Assessment of cardiotoxicity and plasma ropivacaine concentrations after serratus intercostal fascial plane block in an experimental model.

Serratus intercostal fascial plane block (SIFPB) has emerged as an alternative to paravertebral block in breast surgery. It involves the administration of high volumes and doses of local anesthetics (LA) that can potentially reach toxic levels. Ropivacaine is widely used in thoraco-fascial blocks; however, there is no information on the plasma concentrations attained after SIPFB and whether they are associated with cardiotoxicity. Plasma concentrations of ropivacaine and its electrophysiological effects were evaluated in eight pigs after bilateral SIFPB with ropivacaine in doses of 3 mg/kg. Plasma concentrations, electrophysiological and hemodynamic parameters were measured sequentially for the following 180 min until the end of the study. The area under the curve, the maximum plasma concentration (Cmax) and the time to reach Cmax (tmax) were calculated. The median arterial ropivacaine concentration Cmax was, 2.34 [1.40 to 3.74] µg/ml. The time to reach the highest concentration was 15 [10 to 20] min. Twenty-five percent of the animals had arterial concentrations above the lower limit concentration of ropivacaine for LA systemic toxicity (3.4 µg/ml). No alterations were observed in the electrophysiological or electrocardiographic parameters except for a prolongation of the QTc interval, from 489 ± 30 to 544 ± 44 ms (Δ11.38 ± 6%), P = 0.01. Hemodynamic parameters remained in the physiological range throughout the study. SIFPB with ropivacaine in doses of 3 mg/kg has reached potentially toxic levels, however, it has not been associated with adverse electrophysiological or hemodynamic effects.

Sodium bicarbonate reverts electrophysiologic cardiotoxicity of ropivacaine faster than lipid emulsions in a porcine model.

Ropivacaine has been described as a safer local anaesthetic (LA); however, serious cardiotoxic accidents have been reported. Intravenous-lipid-emulsion (ILE) therapy during LA intoxication seems to act as an antidote. Sodium bicarbonate is the standard treatment for sodium channel blocker drug toxicity. We compared both antidotes on the reversion of electrophysiologic toxicity induced by ropivacaine. Ropivacaine 5 mg kg-1 was administered in 24 pigs, and 3 min later, the animals received ILE: 1.5 ml kg-1  + 0.25 ml kg-1  min-1 (ILE group); sodium bicarbonate: 2 mEq kg-1  + 1 mEq kg-1  h-1 (NaHCO3 group); saline solution (CTL group). Electrophysiological parameters were evaluated for 30 min. The area under the curve (AUC) for the first 5 or 30 min was compared between groups. Ropivacaine induced a lengthening of the PR interval by 17% (P = 0.0001), His-ventricle-interval by 58% (P = 0.001), sinus QRS complex by 56% (P = 0.0001), paced QRS at 150 bpm by 257% (P = 0.0001), and at 120 bpm by 143% (P = 0.0001) in all groups. At 5 min after treatment, sinus QRS in the NaHCO3 group was shorter than that in the CTL group (AUCQRS5 , P = 0.003) or ILE group (AUCQRS5 , P = 0.045). During the first minute, seven of the animals in the NaHCO3 group vs. two in the ILE or 0 in the CTL group recovered more than 30% of the sinus QRS previously lengthened by ropivacaine (P = 0.003). Sodium bicarbonate reversed the electrophysiological toxicity of ropivacaine faster than ILE and control groups.

Effects of intravenous lipid emulsions on the reversal of pacing-induced ventricular arrhythmias and electrophysiological alterations in an animal model of ropivacaine toxicity.

