Electrospray Ionization Source Research Articles

Rapid Acid/Base Switching in Flow Injection Analysis and Isocratic Elution Liquid Chromatography with Mass Spectrometric Detection for Improved Sensitivity.

Ion signals in electrospray ionization (ESI) mass spectrometry (MS) are affected by addition of acid or base. Acids or bases are typically added to samples to enhance detection of analytes in positive- or negative-ion mode, respectively. To carry out simultaneous monitoring of analytes with different ionogenic moieties by ESI-MS, a rapid acid/base switching system was developed. The system was further coupled with flow injection analysis (FIA) and liquid chromatography (LC) MS. The two variants enable detection of separated analytes immediately after alternating addition of acid and base. The methods were tested using a set of phospholipids (PLs) as analytes. The rapid acid/base switching enhanced signals of some of the PL analytes in both ion modes of MS. Both FIA-MS and LC-MS with acid/base switching show signal enhancements (∼1.3-23.2 times) of some analyte signals when compared with analysis conducted without acid/base switching. The proposed methods are suitable for simultaneous analysis of cationic and anionic analytes. The FIA-MS and LC-MS methods with acid/base switching were also applied in analysis of lipid extract from real samples (sausage and porcine liver). However, the FIA-MS results were affected by ionization competition and isobaric interference due to the complexity of the sample matrix and diversity of PL species. In contrast, the LC-MS mode provides adequate selectivity to observe signal enhancement for specific analyte ions. Overall, alternating addition of acid and base immediately before the ESI source can improve analytical performance without the need to carry out separate analyses targeting different types of analytes.

Scalable Microfabrication of Multi-Emitter Arrays in Silicon for a Compact Microfluidic Electrospray Propulsion System.

The recent proliferation of SmallSats and their use in increasingly demanding applications require the development of onboard electric propulsion compatible with the power, mass, and volume constraints of these spacecraft. Electrospray propulsion is a promising technology for SmallSats due to its unique high efficiency and scalability across the wide power range of these platforms, for example, from a few watts available in a CubeSat to a few hundred watts in a MiniSat. The implementation of electrospray propulsion requires the use of microfabrication techniques to create compact arrays of thousands of electrospray emitters. This article demonstrates the microfabrication of multi-emitter electrospray sources of a scalable size for electrospray propulsion. In particular, a microfabrication and assembly process is developed and demonstrated by fabricating sources with arrays of 1, 64, and 256 emitters. The electrospray sources are tested in a relevant environment for space propulsion (inside a vacuum chamber), exhibiting excellent propulsive performance (e.g., absence of beam impingement in the extractor electrode, absence of hysteresis in the beam current versus propellant flow rate characteristic, proper operation in the cone-jet electrospraying mode, etc.) and nearly coincident output per emitter. Several design elements contribute to this performance: the even distribution of the propellant among all emitters made possible by the implementation of a network of microfluidic channels in the backside of the emitter array; the small dead volume of the network of microfluidic channels; the accurate alignment between the emitters and extractor orifices; and the use of a pipe-flow configuration to drive the propellant through closed conduits, which protects the propellant.

Developing transmission mode for infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging.

RationaleThe development and characterization of the novel NextGen infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) source catalyzed new advancements in IR‐MALDESI instrumentation, including the development of a new analysis geometry.MethodsA vertically oriented transmission mode (tm)‐IR‐MALDESI setup was developed and optimized on thawed mouse tissue. In addition, glycerol was introduced as an alternative energy‐absorbing matrix for tm‐IR‐MALDESI because the new geometry does not currently allow for the formation of an ice matrix. The tm geom was evaluated against the optimized standard geometry for the NextGen source in reflection mode (rm).ResultsIt was found that tm‐IR‐MALDESI produces comparable results to rm‐IR‐MALDESI after optimization. The attempt to incorporate glycerol as an alternative matrix provided little improvement to tm‐IR‐MALDESI ion abundances.ConclusionsThis work has successfully demonstrated the adaptation of the NextGen IR‐MALDESI source through the feasibility of tm‐IR‐MALDESI mass spectrometry imaging on mammalian tissue, expanding future biological applications of the method.

