Electrospray Interface Research Articles

Miniaturized nanoelectrospray interface for coupling capillary electrophoresis with mass spectrometry detection.

A miniaturized electrospray interface consisting of a microfluidic nanosprayer and nanospray module is reported in the presented short communication. The nanosprayer was fabricated using silicon (Si) technology suitable for cost-efficient high-volume mass production. The nanospray module enabled the positioning of the nanosprayer in front of a mass spectrometry entrance and its coupling with capillary electrophoresis based on the liquid junction principle. A case study of top-down and bottom-up proteomic analyses of intact cytochrome c and its tryptic digest demonstrates the practical applicability of the developed interface.

Highly sensitive detection of fluorescent whitening agents in flour using sheathless capillary electrophoresis-electrospray ionization-tandem mass spectrometry

Fluorescent whitening agents (FWAs) are dyes that emit visible blue or blue-purple fluorescence upon ultraviolet-light absorption. Taking advantage of light complementarity, FWAs can compensate for the yellow color of many substances to achieve a whitening effect; thus, they are used extensively in various applications. FWAs are generally stable, but their presence in the environment can lead to pollution and accumulation in the body through the food chain. Recent studies have revealed that some types of FWAs, such as coumarin-based FWAs, may exhibit photo-induced mutagenic effects that can trigger allergic reactions in humans and even pose carcinogenic risks. Hence, the development of an accurate and highly sensitive method for detecting FWAs in food-related samples is a crucial endeavor. Owing to the high polarity and structural similarity of FWAs, the accurate determination of these substances in complex food samples requires an analytical method that offers both efficient separation and sensitive detection. Capillary electrophoresis (CE) exhibits essential features such as high separation efficiency, short analysis times, very small sample injection requirements, minimal use of organic solvents, and simple operation. Thus, it is often used as an effective alternative to liquid chromatographic techniques. Over the past few decades, electrospray ionization mass spectrometry (ESI-MS) has been utilized as a highly sensitive and accurate detection method in numerous chemical analytical fields because it enables the analysis of molecular structures. By combining the high separation efficiency of CE with the high sensitivity of ESI-MS, a powerful tool for identifying and quantifying trace amounts of FWAs in food samples may be obtained. In this study, we present a method based on sheathless CE coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) for the simultaneous detection of six trace FWAs in flour. In the proposed method, the CE separation device is directly coupled to the mass spectrometer through a sheathless interface without the need for a sheath liquid for electric contact, thereby avoiding the dilution of the analytes and improving detection sensitivity. Various conditions that could affect extraction recovery, separation efficiency, and detection sensitivity were evaluated and optimized. The FWAs were effectively extracted from the sample matrix with reduced matrix effects by ultrasonic-assisted extraction at a temperature of 30 ℃ for 20 min using CHCl3-MeOH (3∶2, v/v) as the extraction solvent. The extract was centrifuged, dried under N2, and reconstituted in CHCl3-MeOH (1∶4, v/v) for subsequent analysis. During the detection process, the CE device was coupled to the ESI-MS/MS instrument via a highly sensitive porous spray needle, which served as the sheathless electrospray interface. The target FWAs were scanned in positive-ion mode (ESI+) to ensure the stability and intensity of the obtained signals. Additionally, multiple-reaction monitoring (MRM) mode and MS/MS analysis were used to simultaneously quantify the six targets with high selectivity. The developed sheathless CE-ESI-MS/MS method detected the FWAs with high sensitivity over wide linear ranges with low method limits of detection (0.04-0.67 ng/g). The recoveries of the six target FWAs at three spiked levels were between 77.5% and 97.2%, with good interday (RSD≤11.5%) and intraday (RSD≤10.2%) precision. Analyses of the six target FWAs in eight commercial flour samples were performed using this method, and four positive samples were identified. These results demonstrate that the proposed CE-ESI-MS/MS method is a promising strategy for the determination of trace FWAs in complex food sample matrices with efficient separation and high sensitivity.

