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%.


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.



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.


Saliva for Cracking Down on Drink-Drivers

Saliva for Cracking Down on Drink-Drivers

The intake of alcohol for leisure is common all around the world, and many of us indulge every now and then (some more so). But we’re all also aware of the effects of alcohol and how it can play a big part in criminal activities, ranging from drunken street brawls to homicide. In fact according to the National Council on Alcoholism and Drug Dependence, in the case of 40% of convicted murderers in the US alcohol was a factor in the crime. But of course most commonly in legal investigations, focus on alcohol consumption is most commonly related to driving.

In the United Kingdom, people suspected of drink-driving are typically pulled over and breathalysed at the roadside. If they fail the test (i.e. they are over the legal limit of 80mg per 100ml of blood), they will be taken to a police station and breathalysed once again. Typical breathalysers work by measuring the concentration of alcohol in a person’s breath (note I say in their breath, not strictly their bloodstream – two very different things). After consumption, a certain amount of alcohol will leave the body via the breath, thus allowing us to pretty accurately calculate a person’s blood alcohol content.


But a primary disadvantage of the breathalyser test is that it cannot be repeated. Once the alcohol is out of the individual’s system, the proof of their blood alcohol content is gone. And as with many types of scientific analysis, a certain margin of error will exist. Furthermore, what if a breathalyser test is simply not plausible? Either because the suspected drink-driver is not able to provide a breath sample or they are making all attempts possible to avoid it (a certain Brighton-based woman who continued to have an alleged panic attack to avoid giving a sample, for instance). In short, there are numerous flaws in the use of breathalysers, and there’s no chance of an accurate retest further down the line if for whatever reason the original sampling comes under scrutiny.

But what if a biological sample could be collected at the time, stored and subjected to future analysis as needed, and even repeat measurements taken if required? A blood sample is surely perfect for this. But on the other hand, this is a fairly invasive procedure that will not necessarily be appropriate in all situations. How about a simple saliva swab?

A number of healthy subjects ingested enough beer to achieve 0.5g ethanol per kg of body weight, after which saliva, urine and breath samples were collected at 10, 30, 60 and 90 minute intervals following alcohol intake. The breath samples were taken using a standard breathalyser, and bodily fluids were subjected to analysis by gas chromatography with a flame ionisation detector (GC-FID). The results correlated well, indicating that the analysis of saliva could well be a suitable alternative for monitoring the alcohol levels in individuals.

The use of saliva to test alcohol levels is not strictly novel, as there are alcohol test strips available for use with saliva. However as with most tests such as these, they are simply presumptive, meaning some additional form of analysis is required for confirmation. But a procedure involving the immediate collection of a sample that can be stored for future analysis along with a confirmatory analytical technique such as gas chromatography can instil more confidence in both drink-driving scenarios and numerous other medico-legal situations.


Bueno, L. H. P. et al. Oral fluid as an alternative matrix to determine ethanol for forensic purposes. Forensic Sci. Int. 2014 (242), pp. 117-222.

National Council on Alcoholism and Drug Dependence. Alcohol and Crime. [Online][Accessed 30 November 2014] Available from: