Pain after combat injury in male UK military personnel deployed to Afghanistan.

BACKGROUND: Chronic pain after injury poses a serious health burden. As a result of advances in medical technology, ever more military personnel survive severe combat injuries, but long-term pain outcomes are unknown. We aimed to assess rates of pain in a representative sample of UK military personnel with and without combat injuries. METHODS: We used data from the ADVANCE cohort study (ISRCTN57285353). Individuals deployed as UK armed forces to Afghanistan were recruited to include those with physical combat injuries, and a frequency-matched uninjured comparison group. Participants completed self-reported questionnaires, including 'overall' pain intensity and self-assessment of post-traumatic stress disorder, anxiety, and depression. RESULTS: A total of 579 participants with combat injury, including 161 with amputations, and 565 uninjured participants were included in the analysis (median 8 yr since injury/deployment). Frequency of moderate or severe pain was 18% (n=202), and was higher in the injured group (n=140, 24%) compared with the uninjured group (n=62, 11%, relative risk: 1.1, 95% confidence interval [CI]: 1.0-1.2, P<0.001), and lower in the amputation injury subgroup (n=31, 19%) compared with the non-amputation injury subgroup (n=109, 26%, relative risk: 0.9, 95% CI: 0.9-1.0, P=0.034). Presence of at least moderate pain was associated with higher rates of post-traumatic stress (RR: 3.7, 95% CI: 2.7-5.0), anxiety (RR: 3.2, 95% CI: 2.4-4.3), and depression (RR: 3.4, 95% CI: 2.7-4.5) after accounting for injury. CONCLUSION: Combat injury, but not amputation, was associated with a higher frequency of moderate to severe pain intensity in this cohort, and pain was associated with adverse mental health outcomes.

Discovery of Biofilm Inhibitors from the Microbiota of Marine Egg Masses.

Biofilms commonly develop in immunocompromised patients, which leads to persistent infections that are difficult to treat. In the biofilm state, bacteria are protected against both antibiotics and the host's immune system; currently, there are no therapeutics that target biofilms. In this study, we screened a chemical fraction library representing the natural product capacity of the microbiota of marine egg masses, namely, the moon snail egg collars. This led to the identification of active fractions targeting both Pseudomonas aeruginosa and Staphylococcus aureus biofilms. Subsequent analysis revealed that a subset of these fractions were capable of eradicating preformed biofilms, all against S. aureus. Bioassay-guided isolation led us to identify pseudochelin A, a known siderophore, as a S. aureus biofilm inhibitor with an IC50 of 88.5 µM. Mass spectrometry-based metabolomic analyses revealed widespread production of pseudochelin A among fractions possessing S. aureus antibiofilm properties. In addition, a key biosynthetic gene involved in producing pseudochelin A was detected on 30% of the moon snail egg collars and pseudochelin A is capable of inhibiting the formation of biofilms (IC50 50.6 µM) produced by ecologically relevant bacterial strains. We propose that pseudochelin A may have a role in shaping the microbiome or protecting the egg collars from microbiofouling.

mtDNAcombine: tools to combine sequences from multiple studies.

BACKGROUND: Today an unprecedented amount of genetic sequence data is stored in publicly available repositories. For decades now, mitochondrial DNA (mtDNA) has been the workhorse of genetic studies, and as a result, there is a large volume of mtDNA data available in these repositories for a wide range of species. Indeed, whilst whole genome sequencing is an exciting prospect for the future, for most non-model organisms' classical markers such as mtDNA remain widely used. By compiling existing data from multiple original studies, it is possible to build powerful new datasets capable of exploring many questions in ecology, evolution and conservation biology. One key question that these data can help inform is what happened in a species' demographic past. However, compiling data in this manner is not trivial, there are many complexities associated with data extraction, data quality and data handling. RESULTS: Here we present the mtDNAcombine package, a collection of tools developed to manage some of the major decisions associated with handling multi-study sequence data with a particular focus on preparing sequence data for Bayesian skyline plot demographic reconstructions. CONCLUSIONS: There is now more genetic information available than ever before and large meta-data sets offer great opportunities to explore new and exciting avenues of research. However, compiling multi-study datasets still remains a technically challenging prospect. The mtDNAcombine package provides a pipeline to streamline the process of downloading, curating, and analysing sequence data, guiding the process of compiling data sets from the online database GenBank.

