Direct Infusion Methods Research Articles

Rapid Analysis of Glucosinolates Using Direct-Infusion Mass Spectrometry.

We developed a rapid, accurate, and quantitative method for analyzing glucosinolates (GSLs) by combining column-free liquid chromatography (LC) with direct-infusion mass spectrometry (MS). Conventional methods for analyzing GSLs take a long time (20-50 min per sample) to perform compound separation on an LC column. We achieved a shortened analysis time of 30 seconds per sample using a direct-infusion method. Samples were continuously injected by a pump and autosampler on an LC system directly into the MS. Orbitrap MS detected 11 types of GSLs in the extracts of turnip hypocotyls. The calibration curve of a GSL standard showed a linear response over a 6-digit concentration range from 1 nM to 1 mM. In addition, no decrease in the detected intensity of GSL ions in 100 continuous analyses of turnip extracts was observed. This method may be applied for rapid analysis of GSLs and other health-functional or bioactive compounds.

Comprehensive Characterization of Endogenous Phospholamban Proteoforms Enabled by Photocleavable Surfactant and Top-down Proteomics.

Top-down mass spectrometry (MS)-based proteomics has become a powerful tool for analyzing intact proteins and their associated post-translational modifications (PTMs). In particular, membrane proteins play critical roles in cellular functions and represent the largest class of drug targets. However, the top-down MS characterization of endogenous membrane proteins remains challenging, mainly due to their intrinsic hydrophobicity and low abundance. Phospholamban (PLN) is a regulatory membrane protein located in the sarcoplasmic reticulum and is essential for regulating cardiac muscle contraction. PLN has diverse combinatorial PTMs, and their dynamic regulation has significant influence on cardiac contractility and disease. Herein, we have developed a rapid and robust top-down proteomics method enabled by a photocleavable anionic surfactant, Azo, for the extraction and comprehensive characterization of endogenous PLN from cardiac tissue. We employed a two-pronged top-down MS approach using an online reversed-phase liquid chromatography tandem MS method on a quadrupole time-of-flight MS and a direct infusion method via an ultrahigh-resolution Fourier-transform ion cyclotron resonance MS. We have comprehensively characterized the sequence and combinatorial PTMs of endogenous human cardiac PLN. We have shown the site-specific localization of phosphorylation to Ser16 and Thr17 by MS/MS for the first time and the localization of S-palmitoylation to Cys36. Moreover, we applied our method to characterize PLN in disease and reported the significant reduction of PLN phosphorylation in human failing hearts with ischemic cardiomyopathy. Taken together, we have developed a streamlined top-down targeted proteomics method for comprehensive characterization of combinatorial PTMs in PLN toward better understanding the role of PLN in cardiac contractility.

Development and validation of analysis method for sennoside B in Cassia angustifolia using UPLC-MRM/MS

Cassia angustifolia is a commonly found wild plant from the family Caesalpinaceae that originates from Yemen and Hadramaut province in Southern Arabia, where it is called Arabian senna. The leaves of the plant have been used to treat a variety of ailments such as constipation, malaria, anemia, loss of appetite, and indigestion. Sennosides A and B are the major glycosides found in the leaves and pods of C. angustifolia and are important ingredients in purgative medicines. These compounds are considered as the major active components of Cassia plants and are responsible for their therapeutic activities. To assess the quality and quantity of sennosides A and B, an appropriate analytical method is required, which must be simple, accurate, precise, and widely used. The UPLC-ESI-MRM/MS method was used in this study to validate the analytical method in determining the contents of Sennoside B in senna leaves extract. The validation parameters included specificity, system suitability, linearity, sensitivity (LOD, LOQ), accuracy, and precision. The results indicated that the optimization of MRM using the direct infusion method provided good separation when eluted using liquid chromatography. The validation parameters for system suitability obtained RSD under 2%. The linearity of sennoside B showed excellent results (R2 = 0.999) in the concentration range of 0.98–62.5 µg/ml. The LOD and LOQ values of sennoside B were 0.011 µg/mL and 0.034 µg/ml, respectively. The accuracy values of sennoside B met the predetermined criteria, with RSD < 2% and % recovery of 97-102%. The quantitative analysis revealed that Cassia angustifolia extract contained 0.43 ±0.06 mg/g of sennoside B.