A UPLC- MS/MS Method to Quantify β-Sitosterol and Ferulic Acid of Pygeum Africanum Extract in Bulk and Pharmaceutical Preparation.

This study uses a liquid chromatography-electrospray ionization-tandem mass spectrometry method to determine β-Sitosterol and Ferulic acid in Pygeum africanum extract. Chromatographic separation of the two analytes was performed on an ACQUITY UPLC H-Class system coupled with Xevo TQD mass spectrometer and HSS T3 C18 column (2.1 X 50mm, 1.8μm). Mobile phase A consisted of an aqueous solution of 0.1% formic acid (v/v), and mobile phase B was 0.1% formic acid (v/v) in methanol pumped through a gradient elution mode. Mass spectrometer parameters were optimized using an electrospray ionization source in the positive and negative ionization modes. The quantification of the two analytes was performed using multiple reaction monitoring transitions. The method was fully validated per (FDA) guidelines regarding linearity, accuracy, precision, carryover and selectivity. The proposed method was applied successfully to determine the two investigated compounds in commercially available pharmaceutical products.

Determination of Ethyl Glucuronide and Ethyl Sulfate in Dried Blood Spots by UHPLC-MS-MS: Method Validation and Assessment of Ethanol Exposure in Postmortem Samples from Road Traffic Victims.

The determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood has been proposed in clinical and forensic applications to identify recent alcohol consumption. Also, there is a growing interest on the use of dried blood spots (DBS) in toxicological analysis, allowing increased stability of the analytes and simplifying sample transportation and storage. This study presents the development and validation of a method for quantifying EtG and EtS in DBS using ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). The DBS samples were extracted with a mixture of methanol and acetonitrile (80:20 v/v) and analyzed using UHPLC-MS-MS with electrospray source in negative mode, after separation with a fluoro-phenyl stationary phase. Validation was performed according to the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, with calibrations ranging from 0.10 to 18 µg/mL for EtG and 0.02 to 6 µg/mL for EtS. The analytes were stable in DBS stored from -20 to 45°C for 21 days. The method was successfully applied to capillary and venous DBS samples from 20 volunteers after ethanol ingestion and to DBS samples from 99 fatal victims of road traffic injuries. Capillary DBS was comparable to venous DBS and fresh whole blood in Passing-Bablok and Bland-Altman analysis, with correlation coefficients >0.91 (P < 0.001) for all comparisons. In postmortem application, the DBS EtG and EtS analysis indicated positive exposure to ethanol in 72.7% of the cases (EtG: 0.10-24.0 µg/mL and EtS: 0.03-4.11 µg/mL). The identification of ethanol consumption from blood alcohol concentrations (BACs) and EtG/EtS in DBS was in agreement in 98.6% of positive and 96.3% of negative cases (kappa 0.877, P < 0.001), indicating a high level of concordance with BAC in assessing alcohol use in postmortem samples.

Fast and Sensitive HPLC-ESI-MS/MS Method for Etoricoxib Quantification in Human Plasma and Application to Bioequivalence Study.

Etoricoxib is a non-steroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation. The objective of the current study was to develop a sensitive, fast and high-throughput HPLC-ESI-MS/MS method to measure etoricoxib levels in human plasma using a one-step methanol protein precipitation technique. A tandem mass spectrometer equipped with an electrospray ionization (ESI) source operated in a positive mode and multiple reaction monitoring (MRM) were used for data collection. The quantitative MRM transition ions were m/z 359.15 > 279.10 and m/z 363.10 > 282.10 for etoricoxib and IS. The linear range was from 10.00 to 4000.39 ng/mL and the validation parameters were within the acceptance limits of the European Medicine Agency (EMA) and Food and Drug Analysis (FDA) guidelines. The present method was sensitive (10.00 ng/mL with S/N > 40), simple, selective (K prime > 2), and fast (short run time of 2 min), with negligible matrix effect and consistent recovery, suitable for high throughput analysis. The method was used to quantitate etoricoxib plasma concentrations in a bioequivalence study of two 120 mg etoricoxib formulations. Incurred sample reanalysis results further supported that the method was robust and reproducible.

