Stability of Lisdexamfetamine in Sampled Whole Blood-Implications of Sampling Tube Additives and Storage Temperature for Interpretation of Impairment.

Lisdexamfetamine (LDX) is a prodrug that is enzymatically converted into dextroamphetamine (d-AMP), a central nervous system stimulant. The stability of LDX in sampled whole blood is an important issue that may be crucial in the assessment of impaired driving caused by d-AMP. This study investigated the stability of LDX in whole blood collected in two different tubes containing a fluoride oxalate (FX) mixture and a fluoride citrate (FC) mixture. Without additives, LDX was unstable. LDX was also unstable in FX blood stored at ambient temperature or 4°C. After 3 days of storage at ambient temperature, an initial LDX concentration of 47 ± 1 ng/g (mean ± SD) was no longer detectable in the samples (n = 3). Instead, 19 ± 0.6 ng/g d-AMP was formed. The stability was improved at 4°C. After 7 days of storage at 4°C, 88 ± 5% of an initial LDX concentration of 50 ± 0.4 ng/g was recovered and 3.8 ± 0.3 ng/g d-AMP was formed. The stability of LDX was greater in FC blood than in FX blood; 79 ± 10% and 93 ± 4% of initial LDX concentrations of 48 ± 2 and 51 ± 0.5 ng/g were recovered from FC blood after 7 days of storage at ambient temperature and 4°C, respectively, and the corresponding formation of d-AMP was 5.8 ± 0.6 and 0.5 ± 0.3 ng/g, respectively. When FX and FC blood were stored at -20°C or -80°C, no detectable degradation of LDX or formation of d-AMP was observed after 3 weeks of storage.

A Retrospective Metabolomics Analysis of Gamma-Hydroxybutyrate in Humans: New Potential Markers and Changes in Metabolism Related to GHB Consumption.

GHB is an endogenous short-chain organic acid presumably also widely applied as a rape and knock out drug in cases of drug-facilitated crimes or sexual assaults (DFSA). Due to the endogenous nature of GHB and its fast metabolism in vivo, the detection window of exogenous GHB is however narrow, making it challenging to prove use of GHB in DFSA cases. Alternative markers of GHB intake have recently appeared though none has hitherto been validated for forensic use. UHPLC-HRMS based screening of blood samples for drugs of abuse is routinely performed in several forensic laboratories which leaves an enormous amount of unexploited data. Recently we devised a novel metabolomics approach to use archived data from such routine screenings for elucidating both direct metabolites from exogenous compounds, but potentially also regulation of endogenous metabolism and metabolites. In this paper we used UHPLC-HRMS data acquired over a 6-year period from whole blood analysis of 51 drivers driving under the influence of GHB as well as a matched control group. The data were analyzed using a metabolomics approach applying a range of advanced analytical methods such as OPLS-DA, LASSO, random forest, and Pearson correlation to examine the data in depth and demonstrate the feasibility and potential power of the approach. This was done by initially detecting a range of potential biomarkers of GHB consumption, some that previously have been found in controlled GHB studies, as well as several new potential markers not hitherto known. Furthermore, we investigate the impact of GHB intake on human metabolism. In aggregate, we demonstrate the feasibility to extract meaningful information from archived data here exemplified using GHB cases. Hereby we hope to pave the way for more general use of the principle to elucidate human metabolites of e.g. new legal or illegal drugs as well as for applications in more global and large scale metabolomics studies in the future.

Vitamin D insufficiency among Danish pregnant women-Prevalence and association with adverse obstetric outcomes and placental vitamin D metabolism.

INTRODUCTION: In pregnancy, vitamin D deficiency is associated with increased risk of fetal growth restriction and preeclampsia. The underlying mechanisms are not known, but placental dysfunction is believed to play a role. In a Danish population, where health authorities recommend a 10 µg/day vitamin D supplement during pregnancy, we explored current use of vitamin D supplements and vitamin D status. In term placentas, alterations in vitamin D metabolism and placental growth, evaluated by the key placental growth factor pregnancy-associated plasma protein-A (PAPP-A), and their relation to vitamin D insufficiency were investigated. MATERIAL AND METHODS: We included 225 randomly selected pregnant women attending a nuchal translucency scan at gestational weeks 11-14. Information on use of vitamin D supplements and body mass index (BMI) at inclusion was obtained using self-reported questionnaires. Plasma 25-hydroxyvitamin D was measured at inclusion and correlated with pregnancy outcomes and placental biology, as judged by expression of PAPP-A and enzymes involved in vitamin D metabolism (CYP24A1, CYP27B1) in term placentas. RESULTS: Vitamin D supplements were used by 92% of the women, but 42% were vitamin D insufficient (plasma 25-hydroxyvitamin D <75 nmol/L). Eleven women with singleton pregnancies developed fetal growth restriction or preeclampsia. In this small subset, first-trimester mean plasma 25-hydroxyvitamin D was lower in women who developed fetal growth restriction (43 ± 33nmol/L; n = 3; P = .006) and there was a tendency towards lower plasma 25-hydroxyvitamin D among women who developed preeclampsia (65 ± 19 nmol/L; n = 8; P = .08) in third trimester compared with uncomplicated pregnancies (79 ± 22 nmol/L; n = 187). In term placentas, PAPP-A expression was lower among participants with first-trimester vitamin D insufficiency (P = .009; n = 30) but no correlation was found between plasma 25-hydroxyvitamin D and mRNA expression of CYP24A1 (P = .67) and CYP27B1 (P = .34). BMI was negatively correlated with plasma 25-hydroxyvitamin D (P = .03) and positively correlated with placental mRNA expression of CYP24A1 (P = .003; n = 30). CONCLUSIONS: Despite high compliance with official guidelines regarding vitamin D supplements, vitamin D insufficiency was frequent and the findings indicate that vitamin D insufficiency may affect placental growth. High BMI was associated with vitamin D insufficiency and increased placental vitamin D turnover, but further investigations are needed.

