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BACKGROUND: The D3-creatine (D3Cr) dilution method provides a direct measure of skeletal muscle. The aim of this study was to compare the association of D3Cr muscle mass with lean body mass (LBM) measured by dual-energy x-ray absorptiometry (DXA) and examine its relation with physical function in postmenopausal women. METHODS: Seventy-four community-dwelling women (mean age 82.3 ± 5.4) participated in this pilot study from the Buffalo, New York clinical site of the Womens Health Initiative (WHI). Participants attended a clinic visit which included anthropometric measures, blood draw, DXA scan, measures of physical function, and initiated the D3Cr protocol. Physical function was evaluated using hand grip strength, short physical performance battery (SPPB), and RAND-36 physical function scale. Descriptive statistics and logistic regression models were used to examine the associations of D3Cr muscle mass with functional outcomes. RESULTS: D3-creatine muscle mass was moderately correlated with DXA LBM (r = 0.50) and DXA appendicular lean mass (ALM) (r = 0.50). Individuals with high D3Cr muscle mass (%) had higher physical function compared to individuals with low muscle mass (%), indicated by high scores on SPPB (odds ratio [OR] = 5.24; 95% confidence interval [CI]: 1.40, 19.58). We observed stronger relationships between high D3Cr and physical function than either DXA LBM (OR = 3.40; 95% CI: 0.88, 13.11) or DXA ALM (OR = 4.15; 95% CI: 1.10, 15.68) and physical function. CONCLUSIONS: Our findings provide strong preliminary data for the associations of D3Cr muscle mass with measures of physical function in older women. These findings support and extend prior work on D3Cr muscle mass in older men.

BackgroundThe retinol isotope dilution (RID) method has been used to evaluate vitamin A (VA) status in healthy adults and children in low- and middle-income countries (LMIC) and to assess the efficacy of various VA interventions.ObjectiveThe study was designed to examine whether dried serum spots (DSS) can be applied to RID when conducting VA total body store (TBS) assessments in community settings.MethodsFour days after an oral dose of 0.4 mg [13C10]retinyl acetate was administered to Filipino children (12-18 mo), a single blood draw was divided to isolate both serum and plasma. Serum (40 μL) was spotted and dried on Whatman 903 cards and shipped at ambient temperature whereas liquid plasma (LP) was frozen at -80°C and shipped on dry ice. The VA tracer to tracee ratio from DSS and LP was quantified by LC-MS/MS. Comparisons between DSS and LP paired samples (n = 72) were made for [13C10]retinol specific activity (SAp) by Pearson's correlation and for VA TBS by Bland-Altman analysis.ResultsThe sum of 3 coextracted DSS were required to consistently detect [13C10]retinol above the LC-MS/MS limit of quantitation (LOQ). [13C10]retinol SAp from DSS was highly correlated with SAp from LP (r = 0.945; P

BackgroundWhether low muscle mass is a risk factor for disability and mortality is unclear. Associations between approximations of muscle mass (including lean mass from dual-energy x-ray absorptiometry [DXA]), and these outcomes are inconsistent.MethodsMuscle mass measured by deuterated creatine (D3Cr) dilution and appendicular lean mass (ALM, by DXA) were assessed at the Year 14 Visit (2014-2016) of the prospective Osteoporotic Fractures in Men study (N = 1,425, age 77-101 years). Disability in activities of daily living (ADLs), instrumental ADLs, and mobility tasks was self-reported at the Year 14 visit and 2.2 years later; deaths were centrally adjudicated over 3.3 years. Relative risks and 95% confidence intervals (CI) were estimated per standard deviation decrement with negative binomial, logistic regression, or proportional hazards models.ResultsIn age- and clinical center-adjusted models, the relative risks per decrement in D3Cr muscle mass/wgt was 1.9 (95% CI: 1.2, 3.1) for incident self-reported ADL disability; 1.5 (95% CI: 1.3, 1.9) for instrumental ADL disability; and 1.8 (95% CI: 1.5, 2.2) for mobility disability. In age-, clinical center-, and weight-adjusted models, the relative risks per decrement in D3Cr muscle mass was 1.8 (95% CI: 1.5, 2.2) for all-cause mortality. In contrast, lower DXA ALM was not associated with any outcome. Associations of D3Cr muscle mass with these outcomes were slightly attenuated after adjustment for confounding factors and the potentially mediating effects of strength and physical performance.ConclusionsLow muscle mass as measured by D3Cr dilution is a novel risk factor for clinically meaningful outcomes in older men.

BackgroundOral biotin supplementation is known to interfere with biotin-streptavidin-based immunoassays, including Roche's fifth-generation cardiac troponin T (cTnT) assay, which plays a critical role in the diagnosis of myocardial infarction (MI). The utility of dilution, a quick and easy method to detect and remove interferences, has not been published for biotin interference.MethodsConcentrations of cTnT were measured in pooled serum from clinical samples. Serum samples were supplemented with biotin to known concentrations, then cTnT concentrations were remeasured to assess for biotin interference. Samples were then diluted to assess for effective removal of biotin interference.ResultsAt cTnT values near the critical reporting range for our institution (100 ng/L) we observed significant interference in measured values with added biotin concentrations above 50 ng/mL. In specimens without added biotin, autodilution at a 1:10 ratio yielded a mean 157% capture of measured cTnT, precluding the use of autodilution for detecting and mitigating biotin interference. A 1:10 dilution with serum containing 20-30 ng/L cTnT yielded a mean capture of 107%, which was suitable for detecting underlying biotin interference in supplemented samples.ConclusionsBiotin interference, at supraphysiologic concentrations, may create an artifactual reduction in measured cTnT to levels that could lead to delayed detection of an MI. Dilution with serum of known cTnT concentration of 20-30 ng/L is a fast and effective method to mitigate the analytical consequences of biotin interference.

