Rapid Extraction and Qualitative Screening of 30 Drugs in Oral Fluid at Concentrations Recommended for the Investigation of DUID Cases.

A rapid, simple extraction method followed by qualitative screening using liquid chromatography-tandem mass spectrometry (LC-MS-MS) for drugs in oral fluid is presented. The decision points were selected to be at, or lower, than those recommended as Tier I compounds by the National Safety Council's Alcohol, Drugs and Impairment Division for toxicological investigation of driving under the influence of drug (DUID) cases and were also at, or lower, than those recommended by Substance Abuse and Mental Health Service Administration and the Department of Transportation for Federal workplace drug testing programs. The method included 30 drugs: delta-9-tetrahydrocannabinol, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine, benzoylecgonine, carisoprodol, meprobamate, zolpidem, alprazolam, clonazepam, 7-aminoclonazepam, diazepam, nordiazepam, lorazepam, oxazepam, temazepam, codeine, morphine, 6-acetylmorphine, buprenorphine, fentanyl, hydrocodone, hydromorphone, oxycodone, oxymorphone, methadone, tramadol and phencyclidine. Phencyclidine was included because it is in the Federal workplace program even though it is considered a Tier II drug for DUID cases. A liquid-liquid extraction method using isopropanol, hexane and ethyl acetate to extract drugs from the oral fluid-buffer mix collected in a Quantisal™ device, followed by LC-MS-MS screening, was developed and validated according to ANSI/ASB 2019 Standard Practices for Method Validation in Forensic Toxicology. Interference studies, limit of detection, precision at the decision point, ionization suppression/enhancement and processed sample stability were determined for each drug. The method was successfully applied to proficiency specimens and routine samples received in the laboratory.

Simultaneous Determination of Paracetamol, Propyphenazone and Caffeine in Presence of Paracetamol Impurities Using Dual-Mode Gradient HPLC and TLC Densitometry Methods.

Two chromatographic methods were validated for the determination of the widely prescribed analgesic and antipyretic drug combination of paracetamol (PC) (recently integrated into the supportive treatment of COVID-19), propyphenazone (PZ) and caffeine (CF) in the presence of two PC impurities, namely 4-aminophenol and 4-nitrophenol. A "dual-mode" gradient high-performance liquid chromatography method was developed, where the separation was achieved via "dual-mode" gradient by changing both the ternary mobile phase composition (acetonitrile: methanol: water) and the flow rate. This enables a good resolution within a relatively shorter analysis time. The analysis was realized using Zorbax Eclipse XDB column C18, 5 µm (250 × 4.6 mm) and the UV detector was set at 220 nm. The other method is a thin-layer chromatography densitometry method, where the separation was achieved using a mobile phase composed of chloroform: toluene: ethyl acetate: methanol: acetic acid (6: 6: 1: 2: 0.1, by volume). Densitometric detection was performed at 220 nm on silica gel 60 F254 plates. The developed methods were fully validated as per the ICH guidelines and proved to be accurate, robust, specific and suitable for application as purity indicating methods for routine analysis of PC in pure form or in pharmaceuticals with PZ and CF in quality control laboratories.

Carisoprodol pharmacokinetics and distribution in the nucleus accumbens correlates with behavioral effects in rats independent from its metabolism to meprobamate.