Simultaneous determination and pharmacokinetic study of six components in beagle dog plasma by UPLC–MS/MS after oral administration of Astragalus Membranaceus aqueous extract

Astragalus Membranaceus (AM) is widely applied in Chinese herbal compound formulas for treating various kinds of diseases. However, relative pharmacokinetics data on AM in nonrodents is still lacking. Here, an UPLC-MS/MS method for determining the six main compounds of AM was developed. The chromatographic separation was carried out by a Waters Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.8μm) with gradient elution of water-formic acid (99.98:0.02, v/v) and acetonitrile-formic acid (99.98:0.02, v/v) at a flow rate of 0.3ml/min within 11 min. Analyses of all compounds were conducted in multiple reaction monitoring mode with a positive/negative ion-switching mode of an electrospray ionization source in a single run. The analytical method was validated in terms of specificity, linearity, accuracy, precision, stability, etc. The method showed excellent linearity (r > 0.999) over certain concentration ranges. The intra-day and inter-day precisions were evaluated, and the RSD values were <12.4%. Furthermore, the validated method was successfully applied to determine the six components in plasma after oral administration of AM aqueous extract to beagle dogs and the pharmacokinetic parameters were obtained. Together, this study provides a reference for medication in the clinical practice of AM.

Simultaneous determination of three naphthoquinones from Arnebia euchroma (Royle) Johnst in beagle plasma by UPLC-MS/MS and application for pharmacokinetics study

Abstract A rapid, simple and efficient ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was established to simultaneous determination of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle plasma and evaluated by using esculetin as internal standard. The electrospray ionization (ESI) source was operated in negative ionization mode. Multi-reaction monitoring (MRM) was used to quantitatively analyzed shikonin m/z 287.0 → 217.9, isobutyryl shikonin m/z 357.0 → 268.9, β, βʹ-dimethylacryl alkanin m/z 370.0 → 270.1 and esculetin m/z 177.0 → 89.0, respectively. The method was validated for selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, matrix effect and stability. All validation parameters met the acceptance criteria according to regulatory guidelines. This method was successfully applied for the pharmacokinetic study of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle dogs plasma after oral administration of A. euchroma extract.

A Sensitive Liquid Chromatography-Mass Spectrometry Method for Determination of 14-Deoxy-12(R)-Sulfo Andrographolide Concentration in Rat Plasma and its Application to a Pharmacokinetic Study.

Andrographolide is a promising natural substance with numerous pharmacotherapy uses. 14-deoxy-12(R)-sulfo andrographolide (SAP) is the main metabolite of andrographolide in the intestine. To investigate the pharmacokinetic properties of SAP, a precise and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of SAP concentration in rat plasma was developed and validated in this study. Chromatographic separation was achieved on an Acpuity UPLC BEH C18 column with gradient elution that consisted of methanol and water at a flow rate of 0.3 mL/min. MS/MS detection was carried out by the multiple reaction monitoring (MRM) mode with negative electrospray ionization (ESI-) source, with the transitions of m/z 413.2→m/z 287.2 for SAP and m/z 269→m/z 133 for genistein [which was used as an internal standard (IS)]. The calibration curve of SAP was linear over the concentration range of 5-120 ng/mL. The selectivity, precision, accuracy, extraction recovery, matrix effect, and stability of the method were within acceptable ranges. This SAP quantification method was then successfully applied to a pharmacokinetic study of SAP. The area under the curve (AUC) of SAP in rats treated with SAP at 60 mg/kg by intravenous administration was 7498.53 ± 2405.02 mg/L·min. The AUC of SAP in rats treated with SAP at 60 mg/kg by oral administration was 97.74 ± 39.56 mg/L·min. Thus, the absolute oral bioavailability of SAP was determined to be 1.40%.