In-depth research into the precise evaluation of enzymatic digestion efficiency and the selection of a suitable deuterium-labelled internal standard remains a gap in the accurate determination of β 2 -agonists in animal-derived food by isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC-MS/MS). In this study, the enzymatic digestion conditions were optimized by monitoring the presence of β 2 -agonist conjugates in positive samples, which proved to be reliable for ensuring complete enzymatic digestion. Comparative analysis of deuterium-labelled internal standards for salbutamol (SAL), ractopamine (RAC), and clenbuterol (CLB) revealed that CLB-D 6 and SAL-D 9 were less effective in compensating for matrix effects due to hydrogen‑deuterium exchange during MS fragment formation. Consequently, SAL-D 3 , RAC-D 3 and CLB-D 9 were chosen for the implementation of ID-LC-MS/MS. The developed method demonstrates high accuracy and precision, with the average recoveries ranging from 93.8% to 107.3% with RSD <6.1%, which can provide higher-order measurement results for β 2 -agonists in pork., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Alternaria toxins (ATs) are produced from Alternaria species that result in crop losses and harmful impacts on human health. A stable isotope dilution LC-MS/MS method was established to quantify four ATs in 15 food commodities: alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), and tenuazonic acid (TeA). Based on systematically optimization of detection conditions and pre-processing steps, the limits of detection and limits of quantification of the four ATs ranged from 0.1 to 10 μg/kg and 0.2 to 30 μg/kg, respectively. The results showed that the recoveries of the four ATs were 72.0%-119.1%. The intra-precision and inter-precision ranged from 0.7% to 11.1% and 1.1% to 13.1%, respectively. The method was successfully applied to the determination of four ATs in 35 food samples, suggesting that this method could provide meaningful occurrence data to support the assessment of emerging ATs in food commodities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pineapple aroma is one of the most important sensory quality traits that influences consumer purchasing patterns. Reported in this paper is a high throughput method to quantify in a single analysis the key volatile organic compounds that contribute to the aroma of pineapple cultivars grown in Australia. The method constituted stable isotope dilution analysis in conjunction with headspace solid-phase microextraction coupled with gas-chromatography mass spectrometry. Deuterium labelled analogues of the target analytes purchased commercially were used as internal standards. Twenty-six volatile organic compounds were targeted for quantification and the resulting calibration functions of the matrix -matched validated method had determination coefficients (R 2 ) ranging from 0.9772 to 0.9999. The method was applied to identify the key aroma volatile compounds produced by popular pineapple cultivars such as 'Aus Carnival', 'Aus Festival', 'Aus Jubilee', 'Aus Smooth (Smooth Cayenne)' and 'Aussie Gold (73-50)', grown in Queensland, Australia. Pineapple cultivars varied in its content and composition of free volatile components, which were predominantly comprised of esters, followed by terpenes, alcohols, aldehydes, and ketones., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Previous research introduced V-PFCRC as an effective spot urinary dilution adjustment method for various metal analytes, including the major environmental toxin arsenic. V-PFCRC normalizes analytes to 1 g/L creatinine (CRN) by adopting more advanced power-functional corrective equations accounting for variation in exposure level. This study expands on previous work by examining the impacts of age and sex on corrective functions., Methods: Literature review of the effects of sex and age on urinary dilution and the excretion of CRN and arsenic. Data analysis included a Data Set 1 of 5,752 urine samples and a partly overlapping Data Set 2 of 1,154 combined EDTA blood and urine samples. Both sets were classified into age bands, and the means, medians, and interquartile ranges for CRN and TWuAs in uncorrected (UC), conventionally CRN-corrected (CCRC), simple power-functional (S-PFCRC), sex-aggregated (V-PFCRC SA), and sex-differentiated V-PFCRC SD modes were compared. Correlation analyses assessed residual relationships between CRN, TWuAs, and age. V-PFCRC functions were compared across three numerically similar age groups and both sexes. The efficacy of systemic dilution adjustment error compensation was evaluated through power-functional regression analysis of residual CRN and the association between arsenic in blood and all tested urinary result modes., Results: Significant sex differences in UC and blood were neutralized by CCRC and reduced by V-PFCRC. Age showed a positive association with blood arsenic and TWuAs in all result modes, indicating factual increments in exposure. Sex-differentiated V-PFCRC best matched the sex-age kinetics of blood arsenic. V-PFCRC formulas varied by sex and age and appeared to reflect urinary osmolality sex-age-kinetics reported in previous research. V-PFCRC minimized residual biases of CRN on TWuAs across all age groups and sexes, demonstrating improved standardization efficacy compared to UC and CCRC arsenic., Interpretation: Sex differences in UC and CCRC arsenic are primarily attributable to urinary dilution and are effectively compensated by V-PFCRC. While the sex and age influence on V-PFCRC formulas align with sex- and age-specific urinary osmolality and assumed baseline vasopressor activities, their impact on correction validity for entire collectives is minimal., Conclusion: The V-PFCRC method offers a robust correction for urinary arsenic dilution, significantly reducing systemic dilution adjustment errors. Its application in various demographic contexts enhances the accuracy of urinary biomarker assessments, benefiting clinical and epidemiological research. V-PFCRC effectively compensates for sex differences in urinary arsenic. Age-related increases in TWuAs are exposure-related and should be additionally accounted for by algebraic normalization, covariate models, or standard range adjustments., (© 2024. The Author(s).)

