Metabolites In Human Serum Research Articles

Critical evaluation of high-resolution and low-resolution mass spectrometry for biomonitoring of human environmental exposure to pesticides

Despite increasing debates about their potential side effects on human health, data concerning the risks and the impacts associated with pesticides remains scarce. Analytical tools allowing the measurement of most pesticides and/or their metabolites to which the population can be exposed are also of need. In the present study, the limits of detection (LODs) of 3 different screening procedures based on either Low-Resolution and High-Resolution Mass Spectrometry (LR-MS and HR-MS) for the determination of pesticides in serum (among which carbamates, dithiocarbamates, neonicotinoids, organochlorines, organophosphates and pyrethroids) were explored. For HR-MS, a quadrupole time-of-flight was used in positive and negative electrospray ionization modes and data were obtained from either a targeted scheduled MSMS acquisition (HR-MSMS) or a data-independent acquisition (HR-DIA). For LR-MS, a triple quadrupole was used and data were acquired with a classical multiple-reaction monitoring (MRM) mode. Depending on the approach, the LOD values varied from 0.05 to 10 ng/mL. For the lowest concentrations, the proportion of molecules detected was systematically greater for the LR-MS approach, while those of HR-MSMS were better than those of HR-DIA. These differences in the LOD values were confirmed in a sample of 174 serums in which LR-MRM detected 89 compounds while HR-MSMS and HR-DIA detected 79 and 75 compounds, respectively. Nevertheless, for environmental and occupational purposes, HRMS approach could probably be efficient to detect most of pesticides and their metabolites in human serum and could be suitable for human biomonitoring studies or fundamental research exploring the impact of exposure to pesticides on human health.

Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study.

Amino acids (AAs) and their metabolites are important building blocks, energy sources, and signaling molecules associated with various pathological phenotypes. The quantification of AA and tryptophan (TRP) metabolites in human serum and plasma is therefore of great diagnostic interest. Therefore, robust, reproducible sample extraction and processing workflows as well as rapid, sensitive absolute quantification are required to identify candidate biomarkers and to improve screening methods. We developed a validated semi-automated robotic liquid extraction and processing workflow and a rapid method for absolute quantification of 20 free, underivatized AAs and six TRP metabolites using dual-column U(H)PLC-MRM-MS. The extraction and sample preparation workflow in a 96-well plate was optimized for robust, reproducible high sample throughput allowing for transfer of samples to the U(H)PLC autosampler directly without additional cleanup steps. The U(H)PLC-MRM-MS method, using a mixed-mode reversed-phase anion exchange column with formic acid and a high-strength silica reversed-phase column with difluoro-acetic acid as mobile phase additive, provided absolute quantification with nanomolar lower limits of quantification within 7.9 min. The semi-automated extraction workflow and dual-column U(H)PLC-MRM-MS method was applied to a human prostate cancer study and was shown to discriminate between treatment regimens and to identify metabolites responsible for discriminating between healthy controls and patients on active surveillance.

Conjugated bisphenol S metabolites in human serum and whole blood

Studies have shown that bisphenol S (BPS) is mainly present as its conjugated metabolites in human blood. However, the distribution of conjugated BPS metabolites in different human blood matrices has not been characterized. In this study, paired human serum and whole blood samples (n = 79) were collected from Chinese participants, and were measured for the occurrence of BPS and 4 BPS metabolites. BPS was detectable in 49% of human serum (<LOD–1.7 ng/mL) and 78% of whole blood (<LOD–2.1 ng/mL) samples. In both human serum and whole blood, BPS-sulfate (BPS–S; 74% and 86%, respectively) and BPS-glucuronide (BPS-G; 68% and 84%, respectively) had higher detection frequency than BPS. Consistently, BPS-S was the predominant BPS metabolite in human serum (mean 0.39 ng/mL) and whole blood (0.41 ng/mL), significantly higher than BPS-G (mean 0.13 and 0.13 ng/mL, respectively). This is contrary to the data from controlled oral exposure studies on model animals and humans, in which BPS-G was the dominant BPS metabolite in serum. BPS-S (mean ± SD, 0.99 ± 0.35) had the highest partitioning coefficient between human serum and whole blood, followed by BPS-G (0.89 ± 0.27) and BPS (0.81 ± 0.28), suggesting their preferential accumulation in the red blood cell fraction. These results represent the first human data on conjugated BPS metabolites in paired human serum and whole blood, which help to elucidate of the occurrence of conjugated BPS metabolites in humans.