Bayesian Skyline Plots disagree with range size changes based on Species Distribution Models for Holarctic birds.

During the Quaternary, large climate oscillations impacted the distribution and demography of species globally. Two approaches have played a major role in reconstructing changes through time: Bayesian Skyline Plots (BSPs), which reconstruct population fluctuations based on genetic data, and Species Distribution Models (SDMs), which allow us to back-cast the range occupied by a species based on its climatic preferences. In this paper, we contrast these two approaches by applying them to a large data set of 102 Holarctic bird species, for which both mitochondrial DNA sequences and distribution maps are available, to reconstruct their dynamics since the Last Glacial Maximum (LGM). Most species experienced an increase in effective population size (Ne , as estimated by BSPs) as well as an increase in geographical range (as reconstructed by SDMs) since the LGM; however, we found no correlation between the magnitude of changes in Ne and range size. The only clear signal we could detect was a later and greater increase in Ne for wetland birds compared to species that live in other habitats, a probable consequence of a delayed and more extensive increase in the extent of this habitat type after the LGM. The lack of correlation between SDM and BSP reconstructions could not be reconciled even when range shifts were considered. We suggest that this pattern might be linked to changes in population densities, which can be independent of range changes, and caution that interpreting either SDMs or BSPs independently is problematic and potentially misleading.

Seed size and its rate of evolution correlate with species diversification across angiosperms.

Species diversity varies greatly across the different taxonomic groups that comprise the Tree of Life (ToL). This imbalance is particularly conspicuous within angiosperms, but is largely unexplained. Seed mass is one trait that may help clarify why some lineages diversify more than others because it confers adaptation to different environments, which can subsequently influence speciation and extinction. The rate at which seed mass changes across the angiosperm phylogeny may also be linked to diversification by increasing reproductive isolation and allowing access to novel ecological niches. However, the magnitude and direction of the association between seed mass and diversification has not been assessed across the angiosperm phylogeny. Here, we show that absolute seed size and the rate of change in seed size are both associated with variation in diversification rates. Based on the largest available angiosperm phylogenetic tree, we found that smaller-seeded plants had higher rates of diversification, possibly due to improved colonisation potential. The rate of phenotypic change in seed size was also strongly positively correlated with speciation rates, providing rare, large-scale evidence that rapid morphological change is associated with species divergence. Our study now reveals that variation in morphological traits and, importantly, the rate at which they evolve can contribute to explaining the extremely uneven distribution of diversity across the ToL.

Population pharmacokinetic model of transdermal nicotine delivered from a matrix-type patch.

AIMS: Nicotine addiction is an issue faced by millions of individuals worldwide. As a result, nicotine replacement therapies, such as transdermal nicotine patches, have become widely distributed and used. While the pharmacokinetics of transdermal nicotine have been extensively described using noncompartmental methods, there are few data available describing the between-subject variability in transdermal nicotine pharmacokinetics. The aim of this investigation was to use population pharmacokinetic techniques to describe this variability, particularly as it pertains to the absorption of nicotine from the transdermal patch. METHODS: A population pharmacokinetic parent-metabolite model was developed using plasma concentrations from 25 participants treated with transdermal nicotine. Covariates tested in this model included: body weight, body mass index, body surface area (calculated using the Mosteller equation) and sex. RESULTS: Nicotine pharmacokinetics were best described with a one-compartment model with absorption based on a Weibull distribution and first-order elimination and a single compartment for the major metabolite, cotinine. Body weight was a significant covariate on apparent volume of distribution of nicotine (exponential scaling factor 1.42). After the inclusion of body weight in the model, no other covariates were significant. CONCLUSIONS: This is the first population pharmacokinetic model to describe the absorption and disposition of transdermal nicotine and its metabolism to cotinine and the pharmacokinetic variability between individuals who were administered the patch.

