Hien P. Nguyen

The advantages of ESI-MS detection in conjunction with HILIC mode separations: Fundamentals and applications.

The analysis of highly hydrophilic, ionic, and polar compounds has been performed by HILIC-ESI-MS for the last few years. The use of low aqueous/high polar organic solvent content in HILIC separation mobile phase is almost ideal for ESI-MS detection in many cases, resulting in increased sensitivity. Although the addition of modifiers such as acids or salts is necessary in some circumstances for a good separation, the optimum concentrations used are still highly amenable for ESI-MS analysis, showing few deleterious effects. In this review, the mechanism of HILIC separation and ESI ion generation will be briefly discussed, followed by a summary of method development and applications in several fields of research including pharmaceutical, biomolecular, food, metabolic, and environmental analysis.

General method for extraction of blueberry anthocyanins and identification using high performance liquid chromatography–electrospray ionization-ion trap-time of flight-mass spectrometry

A systematic approach for optimizing the extraction and identification of anthocyanins from blueberries was explored using HPLC-UV and HPLC-ESI-IT-TOF-MS. Sample homogenization effects, extraction solvent selection, type of acid, and amount used in extraction solvent were investigated. A mixture of methanol:water:trifluoroacetic acid (70:30:1, v/v/v) was found to be the best solvent system for blueberry anthocyanin extraction. Differences in total anthocyanin content due to commercial blueberry processing were explored as an application using the optimized extraction technique and HPLC-UV analysis. A methodical system for anthocyanin identification by HPLC-ESI-IT-TOF-MS without the use of standards was also reviewed and applied. Consideration was given to elution order by chromatographic separation with selective detection at 520nm, high mass accuracy m/z values, tandem MS fragmentation, and previously published literature. Overall, 25 anthocyanins from a wild type highbush blueberry were identified and reported.

The proto-oncometabolite fumarate binds glutathione to amplify ROS dependent signaling

The tricarboxylic acid cycle enzyme fumarate hydratase (FH) has been identified as a tumor suppressor in a subset of human renal cell carcinomas. Human FH-deficient cancer cells display high fumarate concentration and ROS levels along with activation of HIF-1. The underlying mechanisms by which FH loss increases ROS and HIF-1 are not fully understood. Here, we report that glutamine-dependent oxidative citric acid cycle metabolism is required to generate fumarate and increase ROS and HIF-1 levels. Accumulated fumarate directly bonds the antioxidant glutathione in vitro and in vivo to produce the metabolite succinated glutathione (GSF). GSF acts as an alternative substrate to glutathione reductase to decrease NADPH levels and enhance mitochondrial ROS and HIF-1 activation. Increased ROS also correlates with hypermethylation of histones in these cells. Thus, fumarate serves as a proto-oncometabolite by binding to glutathione which results in the accumulation of ROS.

Inhibition of Cancer Cell Proliferation by PPARγ Is Mediated by a Metabolic Switch that Increases Reactive Oxygen Species Levels

The nuclear receptor peroxisome-proliferation-activated receptor gamma (PPARγ), a transcriptional master regulator of glucose and lipid metabolism, inhibits the growth of several common cancers, including lung cancer. In this study, we show that the mechanism by which activation of PPARγ inhibits proliferation of lung cancer cells is based on metabolic changes. We found that treatment with the PPARγ agonist pioglitazone triggers a metabolic switch that inhibits pyruvate oxidation and reduces glutathione levels. These PPARγ-induced metabolic changes result in a marked increase of reactive oxygen species (ROS) levels that lead to rapid hypophosphorylation of retinoblastoma protein (RB) and cell-cycle arrest. The antiproliferative effect of PPARγ activation can be prevented by suppressing pyruvate dehydrogenase kinase 4 (PDK4) or β-oxidation of fatty acids in vitro and in vivo. Our proposed mechanism also suggests that metabolic changes can rapidly and directly inhibit cell-cycle progression of cancer cells by altering ROS levels.

Simultaneous quantification of four native estrogen hormones at trace levels in human cerebrospinal fluid using liquid chromatography-tandem mass spectrometry

Estrogens are known to exhibit neuroprotective effects on the brain. Their importance in this regard and in others has been emphasized in many recent studies, which increases the need to develop reliable analytical methods for the measurement of estrogen hormones. A heart-cutting two-dimensional liquid chromatography separation method coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been developed for simultaneous measurement of four estrogens, including estriol (E3), estrone (E1), 17β-estradiol (17β-E2), and 17α-estradiol (17α-E2), in human cerebrospinal fluid (CSF). The method was based on liquid-liquid extraction and derivatization of estrogens with dansyl chloride to enhance the sensitivity of ESI-based detection in conjunction with tandem mass spectrometry. Dansylated estriol and estrone were separated in the first dimension by an amide-C18 column, while dansylated 17β- and 17α-estradiol were resolved on the second dimension by two C18 columns (175 mm total length) connected in series. This is the first report of a method for simultaneous quantification of all four endogenous estrogen compounds in their dansylated form. The detection limits for E1, 17α-E2, 17β-E2, and E3 were 19, 35, 26, and 61pg/mL, respectively. Due to matrix effects, validation and calibration was carried out in charcoal-stripped CSF. The precision and accuracy were more than 86% for the two E2 compounds and 79% for E1 and E3 while the extraction recovery ranged from 91% to 104%. The method was applied to measure estrogens obtained in a clinical setting, from the CSF of ischemic trauma patients. While 17β-estradiol was present at a significant level in the CSF of some samples, other estrogens were present at lower levels or were undetectable.

