High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

Current strategies for a urine drug screening are expensive to perform for routine analysis of increasingly large panels of drugs of abuse as required for work place testing, as well as therapeutic drug monitoring applications. Additionally, methods based on amino assays are prone to bias and are ultimately limited to target analysis of known drug panels. The main advantage of our method, which is referred to as MSI-CE-MS, is that it allows for the rapid screening of an unlimited drug panel and their metabolites with high accuracy and minimal sample workup.

There is an urgent need for a high-throughput and affordable screening method to improve patient outcomes by confirming adherence and optimizing drug dosage regimes, while revealing illicit drug usage. This multiplexed separation platform can be applied to a variety of other research areas related to targeted or non-targeted metabolite profiling in complex biological samples. Begin by thawing de-identified morning urine samples collected from patients with a known prescription drug history for minus 80 degree Celsius storage on ice and vortexing the thawed samples for 30 seconds.

Next, sediment the samples by centrifugation and combine three 10 microliter aliquots of each sample with 10 microliters of deuterated internal standards, five microliters of a 4-fluoro-L-phenylalanine and 3-chloro-L-tyrosine solution and 25 microliters of deionized water. Then centrifuge the mixtures for one minute and transfer 20 microliters of each sample into individual polypropylene vials. To set the uncoated fused silica capillary conditioning parameters, cut a 135 centimeter long, 50 micrometer inner diameter, and 360 micrometer outer diameter capillary from a spool.

Use a capillary window maker to remove about 7 millimeters of polyamide coating from both ends of each capillary. Next, install the capillary inlets into the capillary electrophoresis, or CE cartridge, by looping each capillary for two 360 degree turns, taking care to protrude about two millimeters of the capillary outlets out of the CE sprayer needle. When all of the capillaries have been installed, carefully load the CE cartridge into the CE system and store the CE sprayer out of the ion source.

Place the liquid chromatography, or LC sprayer, into the ion source and select flush in the system's software to set up the capillary conditioning. Set the duration of methanol, one molar sodium hydroxide, deionized water, and background electrolyte flush to 30 minutes per flush and place the isocratic pump on standby during the capillary conditioning period. After the last flush, clean the capillary electrophoresis mass spectrometer, or CE-MS sprayer, and the CE electrode with methanol soaked wipes.

Clean the CE-MS interface with a one to one isopropanol water solution to remove any residual salt deposits. Replace the LC sprayer with the cleaned CE-MS sprayer and a newly conditioned capillary into the coaxial sheath liquid interface. Then turn on the isocratic pump and apply a voltage of 30 kilovolts for 15 minutes to ensure that a stable CE current profile is achieved prior to the urine analysis.

In the system software, select preconditioning, set the flush to 600 seconds, and specify a background electrolyte file position. Set the injection to hydrodynamically inject the samples at 100 millibars for five seconds and to electrokinetically inject the background electrolyte spacers at 30 kilovolts for 75 seconds. Set the applied voltage to 30 kilovolts, the cartridge temperature to 25 degrees Celsius, and the total run time to 40 minutes.

Then, using the timetable, apply a pressure gradient of two millibars per minute from zero to 40 minutes during the separation. When the sample acquisition is complete, rinse the capillary at 50 millibars of pressure with the background electrolytes overnight while the capillary is in the CE sprayer in the ion source. Set the TOF-MS positive ion detection to span a mass-to-charge ratio of 50 to 1, 700, with a data acquisition rate of 500 milliseconds per spectrum, and confirm that both the profile and the centroid data are stored in D file format.

Set the electrospray ionization conditions to capillary and to nozzle voltages of 2000 volts each, the nebulizer gas to 10 pounds per square inch, and the drying gas delivery to eight liters per minute at 300 degrees Celsius with a sheath gas flow of 3.5 liters per minute at 195 degrees Celsius during acquisition. Then set the MS voltage settings of the fragmenter, skimmer, and octapole one radio frequency to 120, 65, and 750 volts, respectively, and set the instrument control and data acquisition parameters according to the design of the experiment. For data analysis, open the multi-segment CE-MS data in the system software.

Under chromatograms, set the extraction data and the chromatogram and mass spectral data formats to profile mode. Under smoothing, select quadratic cubic Savitzky-Golay and set the function width to 15 points. Then click Integrate MS and set the integrator to agile.

Under peak filters, set the maximum number of peaks to 11. Under view, click integration peak list and select peak number, retention time, peak area, peak height, and signal to noise ratio. Then save the parameters under a unique method name and apply the method to the process for the interpretation of each data set.

In this representative analysis of various isobaric isomeric drugs of abuse and their metabolites, 30 resolved peaks from 10 independent samples of a drug mixture were detected without sample carryover. Two other isobaric opioids could be fully resolved as 20 distinct peaks with the full scan data acquisition. Similarly, two amphetamine positional isomers were fully resolved in this multi-segment CE-MS analysis, to distinguish illicit methamphetamine abuse from the potential misuse of phentermine, a prescribed stimulant that is used as an appetite suppressant for weight loss.

In this representative drug screening analysis, a positive screening test result for methadone was deduced by the detection of a large signal peak co-migrating with the methadone-d3 peak with a low mass error. No other signals were detected in any other urine samples within the same run and the detected methadone concentration exceeded 13 times the recommended cutoff limit compared to it's measured ion response ratio in the sample and corrected by a four fold urine dilution factor, confirming the patients adherence to methadone maintenance therapy. Here, a serial injection configuration in multi-segment CE-MS, comprising five different drug calibrants, was analyzed in duplicate within a single run together with the synthetic urine blank.

The drug metabolites were detected as their potenated molecular ions above their screening cutoff limits. Following this procedure, a large number of polar and ionic metabolites in urine can also be analyzed by MSI CMS in support of discovery-based metabolics research. Additionally, comprehensive surveillance of a wide array of exogenous drugs and their metabolites can be achieved, including non-prescribed medications and illicit drugs prone to abuse.

This optimized method can also be used to routinely screen other drugs of abuse. These could be cannabis or alcohol metabolites and our method can also differentiate between a synthetic or a fake urine matrix and a natural, real, authentically donated urine sample as well. Remember to always use caution when working with biological fluids.