Relationships between structure, ionization profile and sensitivity of exogenous anabolic steroids under electrospray ionization and analysis in human urine using liquid chromatography–tandem mass spectrometry

Abstract

The relationships between the ionization profile, sensitivity, and structures of 64 exogenous anabolic steroids (groups I-IV) was investigated under electrospray ionization (ESI) conditions. The target analytes were ionized as [M + H](+) or [M + H-nH2 O](+) in the positive mode, and these ions were used as precursor ions for selected reaction monitoring analysis. The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The limits of detection (LODs) were 0.05-20 ng/mL for the 64 steroids. The LODs for 38 compounds, 14 compounds and 12 compounds were in the range of 0.05-1, 2-5 and 10-20 ng/mL, respectively. Steroids including the conjugated keto-functional group at C3 showed good proton affinity and stability, and generated the [M + H](+) ion as the most abundant precursor ion. In addition, the LODs of steroids using the [M + H](+) ion as the precursor ion were mostly distributed at low concentrations. In contrast, steroids containing conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H - H2 O](+) or [M + H - 2H2 O](+) ions, and these steroids showed relatively high LODs owing to poor stability and multiple ion formation. An LC-MS/MS method based on the present ionization profile was developed and validated for the determination of 78 steroids (groups I-V) in human urine.

Sensitivity of GC‐EI/MS, GC‐EI/MS/MS, LC‐ESI/MS/MS, LC‐Ag+CIS/MS/MS, and GC‐ESI/MS/MS for analysis of anabolic steroids in doping control

“…The LODs of these steroids obtained with LC‐based methods were mostly distributed across a relatively high concentration range. LODs of steroids that form [M+H] + ions in LC‐ESI/MS/MS analysis were mostly lower than the cut‐off value. LC‐based methods were shown to be more suitable than GC‐based methods for analysis of ATD, GES, THG, FOR‐M, ETHLE, and 3’‐OH‐PROS.…”

“…Except for the injection mode, the conditions were identical for the GC‐based methods. The LC conditions were based on papers published previously by our group . All targeted analytes and their abbreviation are listed in Table .…”

“…Sample preparation was based on our previous reports . Urine (2 mL) samples were loaded onto a PAD‐1 column that had been washed with 2 mL of DW.…”

“…To estimate the limit of detection (LOD) for GC‐EI/MS, GC‐EI/MS/MS, and GC‐ESI/MS/MS techniques, five different urine samples were prepared at each concentration level in the range 0.02 to 200 ng/mL, and analyzed simultaneously with blank urine to determine LOD . The blank urine was a pooled human urine sample (approximately 300 people).…”

“…Liquid chromatography‐electrospray ionization/tandem mass spectrometry (LC‐ESI/MS/MS) has also been used as a complementary analytical tool for general GC‐MS procedures . Many other approaches based on the ionization of steroids using ESI, APCI, APPI, and CIS have been investigated by several research groups . Previously, we reported analytical techniques for the simultaneous ionization and analysis of anabolic steroids using LC‐MS/MS with ESI and liquid chromatography‐silver ion coordination ionspray/triple‐quadrupole mass spectrometry (LC‐Ag + CIS/MS/MS) .…”