Improving Harmonization and Standardization of Expanded Newborn Screening Results by Optimization of the Legacy Flow Injection Analysis Tandem Mass Spectrometry Methods and Application of a Standardized Calibration Approach.

BACKGROUND: Newborn screening (NBS) laboratories in the United Kingdom adhere to common protocols based on single analyte cutoff values (COVs); therefore, interlaboratory harmonization is of paramount importance. Interlaboratory variation for screening analytes in UK NBS laboratories ranges from 17% to 59%. While using common stable isotope internal standards has been shown to significantly reduce interlaboratory variation, instrument set-up, sample extraction, and calibration approach are also key factors. METHODS: Dried blood spot (DBS) extraction processes, instrument set-up, mobile-phase composition, sample introduction technique, and calibration approach of flow injection analysis-tandem mass spectrometry (FIA-MS/MS) methods were optimized. Inter- and intralaboratory variation of methionine, leucine, phenylalanine, tyrosine, isovaleryl-carnitine, glutaryl-carnitine, octanoyl-carnitine, and decanoyl-carnitine were determined pre- and postoptimization, using 3 different calibration approaches. RESULTS: Optimal recovery of analytes from DBS was achieved with a 35-min extraction time and 80% methanol (150 µL). Optimized methodology decreased the mean intralaboratory percentage relative SD (%RSD) for the 8 analytes from 20.7% (range 4.1-46.0) to 5.4% (range 3.0-8.5). The alternative calibration approach reduced the mean interlaboratory %RSD for all analytes from 16.8% (range 4.1-25.0) to 7.1% (range 4.1-11.0). Nuclear magnetic resonance analysis of the calibration material highlighted the need for standardization. The purities of isovaleryl-carnitine and glutaryl-carnitine were 85.13% and 69.94% respectively, below the manufacturer's stated values of ≥98%. CONCLUSIONS: For NBS programs provided by multiple laboratories using single analyte COVs, harmonization and standardization of results can be achieved by optimizing legacy FIA-MS/MS methods, adopting a common analytical protocol, and using standardized calibration material rather than internal calibration.

Validation of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of hydroxychloroquine and metabolites in human whole blood.

Objectives Hydroxychloroquine (HCQ) is an anti-malarial and immunomodulatory drug reported to inhibit the Corona virus, SARS-CoV-2, in vitro. At present there is insufficient evidence from clinical trials to determine the safety and efficacy of HCQ as a treatment for COVID-19. However, since the World Health Organisation declared COVID-19 a pandemic in March 2020, the US Food and Drug Administration issued an Emergency Use Authorisation to allow HCQ and Chloroquine (CQ) to be distributed and used for certain hospitalised patients with COVID-19 and numerous clinical trials are underway around the world, including the UK based RECOVERY trial, with over 1000 volunteers. The validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of HCQ and two of its major metabolites, desethylchloroquine (DCQ) and di-desethylchloroquine (DDCQ), in whole blood is described. Methods Blood samples were deproteinised using acetonitrile. HCQ, DCQ and DDCQ were chromatographically separated on a biphenyl column with gradient elution, at a flow rate of 500 µL/min. The analysis time was 8 min. Results For each analyte linear calibration curves were obtained over the concentration range 50-2000 µg/L, the lower limit of quantification (LLOQ) was 13 µg/L, the inter-assay relative standard deviation (RSD) was <10% at 25, 800 and 1750 µg/L and mean recoveries were 80, 81, 78 and 62% for HCQ, d4-HCQ, DCQ and DDCQ, respectively. Conclusion This method has acceptable analytical performance and is applicable to the therapeutic monitoring of HCQ, evaluating the pharmacokinetics of HCQ in COVID-19 patients and supporting clinical trials.

Automation of tacrolimus measurement on volumetric absorptive microsampling devices by tandem mass spectrometry.

Aims: An automated method for the measurement of blood tacrolimus on volumetric absorptive microsampling (VAMS) devices was developed. Materials & methods: VAMS devices prepared by the automated method were compared with those prepared by the existing manual method (n = 284; mean concentration: 8.0 µg/l; range: 0.6-18.1). Results: The performance of both methods was comparable. Passing-Bablok regression demonstrated an acceptable correlation (y = -0.449 + 1.06x). Bland-Altman analysis demonstrated acceptable agreement (mean bias: -0.007 µg/l; standard deviation: 1.536). Automation reduced operator touch time by 40 min (48-sample batch). Conclusion: Automated preparation of VAMS devices reduced touch time and improved process consistency, facilitating high-throughput testing and transformation of existing laboratory workflows. Automation did not improve precision for VAMS devices but did so for liquid blood samples.