Phenolic Profiles and Antitumor Activity against Colorectal Cancer Cells of Seeds from Selected Ribes Taxa

Seeds from several Ribes taxa were surveyed for phenolic compounds and in vitro antiproliferative activity against HT-29 colorectal cancer cells. Total phenolic compounds were analyzed through the Folin–Ciocalteu procedure, while LC coupled to a single mass spectrometer (MS) Orbitrap using an electrospray interface (ESI) was performed to determine the phenolic profiles. Antitumor effects were established using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Total phenolics ranged from 11.4 in R. alpinum to 94.8 mg of caffeic acid equivalents (CAE)/g in R. nigrum ‘Koksa’. Concerning phenolic compounds, four were hydroxylated benzoic acids, four cinnamic acid derivatives, eight flavonoids, and nine flavonoid glycosides. The growth inhibition against HT-29 cancer cells was exercised much better by R. nigrum ‘Koksa’ and Ribes ‘Erkeeni’ (GI50 37 and 42 µg/mL). All Ribes extracts, except for R. nigrum ‘Hara katarlik’, showed higher activity than R. rubrum (GI50 at 72 h: 99 µg/mL). Interestingly, the extract from Ribes ‘Erkeeni’, which exhibited high bioactivity, contains all detected phenolic compounds, unlike R. nigrum ‘Koksa’, which lacks only populnin. Therefore, the high bioactivity found for such extracts could be due to a synergy of all detected compounds. This work constitutes a comprehensive action for expanding knowledge on the phenolic profiles and antitumor activity of GLA-rich Ribes seeds.

Bottom-up proteomics to investigate the X-ray irradiation effects on soft cheese

Food irradiation is an alternative technological process employed to preserve the hygienic quality of food and to extend its shelf life. In this work, three different types of soft cheese, mozzarella, robiola and primosale, were treated with X-rays at 1.0, 2.0 and 3.0 kGy to evaluate their effect on proteins and peptides using a bottom-up proteomic approach. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF-MS) and liquid chromatography coupled with MS through electrospray interface (LC-ESI-MS) were employed to analyse the peptide mixtures coming from the proteolytic digestion of soft cheese using both trypsin and chymotrypsin enzymes. We discovered 15 peptides most affected by X-rays that were sequenced by tandem MS and identified; ten of these discriminant peptides, generated from the cleavage of β-casein and α-casein (B. taurus), were localized on the proteins' secondary structure, highlighting a heightened susceptibility of random coils to X-ray irradiation. No additional post-translational modifications were found to be induced or emphasized by irradiation. For all studied cheese samples, the major spectral differences were encountered at 1.0 kGy, suggesting that such a dose was the most effective in promoting peptide bond cleavage. Some non-enzymatic and/or half-enzymatic peptides belonging to α-S1-, α-S2- and β-casein were also produced when varying X-ray doses.

The Development, Validation, and Application of a UHPLC-HESI-MS Method for the Determination of 17 Cannabinoids in Cannabis sativa L. var. sativa Plant Material.

Cannabinoids are an important group of secondary metabolites found in the plant Cannabis sativa L. The growing interest in the use of hemp in food production (e.g., hemp teas, hemp cookies) makes it necessary to develop a method for determining these compounds in the plant, both fresh and dried. The selection of a suitable extraction liquid for the extraction of cannabinoids and the development of a method for the determination of 17 cannabinoids is a prelude to the development of an effective method for the extraction of these compounds. In the present study, a novel, simple, and efficient method was developed and validated for the determination of up to 17 cannabinoids in fresh plant parts (inflorescences and leaves) of Cannabis sativa L. and in dried material, including hemp teas. Analyses were performed using ultra-high-performance liquid chromatography-Q-Exactive Orbitrap mass spectrometry setup operating with a heated electrospray interface (UHPLC-HESI-MS). Based on the comparison, methanol was selected as the best for the extraction of cannabinoids from fresh and dried material. The efficiency and validity of the method were assessed using certified reference material (dried Cannabis) and confirmed by z-score from participation in an international proficiency test conducted by ASTM International for dried hemp.

