Ion ESI Research Articles

A novel fast analytical method for the determination of N-nitroso vonoprazan in vonoprazan tablets and raw materials using LC-ESI-MS/MS

ABSTRACT The presence of N-nitrosamine contaminants in pharmaceuticals, particularly N-nitroso vonoprazan (NVP), poses significant health risks due to their genotoxic and carcinogenic properties. This study describes a novel, rapid analytical method for detecting and quantifying NVP in vonoprazan tablets and raw materials. The NVP was separated and quantified using an LC-ESI-MS/MS system with ESI ionisation and an Exion LC Sciex 5500-QTrap. The system utilised an ACE 3 C18-PFP column with dimensions of 4.6 mm × 150 mm and 3.0 µm. It achieved a high correlation coefficient (R2 = 0.9955) and met linearity and accuracy criteria. The method was developed and validated using ICH guidelines and against standard solutions, and it demonstrated high precision, accuracy, and specificity. The method validation results showed that the quantification is accurate, with an RSD of less than 10% and LOD and LOQ values of 0.5 and 1.0 ng/mL, respectively. The analysis of 150 vonoprazan tablets and 100 raw material samples revealed low levels of NVP, mostly falling below the FDA’s Acceptable Daily Intake limit of 96 ng/day. This novel approach provides a powerful and dependable tool for ensuring the safety and quality of vonoprazan-based pharmaceuticals.

Synthesis, Characterization, and Potential Usefulness in Liver Function Assessment of Novel Bile Acid Derivatives with Near-Infrared Fluorescence (NIRBAD).

Conventional serum markers often fail to accurately detect cholestasis accompanying many liver diseases. Although elevation in serum bile acid (BA) levels sensitively reflects impaired hepatobiliary function, other factors altering BA pool size and enterohepatic circulation can affect these levels. To develop fluorescent probes for extracorporeal noninvasive hepatobiliary function assessment by real-time monitoring methods, 1,3-dipolar cycloaddition reactions were used to conjugate near-infrared (NIR) fluorochromes with azide-functionalized BA derivatives (BAD). The resulting compounds (NIRBADs) were chromatographically (FC and PTLC) purified (>95%) and characterized by fluorimetry, 1H NMR, and HRMS using ESI ionization coupled to quadrupole TOF mass analysis. Transport studies using CHO cells stably expressing the BA carrier NTCP were performed by flow cytometry. Extracorporeal fluorescence was detected in anesthetized rats by high-resolution imaging analysis. Three NIRBADs were synthesized by conjugating alkynocyanine 718 with cholic acid (CA) at the COOH group via an ester (NIRBAD-1) or amide (NIRBAD-3) spacer, or at the 3α-position by a triazole link (NIRBAD-2). NIRBADs were efficiently taken up by cells expressing NTCP, which was inhibited by taurocholic acid (TCA). Following i.v. administration of NIRBAD-3 to rats, liver uptake and consequent release of NIR fluorescence could be extracorporeally monitored. This transient organ-specific handling contrasted with the absence of release to the intestine of alkynocyanine 718 and the lack of hepatotropism observed with other probes, such as indocyanine green. NIRBAD-3 administration did not alter serum biomarkers of hepatic and renal toxicity. NIRBADs can serve as probes to evaluate hepatobiliary function by noninvasive extracorporeal methods.

Tuning parameters of single quadrupole mass detector hyphenated to supercritical fluid chromatography for enantioseparation of synthetic cathinones

In our previous paper, we introduced a new method for the enantioseparation of cathinones on chiral zwitterion ion exchangers by SFC-ESI-MS with an inverse make-up solvent gradient. The inverse gradient enabled us to stabilize electrospray ionization while simultaneously increasing MS response. In this study, we fine-tuned our previously optimized SFC-ESI-MS method and compared its efficiency with that of the default ESI-MS method. To enhance analyte detection, we designed a complex series of experiments in which all combinations of the following three parameters were evaluated: cone voltage (5 to 27 V), probe temperature (250 to 550 °C) and capillary voltage for positive ionization (0.3 to 1.5 kV). After optimization, various ratios of the make-up solvent (methanol/water/formic acid) introduced before entering the ESI ion source were also tested. Despite this comprehensive testing regime, there were no significant differences between the results obtained for the tuned and default ESI-MS parameters. Similarly, the most suitable make-up solvent composition was the originally used ratio of 90/10/0.1 (v/v/v). These results reinforce the findings of our previous paper, in which we optimized the make-up solvent flow rate and showed its significant impact on ESI-MS response.

