Determination of buprenorphine, norbuprenorphine, naloxone, and their glucuronides in urine by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), naloxone (NAL), and their glucuronide conjugates BUP-G, NBUP-G, and NAL-G in urine samples was developed. The method, omitting a hydrolysis step, involved non-polar solid-phase extraction, liquid chromatography on a C18 column, electrospray positive ionization, and mass analysis by multiple reaction monitoring. Quantification was based on the corresponding deuterium-labelled internal standards for each of the six analytes. The limit of quantification was 0.5 µg/L for BUP and NAL, 1 µg/L for NAL-G, and 3 µg/L for NBUP, BUP-G, and NBUP-G. Using the developed method, 72 urine samples from buprenorphine-dependent patients were analysed to cover the concentration ranges encountered in a clinical setting. The median (maximum) concentration was 4.2 µg/L (102 µg/L) for BUP, 74.7 µg/L (580 µg/L) for NBUP, 0.9 µg/L (85.5 µg/L) for NAL, 159.5 µg/L (1370 µg/L) for BUP-G, 307.5 µg/L (1970 µg/L) for NBUP-G, and 79.6 µg/L (2310 µg/L) for NAL-G.

Compensation of matrix effects in a standard addition method for metformin in postmortem blood using liquid chromatography-electrospray-tandem mass spectrometry.

This work describes a procedure to evaluate matrix effects in a combined dilution and standard addition method (SAM) using liquid chromatography-electrospray-tandem mass spectrometry. The method was validated and applied to an analysis of metformin in postmortem blood samples. The analytical method included protein precipitation with methanol, followed by liquid chromatographic separation of metformin on Gemini NX-C18 reversed-phase column using a gradient consisting of methanol and ammonium acetate at pH 3.2. The mass spectrometric analysis was performed with a quadrupole-linear ion trap mass spectrometer equipped with a turbo ion spray interface in a positive ion mode using selected reaction monitoring. Quantitation was performed based on an SAM. Validation for metformin revealed a practical limit of quantification of 0.1 mg/L, a linear range from 0.1 to 3.0 mg/L, average precision 10%, accuracy (bias) 9% and reproducibility 10%. Combined matrix effects were evaluated by k-values (slopes) of calibration plots, postextraction addition approach and a comparison of within- and between-sample precision (relative standard deviation). It was demonstrated that the method contained matrix effects which were fully compensated for using dilution and the SAM.

Profiles of pregabalin and gabapentin abuse by postmortem toxicology.

Pregabalin (PRG) and gabapentin (GBP) are used in the treatment of neuropathic pain and epilepsy, and PRG also in generalized anxiety disorder. There is increasing evidence that PRG possesses considerable abuse potential. PRG may have a higher addiction potential than GBP due to its rapid absorption and faster onset of action. Our objective is to estimate the proportion of all PRG- and GBP-related fatalities attributable to PRG and GBP abuse. We investigated all medico-legal death cases in Finland in which PRG or GBP was found in postmortem toxicology during 2010-2011. PRG was found in 316 cases and GBP in 43 cases. Drug abuse was associated with 48.1% of the PRG and 18.6% of the GBP findings. PRG poisoning accounted for 10.1% of all PRG cases and GBP poisoning for 4.7% of all GBP cases. In the drug abuser cases, PRG poisoning represented 19.1%, and GBP poisoning 12.5%. The median blood concentration of PRG was 15 mg/L in the abuser group and 5.8 mg/L in the other cases. For GBP, these concentrations were 12 mg/L and 8.3mg/L, respectively. In the PRG abuser group, 91.4% of cases showed concomitant opioid use, while in the rest of these cases neither alcohol nor opioids were detected, but other central nervous system acting drugs were found in each abuser case. In the GBP abuser group, 87.5% of cases showed concomitant opioid use. PRG abuse with high doses is increasingly common and can be fatal when combined with opioids.

Wastewater analysis reveals regional variability in exposure to abused drugs and opioids in Finland.

