Advanced TOF-MS technology enables fast, sensitive, and reliable detection of drugs of abuse in urine.Advanced TOF-MS technology enables fast, sensitive, and reliable detection of drugs of abuse in urine.

Urine testing is one of the most common screening methods to detect the use of drugs of abuse (DOA) within 72 hours of that use. Gas chromatography-mass spectrometry (GC-MS) has traditionally been used for this type of analysis, but the efficiency of this method can be jeopardized by the high matrix effects and frequent coelution that occur as a result of the chemical complexity of urine.The technique also exhibits limited sensitivity in the analysis of certain drugs while being associated with false positive results. Newly introduced time-of-flight mass spectrometry (TOF-MS) technology offers high sensitivity and superior spectral quality, allowing for increased confidence in results.

This article describes an experiment whereby DOA were extracted from a urine sample and fast GC was performed with detection using the novel TOF-MS technique. Additionally, deconvolution and data-mining software was applied to the TOFMS data. Using this software, a target library of drugs of interest was created and data were processed to automatically identify target compounds within the urine sample.

The analysis of urine for DOA can be used to evaluate possible accidental or intentional overdose or poisoning, to assess the type and amount of prescribed and/or illicit drugs used by a person, or to determine the cause of acute drug toxicity. It is also used to monitor drug dependency or to determine the presence of drugs in the body for medical and legal purposes. Urine drug screening allows for the detection of drugs such as marijuana, cocaine, heroin, and amphetamines as well as their metabolites. In many occupations, urine drug screening has become a required condition of employment.

Urine is a sample that exhibits high chemical complexity, resulting in increased matrix effects and coelution.As a consequence, the analysis of urine is a particularly challenging task, especially when trying to identify trace levels of DOA. Obtaining fast, sensitive, and reliable results from urine analysis is of utmost importance for forensic laboratories. A powerful method is required that is capable of eliminating matrix effects and coelution, as well as facilitating accurate and dependable identification of trace levels of DOA.

GC-MS has been extensively used for this type of analysis, however historical techniques require derivatization to allow the detection of polar compounds. It has also been demonstrated that some GC-MS methods often fail to detect morphine and benzoylecgonine at concentrations above 1.3mg/l and 0.5mg/l respectively. Additionally, there have been cases of false positive results reported due to similar retention times and mass spectra of different compounds.

The forensic industry has been increasingly turning to modern time-of-flight (TOF)-MS instruments as a viable solution for precise screening of urine samples for DOA. These high sensitivity instruments may be used in combination with data-mining and deconvolution software to eliminate matrix effects and co-elution.

TOF-MS Benefits
TOF-MS systems are able to analyze ultra-trace level volatile and semi-volatile organic chemicals in complex real-world samples and over a very wide mass range. These high performance and robust instruments operate at extremely fast scan rates, offering increased sensitivity, compound resolution, and spectral quality. Traditional TOF-MS systems generate spectra that do not often fit existing or commercially available libraries. However, new instruments have been introduced that generate spectra that exactly match classical quadrupole-generated spectra, allowing for confident identification using established libraries, such as NIST.

In addition, the innovative systems operate at speeds which provide compatibility with fast GC,GCxGC, and complex conventional GC applications. Overall, the new instruments facilitate enhanced detection of trace target analytes ensuring fast return on investment.

A urine sample analysis was performed to demonstrate the unique advantages of the latest TOF-MS technology in combination with deconvolution and data-mining software.

Urine samples were collected from a methadone substitution program. Glucuronide separation was followed by solid-phase extraction (SPE) using SPECDAU(Varian Inc.) cation exchangers to extract the organic compounds from the urine. SPE is a highly selective and sensitive technique for sampling volatile compounds from aqueousmatrices. Samples were then evaporated and concentrated in 50μL methanol, and 1μL aliquots of underivatized sample were used for GC and TOF- MS analysis under the conditions below.

A model 6890N GC system was used (Agilent Technologies), along with Optima 5Accent 10m× 0.2mm, 0.35μmdf columns (Agilent).A Bench TOF-dx MS system was used (ALMSCO International),with the transfer line temperature set at 250°C; a mass range m/z 35–635; scan rate/scan set rate 10kHz/2Hz; and ion source 260°C.All samples were analyzed in 10minute runs.

A target library of the compounds of interest was created in the deconvolution and data-mining software package.The GC-MS data were then imported and processed to identify targets.