A methodological approach by capillary electrophoresis coupled to mass spectrometry via electrospray interface for the characterization of short synthetic peptides towards the conception of self-assembled nanotheranostic agents

Nanostructures formed by the self-assembling peptide building blocks are attractive materials for the design of theranostic objects due to their intrinsic biocompatibility, accessible surface chemistry as well as cavitary morphology. Short peptide synthesis and modification are straightforward and give access to a great diversity of sequences, making them very versatile building blocks allowing for the design of thoroughly controlled self-assembled nanostructures. In this work, we developed a new CE-DAD-ESI-MS method to characterize short synthetic amphiphilic peptides in terms of exact sequence and purity level in the low 0.1 mg.mL−1 range, without sample treatment. This study was conducted using a model sequence, described to have pH sensitive self-assembling property. Peptide samples obtained from different synthesis processes (batch or flow, purified or not) were thus separated by capillary zone electrophoresis (CZE). The associated dual UV and MS detection mode allowed to evidence the exact sequence together with the presence of impurities, identified as truncated or non-deprotected sequences, and to quantify their relative proportion in the peptide mixture. Our results demonstrate that the developed CE-DAD-ESI-MS method could be directly applied to the characterization of crude synthetic peptide products, in parallel with the optimization of peptide synthetic pathway to obtain controlled sequences with high synthetic yield and purity, which is crucial for further design of robust peptide based self-assembled nanoarchitectures.

B-174 Simple and Accurate Quantification of Four Direct Oral Anticoagulants in Plasma Using UPLC-MS/MS

Abstract Background Direct oral anticoagulants (DOACs) constitute first-line therapy used for many thromboembolic indications, such as prevention and treatment of venous thromboembolism and stroke prevention in atrial fibrillation. This class of medication consists of the direct thrombin inhibitor (dabigatran) or direct factor Xa inhibitors (apixaban, rivaroxaban, and edoxaban). DOACs quantification may be useful in the case of critical clinical situations, including drug accumulation in long-term treatment, overdosage, thrombotic or bleeding events. This study aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantification of four DOACs: dabigatran, apixaban, rivaroxaban, and edoxaban in plasma. Methods The calibration curve samples were prepared by spiking drug free plasma with DOACs. The stable isotope labeled DOACs was used as an internal standard. After addition of the internal standard and protein precipitation, the supernatant was 10-fold diluted and injected into a chromatography system consisting of a UPLC BEH C18 (2.1 × 50 mm; particle size 1.7 µm) analytical column with gradient made of mobile phase (5 mM ammonium formate pH3.5 in water and methanol). The outlet of the column was connected to a triple quadrupole mass spectrometer with electrospray interface. Ions were detected in the positive multiple reaction monitoring mode. The concentration of analyte was calculated from the calibration curve and ion ratios between the analyte and the internal standard. Results The analytical range was linear with a correlation coefficient of over 0.99 in the range of 0–500 ng/mL for each analyte. Intra- and inter-assay imprecision were less than 3.1% (n = 20) and 6.0% (n = 20), respectively. The accuracy was evaluated by spike recovery and the mean recoveries were between 97.2% and 102.1% for all analytes. This assay showed no ion suppression or enhancement and no carryover. The chromatography run time was 4.5 min. The assay was applied to quantitate DOACs in plasma samples from 93 patients. Our data showed that the DOACs concentrations vary markedly individual patients after equal dose, this therapeutic drug monitoring of DOACs is essential to optimized drug efficacy. Conclusion An accurate, and cost-effective UPLC-MS/MS method for the quantification of DOACs was successfully applied for therapeutic drug monitoring in patients.

