Validation of a quantitative liquid chromatography/hybrid quadrupole-linear ion trap MS/MS method for carfentanil and caffeine in a live human epidermis model.

RATIONALE: Carfentanil, an opioid 10K times more potent than morphine, has no licit clinical use. A powerful CNS depressant, it has been identified increasingly as the cause of overdose death in the United States. Because it is highly lipophilic, the law enforcement and medical communities have been concerned that responding personnel could be percutaneously exposed and that exposure could be enhanced with the use of alcohol-containing hand sanitizers. An LC-MS/MS method was developed to evaluate solvent effects on percutaneous absorption of carfentanil in a live human epidermal model. METHODS: In this study, a quantitative liquid chromatography/hybrid triple quadrupole-linear ion trap method was developed for carfentanil and for caffeine, a molecule routinely used to monitor epidermal cell culture viability. The method employed reverse-phase liquid chromatography coupled with positive electrospray ionization and multiple reaction monitoring (MRM) to quantify carfentanil and caffeine against calibration curves formulated from authentic standards. Limits of detection (LOD) for the two compounds were determined using 10:1 signal-to-noise requirements for all product ions with relative peak areas within ±20% of those observed for a mid-level calibrator. Precision and accuracy were determined by analyzing positive controls formulated in quintuplicate, by a different analyst, at three concentrations bracketing the method dynamic range. Inter-day precision was evaluated using data collected from three separate days of analyses. RESULTS: Calibration curves for seven formulated replicates of the two compounds met linearity requirements over at least four orders of magnitude concentration range. The accuracy of measured concentration results was within ±20% of the actual, precision across results (%CV) was ≤15%, curve coefficients of determination (r) were ≥0.980 (correlation coefficient r > 0.990), and relative ion ratios of all qualifier ions were within ±20% of those for a mid-level standard. Limits of quantification (LOQs) for carfentanil and caffeine were 230 pg/ml and 12 ng/ml, respectively. Intra-day accuracies (mean concentrations) for carfentanil and caffeine ranged from 90.1% to 100.8% and from 87.1% to 108.9%, respectively; inter-day accuracies ranged from 98.7% to 100.4% and from 97.5% to 101.7%, respectively. Intra-day precision (%CV) over the dynamic range ranged from 1.31 to 8.88 and from 1.49 to 6.72 for carfentanil and caffeine, respectively. Inter-day precision ranged from 4.7 to 9.9 %CV for carfentanil and 7.6-121 %CV for caffeine. CONCLUSIONS: The method was used to evaluate the percutaneous absorption kinetics of carfentanil in solution as a function of solvent composition using an in vitro, live human epidermis model. Counterintuitively, as previously reported, the addition of organic solvents to the formulations decreased rather than increased the percutaneous absorption rate of the ultra-potent opioid, carfentanil.

Rapid Denaturing Organic Digestion Method for Targeted Protein Identification and Characterization.

Bottom-up mass spectrometry-based protein analysis methods employing protease digestion are routinely used to identify and characterize proteins with high specificity and sensitivity. Method performance is generally measured by sequence coverage capability and the total number of characteristic peptides identified, when compared to predicted databases. Limitations to commonly used solvent-based digestion methods currently employed include long digestion times (18-24 h or more), leading to protease autolysis, which also precludes automation, decreases sensitivity, and increases both intra- and inter-day performance variability. This report describes the development and validation of a simple, 5 min tryptic denaturing organic digestion (DOD) method for use with tandem mass spectrometry in bottom-up protein identification and characterization. It has been evaluated across select protein toxins and diagnostic clinical protein targets, substantially improving digestion performance when compared to other solution-based and enzyme-immobilized methods. The method was compared to two currently used bottom-up methods, the 24 h filter-aided sample prep (FASP) and Flash Digest (1 and 4 h) methods. Single proteins used to compare the methods included the ricin light chain, ricin heavy chain, ricin holotoxin, serotype A Clostridium botulinum toxin, Staphylococcus enterotoxin B, ribonuclease A, and thyroglobulin. In tests, across the proteins investigated, the 5 min DOD digestion method resulted in sequence coverages ranging from 55 to 100%, with relatively high reproducibility and precision; results were better than or equal to FASP method results and were greatly enhanced when compared to Flash method results. Importantly, DOD method intra- and inter-day precision was much improved as compared to results for both FASP and Flash digestions. These data indicated that the DOD method, when compared to the FASP and Flash Digest methods, dramatically reduced digestion time, while maintaining or improving the ability to detect and characterize targeted proteins, and reduced analytical variability for tryptic digestion, resulting in markedly faster and more precise analyses.

