[Show abstract][Hide abstract] ABSTRACT: Morphine is still the mainstay in treatment of severe pain and is metabolized in the liver mainly by glucuronidation, partly to the pharmacologically active morphine-6-glucuronide (M6G). The sulfation pathway has attracted much less attention but may also form active metabolites. The aim of the present study was to study two sulfate metabolites of morphine in humans. Urine and plasma from newborns, adult heroin addicts, and terminal cancer patients was analyzed for the presence of morphine-3-sulfate (M3S) and morphine-6-sulfate (M6S) by a new liquid chromatography – tandem mass spectrometry (LC-MS/MS) method. In addition, morphine sulfation was studied in vitro in human liver cytosol preparations. M3S was present in urine and plasma from all study groups although at lower concentrations than morphine-3-glucuronide (M3G). The plasma M3S/M3G ratio was 30 times higher in newborns than in adults indicating that the relative sulfation is more important at early stage of life. M6S was measurable in only one plasma sample from a newborn patient, and in one of the urine sample from the drug testing group. The incubation of morphine with liver cytosol extracts resulted in approximately equal rate of formation of both M3S and M6S. In conclusion, sulfation of morphine is catalyzed in human liver but this minor metabolic pathway probably lacks clinical significance. The M6S metabolite is formed at a low rate, making it undetectable in most individuals.
Pharmacology Research & Perspectives. 12/2014; 2(6).
[Show abstract][Hide abstract] ABSTRACT: Exhaled breath has recently been proposed as a matrix for drug testing. This study aims to further explore, develop and validate exhaled breath as a safe and effective non-invasive method for drug testing in a clinical setting. Self-reported drug use was recorded and drug testing was performed by mass spectrometry and immunochemical methods using breath, plasma and urine samples from 45 individuals voluntarily seeking treatment for recreational drug use. Cannabis was the most prevalent drug detected by any method. Urine sampling detected most cases. The exhaled breath technique was less sensitive (73%) than plasma analysis for detection of cannabis uses but captures a more recent drug intake than both plasma and urine. Exhaled breath was the preferred specimen to donate according to interview data of the participants. Testing illicit drugs with the exhaled breath sampling technique is a sufficient, non-invasive and safe alternative and complement to plasma and/or urine sampling.
[Show abstract][Hide abstract] ABSTRACT: A semi-automated method for quantification of budesonide in human plasma was developed, validated, and applied for high-volume analysis of samples in connection with a pharmacokinetic study. Protein and phospholipid removal was performed using an Ostro 96-well filter plate and subsequently combined with C18 solid-phase extraction on a Hamilton Microlab STARlet automation robot. The final extracts were evaporated to dryness and redissolved in 20% acetonitrile/water. The procedure used budesonide-d8 as internal standard and gave a 3.5-fold concentration of plasma to extract. The final extracts (5μL injected) were analyzed with selected reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS/MS) using electrospray ionization in positive mode. The chromatography system used a 100mm ACQUITY BEH UPLC column and a gradient system consisting of aqueous 0.1% formic acid and acetonitrile as organic modifier. Phospholipid removal was found to be needed during method development in order to reduce ion suppression effects from matrix and to increase method sensitivity. The measuring range was 50-5000pg/mL with and LOD 24pg/mL. Calibration response showed good linearity (correlation coefficients<0.99) over the measuring range. The absolute recovery over the sample preparation procedure was estimated to 67%. Total imprecision was <9% at three levels and accuracy was between 98.9 and 103%. The method was successfully applied for analysis of 864 study samples in a short time. The quality control samples at concentration levels 200 and 2000pg/mL gave a total imprecision of 7.4% and 4.2%, respectively, (n=95).