Background: As currently applied, the paired retinol isotope dilution (RID) test, which is used to assess the impact of a vitamin A intervention on vitamin A total body stores (TBS), requires 2 doses of stable isotope-labeled vitamin A., Objectives: The objectives of this study were to evaluate use of a single isotope dose (4 μmol) to assess TBS by RID before and after intervention in theoretical children with low/moderate TBS., Methods: We selected 6 theoretical children with assigned values for TBS ranging from 82 to 281 μmol. Using Simulation, Analysis and Modeling software, we simulated the variable [plasma retinol specific activity (SA p )] and coefficients (Fa and S) used in the RID equation TBS (μmol) = FaS × 1/SA p in both the unsupplemented steady state at day 14 postdosing and during the subsequent 4 mo without or with vitamin A supplementation [2.8 μmol retinol/d (801 μg retinol activity equivalents/d)]., Results: Fraction of dose in plasma on day 150 compared with day 14 was similar in the unsupplemented and supplemented conditions [geometric mean, 32% (range, 20%-48%) and 30% (20%-48%), respectively] and simulated values for FaS were similar under the 2 conditions. After 2 and 4 mo of daily vitamin A supplementation with 2.8 μmol/d, TBS was 78% and 128% higher, respectively, than without supplementation., Conclusions: Results indicate that the paired RID method can successfully be done using a single 4 μmol dose of stable isotope. Furthermore, because values for the RID coefficient FaS were similar in the unsupplemented and vitamin A-supplemented conditions, these results in theoretical children indicate that FaS determined by population ("super-subject") modeling of steady state vitamin A kinetic data could be used to predict TBS by RID after a vitamin A intervention in individuals from the same or a similar group., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

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 (R 2  > 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., (© 2024. The Author(s).)

Introduction: Investigations of responses of animals and humans to changes of plasma volume are usually reported as average responses of groups of individuals. This ignores considerable quantitative variation between individuals. We examined the hypothesis that individual responses follow a common temporal pattern with variations reflecting different parameters describing that pattern., Methods: We illustrate this approach using data of Hahn, Lindahl and Drobin (Acta Anaesthesiol Scand.2011, 55:987-94) who measured urine volume and haemoglobin dilution of 10 female subjects during intravenous Ringer infusions for 30 min and subsequent 3.5 h. The published time courses were digitised and analysed to determine if a family of mathematical functions accounted for the variation in individual responses., Results: Urine excretion was characterised by a time delay (Td) before urine flow increased and a time course of cumulative urine excretion described by a logarithmic function. This logarithmic relation forms the theoretical basis of a family of linear relations describing urine excretion as a function of Td. Measurement of Td enables estimation of subsequent values of urine excretion and thereby the fraction of infused fluid retained in the body., Conclusion: The approach might be useful for physiologists and clinical investigators to compare the response to infusion protocols when both test and control responses can be described by linear relations between cumulative urine volume at specific times and Td. The approach may also be useful for clinicians by complementing strategies to guide fluid therapy by enabling the later responses of an individual to be predicted from their earlier response., (© 2024 The Author(s). Clinical Physiology and Functional Imaging published by John Wiley & Sons Ltd on behalf of Scandinavian Society of Clinical Physiology and Nuclear Medicine.)

Epigenetic modifications are closely related to certain disorders of the organism, including the development of tumors. One of the main epigenetic modifications is the methylation of DNA cytosines, 5-methyl-2'-deoxycycytidine. Furthermore, 5-mdC can be oxidized to form three new modifications, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and 5-carboxy-2'-deoxycytidine. The coupling of liquid chromatography with tandem mass spectrometry has been widely used for the total determination of methylated DNA cytosines in samples of biological and clinical interest. These methods are based on the measurement of the free compounds (e.g., urine) or after complete hydrolysis of the DNA (e.g., tissues) followed by a preconcentration, derivatization, and/or clean-up step. This review highlights the main advances in the quantification of modified nucleotides and nucleosides by isotope dilution using isotopically labeled analogs combined with liquid or gas chromatography coupled to mass spectrometry reported in the last 20 years. The different possible sources of labeled compounds are indicated. Special emphasis has been placed on the different types of chromatography commonly used (reverse phase and hydrophilic interaction liquid chromatography) and the derivatization methods developed to enhance chromatographic resolution and ionization efficiency. We have also revised the application of bidimensional chromatography and indicated significant biological and clinical applications of these determinations., (© 2023 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd.)

Background: The methods of estimating vascular access (VA) flow rates are usually based on the indicator dilution theory by measuring recirculation during dialysis sessions., Methods: This is an observational study comparing the VA flow rates measured by NIKKISO DBB-EXA™ and Transonic®. Sixty-five patients (38 M/27 F, mean age 72 ± 10 years) participated in the study. We measured the VA flow rates during dialysis twice with each method and repeated the procedure 7 days later., Results: In 130 double measurements for each method on the same day, mean flow with Transonic® was 1413±715 ml/min and with DBB-EXA™ 1297 ± 664 ml/min. In Bland-Altman analysis, the mean difference between the two methods was 159 ± 211 ml/min (limits of agreement: -274 and 572 ml/min). Eighty-one out of the 130 DBB-EXA™ measurements were within 25% of the Transonic® measurements (62% accuracy). Regarding reproducibility of each method on different days, mean difference in the Bland-Altman analysis was 29 ± 620 ml/min (limits of agreement: -1186 and 1244 ml/min) for the Transonic® measurements and 132 ± 625 ml/min (limits of agreement: -1092 and 1356 ml/min) for the DBB-EXA™ measurements. The measurements on two different days were within 25% of each other for 52 of the 65 patients (80%) with the Transonic® method, and for 35 of the 65 patients (54%) with the DBB-EXA™ method., Conclusions: In conclusion, the DBB-EXA™ method underestimates VA flow rates compared to the Transonic® technique, resulting in a limited accuracy of 62%. There was poor reproducibility for both methods in different day measurements with better performance of the Transonic® technique. The DBB-EXA™ method could be used as a simple tool for a rough estimate of VA flow rates but cannot replace the Transonic® reference method., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