Development of validated UHPLC–PDA with ESI–MS-MS method for concurrent estimation of magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine and berberine in Berberis aristata

Abstract A validated UHPLC-PDA with an ESI-MS/MS method has been developed for simultaneous estimation of six bioactive alkaloids (magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine and berberine) in the different extracts of the roots of Berberis aristata DC (Family:Berberdiaceae). It is an important medicinal herb native to Northern Himalaya and commonly known as ‘daruharidra’, ‘daruhaldi’, ‘Indian barberry’ or ‘tree turmeric’. An insight into the research literature uncovered reports on isoquinoline alkaloids like magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine, and berberine as major bioactives in B. aristata roots, possessing different pharmacological and therapeutic effects. In the present study, these aforementioned alkaloids were separated on Phenomenex Luna®, 5 µm-C8 analytical column. The HPLC-MS analysis was performed at a flow rate of 0.90 mL min−1. Each alkaloid that is resolved was characterized by precursor ions and fragment ions with electrospray ionization (ESI) source in both positive and negative ionization using scan mode. The limit of detections (LODs) were 0.087, 0.727, 0.035, 0.124, 0.782 and 0.794 μg mL−1 for magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine and berberine, respectively. The proposed UHPLC-PDA method was fully validated according to international (ICH) guidelines and was found to be selective, sensitive and highly accurate for the concomitant estimation of the aforementioned symbolic bio-markers of B. aristata roots.

Antioxidant Activity, Total Phenolic and Flavonoid Content and LC–MS Profiling of Leaves Extracts of Alstonia angustiloba

Plants have a wide range of active compounds crucial in treating various diseases. Most people consume plants and herbals as an alternative medicine to improve their health and abilities. A. angustiloba extract showed antinematodal activity against Bursaphelenchus xylophilus, antitrypanosomal action against Trypanosoma brucei and anti-plasmodial activity against the chloroquine-resistant Plasmodium falciparum K1 strain. Moreover, it has demonstrated growth inhibitory properties towards several human cancer cell lines, such as MDA-MB-231, SKOV-3, HeLa, KB cells and A431. DPPH and ABTS assays were carried out to determine the antioxidant activity of the aqueous and 60% methanolic extract of A. angustiloba leaves. Moreover, total phenolic and flavonoid contents were quantified. The presence of potential active compounds was then screened using liquid chromatography coupled with a Q-TOF mass spectrometer (LC–MS) equipped with a dual electrospray ionisation (ESI) source. The EC50 values measured by DPPH for the 60% methanolic and aqueous extracts of A. angustiloba leaves were 80.38 and 94.11 µg/mL, respectively, and for the ABTS assays were 85.80 and 115.43 µg/mL, respectively. The 60% methanolic extract exhibited the highest value of total phenolic and total flavonoid (382.53 ± 15.00 mg GAE/g and 23.45 ± 1.04 mg QE/g), while the aqueous extract had the least value (301.17 ± 3.49 mg GAE/g and 9.73 ± 1.76 mg QE/g). The LC–MS analysis revealed the presence of 103 and 140 compounds in the aqueous and 60% methanolic extract, respectively. It consists of phenolic acids, flavonoids, alkaloids, amino acids, glycosides, alkaloids, etc. It can be concluded that the therapeutic action of this plant is derived from the presence of various active compounds; however, further research is necessary to determine its efficacy in treating diseases.

Study on metabolites in vivo of Dangefentong Capsules based on UHPLC-Q/Orbitrap-MS/MS