A Brief Review on Hyphenated Techniques

The hyphenated techniques are developed by combining two or three different analytical techniques. Mainly chromatographic techniques are combined with spectroscopic techniques. The separated components obtained from chromatographic techniques are then transmitted to spectroscopic detection technique through an interface. The improvements in hyphenated analytical methods in last two decades have significantly broadened their applications in the analysis of biomolecules especially in natural products. In this review article recent advancement in hyphenated techniques such as GC-MS, LC-MS, LC-NMR, MS-MS, LC-NMR-MS have been highlighted. The term ‘Hyphenated’ refers to separation and identification of the compounds. These techniques also show better analysis of sample compounds with better accuracy, precision and specificity. This review mainly focuses on the instrumental aspects of hyphenated techniques along with their interfaces. The interfaces such as Jet\Orifice separator interface, Electrospray interface, Atmospheric pressure chemical ionization interface have been covered. It has been concluded that these hyphenated techniques have become more advantageous for the analysis of drugs. This technique is useful for quality control, analytical research, impurity profiling and maintenance for human welfare and development.

Analysis of Megadalton-Sized DNA by Charge Detection Mass Spectrometry: Entropic Trapping and Shearing in Nanoelectrospray.

The analysis of nucleic acids by conventional mass spectrometry is complicated by counter ions which cause mass heterogeneity and limit the size of the DNA that can be analyzed. In this work, we overcome this limitation using charge detection mass spectrometry to analyze megadalton-sized DNA. Using positive mode electrospray, we find two dramatically different charge distributions for DNA plasmids. A low charge population that charges like compact DNA origami and a much higher charge population, with charges that extend over a broad range. For the high-charge population, the deviation between the measured mass and mass expected from the DNA sequence is consistently around 1%. For the low-charge population, the deviation is larger and more variable. The high-charge population is attributed to the supercoiled plasmid in a random coil configuration, with the broad charge distribution resulting from the rich variety of geometries the random coil can adopt. High-resolution measurements show that the mass distribution shifts to slightly lower mass with increasing charge. The low-charge population is attributed to a condensed form of the plasmid. We suggest that the condensed form results from entropic trapping where the random coil must undergo a geometry change to squeeze through the Taylor cone and enter an electrospray droplet. For the larger plasmids, shearing (mechanical breakup) occurs during electrospray or in the electrospray interface. Shearing is reduced by lowering the salt concentration.

Assessment and validation of the p-QuEChERS sample preparation methodology for the analysis of >200 veterinary drugs in various animal-based food matrices

The need remains for veterinary multi-residue methods that reliably quantify and identify veterinary drugs in the various animal-based food matrices. Such a method should not only show good method performance parameters (e.g. recoveries of analytes) but must also be fast and cheap. The proposed method focused on the following points: acceptable analyte trueness (recovery) and precision for a large number (200) of diverse veterinary drugs in the relevant animal-based food matrices (egg, muscle, fatty fish, liver, kidney, and honey). The sample preparation method termed p-QuEChERS uses a salt mixture consisting of potassium phosphates to induce phase separation. The avoidance of conventional QuEChERS salts (e.g. magnesium sulphate) significantly improves recoveries of several critical analytes. Analyte recoveries were further improved by adding a centrifugation and a defatting step before initiating the salt-induced phase separation. This combined clean-up removes a large fraction of the potentially interfering matrix compounds. As a result, matrix effects in the electrospray interface were minimized. These factors were the basis for the obtained good validation data. Two types of high-resolution mass spectrometers coupled to liquid chromatography were compared for analysis. In comparison with conventional QuEChERS, the proposed p-QuEChERS concept improved the recovery of polar analytes such as penicillins, tetracyclines and quinolones. The simplicity of the procedure and the low consumable expenses make the method ideal for the routine control of veterinary drugs in all evaluated animal-based food matrices.