Surface-enhanced Raman database of 24 metabolites: Stable measurement of spectra, extraction and analysis of the main features

Surface-enhanced Raman spectroscopy (SERS) has been used in Raman-based metabolomics to provide abundant molecular fingerprint information in situ with extremely high sensitivity, without damaging the sample. However, poor reproducibility, caused by the randomness of the adsorption sites, and the short-range effect of SERS have hindered the development of SERS in metabolomics, resulting in very few SERS reference databases for small-molecule metabolites. In this work, our previously proposed large laser spot-swift mapping SERS method was adopted for the measurement of 24 commercially available metabolite standards, to provide reproducible and reliable references for Raman-based metabolomics study. Among these 24 metabolites, 22 contained no Raman data in PubChem. Other than the SERS spectra data, we extracted and explained the molecular vibration information of these metabolites, and combined with the density functional theory (DFT) calculations, we provided a new possibility for the fast Raman recognition of small-molecule metabolites. Accordingly, a large laser spot-swift mapping SERS database of metabolites in human serum was initially established, which contained not only the original spectral data but also other detailed feature information regarding the Raman peaks. With continuous accumulation, this database could play a promising role in Raman-based metabolomics and other Raman-related research.

Identification of novel metabolites of abiraterone in human serum and their metabolic pathways.

Two novel abiraterone (Abi, 3β-OH-Abi) metabolites in human serum were identified as 3α-OH-Abi and Δ5-Abi (D5A). Both metabolites were confirmed by their retention times on LC/MS and their product-ion mass spectra on LC-MS/MS compared to those of authentic compounds, which were chemically synthesized. The plausible metabolic pathways of these two metabolites are as follows: Abi is first oxidized to D5A by 3β-hydroxysteroid dehydrogenase (3β-HSD) and then irreversibly converted to Δ4-Abi (D4A) by ∆5-∆4 isomerase. Presumably, D5A detection is difficult because of its rapid conversion to D4A and its low concentration in serum samples. In contrast, the low concentration 3α-OH-Abi was generated by reducing the remaining D5A using 3α-hydroxysteroid dehydrogenase (3α-HSD).

2-fluoro-1-methylpyridinium p-toluene sulfonate: a new LC-MS/MS derivatization reagent for vitamin D metabolites

Vitamin D analysis by MS faces several analytical challenges, including inefficient ionization, nonspecific fragmentation, interferences from epimers, isomers, and isobars, as well as very low concentration levels. In this study, we used 2-fluoro-1-methylpyridinium (FMP) p-toluene sulfonate for derivatization of vitamin D3 metabolites to increase detection sensitivity and allow for full chromatographic separation of vitamin D isomers and epimers. UHPLC-MS/MS was used for measurement of five vitamin D3 metabolites in human serum. Compared with Amplifex and 4-phenyl-1,2,4-triazolin-3,5-dion, the FMP p-toluene sulfonate reaction required less time to be performed. The method was optimized and validated to ensure accuracy, precision, and reliability. In-house and commercial quality control samples were used to assure the quality of the results for 25-hydroxyvitamin D3. The method showed very good linearity and intraday and interday accuracy and precision; coefficients of determination (r2) ranged between 0.9977 and 0.9992, relative recovery from 95 to 111%, and coefficient of variation from 0.9 to 11.3. Stability tests showed that the extracted derivatized serum samples were stable for 24 h after storage at −20°C; 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3-FMP derivatives were stable for 1 week at −80°C. The method was applied to samples of healthy individuals for quantitative determination of vitamin D3, the two epimers of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.

Fast gas chromatography-tandem mass spectrometry under milder electron ionization conditions for the assay of vitamin D metabolites in human serum