Shared reference materials harmonize lipidomics across MS-based detection platforms and laboratories

Quantitative MS of human plasma lipids is a promising technology for translation into clinical applications. Current MS-based lipidomic methods rely on either direct infusion (DI) or chromatographic lipid separation methods (including reversed phase and hydrophilic interaction LC). However, the use of lipid markers in laboratory medicine is limited by the lack of reference values, largely because of considerable differences in the concentrations measured by different laboratories worldwide. These inconsistencies can be explained by the use of different sample preparation protocols, method-specific calibration procedures, and other experimental and data-reporting parameters, even when using identical starting materials. Here, we systematically investigated the roles of some of these variables in multiple approaches to lipid analysis of plasma samples from healthy adults by considering: 1) different sample introduction methods (separation vs. DI methods); 2) different MS instruments; and 3) between-laboratory differences in comparable analytical platforms. Each of these experimental variables resulted in different quantitative results, even with the inclusion of isotope-labeled internal standards for individual lipid classes. We demonstrated that appropriate normalization to commonly available reference samples (i.e., “shared references”) can largely correct for these systematic method-specific quantitative biases. Thus, to harmonize data in the field of lipidomics, in-house long-term references should be complemented by a commonly available shared reference sample, such as NIST SRM 1950, in the case of human plasma.

Chemical profiling of bioactive constituents in hop cones and pellets extracts by online comprehensive two-dimensional liquid chromatography with tandem mass spectrometry and direct infusion Fourier transform ion cyclotron resonance mass spectrometry.

Humulus lupulus L. (hop) is highly interesting from a nutraceutical perspective. The hop phytocomplex contains a wide range of bioactive metabolites, and its characterization is challenging. To tackle such a task, for the first time we applied and compared a combined approach consisting of online comprehensive two-dimensional liquid chromatography with tandem mass spectrometry and direct infusion Fourier transform ion cyclotron mass spectrometry. A reversed phase×reversed phase approach with a shifted gradient in the second dimension ensured selectivity and two-dimensional space coverage. Hyphenation with an ion trap time-of-flight analyzer led to the identification of 83compounds in 70min, comprising a novel quercetin derivative and six unknown bitter acids. On the other hand, the direct infusion method was able to identify 40 analytes (except isomers) with high mass accuracy (≤0.1ppm) in less than 1min analysis time. The developed approach can be used in a complementary way, combining the separation capability and high informative spectra of two-dimensional liquid chromatography tandem mass spectrometry with the ultra-high mass accuracy of direct infusion, for potential compound discovery or the accurate profiling of bioactive compounds in different hop cultivars as well as for monitoring processing and storage of hop-based products.

Lipidomic analysis of biological samples: Comparison of liquid chromatography, supercritical fluid chromatography and direct infusion mass spectrometry methods

Lipidomic analysis of biological samples in a clinical research represents challenging task for analytical methods given by the large number of samples and their extreme complexity. In this work, we compare direct infusion (DI) and chromatography – mass spectrometry (MS) lipidomic approaches represented by three analytical methods in terms of comprehensiveness, sample throughput, and validation results for the lipidomic analysis of biological samples represented by tumor tissue, surrounding normal tissue, plasma, and erythrocytes of kidney cancer patients. Methods are compared in one laboratory using the identical analytical protocol to ensure comparable conditions. Ultrahigh-performance liquid chromatography/MS (UHPLC/MS) method in hydrophilic interaction liquid chromatography mode and DI-MS method are used for this comparison as the most widely used methods for the lipidomic analysis together with ultrahigh-performance supercritical fluid chromatography/MS (UHPSFC/MS) method showing promising results in metabolomics analyses. The nontargeted analysis of pooled samples is performed using all tested methods and 610 lipid species within 23 lipid classes are identified. DI method provides the most comprehensive results due to identification of some polar lipid classes, which are not identified by UHPLC and UHPSFC methods. On the other hand, UHPSFC method provides an excellent sensitivity for less polar lipid classes and the highest sample throughput within 10min method time. The sample consumption of DI method is 125 times higher than for other methods, while only 40μL of organic solvent is used for one sample analysis compared to 3.5mL and 4.9mL in case of UHPLC and UHPSFC methods, respectively. Methods are validated for the quantitative lipidomic analysis of plasma samples with one internal standard for each lipid class. Results show applicability of all tested methods for the lipidomic analysis of biological samples depending on the analysis requirements.

