Isotope Dilution Liquid Chromatography Research Articles

An isotope dilution LC–MS/MS-based candidate reference method for the quantification of androstenedione in human serum and plasma

The accurate measurement of androstenedione in human serum and plasma is required for steroid profiling to assure the appropriate diagnosis and differential diagnosis of hyperandrogenism. In this work, we introduce an isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS) candidate reference measurement procedure for the quantification of androstenedione in human serum and plasma. The performance of the procedure enables its use in the evaluation and standardization of routine assays and for the evaluation of patient samples to ensure the traceability of individual patient results. As the primary standard, a certified reference material from NMIA (National Measurement Institute, Australia) was used. Additionally, a quantitative nuclear magnetic resonance (qNMR) method was developed for the value assignment of the primary reference material, which ensures the direct traceability to SI units, as well as the independence from the availability of reference materials. 13C3-labeled androstenedione was used as the internal standard. The introduced method allows the measurement of androstenedione in the range of 0.05–12 ng/mL, and the assay imprecision was found to be <2% between 5 and 12 ng/mL, 3.5% at 1.5 ng/mL, and 5.2% at 0.05 ng/mL, with an accuracy of 95–105% for the serum and 91–103% for the plasma matrix. The transferability to a second laboratory was validated by method comparison based on 112 patient samples. The comparison of the results obtained from the presented method and an LC–MS/MS routine assay, using 150 native patient samples, showed a good correlation with a bias of the routine method of ≤4.0%.

Assessing a method and reference material for quantification of vitamin D binding protein during pregnancy

Vitamin D plays a vital role in successful pregnancy outcomes for both the mother and fetus. Vitamin D is bound to vitamin D binding protein (VDBP) in blood and is carried to the liver, kidneys and other target tissues. Accurate measurements of the clinically measured metabolite of vitamin D, 25-hydroxyvitamin D [25(OH)D], depend on complete removal from the binding protein. It has been found that VDBP concentrations increase in maternal serum during pregnancy, obfuscating the accuracy of 25(OH)D concentration measurements in pregnant women. Additionally, measurements of VDBP concentrations during pregnancy have been performed using immunoassays, which suffer from variations due to differences in antibody epitopes, making clinical comparisons difficult. Quantification of VDBP is also of interest because changes in VDBP expression levels may indicate negative outcomes during pregnancy, such as preterm delivery and restricted fetal growth. To address the need for accurate measurement of VDBP during pregnancy, a method using liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was developed to quantify VDBP using isotopically labeled peptides as internal standards. This method was used to quantify VDBP in Standard Reference Material® (SRM) 1949 Frozen Human Prenatal Serum, which was prepared from separate serum pools of women who were not pregnant and women during each trimester of pregnancy. VDBP concentrations were found to be lowest in the serum pool from non-pregnant women and increased in each trimester. These data had good repeatability and were found to be suitable for reference value assignment of VDBP in SRM 1949.

Evaluation of Tobacco Smoke and Diet as Sources of Exposure to Two Heterocyclic Aromatic Amines for the U.S. Population: NHANES 2013-2014.

Heterocyclic aromatic amines (HAA) are a group of hazardous substances produced during combustion of tobacco or high-temperature cooking of meats. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is a major carcinogenic HAA in tobacco smoke. Urinary AαC, used as a marker of AαC exposure, was analyzed on spot urine samples from adult participants of the 2013-2014 cycle of the National Health and Nutrition Examination Survey (N = 1,792). AαC was measured using isotope-dilution liquid chromatography-tandem mass spectrometry. Exclusive combusted tobacco smokers were differentiated from nonusers of tobacco products through both self-report and serum cotinine data. Among exclusive smokers, sample-weighted median urinary AαC was 40 times higher than nonusers. Sample-weighted regression models showed that urinary AαC increased significantly with serum cotinine among both exclusive tobacco users and nonusers with secondhand smoke exposure. Among nonusers, eating beef cooked at high temperature was associated with a significant increase in urinary AαC, whereas consuming vegetables was associated with decreased AαC. In addition, smoking one-half pack of cigarettes per day was associated with a significant increase of 23.6 pg AαC/mL calculated at geometric mean of AαC, controlling for potential confounders. In comparison, increase in AαC attributable to consuming the 99th percentile of beef cooked at high temperature was 0.99 pg AαC/mL. Both exclusive smokers and nonusers of tobacco in the general U.S. population are exposed to AαC from tobacco smoke, with additional, lesser contributions from certain dietary components. AαC is an important biomarker that is associated with tobacco smoke exposure.