INTRODUCTION: Ropivacaine is considered to have a wider margin of cardiovascular safety. However, several reports of ventricular arrhythmias (VA) due to ropivacaine toxicity have been documented. Intravenous lipid emulsions (ILEs) have recently been used successfully in the treatment of local anesthetic intoxication. The main objective of the present study was to evaluate the efficacy of the ILEs in the prevention of pacing-induced-VA and electrophysiological alterations in an animal model of ropivacaine toxicity. METHODS: Nineteen pigs were anesthetized and instrumentalized. A baseline programmed electrical ventricular stimulation protocol (PEVSP) to induce VA was performed. Ropivacaine (5 mg·kg-1 + 100 µg·kg-1·min-1) followed by normal saline infusion (control group n = 8) or intralipid 20% (1.5 mL·kg-1 + 0.25 mL·kg-1·min-1) for the ILE group (n = 8), were administered three minutes after the ropivacaine bolus. PEVSP was repeated 25 min after the onset of ropivacaine infusion. Pacing-induced VA and electrophysiological abnormalities were assessed in both groups. A sham-control group (n = 3) without ropivacaine infusion was included. RESULTS: Most of the electrophysiological parameters evaluated were affected by ropivacaine: PR interval by 28% (p = 0.001), AV interval by 40% (p = 0.001), sinus QRS by 101% (p = 0.001), paced QRS at a rate of 150 bpm by 258% (p = 0.001), and at 120 bpm by 241% (p = 0.001). Seven animals (87.5%) in the control group and eight animals (100%) in the ILE group developed sustained-VA (p = 0.30). Successful resuscitation occurred in 100% of animals in the ILE group vs. 57% of animals in the control group, p = 0.038. Pacing-induced-VA terminated at the first defibrillation attempt in 75% of the animals in the ILE group vs. 0% in the control group, p = 0.01. CONCLUSION: Ropivacaine strongly altered the parameters of ventricular conduction, thus facilitating the induction of VA. ILEs did not prevent pacing-induced VA. However, facilitated resuscitation and termination of VA were delivered at the first defibrillation attempt compared to the control group.

Genetic identification and subsequent LC-QTOF MS analysis of plant remains (Oenanthe spp.) could prove the cause of an undetermined sudden death.

In recent years, recorded cases related to forensic botany and, in particular, of plant poisoning have become rare. We report on the medicolegal characteristics of an undetermined sudden death (USD) of a woman in which scene there were remnants of a vegetal peeling. After the autopsy, macroscopic findings reported multiorgan failure and requested the investigation of the cause of death. Postmortem blood was firstly investigated on cyanide toxicity presumptively coming from a yucca-like root; however, found cyanide levels were under normality. Because of the lack of morphological features of the encountered plant remains, a genetic nrDNA ITS2 sequence investigation was followed. The resulting DNA sequence could identify the evidence as the water dropwort (Oenanthe spp.) which contains oenanthotoxin, a potent toxin that may be fatal, similar to the more commonly found in hemlock Conium or cowbane Cicuta species. A liquid chromatography-tandem high resolution mass spectrometry (LC-QTOF MS) was later applied to analyse the vegetal extract and stomach content and successfully confirmed the toxin existence. Medicolegal and analytical findings at the forensic laboratory were described, where both biological and chemical techniques could successfully conjugate, as an interdisciplinary research, and explain premortem symptoms and postmortem findings. Present data can be helpful in future investigation on poisoning cases by conjugated polyacetylenes . The present work tries to emphasize the often undervalued plant evidence in legal medicine diagnosis in the context of an unexplained death.

Comparative Effects of Sodium Bicarbonate and Intravenous Lipid Emulsions on Reversing Bupivacaine-Induced Electrophysiological Toxicity in a Porcine Experimental Model.

