Sex Determination with Raman Spectroscopy

Sex Determination with Raman Spectroscopy

The ability to quickly identify a victim or suspect during a criminal investigation is crucial, and the use of fingerprinting and DNA profiling often proves invaluable in this. However, a fingerprint or DNA profile can only be associated with an individual if there is an alternative profile or database match for comparison.

But what can investigators do when comparison profiles are not available, rendering biological fluids found at crime scenes somewhat useless?

The capability of instantly establishing alternative information relating to a suspect – such as sex, age or a phenotypic characteristic – based on the analysis of the evidence could be a substantial benefit to an investigation.

In recent years, the use of both well-established and novel analytical techniques to ascertain information relating to a suspect or victim from bodily fluids has been the focus of a great deal of research. With an increasing number of analytical instruments becoming field portable, the possibility of in situ analysis at crime scenes and instant suspect information is quickly becoming a reality.

Raman Spectroscopy and Sex Determination

Most recently, researchers at the University of Albany (Muro et al, 2016) have highlighted the possibility of using portable Raman Spectroscopy to determine the sex of an individual based only on their saliva in real-time.

The study utilised a total of 48 saliva samples from both male and female donors of multiple ethnicities, depositing the samples onto aluminium foil and drying overnight. Samples were then subjected to Raman analysis and the chemical signatures scrutinised to determine whether or not the saliva of male donors differed from that of female donors.

Raman Spectroscopy is a non-destructive analytical technique used for analyte identification based on molecular vibrations. As a basic explanation, monochromatic light is initially directed towards the sample, some of this light simply passing through the sample and some of it being scattered. A small amount of this scattered light experiences an energy shift due to interactions between the sample and the incident light. These energy shifts are detected and transformed into a visual representation. The resulting Raman spectrum typically plots frequency vs intensity of the energy shifted light. The positions of different bands on this spectrum relate to the molecular vibrations within the sample which, if interpreted correctly, can allow for the identification of analytes.

Raman spectra are somewhat characteristic of the chemical composition of the sample. In the case of the saliva analysed in this study, the features of the spectra were largely caused by amino acids and proteins. When comparing the respective spectra from male and female donors, by eye they appear remarkably similar. However using multivariate data analysis, a statistical technique used to analyse data with multiple variables, the researchers were able to distinguish between the saliva of male donors and that of female donors, reporting the ability to ascertain the sex of the donor with an accuracy of an impressive 94%.

malefemaleramanspectra

Comparison of male and female saliva Raman spectra (Muro et al, 2016)

Although only a proof-of-concept paper, the research demonstrates the possibility of using portable Raman spectroscopy as a method of elucidating donor information, in this case sex, through the analysis of a bodily fluid. The researchers suggest further work will be conducted to include other bodily fluids and donor characteristics.

At this point, the usefulness of the research is limited. Although instantly establishing the sex of the donor of a bodily fluid can aid investigators in developing a suspect or victim profile more efficiently, the pool of potential donors is still huge. The total of 48 saliva donors used in this study is of course not a sufficient representation of the population, thus a much larger sample set would be required to fully evaluate the technique, including non-laboratory setting experiments. Furthermore, there is a wide range of medical conditions and additional factors that can result in changes in the chemical composition of saliva and thus could influence the effectiveness of this technique. Whether or not certain diseases or external influences can hinder gender determination using this method would need to be investigated.

Previous Research

The idea of utilising analytical chemistry to ascertain donor information is not in itself novel, and other researchers have attempted to achieve the same goal through different means.

In 2015, scientists also based at the University of Albany (Huynh et al, 2015) developed a biocatalytic assay approach to the analysis of amino acids in fingerprints to determine the sex of the donor. The study boasted an accuracy of 99%, with the sex differences believed to be due to the higher concentration of amino acids in fingerprints deposited by females.

Research by Takeda et al in 2009 used Nuclear Magnetic Resonance (NMR) Spectroscopy to determine differences between the urine and saliva samples of different donors based on the detection and comparison of different metabolites. Certain compounds, including acetate, formate, glycine and pyruvate, were found in higher concentrations in male samples, allowing for the differentiation between male and female bodily fluids.