Impact of tissue surface properties on the desorption electrospray ionization imaging of organic acids in grapevine stem.

Desorption electrospray ionization (DESI) imaging is a fast analytical technique used to assess spatially resolved biological processes over unmodified sample surfaces. Although DESI profiling experiments have demonstrated that the properties of the sample surface significantly affect the outcomes of DESI analyses, the potential implications of these phenomena in imaging applications have not yet been explored extensively. The distribution of endogenous and exogenous organic acids in pith and out pith region of grapevine stems was investigated by using DESI imaging, ion chromatography and direct infusion methods. Several common normalization strategies to account for the surface effect, including TIC normalization, addition of the internal standard in the spray solvent and deposition of the standard over the sample surface, were critically evaluated. DESI imaging results show that, in our case, the measured distributions of these small organic acids are not consistent with their 'true' localizations within the tissues. Furthermore, our results indicate that the common normalization strategies are not able to completely compensate for the observed surface effect. Variations in the tissue surface properties across the tissue sample can greatly affect the semi-quantitative detection of organic acids. Attention should be paid when interpreting DESI imaging results and an independent analytical validation step is important in untargeted DESI imaging investigations.

A Mass Spectrometric Study of Kratom Compounds by Direct Infusion Electrospray Ionization Triple Quadrupole Mass Spectrometry

Mitragynine (MG) and its major metabolites 7-hydroxymitragynine (7-OH-MG) are two of the major components of the plant extract Kratom, which is a tree planted in Southeast Asia. Kratom has long been used by opioid-dependent individuals as an alternative to their unavailable opioid of choice and chronic pain medication, as a stealth-to-urine drug screening opiate substitute while in opioid recovery treatment and recreationally, alone or as a booster. In this study, a direct infusion method was utilized and electrospray ionization triple quadrupole mass spectrometer was used as the detector for data acquisition. Pharmacokinetic study was conducted to investigate the effect of mitragynine and 7-hydroxymitragynine and major fragments of both compounds were proposed.

Application of electrospray ionization ion trap/time-of-flight mass spectrometry for chemically-synthesized small RNAs

In this study, we have demonstrated an accurate and rapid small RNA analytical method with both sequence determination and detailed modification analysis by electrospray ionization-ion trap/time-of-flight mass spectrometry (ESI-IT/TOFMS). To develop this ideal method, we have examined the performance of ESI-IT/TOFMS using various chemically-synthesized model sequences of modified or unmodified microRNAs (miRNAs). The deconvoluted mass of a 22-nucleotide (nt) miRNA was obtained from a multiply charged precursor ion (MS(1)). The ion exhibited high mass accuracy (< 7 ppm) and high mass resolution (a value of m/Δm=10,000) and was therefore very useful in RNA composition assignment. The optimized MS(2) method using ion trap collision-induced dissociation, as well as automatic annotation analysis of product ions based on the accurate mass information, enabled the precise sequencing determination of intact miRNAs. Further, the detailed structural analysis of 3'-terminal modified nucleic acid in intact methylated miRNA was carried out using the MS(3) capability of the hybrid IT/TOFMS. The direct infusion method also provided a high throughput and good sensitivity because the analytical time and sample concentration needed in a series of experiments with reliable data were only 3 min and 100 nM, respectively. This study provides a novel approach for characterizing the intact chemically-synthesized small RNA without chemical and enzymatic digestions and would be widely applicable for the structural analysis of complicated modified small RNAs.

