Biomarkers of Recent Cannabis Use in Blood, Oral Fluid and Breath.

Proving driving under the influence of cannabis (DUIC) is difficult. Establishing a biomarker of recent use to supplement behavioral observations may be a useful alternative strategy. We determined whether cannabinoid concentrations in blood, oral fluid (OF) or breath could identify use within the past 3 h-likely the period of the greatest impairment. In a randomized trial, 191 frequent (≥4/week) and occasional (<4/week) cannabis users smoked one cannabis (placebo [0.02%], or 5.9% or 13.4% Δ9-tetrahydrocannabinol [THC]) cigarette ad libitum. Blood, OF and breath samples were collected prior to and up to 6 h after smoking. Samples were analyzed for 10 cannabinoids in OF, 8 in blood and THC in breath. Frequent users had more residual THC in blood and were more likely to be categorized as 'recently used' prior to smoking; this did not occur in OF. Per se limits ranging from undetectable to 5 ng/mL THC in blood offered limited usefulness as biomarkers of recent use. Cannabinol (CBN, cutoff = 1 ng/mL) in blood offered 100% specificity but only 31.4% sensitivity, resulting in 100% positive predictive value (PPV) and 94.0% negative predictive value (NPV) at 4.3% prevalence; however, CBN may vary by cannabis chemovar. A 10 ng/mL THC cutoff in OF exhibited the overall highest performance to detect its use within 3 h (99.7% specificity, 82.4% sensitivity, 92.5% PPV and 99.2% NPV) but was still detectable in 23.2% of participants ∼4.4 h post-smoking, limiting specificity at later time points. OF THC may be a helpful indicator of recent cannabis intake, but this does not equate to impairment. Behavioral assessment of impairment is still required to determine DUIC. This study only involved cannabis inhalation, and additional research evaluating alternative routes of ingestion (i.e., oral) is needed.

Neurosteroid analysis.

In this chapter, we review techniques used for the analysis of neurosteroids and discuss the advantages and disadvantages of each method. Because radioimmunoassay (RIA) procedures are well known, we focus more on the relatively recent mass spectrometric methods used for analyzing neurosteroids and their sulfates. We also discuss some promising methods that permit the detection of low levels of neurosteroids in small samples with a minimum number of sample pretreatment procedures. Lowering the limits of detection will enable a better understanding of the physiological function of neurosteroids and the mechanism(s) for neurosteroid regulation of brain function. Moreover, analyzing low levels of neurosteroids more efficiently will increase the throughput, which is important for clinical analysis. Initially, most neurosteroid analyses were performed by RIA. However, many analyses of neurosteroids are now performed by mass spectrometry. To date, the most sensitive, specific, and accurate method for the simultaneous analysis of several neurosteroids is the method of gas chromatography/electron capture/negative chemical ionization mass spectrometry. This method, with its many variants, is described in detail.

Quantification of neurosteroids in rat plasma and brain following swim stress and allopregnanolone administration using negative chemical ionization gas chromatography/mass spectrometry.

A simplified method for the quantitative analysis of neurosteroids in rat plasma and brain is described. The method uses negative chemical ionization gas chromatography/mass spectrometry and involves the synthesis of pentafluorobenzyloxime/trimethylsilyl ether derivatives with excellent chromatographic and electron-capturing properties. Deuterium-labeled analogs of the steroids of interest were synthesized and used as internal standards. The steroids (allopregnanolone, epiallopregnanolone, pregnenolone, testosterone, and dehydroepiandrosterone) were isolated from the plasma or brain matrix by a rapid and straightforward solid-phase extraction procedure. The mass spectrometer was operated in a selective ion monitoring mode, allowing for picograms of neurosteroids to be quantified from biological extracts. The method was linear (typical R(2) = 0.999) over the concentration range (100 to 8000 pg from 0.3 ml plasma and 250 to 8000 pg from 100 mg brain tissue) with good precision and accuracy. In experimental protocols, the procedure was suitable for measuring concentrations of endogenous neurosteroids in rat plasma and brain. Significant elevations (P < 0.001) were observed in the frontal cortex for allopregnanolone and pregnenolone following a swim stress and for allopregnanolone and epiallopregnanolone following allopregnanolone injection (8 mg/kg, sc). The present method allows accurate determination of neurosteroids and will be helpful in elucidating the role of neurosteroids in health and disease.

