Hi everyone,

I’m seeing a broad baseline hump in the TIC after derivatizing acid extracts of biological samples (THC-COOH) with BSTFA + 1% TMCS (Regisil RC-2). This happens only when the acid fraction from urine is included. When I inject the basic extract alone (also derivatized), the chromatogram is clean.

The acid extraction protocol we normally follow is as follows:
	•	Basic hydrolysis: 500 µL 10N KOH, 56 °C for 20 min → neutralized with 1 mL acetic acid.
	•	Extraction: hexane:ethyl acetate (9:1), ultrasound 45 min, centrifugation at 1500 rpm.
	•	Evaporation: vacuum drying at ≤40 °C.
	•	Issue: After derivatization, tiny droplets remain in the tube, and a huge hump appears between 6–17 min in TIC.
	•	Already tried: extended drying, sodium sulfate, and DCM washes — nothing has resolved it.

This elevated baseline interferes with detection, especially when analytes are at low concentrations, as they get masked or lost under the hump.

I suspect residual moisture or stabilized microdroplets in the acid extract are reacting with the BSTFA.
However, we’ve been following exactly the same protocol for several months without this issue, and the hump has only appeared recently.
Also, I do not believe the derivatizing agent is degraded, since it works perfectly well with standard solutions, with blood samples, and with samples processed via basic liquid-liquid extraction from urine (i.e., without going through the acid extraction step).

Has anyone experienced something similar? Would azeotropic drying with toluene be a better approach here?

Baseline hump after acid extraction + BSTFA derivatization (GC-MS)