Carisoprodol (Soma®) is a centrally-acting skeletal-muscle relaxant frequently prescribed for treatment of acute musculoskeletal conditions. Carisoprodol's mechanism of action is unclear and is often ascribed to that of its active metabolite, meprobamate. The purpose of this study was to ascertain whether carisoprodol directly produces behavioral effects, or whether metabolism to meprobamate via cytochrome P450 (CYP450) enzymatic reaction is necessary. Rats were trained to discriminate carisoprodol (100 mg/kg) to assess time course and whether a CYP450 inhibitor (cimetidine) administered for 4 days would alter the discriminative effects of carisoprodol. Additionally, pharmacokinetics of carisoprodol and meprobamate with and without co-administration of cimetidine were assessed via in vivo microdialysis combined with liquid-chromatography-tandem mass spectrometry from blood and nucleus accumbens (NAc). The time course of the discriminative-stimulus effects of carisoprodol closely matched the time course of the levels of carisoprodol in blood and NAc, but did not match the time course of meprobamate. Administration of cimetidine increased levels of carisoprodol and decreased levels of meprobamate consistent with its interfering with metabolism of carisoprodol to meprobamate. However, cimetidine failed to alter the discriminative-stimulus effects of carisoprodol. Carisoprodol penetrated into brain tissue and directly produced behavioral effects without being metabolized to meprobamate. These findings indicate that understanding the mechanism of action of carisoprodol independently of meprobamate will be necessary to determine the validity of its clinical uses.

Unexpected Discoveries Should Be Reconsidered in Science-A Look to the Past?

From the past, we know how much "serendipity" has played a pivotal role in scientific discoveries. The definition of serendipity implies the finding of one thing while looking for something else. The most known example of this is the discovery of penicillin. Fleming was studying "Staphylococcus influenzae" when one of his culture plates became contaminated and developed a mold that created a bacteria-free circle. Then he found within the mold, a substance that proved to be very active against the vast majority of bacteria infecting human beings. Serendipity had a key role in the discovery of a wide panel of psychotropic drugs as well, including aniline purple, lysergic acid diethylamide, meprobamate, chlorpromazine, and imipramine. Actually, many recent studies support a step back in current strategies that could lead to new discoveries in science. This change should seriously consider the idea that to further focus research project milestones that are already too focused could be a mistake. How can you observe something that others did not realize before you? Probably, one pivotal requirement is that you pay a high level of attention on what is occurring all around you. But this is not entirely enough, since, specifically talking about scientific discoveries, you should have your mind sufficiently unbiased from mainstream infrastructures, which normally make you extremely focused on a particular endpoint without paying attention to potential "unexpected discoveries". Research in medicine should probably come back to the age of innocence and avoid the age of mainstream reports that do not contribute to real advances in the curing of human diseases. Max Planck said "Science progresses not because scientists change their minds, but rather because scientists attached to erroneous views die, and are replaced", and Otto Warburg used the same words when he realized the lack of acceptance of his ideas. This editorial proposes a series of examples showing, in a practical way, how unfocused research may contribute to very important discoveries in science.

Medical Devices; Clinical Chemistry and Clinical Toxicology Devices; Classification of the Meprobamate Test System. Final order.

The Food and Drug Administration (FDA or we) is classifying the meprobamate test system into class II (special controls). The special controls that apply to the device type are identified in this order and will be part of the codified language for the meprobamate test system's classification. We are taking this action because we have determined that classifying the device into class II (special controls) will provide a reasonable assurance of safety and effectiveness of the device. We believe this action will also enhance patients' access to beneficial innovative devices, in part by reducing regulatory burdens.

Unique contribution of Ecuadorian kindergartners' spontaneous focusing on numerosity to their early numerical abilities.

Recent evidence indicates that young children's spontaneous focusing on numerosity (SFON) uniquely contributes to their early numerical abilities. This study complements previous findings by validating the relation between young children's SFON and their early numerical abilities in a developing country, namely Ecuador. We analysed 355 Ecuadorian 5- to 6-year-olds' SFON in relation to their early numerical abilities at the start of kindergarten, controlling for children's socio-demographic (socio-economic status, age) and general cognitive (working memory, intelligence) characteristics. Our results evidence the unique contribution of Ecuadorian kindergartners' SFON to their early numerical abilities, controlling for children's working memory, intelligence, socio-economic status, and age. Our findings support the validity of previous findings on the unique contribution of SFON to young children's early numerical abilities in developed countries for developing countries. Additionally, they raise timely questions for further theoretical and methodological studies on young children's numerical development worldwide, in developing and developed countries. Statement of contribution What is already known? SFON uniquely contributes to early numerical abilities. However, this is only documented in Finnish samples. Previously only limited control for domain-general cognitive and socio-demographic characteristics. What does the study add? SFON uniquely contributes to early numerical abilities in Ecuadorian 5- to 6-year-olds. This unique contribution remains after controlling for WM, IQ, SES, and age. Evidence for the universal nature of the association between SFON and early numerical ability.

