Smoker or Non-Smoker? Donor Characteristics from Saliva

Smoker or Non-Smoker? Donor Characteristics from Saliva

The discovery of body fluids during forensic investigation can provide countless clues to a crime. Saliva is often countered during certain investigations, particularly in the investigation of sexual assaults and other violent crimes. The wealth of chemical information housed in the complex matrix could provide clues as to who might have left the substance behind. One example of this is whether or not the donor is a smoker.

A team of researchers at the University at Albany have hit the news multiple times in recent years with their rapid, on-site tools for body fluid analysis. Using Raman spectroscopy, the group have successfully developed methods to identify body fluids, estimate body fluid age, and determine characteristics of the donor, such as race and sex. In the latest paper by Igor Lednev and his colleagues, published in Journal of Biophotonics, Raman spectroscopy has been shown to differentiate between smokers and non-smokers.

Working in collaboration with researchers at Kuwait University, the team applied Raman spectroscopy to dried saliva from smokers and non-smokers, aiming to use chemical differences in the samples to determine whether or not the donor smokes. Raman spectroscopy is a non-destructive technique that enables the rapid, on-site analysis of samples, producing distinctive chemical fingerprints consisting of bands produced by the interaction of light with molecular structures.

One might assume the test would target nicotine, a major chemical component in tobacco. However, nicotine is relatively short-lived in the body, thus is not a suitable target for analytical tests. Instead, the researchers focused on cotinine, a primary metabolite of nicotine with a notably longer half-life. Saliva samples from 32 donors were analysed by Raman spectroscopy and the chemical profiles produced studied for differences. Researchers soon encountered a problem. Raman bands indicative of cotinine overlapped with typical Raman bands produced by saliva, making the detection of cotinine in saliva challenging. The team used machine learning to solve this problem.

First, they identified eight spectral regions that contributed the most variation between the saliva of smokers and non-smokers. Using an artificial neural network, a classification model was constructed for the prediction of smoking habits of a donor. By inputting chemical data from known samples, the network is able to learn from the data in order to predict an output (in this case, whether or not the donor of a saliva sample was a smoker). In laboratory-based studies, the model constructed achieved an impressive accuracy of 100%.

Although this pilot study was based on a very limited sample size, the technique shows great promise in the determination of donor characteristics from dried body fluids.


Al-Hetlani et al. Differentiating smokers and nonsmokers based on Raman spectroscopy of oral fluid and advanced statistics for forensic applications. Journal of Biophotonics, 2019, DOI: 10.1002/jbio.201960123

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.


Raman Spectroscopy & Ink Analysis

Raman Spectroscopy & Ink Analysis

Whether it’s a suspected forgery, a threatening letter or a questionable signature, investigators are always looking for the most effective methods of linking a questioned document to its author. Traditional physical and chemical analyses of inks, such as visual analysis or thin layer chromatography, are undoubtedly not ideal in dealing with the intricate differences in the vast variety of modern inks. But as analytical technology advances, scientists are able to study these samples at a more elemental level, allowing a greater ability to distinguish between different samples or conversely link them.

The inks used in pens every day can vary in composition to a surprising extent, being oil, gel or liquid-based and containing various types and amounts of pigments and dyes, fillers, lubricants, surfactants (to reduce surface tension) and humectants (to prevent unwanted drying). To the naked eye the different inks used to write documents may seem utterly identical. However research has shown that with the right analytical tools in the scientist’s arsenal, it is possible to not only distinguish between inks produced by different manufacturers, but also potentially differentiate between batches produced by the same manufacturer but simply at different times.

A vast range of analytical instrumentation can allow scientists to achieve a colossal collection of things, one of these technique being Raman spectroscopy. Let’s have a brief look into how this technique works, and what it could possibly have to do with ink. Raman spectroscopy is a non-destructive, analytical technique based on the scattering of monochromatic light. Two types of scattering can occur. Primarily this is elastic scattering known as Rayleigh scattering (it is this that is responsible for the blue colour of the sky). But that isn’t what we’re interested in here.  When light encounters particles in the air, there is an exchange of energy between the photon and the molecule, and a scattering of light can occur in which the photon has either a higher or lower energy than the incident photon. Inelastic scattering, known as Raman scattering, is the basis of Raman spectroscopy. The loss or gain of energy during the interaction is referred to as Stokes and Anti-Stokes respectively. The emitted photon can provide information regarding the molecular structure of the sample. An excitation source, typically a laser, directs a beam over the sample, scattered light is then collected with a lens through some form of wavelength selector and a detector, and a Raman spectrum of the sample is obtained. Using this spectrum, it may be possible to differentiate between different substances.


So how can this be applied to ink analysis?

The ability to distinguish between seemingly identical ink samples could play a big part in tracing the ink used to write a document, determining if two documents were written using the same ink, and even helping to establish how long ago a document may have been produced based on when that particular batch of ink was produced. And that is exactly what Raman spectroscopy (and various other techniques) is aiming to achieve.

The research examining inks for purposes of forensic document examination is plentiful. The study under discussion here subjected a wide variety of pen inks to Raman spectroscopy, including pens from different companies, different models from within the same company, and different batches of the same brand and model. The results were promising. Oil-based pen inks were typically very similar, making it difficult to distinguish between different samples. However gel and liquid-based inks were quite a different story, containing such a range of components that the Raman spectral signatures could be used to distinguish between samples. It is one accomplishment to separate the inks produced by different manufacturers, but the use of Raman analysis has even succeeded in distinguishing between inks of the same manufacturer simply produced at different times. Small spectral differences in different batches of the same brand showed slight changes in the chemical composition of the ink over a number of years. These changes in the makeup of the same ink over time could be due to a wide range of factors – manufacturers changing to a different supplier of a particular dye, fluctuations in temperature and other variables during manufacture, and even slight errors during production.


If this application were to be perfected, a Raman spectral signature of a suspected ink sample could theoretically be compared to a wider database of known samples, allowing for the brand of the pen used and potentially even the time in which that pen was manufactured (making dubious assumptions that the ink composition has not changed with time). Perhaps seemingly a benign point to ascertain, but it could very well be a deciding fact in establishing roughly when a document was written or if a ransom note was written with the suspect’s favourite pen (drastic example perhaps!)

Of course the work does not necessarily take into account the fact that thousands of pens produced using the same batch of ink will most likely produce identical Raman spectra or how ink compositions might change over time through various effects, but it’s certainly an interesting application that could prove useful.


Braz, A et al. Studying the variability in the Raman signature of writing pen inks. Forensic Sci. Int. 2014 (245), pp. 38-44.

Georgia State University – Hyper Physics. Raman Scattering. [online][Accessed 10 Dec 2014] Available from:

Penn Arts & Sciences. Raman Spectroscopy. [online][Accessed 10 Dec 2014] Available:

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