Emily C. Lennert
liquid chromatography, mass spectrometry, tandem, quadrupole, LC, MS, LC-MS, MS/MS, dilute, shoot, drug, drugs of abuse, illicit, urine, psychoactive, substances, synthetic cannabinoids, benzodiazepines, metabolites, phenethylamines, synthetic opioids, piperazines, cocaine, LSD, ketamine, amphetamine, fentanyl, norfentanyl, methadone, cathinone
Kong, T.Y.; Kim, J. H.; Kim, J. Y.; In, M. K.; Choi, K. H.; Kim, H. S.; Lee, H. S. Rapid analysis of drugs of abuse and their metabolites in human urine using dilute and shoot liquid chromatography – tandem mass spectrometry. Archives of Pharmacal Research. 2017, 40, 180-196.
The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.
Analysis of a urine sample by liquid chromatography – mass spectrometry (LC-MS) is generally preceded by a sample clean-up procedure. Clean-up procedures are generally used to prepare a urine sample to allow for screening and confirmatory analysis for drugs of abuse and their metabolites. However, in selecting a clean-up procedure, one must consider cost, time, drug characteristics such as pH or solubility, and the analytical objective, i.e. identification or quantification. Clean-up procedures must be properly chosen to ensure that the sample is properly processed for the target compound, a task that may be difficult in a sample in which the target compound is unknown. For example, if a clean-up procedure were employed that worked to extract synthetic cathinones from the sample but did not work for synthetic cannabinoids, then the processed sample could not be used to screen for a full range of potential drugs of abuse. Considering all of these factors, it is desirable to create a method of analysis with simple sample processing that would allow for screening and confirmatory analysis of a wide range of drug compounds. The authors stated that the purpose of this study was to provide a general method for fast, simple, selective, sensitive, and simultaneous screening and analysis of a total of 113 drugs and drug metabolites by LC-MS/MS.
113 drugs and metabolites were selected for this study: 20 benzodiazepines and 6 benzodiazepine metabolites, 24 synthetic cannabinoids, 31 phenethylamines, cocaine and 2 cocaine metabolites, 12 synthetic opioids, 7 piperazines, and 10 other drugs and metabolites, including LSD, ketamine, and others. A full list of the compounds analyzed can be found in table 1 within the study. First, an internal standard mixture was prepared. An internal standard is a known concentration of a compound or mixture of compounds, different from the target compounds, that is added to samples to assist in quantifying the concentration of an unknown target compound within the sample. The unknown concentration can be determined by comparing the signal of the target compound to the signal from the internal standard of known concentration, using the peak area and a calibration curve. Then, a mixed stock solution containing all 113 drugs and metabolites was prepared, with equal concentrations of each compound. Various dilutions of the mixed stock solution were prepared to create working standard mixture solutions. Human urine calibration standards were then prepared at concentrations ranging 1-100 ng/mL by adding 40 μL of the working standard mixture solution required to reach the desired concentration to 360 μL of human urine. The resulting human urine calibration standard solutions were used to make calibrations curves, which are used in quantification of samples of unknown concentration. Finally, quality control standards of various concentrations were prepared from working standard mixture solutions diluted in urine. All human urine samples were stated to be drug free prior to being spiked with the working standard mixtures. To prepare the samples for analysis, 40 μL of the internal standard was added to the chosen sample, i.e. blank urine, urine calibration standard, or quality control standard. The sample was vortex mixed at high speed for 3 minutes, then centrifuged, i.e. spun, at 50,000 g and 4 ˚C for 5 minutes. Following centrifugation, 5 μL of the supernatant, i.e. upper liquid layer, was removed from the sample and injected into the LC-MS/MS.
LC-MS/MS was selected for this study. An electrospray ionization source operated in positive mode was used to ionize the samples. Tandem mass spectrometry, i.e. MS/MS, allowed for multiple stages of mass spectrometric analysis, with some form of fragmentation of the compound in between the stages. Multiple reaction monitoring was used to identify each compound of interest. Multiple reaction monitoring occurs when a precursor ion, e.g. the protonated molecular ion [M+H]+, and selected product ions, i.e. fragment ions, are monitored for each compound. A list of each compound’s precursor and selected product ions can be found in table 1 within the study. In this study, a triple-quadrupole mass spectrometer was used. In this setup, the first and third quadrupoles serve as mass filters that only allowed a selected ion through, with the second serving as the collision cell in which fragmentation of the compound occurs. Precursor ions are selected and analyzed in the first quadrupole, where only the precursor ion goes through and onto the second quadrupole. The precursor ions then enter the second quadrupole, where fragmentation occurs to produce product ions. All of the product ions are then analyzed in the third quadrupole.
Figure 1: Demonstration of a triple quadrupole mass spectrometer.
Blank urine samples indicated no significant interference peaks resulting from the urine matrix present in the samples.
Quality control samples were analyzed for three consecutive days to determine inter-day precision and accuracy of the method. Samples were also analyzed multiple times per day to determine intra-day precision and accuracy. Results were indicative of acceptable accuracy and precision in the method for both inter- and intra-day performance. Additionally, stability of samples after long term storage was evaluated. Quality control samples were analyzed after storage at -80 ˚C for 28 days and compared to samples stored at 4 ˚C for 24 hours. The effect of long term storage was determined to be insignificant.
Calibration curves showed a linear trend for all compounds. This indicates that the signal obtained by the instrument is directly related to the concentration of the analyte, i.e. compound of interest. In other words, as the concentration increased, signal will also increase. Limits of quantification (LOQ) were also determined, which indicate the lowest quantifiable concentration of a given analyte. The LOQ is defined as the lowest concentration that provides a peak with a signal to noise ratio greater than 10; the signal to noise ratio is a number indicating the strength of a signal relative to the background signal, i.e. noise. A number of compounds failed to meet quantification requirements, but were still able to be detected, including cannabidiol, cannabinol, codeine, morphine, delta-9-tetrahydrocannabinol, JWH-018, methcathinone, and others.
The method was then tested on real-world samples obtained from 17 suspected drug abusers by the Narcotics Department at the District Prosecutors’ Offices in the Seoul metropolitan area, South Korea. The method was successfully applied to the samples, and results of each sample are summarized in table 3 within the study. The authors were able to detect and qualify the amount of drugs in the urine samples without significant sample preparation, such as clean-up procedures.
- A simple “dilute and shoot” sample preparation and analysis method for LC-MS/MS was presented; while not all 113 compounds were quantifiable, all were detectable by this method. No significant interference from the urine matrix was observed.
- The method was tested on a mixture of 113 compounds, containing drugs of multiple classes as well as metabolites.
- The method was tested on 17 real world samples obtained from drug abusers, and multiple drugs were detected and quantified in each sample.
In cases of drug abuse, multiple drug use is common. To prepare for LC-MS analysis, urine samples generally require a clean-up procedure; however, due to differences in the compounds, common clean-up procedures may not allow for detection of all drugs within a sample. The method presented provides a simple sample preparation procedure that allows for the detection of 113 drugs and metabolites of multiple classes within a sample.
- The presented method provides a simple, rapid, and accurate procedure for the determination of up to 113 drugs and metabolites within a urine sample.
- The presented method may avoid the potential issues of some clean-up methods, which may not allow for detection of all drugs and metabolites in a sample.