The focus of such research is not limited to sex differentiation, for instance some research has even focused on establishing whether a blood sample belongs to a smoker or non-smoker. Utilising gas chromatography mass spectrometry with a solid phase microextraction pre-concentration step, Mochalski et al (2013) were able to effectively distinguish between the blood and breath of smokers and non-smokers due to the ten-fold increase in levels of benzene and toluene, a conclusion which has been repeated by other researchers.

Looking at just this small handful of studies, it becomes evident that certain analytical techniques have the potential power to ascertain a range of information about the donor of a bodily fluid. However all of these immunoassay and mass spectrometry techniques are typically time-consuming, requiring the transportation of a sample to a laboratory, sometimes extensive sample preparation, followed by a form of analysis that will often destroy the sample. This is evidentially not ideal during a time-sensitive criminal investigation in which sample amount may be limited.

To an extent, the research utilising Raman spectroscopy to determine sex from saliva does alleviate some of these problems. The portability of Raman devices allows for in situ analysis, removing the need for expensive and time-consuming laboratory analysis. As Raman spectroscopy is based on the interaction between the sample analyte and light, it is a non-destructive technique, allowing the sample to be preserved for storage and further analyses is required.

Although these techniques do not hold the power of DNA in almost irrefutably identifying the suspect, they may at least aid investigators in narrowing down their pool of suspects and steering the investigation in the right direction. No doubt further advances in analytical chemistry will allow for more accurate and robust techniques in the future.

 

References

Huynh, C et al. Forensic identification of gender from fingerprints. Anal. Chem. 87(2015), pp11531-11536.

Mochalski, P et al. Blood and breath levels of selected volatile organic compounds in healthy volunteers. Analyst. 7(2013), pp2134-2145.

Muro, C. L et al. Sex determination based on Raman Spectroscopy of saliva traces for forensic purposes. Anal. Chem. 88(2016), pp12489-12493.

Takeda, I et al. Understanding the human salivary metabolome. NMR Biomed. 22(2009), pp577-584.

 

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Fragrance Forensics: Using Perfume to Catch the Culprit

Fragrance Forensics: Using Perfume to Catch the Culprit

 

Every day we apply chemicals to our bodies in the form of perfumes, colognes, deodorants and moisturisers, producing a concoction of pleasant scents that can be quite unique. It is well-known that perfumes and other fragrances can be potent and persistent, lingering on clothes and skin for hours if not days. Furthermore, these aromatic mixtures lend themselves to being easily transferred from one person to another through physical contact.

As the field of forensic science advances, investigators are looking for different ways in which they can identify suspects and connect individuals, and perfume may be an ideal target. What if the fragrance worn by an individual could be identified on a chemical level and used to link that person to a particular person or place? Simona Gherghel and fellow researchers at University College London have aimed to achieve this using analytical chemistry techniques.

LLstructures

Linalool (left) and limonene (right), common components in perfumes and colognes.

Different perfumes and colognes are composed of a variety of volatile organic compounds (VOCs), which provide the products with their powerful and characteristic scents. Compounds commonly detected in such products include linalool, limonene, coumarin, geraniol and eugenol, often in varying quantities and mixed with an assortment of other components. Once applied, these fragrances are absorbed by clothing and skin and can be readily transferred to fabrics and other surfaces.

Using gas chromatography-mass spectrometry (GC-MS), a well-established analytical technique frequently utilised in forensic enquiries, the team analysed fragrances in a number of scenarios to investigate the extent to which chemical components could be transferred between surfaces and what circumstances might affect this transfer.

The research focused on a number of factors relevant to the use of fragrances as a potential form of trace evidence in forensic enquiries, specifically the method of transfer and the time between application of the fragrance and contact with another surface. Experiments involved contact between swatches of fragranced and fragrance-free fabrics, examining transfer of compounds when the fabrics were in contact with no friction, and forcefully rubbed together over periods of time ranging from 1 minute to 60 minutes. After controlled contact, swabs were collected from the fabrics and subjected to GC-MS analysis. Unsurprisingly, extended contact time led to an increase in transferred components. This may have the potential to indicate how long a victim and offender were in physical contact, whether it be fleetingly or for a prolonged period of time, the latter being more likely in the case of an assault.

