Amy Alexander of USDTL presents Demethylated Metabolites of Oxycodone and Hydrocodone in Hair and Nail Specimens at Midwest Association for Toxicology and Therapeutic Drug Monitoring (MATT).
Thank you so i will be discussing the d methylated metabolites of oxycodone and hydrocodone and human hair and nail specimens testing for drugs in hair nail is beneficial because hair and nail are considered reservoir matrices this means that drugs accumulate there so it can lengthen our detection window to about 3 to 6 months as was mentioned earlier drugs are
Incorporated into hair and nail in a few different ways when drugs are ingested they become present in our sweat and our blood sweat from our scalp and our hands deposits drug into our hair and nails while blood carries drugs into the hair nail and it gets incorporated as they grow hair simply grows straight out from the root while nail grows horizontally from the
Nail matrix as well as vertically from the nail bed additionally drugs can be incorporated passively through environmental exposure smoke and fumes in the air can deposit drug into the hair while handling pills and powders with our hands can deposit drug into our nails this however can be a disadvantage of hair nail testing for instance at our laboratory we had a
Client who happened to be a pharmacist who was suspected of abusing oxycodone we tested his hair and we did indeed find that he tested positive for oxycodone however his defense was that he handles these pills every day and so he tested positive not because he took the drug but because he was exposed to it unfortunately we were not able to confirm or refute this
Statement so this type of scenario was the inspiration behind this study we wanted to find a better way to prove that opiates were ingested instead of just being due to environmental exposure so i will be discussing the prescription opioid problem briefs drug metabolism and the results of this study according to the national institute on drug abuse prescription
Opioids account for the greatest amount of prescription drugs of abuse from 2001 to 2014 there was a three point four-fold increase in the number of deaths due to prescription opiates the main consequence of abusing prescription opioids is depressed breathing this causes hypoxia which is reduced oxygen flow to the brain and that causes coma seizures permanent
Brain damage and ultimately death oxycodone is the schedule to narcotic that is available as a prescription such as an oxycontin in the body it has two major metabolic pathways the od methylation pathway as it sounds is the removal of a methyl group from the oxygen and we get oxymorphone the ng methylated pathway is the removal of a methyl group from the nitrogen
And we get noir oxycodone in the body the ng methylation pathway is the major metabolic pathway so we would expect nor oxycodone to be present in higher concentrations than oxymorphone however the presence of either one of these two metabolites along with the presence of the parent drug oxycodone can indicate that the drug was ingested instead of just being due
To environmental exposure oxymorphone however is available as a prescription such as an l panna therefore its presence does not necessarily rule out environmental exposure nor oxycodone is not available as a prescription additionally we have changed an oxycodone tablet and test fit for the presence of no oxycodone on the left we have the drug and internal standard
From the calibrator and on the right we have the drug and internal standard from that oxycodone tablet and as you can see from the upper right hand command graham there was no nor oxycodone present in that oxycodone tablet therefore to the best of our knowledge nor oxycodone only exists when it is the product of metabolism by the human body similarly hydrocodone is
A schedule 2 opiate but is also available as a prescription it also has o and eggs methylated pathways the od methylation pathway gives us hydromorphone and the ng methylated pathway gives us nor hydrophobic once again the ng methylation pathway is the major metabolic pathway so we would expect nor hydrocodone to present in higher concentrations than hydromorphone
But the presence of either metabolite can indicate that the drug was ingested hydromorphone is available as a prescription and nor hydra colon is not unfortunately we were not able to obtain a hydroquinone pill to test for the presence of nor hydrocodone so for this study we took hair and nail specimen powder them once i washed that then powder them then incubated
Them in hydrochloric acid overnight we then subjected them to a solid phase extraction procedure and analyzed them via lc ms/ms this actually is the current method that we use when we test for drugs in hair oh for opiates and hair and now in our current method we already test for hydrocodone hydromorphone oxycodone and oxymorphone what we are looking to do is
Incorporate nor hydrocodone and nor oxycodone into our already existing method so far we have not done any quantitation validation so this is strictly a qualitative study right now a total of 92 hair and nail specimens that had previously been confirmed for opiates were used in this study 51 of those samples were positive for oxycodone and this chart shows which
Metabolites were present in those samples in the blue region we have the specimens that were positive for the parent drug oxycodone but they were negative for both metabolites in the green region we have the specimens positive for the parents oxycodone and positive for the metabolite nor oxycodone but they were negative for oxymorphone and in the purple region we