The proposed research was focused on the development of a gas chromatography-tandem mass spectrometry (GC-QqQ-MS) method under milder electron ionization (EI) conditions for the assay of vitamin D metabolites in human serum. Efficiency of three different silylation agents was evaluated for the conversion of vitamin D species into trimethylsilyl (TMS) derivatives, among which N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) proved to be the most effective. Indeed, the MSTFA reagent was able to convert in TMS ether even the 25-hydroxyl vitamin D derivative that, as known, possesses steric hindrance problems. The separation of vitamin D compounds was obtained in about 11.5 min using a narrow-bore column of dimensions 30 m × 0.25 mm ID × 0.10 μm df with a poly(5% diphenyl/95% dimethyl siloxane) stationary phase. The mass spectrometry ionization of the silylated derivatives was performed under milder EI conditions (20-eV energy) that, respect to common 70-eV energy, generated scan mass spectra with higher relative and absolute intensities of high-mass diagnostic ions, along with a reduced abundance of the low-mass. The signals of the ionized compounds were acquired in multi-reaction-monitoring (MRM) mode, thus enabling the obtainment of highly-sensitive and selective quantitative data. The developed method was validated in term of linearity, accuracy, limits of detection (LoD) and quantification (LoQ). In detail, regression coefficients of the calibration curves were between 0.9959 and 0.9999; LoDs ranged from 0.06 ng mL−1 to 0.73 ng mL−1 and LoQs from 0.16 ng mL−1 to 2.45 ng mL−1. With respect to accuracy, the serum SRM 972a certified reference material (Vitamin D metabolites in frozen human serum) (Levels 1–4) was analyzed.

Determination of valproic acid and its six metabolites in human serum using LC-MS/MS and application to interaction with carbapenems in epileptic patients.

Valproic acid (VPA) is a classic medication for several types of epilepsy and mood disorders, and some of its effectiveness and toxicity is associated with metabolites. Although many reports have reported the drug-drug interactions of VPA, no study has focused on the influence of carbapenems (CBPMs) on VPA's active metabolites. An LC-MS/MS method for determining VPA and its six metabolites (3-hydroxy valproic acid, 4-hydroxy valproic acid, 2-propyl-2-pentenoic acid, 2-propyl-4-pentenoic acid, 3-keto valproic acid, and 2-propylglutaric acid) in human serum was established and applied to evaluate the drug-drug interaction with CBPMs in epileptic patients. The stable isotope valproic acid-d6 was used as an internal standard. Analytes in serum samples (50 μl) were isolated using a Kinetex C18 column (3× 100 mm, 2.6μm) and detected via negative electrospray ionization after protein precipitation. It was linear (r> 0.99) over the calibration range for different analytes. The accuracy was 91.44-110.92%, and the precision was less than 9.98%. The matrix effect, recovery, and stability met the acceptance criteria. According to the data collected from 150 epileptic patients, the concentration-dose ratio for VPA and its metabolites decreased with CBPM polytherapy. This method is simple and rapid with great accuracy and precision. It is suitable for routine clinical analysis of VPA and its metabolites in human serum.

A novel LC-MS/MS method combined with derivatization for simultaneous quantification of vitamin D metabolites in human serum with diabetes as well as hyperlipidemia.

Vitamin D plays an important role in calcium homeostasis. Recent studies indicate that vitamin D deficiency has become a major public health problem. In order to define vitamin D status, many analytical methods were used to quantify 25-hydroxyvitamin D (25OHD), as circulating 25OHD is regarded as the best indicator to evaluate vitamin D status. The current LC-MS/MS technology is internationally recognized as the "gold standard" for the detection of vitamin D and its metabolites. The impediment to the analysis of vitamin D metabolites is the low level of 25OHD and 1,25(OH)2D. Therefore, it is challenging to achieve the desired sensitivity and accuracy in the determination of trace vitamin D compounds in biological liquids. Here, a method based on liquid-liquid extraction in combination with derivatization, followed by liquid chromatography-electrospray/tandem mass spectrometry was developed for determination of the vitamin D metabolites, including 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1α,25-dihydroxyvitamin D2 and 1α,25-dihydroxyvitamin D3. The method was simple and rapid, and it was validated with good linearity (R2 > 0.998), excellent recovery (average value with 81.66-110.31%) and high precision of intra-day and inter-day (0.06-6.38% and 0.20-6.82%). The values of limit of detection (LOD) and limit of quantitation (LOQ) were as low as 0.3 ng mL-1 and 1.0 ng mL-1, respectively. Finally, the developed method was successfully applied to determination of the vitamin D metabolites from the human serum samples of healthy subjects and patients with diabetes as well as hyperlipidemia.

Determination of vitamin D3 conjugated metabolites: a complementary view on hydroxylated metabolites.