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 970C:31-35. · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background. MT-45 (1-cyclohexyl-4-(1,2-diphenylethyl)piperazine) is an opioid analgesic drug candidate developed in the 1970s that has recently been introduced as a new psychoactive substance (NPS) on the "recreational" drug market. We describe a case series of non-fatal intoxications associated with MT-45 within the Swedish STRIDA project. Study design. Observational case series of consecutive patients with admitted or suspected intake of NPS presenting to hospitals in Sweden from November 2013 to February 2014. Patients and methods. Blood and urine samples were collected from intoxicated patients presenting to emergency departments and intensive care units over the country. NPS analysis was performed by an LC-MS/MS multi-component method. Clinical data were collected when caregivers consulted the Poisons Information Centre and also retrieved from medical records. Case series. Among nine intoxications where MT-45 was detected in the biological samples, four cases were indicated to only involve MT-45, while one or several psychoactive substances were found along with MT-45 in the others. All patients were men aged 17-32 years and they commonly presented with opioid-like adverse symptoms, such as unconsciousness and respiratory depression. Naloxone appeared to have utility in the treatment of MT-45 intoxication in several cases. Three patients complained of bilateral hearing loss that in one case persisted after two weeks. Conclusion. MT-45 should be added to the growing list of harmful NPS causing life-threatening poisonings, and rapid actions taken to make it a controlled substance.
[Show abstract][Hide abstract] ABSTRACT: Breath has been investigated as an alternative matrix for detecting recent cocaine intake; however, there are no controlled cocaine administration studies that investigated the drug's disposition into breath. Breath was collected from 10 healthy adult cocaine users by asking them to breathe into a SensAbues device for 3 min before and up to 22 h following 25 mg intravenous (IV) cocaine dosing on days 1, 5, and 10, and assayed with a validated liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method to quantify breath cocaine, benzoylecgonine (BE), ecgonine methyl ester (EME), and norcocaine. The assay was linear from 25 to 1,000 pg/filter, extraction efficiencies were 83.6-126 %, intra- and inter-assay imprecision was <10.6 %, and bias was between -8.5 and 16.8 %. No endogenous or exogenous interferences were observed for more than 75 tested. Analytes were generally stable under short-term storage conditions. Ion suppression was less than 46 %. Of breath specimens collected after controlled cocaine administration, 2.6 % were positive for cocaine (26.1-66 pg/filter, 1-9.5 h), 0.72 % BE (83.3-151 pg/filter, 6.5-12.5 h), and 0.72 % EME (50-69.1 pg/filter, 6.5-12.5 h); norcocaine was not detected. Methanolic extraction of the devices themselves, after filters were removed, yielded 19.2 % positive cocaine tests (25.2-36.4 pg/device, 10 min-22 h) and 4.3 % positive BE tests (26.4-93.7 pg/device, 10 min-22 h), explaining differences between the two extraction techniques. These results suggest that the device reflects the drug in oral fluid as well as lung microparticles, while the filter reflects only drug-laden microparticles. A sensitive and specific method for cocaine, BE, EME, and norcocaine quantification in breath was developed and validated. Cocaine in breath identifies recent cocaine ingestion, but its absence does not preclude recent use.
Analytical and Bioanalytical Chemistry 08/2014; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The advent of LC combined with MS made it possible to design analytical methods for urine drug testing based on the very simple concept of diluting urine with an internal standard as the sole preparation procedure prior to instrumental analysis. The number of publications using this method design increased after the development of high-efficiency LC based on sub-2 μm particles. The success of this method design for drug testing, doping control and toxicological investigations of urine is now well documented and comprise both screening and confirmation methods. The nondiscriminating nature of this method design makes it even more attractive in combination with high-resolution MS for multicomponent target and general unknown analysis applications.
[Show abstract][Hide abstract] ABSTRACT: Background. Therapeutic drug monitoring (TDM) of the antiepileptic drug valproic acid (VPA) is recommended in patients with multiple drug therapy or with concomitant disabilities to ensure treatment efficacy and avoid adverse reactions in both adults and children. The use of sampling techniques compatible with home sampling, such as dried blood spot sampling could potentially facilitate this for patients. Aim. To assess the usefulness of a bioanalytical method for quantification of VPA in dried blood spots. Materials and methods. Quantification was based on liquid chromatography-mass spectrometry (LC-MS), both for the DBS method and the plasma-based reference method. Results. The method was validated in the range 10–1200 μmol/L. Total imprecision ranged from 4.9–8.9 (%CV) and accuracy was within ± 14%. Conclusion. The validated method has potential for evaluation in therapeutic drug monitoring in combination with home sampling of DBS. The impact of spot size can be controlled through acceptance criteria and hematocrit in the range 30–60% can be accepted in sampling. Comparison of VPA levels between plasma and whole blood cannot be done without considering the blood-plasma ratio.
