Ethylene glycol: Evidence of glucuronidation in vivo shown by analysis of clinical toxicology samples.

In the search for improved laboratory methods for the diagnosis of ethylene glycol poisoning, the in vivo formation of a glucuronide metabolite of ethylene glycol was hypothesized. Chemically pure standards of the ß-O-glucuronide of ethylene glycol (EG-GLUC) and a deuterated analog (d4 -EG-GLUC) were synthesized. A high-performance liquid chromatography and tandem mass spectrometry method for determination of EG-GLUC in serum after ultrafiltration was validated. Inter-assay precision (%RSD) was 3.9% to 15.1% and inter-assay %bias was -2.8% to 12.2%. The measuring range was 2-100 µmol/L (0.48-24 mg/L). Specificity testing showed no endogenous amounts in routine clinical samples (n = 40). The method was used to analyze authentic, clinical serum samples (n = 31) from patients intoxicated with ethylene glycol. EG-GLUC was quantified in 15 of these samples, with a mean concentration of 6.5 µmol/L (1.6 mg/L), ranging from 2.3 to 15.6 µmol/L (0.55 to 3.7 mg/L). In five samples, EG-GLUC was detected below the limit of quantification (2 µmol/L) and it was below the limit of detection in 11 samples (1 µmol/L). Compared to the millimolar concentrations of ethylene glycol present in blood after intoxications and potentially available for conjugation, the concentrations of EG-GLUC found in clinical serum samples are very low, but comparable to concentrations of ethyl glucuronide after medium dose ethanol intake. In theory, EG-GLUC has a potential value as a biomarker for ethylene glycol intake, but the pharmacokinetic properties, in vivo/vitro stability and the biosynthetic pathways of EG-GLUC must be further studied in a larger number of patients and other biological matrices.

Patients in medical treatment for attention deficit/hyperactivity disorder (ADHD): Are they at risk in drug screening?

The use of medicines to treat attention deficit/hyperactivity disorder (ADHD) has increased worldwide, including the use of amphetamine-based medicines or prodrugs that metabolise to amphetamine in vivo. At the same time, drugs-of-abuse testing by non-specific, point-of-care immunoassay methods ('quick tests') has increased. This article discusses the risk of 'false positive' results or post-analytical misinterpretations of results when immunoassays are used to analyse biological samples from ADHD patients. A rapid evidence review was conducted to identify studies that have focused on the risk of 'false positive' test results in immunoassay testing of patients treated with atomoxetine, bupropion, clonidine, guanfacine, methylphenidate, and modafinil. There is only evidence to suggest that bupropion should cause 'false positive' immunoassay results. However, there is a lack of systematic, updated evaluations and validations of cross-reactivity patterns for immunoassays in the literature. Advanced laboratory methods can distinguish the use of medicines from illicit amphetamine by stereospecific analysis of dextro- and levoamphetamine; however, these analytical services are not commonly available for routine drug testing. The present situation calls for more awareness, proper education and information on these critical ethical issues in drug testing, both for clinicians, other healthcare professionals involved in drug testing and for patients in medical treatment for ADHD. The pitfalls of immunoassays due to cross-reactivity and insufficient specificity/sensitivity can have serious negative consequences for patients safety with regard to incorrect laboratory drug-testing results. Consequently, confirmatory laboratory analysis should always be performed for 'presumptive' positive immunoassay screening results.

Characteristics of opioid-maintained clients smoking fentanyl patches: The importance of confirmatory drug analysis illustrated by a case series and mini-review.

The increase in opioid prescribing in many European countries over the last decade has raised concerns about associated diversion, overdose, and mortality. Fentanyl is one of these synthetic opioids that is typically prescribed as a transdermal patch for pain that requires continuous pain relief and has been the focus of investigation due to reports of overdose and death. We report a case series of 14 drug addiction treatment entrants, who entered treatment in a service located in the region of Southern Denmark from August 2015 to December 2015 for smoking fentanyl patches. Clients presented with difficulties breathing and pains in the lungs. The clients had a history of past opioid use, including heroin. Relapses resulted in treatment disengagement. Immunoassays for fentanyl were used in the service. In some cases, false negative results occurred. Clients' urine samples were subsequently analysed in a collaborating laboratory. Seven clients tested positive for fentanyl. One client was positive for both fentanyl and heroin. Analyses were also positive for other opioids and metabolites in 6 clients, predominantly codeine and oxycodone. Results from confirmatory analysis contributed to clearer insights into clients' drug histories, which facilitated personalised care plans consisting of opioid agonist therapy informed by confirmed drug use. In Denmark, prescription levels of fentanyl are high, which has been accompanied by observations of diversion and smoking in a smaller population. In addition to revision of inappropriate prescribing to reduce diversion, we recommend increased reliance upon confirmatory drug analysis in the addiction treatment sector in Denmark.