Experts typically define vitamin D deficiency levels by the determination of a circulating 25-hydroxyvitamin D3-calcifediol prohormone. A large part of the population is characterized by deficient vitamin D levels (calcifediol < 20 ng mL-1) despite individuals not being affected by any disorder. Cholecalciferol (vitamin D3) and/or calcifediol supplementation is a common practice for vitamin D-deficient individuals as recommended by international scientific societies and official agencies. In the last few years, several studies have reported the presence of conjugated vitamin D3 metabolites, mainly glucuronidation and sulfation derivatives, although simultaneous quantitative measurements involving phase I and II vitamin D metabolites have not been carried out. A quantitative method based on tandem mass spectrometry detection is proposed here for the combined determination of phase I and phase II vitamin D3 metabolites in human serum. As phase I and phase II metabolites are preferentially ionized in different modes, a switching polarity mode was adopted to determine both groups of compounds in serum at high sensitivity levels (pg mL-1). The validation of this proposal was successfully accomplished by following the Center for Drug Evaluation and Research (CDER) guidelines. Its applicability was tested in a cohort of volunteers with mostly deficient baseline levels. Considering the sulfated form of calcifediol, the sum of its concentrations showed sufficient baseline vitamin D levels in all individuals, suggesting that this could be a novel strategy for vitamin D deficiency definition. Therefore, phase II metabolites are proposed to be included when evaluating the vitamin D status since they provide more information about the overall status of the vitamin D endocrine system. Nevertheless, further studies are required to confirm the biological activity of these conjugated metabolites and the suitability of this strategy for the description of vitamin D deficiency.

Therapeutic drug monitoring in breast cancer therapy – LC-MS/MS method for quantification of the CDK4/6 inhibitors abemaciclib, palbociclib, ribociclib, and major metabolites abemaciclib M20 and M2 in human serum

The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors palbociclib, ribociclib, and abemaciclib were approved by the U.S. Food and Drug Administration (FDA) and European Medicine Agency for the treatment of breast cancer between 2015 and 2018. Oral tumor therapeutics extend the options for cancer therapy, but also challenge physicians and patients. The aim of the present work was to establish a semi-automated liquid-chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of abemaciclib, its active metabolites abemaciclib M20 and M2, palbociclib, and ribociclib in human serum. Detuning of ribociclib enabled the development of a simultaneous quantification method for abemaciclib, M20, M2, palbociclib, and ribociclib in the respective relevant concentration ranges based on semi-automated sample preparation with isotope dilution LC-MS/MS. The method was validated according to the guidance of the FDA. The LC-MS/MS method was successfully validated according to FDA and showed inaccuracies ≤ 10.7% and imprecisions ≤ 8.51%. Linearity was given from 20 to 800 ng/mL for abemaciclib, 15–600 ng/mL for M20, 10–400 ng/mL for M2 and palbociclib, and 100–4000 ng/mL for ribociclib. Normalized matrix factors and process efficiency showed no significant matrix effects regardless of the analytes. To demonstrate the applicability of the method, authentic samples were also analyzed. This novel semi-automated LC-MS/MS method covering all previously approved CDK4/6 inhibitors as well as the similarly pharmacologically active metabolites in human serum simultaneously was developed for potential future use in routine analysis in order to improve personalized therapy, patient safety, and treatment success.

REGULATION OF NEURONAL MITOCHONDRIAL BIOENERGETICS BY LIPID METABOLITES UPREGULATED IN DEMENTIA

Abstract Mitochondrial dysfunction occurs early in Alzheimer’s disease (AD) progression and is evident in the Central Nervous System (CNS) and peripheral circulating cells. While there is evidence indicating that bioenergetic decline can drive the early pathogenesis of AD, little is known about the extracellular factors that alter neuronal bioenergetic capacity. We hypothesized that circulating metabolites contribute to neuronal bioenergetic decline associated with AD. Using a cohort comprised of participants with normal cognition, mild cognitive impairment, and dementia, we show that human serum harbors circulating, non-cellular factors capable of mediating neuronal bioenergetic differences according to the cognitive status of the serum donor. We developed a novel screening-based approach to identify candidate “mito-active” lipid metabolites in human serum that could mediate differences in bioenergetic capacity. Among these, Nervonic Acid and 15-epi-PGA1 were predicted to be mitochondrial inhibitors upregulated in participants with dementia. Neurons exposed to physiologically-relevant ranges of these molecules in-vitro exhibited a dose-dependent reduction in maximal mitochondrial respiration. We found that 500ug/mL of Nervonic Acid and 9ug/mL of 15-epi-PGA1 reduced maximal mitochondrial respiration by 62.3% and 63.3%, respectively, thereby validating our screening and prediction approach. Future experiments may be directed towards investigating if and how these and other mito-active metabolites of interest cross the blood-brain barrier to meaningfully affect AD. Furthermore, identified mito-active molecules can be investigated in other clinical cohorts to examine their role/s in multiple age-related or neurological conditions. This work expands our mechanistic understanding of how extrinsic factors associated with age and cognitive status contribute to neuronal bioenergetic decline.

