Towards adherence monitoring using breath or oral fluid as a matrix in a methadone maintenance treatment program for patients with a chronic heroin use disorder: Issues and interpretation of the results.

Urine has been the preferred matrix for monitoring heroin and methadone adherence due to its large detection window. Drawbacks such as privacy concerns and adulteration however require other matrices. The study aims to determine if oral fluid and exhaled breath are suitable alternatives for heroin and methadone monitoring and to assess the detection time in exhaled breath. Forty-three participants, all on methadone and heroin-assisted treatment, were studied. Participants were monitored after the first and right before the second dosage of heroin. At both time points, oral fluid and exhaled breath samples were collected with urine at the second time point. All samples were screened for opiates, methadone and other drugs using immunoassay and LC-MS-MS. At the second time point, 98% of oral fluid samples and all exhaled breath samples tested positive for 6-monoacetylmorphine (6-MAM). Regarding morphine detection, the findings were reversed (100% in oral fluid, 98% in exhaled breath). Methadone-related results were 100% positive across all matrices, as expected. Notable is the detection of the heroin marker acetylcodeine in oral fluid and exhaled breath samples, which resulted in relatively low negative predictive value (average 54.6%). Oral fluid and exhaled breath are suitable alternatives for heroin and methadone maintenance monitoring. Clinicians should consider ease of collection, adulteration risk, costs, turn-around time and the substance of interest while choosing a matrix. In addition, even in cases when medicinal heroin is used, medical professionals should be aware of the presence of acetylcodeine in these alternate matrices.

Internet-Purchased Sodium Azide Used in a Fatal Suicide Attempt: A Case Report and Review of the Literature.

There has been a significant increase in sodium azide intoxications since the 1980s. Intoxications caused by sodium azide are becoming increasingly prevalent in the Netherlands as a result of its promotion for the purpose of self-euthanasia. The mechanism of toxicity is not completely understood but is dose-dependent. The presented case describes a suicide by sodium azide of a young woman (26 years old) with a history of depression and suicide attempts. The decedent was found in the presence of prescription medicine, including temazepam, domperidone in combination with omeprazole, and the chemical preservative sodium azide. Quantitative toxicology screening of whole blood revealed the presence of 70 µg/L temazepam (toxic range > 1000 µg/L) and 28 mg/L sodium azide (fatal range: 2.6-262 mg/L). Whole blood qualitative analysis revealed the presence of temazepam, temazepam-glucuronide, olanzapine, n-desmethylolanzapine, and acetaminophen. In circles promoting sodium azide, it is recommended to use sodium azide in combination with medications targeting sodium azide's negative effects, such as analgesics, antiemetics, and anti-anxiety drugs. The medicines recovered at the body's location, as well as the results of the toxicology screens, were consistent with the recommendations of self-euthanasia using sodium azide.

Prolonged Ketamine and Norketamine Excretion Profiles in Urine After Chronic Use: A Case Series.

PURPOSE/BACKGROUND: Ketamine (K) is used as a party drug with hallucinogenic properties with a half-life of about 2.5 hours. Data are available with respect to the detection window (ie, when a person is still tested positive for the drug and/or metabolite after use) of K after single use. Nevertheless, no data are available with respect to the detection window of K in urine after chronic use. METHODS/PROCEDURES: This retrospective case series describes 7 patients with K dependency as their main addiction who have been admitted to an addiction center for K detoxification. Their abstinence-oriented care involved routine urinary screening of K and its metabolites, as well as traditional drugs of abuse, such as cocaine and cannabinoids. FINDINGS/RESULTS: Urine samples remained positive for all the cases identified after 22 to 96 days. A peak detection period of 61, 40, and 96 days for K, norketamine, and dehydronorketamine, respectively, measured using liquid chromatography-tandem mass spectrometry at a cutoff concentration of 1.0 ng/mL, is defined. The K/norketamine and K/dehydronorketamine ratios varied over time between 0.33 and 3.06, and 0.01 and 0.36 for all patients, respectively, implying a large interindividual variation in K metabolism. IMPLICATIONS/CONCLUSIONS: Ketamine and its metabolites have a prolonged excretion profile in urine, which requires frequent measurements (at least weekly) to guide abstinence treatment. Further research is needed to develop an algorithm that can differentiate new K use from residual urinary K excretion in urine of chronic daily users.

