Preparation of 18O-labelled azaspiracids for accurate quantitation using liquid chromatography–mass spectrometry

Azaspiracids (AZAs) are a group of polyether marine algal toxins known to accumulate in shellfish, posing a risk to human health and the seafood industry. Analysis of AZAs is typically performed using LC–MS, which can suffer from matrix effects that significantly impact the accuracy of measurement results. While the use of isotopic internal standards is an effective approach to correct for these effects, isotopically labelled standards for AZAs are not currently available. In this study, 18O-labelled AZA1, AZA2, and AZA3 were prepared by reaction with H218O under acidic conditions, and the reaction kinetics and sites of incorporation were studied using LC–HRMS/MS aided by mathematical analysis of their isotope patterns. Analysis of the isotopic incorporation in AZA1 and AZA3 indicated the presence of four exchangeable oxygen atoms. Excessive isomerization occurred during preparation of 18O-labelled AZA2, suggesting a role for the 8-methyl group in the thermodynamic stability of AZAs. Neutralized mixtures of 18O-labelled AZA1 and AZA3 were found to maintain their isotopic and isomeric integrities when stored at −20 °C and were used to develop an isotope-dilution LC–MS method which was applied to reference materials of shellfish matrices containing AZAs, demonstrating high accuracy and excellent reproducibility. Preparation of isotopically labelled compounds using the isotopic exchange method, combined with the kinetic analysis, offers a feasible way to obtain isotopically labelled internal standards for a wide variety of biomolecules to support reliable quantitation. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-023-04868-4.


Table of Contents
The following 16 differential equations describing the changes of each of the 16 isotopologues of the AZA, with entities and constants as defined in Scheme S2.This approach is an extension of that recently applied to 18 O-incorporation into Caribbean ciguatoxins and gambierones.

Figure S6 .
Figure S6.LC-HRMS/MS spectra of: top, AZA3 ([M+H] + m/z 828.5), and; bottom, [21-18 OH]AZA3 ([M+H] + m/z 830.5) after 1.3 h of exchange with H2 18 O in the presence of TFA.Note the absence of 18 O in product-ions except for the neutral loss of H2 18 O from the precursor-ion.

Figure S7 .
Figure S7.Expansion of the LC-HRMS/MS spectra, from Figure S3, of: top, AZA3 ([M+H] + m/z 828.5), and; bottom, [21-18 OH]AZA3 ([M+H] + m/z 830.5) after 1.3 h of exchange with H2 18 O in the presence of TFA.Note the absence of 18 O-label in productions from the [21-18 OH]AZA3 except for the neutral loss of H2 18 O from the precursor ion.

Figure S10 .
Figure S10.LC-HRMS/MS spectra of: top, AZA1 ([M+H] + m/z 842.5), and; bottom, [ 18 O2]AZA1 ([M+H] + m/z 846.5) after 77 h of exchange with H2 18 O in the presence of TFA.Note the presence of two 18 O atoms only in the precursor-ion, and only one 18 O atom in product-ions from the ion cluster associated with retro-Diels-Alder cleavage of the A-ring.

Figure S11 .
Figure S11.LC-HRMS/MS spectra of: top, AZA1 ([M+H] + m/z 842.5), and; bottom, [ 18 O2]AZA1 ([M+H] + m/z 846.5) after 77 h of exchange with H2 18 O in the presence of TFA.Note the presence of 18 O only in the precursor-ion, its water loss ions, and in productions from the ion cluster associated with retro-Diels-Alder cleavage of the A-ring.

Figure S13 .
Figure S13.LC-HRMS/MS spectra of: top, AZA3 ([M+H] + m/z 828.5), and; bottom, [ 18 O2]AZA3 ([M+H] + m/z 832.5) after 77 h of exchange with H2 18 O in the presence of TFA.Note the presence of 18 O only in the precursor-ion, its water loss ions, and in productions from the ion cluster associated with retro-Diels-Alder cleavage of the A-ring.

Figure S14 .
Figure S14.LC-HRMS/MS spectra of: top, AZA3 ([M+H] + m/z 828.5), and; bottom, [ 18 O2]AZA3 ([M+H] + m/z 832.5) after 77 h of exchange with H2 18 O in the presence of TFA.Note the presence of 18 O only in the precursor-ion, its water loss ions, and in productions from the ion cluster associated with retro-Diels-Alder cleavage of the A-ring.

Figure S15 .
Figure S15.LC-HRMS/MS spectra of synthetic AZA3 (lower panels) and its minor contaminating synthetic C-1 alcohol precursor (upper panels) using two CEs, obtained during previous studies to determine the absolute stereochemistry of AZA3.1-2Note the nearly identical relative intensities of corresponding ions from the two compounds when obtained with the same CE, including the production at m/z 125.0597, which would have m/z 109.0648(not observed) in the C-1 alcohol if this ion originated from C-1 to C-7.

Figure S16 .
Figure S16.LC-HRMS/MS spectra of: top, AZA1 ([M+H] + m/z 842.5); middle, [ 18 O2]AZA1 ([M+H] + m/z 846.5, and; bottom, [ 18 O3]AZA1 ([M+H] + m/z 848.5) after 77 h of exchange with H2 18 O in the presence of TFA.Note the presence of three 18 O atoms in the precursor-ion but of only one in the product-ions from the ion cluster associated with retro-Diels-Alder cleavage of the A-ring.

Figure S17 .
Figure S17.LC-HRMS/MS spectra of: top, AZA3 ([M+H] + m/z 828.5); middle, [ 18 O2]AZA3 ([M+H] + m/z 832.5, and; bottom, [ 18 O3]AZA3 ([M+H] + m/z 834.5) after 77 h of exchange with H2 18 O in the presence of TFA.Note the presence of three 18 O atoms in the precursor-ion but of only one in the product-ions from the ion cluster associated with retro-Diels-Alder cleavage of the A-ring.

Figure S18 .
Figure S18.LC-HRMS analysis of 18 O-labelled AZA1 spiked into a mixture of methanol and water (left column) re-analyzed after storage at −20 °C for 18 months (right column), showing chromatograms for 0-4 isotope incorporations into the AZA1 structure (top row), and corresponding isotopic profile for the AZA1 peak (bottom row).
Figure S19.A, Full-scan LC-HRMS chromatogram, extracted at the indicated m/z values, of an extract of FDMT1 spiked with the labelled AZA1 during the isotope-dilution quantitation study, and; B-D, full-scan mass spectra obtained from the chromatogram at the indicated retention times.
Figure S5.Top, the structure of AZA1 with all non-exchangeable alcohol-derived or etheroxygens (blue, with blue atom-numbers), and all carbonyl-equivalent carbon atoms indicated with cyan atom-numbers (comprising the carboxylic acid at C-1, ketals at C-10, C-13, and C-28, a hemiketal at C-21, and a hemiaminal ether at C-36); middle, a purely hypothetical illustrative ring-opened structure in which all the carbonyl-equivalent centres have been exchanged with oxygen-labelled (red) water, and; bottom, the same structure but with the ring closures that establish the carboxylic acid, ketal, and hemiaminal ether groups present in the original AZA1 structure.Note that this figure is not intended to represent a mechanism or intermediate in the exchange reactions, but rather to illustrate why incorporation of labelled oxygen in AZA1 is only possible at C-1 (×2), C-21, and in ring-B.