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Tandem mass spectrometry is an important analytical tool for clinical laboratories, but tests developed and validated in-house (laboratory developed tests, or LDTs) require special consideration. In late 2022, the forecast for United States (U.S.) federal regulation of LDTs changed unexpectedly when the VALID Act was not passed by the U.S. Congress. This Act would have modified the Food and Drug Administration's (FDA's) role to increase regulatory oversight for LDT providers. In this revised context, we review optimization of quantitative mass spectrometry LDT validation and suggest avenues other than an additional FDA mandate to achieve uniform best practice. Common challenges, logistical barriers, and recommendations for easing the burden of best-quality quantitative mass spectrometry LDT method validation are discussed.

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•Policies applied to confirm LC-MS/MS-based results differ between laboratories.•We suggest a systematic approach for validation of individual diagnostic series.•An individual series validation plan can be established with a checklist.

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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.

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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.

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BACKGROUND: The advantage of LC-MS/MS for analyzing serum testosterone in female, pediatric, and hypogonadal male patient samples is well accepted (J Clin Endocrinol Metab 2010;95:4542-8). However, many clinical laboratories still use testosterone immunoassays because of the technical challenges of LC-MS/MS (Clin Chem 2010;56:1234-44). Although LC-MS/MS has been shown to have better accuracy and specificity than immunoassay, better reproducibility has been more elusive because of the complexities of sample preparation (Clin Chem 2008;54:1290-7; Rev Endocr Metab Disord 2013;14:185-205). METHODS: We evaluated replacing automated immunoassay with LC-MS/MS for all testosterone analyses at the University of California San Diego Health System Center for Advanced Laboratory Medicine. We used a novel extraction media, AC Extraction Plate™ (AC Plate), from Tecan Schweiz with automated liquid handling for LC-MS/MS sample preparation. We modified the existing vendor application and validated the method for matrix effect, recovery, precision, trueness [accuracy relative to certified reference material (CRM)], specificity, reportable range, sample stability, and correlation with other methods. RESULTS: Method performance was excellent, with a reportable range of 4-1560 ng/dL (0.14-54.13 nmol/L), between-day CV <6%, mean accuracy for CRM of <4.0% bias, no interference from hemolysis, icterus, lipemia, serum separator tube gel, or common steroids/metabolites, and mean bias of 1.3% vs 4 other LC-MS/MS testosterone methods. A retrospective analysis of calibration stability suggests that sparse and/or historical calibration is feasible for routine use. CONCLUSIONS: We conclude that automated extraction with the AC Plate and a focus on robustness during method development delivered ease of use and method performance consistent with adopting LC-MS/MS for all testosterone testing.

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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.

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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.

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CONTEXT: An index case of a clinically euthyroid woman of South Asian descent was identified with discordant TSH results: undetectable TSH on our routine assay and normal TSH on an alternate assay. Low TSH concentrations due to functionally compromising TSH mutations have been reported. Here we describe a new phenomenon of functional TSH that is undetectable by 4 widely used US Food and Drug Administration (FDA)-approved TSH immunoassays marketed by a single vendor. OBJECTIVE: The purpose of this study was to identify additional cases and investigate the cause of the falsely undetectable TSH. DESIGN: All samples with TSH results of <0.01 µIU/mL were retested with a second TSH assay. Discordant samples were evaluated on up to 8 FDA-approved TSH immunoassays and the TSHß gene was sequenced. Retrospectively, thyroid function tests, diagnoses, and medications from 1.6 million individuals were analyzed. RESULTS: Out of approximately 2 million individuals, we have identified a cohort of 20 hypothyroid and euthyroid patients of shared ethnicity with falsely undetectable TSH (<0.01 µIU/mL) in 4 of 8 commercially available TSH assays. Half of these individuals were initially treated based on repeated falsely undetectable TSH values (7 euthyroid patients were treated with methimazole and 2 hypothyroid patients had doses of levothyroxine decreased). In all cases, a retrospective chart review revealed that clinical assessments and free T4 and total T3 results were inconsistent with the undetectable TSH results. Specific antibodies failing to detect TSH in these cases were identified in the 4 affected assays. A novel TSHß point mutation was identified. CONCLUSIONS: Our data suggest that these individuals have a previously unrecognized, functionally normal, TSH variant to which some monoclonal antibodies fail to bind. To assure appropriate patient management, clinicians and laboratorians need to be aware that certain TSH variants may be undetectable in some hyperselective TSH assays.