Development of a high-throughput liquid chromatography-tandem mass spectrometry platform for the determination of intact natriuretic peptides in human plasma

We present an innovative, reliable, and antibody-free analytical method to determine multiple intact natriuretic peptides in human plasma. These biomolecules are routinely used to confirm the diagnosis and monitor the evolution of heart failure, so that their determination is essential to improve diagnosis and monitor the efficacy of treatment. However, common immunoassay kits suffer from main limitations due to high cross-reactivity with structurally similar species. In our method, we pre-treated the sample by combining salting-out with ammonium sulfate with microextraction by packed sorbent technique. Analyses were then carried out by ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry. The use of 3-nitrobenzyl alcohol as a supercharger reagent enhanced the ESI ionization and improved the signal-to-noise ratio. The analytical protocol showed good linearity over one order of magnitude, recovery in the range of 94–105 %, and good intra- and inter-day reproducibility (RSD<20 %), and the presence of a matrix effect. Limits of detection were in the range of pg/mL for all peptides (0.2–20 pg/mL). Stability study in plasma samples demonstrated that proper protease inhibitors need to be included in blood collection tubes to avoid peptide degradation. Preliminary analyses on plasma samples from heart failure patients allow the quantification of ANP 1–28 as the most abundant species and the detection of ANP 5–28, BNP 1–32, and BNP 5–32. The method could be used to investigate how cross-reactivity issues among structurally similar species impact determinations by ELISA kits.

Simultaneous determination of Cefepime and Tazobactam by using Hyphenated Liquid Chromatography (UPLC-MS/MS)

The present study deals with establishing a hyphenated analytical method and validating the same for the determination of the drug combination of Cefepime and Tazobactam simultaneously by UPLC-MS/MS. A C18 BEH analytical column was employed. A mobile phase of 0.2 percent formic acid in water and acetonitrile were used in a 20:80 v/v ratio. Samples are ionized using +ESI ionization and detected in MRM mode. The retention time of 0.80 min and 0.96 min were observed for Cefepime and Tazobactam respectively. The drugs have linear responses in the investigated range, with r2 = 0.9955 for Cefepime and r2 = 0.9816 for Tazobactam with increased precision and accuracy. The LOD for Cefepime and Tazobactam was 66ng/ml and 8.3ng/ml respectively. The LOQ for Cefepime and Tazobactam was 200ng/ml and 25ng/ml respectively. According to the findings, the hyphenated UPLC-MS/MS method was developed and validated as per ICH regulations to determine Cefepime and Tazobactam simultaneously.

Analysis of Targeted Components in the Combination Extract of Hovenia dulcis and Phyllanthus emblica by UPLC-MS/MS

This study aimed to establish a UPLC-MS/MS method for rapid analysis and identification of target components in the combination extract of Hovenia dulcis and Phyllanthus emblica. The gradient elution was carried out with methanol (A) −0.2% formic acid solution (A) with low rate of 1.0 mL · min−1, column temperature of 30 °C, detection wavelength of 285 nm. The ESI ion source was used to collect data under the negative ion mode. Through analysis and identification, five target components were inferred, namely gallic acid, dihydromyricetin, corilagine, taxifolin, and ellagic acid in the combination extract. Quantitative analysis was conducted on the combination extract. By utilizing the theory of modern supramolecular technology in traditional Chinese medicine, the functional composite product was obtained by selecting the compound extraction of Hovenia dulcis and Phyllanthus emblica as raw materials, and quantitative analysis was conducted on on the combination extract. The results showed that the flavonoid extraction rate and the content of target components were both higher when the mass ratio of Hovenia dulcis to Phyllanthus emblica was 1:1 and 1:2. This indicates that the co-extraction of Hovenia dulcis and Phyllanthus emblica not only simplified the extraction process but also enhances the extraction rate of the flavonoids in Hovenia dulcis. The developed UPLC-MS/MS method is rapid, sensitive, which provides a technical basis for the selection of raw material mass ratios and product development of Hovenia dulcis and Phyllanthus emblica combination.

