HCR/DNAzyme driven cascade signal amplified fluorescent aptasensor for detecting deoxynivalenol in food.

Eosinophilic bronchiectasis is defined by a blood eosinophil count (BEC) ≥300 cells/µL, but blood eosinophils imperfectly reflect airway eosinophilic inflammation. Here, we investigated the relationship between eosinophilic airway inflammation, blood eosinophils and clinical severity in bronchiectasis and explored the phenotype associated with eosinophilic bronchiectasis. Sputum from 180 patients with stable CT-confirmed bronchiectasis was utilised to investigate airway levels of eosinophil proteins (eosinophil peroxidase (EPX), eosinophil derived-neurotoxin (EDN), eosinophil cationic protein (ECP), major basic protein (MBP) and Galectin-10 (Gal-10)) using a novel stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. To profile eosinophilic bronchiectasis, a nested analysis of patients with BEC <150 cells/µL (n=52) and ≥300 cells/µL (n=49) was conducted. Sputum concentrations of Gal-10, ECP and EDN were weakly but significantly associated with radiological severity, FEV1 and sputum culture positivity for Pseudomonas aeruginosa. Airway eosinophil protein concentrations did not associate with exacerbation frequency. Total eosinophil protein concentration moderately correlated with BECs (r=0.33 95% CI 0.14 to 0.49, p=0.0007). Nested analysis revealed increased sputum PCR-positivity for P. aeruginosa (26.7% vs 7.7%, p=0.033) and an increased frequency of patients showing signs of Aspergillus sensitisation (defined as Aspergillus-specific IgE titres >0.35 kUA/L, 24.5% vs 3.8%) in eosinophilic bronchiectasis. Sputum inflammatory biomarkers and clinical parameters did not differ between groups. LC-MS/MS can detect eosinophilic inflammation within bronchiectasis sputum. Weak associations between elevated airway eosinophil proteins, bronchiectasis severity and P. aeruginosa infection were observed. Direct measurement of eosinophilic airway inflammation provides additional information in addition to BECs. Eosinophilic bronchiectasis associated with P. aeruginosa infection and Aspergillus sensitisation.

The AOAC Stakeholder Panel on Strategic Food Analytical Methods approved AOAC INTERNATIONAL Standard Method Performance Requirements (SMPRs®) 2023.003 for per- and polyfluoroalkyl substances (PFAS) in produce, beverages, dairy products, eggs, seafood, meat products, and feed. The U.S. Food and Drug Administration's (FDA's) method is a single laboratory validated (SLV) method for determination of 30 PFAS in food and feed. In response to a call for minimum method performance characteristics and analytical requirements, the FDA's method for detection of PFAS in food and feed was validated in a single laboratory study with comparison to AOAC SMPR 2023.003. Samples were extracted using (Quick, Easy, Cheap, Effective, Rugged, Safe) QuEChERS followed by solid phase extraction (SPE) clean-up and concentration using nitrogen. Analysis was performed using isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated for parameters such as limit of quantification, recovery, repeatability, system suitability, and reference materials were analyzed. A full validation was conducted in European Union (EU)-regulated matrixes (eggs, shrimp, clams, salmon, chicken, beef liver) and other matrixes (produce, milk, baby food, ground coffee, fish oil, protein powder, corn snaplage) with satisfactory performances both in terms of recovery and reproducibility. Recovery percentages at target limits of quantification (LOQ) and two additional levels for perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS) were between 80-120% and repeatability (RSDr) results were ≤20% for EU-regulated matrixes. For other matrixes recovery percentages were between 65-135% and repeatability results were ≤25%. All were within acceptable ranges as per AOAC SMPR 2023.003. The FDA's method for detection of 30 PFAS in food and feed compares favorably with the requirements of AOAC SMPR 2023.003 and should be adopted as a First Action AOAC Official Method. The FDA's single lab validated method for PFAS detection in food and feed meets the requirements of AOAC SMPR 2023.003.

