Isotope Dilution Liquid Chromatography-tandem Mass Research Articles

Quantification of mycophenolic acid in plasma by isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method.

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.

Determination of glycation biomarkers in human fingernails by isotope-dilution liquid chromatography tandem mass spectrometry (LC-MS/MS)

Glycation is a non-enzymatic, post-translational modification of proteins which is elevated in several pathologies, notably diabetes. An early-stage glycation product, glycated hemoglobin (HbA1c), is used in the clinical management of diabetes, and advanced glycation end-products (AGEs) are implicated in the etiology of diabetic complications. Fingernail clippings contain a time-integrated repository of several metabolic processes during the preceding 3–5 months, are easily sampled, and various elements and molecules have been shown to remain stable within them for long periods without refrigeration.Building upon a few underexploited studies, we investigated fingernails as a non-invasive matrix to assess glycation using liquid chromatography–mass spectrometry to quantify ungual biomarkers of early- and advanced glycation (respectively furosine, as a fructose-lysine derivative, and two AGEs (Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL)). The method was appropriately validated and provided accurate and precise measurements of two amino acids and the glycation biomarkers. Sample storage at ± 25 °C for 12 months had no effect upon these analytes, and the method was applied to fingernails from 87 people with diabetes.There was a moderate, linear correlation between ungual furosine concentrations and HbA1c at the time of nail sampling (rs = 0.339, p = 0.0011). Among subjects for whom previous measurements were available, there was no correlation between ungual glycation and HbA1c measured > 3 months before nail sampling, indicating that ungual furosine reflects early-stage glycation over a similar period to HbA1c. This study provides further evidence, using modern analytical techniques, that fingernails offer the possibility to quantitatively and non-invasively assess glycation.

A sterol panel for rare lipid disorders: sitosterolemia, cerebrotendinous xanthomatosis, and Smith-Lemli-Opitz syndrome

Disease-specific sterols accumulate in the blood of patients with several rare lipid disorders. Biochemical measurement of these sterols is important for correct diagnosis and sometimes monitoring of treatment. Existing methods to measure sterols in blood, particularly plant sterols, are often laborious and time consuming. Partly as a result, clinical access to sterol measurements is limited in many parts of the world. A simple and rapid method to extract freesterols from human serum and quantitate theirconcentration using isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization was developed. The method was designed to be compatible with routine workflows (e.g., 96-well format) in a clinical lab and extensively validated. Serum from at least 125 controls were analyzed and used to estimate the upper reference limits for sitosterol, campesterol, stigmasterol, desmosterol, 7-dehydrocholesterol (7DHC), lathosterol, and cholestanol. Serum from patients with the rare lipid disorders sitosterolemia (n=7), Smith-Lemli-Opitz syndrome (SLOS; n=1), and cerebrotendinous xanthomatosis (CTX; n=1) were analyzed. All seven sitosterolemia patients had greatly elevated levels of free plant sterols (sitosterol, campesterol, and stigmasterol) compared to the controls. The SLOS serum contained massively increased concentrations of 7DHC. CTX serum contained greatly increased concentrations of cholestanol, as well as 7DHC and lathosterol. Spiking experiments indicated that the method is likely also useful for the diagnosis of desmosterolosis and lathosterolosis. The reported method is a relatively simple and fast LC-MS/MS method capable of quantitating diagnostically important sterols and differentiated patients with three rare lipid disorders from controls.

An accurate measurement method for serum homocysteine measurement by ID-LC/MS/MS and the application of external quality assessment