REGULATION OF NEURONAL MITOCHONDRIAL BIOENERGETICS BY LIPID METABOLITES UPREGULATED IN DEMENTIA

Abstract Mitochondrial dysfunction occurs early in Alzheimer’s disease (AD) progression and is evident in the Central Nervous System (CNS) and peripheral circulating cells. While there is evidence indicating that bioenergetic decline can drive the early pathogenesis of AD, little is known about the extracellular factors that alter neuronal bioenergetic capacity. We hypothesized that circulating metabolites contribute to neuronal bioenergetic decline associated with AD. Using a cohort comprised of participants with normal cognition, mild cognitive impairment, and dementia, we show that human serum harbors circulating, non-cellular factors capable of mediating neuronal bioenergetic differences according to the cognitive status of the serum donor. We developed a novel screening-based approach to identify candidate “mito-active” lipid metabolites in human serum that could mediate differences in bioenergetic capacity. Among these, Nervonic Acid and 15-epi-PGA1 were predicted to be mitochondrial inhibitors upregulated in participants with dementia. Neurons exposed to physiologically-relevant ranges of these molecules in-vitro exhibited a dose-dependent reduction in maximal mitochondrial respiration. We found that 500ug/mL of Nervonic Acid and 9ug/mL of 15-epi-PGA1 reduced maximal mitochondrial respiration by 62.3% and 63.3%, respectively, thereby validating our screening and prediction approach. Future experiments may be directed towards investigating if and how these and other mito-active metabolites of interest cross the blood-brain barrier to meaningfully affect AD. Furthermore, identified mito-active molecules can be investigated in other clinical cohorts to examine their role/s in multiple age-related or neurological conditions. This work expands our mechanistic understanding of how extrinsic factors associated with age and cognitive status contribute to neuronal bioenergetic decline.

LC-MS/MS methods for the determination of 30 quaternary ammonium compounds including benzalkonium and paraquat in human serum and urine

Benzalkyldimethylammonium (or benzalkonium; BACs), alkyltrimethylammonium (ATMACs), and dialkyldimethylammonium compounds (DDACs) have been widely used for over six decades as disinfectants, especially during the COVID-19 pandemic. Here we describe methods for the determination of 7 BACs, 6 ATMACs, 6 DDACs, 8 BAC metabolites, and the structurally similar quaternary ammonium compound (QAC) herbicides diquat, paraquat, and difenzoquat in human serum and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methods were optimized using isotopically labelled internal standards and solid-phase extraction with weak cation-exchange cartridges. We separated diquat and paraquat chromatographically using a mixed-mode LC column, and BACs, ATMACs, DDACs, difenzoquat, and BAC metabolites using reversed-phase (C8 and C18) LC columns. Method limits of detection (MLODs) and quantification (MLOQs) were 0.002–0.42 and 0.006–1.40 ng/mL, respectively. Recoveries of all analytes fortified at 1, 5, and 20 ng/mL concentrations in serum and urine matrices were 61–129%, with standard deviations of 0–20%. Repeated analysis of similarly fortified serum and urine samples yielded intra-day and inter-day variations of 0.22–17.4% and 0.35–17.3%, respectively. Matrix effects for analytes spiked into serum and urine matrices ranged from –27% to 15.4%. Analysis of real urine and serum samples revealed the presence of several QACs in human serum. Although no parent BACs were found in urine, we detected, for the first time, several ω-hydroxy and ω-carboxylic acid metabolites of BACs at average concentrations in the range of 0.05–0.35 ng/mL. The developed method is suitable for application in large-scale biomonitoring of human exposure to QACs and their metabolites in human serum and urine.