Analytical Method of Multi-Mycotoxins in Table-Ready Foods for a Total Diet Study Using Stable Isotope Dilution Liquid Chromatography–Tandem Mass Spectrometry

Abstract Background: Determining the multi-mycotoxins present in table-ready foods is necessary for a total diet study. However, so far, most methods of analyzing multi-mycotoxins apply to raw foods. Therefore, a reliable method for analyzing multi-mycotoxins in table-ready foods is needed. Objective: We developed and validated methods of multi-mycotoxin analysis that employed stable isotope dilution with LC–tandem MS (LC–MS/MS) using two representative matrices. Methods: Samples were fortified with [13C]-labeled mycotoxins as internal standards and extracted with 50% acetonitrile in water for high-carbohydrate foods and 3% formic acid in acetonitrile for high-protein and/or high-fat foods, cleaned up with n-hexane and the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, and followed up by LC–MS/MS. Results: Validation of the methods was performed, and the results were as follows: the correlation coefficient, R2, was 0.99; method detection limit, 0.01–2.4 μg/kg; recoveries, 83.6–114%; precision, 0.8–10 (intraday) and 3.1–22% (interday); interlaboratory reproducibility, ≤15%; and uncertainty, 3.5–19%error. The applicability of the methods was evaluated by analyzing table-ready foods spiked with standards. Conclusions: These methods were successfully evaluated and deemed appropriate for determining the multi-mycotoxins in table-ready foods. Highlights: This work demonstrates that stable isotope dilution with LC–MS/MS can be effectively used to analyze multi-mycotoxins simultaneously in a total diet study.

Development of an Isotope Dilution Mass Spectrometry Method for Accurate Determination of Thiamine in Diverse Food Matrices

Many foods contain thiamine as a natural or fortified nutrient. In order to realize accurate measurement of thiamine in various food matrices, analytical methods should be developed on the basis of careful examination of the matrix and the analytes in the matrix. An isotope dilution liquid chromatography–mass spectrometry (ID-LC-MS/MS) method was developed for the analysis of thiamine in various samples such as multivitamin tablets, infant formula, rice, and wheat flour. After using spiked isotope-labeled thiamine HCl (13C4-thiamine HCl) as an internal standard, samples were analyzed by LC/MS in a selected reaction monitoring mode to detect ions at m/z 265 → 122 and m/z 269 → 122 for thiamine and 13C4-thiamine, respectively. To improve the accuracy and precision of this method, the preparation procedure was optimized depending on the chemical form of thiamine present in each sample. Thiamine in multivitamin tablets, infant formula, rice, and wheat flour was analyzed after buffer extraction, acid hydrolysis, and continuous acid and enzyme hydrolyses, respectively. The instrumental analyses were also optimized for individual samples, facilitating excellent repeatability (0.16–0.81%) and reproducibility (0.1–4%). The accuracy of the developed method was verified by comparing the experimental data with the certified values of certified reference materials. Thus, optimization of the preparation procedures and ID-LC-MS/MS method allowed the accurate determination of thiamine in diverse food matrices.

Simple and accurate candidate reference measurement procedure for total testosterone in human serum by one-step liquid-liquid extraction coupled with isotope dilution mass spectrometry.