Similar social attention, physiological arousal, and familiarity effect in autistic and neurotypical children: A real-life recreational eye-tracking paradigm.

Social attention is reported to be crucial for the development of social skills, and, according to the social cognitive developmental theory, is fostered by social interactions. Autism is of central importance to the study of social attention, as autism is characterized by atypical social interactions and low social attention, both linked according to the social motivation theory to diminished social interest. Much evidence for positing low social interest in autism comes from eye-tracking studies, which, however, lack ecological validity. Our study documents social attention and physiological arousal, within close to real-life settings, in autistic children, as well as in their neurotypical peers, matched on gender and mental or chronological age. To explore the potential influence of partner familiarity or of the conversational topic, children gaze and electrodermal activity were recorded while they engaged in watercolor activities with, first a familiar and, next, an unfamiliar adult experimenter, who both introduced various topics. Autistic and neurotypical children exhibited comparable attention to their partners' eyes. Notably, across all groups, heightened visual attention was directed to familiar rather than unfamiliar partners. Moreover, parallel arousal patterns emerged, with all children displaying increased skin conductance responses during more engaging topics and when looking at their interactional partner's eyes. These findings underscore the task- and context-dependent nature of social attention and highlight the role of familiarity in an ecologically valid context. The absence of group differences challenges the universality of the social cognitive developmental theory and questions the scope of the social motivation theory of autism. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Navigating Transitions in Oncology Care: From Emergency Department to Outpatient Clinic.

OBJECTIVE: This quality improvement project was a collaborative effort with Penn Medicine's emergency department (ED) and oncology nurse navigators (ONNs). The goal of the project was to streamline patient transitions from the ED to the outpatient oncology clinic by developing a standardized referral process. The main objectives were to simplify and automate the referral process using the electronic medical record, improve multidisciplinary communication across the care continuum, ensure timely follow-up, and address barriers to oncology care. METHODS: The ED providers placed a consult to ONNs. The ONNs reached out to the patient within 48 hours of the consult. They maintained a database of patient referrals and collected information such as patient demographics, reason for referral, insurance, and patient outcomes. RESULTS: The ED providers referred 204 patients to the ONNs from April 2022 to September 2023. The development of a standardized referral process from the ED to the outpatient oncology clinic proved successful. Of the patients referred, the ONNs facilitated 98 cancer diagnoses and 80 of those patients are receiving oncology care at Penn Medicine. The median time to the patient's first appointments was seven days, diagnosis was 15 days, and treatment initiation occurred within 32 days. CONCLUSION: The project team achieved their goal of facilitating timely access to oncology care, ensuring continuity, and addressing patient-specific barriers. IMPLICATIONS FOR NURSING PRACTICE: This quality improvement initiative highlights the ONNs' role in enhancing access and equity in cancer care delivery. The success of the project underscores the ONN's expertise and leadership in addressing healthcare disparities in oncology care. Collaboratively, the teams created a new referral workflow improving care transitions from the ED to the outpatient oncology clinic. The project sets a precedent for optimizing patient care transitions, demonstrating the positive impact of ONNs as key members of the multidisciplinary healthcare team.

Academic Nurse Navigation: Unique Aspects and Strategies for Success.

The role and competencies of the oncology nurse navigator (ONN) are well defined and emphasize education, communication, and collaboration to support patients, families, and caregivers in overcoming barriers within healthcare systems. The authors describe the operationalization of the ONN role within a large academic health system and present a case study that illustrates the work of a team of navigators. The importance of co-navigating individuals with their community-based navigators and healthcare teams is presented, emphasizing the significance of interprofessional collaboration to support continuity of care for individuals with cancer.

Validation of an enzyme-linked immunosorbent assay screening method and a liquid chromatography-tandem mass spectrometry confirmation method for the identification and quantification of ketamine and norketamine in urine samples from Malaysia.

