LC-MS-MS vs ELISA: Validation of a Comprehensive Urine Toxicology Screen by LC-MS-MS and a Comparison of 100 Forensic Specimens

2019 ◽  
Vol 43 (9) ◽  
pp. 734-745 ◽  
Author(s):  
Kristin W Kahl ◽  
Joshua Z Seither ◽  
Lisa J Reidy
Keyword(s):  
Mass Spectrometry ◽  
Limit Of Detection ◽  
Screening Method ◽  
Driving Under The Influence ◽  
Cost Comparison ◽  
Enzyme Linked Immunosorbent Assay ◽  
Screening Methods ◽  
Limits Of Detection ◽  
The Cost ◽  
Urine Specimens

Abstract Toxicology laboratories commonly employ immunoassay methodologies to perform an initial drug screen on urine specimens to direct confirmatory testing. Due to limitations of immunoassay testing and the need to screen for a broader range of drugs with lower limits of detection at a lower cost, mass spectrometry screening techniques have gained favor in the toxicology field. A liquid chromatography–tandem mass spectrometry (LC-MS-MS) urine screening panel was developed and validated for 52 drugs and metabolites. A simple dilute-and-shoot with enzymatic hydrolysis technique was utilized to prepare the urine specimens for analysis. Limit of detection, interference, ionization suppression/enhancement, carryover and stability of processed specimens were assessed during validation. To evaluate the toxicological results obtained from utilizing the LC-MS-MS in comparison with the laboratory’s current enzyme-linked immunosorbent assay (ELISA) panel, 100 authentic urine specimens from suspected driving under the influence and drug-facilitated crime cases were analyzed using both methodologies and the results were compared. In addition, the cost of each methodology was evaluated and compared. The validated LC-MS-MS method had limits of detection that were equal to or lower than the concentrations validated for ELISA cutoffs, had fewer exogenous interferences, and the cost of screening per specimen was reduced by ~70% when compared to ELISA. Comparing the toxicology results of forensic urine specimens demonstrated that by only using ELISA, the laboratory was unable to detect benzoylecgonine in 26%, lorazepam in 33% and oxymorphone in 60% of the positive specimens. Additional analytes detected using the LC-MS-MS method were zolpidem and/or metabolite, gabapentin, tramadol and metabolite, methadone and metabolite, meprobamate and phentermine. The results of the validation, the toxicological result comparison and the cost comparison showed that the LC-MS-MS screening method is a simple, sensitive and cost-effective alternative to ELISA screening methods for urine specimens.

scholarly journals DEVELOPMENT AND APPLICATION OF A RAPID SCREENING METHOD FOR DETERMINATION OF NEW PSYCHOACTIVE SUBSTANCES AND THEIR METABOLITES IN URINE

2019 ◽  
Vol 16 (33) ◽  
pp. 206-224
Author(s):  
K. M. SHESTAKOVA ◽  
S. A. SAVCHUK ◽  
N. V. MESONZHNIK ◽  
A. V. KUHARENKO ◽  
S. A. APPOLONOVA
Keyword(s):  
Mass Spectrometry ◽  
Limit Of Detection ◽  
Screening Method ◽  
Mass Spectrometry Analysis ◽  
Rapid Screening ◽  
New Psychoactive Substances ◽  
Preparation Technique ◽  
Psychoactive Substances ◽  
Urine Specimens

The ongoing appearance of new psychoactive substances on the black market of illegal drugs, as well as the lack of information on their influence on the human body, faces several challenges in their determination by standard analytical techniques. Moreover, the rapid metabolism of new psychoactive substances reveals in the absence of possibility in the identification of their native structures in biological fluids. This study presents a new screening method for determination 137 psychoactive substances including their metabolites. 'Dilute-and-shoot' method was chosen as the preferable sample preparation technique, and consisted of 1:5 dilution of urine specimens with the solution of acetonitrile and water (30:70) followed by electrospray ionization – liquid chromatography-tandem mass spectrometry analysis. The developed qualitative method was validated according to United Nations Office on Drugs and Crime requirements that included assessment of selectivity, limits of detection, precision, and stability. In addition, the presented method was tested on 50 certified positive urine specimens containing different drugs of abuse. The confirmatory analysis was performed using a high-resolution mass-spectrometry approach. The presented screening method provides the possibility of simultaneous determination of synthetic cannabinoids (96), opioid analgesics (16), stimulators (13), hallucinogens (5), benzodiazepines (5) and non-classified drugs (10) during one run. The validation assessments of the novel method have shown high rates of its specificity, selectivity, intra- and inter-day precision and stability with the limit of detection ranged from 1 to 5 ng?mL-1. At the same time, tests of 50 positive samples showed excellent applicability of the developed screening method for routine preliminary screening analysis in toxicological laboratories.