Testosterone in human serum is commonly tested in clinical laboratories using immunoassay methods as well as liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. To standardize and ensure the accuracy of the measurement results, reference procedures with higher metrological order are required. A simple measurement procedure based on one-step liquid-liquid extraction (LLE) and liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) was developed for total testosterone in human serum. The procedure involved serum spiked with 13C3-testosterone, equilibration for 2h, and extraction with an organic solvent. Testosterone certified reference material (CRM) was used as the calibration standard to ensure the traceability to the International System of Units (SI). Testosterone in serum CRMs from the National Institute for Standards and Technology (NIST) and LGC were used to validate the accuracy of the newly developed method. The deviations of the obtained values from the NIST and LGC certified values ranged from -0.55% to 0.45%. Similarly, the coefficient of variations (CVs) of the replicate measurements were in the range of 0.55% and 0.78%, respectively. The relative expanded uncertainties were comparable with those of the certified materials. The newly developed LC-IDMS/MS procedure demonstrated adequate trueness and precision, and was simple to perform. The method can be used for value assignment of testosterone in external quality assessment (EQA) materials as well as certification of CRMs in the future. Graphical abstract.

Comparison of bracketing calibration and classical calibration curve quantification methods in establishing a candidate reference measurement procedure for human serum 17β-estradiol by isotope dilution liquid chromatography tandem mass spectrometry

A highly accurate liquid chromatography tandam mass spectrometry (LC–MS/MS) based candidate reference measurement procedure (cRMP) for the determination of human serum 17β-estradiol (E2) was developed. A bracketing calibration method (BCM) based on internal standard (IS) concentrations for each sample used to best match the analyte concentration was compared to the classical calibration curve-based method with a constant IS concentration. Precision was analyzed according to the inter-assay reproducibility (n = 15) and intra-assay repeatability (n = 9). Accuracy was confirmed using certified reference materials (CRMs) and further validated by comparison to the established RMPs. Sixty patient serum samples were collected, and the E2 levels were measured by the BCM isotope dilution (ID) procedure coupled with the LC–MS/MS method to evaluate three immunoassays commonly used in clinical laboratories in China. Limits of quantification (LoQ) as low as 5 pg/mL (18 pmol/L) were achieved. The isomer of E2 (17α-estradiol) was successfully separated by optimizing the LC method. The results of method validation showed that the intra- and inter-assay imprecisions were 1.84 ∼ 2.91% vs 2.08 ∼ 9.53%, while the analytical recoveries were 98.73 ∼ 100.77% vs 93.72 ∼ 102.39% for the BCM and classical calibration curve quantification method, and the BCM demonstrated a higher agreement with CRMs and established RMPs. Bland-Altman plots of the E2 results revealed concentration-dependent biases. In conclusion, a precise, facile, and reliable ID-LC–MS/MS method is developed in this study. It is demonstrated that the use of the BCM could achieve higher precision and accuracy, which better meet the needs of RMPs.

106Plasma trimethylamine N-oxide is associated with culprit plaque characterization as assessed by optical coherence tomography in patients with acute myocardial infarction