Abused drug concentrations were determined in nine Finnish wastewater treatment plants (WWTPs), representing the metropolitan area, university cities and rural towns. In an eight-day study period in August 2012, 24-hour composite influent wastewater samples were collected. Biological markers and census-based information were used to estimate the size of the population served. The analytical method included solid phase extraction, liquid chromatographic separation, tandem mass spectrometric identification, and quantification using isotope-labeled internal standards. The study covered amphetamines, cannabis and cocaine. The levels of some opioids used in treatment and their metabolites were also determined. Amphetamine was the most prevalent drug of abuse, the median loads varying between the cities from 4.16 to 29.6 mg/1000 inhabitants/d. In three western cities methamphetamine was detected in even higher amounts, ranging from 0.87 to 47.5mg/1000 inhabitants/d. Ecstasy (MDMA) and cocaine (as benzoylecgonine, BE) were found in higher concentrations during weekends compared to weekdays, the difference being statistically significant. The concentration of tetrahydrocannabinol-9-carboxylic acid (THCA) was below the limit of quantification in the two rural towns, while in the other cities the load varied between 3.77 and 20.7 mg/1000 inhabitants/d. The average variation in BE load was 0.05-6.82 and that of MDMA 0-20.6 mg/1000 inhabitants/d. While the metropolitan area showed the highest loads of abused drugs, the substances were continuously detected at all WWTPs included in the study. The median concentration of codeine ranged from 164 to 325 mg/1000 inhabitants/d and that of morphine from 18.8 to 31.5mg/1000 inhabitants/d. The methadone load was below the level of detection in two towns, and at the other locations were 1.22-9.46 mg/1000 inhabitants/d. The first metabolite of heroin, 6-monoacetylmorphine (6-MAM), was not detected at all. Although the method has limitations, wastewater analysis gives additional information for assessing the degree of drug abuse and range of drugs abused in a society.

Second-generation antihistamines exhibit a protective effect on drivers in traffic-a preliminary population-based case-control study.

INTRODUCTION: Although there have been experimental studies concerning driving and drugs, studies on the risk of antihistamines are not numerous. This is the first population-based epidemiological study concerning the association of sedating/nonsedating antihistamines and fatal traffic accidents. METHODS: Car drivers (n = 428) who died in accidents before reaching the hospital and controls (n = 688) matched for accident area and driving season were studied for antihistamines in blood. At the time of the fatal road traffic accident, 6 drivers had a detectable amount of sedating antihistamines in blood, and the corresponding number for controls was 4; nonsedating antihistamines in blood were detected in 12 accident cases and 28 controls. The fatal accidents occurred between 1998 and 2002 and the information on the controls was collected between 2000 and 2002 in Finland. RESULTS: Regarding fatal traffic accident causality, the nonsedating antihistamines proved to have a protective effect after adjusting for age and gender (relative risk = 0.40, 95% confidence interval [CI], 0.20 to 0.82; P =.01). The risk of fatal traffic accident of those driving under the influence of sedating antihistamines was 1.61 (0.38 to 6.77, P =.51) times the risk of those without medication. DISCUSSION: This preliminary study supports the protective effect of second-generation antihistamines with respect to fatal traffic accidents. Due to the small sample size the results are not conclusive.

Urine naloxone concentration at different phases of buprenorphine maintenance treatment.

In spite of the benefits of buprenorphine-naloxone co-formulation (BNX) in opioid maintenance treatment, the naloxone component has not prevented parenteral use of BNX. Current laboratory methods are not sufficient to differentiate between therapeutic and illicit use of buprenorphine, and little is known about urine naloxone concentrations. Measurement of urine naloxone, together with buprenorphine and norbuprenorphine, might help to determine the naloxone source and administration route. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for this purpose. Naloxone, buprenorphine, and norbuprenorphine total concentrations were measured in urine samples from opioid-dependent patients before and during stable and unstable phases of maintenance treatment with BNX. The limit of quantification in urine was 1.0 µg/L for naloxone, buprenorphine and norbuprenorphine. Before treatment, all samples contained buprenorphine but the median naloxone concentration was 0 µg/L. During the maintenance treatment with BNX all urine samples were positive for naloxone, buprenorphine and norbuprenorphine. The naloxone concentration at a stable phase of treatment (median 60 µg/L, range 5-200 µg/L) was not different from the naloxone concentration at an unstable phase (70 µg/L, 10-1700 µg/L). Applying an upper limit of 200 µg/L to the sample, the median naloxone/buprenorphine ratio was higher in the high than in the low naloxone concentration group (0.9 vs 0.3, respectively). This study suggests that naloxone in urine can act as an indicator of compliance with BNX. Parenteral use of BNX was associated with a high naloxone/buprenorphine ratio. Negative naloxone with positive buprenorphine suggests the use/abuse of buprenorphine alone.

Combined drug screening and confirmation by liquid chromatography time-of-flight mass spectrometry with reverse database search.