Dangefentong Capsules is a new traditional Chinese medicine preparation for the treatment of diabetic peripheral neuropathy. It is based on the Salviae Miltiorrhizae Radix et Rhizoma-Puerariae Lobatae Radix herb pair with salvianolic acids, tanshinones and pueraria flavonoids as main components. Studying the chemical composition in vivo of Dangefentong Capsules and its metabolites is of great significance for making clear its pharmacodynamic material basis and the action mechanism. The UHPLC-Q/Orbitrap-MS/MS was applied to rapidly analyze the metabolites and metabolic pathways of Dangefentong Capsules in Beagle dogs after gavage. Eclipse plus C_(18) column(2.1 mm×50 mm, 1.8 μm) was used, and gradient elution was performed with 0.1% formic acid aqueous solution(A)-formic acid acetonitrile solution(B). A heated electrospray ion source(HESI) was employed. The scanning mode was set as the positive and negative ion mode, and the mass scanning range was m/z 100-1 000. The plasma, urine and feces samples were collected after male Beagle dogs were administered with Dangefentong Capsules. The prototype components and metabolites were identified by UHPLC-Q/Orbitrap-MS/MS analysis combined with reference substances and references. The results showed that 107 chemical components were identified, including 58 prototype components and 49 metabolites. The identified prototype components included 42 components from Salviae Miltiorrhizae Radix et Rhizoma and 16 components from Puerariae Lobatae Radix. The metabolites consist of 21 and 28 metabolites of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix, respectively. They are mainly derived from the methylation, hydroxylation, sulfation and glucuronidation of salvianolic acids, tanshinones and pueraria flavonoids. This research rapi-dly analyzes the chemical components in vivo of Beagle dogs administered with Dangefentong Capsules, laying a basis for illustrating the pharmacodynamic material basis and mechanism of Dangefentong Capsules.

Advancing Cyclic Ion Mobility Mass Spectrometry Methods for Studying Biomolecules: Toward the Conformational Dynamics of Mega Dalton Protein Aggregates.

Native mass spectrometry is a powerful tool for the analysis of noncovalent complexes. When coupled with high-resolution ion mobility, this technique can be used to investigate the conformational changes induced in said complexes by different solution or gas-phase conditions. In this study, we describe how a new-generation high-resolution ion mobility instrument equipped with a cyclic ion mobility cell can be utilized for the analysis of large biomolecular systems, including temperature-induced protein aggregates of masses greater than 1.5 MDa, as well as a 63 kDa oligonucleotide complex. The effects of and the interplay between the voltages applied to the different components of the cyclic ion mobility spectrometry system on ion transmission and arrival time distribution were demonstrated using biomolecules covering the m/z range 2000-10,000. These data were used to establish a theoretical framework for achieving the best separation in the cyclic ion mobility system. Finally, the cyclic ion mobility mass spectrometer was coupled with a temperature-controlled electrospray ionization source to investigate high-mass protein aggregation. This analysis showed that it was possible to continuously monitor the change in abundance for several conformations of MDa aggregates with increasing temperature. This work significantly increases the range of biomolecules that can be analyzed by both cyclic ion mobility and temperature-controlled electrospray ionization mass spectrometry, providing new possibilities for high-resolution ion mobility analysis.

Simultaneous Determination and Pharmacokinetics Study of Three Triterpenes from Sanguisorba officinalis L. in Rats by UHPLC–MS/MS

A selective and rapid ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was established and validated for the determination of ziyuglycoside I, 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-d-glucopyranosyl ester, and pomolic acid in rats after the oral administration of ziyuglycoside I, 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-d-glucopyranosyl ester, pomolic acid, and Sanguisorba officinalis L. extract. The separation was carried out on an ACQUITY UPLC®HSS T3 column (2.1 mm × 100 mm, 1.8 μm), using methanol and 5 mmol/L ammonium acetate water as the mobile phase. The three compounds were quantified using the multiple reaction monitoring mode with the electrospray ion source in both the positive and negative mode. Liquid-liquid extraction was applied to the plasma sample preparation. Bifendate was selected as the internal standard. The intra-day and inter-day precision and the accuracy of the method were all within receivable ranges. The lower limit of quantification of ziyuglycoside I, 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-d-glucopyranosyl ester, and pomolic acid were 6.50, 5.75, and 2.63 ng/mL, respectively. The extraction recoveries of analytes in rat plasma ranged from 83 to 94%. The three components could be rapidly absorbed into the blood (Tmax, 1.4–1.6 h) both in the single-administration group or S. officinalis extract group, but the first peak of PA occurred at 0.5 h and the second peak at 4–5 h in the S. officinalis extract. Three compounds were eliminated relatively slowly (t1/2, 7.3–11 h). The research was to establish a rapid, sensible, and sensitive UHPLC–MS/MS method using the multi-ion mode for multi-channel simultaneous mensuration pharmacokinetics parameters of three compounds in rats after oral administration of S. officinalis extract. This study found, for the first time, differences in the pharmacokinetic parameters of the three compounds in the monomer compounds and S. officinalis extract administration, which preliminarily revealed the transformation and metabolism of the three compounds in vivo.

