Dilution Liquid Chromatography-tandem Mass Spectrometry Research Articles

A candidate reference measurement procedure for quantification of glycocholic acid in human serum based on isotope dilution liquid chromatography-tandem mass spectrometry.

Accurate measurement of serum glycocholic acid (GCA) is crucial for evaluating the activity of chronic hepatitis. Moreover, GCA is a novel identified biomarker for hepatocellular carcinoma. Although some laboratories have used theliquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure GCA in recent years, the problem of potential interference of GCA analogues has not been solved well yet. Neither reference measurement procedures nor reference materials for GCA have been listed in the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database. For standardization of GCA, it is urgent to establish a candidate measurement procedure for GCA. In this study, a candidate reference measurement procedure for the quantification of GCA in human serum based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) by a two-step sample pretreatment of protein precipitation and MAX solid-phase extraction was developed and validated. GCA can be completely separated from its structural analogues with gradient elution in 9 min compared with short time gradients published in previous literature by Huang's group. Method validation indicated perfect quantitation precision with intra-day and inter-day values that were ≤1.30% and ≤1.80%, respectively. The method showed excellent linearity with high regression coefficients (R2 > 0.999) over a range of 0.92 ng/g-38.38 μg/g and perfect recoveries at three spiked levels (99.87-100.43%). No interference, matrix effect, and carryover were observed. Moreover, the cRMP was successfully applied to measure GCA in serum samples and compared with two immunoassays in aclinical laboratory. As a candidate reference method, this method can promote aGCA standardization program.

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.

Serum S-adenosylhomocysteine levels are associated with first stroke in Chinese adults with hypertension

BackgroundThe association between S-adenosylhomocysteine (SAH) and stroke has not been confirmed due to the specialized equipment and time requirements necessary for S-adenosylhomocysteine testing. We aimed to explore the association between SAH and stroke. MethodsA nested, case-control study drawn from the China Stroke Primary Prevention Trial of rural adults with hypertension, including 557 first stroke cases and 557 matched controls was conducted. Serum SAH was measured by stable-isotope dilution liquid chromatography-tandem mass spectrometry using 4500MD. Multiple conditional logistic regression models were used to evaluate the association between SAH and first stroke. ResultsIn females, SAH levels were significantly higher in the stroke population than in the control group (16.0 ng/mL vs. 14.6 ng/mL). When SAH was assessed as quartiles, the odds of stroke were 1.78 (95 % CI: 1.02–3.09) in Quartile 2, 1.31 (95 % CI: 0.73–2.33) in Quartile 3, and 1.93 (95 % CI: 1.03–3.62) in Quartile 4, compared to Quartile 1. When Quartiles 2–4 were combined, the adjusted odds ratio of first stroke was 1.64 (95 % CI: 1.03–2.62) compared with Quartile 1. In subgroup analysis, a significant SAH-stroke association was observed in the lower vitamin D3 group (OR = 3.35, 95 % CI:1.72–6.53; P interaction, 0.035). In males, higher levels of SAH were associated with an increased risk of stroke in those under age 60. Compared with the reference group, the adjusted odds ratio of total stroke was 2.40 (95 % CI: 1.02–5.91) in the combined group (Quartile 2–4). In contrast, no significant association between SAH and stroke was found in males aged 60 or older. ConclusionsThis study reveals that SAH is associated with a higher risk of stroke independently of homocysteine, especially in females. SAH may be a second predictor of stroke in the metabolic pathway of methionine, after homocysteine.

Determination of six volatile fatty acids in human serum, urine and faeces by low temperature derivatisation combined with HPLC–MS/MS

A stable isotope dilution-liquid chromatography tandem mass spectrometry method based on a low-temperature derivatization strategy with 3-nitrophenylhydrazine (3-NPH) was developed for the determination of six volatile fatty acids (VFAs) in serum, urine, and feces. Ice acetonitrile was used to precipitate proteins and extract the target analytes. The extract was derivatized with 3-NPH methanol solution at 4 °C. BEH C8 (1.7 μm, 2.1 × 100 mm) column was used for chromatographic separation, and acetonitrile–water (both containing 0.01 % formic acid) were used as the mobile phase with a gradient elution of 10 min. Electrospray ionization source (ESI) in negative ion multiple reaction monitoring (MRM) mode were used for analyte detection. The regression coefficients R2 of the calibration curves for the six VFAs were in the range of 0.9963–0.9994, and the LOQs were in the range of 0.02–0.5 μg mL−1, with the recoveries in the range of 85.3–104.3 %, and the intra- and inter-day precision in the range of 1.8–9.1 %. The method is simple, accurate and reliable, and has been applied in the sensitive determination of VFAs in complex biological samples.

