Pittcon 2022 Conference Highlights - Session 3

For the first time, a simple and robust quantitative reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed for dissolution profile studies for cannabidiol (CBD) in hemp oil infused products. The RP-HPLC method was developed and validated for its efficiency to quantitate CBD in hemp oil infused products and simultaneously applied for dissolution profiling. The chromatographic parameters used involves a mobile phase of 75/25 acetonitrile/water v/v with an isocratic elution and utilizes a stationary phase of SOLAS™ C18 150mm x 4.6mm, 100A°, 5µm column where the flow rate was 1.5mL/minutes and CBD was detected at 214 nm. Dissolution media to be used for the testing was optimised and performed the studies using 500 mL of simulated gastric and intestinal fluids separately where equivalent of 5mg and/or 10mg of CBD was introduced into the media. The dissolution was conducted using paddles at 50 rpm for 180 minutes. A comparison of dissolution profile was made for the hemp oil infused products purchased from the United states and Europe. Moreover, dissolution studies were performed at increase agitation speed of 75 and 100 rpm. An extended runtime for dissolution of 240 minutes also recorded. As the hemp industry now focuses on hemp oil-infused products with bioavailable CBD, the proposed dissolution test can be considered as a standard quality control test in future.

Sandhyarani Analakkattillam

University College Cork, Ireland

Determining fingerprint (FP) age is critical to suggest whether evidence is relevant to a crime timeline. So far, there has been no definitive way to determine the FP age. We have recently demonstrated ambient ozonolysis of unsaturated triacylglycerols has the potential to determine the age of latent FPs (Hinners et al., Anal. Chem. 2020, 92, 3125-3132). Additionally, the laser desorption/ionization mass spectrometry technique we use is ideal for FP analysis due to minimal FP detail destruction during analysis, unlike GC-MS or LC-MS. However, without chromatographic separation, spectra can become convoluted during the FP aging process. By employing Kendrick mass defect (KMD) plots for deconvolution, one can readily assign compound classes, assess degradation pathways, and have guidance in the selection of reliable spectral features to monitor during FP aging.

Groomed sebaceous FPs were acquired from a single individual onto precleaned glass slides. FPs were aged in the ambient laboratory environment for 0, 3, and 7 days. Sodium additive and sputtered gold were deposited onto the samples to assist the desorption/ionization. Samples were then analyzed using a QExactive HF (Thermo) with a MALDI source (Spectroglyph). The results corroborate that triacylglycerols, diacylglycerols, and wax esters degrade primarily by ozonolysis, and multiply unsaturated species can undergo multiple ozonolysis processes. Though these trends are intuitive based on previous work, the KMD plot analysis suggests that there should be a consideration when selecting compounds to monitor during degradation. Specifically, challenging to resolve isobars and isomers that arise from degradation manifest as overlapping series in KMD plots. Once identified, challenging spectral regions can be avoided while using lower resolution mass analyzers, which are more likely to be adopted in a forensic lab. With these considerations in mind, better fingerprint aging models can be developed in the future.

Andrew E. Paulson

Iowa State University, Department of Chemistry

In the analysis of North American pottery, a general inquiry made was if there was a way to organize and group pottery based on their elemental compositions. An approach to the problem included researching the distinct kinds of minerals these artifacts were composed of to determine their site of origin, though there was no conclusive method of doing so by using X-ray Fluorescence (XRF) alone. Using XRF to determine elemental compositions, it was found that there are differences between painted pottery and non-painted pottery. Additionally, elemental percentages varied between using XRF destructively and non-destructively. To organize data acquired by the XRF readings of these artifacts, Principal Component Analysis (PCA) was utilized for creating clusters of similar artifact groups via dimension reduction of the many elements measured. This approach proved a bit tricky since the pottery dug up from certain locations could point to origination, or the pottery simply passed by the area through trading. With this approach, certain pots from the same local region did not cluster. Significance tests including t-tests and ANOVA tests were utilized and revealed significant differences between elements, pinpointing where a sample had originated in the United States or possibly could have been traded to that region, which explains the strange clustering effect observed.

