Chromatography in action

Andrew Williams reports on chromatography in forensic science

Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) technologies are increasingly used across a wide range of forensic science disciplines. So, what are the main applications? And what technology and hardware is used to carry out analyses?

ANCIENT CHEESE
One interesting recent application of LC-MS techniques took place at the University of Catania in Sicily, where researchers carried out proteomic analyses on a sample of ancient Egyptian cheese using an Orbitrap Fusion Tribrid (Q-OT-qIT) mass spectrometer from ThermoFisher Scientific, coupled on-line with a Dionex UltiMate 3000 RSLC nano-liquid chromatography system.

As Vincenzo Cunsolo, researcher at the Laboratory of Organic Mass Spectrometry (LOMS) at the University of Catania, explains, survey scans of peptide precursors in the m/z range 400–1600 were performed at resolution of 120,000 (@200m/z). Tandem MS was also performed by isolation at 1.6 Th with the quadrupole, and isolated peptide ions were subjected to high energy collisional dissociation (HCD). LC-MS-MS data was then analysed and searched against the comprehensive (all species) UniProt protein sequences database using integrated PEAKS de novo sequencing software and the Mascot algorithm.

Although keen to point out that such studies tend to use ‘multiple technologies’ aimed at the deep characterisation of the sample under investigation, and to provide complementary results, Cunsolo admits there is ‘no doubt’ that current LC-ESI-MS (liquid chromatography-electrospray ionisation-mass spectrometry) technology is the ‘most used MS-based method for food analysis, and in more particular in proteomics.’ LC-ESI-MS methodologies couple high-performance liquid chromatography [HLPC], a very powerful separation method, with MS, one of the stronger and most sensitive identification and structural characterisation analytical tools currently available.

“This approach allows confident identification of components in extremely complex matrices, such as a protein mixture extracted from a food, using sample amounts in the order of nanograms. Moreover, the results obtained show how proteomic investigation of ancient materials may provide valuable contributions for their characterisation,” says Cunsolo.

“More extensively, these results demonstrate that proteins can be identified in very ancient samples even if they have been exposed for a long time to harsh environmental conditions, because some peptide sequences, in contrast to what is commonly assumed, are intrinsically extremely stable. This feature opens new possibilities for the application of the methodology to other fields, such
as forensic science,” he adds.

DRUG SEIZURES
Meanwhile, Eleanor Miller, forensics market specialist at Cambridge, UK-based GC-MS specialist Anatune, points out that GC-MS has a wide-range of applications in forensic science, including the analysis of alcohol and ‘drugs of abuse’ – either following bulk drug seizures or as part of post mortem analyses of items such as blood, urine or hair – as well as for the analysis of fire debris samples, explosives, gunshot residues, FAME (fatty acid methyl ester) profiles in fingerprints and soil samples.

“At the moment, we currently have a Gerstel-Agilent GC-MS system at a hospital in England that runs forensic and clinical toxicology samples. The fast and accurate turnaround time is crucial for the clinical application, which could be a life or death situation if not treated fast enough,” she says.

“Many forensic labs are keen to cover some of the above applications with Gerstel and GC-MS as they can appreciate the many benefits like quality of data, reduced turnaround time and reduced cost per sample. Unfortunately however, funding for any forensic science project is currently an issue as there are very limited funding routes for forensic projects,” she adds.

For Miller, the key benefits of using GC-MS include its ability to analyse complex mixtures of compounds, to provide versatility and high peak resolution and to ‘accurately and reliably detect targeted and non-targeted analytes’ – as well as the high sensitivity of the technology in the determination of trace quantities and its ‘high specificity for unequivocal identification of forensically relevant compounds.’

“These have many profound applications. For example, trace determination of things such as single-dose drug administration in sexual assault cases, analysis of pictogram quantities of drugs and metabolites in hair samples, trace levels of ignitable fluids from arson scenes, minute quantities of FAMES in fingerprint profiling, trace levels of gunshot residues and explosives on swabs taken from hands to name but a few,” she says.

“I think the frequent introduction of newly modified drugs on the ‘market’ will drive an increased interest in the use of GC-QTOF and the powerful advantages it affords in analysing unknown compounds,” she adds.

