Detection of cocaine and its metabolites in breast milk.

A method was developed for measuring cocaine and its metabolites, benzoylecgonine, ecgonine methyl ester, norcocaine, ecgonine ethyl ester, cocaethylene, and m-hydroxybenzoylecgonine, in breast milk by gas chromatography/mass spectrometry. Limits of detection for this method ranged from 2.5 to 10 ng/mL, and limits of quantitation ranged from 5 to 50 ng/mL. For each of the compounds measured by this method, linear response was demonstrated to 750 ng/mL. Breast milk was collected from 11 mothers who admitted to drug use during pregnancy and ten drug-free volunteers serving as control subjects. Cocaine was detected in six of the specimens obtained from drug-exposed subjects, and in none of the drug-free control subjects. In breast milk specimens where cocaine and one or more of its metabolites were detected, the concentration of parent compound was greater than any of the metabolites. The highest cocaine concentration found was over 12 microg/mL. Breast-fed infants of cocaine abusing mothers may be exposed to significant amounts of drug orally.

Protecting human research subjects.

Respect for persons, beneficence, and justice are the cardinal principles that guide the ethical conduct of research on humans. Past abuses of human research subjects prompted medical organizations and governmental agencies to develop guidelines that ensure the protection of human research subjects. Human research funded by the U.S. government is strictly regulated, and Institutional Review Board approval of the experimental protocol is required prior to the award. Under limited circumstances, human research may be exempted from review, or review may be expedited. Research involving specimens submitted for pathological examination or diagnostic studies sometimes qualifies for these special categories of limited review. Academic pathologists and laboratorians should be aware of the regulations that apply to research on human subjects.

Discordant results of CK-MB and troponin I measurements: a review of 14 cases.

In the course of a clinical comparison involving 204 parallel total creatine kinase (CK), creatine kinase-MB isoenzyme (CK-MB), and cardiac troponin I (cTnI) measurements, 12 patients were identified in whom cTnI was elevated while total CK was normal, as well as 2 patients in whom CK-MB was elevated while cTnI was normal. CK-MB relative index was elevated in 6 of the twelve cTnI-positive patients with normal total CK; only 2 of these patients had a discharge diagnosis of acute myocardial infarction (AMI). All of the 12 patients in this group had medical conditions that are associated with greater risk for acute cardiac events. Both patients with normal cTnI but elevated total CK and CK-MB index had chronic renal insufficiency; one of these patients had a positive stress test and a diagnosis of AMI. The other cTnI-negative patient died 2 days after admission, and autopsy revealed evidence of ischemic changes, but not acute infarction. Significant differences were apparent between traditional CK-MB results and cTnI measurements. Using total CK elevation as a prerequisite for subsequent CK-MB measurement may limit the clinical sensitivity of this enzyme marker for detecting subacute ischemic damage to the myocardium. Elevated total CK and CK-MB isoenzyme without corresponding elevations in cTnI, on the other hand, may reflect changes in enzyme elimination kinetics due to renal failure, or cross-reactivity of the cTnI assay with non-cardiac antigens.

Modification of screening immunoassays to detect sub-threshold concentrations of cocaine, cannabinoids, and opiates in urine: use for detecting maternal and neonatal drug exposures.

Testing for drugs of abuse in urine is commonplace in emergency departments and neonatal units. However, the clinical sensitivity of immunochemical screening methods is limited by the threshold concentrations used to distinguish between positive and negative specimens. Immunochemical screening methods for cocaine metabolite (benzoylecgonine), cannabinoids, and opiates in urine were recalibrated to detect drugs at lower threshold concentrations. The precision and linearity of the signals at the modified thresholds were verified by diluting drug-positive urine specimens to concentrations below the conventional cutoff concentration and measuring the rate signals in triplicate. To assess the clinical performance of the modified methods, specimens that tested negative using the unmodified assays were re-screened at the lower threshold, and specimens that re-screened positive were submitted for gas chromatographic/mass spectrometric (GC/MS) confirmation. Reproducibility of sub-threshold measurements was comparable to the unmodified assays, and rate separations between successive dilutions were sufficient to give semi-quantitative results. Using the lower thresholds, drugs were detected in 4-5% of the subjects that had screened negative at the conventional threshold concentration. GC/MS analysis confirmed the presence of cannabinoids and cocaine metabolite in 74% and 84%, respectively, of urine specimens that re-screened positive. Morphine, codeine, hydromorphone, or hydrocodone was detected by GC/MS analysis in 31% of opiate-positive re-screens.

