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.

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

OBJECTIVES: To describe and validate a reference measurement procedure (RMP) for gabapentin, employing quantitative nuclear magnetic resonance (qNMR) spectroscopy to determine the absolute content of the standard materials in combination with isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) to accurately measure serum and plasma concentrations. METHODS: A sample preparation protocol based on protein precipitation in combination with LC-MS/MS analysis using a C8 column for chromatographic separation was established for the quantification of gabapentin. Assay validation and determination of measurement uncertainty were performed according to guidance from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. ID-LC-MS/MS parameters evaluated included selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement. RESULTS: The use of qNMR provided traceability to International System (SI) units. The chromatographic assay was highly selective, allowing baseline separation of gabapentin and the gabapentin-lactam impurity, without observable matrix effects. Variability between injections, preparations, calibrations, and days (intermediate precision) was <2.3%, independent of the matrix, while the coefficient of variation for repeatability was 0.9-2.0% across all concentration levels. The relative mean bias ranged from -0.8-1.0% for serum and plasma samples. Passing-Bablok regression analysis indicated very good inter-laboratory agreement; the slope was 1.00 (95% confidence interval [CI] 0.98 to 1.03) and the intercept was -0.05 (95% CI -0.14 to 0.03). Pearson's correlation coefficient was ≥0.996. Expanded measurement uncertainties for single measurements were found to be ≤5.0% (k=2). CONCLUSIONS: This analytical protocol for gabapentin, utilizing traceable and selective qNMR and ID-LC-MS/MS techniques, allows for the standardization of routine tests and the reliable evaluation of clinical 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 a candidate reference measurement procedure by ID-LC-MS/MS for total tau protein measurement in cerebrospinal fluid (CSF).

OBJECTIVES: In clinical pratice, tau protein measurement generally relies on immunoassays (IAs), whose major drawback is the lack of results comparability due to differences in selectivity and/or calibration. This underlines the importance of establishing a traceability chain for total tau (t-tau) measurements. The objective of this work is to develop a higher order candidate reference measurement procedure (RMP) for the absolute quantification of t-tau in cerebrospinal fluid (CSF). METHODS: To calibrate the candidate RMP and establish metrological traceability to the SI units, a primary calibrator consisting in a highly purified recombinant protein was sourced. Its purity was evaluated by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) and the protein mass fraction in solution was certified by amino acid analysis (AAA). An isotopically-labelled homologue was obtained to develop a candidate RMP by isotope dilution mass spectrometry (IDMS) for t-tau absolute quantification in CSF. Calibration blends and quality control (QC) materials were gravimetrically prepared and subjected to the same preparation workflow as CSF samples, followed by LC-HRMS analysis in Parallel Reaction Monitoring (PRM) mode. RESULTS: A primary calibrator has been developed and an IDMS candidate RMP has been validated for CSF t-tau. The candidate RMP was used to certify t-tau concentration in three pools of CSF (low, medium, high). CONCLUSIONS: The candidate RMP will pave the road towards global standardization of CSF t-tau measurements. Together with commutable Certified Reference Materials (CRMs), it will allow evaluating and improving the accuracy and comparability of results provided by IAs.

Development of matrix-based reference materials for 17 beta-estradiol by the recommended reference method of ID-LC-MS/MS.

We developed and evaluated two-level, namely 2017011 and 2017012, serum-based reference materials (RMs) for 17 beta-estradiol (17 ß-E2) by the reference method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) from the remaining serum samples after routine clinical tests, to help improve clinical routine testing and provide the traceability of results. This paper describes the development process of these RMs. The National Metrology Institute of Japan (NMIJ) certified reference material (CRM) 6004-a was used as the primary RM for the measurement of 17 ß-E2. These serum-based RMs showed satisfactory homogeneity and stability. They also assessed the commutability between the reference method and the three routine clinical immunoassay systems. Besides, a collaborative study was carried out in five reference laboratories, all of which had been accredited by the China National Accreditation Service for Conformity Assessment (CNAS) in accordance with ISO/WD 15725-1. Statistical analysis of raw results and uncertainty assessment obtained certified values: 2017011 was 445.2 ± 39.0 pmol/L, and 2017012 was 761.9 ± 35.5 pmol/L.