BackgroundMeasurements of maternal fat mass (FM) are important for studies of maternal and fetal health. Common methods of estimating FM have not been previously compared in pregnancy with measurements using more complete body composition models.ObjectivesThe goal of this pilot study was to compare multiple methods that estimate FM, including 2-, 3- and 4-compartment models in pregnant women at term, and to determine how these measures compare with FM by dual-energy X-ray absorptiometry (DXA) 2 wk postpartum.DesignForty-one healthy pregnant women with prepregnancy body mass index (in kg/m(2)) 19 to 46 underwent skinfold thickness (SFT), bioelectrical impedance analysis (BIA), body density (Db) via air displacement plethysmography (ADP), and deuterium dilution of total body water (TBW) with and without adjustments for gestational age using van Raaij (VRJ) equations at 37-38 wk of gestation and 2 wk postpartum to derive 8 estimates of maternal FM. Deming regression analysis and Bland-Altman plots were used to compare methods of FM assessment.ResultsSystematic differences in FM estimates were found. Methods for FM estimates from lowest to highest were 4-compartment, DXA, TBW(VRJ), 3-compartment, Db(VRJ), BIA, air displacement plethysmography body density, and SFT ranging from a mean ± SD of 29.5 ± 13.2 kg via 4-compartment to 39.1 ± 11.7 kg via SFT. Compared with postpartum DXA values, Deming regressions revealed no substantial departures from trend lines in maternal FM in late pregnancy for any of the methods. The 4-compartment method showed substantial negative (underestimating) constant bias, and the air displacement plethysmography body density and SFT methods showed positive (overestimating) constant bias. ADP via Db(VRJ)and 3-compartment methods had the highest precision; BIA had the lowest.ConclusionsADP that uses gestational age-specific equations may provide a reasonable and practical measurement of maternal FM across a spectrum of body weights in late pregnancy. SFT would be acceptable for use in larger studies. This trial was registered at clinicaltrials.gov as NCT02586714.

17α-Hydroxyprogesterone (17α-OHP) quantification in dried blood spots (DBS) is essential for newborn screening for congenital adrenal hyperplasia (CAH), which is challenging due to its low physiological concentration. The high false-positive rates of immunoassays necessitate the development of more accurate methods. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers increased specificity and sensitivity, yet standardized procedures for 17α-OHP measurement are required for clinical application. A candidate reference measurement procedure (cRMP) using isotope dilution LC-MS/MS was developed for 17α-OHP quantification in DBS. By utilizing stable isotope-labeled D8-17α-OHP as an internal standard, the cRMP was optimized, covering sample preparation, calibration, and LC-MS/MS analysis. The method performance was validated across several parameters, including precision, accuracy, specificity, detection limits, and matrix effects. Clinical applicability was further assessed through the establishment of reference intervals for healthy newborns. The developed cRMP exhibited a linear range of 1.00 to 80.00 ng/mL for 17α-OHP, with detection and quantification limits of 0.14 ng/mL and 0.52 ng/mL, respectively. Inter- and intraday precision demonstrated coefficients of variation within 1.27 to 5.69%. The recovery rates and matrix effects were well within acceptable limits, ensuring method reliability. Clinical application showed distinct reference intervals for healthy newborns that were unaffected by sex but influenced by weight and gestational age. This method significantly enhances CAH diagnostic accuracy in newborns, providing a valuable tool for clinical laboratories and improving newborn screening program standardization and traceability., (© 2024. The Author(s).)

Competing Interests: Conflicts of Interest: See Disclosures at the end of the article.

N-Terminal pro-B-type natriuretic peptide (NT-proBNP) is a pivotal biomarker for the diagnosis and prognosis of heart failure (HF). However, no SI-traceable certified reference material (CRM) or reference measurement procedure (RMP) is available for this biomarker, and so clinical testing results obtained in different laboratories cannot be traced to a higher-order standard, leading to incomparable measurements. Protein hydrolysis and protein cleavage isotope dilution mass spectrometry (AAA-IDMS and PepA-IDMS) were used to develop a CRM. Structurally related impurities were identified by high-resolution mass spectrometry. The quantitative AAA-IDMS results were corrected according to the amino acid compositions of the impurities. Using PepA-IDMS, two peptides from the proteolyzed product were confirmed as signature peptides. To obtain traceable and accurate results, the signature peptides were quantified using impurity-corrected AAA-IDMS. The candidate NT-proBNP solution was denatured and enzymatically digested using the Glu-C endoproteinase. The released signature peptides were measured using an isotopic dilution approach. The homogeneity and stability of the candidate CRM were characterized, and their uncertainties were combined with the value assignment process. The developed CRM can be considered a unique SI-traceable NT-proBNP reference material and is expected to be used as a primary calibrator for matrix NT-proBNP CRM development., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

In this study, we demonstrate the applicability of nitrogen microwave inductively coupled atmospheric pressure mass spectrometry (MICAP-MS) for Ca, Fe, and Se quantification in human serum using isotope dilution (ID) analysis. The matrix tolerance of MICAP-MS in Na matrix was investigated, revealing that high Na levels can suppress the signal intensity. This suppression is likely due to the plasma loading and the space charge effect. Moreover, 40 Ca and 44 Ca isotopic fractionation was noted at elevated Na concentration. Nine certified serum samples were analyzed using both external calibration and ID analysis. Overestimation of Cr, Zn, As, and Se was found in the results of external calibration, which might result from C-induced polyatomic interference and signal enhancement, respectively. Further investigations performed with methanol showed a similar enhancement effect for Zn, As, and Se, potentially supporting this assumption. The mass concentrations determined with ID analysis show metrological compatibility with the reference values, indicating that MICAP-MS combined with ID analysis can be a promising method for precise Ca, Fe, and Se determination. Moreover, this combination reduces the influence of matrix effects, broadening the applicability of MICAP-MS for samples with complex matrixes., (© 2024. The Author(s).)