A method based on in-source collision-induced dissociation (ISCID) liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) and reverse target database search was developed and evaluated for drug screening and confirmation in analytical toxicology context. An established LC-TOFMS screening method, in which identification relies solely on protonated molecule accurate mass measurement, isotopic pattern fit, and retention time (RT), was completed to include 1-3 qualifier ions for each analyte in the database. The qualifier ions for 431 compounds were selected from the experimental ISCID spectra, and their molecular formulae were assigned by applying SmartFormula3D and MSFragmenter software. Three qualifier ions were assigned for 64.5%, two or three for 81.4%, one for 14.8%, and none for 3.7% of the compounds studied. Comparison between ISCID LC-TOFMS and LC-TOFMS with 25 authentic autopsy urine samples showed an improved confidence level with the ISCID method, as isomeric interferences were excluded in most cases. However, some false negative (FN) results were obtained at low concentration levels close to the reporting criteria. The cut-off concentration of the ISCID method was 10-100 ng/mL with 80% of the 49 representative compounds tested, and the level was approximately two times higher than that obtained by LC ion trap MS. The presented method enables simultaneous screening and confirmation whenever at least one qualifier ion is available, as applying an accurate mass precursor ion and one product ion surpasses the standard of four identification points that is required by the current EU protocol.

Broad-spectrum drug screening of meconium by liquid chromatography with tandem mass spectrometry and time-of-flight mass spectrometry.

Analysis of the major drugs of abuse in meconium has been established in clinical practice for detecting fetal exposure to illicit drugs, particularly for the ready availability of the sample and ease of collection from diapers, compared with neonatal hair and urine. Very little is known about the occurrence and detection possibilities of therapeutic and licit drugs in meconium. Meconium specimens (n = 209) were collected in delivery hospitals, from infants of mothers who were suspected to be drug abusers. A targeted analysis method by liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) was developed for abused drugs: amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, morphine, codeine, 6-monoacetylmorphine, oxycodone, methadone, tramadol, buprenorphine, and norbuprenorphine. A separate LC-MS/MS method was developed for 11-nor-∆(9)-tetrahydrocannabinol-9-carboxylic acid. A screening method based on LC coupled to time-of-flight MS was applied to a broad spectrum of drugs. As a result, a total of 77 different compounds were found. The main drug findings in meconium were as follows: local anesthetics 82.5% (n = 172), nicotine or its metabolites 61.5% (n = 129), opioids 48.5% (n = 101), stimulants 21.0% (n = 44), hypnotics and sedatives 19.0% (n = 40), antidepressants 18.0% (n = 38), antipsychotics 5.5% (n = 11), and cannabis 3.0% (n = 5). By revealing drugs and metabolites beyond the ordinary scope, the present procedure helps the pediatrician in cases where maternal denial is strong but the infant seems to suffer from typical drug-withdrawal symptoms. Intrapartum drug administration cannot be differentiated from gestational drug use by meconium analysis, which affects the interpretation of oxycodone, tramadol, fentanyl, pethidine, and ephedrine findings.

ESI-MS/MS library of 1,253 compounds for application in forensic and clinical toxicology.

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) library which contains over 5,600 spectra of 1,253 compounds relevant in clinical and forensic toxicology has been developed using a hybrid tandem mass spectrometer with a linear ion trap. Pure compound solutions-in some cases solutions made of tablets-were prepared and 1 to 2,000 ng of each compound were injected into the system using standard reversed-phase analytical columns with gradient elution. To obtain maximum mass spectral information enhanced product ion spectra were acquired with positive and/or negative ionization at low, medium, and high collision energies and additionally applying collision energy spread. In this mode, all product ions generated by the different collision energies are trapped in the linear ion trap prior to their detection. The applicability of the library for other types of hybrid tandem mass spectrometers with a linear ion trap of the same manufacturer as well as a standard triple-quadrupole tandem mass spectrometer has been investigated with a selection of compounds. The spectra of the developed library can be used to create methods for target analysis, either screening methods or quantitative procedures by generating transitions for multiple reaction monitoring. For those procedures, suitable transitions and convenient collision energies are selected from the library. It also has been utilized to identify compounds with a multi target screening approach for clinical and forensic toxicology with a standardized and automated system. The novel aspects compared to our former library produced with a standard triple-quadrupole mass spectrometer are the enlargement of the ESI-MS/MS library and the additional acquisition of spectra with collision energy spread.

Transdermal fentanyl in cachectic cancer patients.