BACKGROUND: Bupivacaine cardiotoxicity mainly manifests as inhibition of the cardiac sodium channel, which slows conduction, particularly at the ventricular level. Experimental studies have demonstrated that intravenous lipid emulsions (ILEs) can reduce the cardiotoxic effects of bupivacaine, but the extent of these effects is controversial. Sodium bicarbonate (B) represents the standard treatment of toxicity related to sodium channel-blocking drugs. The aim of this study was to compare the effects of ILEs and B on the speed of recovery from bupivacaine-induced effects on the electrocardiographic parameters. METHODS: Bupivacaine 4 mg/kg was administered to 24 anesthetized pigs. Three minutes after delivering the bupivacaine bolus, the animals were given the following: ILE 1.5 mL/kg followed by 0.25 mL/kg/min (ILE group) and B 2 mEq/kg followed by 1 mEq/kg/h (B group). Controls (C group) were given saline solution, 50 mL followed by 1 mL/kg/h. Electrophysiological parameters were evaluated in sinus rhythm and during right ventricular pacing at several time intervals up to 30 minutes. Data were analyzed as the area under the curve (AUC) for the first 10 minutes (AUC10) or 30 minutes (AUC30). RESULTS: Bupivacaine increased the sinus cycle length, PR interval, and QRS duration. AUC30 of the sinus rhythm QRS duration after antidote administration was significantly different among the 3 groups (P = .003). B group experienced faster recovery from intoxication than the C group (AUC10, P = .003; AUC30, P = .003) or the ILE group (AUC10, P = .018). During the first minute, 50% of the B group (versus 0% of the ILE and C groups) had recovered >30% of QRS duration (P = .011). The trend toward faster recovery in the ILE group than in the C group did not reach significance (AUC10, P = .23; AUC30, P = .06). Effects on the paced QRS duration at a rate of 150 bpm were more intense but with similar results (B versus C group: AUC10, P = .009; AUC30, P = .009; B versus ILE: AUC10, P = .015; AUC30, P = .024). The recovery process of the paced QRS tended to be slower for all antidotes. CONCLUSIONS: In a closed-chest swine model, B was an effective treatment for electrophysiological alterations caused by established bupivacaine toxicity. At clinical doses, B ameliorated bupivacaine electrocardiographic toxicity faster than ILE. Use-dependent effects of bupivacaine are prominent and delay the effects of both antidotes, but B produces faster recovery than ILE.

ABCC3 Polymorphisms and mRNA Expression Influence the Concentration of a Carboxylic Acid Metabolite in Patients on Clopidogrel and Aspirin Therapy.

Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism. These results suggest that up-regulation of ABCC3 mRNA expression leads to increased plasma CAM levels through MRP3-mediated cell efflux. The ABCC3 rs757421 polymorphism may contribute to gene expression. Therefore, ABCC3 may be a potential biomarker for the response to clopidogrel.

ABCC3 Polymorphisms and mRNA Expression Influence the / Concentration of a Carboxylic Acid Metabolite in Patients on / Clopidogrel and Aspirin Therapy

Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism. These results suggest that up-regulation of ABCC3 mRNA expression leads to increased plasma CAM levels through MRP3-mediated cell efflux. The ABCC3 rs757421 polymorphism may contribute to gene expression. Therefore, ABCC3 may be a potential biomarker for the response to clopidogrel.

ABCC3 Polymorphisms and mRNA expression influence the concentration of a carboxylic acid metabolite in patients on clopidogrel and aspirin therapy

Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism...

The determination of cannabinoids using liquid chromatography with mass spectrometric detection.

Because of their prevalence in drugged driving and other medicolegal investigations, cannabinoids are routinely analyzed by forensic laboratories. Until relatively recently, these analyses were performed by gas chromatography coupled to mass spectrometry (GC-MS). However, the need for derivatization and extensive sample preparation made GC-MS approaches tedious and time consuming. As a consequence, many laboratories have explored alternative analysis techniques. The advent of more affordable liquid chromatography-mass spectrometry (LC-MS) instruments and the utility of atmospheric pressure ionization sources have made LC-MS a promising alternative to GC-MS for the detection and quantitation of cannabinoids in forensic applications.

Target screening and confirmation of 35 licit and illicit drugs and metabolites in hair by LC-MSMS.