An ELISA and a liquid chromatography-tandem mass spectrometry (LC-MS-MS) confirmation method were developed and validated for the identification and quantitation of ketamine and its major metabolite norketamine in urine samples. The Neogen ketamine microplate ELISA was optimized with respect to sample and enzyme conjugate volumes and the sample preincubation time before addition of the enzyme conjugate. The ELISA kit was validated to include an assessment of the dose-response curve, intra- and interday precision, limit of detection (LOD), and cross-reactivity. The sensitivity and specificity were calculated by comparison to the results from the validated LC-MS-MS confirmation method. An LC-MS-MS method was developed and validated with respect to LOD, lower limit of quantitation (LLOQ), linearity, recovery, intra- and interday precision, and matrix effects. The ELISA dose-response curve was a typical S-shaped binding curve, with a linear portion of the graph observed between 25 and 500 ng/mL for ketamine. The cross-reactivity of 200 ng/mL norketamine to ketamine was 2.1%, and no cross-reactivity was detected with 13 common drugs tested at 10,000 ng/mL. The ELISA LOD was calculated to be 5 ng/mL. Both intra- (n = 10) and interday (n = 50) precisions were below 5.0% at 25 ng/mL. The LOD for ketamine and norketamine was calculated statistically to be 0.6 ng/mL. The LLOQ values were also calculated statistically and were 1.9 ng/mL and 2.1 ng/mL for ketamine and norketamine, respectively. The test linearity was 0-1200 ng/mL with correlation coefficient (R(2)) > 0.99 for both analytes. Recoveries at 50, 500, and 1000 ng/mL range from 97.9% to 113.3%. Intra- (n = 5) and interday (n = 25) precisions between extracts for ketamine and norketamine were excellent (< 10%). Matrix effects analysis showed an average ion suppression of 5.7% for ketamine and an average ion enhancement of 13.0% for norketamine for urine samples collected from six individuals. A comparison of ELISA and LC-MS-MS results demonstrated a sensitivity, specificity, and efficiency of 100%. These results indicated that a cutoff value of 25 ng/mL ketamine in the ELISA screen is particularly suitable and reliable for urine testing in a forensic toxicology setting. Furthermore, both ketamine and norketamine were detected in all 34 urine samples collected from individuals socializing in pubs by the Royal Malaysian Police. Ketamine concentrations detected by LC-MS-MS ranged from 22 to 31,670 ng/mL, and norketamine concentrations ranged from 25 to 10,990 ng/mL. The concentrations of ketamine and norketamine detected in the samples are most ikely indicative of ketamine abuse.

Simultaneous detection and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates in hair by LC-ESI-MS-MS using a single extraction method.

A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous identification and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates from hair using a single extraction method. As part of the method development, Gemini C18, Synergi Hydro RP, and Zorbax Stablebond-Phenyl LC columns were tested with three different mobile phases. Analyte recovery and limit of detection were evaluated for two different solid-phase extraction methods that used Bond Elut Certify and Clean Screen cartridges. Phosphate buffer (pH 5.0) was chosen as the optimum hair incubation medium because of the high stability of cocaine and 6-monoacetylmorphine using this method and faster sample preparation. The optimized method was fully validated. Linearity was established over the concentration range 0.2-10 ng/mg hair, and the correlation coefficients were all greater than 0.99. Total extraction recoveries were greater than 76%, detection limits were between 0.02 and 0.09 ng/mg, and the intra- and interday imprecisions were generally less than 20% in spiked hair. The intra- and interbatch imprecision of the method for a pooled authentic hair sample ranged from 1.4 to 23.4% relative standard deviation (RSD) and 8.3 to 25.4% RSD, respectively, for representative analytes from the different drug groups. The percent matrix effect ranged from 63.5 to 135.6%, with most analytes demonstrating ion suppression. Sixteen postmortem samples collected from suspected drug-related deaths were analyzed for the 17 drugs of abuse and metabolites included in the method. The method was sufficiently sensitive and specific for the analysis of drugs and metabolites in postmortem hair samples. There is scope for the inclusion of other target drugs and metabolites in the method.

