Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use.

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.

Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis.

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

Analytical Evaluation of the Ideal Strategy for High-Throughput Flow Injection Analysis by Tandem Mass Spectrometry in Routine Newborn Screening.

The introduction of tandem mass spectrometry (MS/MS) to clinical laboratories and the advent of expanded newborn screening (NBS) were crucial changes to public health programs worldwide. Speed, robustness, accuracy, selectivity, and specificity of analysis are all requirements of expanded NBS and are needed to minimize false positive results risks, to possibly eliminate false negatives, and to improve the positive predictive value of NBS. In this study, we firstly evaluated the analytical performances of the RenataDX Screening System, a fully integrated flow-injection MS/MS (FIA-MS/MS) IVD system for high-throughput dried blood spot (DBS) analysis in a routine NBS laboratory. Since a choice of several commercial NBS kits is available, we sought to compare NeoBaseTM 2 (PerkinElmer®) and MassChrom® (Chromsystems) non-derivatized kits on the RenataDX platform by evaluating their analytical performances. Moreover, we verified the degree of correlation between data obtained by the two different NBS MS/MS kits by FIA-MS/MS of over 500 samples. Our data suggest that both methods correlate well with clinically insignificant differences that do not impact the NBS result. Finally, while NeoBase™ 2 offers an easier and faster sample preparation, MassChrom® provides a cleaner sample extract which empirically should improve instrument reliability.

Standardization of LC-MS for therapeutic drug monitoring of tacrolimus.

BACKGROUND: LC-MS is increasingly used for therapeutic drug monitoring of tacrolimus. A recent summary from an international proficiency-testing scheme demonstrated that the mass spectrometry respondents were the largest method group. However, these methods lack standardization, which may explain the relatively poor interlaboratory agreement for such methods. This study aimed to provide one path toward the standardization of tacrolimus quantification by use of LC-MS. METHODS: A 40-member whole blood tacrolimus proficiency panel was circulated to 7 laboratories, 4 in the US and 3 in Europe, offering routine LC-MS-based quantification of tacrolimus. All laboratories used a common LC-MS platform and followed the manufacturer's instructions that accompanied an LC-MS reagent kit intended for tacrolimus quantification in whole blood samples. Four patient pools were prepared that had sufficient volume to allow comparison with a tacrolimus reference measurement procedure. RESULTS: For the 40-member panel, the standardized MassTrak LC-MS assay demonstrated excellent agreement with a validated LC-MS method used by Analytical Services International (y = 1.02x - 0.02; r = 0.99). The CVs for the pooled patient samples ranged from 2.0% to 5.4%. The mean difference from the reference measurement procedure ranged from 0.4% to 4.4%. CONCLUSIONS: Tacrolimus assay standardization, which must include all facets of the analysis, is necessary to compare patient results between laboratories and to interpret consensus guidelines. LC-MS can provide accurate and precise measurement of tacrolimus between laboratories.

Confirmation of congenital adrenal hyperplasia by adrenal steroid profiling of filter paper dried blood samples using ultra-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: The specificity of screening for congenital adrenal hyperplasia by direct measurement of 17-hydroxyprogesterone in filter paper dried blood spot samples by immunoassay is low and has a high false-positive rate. In order to reduce the false-positive rate of this test, we developed a rapid, robust, specific confirmatory procedure in which cortisol, 4-androstene-3,17-dione and 17-hydroxyprogesterone were measured simultaneously by ultra-performance liquid chromatography-tandem mass spectrometry. METHODS: After extraction, samples were analysed by ultra-performance liquid chromatography-tandem mass spectrometry and 17-hydroxyprogesterone was quantified accurately. Other steroids were determined using stable deuterated internal standards. In total, 25 patient blood spot samples and 92 control samples were analysed. RESULTS: The assay was linear for 17-hydroxyprogesterone, with a coefficient of determination >0.997 and imprecision ≤ 6.5%. An upper limit of normal for 17-hydroxyprogester-one of 4.45 nmol/L was established by analysing a cohort of samples from unaffected newborns. In addition, a cut-off of 3.5 for the peak areas ratio (17-hydroxyprogesterone+4-androstene-3,17-dione)/cortisol, allows confirmation of the affected steroidogenic enzyme. CONCLUSIONS: A high throughput method for the detection of steroids related to congenital adrenal hyperplasia has been developed, allowing the false-positive rate associated with screening for 17-hydroxyprogesterone by immunoassay to be determined.

Serum steroid profiling for congenital adrenal hyperplasia using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Diagnosis of Congenital Adrenal Hyperplasia (CAH) is based on the quantification of 17-hydroxyprogesterone (17-OHP), usually by immunoassay. During the neonatal period the specificity of screening for CAH by blood spot 17-OHP immunoassay is low. High false-positive rates result in a relatively high demand for a second-tier serum confirmation test. A robust, specific and selective method for measurement of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstene-3,17-dione (A4) and 17-OHP in serum has been developed. The method involves a simple extraction procedure and a fast analysis using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS). METHODS: The steroids were extracted from 50microl of serum using methyl-tert-butyl-ether. Analysis was performed on a UPLC tandem quadrupole mass spectrometer system in positive mode electrospray ionization and multiple reaction monitoring acquisition. RESULTS: The assay was linear over each analyte concentration range with all correlation coefficients (r(2))>0.996. Inter- and intra-day CVs were

A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Measurement of 25-hydroxyvitamin D(3) (25OHD(3)) and D(2) (25OHD(2)) is challenging. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been described but they are often complex and difficult to automate. We have developed a simplified procedure involving an automated solid-phase extraction (SPE). METHODS: Internal standard (hexadeuterated 25-hydroxyvitamin D(3)) was added to serum or plasma followed by protein precipitation with methanol. Following centrifugation, a robotic instrument (CTC PAL [Presearch] for ITSP SPE [MicroLiter Analytical Supplies, Inc]) performed a six-step SPE procedure and the purified samples were injected into the LC-MS/MS. Quantification of 25OHD(3) and 25OHD(2) was by electrospray ionization MS/MS in the multiple-reaction monitoring mode. RESULTS: The lower limit of quantitation was 4.0 nmol/L for 25OHD(3) and 7.5 nmol/L for 25OHD(2). Within- and between-assay precision was below 10% over the concentration range of 22.5-120 nmol/L for D(3) and 17.5-70 nmol/L for D(2) (n = 10). The calibration was linear up to 2500 nmol/L (r = 0.99). Recoveries ranged between 89% and 104% for both metabolites and no ion suppression was observed. The results obtained compared well (r = 0.96) with the IDS-OCTEIA 25-hydroxyvitamin D enzyme immunoassay for samples containing less than 125 nmol/L, at higher concentrations the immunodiagnostic system (IDS) method showed positive bias. CONCLUSIONS: Our simplified sample preparation and automated SPE method is suitable for the measurement of 25OHD(3) and D(2) in a routine laboratory environment. The system can process up to 300 samples per day with no cumbersome solvent evaporation step and minimal operator intervention.