Impact of Expanding ELISA Screening in DUID Investigations to Include Carisoprodol/Meprobamate and Zolpidem.

In 2013, the National Safety Council's Alcohol Drugs and Impairment Division added zolpidem and carisoprodol and its metabolite meprobamate to the list of Tier 1 drugs that should be tested for in all suspected drug impaired driving and motor vehicle fatality investigations. We describe the validation of an enzyme linked immunosorbent assays (ELISA) for both drugs in whole blood, and the utilization of the ELISA to assess their positivity in a sample of 322 suspected impaired driving cases that was retrospectively screened using the validated assays. The occurrence of carisoprodol/meprobamate was found to be 1.2%, and for zolpidem, 1.6%. In addition, we analyzed a large dataset (n = 1,672) of Driving Under the Influence of Drugs (DUID) test results from a laboratory performing high volume DUID testing to assess the frequency of detection of both drugs after implementing the expanded NSC scope. Carisoprodol or meprobamate were found positive in 5.9% (n = 99) of these samples, while zolpidem was found positive in 5.3% (n = 89) in drivers who in many cases had been found to be negative for other drugs. Carisoprodol and zolpidem are both potent CNS depressants and are appropriate additions to the recommended NSC scope of testing.

Pharmacokinetic modeling of carisoprodol and meprobamate disposition in adults.

Carisoprodol is a widely prescribed muscle relaxant and is also a drug known to be a subject to abuse. Despite the fact that carisoprodol has been available for prescription since 1959, a number of gaps in our knowledge of the toxicokinetics of this common drug exist. For example, the volume of distribution (Vd) for carisoprodol in humans has not been reported. A two-compartment pharmacokinetic model describing carisoprodol metabolism and that of the primary metabolite, meprobamate, was developed to better understand the pharmacokinetics of this drug. The model accounts for first pass metabolism of carisoprodol and was able to replicate the data from several previously reported data sets. Based on an analysis of four different data sets, the Vd for carisoprodol ranged from 0.93 to 1.3 L/kg, while that for meprobamate ranged from 1.4 to 1.6 L/kg. The model was also used to estimate the probable dose of this drug in an individual where questions concerning the drug's role in her death had been posed. The model may, therefore, have significant utility for estimating doses of carisoprodol in medicolegal cases.

Mass spectrometric analysis of carisoprodol and meprobamate in rat brain microdialysates.

We report the evaluation of several mass spectrometry-based methods for the determination of carisoprodol and meprobamate in samples obtained from the rat brain by in vivo intracranial microdialyis. Among the techniques that aspire to perform analyses without chromatographic separation and thereby increase throughput, chip-based nanoelectrospray ionization and the use of an atmospheric pressure solids analysis probe fell short of requirements because of insufficient detection sensitivity and hard ionization, respectively. Although direct analysis in real time provided the required soft ionization, shortcomings of a tandem mass spectrometry-based assay also included inadequate detection sensitivity and, in addition, poor quantitative reproducibility. Therefore, liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry was developed to determine carisoprodol and meprobamate from artificial cerebrospinal fluid as the medium. No desalting and/or extraction of the samples was necessary. The assay, combined with in vivo sampling via intracranial microdialyis, afforded time-resolved concentration profiles for the drug and its major metabolite from the nucleus accumbens region of the brain in rats after systemic administration of carisoprodol. Copyright © 2016 John Wiley & Sons, Ltd.

Something old, something new: a successful case of meprobamate withdrawal.