Accurate quantification of aldosterone is crucial for screening and diagnosing primary aldosteronism. In clinical laboratories, chemiluminescence is commonly used for aldosterone measurement. However, significant variability exists between different measurement systems. A complete reference system is needed to achieve comparable results across laboratories. Therefore, we developed a candidate reference measurement procedure based on liquid chromatography-tandem mass spectrometry for the quantitative determination of aldosterone in serum and urine. In this study, aldosterone-d8 was used to prepare standard solutions. Serum and urine aldosterone samples were extracted via liquid-liquid extraction and solid-phase extraction, and the steps in the pre-treatment process were optimised. Chromatographic separation was performed on a ZORBAX Eclipse XDB-C18 column, and quantitative detection was carried out in negative mode via an electrospray ionization source. The performance of the method was also evaluated. Serum aldosterone exhibited linearity in the range of 4.5-3902 pg mL-1, with a limit of quantification (LOQ) of 3.1 pg mL-1. The coefficient of variation (CV) ranged from 0.5% to 1.2%. Urine aldosterone showed linearity in the range of 10-998 pg mL-1, with a LOQ of 9.9 pg mL-1. The CV ranged from 1.1% to 1.4%. The recoveries of both samples were within the range of 95-105%. This method meets the analytical requirements of a reference measurement procedure, reduces the use of hazardous chemicals during sample preparation, and may serve as a candidate reference method for the accurate quantification of aldosterone in serum and urine samples in the laboratory.

Development of an SI-traceable magnetic solid-phase extraction coupled with isotope dilution LC-MS/MS method for accurate quantification of alpha-fetoprotein in human serum.

Purpose: This study aimed to investigate the associations between serum per- and polyfluoroalkyl substances (PFAS) and reproductive hormones, including follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), estradiol, and progesterone, in U.S. women. Approach and Results: We conducted a cross-sectional study using data from the National Health and Nutrition Examination Survey (NHANES) 2017–2018. The study included 612 women aged ≥18 years with available PFAS and sex hormone measurements. Serum concentrations of four major PFASs (linear perfluorooctanoic acid [n-PFOA], perfluorooctane sulfonic acid [PFOS], perfluorononanoic acid [PFNA], and perfluorohexane sulfonic acid [PFHxS]) were analyzed, along with serum levels of FSH, AMH, estradiol, and progesterone measured by isotope dilution liquid chromatography–tandem mass spectrometry. Higher serum PFAS concentrations were associated with increased FSH and decreased AMH, estradiol, and progesterone. For example, each interquartile range (IQR) increase in ln-PFNA was associated with a 42.0% increase in ln-FSH (p = 0.01) and 32.2% lower ln-AMH (p < 0.001), 33.0% lower ln-estradiol (p = 0.004), and 40.9% lower ln-progesterone (p = 0.02). A PFAS exposure index was related to higher FSH and lower AMH, estradiol, and progesterone, with stronger effects in premenopausal women. Conclusions: PFAS exposure was linked to broad endocrine disruption in women, with consistent alterations across gonadotropins and sex steroids. These findings suggest that PFAS exposure was associated with hormonal patterns consistent with diminished ovarian reserve and potential changes in reproductive function, underscoring the need for longitudinal studies and regulatory actions to mitigate exposure.

Development of certified reference materials for measuring perfluorooctanoic acid and perfluorooctane sulfonate concentrations in soil.