Comparing ELISA and LC-MS/MS: A Simple, Targeted Postmortem Blood Screen

2021 ◽  
Author(s):  
Dina M Swanson ◽  
Julia M Pearson ◽  
Theresa Evans-Nguyen
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Limit Of Detection ◽  
Screening Method ◽  
Chemical Properties ◽  
Forensic Toxicology ◽  
Enzyme Linked Immunosorbent Assay ◽  
Minimal Sample ◽  
Elisa Testing ◽  
Postmortem Blood

Abstract A comprehensive screening method that is specific, accurate, and customizable is necessary in any forensic toxicology laboratory. Most laboratories utilize some form of immunoassay testing as it is reliable and sensitive with minimal sample preparation and is relatively inexpensive to simultaneously screen for multiple classes of drugs with different chemical properties. However, accessibility to more specific technology and instrumentation such as mass spectrometry has increased and therefore using immunoassay as the screening method of choice may be revisited. A screening method for 42 drugs in postmortem blood was developed and validated following the Organization of Scientific Area Committees for Forensic Science (OSAC) guidelines for toxicology method validation. The method was developed using minimal sample preparation of postmortem blood consisting only of a protein precipitation. Only two internal standards were used which greatly reduces the cost of implementing this method. Limit of detection (LOD), interference studies, processed sample stability and ion suppression/enhancement were examined. Additionally, over 100 case samples were analyzed by both the current enzyme linked immunosorbent assay (ELISA) testing procedure and the proposed liquid chromatography tandem mass spectrometry (LC-MS/MS) screening method. The comparison determined that the LC/MS-MS method performed as well as or better than the ELISA in nearly all cases. The ability to add additional target drugs increases the laboratory’s scope of analysis as well. This method is ideal for forensic laboratories wishing to improve screening while working within budget constraints.

scholarly journals Validation of the method of quantitative determination of florphenicol in muscle samples by the method of immuno-enzyme analysis

2020 ◽  
pp. 40-45
Author(s):  
M. V. Kostyuk ◽  
◽  
K. S. Myagka ◽  
G. S. Kochetova ◽  
◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Screening Method ◽  
Enzyme Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Laboratory Diagnostics ◽  
Screening Methods ◽  
Detection Ability ◽  
Wide Range ◽  
Control Samples

Introduction. Amphenicols are a group of chemical compounds with antibacterial activity, including chloramphenicol (HAF), thiamphenicol (TAF), florfenicol (FF) and their derivatives. Florfenicol (FF) is a synthetic antimicrobial agent with a broad spectrum of action and is one of the most commonly used drugs in poultry, and was developed specifically for veterinary medicine. Given the wide range of activity of florfenicol, the high therapeutic effect combined with low toxicity makes it important for use in animal husbandry. The known method of enzyme-linked immunosorbent assay (ELISA) differs favorably from other screening methods by high sensitivity, specificity, simplicity and speed of performance, availability and stability of reagents, the ability to computer processing of measurement results and automation of test steps, which provides high test efficiency. The aim of the work. To validate the method of enzyme-linked immunosorbent assay to determine the residues of florfenicol in the samples of months of different species of animals (cattle, pigs, chickens, geese, turkeys, rabbits) and fish. Materials and methods. The research was conducted on the basis of the State Research Institute for Laboratory Diagnostics and Veterinary Sanitary Expertise. The material for the study was a solution of florfenicol with a concentration of 1 mg/liter. Results of research and discussion. It was found that the highest value (highest response) for control samples is 0.24 μg/kg and the lowest value for enriched samples (lowest response) - 3.62 μg/kg. According to the obtained results, none of the answers for the enriched samples coincides with the range of answers for the control samples. It follows that the detection ability (CCβ) for this screening method reduces or decreases 5.0 μg/kg The cut-off rate of this test is 3.62 μg/kg. Conclusions and prospects for further research. Validation characteristics have been established for the determination of florfenicol residues in muscle samples, such as: detection ability (CCβ) is 5.0 μg/kg, cut-off level is 3.62 μg/kg. The lowest content of florfenicol that can be determined is 0.2 μg/kg. The percentage of return for enriched samples of both groups is 93 %, which corresponds to the specified production.

scholarly journals Adaptation of ELISA Detection of Plasmodium Falciparum and Plasmodium Vivax Circumsporozoite Proteins in Mosquitoes to a Multiplex Bead-Based Immunoassay

2021 ◽  
Author(s):  
Alice Sutcliffe ◽  
Seth R. Irish ◽  
Eric Rogier ◽  
Micaela Finney ◽  
Sarah Zohdy ◽  
...  
Keyword(s):  
Lower Limit ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Limits Of Detection ◽  
Detection Range ◽  
Cutoff Value ◽  
Sporozoite Rate ◽  
The Impact ◽  
Lower Limits ◽  
Multiplex Bead

Abstract Background: Plasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of P. falciparum (Pf), P. vivax VK210 (Pv210) or P. vivax VK247 (Pv247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites.Methods: Recombinant positive controls for Pf, Pv210 and Pv247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cutoff measures were applied to demonstrate how cutoff criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins. Results: Following optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for Pf, Pv and Pv247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for Pf, 425 for Pv210 and 1650 for Pv247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4% and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decreased in the Madagascar sporozoite rate to 2.8-4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cutoff value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cutoff value is determined.Conclusions: The csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to Pf, Pv210 and Pv247 can be added following optimization.