After a kidney transplant, many patients take a drug called tacrolimus to help prevent their new kidney from being rejected. Blood levels of tacrolimus are checked regularly to ensure each patient is receiving the right dose. This means regular visits to the hospital for blood tests, which can be inconvenient and time-consuming for the patient. Microsampling devices are now available that would enable patients to collect blood from a finger prick sample, at home, and post it back to the lab for testing. However, to date, access to home sampling is limited because measuring tacrolimus from blood collected on a microsampling device relies on a manual laboratory process that is difficult to do and takes a long time. Measurement of tacrolimus from blood collected on a microsampling device can be successfully automated with a Gerstel MPS robot. The robot extracts the tacrolimus from the blood on the microsampling device and injects the resulting sample into a mass spectrometer for measurement. Two sets of microsamples were prepared. One set of samples was extracted by the robot and one set of VAMS samples was extracted manually. Tacrolimus was measured by mass spectrometry for both sets of samples and the results compared well. The automated method requires less operator input than the manual method, which will make it easier to measure large numbers of microsamples quickly and safely, increasing the number of patients who can benefit from the advantages of remote sampling.

Evaluation of a Common Internal Standard Material to Reduce Inter-Laboratory Variation and Ensure the Quality, Safety and Efficacy of Expanded Newborn Screening Results When Using Flow Injection Analysis Tandem Mass Spectrometry with Internal Calibration.

In 2015, the newborn screening (NBS) programmes in England and Wales were expanded to include four additional disorders: Classical Homocystinuria, Isovaleric Acidemia, Glutaric Aciduria Type 1 and Maple Syrup Urine Disease, bringing the total number of analytes quantified to eight: phenylalanine, tyrosine, leucine, methionine, isovalerylcarnitine, glutarylcarnitine, octanoylcarnitine and decanoylcarnitine. Post-implementation, population data monitoring showed that inter-laboratory variation was greater than expected, with 90th centiles varying from 17 to 59%. We evaluated the effect of stable isotope internal standard (IS) used for quantitation on inter-laboratory variation. Four laboratories analysed routine screening samples (n > 101,820) using a common IS. Inter-laboratory variation was determined for the eight analytes and compared with results obtained using an in-house common IS (n > 102,194). A linear mixed-effects model was fitted to the data. Using a common IS mix reduced the inter-laboratory variation significantly (p < 0.05) for five analytes. For three analytes, the lack of significance was explained by use of individual laboratory "calibration factors". For screening programmes where laboratories adhere to single analyte cut-off values (COVs), it is important that inter-laboratory variation is minimised, primarily to prevent false positive results. Whilst the use of a common IS helps achieve this, it is evident that instrument set-up also contributes to inter-laboratory variation.

Challenging the status quo: A comparison of ion exchange chromatography with liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods for the measurement of amino acids in human plasma.

BACKGROUND: Plasma amino acid analysis is key to the diagnosis and monitoring of inherited disorders of amino acid synthesis, catabolism and transport. Ion exchange chromatography (IEC) is widely accepted as the gold standard method of analysis, but with the introduction of liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography mass spectrometry (LC-MS) methods, this should now be questioned. METHODS: The analytical performance of three commercially available reagent kits, Waters AccQ Tag™ ULTRA LC-MS, SpOtOn Amino Acids LC-MS/MS and Chromsystems MassChrom® Amino Acid Analysis LC-MS/MS, were evaluated and compared with Biochrom Physiological Amino Acids ion exchange chromatography. Correlation with IEC was assessed by Passing-Bablok regression, concordance correlation coefficients (CCC) and Bland-Altman analysis for 21 common amino acids. Calculation of the total error from imprecision and bias was also used to benchmark performance. RESULTS: The MassChrom® and SpOtOn kits demonstrated acceptable inter-batch imprecision (CV < 10%) and accuracy (mean bias < 10%), whereas the AccQ Tag™ ULTRA kit did not. Good correlation (CCC > 0.95) with Biochrom IEC was demonstrated for 10/21 analytes in both the MassChrom® and SpOtOn kits and 6/21 in the AccQ Tag™ ULTRA kit. CONCLUSIONS: The LC-MS assay demonstrated variable analytical performance and correlated poorly with ion exchange chromatography. Both LC-MS/MS assays demonstrated comparable analytical performance and reasonable correlation with ion exchange chromatography. They also confer practical advantages which cannot be realized by ion exchange chromatography, superior specificity and significantly faster analysis time, suggesting that ion exchange chromatography should no longer be described as the gold standard method for plasma amino acid analysis.