A candidate reference measurement procedure (RMP) based on isotope dilution (ID) liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated to accurately measure serum and plasma concentrations of mycophenolic acid glucuronide (MPAG). Quantitative nuclear magnetic resonance (qNMR) spectroscopy was utilized for determining the absolute content (mass fraction; g/g) of the reference material, thereby, establishing the traceability to SI units. Separation of MPAG from potential interferences, whether known or unknown, was accomplished by using a Phenomenex Luna C18(2) column. For sample preparation, a protocol based on protein precipitation followed by a high-dilution step was established. A multi-day validation experiment evaluated precision and accuracy. Reproducibility was determined by comparing the results of the procedure between two independent laboratories. Measurement uncertainty (MU) was assessed in accordance with current guidelines. The RMP demonstrated high selectivity and specificity enabling the quantification of MPAG in the range between 0.750 and 600 μg/mL. The intermediate precision and repeatability (n=60, measurements) were found to be in the range from 0.9 to 3.7 % for serum samples and from 1.2 to 4.6 % for plasma samples. The repeatability was less than 3.5 % for serum samples and less than 4.0 % for plasma samples. The relative mean bias ranged from-0.9 to 3.2 % for serum samples and from-0.3 to 2.9 % for plasma samples. The expanded measurement uncertainties (k=2) for single measurements ranged between 2.4 and 7.7 % and were further reduced performing a target value assignment (n=6) resulting in expanded measurement uncertainties between 1.8 and 3.3 % (k=2), respectively. We herein present a new LC-MS/MS-based candidate RMP for MPAG in human serum and plasma which offers 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 (cRMP) was developed and validated to measure serum and plasma concentrations of the total and free form of valproicacid. Quantitative nuclear magnetic resonance spectroscopic methodology was used to determine the absolute content (g/g) of the reference material, ensuring traceability to SI units. Separation of valproic acid from potential unknown interferences was achieved with reversed-phase chromatography. A protein precipitation protocol was established for sample preparation for total valproic acid, while the free form was separated by ultrafiltration. Assay validations and measurement uncertainties were aligned with guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. The cRMPs were highly selective and specific with no evidence of matrix effects, allowing quantifying total andfree valproic acid in a range of 2.40-145 μg/mL and 1.60-42.0 μg/mL, respectively. Intermediate precision was <4.0 % and repeatability CV ranged from 0.9 to 3.5% for all concentrations of free and total valproic acid. The relative mean bias ranged from-0.4 to 4.1 % for native serum and from-0.3 to 3.5 % for Li-heparin plasma levels for total valproic acid. Free valproic acid showed mean biases between-2.9 and 4.0 % for native serum and ultrafiltrates. Measurement uncertainties for single measurements and target value assignment ranged from 1.7 to 3.4 % and 0.9-1.3 %, respectively, for total valproic acid. Free valproic acid ranged from 2.0 to 4.1 % and from 0.8 to 1.5 % for single measurements and target value assignment, respectively. We present novel ID-LC-MS/MS-based cRMPs for total and free valproic acid in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

A candidate reference measurement procedure (RMP) based on isotope dilution (ID) liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated to accurately measure serum and plasma concentrations of mycophenolic acid (MPA). Quantitative nuclear magnetic resonance (qNMR) spectroscopy was utilized for determining the absolute content of the reference material, guaranteeing its traceability to SI units. Separation of MPA from potential interferences, whether known or unknown, was accomplished by using a C18 column. For sample preparation, a protocol for protein precipitation followed by a high dilution step was established. Assay validation and measurement uncertainty determination were conducted following the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement guidelines. The RMP demonstrated high selectivity and specificity without any indication of a matrix effect, enabling the quantification of MPA in the range between 0.240 and 18.0 mg/L. The intermediate precision and repeatability (n=60, measurements) were found to be <3.7 % and <3.0 %, respectively, across all concentration levels and independent from the matrix. The relative mean bias ranged from-1.9 to 2.1 % for serum and from-0.5 to 1.8 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <6.4 % and <3.0 % (k=2), respectively. We are pleased to present a new LC-MS/MS-based candidate RMP for MPA in human serum and plasma which offers a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

