Chromatographic column Kromasil-C 18 (250× 4.6 mm, 5 μ m), mobile phase A(0. 1%TFA), mobile phase b (methanol) gradient elution: mobile phase b is 40% ~ 80%, 0~ 105min. NTB standard curve y = 3.67059x+0.14123, recovery10/0.9%, RSD=l. 17%, thus establishing a highly sensitive sulfhydryl detection method.
2. Using molecular fluorescence spectrometry as the reaction condition, the sulfhydryl group was determined by reversed-phase high performance liquid chromatography gradient elution method.
OPA, dansyl chloride and ninhydrin reacted with cysteine, and their visible ultraviolet absorption spectra and fluorescence spectra were determined. Under different PH, temperature and reaction time, the fluorescence was determined by the reaction between OPA and cysteine. Suck cysteine solutions of 0, 20, 40, 60, 80, 100μL 0. 1 mmol/L respectively, add10μ l lh2o2 respectively, react at room temperature for 30min, then heat and evaporate, and the residue is diluted to 5 ml with 200μl OPA derivative solution, after 4 min. Take 5 μ l boric acid buffer solution with pH 8.4 and mix it 10 times; Add 2μl OPA derivative solution, and mix the samples for HPLC. Gradient conditions: the ratio of eluent B was 0 at 0 min, linearly increased to 60% at 17 min, linearly increased to 100% at 17.5 min, and the elution ended at 20min. The excitation wavelength is 340nm and the fluorescence detection wavelength is 450nm.
3. Pre-column derivatization HPLC-UV detection method
Tris (2- carboxyethyl)-phosphine (TCEP) was used as reducing agent, 7- fluorobenzene -2-OXA- 1, 3- oxadiazole -4- sulfonate (SBD-F) was used as derivative, N- acetylcysteine was used as internal standard, and methanol was used as mobile phase. The linear range is 8. 3 ~ 1042.6 μ mol/L, and the minimum detection limit is 0. The intra-day precision was 1.67% ~ 1.86%, and the inter-day precision was 2. 08% ~ 3.06%, and the average recovery rate is 90.
4. Electrochemical desorption combined with fluorescence technology.
The sample was fixed on the surface of gold electrode modified by alkyl mercaptan self-assembled film, and GSH was labeled in situ by the reaction of fluorescent reagent maleimide with free sulfhydryl group. The adsorbate on the electrode surface was desorbed at constant potential, and the fluorescence intensity of the desorbed substance in 0.1mol l KOH solution was detected.