Fadi Alhalabi <falhalabi1@live.maryville.edu>

Maryville University of St. Louis

The postmortem interval (PMI) is a critical piece of information to determine when human remains are discovered. An accurate determination of PMI can facilitate the identification of an unknown individual and help to reconstruct the events around the time of death. A major weakness with the current state of the research is the lack of a reference dataset with a large number of cases from which research questions regarding factors impacting the rate of decomposition can be addressed. This presentation will be used to demonstrate an application that can be used by practitioners using crowdsourced data to collect information from scenes where human remains are found. This research utilizes a spatially coded, GIS application that is accessible from mobile devices and tablets among other devices. The decomposition process is influenced by a wide range of factors both intrinsic to the individual and extrinsic environmental variables. The only way to develop accurate models of this complex system is to have sufficiently large sample sizes. In the absence of a large dataset, a scientific investigation of PMI is severely limited. The small sample sizes and dependence on case studies has limited research in PMI. Previous research has demonstrated that there are multiple factors that impact the rate of decomposition and to develop a robust and multifaceted model a representative and large dataset is needed. In order for the field to move forward and to develop models that are reliable, useful, and with known error rates, then a large dataset is necessary and currently missing from the field. The forensic community can work together to construct a reference set to build models of decomposition and improving methods for determining PMI when remains are discovered.

Katherine Weisensee

Clemson University, Department of Sociology, Anthropology and Criminal Justice

Significant losses in peak resolution are encountered in ultra-high performance liquid chromatography (UHPLC) coupled to mass spectrometry (MS) detection. This problem is caused by the excessive post-column dispersion from the column outlet to the direct/infusion valve, and to the ionization probe. This issue is of great concern for short (2-5 cm long) narrow-bore (2.1 mm i.d.) columns packed with sub-2 m particles. The loss in peak capacity can then be as large as 50 to over 100% depending on the dimensions of the post-column tubing [1].

In this presentation, a solution that consists in deploying the column very close to the point of sample ionization while running the column in absence of a LC oven is proposed and described. First, a vacuum jacket is placed around the column in order to maintain the column temperature nearly uniform along its length. The vacuum jacket column (VJC) prevents most of the heat exchange between the lab air environment and the column wall. Secondly, a Joule heater is placed locally at the column outlet to 1) compensate for possible heat leaks at both column extremities and 2) reduce the nefarious impact of radial temperature gradients expected in UHPLC columns at high speeds/pressure [2].

Finally, the advantage of the VJC directly connected to the ionization probe of the MS detector over classical columns mounted in the oven of standard UHPLC-MS systems is demonstrated for a few applications including the improved baseline separation of drug compounds (RPLC CORTECS-C18, 2.1 x 100 mm column), 20 metabolites (RPLC CORTECS-C18, 2.1 x 50 mm column), and 6 substrates / 6 metabolite mixture (RPLC XBRIDGE-C18, 2.1 x 30 mm column). The gain in peak capacity (+130%) is explained quantitatively from the reduction of the post-column dispersion (from 13 to 0.3 uL^2), the thermal insulation of the column (using a vacuum jacket), and the integrity of the peak shape after optimizing the outlet endnut temperature.



[1]. Gritti F., Vacuum-Jacketed Columns: Maximum Efficiency, Easy Deployment Without Oven, and Improved LC-MS Performance. LC GC North America 2019;32(5):8-13. [2]. Gritti F.; Gilar M.; Jarrell J.A., Achieving quasi-adiabatic thermal environment to maximize resolution power in very high-pressure liquid chromatography: Theory, models, and experiments. J. Chromatogr. A 2016;1444:86-98. [3]. Plumb R., McDonald T., Rainville P., Hill J., Gethings L., Johnson K.; Wilson I., High-Throughput UHPLC/MS/MS-Based Metabolic Profiling Using a Vacuum Jacketed Column. Anal. Chem. 2021; doi.org/10.1021/acs.analchem.1c01982.

Fabrice Gritti <fabrice_gritti@waters.com>

Waters Corporation