This article describes the need for, stratifies the complexity of, and proposes detailed lists of training competencies for diagnostic laboratory personnel using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) for patient care. Although quantitative LC-MS/MS is evolving toward greater automation with less need for technical expertise, gaps remain in resources for training and assessment.

Background

Historically the success of mass spectrometry in the clinical laboratory has focused on drugs of abuse confirmations, newborn screening, and steroid analysis. Clinical applications of mass spectrometry continue to expand, and mass spectrometry is now being used in almost all areas of laboratory medicine.

Content

A brief background of the evolution of mass spectrometry in the clinical laboratory is provided with a discussion of future applications. Prominent examples of mass spectrometry are covered to illustrate how it has improved the practice of medicine and enabled physicians to provide better patient care. With increasing economic pressures and decreasing laboratory test reimbursement, mass spectrometry testing has been shown to provide cost-effective solutions. In addition to pointing out the numerous benefits, the challenges of implementing mass spectrometry in the clinical laboratory are also covered.

Summary

Mass spectrometry continues to play a prominent role in the field of laboratory medicine. The advancement of this technology along with the development of new applications will only accelerate the incorporation of mass spectrometry into more areas of medicine.

Alternative specimens have been occasionally considered as substitutes for whole blood for postmortem toxicology testing. We studied the applicability of vitreous humor, and evaluated whether it would be suitable to replace (or augment) whole blood for routine drug screening. Results showed that from 51 autopsy cases, we were able to identify an aggregate of 209 findings in whole blood compared with 169 in vitreous. The total number of compounds identified was 71 for whole blood and 60 for vitreous humor. Quantitative analysis showed that whole-blood concentrations of trazodone were several fold higher than vitreous humor concentrations (1.42 ± 0.57 vs. 0.15 ± 0.05 mg/L, respectively) and similar results were also obtained for diazepam (0.37 ± 0.06 vs. 0.13 ± 0.01, respectively). For other drugs such as oxycodone, hydrocodone and doxylamine, a trend suggesting higher concentrations in vitreous humor vs. whole blood was observed; however, this was not significant. Our results are consistent with the limited work of other investigators, and suggest that vitreous humor could be an appropriate matrix for drug screening in postmortem toxicology.

Urine drug screen (UDS) immunoassays are a quick and inexpensive method for determining the presence of drugs of abuse. Many cross-reactivities exist with other analytes, potentially causing a false-positive result in an initial drug screen. Knowledge of these potential interferents is important in determining a course of action for patient care. We present an inclusive review of analytes causing false-positive interferences with drugs-of-abuse UDS immunoassays, which covers the literature from the year 2000 to present. English language articles were searched via the SciFinder platform with the strings 'false positive [drug] urine' yielding 173 articles. These articles were then carefully analyzed and condensed to 62 that included data on causes of false-positive results. The discussion is separated into six sections by drug class with a corresponding table of cross-reacting compounds for quick reference. False-positive results were described for amphetamines, opiates, benzodiazepines, cannabinoids, tricyclic antidepressants, phencyclidine, lysergic acid diethylamide and barbiturates. These false-positive results support the generally accepted practice that immunoassay positive results are considered presumptive until confirmed by a second independent chemical technique.

Quantitative serum alcohol concentrations from regional hospitals (from specimens collected at time of hospital admission) were compared to results from whole blood (from specimens collected at the time of hospital admission) concentrations measured at the San Diego County Medical Examiner's Office (SDCMEO). Over a 15 month period (January 2012 to March 2013), the postmortem forensic toxicology laboratory analyzed a total of 2,321 cases. Of these, 280 were hospital cases (antemortem) representing 12% of the overall Medical Examiner toxicology casework. 59 of the 280 hospital cases (or 21%) screened positive for alcohol (ethanol). 39 of these 59 cases were included in the study based on available specimens for quantitative analyses. This investigation indicated that serum hospital ethanol concentrations correlated well (R(2) = 0.942) with ethanol values determined at SDCMEO (generally measured in whole blood). There was an observed negative bias with an average of -14.1%. A paired t-test was applied to the data and it was shown that this observed bias is statistically significant. These differences in ethanol concentrations could result from differences in specimen, analytical techniques, and/or calibration. The potential for specimen contamination is also discussed.

Background

In the last decade there has been a worldwide surge in the recreational abuse of novel psychoactive substances, particularly amphetamine derivatives and synthetic cannabinoids. Synthetic opioids such as AH-7921, MT-45, and U-47700, with structures distinct from those ever used therapeutically or described recreationally, have also recently emerged.

Case details

We report a patient who suffered respiratory failure and depressed level of consciousness after recreationally using a novel synthetic opioid labeled U-47700. A single dose of naloxone administered by paramedics completely reversed his opioid poisoning. Comprehensive laboratory analysis confirmed the presence of a novel synthetic opioid and excluded other drugs. The drug used appeared to have caused a false positive benzodiazepine result on the initial urine drugs of abuse panel.