In vitro dermal absorption of carfentanil.

There is growing concern regarding potential occupational exposures to the ultra-potent synthetic opioid carfentanil. However, little data are available on the toxicity of carfentanil in humans, particularly for dermal exposures. To begin to address this, permeation of carfentanil formulated in three vehicles, water, ethanol, and hand sanitizer was measured under infinite-dose conditions in an in vitro static diffusion cell system using the EpiDerm™ (EPI-606-X) RhE model. The permeation rate was fastest for carfentanil in water (3.9 × 10-3 cm/h), followed by hand sanitizer (1.2 × 10-3 cm/h), and slowest for carfentanil in ethanol (0.2 × 10-3 cm/h). In both ethanol and hand sanitizer, a lag-time between exposure and permeation of approximately 1.5 h was observed, while lag-time in water was approximately half an hour. Flux at steady-state was greater at 50.6 µg/ml than at 5.3 µg/ml for both water and ethanol; however, the percent of dose absorbed did not differ between doses for either vehicle. Slight differences in percutaneous permeation of carfentanil were observed between two brands of hand sanitizer, likely due to differences in relative proportion of alcohol and skin penetration enhancers. These data indicate that small skin exposures may not result in rapid, significant toxicity as previously reported.

Engineering Botulinum Neurotoxin C1 as a Molecular Vehicle for Intra-Neuronal Drug Delivery.

Botulinum neurotoxin (BoNT) binds to and internalizes its light chain into presynaptic compartments with exquisite specificity. While the native toxin is extremely lethal, bioengineering of BoNT has the potential to eliminate toxicity without disrupting neuron-specific targeting, thereby creating a molecular vehicle capable of delivering therapeutic cargo into the neuronal cytosol. Building upon previous work, we have developed an atoxic derivative (ad) of BoNT/C1 through rationally designed amino acid substitutions in the metalloprotease domain of wild type (wt) BoNT/C1. To test if BoNT/C1 ad retains neuron-specific targeting without concomitant toxic host responses, we evaluated the localization, activity, and toxicity of BoNT/C1 ad in vitro and in vivo. In neuronal cultures, BoNT/C1 ad light chain is rapidly internalized into presynaptic compartments, but does not cleave SNARE proteins nor impair spontaneous neurotransmitter release. In mice, systemic administration resulted in the specific co-localization of BoNT/C1 ad with diaphragmatic motor nerve terminals. The mouse LD50 of BoNT/C1 ad is 5 mg/kg, with transient neurological symptoms emerging at sub-lethal doses. Given the low toxicity and highly specific neuron-targeting properties of BoNT/C1 ad, these data suggest that BoNT/C1 ad can be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm.

The Pesticide Metabolites Paraoxon and Malaoxon Induce Cellular Death by Different Mechanisms in Cultured Human Pulmonary Cells.

Organophosphorus (OP) pesticides are known to induce pulmonary toxicity in both humans and experimental animals. To elucidate the mechanism of OP-induced cytotoxicity, we examined the effects of parathion and malathion and their respective metabolites, paraoxon and malaoxon, on primary cultured human large and small airway cells. Exposure to paraoxon and malaoxon produced a dose-dependent increase in cytotoxicity following a 24-hour exposure, while treatment with parathion or malathion produced no effects at clinically relevant concentrations. Exposure to paraoxon-induced caspase activation, but malaoxon failed to induce this response. Since caspases have a major role in the regulation of apoptosis and cell death, we evaluated OP-induced cell death in the presence of a caspase inhibitor. Pharmacological caspase inhibition protected against paraoxon-induced cell death but not malaoxon-induced cell death. These data suggest that caspase activation is a key signaling element in paraoxon-induced cell death, but not malaoxon-induced cellular death in the pulmonary epithelium.