A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.

Nonphysiological truncations of apolipoprotein (apo) B-100 cause familial hypobetalipoproteinemia (FHBL) in humans and mice. An elucidation of the mechanisms underlying the FHBL phenotypes may provide valuable information on the metabolism of apo B-containing lipoproteins and the structure-function relationship of apo B. To generate a faithful mouse model of human FHBL, a subtle mutation was introduced into the mouse apo B gene by targeting embryonic stem cells using homologous recombination followed by removal of the selection marker gene by Cre-loxP-mediated site-specific recombination. The engineered mice bear a premature stop codon at residue 1767 and a 42-base pair loxP inserted into intron 24 of the apo B gene, thus closely resembling the apo B-38.9-producing mutation in humans. Apo B-38.9 was the sole apo B protein in homozygote (apob(38.9/38.9)) plasma. In heterozygotes (apob(+/)(38. 9)), apo B-100 and apo B-48 were reduced by 75 and 40%, respectively, and apo B-38.9 represented 20% of total circulating apo B. Hepatic apo B-38.9 mRNA levels were reduced by 40%. In cultured apob(+/)(38. 9) hepatocytes, apo B-100 was produced in trace quantities, and the synthesis rate of apo B-38.9 relative to apo B-48 was reduced by 40%. However, almost equimolar amounts of apo B-38.9 and apo B-48 were secreted into the media. Pulse-chase studies revealed that apo B-38. 9 was secreted at a faster rate and more efficiently than apoB-48. Nevertheless, both apob(+/)(38.9) and apob(38.9/38.9) mice had reduced hepatic triglyceride secretion rates and fatty livers. Thus, low mRNA levels or defective secretion of apo B-38.9 may not be responsible for the FHBL phenotypes caused by the apo B-38.9 mutation. Rather, a reduced capacity of apo B-38.9 for triglyceride transport may account for the fatty livers in these mice.

Broad spectrum drug identification directly from urine, using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Currently the rate-limiting step for mass spectrometric analysis of drugs in biological samples is sample preparation. Many gas chromatography/mass spectrometry (GC/MS) methods are specific for a certain class of compounds, requiring extraction and/or derivatization before analysis. The purpose of this study was to develop a broad spectrum liquid chromatography/mass spectrometry (LC/MS) procedure that allowed for direct analysis of urine specimens with potential for quantitative analysis. METHODS: We modified a commercially available column-switching instrument, the REMEDi HS from Bio-Rad Diagnostics, to make it compatible with atmospheric pressure ionization. The system we developed was based on electrospray ionization and used three LC columns to extract, purify, and separate drugs directly from urine specimens. Drugs and metabolites were tentatively identified on the basis of retention times and (M+H)(+) ions. Tandem mass spectrometry (MS/MS) was used to confirm the qualitative identification of suspected drugs, using data-dependent acquisition. For quantitative analysis, the cocaine metabolite benzoylecgonine was analyzed using isotope dilution and selected reaction monitoring. RESULTS: Seventeen basic drugs from a variety of classes of compounds were identified directly from urine without the need for prior sample extraction, using LC and MS/MS. Quantitative analysis was demonstrated for benzoylecgonine. When benzoylecgonine-d(3) was used as the internal standard, the method was linear from 30 to 10 000 microgram/L (range tested). At these concentrations, the within-run accuracy was +/- 10% of the target concentration, with CVs <10%. Analytical results by LC/MS/MS compared favorably with GC/MS values for 50 benzoylecgonine-containing specimens and for 25 negative specimens. CONCLUSIONS: The ability to directly analyze urine for a wide variety of drug classes, combined with the sensitivity and specificity of LC/MS/MS makes this technique attractive for many clinical, forensic, and biotechnology applications.

