Reversed-phase HPLC based assay for selective and sensitive endotoxin quantification - part II.

The impact of naturally occurring 3-deoxy-d-manno­oct-2-ulsonic acid (Kdo) derivatives on endotoxin (ET) quantification was investigated for six ET standards. In our recently published chemical Kdo-DMB-LC ET assay (Bucsella et al., Anal. Methods, 2020, 12,4621) [1], the rare, ET specific sugar acid Kdo is used for ET quantification of S-type ETs. The ET content is calculated based on an external Kdo standard or a representative ET standard. In absence of a specific ET standard, the calculation is based on the reference standard ET (RSE) structure or on a worst-case scenario. This scenario overestimates the total ET content of typical S-type ET preparations by a factor of four. Mainly R-type ETs contain in addition to Kdo also Kdo-s non-stoichiometrically modified with phosphoethanolamine (PEtN), galactose (Gal) or L­glycero-d-manno-heptose (Hep) in substantial quantities. These Kdo species are separated from the unmodified Kdo. All Kdo and Kdo species follow an exponential hydrolytic release from the ET core in dependence on the hydrolysis time. Hydrolysis kinetics for identical Kdo species are the same for all ET standards. Kdo-Gal was released fastest followed by unsubstituted Kdo, Kdo-PEtN, and Kdo-Hep. Between 90 and 150 min a plateau of maximum content is obtained for all Kdo-s. That allows in case of a representative ET standard, ET quantification based on the most present Kdo derivative, here mainly unsubstituted Kdo. If no representative ET standard is available Kdo and all Kdo species must be considered for ET quantification. With that the Kdo-DMB-LC assay is applicable for R- and S-type ETs.