The data obtained for one urine sample can be seen in Figure 1.These data are affected by high levels of background interference,which is to be expected due to the nature of the sample.This makes confident identification of compounds, particularly those at trace-level, difficult.

The TOF-MS data were processed by the associated deconvolution/ data-mining software,which initially applies a background compensation algorithm to the matrix-affected total ion chromatogram (TIC) (Figure 2).This removes interfering ions, flattening the baseline and producing “cleaner” spectra, allowing more reliable identification of compounds down to the lowest concentrations. In addition, the TOF-MS system used is able to produce “classical” electron ionization (EI) spectra for compound peaks. These spectra match those of established or commercial libraries, allowing simple and reliable identification and removing the need to create new libraries.

In order to address the problem of compounds coeluted within the TIC, the next step is the deconvolution of background-free peak spectra. The speed of data acquisition by the TOF-MS system allows the summation of many single scans to scan sets. The scan sets provide high S/N values and spectra that are unaffected by the phenomenon of spectral skew, which in turn enables effective deconvolution.

Deconvolution is followed by chemometric data analysis of the distinct spectra.When a target compound is detected, the software derives a match coefficient (0–1).Ultimately, a report is generated listing all positive compound identifications.

The background-compensated TIC of the urine sample within the deconvolution software can be seen in Figure 3. The software has interrogated the TIC for compounds listed in the drug library created. Detected target compounds are represented by red bars and the heights of the bars represent their peak sum. A section of the report of targets found can be seen in Table 1.

A number of compounds that would have been difficult or even impossible to identify from quadrupole data or using traditional compound identification software have been identified in the sample. In particular, it has been difficult historically to detect benzodiazepines using GC-MS due to the polarity of the non derivatized analytes. The high sensitivity and non skewed classical spectra provided by the TOF-MS system in combination with the advanced deconvolution software used has resulted in the confident identification of this class of compounds in their non-derivatized form.

Figure 4 illustrates the capabilities of the system, showing a saturated amisulpride peak that is overlapping a small concentration of alprazolam completely. By limiting the mass range between 100 and 400amu, alprazolamis positively identified, showing good spectral similarity to the library spectrum (Figure 5) and, as a result, a high match coefficient (0.88). The deconvolved spectrum versus the library spectrum (green) is shown in Figure 5. The quality of spectra is excellent despite the large matrix interference. Using the advanced software, it is also possible to library search unknown peaks in the NIST database for confident and comprehensive profiling of the sample.

Figure 1: TIC of raw GC-MS data from a urine sample.

Figure 2: Background-compensated TIC of the urine sample.

Figure 3: Software interface showing background-compensated TIC and specific identification of diazepam.

Figure 4: Overlaid chroatograms showing amisulpride and alprazolam coeluted.

Figure 5: Comparison of sample and library spectra for alprazolam showing strong match.

Table 1: Section of report listing drugs of abuse found in the urine sample.

The analysis of urine for DOA is traditionally performed using GC MS methods. However, historical techniques have been associated with a number of shortcomings that prevent the accurate and reliable detection of DOA in urine samples.New TOF-MS offers a viable alternative to conventional MS techniques, achieving fast, sensitive, and dependable results. The latest technological advancements have seen the introduction of novel TOF-MS instrumentation designed to offer the benefits of traditional TOF-MS systems while at the same time delivering NIST-searchable spectra meaning that proprietary libraries can be employed without modification. Even when a high matrix background exists, the TOF-MS data generated using a unique deconvolution algorithm allows for the identification of DOA at trace levels with a high similarity to commercial libraries.At the same time, the new TOF-MS instruments are able to record high-quality mass spectra over a wide concentration range.

Petra Gerhards is the Business and Development Manager for DACH for ALMSCO International. She has an MS degree in analytical chemistry and over 20 years experience in the interfacing of chromatographic techniques with mass spectrometry.

Dr. Pierre Schanen is principal scientist of five technologies, Munich, Germany, and head of a time-of-flight mass spectrometer design group.His main interests are ion and electron optics, spectral data processing, data compression, and instrument tuning algorithms.

Dr.Gerhard Horner is managing director of five technologies, Munich, Germany, and associated technical director of ALMSCO International, Llantrisant,UK.His main interest is multivariate signal processing for GC-MS data. Gerhard Horner is project coordinator of the joint research project EXAKT for the near-real time detection of hazardous substances funded by the German Federal Ministry of Education and Research.