BackgroundSerum homocysteine (Hcy) measurement accuracy is essential for detecting and diagnosing cardio-cerebrovascular illnesses. Although many different methods have been developed to determine the concentration of Hcy, those different assays showed nonequivalent results. This study aimed to develop an accurate and precise isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS) method for serum homocysteine (Hcy) and assign values for reference materials used in external quality assessment (EQA) programs. MethodsDifferent concentrations of homocysteine calibration solutions were prepared with d4-Hcy as the internal standard. This method employed DTT to reduce protein-bound Hcy, followed by protein precipitation, and gradient elution on a Supelcosil LC-CN column for chromatographic separation, and multiple reaction monitor (MRM) mode with electrospray ionization (ESI) for mass spectrometric detection. After optimized ID-LC/MS/MS parameters, imprecision, trueness, the limit of quantification (LoQ), the limit of detection (LoD), and measurement uncertainty were evaluated to check the methodological performance. The established method was employed in assigning values to EQA samples, which were sent to 63 clinical laboratories in Beijing to measure Hcy. ResultsAt 10.69,15.99, and 37.80 μmol/L levels, the present method demonstrated analytical imprecision of 0.57 %, 0.65 %, and 0.57 %, and recoveries of 99.67 % to 100.21 %, respectively. The bias between the target values of GBW (Guojia Biaozhun Wuzhi) were − 0.79 % to 0.62 %. The LoQ and LoD for Hcy were 0.36 nmol/L and 0.27 nmol/L. The method had an uncertainty (U 95 %) of 1.34 % to 1.48 %. For the three levels of EQA samples, the percentage of laboratories meeting the trueness evaluation criteria (±10 %) was 81.67 %, 83.33 %, and 71.67 % respectively. ConclusionsWith optimal method precision and trueness, the ID-LC/MS/MS method to measure serum Hcy can be used for value assignment of EQA samples, which can provide reliable data for monitoring the accuracy of clinical laboratory for Hcy measurement.

Aflatoxins in dried chilli products in Gansu Province and health risk.

In order to understand the status of aflatoxin contamination in dried chilli products in Gansu Province and the risk of dietary exposure, a total of 106 samples of dried chilli products from farmers' markets and supermarkets in 14 prefecture-cities of Gansu Province were collected and analysed by isotope dilution liquid chromatography-tandem mass spectrometry. The results showed that the detection rate of aflatoxin in dried chilli products in Gansu Province was 30.2%, and the average level was 1.57 μg/kg. The detection rates of dried chillies, paprika, and chilli powders were 16.7%, 43.6%, and 46.2%, respectively. The detection rates of aflatoxin in dried chilli products from shops and farmers' markets were 22.5% and 40.0%, respectively. The dietary exposure of AFB1 was 0.0001 μg/kg bw/day, and the MOE calculated from its average concentration was 305.

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 the liquid 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 a clinical laboratory. As a candidate reference method, this method can promote a GCA standardization program.Graphical

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.

A Cross-Sectional Exploratory Study of Cardiovascular Risk Biomarkers in Non-Obese Women with and without Polycystic Ovary Syndrome: Association with Vitamin D.

Vitamin D is proposed to have a protective effect against cardiovascular disease, though the mechanism is unclear. Vitamin D deficiency is common in polycystic ovary syndrome (PCOS), where it is strongly related to obesity, insulin resistance (IR) and risk of cardiovascular disease. To determine if the inherent pathophysiology of PCOS or vitamin D levels are linked to dysregulation of cardiovascular risk proteins (CVRPs), a study in non-obese women with PCOS and without IR was undertaken. Our hypothesis was that the levels of vitamin D3 and its active metabolite would be associated with CVRPs comparably in women with and without PCOS. In women with PCOS (n = 29) and controls (n = 29), 54 CVRPs were determined by Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement and correlated to 25-hydroxyvitamin D3 (25(OH)D3) and the active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) measured by gold standard isotope-dilution liquid chromatography tandem mass spectrometry. Women with PCOS had comparable IR and systemic inflammation (normal C-reactive protein) to control women, though had higher free androgen index and anti-Mullerian hormone levels. 25(OH)D3 and 1,25(OH)2D3 levels did not differ between groups. Nine CVRPs were higher in PCOS (p < 0.05) (Galectin-9, Brother of CDO, C-motif chemokine 3, Interleukin-18 receptor-1, Thrombopoietin, Interleukin-1 receptor antagonist protein, Programmed cell death 1 ligand-2, Low-affinity immunoglobulin gamma Fc-region receptor II-b and human growth hormone), whilst 45 CVRPs did not differ. 25(OH)D3 correlated with five CVRPs in PCOS and one in controls (p < 0.05). Despite the women with PCOS not exhibiting overt systemic inflammation, 9 of 54 CVRPs were elevated, all relating to inflammation, and 5 of these correlated with 25(OH)D3, suggesting an ongoing underlying inflammatory process in PCOS even in the absence of obesity/IR.