Conclusion

The case we describe of toxicity from the synthetic opioid labeled U-47700 highlights the emerging trend of novel synthetic opioid abuse.

Background

The VALID Act is a legislative effort that, if enacted, would alter the regulatory requirements of laboratory developed tests (LDTs) used for clinical testing in the United States. Benzodiazepines, which are primarily excreted into urine as glucuronidated metabolites such as lorazepam, cross-react poorly with FDA-cleared immunoassays, leading to false-negatives. This shortfall can be addressed with LDTs created by adding glucuronidase to the immunoassay reagents producing "high sensitivity" assays that detect glucuronidated metabolites.

Methods

Precision and stability of two high-sensitivity (HS) benzodiazepine immunoassays from Roche and Thermo Scientific were evaluated using manufacturer-supplied quality control (QC) material and glucuronidated QC material. The immunoassays were directly compared to an LC-MS/MS LDT benzodiazepine assay to determine clinical sensitivity/specificity using urine specimens (n = 82 for Thermo Scientific; n = 265 for Roche). The clinical impact of the HS LDT immunoassay was determined by analyzing clinical testing results 60 days before and after its implementation.

Results

The precision and clinical sensitivity/specificity of the HS-Thermo Scientific and HS-Roche benzodiazepine assays were acceptable. The reagent stability of the HS-Thermo Scientific immunoassay was poor, whereas the HS-Roche immunoassay was stable. After implementation of the HS-Roche benzodiazepine immunoassay as an LDT, there was a 30-fold increase (p-value: < 0.00001) in the percentage of lorazepam confirmations.

Conclusions

We demonstrate the development and validation of an immunoassay LDT with improved sensitivity for glucuronidated benzodiazepines. This LDT can detect glucuronidated benzodiazepines in clinical urine specimens and is stable for 60 days. Importantly, we were able to validate the immunoassay as an LDT by utilizing an LC-MS/MS LDT.

Background

Oral biotin supplementation is known to interfere with biotin-streptavidin-based immunoassays, including Roche's fifth-generation cardiac troponin T (cTnT) assay, which plays a critical role in the diagnosis of myocardial infarction (MI). The utility of dilution, a quick and easy method to detect and remove interferences, has not been published for biotin interference.

Methods

Concentrations of cTnT were measured in pooled serum from clinical samples. Serum samples were supplemented with biotin to known concentrations, then cTnT concentrations were remeasured to assess for biotin interference. Samples were then diluted to assess for effective removal of biotin interference.

Results

At cTnT values near the critical reporting range for our institution (100 ng/L) we observed significant interference in measured values with added biotin concentrations above 50 ng/mL. In specimens without added biotin, autodilution at a 1:10 ratio yielded a mean 157% capture of measured cTnT, precluding the use of autodilution for detecting and mitigating biotin interference. A 1:10 dilution with serum containing 20-30 ng/L cTnT yielded a mean capture of 107%, which was suitable for detecting underlying biotin interference in supplemented samples.

Conclusions

Biotin interference, at supraphysiologic concentrations, may create an artifactual reduction in measured cTnT to levels that could lead to delayed detection of an MI. Dilution with serum of known cTnT concentration of 20-30 ng/L is a fast and effective method to mitigate the analytical consequences of biotin interference.

Background

Tacrolimus (Prograf, Advagraf, and FK-506) is the most commonly prescribed calcineurin inhibitor after kidney and liver transplantation. The use of tacrolimus (in conjunction with other drugs) has successfully contributed to the maintenance of solid organ allografts; however, it also exhibits toxic side effects. Therapeutic drug monitoring of tacrolimus is used as an aid to achieve drug concentrations within a narrow therapeutic window.

Methods

The Waters MassTrak Immunosuppressants assay (LC-MS/MS) for the quantification of tacrolimus in whole blood was evaluated for precision, linearity, lower limit of quantification, matrix effects, and accuracy. A method comparison with the Abbott Architect Tacrolimus immunoassay was also performed.

Results

The mean concentration (nanograms per milliliter) and coefficient of variation for low, mid, and high patient pools were 0.6% ± 19.9%, 16.0% ± 5.4%, and 31.2% ± 5.8%, respectively. The MassTrak assay was linear from 0.5 to 30.0 ng/mL. Although the MassTrak and Architect assays correlated well (R = 0.97) for patient samples, the MassTrak assay displayed an average negative bias of 18.5% versus Architect (range of 0.0%-36.7%). Analysis of a certified tacrolimus reference material in human whole blood [European Reference Materials (ERM)-DA110a, LGC Standards] on both platforms failed to completely explain the observed difference for patient samples.

Conclusions

Two widely used assays for therapeutic drug monitoring of tacrolimus are not in agreement with one another. Care should be exercised when interpreting results generated on these 2 assay platforms.