To begin, use a pre-chilled spatula to add 100 milligrams of plant tissue powder into a tiered polypropylene microcentrifuge tube. Then immediately add one milliliter of 20%methanol to the tube. Place the capped tube on a rocking shaker for three hours at room temperature.
Next, centrifuge the tube at 9, 464 G for 10 minutes, and collect the supernatant into an LC-MS autosampler vial. Set up an LC-MS instrument with an electrospray ionization source and a reverse phase HPLC column. For HPLC, use 0.1%formic acid in water as solvent A and 0.1%formic acid in acetonitrile as solvent B.Adjust the column oven temperature to 45 degrees Celsius and the flow rate to 0.5 milliliters per minute.
Equilibrate the column with 99%A and 1%B. Set up a 30-minute gradient for solvent B concentration to increase from 1%to 50%over 26 minutes, then rapidly return to 1%B at 26.01 minutes and hold for four minutes. Configure the mass spectrometer's operating parameters.
Set the interface voltage to four kilovolts, the nebulizing gas flow to three liters per minute, and the heating gas flow to 10 liters per minute. Adjust the desolvation line temperature to 250 degrees Celsius, heat block temperature to 400 degrees Celsius, and interface temperature to 300 degrees Celsius. Set drying gas flow to 10 liters per minute and collision-induced dissociation gas pressure to 17 kilopascals.
Build an MS method in electrospray ionization source positive mode, include both Q3 and Q1 scans covering the mass range of 100 to 1, 000 daltons with the Q1 scan serving as a survey event with automatic isotope exclusion. Add a product ion scan link to the Q1 scan with a mass window of 50 to 1, 000 daltons, collision cell energy of minus 20 volts, and an event time of less than 0.2 seconds. Next, adjust the MS method to add positive mode precursor ion scans equal to the length of the LC method with collision cell energies of minus 20 volts and event times of 0.75 seconds.
Ensure that in all cases, automatic isotope exclude or deisotoping functions are enabled. Set the master charge values for monosubstituted, disubstituted, and trisubstituted tropane alkaloid fragments. Next, to the MS-MS method, add positive mode neutral loss scans equal to the length of the LC method with collision cell energies of minus 20 volts and event times of 0.75 seconds.
Ensure that in all cases, automatic isotopic exclude or deisotoping functions are enabled. Set up the neutral loss masses of interest for esters on tropane alkaloids for esters derived from tiglic acid, acetyl groups, and phenyllactic or tropic acid esters. Download the LC-MS method and create data files for the samples of interest.
Once the HPLC column is equilibrated at 45 degrees Celsius, inject 10 to 20 microliters of sample.