Bioelectrical Impedance Measurements for Assessment of Kidney Function in Critically Ill Patients.

OBJECTIVES: To evaluate the use of multifrequency bioelectrical impedance analysis to predict creatinine/urea clearance based on 24 hours urine collection. A practical formula was developed, and its performance was compared with that of established formulas such as Cockcroft-Gault, Modification of Diet in Renal Disease, and Jelliffe's. DESIGN: An open-label prospective observational cohort study. SETTING: A 12-bed ICU at a nonuniversity major teaching hospital (Gelre ziekenhuizen Apeldoorn/Zutphen, The Netherlands). PATIENTS: Adult critical care patients with an expected ICU length of stay at admission of at least 48 hours. INTERVENTIONS: Each patient's body composition was assessed using a validated Quadscan 4000 analyzer (Bodystat, Isle of Man, British Isles). Twenty-four hours urine was collected, and laboratory variables in serum including creatinine, urea, and albumin were obtained at the beginning and end of the collection period. MEASUREMENTS AND MAIN RESULTS: A total of 151 patients, stratified to an acute and nonacute ICU-group, were enrolled in the study over a 2-year period. A formula to predict creatinine/urea clearance based on 24 hours urine collection was developed using stepwise linear regression using a training data set of 75 patients. This formula was subsequently tested and compared with other relevant predictive equations using a validation data set of 76 patients. Serum creatinine values ranged from 40 to 446 µmol/L. With the predictive model based on estimated body cell mass and a "prediction marker" more than 71% of the observed variance in creatinine/urea clearance based on 24 hours urine collection could be explained. Predictive performance was superior to the other eight evaluated models (R = 0.39-0.55) and demonstrated to be constant over the whole range of creatinine/urea clearance based on 24 hours urine collection values. CONCLUSIONS: Multifrequency bioelectrical impedance analysis measurements can be used to predict creatinine/urea clearance based on 24 hours urine collection with superior performance than currently established prediction models. This rapid, noninvasive method enables correction for influences of a patient's actual body composition and may prove valuable in daily clinical practice.

The risk of emerging new psychoactive substances: The first fatal 3-MeO-PCP intoxication in The Netherlands.

Structural analogs of classic drugs, also called designer drugs, are a booming market due to the easy accessibility on the internet and their legal status. One of those 'legal highs' is an analog of phencyclidine, namely 3-methoxyphencyclidine (3-MeO-PCP). Very few fatalities have been reported where 3-MeO-PCP contributed to the death of an individual. We present the first fatal case in the Netherlands and one of the few worldwide. Postmortem biological samples and the presumed abused unknown substance, sold as ant poison, were obtained. 3-MeO-PCP was detected, and the resulting concentration was 152 µg/l in whole blood. The presumed taken unknown sample was identified as 3-MeO-PCP and thus linked to the victim. The cause of death was a combination of 3-MeO-PCP, amphetamine, and alcohol. Improved diagnostic skills are necessary to face these emerging novel psychoactive substances also in light of public health and social risks.

Assessment of medication use during pregnancy by Web-based questionnaires, pharmacy records and serum screening.

OBJECTIVE: To compare assessment of early pregnancy medication exposure using three methods of data collection. METHODS: Serum samples were obtained from 752 women participating in the PRegnancy and Infant DEvelopment (PRIDE) Study before gestational week 17. For 52 women using medication at the date of blood sampling according to Web-based questionnaires or pharmacy records, we analysed serum samples using untargeted liquid chromatography time-of-flight spectrometry. RESULTS: Medication was detected in 18 serum samples (35%). Medications taken orally for chronic conditions reported in the questionnaire were detected in serum and vice versa. Pharmacy records did not identify additional exposed women, but missed exposure in 5 women mainly due to unavailability. We observed substantial discordance between the three methods for inhaled medication, dermatological preparations, and medications for short-term use, which went often undetected in serum. CONCLUSIONS: It remains challenging to assess medication use in large-scale studies as no 'gold standard' is currently available.