Multisite study of a second generation whole blood rapid assay for cardiac troponin T.

We evaluated a second generation, qualitative, whole blood rapid assay for cardiac troponin T (cTnT), which employs a more cardio-specific troponin T mouse monoclonal capture antibody. Using quantitative cTnT enzyme-linked immunosorbent assay (ELISA) results as the benchmark for accuracy, we compared the performance of the second generation and the original whole blood rapid assays in 445 samples from patients with the following diagnoses, determined by medical record review: myocardial infarction, coronary bypass surgery, ischaemic heart disease, musculoskeletal disease, renal failure or other noncardiac conditions. Overall, concordance between the second generation cTnT Rapid Assay and the quantitative cTnT ELISA, compared using the McNemar test and a cut-off concentration of 0.1 microgram/L, was in the range 76-94% for each patient group. Using a receiver operating characteristic plot, the cut-off for the second generation cTnT Rapid Assay was in the range 0.06-0.08 microgram/L. We conclude that the second generation cTnT whole blood assay has a 2.5-fold lower analytical cut-off than the original rapid assay (0.2 microgram/L) and may represent a more sensitive clinical tool for the rapid triage and risk stratification of cardiac patients.

Utility of insufflation filters in laparoscopic surgery.

OBJECTIVES: To determine whether filters, regularly used as part of the insufflator tubing during laparoscopic surgery, trap microbial and particulate matter from CO2 tanks, thus preventing passage from one patient to another. METHODS: A total of 67 used filters were collected from 17 CO2 tanks and six insufflation machines at three local hospitals, and sterile unused filters were used as controls. The used filters were distributed equally and sequentially into three groups: Group I-viewed under a dissecting microscope for particulate matter; group II-examined by mass spectrometry for contamination with oils and other impurities; group III-incubated on sheep blood agar plates and evaluated for growth of microorganisms. RESULTS: Negative. Used filters were indistinguishable by all parameters from controls. CONCLUSIONS: This limited study suggests filters now used in laparoscopic surgery fail to trap microbes or particulate matter. The question remains whether tank waste is absent or these filters fail to trap waste matter.

Comparison of an ion-trap and a quadrupole mass spectrometer using diazepam as a model compound.

Recent innovations in mass spectrometry (MS) have led to the development of instruments with increased capabilities, smaller footprints, and relatively low cost. The traditional MS in most toxicology laboratories is a quadrupole system equipped with electron impact ionization. Recently, an ion trap with electron impact, positive chemical ionization, negative chemical ionization, and tandem MS capabilities was introduced by Finnigan MAT. This paper compares the sensitivity and precision of ion-ratio measurements between a Finnigan GCQ ion-trap mass spectrometer (ITMS) and a Hewlett Packard quadrupole mass spectrometer (QMS) using electron impact ionization with diazepam as the model compound. Additionally, the sensitivity and precision of ion ratio measurements are evaluated for the ITMS using positive chemical ionization, negative chemical ionization and tandem MS modes of analysis. In the full scan mode (m/z 50-650, 1 Hz), the ITMS had an average signal-to-noise ratio (S/N) of 1400 for a 2-ng injection of diazepam (10 injections per day for 5 days), within-run ion ratio precision had coefficients of variation from 5 to 11%. Using similar full scan conditions, a 10-ng injection of diazepam on the QMS had an average S/N ratio of 160, and precision of ion ratio measurements varied from 5 to 13%. In the selected ion mode (SIM) of analysis (three ions, 2 Hz), the ITMS had an average S/N of 14,000 for a 2-ng injection and ion-ratio precision ranging from 6 to 15%. Using similar SIM conditions, a 2-ng injection in the QMS had an average S/N of 3000 with ion ratio standard deviations of 0.67 to 2.9%. Overall, the ITMS provided at greater S/N, equivalent precision in full scan, but was 5- to 10-fold less precise in measuring ion ratios in the SIM mode as compared with the QMS.

Comparison of assay methods for benzodiazepines in urine. A receptor assay, two immunoassays, and gas chromatography-mass spectrometry.