Fragmentation pathways and structural characterization of 14 nerve agent compounds by electrospray ionization tandem mass spectrometry.

Organophosphate nerve agents (OPNAs) are some of the most widely used and proliferated chemical warfare agents. As evidenced by recent events in Syria, these compounds remain a serious military and terrorist threat to human health because of their toxicity and the ease with which they can be used, produced and stored. There are over 2,000 known, scheduled compounds derived from common parent structures with many more possible. To address medical, forensic, attribution, remediation and other requirements, laboratory systems have been established to provide the capability to analyze 'unknown' samples for the presence of these compounds. Liquid chromatography/mass spectrometric methods have been validated and are routinely used in the analysis of samples for a very limited number of these compounds, but limited data exist characterizing the electrospray ionization (ESI) and mass spectrometric fragmentation pathways of the compound families. This report describes results from direct infusion ESI/MS, ESI/MS(2) and ESI/MS(3) analysis of 14 G and V agents, the major OPNA families, using an AB Sciex 4000 QTrap. Using a range of conditions, spectra were acquired and characteristic fragments identified. The results demonstrated that the reproducible and predictable fragmentation of these compounds by ESI/MS, ESI/MS(2) and ESI/MS(3) can be used to describe systematic fragmentation pathways specific to compound structural class. These fragmentation pathways, in turn, may be useful as a predictive tool in the analysis of samples by screening and confirmatory laboratories to identify related compounds for which authentic standards are not readily available.

Chemical warfare agent and biological toxin-induced pulmonary toxicity: could stem cells provide potential therapies?

Chemical warfare agents (CWAs) as well as biological toxins present a significant inhalation injury risk to both deployed warfighters and civilian targets of terrorist attacks. Inhalation of many CWAs and biological toxins can induce severe pulmonary toxicity leading to the development of acute lung injury (ALI) as well as acute respiratory distress syndrome (ARDS). The therapeutic options currently used to treat these conditions are very limited and mortality rates remain high. Recent evidence suggests that human stem cells may provide significant therapeutic options for ALI and ARDS in the near future. The threat posed by CWAs and biological toxins for both civilian populations and military personnel is growing, thus understanding the mechanisms of toxicity and potential therapies is critical. This review will outline the pulmonary toxic effects of some of the most common CWAs and biological toxins as well as the potential role of stem cells in treating these types of toxic lung injuries.

Measurement of trace explosive residues in a surrogate operational environment: implications for tactical use of chemical sensing in C-IED operations.

A campaign to measure the amount of trace explosive residues in an operational military environment was conducted on May 27-31, 2007, at the National Training Center at Fort Irwin, CA, USA. The objectives of this campaign were to develop the methods needed to collect and analyze samples from tactical military settings, to use the data obtained to determine what the trace explosive signatures suggest about the potential capabilities of chemical-based means to detect IEDs, and, finally, to present a framework whereby a sound understanding of the signature science can be used to guide development of new sensing technologies and sensor concepts of operation. Through our use of combined background and threat signature data, we have performed statistical analyses to estimate upper limits of notional sensor performance that is limited only by the spatial correlation of the signature chemicals to the threats of interest.

Positive identification of the principal component of a white powder as scopolamine by quantitative one-dimensional and two-dimensional NMR techniques.