A novel [4 + 2] cycloaddition reaction involving Lawesson’s reagent. Structure and specific fragmentations of a new cyclic 1,2-thiaphosphinane-4-one

A crude morpholine enamine of acetone treated with Lawesson’s reagent unexpectedly yielded a six-membered thiaphosphinane-4-one. This compound is the first example of a new class of heterocycles. It has been proven that it is formed from 4-methyl-2-morpholino-1,3-pentadiene which is usually present in crude morpholine enamine batches. A mechanism of this regioselective reaction was postulated and a characteristic chair-like conformation of the product was examined in detail. Additionally, some unusual primary fragmentations of the product with the loss of H2S and isobutylene were observed for positive and negative ESI ionization mode, respectively.

Untargeted Metabolomics Reveals Alterations of Rhythmic Pulmonary Metabolism in IPF.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive condition characterized by the impairment of alveolar epithelial cells. Despite continued research efforts, the effective therapeutic medication is still absent due to an incomplete understanding of the underlying etiology. It has been shown that rhythmic alterations are of significant importance in the pathophysiology of IPF. However, a comprehensive understanding of how metabolite level changes with circadian rhythms in individuals with IPF is lacking. Here, we constructed an extensive metabolite database by utilizing an unbiased reference system culturing with 13C or 15N labeled nutrients. Using LC-MS analysis via ESI and APCI ion sources, 1300 potential water-soluble metabolites were characterized and applied to evaluate the metabolic changes with rhythm in the lung from both wild-type mice and mice with IPF. The metabolites, such as glycerophospholipids and amino acids, in WT mice exhibited notable rhythmic oscillations. The concentrations of phospholipids reached the highest during the fast state, while those of amino acids reached their peak during fed state. Similar diurnal variations in the metabolite rhythm of amino acids and phospholipids were also observed in IPF mice. Although the rhythmic oscillation of metabolites in the urea cycle remained unchanged, there was a significant up-regulation in their levels in the lungs of IPF mice. 15N-ammonia in vivo isotope tracing further showed an increase in urea cycle activity in the lungs of mice with IPF, which may compensate for the reduced efficiency of the hepatic urea cycle. In sum, our metabolomics database and method provide evidence of the periodic changes in lung metabolites, thereby offering valuable insights to advance our understanding of metabolic reprogramming in the context of IPF.

Determination of Ten Plant Growth Regulators in Bean Sprouts by Mixed Solid Phase Extraction Columns Separation and LC-MS/MS

(1) Background: Plant growth regulators (PGRs) can accelerate growth or improve the quality and quantity of bean sprouts but are forbidden to use in bean sprout cultivation, as the sprouting process’s increased chemicals will disturb the PGRs analysis. This article aimed to increase the accuracy and level of sensitivity of the LC-MS/MS method for the simultaneous analysis of 10 PGRs after mixed solid phase extraction (SPE) purification. (2) Methods: An LC-MS/MS detection method for 10 kinds of PGRs was established based on ESI ionization in the positive ion mode for 6-furfurylaminopurine (6-KT), paclobutrazol (PBZ), indole-2-acetic acid (IAA), and indole-3-butyric acid (IBA) and in the negative ion mode for gibberellin A3 (GA3), 2,4-dichlorophenoxy acetic acid (2,4-D), 4-chlorophenoxyacetic acid (4-CPA), forchlorfenuron (FCF), thidiazuron (TDZ), and 6-benzyl adenine (6-BA). (3) Results: The 10 PGR compounds were detected within a concentration range of 1.0–50 ng/mL. The average recovery was 68.3–97.3% with relative standard deviations (RSD) of 4.6–15.2% (n = 6); the limit of detection (LOD) and limit of quantification (LOQ) were found to be 2 and 5 ng/g, respectively. PGRs were surveyed in 36 soybean sprouts and 33 mungbean sprouts; the results showed that 4-CPA and IAA were detected in 10 soybean sprouts and 10 mungbean sprouts, respectively. Five samples contained both 4-CPA and IAA. (4) Conclusions: The established method is simple, rapid, accurate, and highly sensitive for the detection of PGR residues in bean sprout products.