This research also investigated the effects of time passed between application of a fragrance product and contact between two surfaces on the transfer and persistence of VOCs. Contact between a fragranced piece of fabric and a fragrance-free swatch was investigated at a number of time points ranging from contact occurring 5 minutes after fragrance use and to 7 days after use. As was expected, the number of chemical compounds transferred between the fabric swatches decreased with time, with larger-sized, less volatile molecules persisting for longer. When only 5 minutes had passed before contact occurred, an average of 24 volatile components were transferred from the perfumed fabric. However after 6 hours only 12 components were detected, and this decreased to only 6 components after 7 days. Although this shows that certain transferred chemical compounds can persist for days, there is a discernible decrease in their presence which ultimately makes the sample less detectable and less unique, as a smaller mixture of chemicals are available for identification and comparison.

Although this is the first published work demonstrating the transfer of fragrance between garments in a forensic setting, the possibility of identifying perfumes based on their chemical composition for forensic purposes has been previously examined by experts at Staffordshire University in the UK. Led by PhD student Alison Davidson, the team has been compiling chemical profiles of popular perfumes and colognes with the hope of distinguishing between brands of difference fragrances and ultimately using this information to aid criminal investigations.

The ability to identify perfumes and establish physical contact between two individuals based on VOCs could be of particular use in the investigation of sexual assaults and other violent crimes in which the victim and offender were in close contact. For instance, the contact between a victim’s perfumed clothing and the clothing of the offender could cause the transfer of volatile organic compounds to the offender’s clothing (or vice versa). Later analysis of a suspect’s clothing may then result in the identification of chemical compounds originating from the victim’s perfume, indicating physical contact and thus potentially supporting an accusation.

Although the research conducted has supported the possibility of utilising transferred VOCs in perfume and possible affecting factors to aid legal investigations, it is vital to consider that a greater range of variables must be taken into account if such analyses were to be utilised in real life scenarios. The degree of activity by the victim and offender and the time passed between the offense and forensic analysis must be considered, as should how unique the mixture of chemical components detected really is. Furthermore, if the transfer of perfume between fabrics can occur so easily, there is a distinct possibility that such a transfer could occur in entirely innocent circumstances, highlighting the importance of such analysis only being utilised alongside alternative sources of evidence.

The concept of studying the chemical composition of perfumes and fragrances to aid legal investigations is very much in its infancy, but with further research this technique may have the potential to offer investigators an additional tool to sniff out suspects.

 

References

S. Gherghel, et al., Analysis of transferred fragrance and its forensic implications, Sci. Justice (2016), http://dx.doi.org/ 10.1016/j.scijus.2016.08.004

 

Killer Cocktails: The Chemistry Behind the Lethal Injection

Killer Cocktails: The Chemistry Behind the Lethal Injection

In many countries worldwide, including the United States, lethal injection is used as a humane method of executing a death row inmate. With the lethal injection, the life of the inmate can theoretically be cleanly and swiftly ended through administering a number of drugs, with no pain and minimal trauma.

The debate over the lethal injection hit the news again last month when the U.S. Supreme Court ruled against claims that the use of a drug used in lethal injections (midazolam hydrochloride) violates the Eighth Amendment (relating to prohibiting cruel and unusual punishment). Despite this method of capital punishment largely replacing supposedly less humane forms of death such as the electric chair and hanging, there is still great debate over the ethics of certain drugs used, and whether they actually do provide a swift and pain-free death.

But what drugs are involved in this lethal cocktail, and how do these end life in an apparently ethical manner?

The procedure for lethal injection can vary across different countries and even different states. In the United States, execution by lethal injection is typically achieved through the intravenous use of three drugs in succession, each with a different purpose, though in some instances a single-drug method is used, usually involving a lethal dose of anaesthetic.