The expanding role of molecular biology in clinical chemistry.

Technical improvements in the application of molecular biology methods to detection and identification of specific nucleic acid sequences have resulted in more widespread incorporation of these techniques into clinical laboratory services. Gene amplification techniques have been refined and automated, making possible the rapid and economical detection of attomolar and smaller quantities of genetic material. Of particular benefit to clinical chemistry applications is the remarkable specificity of a DNA probe for its complementary sequence. Applications of molecular biology techniques in clinical chemistry include diagnosis of infectious, neoplastic, genetic, and hematological diseases. In addition, the use of oligonucleotides as molecular recognition probes may provide new analytical strategies for a wide range of diagnostic applications that currently rely on antibodies.

Detection of cocaine and its metabolites in amniotic fluid and umbilical cord tissue.

The increased use of cocaine by women of child-bearing age has left many health care scientists searching for improved methods of detecting prenatal cocaine exposure. To that end, a study of the determination of cocaine and its metabolites in amniotic fluid and umbilical cord tissue was undertaken. Amniotic fluid (n = 32) and umbilical cord tissue (n = 70) specimens were collected from pregnant subjects admitted to labor and delivery at Shands Hospital at the University of Florida (Gainesville, FL). Subjects were interviewed regarding drug use during each trimester. Subjects reporting cocaine use were designated as target subjects, and those denying use were control subjects. The specimens were subjected to solid-phase extraction and analyzed for cocaine and its metabolites by gas chromatography-mass spectrometry. Cocaine analytes (predominantly benzoylecgonine) were detected in 28.1 and 18.5% of the amniotic fluid and umbilical cord tissue specimens, respectively. Other cocaine analytes frequently detected included ecgonine methyl ester and m-hydroxy-benzoylecgonine in amniotic fluid specimens and ecgonine methyl ester, norcocaine, and m-hydroxybenzoylecgonine in umbilical cord tissue specimens. This study has shown that cocaine and its metabolites are readily detected in specimens of maternal and fetal origin.

Comparison of HPLC and GC-MS for measurement cocaine and metabolites in human urine.

High-performance liquid chromatography (HPLC) was compared with gas chromatography-mass spectrometry (GC-MS) for quantitation of cocaine, benzoylecgonine, norcocaine, and cocaethylene in urine. Calibration standards were prepared in human urine, and bupivacaine was added as the internal standard for quantitation. After solid-phase extraction, the reconstituted samples were divided into aliquots for analysis by HPLC and GC-MS. The analytical performance of the two methods were compared with regard to sensitivity, precision, and dynamic range. Results of GC-MS and HPLC analyses of nine urine specimens previously confirmed as positive for benzoylecgonine were compared. Analytical results by HPLC were comparable to GC-MS. Therefore, for many laboratories, HPLC is a useful alternative to GC-MS for measuring cocaine and metabolites in urine.

Detection of cocaine exposure in the neonate. Analyses of urine, meconium, and amniotic fluid from mothers and infants exposed to cocaine.

Cocaine and its metabolites were measured in urine, meconium, and amniotic fluid specimens collected from 30 maternal-infant pairs with histories of prenatal cocaine use. Cocaine, benzoylecgonine, and ecgonine methyl ester were measured by isotope dilution gas chromatography-mass spectrometry. Mothers were interviewed at delivery regarding their cocaine use during pregnancy. There was qualitative agreement between the results of drug determinations in maternal urine, amniotic fluid, infant urine, and meconium. Although all of the mothers in this study admitted to using cocaine during their pregnancy, cocaine or its metabolites were detected only in the 20 cases in which cocaine was used within 3 weeks before delivery. We conclude that when sufficiently sensitive analytic methods are used, maternal urine, infant urine, and meconium analyses yield equivalent results for detection of prenatal cocaine exposure. Importantly, neither meconium nor urinary drug measurements detected cocaine exposure when the last reported use was prior to 3 weeks before delivery.