Scandinavian Journal of Clinical & Laboratory Investigation. 07/2014;
[Show abstract][Hide abstract] ABSTRACT: Aim. Products for on-site urine drug testing offer the possibility to perform screening for drugs of abuse directly at the point-of-care. This is a well-established routine in emergency and dependency clinics but further evaluation of performance is needed due to inherent limitations with the available products. Methods. Urine drug testing by an on-site product was compared with routine laboratory methods. First, on-site testing was performed at the laboratory in addition to the routine method. Second, the on-site testing was performed at a dependency clinic and urine samples were subsequently sent to the laboratory for additional analytical investigation. Results. The on-site testing products did not perform with assigned cut-off levels. The subjective reading between the presence of a spot (i.e. negative test result) being present or no spot (positive result) was difficult in 3.2% of the cases, and occurred for all parameters. The tests performed more accurately in drug negative samples (specificity 96%) but less accurately for detecting positives (sensitivity 79%). Of all incorrect results by the on-site test the proportion of false negatives was 42%. The overall agreement between on-site and laboratory testing was 95% in the laboratory study and 98% in the clinical study. Conclusion. Although a high degree of agreement was observed between on-site and routine laboratory urine drug testing, the performance of on-site testing was not acceptable due to significant number of false negative results. The limited sensitivity of on-site testing compared to laboratory testing reduces the applicability of these tests.
Scandinavian journal of clinical and laboratory investigation. 07/2014;
[Show abstract][Hide abstract] ABSTRACT: Context. 5-(2-aminopropyl)indole (5-IT) is a new psychoactive substance (NPS; "legal high" or "research chemical") structurally related to indoleamines and substituted phenethylamines and implicated in several fatalities. We describe the clinical characteristics and results of laboratory investigations of 14 analytically confirmed nonfatal cases of 5-IT intoxication within the Swedish STRIDA project. Study design. Observational case series of consecutive patients with admitted or suspected intake of NPS presenting to hospitals in Sweden in 2012. Patients and methods. Blood and/or urine samples were collected from intoxicated patients presenting to emergency departments and intensive care units over the country. Analysis of NPS was performed using an LC-MS/MS multi-component method. Clinical data were collected when caregivers consulted the Poisons Information Centre and also retrieved from medical records. The severity of poisoning was graded retrospectively using the Poisoning Severity Score (PSS). Results. Eleven male and three female patients (age: 21-53 years, median: 27) tested positive for 5-IT in 2012, all cases appearing in April-July. The 5-IT concentration in serum ranged between 0.015 and 0.59 μg/mL (median: 0.22; n = 8) and in urine between 0.005 and 24.7 μg/mL (median: 5.95; n = 12). Five intoxications were indicated to be caused by 5-IT alone, whereas additional psychoactive substances were detected in the other nine cases. Six (43%) of fourteen cases were graded as severe (PSS 3), five (36%) as moderate (PSS 2), and three (21%) as minor (PSS 1) poisonings. In the severe cases, agitation, hallucinations, dilated pupils without light reaction, tachycardia, hypertension, hyperthermia, myoclonus, muscle rigidity, arrhythmias, seizures, rhabdomyolysis, and/or renal failure were noted. Conclusions. The results demonstrated that severe clinical toxicity was commonly present in patients with analytically confirmed 5-IT exposure. The clinical features are consistent with a sympathomimetic toxidrome, and some patients also displayed symptoms associated with serotonin toxicity.
[Show abstract][Hide abstract] ABSTRACT: It has been discovered recently that exogenous substances are detectable in exhaled breath after intake. Exhaled breath therefore constitutes a new possible matrix in clinical pharmacology and toxicology. The present work was aimed at exploring this possibility further by a study on patients treated for attention-deficit/hyperactivity disorder with D-amphetamine and methylphenidate.
