Evaluation and clinical application of a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for serum theophylline.

A candidate reference measurement procedure (RMP) for serum theophylline via isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. With a single-step precipitation pretreatment and a 6-min gradient elution, the method achieved baseline separation of theophylline and its analogs on a C18-packed column. A bracketing calibration method was used to ensure repeatable signal intensity and high measurement precision. The intra-assay and inter-assay imprecisions were 1.06%, 0.84%, 0.72% and 0.47%, 0.41%, 0.25% at concentrations of 4.22 µg/mL (23.40 µmol/L), 8.45 µg/mL (46.90 µmol/L), and 15.21 µg/mL (84.43 µmol/L), respectively. Recoveries ranged from 99.35 to 102.34%. The limit of detection (LoD) was 2 ng/mL, and the lowest limit of quantification (LLoQ) was 5 ng/mL. The linearity range extended from 0.47 to 60 µg/mL (2.61-333.04 µmol/L). No ion suppression and carry-over (< 0.68%) were observed. The relative bias for this candidate RMP that participated in 2023 External Quality Control for Reference Laboratories (RELA) conducted by the International Federation of Clinical Chemistry (IFCC) was within a range of 0.17 to 0.93%. Furthermore, two clinical immunoassay systems were compared with this candidate RMP, demonstrating good correlations. The results of the Trueness Verification Plan indicate significant differences among routine systems, highlighting the need for standardization efforts. The developed candidate RMP for serum theophylline serves as a precise reference baseline for standardizing clinical systems and assigning values to reference materials.

Establishment and clinical application of a candidate reference measurement procedure for quantification of urinary vanillylmandelic acid and homovanillic acid using ID-LC-MS/MS method.

Neuroblastoma (NB), an embryonic tumor of the autonomous nervous system, poses a significant threat to the health and lives of children. Accurate measurement of vanillylmandelic acid (VMA) and homovanillic acid (HVA) in human urine is crucial for screening and diagnosis of NB. Although various laboratories have developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect VMA and HVA, the comparability between the results obtained from different laboratories and methods was poor. The absence of reference method for VMA and HVA hinders the standardization of their measurements. Therefore, a candidate reference measurement procedure (cRMP) based on isotope dilution LC-MS/MS (ID-LC-MS/MS) for the detection of VMA and HVA in human urine was established. Urine samples were spiked with VMA-d3 and HVA-d5 as internal standards and extracted using a protein precipitation method. The cRMP exhibited desirable precision with the total imprecision below 5 %. The accuracy of this cRMP was demonstrated by the high analytical recovery (98.64 % - 102.22 % and 98.41 % - 100.97 % for VMA and HVA, respectively), and comparability between different reference systems. The limit of detection for HVA and VMA were 15.625 ng/mL and 3.906 ng/mL, respectively; the quantification limits were 62.5 ng/mL and 7.813 ng/mL, respectively, which can meet the clinical detection requirements. The linear range was from 78.125 ng/mL to 20 µg/mL. Specificity evaluations showed no corresponding interference from structurally similar analogs. In conclusion, we have established a cRMP based on ID-LC-MS/MS for the measurement of VMA and HVA in urine samples, demonstrating well-defined method performance including accuracy, precision, and specificity. This newly established cRMP is suitable for routine assay standardization and evaluation of clinical samples. Furthermore, this method has the potential to significantly enhance the diagnostic accuracy for neuroblastoma.

Enhancing the understanding of SARS-CoV-2 protein with structure and detection methods: An integrative review.

As the rapid and accurate screening of infectious diseases can provide meaningful information for outbreak prevention and control, as well as owing to the existing limitations of the polymerase chain reaction (PCR), it is imperative to have new and validated detection techniques for SARS-CoV-2. Therefore, the rationale for outlining the techniques used to detect SARS-CoV-2 proteins and performing a comprehensive comparison to serve as a practical benchmark for future identification of similar viral proteins is clear. This review highlights the urgent need to strengthen pandemic preparedness by emphasizing the importance of integrated measures. These include improved tools for pathogen characterization, optimized societal precautions, the establishment of early warning systems, and the deployment of highly sensitive diagnostics for effective surveillance, triage, and resource management. Additionally, with an improved understanding of the virus' protein structure, considerable advances in targeted detection, treatment, and prevention strategies are expected to greatly improve our ability to respond to future outbreaks.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of primidone in human serum and plasma.