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.

Comparison of four different immunoassays and a rapid isotope-dilution liquid chromatography-tandem mass spectrometry assay for serum folate.

OBJECTIVES: Accurate measurement of serum folate is essential for the diagnosis and management of various disorders. This study aims to investigate the between-method differences of four immunoassays and a rapid isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method. METHODS: Roche Cobas (USA), Abbott Alinity i2000 (USA), Beckman Coulter Access (USA), Mindray CL-6000i (China), and the ID-LC-MS/MS method were compared using 46 human serum samples. The results were analysed by Passing-Bablok regressions and Bland-Altman plots. A bias of 13.31% based on biological variation was used as the bias criterion. RESULTS: All the within-run and total coefficients of variation (CVs) met the specification. The folate concentrations determined by all the assays were significantly different (p=0.0028). All assays had correlation coefficients over 0.97 with each other. The 95% confidence intervals (CIs) for the slope seldom contained 1 and few 95% CIs for the intercept contained 0 in the regression equations. Compared to ID-LC-MS/MS, the biases of all assays ranged from -20.91 to 13.56 nmol/L, and the mean relative biases ranged from -9.85 to 40.33%. The predicted mean relative biases at the medical decision levels rarely met the criterion. CONCLUSIONS: Assays for serum folate had good correlations with each other but lacked good agreement. The accuracy and consistency of assays for serum folate should be measured and assessed routinely. Standardization work to improve the accuracy of serum folate assays, such as the extension of traceability to reference methods or materials, calibration standardization efforts, and assay-adjusted cut-offs should be promoted.

Isotope dilution LC-MS/MS quantification of the cystic fibrosis transmembrane conductance regulator (CFTR) modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their major metabolites in human serum.

OBJECTIVES: Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators have revolutionized the therapeutic landscape in CF treatment. These vital drugs are extensively metabolized via CYP3A, so caution must be exercised in multimodal CF therapy because of the risk of adverse drug interactions. Our goal was to develop a highly sensitive assay for the purpose of therapeutic drug monitoring (TDM) in diagnostic laboratories. METHODS: After protein precipitation, the CFTR modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their metabolites ivacaftor-M1, ivacaftor-M6, and tezacaftor-M1 were separated with a two-dimensional chromatography setup within 5 min, and quantified with stable isotope-labeled internal standards. The method was validated according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and applied to CF patient samples. RESULTS: Inaccuracy was ≤7.0% and the imprecision coefficient of variation (CV) was ≤8.3% for all quality controls (QCs). The method consistently compensated for matrix effects, recovery, and process efficiency were 105-115 and 96.5-103%, respectively. Analysis of CF serum samples provided concentrations comparable to the pharmacokinetic profile data reported in the EMA assessment report for the triple combination therapy Kaftrio. CONCLUSIONS: We hereby present a robust and highly selective isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) assay for the simultaneous quantification of the so far approved CFTR modulators and their metabolites in human serum. The assay is suitable for state-of-the-art pharmacovigilance of CFTR modulator therapy in CF patients, in order to maximize safety and efficacy, and also to establish dose-response relationships in clinical trials.

Measurement of serum progesterone by isotope dilution liquid chromatography tandem mass spectrometry: a candidate reference method and its application to evaluating immunoassays.

Measurement of serum progesterone is important in determining ovarian function. Most progesterone measurements are performed with immunoassays and results are often variable. Standardization of the measurements requires a reliable reference method. An isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for the measurement of serum progesterone was developed. Serum samples were spiked with 13C3-progesterone, extracted with a three-step liquid-liquid extraction, and analyzed by LC/MS/MS. A bracketing calibration was used for the analysis and samples were prepared gravimetrically. The developed method showed intra-run and total relative standard deviations (RSDs) of 0.50~0.58% and 0.71~1.33%, respectively. The analytical recoveries were 99.08~101.50%. Measurement results on certified reference materials obtained with this method agreed with the certified values within the stated measurement uncertainties. The method was applied to evaluate immunoassays through split-sample comparisons. A panel of 48 fresh frozen individual samples were measured with the ID-LC/MS/MS method, and six immunoassays and results were compared. Significant calibration biases and sample-specific deviations were observed on some of the immunoassays.

Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples.

Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40 µg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).

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.

A suggested protocol for value assignment using isotope-dilution-LC-MS/MS in reference measurement procedures - Exemplary application for ciprofloxacin in serum.

BACKGROUND: Reference measurement procedures for assigning values to calibration materials play a crucial role in the concept of metrological traceability in laboratory medicine. ISO standard 15,193 specifies the requirements for such measurement procedures, albeit in a very general terms. MATERIALS AND METHODS: A standard structure of analysis series for value assignment by LC-MS/MS was developed and tested,this structure was complemented by a spreadsheet file for result calculation, metadata evaluation, and finally validation and confirmation of individual analysis runs and individual results, and measurement uncertainty evaluation. This framework was applied to a procedure for the quantification of ciprofloxacin in serum as an example. RESULTS: The approach of a detailed description of the analytical procedures of isotope dilution LC-MS/MS reference measurement methods together with a highly standardized spreadsheet-based method for data processing was found to be practical and efficient. The described measurement procedure for the quantification of ciprofloxacin in serum was found to be fit for purpose. CONCLUSION: A standardized, detailed procedure for the application of isotope dilution LC-MS/MS in reference measurement procedures can complement the ISO 15193 standard with respect to this particular analytical technique, which is now widely used in the context of metrological traceability in laboratory medicine.

A sensitive, robust and high-throughput isotope dilution LC-MS/MS method for quantifying three folate forms in serum.

This study aimed to establish an isotope dilution LC-MS/MS method for the determination of folic acid, 5-formyltetrahydrofolate and 5-methyltetrahydrofolate in human serum. This method was then used to quantify these three folate forms in the healthy adult population and supplement users. A stable 96-well solid-phase extraction system was used to prepare serum samples. The highly sensitive method was established using a Shimadzu LCMS-8060NX. The linearity was good in the range of 0.1-10 nmol/l for folic acid and 5-formyltetrahydrofolate and 1.0-100 nmol/l for 5-methyltetrahydrofolate. The accuracy and precision were good. The method was sensitive, robust and high-throughput and could be used for the routine clinical monitoring of these three folate forms in the Chinese population.

A Study of a New Certified Reference Material for Accurate Determination of the Main Fusarium Mycotoxins in Whole-Wheat Flour.

Matrix certified reference materials (CRMs) play a critical role in analytical method validation and the assurance of reliable measurement results. A certified reference material (GBW(E)100813) for whole-wheat flour was developed to ensure an accurate and reliable measurement of the main Fusarium mycotoxins (deoxynivalenol (DON), nivalenol (NIV), deoxynivalenol-3-glucoside (DON-3G), and zearalenone (ZEN)). CRM candidates were prepared using sun-drying, grinding, sieving, homogenising, packaging, and gamma irradiation. The final produced CRM was packaged at 50 g per unit and stored at 20 °C. Certification was performed using isotope dilution-liquid chromatography-tandem mass spectrometry. CRM characterization was performed in eight laboratories in accordance with the requirements of ISO Guide 35. The certified values and expanded uncertainties (at a confidence of 95%, k = 2) for DON, NIV, DON-3G, and ZEN were determined to be 0.98 ± 0.12 mg/kg, 1.37 ± 0.20 mg/kg, 242 ± 35 µg/g, and 382 ± 50 µg/g. The CRM was sufficiently homogeneous between and within bottles, and remained stable for up to 12 months at 20 °C and 9 days below 40 °C for transportation. Thus, CRM can be used for quality control and method validation to ensure the accurate and reliable quantification of the main Fusarium mycotoxins in whole-wheat flour.

Isotope-dilution liquid chromatography-tandem mass spectrometry method for serum beta 2-microglobulin quantification.