We analyzed 127 urine samples for benzodiazepines by a radioreceptor assay and two immunoassays (cloned enzyme donor immunoassay [CEDIA, Boehringer Mannheim, Indianapolis, Ind] and agglutination immunoassay [ONLINE, Roche Diagnostic Systems, Branchburg, NJ]). Sixty samples that were positive by at least one of these assays were submitted to gas chromatography-mass spectrometry, which detected benzodiazepines such as oxazepam, nordiazepam, and lorazepam in 44 samples. Diagnostic sensitivity and specificity of the receptor assay were almost equal or superior to those of the two immunoassays, although discrepant results were obtained in some samples. The discrepancies resulted from differences in the cross-reactivity of these assays. For example, the receptor assay could detect lorazepam, while the two immunoassays could not, and CEDIA was interfered with by a metabolite of sertraline while the others were not. This is the first report showing the utility of a receptor assay for screening benzodiazepines in urine.

CTCL in patients under 20 years of age: a series of five cases.

The diagnosis of cutaneous T-cell lymphoma in patients under 20 years of age is extremely rare. We report five patients diagnosed before 20 years of age who illustrate the striking variations in clinical and histologic features as well as disease progression. We feel this information stresses the importance of multiple biopsies in young patients with chronic dermatoses.

Characteristics of a 20-minute whole blood rapid assay for cardiac troponin T.

OBJECTIVES: A qualitative whole blood rapid assay for cardiac troponin T (cTnT) was examined. The assay uses the same antibodies as for a benchmark ELISA, but the capture and detection roles were switched to enhance specificity. DESIGN AND METHODS: The cTnT Rapid Assay and ELISA were compared in 643 samples from patients having myocardial infarction, coronary artery bypass surgery, ischemic heart disease, musculoskeletal disease, renal failure, or other noncardiac conditions. Concordance between the methods was compared using the McNemar Test and cTnT cutoff of 0.2 micrograms/L. RESULTS: For the "cutoff" of 0.2 micrograms/L, concordance between the cTnT Rapid Assay and ELISA was in the range of 90-95% for each group except the renal failure patients, where concordance was 77.9%. There was no significant difference between the cTnT Rapid Assay and ELISA, except in the renal failure and ischemic heart disease patients, where there was a greater number (p < 0.05) of cTnT Rapid Assay negative results when the ELISA was > 0.2 micrograms/L. Overall concordance between the cTnT Rapid Assay and CK-MBmass was 78%. CONCLUSION: The McNemar test indicated that the cutoff for the cTnT Rapid Assay was 0.2 micrograms/L. Evidently the lower concordance among renal failure and ischemic heart disease patients reflects higher cTnT specificity for the Rapid Assay that was conferred by switching the capture and detection antibodies.

Improved CEDIA benzodiazepine assay eliminates sertraline crossreactivity.

Initial experiments demonstrated that the original CEDIA (cloned enzyme donor immunoassay) benzodiazepine assay crossreacted with setraline and sertraline metabolites. In response to this phenomenon, Boehringer Mannheim Corporation developed an improved CEDIA benzodiazepine assay in order to eliminate sertraline crossreactivity. The improved CEDIA assay was evaluated against the original CEDIA product, EMIT II (enzyme multiplied immunoassay technique) benzodiazepine assay, and electron capture negative chemical ionization (ECNCI) gas chromatography-mass spectrometry (GC-MS). Five hundred and thirty-one urine drug screens were tested by the immunoassays. Sensitivity and specificity of these immunoassays for the 5-aryl-7-chloro-1,4-benzodiazepine compounds were 92 and 98%, respectively, for the improved CEDIA assay; 92 and 93%, respectively, for the current CEDIA assay; and 87 and 98%, respectively, for EMIT II. The improved CEDIA assay performed almost identically to the EMIT II assay, both of which had a significant advantage over the original CEDIA product, which was subject to crossreactivity because of sertraline metabolites. The alpha-hydroxy ketone metabolites of sertraline are identified in human urine specimens for the first time using ECNCI GC-MS.