Development of a multiplex non-radioactive receptor assay: the benzodiazepine receptor, the serotonin transporter and the beta-adrenergic receptor.

Binding assays still form a fundamental part of modern drug development. Receptor binding assays are mostly based on radioactivity because of their speed, ease of use and reproducibility. Disadvantages, such as health hazards and production of radioactive waste, have prompted the development of non-radioactive receptor binding assays. This application therefore focuses on measuring receptor-ligand interactions using mass spectrometry. Moreover, the novelty of this approach originates in determining multiple analytes in a single assay (multiplexing). The proof of principle of a non-radioactive multiplex receptor assay is demonstrated using a pool of receptors from rat cortical tissue with flunitrazepam, MADAM and pindolol in one vial with or without their respective displacers. Flunitrazepam, MADAM and pindolol bound specifically at 73%, 30% and 40% to their respective receptors. This corresponds to specific binding sites of 0.61 pmol/mg protein, 0.07 pmol/mg protein and 0.06 pmol/mg protein, respectively. We propose to measure the bound fraction instead of the free fraction in order to reach a significant difference in measured signals (total binding versus non-specific binding). The bound fraction can be obtained after dissociating the ligand from the receptor-ligand complex using 50% methanol in water. The current setup of the assay calls for further improvement with respect to the measurement of binding constants for a multitude of receptors in one assay with sufficient accuracy and precision.

Improving the performance of glutamate microsensors by purification of ascorbate oxidase.

Enzyme-based biosensors have the potential to directly detect extracellular concentrations of glutamate in brain tissue with a high spatial and temporal resolution. To optimize their analytical performance, much attention has been paid to the architectural construction of these biosensors. In particular, the coupling of enzymes to the electrode surface has received much interest, which has resulted in many (derivatives of) first-, second-, and third-generation type of biosensors. However, it is remarkable that in the literature little attention, if any, has been paid to the influence of the quality of the enzyme itself on the analytical performance of a biosensor. Previously we have reported that different batches of ascorbate oxidase significantly altered the performance of our glutamate microsensor.(1) In this note, it is shown that a simple enzyme purification procedure as buffer exchange leads to a more uniform enzyme quality and also significantly improves the reproducibility and performance of the microsensor. In our opinion, this is an important observation and of general interest for the construction of enzyme-based biosensors.

Receptor-ligand binding assays: technologies and applications.

Receptor-ligand interactions play a crucial role in biological systems and their measurement forms an important part of modern pharmaceutical development. Numerous assay formats are available that can be used to screen and quantify receptor ligands. In this review, we give an overview over both radioactive and non-radioactive assay technologies with emphasis on the latter. While radioreceptor assays are fast, easy to use and reproducible, their major disadvantage is that they are hazardous to human health, produce radioactive waste, require special laboratory conditions and are thus rather expensive on a large scale. This has led to the development of non-radioactive assays based on optical methods like fluorescence polarization, fluorescence resonance energy transfer or surface plasmon resonance. In light of their application in high-throughput screening environments, there has been an emphasis on so called "mix-and-measure" assays that do not require separation of bound from free ligand. The advent of recombinant production of receptors has contributed to the increased availability of specific assays and some aspects of the expression of recombinant receptors will be reviewed. Applications of receptor-ligand binding assays described in this review will relate to screening and the quantification of pharmaceuticals in biological matrices.

Development and validation of a radioreceptor assay for the determination of morphine and its active metabolites in serum.

