We present three fatal intoxications of methylone, a cathinone derivative. Blood was analyzed with a routine alkaline liquid-liquid extraction and analyzed by gas chromatography coupled with a mass spectrometer (GC-MS). Methylone was identified by a full scan mass spectral comparison to an analytical standard of methylone. For a definitive and conclusive confirmation and quantitation, methylone was also derivatized with heptafluorobutyric anhydride and analyzed by GC-MS. In all three fatalities, the deceased exhibited seizure-like activity and elevated body temperatures (103.9, 105.9 and 107°F) before death. Two of the three cases also exhibited metabolic acidosis. One of the three cases had prolonged treatment and hospitalization before death with symptoms similar to sympathomimetic toxicity, including metabolic acidosis, rhabdomyolysis, acute renal failure and disseminated intravascular coagulation. The laboratory results for this patient over the 24 h period of hospitalization were significant for increased lactate, liver transaminases, creatinine, myoglobin, creatine kinase and clotting times, and decreased pH, glucose and calcium. Peripheral blood methylone concentrations in the three fatal cases were 0.84, 3.3 and 0.56 mg/L. In conlusion, peripheral blood methylone concentrations in excess of 0.5 mg/L may result in death due to its toxic properties, which can include elevated body temperature and other sympathomimetic-like symptoms.

The illicit drug market has rapidly evolved from synthetic cannabinoids to cathinone derivatives and now a new emerging threat of synthetic opioids. These compounds were mostly developed by pharmaceutical companies during drug discovery. The new psychoactive substances are not routinely covered in drug screening and may go undetected. Recently fentanyl analogous, AH-7921, MT-45 and now U-47700 have been encountered in clinical and forensic casework. U-47700 is gaining popularity on drug user forms as a legal alternative to heroin. It is a µ-receptor agonist that is part of the trans-1-2-diamine opioid analgesic drug class developed by The Upjohn Company in an attempt to develop a non-addicting analgesic. A LC-MS-MS method was developed and validated to detect and quantify U-47700. Additional analysis was conducted with an LC-QToF to identify the presence of the parent drug and metabolites. A total of four cases have been evaluated by the LC-MS-MS methodology which has an analytical range of 1-1,250 ng/mL and limit of detection of 1 ng/mL. The concentration of U-47700 in urine specimens ranged from below the limit of quantification to 224 ng/mL. The ToF analysis detected the presence of suspected phase I demethylated metabolites that may assist future analysis of this compound. The prevalence of designer opioids in casework highlights the importance of analysis for new psychoactive substances. Traditional opiates/opioids were not detected in the presented cases, but the available case histories revealed an opioid toxidrome. These findings suggest that U-47700 drug may cause significant morbidity and mortality within the United States as an emerging drug threat.

These cases are concerning because U-47700 is a relatively new agent that is easy to obtain over the internet and has the potential to cause significant morbidity and mortality.

Phenytoin (diphenylhydantoin) is an anticonvulsant drug that has been used for decades for the treatment of many types of seizures. The drug is highly protein bound and measurement of free-active form of the drug is warranted particularly in patients with conditions that can affect drug protein binding. Here, we describe a LC/MS/MS method for the measurement of free phenytoin. Free drug is separated by ultrafiltration of serum or plasma. Ultrafiltrate is treated with acetonitrile containing internal standard phenytoin d-10 to precipitate proteins. The mixture is centrifuged and supernatant is injected onto LC-MS-MS, and analyzed using multiple reaction monitoring. This method is linear from 0.1 to 4.0 μg/mL and does not demonstrate any significant ion suppression or enhancement.

A simple LC-MS-MS method was developed for the assay of free phenytoin. The method does not require laborious liquid-liquid or solid-phase extraction. The method has high analytical sensitivity, low imprecision, and a wide analytical measurement range.

Vitamin D plays a vital role not only in bone health but also in pathophysiology of many other body functions. In recent years, there has been significant increase in testing of 25-hydroxyvitamin D (25-OH vitamin D), a marker of vitamin D deficiency. The most commonly used methods for the measurement of 25-OH vitamin D are immunoassays and liquid chromatography tandem mass spectrometry (LC-MS-MS). Since immunoassays suffer from inaccuracies and interferences, LC-MS-MS is a preferred method. In LC-MS-MS methods, 25-OH vitamin D is extracted from serum or plasma by solid-phase or liquid-phase extraction. Because these extraction methods are time consuming, we developed an easy method that uses simple protein precipitation followed by injection of the supernatant to LC-MS-MS. Several mass-to-charge (m/z) ratio transitions, including commonly used transitions based on water loss, were evaluated and several tube types were tested. The optimal transitions for 25-OH vitamin D2 and D3 were 395.5 > 269.5 and 383.4 > 257.3, respectively. The reportable range of the method was 1-100 ng/mL, and repeatability (within-run) and within-laboratory imprecision were <4% and <6%, respectively. The method agreed well with the solid-phase extraction methods.

Marijuana, which is made from crushing the leaves, flowers, and sometimes the stems of the plant Cannabis sativa, contains more than 30 cannabinoids. The major psychoactive cannabinoid is delta-9-tetrahydrocannabinol (THC). The major metabolite of THC, 11-nor-delta 9-carboxy-tetrahydrocannabionol (THC-COOH), is excreted in the urine primarily as a glucuronide conjugate and is commonly analyzed in biological specimens for detecting marijuana usage. The procedure described here involves the addition of deuterated internal standard THC-COOH-d9 into the sample followed by hydrolysis of conjugated THC-COOH by alkali. THC-COOH is extracted from urine or blood using liquid-liquid extraction followed by preparation of its trimethylsilyl derivatives. The analysis of derivatized THC-COOH is performed using gas-chromatography/mass spectrometry (GC/MS). Quantification of the drug in a sample is achieved by comparing the responses of the unknown sample to the responses of the calibrators using selected ion monitoring.

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