Development of liquid chromatography-mass spectrometry method to determine biotin content in nutritional products and supplements

The study was carried out to develop a method for determining the biotin content in nutritional products and supplements by liquid chromatography-mass spectrometry (LC-MS/ MS) combined with a fast and simple pretreatment. The analyzed sample was hydrolyzed in acetate buffer (50 mM, pH 4) with the presence of ascorbate solution (10%) at 120 &amp;plusmn; 1&amp;deg;C. After adding the internal standard (2H4-biotin), the mixture was analyzed on LC-MS/ MS system using C18 (100 mm &amp;times; 2.1 mm; 1.7 &amp;micro;m) column and 0.1% formic acid and MeOH as mobile phases. The analyte was detected on an MS/MS system with an ESI (+) ionization source. The method was validated following the AOAC criteria. The method detection limit was 0.15 - 4.20 &amp;micro;g/100g, the method quantitative limit was in the range of 0.5 - 14.0 &amp;micro;g/100g, the recovery range was 81.3 - 107.3%, the repeatability was in the range of 2.00 - 7.30 %, the reproducibility was 2.50 - 7.70%, which were in accordance with AOAC requirements. The method has been applied to analyze biotin content in 10 nutritional products and 10 supplements purchased in the market.

LC-MS Analysis of Kigelia Pinnata (JACQ) DC. Root Bark- A Multi-Potent Medicinal Plant

Phytochemicals of the plants have significant role in drug development for treatment of human diseases. Majority population in Africa, Asia and Latin America use plant based medicines. Kigelia pinnata DC. is a reputed African folklore drug and now abundantly found throughout India. It belongs to the family Bignoniaceae and used in different disease conditions traditionally. Out of various parts used, root bark owns medicinal properties like gynaecological complaints for fibroid, cancer of uterus and also reported biological activities such as antimalarial activity, antiprotozoal activity, antioxidant activity etc. The study was carried out for detailed exploration of biological active components in root bark extract of K. pinnata through Liquid chromatography/Mass Spectroscopy (LC/MS). It was performed on ESI ionization mode using both polarities. Total 63 known compounds are found from positive and negative mode. The molecules are derived from class group as terpenoid, flavonoids, phenols, glycoside and others which possess antioxidant, anti-inflammatory, antimicrobial, antihypertensive, anti-diabetic type of biological activities. This work therefore uncovers the phytochemical profile of K.pinnata root bark emphasizing their medicinal properties against important human diseases.

The delivery of nanoparticles improves the pharmacokinetic properties of celecoxib to open a therapeutic window for oral administration of insoluble drugs.

A sensitive and reliable LC-MS/MS method is established and validated to determine the concentration of celecoxib, in the serum of cynomolgus monkey, using celecoxib-D7 as an internal standard. The pharmacokinetic process was investigated after giving Celebrex, celecoxib nanoparticles (CXB-NPs) and hyaluronic acid celecoxib nanoparticles (HA-CXB-NPs) by intragastric (i.g.) administration. Chromatographic separation was performed with a C18 column (2.1 × 100 mm, 2.6 μm) at 40°C with a mobile phase of 2‰ HCOOH in water and acetonitrile. The mass spectral acquisition was then performed in the multiple reaction monitoring mode, with negative ESI ion at m/z 380.0 → 316.0 and m/z 387.1 → 323.1 for celecoxib and celecoxib-D7, respectively. Good linearity was observed over the concentration range from 3 to 2,000 ng/ml (R2 = 0.9954). The intra- and inter-day precision and accuracy, matrix effect and extraction recovery, as well as stability, all met the determination requirements of biological samples. The pharmacokinetic parameters of Celebrex, CXB-NPs and HA-CXB-NPs were determined as: area under the curve, 1,855.98 ± 346.59, 1,908.00 ± 1,130.24 and 2,164.48 ± 657.47 h·ng/ml; peak concentration, 261.08 ± 113.26, 261.12 ± 94.67 and 263.34 ± 151.78 μg/L; time to peak concentration, 2.00 ± 1.22, 4.00 ± 0.00 and 3.60 ± 0.89 h; half-life, 4.39 ± 1.26, 2.33 ± 0.94 and 4.92 ± 3.13 h; relative bioavailability, 102.80 ± 49.62 and 116.63 ± 25.55%. The validated method was successfully applied to the pharmacokinetic study of celecoxib in cynomolgus monkey, after i.g. administration. The preparation of the nanoparticles of celecoxib and the modification of hyaluronic acid on the surface of nanoparticles could improve the bioavailability and prolong the circulation of celecoxib in vivo, which could lay the foundation for further development of celecoxib nanoparticles.

Plasma-Droplet Reaction Systems: A Direct Mass Spectrometry Approach for Enhanced Characterization of Lipids at Multiple Isomer Levels.