An unidentified white powder collected as evidence in an intelligence investigation was characterized exclusively by nuclear magnetic resonance (NMR) analysis. A small fraction of the powder dissolved in D2O was subjected to a series of one- and two-dimensional techniques which were used to elucidate the molecular structure of the powder's major component and positively identify it as the scopolamine biotoxin. Quantitative one-dimensional experiments identified individual proton and carbon atom sites, and conventional 14N spectroscopy detected a single nitrogen atom site. Heteronuclear single quantum coherence data correlated all protons to their directly bonded carbon atom, and together with the quantitative spectra, were used to determine the number of protons directly bonded to each carbon atom. The presence of a methyl, carboxyl, and a benzyl group was also identified from these data. Correlation spectroscopy detected a three proton and a nine proton JH,H network, representing a CH2CH moiety and seven carbon atom ring, respectively. These five elements were assembled into an almost complete molecular structure by using long-range, J-coupled, 1H-13C pairs detected by heteronuclear multiple bond correlation (HMBC) spectroscopy and 1H-1H dipolar-coupled pairs found from nuclear Overhauser effect spectroscopy (NOESY) data. Additional oxygen atom sites were inferred from 1H-13C correlation intensities in the HMBC spectra along with 1H and 13C chemical shift values, or directly from NOESY correlations. Only a single oxygen atom site could not be inferred from NMR data, but its presence was inferred from comparisons to target analyte structures to complete the structure of the scopolamine molecule. To confirm these results, an ethanol/H2O solution of the powder was analyzed by direct infusion into an ion trap mass spectrometer. A prominent base signal was observed at m/z 304.1 amu, corresponding to the protonated molecular ion of scopolamine. Subsequently, the ion was selected and subjected to collision-induced dissociation, producing characteristic major MS/MS fragments at m/z 138.1 and 156.1. Comparisons of 1H and 13C chemical shift values and JH,H values measured from our NMR data were found to agree very favorably with previously reported values for scopolamine in D2O.

Urinary pharmacokinetics of methamphetamine and its metabolite, amphetamine following controlled oral administration to humans.

Methamphetamine is widely abused for its euphoric effects. Our objectives were to characterize the urinary pharmacokinetics of methamphetamine and amphetamine after controlled methamphetamine administration to humans and to improve the interpretation of urine drug test results. Participants (n = 8) received 4 daily 10-mg (low) oral doses of sustained-release (d)-methamphetamine hydrochloride within 7 days. After 4 weeks, 5 participants received 4 daily 20-mg (high) oral doses. All urine specimens were collected during the study. Methamphetamine and amphetamine were measured by GC-MS/PCI. Maximum excretion rates ranged from 403 to 4919 microg/h for methamphetamine and 59 to 735 microg/h for amphetamine with no relationship between dose and excretion rate. The mean molar percentage of dose in the urine as total methamphetamine and amphetamine were 57.5 +/- 21.7% (low dose) and 40.9 +/- 8.5% (high dose). Mean urinary terminal elimination half-lives across doses were 23.6 +/- 6.6 hours for methamphetamine and 20.7 +/- 7.3 hours for amphetamine. Methamphetamine renal clearance across doses was 175 +/- 102 mL/min. The mean amphetamine/methamphetamine percentage ratio based on the area under the urinary excretion-time curve increased over time from 13.4 +/- 6.5% to 35.7 +/- 26.6%. Slow urinary excretion results in drug accumulation and increases in detection time windows. Our findings also support the presence of an active renal excretion mechanism for methamphetamine.

Urine testing for cocaine abuse: metabolic and excretion patterns following different routes of administration and methods for detection of false-negative results.