The limit of detection (LOD) is a crucial measure in analytical methods, representing the smallest amount of a substance that can be distinguished from background noise. In the realm of gas chromatography (GC), however, determining LOD can be quite subjective, leading to significant variability among researchers. In this study, we validate the Hubaux-Vos method, an International Standards Organization(ISO)-approved approach for determining LOD in gas concentration measurements, using a GC equipped with a discharge ionization detector (DID) and a dynamic dilution system. We employ a gas mixture certified reference material (CRM) of CO, CH 4 , and CO 2 at various concentrations to generate calibration curves for each gas. Subsequently, we estimate the LODs for each gas using the Hubaux-Vos method. Surprisingly, our findings indicate a notable difference between the LODs calculated using the Hubaux-Vos method and those confirmed through experiments. This highlights the importance of critically examining the theoretical foundations of LOD determination. We strongly recommend researchers to scrutinize the principles guiding LOD determination. The method proposed in this study offers an effective way to rigorously validate theoretical approaches for estimating LODs in gas concentration measurements using GC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: The D3-creatine (D3-Cr) dilution method is of emerging interest for estimating total-body skeletal muscle mass. This review explores the association of muscle mass estimated via D3-Cr with various clinical outcomes and provides a summary of the literature comparing D3-Cr with other body composition techniques., Methods: A literature search was conducted on PubMed/MEDLINE and Web of Science for studies using D3-Cr to measure muscle in adult populations (ie, ≥18 years old) from inception until September 2023., Results: Out of the 23 included studies, 15 investigated the correlation between D3-Cr and clinical outcomes. More consistent associations were reported for mortality (100%, n = 2), mobility disability (100%; n = 5), falls and fractures (100%; n = 3), physical performance (63.3%; n = 11), muscle strength (44.4%; n = 9), and muscle composition (33.3%; n = 3). However, conflicting findings were also reported for such correlations. Among the 23 studies, 14 compared D3-Cr-estimated muscle with other body composition techniques, including magnetic resonance imaging (MRI) as a reference method. Strong and positive correlations were found between D3-Cr and MRI. Nonetheless, variations in muscle measurements were noted, with differences in D3-Cr values ranging from 0.62 kg lower to 13.47 kg higher compared to MRI., Conclusions: D3-Cr-estimated muscle mass may be a valuable predictor of clinical outcomes showing consistent associations with falls and fractures, mobility disability, and mortality. However, less consistent associations were found with muscle strength and composition, and physical performance. Although a strong correlation exists between D3-Cr-estimated muscle mass and MRI measurements, under- or overestimation may occur., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Gerontological Society of America.)

Objectives: Primidone is an anticonvulsive drug used in the treatment of epilepsy and essential tremor. It offers beneficial effects in controlling seizures, but its usage is also associated with possible side effects. To ensure optimal therapy, it is crucial to measure its concentration through accurate quantification methods. Therefore, our main goal was to develop and validate a new reference measurement procedure (RMP) for accurately measuring primidone levels in human serum and plasma., Methods: In our study, we focused on the separation of primidone from both known and unknown interferences using a C18 column. To achieve accurate sample preparation, we developed a protocol involving protein precipitation followed by a high dilution step. The validation of the assay and determination of measurement uncertainty were carried out following guidelines from organizations such as the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. These rigorous validation processes ensure the reliability and accuracy of our method for quantifying primidone levels in human serum and plasma samples., Results: The RMP was shown to be highly selective and specific, with no evidence of matrix interference. It can be used to quantify primidone in the range of 0.150-30.0 μg/mL. Intermediate precision was less than 4.0 %, and repeatability CV ranged from 1.0 to 3.3 % across all concentration levels. The relative mean bias ranged from 0.1 to 3.9 % for native serum levels, and from -2.6 to 2.8 % for lithium-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were 1.5-4.1 % and 0.9-1.0 %, respectively., Conclusions: In this study, we introduce an innovative LC-MS/MS-based candidate RMP specifically designed for primidone in human serum and plasma. Our RMP offers a traceable platform, facilitating the standardization of routine assays and enabling the evaluation of clinically relevant samples. With this novel approach, we aim to enhance the accuracy and reliability of primidone measurements, ultimately benefiting the field of clinical research and patient care., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Objectives: A reference measurement procedure (RMP) using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated with the aim of accurately measuring carbamazepine-10,11-epoxide concentrations in human serum and plasma., Methods: To establish traceability to SI units, the absolute content of the reference material was determined using quantitative nuclear magnetic resonance (qNMR) spectroscopy. As sample preparation a protein precipitation protocol followed by a high dilution step was established. Chromatographic separation from carbamazepine and potential metabolites was achieved using a C18 stationary phase. Selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement were evaluated based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement., Results: The RMP demonstrated very good selectivity and specificity, showing no evidence of a matrix effect. This enabled accurate quantification of carbamazepine-epoxide in the concentration range of 0.0400-12.0 μg/mL. The intermediate precision was found to be less than 2.1 %, and the repeatability coefficient of variation (CV) ranged from 1.2 to 1.8 % across all concentration levels. Regarding accuracy, the relative mean bias varied from 1.4 to 2.5 % for native serum levels and from 1.4 to 3.5 % for Li-heparin plasma levels. The measurement uncertainty for single measurements ranged from 1.6 to 2.1 %., Conclusions: In this study, we introduce a new LC-MS/MS-based candidate RMP for accurately measuring carbamazepine-10,11-epoxide in human serum and plasma. This novel method offers a traceable and dependable platform, making it suitable for standardizing routine assays and assessing clinically relevant samples., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Physicochemical examinations of very high dilution (UHD) solutions subjected to certain physical factors (such as shaking) are becoming more frequent and are increasingly producing conclusive results. A much less studied phenomenon is the transfer of molecular information (i.e., UHD signals of dilute substances) from one liquid to another without an intermediate liquid phase. The aim of this study was to investigate the possibility of such a transfer of the UHD signal from the UHD solutions to the receiver solution, in particular, if the molecular source used in the donor solutions was the biologically active antibodies to interferon-gamma molecule. We were especially interested in how the transfer of the UHD signal is affected by the time of exposure of the receiver to the donor, the distance between the two, and how the transfer is affected by activation (striking) versus exposure alone. Signal transfer was evaluated by differential measurements of electrical conductivity, ORP, pH, and UV/VIS spectroscopy of the exposed liquid. The results showed that activation strongly influences signal transfer and that this can be compensated to some extent by prolonged direct exposure. In principle, exposure time has a positive effect on signal transfer. Interestingly, the results of different distances between the donor and receiver showed similar changes in the parameters in the range of 0-4 cm, as estimated in this study. While the study mainly confirms the two hypotheses, it also raises a number of new questions and provides clues for further research.