Neuro-Oncology Nurse Navigation: Developing the Role for a Unique Patient Population.

Navigation seeks to assess and address barriers that hinder a patient's access to care. With the success of the nurse navigation program at Penn Medicine's Abramson Cancer Center, leadership expanded navigation to neuro-oncology. The purpose of this article is to describe this population's unique needs and the effect of nurse navigation. Although the navigation role maintains integrity with regard to scope of practice, specialized navigation strategies are tailored to the neuro-oncology population and are different from other disease sites.

Global demographic history of human populations inferred from whole mitochondrial genomes.

The Neolithic transition has led to marked increases in census population sizes across the world, as recorded by a rich archaeological record. However, previous attempts to detect such changes using genetic markers, especially mitochondrial DNA (mtDNA), have mostly been unsuccessful. We use complete mtDNA genomes from over 1700 individuals, from the 1000 Genomes Project Phase 3, to explore changes in populations sizes in five populations for each of four major geographical regions, using a sophisticated coalescent-based Bayesian method (extended Bayesian skyline plots) and mutation rates calibrated with ancient DNA. Despite the power and sophistication of our analysis, we fail to find size changes that correspond to the Neolithic transitions of the study populations. However, we do detect a number of size changes, which tend to be replicated in most populations within each region. These changes are mostly much older than the Neolithic transition and could reflect either population expansion or changes in population structure. Given the amount of migration and population mixing that occurred after these ancient signals were generated, we caution that modern populations will often carry ghost signals of demographic events that occurred far away from their current location.

Validation of the Immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine.

The purpose of this study was to validate the Immunalysis Buprenorphine Microplate enzyme-linked immunosorbent assay (ELISA) for the detection of buprenorphine in urine samples. Sixty-nine urine samples were obtained from volunteers on the Subutex treatment program and from routine samples submitted to the laboratory for buprenorphine testing. For ELISA analysis, samples were diluted 1:10 with K(2)HPO(4) (0.1M, pH 7.0). The limit of detection was calculated as 0.5 ng/mL buprenorphine. The intra-assay and interday precision was 3.8% (n = 10) and 8.6% (n = 50) respectively at 1 ng/mL buprenorphine. At a low concentration of norbuprenorphine (1 ng/mL), the immunoassay demonstrated a cross-reactivity of 78%. A higher cross-reactivity of 116% was observed at a higher concentration of norbuprenorphine (10 ng/mL). Dihydrocodeine, codeine, tramadol, morphine, propoxyphene, methadone, and EDDP were tested at concentrations of 10 ng/mL and 10,000 ng/mL and demonstrated no cross-reactivity with the assay. For liquid chromatography-tandem mass spectrometry (LC-MS-MS), deuterated internal standard mixture, 1M acetate buffer (pH 5.0), and b-glucuronidase were added to the standards and samples, which were then incubated for 3 h at 60 degrees C. After incubation, 3 mL K(2)HPO(4) (0.1M, pH 6.0) was added and the pH altered to pH 6.0 using 1M KOH. Buprenorphine and norbuprenorphine were subsequently extracted by solid-phase. Twenty-one samples were confirmed positive and 48 samples were confirmed negative by LC-MS-MS. Using a cut-off value of 0.5 ng/mL buprenorphine, the immunoassay demonstrated a sensitivity and specificity of 100%.

Detection of benzodiazepines in hair using ELISA and LC-ESI-MS-MS.

This study was designed to validate an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of nine benzodiazepines in hair. Sixteen hair case samples were tested from drug-related deaths where a positive benzodiazepine blood result was obtained. The case samples were decontaminated with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h and then neutralized with dibasic phosphate buffer. They were diluted 1:5 with phosphate buffer saline (PBS) prior to analysis. For LC-MS-MS, the samples were incubated overnight in methanol/25% ammonium hydroxide (20:1). The benzodiazepines were extracted by solid phase. Thirteen samples were confirmed positive by LC-MS-MS. The benzodiazepines detected included diazepam, nordiazepam, temazepam, oxazepam, nitrazepam, and lorazepam. Using a cut-off concentration of 0.1 ng/mg oxazepam, the Immunalysis Benzodiazepine Microplate ELISA demonstrated a sensitivity and specificity of 100% and 81%, respectively, compared with LC-MS-MS results.

