Date of Award

12-2015

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Bioengineering

Advisor

Harman, Melinda K

Committee Member

Schlautman, Mark

Committee Member

Weisensee, Katherine

Abstract

Approximately 1.5 wt. % of bone is comprised of citrate molecules bound to the surface of apatite crystals. Furthermore, 80 to 90% of physiological citrate is contained within bone. Recently, interest in citrate has increased due to the potential of a citrate based method for estimation of postmortem intervals, time since death, of skeletal remains. The broad objective of this research was to develop and validate a High-Performance Liquid Chromatography (HPLC) method for quantitative and qualitative analysis of citrate in bone. An appropriate HPLC column and operating conditions for the detection of citrate were selected and subsequently used to analyze standard solutions consisting of sodium citrate dihydrate in water from 0 to 2.5 mM, a physiological range, for linearity, impact of storage time, and intra-day test method precision. After testing on simple matrices, an existing bone processing protocol was optimized by determining the optimal mass of sample, presence of matrix effects, and most suitable bone type to sample (cortical or trabecular). Finally, the HPLC method and optimized processing method were combined to analyze the impact of short term (14 day) storage conditions, intra-bone factors associated with sampling at different anatomical locations, and applicability to a model for the determination of postmortem interval (PMI). The HPLC method developed in this study demonstrated acceptable linearity over a physiological range of standards (0 to 2.5 mM) in all sets of standards analyzed (n =11). Also, the method provided repeatable results under normal operating conditions. Analysis of three bone masses (50, 75, and 100 mg) and different bone types (cortical and trabecular) demonstrated 75 mg, cortical only bone samples were most suitable for analysis. Furthermore, standard addition and recovery tests indicated the presence of matrix effects within the processing protocol of the bones samples. Finally, a total of 36 bone specimens were prepared with the optimized processing protocol and analyzed for citrate. Chromatograms of each sample were analyzed for peak resolution and separation, as well as used to calculate the normalized concentration of citrate (wt. %.). Postmortem intervals were then calculated using a previously published citrate degradation model by Schwarcz et al. in order to assess precision. The mean concentration of citrate in all samples analyzed in this thesis was 1.12 ± 0.6 wt. %, which translated into a mean calculated PMI of 7.93 ± 13.72 years. The results from this thesis demonstrate the need for a better model of PMI estimation, as well as further research on the distribution of citrate in bone.

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