This article describes the development and validation of a radioreceptor assay for the determination of morphine and morphine-6-beta-glucuronide (M6G) in serum. The assay is based on competitive inhibition of the mu-opioid-selective radiolabeled ligand [3H]-DAMGO by opioid ligands (e.g. M6G) for binding to the striatal opioid receptor. The assay has been validated according to the Washington Conference Report on Analytical Method Validation. The radioreceptor assay can be performed in serum without prior pre-treatment of the sample. Direct addition of the sample results in no significant loss in maximal binding sites, and therefore, no loss in sensitivity. The assay proves to be selective for a multitude of opioid agonists and antagonists (e.g. morphine IC50 = 4.1 nM and M6G IC50 = 12.8 nM). Moreover, morphine-3-glucuronide (M3G) displays a low affinity (IC50 = 1100 nM) for the mu-opioid receptor and according to the literature demonstrates no analgesic activity. This makes discrimination, in relation to the analgesic effect, of the two metabolites of morphine possible. The assay is fast (assay time <4h, analysis 5 min/sample), easy and the sensitivity (limit of detection (LOD) = 1.6 nM M6G-equivalents) is such that very potent agonists, like morphine and M6G, can be measured at the desired serum levels. The assay is accurate (<18%), but precision is limited if measured over several days (>35%). The assay is most accurate and precise if measured over a range from 3.5 to 40 nM M6G-equivalents. Based on the limited inter-assay precision, we propose to use this receptor assay mainly as a screening tool for neonates treated with morphine.

Determination of the benzodiazepine plasma concentrations in suicidal patients using a radioreceptor assay.

Impairments in memory and psychomotor function appear to be induced by benzodiazepines not only after long-term use, but also after administration of a single dose. Because it is known on which neurotransmitter system the benzodiazepines exert their action, the use of a quantitative radioreceptor assay (RRA) can be a useful tool in studying the interrelationship between the neurochemical and memory processes. The RRA measures the sum of the main compound(s) and all active metabolites present, where it relates the biological activity to the pharmacodynamic effect instead of relating it to the plasma levels of the individual compounds. To correlate the loss of memory with the benzodiazepine concentration, plasma concentrations were determined in suicidal patients. From suicidal patients (n = 84), the benzodiazepines in plasma were measured with a direct radioreceptor assay using tritiated flunitrazepam as the labelled ligand. The receptor material was a lyophilized preparation from calf cortex. Furthermore, the samples were subjected to high-performance liquid chromatographic (HPLC) analysis, and the HPLC data were converted to diazepam equivalents using cross-reactivities of the individual compounds. Patients who had ethanol residues in their plasma were excluded from this correlation experiment. The data (n = 40) obtained with the two analytical techniques were compared and correlated to assess the validity of the radioreceptor assay in establishing the relationship between the loss of memory and the total amount of benzodiazepines present. The cumulative amount of diazepam determined with the RRA and the sum of compounds determined with the HPLC method, after correction using the cross-reactivities, were plotted and correlated using regression analysis. Regression analysis showed an x variable of 0.75 and a correlation coefficient of 0.67. The intercept was not significantly different from zero (P = 0.49, t-test), whereas the slope was significantly different from zero (P < 0.01). Benzodiazepines can be directly determined in plasma using this radioreceptor assay. The data obtained from HPLC analysis were easily converted to diazepam equivalents using the cross-reactivities. A discrepancy between the data obtained from the two analytical techniques, however, indicates that certain metabolites are present, which were not quantitated in the HPLC analysis, but were measured in the radioreceptor assay. Therefore, the radioreceptor assay proved to be a valuable tool for the assessment of clinical effects, such as the demonstration of the loss of memory in suicidal patients after a benzodiazepine overdose.

Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris.

The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40kDa exhibited specific and saturable binding properties. The dopamine antagonist [3H]spiperone showed an average dissociation constant K(d) of 0.6+/-0.17 nM for the dopamine D2S receptor. The receptor was solubilized using the non-ionic detergent dodecylmaltoside and purified by affinity chromatography using a Ni(2+) chelate (His-Trap) column or by batch extraction with an anti-FLAG M1 affinity resin. The receptor maintained its biological activity after solubilization and purification from the membrane protein fraction. A 244- or 185-fold enrichment, as judged by an increase in specific binding, was obtained after adsorption to the His-Trap or anti-FLAG materials, respectively.