Abstract Background Previous study have demonstrated that plasma trimethylamine N-oxide (TMAO) is associated with vulnerable plaque characteristics as assessed by optical coherence tomography (OCT) in patients with coronary artery disease. However, the relation between TMAO and the culprit plaque characteristics as assessed by OCT in patients with acute myocardial infarction (AMI) exhibiting plaque rupture at the site of the culprit stenosis is unknown. Objective To explore the relation between plasma TMAO and coronary culprit plaque characterization assessed by OCT in AMI patients exhibiting plaque rupture. Method We prospectively enrolled 90 AMI patients with plaque rupture identified by OCT and collected demographic data, risk factors, coronary angiography and OCT data, medical history and laboratory findings of all patients. Plasma TMAO levels were detected by stable isotope dilution liquid chromatography tandem mass spectrometry. Macrophage presence in coronary culprit plaque was quantified by normalized standard deviation (NSD). Result All patients were divided into two groups (high TMAO group and low TMAO group) according to the median plasma TMAO level (3.22uM). The culprit plaques in the high TMAO group exhibited a thinner fibrous cap thickness (60um [60–100um] versus 90um [70–110um], P=0.013]), higher frequency of thin-cap fibroatheroma (TCFA) (15.6% versus 55.6%, P&lt;0.001), microvessel (24.4% versus 4.4%, P=0.014) and macrophage infiltration (66.7% versus 26.7%, P&lt;0.001) compared with the low TMAO group. Moreover, the level of TMAO was significantly positively associated with NSD (Pearson's correlation coefficient: r=0.766, P&lt;0.001). Conclusion Plasma TMAO levels are associated with coronary plaque vulnerability and inflammation in patients with AMI exhibiting plaque rupture. Acknowledgement/Funding Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2016-I2M-1-009)

Promising Tropical Fruits High in Folates.

As the popularity of tropical fruits has been increasing consistently during the last few decades, nutritional and health-related data about these fruits have been gaining more and more interest. Therefore, we analyzed 35 samples of tropical fruits and vegetables with respect to folate content and vitamer distribution in this study. The fruits and vegetables were selected by their availability in German supermarkets and were grouped according to their plant family. All fruits and vegetables were lyophilized and analyzed by stable isotope dilution assay (SIDA) and liquid chromatography mass spectrometry (LC-MS/MS). The results vary from 7.82 ± 0.17 µg/100 g in the horned melon to 271 ± 3.64 µg/100 g in the yellow passion fruit. The yellow passion fruit is a good source for meeting the recommended requirements, as just 110 g are needed to cover the recommended daily intake of 300 µg folate for adults; however, longan fruits, okras, pete beans, papayas, mangos, jack fruits, and feijoas are also good sources of folates. In conclusion, the study gives a good overview of the total folate content in a broad range of tropical fruits and vegetables and shows that some of these fruits definitely have the potential to improve the supply of this critical vitamin.

Quantification of serum apolipoprotein E and patient phenotyping using isotope dilution liquid chromatography-tandem mass spectrometry

Objective To establish an isotope dilution liquid chromatography tandem mass spectrometry method (ID-LC-MS) for quantification of serum apolipoprotein E and phenotyping. Methods Method establishment. Samples underwent denaturing, alkylation and trypsin digestion with addition of internal standards as isotope labelling arginine. SB-C18 column was used for the liquid chromatographic separation and mass spectrometry positive ion mode and multiple reaction monitoring were employed for quantification and phenotyping. Precision, accuracy and linearity were investigated for method evaluation. 40 serum samples from Shanghai Dongfang Hospital during Oct. to Dec., 2018 were used for method comparison between ID-LC-MS and immunoassay. Deming regression and Bland-Altman were used for method comparison analysis and SPSS 24 for linearity. Results Target peptides reached their releasing maximum within 4 hours and SE did at 3 hours. 3 phenotyping of ApoE were observed, such as E3/E3, E2/E3 and E3/E4. The imprecision of IQC was 5.2%. The relative bias for low and high levels of accuracy-based samples was 7.6% and 3.6%, respectively. Deming regression showed the intercept with 95% confidence interval (CI) was 6.44-11.44 (P<0.05 and the 95% confidence interval for the slopewas 0.77-0.89 (P<0.05). The coefficient was r=0.97. The mean difference was -2.95 mg/L with 95% CI -4.26--1.65 mg/L. The linearity covered from 16.9 to 58.5 mg/L. Conclusion ID-LC-MS can be used to quantify serum apolipoprotein E and simultaneously detect its phenotyping. Key words: Apolipoproteins E; Phenotype; Chromatography, liquid; Tandem mass spectrometry

Complete validation according to current international criteria of a confirmatory quantitative method for the determination of nitrofuran metabolites in seafood by liquid chromatography isotope dilution tandem mass spectrometry