Stability and Dissociation of Adeno-Associated Viral Capsids by Variable Temperature-Charge Detection-Mass Spectrometry.

Adeno-associated viral (AAV) vectors have emerged as gene therapy and vaccine delivery systems. Differential scanning fluorimetry or differential scanning calorimetry is commonly used to measure the thermal stability of AAVs, but these global methods are unable to distinguish the stabilities of different AAV subpopulations in the same sample. To address this challenge, we combined charge detection-mass spectrometry (CD-MS) with a variable temperature (VT) electrospray source that controls the temperature of the solution prior to electrospray. Using VT-CD-MS, we measured the thermal stabilities of empty and filled capsids. We found that filled AAVs ejected their cargo first and formed intermediate empty capsids before completely dissociating. Finally, we observed that pH stress caused a major decrease in thermal stability. This new approach better characterizes the thermal dissociation of AAVs, providing the simultaneous measurement of the stabilities and dissociation pathways of different subpopulations.

Quantification of 3-bromopyruvate in rat plasma by HPLC-MS/MS employing precolumn derivatization and the application to a pharmacokinetics study.

A simple high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed for the determination of 3-bromopyruvate (3-BrPA) in rat plasma for the first time. The analytes were separated on a C18 column (100 × 2.1mm, 1.7μm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization source was applied for detection. 3-BrPA was extracted from rat plasma with protein precipitation, and then derivatized with 4-nitro-1,2-phenylenediamine to obtain the mass signal because 3-BrPA could not be detected in mass spectrometry. The method was fully validated according to the US Food and Drug Administration guidance. The method was linear over the concentration range 0.5-1,000.0ng/ml for 3-BrPA. The precision and accuracy, extraction recovery, and matrix effect were within acceptable limits. The method was then applied to support a pharmacokinetic study after 3-BrPA and 3-BrPA-l-Val-l-Ile (a dipeptide prodrug of 3-BrPA, 3-BrPA-l-Valine-l-isoleucine) had been administered to the Sprague-Dawley rats, respectively.

Enantioseparation and quantitative analysis of three alkaloids in Rhizoma Corydalis by chiral LC-MS/MS

A chiral liquid chromatography-mass spectrometry (LC-MS/MS) approach was developed and verified for the first time in order to quantify the three alkaloids, namely tetrahydropalmatine (THP), tetrahydroberberine (THB) and tetrahydrocoptisine (THC) in Rhizoma Corydalis. Solid-phase extraction was used to prepare sample solution. Enantiomeric separation was obtained on a Chiralpak IC column using acetonitrile-water-ammonia (90:10: 0.1, v/v/v) as mobile phase. The detection was carried out in multiple reaction monitoring (MRM) mode with positive electrospray ionization source. Transitions of m/z 356.0→192.0, m/z 340.0→176.0 and m/z 324.1→176.0 were monitored for THP, THB and THC respectively. All calibration curves showed good linearity (r2 ≥0.9994) within the test ranges. The lower limit of quantification (LLOQ) was 1.0 ng mL−1 and 1.5 ng mL−1 for (+)- and (−)-THP, 1.0 ng mL−1 and 3.0 ng mL−1 for (+)- and (−)-THB, 1.5 ng mL−1and 1.8 ng mL−1 for (+)- and (−)-THC, respectively. The precision, repeatability, and stability tests all meet the requirements. The average recoveries of the analytes ranged from 98.9 % to 101.3 %. Ultimately, the established method was successfully applied to the quantitative analysis of these alkaloid enantiomers extracted from Rhizoma Corydalis of different habitats.

