Isotope Dilution Liquid Chromatography Research Articles

Inflammatory Markers in Non-Obese Women with Polycystic Ovary Syndrome Are Not Elevated and Show No Correlation with Vitamin D Metabolites.

Introduction. Chronic low-grade inflammation is a characteristic of women with polycystic ovary syndrome (PCOS), although this may be obesity-driven rather than an intrinsic facet of PCOS; furthermore, vitamin D deficiency, another common feature of PCOS, is reported to have an association with increased inflammation. Therefore, circulating inflammatory protein levels and circulating levels of vitamin D may be linked in PCOS, though it is unclear which vitamin D metabolites may be important. Methods. We measured plasma levels of 24 inflammatory proteins and 12 matrix metalloproteinases (proteins modulated by the inflammatory process) by slow off-rate modified aptamer (SOMA)-scan plasma protein measurement in weight and aged-matched non-obese non-insulin resistant PCOS (n = 24) and control (n = 24) women. Inflammatory proteins and matrix metalloproteinases were correlated to 25-hydroxy vitamin D3 (25(OH)D3), its epimer 25-hydroxy-3epi-vitamin D (3epi25(OH)D) and the active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) as measured by gold standard isotope-dilution liquid chromatography tandem mass spectrometry. Results. PCOS women had both an elevated free androgen index and circulating anti-mullerian hormone, though insulin resistance was comparable to controls. C-reactive protein, as a standard circulatory marker of inflammation, was comparable between cohorts. Levels of circulating inflammatory proteins and matrix metalloproteinases were not different between the PCOS and control women, with no correlation of 25(OH)D3, 1,25(OH)2D3 or 3epi25(OH)D with any of the inflammatory proteins. Conclusion. In a non-obese PCOS population matched for age and insulin resistance, circulating inflammatory proteins and matrix metalloproteinases were not elevated and did not correlate with 25(OH)D3, its epimer 3epi25(OH)D or 1,25(OH)2D3 in either control or PCOS women, indicating that the inflammatory response is absent and the vitamin D-metabolite independent in non-obese women with PCOS.

Measurement of serum uric acid by isotope dilution liquid chromatography tandem mass spectrometry: Modification of a candidate reference measurement method and its clinical application

Uric acid is a precipitating factor for gout and renal disease, and regular monitoring of uric acid concentration is essential for health management. Hence, accurate measurement is indispensable for clinical diagnosis. Reference measurement procedure (RMP) is an essential element to ensure accuracy and comparability of tested results. In this study, the optimization of a candidate RMP (cRMP) adopting isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for human serum uric acid measurement has been developed. The accuracy of this cRMP was validated by measuring certified reference materials (CRMs) and participating in the external quality assessment (EQA) scheme initiated by International Federation of Clinical Chemistry (IFCC) and National Center for Clinical Laboratory (NCCL). Biases were −0.64-1.51%, which were within the required equivalent limit. Excellent precision was obtained with intra- and inter-assay CVs of 0.42–0.73% and 0.20–0.43%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) was determined to be 0.24 and 1.19 μmol/L, respectively. A good liner response was obtained ranged from 30.0 to 895 μmol/L. Compared with the previous RMP for uric acid, the much easier one-step protein precipitation method without further evaporation, the simplified isocratic elution of chromatographic separation condition, and the shorter analysis running time in only 5 min allowed simpler and faster analysis. Moreover, this optimized cRMP was also applied for value assignment for clinical serum samples covered a wide concentration range of uric acid and obtained a decent calibration and measurement capability (CMC).

Absolute Quantification of Total Hemoglobin in Whole Blood by High-Performance Liquid Chromatography Isotope Dilution Inductively Coupled Plasma-Mass Spectrometry.