Rapid Screening Method for Aflatoxin M1 in Milk

1981 ◽  
Vol 64 (5) ◽  
pp. 1064-1066
Author(s):  
Charles E Holaday
Keyword(s):  
Limit Of Detection ◽  
Screening Method ◽  
Rapid Screening ◽  
Aflatoxin M1 ◽  
Fluorescent Band ◽  
Neutral Alumina ◽  
Rapid Screening Method ◽  
The Cost

Abstract A rapid screening method for detecting aflatoxin M1 in milk has been developed, based on minicolumn chromatography and requiring 8-10 min for each test. The minicolumn is packed with dry Florisil (100-200 mesh) on the bottom, anhydrous Na2S04 as the next layer, topped with neutral alumina (70-200 mesh) to which 8% water (wet basis) has been added. A blue fluorescent band at the Florisil-Na2SO4 interface indicates the presence of aflatoxin M1. The limit of detection is estimated to be about 0.2 μg/kg. Because several items are disposable, both the time to maintain glassware and the cost per determination are reduced.

Simultaneous Quantitation of Seven Phenethylamine-Type Drugs in Forensic Blood and Urine Samples by UHPLC–MS-MS

2021 ◽  
Author(s):  
Chu-An Yang ◽  
Hsiu-Chuan Liu ◽  
Ray H Liu ◽  
Dong-Liang Lin ◽  
Shu-Pao Wu
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Limit Of Detection ◽  
Multiple Reaction Monitoring ◽  
Analytical Data ◽  
Tandem Mass ◽  
Limit Of Quantitation ◽  
Routine Identification ◽  
Urine Specimens

Abstract Abuse of new psychoactive substances (NPS) has become a health and social issue of global concern. p-Methoxyamphetamine (PMA)/p-methoxymethamphetamine (PMMA) with fluoro- or chloro-derivatives of amphetamine and methamphetamine were among the most common drugs found in specimens from fatal cases in Taiwan during the January 2011 to December 2018 period. A liquid–liquid extraction sample preparation protocol with highly sensitive ultra-high performance liquid chromatography–tandem mass spectrometry approach was developed for the simultaneous analysis of seven phenethylamine-type drugs—PMA, PMMA, p-methoxyethylamphetamine, 4-fluoroamphetamine (4-FA), 4-fluoromethamphetamine (4-FMA), 4-chloroamphetamine (4-CA) and 4-chloromethamphetamine (4-CMA)—in postmortem blood and urine specimens. Separation by liquid chromatography was performed by Agilent Zorbax SB-Aq column. Tandem mass spectrometry was operated in Agilent Jet Stream Technology electrospray ionization in positive-ion multiple reaction monitoring mode. An analytical methodology was evaluated using drug-free blood and urine after fortification with 100–2,000 ng/mL of the seven target analytes. Average extraction recoveries were >80%; slightly higher ion suppression was observed for PMA and 4-CA; intra-/inter-day precision (% coefficient of variation) and accuracy were in the ranges of 0.52–12.3% and 85–110%, respectively. Limit of detection and lower limit of quantitation for these seven analytes were both in the 0.5–5 ng/mL range. Interference and carryover were not significant. This relatively simple methodology was found effective and reliable for routine identification and quantitation of these seven analytes in postmortem and antemortem blood and urine specimens received in 2018. Analytical data obtained from these actual cases indicated the following: (i) compared to findings reported during the 2007–2011 period, the use of substituted phenethylamine-type drugs decreased in 2018; (ii) ketamine and 7-aminonimetazepam (the main metabolite of nimetazepam) were the most common co-ingested substances in specimens containing PMA/PMMA, 4-FA/4-FMA, or 4-CA/4-CMA; and (iii) in drug fatalities, the concentration of PMA was significantly higher than the concentration of PMMA in both urine and blood, while the reverse was true in urine specimens from antemortem cases.

scholarly journals Implications of Limits of Detection of Various Methods for Bacillus anthracis in Computing Risks to Human Health

2009 ◽  
Vol 75 (19) ◽  
pp. 6331-6339 ◽  
Author(s):  
Amanda B. Herzog ◽  
S. Devin McLennan ◽  
Alok K. Pandey ◽  
Charles P. Gerba ◽  
Charles N. Haas ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Bacillus Anthracis ◽  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Risk Assessment ◽  
Detection Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Environmental Matrices ◽  
Limits Of Detection