Sodium Thiopental (Source: Chemspider)

Sodium Thiopental (Source: Chemspider)

But let’s look at the three-part cocktail. The first drug to be administered is usually a barbiturate to act as an anaesthetic (painkiller), used to ensure the remaining steps in the procedure do not cause any pain. Traditionally sodium thiopental is used, a fast-onset but short-acting barbiturate. Barbiturates are compounds which can ultimately produce anaesthetic effects. They act as agonists of gamma-aminobutyric acid (GABA) receptors, which are inhibitory neurotransmitters in the central nervous system. By binding to this receptor, the activity of the central nervous system is depressed, bringing about effects ranging from mild sedation to general anaesthesia. In this instance, a sufficient dosage is administered to render the inmate unconscious, thus ensuring a painless procedure. However some have argued that the fast-acting effects of sodium thiopental can wear off before the execution procedure is complete.

Succinylcholine Chloride (Source: Chemspider)

Succinylcholine Chloride (Source: Chemspider)

Once the inmate is unconscious, a neuromuscular-blocking drug is then administered, generally succinylcholine (also known as suxamethonium chloride) or pancuronium bromide. Compounds such as succinylcholine bind to acetylcholine receptors, blocking the action of acetylcholine, a neurotransmitter essential in the proper functioning of skeletal muscle. When succinylcholine binds to this receptor, a cation channel in the receptor opens and depolarisation of the neuromuscular junction occurs. Normally when acetylcholine binds to this receptor, it soon dissociates following depolarisation and the muscle cell will be ready for the next signal. However compounds such as succinylcholine have a significantly longer duration, ultimately resulting in paralysis. In short, administering a drug such as succinylcholine prevents acetylcholine from communicating with the muscles and thus paralyses the inmate’s muscles, including those used to breathe. Other drugs such as pancuronium bromide can also be used, which have a different mechanism of action but ultimately achieve the same final result of muscle paralysis.

Finally the salt potassium chloride is administered. Within the body a variety of salts are vital for brain function, transmission of nerve signals and the beating of the heart, and these salt levels are tightly regulated by the body. In the normal functioning of the body, the majority of potassium is confined to the cells, with very little being present in the bloodstream at any one time. The introduction of a large amount of potassium chloride disrupts this electrochemical balance as the body’s cell are not able to equilibrate, rendering the cells unable to function, leading to cardiac arrest. In simpler terms, the overdose of potassium chloride brings about a condition known as hyperkalemia, in which the potassium concentration in the body is too high, causing the heart to fail. The inmate is officially declared dead when a cardiac monitor indicates the heart has stopped.

Recently, the drug used to initially render the inmate unconscious, sodium thiopental, has been difficult to obtain for a number of reasons, thus some states in the U.S. have used midazolam hydrochloride, a drug which has ultimately caused a great deal of controversy in recent years, such as in the Clayton Lockett case. This benzodiazepine is commonly used as a sedative, but when used during the lethal injection procedure, it is generally combined with an opiate. This is because midazolam itself has no analgesic (painkilling) effect, thus an additional drug is required to achieve this. Despite its recent use, claims have been made that a number of executions using this drug resulted in the prisoners showing signs of consciousness and gasping, suggesting that they were not quite as unconscious as intended. If the inmate is not unconscious when the muscle paralyser and electrolytes are administered, they may experience suffocation due to the muscle paralysing agent and burning caused by the potassium chloride.

So there we have it – some of the primary drugs administered during the lethal injection procedure and how they react within the body to bring about death. For more information on the death penalty (namely in the U.S), visit the Death Penalty Information Center.

References

Johnson, B. A. 2011. Addiction Medicine: Science and Practice Volume 1. New York: Springer.

Kroll, D. 2014. The Drugs Used in Execution by Lethal Injection. [online] Available from: http://www.forbes.com/sites/davidkroll/2014/05/01/the-pharmacology-and-toxicology-of-execution-by-lethal-injection

Kemsley, J. 2015. Sedative for Lethal Injections Affirmed. [online] Available from: http://cen.acs.org/articles/93/i27/Sedative-Lethal-Injections-Affirmed.html

Cover Image Credit: Thomas Boyd (The Oregonian)