Thirteen patients (age range: 32-61 years; 5 women) were included in the study, and breath and urine samples were collected at different times in the dose interval. Analyses of breath and urine samples were done with liquid chromatography-mass spectrometry methods. Urine was examined for amphetamine, methylphenidate, and its metabolite ritalinic acid.
Among the 9 patients who received D-amphetamine medication in daily doses of 20-100 mg, amphetamine was detected in all subjects in amounts ranging from 1200 to 30,800 picogram per filter. Among 8 patients receiving methylphenidate medication in daily doses of 80-400 mg, it was detected and quantified in 7 of the cases in amounts ranging from 150 to 10,400 picogram per filter and ritalinic acid was detected and quantified in 3 of the cases ranging from 35 to 360 picogram per filter. In 1 case, methylphenidate was only detectable in breath and urine, whereas ritalinic acid was quantifiable in urine, which could indicate noncompliance, with the 4 hours of dose regimen prescribed. In a number of cases, the sampling was performed 24 hours after the last dose intake. Identification of amphetamine and methylphenidate was based on correct chromatographic retention time and correct product ion ratio with detection performed in selected reaction monitoring mode.
The results confirm that amphetamine is present in exhaled breath after intake and demonstrate for the first time the presence of methylphenidate and ritalinic acid after its intake. This gives further support to the potential use of exhaled breath for detecting drug intake.
Therapeutic drug monitoring 01/2014; · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A semi-automated method for quantification of budesonide in human plasma was developed, validated, and applied for high-volume analysis of samples in connection with a pharmacokinetic study. Protein and phospholipid removal was performed using an Ostro 96-well filter plate and subsequently combined with C18 solid-phase extraction on a Hamilton Microlab STARlet automation robot. The final extracts were evaporated to dryness and redissolved in 20% acetonitrile/water. The procedure used budesonide-d8 as internal standard and gave a 3.5-fold concentration of plasma to extract. The final extracts (5 μL injected) were analyzed with selected reaction monitoring liquid chromatography–tandem mass spectrometry (LC–MS/MS) using electrospray ionization in positive mode. The chromatography system used a 100 mm ACQUITY BEH UPLC column and a gradient system consisting of aqueous 0.1% formic acid and acetonitrile as organic modifier. Phospholipid removal was found to be needed during method development in order to reduce ion suppression effects from matrix and to increase method sensitivity. The measuring range was 50–5000 pg/mL with and LOD 24 pg/mL. Calibration response showed good linearity (correlation coefficients < 0.99) over the measuring range. The absolute recovery over the sample preparation procedure was estimated to 67%. Total imprecision was <9% at three levels and accuracy was between 98.9 and 103%. The method was successfully applied for analysis of 864 study samples in a short time. The quality control samples at concentration levels 200 and 2000 pg/mL gave a total imprecision of 7.4% and 4.2%, respectively, (n = 95).
[Show abstract][Hide abstract] ABSTRACT: The “STRIDA” project monitors the occurrence and trends of new psychoactive substances (NPS; “Internet drugs/designer drugs/legal highs”) in Sweden, and collects information about their clinical symptoms, toxicity and associated health hazards. The initial results of the project documented a widespread use of many different NPS by mainly adolescents and young (age range 13–63 years, median 20), male (79%) adults, among cases of drug intoxications presenting at emergency departments and intensive care units across the country. The new substances were identified in samples of urine and blood by a multi-component LC-MS/MS method, and the severity of clinical symptoms were graded by the Poisoning Severity Score (PSS). Of the initial 189 samples submitted for laboratory investigation, 156 (83%) tested positive for at least one drug. Besides classical substances such as ethanol, cannabis and amphetamines, many NPS were detected comprising synthetic cannabinoid receptor agonists (“Spice”), piperazines, substituted phenethylamines, synthetic cathinones, hallucinogenic tryptamines, piperidines, opioid related substances, ketamine and related substances, and GABA analogues (in total more than 50 substances). About half of the cases were demonstrated to be multiple drug intoxications, sometimes making it hard to associate the clinical presentations with one specific substance. In conclusion, the STRIDA project has documented use of a broad variety of NPS among mainly young people all over Sweden.