Development and application of a sensitive liquid chromatography-tandem mass spectrometry method for the quantitative analysis of 11 free fatty acids in human serum using a derivatisation strategy

A stable isotope dilution–liquid chromatography–tandem mass spectrometry method based on a derivatisation strategy involving an N,N'-carbonylimidazole solution (CDI) with 4-(dimethylamino)-benzenemethanamine was developed for the determination of 11 free fatty acids (FFAs) in human blood samples. Serum samples were subjected to liquid‒liquid extraction and centrifuged, and the supernatant was collected for a two-step derivatisation reaction with a CDI and 4-(dimethylamino)-aniline acetonitrile solution. The derivatised solution was separated on a ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) column with a mobile phase consisting of water–acetonitrile in gradient elution and then detected by tandem mass spectrometry using electrospray ionisation (ESI) and multiple reaction monitoring (MRM) in positive ion mode and quantified using the isotope internal standard method. The effects of the derivatisation reaction time, temperature and concentration of derivatisation reagents on the response values of the analytes were investigated. The optimal conditions were as follows: 1.0 mg mL−1 CDI acetonitrile solution at 25 °C for 25 min, followed by a reaction with a 1.0 mg mL−1 4-(dimethylamino)-benzenemethanamine acetonitrile solution at 70 °C for 30 min. Under the optimal conditions, the limits of detection (LODs) of the 11 FFAs were in the range of 3.0–14.0 ng mL−1; the limits of quantification (LOQs) were in the range of 8.0–45.0 ng mL−1; and the mean recoveries ranged from 83.4 to 112.8%, with intraday and interday precisions ranging from 0.7 to 9.1% and 3.7–9.5%, respectively. The experimental method is simple in terms of the pretreatment operation, accurate and reliable, and can be applied to the sensitive determination of FFAs in human blood samples.

Association between visceral adipose tissue and total testosterone among the United States male adults: a cross-sectional study.

Visceral adipose tissue (VAT) is regarded as an important risk factor for obesity-related diseases. The results of the association between VAT and total testosterone (TT) are controversial and whether this association is nonlinear is still unknown. 3971 male participants who were aged 20-59 years from the National Health and Nutrition Examination Surveys 2011-2016 were included. VAT area was measured by dual-energy x-ray absorptiometry. TT in serum was assessed utilizing the isotope dilution liquid chromatography-tandem mass spectrometry technique. Linear regression models assessed the associations between VAT area and TT. A restricted cubic spline model was employed to investigate nonlinear relationships. A two-piecewise linear regression model was applied to determine the threshold effect. Subgroup analyses were conducted. The weighted methods were utilized in all analyses. VAT area was inversely associated with TT in the crude and adjusted models. In the fully adjusted model, VAT area was associated with TT (β = -0.59, 95% confidence interval [CI] = -0.74, -0.43) and compared to the first tertile of VAT area, the second and the third tertile had a lower TT level, the β and 95% CI = -65.49 (-83.72, -47.25) and -97.57 (-121.86, -73.27) respectively. We found these inverse associations were nonlinear. The cutoff point of the VAT area was 126 cm2. When the VAT area was <126 cm2, VAT area was significantly associated with a lower TT level (β = -1.55, 95% CI = -1.93 to -1.17, p < 0.0001). However, when the VAT area was ≥126 cm2, this association was less apparent (β = -0.26, 95% CI = -0.52 to 0.01, p = 0.06). No significant interactions among different ages (<50 or ≥50 years), marital, and physical activity status were found. These findings underscore the potential for VAT area as a modifiable indicator for improving testosterone deficiency.

Targeted metabolomic profiles of serum amino acids are independently correlated with malnutrition in older adults.

