Review: Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry Screening Procedure for Urine Samples in Forensic Casework Compared to Gas Chromatography-Mass Spectrometry

Emily C. Lennert





drugs of abuse, urine, gas chromatography, liquid chromatography, mass spectrometry, GC-MS, LC-MS, time-of-flight, TOF, quadrupole

Article Reviewed

Fels, H.; Dame, T.; Sachs, H.; Musshoff, F. Liquid chromatography-quadrupole-time-of-flight mass spectrometry screening procedure for urine samples in forensic casework compared to gas chromatography-mass spectrometry. Drug Testing and Analysis. 2016, 9, 824-830.


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.


Forensic toxicology screening generally involves two types of screening procedures: general unknown screening and multi-target screening. General unknown screening is the screening of a sample for an unlimited amount of possible compounds, whereas multi-target screening is the screening of a sample for a defined set of compounds. Gas chromatography – mass spectrometry (GC-MS) is a common general screening method, considered a “gold standard” in forensic analysis. However, liquid chromatography – mass spectrometry (LC-MS) is often used for targeted analysis, due to the technique’s highly sensitive and specific screening ability that allow for the detection of non-volatile, thermolabile (i.e. compounds that breakdown easily at high temperatures), and polar compounds without the need for derivatization steps. For GC-MS, derivatization may be required to detect these types of samples. Derivatization is a reaction that transform the compound of interest into a form that is more amenable to GC-MS analysis, e.g. more volatile. LC-time-of-flight-MS has been reported for the analysis of toxicological samples. By this method, samples are identified based on exact mass, isotopic pattern, and retention time, if retention time data is available. LC- quadrupole TOF-MS (LC-QTOF-MS) adds another dimension to the analysis by allowing for the fragmentation of molecules by collision induced dissociation, which allows for the matching of library fragmentation spectra. In this study, urine samples were screened using LC-QTOF-MS and analytes were identified based on accurate mass, isotopic pattern, retention time, and library search. Analytes identified were also compared to those identified by GC-MS.

Urine samples were obtained from autopsies performed at the Institute of Forensic Medicine, Ludwig-Maximilians-University of Munich. Blank urine samples were collected from healthy volunteers. Samples were prepared separately for LC-MS and for GC-MS. Details of the sample preparation can be found in the study under Sample preparation. Samples were qualitatively analyzed by LC-MS/MS, followed by semi-quantitative analysis. Semi-quantitative analysis was performed using LC-MS/MS for targeted screening and LC-QTOF-MS for untargeted screening. Limits of detection (LOD) and limits of quantitation (LOQ) were calculated for 34 substances. Confirmatory analysis was performed by GC-MS.

LODs and LOQs were determined for 34 compounds of interest. For LC-MS/MS and LC-QTOF-MS, LODs were determined to be less than 10 ng/mL for 91% of the compounds examined, displayed in Figure 1 within the study. LSD showed a LOD of less than 0.05 ng/mL for both techniques. However, MDA (3,4-Methylenedioxyamphetamine) had higher LODs of 4.0 ng/mL by LC-MS/MS and 13.7 ng/mL by LC-QTOF-MS. For diazepam, the LOD by LC-MS/MS was determined to be 4.1 ng/mL, whereas the LOD was determined to be 118.9 ng/mL by LC-QTOF-MS. By LC-MS/MS, LOQ was determined to be less than 20 ng/mL for 30 of 34 compounds, as shown in Figure 2 within the study. Four compounds had LOQs slightly above 20 ng/mL. LSD had a LOQ of 0.13 ng/mL by LC-MS/MS. Similarly, 31 of 34 compounds had LOQs less than 20 ng/mL by LC-QTOF-MS. Diazepam and methamphetamine had much higher LOQs, greater than 40 ng/mL, showing the greatest difference between techniques. Similar to LC-MS/MS, the LOQ of LSD was 0.12 ng/mL for LC-QTOF-MS.

A total of 86 post-mortem urine samples were analyzed for qualitative comparison between LC-MS/MS and LC-QTOF-MS. Multiple drugs were identified in several samples, resulting in 262 findings consisting of 76 different compounds for LC-MS/MS. Similarly, multiple drugs were identified in several samples analyzed by LC-QTOF-MS, with 71 compounds resulting in 247 total findings. Five substances that were identified by LC-MS/MS but not by LC-QTOF-MS were benzylpiperazine, duloxetine, risperidone, sufentanil, and triazolam. A comparison of 94.3% was reported, with 94.3% of compounds detected by LC-MS/MS being detected by LC-QTOF-MS.

Compound identification was performed using PeakView 2.2 Software with the integrated MasterView 1.1 Software. Identification criteria included mass error, retention time, isotope ratio difference between the observed and reference, and library search results. When all four criteria were fulfilled within the narrowest tolerance range, compounds were considered identified. If three of four were fulfilled, it was considered a strong indication of the compound’s identity. Two of four criteria being fulfilled was indicative of a slight indication of the compound. To allow for screening, two in-house search lists were prepared containing approximately 480 total substances. Other screening methods exist and are mentioned, such as “extended screening” and Non-Targeted Peak Finding, however, these methods are more time intensive.

Finally, LC-QTOF-MS results were compared to those obtained by GC-MS. The authors state that, in most instances, findings between the two techniques were consistent. LC-QTOF-MS, in some instances, allowed for the identification of more compounds than GC-MS. However, some compounds were preferentially detected by GC-MS, i.e. atracurium or propofol. Some compounds were identified by GC-MS but not by LC-QTOF-MS due to the compound not being present in the search list. After entering the compound into the search list, these compounds could be identified for LC-QTOF-MS, see Table 2 within the study. Additionally, some compounds were identified based on medication regime and were added to the search list prior to processing the LC-QTOF-MS data, allowing for subsequent identification of these compounds in the urine samples, see Table 2 within the study.

Scientific Highlights

  • LC-QTOF-MS provided a fast and specific technique for the identification of drugs in urine.
  • Identification of compounds was based on retention time, isotopic pattern, exact mass, and library search results.
  • LC-QTOF-MS provided comparable results to GC-MS, which is considered the “gold standard” in forensic analysis.
  • LC-QTOF-MS allows for targeted or untargeted data analysis.


Drug screening methods that are reliable, as well as rapid, are desirable in forensic toxicology.

Potential Conclusions

LC-QTOF-MS may be a suitable technique for the forensic analysis of urine samples.