OBJECTIVES: Primidone is an anticonvulsive drug used in the treatment of epilepsy and essential tremor. It offers beneficial effects in controlling seizures, but its usage is also associated with possible side effects. To ensure optimal therapy, it is crucial to measure its concentration through accurate quantification methods. Therefore, our main goal was to develop and validate a new reference measurement procedure (RMP) for accurately measuring primidone levels in human serum and plasma. METHODS: In our study, we focused on the separation of primidone from both known and unknown interferences using a C18 column. To achieve accurate sample preparation, we developed a protocol involving protein precipitation followed by a high dilution step. The validation of the assay and determination of measurement uncertainty were carried out following guidelines from organizations such as the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. These rigorous validation processes ensure the reliability and accuracy of our method for quantifying primidone levels in human serum and plasma samples. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interference. It can be used to quantify primidone in the range of 0.150-30.0 µg/mL. Intermediate precision was less than 4.0 %, and repeatability CV ranged from 1.0 to 3.3 % across all concentration levels. The relative mean bias ranged from 0.1 to 3.9 % for native serum levels, and from -2.6 to 2.8 % for lithium-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were 1.5-4.1 % and 0.9-1.0 %, respectively. CONCLUSIONS: In this study, we introduce an innovative LC-MS/MS-based candidate RMP specifically designed for primidone in human serum and plasma. Our RMP offers a traceable platform, facilitating the standardization of routine assays and enabling the evaluation of clinically relevant samples. With this novel approach, we aim to enhance the accuracy and reliability of primidone measurements, ultimately benefiting the field of clinical research and patient care.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine-10,11-epoxide in human serum and plasma.

OBJECTIVES: A reference measurement procedure (RMP) using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated with the aim of accurately measuring carbamazepine-10,11-epoxide concentrations in human serum and plasma. METHODS: To establish traceability to SI units, the absolute content of the reference material was determined using quantitative nuclear magnetic resonance (qNMR) spectroscopy. As sample preparation a protein precipitation protocol followed by a high dilution step was established. Chromatographic separation from carbamazepine and potential metabolites was achieved using a C18 stationary phase. Selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement were evaluated based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. RESULTS: The RMP demonstrated very good selectivity and specificity, showing no evidence of a matrix effect. This enabled accurate quantification of carbamazepine-epoxide in the concentration range of 0.0400-12.0 µg/mL. The intermediate precision was found to be less than 2.1 %, and the repeatability coefficient of variation (CV) ranged from 1.2 to 1.8 % across all concentration levels. Regarding accuracy, the relative mean bias varied from 1.4 to 2.5 % for native serum levels and from 1.4 to 3.5 % for Li-heparin plasma levels. The measurement uncertainty for single measurements ranged from 1.6 to 2.1 %. CONCLUSIONS: In this study, we introduce a new LC-MS/MS-based candidate RMP for accurately measuring carbamazepine-10,11-epoxide in human serum and plasma. This novel method offers a traceable and dependable platform, making it suitable for standardizing routine assays and assessing clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of phenobarbital in human serum and plasma.