Accurate and traceable measurement of hemoglobin (HGB) is of great importance in clinical testing. Although the HiCN method is the internationally accepted conventional reference method for this biomarker and frequently used in clinical routine diagnostics, the HiCN method cannot be traceable to the International System of Units (SI) and thus does not meet highest metrological demands. In this study, an absolute quantitative approach for total HGB in a whole blood sample is proposed based on the determination of natural Fe and S present in the heme-group of HGB by HPLC isotope dilution ICP-MS. IRMM/IFCC-467 is used for method validation, and then clinical blood samples are measured by the established strategy and HiCN method. The measurable ranges of total HGB were 10.0-240.0 g L-1. Limits of detection via Fe and S were 0.01 and 0.07 g L-1, respectively. The intra-assay imprecision CVs via Fe and S were 0.89-1.35 and 0.99-1.56%, and the interassay CVs were 1.19-2.15 and 1.55-2.55%, respectively. Good agreement was achieved in the method validation. In the comparison with HiCN experiments, the ID-ICP-MS assays via Fe and S showed correlations of r2 = 0.991 and 0.970 against HiCN methods. Moreover, the concentration of transferrin (Tf) was also simultaneously measured. This strategy has potential to serve as a reference measurement procedure for total HGB in whole blood, which could be traceable to SI and does not require toxic derivation reagent.

Increased risk of testosterone deficiency is associated with the systemic immune-inflammation index: a population-based cohort study.

PurposeThis study aimed to explore the relationship between serum testosterone levels and systemic immune-inflammation index (SII).MethodsComplete SII and serum testosterone data of men over 20 years of age were retrieved from the 2011–2016 National Health and Nutrition Examination Survey to conduct a prevalence survey. To calculate SII, the platelet count was multiplied by the neutrophil-to-lymphocyte count ratio. Isotope dilution liquid chromatography and tandem mass spectrometry were employed to measure serum testosterone concentration. Testosterone deficiency (TD) was defined as a serum testosterone level ≤ 300ng/dl. Weighted proportions and multivariable regression analyses were used to analyze the association between SII and TD.ResultsOverall, the data of 7389 participants were analyzed, The SII ranged from 1.53 - 6297.60. Of the participants, 28.42% had a low serum testosterone level (≤ 300 ng/dl). In the fully adjusted multivariable logistic model, the second quartile (OR: 1.27, p = 0.0737), the third quartile (OR: 1.43, p = 0.0090), and the fourth quartile (OR:1.48, p = 0.0042) of SII significantly increased the TD incidence rate, with the lowest quartile of the SII as a reference. For subgroup analysis, statistically significant associations were observed in participants aged 20-40, obese, non-hypertensive, and non-diabetic. The interaction test revealed no significant effect on this connection.ConclusionsThere was a positive relationship between a high SII and an increased prevalence of TD in a nationwide sample of adult men in the United States. Further prospective studies on a larger scale are warranted to confirm the causality between SII and TD.

Perfluorooctanesulfonate Can Cause Negative Bias in Creatinine Measurement in Hemodialysis Patients Using Polysulfone Dialysis Membranes.

Serum creatinine is an important clinical marker for renal clearance. However, two conventional methods (Jaffe and enzymatic) are prone to interferences with organic compounds as compared to the standard method (isotope dilution–liquid chromatography–mass spectrometry) and can cause a significant negative bias. Perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) are two common perfluorochemicals (PFCs) that can easily be accumulated in humans. We aimed to verify whether this bias is the result of an accumulation of PFCs. The serum creatinine values of 124 hemodialysis patients were analyzed using the three methods. We also aimed to evaluate which biochemical parameters will influence the difference between the conventional methods and the standard method. We found that a significant underestimation occurred when using the conventional methods. Albumin is an independent factor associated with negative bias, but it loses this correlation after dialysis, likely due to the removal of protein-bound uremic toxins. PFOS can cause negative bias when using the enzymatic method. Furthermore, this linear correlation is more significant in patients who used polysulfone-based dialysis membranes, possibly due to the better clearance of other uremic toxins. The serum creatinine of uremic patients can be significantly underestimated when using conventional methods. PFCs, as well the type of dialysis membrane being used, can be influencing factors.

Study of a new matrix certified reference material for accurate measurement of pefloxacin in fish meat powder

Matrix certified reference materials (CRMs) have played a critical role in analytical method validation and assurance of reliable measurement results. A matrix CRM of pefloxacin in fish meat powder was newly studied and developed for fishery food quality and safety monitoring. The CRM candidates were prepared from incurred samples by procedures of freeze-drying, grounding, sieving, homogenizing, secondary freeze-drying, bottling and gamma-irradiating. Isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) was established and applied to value assignment. The certified value was 45.3 μg/kg for mass fraction of pefloxacin in fish meat powder with expanded uncertainty of 3.7 μg/kg (k = 2, at a confidence of 95 %). Homogeneity, long-term stability of six months at −20 °C for storage, and short-term stability of nine days at 4 °C, 20 °C and 40 °C for transportation were investigated. In addition, uncertainty of the certified value originating from value assignment, homogeneity testing and stability monitoring were evaluated.

