conventionally, amino acid analysis by isotope dilution tandem mass spectrometry (LC-MS/MS) following peptide hydrolysis is employed as a reference method. By contrast, quantitative nuclear magnetic resonance (qNMR) spectroscopy allows for quantitation of intact peptides, thus eliminating potential bias due to hydrolysis. Both methods are susceptible to interference from related peptide impurities, which need to be accurately measured and accounted for. The mass balance approach has also been employed for peptide purity measurements, whereby the purity is defined by the sum of the mass fraction of all impurities identified. Ideally, results from these three orthogonal methods can be combined for final purity assignment of peptide reference standards. Here we report a novel strategy for correcting both LC-MS/MS and 1H-qNMR results for related peptide impurities and combining results from both methods using a Bayesian statistical approach using mass balance results as prior knowledge. The mass balance method relied on a validated 19F-qNMR method to measure the trifluoroacetic acid (TFA) counter-ion, considered an impurity in this case at nearly 25% by mass. Using a candidate certified reference material (CRM) for angiotensin II, excellent agreement was achieved with the three methods. The final purity value assignment of the candidate CRM was 691 ± 9 mg/g (k = 2).

Compliance and persistence with newer antihypertensive agents.

Individuals with hypertension need to stay on therapy with antihypertensive medication to obtain the full benefits of blood pressure reduction. There are important differences in tolerability across antihypertensive drug classes, and these differences influence the extent to which patients are willing to continue taking their drugs. Three separate sources of evidence--postmarket surveillance studies, medical/prescription database studies, and discontinuation of study medication in long-term endpoint clinical trials--support the proposition that angiotensin II antagonists, the newest class of antihypertensives, are well tolerated, and that patients whose initial treatment is an angiotensin II antagonist are more likely to persist with therapy than patients who use other classes of antihypertensives. Recent landmark trials with losartan in hypertensive patients with left ventricular hypertrophy (Losartan Intervention For Endpoint reduction [LIFE]) and in diabetes (Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan [RENAAL]) demonstrated excellent tolerability, a high level of persistence, and clinical benefits exceeding those provided by blood pressure control alone for the prototype angiotensin II antagonist in clinical settings.

Measurement of angiotensin II: use of two injectors to minimize HPLC shadowing.

We investigated the use of two HPLC injectors, one reserved for standards and the other for blanks or biological samples, to minimize shadowing in the measurement of angiotensin II (ANGII). HPLC carryover of standard ANGII to blank with a one-injector and a two-injector system were 47.0 +/- 5.0 and 2.4 +/- 0.5 fmol/ml, respectively, a 19.6-fold reduction. Measured normal canine left ventricular myocardium ANGII level by the two-injector HPLC-RIA system was 22.3 +/- 2.4 fmol/g, with a signal-to-noise ratio of 11.7, an improved signal-to-noise ratio of 29.3 fold vs. the one injector. This innovation reduced the incidence of false-positive ANGII results, and thus can be applied to other compounds that exhibit HPLC-derived shadowing.