Despite the ban of nitrofurans (NFs) for use in food production in many countries in the 1990s, NF metabolites in food are still regularly detected during import control testing. We have developed a confirmatory routine method for the detection and quantification of NF metabolites in seafood using LC-MS/MS and validated the method according to the strict criteria in European legislation and Codex Alimentarius. Method characteristics were found to fulfill the criteria. We report for the first time a new false positive for 1-amino-2,4-imidazolidinedione (AHD), the metabolite of Nitrofurantoin (NFT). By using optimized washing procedures, the non tissue bound false positives can be minimized. The results from the validation on both lean and fatty fish and crustaceans, results from proficiency tests and routine use over many years, demonstrates that the method is fit for purpose to determine NF metabolites in the seafood category.

Abstract 587: Highly sensitive and accurate method for measuring serum estradiol in postmenopausal women and other population subgroups

Abstract Meta-analyses consistently show that postmenopausal women with elevated levels of estradiol have an increased risk for certain cancers, such as breast cancer. At the same time, estradiol therapy is used to treat menopausal symptoms. Data on blood levels of estradiol are highly variable, especially in postmenopausal women, which prevents the formulation of generally recognized normal ranges and the consistent treatment of postmenopausal women. Similarly, no generally recognized reference ranges for estradiol exist for men and children. One reason for this variability in estradiol blood levels is the lack of appropriate analytical methods. To overcome these challenges, new, highly sensitive, specific, and standardized methods for measuring estradiol are needed. This study describes a new analytical method for measuring estradiol in serum from post and premenopausal women, men and children, which is standardized to the CDC Hormone Standardization Program (HoSt). Data produced by this analytical method can be compared with data produced by other methods standardized to CDC HoSt. The analytical method optimizes the extraction of estradiol from serum using two liquid-liquid extractions with pH and polarity adjustments prior to LC-MS/MS analysis. All sample handling and extraction procedures are automated using 96-well plates. Chromatographic separation is carried out using a phenyl-hexyl HPLC column and a gradient of methanol and methanol:water. E2 and its C13 internal standard were analyzed by selected reaction monitoring (SRM) in the negative ion mode with transitions of m/z 271 to 145 and 274 to 148, respectively. The mean biases of this method to certified reference materials ranged between −1.3% and −1.7% and were statistically not significant. No significant difference to established metrological reference methods was determined. The limit of detection for estradiol using 200 µL of serum is 11.0 pM (2.99 pg/mL). The method is highly precise with a within-run, among-day (determined over 68 days), and total within-laboratory imprecision ranging between 2.9-5.0%, 1.5-1.8%, and 3.3-5.3% CV, respectively [1]. The sensitivity of the method was found to be suitable for determining estradiol levels in postmenopausal women, men, and children, and is being applied to measure estradiol in postmenopausal women in NHANES 2013-2014. Data generated by this method are comparable to those of other analytical methods that are standardized to the CDC HoSt program. This method allows for the development of generally recognized normal ranges of estradiol in postmenopausal women and other population subgroups.

Sources of variation evaluation of 24,25(OH)2D levels and the ratio of 25OHD to 24,25(OH)2D in apparently healthy Chinese adults: a multicenter cross-sectional study