Validation of the immunalysis microplate ELISA for the detection of methamphetamine in hair.

The object of this study was to validate the Immunalysis Methamphetamine Microplate ELISA for detecting methamphetamine in hair. Twenty-nine scalp hair samples were obtained as routine cases submitted to the National Institute of Scientific Investigation in Seoul by the police. The hair samples were washed with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. The samples were screened using the Immunalysis Methamphetamine Microplate ELISA and confirmed using gas chromatography-mass spectrometry (GC-MS). Twenty-eight hair samples were screened and confirmed as positive for methamphetamine. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h at 60 degrees C. For GC-MS, the samples were extracted for 20 h in methanol containing 1% hydrochloric acid. The methanol/acid solution was evaporated to dryness and the resulting residue was derivatized with trifluoroacetic anhydride. Methamphetamine and amphetamine were detected using selective ion monitoring (SIM) mode. The Immunalysis Methamphetamine Microplate ELISA demonstrated a sensitivity and specificity of 97% and 100%, respectively, using a cut-off concentration of 0.5 ng/mg d-methamphetamine. The ELISA kit showed 63% cross-reactivity with d,l-methamphetamine and did not cross-react to any significant extent with the licit l-methamphetamine isomer. The intra- and interassay precisions were 2.5% and 3.7%, respectively.

A comparative evaluation of self-report and biological measures of cigarette use in nondaily smokers.

A large subset of individuals who smoke cigarettes do not smoke regularly, but the assessments used to collect data on cigarette consumption in nondaily smokers have not been rigorously evaluated. The current study examined several self-report and biomarker approaches to the assessment of cigarette use in a sample of nondaily smokers (n = 176). Participants were randomly assigned to a daily monitoring condition (n = 89), requiring a daily report of the number of cigarettes smoked in the previous 24 hours, or a no monitoring condition (n = 87). Number of cigarettes smoked over the first 28 days of the study was assessed using 2 quantity frequency measures, a graduated frequency measure, and a timeline follow back (TLFB) interview at the Session 5 study visit. Hair nicotine (NIC), hair cotinine (COT), and expired-air carbon monoxide (CO) were collected from each participant. Total cigarettes reported via daily report were strongly correlated with all Session 5 measures of total cigarettes, but were most strongly associated with TLFB total cigarettes. Collapsed CO across 5 sessions was the biomarker most strongly correlated with daily report total cigarettes. The results support the use of daily report and TLFB methods of assessing cigarette use in nondaily smokers. Results also support the use of CO as appropriate biological markers of exposure in nondaily smokers, and point to some limitations in the use of hair biomarkers in this population. (PsycINFO Database Record