OBJECTIVES: Phenobarbital serves as an antiepileptic drug (AED) and finds application in the treatment of epilepsy either as monotherapy or adjunctive therapy. This drug exhibits various pharmacodynamic properties that account for its beneficial effects as well as potential side effects. Accurate measurement of its concentration is critical for optimizing AED therapy through appropriate dose adjustments. Therefore, our objective was to develop and validate a new reference measurement procedure (RMP) for the accurate quantification of phenobarbital levels in human serum and plasma. METHODS: A sample preparation protocol based on protein precipitation followed by a high dilution step was established in combination with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a C8 column to separate target analytes from known and unknown interferences. Assay validation and determination of measurement uncertainty were performed based on current guidelines. Selectivity and Specificity were assessed using spiked serum and plasma samples; to investigate possible matrix effects (MEs) a post-column infusion experiment and a comparison of standard line slopes was performed. Precision and accuracy were determined within a multiday precision experiment. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interferences. It can be used to quantify phenobarbital in the range of 1.92 to 72.0 µg/mL. Intermediate precision was less than 3.2 %, and repeatability coefficient of variation (CV) ranged from 1.3 to 2.0 % across all concentration levels. The relative mean bias ranged from -3.0 to -0.7 % for native serum levels, and from -2.8 to 0.8 % for Li-heparin plasma levels. The measurement uncertainties (k=1) for single measurements and target value assignment were 1.9 to 3.3 % and 0.9 to 1.6 %, respectively. CONCLUSIONS: A novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma is presented which can be used for the standardization of routine assays and the evaluation of clinically relevant samples.

Evolution and impact of Standard Reference Materials (SRMs) for determining vitamin D metabolites.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health, Office of Dietary Supplements (NIH ODS), introduced the first Standard Reference Material® (SRM) for determining vitamin D metabolites in 2009 motivated by significant concerns about the comparability and accuracy of different assays to assess vitamin D status. After 14 years, a suite of five serum matrix SRMs and three calibration solution SRMs are available. Values were also assigned for vitamin D metabolites in five additional SRMs intended primarily to support measurements of other clinical diagnostic markers. Both the SRMs and the certification approach have evolved from significant exogenous serum content to primarily endogenous content and from value assignment by combining the results of multiple analytical methods to the use of measurements exclusively from reference measurement procedures (RMPs). The impact of the availability of these SRMs can be assessed by both the distribution information (sales) and by reports in the scientific literature describing their use for method validation, quality control, and research. In this review, we describe the development of these SRMs, the evolution in design and value assignment, the expansion of information reported, and SRM use in validating analytical methods and providing quality assurance within the vitamin D measurement community.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of zonisamide in human serum and plasma.

OBJECTIVES: To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations. METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was employed to determine the absolute content of the reference material used in order to establish traceability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of zonisamide within the range of 1.50-60.0 µg/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8-1.0 %, respectively. CONCLUSIONS: We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine in human serum and plasma.

OBJECTIVES: An isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed and validated to accurately measure serum and plasma concentrations of carbamazepine. METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was used to determine the absolute content of the reference material, ensuring its traceability to SI units. The separation of carbamazepine from potential interferences, whether known or unknown, was achieved using a C18 column. A protein precipitation protocol followed by a high dilution step was established for sample preparation. Assay validation and determination of measurement uncertainty were performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute, the International Conference on Harmonization (ICH), and the Guide to the Expression of Uncertainty in Measurement (GUM). In order to demonstrate equivalence to the already existing RMP a method comparison study was performed. RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of carbamazepine within the range of 0.800-18.0 µg/mL. Intermediate precision and repeatability (n=60 measurements) was found to be <1.6 % and <1.3 % over all concentration levels and independent from the matrix. The relative mean bias ranged from -0.1 to 0.6 % for native serum and from -0.3 to -0.1 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <1.8 % and <1.3 %, respectively. Method comparison showed a good agreement between the Joint Committee of Traceability in Laboratory Medicine (JCTLM) listed RMP and the candidate RMP resulting in a Passing-Bablok regression equation with a slope of 1.01 and an intercept of -0.01. The bias in the patient cohort was found to be 0.9 %. CONCLUSIONS: We present a novel LC-MS/MS-based candidate RMP for carbamazepine in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Development of an ID-LC-MS/MS method using targeted proteomics for quantifying cardiac troponin I in human serum.