24,25(OH)2D and the 25OHD/24,25(OH)2D ratio have recently been shown to be useful for screening for hypercalcemia caused by CYP24A1 mutations. However, no population-based data on 24,25(OH)2D have been published for Chinese populations, and few studies evaluated the sources of variation on 24,25(OH)2D levels and the 25OHD/24,25(OH)2D ratio. Hence, in 2018, we enrolled 1211 apparently healthy adults without systematic diseases from six representative cities in China (Beijing, Dongying, Guiyang, Urumqi, Shenzhen, and Qiqihar). 24,25(OH)2D and 25OHD levels were measured using isotope dilution liquid chromatography tandem mass spectrometry. Multiple regression analysis showed that sex contributed the most to variations in 24,25(OH)2D, 25OHD, and 25OHD/24,25(OH)2D (rp= -0.255, -0.253, and 0.141, respectively), and age also contributed to variations in 25OHD but not 24,25(OH)2D or 25OHD/24,25(OH)2D. Men had significantly higher 24,25(OH)2D and 25OHD levels than women, but had significantly lower 25OHD/24,25(OH)2D values. Individuals from Dongying had the highest 24,25(OH)2D and 25OHD values, whereas individuals from Urumqi had the lowest values. The median(2.5–97.5%) values for 24,25(OH)2D, 25OHD, and 25OHD/24,25(OH)2D were 1.2(0.36–2.65) ng/mL, 18.9(8.6–32.5) ng/mL, and 16.0 (9.8–30.8), respectively. 24,25(OH)2D was significantly correlated with 25OHD (r = 0.838, p < 0.001), and 25OHD/24,25(OH)2D was significantly negatively correlated with 24,25(OH)2D (r = -0.758, P < 0.001) and 25OHD (r = -0.310, P < 0.001). In conclusion, in this nationwide, multicenter, cross-sectional study, we evaluated the levels of 24,25(OH)2D and the 25OHD/24,25(OH)2D ratio in the Chinese population. Sex contributed the most to variations in 24,25(OH)2D, 25OHD, and 25OHD/24,25(OH)2D.

Accuracy-Based Vitamin D Survey: Six Years of Quality Improvement Guided by Proficiency Testing.

The goal of the College of American Pathologists Accuracy-Based Proficiency Testing Program is to promote the quality, standardization, and harmonization of clinical laboratory results through proficiency testing specimens that are free from matrix effects, have target values that are traceable to reference methods, and that probe the limitations of current methods. To summarize the first 6 years of the Accuracy-Based Vitamin D Survey and highlight key insights from the data generated as it relates to assay performance. Accuracy-based challenges were created by using pooled human serum samples. Certain samples were derived from participants in an institutional review board-approved protocol in which vitamin D-deficient participants were treated with ergocalciferol (vitamin D2). Reference targets for the survey were set by the Centers for Disease Control and Prevention using isotope-dilution liquid chromatography-tandem mass spectrometry. Each method was compared with the reference method procedure over the course of the program (n = 43 proficiency testing samples). Linear regression versus the reference method procedure revealed proportional biases across the methods, ranging from 0.0% to 16.7%. Pearson correlation coefficients (r2) ranged from 0.902 to 0.996. Results were influenced by the concentration of 25-hydroxyvitamin D2 as well as the C-3 epimer of 25-hydroxyvitamin D3. During the 6 years, 2 manufacturers altered their assays to match the reference method procedure more closely. There is considerable bias, both proportional bias and sample-specific matrix effects, affecting many assays. This ongoing accuracy-based proficiency testing program for vitamin D will provide the data needed for laboratories and manufacturers to improve their assays and thereby patient care.

Commutable whole blood reference materials for hemoglobin A1c validated on multiple clinical analyzers.

Background The measurement of hemoglobin A1c (HbA1c) is important for diagnosing diabetes mellitus as well as assessing glycemic control in diabetic patients. Commutable whole blood certified reference materials (CRMs) are needed in the measurement of HbA1c for method validation and/or as quality controls. Methods We developed three levels of hemolyzed whole blood CRMs for HbA1c. The certified values were determined using liquid chromatography-isotope dilution tandem mass spectrometry method (LC-IDMS/MS) where two "signature" hexapeptides of HbA1c and hemoglobin A0 (HbA0) were used as the calibration standards. The concentrations of the hexapeptide solutions were determined by amino acid analysis by the LC-IDMS/MS method using amino acid CRMs as the calibration standards. The commutability study was conducted by measuring 25 patient specimens and the whole blood CRMs by both LC-IDMS/MS method and various routine methods using six different clinical analyzers. Results The certified values were determined to be 35.1±2.0, 50.3±1.9 and 65.8±2.6 mmol/mol, respectively. These CRMs showed good commutability on five of the six clinical analyzers but showed poor commutability on one of the clinical analyzers that used similar method as two other analyzers where good commutability was observed. Conclusions With certified target values based on metrological traceability and good commutability on most of the clinical analyzers, the developed whole blood CRMs can be used for method validation or as quality control materials in the measurement of HbA1c. The commutability study results also underscored the need of commutability testing of clinical CRMs using various clinical analyzers.