[New psychoactive substances require a paradigm shift in drug testing in Denmark].

The emergence of an increasing number of new psychoactive substances (NPS) on the drug market requires a paradigm shift in drug testing. Immunoassay screening needs to be replaced with highly specific and sensitive analytical methods based on chromatography and mass spectrometry to produce accurate results, promote health and patient safety and collect data on the prevalence of NPS use, impact on public health and clinical aspects of NPS in Denmark. Development and implementation of new analytical methods currently present a major challenge for both clinical and forensic laboratories.

Identification of a new psychoactive substance in seized material: the synthetic opioid N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]prop-2-enamide (Acrylfentanyl).

Among the new psychoactive substances (NPS) that have recently emerged on the market, many of the new synthetic opioids have shown to be particularly harmful. A new synthetic analogue of fentanyl, N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]prop-2-enamide (acrylfentanyl), was identified in powder from a seized capsule found at a forensic psychiatric ward in Denmark. Gas chromatography with mass spectrometry (GC-MS) identified a precursor to synthetic fentanyls, N-phenyl-1-(2-phenylethyl)piperidin-4-amine; however, the precursor 1-(2-phenethyl)piperidin-4-one, was not detected. Analysis of the electron impact mass spectrum of the main, unknown chromatographic peak (GC) tentatively identified an acryloyl analogue of fentanyl. Further analyses by quadrupole time-of-flight high resolution mass spectrometry (QTOF-MS), matrix-assisted laser ionization Orbitrap mass spectrometry (MALDI-Orbitrap-MS), nuclear magnetic resonance spectroscopy (NMR), and infra-red spectroscopy (IR) confirmed the presence of acrylfentanyl (also known as acryloylfentanyl). Quantitative analysis with liquid chromatography and triple quadrupole mass spectrometry (LC-MS/MS) determined the content of acrylfentanyl in the powder, equal to 88.3 mass-% acrylfentanyl hydrochloride. An impurity observed by NMR was identified as triethylamine hydrochloride. Acrylfentanyl is sold on the Internet as a 'research chemical'. Like other synthetic fentanyls, such as acetylfentanyl, it poses a serious risk of fatal intoxication. Copyright © 2016 The Authors. Drug Testing and Analysis Published by John Wiley & Sons Ltd.

How Resistant to Tampering are Codeine Containing Analgesics on the Market? Assessing the Potential for Opioid Extraction.

INTRODUCTION: Misuse of opioid analgesics, in combination with diversion, dependence, and fatal overdoses, presents a serious problem for public health, which affects many countries worldwide. Within this context, tampering with opioids has been associated with serious harm. The aim of the present study was to assess the tampering potential of codeine combination analgesics on the market (containing codeine/non-opioid analgesics) by the extraction of codeine. METHODS: Codeine was extracted from three combination formulations sold lawfully from licensed pharmacies without a medical prescription in Denmark and the UK. Extraction of codeine followed tampering procedures available on the Internet. The amounts of codeine and accompanying non-opioid analgesics in tampering products were analysed with liquid chromatography and tandem mass spectrometry (LC-MS/MS). RESULTS: LC-MS/MS showed recoveries of the total amounts of codeine in tampering products of 81-84% from Product 1 (codeine/acetylsalicylic acid); 61-67% from Product 2 (codeine/ibuprofen); and 42-71% from Product 3 (codeine/paracetamol). Recoveries of non-opioid analgesics ranged between: 57-73% acetylsalicylic acid; 5.5-8.5% ibuprofen, and 5.0-9.2% paracetamol. CONCLUSION: With the tampering procedures used, high amounts of codeine were separated from the accompanying analgesics in some, but not in all of the codeine containing formulations. Evidence-based medicine regulation, treatment for opioid dependence, and information to minimise risks to the public are essential components of an effective public health strategy to address the harms of tampering and misuse. FUNDING: Marie Pedersen and Jensine Heiberg Foundation.

Gamma-hydroxybutyrate and cocaine intoxication in a Danish child.

GHB intoxication must be considered in children with coma and a suspicion of drug intoxication. Furthermore, mixed intoxication with several substances and the possibility of unpredictable symptom profiles should be anticipated to ensure optimal symptomatic treatment of patients.

NT-proBNP on Cobas h 232 in point-of-care testing: Performance in the primary health care versus in the hospital laboratory.