Malnutrition is a common geriatric syndrome that is closely associated with adverse clinical outcomes and poses significant harm to older adults. Early assessment of nutritional status plays a crucial role in preventing and intervening in cases of malnutrition. However, there is currently a lack of measurable methods and biomarkers to evaluate malnutrition in older adults accurately. The aim of this study is to investigate the independent correlation between serum levels of amino acids and malnutrition in older adults, and to identify effective metabolomics biomarkers that can aid in the early detection of geriatric malnutrition. A total of 254 geriatric medical examination participants from Beijing Hospital were included in the study, consisting of 182 individuals with normal nutritional status (Normal group) and 72 patients at risk of malnutrition or already malnourished (MN group). Malnutrition was assessed using the Mini-Nutritional Assessment Short-Form (MNA-SF). Demographic data were collected, and muscle-related and lipid indexes were determined. Serum amino acid concentrations were measured using isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). The correlation between serum amino acid levels and malnutrition was analyzed using non-parametric tests, partial correlation analysis, linear regression, and logistic regression. The geriatric MN group exhibited significantly lower serum aromatic amino acid levels (P < 0.05) compared to the normal group. A positive correlation was observed between serum aromatic amino acid levels and the MNA-SF score (P = 0.002), as well as with known biomarkers of malnutrition such as body mass index (BMI) (P < 0.001) and hemoglobin (HGB) (P = 0.005). Multivariable logistic or linear regression analyses showed that aromatic amino acid levels were negatively correlated with MN and positively correlated with the MNA-SF score, after adjusting for some confounding factors, such as age, gender, BMI, smoking status, history of dyslipidemia, diabetes mellitus and frailty. Stratified analyses revealed that these trends were more pronounced in individuals without a history of frailty compared to those with a history of frailty, and there was an interaction between aromatic amino acid levels and frailty history (P = 0.004). Our study suggests that serum aromatic amino acids are independently associated with malnutrition in older adults. These results have important implications for identifying potential biomarkers to predict geriatric malnutrition or monitor its progression and severity, as malnutrition can result in poor clinical outcomes.

Association between secondhand smoke exposure and serum sex hormone concentrations among US female adults: a cross-sectional analysis using data from the National Health and Nutrition Examination Survey, 2013–2016

ObjectiveTo estimate the association between secondhand smoke (SHS) exposure and serum sex hormone concentrations in female adults (never smokers and former smokers).DesignCross-sectional analysis.SettingUS National Health and Nutrition Examination Survey, 2013–2016.Outcome...

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.

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. 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. 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 %. 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 itsuitable for standardizing routine assays and assessing clinically relevant samples.

Quantification of midostaurin in plasma and serum by stable isotope dilution liquid chromatography-tandem mass spectrometry: Application to a cohort of patients with acute myeloid leukemia.

Midostaurin is an oral multitargeted tyrosine kinase inhibitor for the treatment of acute myeloid leukemia (AML). Therapeutic drug monitoring of midostaurin may support its safe use when suspecting toxicity or combined with strong CYP3A4 inhibitors. A stable isotope dilution liquid chromatography-tandem mass spectrometry method was developed and validated for the determination and quantification of midostaurin in human plasma and serum. Midostaurin serum concentrations were analyzed in 12 patients with FMS-like tyrosine kinase 3 (FLT3)-mutated AML during induction chemotherapy with cytarabine, daunorubicin, and midostaurin. Posaconazole was used as prophylaxis of invasive fungal infections. Linear quantification of midostaurin was demonstrated across a concentration range of 0.01-8.00 mg/L. Inter- and intraday imprecisions of the proposed method were well within ±10%. Venous blood samples were taken in nine and three patients in the first and second cycle of induction chemotherapy. Median (range) midostaurin serum concentration was 7.9 mg/L (1.5-26.1 mg/L) as determined in 37 independent serum specimens. In a real-life cohort of AML patients, interindividual variability in midostaurin serum concentrations was high, highlighting issues concerning optimal drug dosing in AML patients. A personalized dosage approach may maximize the safety of midostaurin. Prospective studies and standardization of analytical methods to support such an approach are needed.

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.

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. 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. 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 %. 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.

