C. Clinton Frazee

Three Fatal Intoxications Due to Methylone

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.

Two cases of intoxication with new synthetic opioid, U-47700

Abstract Background: Novel substances often referred to as “designer drugs” have emerged as drugs of abuse, and recognition of these is difficult as routine blood and urine screening tests do not detect these agents. U-47700 is a synthetic selective μ-opioid agonist that can be bought online for as little as

Analysis of U-47700, a Novel Synthetic Opioid, in Human Urine by LC–MS–MS and LC–QToF

40 per gram. We report two patients presenting after insufflation of U-47700, with subsequent confirmation of this substance in urine samples. Case details: A 26-year-old man and 24-year-old woman insufflated a substance they believed to be “synthetic cocaine.” The man was found down with cyanosis and agonal respirations. He was intubated and taken to hospital where he recovered well with supportive care. The woman presented with anxiety, tremors and drowsiness and was admitted for observation. Urine samples from both patients were analyzed using GC/MS/MS and LC/QToF, and U-47700 was isolated in both cases. No other opioids were detected. Discussion: 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.

Quantitation of Total 11-Nor-9-Carboxy-Delta 9-Tetrahydrocannabinol in Urine and Blood Using Gas Chromatography-Mass Spectrometry (GC-MS)

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.

Quantification of Free Phenytoin by Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS).

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.

Comprehensive Urine Drug Screen by Gas Chromatography/Mass Spectrometry (GC/MS)

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.

Simultaneous Determination of Cyclosporine, Sirolimus, and Tacrolimus in Whole Blood Using Liquid Chromatography–Tandem Mass Spectrometry

Drug screening is an essential component of clinical toxicology laboratory service. Some laboratories use only automated chemistry analyzers for limited screening of drugs of abuse and few other drugs. Other laboratories use a combination of various techniques such as immunoassays, colorimetric tests, and mass spectrometry to provide more detailed comprehensive drug screening. Mass spectrometry, gas or liquid, can screen for hundreds of drugs and is often considered the gold standard for comprehensive drug screening. We describe an efficient and rapid gas chromatography/mass spectrometry (GC/MS) method for comprehensive drug screening in urine which utilizes a liquid-liquid extraction, sample concentration, and analysis by GC/MS.

Comprehensive Toxicology Drug Screening Data in a Pediatric Population

A multiple reaction monitoring, positive ionization electrospray, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous quantification of cyclosporine, sirolimus, and tacrolimus in human whole blood. Proteins in the samples are precipitated with a mixture of methanol and zinc sulfate. The supernatant is injected into the LC-MS/MS for analysis. Chromatography involves the use of a C18 column and ammonium acetate/water/methanol-containing mobile phases. The MS/MS is operated in positive ion electrospray mode. Quantification is achieved by comparing peak area ratios of MRMs of analytes and internal standards with that of calibrators. Calibration curves are constructed from peak area ratios of MRMs of calibrators and internal standards versus concentrations. MRMs used are ascomycin (m/z 809.5 → 756.5), cyclosporine A (m/z 1,219.9 → 1,203.1), cyclosporine D (m/z 1,234.0 → 1,217.0), sirolimus (m/z 931.6 → 864.5), and tacrolimus (m/z 821.5 → 768.4).

Quantification of Dehydroepiandrosterone, 11-Deoxycortisol, 17-Hydroxyprogesterone, and Testosterone by Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS)

The paper by Kyle et al. (1) intrigued us to analyze our pediatric experience with comprehensive drug screens from our hospital and many area hospitals for which we serve as a referral laboratory. Similar to Kyle et al. we use a combination of enzyme immunoassays and gas chromatography-mass spectrometry (GC-MS) from Agilent Technologies (Palo Alto, CA) for comprehensive drug screens. We also frequently use Varian Inc. (Palo Alto, CA) thin-layer chromatography system for drug screening. Other tests in our comprehensive drug screen include salicylate screen by Trinder’s reagent and volatile screen by microdiffusion. If microdiffusion is positive, confirmation of acetone, ethanol, isopropanol, and methanol is done by gas chromatography with flame ionization detector. Enzyme immunoassays for amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine metabolites, methadone, opiates, phencyclidine, and propoxyphene are by Beckman-Coulter Inc. (Brea, CA). In 2 yrs, 2002 and 2003, we screened 1248 samples for comprehensive drug screens. Based on age, we divided our data in three groups (<6, 6– <12, and 12– <18 years). These groups constituted 40, 16, and 44%, respectively. As reported by Kyle et al., our smallest group was also the 6– <12-yr-olds. Unlike Kyle et al. we analyzed the drug data for different age groups and looked at the differences in these age groups. Table 1 summarizes the immunoassay data on drugs of abuse. The major difference between Kyle et al. and our data was a significantly higher rate of benzodiazepines and cannabinoids in our pediatric population. This higher rate of benzodiazepine positives in our population may be due to their frequent use in our hospital or may be due to methodological

A Death Involving Hydrogen Sulfide Exposure from a Domestic Sink Drain

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders due to enzymatic defects in the biosynthetic pathway of cortisol and/or aldosterone. The analysis of cortisol, 17-hydroxyprogesterone (OHPG), dehydroepiandrosterone (DHEA), 11-deoxycortisol, and testosterone is generally performed in the diagnosis and/or follow-up of CAH. Cortisol is generally analyzed by immunoassays whereas other hormones are preferably assayed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). A multiple reaction monitoring, positive mode atmospheric pressure chemical ionization, LC/MS/MS method is described for the simultaneous quantification of 17-hydroxyprogesterone, DHEA, 11-deoxycortisol, and testosterone. Stable-isotope labeled internal standards are added to serum samples and steroids are extracted by liquid-liquid extraction using methyl tert-butyl ether. The extract is evaporated under stream of nitrogen and the residue is reconstituted in methanol and analyzed by LC/MS/MS.