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.

Microdroplet mass spectrometry: Accelerating reaction and application.

Electrospray ionization (ESI) and desorption electrospray ionization (DESI) are common soft ionization method of mass spectrometry (MS). However, recent studies revealed that some chemical reactions can be induced or greatly accelerated in the sprayed microdroplets compared to the same reaction in the bulk. These open a new area in using microdroplet MS to explore new chemistry and develop new applications. This minireview will introduce microdroplet chemistries and explore various microdroplet techniques most of which are ESI- or DESI-based extensions by incorporating transfer tube, supersonic nebulizing gas, droplet fusion, spray extraction, laser irradiation, or laser ablation for online/offline MS analysis. Potential applications associated with new techniques, including real-time reaction monitoring, high-throughput reaction screening, protein identification, and protein characterization, are also described. Future outlook, such as coupling microdroplet MS with separation techniques, is proposed and discussed.

Quantification of the Endogenous Adduction Level on Hemoglobin and Correlation with Albumin Adduction via Proteomics: Multiple Exposure Markers of Catechol Estrogen.

Catechol estrogens (CEs) are genotoxic metabolites whose detection is challenging due to their low concentrations and high variability in the blood. By intact protein and free CE measurement of the spiked hemolysate, endogenous CEs were revealed to mainly (>99%) exist as hemoglobin (Hb) adducts in red blood cells. In order to detect endogenous CE-Hb adducts, we developed a two-step method that involved protein precipitation and solid phase extraction to purify Hb from red blood cells, and the method was coupled with proteomics using liquid chromatography-tandem mass spectrometry. Using bottom-up proteomics and standard additions, we identified C93 and C112 of Hb-ß as the main adduction sites of Hb, and this accounted for CE-induced oxidization of adducted peptides by sample preparation. The non-adducted, adducted, and oxidized tryptic peptides that covered the same Hb-ß sequences were targeted by parallel reaction monitoring to determine the adduction level in red blood cells. A quantification limit (S/N < 8) below the endogenous CE-Hb adduction level with relative standard errors that ranged from 5 to 22% was achieved and applied to clinical samples. The human serum albumin (HSA) adduction levels from the same patient were also determined using a previously developed method (Anal. Chem.2019,91, 15922-15931). A positive correlation (R2 = 0.673) between the CE-HSA and CE-Hb adduction level was obtained from all clinical samples, and both levels were significantly (p < 0.005) higher for patients with breast cancer compared to healthy controls. However, double indexes derived from the red blood cell and the serum, respectively, provide higher precision and confidence in predicting cancer risk than the single index. This study reported an efficient sample preparation for proteomics-based Hb adducts and revealed the potential of using multiple blood proteins for developing more reliable and specific markers based on protein adductomics.

Effect of Mobile Phase pH on the Electrospray Ionization Efficiency and Qualitative Analysis of Pharmaceuticals in ESI + LC-MS/MS.

The effect of mobile phase pH on positive ionization process and retention time of nine pharmaceuticals on ultra-performance liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS) was discussed. The effective use of high and low mobile phase pH in LC-MS/MS qualitative analyses method was also evaluated by comparing the instrument detection limit, quantification limit, precision, linearity and signal to noise (S/N) under low and high mobile phase pH. In this work, six mobile phase pH that ranged between pH 2 and pH 10 were used to evaluate the effect of the mobile phase pH changes on the ionization process in electrospray ionization. Results revealed that high mobile phase pH ionized more pharmaceuticals molecules and gave a higher signal than low mobile phase pH in positive ionization mode. The results proved that ammonium ion was better as a proton donor in high pH mobile phase than the hydronium ion in acidic mobile phase. The results revealed that the qualitative LC-MS/MS analyses method by using high mobile phase pH has better performance for most analytes in terms of sensitivity, precision, linearity and S/N than the low mobile phase pH.

Disk solid-phase extraction of multi-class pharmaceutical residues in tap water and hospital wastewater, prior to ultra-performance liquid chromatographic-tandem mass spectrometry (UPLC-MS/MS) analyses.

In this work, a new clean-up and pre-concentration method based on disk solid-phase extraction (SPE) was developed to determine multi-class pharmaceutical residues covering a wide range of polarities (log K ow values from -0.5 to 5.1) in water systems, prior to ultra-performance liquid chromatographic-tandem mass spectrometry (UPLC-MS/MS) analyses. Electrospray ionisation in positive and negative modes was used for the simultaneous determination of both acidic and basic pharmaceuticals. The performances of disk SPE and cartridge SPE were compared. The targeted pharmaceutical compounds list included bronchodilators, antidiabetic drugs, antihypertensive drugs, a lipid-lowering agent, analgesics, and anti-inflammatory drugs. Based on our results, the disk SPE demonstrated a higher sensitivity and recovery value and less analysis time as compared to the cartridge SPE method. The limits of detection (LOD) for the new method ranged from 0.02-3.2 ng L-1, 0.02-3.1 ng L-1 and 0.02-4.7 ng L-1 for tap, effluent and influent wastewater, respectively. The method's absolute recovery values ranged from 70% to 122% for tap water, 62% to 121% for effluent wastewater and 62% to 121% for influent wastewater, except for metformin in which the absolute recovery value was approximately 48% for all samples. Intra-day precision for tap water, effluent and influent wastewater ranged from 3-12%, 4-9% and 2-8%, respectively. The method developed was applied for the determination of targeted pharmaceuticals in tap, effluent, and influent wastewater from one hospital treatment plant in Malaysia. The results revealed that the highest concentrations of certain pharmaceuticals were up to 49 424 ng L-1 (acetaminophen) and 1763 ng L-1 (caffeine) in the influent and effluent wastewater, respectively. The results also showed a variation in the treatment efficiencies for the hospital treatment plant from one compound to another. Nevertheless, the removal efficiencies ranged from 0-99%.