Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

In the simplest form of our research, we are trying to discover new molecules in plants. Plants are chemically complex and some of these chemicals may be medicinally useful. We're especially interested in finding new alkaloids, which are certain molecules that contain nitrogen.

Recently, we have so many new and different alkaloid instruments and techniques available, so people discover more and more new molecules every day. With these instruments, we can answer questions like what chemicals are present, when are they present, in what part of the plant can they be found. Mass spectrometry, as an analytical technique, has really improved over the past 50 years.

Instruments are more powerful, less expensive, and have higher resolution. And these techniques allow us to not only identify more plant molecules, like alkaloids, but also be more sure in our identification. This method is fast and sensitive.

We were able to collect a full set of data on nightshade plants containing many tropane alkaloids in 60 to 90 minutes, and assign proposed structures to even the low-abundance alkaloids. We're very interested in developing tandem mass spectrometry-based filtering methods for other classes of alkaloids, and using methods like these to study how plant chemistry differs between different species, different plant parts, and different growth stages. 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 reversed-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 linked to the Q1 scan with a mass window of 50 to 1, 000 daltons, collision cell energy of 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 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 20 volts and event times of 0.75 seconds. Ensure that in all cases, automatic isotope 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. After the LC-MS run of plant root extracts, examine the total ion chromatogram of the Q1 and Q3 scans, including the data-dependent product ion scan.

Observe for apparent mass of abundant ions with tropane alkaloid-like features, including a mass under 500 daltons for the positively charged M+H ion, typically an even number, retention times, between two to 22 minutes, and fragments matching master charge values consistent with tropane alkaloids. Examine the precursor ion scan chromatogram or channel, specifically for master charge 124, and determine which peaks or ions at which retention times produce this specific fragment. Click scan by scan through the chromatogram and review the full MS-MS spectra from the data-dependent product ion scan, especially for lower abundant species.

Next, analyze the precursor ion scan chromatograms together for master charge 122 and 140, which indicate disubstituted tropane alkaloids, and master charge 138 and 156, indicative of trisubstituted tropane alkaloids. Examine the neutral loss scan chromatogram or channels for master charge 160 and 166. Identify which peaks or ions produce neutral losses and note their retention times.

Click scan by scan through the chromatogram and compare with the data-dependent product ion scan fragmentation, especially for lower abundant species. Use the combination of precursor ion scan and neutral loss scan data, supported by data-dependent product ion scan results, to make putative annotations of the observed alkaloids. Start with the smallest tropane mass, then add the neutral loss and account for any remaining mass.

Compare the alkaloid annotations against those reported in the literature to determine the tropane alkaloid substitution pattern. Additionally, use commercially available standards of common tropane alkaloids for confirmation. The full Q1 scan chromatogram of Datura metel root extract revealed diverse tropane alkaloids with varying abundances.

Features for master charge 124, 122, 140, 138, and 156 indicated the presence of tropane alkaloids. Many of these tropane alkaloids were acetylated, tigloylated, or derived from phenyllactic or tropic acid, as evidenced by the signals in the neutral loss scan chromatograms. Spectral data utilization enabled the annotation of specific alkaloids, notably identifying a compound with apparent mass of master charge 224.

The method allowed for the deduction of a compound structure through its fragmentation pattern and neutral loss, confirming the presence of tigloyl groups as substitutions. The examination of a methanol water extract from Datura stramonium seeds yielded a full Q1 scan base peak chromatogram, identifying a highly-abundant monosubstituted tropane alkaloid, likely hyoscyamine. The high resolution MS-MS spectrum also allows the identification of new alkaloids from Datura stramonium seeds.