Determination of Vitamin B12 in Milk and Dairy Products by Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry

An isotope-dilution liquid chromatography tandem mass spectrometry method was established for the determination of vitamin B12 in milk and dairy products. The samples were spiked with stable isotope-labeled vitamin B12 and digested by pepsin and amylase. The various forms of cobalamin were transformed to cyanocobalamin by potassium cyanide after they were released from the enzymatically digested samples. Cyanocobalamin was extracted and purified by an immunoaffinity SPE cartridge and then measured in multiple reaction monitoring mode (MRM). The linear correlation coefficient (R2) of this method was greater than 0.999 in the range of 2–100 ng/mL. The detection limit and the quantification limit were 0.5 μg/kg and 1.0 μg/kg, respectively. The spiked recoveries ranged from 92.0% to 99.4% at the three spiked levels with the relative standard deviation (RSD) between 1.89% and 4.51%. The measured results of NIST SRM1849a and NIST SRM1869a by the current method are all within the reference value range. The Z value was 0.8 during participating in the FAPAS proficiency test using the developed method in 2021. The method is simple, rapid, accurate, and sensitive, and it is suitable for the determination of vitamin B12 in different types of milk and dairy products such as whey powder, whole milk powder, pure milk, fermented milk, infant formula, and prescription food for special medical purposes.

A multicenter performance evaluation of the new Elecsys Vitamin D total III assay versus reference isotope dilution liquid chromatography tandem mass spectrometry and commercially available comparators.

BackgroundVitamin D deficiency/insufficiency and toxicity are worldwide issues; thus, accurate diagnostic assays are required to measure vitamin D. We evaluated the performance of the new Elecsys® Vitamin D total III assay (Roche Diagnostics International Ltd).MethodsRepeatability and intermediate precision of the Elecsys Vitamin D total III assay (cobas e 601 analyzer) were evaluated at three sites using five human serum pools (HSPs) and two PreciControls (five‐day model, one reagent lot [CLSI‐EP05‐A3]) and compared against prespecified acceptance criteria. A serum verification panel, with reference isotope dilution liquid chromatography tandem mass spectrometry (ID‐LC–MS/MS) values, was used for comparator assay/concordance studies at two sites, assessed using unweighted Deming regression. Testing of serum vs. plasma on the Elecsys assay was conducted at one site using samples from healthy adults; assessed using Passing‐Bablok regression.ResultsRepeatability (HSP1 [16.8–18.4 ng/ml], SD 0.87–1.07; HSP5 [94.5–98.0 ng/ml], CV 1.58%–2.76%) and intermediate precision (HSP1, SD 1.14–1.77; HSP5, CV 2.00%–4.13%) met acceptance criteria across sites. Agreement was observed between the Elecsys assay and (i) the ID‐LC–MS/MS verification panel (slope, 0.936–1.01; Pearson's r, 0.960–0.986) and (ii) comparator assays (slope, 0.921–1.15; Pearson's r, 0.958–0.982). The Elecsys assay correctly assigned the highest combined percentage of samples to deficient (100%) and insufficient (89.5%) vitamin D categories vs. comparator assays and demonstrated comparable performance in serum and plasma (y = 0.103 + 0.984x).ConclusionsThe Elecsys Vitamin D total III assay demonstrated good analytical performance and compared favorably with other assays, supporting its use in clinical practice.

Development of Certified Reference Material for Amino Acids in Dried Blood Spots and Accuracy Assessment of Disc Sampling.

To achieve the measurement reliability of amino acids used as diagnostic markers in clinical fields, establishing a reference measurement system is required, in which certified reference materials (CRMs) are an essential step in the hierarchy of measurement traceability. This study describes the development of dried blood spot (DBS) CRMs for amino acid analysis with complete measurement traceability to the International System of Units (SI). Six essential amino acids—proline, valine, isoleucine, leucine, phenylalanine, and tyrosine—were analyzed using isotope-dilution liquid chromatography–mass spectrometry (ID-MS). For minimizing measurement bias and uncertainty overestimation, whole spots with 50 μL of whole blood were adopted in the certification. The between-spot homogeneities by whole spot sampling were lower than 2.1%. The relative expanded uncertainties of the six amino acids in the developed DBS CRMs were lower than 5.7% at 95% confidence. The certified values are traceable to SI through both gravimetric preparation and the primary method in certification, ID-MS. Comparison among DBS testing laboratories revealed discrepancies between the whole spot and disc sampling methods. The actual sampling volume was accurately estimated by weighing, which revealed the possibility of underestimation in routine DBS testing. The candidate CRMs can support the standardization of DBS testing for amino acids through the qualification and validation of many kinds of measurement procedures to compensate the measurement bias caused by matrix-specific sampling error.

