Succinylmonocholine analytics as an example for selectivity problems in high-performance liquid chromatography/tandem...

The determination and quantitation of drugs in biological matrices using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) is becoming increasingly popular in analytical toxicology, while at the same time a growing awareness for the limits of this technique can be observed. Our group previously developed a rapid HPLC/ESI-MS/MS method for the detection and quantitation of succinylcholine (SUX) and succinylmonocholine (SMC) using ion-pairing extraction of samples with subsequent separation by gradient chromatography on a Synergi Hydro RP C18 column (4 microm, 150 x 2 mm). Identification of analytes was achieved in the multiple reaction monitoring (MRM) mode, using two characteristic ion transitions each, the respective analytes' retention time as well as co-elution of stable isotopic analogues. In both native serum as well as urine an interference with the main MRM transition of SMC was found to co-elute with this analyte, thus severely compromising the identification and quantitation of this target analyte. The interference was further shown to be eliminated from serum and urine by exposure to alkaline conditions and hence proven to share a key physicochemical property with SMC. The observed absence of the second and third most intense ion transitions of SMC in the unknown substance was the only useful distinction between both compounds.The detailed presentation of selectivity problems encountered during method development is intended to initiate further discussion on this yet underrepresented issue in HPLC/MS/MS. The present work emphasizes the need to monitor more than just one ion transition to confidently rule out signal interferences, ensure correct analyte identification as well as quantitation, and thus avoid false-positive results. In this context, the employment of minor MRM transitions for the quantitation and identification of a given analyte is presented as a satisfactory solution to HPLC/MS/MS selectivity problems, and proposed as a possible alternative to previously published approaches.