A liquid chromatography-tandem mass spectrometry (LC-MSMS) target screening in 50mg hair was developed and fully validated for 35 analytes (Δ9-tetrahidrocannabinol (THC), morphine, 6-acetylmorphine, codeine, methadone, fentanyl, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, benzoylecgonine, cocaine, lysergic acid diethylamide, ketamine, scopolamine, alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, 7-aminoflunitrazepam, lorazepam, lormetazepam, nordiazepam, oxazepam, tetrazepam, triazolam, zolpidem, zopiclone, amitriptyline, citalopram, clomipramine, fluoxetine, paroxetine and venlafaxine). Hair decontamination was performed with dichloromethane, and incubation in 2 mL of acetonitrile at 50°C overnight. Extraction procedure was performed in 2 steps, first liquid-liquid extraction, hexane:ethyl acetate (55:45, v:v) at pH 9, followed by solid-phase extraction (Strata-X cartridges). Chromatographic separation was performed in AtlantisT3 (2.1 mm × 100 mm, 3 µm) column, acetonitrile and ammonium formate pH 3 as mobile phase, and 32 min total run time. One transition per analyte was monitored in MRM mode. To confirm a positive result, a second injection monitoring 2 transitions was performed. The method was specific (no endogenous interferences, n=9); LOD was 0.2-50 pg/mg and LOQ 0.5-100 pg/mg; linearity ranged from 0.5-100 to 2000-20,000 pg/mg; imprecision <15%; analytical recovery 85-115%; extraction efficiency 4.1-85.6%; and process efficiency 2.5-207.7%; 27 analytes showed ion suppression (up to -86.2%), 4 ion enhancement (up to 647.1%), and 4 no matrix effect; compounds showed good stability 24-48 h in autosampler. The method was applied to 17 forensic cases. In conclusion, a sensitive and specific target screening of 35 analytes in 50mg hair, including drugs of abuse (THC, cocaine, opiates, amphetamines) and medicines (benzodiazepines, antidepressants) was developed and validated, achieving lower cut-offs than Society of Hair Testing recommendations.

Methamphetamine transiently increases the blood-brain barrier permeability in the hippocampus: role of tight junction proteins and matrix metalloproteinase-9.

Methamphetamine (METH) is a powerful stimulant drug of abuse that has steadily gained popularity worldwide. It is known that METH is highly neurotoxic and causes irreversible damage of brain cells leading to neurological and psychiatric abnormalities. Recent studies suggested that METH-induced neurotoxicity might also result from its ability to compromise blood-brain barrier (BBB) function. Due to the crucial role of BBB in the maintenance of brain homeostasis and protection against toxic molecules and pathogenic organisms, its dysfunction could have severe consequences. In this study, we investigated the effect of an acute high dose of METH (30mg/kg) on BBB permeability after different time points and in different brain regions. For that, young adult mice were sacrificed 1h, 24h or 72h post-METH administration. METH increased BBB permeability, but this effect was detected only at 24h after administration, being therefore a transitory effect. Interestingly, we also found that the hippocampus was the most susceptible brain region to METH, comparing to frontal cortex and striatum. Moreover, in an attempt to identify the key players in METH-induced BBB dysfunction we further investigated potential alterations in tight junction (TJ) proteins and matrix metalloproteinase-9 (MMP-9). METH was able to decrease the protein levels of zonula occludens (ZO)-1, claudin-5 and occludin in the hippocampus 24h post-injection, and increased the activity and immunoreactivity of MMP-9. The pre-treatment with BB-94 (30mg/kg), a matrix metalloproteinase inhibitor, prevented the METH-induced increase in MMP-9 immunoreactivity in the hippocampus. Overall, the present data demonstrate that METH transiently increases the BBB permeability in the hippocampus, which can be explained by alterations on TJ proteins and MMP-9.

Direct surface plasmon resonance immunosensor for in situ detection of benzoylecgonine, the major cocaine metabolite.