We developed a candidate reference measurement procedure (cRMP) based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for the accurate quantification of clozapine (CLO) in human plasma. After systematic optimization of chromatographic separation conditions for CLO, we evaluated various sample pretreatment methods. The analytical performance of the cRMP was rigorously validated, encompassing specificity, recovery, precision, linearity, limit of quantitation (LoQ), limit of detection (LoD), carryover, stability, and method comparison. Measurement uncertainties were assessed in accordance with GUM, taking CLO purity, balance weighing, calibration curve, measurement imprecision, recovery, and carryover into account. Under optimized conditions, CLO showed effective separation from potential interferents in human plasma. The method exhibited excellent linearity (R2 = 0.9988) across a concentration range of 5.65-1693.51 ng/g. The total coefficients of variation (CVs) were 2.04%, 0.97%, and 0.65% at concentrations of 24.53, 98.06, and 987.02 ng/g, respectively. Average recoveries ranged from 97.80% to 99.28%, with a LoQ of 2.73 ng/g and a LoD of 0.91 ng/g. The expanded measurement uncertainties (U) were 1.3 ng/g (k = 2) at 24.53 ng/g, 3.8 ng/g (k = 2) at 98.06 ng/g, and 35.3 ng/g (k = 2) at 987.02 ng/g. The developed cRMP for CLO quantification demonstrates excellent specificity, accuracy, precision, and traceability, meeting all the criteria for cRMPs. This method has the potential to standardize therapeutic drug monitoring (TDM) for CLO, improving clinical outcomes and patient safety.

In order to understand the current situation of mycotoxin contamination and risk assessment of dietary exposure of coix seed (Coix lacryma-jobi) sold in Gansu Province, China, a total of 67 coix seed samples were collected from the local markets and supermarkets in 14 prefectures and cities in Gansu Province. Sixteen mycotoxins were determined by isotope dilution liquid chromatography-tandem mass spectrometry. The results showed that 37.3% of coix seed was contaminated by mycotoxins. Among them, fumonisin B1 (FB1), fumonisin B2(FB2), fumonisin B3 (FB3) and zearalenone (ZEN) had the highest prevalence in coix seed, 10.5%, 11.9%, 17.9% and 10.5%, respectively. Coix seed was susceptible to mixed contamination of various mycotoxins, which was often detected in the forms of an FB1, FB2, and FB3 combination (35.7%) and an ZEN-FBs combination (21.5%). In this study, the exposure assessment was evaluated separately according to the use of coix seed for food or clinical use. It was found that the EDI values of ZEN, FB1, FB2, FB3, AME (alternariol monomethyl ether), TeA (tenuazonic acid), and TEN (tentoxin) calculated from the intake of coix seed for both medicinal and edible purposes were less than their TDIs, indicating that the health risk of exposure to the above toxins caused by the consumption of coix seed was within an acceptable range.

In clinical practice, therapeutic drug monitoring (TDM) typically relies on specialized immunoassays or chromatography-based methods. These approaches require drug-specific setups and are limited to batch measurements, which can restrict their availability. Recent advances in quantitative nuclear magnetic resonance spectroscopy (qNMR) have made it suitable for rapid and reliable quantification of small molecules in human plasma. However, no qNMR-based method has yet been developed specifically for TDM. A qNMR method for quantification of piperacillin (PIP) in human plasma was developed. Plasma samples were collected from 15 patients receiving PIP treatment in an intensive care unit. A total of 126 samples were subjected to analysis using 1H-NMR spectroscopy and isotope dilution liquid chromatography-tandem mass spectrometry. Measurements were compared within the common quantifiable range of 10 to 100mg/L. The degree of agreement between the methods was assessed using linear regression with post-hoc bootstrapping and Bland-Altman analysis. The concordance correlation coefficient between methods was 0.895, suggesting good alignment. Linear regression showed agreement with an intercept of -0.50mg/L and a slope of 1.03. Bland-Altman analysis revealed a mean bias of -0.85mg/L, with limits of agreement from -18.67mg/L to +16.97mg/L. This study demonstrates the potential of qNMR as a viable alternative for therapeutic drug monitoring, with a promising level of agreement supporting further validation in larger patient cohorts.

