Development and Validation of Toxicological Methods for Cognitive Stimulants in Traditional and Alternative Matrices

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2021-12-01T06:00:00.000Z
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Abstract

Attention-deficit/hyperactivity disorder (ADHD) is a neurological disorder that arises from a lack of dopamine in the brain. Patients with this disorder have an increased level of dopamine reuptake transporters in the brain which leads to a lack of dopamine in the synapse. The dopamine deficiency leads to inability to pay attention, lack of focus and boredom. Medications work to combat this disorder by blocking dopamine reuptake transporters thus increasing dopamine and speeding up brain activity. One of the main medications prescribed to treat ADHD is methylphenidate. Methylphenidate has two chiral centers which gives rise to four stereoisomers: the threo- and erythro- configurations of the dextro- and levo-methylphenidate enantiomers. The threo-methylphenidate configuration is known to be responsible for the pharmaceutical effects, specifically the d enantiomer as the l enantiomer has been proven to be toxic. Methylphenidate is typically sold as a racemic mixture of threo-methylphenidate with both the d and l enantiomer present. Due to the differing effects of the enantiomers, it is important to separate the enantiomers to better understand the pharmacodynamic and pharmacokinetics (PD/PK) of these medications. Methylphenidate is metabolized to ritalinic acid and, in the presence of ethanol, can break down to ethylphenidate. There are minimal comprehensive methods that separate the enantiomers of methylphenidate and its metabolites. To bridge the gap in knowledge, this study aims to analyze these cognitive stimulants in traditional and alternative matrices across multiple analytical platforms. Additionally, stability of these analytes needs to be assessed to better understand proper handling conditions of forensic toxicology specimens.

To better understand cognitive stimulants, such as methylphenidate, this study sought to develop analytical methods that can be used for the quantification of these analytes. Additionally, proof of applicability was conducted to demonstrate method validity. The main goals of this study were to 1) develop and validate a method for the chiral separation and analysis of d,l-methylphenidate, d,l-ethylphenidate and ritalinic acid in blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS); 2) develop and validate an achiral method for d,l-methylphenidate, d,l-ethylphenidate, lisdexamfetamine, and amphetamine in oral fluid using LC-MS/MS with application of the method to authentic oral fluid samples; 3) develop and validate a method for the chiral separation and analysis of d,l-methylphenidate, d,l-ethylphenidate and d,l-ritalinic acid in blood using supercritical fluid chromatography (SFC) coupled to LC-MS/MS and apply the method to authentic postmortem blood samples; and 4) assess long- and short-term stability of d,l-methylphenidate, d,l-ethylphenidate and ritalinic acid in blood.

A method was developed, optimized and validated for quantification of d,l-methylphenidate, d,l-ethylphenidate, and ritalinic acid in blood using LC-MS/MS. Chiral separation of the enantiomers was achieved using an Agilent Chiral-V column and this method was considered acceptable per validation guidelines with the exception of ion suppression/enhancement. However, the deuterated internal standards compensated for this as well as reproducibility of the effects. This method proved to be suitable for chiral separation without the need for hazardous and costly derivatizing agents traditionally used for separating enantiomers.

A method was developed, optimized, and validated for quantification of methylphenidate, ethylphenidate, lisdexamfetamine and amphetamine in oral fluid using LC-MS/MS. This method was considered sensitive and acceptable per validation guidelines except for ion suppression/enhancement. Similar to the blood method, the deuterated internal standard compensated for this phenomenon. For proof of applicability, this method was applied to 4 authentic oral fluid samples collected from college students alongside self-reported medication use. Both lisdexamfetamine and amphetamine were detected in the samples, as expected from subject surveys. This method demonstrates that oral fluid can be used as an alternative forensic toxicology matrix for detection of cognitive stimulants.

A method was developed and optimized for quantification of d,l-methylphenidate, d,l-ethylphenidate and d,l-ritalinic acid in blood using SFC-MS/MS. Method validation was conducted and was deemed acceptable. This method was applied to 49 authentic postmortem samples in which the enantiomers of the analytes were quantified and compared to results achieved from an achiral assay. Of the 49 samples, d,l-ritalinic acid was detected in all 49 samples, d-methylphenidate was detected in 29 samples, l-methylphenidate was detected in 15 samples, d-ethylphenidate was detected in 5 samples and l-ethylphenidate was detected in 1 sample. This technique offers an alternative way to achieve chiral separation of analytes.

Lastly, the stability of d,l-methylphenidate, d,l-ethylphenidate, and ritalinic acid were assessed over a 9 month period. Storage under frozen temperatures (-20ºC) was the only condition in which all analytes remained stable. A follow up study was conducted to assess methylphenidate degradation and determined that methylphenidate degrades to ritalinic acid under non-frozen conditions. This study demonstrates the importance of understanding proper sample handling and storage conditions as well as time of analysis for unstable drugs where quantification may be of important toxicological value.

The developed analytical methods herein offer chiral separation and quantification of methylphenidate and other cognitive stimulants in blood and oral fluid through various analytical techniques. As the potential for cognitive stimulant abuse and misuse is rising, it is important to analyze these analytes in forensic toxicology samples. Data from these studies can be useful for laboratories to better understand chiral analysis, alternative matrices and stability of these analytes for proper detection, quantification, and interpretation.

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Chemistry, Analytical
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