Fentanyl is an opioid with high lipid solubility, suitable for intravenous, spinal, transmucosal and transdermal administration. The transdermal fentanyl patch has become widely used in the treatment of both malignant and non-malignant chronic pain. The absorption of fentanyl from the patch is governed by the surface area of the patch, by skin permeability and by local blood flow. The aim of this study is to find out whether absorption of fentanyl in cachectic patients with cancer-related pain is different from that of normal weight cancer patients. We recruited ten normal weight (mean body mass index (BMI) 23 kg/m(2)) and ten cachectic (mean BMI 16 kg/m(2)) cancer pain patients. A transdermal fentanyl patch with a dose approximately equianalgesic to the patients' previous opioid dose was administered to the upper arm of the patient for 3 days. Prior to patch application, the height, weight and BMI of the patient, as well as upper arm skin temperature, local sweating, thickness of skin fold and local blood flow were measured. Plasma fentanyl concentrations were analyzed from blood samples taken at baseline, 4, 24, 48 and 72 h. Plasma fentanyl concentrations adjusted to dose were significantly lower at 48 and 72 h in cachectic patients than normal weight patients. The cachectic patients had a significantly thinner upper arm skin fold, but no differences were found in local blood flow, sweating, or skin temperature. Absorption of transdermal fentanyl is impaired in cachectic patients compared with that of normal weight cancer pain patients.

An epidemic of fatal 3-methylfentanyl poisoning in Estonia.

An exceptional epidemic of poisonings due to the highly potent opioid designer drug 3-methylfentanyl (TMF) was revealed among Estonian drug users in 2005-2006 by post-mortem forensic toxicology. Quantitative analysis of cis-TMF, trans-TMF, and fentanyl was performed by liquid chromatography-tandem mass spectrometry. Comprehensive toxicological analysis was performed using a multi-technique approach. The number of TMF-related fatal accidental poisonings identified was 46 and 71 for 2005 and 2006, respectively. The proportion of male victims was 91.5% and the mean age of all victims was 26 years at death. TMF was used predominantly by intravenous injection. There was no significant difference in the blood concentrations of cis-TMF and trans-TMF between pure TMF poisonings and mixed TMF poisonings. The mean combined concentration of TMF stereoisomers among pure TMF cases (1.9 microg/l) was more than ten times lower than the mean fentanyl concentration in fentanyl-related fatalities. Concomitant use of other drugs involved alcohol, amphetamines, benzodiazepines, and cannabis, but very rarely other opioids.

Does coronary artery bypass surgery affect metoprolol bioavailability.

BACKGROUND: beta-blockers are commonly administered in patients with coronary artery bypass surgery (CABG). Despite this therapy, however, the incidence of postoperative atrial fibrillation (AF) is high (9-19%), and it is unknown why the beta-blockers do not reduce the incidence of AF more efficiently. In this pharmacokinetics study, in which the patients acted as their own controls, we have evaluated the bioavailability of perioperative metoprolol tablets in CABG surgery patients. METHODS: Twelve male patients, aged 45-64 years, scheduled for CABG surgery were administered an initial 50 mg metoprolol tartrate tablet orally on the morning of the preoperative day and thereafter at 12-h intervals. Regular blood samples were collected up to 12 h after the first administration of the drug on the preoperative day as well on the first and third postoperative days. The plasma concentration for metoprolol was analyzed (limit of quantification = 0.001 mg/L) using liquid chromatography-tandem mass spectrometry. RESULTS: The bioavailability of the metoprolol was significantly less on the first postoperative day, with AUC(0-12) values ranging from 0.7 to 17.1 (median: 7.2) mg min/L, than on the preoperative day, with AUC(0-12) values of 5.1-26.7 (12.6) mg min/L; however, it returned to the preoperative values on the third postoperative day, with AUC(0-12) values of 3.5-25.2 (15.2) mg min/L. Similar changes were observed in C(max) values: preoperative C(max) ranged between 0.026 and 0.123 (0.060) mg/L, on the first postoperative day, the C(max) ranged between 0.003 and 0.093 (0.025) mg/L, and on the third postoperative day, the C(max) ranged between 0.009 and 0.136 (0.061) mg/L. There was no correlation between the pharmacokinetic parameters and patient characteristics, but both the preoperative C(max) and C(60) correlated significantly with the postoperative C(max) (Pearson correlation coefficient: 0.61-0.72). One patient with one of the lowest rates and extent of metoprolol absorption developed AF. CONCLUSION: This study indicates that the bioavailability of metoprolol is markedly reduced when administered in tablet form during the early phase after CABG.

Blood levels of 3-methylfentanyl in 3 fatal poisoning cases.