Changes in cytokinins and auxins levels in the rind of ‘Nules Clementine’ mandarin as related to the fruit position on the tree and the susceptibility to non-chilling rind breakdown disorder

• Phytohormonal changes in relation to citrus rind disorder was examined postharvest. • Fruit were harvested from inside and outside canopy position of the tree. • Endogenous levels of cytokinins (CK), auxins, isomers and conjugates were analysed. • Cis -zeatin-type CK concentrations were higher in outside canopy fruit postharvest. The role of phytohormones in mitigating or enhancing the incidence of non-chilling rind physiological disorder of citrus fruit is largely unknown. Therefore, this study attempted to elucidate cytokinins (CK) and auxin changes in ‘Nules Clementine’ mandarin fruit rind from different canopy positions during postharvest non-chilling cold storage regarding rind breakdown disorder (RBD). Fruit from the inside canopy (IC) and outside canopy (OC) positions of citrus trees were harvested at commercial maturity from Eastern Cape (EC) and Western Cape (WC) provinces of South Africa, respectively. Endogenous levels of CK, auxins, their isomers, and conjugates were analysed using ultra-performance liquid chromatography coupled with a triple quadrupole mass spectrometer (MS/MS) equipped with an electrospray interface. Cis -zeatin-type cytokinins were significantly higher in the OC fruit than in the IC fruit from both provinces during cold storage. Whereas dihydrozeatin concentration (the most abundant CK) was significantly higher in fruit from IC (326.10 and 29.38 nmol kg −1 ) than in OC (65.75 and 17.72 nmol kg −1 ) fruit before cold storage, respectively. Fruit without rind disorder had lower CK - conjugates than fruit with the disorder. The IAA concentration was higher in the IC fruit (344.15 nmol kg −1 ) than in the OC fruit (194.20 nmol kg −1 ) from EC at week 0 while the IAA concentration of OC fruit from WC was below the limit of detection and IC fruit had 53.20 nmol kg −1 at week 0. Fruit without the disorder had more IAA concentration than fruit with RBD while indole-3-acetylaspartic acid (IAAsp) was higher in fruit with RBD than fruit without the disorder, indicating the preventive role of IAA in the incidence of the disorder. This study is the first to record the negative effect of IAAsp in RBD and revealed the crucial role and synergistic role of CK, IAA and their isomers in RBD of ‘Nules Clementine’ fruit.

Coupling Droplet Microfluidics with Ion Mobility Spectrometry for Monitoring Chemical Conversions at Nanoliter Scale.

We introduce the coupling of droplet microfluidics and ion mobility spectrometry (IMS) to address the challenges of label-free and chemical-specific detection of compounds in individual droplets. In analogy to the established use of mass spectrometry, droplet-IMS coupling can be also achieved via electrospray ionization but with significantly less instrumental effort. Because IMS instruments do not require high-vacuum systems, they are very compact, cost-effective, and robust, making them an ideal candidate as a chemical-specific end-of-line detector for segmented flow experiments. Herein, we demonstrate the successful coupling of droplet microfluidics with a custom-built high-resolution drift tube IMS system for monitoring chemical reactions in nL-sized droplets in an oil phase. The analytes contained in each droplet were assigned according to their characteristic ion mobility with limit of detections down to 200 nM to 1 μM and droplet frequencies ranging from 0.1 to 0.5 Hz. Using a custom sheath flow electrospray interface, we have further achieved the chemical-specific monitoring of a biochemical transformation catalyzed by a few hundred yeast cells, at single droplet level.

Metabolite mass spectrometry profiling of cacao genotypes reveals contrasting resistances to Ceratocystis cacaofunesta phytopathogen.

