Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

The overall goal of this experiment is to identify the species of botanical material using ambient ionization mass spectrometry. In conjunction with multivariate statistical analysis. This method can help to answer key questions in the chemistry and biology fields such as, for example, what species of plant you have based on its chemical fingerprint.

And you can also get information on the bioactive molecules that are in the plant tissue. The main advantage of this technique is that very little sample preparation is needed for the analysis and the data can be acquired within a few minutes. This method can also be used to solve other problems in chemistry.

For example tracking reaction intermediates and products in organic synthesis analyzing complex matrices of different types and doing headspace analysis. To prepare the kratom fresh leaf material use a six millimeter diameter hole punch to create uniform chads of kratom leaf material from M.speciosa plant. Repeat this process five times.

To extract the kratom powder work in a 1.5 milliliter microcentrifuge tube and suspend a small amount of kratom Bali powder in one milliliter of a one to one ethanol to water solvent mixture. Repeat this process five times. Sonicate the kratom Bali powder extract samples in an ultrasonic bath for 30 minutes at ambient temperature.

Then, centrifuge the kratom Bali powder extract samples for two minutes at 750 times G at ambient temperature. Decant the solvent from residual powder for subsequent analysis. To prepare the datura seeds slice a D.stramonium seed in half across the transverse plane using a razor blade.

Repeat this process using five different seeds. After setting up the mass spectrometer or MS parameters as described in the text protocol press Start Run in the mass spectrometer control software to analyze the kratom leaf. Suspend the chad of kratom leaf plant material between the ion source and mass spectrometer inlet with tweezers until a spectrum is obtained.

Repeat this procedure five times with separate chads of plant material. To calibrate the spectrum with polyethyline glycol 600 abbreviated PEG dip the closed end of a melting point capillary tube into the peg standard. Suspend the coated capillary between the ion source and mass spectrometer inlet.

Select the Stop button to end the analytical run. Press Start Run in the mass spectrometer control software to analyze the dried kratom leaf. Suspend a small amount of dried leaf material between the ion source and mass spectrometer inlet with tweezers until a spectrum is obtained.

Repeat acquisition five times analyzing new plant material each time. Calibrate the spectrum with PEG as before and then select the Stop button to end the analytical run. To analyze the kratom powder press Start Run in the mass spectrometer control software.

Dip the closed end of a melting point capillary into the kratom powder. Suspend the coated capillary between the ion source and mass spectrometer inlet until a spectrum is obtained. Repeat the analysis five times with a new capillary each time, followed by PEG calibration.

To analyze the kratom extract immerse the end of a capillary tube into the extract. Suspend the capillary tube in the 12 sample holder on the linear rail of the mass spectrometer. Repeat five times with a different extract each time.

Press Start Run in the mass spectrometer control software. Using the control panel, select the forward advance button to advance the linear rail through the ion stream at a rate of one millimeter per second to collect spectra. Calibrate the spectrum with PEG as before and press Stop to end the analytical run.

To analyze the datura seeds press Start Run in the mass spectrometer control software. Suspend the datura seed half between the ion source and mass spectrometer inlet with tweezers until a spectrum is collected. Ensure that the cut side is oriented to face the ion source.

Repeat this procedure five times analyzing a new seed half each time. Complete the analysis with PEG calibration and press Stop to end the analytical run. After performing data processing as described in the text protocol perform the principle component analysis by navigating to the Classify section of the spectral analysis software under the Setup tab and creating classes for data processing by selecting Add Class.

Import the text files of data by selecting Add Files. Assign data files to the appropriate class of plant by selecting Text Files and Set Class for Selected Files. Select feature masses for discrimination from MS from training set and set threshold percent to 1%Next, set a mass tolerance to 10 and select Build Vectors from Data Files.

In the Compute section perform principle component analysis by checking the box for 3D PCA Graph and select Calculate. Perform leave-one-out cross validation by selecting Validate. Then, generate the heat map of the data by navigating to the Frequency Plot tab of the spectral analysis software and selecting Heat Map.

Select Threshold saved data to set the abundance threshold to 1%Export the heat map to a spread sheet by selecting Save Heat Map to Excel. In the spreadsheet program save the exported heat map as a text file. Then, to perform the hierarchical clustering analysis import the heat map as a text file into Cluster 3.0 software.

In the Hierarchical tab of Cluster 3.0 under Genes and Arrays check boxes Cluster and Calculate weights. Set the cutoff at 0.1 and the exponent to one. Select single linkage clustering to perform analysis.

Finally, view the generated cdt data file in Java tree view. Representative soft ionization positive ion mode spectra of kratom products are shown here. Compounds previously isolated from M.speciosa were identified.

Including psychoactive biomarkers Mitragynin and 7-Hydroxy-mitragynine. Representative soft ionization positive ion mode spectra of datura seeds are shown here. Compounds previously isolated from datura were identified including psychoactive biomarkers Atropine and Scopolamine.

Heat map renderings of the mass spectrometric data are illustrated here. A principle component analysis or PCA plot of the kratom and datura data is shown. The PCA plot clearly shows that the kratom data and datura data are well resolved from one another.

PCA analysis also revealed that the individual varieties of kratom and the different species of datura can be identified and distinguished from one another. Hierarchical clustering analysis or HCA results of the kratom and datura mass spectral data are shown. The results of HCA revealed class, species, and variety differentiation solely based on direct analysis in real-time high resolution mass spectrometry derived data and confirmed the results of the PCA analysis.

Once mastered, this technique can be done in less than an hour depending upon the number of samples you have if done properly. After watching this video you should have a good understanding of how to use direct analysis and real time mass spectometry to analyze complex samples and to use statistical analysis processing of the data to get species information. While attempting this procedure it is important to hold the sample in the ion stream for an appropriate amount of time in order to acquire a complete mass spectrum of the sample.

This technique has allowed researchers in many fields including Plant Omics, Forensics, Organic and Analytical Chemistry to explore analysis of volatiles emitted by plants identify legal alternatives to elicit drugs and monitor reaction products in real-time.