BACKGROUND: NT-proBNP may be useful for ruling out heart failure in primary health care. In this study we examined the analytical quality of NT-proBNP in primary health care on the Cobas h 232 point-of-care instrument compared with measurements performed in a hospital laboratory. MATERIALS AND METHODS: Blood samples requested for NT-proBNP were collected in primary health care (n = 95) and in a hospital laboratory (n = 107). NT-proBNP was measured on-site on Cobas h 232 instruments both in primary health care centres and at the hospital laboratory and all samples were also analyzed with a comparison method at the hospital. Precision, trueness, accuracy, and lot-variation were determined at different concentration levels and evaluated according to acceptance criteria. Furthermore user-friendliness was assessed by questionnaires. RESULTS: For Cobas h 232 repeatability CV was 8.5-10.7% in the hospital setting and 5.3-10.0% in the primary health care and within the analytical quality specifications, but higher than with the comparison method (< 4%). NT-proBNP results obtained in primary health care were significantly higher than by the hospital comparison method (bias ranged from 14.3-23.7%), whereas there was no significant bias when Cobas h 232 was used in the hospital setting (bias ranged from - 4.9 to 7.0%). User-friendliness of Cobas h 232 was overall acceptable. CONCLUSION: Cobas h 232 point-of-care instrument for measurement of NT-proBNP performed satisfactorily with regard to precision, user-friendliness, and lot-variation. A decrease in NT-proBNP levels observed in samples transported to a central laboratory needs further attention and investigation.

Analysis of Urinary Biomarkers for Smoking Crack Cocaine: Results of a Danish Laboratory Study.

Crack cocaine (free-base cocaine) smokers belong to a subgroup of marginalized drug users exposed to severe health risks and great social harm. Detection of the urinary, pyrolytic biomarker methylecgonidine (MED) and its metabolite ecgonidine (ED) secures an unambiguous confirmation of crack cocaine smoking. Although prevalence studies of cocaine based upon self-reporting may not be accurate, laboratory analysis is seldom used for neither diagnostic purpose nor early identification of crack cocaine smoking, which is far more severe than snorting cocaine. A new analytical method was validated for MED, ED and other relevant cocaine metabolites using automated liquid handling and column switching coupled to liquid chromatography and tandem mass spectrometry. Limit of quantification was 30 ng/mL for ED and MED. This method was applied in a laboratory study of urine samples (n = 110) from cocaine users in Denmark subjected to routine drugs-of-abuse testing. Crack cocaine smoking was confirmed by the presence of MED and/or ED. Eighty-four samples (76.4%) were found positive for crack cocaine smoking in this group of problematic cocaine users. MED was only detected in 5.9% of the positive samples. The study shows a prevalence 3-fold higher to that recently suggested by European Monitoring Centre for Drugs and Drug Addiction. We therefore advocate that the urinary biomarkers MED and ED are included in routine testing methods for clinical toxicology. This may lead to an earlier identification of crack cocaine smoking and possibly prevent a more severe drug use.

Identification and characterization by LC-UV-MS/MS of melanotan II skin-tanning products sold illegally on the Internet.

New methods were developed and validated to determine the identity, contents, and purity of samples of melanotan II, a synthetic melanocortin receptor agonist, sold in vials as injectable skin-tanning products that were purchased from three online shops. Methods were based on liquid chromatography with ultra-violet detection (LC-UV) at wavelength 218 nm, and tandem mass spectrometric detection (MS/MS) after collision-induced fragmentation of the double charged [M+2H](2+) precursor ion (m/z 513). Identification of melanotan II was verified by correct chromatographic retention time, and relative abundance ratios of five qualifying fragment ions. LC-UV was used to quantify melanotan II as well as impurities. Method validation was performed with reference to guidelines for assessing active substances in authorized medicinal products to reach acceptable accuracy and precision. Vials from two shops contained unknown impurities ranging from 4.1 to 5.9%; impurities from one shop were below the quantification limit. The total amount of melanotan II in vials ranged between 4.32 and 8.84 mg, although each shop claimed that vials contained 10 mg melanotan II. A broad range of drugs used for enhancement purposes can be obtained from the illicit market. However, users of these drugs may be exposed to a range of potential harms, as shown in this study, given that these products are manufactured, distributed and supplied from an illicit market.

Identification of a new metabolite of GHB: gamma-hydroxybutyric acid glucuronide.