Forensic science international 01/2014; · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction
The oral direct thrombin inhibitor dabigatran is increasingly used to prevent thromboembolic stroke in patients with atrial fibrillation (AF). Routine laboratory monitoring is currently not recommended, but measurements of dabigatran and/or its effect are desirable in certain situations. We studied dabigatran exposure and compared different tests for monitoring of dabigatran in a real-life cohort of AF patients.
Material and methods
Ninety AF patients (68 ± 9 years, 67% men, mean CHADS2 score 1.5) were treated with dabigatran 150 (n = 73) or 110 mg BID (n = 17). Trough plasma concentrations of total and free dabigatran by liquid chromatography-tandem mass-spectrometry (LC-MS/MS) were compared to indirect measurements by Hemoclot thrombin inhibitors (HTI) and Ecarin clotting assay (ECA), as well as PT-INR and aPTT.
Total plasma dabigatran varied 20-fold (12–237 ng/mL with 150 mg BID) and correlated well with free dabigatran (r2 = 0.93). There were strong correlations between LC-MS/MS and HTI or ECA (p < 0.001) but these assays were less accurate with dabigatran below 50 ng/mL. The aPTT assay was not dependable and PT-INR not useful at all. There were weak correlations between creatinine clearance (Cockcroft-Gault) and LC-MS/MS, HTI and ECA (p < 0.001 for all). A high body weight with normal kidney function was associated with low dabigatran levels.
HTI and ECA reflect the intensity of dabigatran anticoagulation, but LC-MS/MS is required to quantify low levels or infer absence of dabigatran. Most real life patients with a normal creatinine clearance had low dabigatran levels suggesting a low risk of bleeding but possibly limited protection against stroke.
[Show abstract][Hide abstract] ABSTRACT: To test the efficacy and safety of osmotic release oral system (OROS) methylphenidate (MPH) in doses up to 180 mg/day to treat attention deficit hyperactivity disorder (ADHD) and prevent any drug relapse in individuals with a co-diagnosis of ADHD and amphetamine dependence.
Randomized placebo-controlled 24-week double-blind trial with parallel groups design.
Participants were recruited from medium security prisons in Sweden. The medication started within two weeks before release from prison and continued in outpatient care with twice weekly visits including once weekly cognitive behaviour therapy.
Fifty-four men with a mean age of 42 years, currently incarcerated, meeting DSM-IV criteria for ADHD and amphetamine dependence.
Change in self-reported ADHD symptoms, relapse to any drug use (amphetamine and other drugs) measured by urine toxicology, retention to treatment, craving, and time to relapse.
The MPH treated group reduced their ADHD symptoms during the trial (P = 0.011) and had a significantly higher proportion of drug negative urines compared with the placebo group (P= 0.047), including more amphetamine negative urines (P = 0.019) and better retention to treatment (P = 0.032).
Methylphenidate treatment reduces attention deficit hyperactivity disorder (ADHD) symptoms and the risk for relapse to substance use in criminal offenders with ADHD and substance dependence.
[Show abstract][Hide abstract] ABSTRACT: Δ(9)-Tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC (THCCOOH), and cannabinol (CBN) were measured in breath following controlled cannabis smoking to characterize the time course and window of detection of breath cannabinoids.METHODS: Exhaled breath was collected from chronic (≥4 times per week) and occasional (<twice per week) smokers before and after smoking a 6.8% THC cigarette. Sample analysis included methanol extraction from breath pads, solid-phase extraction, and liquid chromatography-tandem mass spectrometry quantification.RESULTS: THC was the major cannabinoid in breath; no sample contained THCCOOH and only 1 contained CBN. Among chronic smokers (n = 13), all breath samples were positive for THC at 0.89 h, 76.9% at 1.38 h, and 53.8% at 2.38 h, and only 1 sample was positive at 4.2 h after smoking. Among occasional smokers (n = 11), 90.9% of breath samples were THC-positive at 0.95 h and 63.6% at 1.49 h. One occasional smoker had no detectable THC. Analyte recovery from breath pads by methanolic extraction was 84.2%-97.4%. Limits of quantification were 50 pg/pad for THC and CBN and 100 pg/pad for THCCOOH. Solid-phase extraction efficiency was 46.6%-52.1% (THC) and 76.3%-83.8% (THCCOOH, CBN). Matrix effects were -34.6% to 12.3%. Cannabinoids fortified onto breath pads were stable (≤18.2% concentration change) for 8 h at room temperature and -20°C storage for 6 months.CONCLUSIONS: Breath may offer an alternative matrix for testing for recent driving under the influence of cannabis, but is limited to a short detection window (0.5-2 h).