Use of stimulants, over-the-counter and prescription drugs among Danish pregnant women.

Using self-reports and blood samples from 225 unselected Danish first trimester pregnant women, the purpose of this study was to assess their use of stimulants, for example caffeine and nicotine as well as over-the-counter (OTC) and prescription drugs. According to self-reported information, 24% had used prescription drugs and 48% had used OTC drugs mainly acetaminophen (42%), 9.3% were habitual smokers, 44% stated a daily use of caffeinated beverages, and 1.3% used illegal drugs. Ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC-HR-TOFMS) analysis was performed on corresponding blood samples applying golden standards for use of UPLC-HR-TOFMS in forensic medicine. Traces of prescription drugs were detected in 5.3% of the samples and 8.9% contained OTC drugs (acetaminophen 7.1%). Traces of smoking were identified in 8.0%, caffeine in 83% and illegal drugs in 0.9%. These results indicate a substantial use of OTC drugs and caffeine among Danish pregnant women. Blood analysis indicated that many women could be unaware of their caffeine intake. As common substances may be associated with adverse pregnancy outcomes, healthcare professionals should inquire about such habits during pregnancy. The results also underline the need for more research into the molecular effects of such drugs on placental function and foetal development.

A Metabolomics Study of Retrospective Forensic Data from Whole Blood Samples of Humans Exposed to 3,4-Methylenedioxymethamphetamine: A New Approach for Identifying Drug Metabolites and Changes in Metabolism Related to Drug Consumption.

The illicit drug 3,4-methylenedioxymethamphetamine (MDMA) has profound physiological cerebral, cardiac, and hepatic effects that are reflected in the blood. Screening of blood for MDMA and other narcotics are routinely performed in forensics analysis using ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC-HR-TOFMS). The aim of this study was to investigate whether such UPLC-HR-TOFMS data collected over a two-year period could be used for untargeted metabolomics to determine MDMA metabolites as well as endogenous changes related to drug response and toxicology. Whole blood samples from living Danish drivers' positive for MDMA in different concentrations were compared to negative control samples using various statistical methods. The untargeted identification of known MDMA metabolites was used to validate the methods. The results further revealed changes of several acylcarnitines, adenosine monophosphate, adenosine, inosine, thiomorpholine 3-carboxylate, tryptophan, S-adenosyl-l-homocysteine (SAH), and lysophospatidylcholine (lysoPC) species in response to MDMA. These endogenous metabolites could be implicated in an increased energy demand and mechanisms related to the serotonergic syndrome as well as drug induced neurotoxicity. The findings showed that it was possible to extract meaningful results from retrospective UPLC-HR-TOFMS screening data for metabolic profiling in relation to drug metabolism, endogenous physiological effects, and toxicology.

A PET study of effects of chronic 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") on serotonin markers in Göttingen minipig brain.

The psychostimulant 3,4-methylendioxymethamphetamine (MDMA, "ecstasy") evokes degeneration of telencephalic serotonin innervations in rodents, nonhuman primates, and human recreational drug users. However, there has been no alternative to nonhuman primates for studies of the cognitive and neurochemical consequences of serotonin depletion in a large-bodied animal. Therefore, we used positron emission tomography (PET) with [(11)C]DASB to map the distribution of plasma membrane serotonin transporters in brain of Göttingen minipigs, first in a baseline condition, and again at 2 weeks after treatment with MDMA (i.m.), administered at a range of doses. In parallel PET studies, [(11)C]WAY-100635 was used to map the distribution of serotonin 5HT(1A) receptors. The acute MDMA treatment in awake pigs evoked 1 degrees C of hyperthermia. MDMA at total doses greater than 20 mg/kg administered over 2-4 days reduced the binding potential (pB) of [(11)C]DASB for serotonin transporters in porcine brain. A mean total dose of 42 mg/kg MDMA in four animals evoked a mean 32% decrease in [(11)C]DASB pB in mesencephalon and diencephalon, and a mean 53% decrease in telencephalic structures. However, this depletion of serotonin innervations was not associated with consistent alterations in the binding of [(11)C]WAY-100635 to serotonin 5HT(1A) receptors. Stereological cell counting of serotonin-positive neurons, which numbered 95,000 in the dorsal raphé nucleus of normal animals, was unaffected in MDMA-treated group. group.