BACKGROUND: Cardiac troponin is a complex protein consisting of the three subunits I, T and C located in heart muscle cells. When the heart muscle is damaged, it is released into the blood and can be detected. Cardiac troponin I (cTnI) is considered the most reliable and widely accepted test for detecting and confirming acute myocardial infarction. However, there is no current standardization between the commercial assays for cTnI quantification. Our work aims to create a measurement procedure that is traceable to the International System of Units for accurately measuring cardiac cTnI levels in serum samples from patients. METHODS: The workflow begins with immobilizing anti-cTnI antibodies onto magnetic nanoparticles to form complexes. These complexes are used to isolate cTnI from serum. Next, trypsin is used to enzymatically digest the isolated cTnI. Finally, the measurement of multiple cTnI peptides is done simultaneously using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). RESULTS: The maximum antibody immobilization was achieved by combining 1 mg of nanoparticles with 100 µg of antibody, resulting in an average of 59.2 ± 5.7 µg/mg of immobilized antibody. Subsequently, the anti-cTnI-magnetic nanoparticle complex was utilized to develop and validate a method for quantifying cTnI in human serum using ID-LC-MS/MS and a protein calibration approach. The analytical method was assessed regarding linearity and recovery. The developed method enables the quantification of cTnI from 0.7 to 24 µg/L (R > 0.996). The limit of quantification was 1.8 µg/L and the limit of detection was 0.6 µg/L. Intermediate precision was ≤ 9.6% and repeatability was 2.0-8.7% for all quality control materials. The accuracy of the analyzed quality control materials was between 90 and 110%. Total measurement uncertainties for target value assignment (n = 6) were found to be ≤ 12.5% for all levels. CONCLUSIONS: The analytical method demonstrated high analytical performance in accurately quantifying cardiac troponin I levels in human serum. The proposed analytical method has the potential to facilitate the harmonization of cTnI results between clinical laboratories, assign target values to secondary certified reference materials and support reliable measurement of cTnI.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of topiramate in human serum and plasma.

OBJECTIVES: Topiramate is an antiepileptic drug (AED) used for the monotherapy or adjunctive treatment of epilepsy and for the prophylaxis of migraine. It has several pharmacodynamic properties that contribute to both its clinically useful properties and observed adverse effects. Accurate measurement of its concentration is therefore essential for dose adjustment/optimisation of AED therapy. Our aim was to develop and validate a novel reference measurement procedure (RMP) for the quantification of topiramate in human serum and plasma. METHODS: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method in combination with a protein-precipitation-based sample preparation allows for quantification of topiramate in human serum and plasma. To assure traceability to SI units, quantitative nuclear magnetic resonance (qNMR) was applied to characterize the reference material used as primary calibrator for this RMP. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Accuracy and precision was evaluated performing an extensive five day precision experiment and measurement uncertainty was evaluated according Guide to the Expression of Uncertainty in Measurement (GUM). RESULTS: The method enabled topiramate quantification within the range of 1.20-36.0 µg/mL without interference from structurally related compounds and no evidence of a matrix effect. Intermediate precision was ≤3.2 % and repeatability was 1.4-2.5 % across all concentration levels. The relative mean bias was -0.3 to 3.5 %. Expanded measurement uncertainties for target value assignment (n=6) were found to be ≤2.9 % (k=2) independent of the concentration level and the nature of the sample. CONCLUSIONS: In human serum and plasma, the RMP demonstrated high analytical performance for topiramate quantification and fulfilled the requirements on measurement uncertainty. Traceability to SI units was established by qNMR content determination of the topiramate, which was used for direct calibration of the RMP. This RMP is, therefore, fit for purpose for routine assay standardization and clinical sample evaluation.

Developing an SI-traceable Lp(a) reference measurement system: a pilgrimage to selective and accurate apo(a) quantification.