Simultaneous quantification of nicotine and metabolites in rat brain by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of nicotine (NIC), cotinine (COT), nornicotine (NNIC), norcotinine (NCOT), nicotine-N-ß-D-glucuronide (NIC GLUC), cotinine-N-ß-D-glucuronide (COT GLUC), nicotine-1'-oxide (NNO), cotinine-N-oxide (CNO), trans-3'-hydroxycotinine (3-HC), anabasine (AB) and anatabine (AT) was modified and validated for quantification of these selected analytes in rat brain tissue. This analytical method provides support for preclinical NIC pharmacokinetic and toxicological studies after controlled dosing protocols. After brain homogenization and solid-phase extraction, target analytes and corresponding deuterated internal standards were chromatographically separated on a Discovery(®) HS F5 HPLC column with gradient elution and analyzed by LC-MS/MS in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. Method linearity was assessed and calibration curves were determined over the following ranges: 0.1-7.5 ng/mg for NIC, COT GLUC and AB; and 0.025-7.5 ng/mg for COT, NNIC, NCOT, NIC GLUC, NNO, CNO, 3-HC and AT (R(2)≥0.99 for all analytes). Extraction recoveries ranged from 64% to 115%, LC-MS/MS matrix effects were ≤21%, and overall process efficiency ranged from 57% to 93% at low and high quality control concentrations. Intra- and inter-assay imprecisions and accuracy for all analytes were ≤12.9% and ≥86%, respectively. The method was successfully applied to quantification of NIC and metabolites in the brain of post-natal day 90 rats that were sacrificed 2-h after a single 0.8 mg/kg s.c. administration of (-)NIC. In these tissues, striatal concentrations were 204.8±49.4, 138.2±14.2 and 36.1±6.1 pg/mg of NIC, COT and NNIC, respectively. Concentrations of NIC, COT and NNIC in the remaining whole brain (RWhB) were 183.3±68.0, 130.0±14.1 and 46.7±10.3 pg/mg, respectively. Quantification of these same analytes in plasma was also performed by a previously validated method. NIC, COT, NNIC, NCOT, NNO and CNO were detected in plasma with concentrations comparable to those reported in previous studies. However, and in contrast to brain tissues, COT concentrations in plasma were significantly higher than were those of NIC (194.6±18.6 ng/mL versus 52.7±12.9 ng/mL). Taken together, these results demonstrate that a sensitive and selective method has been developed for the determination of NIC biomarkers in rat brain.

Validation of a liquid chromatography-tandem mass spectrometry method for the detection of nicotine biomarkers in hair and an evaluation of wash procedures for removal of environmental nicotine.

The aim of this exploratory study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the quantification of nicotine, eight nicotine metabolites, and two minor tobacco alkaloids in fortified analyte-free hair and subsequently apply this method to hair samples collected from active smokers. An additional aim of the study was to include an evaluation of different wash procedures for the effective removal of environmentally deposited nicotine from tobacco smoke. An apparatus was designed for the purpose of exposing analyte-free hair to environmental tobacco smoke in order to deposit nicotine onto the hair surface. A shampoo/water wash procedure was identified as the most effective means of removing nicotine. This wash procedure was utilized for a comparison of washed and unwashed heavy smoker hair samples. Analytes and corresponding deuterated internal standards were extracted using a cation-exchange solid-phase cartridge. LC-MS-MS was carried out using an Acquity™ UPLC(®) system (Waters) and a Quattro Premier XE™ triple quadrupole MS (Waters) operated in electrospray positive ionization mode, with multiple reaction monitoring data acquisition. The developed method was applied to hair samples collected from heavy smokers (n = 3) and low-level smokers (n = 3) collected through IRB-approved protocols. Nicotine, cotinine, and nornicotine were quantified in both the washed and unwashed hair samples collected from three heavy smokers, whereas 3-hydroxycotinine was quantified in only one unwashed sample and nicotine-1'-oxide in the washed and unwashed hair samples from two heavy smokers. In contrast, nicotine-1'-oxide was quantified in one of the three low-level smoker samples; nicotine was quantified in the other two low-level smoker samples. No other analytes were detected in the hair of the three low-level smokers.

A novel validated procedure for the determination of nicotine, eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry.

A novel validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-beta-D-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1'-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-beta-D-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC-MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R(2)) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged between 52-88% in plasma and 51-118% in urine. The limits of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL, respectively, with the exception of cotinine-N-beta-D-glucuronide, which was 50 ng/mL. Intra-day and inter-day imprecision were < or = 14% and < or = 17%, respectively. Matrix effect (%) was sufficiently minimized to < or = 19% for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze-thaw cycles, 24 h at room temperature, 24 h in the refrigerator (4 degrees C) and 1 week in the freezer (-20 degrees C). Reconstituted plasma and urine extracts were stable for at least 72 h storage in the liquid chromatography autosampler at 4 degrees C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1'-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrollment) and on the pharmacokinetic study day.