With the on-going emergence of unlisted designer androgens/SARMs and masking tactics, firsFlagX (FFX) closes the gap by detecting unknown, biologically active androgenic agents in biological matrices and products ahead of targeted confirmation. We have updated FFX to cut cost and reaction time to make robot use a practical next step. Key changes include: (1) reduced reaction volumes to match common liquid-handler ranges; (ii) step consolidation by adding detection buffer directly to the mastermix (single mastermix setup); (iii) shortened incubation times to accelerate turnaround; and (iv) a rapid liquid-liquid extraction (LLE) that mitigates matrix interference. To ensure recovery of designer steroids and SARMs regardless of scaffold, we evaluated orthogonal extraction strategies- LLE (e.g. IPA_EtOAc with pH-shift and salting-out) alongside complementary solid-phase extraction chemistries. Using administered equine samples together with neat solutions, we demonstrated broad, catch-all capture into the extract.
We now demonstrate detection of androgenic bioactivity in plasma and serum from horses, camels, and dogs. The extraction step reduced matrix interference, increased the dynamic range, and improved sensitivity. This workflow now provides a clearer path to hair and urine applications where pre-concentration and cleanup are essential. The same extraction and patented technology is also now being applied to sports supplements (for functional flagging of androgenic activity) and to water and consumer products after simple concentration and extraction steps, enabling triage before targeted chemical confirmation.
FFX performs effect-based screening via receptor activation. Only the positives are escalated to confirmatory analytics (e.g. LC-MS/MS) linking a high-throughput triage to hard chemistry for unambiguous identification.
Together, these improvements position FFX as an automation-ready, cost-efficient screen for unwarranted androgenic agents in biological, product and environmental matrices- delivering rapid no/no-go decisions that seamlessly feed into downstream identification workflows.