Neutral triacylglyceride (TG) lipids are critical in cellular function, signaling, and energy storage. Multiple molecular pathways control TG structure via nonselective routes making them structurally complex and analytically challenging to characterize. The presence of C=C bond positional isomers exacerbates this challenge as complete structural elucidation is not possible by conventional tandem mass spectrometric methods such as collision-induced dissociation (CID), alone. Herein, we report a custom-made coaxial contained-electrospray ionization (ESI) emitter that allows the fusion of plasma discharge with charged microdroplets during electrospray (ES). Etched capillaries were incorporated into this contained-ES emitter, facilitating the generation of reactive oxygen species (ROS) at low (3 kV) ESI voltages and allowing stable ESI ion signal to be achieved at an unprecedented high (7 kV) spray voltage. The analytical utility of inducing plasma discharge during electrospray was investigated using online ionization of neutral TGs, in situ epoxidation of unsaturation sites, and C=C bond localization via conventional CID mass spectrometry. Collisional activation of the lipid epoxide generated during the online plasma-droplet fusion experiment resulted in a novel fragmentation pattern that showed a quadruplet of diagnostic ions for confident assignment of C=C bond positions and subsequent isomer differentiation. This phenomenon enabled the identification of a novel TG lipid, composed of conjugated linoleic acid, that is isomeric with two other TG lipids naturally found in extra virgin olive oil. To validate our findings, we analyzed various standards of TG lipids, including triolein, trilinolein, and trilinolenin, and isomeric mixtures in the positive-ion mode, each of which produced the expected quadruplet diagnostic fragment ions. Further validation was obtained by analyzing standards of free fatty acids expected from the hydrolysis of the TG lipids in the negative-ion mode, together with isomeric mixtures. The chemistry governing the gas-phase fragmentation of the lipid epoxides was carefully elucidated for each TG lipid analyzed. This comprehensive shotgun lipidomic approach has the potential to impact biomedical research since it can be accomplished on readily available mass spectrometers without the need for instrument modification.

The UHPLC-MS/MS determination of ostarine in food supplements for athletes

Ostarine, also called enobosarm or MK-2866, is one of the Selective Androgen Receptor Modulators (SARMs). A number of preclinical data suggest the beneficial use of these substances to increase muscle mass growth and bone density. Unlike anabolic steroids, they have suppressed androgenic side effects and are therefore thought to be useful in the treatment of health conditions associated with insufficient muscle growth. For the same reason, substances from the SARMs group are abused in sports and bodybuilding. They have been added on the World Anti-Doping Agency (WADA) list in 2008 and their use in sports is prohibited. In many countries, products containing these substances are available through a number of e-shops offering supplements for athletes. The positive effects on muscle growth and body fat loss and the absence of side effects are emphasized. Because SARMs are banned in sports and are not approved for any therapeutic use, they are marketed as “Research Only”. However, the products are sill quite popular among athletes. As with anabolic steroids, there are a variety of dosing regimens for both beginners and advanced athletes, which end up recommending a combination of three to four SARMs over several weeks. Although more extensive clinical data are lacking, case studies are beginning to appear describing liver damage or heart attack in people taking ostarine or other SARMs products. However, due to the lack of clinical data, no positive or negative effect on the organism is objectively confirmed. For these reasons, it is very appropriate to have an analytical method available that allows the rapid and correct determination of the ingredients in preparations that have ostarine declared in the list of ingredients. Equally important is the possibility of identifying this substance in preparations that should not contain any active substance and appear as purely natural products. For the ostarine determination a simple and fast UHPLC-MS/MS method was developed. For extraction of ostarine the content of the studied capsule was weighted and transferred to a volumetric flask, which was then made up with methanol to 100 mL. The mixture was shaken for one hour. After filtration 10 μL of extract was transferred to a new flask and made up to 5 mL with 10% methanol with 0,1% acetic acid and 10 mM ammonium acetate. This solution was used for UHPLC-MS/MS analysis. An UHPLC system Infinity 1290 and chromatographic column Eclipse Plus C18 (50 × 2.1 mm; 1.8 μm) (both Agilent Technologies, USA) were used for the separation. 0.1% acetic acid and 10 mM ammonium acetate in water and methanol were chosen as mobile phases for gradient elution. For the MS/MS detection 6460 Triple Quadrupole mass spectrometer (Agilent Technologies, USA) with negative ESI ionization were used. For dynamic MRM mode two transitions of m/z were chosen for the detection: 388.1 → 117.9 and 269.0. Limit of detection was 0.2 ng/mL and limit of quantification 0.5 ng/mL. The method was used for quantitation of ostarine in dietary supplements for athletes. The content of ostarine in one capsule ranged from 10 to 15 mg and did not differ from the quantity indicated on the label. The described method is suitable for the determination of ostarine and can be used in toxicological and forensic practice.