The Triglycerides and Glucose Index is not superior to HOMA-IR in predicting testosterone deficiency among adultmales.

Testosterone decline and deficiency in males have aroused increased attention in male health management, which might have a close relationship with insulin resistance (IR). We utilized a novel and practical IR indicator, the triglyceride-glucose (TyG) index, to investigate the association between the TyG index and serum testosterone in US adult males. This was a cross-sectional study based on data from the National Health and Nutrition Examination Survey (NHANES) 2013-2014 and 2015-2016. The TyG index was calculated from fasting plasma glucose and serum triglyceride, and serum testosterone was measured by isotope dilution liquid chromatography tandem mass spectrometry in NHANES. A total of 2186 male participants aged more than 20 years old were included, and the mean TyG index was 8.72 ± 0.71. Participants with a higher TyG index showed a lower level of total testosterone (β=-45.83, 95% CI: -58.50, -33.15, p<0.0001) and a higher risk of testosterone deficiency (OR=1.80, 95% CI: 1.46, 2.21, p<0.0001) after we adjusted for all potential cofounders. Males in TyG index tertile 3 had a 113% (95% CI: 1.50, 3.02, p<0.0001) higher risk of testosterone deficiency than those in tertile 1. Subgroup analysis stratified by diabetes condition indicated that the TyG index might be a good predictor of testosterone decline or deficiency in either people with or without diabetes. However, ROC cure analysis indicated that a larger area under the curve was found in the homeostasis model assessment of insulin resistance (HOMA-IR) (0.71, 95% CI: 0.68,0.74) than in the TyG index (0.67, 95% CI: 0.64, 0.70). Males with a higher TyG index tended to have a higher risk of testosterone decline or even testosterone deficiency. However, the predictability of the TyG index for testosterone deficiency was not better than that of HOMA-IR. More well-designed studies are still needed to validate this relationship.

Prenatal alcohol exposure can be determined from baby teeth: Proof of concept.

Prenatal alcohol exposure (PAE), leading to fetal alcohol spectrum disorders (FASD), is a serious public health issue in the United States and globally. Diagnosis of FASD is crucial in obtaining appropriate care, but it is not always possible when PAE cannot be documented. Deciduous teeth from a child with known PAE and a child with known absence of PAE were analyzed using liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) in a multiple-reaction monitoring mode for direct markers and LC-high resolution MS in positive and negative mode with hydrophilic interaction liquid chromatography and reverse-phase chromatography, respectively, for indirect markers. Direct markers of PAE (ethyl glucuronide and ethyl sulfate) were detected in prenatal and postnatal dentine from a case tooth but not from a control tooth. Indirect biomarker analysis indicated a dysregulation of amino acids and an increase in cholesterol sulfate in the case compared to the control tooth. This proof-of-concept study demonstrates for the first time that direct biomarkers of PAE are detectable and measurable in deciduous teeth which begin forming in utero and are typically naturally shed between 5 and 12 years of age. Further examination of these novel biomarkers may allow diagnosis of FASD where documentation of PAE is otherwise unavailable. Furthermore, because teeth grow incrementally, defined growth zones can be sampled allowing for identification of gestational timing of PAE to help better understand mechanisms underlying alcohol's disruption of perinatal development.

Number of sexual partners and serum testosterone levels in a population-based sample of unpartnered heterosexual men.

