Sensitive detection of anionic metabolites of drugs by positive ion mode HPLC-PIESI-MS

• Detection of the anionic drug metabolites in positive ion mode by using the paired ion electrospray ionization (PIESI) technique.
• The detection sensitivity for the drug metabolites has been improved by 3 to 48 folds, as compared to the results obtained from the negative ion mode.
• A method based on solid phase extraction and HPLC–MS was developed for simultaneous determination of drug metabolites in urine.

The detection window for drugs of abuse, including performance-enhancing drugs, is limited by the sensitivity of analytical methodologies. Herein, paired ion electrospray ionization mass spectrometry (PIESI-MS) was employed for sensitive analysis of performance-enhancing drugs and drugs of abuse by detecting their glucuronide and sulfate conjugates. The proposed approach provides enhanced sensitivity for these drug metabolites, and overcomes the drawbacks of the less sensitive negative ion mode ESI-MS by detecting the anionic metabolites in the positive ion mode at higher m/z where the background noise is less. Absolute LODs down to sub-pg levels were obtained with the use of the optimal symmetrical or unsymmetrical ion pairing reagents. One to three orders of magnitude improvement were obtained compared to other reported methods performed in the negative ion mode. Structurally similar steroid conjugates were chromatographically separated and detected by HPLC coupled with PIESI-MS. Finally, an off-line solid phase extraction (SPE) protocol was successfully developed to eliminate any matrix effects in the analysis of human urine samples.

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