FORENSIC TOXICOLOGY
Elsewhere, in the USA, the Alabama Department of Forensic Sciences (ADFS) uses a battery of mass spectrometry techniques and applications in both forensic toxicology – which focuses on the analysis of biological specimens, such as blood, for the presence of drugs and alcohol – and drug chemistry disciplines. As Curt Harper, Toxicology Discipline Chief at the ADFS, explains, most forensic toxicology laboratories screen cases by immunoassay and confirm or quantify them using a specific analytical platform such as GC-MS or liquid chromatography tandem mass spectrometry (LC-MS-MS).
 
“Mass spectrometry is often characterised as the ‘gold standard’ for confirmation testing, allowing for a positive identification based on a unique chemical ‘fingerprint’ for each drug,” he says.

In drug chemistry labs, investigators analyse submissions, for example related to drug trafficking cases, by testing the raw substance, such as leaf material, powders or pills. According to Harper, the ADFS has also begun using a direct analysis in real time (DART) ion source coupled to a time-of-flight (TOF) mass spectrometer (MS) instrument for the preliminary screening of chemistry cases – providing a presumptive result based on a molecular weight, which can then be used to batch cases for confirmation analysis with GC-MS.  

“This process has proven particularly useful in the analysis of emerging drugs of interest including synthetic cannabinoids, synthetic cathinones and fentanyl analogues,” he says.

The toxicology department at ADFS currently possesses seven Agilent 6890/5973 or 5975 GC/MS instruments, one Agilent 6430 LC/MS/MS, one Agilent 6460 LC/MS/MS, and one ABSciex 3200 QTrap – which are used to ‘quantitate’ drugs such as cocaine, methamphetamine, THC or morphine in biological specimens. The lab also houses an Agilent 6545 quadrupole-time of flight liquid chromatography mass spectrometer (Q-TOF LC/MS),which is used to identify novel psychoactive substances, such as fentanyl analogues and designer benzodiazepines, stimulants and hallucinogens. This is accomplished by comparing the ‘unknown target’ accurate mass and retention time to a known standard, before a score is generated by an algorithm within the software.

Harper also reveals that, statewide, the drug chemistry arm at ADFS currently uses five DART ion sources coupled to one Agilent 6224 TOF MS instrument, two Agilent 6230 TOF MS instruments, one Jeol AccuTOF MS instrument, and one ABSciex 3200 Qtrap. The four TOF MS instruments are used for preliminary screening of drug chemistry cases, with the Qtrap used for confirmation analysis of selected analytes. The drug chemistry lab also has 26 Agilent 6890/5973 or 5975 GC-MS instruments statewide, which are used for confirmation analyses. The screening instruments detect a molecular ion that is searched against a database with potential matches determined within 5 amu of a known mass.  

“For qualitative confirmation, an unknown sample is run in batch with controls, and the retention time must match one of the controls within 0.1 min, the library match must be above 80%, and ion ratios must match within 20% of the control,” says Harper.

In Harper’s view, the primary advantage and benefit of using LC-MS over GC-MS ‘lies in the sample preparation component as well as instrument run time.’ For forensic science applications, he points out that there may be a variety of biological matrices that are submitted for analysis and, while GC-MS ‘remains a powerful methodology for molecular identification,’ when dealing with biological matrices the sample preparation aspects ‘can be cumbersome.’  

“Often, derivatisation is required and extended run times to ensure complete resolution of interferences. Modern LC-MS instrumentation coupled with electrospray mass spectrometry eliminates the need for derivatisation and less complex sample preparation techniques may be employed,” he says.

“In addition, using LC-MS systems such as a triple quadrupole mass spectrometer (LC-QQQ) significantly reduces the background noise, interferences, and in turn increases sensitivity. The flexibility that the user has for selection of mobile phases and stationary phases with LC-MS systems is also advantageous because the molecular properties of the analytes of interest can be further exploited to ensure optimal chromatography,” he adds.

As far as toxicology goes, Harper believes the future lies in the ‘combined utility’ of LC-MS-MS targeted analysis and unknown screening by Q-TOF, which together form a useful ‘multi-tier approach.’  

“In drug chemistry, we believe that GC-MS will continue to remain the gold standard for the identification of controlled substances. DART-TOF MS instrumentation as a primary screening tool is both robust and efficient, and ultimately we would like to have one located in every laboratory in our system to streamline drug chemistry casework,” he says.

“LC-MS instrumentation could potentially provide a useful tool in the analysis of compounds that are not heat labile, or compounds that require specific columns for separation,” he adds.

 

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