Use of urine cotinine to validate smoking self-reports in U.S. Navy recruits.

Cotinine analysis of urine has been used by many researchers to determine validity of smoking self-reports. This technique is easy and inexpensive, but has not been used previously in military smoking studies. This study incorporated a random validation of self-reported smoking by U.S. Navy recruits participating in a smoking relapse program (N = 496). Results of cotinine analysis indicate only a 1% misrepresentation of actual smoking status. These results suggest smoking self-reports from U.S. Navy recruits are very good indicators of actual smoking status.

Measurement of cocaine and metabolites in urine, meconium, and diapers by gas chromatography/mass spectrometry.

Analytical methods were evaluated for measuring cocaine (CO), benzoylecgonine (BE), and ecgonine methyl ester (EME) in urine and methanolic extracts from meconium and diapers by isotope dilution gas chromatography/mass spectrometry (GC/MS). Volatile derivatives of the extracted drugs were generated before GC/MS analysis. Methanolic extracts from meconium and diapers were reconstituted in drug-free urine and treated as above. The limit of detection for the GC/MS method was calculated to be approximately 11 ng per mL. Within-run coefficients of variation (CVs) for urinary CO, BE, and EME were 5.7, 5.3, and 11.4 percent, respectively (N = 10); corresponding CVs for meconium 6.4, 10.7, and 21.9 percent (N = 8). Quantitative results were linear from 25 to 10,000 ng per g of meconium and 25 to 5,000 ng per mL of urine. Day-to-day precision varied from eight percent (CV) for BE in refrigerated or frozen urine to 34 percent for EME in refrigerated meconium. Recoveries of CO, BE, and EME from urine were 63, 19, and 42 percent, respectively; corresponding recoveries from meconium were 64, 21, and 25 percent. Cocaine and metabolites were extracted from wet but meconium-free diapers into methanol, which was evaporated before reconstituting in drug-free urine and extraction on a solid phase column. The CO, BE, and EME were detected in previously drug-free meconium after portions were deposited in a diaper which was wet with drug-positive urine. Unless precautions are taken to prevent extracorporeal contamination of meconium with urine, concentrations of CO and metabolites in meconium may be substantially augmented by contamination with urine. Analysis by GC/MS of CO and metabolites extracted from diapers provides an attractive alternative to collection of urine, which is difficult and may cause discomfort for the neonate.

Ionized calcium buffering in the transfused anhepatic patient: Ab initio calculations of calcium ion concentrations.

Homeostatic control of ionized calcium can be volatile during liver transplantation, particularly during the anhepatic stage. Recently, an opportunity arose to evaluate an 11-year-old girl who developed persistent ionic hypocalcemia during a prolonged anhepatic period subsequent to the failure and removal of the graft. The patient was remarkable for having survived a 34-hour anhepatic interval before a second and successful orthotopic liver transplant. Ionic hypocalcemia (ionized calcium less than 1 mmol per L) coexisted with significant hypercalcemia (total calcium greater than 5 mmol per L) during this anhepatic interval. The discrepancy was due to high concentrations of citrate, which accumulated from the multiple transfusions of citrated blood, and the inability to metabolize citrate in the anhepatic state. Using a mathematical model to solve for free calcium ion concentration in the presence of multiple ligands, it is demonstrated that prolonged hypercitricemia markedly alters the calcium ion buffering properties of blood, and these changes must be recognized in order to prevent adverse clinical consequences of ionic hypocalcemia.

Ultrastructural localization of aluminium in liver of aluminium maltol-treated rabbits by laser microprobe mass analysis.

By means of laser microprobe mass analysis (LAMMA), we have studied the ultrastructural localization of aluminium in livers of aluminium maltol-treated rabbits. This animal model was developed to study long-term aluminium toxicity using systemic (intravenous) administration of aluminium. We could only detect aluminium in electron-dense inclusion bodies found in large, sometimes multinucleated cells. These results prove that the actual observation of aluminium deposits in liver with LAMMA gives more information than bulk analysis and can be very useful to explore mechanisms of toxicity.