Gamma-hydroxybutyric acid (GHB) is an important analyte in clinical and forensic toxicology with a narrow detection window of 3-6 h. In the search of improved detection methods, the existence in vivo of a glucuronated GHB metabolite (GHB-GLUC) was hypothesized. Chemically pure standards of GHB-GLUC and a deuterated analogue for chromatography were synthesized. Liquid chromatography and tandem mass spectrometry were used for targeted analysis in anonymous clinical urine samples (n = 50). GHB-GLUC was found in concentrations ranging from 0.11 to 5.0 µg/mL (mean: 1.3 ± 1.2 µg/mL). Thus far, this is the first report of a GHB glucuronide detected in biological samples. Given that glucuronides generally have longer half-life values than their corresponding free drugs, GHB-GLUC should theoretically be a biomarker of GHB intoxication. It is also proposed that the hitherto unexplained reports of elevated GHB concentrations in some biological samples, which has caused the setting of a relatively high cutoff value (10 µg/mL), represent total GHB measurements (sum of free GHB and actively chemically hydrolyzed GHB-GLUC). To address these challenges, the present study must be followed by comprehensive pharmacokinetic and stability studies after the controlled administration of GHB.

Synthesis and stability study of a new major metabolite of γ-hydroxybutyric acid.

γ-Hydroxybutanoic acid (GHB) is used as a date-rape drug, which renders the victims unconscious and defenceless. Intoxications are very difficult to detect for forensic scientists due to rapid metabolism to endogenous levels of GHB. We recently discovered a new major metabolite, 2, of GHB (1) that could potentially extend the analytical detection window for GHB intoxications. Herein we disclose synthetic procedures based on a Koenigs-Knorr glucuronidation approach that provides GHB glucuronide 2 and a deuterium-labelled analogue d 4-2 of high purity suitable for analytical chemistry. In addition, we have assessed the stability of GHB glucuronide 2 by mimicking the natural pH range for urine, which is of importance in the development of new analytical methods. Using NMR we show that GHB glucuronide 2 is highly stable towards aqueous hydrolysis within the pH range normally observed for urine even at elevated temperature.

Autologous blood transfusion after local infiltration analgesia with ropivacaine in total knee and hip arthroplasty.

Aims. To study the safety of autotransfusion following local infiltration analgesia (LIA) with ropivacaine. Background. Knowledge of blood concentrations of ropivacaine after LIA and autotransfusion is crucial. However, very limited data are available for toxicological risk assessment. Methods. Autotransfusion was studied in patients after total knee arthroplasty (TKA: n = 25) and total hip arthroplasty (THA: n = 27) with LIA using 200 mg ropivacaine, supplemented with two postoperative bolus injections (150 mg ropivacaine). Drainage blood was reinfused within 6 h postoperatively. Results. Reinfusion caused a significant increase in the serum concentration of total ropivacaine for TKA from 0.54 ± 0.17 (mean ± SD) to 0.79 ± 0.20 µg/mL (P < 0.001) and a nonsignificant increase for THA from 0.62 ± 0.17 to 0.63 ± 0.18 µg/mL. The maximum free (unbound) concentration after reinfusion was 0.038 µg/mL. Peak total and free venous ropivacaine concentrations after 8 h and 16 h postoperative bolus injections were 2.6 µg/mL and 0.11 µg/mL, respectively. All concentrations observed were below the threshold for toxicity and no side effects were observed. Conclusion. Autotransfusion of patients undergoing knee or hip arthroplasty after local infiltration analgesia with 200 mg ropivacaine can be performed safely, even supplemented with 8 h and 16 h postoperative bolus injections.

Potentially toxic concentrations in blood of total ropivacaine after bilateral transversus abdominis plane blocks; a pharmacokinetic study.

CONTEXT: Elevated blood levels of lidocaine and ropivacaine have been described after transversus abdominis plane (TAP) block. OBJECTIVE: To investigate the pharmacokinetic profile of ropivacaine after bilateral TAP blocks. DESIGN: Prospective observational pharmacokinetic study. SETTING: University teaching hospital in Copenhagen, Denmark. PATIENTS: Twenty-one adult patients presenting for abdominopelvic surgery with bilateral TAP blocks were enrolled. PROCEDURES: Ultrasound-guided TAP blocks with bilateral injections of 20 ml ropivacaine 0.5% w/v (total dose 200 mg). Blood was sampled at 0, 10, 30 and 60 min after TAP blocks. MEASURES: Total and free peak blood concentrations (Cmax) of ropivacaine. RESULTS: Data were analysed from N = 18 patients. The median dose of ropivacaine was 2.7 mg kg(-1) (range: 1.9-4.2 mg kg(-1)). Median total ropivacaine concentrations were 1.0, 1.6 and 1.7 µg ml(-1) at 10, 30 and 60 min, respectively. Six patients (33%) had Cmax values above 2.2 µg ml(-1) and the highest concentration measured was 5.1 µg ml(-1). One patient had a 33% drop in mean arterial blood pressure. CONCLUSION: TAP blocks with bilateral injections of 20 ml ropivacaine 0.5% w/v gave rise to potentially toxic peak blood concentrations of total ropivacaine in one-third of the patients.