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Dabigatran is an oral direct thrombin inhibitor for which routine laboratory monitoring is currently not recommended. However, there are situations in which measurements of the drug and its effect are desirable. We therefore compared and validated different coagulation methods for assessments of dabigatran in clinical samples in relation to measurements of plasma dabigatran, without the purpose of establishing effective and safe concentrations of dabigatran in plasma. METHODS: Samples were obtained from 70 atrial fibrillation patients treated with dabigatran etexilate. Plasma concentrations were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and were compared with coagulation methods Hemoclot thrombin inhibitors (HTI) and Ecarin clotting assay (ECA), as well as with prothrombin time-international normalized ratio (PT-INR) and activated partial thromboplastin time (aPTT). RESULTS: A wide range of dabigatran concentrations was determined by LC-MS/MS (<0.5-586 ng/mL). Correlations between LC-MS/MS results and estimated concentrations were excellent for both HTI and ECA overall (r(2) = 0.97 and 0.96 respectively, p < 0.0001), but the precision and variability of these assays were not fully satisfactory in the low range of dabigatran plasma concentrations, in which ECA performed better than HTI. aPTT performed poorly, and was normal (<40 s) even with dabigatran levels of 60 ng/mL. PT-INR was normal even at supratherapeutic dabigatran concentrations. CONCLUSION: LC-MS/MS is the gold standard for measurements of dabigatran in plasma. Alternatively, either HTI or ECA assays may be used, but neither of these assays is dependable when monitoring low levels or to infer total absence of dabigatran. The aPTT assay is relatively insensitive to dabigatran, and normal aPTT results may be observed even with therapeutic dabigatran concentrations.
European Journal of Clinical Pharmacology 06/2013; · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aim. The study aimed to collect information concerning the increasing use of new psychoactive substances, commonly sold through online shops as 'Internet drugs' or 'legal highs', or in terms of masked products such as 'bath salts' and 'plant food'. Methods. The Karolinska Institutet and Karolinska University Laboratory and the Swedish Poisons Information Centre have initiated a project called 'STRIDA' aiming to monitor the occurrence and trends of new psychoactive substances in Sweden, and collect information about their clinical symptoms, toxicity and associated health risks. A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-component method has been developed, currently allowing for the determination of > 80 novel psychoactive compounds or metabolites thereof. This study focused mainly on the particular drug substances identified and the population demographics of the initial STRIDA cases. Results. In urine and/or blood samples obtained from 103 consecutive cases of admitted or suspected recreational drug intoxications in mostly young subjects (78% were ≤ 25 years, and 81% were males) presenting at emergency departments all over the country, psychoactive substances were detected in 82%. The substances comprised synthetic cannabinoids ('Spice'; JWH analogues), substituted cathinones ('bath salts'; e.g. butylone, MDPV and methylone) and tryptamines (4-HO-MET), plant-based substances (mitragynine and psilocin), as well as conventional drugs-of-abuse. In 44% of the cases, more than one new psychoactive substance, or a mixture of new and/or conventional drugs were detected. Conclusion. The initial results of the STRIDA project have documented use of a broad variety of new psychoactive substances among mainly young people all over Sweden.
Scandinavian journal of clinical and laboratory investigation 05/2013; · 1.38 Impact Factor