A long-term intravenous model of aluminum maltol toxicity in rabbits: tissue distribution, hepatic, renal, and neuronal cytoskeletal changes associated with systemic exposure.

We studied the toxicity of an intravenously injected, water-soluble aluminum complex (aluminum maltol) in 20 young adult male New Zealand white rabbits over a period of 8 to 30 weeks. Sixteen rabbits injected with aluminum-free maltol and 15 untreated rabbits served as controls. Rabbits were injected three times per week with 75 mumol of aluminum maltol per injection, or a molar equivalent amount of maltol alone, through an indwelling jugular catheter. Liver contained the highest concentrations of aluminum among the aluminum maltol-treated rabbits, and aluminum accumulation was correlated with the appearance of periportal multinucleated giant cells in 13 of 20 rabbits. These cells stained positively for aluminum when a fluorescent (Morin) stain was applied to tissue from rabbits with a high concentration of aluminum in the liver. Proximal renal tubular necrosis or atrophy was found in 15 of 20 aluminum maltol-treated rabbits but not in maltol-treated and untreated controls. Renal tubules in rabbits with acute proximal renal necrosis stained positively for aluminum. Neurofibrillary tangles, immunoreactive with a monoclonal antibody to the 200-kDa subunit of neurofibrillary protein, were observed in the oculomotor nucleus of 3 aluminum maltol-treated rabbits (treated for 12, 20, and 29 weeks), but in none of the two groups of controls. These tangles were present in 3 of 10 aluminum-treated rabbits in which the nucleus was located. None of the 17 animals in both control groups in which the nucleus was found demonstrated tangles. A slight increase in brain tissue aluminum concentration was confirmed by an electrothermal atomic absorption spectrophotometric method. There were no specific findings in heart or lung tissue from aluminum-treated rabbits, although the aluminum content of these tissues was 10 to 20 times greater than control values. This model should be useful for investigating the effects of systemic exposure to high concentrations of solubilized aluminum.

Lithium determined in serum with an ion-selective electrode.

We evaluated the performance of the lithium ion-selective electrode (ISE) in the Du Pont Na/K/Li analyzer. Lithium concentrations in 106 serum samples from patients being treated with lithium were measured in duplicate with the ISE and by flame photometry. The slope of the regression line for the two methods was 1.004 with a standard error of the estimate of 0.049 mmol/L (x = flame photometry, y = ISE). Lithium measurements by the ISE method in serum or aqueous standards were linear to greater than 2.0 mmol/L. Within-run CVs for low (0.31 mmol/L) and high (1.15 mmol/L) lithium controls were 5.9% and 1.7%, respectively (n = 20). Day-to-day CVs for the same controls were 9.8% and 3.3%, respectively (n = 20). There was no significant interference when the concentrations of sodium, potassium, calcium, or magnesium were varied, nor did intervening urinary lithium analyses affect the measurement of serum lithium. Results for lithium measurement in four serum-based survey materials compared well with results by isotope dilution/mass spectrometry.

Amylase in urine as measured by a single-step chromolytic method.

We studied a new single-step direct chromolytic method (Behring D.A.T.) for measuring urinary amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) activity, comparing results with those by a similar, but two-step, procedure that requires an auxiliary (coupling) enzyme. The two methods gave virtually identical relative responses to purified human pancreatic and salivary amylases. Assay of four quality-control materials to evaluate the total (day-to-day) precision of the new method yielded CVs of 4 to 7%, similar to those of the comparison method for each of the four quality-control samples. Amylase activity was measured by both methods in 110 random (i.e., untimed) urine specimens. Linear regression analysis provided a slope and y-intercept of 0.947 and 4 U/L (x = comparison method, y = direct method), respectively, and a standard error of the estimate of 25 U/L for specimens in which the amylase activities ranged from 11 to 1465 U/L (mean = 358 U/L) by the comparison method. The mean rate of amylase excretion in 2-h timed urine specimens from 95 healthy volunteers, as measured by the new method, was 7.18 (SD = 3.18) U/h, and the nonparametric (95% confidence interval) reference interval was 1.6 to 15.2 U/h. We consider the new method a promising alternative to other kinetic assays that require the use of auxiliary enzymes.