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.

Comparative Pharmacokinetics of Cinobufacini Capsule and Injection by UPLC-MS/MS.

Cinobufacini capsule and injection are two different formulations from the same source, obtained from the extraction of the skin of Bufo bufo gargarizans Cantor, which have been approved by the Chinese State Food and Drug Administration (CFDA) for the treatment of various cancers. Our previous study has found that the cinobufacini capsule and injection exhibited different anticancer effects, but their different pharmacokinetic behaviors, which could give a cause of that, have never been reported. So a sensitive and selective method for the simultaneous quantitation of 13 compounds in the rat plasma, including bufothionine, hellebrigenin, bufalin, gamabufotalin, telocinobufagin, cinobufagin, arenobufagin, cinobufotalin, desacetylcinobufotalin, bufotalin, pseudobufarenogin, resibufogenin, and desacetylcinobufagin, was established by using the Agilent 6460 mass spectrometer equipped with an ESI ion source in a multiple-reaction monitoring (MRM) mode. Chromatographic analysis was accomplished in 6 min by using an Agilent SB-C18 column and a mobile phase consisting of 0.1% formic acid in water and acetonitrile in an optimized gradient program at a flow rate of 0.3 ml/min. The correlation coefficients (r) of all analytes ranged from 0.9967 to 0.9996, while their lower limits of quantification ranged from 0.20 to 4.84 ng/ml. The method has been fully verified and applied for the pharmacokinetic difference study of the Cinobufacini capsule and injection in rats. The results showed that nine components could be quantitated in rat plasma samples after the administration of the cinobufacini capsule, while only bufothionine, bufalin, arenobufagin, and pseudobufarenogin could be detected in the cinobufacini injection group. Their pharmacokinetic studies indicated telocinobufagin, bufalin, desacetylcinobufagin, and arenobufagin were predicted as the potential active substances of the Cinobufacini capsule, while bufothionine was considered as a major ingredient in the cinobufacini injection due to its relatively high blood drug exposure. Also, the AUC of the nine components in cinobufacini capsule groups with three different doses showed a similar trend with significant differences, and the exposure increased with the increase of the dose. The pharmacokinetic characteristics of all major ingredients in cinobufacini capsules and injection were of wide variation, which could be used to explain differences in the efficacy of the cinobufacini capsule and injection and infer the pharmacodynamic ingredients of various cinobufacini preparations.

Further transformation of the primary ozonation products of tramadol- and venlafaxine N-oxide: Mechanistic and structural considerations

Ozonation has been used to effectively remove micropollutants from the secondary effluent in several wastewater treatment plants. It is known that ozonation transforms tertiary amine compounds into their respective N-oxides, however in an earlier study a mass balance could not be closed at elevated ozone concentrations, leading to the assumption that more ozonation products are possible. This study was conducted to elucidate which (hitherto unknown) ozonation products can be formed from venlafaxine and tramadol when ozonating wastewater. Ozonation experiments were performed with tramadol and venlafaxine N-oxide in two different set-ups. Both tramadol- and venlafaxine N-oxide degraded during ozonation in pure (deionized) water in both semi-continuous and batch mode ozonation set-ups. 13 and 17 new transformation products were detected from tramadol- and venlafaxine N-oxide respectively, using high resolution mass spectrometry with ESI(+) ionization. Empirical chemical formulas were proposed based on the determination of the exact masses and interpretation of the product ion spectra. These transformation products result from the addition of one to three oxygen atoms and removal of C, –CH2, C2H2, C3H6, etc., from the parent molecule, respectively. Quenching experiments suggested that most of the transformation products originated from the direct reaction with ozone (eight for tramadol N-oxide and ten for venlafaxine N-oxide), whereas fewer products originated from the reaction with OH radicals (three for tramadol N-oxide and three for venlafaxine N-oxide). Reaction mechanisms and chemical structures of products are proposed, based on the available active sites and past literature on ozone reaction mechanisms. The experimental results are compared to theory and literature on ozone reactive sites and ozone reaction mechanisms. All in all this shows that there can be multiple ozonation products, and ozonation pathways can be complex, even if initially only one ozonation product is formed.