Omicron variant (B.1.1.529) is able to escape from naturally acquired and vaccine-induced immunity, which mandates updating the current COVID-19 vaccines. Here, we investigated and compared the neutralising antibody induction of the ancestral variant-based BIV1-CovIran vaccine, the Omicron variant-based BIV1-CovIran Plus vaccine, and the novel bivalent vaccine candidate, BBIV1-CovIran, against the Omicron and ancestral Wuhan variants on the rat model. After inactivating the viral particles, the viruses were purified and formulated. Bivalent vaccines were a composition of 2.5 µg (5 µg total) or 5 µg (10 µg total) doses of each ansectral-based and Omicron-based monovalent vaccine. Subsequently, the potency of the monovalent and bivalent vaccines was investigated using the virus neutralisation test (VNT). The group that received three doses of the Omicron-specific vaccine demonstrated neutralisation activity against the Omicron variant with a geometric mean titer of 337.8. However, three doses of the Wuhan variant-specific vaccine could neutralise the Omicron variant at a maximum of 1/32 serum dilution. The neutralisation activity of the Omicron-specific vaccine, when administered as the booster dose after two doses of the Wuhan variant-specific vaccine, was 100% against the Omicron variant and the Wuhan variant at 1/64 and 1/128 serum dilution, respectively. Three doses of 5 µg bivalent vaccine could effectively neutralise both variants at the minimum of 1/128 serum dilution. The 10 µg bivalent vaccine at three doses showed even higher neutralisation titers: the geometric mean of 388 (95% CI 242.2-621.7) against Omicron and 445.7 (95% CI 303.3-655.0) against Wuhan. It is shown that the candidate bivalent and Omicron-specific vaccines could elicit a potent immune response against both Wuhan-Hu-1 and Omicron BA.1 variants., (© 2024. The Author(s).)

In LC-MS bioanalysis, sample dilution plays various roles, including bringing analyte concentrations within the validated/qualified dynamic range or alleviating matrix effect for accurate determination of the target analyte(s) in the intended study samples. Adherence to health authority requirements, incorporating good dilution practices, and timely demonstration of dilution integrity whenever samples are diluted in an analytical run are essential to ensure the reliability of bioanalytical results.

Advances in inductively coupled plasma mass spectrometry and the methods used to prepare isotopically enriched standards, allow for the high accuracy measurement of metalloproteins by isotope dilution mass spectrometry. This technique has now reached a level of maturity whereby a step change in the accuracy, precision, and traceability of, in particular, clinical, and biomedical measurements is achievable. Current clinical measurements, which require low limits of detection in the presence of complex sample matrices, use indirect methods based on immunochemistry for the study of human disease. However, this approach suffers from poor traceability, requiring comparisons based on provision of matrix-based reference materials, used as analytical standards. This leads to difficulty when changes in the reference material are required, often resulting in a lack of interlaboratory and temporal comparability in clinical results and reference ranges. In this review, we focus on the most important metalloproteins for clinical studies, to illustrate how the attributes of chromatography coupled to inorganic mass spectrometry can be used for the direct measurement of metalloproteins such as hemoglobin, transferrin, and ceruloplasmin. By using this approach, we hope to demonstrate how isotope dilution analysis can be used as a reference method to improve traceability and underpin clinical, biomedical, and other biological measurements.

ObjectiveTo validate a recently developed commercially available leptin radioimmunoassay (RIA) for use with feline serum and evaluate the relationship between serum leptin concentrations and body fat mass in domestic cats.Animals19 sexually intact male specific-pathogen-free domestic cats that weighed 3.8 to 7.1 kg and were 1.1 to 3.5 years old.ProcedureSpecificity for feline leptin was evaluated by use of gel filtration chromatography and reverse-phase high-performance liquid chromatography fractionation of serum. Body fat mass was determined by use of the deuterium oxide (D2O) dilution method. Serum water D2O enrichment was measured by use of gas-phase Fourier transform infrared spectroscopy.ResultsBody fat mass and percentage body fat ranged from 0.3 to 2.3 kg and 7.5 to 34.9%, respectively. Serum leptin concentrations were lower in the unfed versus the fed state and ranged between 1.6 and 4.9 ng/ml human equivalent (HE); mean +/- SD value was 2.9 +/- 0.2 ng/ml HE. Leptin concentrations increased with increasing body fat mass and percentage of body fat.ConclusionsLeptin is in the serum of domestic cats in free (> 78%) and apparently bound forms. The relationship between body fat and serum leptin concentration was similar to that observed in humans and rodents and indicative of a lipostatic role for leptin in cats. Cats that have an overabundance of body fat appear to be less sensitive to the weight-normalizing action of leptin than cats of ideal body condition.