In clinical practice, glycated albumin (GA), as a key biomarker reflecting the level of blood glucose control in the short and medium term, has been widely used in the diagnosis and efficacy monitoring of diabetes. However, the consistency of GA results between different detection systems remains one of the current challenges in laboratory medicine. we evaluated the commutability of 10 evaluated samples to identify candidate EQA materials for GA measurement. According to the Clinical and Laboratory Standards Institute (CLSI) document EP14-A3 and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) commutability evaluation program, we used the established isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) method for serum glycated albumin determination as the comparative method, and six different commercial kits used enzyme assay as the evaluating method. A total of 40 clinical sample serums, 9 pregnancy sample serums, and 10 evaluated samples were analyzed. For the CLSI approach, pooled serum samples (cRM1-3) at medium concentrations (cRM2) are commutable in 5/6 kits. For the IFCC approach, none of samples commutable. The commutability evaluation results of the two approaches were different, and the cRM2 has commutability in most enzymatic kits. It can be a commutable material for the EQA project.

Testosterone is a crucial hormone for male health, influencing metabolism, cardiovascular function, bone density, and cognitive abilities. Elevated non-HDL cholesterol to HDL cholesterol ratio (NHHR) has been implicated in lipid metabolism disorders, which may adversely affect testosterone levels. This study investigates the association between NHHR and testosterone levels in adult males, utilizing data from the National Health and Nutrition Examination Survey (NHANES). This cross-sectional study analyzed data from 2,859 adult males from the NHANES cycles 2011-2016. Total testosterone levels were measured using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). NHHR was calculated and analyzed as both a continuous variable and in quartiles. Multivariable linear and logistic regression models, adjusted for demographic, biochemical, lifestyle factors, and medical comorbidities, were used to assess the relationship between NHHR and total testosterone levels and the risk of testosterone deficiency (TD). Higher NHHR was significantly associated with lower total testosterone levels and increased risk of TD. In fully adjusted models, each unit increase in NHHR was associated with a decrease in total testosterone levels (β = -16.31, 95% CI: -26.58 to -6.04, P = 0.003) and an increased risk of TD (OR = 1.24, 95% CI: 1.07 to 1.44, P = 0.01). When NHHR was analyzed in quartiles, participants in the highest quartile (Q4) had significantly lower testosterone levels (β = -54.98, 95% CI: -86.21 to -23.74, P = 0.001) and a higher risk of TD (OR = 2.04, 95% CI: 1.20 to 3.49, P = 0.01) compared to those in the lowest quartile (Q1). Subgroup analyses confirmed these findings across different age groups, BMI categories, smoking status, and presence of comorbidities. Smooth curve fitting demonstrated a linear relationship among them. Our study is the first to identify a significant association between elevated NHHR and both reduced total testosterone levels and increased risk of TD in a large, representative sample of adult American males. These findings suggest that NHHR could serve as a valuable marker for early identification of individuals at risk for testosterone decline and TD, enabling timely and targeted clinical interventions.

The gut microbiome produces short-chain fatty acids (SCFAs), which serve as a substantial energy source and provide a link between the microbiome and (cardiac) metabolism. It has been demonstrated that the composition of the microbiome is altered in patients with heart failure (HF), but whether circulating levels of SCFAs are altered in HF is unknown. Serum concentrations of the SCFAs acetate, propionate, and butyrate were measured in 205 patients with HF and in 54 healthy controls, using isotope dilution liquid chromatography-tandem mass spectrometry. Of the patients with HF, 99 had HF with a reduced ejection fraction (HFrEF) and 106 had HF with mildly-reduced or preserved ejection fraction (HFmrEF/HFpEF). Healthy controls were age and sex matched to the HFrEF patients. Serum concentrations of acetate and propionate were significantly lower in patients with HF than in healthy controls, whereas butyrate levels were higher in patients with HF. Analyses by HF type revealed that acetate and propionate levels were lower in both HFrEF and HFpEF/HFmrEF patients in comparison to healthy controls. However, butyrate levels were observed to be lower in patients with HFmrEF/HFpEF in comparison to healthy controls, while they were higher in patients with HFrEF. In patients with HF, serum levels of acetate and propionate are lower across the HF spectrum, whereas serum butyrate levels are elevated in HFrEF, but lower in HFmrEF/HFpEF. These alterations in SCFA profiles suggest a microbiome-driven metabolic dysregulation, which appears to differ between HF subtypes.