Meprobamate, a benzodiazepine-like drug, was commonly prescribed for anxiety in the 1960s and 1970s, but fell out of favour, at least in part, due to the risk of dependence, for which there is little published evidence to guide clinical management. We discuss a 70-year-old man with a 45-year history of meprobamate dependency and multiple failed previous withdrawal attempts who was successfully withdrawn from meprobamate using diazepam during a 2-week inpatient stay on a specialist Addictions ward. An appropriate diazepam dose was established using the Clinical Institute Withdrawal Assessment scale for benzodiazepines (CIWA-B). This dose was then slowly reduced over 12 days. Multidisciplinary input, especially psychological therapy tackling his underlying anxiety disorder during his admission, was thought to be particularly helpful.

[What happened after meprobamate's withdrawal? Survey in two nursing homes]. / Enquête dans deux Ehpad sur le retrait du méprobamate.

We have conducted in two nursing homes a survey to study the impact of meprobamate's withdrawal, at the beginning of 2012, in terms of extent of prescribing to others psychotropic drugs and occurrence of adverse events. After meprobamate's withdrawal, 65 % of residents did not receive alternative medication and within three months after meprobamate stopping, adverse events (drowsiness, falls and hospitalization) decreased while agitation did not increase.

Assessment of direct gating and allosteric modulatory effects of meprobamate in recombinant GABA(A) receptors.

Meprobamate is a schedule IV anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (γ-aminobutyric acid Type A) receptors, and has barbiturate-like activity. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In αxßzγ2 GABAA receptors (where x=1-6 and z=1-3), the ability to enhance GABA-mediated current was evident for all α subunit isoforms, with the largest effect observed in α5-expressing receptors. Direct gating was present with all α subunits, although attenuated in α3-expressing receptors. Allosteric and direct effects were comparable in α1ß1γ2 and α1ß2γ2 receptors, whereas allosteric effects were enhanced in α1ß2 compared to α1ß2γ2 receptors. In "extrasynaptic" (α1ß3δ and α4ß3δ) receptors, meprobamate enhanced EC20 and saturating GABA currents, and directly activated these receptors. The barbiturate antagonist bemegride attenuated direct effects of meprobamate. Whereas pentobarbital directly gated homomeric ß3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric ρ1 receptors, pentobarbital and meprobamate were ineffective in direct gating; a mutation known to confer sensitivity to pentobarbital did not confer sensitivity to meprobamate. Our results provide insight into the actions of meprobamate and parent therapeutic agents such as carisoprodol. Whereas in general actions of meprobamate were comparable to those of carisoprodol, differential effects of meprobamate at some receptor subtypes suggest potential advantages of meprobamate may be exploited. A re-assessment of previously synthesized meprobamate-related carbamate molecules for myorelaxant and other therapeutic indications is warranted.

Quantitation of Carisoprodol and Meprobamate in Urine and Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Carisoprodol and meprobamate are centrally acting muscle relaxant/anxiolytic drugs that can exist in a parent-metabolite relationship (carisoprodol → meprobamate) or as a separate pharmaceutical preparation (meprobamate aka Equanil, others). The monitoring of the use of these drugs has both clinical and forensic applications in pain management applications and in overdose situations. LC-MS/MS is used to analyze urine or plasma/serum extracts with deuterated analogs of each analyte as internal standards to ensure accurate quantitation and control for any potential matrix effects. Positive ion electrospray is used to introduce the analytes into the mass spectrometer. Selected reaction monitoring of two product ions for each analyte allows for the calculation of ion ratios which ensures correct identification of each analyte, while a matrix-matched calibration curve is used for quantitation.

Photochemical degradation of atenolol, carbamazepine, meprobamate, phenytoin and primidone in wastewater effluents.