Ceratocystis wilt is a lethal disease of cacao, and the search for resistant genotypes may provide the best way to deal with the disease. Resistance or susceptibility behavior of some cacao genotypes when infected by Ceratocystis cacaofunesta is not yet understood. Herein, we report an LC-MS metabolomic screening analysis based on high-resolution MS to obtain comprehensive metabolic profile associated with multivariate data analysis of PLS-DA, which was effective to classify CCN-51 and TSH-1188 as resistant genotypes to C. cacaofunesta fungus, while CEPEC2002 was classified as a susceptible one. Using reversed-phase LC method, electrospray interface, and high-resolution tandem MS by the quadrupole-TOF analyzer, the typical profiles of metabolites, such as phenylpropanoids, flavonoids, lipids, alkaloids, and amino acids, were obtained. Untargeted metabolite profiles were used to construct discriminant analysis by partial least squares (PLS-DA)-derived loading plots, which placed the cacao genotypes into two major clusters related to susceptible or resistant groups. Linolenic, linoleic, oleic, stearic, arachidonic, and asiatic acids were annotated metabolites of infected, susceptible, and resistant genotypes, while methyl jasmonate, jasmonic acid, hydroxylated jasmonic acid, caffeine, and theobromine were annotated as constituents of the resistant genotypes. Trends of these typical metabolites levels revealed that CCN51 is susceptible, CEPEC2002 is moderately susceptible, and TSH1188 is resistant to C. cacaofunesta. Therefore, profiles of major metabolites as screened by LC-MS offer an efficient tool to reveal the level of resistance of cacao genotypes to C. cacaofunesta present in any farm around the world.

Structure-Indicated LC-MS/MS Bioanalysis of Therapeutic Antibodies.

Monoclonal antibodies bind to Protein A/G resin with 100nm-diameter pores, which orients the Fab toward the reaction solution. Then, they can be proteolyzed using trypsin immobilized on the surface of 200nm-diameter nanoparticles. The difference between the two particle diameters allows Fab-selectiveproteolysis by limiting trypsin access to the antibody substrate. The specific signature peptide of monoclonal antibody is collected, which comprises the complementarity-determining regions (CDRs). Excess trypsin protease and peptide fragments from common sequences in Fc that inhibit the analysis can then be separated and removed. The resulting peptide samples are separated through high performance liquid chromatography on a 20nm-diameter pore-size reversed-phase C18 column. These are then sequentially ionized with an electrospray interface and subjected to mass spectrometry (MS). In MS, peptide ions are trapped and fragment ions are generated by the collision-induced dissociation with argon gas. These are detected with multiple reaction monitoring measurements to perform a highly sensitive and accurate quantitative analysis.By focusing on various physicochemical features at each analytical scene, such as characteristic structure and orientation of antibody, control of trypsin reaction field, carry-over on HPLC column, ionization suppression effect from endogenous proteins, and detection of amino acid sequence specificity of antibody, we optimized the overall conditions from the sample processing up to MS detection and developed analytical validation and clinical application of many therapeutic antibodies using our Fab-selective proteolysis technology that is based on the structure-indicated approach.

A Comparative Pharmacokinetic Study of Schisandrol B After Oral Administration of Schisandrol B Monomer and Schisandra chinensis Extract

Background: Schisandra chinensis Turcz. (Baill.) is a perennial deciduous woody vine plant, which is beneficial to all systems of the body. Objective: The goals of the present study were to compare the pharmacokinetics of schisandrol B in rats after the oral administration of schisandrol B monomer (10 mg/kg) and S. chinensis extract (equivalent to 10 mg/kg schisandrol B) and to explore interactions among the components in S. chinensis extract. Methods: Twelve Sprague-Dawley rats of SPF grade were randomly divided into the monomer and S.chinensis extract groups. Plasma samples were extracted with methyl tert-butyl ether, and chromatographic separation was performed on an Agilent ZORBAX Eclipse XDB-C18 (4.6 × 150 mm, 5 μm) column with the mobile phase consisting of methanol (containing 0.1% formic acid)-water (containing 0.1% formic acid and 5 mmol ammonium acetate). This analysis was achieved by multiple reaction monitoring modes in an electrospray interface. Results: The seven lignans had a good linear relationship within the determination range (r>0.9950); the intra- and inter-day precision was <12.08% and accuracy was 88.64%-111.61%. The pharmacokinetic parameters (T1/2, Tmax, MRT0-∞, CL, AUC0-t, and AUC0-∞) of schisandrol B showed significant differences between the two groups (P<0.05). Conclusion: The validated method has been successfully applied to the pharmacokinetics of schisandrin, schisandrol B, schisandrin A, schisandrin B, schisandrin C, schisanhenol, and schisantherin A. The pharmacokinetic differences indicate that other components in the extract may increase the absorption of schisandrol B, decrease the rate of elimination, and improve the bioavailability of schisandrol B.