In the past decade a remarkable rebirth of serum/plasma lipoprotein(a) (Lp(a)) as an independent risk factor of cardiovascular disease (CVD) occurred. Updated evidence for a causal continuous association in different ethnic groups between Lp(a) concentrations and cardiovascular outcomes has been published in the latest European Atherosclerosis Society (EAS) Lp(a) consensus statement. Interest in measuring Lp(a) at least once in a person's lifetime moreover originates from the development of promising new Lp(a) lowering drugs. Accurate and clinically effective Lp(a) tests are of key importance for the timely detection of high-risk individuals and for future evaluation of the therapeutic effects of Lp(a) lowering medication. To this end, it is necessary to improve the performance and standardization of existing Lp(a) tests, as is also noted in the Lp(a) consensus statement. Consequently, a state-of-the-art internationally endorsed reference measurement system (RMS) must be in place that allows for performance evaluation of Lp(a) field tests in order to certify their validity and accuracy. An ELISA-based RMS from Northwest Lipid Research Laboratory (University of Washington, Seattle, USA) has been available since the 1990s. A next-generation apo(a)/Lp(a) RMS is now being developed by a working group from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). The envisioned apo(a) RMS is based on the direct measurement of selected proteotypic fragments generated after proteolytic digestion using quantitative protein mass spectrometry (MS). The choice for an MS-based RMS enables selective measurement of the proteotypic peptides and is by design apo(a) isoform insensitive. Clearly, the equimolar conversion of apo(a) into the surrogate peptide measurands is required to obtain accurate Lp(a) results. The completeness of proteolysis under reaction conditions from the candidate reference measurement procedure (RMP) has been demonstrated for the quantifying apo(a) peptides. Currently, the candidate apo(a) RMP is endorsed by the IFCC and recommendations for suitable secondary reference materials have been made in a recent commutability study paper. Ongoing efforts toward a complete apo(a) RMS that is listed by the Joint Committee on Traceability in Laboratory Medicine (JCTLM) are focused on the peptide-based calibration and the establishment of a network of calibration laboratories running the apo(a) RMS in a harmonized way. Once completed, it will be the holy grail for evaluation and certification of Lp(a) field methods.

Determination of 24,25-dihydroxyvitamin D3 in Vitamin D External Quality Assessment Scheme samples using a reference measurement procedure.

Ninety archived human serum samples from the Vitamin D External Quality Assessment Scheme (DEQAS) were analyzed using a reference measurement procedure (RMP) based on isotope dilution liquid chromatography - tandem mass spectrometry (ID LC-MS/MS) for the determination of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3]. These 24,25(OH)2D3 results, in conjunction with concentration values assigned using RMPs for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3], provide a valuable resource for assessing the accuracy of measurements for 24,25(OH)2D3 and for investigating the relationship between 24,25(OH)2D3 and 25(OH)D3. Results for 24,25(OH)2D3 using the RMP were compared to DEQAS consensus values demonstrating that the consensus values were not sufficient to assess the accuracy of measurements among different laboratories and methods. A multivariable regression analysis approach using historical DEQAS consensus values for various total 25(OH)D assays was used to assess the contribution of 24,25(OH)2D3 concentration on the assay response. The response of several ligand binding assays for total 25(OH)D was shown to be impacted by the presence of 24,25(OH)2D3.

Estimation of measurement uncertainty for the quantification of protein by ID-LC-MS/MS.

The emergence of mass spectrometry (MS)-based methods to quantify proteins for clinical applications has led to the need for accurate and consistent measurements. To meet the clinical needs of MS-based protein results, it is important that the results are traceable to higher-order standards and methods and have defined uncertainty values. Therefore, we outline a comprehensive approach for the estimation of measurement uncertainty of a MS-based procedure for the quantification of a protein biomarker. Using a bottom-up approach, which is the model outlined in the "Guide to the Expression of Uncertainty of Measurement" (GUM), we evaluated the uncertainty components of a MS-based measurement procedure for a protein biomarker in a complex matrix. The cause-and-effect diagram of the procedure is used to identify each uncertainty component, and statistical equations are derived to determine the overall combined uncertainty. Evaluation of the uncertainty components not only enables the calculation of the measurement uncertainty but can also be used to determine if the procedure needs improvement. To demonstrate the use of the bottom-up approach, the overall combined uncertainty is estimated for the National Institute of Standards and Technology (NIST) candidate reference measurement procedure for albumin in human urine. The results of the uncertainty approach are applied to the determination of uncertainty for the certified value for albumin in candidate NIST Standard Reference Material® (SRM) 3666. This study provides a framework for measurement uncertainty estimation of a MS-based protein procedure by identifying the uncertainty components of the procedure to derive the overall combined uncertainty.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of levetiracetam in human serum and plasma.