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.

Relationship between endocrine disrupting chemicals (phthalate metabolites, triclosan and bisphenols) and vitamin D in female subjects: An exploratory pilot study

IntroductionEvidence suggests that endocrine disrupting chemicals (EDCs), commonly used in plastics and personal care products, may be associated with reduced levels of vitamin D. Therefore, this study examined the relationship between phthalate metabolites, 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan; TCS) and bisphenols (BPs) with vitamin D3 (25(OH)D3) and active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and their relationship to calcium homeostasis. Methods57 female participants (age 31.8 ± 4.6 years; BMI 25.6 ± 3.7 kg/m2) were analyzed for urinary levels of phthalate metabolites, TCS and BPs, and serum levels of 25(OH)D3 and 1,25(OH)2D3, determined by isotope-dilution liquid chromatography tandem mass spectrometry. Serum calcium/calmodulin-dependent (CaM) associated proteins were determined by Slow Off-rate Modified Aptamer (SOMA)-scan. ResultsIn the study cohort, 25(OH)D3 and 1,25(OH)2D3 levels were 22.9 ± 11.2 ng/mL and 0.05 ± 0.02 ng/mL, respectively: mono-3-carboxypropyl-phthalate (MCPP) correlated negatively with 25(OH)D3 (ρ = −0.53, p = 0.01). 28 of the 57 women recruited were 25(OH)D3 deficient, <20 ng/mL (50 nmol/L): in this group, mono-iso-butylphthalate (MiBP) and mono-butylphthalate (MBP) negatively correlated with 25(OH)D3; (ρ = −0.47, p = 0.049) and (ρ = −0.64, p = 0.005), respectively. EDCs did not correlate with 1,25(OH)2D3, measures of renal function or CaM proteins. ConclusionThese putative data indicate that MCPP is related to 25(OH)D3, while MiBP and MBP were related to vitamin D deficiency; however, no correlations were observed with TCS and BPs. No phthalate metabolites correlated with 1,25(OH)2D3, CaM associated proteins or renal function, suggesting that effects occur earlier in the vitamin D pathway and not through modulation of cellular calcium flux. The observed correlations are surprisingly strong compared to other predictors of 25(OH)D3, and larger studies adjusting for potential confounders are warranted.

Simultaneous determination of ergothioneine, selenoneine, and their methylated metabolites in human blood using ID-LC-MS/MS.

Ergothioneine and selenoneine are structurally related dietary antioxidants and cytoprotectants that may help prevent several chronic diseases associated with inflammation and aging. Both compounds share pharmacokinetic characteristics such as cellular uptake through the ergothioneine transporter, accumulation in red blood cells, and biotransformation to methylated metabolites. A rapid, sensitive, specific, precise, and cost-effective analytical method is required to further investigate the potential health benefits of these compounds, individually or combined, in large epidemiological studies. We developed and validated an isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous specific quantification of these analytes in human blood following a simple sample preparation consisting of dilution in aqueous dithiothreitol followed by centrifugal filtration. Chromatographic separation of the analytes is achieved using a reversed-phase chromatography within an 8-min run. Analyte detection is performed using triple quadrupole mass spectrometry in multiple reaction monitoring mode. Each analyte is quantified against its corresponding isotopically labeled internal standard either commercially available or synthesized in-house (77Se-labeled selenoneine compounds). The validated method demonstrates excellent linearity and very good precision (all CV < 10%). Matrix effects are minimal, suggesting that this method could easily be adapted to other matrices. Freeze/thaw cycles have little effect on methylated metabolites but significantly reduced concentrations of the parent compounds. The method was successfully applied to a small set of volunteer blood samples containing low levels of the analytes. The developed ID-LC-MS/MS method opens new avenues for exploring the roles of these bioactive compounds and their metabolites in human health and disease.

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