Determination of fentanyl qualification and quantification in Phormia regina (Meigen) (Calliphoridae) using LC-MSMS
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As a primary colonizer of carrion, Phormia regina plays a vital role in both nutrient recycling and ecosystems via carrion decomposition. They are currently used by forensic scientists in establishing minimum post mortem interval estimation during criminal cases but recent research has that larvae can be further used for detection of drugs and toxins when traditional matrices are unavailable. In addition, the presence of toxins has been shown to increase or delay development of entomological specimens, which can be critical in a criminal investigation. It has become critical to understand the detection and effects of fentanyl on different matrices due to its increased use and high contribution to overdosing. The purpose of this study was to further analyze the question currently poised for forensic entomologists/toxicologists: if there is any correlation between concentrations of toxins in human postmortem tissue to those concentrations detected in blowfly larvae and can they be used reliably as samples for toxicological investigations. Using Phormia regina, this study evaluated LC-MSMS to quantify and qualify fentanyl accurately as well as obtain some preliminary information pertaining to the effects of fentanyl on larvae development. A maximum of 100 individual pupae received from the University of New Haven (West Haven, Connecticut, USA) and University College of St. Mary (Omaha, Nebraska, USA) were placed inside a mesh enclosure with an ambient temperature of 26-28 °C. Chunks of beef liver were provided to the adults to facilitate oviposition. Eggs were collected and placed on homogenized ground liver pre-dosed with fentanyl. The doses for trial 1 were low end therapeutic levels (1.5 ng/mL and 3.0 ng/mL). The doses for trial 2 were a high end therapeutic level (50 ng/mL), a toxic level (125 ng/mL), a lethal level (250 ng/mL) and beyond lethal level (500 ng/mL). The larvae were monitored, and sample larvae were taken every day to confirm the lifecycle stage. Triplicate larvae samples were collected at the 2nd instar, 3rd instar, post-feeding, pupae and adult life stages. The larvae were homogenized, extracted and analyzed using liquid chromatograph tandem mass spectrometry (Shimadzu, Kyoto; Japan). Fifteen random specimens were selected and used as a representative set every day for each dose to monitor life stage duration. These larvae were also measured to obtain a growth curve. During trial 1 where larvae were fed liver substrate with low-level amounts of fentanyl, the detection of the drug during 2nd/3rd instar, pupae and adult was either minimal or nonexistent. Most of the results did not meet the criteria of acceptability for LOD and LOQ (0.5 ng/mL) for the method used for the LC-MSMS except for one pupae from the 3.0 ng/mL dosed liver. False positives were seen in trial 1 of the adult control sample set. As preliminary results remain inconclusive, recovery involving chitinized empty puparial cases should be re-analyzed utilizing a separate validated method for the empty puparial casings for optimum recovery. During trial 1 the larvae went through life stages but only the control group successfully emerged into adults. The development and survival rates of the larvae were noted but ultimately deemed inconclusive due to inconsistent temperatures forcing the larvae into diapause. During the trial 2 fentanyl was detected in 2nd and 3rd instar larvae fed on toxic and lethal level dosed meat while detection remained minimal or nonexistent in the post-feeding larvae of any dose. There was no correlation found between the feeding substrate and the larvae samples of any stage of the life cycle. The development rate to 2nd instar was delayed and correlated with the increase of fentanyl thus prolonging the total time for development from 2 to 4 days. This study demonstrated that fentanyl was detectable in 2nd and 3rd instar larvae in toxic and lethal doses. Further, it demonstrated that fentanyl delayed development in Phormia regina by up to 4 days when exposed to high levels of lethal dosing, effecting accurate post mortem interval estimation. It was also concluded that there is no correlation between spiked food substrates and the concentration found in the analyzed insect. Factors such as the considerable variation in toxins found in a corpse, decomposition chemicals, and different carrion insects during different times of the year, as well as more research are needed in the field before correlation can be confirmed. As for development and survival rates, this remains to be the most important to note when studying toxins in insects.