OBJECTIVES: To develop an isotope dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for levetiracetam quantification in human serum and plasma. METHODS: Quantitative nuclear magnetic resonance spectroscopy (qNMR) was used to characterize the RMP material to ensure traceability to SI units. To quantify levetiracetam, an LC-MS/MS method was optimized using a C8 column for chromatographic separation following protein-precipitation-based sample preparation. Spiked matrix samples of serum and plasma were used to test selectivity and specificity. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Precision and accuracy were evaluated over 5 days. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM). RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of levetiracetam within the range of 1.53-90.0 µg/mL. Intermediate precision was <2.2% and repeatability was 1.1-1.7% across all concentrations. The relative mean bias ranged from -2.5% to -0.3% across all levels and matrices within the measuring range. Diluted samples were found with a mean bias ranging from -0.1 to 2.9%. The predefined acceptance criterion for measurement uncertainty was met and determined for individual measurements independently of the concentration level and sample type to be ≤4.0% (k=2). CONCLUSIONS: We present a novel LC-MS/MS)-based candidate RMP for levetiracetam in human serum and plasma. Its expanded measurement uncertainty of ≤4.0% meets the clinical needs in levetiracetam monitoring. Utilizing qNMR to characterize levetiracetam reference materials allowed metrological traceability to SI units.

Development of an equilibrium dialysis ID-UPLC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

BACKGROUND: Accurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. METHODS: Serum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. RESULTS: The described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. CONCLUSION: Our ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays.

Description and validation of an equilibrium dialysis ID-LC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

OBJECTIVES: Free thyroxine (FT4) in serum is routinely measured in clinical practice to diagnose and monitor thyroid disease. Due to its concentration in picomolar range and the delicate equilibrium of free and protein-bound T4, accurate measurement is challenging. As a consequence, large inter-method differences in FT4 results exists. Optimal method design and standardization of the FT4 measurement is therefore necessary. The IFCC Working Group for Standardization of Thyroid Function Tests proposed a reference system with a conventional reference measurement procedure (cRMP) for FT4 in serum. In this study, we describe our FT4 candidate cRMP and its validation in clinical samples. METHODS: This candidate cRMP is based on equilibrium dialysis (ED) combined with determination of T4 with an isotope-dilution liquid chromatography tandem mass-spectrometry (ID-LC-MS/MS) procedure and was developed according to the endorsed conventions. Its accuracy, reliability, and comparability was investigated using human sera. RESULTS: It was shown that the candidate cRMP adhered to the conventions and its accuracy, precision, and robustness were adequate in serum of healthy volunteers. CONCLUSIONS: Our candidate cRMP measures FT4 accurately and performs well in serum matrix.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of aldosterone in human serum and plasma.

OBJECTIVES: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) for aldosterone quantification in human serum and plasma is presented. METHODS: The material used in this RMP was characterized by quantitative nuclear magnetic resonance (qNMR) to assure traceability to SI Units. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis a two-dimensional heart cut LC approach, in combination with an optimal supported liquid extraction protocol, was established for the accurate analysis of aldosterone in human serum and plasma in order to minimize matrix effects and avoid the co-elution of interferences. Assay validation was performed according to current guidelines. Selectivity and specificity were assessed using spiked serum; potential matrix effects were examined by a post column infusion experiment and the comparison of standard line slopes. An extensive protocol over 5 days was applied to determine precision, accuracy and trueness. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), for which three individual sample preparations were performed on at least two different days. RESULTS: The RMP allowed aldosterone quantification within the range of 20-1,200 pg/mL without interference from structurally-related compounds and no evidence of matrix effects. Intermediate precision was ≤4.7% and repeatability was 2.8-3.7% for all analyte concentrations. The bias ranged between -2.2 and 0.5% for all levels and matrices. Total measurement uncertainties for target value assignment (n=6) were found to be ≤2.3%; expanded uncertainties were ≤4.6% (k=2) for all levels. CONCLUSIONS: The RMP showed high analytical performance for aldosterone quantification in human serum and plasma. The traceability to SI units was established by qNMR content determination of aldosterone, which was utilized for direct calibration of the RMP. Thus, this candidate RMP is suitable for routine assay standardization and evaluation of clinical samples.