The relationship between men's testosterone levels and their sexual behaviors is unclear as existing studies find mixed results. The key objective of this study is to assess whether men's testosterone levels are affected by sexual behaviors and to explore whether this relationship varies by age. Specifically, this study addresses the question: Are men's testosterone levels affected by the number of sexual partners they accrue? This analysis was based on a nationally representative sample of 977 unpartnered heterosexual men between the ages of 20 and 65 in the United States. Serum testosterone levels were measured from blood samples using isotope dilution liquid chromatography tandem mass spectrometry and the number of sexual partners in the past 12months was ascertained from an audio computer-assisted personal self-interview. Using ordinary least squares regression, serum testosterone levels were predicted as a function of the number of sexual partners that men reported, sociodemographic characteristics, and factors known to affect endocrine regulation. An interaction term between the number of sexual partners in the past 12months and age was included. The correlation between the number of sexual partners in the past 12months and testosterone levels is contingent upon age: The relationship is positive for younger men but negative for older men. Among 20-year-old unpartnered heterosexual men, an additional sexual partner in the past 12months is associated with an increase of 5.55ng/dl of testosterone (95% confidence interval (CI): 2.92, 8.19). This initial positive effect at age 20 is reduced by 0.25ng/dl of testosterone (95% CI: -0.44, -0.06) with each year that men age. Testosterone levels in men are related to reproductive behaviors, but the direction of this relationship changes as men age.

Online In-Tube Solid-Phase Microextraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry for Automated Analysis of Four Sulfated Steroid Metabolites in Saliva Samples.

Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17β-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC–MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01–2 ng mL−1 for PREGS, DHEAS, and CRTS and of 0.05–10 ng mL−1 for E2S. The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL−1, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6–112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 μL saliva samples.

Conversion of Classical and 11-Oxygenated Androgens by Insulin-Induced AKR1C3 in a Model of Human PCOS Adipocytes.

Polycystic ovary syndrome (PCOS) is the most prevalent endocrinopathy in women. A common symptom of PCOS is hyperandrogenism (AE); however, the source of these androgens is uncertain. Aldo-keto reductase family 1 member C3 (AKR1C3) catalyzes the formation of testosterone (T) and 5α-dihydrotestosterone (DHT) in peripheral tissues, which activate the androgen receptor (AR). AKR1C3 is induced by insulin in adipocytes and may be central in driving the AE in PCOS. We elucidated the conversion of both classical and 11-oxygenated androgens to potent androgens in a model of PCOS adipocytes. Using high-performance liquid chromatography (HPLC) discontinuous kinetic assays to measure product formation by recombinant AKR1C3, we found that the conversion of 11-keto-Δ4-androstene-3,17-dione (11K-4AD) to 11-ketotestosterone (11K-T) and 11-keto-5α-androstane-3,17-dione (11K-5AD) to 11-keto-5α-dihydrotestosterone (11K-DHT) were superior to the formation of T and DHT. We utilized a stable isotope dilution liquid chromatography high resolution mass spectrometric (SID-LC-HRMS) assay for the quantification of both classical and 11-oxygenated androgens in differentiated Simpson-Golabi-Behmel syndrome adipocytes in which AKR1C3 was induced by insulin. Adipocytes were treated with adrenal derived 11β-hydroxy-Δ4-androstene-3,17-dione (11β-OH-4AD), 11K-4AD, or Δ4-androstene-3,17-dione (4AD). The conversion of 11β-OH-4AD and 11K-4AD to 11K-T required AKR1C3. We also found that once 11K-T is formed, it is inactivated to 11β-hydroxy-testosterone (11β-OH-T) by 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1). Our data reveal a unique role for HSD11B1 in protecting the AR from AE. We conclude that the 11-oxygenated androgens formed in adipocytes may contribute to the hyperandrogenic profile of PCOS women and that AKR1C3 is a potential therapeutic target to mitigate the AE of PCOS.

Validation of a high-throughput method for simultaneous determination of areca nut and tobacco biomarkers in hair using microwave-assisted extraction and isotope dilution liquid chromatography tandem mass spectrometry

