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.