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

Abstract

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 H-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 validatedF-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).

Metrologically traceable quantification of trifluoroacetic acid content in peptide reference materials by19F solid-state NMR

“…From this distribution we determine the average mean TFA mass fraction to be w ana = 246.8 mg g −1 with and extended uncertainty of U = 3.7 mg g −1 . This value is in excellent agreement with the TFA mass fraction of 248.3 mg g −1 (U = 2.3 mg g −1 ) in a sample of ANGII-1 determined recently by 19 F-qNMR in solution using BTFMBA as internal calibrant [7]. It should be noted that this material has also been fully characterized by the mass balance approach using various other analytical techniques, and beyond TFA, no other fluorine-containing impurities were identified [7].…”

“…This value is in excellent agreement with the TFA mass fraction of 248.3 mg g −1 (U = 2.3 mg g −1 ) in a sample of ANGII-1 determined recently by 19 F-qNMR in solution using BTFMBA as internal calibrant [7]. It should be noted that this material has also been fully characterized by the mass balance approach using various other analytical techniques, and beyond TFA, no other fluorine-containing impurities were identified [7]. The non-parametric bootstrap re-sampling of Ī norm ana and Ī norm cal allows the estimation of the worst-case true experimental error for w ana , as all results obtained on the analyte and calibrant listed in tables 2 and 3 are randomly combined.…”

“…In addition, strong acids such as trifluoroacetic acid (TFA) are used to cleave the peptide from the solid support and to remove all protecting groups, which can then co-crystallize with the peptide [6]. Depending on the number of basic sites of the peptide, large amounts of TFA counter-ion can result, in some cases up to 25% by weight [7]. If determining peptide purity by conventional means, such as by LC-UV, the amount of these salts can be greatly underestimated or completely undetected [6], thereby significantly over-estimating the peptide purity level.…”

“…For more accurate peptide purity measurements, specific methods to detect peptide counter-ions such as TFA would be valuable. Accurate determination of TFA levels in a peptide reference material has been demonstrated using 19 F quantitative nuclear magnetic resonance spectroscopy ( 19 F-qNMR) in solution using an internal calibrant [7]. This approach has been successfully validated against ion chromatography methods in an international comparison exercise [8].…”

Preparation and Certification of a New Salvianolic Acid A Reference Material for Food and Drug Research

“…Nowadays, qNMR has been adopted as the standard method by the pharmacopeias of many countries including the USA, UK, EU, JP and CHN, etc. [18]. In this work, butylparaaben (BPB) was chosen as internal standard for qNMR analysis.…”