Analytical Method of Multi-Mycotoxins in Table-Ready Foods for a Total Diet Study Using Stable Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry.

Background: Determining the multi-mycotoxins present in table-ready foods is necessary for a total diet study. However, so far, most methods of analyzing multi-mycotoxins apply to raw foods. Therefore, a reliable method for analyzing multi-mycotoxins in table-ready foods is needed. Objective: We developed and validated methods of multi-mycotoxin analysis that employed stable isotope dilution with LC-tandem MS (LC-MS/MS) using two representative matrices. Methods: Samples were fortified with [13C]-labeled mycotoxins as internal standards and extracted with 50% acetonitrile in water for high-carbohydrate foods and 3% formic acid in acetonitrile for high-protein and/or high-fat foods, cleaned up with n-hexane and the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, and followed up by LC-MS/MS. Results: Validation of the methods was performed, and the results were as follows: the correlation coefficient, R², was 0.99; method detection limit, 0.01-2.4 μg/kg; recoveries, 83.6-114%; precision, 0.8-10 (intraday) and 3.1-22% (interday); interlaboratory reproducibility, ≤15%; and uncertainty, 3.5-19%error. The applicability of the methods was evaluated by analyzing table-ready foods spiked with standards. Conclusions: These methods were successfully evaluated and deemed appropriate for determining the multi-mycotoxins in table-ready foods. Highlights: This work demonstrates that stable isotope dilution with LC-MS/MS can be effectively used to analyze multi-mycotoxins simultaneously in a total diet study.

Determination of microcystins, nodularin, anatoxin-a, cylindrospermopsin, and saxitoxin in water and fish tissue using isotope dilution liquid chromatography tandem mass spectrometry

Cyanobacteria can form dense blooms under specific environmental conditions, and some species produce secondary metabolites known as cyanotoxins, which present significant risks to public health and the environment. Identifying toxins produced by cyanobacteria present in surface water and fish is critical to ensuring high quality food and water for consumption, and protectionn of recreational uses. Current analytical screening methods typically focus on one class of cyanotoxins in a single matrix and rarely include saxitoxin. Thus, a cross-class screening method for microcystins, nodularin, anatoxin-a, cylindrospermopsin, and saxitoxin was developed to examine target analytes in environmental water and fish tissue. This was done, due to the broad range of cyanotoxin physicochemical properties, by pairing two extraction and separation techniques to improve isolation and detection. For the first time a zwitterionic hydrophilic interaction liquid chromatography column was evaluated to separate anatoxin-a, cylindrospermopsin, and saxitoxin, demonstrating greater sensitivity for all three compounds over previous techniques. Further, the method for microcystins, nodularin, anatoxin-a, and cylindrospermopsin were validated using isotopically labeled internal standards, again for the first time, resulting in improved compensation for recovery bias and matrix suppression. Optimized extractions for water and fish tissue can be extended to other congeners in the future. These improved separation and isotope dilution techniques are a launching point for more complex, non-targeted analyses, with preliminary targeted screening.

