PAPER CRIMINALISTICS Julie A. Barrett, 1 M.S.; Jay A. Siegel, 2 Ph.D.; and John V. Goodpaster, 2 Ph.D. Forensic Discrimination of Dyed Hair Color: I. UV-Visible Microspectrophotometry*   ABSTRACT: Current protocols for examining hair do not attempt to differentiate hair color using instrumental analysis. In this study, hair sam- ples treated with 55 different red hair dyes were analyzed using UV-visible microspectrophotometry between 200 and 700 nm. Using air as a back- ground reference gave the best results, although mounting media such as glycerin could also be used. The contribution of the hair substrate is predominantly observed in the range of 300–400 nm while the dye peak is evident in the range of 425–550 nm. It was found that the presence of hair dye reduces the overall intrasample variability of the hair color. In addition, visual inspection and spectral interpretation showed that dyed hair exhibits distinct and discernable shades. The color of all samples was stable during storage and while all hair dyes fade with washing, significant fading of the color was only evident after daily washing for 3 weeks. KEYWORDS: forensic science, hair dyes, microspectrophotometry, hair examination, spectral comparison, cosmetic modifications Although cosmetic modifications such as hair bleaching and ⁄ or dyeing occur with significant frequency, insufficient research has been performed to distinguish hair samples based on their color. Hence, this research is intended to assess the ability of UV-visible microspectrophotometry (MSP) to differentiate the color of dyed hair in the context of forensic hair comparisons. Microscopic examination of hair does not provide a definitive link between an unknown speci- men and the suspected source, and the proposed research will not claim to do so; however, extending the current hair analysis protocol to include forensic characterization of dyed hair color may ultimately provide a higher degree of association between the sample and the source, as well as reducing false positive conclusions by revealing additional data which has been previously overlooked. Microscopic hair examination provides valuable evidence and insight into civil and criminal investigations. For example, the mor- phology and microscopic features of human hair provide a wealth of information such as species of origin, area of the body from which the hair originated, ethnicity, removal from the body, either forcible or naturally shed, in addition to disease states, thermal damage, and cosmetic modifications (1,2). In cases where the root of the hair is absent, and thus nucleated DNA (nDNA) is unavail- able, mitochondrial DNA (mtDNA) analysis can be utilized, if available. Microscopic hair examination is particularly critical in circumstances where either insufficient DNA is present in the sam- ple to carry out the mtDNA analysis or the suspects are maternally related (1,2). These cases support the necessity of exploiting all potential data present in each hair sample; thus, it is important to explore alternative analytical techniques. Hair dyes can be classified as either nonoxidative or oxidative based on the reactivity of the dyes (3). Nonoxidation dyes only physi- cally coat the surface of the hair and contain a single component. Oxidative dyes represent approximately 80% of the hair color market and are favored for providing a more consistent overall appearance of the hair as well as lasting for longer periods of time (3). All of the dyes presented in this study are oxidative dyes; thus, only the chemi- cal reactivity of this type of dye will be discussed in detail. Two com- ponent mixtures, referred to as the base colorant and developer oxidant, are characteristic of oxidative dyes and are stored separately until the dyeing process is initiated. Generally, the developer is a lotion or emulsion, while the base colorant is either a gel or a cream (4). Oxidative dyes are generally comprised of an oxidant, alkalizer, primary intermediates, couplers, solvents, thickeners, surfactants, buf- fers, a chelating agent, as well as an antioxidant. Primary intermedi- ates, also called precursors, and couplers diffuse into the cuticle independently as low molecular weight species and react with one another to create larger dye molecules within the hair. Demi-perma- nent, or long-lasting semi-permanent, and permanent dyes are two types of oxidative dyes. Although demi-permanent dyes are oxidative in nature, the mild compositions of alkalizing and oxidizing agents do not lighten the natural hair color, but only deposit the color. Per- manent colors both lighten the natural hair color and effectively deposit color in the cortex of the hair (4). The final shade of color produced for hair dyes relies primarily on three factors: the natural or preexisting color of the hair substrate, the interaction of the dye com- ponents with the hair, and the resulting color molecules deposited during the reaction process. The intensity of the natural hair color is indicated numerically, on a scale of 1–10, with level one representing black and level ten signifying the lightest shade of blonde hair. Light- ening of the natural hair color is directly proportional to the concen- tration of hydrogen peroxide in the developer along with the pH of the base colorant (5–7). 1 Department of Chemistry, Butler University, Indianapolis, IN. 2 Forensic and Investigative Sciences Program, Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN. *Presented in part at the 61st Annual Meeting of the American Academy of Forensic Sciences, February 16–21, 2009, in Denver, CO.   Funded by the Forensic Sciences Foundation Jan S. Bashinski Criminal- istics Graduate Thesis Grant. Received 26 Nov. 2008; and in revised form 27 Feb. 2009; accepted 29 Mar. 2009. J Forensic Sci, March 2010, Vol. 55, No. 2 doi: 10.1111/j.1556-4029.2009.01306.x Available online at: interscience.wiley.com Ó 2010 American Academy of Forensic Sciences 323