For people with habits of chewing betel nuts and smoking, the probability of suffering from oral cancer is ten to a hundred times higher than others. Due to the serious health consequences of areca nut and tobacco, a reliable cessation program is needed. Hair is the best option to document long-term exposure. Unfortunately, the research on betel nut in hair did not attract much attention. In this study, a high-throughput method based on microwave-assisted extraction (MAE) and isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed to measure the four biomarkers of betel nuts and cigarettes, including areca alkaloids (arecoline), tobacco alkaloids (nicotine), and their metabolites (arecaidine and cotinine). The hair sample was washed, cut, weighed, and incubated for 3 min MAE with methanol/trifluoroacetic acid, then evaporated and reconstituted for LC-MS/MS analysis. The total experiment time was 50 min. The lower limits of quantification (LOQ) were 5–10 pg/mg. The intra-day and inter-day precision were 2.2–7.6%. Intra-day and inter-day accuracy were − 6.1–8.2%. The method showed good linearity (r2 > 0.995) over LOQ - 1000 pg/mg concentration ranges. It was successfully applied to analyze 11 subjects of regular areca nut chewers, also smokers. Eight samples were black hair; three samples were naturally black hair with partially gray hair. Measured concentrations in black hair were in the range 56.9 pg/mg to 3.2 ng/mg for arecoline, 12.8 pg/mg to 222.2 pg/mg for arecaidine, 3.8 ng/mg to 33.4 ng/mg for nicotine and 1.1 ng/mg to 6.1 ng/mg for cotinine. The results showed lower levels in gray hair. This method was utilized successfully to analyze pg/mg levels of arecoline, arecaidine, nicotine, and cotinine, and good recoveries were obtained. The mean concentration of arecaidine and cotinine in hair was 15% and 20% of arecoline and nicotine, respectively. A good positive correlation was found between the concentrations of these compounds and self-report. This method improved extraction speed, concentration, and analysis of samples and is useful for monitoring betel nut and smoking cessation programs.

Peptide purity assignment for antibody quantification by combining isotope dilution mass spectrometry and liquid chromatography

AbstractPeptide purity assignment is necessary for quantification of antibody. Recently, isotope dilution mass spectrometry (IDMS) has been proposed as a primary method for an accurate antibody measurement with traceability. When using the IDMS method, antibodies are measured using their signature peptide as the calibration standard. Therefore, the purity assignments of their signature peptide are essential for the standardization and harmonization of the antibody measurement. In this study, SI‐traceable purity assignments of the signature peptides of an antibody were investigated by combining the IDMS and high performance liquid chromatography (HPLC) method. The final purity was calculated from the mass fraction of the total peptide determined by IDMS and the relative fraction of the target peptide determined by HPLC. The uncertainty was evaluated within the 95% confidence range. These peptides with the certified purity value can also be used for the quantification of antibodies for SARS‐CoV‐2.

Unexpected sensitivity enhancement in analysing alfatoxin M1 using LC-IDMS

Aflatoxin M1 (AFM1) and patulin (PAT) are regulated mycotoxins, which are generally analyzed in food samples alone (“one analyte at a time”). This paper presents the development of a liquid chromatography isotope dilution tandem mass spectrometric (LC-IDMS) method for the determination of AFM1 in milk-based products, which method was then successfully applied to PAT in apple-based food samples. The method involved a selective solid-phase extraction clean-up at acidic pH employing mixed-mode cation exchange cartridges. The purification allowed the adequate adsorption of neutral target compounds and basic matrix constituents of the sample to the reversed-phase (RP) and to the cation exchange phase of the cartridge, respectively. The sample elution with pure methanol yielded an extract free from basic matrices. The remaining acidic and neutral matrix constituents of the samples were separated from the analytes using mixed-mode anion exchange HPLC column. Mobile phases without any modifiers were used for the highest sensitivity and better selectivity. The intensity enhancement obtained by neutral eluent composition was inter-laboratory tested and confirmed on same type of instruments. The sample preparation requires low amount of organic solvent, thus adhered to green analytical chemistry. The developed method for analyzing AFM1 resulted in low matrix effect of LC-IDMS separation −4.9-(−1.5%) and high precision (2.7–5.9%), calculated under method validations at spiking levels between 0.005 and 0.050 µg/kg. Method performance characteristics for analyzing PAT were also evaluated at 10 and 25 µg/kg levels. Finally, the advantageous features of the newly developed method were briefly compared and discussed with the existing approaches.

Comparison between Egg Intake versus Choline Supplementation on Gut Microbiota and Plasma Carotenoids in Subjects with Metabolic Syndrome.