Objectives: To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations., Methods: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was employed to determine the absolute content of the reference material used in order to establish traceability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement., Results: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of zonisamide within the range of 1.50-60.0 μg/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8-1.0 %, respectively., Conclusions: We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Objectives: An isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed and validated to accurately measure serum and plasma concentrations of carbamazepine., Methods: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was used to determine the absolute content of the reference material, ensuring its traceability to SI units. The separation of carbamazepine from potential interferences, whether known or unknown, was achieved using a C18 column. A protein precipitation protocol followed by a high dilution step was established for sample preparation. Assay validation and determination of measurement uncertainty were performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute, the International Conference on Harmonization (ICH), and the Guide to the Expression of Uncertainty in Measurement (GUM). In order to demonstrate equivalence to the already existing RMP a method comparison study was performed., Results: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of carbamazepine within the range of 0.800-18.0 μg/mL. Intermediate precision and repeatability (n=60 measurements) was found to be <1.6 % and <1.3 % over all concentration levels and independent from the matrix. The relative mean bias ranged from -0.1 to 0.6 % for native serum and from -0.3 to -0.1 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <1.8 % and <1.3 %, respectively. Method comparison showed a good agreement between the Joint Committee of Traceability in Laboratory Medicine (JCTLM) listed RMP and the candidate RMP resulting in a Passing-Bablok regression equation with a slope of 1.01 and an intercept of -0.01. The bias in the patient cohort was found to be 0.9 %., Conclusions: We present a novel LC-MS/MS-based candidate RMP for carbamazepine in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Changes in flavor quality of thermal process flavorings with beef-like odor (TPFB) affected sensory properties upon storage. The changes in sensory quality and odorants of TPFB stored at 50 ℃ for 168 days were evaluated using sensomics approach. The aroma profiles of TPFB gradually changed from stronger meaty notes to stronger burnt and soybean paste-like notes during storage. Forty-two quantified odor-active compounds with flavor dilution ≥ 27 were assessed using the odor activity value concept. Correlation analysis indicated that a decreasing trend of meaty note was closely associated with 5-methyl furfural, dimethyl disulfide, dimethyl trisulfide, furfuryl methyl sulfide and furfuryl thioacetate, which all enriched with time. Omission and addition tests showed that dimethyl disulfide, dimethyl trisulfide and furfuryl thioacetate with the concentration increasing considerably reduced the intensity of meaty note, particularly for dimethyl trisulfide. Therefore, the formation of dimethyl trisulfide should be limited to produce high-quality TPFB during storage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Bioaugmenting Daqu is an effective strategy to improve the quality of Baijiu, but its effect on overall flavor profiles and aroma-active compounds is unknown. Here, 168 volatiles were determined from fresh strong-flavor Baijiu (SFB) and bioaugmented Daqu increased their diversity and altered the flavor characteristics. Among 49 odorants identified by aroma extraction dilution analysis, 29 aroma-active compounds had odor activity values ≥1, of which 18, 8, and 3 components exhibited the highest content in the SFB fermented by fortified-, space- and conventional-Daqu, respectively. The contribution of increasing ethyl hexanoate and decreasing ethyl lactate of fresh SFB by bioaugmented Daqu was confirmed, and their content changed from 4650 and 1890 mg/L (conventional-SFB) to 6680 and 1760 mg/L (fortified-SFB) and 6130 and 1710 mg/L (space-SFB). Meanwhile, the discriminators among different samples were determined by multivariate statistical analysis. These findings are beneficial for the optimization and improvement of Baijiu aroma., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Real-time and accurate estimation of surgical hemoglobin (Hb) loss is essential for fluid resuscitation management and evaluation of surgical techniques. In this study, we aimed to explore a novel surgical Hb loss estimation method using deep learning-based medical sponges image analysis. Whole blood samples of pre-measured Hb concentration were collected, and normal saline was added to simulate varying levels of Hb concentration. These blood samples were distributed across blank medical sponges to generate blood-soaked sponges. Eight hundred fifty-one blood-soaked sponges representing a wide range of blood dilutions were randomly divided 7:3 into a training group (n = 595) and a testing group (n = 256). A deep learning model based on the YOLOv5 network was used as the target region extraction and detection, and the three models (Feature extraction technology, ResNet-50, and SE-ResNet50) were trained to predict surgical Hb loss. Mean absolute error (MAE), mean absolute percentage error (MAPE), coefficient (R 2 ) value, and the Bland-Altman analysis were calculated to evaluate the predictive performance in the testing group. The deep learning model based on SE-ResNet50 could predict surgical Hb loss with the best performance (R 2  = 0.99, MAE = 11.09 mg, MAPE = 8.6%) compared with other predictive models, and Bland-Altman analysis also showed a bias of 1.343 mg with narrow limits of agreement (- 29.81 to 32.5 mg) between predictive and actual Hb loss. The interactive interface was also designed to display the real-time prediction of surgical Hb loss more intuitively. Thus, it is feasible for real-time estimation of surgical Hb loss using deep learning-based medical sponges image analysis, which was helpful for clinical decisions and technical evaluation., (© 2023. Springer Nature Limited.)