Identification and quantification of isoguanosine in humans and mice

Isoguanine (2-hydroxyadenine), considered to be a non-natural nucleobase has, however, been shown to occur in the croton bean, butterfly wings and a mollusk. For the first time, to the best of our knowledge, we report the identification of isoguanosine (2-hydroxyadenosine), the ribonucleoside, in humans and mouse. Isoguanosine is identified and quantified in RNA from mouse liver samples and in human urine and cerebrospinal fluid. Isoguanine could not be detected as the 2′-deoxyribonucleoside in mouse liver DNA. It could be speculated that the source of isoguanosine was formation from adenosine during oxidative stress in the body. However, the urinary concentrations of isoguanosine and the levels in the liver found here by using isotope dilution liquid chromatography–tandem mass spectrometry are identical to or exceed those of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine. Guanine is the nucleobase that is oxidized the easiest, so it appears spectacular that the levels of isoguanosine are higher than the levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine. It also appears intriguing that it was only possible to detect the ribonucleoside isoguanosine and not the 2′-deoxyribonucleoside. These observations could indicate that the isoguanosine found is not formed by oxidative stress and could have biological functions.

Measurement of serum progesterone by isotope dilution liquid chromatography tandem mass spectrometry: a candidate reference method and its application to evaluating immunoassays.

Measurement of serum progesterone is important in determining ovarian function. Most progesterone measurements are performed with immunoassays and results are often variable. Standardization of the measurements requires a reliable reference method. An isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for the measurement of serum progesterone was developed. Serum samples were spiked with 13C3-progesterone, extracted with a three-step liquid-liquid extraction, and analyzed by LC/MS/MS. A bracketing calibration was used for the analysis and samples were prepared gravimetrically. The developed method showed intra-run and total relative standard deviations (RSDs) of 0.50~0.58% and 0.71~1.33%, respectively. The analytical recoveries were 99.08~101.50%. Measurement results on certified reference materials obtained with this method agreed with the certified values within the stated measurement uncertainties. The method was applied to evaluate immunoassays through split-sample comparisons. A panel of 48 fresh frozen individual samples were measured with the ID-LC/MS/MS method, and six immunoassays and results were compared. Significant calibration biases and sample-specific deviations were observed on some of the immunoassays.

Retrospective nationwide occurrence of fipronil and its degradates in U.S. wastewater and sewage sludge from 2001 - 2016

The insecticide fipronil is under regulatory scrutiny worldwide for its toxicity to pollinators and aquatic invertebrates. We conducted the first U.S. nationwide, longitudinal study of sewage sludges for fiproles, i.e., the sum of fipronil and its major degradates (fipronil sulfone, sulfide, amide, and desulfinyl). Archived sludges (n = 109) collected in three campaigns over 15 years were analyzed by isotope dilution liquid chromatography tandem mass spectrometry, revealing ubiquitous fiprole occurrence (0.2–385.3 μg/kg) since 2001 and a significant increase (2.4 ± 0.3 fold; p < 0.005) both from 2001 to 2006/7 and from 2001 to 2015/6, but not a significant increase from 2006/7 to 2015/6 (p = 0.275). A geospatial analysis showed fiprole levels in municipal sludges to be uncoupled from agricultural use of fipronil on cropland surrounding sampled municipalities, thus pointing to non-agricultural uses (i.e., spot-on treatment and urban pest control) as a major source of fiprole loading to wastewater. Whereas anaerobic digestion was correlated with increases in fipronil sulfide at the expense of parental fipronil (p < 0.001), total fiprole levels in sewage sludges were similar regardless of the solids treatment approach applied (p = 0.519). Treatment plant effluent available from 12 facilities in 2015/6 contained fiproles at 0.3–112.9 ng/L, exceeding the United States Environmental Protection Agency (USEPA) aquatic invertebrate life benchmark for chronic fipronil exposure (11 ng/L) in 67% of cases. Whereas the USEPA identified fipronil in sludge only recently (2015), retrospective analyses and modeling conducted here show contaminant ubiquity and nationwide increases of fiprole mass (compared to 2001 levels) in U.S. municipal sludge (1140 ± 230 kg in 2015/6), and treated effluent nationwide (1970 ± 390 kg in 2015/6) over the past 15 years.