ABSTRACT Used for decades for biological warfare, Bacillus anthracis (category A agent) has proven to be highly stable and lethal. Quantitative risk assessment modeling requires descriptive statistics of the limit of detection to assist in defining the exposure. Furthermore, the sensitivities of various detection methods in environmental matrices are vital information for first responders. A literature review of peer-reviewed journal articles related to methods for detection of B. anthracis was undertaken. Articles focused on the development or evaluation of various detection approaches, such as PCR, real-time PCR, immunoassay, etc. Real-time PCR and PCR were the most sensitive methods for the detection of B. anthracis, with median instrument limits of detection of 430 and 440 cells/ml, respectively. There were very few peer-reviewed articles on the detection methods for B. anthracis in the environment. The most sensitive limits of detection for the environmental samples were 0.1 CFU/g for soil using PCR-enzyme-linked immunosorbent assay (ELISA), 17 CFU/liter for air using an ELISA-biochip system, 1 CFU/liter for water using cultivation, and 1 CFU/cm2 for stainless steel fomites using cultivation. An exponential dose-response model for the inhalation of B. anthracis estimates of risk at concentrations equal to the environmental limit of detection determined the probability of death if untreated to be as high as 0.520. Though more data on the environmental limit of detection would improve the assumptions made for the risk assessment, this study's quantification of the risk posed by current limitations in the knowledge of detection methods should be considered when employing those methods in environmental monitoring and cleanup strategies.

Hog Charm II Tetracycline Test Screening Results Compared with a Liquid Chromatography Tandem Mass Spectrometry 10-μg/kg Method

2012 ◽  
Vol 75 (2) ◽  
pp. 405-407 ◽  
Author(s):  
ROBERT SALTER ◽  
STEVEN HOLMES ◽  
DAVID LEGG ◽  
JOEL COBLE ◽  
BRUCE GEORGE
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Limit Of Detection ◽  
Screening Method ◽  
Tandem Mass ◽  
Tissue Samples ◽  
Plant Laboratory ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Pork tissue samples that tested positive and negative by the Charm II tetracycline test screening method in the slaughter plant laboratory were tested with the modified AOAC International liquid chromatography tandem mass spectrometry (LC-MS-MS) method 995.09 to determine the predictive value of the screening method at detecting total tetracyclines at 10 μg/kg of tissue, in compliance with Russian import regulations. There were 218 presumptive-positive tetracycline samples of 4,195 randomly tested hogs. Of these screening test positive samples, 83% (182) were positive, >10 μg/kg by LC-MS-MS; 12.8% (28) were false violative, greater than limit of detection (LOD) but <10 μg/kg; and 4.2% (8) were not detected at the LC-MS-MS LOD. The 36 false-violative and not-detected samples represent 1% of the total samples screened. Twenty-seven of 30 randomly selected tetracycline screening negative samples tested below the LC-MS-MS LOD, and 3 samples tested <3 μg/kg chlortetracycline. Results indicate that the Charm II tetracycline test is effective at predicting hogs containing >10 μg/kg total tetracyclines in compliance with Russian import regulations.

scholarly journals Cost-Effectiveness of Active Screening for Early Identification of HIV in Injection Drug Users

2021 ◽  
Vol 22 (2) ◽  
Author(s):  
Shima Bordbar ◽  
Hassan Joulaei ◽  
Abdosaleh Jafari ◽  
Mehrdad Askarian ◽  
Charles John Palenik ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Injection Drug Users ◽  
Drug Users ◽  
Screening Method ◽  
Cost Effective ◽  
Injection Drug ◽  
Behavioral Disorder ◽  
Screening Methods ◽  
Active Screening ◽  
The Cost

Background: Acquired immunodeficiency syndrome is a behavioral disorder that can be detected via two methods, including active and passive screening. Objectives: This study aimed to evaluate the cost-effectiveness of screening strategies of HIV/AIDS among injection drug users (IDUs) referring to the voluntary counseling and testing (VCT) center and drop-in center (DIC) of Shiraz University of Medical Sciences. Methods: This was a cross-sectional cost-effectiveness analysis to compare the cost-effectiveness of the two active and passive screening methods in 2015. The decision tree model, along with the TreeAge11 software, was used to analyze the data. Results: The averages of cost and effectiveness were $989 and 987 subjects in the active screening method while they were $1,767 and 209 subjects in the passive screening method, respectively. The incremental cost-effectiveness ratio (ICER) to early-diagnosed and averted cases was $855/39 for the active screening method and $1528/90 for the passive screening method. According to the findings of the study, the active screening method is more cost-effective than its passive counterpart. Conclusions: According to the findings of the study, the active screening method is more cost-effective than its passive counterpart, and it is recommended to be used in these cases.

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