Oxidative/nitrosative stress increased the risk of recurrent pregnancy loss–Taiwan Recurrent Pregnancy Loss and Environmental Study (TREPLES)

ObjectiveOxidative stress biomarkers (OSBs) may be strongly associated with disease progression and recurrent pregnancy loss (RPL). However, the research on associations of most OSBs (e.g., 8-nitroguanine [8-NO2Gua] and 4-hydroxy-2-nonenal-mercapturic acid [HNE-MA]) with RPL is limited. Therefore, we aimed to investigate the effect of OSBs exposure on RPL risk by performing a case-control study. Material and methodsWe use our established dataset, Taiwan Recurrent Pregnancy Loss and Environmental Study (TREPLES), which included 514 Taiwanese reproductive age women (aged 20–50 years; 397 cases and 117 controls) from National Cheng Kung University Hospital. RPL is clinically defined by a history of two or more consecutive miscarriages, where a miscarriage is defined as the termination of pregnancy before 20 weeks of gestation. The urinary levels of several OSBs (e.g., 8-hydroxy-2′-deoxyguanosine [8-OHdG], 8-NO2Gua, 8-isoprostaglandin F2α [8-isoPGF2α], and HNE-MA) and malondialdehyde (MDA) were measured using isotope dilution liquid chromatography-tandem mass spectrometry and thiobarbituric acid reactive substances, respectively. ResultsThe median levels of 8-NO2Gua (6.15 vs. 3.76 ng/mL) and HNE-MA (30.12 and 21.54 ng/mL) were significantly higher in the RPL group than in the control group. By categorizing the OSBs data into tertiles, after we adjusted for age and urine creatinine levels discovered that the RPL risk associated with 8-NO2Gua and HNE-MA levels in the third tertile were approximately 2 times higher than those in the first tertile (8-NO2Gua, adjusted OR = 3.27, 95 % CI = 1.66–6.43; HNE-MA, adjusted OR = 1.96, 95 % CI = 1.05–3.64; p < 0.05). These findings suggest that the oxidative stress biomarkers of 8-NO2Gua and HNE-MA are risk factors for RPL. ConclusionOur findings indicate that specific OSBs are associated with an increased RPL risk, suggesting that reducing OSB levels can improve RPL risk. Nevertheless, more studies on preventive medicine are required to understand the exposure sources and adverse outcome pathways of OSBs associated with RPL.

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

BackgroundCardiac 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.MethodsThe 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).ResultsThe 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.ConclusionsThe 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.Graphical

Development of isotope dilution-liquid chromatography tandem mass spectrometry for the accurate determination of vitamin K1 in spinach and kimchi cabbage

In the human body, vitamin K1 is important for bone and cardiovascular health and blood coagulation. To assess the correlation between vitamin K1 intake and health outcomes, the accurate determination of the amounts of vitamin K1 in green leafy vegetables, which are its major source in the diet, is needed. In this study, an accurate method for quantifying naturally occurring trans- and cis-vitamin K1 in spinach and kimchi cabbage was developed on the basis of isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS). A C30 column was employed for proper separation of trans- and cis-vitamin K1 isomers, and vitamin K1-d7 was used as an internal standard. The developed method was validated by measuring gravimetrically fortified samples, and its performance parameters were evaluated. The measured results agreed with the gravimetric results with a difference of less than 3%. The repeatability and reproducibility of the vitamin K1 analysis were less than 2% relative standard deviation, indicating that the method had a higher-order metrological quality as a reference method.

Novel Isotope Dilution High-Performance Liquid Chromatography Quadrupole–Linear Ion Trap Tandem Mass Spectrometry Method for the Determination of Five Steroid Hormones in Human Serum

Accurate measurement of endogenous steroid hormones in serum is a powerful tool for understanding the status of human adrenal diseases. Isotope dilution–liquid chromatography–tandem mass spectrometry is the most commonly used method for determination of steroids in serum. In this study, we used a laboratory-constructed quadrupole-linear ion trap (Q-LIT) tandem mass spectrometer to develop a new method for the quantification of five steroid hormones in serum. A simple and efficient pretreatment was improved, wherein the serum was extracted once by a mixed solvent of 1.0 mL of ethyl acetate and n-hexane. Using the optimized method, the regression coefficients of the calibration curves were all higher than 0.99, the limits of detection were from 0.31 to 1.25 ng mL−1, and the limits of quantification were between 1.00 and 2.50 ng mL−1. The intra-day (n = 3) relative standard deviations ranged from 4.24% to 13.79%, and the inter-day (n = 3) relative standard deviations from 3.99% to 11.43%. After internal standard correction, the relative recoveries of all steroids were from 86.08% to 106.60%. Twenty-two serum samples were analyzed by Q-LIT, which was compared with commercial triple quadrupole mass spectrometer. The results of the instruments had high consistency. Therefore, Q-LIT accurately quantified steroid hormones in human samples and may be used for clinical diagnosis and monitoring of hormone-dependent diseases.