Although cocaine is typically the second-most identified drug of abuse in drug-testing programs, there is surprisingly little quantitative information on excretion patterns following different routes of administration. This report details the urinary excretion and terminal elimination kinetics for cocaine and eight metabolites [benzoylecgonine (BZE), ecgonine methylester (EME), norcocaine (NCOC), benzoylnorecgonine (BNE), m-hydroxy-BZE (m-HO-BZE), p-hydroxy-BZE (p-HO-BZE), m-hydroxy-COC (m-HO-COC), and p-hydroxy-COC (p-HO-COC)]. Six healthy males were administered approximately equipotent doses of cocaine by the intravenous (IV), smoking (SM), and inhalation (IN) routes of administration. Urine specimens were collected for a minimum of three days after drug administration, screened by immunoassay (EMIT and TDX, 300 ng/mL), and analyzed by GC-MS. Mean Cmax values were generally as follows: BZE > EME > COC > BNE approximately p-HO-BZE > m-HO-BZE > m-HO-COC > NCOC > p-HO-COC. Elimination half-lives for cocaine and metabolites were generally shorter following s.m., intermediate after i.v., and longest following i.n. administration. m-HO-BZE demonstrated the longest half-life (mean range 7.0-8.9 h), and cocaine displayed the shortest (2.4-4.0 h). Mean detection times were extended progressively by lowering cutoff concentrations. The maximum increases were approximately 55% at 50 ng/mL for the TDx assay (e.g., the detection time for the last consecutive positive changed from 32.8 h to 50.6 h for i.v. cocaine) and up to 39% for GC-MS at a cutoff concentration of 40 ng/mL (e.g., the detection time for the last consecutive positive changed from 34.8 h to 48.1 h for i.v. cocaine). Sensitivity, specificity, and predictive values for EMIT and TDx were comparable at the 300-ng/mL cutoff concentration; but at lower cutoff concentrations, predictive values of positive results for TDx were diminished indicating a higher risk of false-positive results, that is, positive results that failed to meet administrative cutoff criteria. Detection of positive results was significantly enhanced through the use of the "Zero Threshold Criteria Method", a method developed by the authors to differentiate false-negatives from true-negatives. The method was based on establishing mean immunoassay response (MIR) baselines and variance (SD) in assays of drug-free specimens. Arbitrary thresholds (MIR + 0.5 SD, MIR + 1 SD, MIR + 2 SD) were utilized to evaluate all negative specimens. Apparent true positives were identified by the presence of BZE at or above 40% GC-MS cutoff concentrations. With these criteria, up to 111 false-negative specimens were confirmed as true-positive specimens; this was in addition to the 208 true positives detected at recommended cutoff concentrations. This represents a 50% increase in positive detection rates through the use of this methodology. Such methodology is recommended for further evaluation by drug-testing programs for enhancement of positive detection rates and as an alternative to creatinine testing for dealing with dilute specimens that test negative by initial tests, but contain quantifiable concentrations of drugs of abuse.

Methamphetamine and amphetamine pharmacokinetics in oral fluid and plasma after controlled oral methamphetamine administration to human volunteers.

BACKGROUND: Methamphetamine (METH) and amphetamine (AMP) concentrations in 200 plasma and 590 oral fluid specimens were used to evaluate METH pharmacokinetics and pharmacodynamics after oral administration of sustained-release METH. METHODS: Eight participants received four oral 10-mg S-(+)-METH hydrochloride sustained-release tablets within 7 days. Three weeks later, five participants received four oral 20-mg doses. Blood samples were collected for up to 24 h and oral fluid for up to 72 h after drug administration. RESULTS: After the first oral dose, initial plasma METH detection was within 0.25-2 h; c(max) was 14.5-33.8 micro g/L (10 mg) and 26.2-44.3 micro g/L (20 mg) within 2-12 h. In oral fluid, METH was detected as early as 0.08-2 h; c(max) was 24.7-312.2 micro g/L (10 mg) and 75.3-321.7 micro g/L (20 mg) and occurred at 2-12 h. The median oral fluid-plasma METH concentration ratio was 2.0 across 24 h and was highly variable. Neutral cotton swab collection yielded significantly higher METH and AMP concentrations than citric acid candy-stimulated expectoration. Mean (SD) areas under the curve for AMP were 21% +/- 25% and 24% +/- 11% of those observed for METH in plasma and oral fluid, respectively. After a single low or high dose, plasma METH was >2.5 micro g/L for up to 24 h in 9 of 12 individuals (mean, 7.3 +/- 5.5 micro g/L at 24 h); in oral fluid the detection window was at least 24 h (mean, 18.8 +/- 18.0 micro g/L at 24 h). The plasma and oral fluid 24-h METH detection rates were 54% and 60%, respectively. After four administrations, METH was measurable for 36-72 h (mean, 58.3 +/- 14.5 h). CONCLUSIONS: Perceived advantages of oral fluid for verifying METH exposure compared with urine include simpler specimen collection and reduced potential for adulteration, but urine offers higher analyte concentrations and a greater window of detection.