Three fatal poisoning cases due to 3-methylfentanyl are described. In each case, the death was accidental and occurred after injection of the opioid combined with amphetamine, heroin, or other drugs. The victims' ages, ranging from 30 to 41 years, were higher than those typically found in heroin poisonings in Finland. The blood concentrations of cis-3-methylfentanyl, measured here for the first time by a specific tandem mass spectrometric method, ranged from 0.3 to 0.9 microg/L (mean 0.5 microg/L). These values are significantly lower than the levels reported for alpha-methylfentanyl and fentanyl in fatal poisonings. Repeated seizures of fentanyl and its analogs have been reported in Europe close to the Russian border.

CYP2D6 and CYP2C19 genotypes and amitriptyline metabolite ratios in a series of medicolegal autopsies.

In a series of 202 postmortem toxicology cases, the CYP2D6 and CYP2C19 genes were genotyped, and the concentrations of amitriptyline (AT) and six metabolites were analyzed. The polymorphic CYP2D6 and CYP2C19 genes encode enzymes participating in the metabolism of several potentially toxic drugs, and mutations in these genes may lead to adverse drug reactions, possibly even intoxications. AT was chosen as the substrate of interest because it is mainly metabolized by these enzymes, is considered relatively toxic, and ranks among the major causes of fatal drug poisoning in Finland. Our objective was to evaluate genetically determined interindividual variation in conjunction with metabolite ratios of drugs found in toxicological analysis in a series of medicolegal autopsies. Positive correlations were found between the proportion of trans-hydroxylated metabolites and the number of functional copies of CYP2D6 and between the proportion of demethylated metabolites and the number of functional copies of CYP2C19. None of the accidental or undetermined AT poisonings coincided with the CYP2D6 or CYP2C19 genotype which predicts a poor metabolizer phenotype. However, an unusually high femoral blood concentration of AT, 60mg/l, was found in one suicide case with no functional CYP2D6 genes. Our study shows a concordance of AT metabolite patterns with CYP2D6 and CYP2C19 genotypes in the presence of confounding factors typical for postmortem material. This result demonstrates the feasibility of postmortem pharmacogenetic analysis and supports the dominant role of genes in drug metabolism.

Application of accurate mass measurement to urine drug screening.

Poor availability of reference standards for designer drugs, metabolites, and new substances prevents toxicology laboratories from rapidly responding to the changing analytical challenges of drug abuse. A novel screening approach comprising determination of accurate masses of sample components and comparison of these with databases of theoretical monoisotopic masses is described. Using liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS), a routine mass search window of 20-30 ppm was applied to urine samples. The ultimate reference technique, liquid chromatography-Fourier transform mass spectrometry (LC-FTMS), was capable of confirming the findings within a 3 ppm mass accuracy. Using a target database of 7640 compounds, the number of potential elemental formulas ranged from one to three with LC-TOFMS, and it was always one with LC-FTMS. In contrast to ordinary techniques requiring primary reference standards, the formula-based databases can be updated instantly with fresh numeric data from scientific literature and authority sources.

Comparison of product ion spectra obtained by liquid chromatography/triple-quadrupole mass spectrometry for library search.

Reproducibility of product ion spectra acquired using a liquid chromatography/triple-quadrupole mass spectrometry (LC/MS/MS) instrument over a 4-year period, and with three other LC/MS/MS instruments, one from the same manufacturer and two from a different manufacturer, was examined. The MS/MS spectra of 30 drug substances were generated in positive electrospray ionization mode at low, medium, and high collision energies (20, 35, and 50 eV). Purity and Fit score percentages against a 400-compound LC/MS/MS spectral library were calculated using an algorithm in which fragment intensity ratios and weighting factors were included. The long-term reproducibility study was conducted using a brand A instrument; after 4 years the reproducibility of the product ion spectra was still 94%, expressed as average Purity score. The inter-laboratory study involved two parts. Firstly, two LC/MS/MS spectral libraries, created independently in separate laboratories using brand A instruments, were compared with each other. The average Fit and Purity scores of spectra from one library against the other were better than 93 and 91%, respectively, when the same collision energies were used. Secondly, for the comparison of product ion spectra between brand A and brand B instruments, fragmentation conditions were first standardized for amitriptyline as the standard analyte. The average Fit scores of brand B spectra against the brand A spectral library varied between 79 and 85% at all three collision energies. These results indicate that, after standardizing the instrumental conditions, LC/MS/MS spectral libraries of drug substances are suitable for inter-laboratory use.