The photochemical degradation of five pharmaceuticals was examined in two secondary wastewater effluents. The compounds, which included atenolol, carbamazepine, meprobamate, phenytoin and primidone, were evaluated for both direct and sensitized photolysis. In the two wastewaters, direct photolysis did not lead to significant compound degradation; however, sensitized photolysis was an important removal pathway for the five pharmaceuticals. Upon solar irradiation, hydroxyl radical (HO) was quantified using the hydroxylation of benzene and singlet oxygen ((1)O2) formation was monitored following the degradation of furfuryl alcohol. Degradation via sensitized photolysis was observed following five-day exposures for atenolol (69-91%), carbamazepine (67-98%), meprobamate (16-52%), phenytoin (44-85%), and primidone (34-88%). Varying removal is likely a result of the differences in reactivity with transient oxidants. Averaged steady state HO concentrations ranged from 1.2 to 4.0×10(-16)M, whereas the concentrations of (1)O2 were 6.0-7.6×10(-14)M. Partial removal due to presence of HO indicates it was not the major sink for most compounds examined. Other transient oxidants, such as (1)O2 and triplet state effluent organic matter, are likely to play important roles in fates of these compounds.

Multidrug toxicity involving sumatriptan.

A multidrug fatality involving sumatriptan is reported. Sumatriptan is a tryptamine derivative that acts at 5-HT(1B/1D) receptors and is used for the treatment of migraines. The decedent was a 21-year-old white female found dead in bed by her spouse. No signs of physical trauma were observed and a large number of prescription medications were discovered at the scene. Toxicological analysis of the central blood revealed sumatriptan at a concentration of 1.03 mg/L. Following therapeutic dosing guidelines, sumatriptan concentrations do not exceed 0.095 mg/L. Sumatriptan was isolated by solid-phase extraction and analyzed using liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode. A tissue distribution study was completed with the following concentrations measured: 0.61 mg/L in femoral blood, 0.56 mg/L in iliac blood, 5.01 mg/L in urine, 0.51 mg/kg in liver, 3.66 mg/kg in kidney, 0.09 mg/kg in heart, 0.32 mg/kg in spleen, 0.01 mg/kg in brain, 15.99 mg/kg in lung and 78.54 mg/45 mL in the stomach contents. Carisoprodol, meprobamate, fluoxetine, doxylamine, orphenadrine, dextromethorphan and hydroxyzine were also present in the blood at the following concentrations: 3.35, 2.36, 0.63, 0.19, 0.06, 0.55 and 0.16 mg/L. The medical examiner ruled the cause of death as acute mixed drug toxicity and the manner of death as accident.

Quantification of five compounds with heterogeneous physicochemical properties (morphine, 6-monoacetylmorphine, cyamemazine, meprobamate and caffeine) in 11 fluids and tissues, using automated solid-phase extraction and gas chromatography-tandem mass spectrometry.

An automated solid-phase extraction (SPE) protocol followed by gas chromatography coupled with tandem mass spectrometry was developed for quantification of caffeine, cyamemazine, meprobamate, morphine and 6-monoacetylmorphine (6-MAM) in 11 biological matrices [blood, urine, bile, vitreous humor, liver, kidney, lung and skeletal muscle, brain, adipose tissue and bone marrow (BM)]. The assay was validated for linearity, within- and between-day precision and accuracy, limits of quantification, selectivity, extraction recovery (ER), sample dilution and autosampler stability on BM. For the other matrices, partial validation was performed (limits of quantification, linearity, within-day precision, accuracy, selectivity and ER). The lower limits of quantification were 12.5 ng/mL(ng/g) for 6-MAM, morphine and cyamemazine, 100 ng/mL(ng/g) for meprobamate and 50 ng/mL(ng/g) for caffeine. Analysis of real-case samples demonstrated the performance of the assay in forensic toxicology to investigate challenging cases in which, for example, blood is not available or in which analysis in alternative matrices could be relevant. The SPE protocol was also assessed as an extraction procedure that could target other relevant analytes of interest. The extraction procedure was applied to 12 molecules of forensic interest with various physicochemical properties (alimemazine, alprazolam, amitriptyline, citalopram, cocaine, diazepam, levomepromazine, nordazepam, tramadol, venlafaxine, pentobarbital and phenobarbital). All drugs were able to be detected at therapeutic concentrations in blood and in the alternate matrices.