Exploration of suitable external quality assessment materials for serum C-peptide measurement.

To find suitable external quality assessment (EQA) materials for serum C-peptide, we evaluated the commutability of five types of processed materials. Seventy-four individual serum samples and 12 processed samples including three EQA samples currently in use, frozen human serum pools (FHSP), and three other kinds of processed samples were prepared by dissolving WHO International Standard Reagent for C-peptide (WHO ISR 13/146) in three different matrixes: 0.05 % bovine serum albumin, fetal bovine serum and human serum pools. Samples were analyzed using the isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method and six widely used immunoassays. The commutabilities of processed materials were assessed according to the difference in bias approach recommended by the IFCC. And the short- and long-term stability of FHSP samples at different temperatures were also evaluated. Out of the five kinds of processed materials, FHSP samples were commutable on most assays. In contrast, the EQA materials currently in use were only commutable on a few immunoassays. Additionally, processed materials derived from WHO ISR 13/146 were found to be un-commutable on over half of immunoassays. The FHSP samples could be stably stored at 4 and-20 °C for at least 16days, and at-80 °C for at least 1 year, but at room temperature only for 12 h. With clarified commutability and stability information, the human serum pool samples along with the developed ID-LC-MS/MS method could be used in the EQA program to promote the comparability among laboratories for C-peptide measurement in China.

An Accurate Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry Method for Serum C-Peptide and Its Use in Harmonization in China.

Serum C-peptide results from various routine methods used in China are highly variable, warranting well-performing methods to serve as an accuracy base to improve the harmonization of C-peptide measurements in China. We developed an accurate isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for serum C-peptide measurement and explored its use in harmonization. After protein precipitation with ZnSO4 solution, C-peptide was extracted from serum samples by anion-exchange solid-phase extraction and quantified by ID-LC-MS/MS in positive ion mode. The precision and analytical recovery of the ID-LC-MS/MS method were assessed. Seventy-six serum samples were analyzed using the ID-LC-MS/MS method and six routine immunoassays. Ordinary linear regression (OLR) and Bland-Altman (BA) analyses were conducted to evaluate the relationship between the ID-LC-MS/MS method and routine immunoassays. Five serum pool samples assigned using the ID-LC-MS/MS method were used to recalibrate the routine assays. OLR and BA analyses were re-conducted after recalibration. The within-run, between-run, and total precision for the ID-LC-MS/MS method at four concentrations were 1.0%-2.1%, 0.6%-1.2%, and 1.3%-2.2%, respectively. The analytical recoveries for the ID-LC-MS/MS method at three concentrations were 100.3%-100.7%, 100.4%-101.0%, and 99.6%-100.7%. The developed method and the immunoassays were strongly correlated, with all R2 >0.98. The comparability among the immunoassays was substantially improved after recalibration. The performance of the ID-LC-MS/MS method was carefully validated, and this method can be used to improve the harmonization of serum C-peptide measurements in China.

Reduced Steroid Synthesis in the Follicular Fluid of MTHFR 677TT Mutation Carriers: Effects of Increased Folic Acid Administration.

Objective To compare steroid profiles in the follicular fluid (FF) from women homozygous for the methylenetetrahydrofolate reductase (MTHFR) 677C>T mutation and wildtype controls and to correlate it with the folic acid administration scheme applied at the time of oocyte retrieval. Design Retrospective single center study. Subjects and Methods Infertile patients treated by using assisted reproductive techniques were genotyped routinely for the MTHFR 677C>T mutation. In 2006 they had received folic acid supplementation doses of 400 µg daily per os. This group was designated Group-400 (n = 10). From 2008 onwards, all of our infertility patients received a daily dose of 800 µg folic acid per os. Women from this group were designated Group-800 (n = 28). FF were collected and a panel of steroid hormones (estradiol, estrone, estriol, cortisol, progesterone, 17-OH progesterone, testosterone, androstenedione, aldosterone, DHEA, and DHEA-S) was measured by isotope dilution liquid chromatography-tandem mass spectrometry employing atmospheric pressure photo ionization (APPI). Results In Group-400, the FF hormone profile confirmed a significant reduction of estradiol in homozygous 677TT carriers (0.52 ± 0.08-fold, exact p = 0.032) and for the first time also revealed significantly reduced estriol concentrations in these individuals (0.54 ± 0.05-fold, p = 0.016), as compared to wildtype controls. In Group-800, no significant differences were found for concentrations of any of the steroid hormones between homozygous 677TT carriers and wildtype controls. Conclusions The current findings support and extend previous reports on reduced concentrations of specific steroid hormones in follicular fluids of homozygous MTHFR 677C>T mutation carriers. The restoration of the FF hormone profile by elevated-dose folic acid supplementation might impact performing ART in infertile women with the MTHFR 677TT-genotype. Further adequately powered studies are necessary to verify our finding and to demonstrate the clinical effect of enhanced folic supplementation on ovarian function.