Comparison of electrospray and UniSpray™ ion sources with post-column hydrochloric acid infusion for 119 abuse relevant substances

Comparing the sensitivity of the Waters UniSpray™ ion source (USI) with the Waters electrospray ion source (ESI) on a Waters Xevo TQ-XS mass spectrometer with and without additional post-column infusion of low concentrated hydrochloric acid (HCl). To reduce sample volumes or increase analytical sensitivity for drugs of abuse detection in human body fluids, technological advances in mass spectrometry often target an increase in ionization efficiency. One example for this development is the USI, which claims higher ion yields and therefore higher sensitivity due to a unique geometry. Another possibility to increase sensitivity through increased ion yield is the post-column infusion of low concentrated HCl. For sensitivity comparison, we choose the, in our opinion, 119 most relevant medications and drugs of abuse. These were prepared in eleven acetonitrile solutions containing 10 analytes each and one with 9 analytes at a concentration of 1 μg/mL for each substance. For analysis, every solution was diluted 1:1000 in mobile phase A/ethylene glycol (70:30 v/v) to a concentration of 1.0 ng/mL. Solutions were analysed in triplicate on the same day interrupted by the ion source change, to decrease inter-day variations in sensitivity. Chromatographic separation was performed on a Waters 2.1 × 150 mm, 1.8 μm BEH Phenyl column kept at 60 °C on an Acquity UPLC connected to a Xevo TQ-XS detector (Waters). The standard ESI ion source or the USI ion source was mounted on the detector. Mobile phase A consisted of 20 mM ammonium formate and 0.1% formic acid (pH 3) and mobile phase B was 0.1% formic acid in methanol. Gradient elution was conducted within 6.0 min at a flow rate of 0.5 mL/min with 85% A and 15% B at the beginning and 100% B at the end. Data were acquired with the ion sources operating in the positive ionization MRM mode. Two transitions for each analyte were monitored at a constant dwell time of 10 ms over the full chromatographic run time. In an additional experiment, both ion sources were compared with additional post-column infusion of 0.0037% HCl at a flow rate of 5.0 μL/min. Injection volume was 2.0 μL in all experiments. USI provided a general gain in signal intensity (peak area): 12% ( n = 14) of the analytes showed a peak area increase up to 2-fold, 85% ( n = 101) increased between 2- and 4-fold and 3% ( n = 4) increased above 4-fold in peak area. Post-column HCl infusion with the ESI source mounted, resulted in an analyte dependent additional gain in signal intensity for 15% ( n = 18) of the analytes ranging from 2- to 21-fold. 78% ( n = 93) ranged from 0.8-fold to 2-fold. In contrast 7% ( n = 8) showed a signal decrease between 0.4-fold and 0.8-fold. The combination of USI and post-column HCl infusion resulted in an increase in peak area from 2-fold up to 62-fold compared to ESI without infusion for 91% (n = 108) of the analytes. 8% ( n = 9) where between 0.8-fold and 2-fold and 1% ( n = 2) ranged from 0.4-fold to 0.8-fold. With USI, a general improvement in analytical sensitivity under identical conditions could be achieved for all of our 119 “favorite analytes” separated in one chromatographic run. Additional post-column infusion of HCl increased sensitivity for many substances. This approach can be used very selectively when applied to certain retention time windows only. Further experiments are needed to understand how different adjustments of ESI and USI and different matrices will affect ionization. USI, especially in combination with post-column HCl infusion, is a promising tool to increase sensitivity in toxicological multi-target LC-MS/MS analysis.

Human methylone, MDMA and metabolites’ plasma concentrations by a new LC-MS/MS method after controlled administration of methylone and MDMA