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Phencyclidine is one of the drugs of abuse included in qualitative urine drug screens that are frequently ordered in the emergency department despite concerns about specificity and clinical utility. Many drugs have been described to cause false-positive results for phencyclidine. We present 2 cases of false-positive phencyclidine qualitative urine drug screen results in patients with seizures from tramadol misuse or abuse. The involvement of tramadol and its active metabolite, N-desmethyltramadol, was confirmed by in vitro testing. These cases illustrate that tramadol and its metabolites can trigger a false-positive phencyclidine urine drug screen result in nonfatal cases and highlight the lack of specificity of the phencyclidine qualitative urine drug screen.

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Confirmation of opioids in urine samples of clinical patients requires liberation of opioids from their glucuronide conjugates. Both acid hydrolysis and enzyme hydrolysis using beta-glucuronidase from various sources have been reported, with the latter approach prevailing in most clinical toxicology laboratories. The goal of this study was to compare the efficiency of acid versus different enzyme hydrolysis methods in recovering morphine and common semisynthetic opioids from glucuronide standards and 78 patient urine samples that were screened positive for opioids as a class. Specimens were analyzed with a validated gas chromatography-mass spectrometry (GC-MS) procedure. With the exception of oxycodone, the results indicated that the majority of opioids tested were extensively glucuronide-conjugated in urine. Significantly, acid hydrolysis liberated > 90% of morphine and hydromorphone from their glucuronide standards but enzyme hydrolysis had lower and variable efficiency, depending on the opiate type and the enzyme source. In patient specimens, much higher concentrations of free codeine, morphine, hydromorphone, and oxymorphone were obtained with acid hydrolysis than with various enzyme methods. Incomplete hydrolysis using beta-glucuronidase could lead to false-negative results for many opioids when urine is tested for drugs of abuse. We conclude that acid hydrolysis is the method of choice for GC-MS confirmation of urine opioids.

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A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS-MS) method has been developed to measure the levels of the HIV-1 non-nucleoside reverse transcriptase inhibitor nevirapine (NVP) in human plasma. The analyte and internal standard (IS) are isolated from plasma by a simple perchloric acid precipitation of plasma proteins followed by centrifugation. LC-MS-MS in positive mode used pairs of ions at m/z of 267/226 for NVP and 628/421 for the IS, respectively. Two linear calibration curves were established for quantitation of NVP with the low curve ranging from 25 to 1000 ng/ml and the high curve ranging from 1000 to 10,000 ng/ml. Mean inter- and intra-assay coefficients of variation (CVs) over the ranges of the two standard curves were less than 10%. The overall recovery of NVP was 92.4%.

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A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS-MS) method has been developed to measure the levels of five HIV protease inhibitors nelfinavir (NFV), indinavir (IDV), ritonavir (RTV), saquinavir (SQV) and amprenavir (APV) in human plasma. The analytes and internal standard are isolated from plasma by a simple acetonitrile precipitation of plasma proteins followed by centrifugation. LC-MS-MS in positive mode used pairs of ions at m/z of 568.4/330.0, 614.3/421.2, 720.9/296.0, 671.1/570.2 and 505.9/245.0 for NFV, IDV, RTV, SQV and APV, respectively and 628/421 for the internal standard. Two 1/x weighted linear calibration curves for each analyte were established for quantitation with the low curve ranging from 5 to 1000 ng/ml and while the high curve ranging from 1000 to 10,000 ng/ml. Mean inter- and intra-assay coefficients of variation (CVs) over the ranges of the standard curves were less than 10%. The overall recovery of NFV, IDV, RTV, SQV and APV were 88.4, 91.4, 92.2, 88.9 and 87.6%, respectively.

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A method was developed and validated for measuring the free fraction of nelfinavir in plasma employing equilibrium dialysis for the separation of free (unbound) drug and liquid chromatography/tandem mass spectrometry for quantitation. Nelfinavir, widely used to treat HIV infection, is a highly bound HIV protease inhibitor with the fraction bound in plasma being greater than 98%. Thus variations in the free fraction may be clinically important when interpreting total drug concentrations. Optimization of the method was carried out considering the influence of sample matrix and physicochemical and absorptive properties of nelfinavir. Nelfinavir free fraction averaged 0.41 +/- 0.094, 0.43 +/- 0.087 and 0.41 +/- 0.063% at nelfinavir plasma concentrations of 1000, 2000 and 3000 ng/ml, respectively. Free nelfinavir concentrations were underestimated with this assay by approximately 25% because of unavoidable losses to adsorption. However, the adsorptive loss was reproducible and consistent across the concentration range of the assay. Within-day and between-day precisions ranged from 6.0 to 9.4% and 15.2 to 27.3%, respectively. The lower limit of quantitation of the unbound concentration of nelfinavir was 1.0 ng/ml, permitting analysis of samples with total concentrations of nelfinavir in plasma that are > or = 400 ng/ml. This developed method proves reproducible and sensitive and its application to patient plasma samples is also reported.

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