Beta 2-microglobulin (B2M) is a commonly used detection index in clinical laboratories. Currently, it is used as a sensitive indicator for the early detection of kidney disease. Immunoassay is the most commonly used method for B2M detection in clinical practice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has changed the face of laboratory testing by providing high-throughput analysis with better specificity than detection methods using only antibodies. An isotope-dilution LC-MS/MS (ID-LC-MS/MS) method was developed for serum B2M quantification. After B2M denaturation, reduction and alkylation of cysteine residues, trypsin was added for digestion of B2M. Then, it was purified by a solid-phase extraction column, and the sample was injected into a high-performance LC-MS/MS for measurement. A signature peptide (VNHVTLSQPK) was selected as a surrogate for B2M. A stable isotope-labeled peptide (VNHVT[13C615N]LSQP) was used as the internal standard to quantify B2M based on the calibration curve method. The linear range of serum B2M calibration curve was from 0.25 to 40 mg/L. The limit of quantification was 0.25 mg/L, and limit of detection was 0.06 mg/L. At different concentrations of serum B2M, the precision (coefficients of variation, CV%) ranged from 1.47% to 3.97%, and accuracy (relative error, RE%) was within -3.15% and 6.80% of nominal values. The applicability and reliability of the method were verified by measuring B2M in the ERM-DA470k/IFCC and serum samples. The validated LC-MS/MS method was successfully applied to a clinical study involving quantification of serum B2M in patients with acute renal insufficiency and healthy individuals. Deming analysis showed that the ID-LC-MS/MS and immunoassay were in good agreement. The LC-MS/MS method we developed is sensitive and reliable for the absolute quantification of serum B2M.

Development of isotope dilution-liquid chromatography/tandem mass spectrometry for the accurate determination of zearalenone and its metabolites in corn.

A method using isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS) was developed for the accurate determination of zearalenone (ZEN) and its five major metabolites in corn. 13C- or 2H-labeled analogues of the target mycotoxins were used as internal standards. As the immunoaffinity columns used demonstrated selectivity to a specific chiral isomer of a racemic mixture of zearalanone-d6, a clean-up cartridge without stereoselectivity (Mycosep 226 column) was selected for the same recovery of the analyte and its internal standard with adequate elimination of matrix interferences. The method demonstrated sufficient selectivity, sensitivity, accuracy and precision over a concentration range of 20-400 µg/kg. The limit of detections and limit of quantifications were 0.14-0.33 µg/kg and 0.45-1.11 µg/kg, respectively. The accuracy values were 96.7%-103.6%, with intra and inter-day precisions of less than 3% and 4%, respectively. The expanded measurement uncertainty was less than 7% (with a 95% confidence level).

Correlation between endemic chlordecone concentrations in three bovine tissues determined by isotopic dilution liquid chromatography-tandem mass spectrometry.

Chlordecone (CLD) is an organochlorine pesticide widely used from the 1970s to the 1990s in the French West Indies that induced long-term pollution of the ecosystem. Due to involuntary soil ingestion, some species bred in open-air areas can be contaminated. As CLD is distributed in various tissues depending on the breeding species, this study focuses on the distribution of CLD in bovines. For this purpose, three tissues, i.e. fat, muscle, and liver, from 200 bovines originating from Martinique and Guadeloupe were sampled in 2016 to determine their endemic contamination levels. Analyses were performed with the official method for veterinary controls, isotopic dilution liquid chromatography-tandem mass spectrometry, which has been fully validated and which reaches a limit of quantification of 3 µg.kg-1 fresh weight (fw). Irrespective of the matrices, CLD was detected in 68% of samples (404 samples above the LOD) and quantified in 59% of samples (332 samples above the LOQ). Regarding contamination levels, the liver had a broader range of concentrations (LOQ up to 420.6 µg.kg-1 fw) than fat (LOQ up to 124.6 µg.kg-1 fw) and muscle (LOQ up to 67.6 µg.kg-1 fw). This confirms the atypical behaviour of CLD compared to other persistent organochlorine pollutants. Statistical processing demonstrated a correlation between CLD concentrations among the three studied tissues. The CLD concentration ratios were 0.54 for muscle/fat, 3.75 for liver/fat, and 0.14 for muscle/liver.