Factors affecting carisoprodol metabolism in pain patients using urinary excretion data.

Carisoprodol is a skeletal muscle relaxant prescribed to treat pain. Carisoprodol is metabolized to meprobamate, an active metabolite with anxiolytic effects, by the genetically polymorphic CYP2C19 enzyme. Concomitant use of CYP2C19 substrates or inhibitors may alter carisoprodol metabolism, with therapeutic and/or toxic implications for effectively treating patients with pain. This was a retrospective analysis of urinary excretion data collected from patients with pain from March 2008 to May 2011. Carisoprodol and meprobamate urine concentrations were measured by liquid chromatography-tandem mass spectrometry, and the metabolic ratio (MR) of meprobamate to carisoprodol concentrations was determined in 14,965 subjects. The MR geometric mean and 95% confidence interval (95% CI) of the young group (105, 95% CI = 99.1-113) were ∼47.4% higher than the middle-aged group (71.9, 95% CI = 70-73.8) and nearly two times higher than the elderly group (54.4, 95% CI = 51.3-57.6). Females had a 20.7% higher MR compared with males. No significant change in the MR was observed with overall CYP2C19 inhibitor or substrate use. However, evaluation of individual inhibitors showed co-administration with esomeprazole or fluoxetine was associated with a 31.8 and 24.6% reduction in MR, respectively, compared with controls (P < 0.05). Omeprazole did not significantly affect the MR. Patient-specific factors such as age, sex and co-medications may be important considerations for effective carisoprodol therapy.

Meprobamate-induced fixed drug eruption.

Meprobamate is usually a safe drug prescribed for anxiety disorders. Fixed drug eruption (FDE) is an exceptional cutaneous adverse effect of this drug. We report a case of FDE induced by meprobamate with positive patch test. A 22-year-old woman was prescribed for depression meprobamate, aceprometazine, valpromide and lorazepam. On the second day of treatment, the patient presented red erythematous and pruriginous plaques in the limbs and the face. After stopping the previous treatment, the patient's lesions resolved completely within 3 weeks with residual pigmentation. One month later, patch tests were performed and were positive to meprobamate. Exceptional cases of FDE were reported in literature with meprobamate. None has reported the use of patch test to confirm the diagnosis.

Comparative uptake and translocation of pharmaceutical and personal care products (PPCPs) by common vegetables.

Reuse of treated wastewater to irrigate agricultural crops is increasing in many arid and semi-arid areas around the world. The presence of numerous pharmaceutical and personal care products (PPCPs) in treated wastewater and their potential transfer into food produce such as vegetables poses an unknown human health risk. The goal of this study was to identify PPCPs that have a comparatively high potential for plant uptake and translocation. A total of 20 frequently-occurring PPCPs were compared for their accumulation into four staple vegetables (lettuce, spinach, cucumber, and pepper) grown in nutrient solutions containing PPCPs at 0.5 or 5µgL(-1). Triclocarban, fluoxetine, triclosan, and diazepam were found at high levels in roots, while meprobamate, primidone, carbamazepine, dilantin, and diuron exhibited more active translocation from roots to leaves. Root uptake of neutral PPCPs was positively correlated with the pH adjusted log Kow(i.e., log Dow), and was likely driven by chemical adsorption onto the root surfaces. In contrast, translocation from roots to leaves was negatively related to log Dow, suggesting hydrophilicity-regulated transport via xylems. Compounds preferentially sorbed to roots should be further evaluated for their uptake in tuber vegetables (e.g., carrot, radish) under field conditions, while those easily translocated into leaves (e.g., carbamazepine, dilantin) merit focused consideration for leafy and other vegetables (e.g., lettuce, cucumber). However, estimation of dietary intake by humans suggested the implied risks from exposure to PPCPs via wastewater irrigation to be negligible.