Duration of detectable methamphetamine and amphetamine excretion in urine after controlled oral administration of methamphetamine to humans.

BACKGROUND: Confirmation of a workplace drug test requires urinary methamphetamine (MAMP) and amphetamine (AMP) concentrations > or = 500 and 200 micro g/L, respectively, but cutoffs at half those values (250/100 micro g/L) have been proposed. We determined the urinary excretion of MAMP after oral ingestion and examined the effect of using lower cutoffs on detection of exposure. METHODS: Volunteers (n = 8) ingested four 10-mg doses of MAMP. HCl daily over 7 days, and five of them ingested four 20-mg doses 4 weeks later. After ingestion, the volunteers collected all urine specimens for 2 weeks. After solid-phase extraction, MAMP and AMP were measured by gas chromatography-positive chemical ionization mass spectrometry with dual silyl derivatization. RESULTS: MAMP and AMP were generally detected in the first or second void (0.7-11.3 h) collected after drug administration, with concentrations of 82-1827 and 12-180 micro g/L, respectively. Peak MAMP concentrations (1871-6004 micro g/L) after single doses occurred within 1.5-60 h. MAMP > or = 500 micro g/L was first detected in the first or second void (1-11 h) at 524-1871 micro g/L. Lowering the MAMP cutoff to 250 micro g/L changed the initial detection time little. AMP > or = 200 micro g/L was first detected in the 2nd-13th (7-20 h) post-administration voids. At a cutoff of 100 micro g/L, AMP was first confirmed in the second to eighth void (4-13 h). Reducing the cutoff to 250/100 micro g/L extended terminal MAMP detection by up to 24 h, increased total detection time by up to 34 h, and increased the total number of positive specimens by 48%. CONCLUSIONS: At the lower cutoff, initial detection times are earlier, detection windows are longer, and confirmation rates are increased. Elimination of the AMP requirement would increase detection rates and allow earlier detection.

Plasma and oral fluid pharmacokinetics and pharmacodynamics after oral codeine administration.

BACKGROUND: The ease, noninvasiveness, and safety of oral fluid collection have increased the use of this alternative matrix for drugs-of-abuse testing; however, few controlled drug administration data are available to aid in the interpretation of oral fluid results. METHODS: Single oral codeine doses (60 and 120 mg/70 kg) were administered to 19 volunteers. Oral fluid and plasma were analyzed for free codeine, norcodeine, morphine, and normorphine by solid-phase extraction combined with gas chromatography-mass spectrometry (SPE/GC-MS). Physiologic and subjective effects were examined. RESULTS: Mean (SE) peak codeine concentrations were 214.2 +/- 27.6 and 474.3 +/- 77.0 micro g/L in plasma and 638.4 +/- 64.4 and 1599.3 +/- 241.0 micro g/L in oral fluid. The oral fluid-to-plasma ratio for codeine was relatively constant ( approximately 4) from 1 to 12 h. The mean half-life (t(1/2)) of codeine was 2.2 +/- 0.10 h in plasma and 2.2 +/- 0.16 h in oral fluid. Significant dose-related miosis and increases in sedation, psychotomimetic effect, and "high" occurred after the high dose. Mean codeine oral fluid detection time was 21 h with a 2.5 microg/L cutoff, longer than that of plasma (12-16 h). Detection times with the proposed Substance Abuse and Mental Health Services Administration cutoff (40 microg/L) were only 7 h. Norcodeine, but not morphine or normorphine, was quantified in both plasma and oral fluid. CONCLUSIONS: The disposition of codeine over time was similar in plasma and oral fluid, but because of high variability, oral fluid codeine concentrations did not reliably predict concurrent plasma concentrations. Oral fluid testing is a useful alternative matrix for monitoring codeine exposure with a detection window of 7-21 h for single doses, depending on cutoff concentrations. These controlled drug administration data should aid in the interpretation of oral fluid codeine results.