Development and Implementation of One-Step, Broad-Spectrum, High-Sensitivity Drug Screening by Tandem Mass Spectrometry in a Pediatric Population.

BACKGROUND: Drug screening by immunoassay is common in pediatric populations. However, false-positive and -negative results due to antibody cross-reactivity and dilute urine are frequent and underappreciated. Accurate ascertainment of drug exposure in children has significant clinical and medico-legal consequences. DESIGN AND METHODS: We developed and characterized an LC-MS/MS drug screening assay to supplant immunoassay and detect 38 compounds at the lowest concentrations distinguishable from analytic noise. Once implemented, we conducted a retrospective analysis of 3985 pediatric urine drug screens performed a year before (n = 1663) and after (n = 2322) implementation to examine the frequency and breadth of drug detection in our pediatric population. RESULTS: Using immunoassay, 23% (293/1269) of samples from the general pediatric and 37% (147/394) of nursery populations had presumptively positive results. Of the presumptive positive compounds, 85% (288/338) from the general pediatric population and 40% (65/162) from the nursery cohort were confirmed by mass spectrometry. After LC-MS/MS implementation, 31% (628/2052) of general pediatric, and 18% (48/270) of the nursery samples were positive for 1 or more compounds. In the nursery population, immunoassays over-detected the presence of THC but under-detected exposure to cocaine. CONCLUSION: A broadly targeted, analytically sensitive LC-MS/MS drug screening assay detects a larger number and variety of compounds in a single step compared to a screen-then-confirm approach initiated by immunoassay in our pediatric population. Rapid delivery of accurate results enables timely, appropriate disposition of patients in a variety of settings including the emergency department and labor/delivery.

Impact of premature birth and critical illness on neonatal range of plasma amino acid concentrations determined by LC-MS/MS.

BACKGROUND: The number of newborns and the number of disorders detected by large-scale screening programs has increased dramatically in the last decade. Newborn screening is a multi-step process requiring confirmatory testing to establish and refine a diagnosis. Whereas screening cutoffs are established to detect all cases of a specific disease, confirmatory testing is performed against a backdrop of many co-morbid conditions and interpretation of results is often not straightforward. The goal of the current study was to define the range of amino acid concentrations encountered in normal, premature, and acutely ill newborns as an aid to the interpretation of follow-up testing for suspicious newborn screens. MATERIALS AND METHODS: Residual plasma samples from 354 neonates (age 1-10 days) were utilized. 206 samples were from neonates with uncomplicated birth histories and prompt hospital discharge. 98 specimens were derived from a population of children receiving intensive care with diagnoses that included sepsis, respiratory insufficiency, cardiac malfunction/malformation and gastrointestinal complications. 50 samples were from infants born after 32 but before 37 full weeks gestation that were discharged following uneventful hospital courses. 25 amino acids were quantitated by LC-MS/MS and reference intervals determined by non-parametric statistical methods. Distributions were compared using Kruskal-Wallis analyses for independent samples. RESULTS: The distributions of multiple amino acids in premature and critically ill infants were significantly different than those observed in healthy newborns. Differing distributions were found for phenylalanine, the branched chain amino acids, methionine, glutamine, glutamate, arginine, lysine, α-aminoadipic acid, and ß-aminoisobutyric acid. In most cases median values were increased and distributions more positively skewed in premature or ill newborns compared to healthy newborns. In addition, we report neonatal homocitrulline reference intervals for these newborn populations determined by an LC-MS/MS technique that is not confounded by methionine interference. CONCLUSION: These data provide a basis for improved detection of genetic metabolic disorders in newborns, particularly those born prematurely or with a variety of critical co-morbid conditions.

Differentiation of amphetamine/methamphetamine and other cross-immunoreactive sympathomimetic amines in urine samples by serial dilution testing.

BACKGROUND: Immunoassay-based screening for amphetamines has a variable positive predictive value (PPV) for detecting amphetamine abuse. The lack of immunoassay specificity necessitates confirmatory testing by gas chromatography-mass spectrometry (GC/MS), but the technical complexity and expense of GC/MS limit its availability. Physicians may make decisions regarding patient disposition based on unverified results. In this study we assessed the utility of using dose-response properties to distinguish urine samples containing amphetamines from samples containing cross-immunoreactive species. METHODS: Urine was supplemented with known concentrations of amphetamine, methamphetamine, methylenedioxymethamphetamine (MDMA), or pseudoephedrine. Using a series of dilutions, we determined the maximum change in rate over the fractional change in concentration for each compound in the Emit II amphetamine/methamphetamine immunoassay. Patient urine samples that screened positive for amphetamines were diluted 1:1, 1:10, and 1:20, and maximum slope estimates within the dynamic assay range were determined. An optimal slope cutoff that differentiated samples containing (meth)amphetamine from those containing cross-reacting species was determined by ROC analysis. RESULTS: The slope of the dose response was largest for amphetamine and methamphetamine, followed by MDMA and pseudoephedrine. The optimum slope cutoff for identifying patient specimens containing (meth)amphetamine was 320 (sensitivity, 96%; specificity, 90%; PPV, 92%). High concentrations of less reactive compounds may mask low concentrations of amphetamines. CONCLUSIONS: Use of the slope of the dose-response relationship in patient urine specimens can enhance the PPV of presumptive positive immunoassay results but does not exclude the presence of low amphetamine concentrations in samples containing high concentrations of cross-reactive species.

Analytic performance of immunoassays for drugs of abuse below established cutoff values.

BACKGROUND: The analytic performance and accuracy of drug detection below Substance Abuse and Mental Health Services Administration (SAMHSA) cutoffs is not well known. In some patient populations, clinically significant concentrations of abused drugs in urine may not be detected when current SAMHSA cutoffs are used. Our objectives were to define the precision profiles of three immunoassay systems for drugs of abuse and to evaluate the accuracy of testing at concentrations at which the CV was <20%. METHODS: Drug-free urine was supplemented with analytes to assess the precision in three commercial drugs-of-abuse immunoassay systems below the SAMHSA-dictated cutoffs for amphetamines, opiates, benzoylecgonine, phencyclidine, and cannabinoids. Consecutive urine samples with signals associated with a CV <20% by Emit immunoassay and below SAMHSA cutoffs were then subjected to confirmatory analysis. RESULTS: The CV of all immunoassay systems tested remained <20% to drug concentrations well below SAMHSA cutoffs. The accuracy of urine drug-screening results between the SAMHSA-specified cutoffs and the precision-based cutoffs was less than accuracy for specimens above the SAMHSA cutoffs, but the use of the precision-based cutoff produced a 15.6% increase in the number of screen-positive specimens and a 7.8% increase in the detection of specimens that yielded positive results on confirmatory testing. CONCLUSION: The precision of three commercial immunoassay systems for drugs-of-abuse screening is adequate to detect drugs below SAMHSA cutoffs. Knowledge of the positive predictive values of screening immunoassays at lower cutoff concentrations could enable efficient use of confirmatory testing resources and improved detection of illicit drug use.