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

High-resolution mass spectrometry (HRMS) has the potential to supplement other drug screening platforms used in toxicology laboratories. HRMS offers high analytical specificity, which can be further enhanced by incorporating a fragment ion for each analyte. The ability to obtain precursor ions and fragment ions using elevated collision energies (MS(E)) can help improve the specificity of HRMS methods.

Methods

We developed a broad-spectrum screening method on an ultraperformance liquid chromatography TOF mass spectrometer (UPLC-TOF-MS) using the MS(E) mode. A diverse set of patient samples were subjected to a simple dilute, hydrolyze, and shoot protocol and analyzed in a blind manner. Data were processed with 3 sets of criteria with increasing stringency, and the results were compared with the reference laboratory results.

Results

A combination of retention time match (±0.2 min), a protonated analyte, and fragment ion mass accuracy of ±5 ppm produced zero false-positive results. Using these criteria, we confirmed 92% (253/275) of true positives. The positive confirmation rate increased to 98% (270/275) when the requirement for a fragment ion was dropped, but also produced 53 false positives. A total of 136 additional positive drug findings not identified by the reference methods were identified with the UPLC-TOF-MS.

Conclusions

MS(E) provides a unique way to incorporate fragment ion information without the need of precursor ion selection. A primary limitation of requiring a fragment ion for positive identification was that certain drug classes required high-energy collisions, which formed many fragment ions of low abundance that were not readily detected.

Currently, the use of time of flight (TOF)-mass spectrometry (MS) in quantitative analysis of small molecules is rare. Recently, the quantitative performance of TOF mass analyzers has improved due to the advancements in TOF technology. We evaluated a Q-TOF-MS in different modes, i.e., Q-TOF-full scan (Q-TOF-FS), Q-TOF-enhanced-full scan (Q-TOF-En-FS), MS(E), Q-TOF-targeted (Q-TOF-TGT), Q-TOF-enhanced-targeted (Q-TOF-En-TGT), and compared their quantitative performance against a unit resolution LC-MS-MS (tandem quadrupole) platform. The five modes were investigated for sensitivity, linearity, signal-to-noise ratio, recovery and precision using 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) as a model compound in electrospray ionization (ESI) with negative polarity. Preliminary studies indicated that Q-TOF-FS mode was the least linear and precise; hence, it was eliminated from further investigation. Total imprecision in remaining four modes was <10%. The Q-TOF-En-FS and Q-TOF-En-TGT showed better signal intensity than their respective modes without enhancement. Overall, peak signal intensity was the highest in MS(E) mode, whereas the signal-to-noise ratio was the best in the Q-TOF-En-TGT mode. Relatively, MS(E) and Q-TOF-En-TGT modes were the best overall performers compared with the other modes. Both MS(E) and Q-TOF-En-TGT modes showed excellent precision (coefficient of variation <6%), patient correlation (r > 0.99) and linearity (range, 5-455 ng/mL) for THC-COOH analysis when compared with LC-MS-MS. We also investigated the performance of the same four modes using methamphetamine in positive ESI. Quantitative data obtained by Q-TOF-En-TGT and MS(E), using both positive and negative ESI, suggest that these modes performed better than the other modes. While unit resolution LC-MS-MS remains the optimal technique for quantification, our data showed that Q-TOF-MS can also be used to quantify small molecules in complex biological specimens.

Introduction

Immunoassays and liquid chromatography-tandem mass spectrometry assays are commonly employed in clinical laboratories for measurement of total testosterone in serum. Results obtained from either of these methodologies compare poorly due to differences in calibration and/or inadvertent detection of interfering substances by the immunoassays. Standardization efforts are underway, but recent studies indicate that accuracy remains an issue.

Methods

This study compares the results from four independently developed and validated LC-MS/MS assays for total testosterone. The calibration for each assay was verified using National Institute of Standards and Technology Standard Reference Material 971.

Results

Initially, one of the four assays had a mean percent difference of +11.44%, compared to the All Method Mean, but following re-verification of all five non-zero calibrator concentrations with the NIST SRM 971, the mean percent difference decreased to -4.88%. Subsequently, the agreement between all four assays showed a mean bias of <5% across the range of all testosterone concentrations (0.13-38.10 nmol/L; 3.7-1098 ng/dL), including at low concentrations of <1 nmol/L (<29 ng/dL).

Conclusions

Excellent agreement between four independently developed LC-MS/MS assays demonstrates that harmonization using standard reference material is attainable. However, as we found in this study, to ensure accurate calibration it is critical to validate the concentrations of new lots of calibrators.

Background The widespread availability of cannabis raises concerns regarding its effect on driving performance and operation of complex equipment. Currently, there are no established safe driving limits regarding ∆9-tetrahydrocannabinol (THC) concentrations in blood or breath. Daily cannabis users build up a large body burden of THC with residual excretion for days or weeks after the start of abstinence. Therefore, it is critical to have a sensitive and specific analytical assay that quantifies THC, the main psychoactive component of cannabis, and multiple metabolites to improve interpretation of cannabinoids in blood; some analytes may indicate recent use. Methods A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to quantify THC, cannabinol (CBN), cannabidiol (CBD), 11-hydroxy-THC (11-OH-THC), (±)-11-nor-9-carboxy-Δ9-THC (THCCOOH), (+)-11-nor-Δ9-THC-9-carboxylic acid glucuronide (THCCOOH-gluc), cannabigerol (CBG), and tetrahydrocannabivarin (THCV) in whole blood (WB). WB samples were prepared by solid-phase extraction (SPE) and quantified by LC-MS/MS. A rapid and simple method involving methanol elution of THC in breath collected in SensAbues® devices was optimized. Results Lower limits of quantification ranged from 0.5 to 2 μg/L in WB. An LLOQ of 80 pg/pad was achieved for THC concentrations in breath. Calibration curves were linear (R2>0.995) with calibrator concentrations within ±15% of their target and quality control (QC) bias and imprecision ≤15%. No major matrix effects or drug interferences were observed. Conclusions The methods were robust and adequately quantified cannabinoids in biological blood and breath samples. These methods will be used to identify cannabinoid concentrations in an upcoming study of the effects of cannabis on driving.