We previously demonstrated that intake of three eggs/d for 4 weeks increased plasma choline and decreased inflammation in subjects with metabolic syndrome (MetS). The purpose of the current study was to further explore the effects of phosphatidylcholine (PC) provided by eggs versus a choline bitartrate (CB) supplement on the gut microbiota, trimethylamine N-oxide (TMAO) formation, and plasma carotenoids lutein and zeaxanthin in MetS. This randomized, controlled crossover clinical trial included 23 subjects with MetS. Following a washout period of 2 weeks without consuming any choline-containing foods, subjects were randomly allocated to consume either three eggs/d or a CB supplement for 4 weeks (both diets had a choline equivalent of 400 mg/day). DNA was extracted from stool samples to sequence the 16S rRNA gene region for community analysis. Operational taxonomic units (OTUs) and the α-diversity of the community were determined using QIIME software. Plasma TMAO, methionine, betaine, and dimethylglycine (DMG) were quantified by stable isotope dilution liquid chromatography with tandem mass spectrometry. Plasma carotenoids, lutein, and zeaxanthin were measured using reversed-phase high-performance liquid chromatography. There were significant increases in plasma lutein and zeaxanthin after egg intake compared to the baseline or intake of CB supplement (p < 0.01). In contrast, TMAO was not different between treatments compared to the baseline (p > 0.05). Additionally, while diet intervention had no effects on microbiota diversity measures or relative taxa abundances, a correlation between bacterial biodiversity and HDL was observed. Following egg intake, the observed increases in plasma lutein and zeaxanthin may suggest additional protection against oxidative stress, a common condition in MetS.

Creatinine standardization: a key consideration in evaluating whole blood creatinine monitoring systems for CKD screening

Early detection of CKD using point of care creatinine and eGFR testing improves patient management outcomes. We undertook a field study to evaluate the use of a whole blood creatinine/eGFR device to screen a rural Nicaraguan population to determine the variability between creatinine methods and specimen types. All specimens including capillary and venous dried blood spots (DBS) were tested with an isotope dilution liquid chromatography mass spectrometry (ID-LCMS) gold standard method. This is to our knowledge the first time a capillary whole blood (POC) method has been directly compared to the gold standard IDMS method, through the novel approach of using dried blood spots. Capillary and venous whole blood specimens were obtained and tested directly with the BCMS method, and then, DBS samples were prepared. Venous plasma specimens were tested using three laboratory analyzer creatinine methods. DBS were sent to the site performing ID-LCMS. Control samples were also prepared to assess the stability of shipment and storage of DBS. The ID-LCMS method was aligned using primary and secondary standards. Sixty-six (66) patients participated in the study, and the CKD prevalence rate was 7.8%. While all creatinine methods showed a good correlation to ID-LCMS, there was a positive bias (mean absolute bias range: 0.21–0.63 mg/dL). All methods used were 100% sensitive, but specificity varied from 62.7 to 94.9% with PPV ranging from 25 to 62.5%. A correction factor was used to align the values from each method to ID-LCMS which improved the specificity of each method. This study used a unique DBS approach to align capillary whole blood creatinine to ID-LCMS. To ensure reliability of BCMS for identifying screened patients with CKD, it is important to establish IDMS traceability and alignment prior to undertaking CKD studies.

Characterization of a SARS-CoV-2 spike protein reference material

Development of diagnostic testing capability has advanced with unprecedented pace in response to the COVID-19 pandemic. An undesirable effect of such speed is a lack of standardization, often leading to unreliable test results. To assist the research community surmount this challenge, the National Research Council Canada has prepared a SARS-CoV-2 spike protein reference material, SMT1-1, as a buffered solution. Value assignment was achieved by amino acid analysis (AAA) by double isotope dilution liquid chromatography–tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in combination with ultraviolet–visible spectrophotometry (UV–Vis) based on tryptophan and tyrosine absorbance at 280 nm. Homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. Transportation and long-term storage stabilities were assessed by monitoring relative changes in oligomeric state by size-exclusion liquid chromatography (LC-SEC) with UV detection. The molar concentration of the spike protein in SMT1-1 was 5.68 ± 0.22 µmol L−1 (k = 2, 95% CI), with the native trimeric form accounting for ~ 94% of the relative abundance. Reference mass concentration and mass fraction values were calculated using the protein molecular weight and density of the SMT1-1 solution. The spike protein is highly glycosylated which leads to analyte ambiguity when reporting the more commonly used mass concentration. After glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection, we thus reported mass concentration values for both the protein-only portion and intact glycoprotein as 0.813 ± 0.030 and 1.050 ± 0.068 mg mL−1 (k = 2), respectively.