Retention behavior of estrogen metabolites on hydrophilic interaction chromatography stationary phases

Estrogens and estrogen metabolites are important biological mediators of the endocrine system. They have also been implicated in detrimental carcinogenesis and beneficial neuroprotective processes. The retention behavior of estrogen metabolites was investigated on five polar stationary phases, used for hydrophilic interaction chromatography, and coupled with ESI-MS. Data were fit to partitioning and surface adsorption models. Retention of the compounds, especially estrogen glucuronides, on the amide- and diol-bonded stationary phases, could be best described by the surface adsorption model; however, mixed modes of retention were observed on most stationary phases. Retention time increased while the peak efficiency decreased proportional to the number of hydroxyl groups in the analytes. The effects of salt concentration and salt type were also investigated. The presence of solvated salt ions, which interact with the stationary phase and the analyte, enhanced retention of the analytes. This was believed to be due to two effects. The increased ionic strength reduced the contribution of secondary electrostatic interactions (mixed-mode effects). It also enhanced hydrogen-bonding and partitioning (hydrophilic interaction) between the analyte and the stationary phase, likely facilitated by the associated solvated salt ions.

Evaluation of matrix effects in analysis of estrogen using liquid chromatography-tandem mass spectrometry.

Matrix effects of different biological samples, including phosphate-buffered saline-bovine serum albumin (PBS-BSA), gelded horse serum, mouse serum, and mouse brain, were investigated for the determination of 17α- and β-estradiol using derivatization with dansyl chloride prior to LC-MS/MS. Matrix effects were evaluated based on the slopes of regression lines plotted from results obtained in biological matrices versus pure standard solutions. Such plots indicate the enhancement or suppression of signal based on the presence of a particular biological fluid for a particular method. The matrix effects from PBS-BSA were similar to those of mouse serum. In contrast, analyses performed from horse serum and mouse brain yielded significant ion suppression, especially for 17β-estradiol. Precipitation during derivatization was observed when pre-concentrated samples were processed with ethyl acetate as an extraction solvent. This was overcome with the use of methyl tert-butyl ether; however, matrix effects from this preparation were still present, evidenced by signal suppression and poor linearity in the standard curve. This work affirms that caution should be taken in the transfer of methods for use with different biological matrices, especially in the case where surrogate matrices are necessary for calibration purposes.

Quantitative determination of Bisphenol A from human saliva using bulk derivatization and trap‐and‐elute liquid chromatography coupled to electrospray ionization mass spectrometry

Endocrine disruptors cause adverse health effects as a result of their ability to shift the hormonal balance that is essential to the body. Bisphenol A (BPA) is an endocrine disruptor that has garnered much attention because of its presence in many consumer materials, which generates a significant risk for exposure. A method is presented for rapid detection of oral exposure to BPA directly from human saliva. Saliva was chosen because it serves as a noninvasive sampling route to detect BPA exposure; however, it is one of many complex biological matrices that have traditionally posed problems in quantitative analysis. Such analyses usually require extensive sample preparation to reduce interferences contributed by the sample matrix. Three validated methods are presented here that feature a streamlined sample-preparation strategy (bulk derivatization) prior to accurate and sensitive analysis by trap-and-elute liquid chromatography coupled to electrospray ionization mass spectrometry. Validated methods include standard addition calibration with variable injection volumes and multiple injection loading, as well as with incorporation of an internal standard. Reported limits of detection reached as low as 49.0 pg/ml (2.9 pg loaded on-column; equivalent to parts per trillion in saliva) among the presented methods with good accuracy and precision throughout. A proof-of-concept study is demonstrated to show that the final validated method has potential application to specific studies for trace-level BPA detection from real samples.

Matrix-assisted laser desorption/ionization-mass spectrometry of cuticular lipid profiles can differentiate sex, age, and mating status of Anopheles gambiae mosquitoes

Malaria is a devastating mosquito-borne disease, which affects hundreds of millions of people each year. It is transmitted predominantly by Anopheles gambiae, whose females must be >10 days old to become infective. In this study, cuticular lipids from a laboratory strain of this mosquito species were analyzed using a mass spectrometry method to evaluate their utility for age, sex and mating status differentiation. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), in conjunction with an acenaphthene/silver nitrate matrix preparation, was shown to be 100% effective in classifying A. gambiae females into 1, 7-10, and 14 days of age. MALDI-MS analysis, supported by multivariate statistical methods, was also effective in detecting cuticular lipid differences between the sexes and between virgin and mated females. The technique requires further testing, but the obtained results suggest that MALDI-MS cuticular lipid spectra could be used for age grading of A. gambiae females with precision greater than with other available methods.

Laser desorption/ionization mass spectrometry fingerprinting of complex hydrocarbon mixtures: Application to crude oils using data mining techniques

Crude oil fingerprints were obtained from four crude oils by laser desorption/ionization mass spectrometry (LDI-MS) using a silver nitrate cationization reagent. Replicate analyses produced spectral data with a large number of features for each sample (>11,000 m/z values) which were statistically analyzed to extract useful information for their differentiation. Individual characteristic features from the data set were identified by a false discovery rate based feature selection procedure based on the analysis of variance models. The selected features were, in turn, evaluated using classification models. A substantially reduced set of 23 features was obtained through this procedure. One oil sample containing a high ratio of saturated/aromatic hydrocarbon content was easily distinguished from the others using this reduced set. The other three samples were more difficult to distinguish by LDI-MS using a silver cationization reagent; however, a minimal number of significant features were still identified for this purpose. Focus is placed on presenting this multivariate statistical method as a rapid and simple analytical procedure for classifying and distinguishing complex mixtures.