Synthetic cathinones are a class of new psychoactive substances (NPS) with structural and pharmacological similarity to amphetamines. Methylone, a synthetic cathinone NPS, is the β-keto analog of MDMA. There are no controlled administration data for methylone in humans. The aim of this research was development of an LC-MS/MS method to simultaneously quantify methylone, MDMA and their metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymeth-amphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) in human plasma and application of the method to the first controlled oral methylone administration study. Briefly, 10 μL 100 ng/mL internal standard mixture (methylone-d3, MDA-d5 and MDMA-d5) and 2 μL NH2 2% in H2O (pH 9) were added to 100 μL plasma, tubes were stirred in a roller mixer for 10 min and centrifuged at 3500 rpm for 5 min. Supernatants were collected into clean tubes, 100 μL acidic methanol (1% HCl) was added to prevent evaporative losses, and samples dried under nitrogen for approximately 30 min. Samples were reconstituted in 100 μL mobile phase A:B (95:5) (0.1% formic acid in water:acetonitrile) and transferred into autosampler vials for LC–MS/MS analysis. Analytes separation was obtained on a UHPLC 1290 Infinity II (Agilent Technologies Italia S.p.a.) coupled to a mass spectrometer (6470A Triple Quadrupole LC-MS) equipped with an electrospray ionization source operating in positive-ion mode (ESI+). Limits of quantification (LLOQ) were 5 (methylone) to 500 (MDMA) μg/L and 0.5 to 50 μg/L for their metabolites. A randomized double blinded study was conducted in 12 male volunteers (aged 18–45 years old; weight ranging from 50 to 90 kg) and divided into 3 sessions, with single oral doses of 50, 100, 150 or 200 mg methylone, 100 mg MDMA or placebo in each session. Plasma samples were collected before and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24 h after dosing. The analytical method was validated over five successive days in plasma following the most recent criteria for bioanalytical method development and validation. The method was linear for all analytes over the calibration range. Intra- and inter-assay accuracies were ±15%, while intra- and inter-assay precision were ±10%. Methylone plasma concentrations increased in a dose-related manner. Methylone Cmax were 138, 243, 320 and 530 μg/L following the 50, 100, 150 and 200 mg doses, respectively, while corresponding area under the curve (AUC) were 1065, 2486, 3511 and 5489 min ×μg/L. Methylone Tmax were 1 h for the lowest 50 mg dose and 2 h for the remaining doses. The last detected methylone concentrations (Clast) at 24 h were 10.5, 18.8, 36.4 and 51.8 μg/L (for 50, 100, 150 and 200 mg respectively). For methylone's HMMC metabolite, Cmax were 6.6, 16.9, 28.3, and 32.7 μg/L, while AUC were 36.9, 90.1, 129 and 173 min ×μg/L. HMMC exhibited rapid kinetics with Tmax of 1–1.25 h for all doses. As for Clast, HMMC concentrations were 0.5, 0.4, 1.3 and 0.8 μg/L (for 50, 100, 150 and 200 mg, respectively) 24 h after drug administration. Methylone is a popular drug of abuse, yet no studies addressed its metabolism and pharmacokinetics in humans. A validated LC-MS/MS method for simultaneous quantification of methylone, MDMA and their metabolites (HMMC, MDA, HMMA and HMA) in human plasma is presented. Considering the pharmacokinetic study for methylone and HMMC, concentrations following the 100 mg dose were approximately doubled compared to the 50 mg dose, and after the 200 mg dose were about four times higher than the lowest dose (within ±20%), suggesting linear pharmacokinetics for both analytes.

Aqueous Extracts of Fish Roe as a Source of Several Bioactive Compounds

Regular consumption of seafood and, in particular, fish has been associated with important health benefits. A fish product that has been increasingly included in the human nutrition is roe. Despite its nutritional value has been established (fatty acid profile and protein content), the knowledge of the composition of its aqueous extracts is still limited. This work describes the bioactive compounds profile in the roe-derived aqueous extracts of three different marine species (sardine, horse mackerel and sea bass) using a method based on liquid chromatography coupled to high-resolution mass spectrometry with an electrospray ionisation source (LC-ESI/HRMS). The presence of substances with well-known nutritional and functional properties (e.g., antioxidant and anti-inflammatory properties) was demonstrated, namely essential amino acids (e.g., taurine), peptides (e.g., anserine and carnosine), B-group vitamins (e.g., nicotinamide) and gadusol. Therefore, roe-derived aqueous extracts are excellent sources of bioactive compounds and may be used as a font of functional components for several medical and veterinary applications.