High performance liquid chromatography–tandem mass spectrometry quantification of tryptophan metabolites in human serum and stool – Application to clinical cohorts in Inflammatory Bowel Diseases

Tryptophan, an essential amino acid, and its metabolites are involved in many physiological processes including neuronal functions, immune system, and gut homeostasis. Alterations to tryptophan metabolism are associated with various pathologies such as neurologic, psychiatric disorders, inflammatory bowel diseases (IBD), metabolic disorders, and cancer. It is consequently critical to develop a reliable, quantitative method for the analysis of tryptophan and its downstream metabolites from the kynurenine, serotonin, and indoles pathways. An LC-MS/MS method was designed for the analysis of tryptophan and 20 of its metabolites, without derivatization and performed in a single run. This method was validated for both serum and stool. The comparisons between serum and plasma, collected with several differing anticoagulants, showed significant differences only for serotonin. References values were established in sera and stools from healthy donors. For stool samples, as a proof of concept, the developed method was applied to a healthy control group and an IBD patient group. Results showed significant differences in the concentrations of tryptophan, xanthurenic acid, kynurenic acid, indole-3-lactic acid, and picolinic acid. This method allowed an extensive analysis of the three tryptophan metabolic pathways in two compartments. Beyond the application to IBD patients, the clinical use of this method is wide-ranging and may be applied to other pathological conditions involving tryptophan metabolism, such as neurological, psychiatric, or auto-inflammatory pathologies.

Serum concentrations of phthalate metabolites in pregnant women and their association with gestational diabetes mellitus and blood glucose levels

Phthalate metabolites are widely present in humans and can have many adverse effects on pregnant women. To date, many studies on the effects of phthalate metabolites on the risk of gestational diabetes mellitus (GDM) have been published, but the findings of these studies are controversial. We conducted a case-control study to quantify the concentrations of seven phthalate metabolites in the serum of pregnant women and to investigate their association with the risk of GDM and blood glucose levels in pregnant women. Therefore, 201 serum samples (139 pregnant women with GDM and 62 control serum samples) were collected from Hangzhou, China, between 2011 and 2012. The results showed that mono butyl phthalate (MBP; mean = 4.08 ng/mL) was the most abundant phthalate metabolites in human serum, followed by mono (2-ethylhexyl) phthalate (MEHP; mean = 1.28 ng/mL) and mono isobutyl phthalate (MiBP; mean = 1.20 ng/mL). The other results indicated significant associations between MBP (β = 2.24, 95 % confidence interval (CI): 1.02, 5.07, P = 0.050) and MiBP (β = 1.84, 95 % CI: 1.03, 3.31, P = 0.041) concentrations in human serum and the incidence of GDM. Moreover, serum MBP (β = 0.40, 95 % CI: 0.10, 0.70, P = 0.010) and MiBP levels (β = 0.18, 95 % CI: 0.010, 0.35, P = 0.047) in humans were positively associated with 2-hour blood glucose levels. Our study provides affirmative evidence on previously inconsistent findings that MBP and MiBP exposure may increase the risk of GDM in pregnant women.

Label-free and de-conjugation-free workflow to simultaneously quantify trace amount of free/conjugated and protein-bound estrogen metabolites in human serum

Different chemical forms of sex hormones including free/conjugated metabolites as well as their protein/DNA adducts in human serum are a panel of important indicators of health conditions. It is, however, hard to quantify all species simultaneously due to the lack of general extraction, derivatization, and de-conjugation methods. Here we developed a label-free and de-conjugation-free workflow to quantify 11 free/conjugated estrogen metabolites including depurinating DNA and protein adduct forms of 4-hydroxyestradiol (4OHE2) in human serum. Acetonitrile acts as an excellent solvent to purify adducted and non-adducted human serum albumin (HSA) by precipitation as well as to extract free/conjugated metabolites and depurinating DNA adducts from the supernatant by salting-out effect. The adduction level of 4OHE2 on HSA was determined by proteomics; free/conjugated metabolites were quantified by a newly developed microflow liquid chromatography (microflow LC)-nanoelectrospray ionization (nanoESI)-multiple reaction monitoring (MRM) method with high reproducibility (7–22% RSD, n > 3) and sub-picogram levels (0.6–20 pg/mL) of quantification limits (S/N = 8) by using non-pulled capillary as nano-ESI emitter. This workflow was demonstrated to reveal endogenous adduction level of 4OHE2 on HSA as well as circulation levels of free/conjugated metabolites in clinical samples. 4OHE2 in human serum were solely detected as protein-bound form, indicating the merit of such integrated platform covering unstable or active metabolites. Compared to traditional methods using labeling or de-conjugation reaction, this workflow is much simplier, more sensitive, and more specific. Moreover, it can be widely applied in omics to concurrently access various bio-transformed known and un-known markers or drugs.