Automated Chiral Analysis of Amino Acids Based on Chiral Derivatization and Trapped Ion Mobility-Mass Spectrometry.

A fast and fully automated method for chiral analysis has been developed by combining a chiral derivatization approach with high-resolution trapped ion mobility separation. Although the presented approach can be potentially applied to diverse types of chiral compounds, several benchmark amino acids were used as model compounds, focusing on the smallest amino acid alanine. An autosampler with an integrated chromatography system was used for inline chiral derivatization with (S)-naproxen chloride and subsequent preseparation. Afterwards, derivatized amino acids were directly introduced into the electrospray interface of a trapped ion mobility-mass spectrometer for rapid diastereomer separation in the gas phase. This unique combination of preseparation and trapped ion mobility spectrometry separation in the negative ion mode enabled rapid chiral analysis within 3 min per sample. Furthermore, the diastereomer separation proved to be independent of alkali salts or other metal ions, offering robustness with regard to samples containing high amounts of salts. Highly sensitive detection of amino acid diastereomers was possible down to the lower nanomolar concentration range, and enantiomeric ratios could be readily determined from the recorded mobilograms with excellent reproducibility and precision. To demonstrate the general applicability of our method, alanine and other amino acids were analyzed from soy sauces and seasonings, which revealed extraordinarily high d-Ala contents of up to 99% in all samples.

Quantification of the bisphosphonate alendronate using capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing.

A quantitative method was developed for the direct identity confirmation and quantification of alendronate using CE-MS combined with a pH-assisted focusing technique, dynamic pH barrage junction focusing. A pH-induced variation in electrophoretic mobility led to online focusing of alendronate at the sample/pH barrage boundary, significantly improving the detection sensitivity. In addition, the use of a flow-through microvial CE electrospray interface and the multiple reaction monitoring mode of MS further improved the specificity and quantification capability of this technology. This quantitative method presented a wide linear dynamic range over 8-2000ng/mL and an LOD of 2ng/mL. A 460-fold improvement in sensitivity was obtained when pH barrage junction focusing was applied during the CE process, in comparison to when normal CE was conducted without online sample stacking. The superior detection sensitivity over previously reported methods enables direct analysis of bisphosphonate compounds, eliminating tedious pre-column sample enrichment and derivatization. Validation of alendronate content in a commercial drug tablet further proved the reliability and power of this method. This simple method with no sample derivatization, superior sensitivity, and short run time (<8min) is a promising alternative for accurate quantification of alendronate and other types of bisphosphonate compounds in both drug formulations and plasma samples.

Quantification of mycophenolic acid in human plasma by liquid chromatography with time-of-flight mass spectrometry for therapeutic drug monitoring.

This study presents, for the first time, the development and validation of a liquid chromatography and time-of-flight mass-spectrometry (LC-TOF-MS) based assay to quantify mycophenolic acid (MPA) in patient samples as part of a routine therapeutic drug monitoring service. MPA was extracted from 50 μl human plasma by protein precipitation, using sulindac as internal standard (IS). Separation was obtained on a Luna™ Omega polar C18 column kept at 40°C. The mobile phase consisted of a mixture of acetonitrile-deionized water (50:50, v/v) with 0.1% formic acid at a flow rate of 350 μl/min. Analyte and IS were monitored on a TOF-MS using a Jet-Stream™ (electrospray) interface running in positive mode. Assay performance was evaluated by analysing patient plasma (N = 69) and external quality assessment (N = 6) samples. The retention times were 2.66 and 2.18 min for MPA and IS, respectively. The lower limit of quantification of MPA was 0.1 μg/ml. The within- and between-assay reproducibility results ranged from 1.81 to 10.72%. Patient and external quality assessment sample results were comparable with those obtained previously by an in-house validated LC-MS/MS method. This method showed satisfactory analytical performance for the determination of MPA in plasma over the calibration range of 0.1-15.0 μg/ml.