Aging aroma is an intriguing but an understudied phenomenon in baijiu. The aromatic characteristics of Qingxiangxing (QXX) baijiu stored for 45 years (aged) were investigated using the sensomics approach and were compared with those of young baijiu (0 year-old). Aroma extract dilution analysis revealed 59 odorants, with many long-retained components exhibiting greater flavor dilution values in the aged than the young sample. The analysis of variance of the quantitative data showed significant differences between the young and aged baijiu for 26 compounds. Based on the variations in the content of these compounds, QXX baijiu aged for 0, 5, 15, 20, and 45 years was analyzed and twelve compounds were significantly correlated with aging duration. Finally, eight positively correlated compounds were omitted from the recombinant model, and sotolon, methional, vanillin, dimethyl trisulfide, benzaldehyde, and 3-hydroxy-2-butanone were confirmed to primarily contribute toward the aging aroma of QXX baijiu., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

We developed and evaluated two-level, namely 2017011 and 2017012, serum-based reference materials (RMs) for 17 beta-estradiol (17 β-E2) by the reference method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) from the remaining serum samples after routine clinical tests, to help improve clinical routine testing and provide the traceability of results. This paper describes the development process of these RMs. The National Metrology Institute of Japan (NMIJ) certified reference material (CRM) 6004-a was used as the primary RM for the measurement of 17 β-E2. These serum-based RMs showed satisfactory homogeneity and stability. They also assessed the commutability between the reference method and the three routine clinical immunoassay systems. Besides, a collaborative study was carried out in five reference laboratories, all of which had been accredited by the China National Accreditation Service for Conformity Assessment (CNAS) in accordance with ISO/WD 15725-1. Statistical analysis of raw results and uncertainty assessment obtained certified values: 2017011 was 445.2 ± 39.0 pmol/L, and 2017012 was 761.9 ± 35.5 pmol/L., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

The optical properties of indocyanine green (ICG) as a near-infrared (NIR) fluorescence dye depend on the nature of the solvent medium and the dye concentration. In the ICG absorption spectra of water, at high concentrations, there were absorption maxima at 700 nm, implying H-aggregates. With ICG dilution, the main absorption peak was at 780 nm, implying monomers. However, in ethanol, the absorption maximum was 780 nm, and the shapes of the absorption spectra were identical regardless of the ICG concentration, indicating that ICG in ethanol exists only as a monomer without H-aggregates. We found that emission was due to the monomer form and decreased with H-aggregate formation. In the fluorescence spectra, the 820 nm emission band was dominant at low concentrations, whereas at high concentrations, we found that the emission peaks were converted to 880 nm, suggesting a new form via the twisted intramolecular charge transfer (TICT) process of ICG. The NIR fluorescence intensity of ICG in ethanol was approximately 12- and 9-times brighter than in water in the NIR-I and -II regions, respectively. We propose an energy diagram of ICG to describe absorptive and emissive transitions through the ICG structures such as the monomer, H-aggregated, and TICT monomer forms.

Here we proposed a method for peptide purity analysis using gas chromatography-isotope dilution infrared spectroscopy. The principle and feasibility of the proposed measurement method were investigated. The derivatization, separation, and infrared detection conditions for amino acids were optimized, and the performance of the method was investigated. Then, the proposed method was used for assessment of [Glu 1 ]-fibrinopeptide B purity, and the results were compared with those obtained by high performance liquid chromatography-isotope dilution mass spectrometry. The average purity of six sub-samples using the proposed method was (0.755 ± 0.017) g/g, which agreed well with that obtained by isotope dilution mass spectrometry (0.754 ± 0.012) g/g. The repeatability of the proposed method was 2.2%, which was similar to that of isotope dilution mass spectrometry (1.7%). The proposed method has a similar principle and had similar accuracy, precision, and linearity to isotope dilution mass spectrometry; however, the developed method had higher limit of detection (LOD) and limit of quantitation (LOQ) values because of the low sensitivity of infrared detection. The results were also Système International d'Unités (SI) traceable. The developed method has the advantage of lower cost compared with isotope dilution mass spectrometry because only one isotope-labeled atom in an analog is required, and several infrared spectra can be extracted, averaged, and used for an amino acid calculation during one run, potentially leading to higher accuracy. This method could be easily expanded to the accurate quantitation of other organic compounds, including proteins. It is expected that the proposed method will be widely used in chemical and biological measurements as a new primary method., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: Topiramate is an antiepileptic drug (AED) used for the monotherapy or adjunctive treatment of epilepsy and for the prophylaxis of migraine. It has several pharmacodynamic properties that contribute to both its clinically useful properties and observed adverse effects. Accurate measurement of its concentration is therefore essential for dose adjustment/optimisation of AED therapy. Our aim was to develop and validate a novel reference measurement procedure (RMP) for the quantification of topiramate in human serum and plasma., Methods: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method in combination with a protein-precipitation-based sample preparation allows for quantification of topiramate in human serum and plasma. To assure traceability to SI units, quantitative nuclear magnetic resonance (qNMR) was applied to characterize the reference material used as primary calibrator for this RMP. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Accuracy and precision was evaluated performing an extensive five day precision experiment and measurement uncertainty was evaluated according Guide to the Expression of Uncertainty in Measurement (GUM)., Results: The method enabled topiramate quantification within the range of 1.20-36.0 μg/mL without interference from structurally related compounds and no evidence of a matrix effect. Intermediate precision was ≤3.2 % and repeatability was 1.4-2.5 % across all concentration levels. The relative mean bias was -0.3 to 3.5 %. Expanded measurement uncertainties for target value assignment (n=6) were found to be ≤2.9 % (k=2) independent of the concentration level and the nature of the sample., Conclusions: In human serum and plasma, the RMP demonstrated high analytical performance for topiramate quantification and fulfilled the requirements on measurement uncertainty. Traceability to SI units was established by qNMR content determination of the topiramate, which was used for direct calibration of the RMP. This RMP is, therefore, fit for purpose for routine assay standardization and clinical sample evaluation., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Background: 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., Methods: After protein precipitation with Z n SO 4 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., Results: 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 R 2 >0.98. The comparability among the immunoassays was substantially improved after recalibration., Conclusions: 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.