Rapid Identification of Constituents in Cephalanthus tetrandrus (Roxb.) Ridsd. et Badh. F. Using UHPLC-Q-Exactive Orbitrap Mass Spectrometry.

Cephalanthus tetrandrus (Roxb.) Ridsd. et Badh. F. (CT) belongs to the Rubiaceae family. Its dried leaves are widely used in traditional Chinese medicine to treat enteritis, dysentery, toothache, furuncles, swelling, traumatic injury, fracture, bleeding, and scalding. In order to further clarify the unknown chemical composition of CT, a rapid strategy based on UHPLC-Q-exactive orbitrap was established for this analysis using a Thermo Scientific Hypersil GOLDTM aQ (100 mm × 2.1 mm, 1.9 µm) chromatographic column. The mobile phase was 0.1% formic acid water–acetonitrile, with a flow rate of 0.3 mL/min and injection volume of 2 µL; for mass spectrometry, an ESI ion source in positive and negative ion monitoring modes was adopted. A total of 135 chemicals comprising 67 chlorogenic acid derivatives, 48 flavonoids, and 20 anthocyanin derivatives were identified by comparing the mass spectrum information with standard substances, public databases, and the literature, which were all discovered for the first time in this plant. This result broadly expands the chemical composition of CT, which will contribute to understanding of its effectiveness and enable quality control.

Comprehending Cardiac Dysfunction by Oxidative Stress: Untargeted Metabolomics of In Vitro Samples.

Cardiovascular diseases (CVDs) are noncommunicable diseases known for their complex etiology and high mortality rate. Oxidative stress (OS), a condition in which the release of free radical exceeds endogenous antioxidant capacity, is pivotal in CVC, such as myocardial infarction, ischemia/reperfusion, and heart failure. Due to the lack of information about the implications of OS on cardiovascular conditions, several methodologies have been applied to investigate the causes and consequences, and to find new ways of diagnosis and treatment as well. In the present study, cardiac dysfunction was evaluated by analyzing cells’ alterations with untargeted metabolomics, after simulation of an oxidative stress condition using hydrogen peroxide (H2O2) in H9c2 myocytes. Optimizations of H2O2 concentration, cell exposure, and cell recovery times were performed through MTT assays. Intracellular metabolites were analyzed right after the oxidative stress (oxidative stress group) and after 48 h of cell recovery (recovery group) by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in positive and negative ESI ionization mode. Significant alterations were found in pathways such as “alanine, aspartate and glutamate metabolism”, “glycolysis”, and “glutathione metabolism”, mostly with increased metabolites (upregulated). Furthermore, our results indicated that the LC-MS method is effective for studying metabolism in cardiomyocytes and generated excellent fit (R2Y > 0.987) and predictability (Q2 > 0.84) values.

Quantification of ceftazidime/avibactam in human plasma and dried blood spots: Implications on stability and sample transport

Ceftazidime is an established third-generation cephalosporin antibiotic frequently administered to intensive care patients. To overcome drug resistance of pathogens, it is combined with the newly developed non-ß-lactam ß-lactamase inhibitor avibactam under the brand name Zavicefta®. To facilitate therapeutic drug monitoring (TDM), we developed a method for the simultaneous quantification of these substances by LC-MS/MS. A problem for TDM is the low stability of the analytes in plasma, requiring transport times of less than 6 h at 23 °C. Thus, we evaluated dried blood spots (DBS) as matrix for better stability. For both analytes, stable isotope labelled internal standards were applied. Plasma samples were prepared by protein precipitation, DBS by liquid extraction. The chromatographic separation took place on a polar-modified C18 column, and detection was achieved by tandem mass spectrometry with ESI ionization in positive mode for ceftazidime and negative mode for avibactam. Calibration was linear in the ranges of 5 – 100 µg/mL for ceftazidime and 1.25 – 25 µg/mL for avibactam in plasma and 2.5 – 50 µg/mL and 0.625 – 12.5 µg/mL in DBS, respectively. Precision was better than 7 % and accuracy better than 10% for plasma as well as for DBS. The stability of ceftazidime and avibactam was better in DBS than in plasma or full blood, extending maximal transport times at 23 °C from 6 h in plasma or full blood to 24 h for DBS samples. However, robust estimation of plasma concentrations from DBS measurements requires validation by future clinical studies.