Background and AimsTherapeutic drug monitoring (TDM) of methotrexate (MTX) is challenging due to its pharmacokinetics and short plasma half-life. Intracellular MTX–polyglutamates (PG1–5), which accumulate over time, have not been assessed in pediatric inflammatory bowel disease (IBD). This study aimed to evaluate erythrocyte MTX-PG as a potential TDM tool in pediatric IBD.MethodsIn this cross-sectional study, MTX-PG concentrations were measured in erythrocytes of children with IBD on stable low-dose MTX for at least 12 weeks using stable-isotope dilution liquid chromatography–tandem mass spectrometry. The influence of administration route, MTX dosage, and anthropometrics on MTX-PG concentrations was examined.ResultsSeventy-eight patients were included, showing MTX-PG3 as the predominant subspecies (median 27.0 nmol/L) with a median MTX-PGtotal of 74.8 nmol/L. A higher MTX dose correlated significantly with elevated levels of MTX-PG3, MTX-PG4, MTX-PG5, and MTX-PGtotal (P < .01). Adjusted for body surface area, MTX dose remained significantly associated with higher MTX-PG concentrations (P < .01). However, comparison by administration route was limited due to a few patients on subcutaneous MTX (n = 4).ConclusionsWe observed high interindividual variability in the reached erythrocyte MTX-PG concentrations. Body surface adjusted or unadjusted MTX dosage showed a positive linear correlation with erythrocyte MTX-PG concentrations in children with IBD. This is a prerequisite for TDM and provides a strong basis for further research into the relation between TDM of MTX, efficacy, and toxicity.

Accurate measurements of plasma mycophenolic acid (MPA) are essential for therapeutic drug monitoring in transplant recipients and autoimmune diseases. The performance of plasma mycophenolic acid routine methods remains highly variable that calls for a candidate reference measurement procedure (cRMP) to improve the standardization of plasma mycophenolic acid measurements. In this study, sample preparation was based on protein precipitation with methanol followed by further dilution. The mycophenolic acid was quantified by the isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with electrospray ionization in positive ion mode. According to the Clinical and Laboratory Standards Institute (CLSI) documents C62-A and C50-A, the basic analytical performance of the candidate reference method was verified, such as linearity, limit of quantification, matrix effect, precision, accuracy, and uncertainty. Moreover, the candidate reference measurement procedure was compared with the routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a clinical laboratory. Based on the data, the mycophenolic acid in human plasma was well detected by ID-LC-MS/MS. No apparent interferences were found with the mycophenolic acid measurement. The calibration curve for the mycophenolic acid was linear in the concentration range of 0.1-50μg/mL with a correlation coefficient of 0.9999 under the optimum experimental conditions. This method was sensitive because the low limit of quantitation (LOQ) was 0.05μg/mL. The recoveries of MPA were 98.11-98.95%. The intra-day and inter-day coefficients of variations (CV) of our method were ≤ 1.53% and ≤ 0.51%, respectively. No obvious matrix effect was observed. There was a good correlation between this method and the clinical routine LC-MS/MS method. To sum up, we established and validated a reliable plasma MPA method using ID-LC/MS/MS. The desirable accuracy and precision of this method enable it to serve as a promising cRMP to improve the standardization for plasma MPA routine measurements.