Determination of multiple bisphenol analogues and their metabolites in human serum by liquid chromatography tandem mass spectrometry

To date, knowledge of internal human exposure to BPA and its analogues (particularly bisphenol S and bisphenol F, etc.) remains limited. In the present study, a method involving dispersive solid-phase extraction and LC/MS was proposed to investigate the contamination levels of 28 precursor bisphenols and 9 major metabolites in serum. The critical variables of preparation method were screened out by Plackett-Burman design and further optimized by central composite design. Left in optimal conditions, a total of 286 samples consisting of 153 males and 133 females were analyzed. The results showed that BPA dominated over all the cases with the highest positive rate (82.2% of all the surveyed people), and totally four metabolites (BPA β-D-glucuronide, BPA monosulfate, BPA bis-(β-D-glucuronide) and BPS monosulfate) were detectable. The occurrence of BPA bis-(β-D-glucuronide) in serum is reported for the first time and its higher positive rate and contamination concentrations suggested that it may be a more important metabolite of BPA than others. Negligible potential risk of health effects to blood donors was observed, since the estimated exposure levels (mean 32.1 ng/kg bw/day, 95th 123.2 ng/kg bw/day) were well below far less than the temporary tolerable reference dose of BPA that recommended by the European Food Safety Authority (4 μg/kg bw/day by). The reference level of BPA for healthy population was determined to be 4.09 μg/L via the percentile method.

Rapid and simultaneous determination of multiple endocrine-disrupting chemicals and their metabolites in human serum and urine samples

Bisphenols, parabens, and their metabolites are a group of chemical compounds with a wide range of polarities but similar chemical structures, which presents a challenge for the simultaneous determination of these compounds in complex biological samples. In this study, a rapid and sensitive method for simultaneous quantification of free bisphenol A (BPA), conjugated BPA, bisphenols, and parabens analogs was developed using solid-phase extraction (SPE) tandem liquid–liquid extraction (LLE). We compared the effects of different types of SPE cartridges, diluents, and LLE solvents on the analyte recovery. Utilizing the direct and indirect determination methods (enzyme hydrolysis), we confirmed the accuracy of the direct method for measuring BPA glucuronide and BPA disulfate. The method enabled the analysis of 24 endocrine-disrupting chemicals (EDCs) in one injection through UHPLC-MSMS measurements, with satisfactory recovery (mean: 91.8–98.6% for urine, 80.2%–96.8% for serum) and precision (RSD <15%). The LOD and LOQ values were 0.003 and 0.01 ng/mL for serum, and 0.002 and 0.006 ng/mL for urine samples, respectively. For real sample analysis, the median concentration of analytes in serum and urine samples ranged from 0.04 ng/mL (BPS) to 56.4 ng/mL (4-HB) and 0.11 ng/mL (BPA) to 136 ng/mL (4-HB), respectively. This method provides a new strategy to simultaneously identify compounds with a wide range of polarities from complicated biological matrices.

Isotope dilution LC-MS/MS quantification of the cystic fibrosis transmembrane conductance regulator (CFTR) modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their major metabolites in human serum.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators have revolutionized the therapeutic landscape in CF treatment. These vital drugs are extensively metabolized via CYP3A, so caution must be exercised in multimodal CF therapy because of the risk of adverse drug interactions. Our goal was to develop a highly sensitive assay for the purpose of therapeutic drug monitoring (TDM) in diagnostic laboratories. After protein precipitation, the CFTR modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their metabolites ivacaftor-M1, ivacaftor-M6, and tezacaftor-M1 were separated with a two-dimensional chromatography setup within 5min, and quantified with stable isotope-labeled internal standards. The method was validated according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and applied to CF patient samples. Inaccuracy was ≤7.0% and the imprecision coefficient of variation (CV) was ≤8.3% for all quality controls (QCs). The method consistently compensated for matrix effects, recovery, and process efficiency were 105-115 and 96.5-103%, respectively. Analysis of CF serum samples provided concentrations comparable to the pharmacokinetic profile data reported in the EMA assessment report for the triple combination therapy Kaftrio. We hereby present a robust and highly selective isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) assay for the simultaneous quantification of the so far approved CFTR modulators and their metabolites in human serum. The assay is suitable for state-of-the-art pharmacovigilance of CFTR modulator therapy in CF patients, in order to maximize safety and efficacy, and also to establish dose-response relationships in clinical trials.