Internal Hydrolysis Indicator for Sample Specific Monitoring of ß-Glucuronidase Activity.

Metabolized forms of benzodiazepines (benzos) can cause issues with mass spectrometry identification. Benzodiazepines undergo a process called glucuronidation during metabolism that attaches a glucuronic acid for increased solubility. Often in clinical testing an enzymatic hydrolysis step is implemented to increase the sensitivity of benzodiazepines by hydrolyzing ß-D-glucuronic acid from benzodiazepine-glucuronide conjugates in urine samples using the ß-Glucuronidase enzyme. In this study resorufin ß-D-glucuronide, a substrate of the ß-Glucuronidase enzyme, was added to patient samples to determine if proper hydrolysis had occurred. The presence of resorufin as an Internal Hydrolysis Indicator (IHI) shows the activity and efficiency of the enzyme in each patient sample. Synthetic/patient urine samples were obtained and mixed with hydrolysis buffer containing resorufin ß-D-glucuronide. The ß-Glucuronidase enzyme was used to hydrolyze the benzodiazepine analytes as well as resorufin ß-D-glucuronide. The enzymatic hydrolysis addition increased the positivity rate of benzodiazepines by 42.5%. The ß-Glucuronidase substrate resorufin (IHI) displayed variability in area counts between patient samples. Comparative studies with internal standards and resorufin (IHI) showed no correlation between recovery and analyte variability. Hydrolysis reactions greatly improved the sensitivity of benzodiazepines by liquid chromatography time-of-flight mass spectrometry analysis. The large variation in resorufin (IHI) area counts amongst patient samples indicates possible variability in enzymatic hydrolysis activity. The enzymatic hydrolysis step is a part of the extraction procedure and should be controlled for in each patient sample.

Detection of neonatal drug exposure using umbilical cord tissue and liquid chromatography time-of-flight mass spectrometry.

BACKGROUND: A method for qualitative detection of 57 drugs and metabolites in umbilical cord tissue using liquid chromatography time-of-flight (TOF) mass spectrometry is described. METHODS: Results from 32 deidentified positive specimens analyzed by an outside laboratory using "screen with reflex to confirmation" testing were compared with TOF results. In addition, 57 umbilical cord tissue specimens paired with corresponding chart review data and 37 with meconium test results were analyzed by TOF. Urine drug test results from mother (n = 18) and neonate (n = 30) were included if available. Cutoff concentrations, recovery, and matrix effects were determined by analyzing fortified drug-free cord tissue and negative specimens. Cutoffs (in nanograms per gram) ranged from 1 to 10 for opioids and opioid antagonists, 5-10 for benzodiazepines and nonbenzodiazepine hypnotics, 20-40 for barbiturates, 8 for stimulants, and 4 for phencyclidine. Adequate sensitivity for the detection of cannabis exposure could not be realized with this method. CONCLUSIONS: Liquid chromatography time-of-flight mass spectrometry can provide accurate and sensitive detection of in utero drug exposure using umbilical cord tissue.

Rapid screening for 67 drugs and metabolites in serum or plasma by accurate-mass LC-TOF-MS.

Sixty-seven drugs and metabolites were detected in serum or plasma using a fast (7.5 min) liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) method. This method was developed as a blood drug screen, with emphasis on the detection of common drugs of abuse and drugs used to manage chronic pain. Qualitative drug detection may identify a drug exposure, assure patient adherence with prescribed therapy and document abstinence from non-prescribed medications. Compound identification is based on chromatographic retention time, mass, isotope spacing and isotope abundance. Data analysis software (Agilent) generates a compound score based on how well these observed criteria matched theoretical and empirical values. The method was validated using fortified samples and 299 residual patient specimens (920 positive results). All results were confirmed by gas chromatography-MS or LC-tandem MS. The accuracy of positive results (samples meeting all qualitative criteria for retention time, mass and compound score) was >90% for drugs and/or metabolites, except for two benzodiazepines. There were 35 false positive results (seven compounds, 3.8%) that could be distinguished by retention time and/or absence of metabolites. The most frequent was 6-acetylmorphine in the absence of morphine. The LC-TOF-MS targeted screening method presented represents a sensitive and specific technology for drug screening of serum or plasma.

Method comparison of the Ortho Vitros Fusion 5,1 chemistry analyzer and the Roche COBAS Integra 400 for urine drug screen testing in the emergency department.

Exposure to drugs and toxins is a major cause for the rising number of emergency department visits each year. Immunoassays are commonly used in the emergency department to provide rapid turnaround time for acute care. The purpose of this study was to compare two automated immunoassay chemistry analyzers to determine which platform produced the fewest number of false positive/negative results. Residual patient urine samples were were collected for each of the following drugs/drug classes: cocaine (n = 40), opiates (n = 45), and amphetamines (n = 54) and confirmed either positive or negative by mass spectrometry. Split sample analyses of these specimens were performed on both the Roche COBAS INTEGRA 400 plus and Ortho Vitros 5,1 FS instruments. The results from the two chemistry analyzers were compared to confirmed results. Both immunoassays were prone to false positive results for cocaine and false negative results for opiates and amphetamines. The Vitros Fusion analyzer generated fewer false positive and false negative results for opiate and amphetamine testing than the Roche Integra, but the platforms performed comparably for cocaine.

Nicotine and metabolites in paired umbilical cord tissue and meconium specimens.

Umbilical cord tissue was studied as a means of detecting prenatal exposure to nicotine. This was accomplished by comparing the presence and concentration of nicotine as well as nicotine metabolites in both umbilical cord tissue and paired meconium samples with maternal smoking histories obtained by self-report. Nicotine and metabolites (cotinine, 3-hydroxycotinine, nornicotine, and anabasine) were detected and quantitated using liquid chromatography-tandem mass spectroscopy. Between June and September 2009, 19 women with a tobacco exposure history (either first- or second-hand tobacco smoke exposure during pregnancy) were consented for the study. A questionnaire was completed to document nicotine exposure during each trimester of pregnancy. All infants were delivered at term (38 weeks or greater) and paired umbilical cord tissue (10-cm segment or greater) and meconium were obtained. Nicotine and 3-hydroxycotinine were most prominent in meconium, whereas cotinine and 3-hydroxycotinine were most prominent in the umbilical cord. Concentrations of all three analytes were generally higher in meconium. Nornicotine was detected only in meconium, at very low concentrations, and anabasine was not detected in either specimen. All analyte concentrations were lowest when the mother stated she quit smoking early in pregnancy or had only second-hand exposure, and detection was poor if exposure was limited to the first or second trimesters. Although different nicotine and metabolite patterns exist in meconium versus umbilical cord tissue, this work indicates that either specimen can be used to detect third-trimester fetal nicotine exposure.