Comparison of troponin-T with other cardiac markers in a VA hospital.

Measuring protein markers of cardiac damage is important for the diagnosis of myocardial infarction (MI). This study accessed the positive and negative predictive values of cardiac markers for detecting MI and perioperative MI in cardiac surgery by evaluating: creatine kinase (CK); creatine kinase MB isoenzyme mass assay (CK-MB); lactate dehydrogenase (LDH); lactate dehydrogenase isoenzyme-1 (LDH-1); myoglobin; and cardiac troponin T (cTnT) in a Veterans Affairs Medical Center. Inclusion criterion was any patient who had a CK ordered over a 6-month period. Patient history and diagnosis were obtained from discharge summaries. The two groups of patients studied either presented with symptoms of MI (n = 370), or underwent open heart surgery (n = 63). In the patients evaluated for MI, there were 433 suspected cardiac events with 48 MIs diagnosed. Cardiac marker sensitivities and specificities were cTnT (98% and 73%), CK-MB mass (81% and 97%), CK (73% and 78%), LDH (67% and 80%), LDH-1 (33% and 95%), and myoglobin (79% and 66%). For detecting MI, the marker that provided the optimum specificity was CK-MB mass, but cTnT had the highest negative predictive value. There was one perioperative MI in the 63 cardiac surgery patients. Surgical duration and aortic cross clamp time correlated with peak cTnT and CK-MB mass concentrations, but there was a wide degree of variability for any given time period.

Cardiac troponin T is elevated in asymptomatic patients with chronic renal failure.

Patients with chronic renal failure (CRF) are at increased risk for myocardial events that are difficult to evaluate due to atypical symptoms and chronically elevated protein markers of cardiac damage. This study evaluated cardiac troponin T (cTnT), a sensitive marker of cardiac injury, in patients with CRF without myocardial infarction symptoms, and assessed potential causes for elevated cTnT. Blood was obtained from 38 patients with CRF immediately before hemodialysis and from 16 of them post-dialysis, from 21 peritoneal dialysis patients, 10 patients with CRF not on dialysis, 11 patients with cardiomyopathy, and 10 adolescent patients with CRF undergoing hemodialysis. Samples were analyzed for myoglobin, creatine kinase, creatine kinase isoenzyme-MB (CK-MB), lactate dehydrogenase, lactate dehydrogenase isoenzyme-1 (LD-1), and cTnT. Cardiac TnT was elevated in: 71% of patient with CRF undergoing hemodialysis with no significant differences between pre- and post-dialysis values, 57% of patients with CRF on peritoneal dialysis, 30% of patients with CRF without dialysis, 18% of patients with cardiomyopathy, and 20% of adolescent patients with CRF undergoing hemodialysis. Myoglobin was elevated in almost all patients with CRF undergoing hemodialysis and without dialysis, whereas CK-MB and LD-1 were rarely elevated. Cross-reacting dialyzable substances and myocardial stretch were not major causes for elevated cTnT. Until future studies clarify the etiology of elevated cTnT in patients with CRF, results should be interpreted cautiously.

Serum total testosterone: immunoassay compared with negative chemical ionization gas chromatography-mass spectrometry.

We have developed an electron capture negative chemical ionization gas chromatography-mass spectrometry (GC-MS) procedure to quantify serum testosterone in the clinically relevant range 0.69-69.3 nmol/L and used this procedure to assess Ciba Corning Diagnostics ACS:180 testosterone immunoassay. The GC-MS method involves liquid-liquid extraction of serum samples and synthesis of a pentafluorobenzyloxime/silyl ether derivative of testosterone with excellent chromatographic and electron capturing properties. The ACS testosterone assay is the first fully automated nonradioactive testosterone immunoassay approved by the US Food and Drug Administration. Patients' specimens (101, 57 males, 44 females) were analyzed by both techniques. A plot of the GC-MS (x) vs ACS (y) testosterone concentrations for men was linear (y = 1.07x + 0.19 nmol/L), showing excellent correlation (r2 = 0.98) between the two assays. Agreement of the two assays for female specimens was poor (y = 0.72x + 1.2 nmol/L), with a poor correlation (r2 = 0.31).

