Fentanyl Analog Screening using LC-TIMS-TOF MS/MS

The scope of our research is to develop a method to discriminate and characterize isomeric analogs. This research aims to investigate if a method can be developed to confidently differentiate a large quantity, especially structural isomers. This protocol incorporates four modes of characterization that have previously not been coupled.

Using four orthogonal methods of identification for the analogs should improve the ability to differentiate isomers that are difficult to differentiate with current standard procedures. Titanium is a big societal concern in the opioid epidemic. New analogs creating laser labs are being found every day and having a methodology that quickly and confidentially helps determine each analog variant in the sample could improve the efficiency and drug law enforcement.

Begin by preparing the mobile phases for high performance liquid chromatography or HPLC. For mobile phase A or MPA, use five millimolar ammonium formate in water with 0.05%formic acid. For mobile Phase B or MPB use 0.05%formic acid in a one-to-one methanol acetonitrile mixture.

To begin the HPLC method development, open the liquid chromatography or LC software and create a new protocol. Then click the edit method button to edit the binary gradient. Under the binary gradient tab, set the timestamps and concentrations of MPA and MPB.

Set the stop time to 18 minutes, the flow rate to 0.400 milliliters per minute, and the minimum and maximum pressure limits to zero and 6, 000 PSI respectively. To initialize the HPLC, insert the LC column and column guards in the HPLC column section. Carefully observe the minimum and maximum column pressures.

To begin, TIMS-TOF MS/MS method development open the TIMS control application, under MS settings set the scan beginning and ending values to 50 mass-to-charge ratio and 1, 800 mass-to-charge ratio respectively. Select the ion polarity mode as positive and the scan mode as parallel accumulation serial fragmentation or PASEF. Next, move to the tabs under source and set the end plate offset to 500 volts, capillary to 4, 500 volts, nebulizer to 3.0 bar, dry gas to 10.0 liters per minute and dry temp to 200 degrees Celsius In the syringe pump settings assign the syringe as Hamilton one milliliter.

Enable the option active and set the flow rate to 80.0 microliters per hour. In the MS/MS tab under precursor ions, set the number of PASEF ramps to eight, minimum charge to one, and maximum charge to five. In scheduling settings, enable the precursor repetitions and under active exclusion check the box to enable and set the release to 0.40 minutes after.

Do not adjust the collision energy settings and isolation width settings. Then click to enable the TIMS stepping box. To perform calibration for both mass-to-charge ratio and mobility domains, under the mass-to-charge ratio calibration settings select one of the preloaded tuning mix profiles in the reference list box.

To calibrate, load the syringe for the TOF with a tuning mix solution. In the calibration mode on the right side of the window, select between linear, quadratic and enhanced quadratic to achieve the best score. And click calibrate to get as close to 100%as possible.

Next, calibrate for mobility following the steps demonstrated previously for calibrating mass-to-charge ratio. Once calibrated, save the MS method by selecting the method tab on the top bar. In the dropdown menu, select save as to generate a new MS method file.

Once a TIMS-TOF MS/MS data set has been collected in the data dependent acquisition or DDA PASEF mode, run the data independent acquisition or DIA on the ion mobility software application. To do so, first load the previously saved DDA method. Change the mass spectrometry to DIA passive fragmentation.

Leave all other settings the same. Under the MS/MS tab, select edit windows then use the open analysis button located at the top of the DIA pop-up window and load the previously saved DDA data set. When a heat map appears at the bottom left, displaying a polygon of windows running diagonally across the graph, click and drag to resize the polygon so that it fits the data in the heat map.

Set the number of mobility windows in the per mass width and the overlap. Click calculate windows on the bottom right of the pop-up to see the windows displayed with the new settings. Once suitable, click on apply.

To process the HPLC ion mobility TOF data of fentanyl analog screening, begin by checking the calibration in the data analysis software. To do so, right click on the file name under the analysis box and select properties. When the window labeled filename_analysis properties appears select calibration status.

From the dropdown box select instrument calibration for the mass spectrometer and confirm that the error is no greater than one PPM. Then select initial mobility calibration and confirm that the error is no greater than 1%To prepare a chromatogram in the data analysis software, right click on chromatogram under the file name and select edit chromatogram. In type, click the dropdown box and select extracted ion chromatogram.

Under filter, select all MS, and for scan mode, select all. If using masses to extract an ion, insert the theoretical mass-to-charge ratio of the molecule of interest. Set the polarity to positive mode and add the ion to the list.

If using a formula to extract an ion, insert the formula for the molecule as well as the ion forms of interest for the chromatogram. For the mass spectrum, make sure the chromatogram is selected and right click at the baseline of one edge of the compound peak and drag to the other edge of the peak. To generate a mobiligram right click on the left tab titled mobiligram and select edit mobiligram.

When the window titled edit mobiligram traces appears, begin editing the chromatogram. In the retention time input, add the retention time range of the peak of interest. Once the parameters are selected, click add, followed by okay at the top right.

To generate the compound spectra, at the bottom of the spectrum view window sequentially select profile MS and fragment MS.At spectrum view, right click and select copy compound spectra. To process the data, click on find then select compounds manually, chromatogram or mobiligram. Left click and drag to highlight the desired peak to yield important information on the molecule of interest.

Screening of the four isomers in a particular fentanyl analog screening kit revealed that the methoxyacetyl fentanyl and fentanyl carbamate separated well in the LC and mobility domains from the para-methoxy acetyl fentanyl, and B-hydroxyl fentanyl. However, a clear separation of the latter two was achieved in their fragmentation patterns. Screening of three isomers in another kit showed that alpha-methyl thiofentanyl and trans-3-methyl thiofentanyl separated in the LC domain, but not in the mobility domain.

However, trans-3-methyl thiofentanyl and cis-3-methyl thiofentanyl separated in the mobility domain and not in the LC domain. This data indicated the complementarity of the orthogonal LC and mobility domains in improving the identification of the isometric species.