BackgroundMetabolic health is closely related to testosterone levels, and the cardiometabolic index (CMI) is a novel metabolic evaluation metric that encompasses obesity and lipid metabolism. However, there is currently a lack of research on the relationship between CMI and testosterone, which is the objective of this study.MethodsThis study utilized data from the National Health and Nutrition Examination Survey (NHANES) cycles from 2011 to 2016. Only adult males who completed physical measurements, lipid metabolism assessments, and testosterone measurements were included in the final analysis. The exposure variable CMI was analyzed both as a continuous variable and a categorical variable divided into quartiles. Testosterone was measured using the isotope dilution liquid chromatography-tandem mass spectrometry technique. Linear and logistic regression analyses were used to explore the relationship between CMI and total testosterone (TT) levels, as well as the risk of testosterone deficiency (TD). Smooth curve fittings were employed to visualize their linear relationships. Subgroup analyses were conducted to evaluate the stability of our results across different participant characteristics. Finally, ROC analysis was used to assess the performance of CMI in predicting TD.ResultsA total of 2,747 participants were included in the analysis, including 552 with TD (20.10%). The average CMI of the sample was 1.59 ± 0.03, with TD participants having a higher CMI of 2.18 ± 0.08 compared to non-TD participants at 1.46 ± 0.03. Corresponding testosterone levels were 223.79 ± 3.69 ng/dL and 508.36 ± 5.73 ng/dL, respectively. After adjusting for all covariates, participants with higher CMI showed lower TT (β = -23.84, 95% CI: -33.94, -13.74, p < 0.0001) and a higher risk of TD (OR = 1.26, 95% CI: 1.08, 1.48, p = 0.01). When CMI was categorized into quartiles with Q1 as the reference, participants in Q4 exhibited significantly lower TT (β = -74.04, 95% CI: -106.01, -42.08, p < 0.0001) and a higher risk of TD (OR = 2.34, 95% CI: 1.18, 4.64, p = 0.02). Smooth curve fittings indicated a linear relationship between these variables. Subgroup analyses confirmed the stability of these associations across different population characteristics. ROC curve analysis demonstrated that CMI had good predictive performance for TD with a cut-off value of 1.126 and an AUC (95% CI) of 0.673 (0.649, 0.700).ConclusionCMI is associated with lower TT and a higher risk of TD, and it can predict the risk of TD. Using CMI for early detection and timely intervention could reduce the disease burden and promote reproductive health. Further prospective studies with large sample sizes are needed to validate these findings.

An analytical protocol based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), which includes a peptide-based calibration strategy, was developed and validated for the determination of cardiac troponin I (cTnI) levels in clinical samples. Additionally, thedeveloped method was compared with a protein-based calibration strategy, using cTnI serving as a model for low-abundant proteins. The aim is to evaluate new approaches for protein quantification in complex matrices, supporting the metrology community in implementing new methods and developing fit-for-purpose SI- traceable peptide or protein primary calibrators. To establish traceability to SI units, peptide impurity correction amino acid analysis (PICAA) was conducted to determine the absolute content of signature peptides in the primary standards. Immunoaffinity enrichment was used to capture cTnI from human serum, with a comparison between microbeads and nanobeads to improve enrichment efficiency. Parallel reaction monitoring was used to monitor two signature peptides specific to cTnI. Various digestion parameters were optimized to achieve complete digestion. The analytical method demonstrated selectivity and specificity, allowing the quantification of cTnI within 0.9-22.0 μg/L. The intermediate precision RSD was below 28.9 %, and the repeatability RSD was below 5.8 % at all concentration levels, with recovery rates ranging from 87 % to121 %. The comparison of calibration strategies showed similar LOQ values, but the peptide-based calibration exhibited significant quantitative bias in recovery rates. The data are available via ProteomeXchange (PXD055104). This isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method, based on peptide calibration, successfully quantified cTnI in human serum. Comparing this with protein-based calibration highlighted both the strengths and potential limitations of peptide-based strategies.