Current techniques in mycobacterial detection and speciation.

Tuberculosis, a health concern so well controlled in recent decades that eradication seemed imminent, is once again reaching epidemic proportions following the increasing prevalence of AIDS. One important means of curbing this resurgence is a robust method that has the capability of identifying and speciating mycobacterial infections in a matter of days. Classic biochemical techniques, which require 4 to 8 weeks to identify and speciate tuberculosis infections, are in the process of being replaced by newer methods, including BACTEC, gene probes, nucleic acid amplification, amplification of ribosomal RNA, high-performance liquid chromatography, and gas chromatography-mass spectrometry. This review is intended to give the reader a synopsis of the current literature and research on these methods, including reliability, approximate time required for detection and speciation, and clinical utility.

Determination of blood lead by electron-capture negative chemical ionization gas chromatography-mass spectrometry.

An electron-capture negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for determination of lead (Pb) in blood samples is described. Extraction of Pb from the sample does not involve hot digestion but is based on treatment at ambient temperature. The blood sample is supplemented with a known amount of internal standard (204Pb) for isotope dilution and is treated with concentrated nitric acid. After adjusting the pH to 7, the Pb is extracted into toluene as the pyrrolidine-dithiocarbamate chelate. Samples are then derivatized with 4- fluorophenylmagnesium bromide to form Pb(FC6H4)4. The use of NCI offers enhanced sensitivity (by 75-fold better than previously used electron ionization), gives good precision and accuracy, and has no observable memory effect. The isotope dilution GCoff methodology typically agreed within 2-3% of expected values for the College of American Pathologists blood Pb specimens and the National Institute of Standards and Technology Standard Reference Material 955a.

Intracoronary versus intravenous effects of cocaine on coronary flow and ventricular function.

BACKGROUND: Cocaine use has been associated with cardiomyopathy and ischemic coronary syndromes. However, the pathophysiological mechanisms responsible for these syndromes are not clear and have been suggested to involve direct effects of cocaine on myocyte contractility and coronary resistance as well as indirect effects via altered autonomic tone, secondary mediators, and myocardial metabolism. We sought to distinguish direct from indirect effects of cocaine on ventricular function and coronary resistance by comparison of the administration of intracoronary cocaine (0.12 to 0.36 mg/min constant infusion) with intravenous cocaine (5 mg/kg bolus infusion) in an in vivo anesthetized pig preparation. METHODS AND RESULTS: To control for changes in coronary resistance secondary to autoregulation and myocardial metabolism, the left anterior descending coronary artery was perfused at constant coronary pressure and the interventricular vein was cannulated for coronary venous oxygen saturation measurement. Coronary blood flow, regional percent segment shortening, myocardial oxygen consumption, and serum cocaine concentrations were measured. Intracoronary cocaine produced a dose-dependent decrease in percent segment shortening in the absence of significant changes in coronary flow or systemic hemodynamics. In contrast, intravenous cocaine had mild biphasic effects on coronary resistance with an initial brief vasodilation (30.0 +/- 5% increase in flow from control) followed by more prolonged vasoconstriction (17.0 +/- 3.3% decrease in flow from control), which were independent of autoregulation or myocardial metabolism. In addition, intravenous cocaine caused an early 48% decrease in percent segment shortening, at which time the measured cocaine concentration was 20.1 micrograms/mL blood. This was comparable to the intracoronary cocaine concentration of 17.1 micrograms/mL blood, which produced a similar 48% decrease in percent segment shortening. CONCLUSIONS: We conclude that the effects of acute cocaine exposure on ventricular function are predominantly direct but of brief duration and therefore probably not clinically relevant. The effects of cocaine on coronary tone are predominantly indirect and biphasic, with early vasodilation followed by mild and more prolonged vasoconstriction. In the absence of coronary stenosis or ventricular hypertrophy, this small amount of vasoconstriction is unlikely to cause ischemia.