In this paper the development of the first direct surface plasmon resonance (SPR) immunoassay for the detection of benzoylecgonine (BZE) is described. Immunosensor chips consisting of a high affinity monoclonal anti-BZE-antibody (anti-BZE-Ab) immobilized at high density to a sensor chip were prepared. First, BZE detection in Hepes buffer was achieved by direct, real time monitoring of the binding between BZE in solution and the surface bound antibody. The detection protocol was based on calibration curves obtained from reaction rate data and end point data analysis of sensorgrams registered after injection of a series of known BZE concentrations over the chips. Moreover, immunosensor accuracy, reproducibility, stability and robustness were tested to demonstrate their good performance as reusable devices. The immunosensor was used for BZE detection in oral fluid (OF) showing that, within 180 s, our immunoassay detects BZE concentrations as low as 4 µg/L in filtered OF-buffer (1:4) samples. This value is remarkably lower than current cut off levels established by the Substance Abuse and Mental Health Services Administration. These results manifest the potential use of this direct SPR immunoassay for the in situ sensitive detection of recent cocaine abuse, of utility in roadside drug OF testing. Moreover, it exemplifies the high potential of direct SPR immunoassays for the rapid, sensitive detection of small molecules in contrast with the more established indirect methods.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) determination of cocaine and its metabolites benzoylecgonine, ecgonine methyl ester, and cocaethylene in hair samples.

This study reports the development and validation of a method using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) for the analysis of cocaine and its metabolites benzoylecgonine (BE), ecgonine methyl ester (EME), and cocaethylene (CE) in hair samples. Decontamination was performed as follows: Firstly, the aliquot of hair was briefly rinsed with 2 mL dichloromethane, then was washed three times with 10 mL 0.01 M phosphate buffer, pH 6, for 15 min, followed by 2 mL 2-propanol for less than 2 min, and, finally, a last rinse with 2 mL dichloromethane was again done. Cocaine compounds were extracted from 10 mg of hair by incubation with 2 mL 0.1 M HCl at 50 °C for 12 h and purified by solid phase extraction with Oasis MCX cartridges. Analysis was performed by LC-MS/MS using an Atlantis HILIC silica chromatographic column. The method was fully validated. Linearity was established over the concentration range 0.020-10.0 ng/mg for cocaine (COC), 0.010-10.0 ng/mg for BE and CE, and 0.005-2.0 ng/mg for EME, and the correlation coefficients were all >0.99. Extraction efficiency was >70% for all analytes. Limits of detection were 0.0005 ng/mg for CE and 0.001 ng/mg for the other analytes (COC, BE, and EME). Lower limits of quantification were the lowest points of the calibration curves with acceptable accuracy and precision (coefficient of variation ≤20%). Intra- and inter-day imprecision ranged between 1.5% and 9.5% and 0.7% and 12.6%, respectively. Intra- and inter-day inaccuracy ranged from 0.5% to 12.3% and from 0.7% to 7.1%, respectively. With regard to matrix effects, suppression was <27.5% in all cases. The method was applied to the analysis of several samples derived from forensic cases.

Ethylglucuronide determination in urine and hair from alcohol withdrawal patients.

Two methods for the determination of ethylglucuronide (EtG) in urine and in hair have been developed by liquid chromatography- tandem mass spectrometry. These two methods were fully validated, including linearity (0.25-100 microg/mL in urine; 0.05-5 ng/mg in hair; r(2) > 0.99, n = 5), limits of detection (0.1 microg/mL in urine, 0.025 ng/mg in hair) and quantitation (lowest level of the calibration curve), extraction efficiency (> 55%), within-day and between-day imprecision and bias (CV and mean relative error < 15%), matrix effect, and relative ion intensity. These methods have been applied to 541 urine samples and 17 hair specimens collected from 156 alcohol withdrawal patients. The determination of ethanol versus EtG in urine was compared, and also the convenience of EtG determination in hair. EtG in urine and in hair proved to be a powerful tool for monitoring abstinence in these patients.

Determination of illicit and medicinal drugs and their metabolites in oral fluid and preserved oral fluid by liquid chromatography-tandem mass spectrometry.