Assessment of the impact of hydrolysis on bound sulfonamide residue determination in honey using stable isotope dilution ultrahigh performance liquid chromatography tandem mass spectrometry.

In this study, an analytical method based on isotope dilution-liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) was developed as a candidate reference method for the determination of sulfonamides (SAs) in honey. To guarantee the accuracy and authenticity, the impact of hydrolysis on bound SA residues was first investigated by enabling (i) identification of sugar-bound SAs, (ii) clarifying the binding reaction rule between the SAs and sugar, (iii) detection of free SAs and sugar-bound SAs, and (iv) preparation of SA-contaminated honey. Thus, the efficiency of different hydrolysis conditions was assessed by comparing the bound SA content before and after hydrolysis. In addition, optimization of the sample pretreatment procedures and LC conditions to minimize matrix effects by separation from significant matrix interferences was also performed. Satisfactory results in terms of hydrolysis efficiency (approximately 88.3%-99.2%), extraction efficiency (84.2%-105.3%), recovery (95.9%-103.1%), and limit of quantification (0.6-1.5 µg·kg-1) were obtained.

The Role of Non-Enzymatic Degradation of Meropenem-Insights from the Bottle to the Body.

Several studies have addressed the poor stability of meropenem in aqueous solutions, though not considering the main degradation product, the open-ring metabolite (ORM) form. In the present work, we elucidate the metabolic fate of meropenem and ORM from continuous infusion to the human bloodstream. We performed in vitro infusate stability tests at ambient temperature with 2% meropenem reconstituted in 0.9% normal saline, and body temperature warmed buffered human serum with 2, 10, and 50 mg/L meropenem, covering the therapeutic range. We also examined meropenem and ORM levels over several days in six critically ill patients receiving continuous infusions. Meropenem exhibited a constant degradation rate of 0.006/h and 0.025/h in normal saline at 22 °C and serum at 37 °C, respectively. Given that 2% meropenem remains stable for 17.5 h in normal saline (≥90% of the initial concentration), we recommend replacement of the infusate every 12 h. Our patients showed inter-individually highly variable, but intra-individually constant molar ORM/(meropenem + ORM) ratios of 0.21-0.52. Applying a population pharmacokinetic approach using the degradation rate in serum, spontaneous degradation accounted for only 6% of the total clearance.

Simultaneous quantification of seven repurposed COVID-19 drugs remdesivir (plus metabolite GS-441524), chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin by a two-dimensional isotope dilution LC-MS/MS method in human serum.

BACKGROUND: The present COVID-19 pandemic has prompted worldwide repurposing of drugs. The aim of the present work was to develop and validate a two-dimensional isotope-dilution liquid chromatrography tandem mass spectrometry (ID-LC-MS/MS) method for accurate quantification of remdesivir and its active metabolite GS-441524, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in serum; drugs that have gained attention for repurposing in the treatment of COVID-19. METHODS: Following protein precipitation, samples were separated with a two-dimensional ultra-high performance liquid chromatography (2D-UHPLC) setup, consisting of an online solid phase extraction (SPE) coupled to an analytical column. For quantification, stable isotope-labelled analogues were used as internal standards for all analytes. The method was validated on the basis of the European Medicines Agency bioanalytical method validation protocol. RESULTS: Detuning of lopinavir and ritonavir allowed simultaneous quantification of all analytes with different concentration ranges and sensitivity with a uniform injection volume of 5 µL. The method provided robust validation results with inaccuracy and imprecision values of ≤ 9.59 % and ≤ 11.1 % for all quality controls. CONCLUSION: The presented method is suitable for accurate and simultaneous quantification of remdesivir, its metabolite GS-441525, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in human serum. The quantitative assay may be an efficient tool for the therapeutic drug monitoring of these potential drug candidates in COVID-19 patients in order to increase treatment efficacy and safety.