Production and Characterization of a SARS-CoV-2 Nucleocapsid Protein Reference Material.

Rapid antigen tests have become a widely used COVID-19 diagnostic tool with demand accelerating in response to the highly contagious SARS-CoV-2 Omicron variant. Hundreds of such test kits are approved for use worldwide, predominantly reporting on the presence of the viral nucleocapsid (N) protein, yet the comparability among manufacturers remains unclear and the need for reference standards is recognized. To address this lack of standardization, the National Research Council Canada has developed a SARS-CoV-2 nucleocapsid protein reference material solution, NCAP-1. Reference value determination for N protein content was realized by amino acid analysis (AAA) via double isotope dilution liquid chromatography-tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in conjunction with UV spectrophotometry based on tryptophan and tyrosine absorbance at 280 nm. The homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. The molar concentration of the N protein in NCAP-1 was 10.0 ± 1.9 μmol L-1 (k = 2, 95% confidence interval). Reference mass concentration and mass fraction values were subsequently calculated using the protein molecular weight and density of the NCAP-1 solution. Changes to protein higher-order structure, probed by size-exclusion liquid chromatography (LC-SEC) with UV detection, were used to evaluate transportation and storage stabilities. LC-SEC revealed nearly 90% of the N protein in the material is present as a mixture of hexamers and tetramers. The remaining low molecular weight species (<30 kDa) were interrogated by top-down mass spectrometry and determined to be autolysis products homologous to those previously documented for N protein of the original SARS-CoV [Biochem. Biophys. Res. Commun.2008t, 377, 429-433].

The associations between TMAO-related metabolites and blood lipids and the potential impact of rosuvastatin therapy

BackgroundTrimethylamine N-oxide (TMAO)-related metabolites are associated with the pathogenesis of atherosclerotic cardiovascular disease (ASCVD) and are known to disrupt lipid metabolism. The aims of this study were to evaluate the associations between TMAO-related metabolites and blood lipids and determine how lowering the lipid profile via rosuvastatin therapy influences TMAO-related metabolites.MethodsA total of 112 patients with suspected ASCVD were enrolled in this study. The levels of plasma TMAO-related metabolites, including TMAO, choline, carnitine, betaine, and γ-butyrobetaine (GBB), were analyzed by stable isotope dilution liquid chromatography-tandem mass spectrometry (LC/MS/MS) before and after rosuvastatin therapy in all patients. Statistical methods were used to detect the associations between TMAO-related metabolites and blood lipids and determine how rosuvastatin therapy alters the levels of these metabolites.ResultsA significant positive correlation was found between TMAO and triglycerides (TG) (r = 0.303, P < 0.05). Furthermore, significant negative correlations were found between TMAO and high-density lipoprotein cholesterol (HDL-c) and between betaine and low-density lipoprotein cholesterol (LDL-c) (r = − 0.405 and − 0.308, respectively, both P < 0.01). Compared to baseline, significantly lower TMAO levels and higher carnitine, betaine and GBB levels were observed after rosuvastatin therapy, while the lipids decreased significantly (P < 0.05). The significant correlation between TMAO and TG or between betaine and LDL-c disappeared after rosuvastatin therapy (r = 0.050 and − 0.172, respectively, both P > 0.05). However, a significantly positive association between carnitine and TC and a negative association between carnitine and LDL-c or between betaine and TG were found after adjustment for sex, age, body mass index (BMI) and lipids (P < 0.05).ConclusionsThis study suggests that TMAO-related metabolites are significantly associated with blood lipids, although some of them are changed postrosuvastatin therapy. Lower TMAO and higher TMAO precursors were observed after rosuvastatin therapy compared to baseline. This study indicates that elevated TMAO precursors after rosuvastatin therapy and their potential impact on ASCVD should be considered in the clinic.