An LC-MS/MS method using 0.5 ml of oral fluid was developed for the determination of morphine, codeine, 6-monoacetylmorphine, methadone, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, benzoylecgonine, cocaine, delta-9-tetrahydrocannabinol, zolpidem, zopiclone, alprazolam, clonazepam, oxazepam, nordiazepam, lorazepam, flunitrazepam, diazepam, diphenhydramine and amitriptyline. The method was fully validated in terms of linearity (the method was linear between 1-5 microg/L and 100-200 microg/L) recoveries (7.5-82.6%), within-day and between-day precisions and accuracies (CV and MRE, both <15%), limits of detection (0.5 microg/L) and quantitation (the lowest point on the calibration curve), relative ion intensities, freeze-and-thaw stability and matrix effect. The method was applied to preserved oral fluid collected by a special commercial device, the StatSure Saliva Sampler.

Laboratory evaluation and field application of roadside oral fluid collectors and drug testing devices.

This study was a part of a collaborative U.S./E.U. international research effort (Roadside Testing Assessment, ROSITA II) to assess illegal drug use among motor vehicle operators suspected of driving while under the influence of drugs and to evaluate the effectiveness of point-of-collection oral fluid drug detection technologies. A goal of the study was to assess commercial oral fluid drug testing devices for potential use in law enforcement. Ten devices were evaluated in the laboratory for their ability to meet manufacturers' claimed (and proposed) cutoff concentrations for the detection of amphetamine(s), cocaine/metabolite, opiates, cannabinoids, and benzodiazepines (2 devices). The field study portion of the research was conducted in major cities in the United States and Western Europe by teams of scientists working in collaboration with the local police. In Salt Lake City, Utah, the Drugwipe, Securetec, Ottobrunn, Germany (Securetec) oral fluids drug testing device was also evaluated in the field by testing suspected drug-impaired drivers. During the initial phase of the field study, 40 subjects were recruited. Drugwipe results were compared with laboratory-based immunoassay and mass spectrometry results and demonstrated that calculated sensitivities were between 75% and 100% depending on drug class. Specificities varied from 36% for cannabinoids to over 95% for opiates. During the second phase of the field study, 267 subjects were recruited. The Drugwipe sensitivities were 36.4%, 35.9%, 42.9%, and 7.7%, respectively, for amphetamine(s), cocaine, opiates, and cannabinoids. The Drugwipe specificities were 99.2%, 97.4%, 99.6%, and 99.6%, respectively, for amphetamine(s), cocaine, opiates, and cannabinoids. Drugwipe failed to meet the study criteria for acceptable device performance, required performance sensitivities, and specificities 90% or greater.

Determination of illicit drugs and their metabolites in human urine by liquid chromatography tandem mass spectrometry including relative ion intensity criterion.

A method, using 0.5 mL of urine, was developed for the simultaneous determination of ecgonine methyl ester, benzoylecgonine, morphine, codeine, 6-acetylmorphine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), and d-lysergic acid diethylamide (LSD). The analysis was performed by liquid chromatography with tandem mass spectrometry, after solid-phase extraction in the presence of their deuterated analogues. Reversed-phase separation on an Atlantis dC18 column was achieved in 12.5 min, under gradient conditions. The method was fully validated, including linearity (1-2000 microg/L for ecgonine methyl ester, benzoylecgonine, 6-acetylmorphine, methamphetamine, MDMA, and EDDP; 2-2000 microg/L for morphine, codeine, MDA, and methadone; 2-1000 microg/L for amphetamine, and 0.2-100 microg/L for LSD; r(2)>0.99); recovery (>65%), within-day and between-day precision, and accuracy (CV and MRE<15%); limit of detection (0.1 microg/L for LSD, 0.5 microg/L for ecgonine methyl ester, benzoylecgonine, methamphetamine, MDMA, 6-monoacetylmorphine, and EDDP, and 1 microg/L for amphetamine, MDA, morphine, and methadone); quantitation (lowest level of the calibration curve); relative ion intensities, freeze-and-thaw stability, and matrix effect. The procedure showed to be sensitive and specific, and was applied to real cases and quality control samples from a quality control program.

Confirmation by LC-MS of drugs in oral fluid obtained from roadside testing.