Lipids of human meibum: mass-spectrometric analysis and structural elucidation

The purpose of this study was to structurally characterize the major lipid species present in human meibomian gland secretions (MGS) of individual subjects by means of ion trap atmospheric pressure ionization mass spectrometry analysis (API MS(n)). The samples of MGS and authentic lipid standards were analyzed in direct infusion and high-pressure liquid chromatography (HPLC) experiments with API MS(n) detection of the analytes (HPLC API MS(n)). The major precursor ions were isolated and subjected to further sequential fragmentation in MS(n) experiments, and their fragmentation patterns were compared with those of authentic lipid standards. Multiple precursor ions were observed in the positive-ion mode. Among those, previously identified cholesterol (Chl; m/z 369; [M - H(2)O + H](+)) and oleic acid (OA; m/z 283; [M + H](+)) were found. The other major compounds of the general molecular formula C(n)H(2n-2)O(2) were consistent with wax esters (WEs), with OA as fatty acyl component. Accompanying them were two homologous series of compounds that fit the molecular formulas C(n)H(2n-4)O(2) and C(n)H(2n)O(2). Subset 2 was found to be a homolog series of linoleic acid-based WEs, whereas subset 3 was, apparently, a mixture of stearic acid-based WEs. HPLC API MS(n) analysis revealed the presence of large quantities of cholesteryl esters (Chl-Es) in all of the tested samples. Less than 0.1% (w/w) of oleamide was detected in human MGS. In the negative-ion mode, three major compounds with m/z values of 729, 757, and 785 that were apparently related to anionogenic lipids of the diacylglyceryl family were found in all of the samples. Common phospholipids and ceramides (Cers) were not present among the major MGS lipids. Phosphocholine-based lipids were found in MGS in quantities less than 0.01% (w/w), if at all. This ratio is two orders of magnitude lower than reported previously. These observations suggest that MGS are a major source of nonpolar lipids of the WE and Chl-E families for the tear film lipid layer, but not of its previously reported (phospho)lipid, Cer, and fatty acid amide components.

Cell Saver, Ultrafiltration and Direct Transfusion: Comparative Study of Three Blood Processing Techniques

Intraoperative blood salvage is an important part of blood conservation efforts in cardiac surgery. The purpose of this study is to examine the effects of three different circuit blood-salvaging techniques: centrifugation, ultrafiltration and direct infusion. Sixty patients undergoing elective coronary bypass graft procedures were randomly assigned in a prospective manner to one of the three blood-salvaging methods. Intra- and postoperative blood samples demonstrated increased hemoglobin values in the direct infusion group and higher platelet count in the ultrafiltration group. There were no significant differences in these results. The analysis of coagulation parameters revealed a similar prolongation of partial thromboplastin time and activated clotting time in all groups. The amount of blood loss was not significantly different between the three blood conservation methods. The effect of direct infusion method does not result in either major disturbance of coagulation parameters or in increased blood loss. In a sample of adult patients undergoing coronary artery bypass grafting, direct transfusion is, in consideration of the cost-effective factor, an appropriate approach for returning cardiopulmonary bypass circuit blood.

Mass spectrometry monitoring of rhamnolipids from a growing culture of Pseudomonas aeruginosa strain 57RP

Two rapid and simple methods for the characterisation and quantification of rhamnolipids produced by a growing culture of the Pseudomonas aeruginosa strain 57RP were developed. Two rhamnolipids were purified and their response factors determined. The various rhamnolipids produced were then measured using liquid chromatography/mass spectrometry. The culture supernatants were injected directly, without prior purification, in a HPLC equipped with a C 18 reverse-phase column. The complete profile of rhamnolipid congeners produced during a 2 week cultivation period was monitored. In order to shorten the analysis time, another method was developed which did not require chromatographic separation of the rhamnolipids prior to their detection. Quantification of rhamnolipids using the direct infusion method gave results very similar to those obtained with HPLC separation. These two methods were very well correlated with the standard colorimetric orcinol method. The rhamnolipid profiles obtained show that the various rhamnolipid congeners are secreted simultaneously, and that their relative proportion remained unchanged throughout the cultivation period.