Have the specimens positive for the parent oxycodone and they were positive for both metabolites there were no specimen that were positive for oxymorphone but negative for nor oxycodone so if we wanted to look at the specimens that were positive for nor oxycodone it would be the green region combined with the purple region but assessments positive for oxymorphone is
Just the purple region therefore there are a greater number of samples positive for an or oxycodone event for oxymorphone additionally we looked at the parent drug concentration for each group and what we found was that the lowest parent drug concentration occurred in a blue region which is where no metabolites were detected an intermediate parent drug concentration
Occurred when only nour oxycodone was detected and the highest parent drug concentration occurred when both metabolites were detected so this hand suggest to us that in order to be able to attacked nour oxycodone we need an intermediate parent drug concentration however in order to detect oxymorphone we tended to meet higher parent drug concentrations though same
Specimen were also tested for hydrocodone and 63 samples were positive in the blue region we have the specimens positive only for the parent drug hydrocodone and they were negative for both metabolites in the green region we have the specimens positive for the parent hydrocodone and the metabolite nor hydrocodone but they were negative for hydromorphone in the
Purple region we have the specimens positive for the parent as well as both metabolites and in the red region we have the specimens positive for the parent hydrocodone and the metabolite hydromorphone but they were negative for nour hydrocodone and they were two specimen that fell into this group so once again if we wanted to look at the specimens positive for nour
Hydrocodone it would be the green region combined with the purple region and the specimens positive for hydromorphone is the purple region combined with the red region so once again you see that the nd methylated metabolite gives us a greater number of positive samples we also looked at the parent drug concentration for each group and found a very similar trend
The lowest parent drug concentration occurred when no metabolites were detected an intermediate errant drug concentration occurred when only noir hydrocodone was detected and the highest parent drug concentrations occurred when haddem when both metabolites were detected so once again this can indicate that in order to be able to detect noir hydrocodone we need an
Intermediate parent drug concentration but in order to detect hydromorphone we tended to need higher parent drug concentrations this red region however does not follow this trends because we see hydromorphone at pretty low concentrations of the parent drug this discrepancy can be explained through environmental exposure since both hydrocodone and hydromorphone are
Available as prescriptions and since there were only two samples and the concentrations were relatively low it’s very possible that these specimens were due to environmental exposure so in addition to there being a greener number of samples positive for the nz methylated metabolite both noir oxycodone and noir hydrocodone had greater concentrations than oxymorphone
And hydromorphone on this chart the vertical lines represent the ranges of concentrations for each analyte the filled in boxes represent the interquartile ranges the horizontal line through each box indicates the median and the tan dots indicate the average concentrations i also zoomed in on the oxymorphone peak and that’s what you see in the other right-hand
Corner so we compared the noir oxycodone concentrations to the oxymorphone concentrations using the wilcoxon test and we discovered that the p-value was less than point zero zero one this indicates a strong statistical significance that noir oxycodone existed at higher concentrations than oxymorphone now this chart includes all the specimen that were positive for
Oxycodone however there were specimens that were positive for oxycodone but negative for both metabolites in this scenario we are not sure whether these samples tested positive due to environmental exposure or due to ingestion so if we remove these ambiguous samples we are left with data that only comes from samples we know we’re due to ingestion and as you can see
The data does not change that much the same analysis was done for hydrocodones and we compared the noir hydrocodone concentrations to the hydromorphone concentrations using a mock accent test and once again we found that the p-value was less than point zero zero one indicating the strong statistical significance that noir hydrocodone exists at higher concentrations
Than hydromorphone and once again if we remove those ambiguous samples we are left with samples that we know for sure were due to ingestion and once again the data looks essentially the same so in conclusion nor hydrocodone and nor oxycodone were detected in a greater number of samples than hydromorphone and oxymorphone also nor hydrocodone and nor oxycodone had
Higher concentrations than hydromorphone and oxymorphone therefore when determining exposure over ingestion the mg methylated metabolites are a better indicator and give us greater confidence than the ohs methylated metabolites so all of this basically has been a proof of concept and so we do plan to continue to fully validate this method
Transcribed from video
Demethylated Metabolites of Oxycodone and Hydrocodone in Hair and Nail Specimens By United States Drug Testing Laboratories (USDTL)