The aim of this study was to assess the effectiveness of two current on-site oral fluid (OF) drug detection devices (OraLab and Dräger), as part of the Spanish participation in the Roadside Testing Assessment Project (ROSITA Project). The study was done in collaboration with the Spanish Traffic Police, in Galicia (NW Spain), during 2004 and 2005. A total of 468 drivers selected at the police controls agreed to participate through informed consent. In addition, saliva samples were collected and sent to the laboratory to confirm the on-site results. For this purpose, two different analytical liquid chromatography-mass spectrometry (LC-MS) methods were used to detect 11 drugs or metabolites in a 300 microL sample. Simultaneous analysis of morphine, 6-acetylmorphine, amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB, cocaine and benzoylecgonine was carried out using 100 microL of oral fluid, after an automated solid phase extraction. A different LC-MS method was performed to detect Delta(9)-THC in 200 microL of oral fluid using liquid-liquid extraction with hexane at pH 6. Both methods were fully validated, including linearity (1-250 ng/mL, 2-250 ng/mL) recovery (>50%), within-day and between-day precision (CV<15%), accuracy (mean relative error<15%), limit of detection (0.5 and 1 ng/mL), quantitation (1 and 2 ng/mL) and matrix effect. All of the positive cases and a random selection of 30% of the negatives were analyzed for confirmation analysis. Good results (sensitivity, specificity, accuracy, positive predictive value and negative predictive value>90%) were obtained for cocaine and opiates by OraLab, and for cocaine by Dräger. However, the results for the other compounds could be improved for both detection devices. Differences in the ease of use and in the interpretation mode (visual or instrumental) were observed.

A validated method for the detection of Delta 9-tetrahydrocannabinol and 11-nor-9-carboxy- Delta 9-tetrahydrocannabinol in oral fluid samples by liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.

A quantitative liquid chromatography-electrospray ionization-quadrupole-time-of-flight (TOF) mass spectrometry (MS) method for simultaneous determination of Delta(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in oral fluid samples was developed and fully validated. The analytes were isolated from the oral fluid by classic liquid-liquid extraction. The two compounds were separated in 9.5 min with a total analysis time of 19 min. The linear dynamic range was established from 0.1 to 100 ng/mL for THC and 0.5-100 ng/mL for the THC-COOH. The method had a good intra- and interassay accuracy and precision at each measured concentration. No matrix effects were observed for the analytes or the labeled internal standards. Accurate mass measurement was achieved to +/- 5 ppm. The sensitivity of the method results from a combination of unique chromatographic retention time, the use of MS-MS, and the "exact mass" measurement of the target ions by a TOF analysis. The method was successfully applied to two types of oral fluid samples. One type was collected from roadside driving-under-the-influence studies. The second was used to assess the recovery of the THC from an oral fluid collection device under laboratory conditions. Although the use of the TOF MS in the forensic and toxicology fields is not as widespread as the triple-quadrupole technology, it offers some significant analytical advantages that are demonstrated by this method.

Fast LC-MS/MS method for the determination of amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB and PMA in urine.

A fast method was designed for the simultaneous determination of amphetamine (A), methamphetamine (MA), PMA, MDA, MDMA, MDEA and MBDB in urine. The drugs were analysed by LC (ESI)-MS/MS, after a simple liquid-liquid extraction in the presence of the deuterated analogues. Reverse phase separation on an Atlantis dC18 Intelligent Speed column was achieved in less than 4 min under gradient conditions, and the total run time was 8 min. The method was fully validated, including linearity (1-1000 ng/mL for A, MDMA, MDEA and MBDB; 2-1000 ng/mL for MDA and PMA; 1-200 ng/mL for MA; r2>0.99 for all compounds), recovery (>80%), within-day and between-day precision and accuracy (CV and MRE<12.7% for intermediate level and ULOQ, and <17.2% for LLOQ), limit of detection (0.2 ng/mL for MDMA, MDEA and MBDB; 0.5 ng/mL for A, MA and PMA; 1 ng/mL for MDA) and quantitation (1 ng/mL for A, MA, MDMA, MDEA and MBDB; 2 ng/mL for MDA and PMA) and relative ion intensities. No matrix effect was observed. The procedure proved to be sensitive, specific and rapid, and was applied to real forensic cases.