Spray-Drying Microencapsulation of Bioactive Compounds from Lemon Verbena Green Extract.

Lippia citriodora has been demonstrated to have a wide variety of phytochemicals which provide benefits to human health acting as antioxidants or anti-obesogenics. In this study, these phytochemicals were recovered using a microwave-assisted technology and applying optimal conditions and microencapsulated using spray drying. In this study, two different carbohydrates, maltodextrin (MD) and inulin (IN), were compared as carriers in the encapsulation procedure. The spray drying process was optimized by using a response surface methodology (RSM) based on a central composite design 22, where air inlet temperature and the sample:encapsulating agent ratio (S:EA) were selected as independent variables. Both designs were analyzed equally to evaluate differences between each carrying agent on polar compounds’ encapsulation (process yield (Y%), encapsulation efficiency (EE%) and recovery of compounds (R%)) during the spray drying. The EE% and R% of each polar compound was monitored by High Performance Liquid Chromatography coupled to Time-of-Flight mass spectrometer by electrospray interface (HPLC-ESI-TOF-MS). The results showed that the use of IN as a carrier increased the powder recovered and the recovery of polar compounds after the spray dry process, whereas MD achieved a higher encapsulation efficiency.

An improved strategy to analyse strigolactones in complex sample matrices using UHPLC\u2013MS/MS

BackgroundStrigolactones represent the most recently described group of plant hormones involved in many aspects of plant growth regulation. Simultaneously, root exuded strigolactones mediate rhizosphere signaling towards beneficial arbuscular mycorrhizal fungi, but also attract parasitic plants. The seed germination of parasitic plants induced by host strigolactones leads to serious agricultural problems worldwide. More insight in these signaling molecules is hampered by their extremely low concentrations in complex soil and plant tissue matrices, as well as their instability. So far, the combination of tailored isolation—that would replace current unspecific, time-consuming and labour-intensive processing of large samples—and a highly sensitive method for the simultaneous profiling of a broad spectrum of strigolactones has not been reported.ResultsDepending on the sample matrix, two different strategies for the rapid extraction of the seven structurally similar strigolactones and highly efficient single-step pre-concentration on polymeric RP SPE sorbent were developed and validated. Compared to conventional methods, controlled temperature during the extraction and the addition of an organic modifier (acetonitrile, acetone) to the extraction solvent helped to tailor strigolactone isolation from low initial amounts of root tissue (150 mg fresh weight, FW) and root exudate (20 ml), which improved both strigolactone stability and sample purity. We have designed an efficient UHPLC separation with sensitive MS/MS detection for simultaneous analysis of seven natural strigolactones including their biosynthetic precursors—carlactone and carlactonoic acid. In combination with the optimized UHPLC–MS/MS method, attomolar detection limits were achieved. The new method allowed successful profiling of seven strigolactones in small exudate and root tissue samples of four different agriculturally important plant species—sorghum, rice, pea and tomato.ConclusionThe established method provides efficient strigolactone extraction with aqueous mixtures of less nucleophilic organic solvents from small root tissue and root exudate samples, in combination with rapid single-step pre-concentration. This method improves strigolactone stability and eliminates the co-extraction and signal of matrix-associated contaminants during the final UHPLC–MS/MS analysis with an electrospray interface, which dramatically increases the overall sensitivity of the analysis. We show that the method can be applied to a variety of plant species.