A mussel tissue certified reference material for multiple phycotoxins. Part 5: profiling by liquid chromatography–high-resolution mass spectrometry

Identification of 24-O-β-d-Glycosides and 7-Deoxy-Analogues of Okadaic Acid and Dinophysistoxin-1 and -2 in Extracts from Dinophysis Blooms, Dinophysis and Prorocentrum Cultures, and Shellfish in Europe, North America and Australasia

“…LC-MS n analyses showed that a compound with the expected characteristics was present at a low level, along with a higher level of 3, in the corresponding aqueous fraction obtained after pumping a Dinophysis bloom at Sogndal, Norway, in November 2005. The positive and negative ion MS 2 and MS 3 fragmentation of 6 was similar to those of 4 and 5, other than a 14 Da increase in the mass of some of the product ions due to the presence of methyl groups at both C- 31 4) based on the modelled structure of 1 that Larsen et al [19] derived from the X-ray crystal structure of Tachibana et al [28], showing the glucoside-containing region. The arrows show structurally diagnostic through-space correlations relating to the glucosyl moiety observed in SELROESY NMR experiments.…”

“…Because LC–HRMS/MS was not available in our laboratories at the time that this extract was available, we sought to confirm the identification of 6 by LC–HRMS/MS analysis of extracts of shellfish naturally contaminated with 3 from Dinophysis blooms ( Tables S1 and S2 ). CRM-FDMT1 is a freeze-dried certified reference material that contains Norwegian mussels as its sole source of 3 [ 29 , 30 ], and was recently reported to contain low levels of 4 and 5 and a compound with some of its mass spectral characteristics consistent with those expected for 6 [ 31 ]. We therefore undertook a detailed LC–HRMS/MS analysis of an SPE-concentrated extract of FDMT1 ( Figure 3 and Figure S2 ), as well as of several extracts of shellfish naturally contaminated with 3 harvested in Atlantic Canada and Norway ( Figure 4 , Tables S1 and S2 ), in both positive and negative ionization modes, to determine whether the characteristics of this compound paralleled those of 4 and 5 and were fully consistent with 6 .…”

“…This prompted an examination of shellfish samples known to contain 1 – 3 . A recent study [ 31 ] suggested that an SPE-concentrated extract of NRC CRM-FDMT1, a composite freeze-dried reference material containing 1 – 3 from mussels harvested in Ireland and Norway [ 30 ], also appeared to contain compounds with properties consistent with 4 – 9 . A comparison of retention times, accurate masses ( Figure S35 ), and HRMS/MS spectra of these compounds in the sample, using LC–HRMS/MS (method B), with those obtained in the studies described above was entirely consistent with the presence of low levels of 4 – 9 , together with much higher levels of 1 – 3 .…”

“…Prior to analysis, samples were filtered through spin filters (0.45 µm PVDF, Millipore Corp., Billerica, MA, USA) by centrifugation (1400 g , 5 min). Extracts of CRM-FDMT1 and CRM-DSP-Mus were prepared according to the liquid–solid extraction method of McCarron et al [ 32 , 44 ], and a solid-phase extraction (SPE) concentrate of CRM-FDMT1 was prepared as described by Wright and McCarron [ 31 ]. The CRM-FDMT1 SPE-concentrate (100 µL) was spiked with the stock solutions of 4 , 5, and 7 using volumes (~1–2 µL) estimated by LC–HRMS (method C) to add approximately equimolar amounts of each compound.…”

In Vitro Metabolism of Azaspiracids 1–3 with a Hepatopancreatic Fraction from Blue Mussels (Mytilus edulis)

“…NRC CRM-AZA-Mus and NRC CRM-FDMT1 were from National Research Council Canada (Halifax, NS, Canada), and mussels were harvested at Bruckless (Donegal Bay, Ireland) in 2005 . NRC CRM-FDMT1 was extracted and concentrated by SPE as described by Wright and McCarron . NRC CRM-AZA-Mus was extracted using a four-step extraction procedure (method B of McCarron et al).…”