Gas Chromatography–Mass Spectrometry Based Approach for the Determination of Methionine-Related Sulfur-Containing Compounds in Human Saliva

Gas chromatography–mass spectrometry technique (GC-MS) is mainly recognized as a tool of first choice when volatile compounds are determined. Here, we provide the credible evidence that its application in analysis can be extended to non-volatile sulfur-containing compounds, to which methionine (Met), homocysteine (Hcy), homocysteine thiolactone (HTL), and cysteine (Cys) belong. To prove this point, the first method, based on GC-MS, for the identification and quantification of Met-related compounds in human saliva, has been elaborated. The assay involves simultaneous disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP) and acetonitrile (MeCN) deproteinization, followed by preconcentration by drying under vacuum and treatment of the residue with a derivatizing mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA), and trimethylchlorosilane (TMCS). The validity of the method was demonstrated based upon US FDA recommendations. The assay linearity was observed over the range of 0.5–20 µmol L⁻¹ for Met, Hcy, Cys, and 1–20 µmol L⁻¹ for HTL in saliva. The limit of quantification (LOQ) equals 0.1 µmol L⁻¹ for Met, Hcy, Cys, while its value for HTL was 0.05 µmol L⁻¹. The method was successfully applied to saliva samples donated by apparently healthy volunteers (n = 10).     

Development of a Sensitive Screening Method for Simultaneous Determination of Nine Genotoxic Nitrosamines in Active Pharmaceutical Ingredients by GC-MS

A worldwide crisis with nitrosamine contamination in medical products began in 2018. Therefore, trace-level analysis of nitrosamines is becoming an emerging topic of interest in the field of quality control. A novel GC-MS method with electron ionization and microextraction was developed and validated for simultaneous determination of nine carcinogenic nitrosamines (NDMA, NMEA, NDEA, NDBA, NMOR, NPYR, NPIP, NDPA, and N-methyl-npz) in active pharmaceutical ingredients (APIs): cilostazol, sunitinib malate, and olmesartan medoxomil. The method was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, demonstrating good linearity in the range of LOQ up to 21.6 ng/mL (120% of specification limit). The limits of detection for the nine nitrosamines were determined to be in the range 0.15–1.00 ng/mL. The developed trace level GC-MS method turned out to be specific, accurate, and precise. The accuracy of all the tested APIs ranged from 94.09% to 111.22% and the precision evaluated by repeatability, intermediate precision, and system precision was RSD ≤ 7.65%. Nitrosamines were not detected in cilostazol and sunitinib, whereas in olmesartan medoxomil NDEA was detected at the level of LOQ. The novel protocol was successfully applied for nitrosamines determination in selected APIs and can be used for the routine quality control of APIs under Good Manufacturing Practices rules, ensuring the safety and effectiveness of pharmaceutical products.     

Pyrrolidinyl Synthetic Cathinones α-PHP and 4F-α-PVP Metabolite Profiling Using Human Hepatocyte Incubations

For more than ten years, new synthetic cathinones (SCs) mimicking the effects of controlled cocaine-like stimulants have flooded the illegal drug market, causing numerous intoxications and fatalities. There are often no data on the pharmacokinetics of these substances when they first emerge onto the market. However, the detection of SC metabolites is often critical in order to prove consumption in clinical and forensic settings. In this research, the metabolite profile of two pyrrolidinyl SCs, α-pyrrolidinohexaphenone (α-PHP) and 4′′-fluoro-α-pyrrolidinovalerophenone (4F-α-PVP), were characterized to identify optimal intake markers. Experiments were conducted using pooled human hepatocyte incubations followed by liquid chromatography–high-resolution tandem mass spectrometry and data-mining software. We suggest α-PHP dihydroxy-pyrrolidinyl, α-PHP hexanol, α-PHP 2′-keto-pyrrolidinyl-hexanol, and α-PHP 2′-keto-pyrrolidinyl as markers of α-PHP use, and 4F-α-PVP dihydroxy-pyrrolidinyl, 4F-α-PVP hexanol, 4F-α-PVP 2′-keto-pyrrolidinyl-hexanol, and 4F-α-PVP 2′-keto-pyrrolidinyl as markers of 4F-α-PVP use. These results represent the first data available on 4F-α-PVP metabolism. The metabolic fate of α-PHP was previously studied using human liver microsomes and urine samples from α-PHP users. We identified an additional major metabolite (α-PHP dihydroxy-pyrrolidinyl) that might be crucial for documenting exposure to α-PHP. Further experiments with suitable analytical standards, which are yet to be synthesized, and authentic specimens should be conducted to confirm these results.     

Exploratory Metabolomic Analysis Based on Reversed-Phase Liquid Chromatography–Mass Spectrometry to Study an In Vitro Model of Hypoxia-Induced Metabolic Alterations in HK-2 Cells

Oxygen deficiency in cells, tissues, and organs can not only prevent the proper development of biological functions but it can also lead to several diseases and disorders. In this sense, the kidney deserves special attention since hypoxia can be considered an important factor in the pathophysiology of both acute kidney injury and chronic kidney disease. To provide better knowledge to unveil the molecular mechanisms involved, new studies are necessary. In this sense, this work aims to study, for the first time, an in vitro model of hypoxia-induced metabolic alterations in human proximal tubular HK-2 cells because renal proximal tubules are particularly susceptible to hypoxia. Different groups of cells, cultivated under control and hypoxia conditions at 0.5, 5, 24, and 48 h, were investigated using untargeted metabolomic approaches based on reversed-phase liquid chromatography–mass spectrometry. Both intracellular and extracellular fluids were studied to obtain a large metabolite coverage. On the other hand, multivariate and univariate analyses were carried out to find the differences among the cell groups and to select the most relevant variables. The molecular features identified as affected metabolites were mainly amino acids and Amadori compounds. Insights about their biological relevance are also provided.     

Magnetic Core–Shell Molecularly Imprinted Nano-Conjugates for Extraction of Antazoline and Hydroxyantazoline from Human Plasma—Material Characterization,Theoretical Analysis and Pharmacokinetics

The aim of this study was to develop magnetic molecularly imprinted nano-conjugate sorbent for effective dispersive solid phase extraction of antazoline (ANT) and its metabolite, hydroxyantazoline (ANT-OH) in analytical method employing liquid chromatography coupled with mass spectrometry method. The core–shell material was characterized in terms of adsorption properties, morphology and structure. The heterogeneous population of adsorption sites towards ANT-OH was characterized by two K_d and two B_max values: K_d (1) = 0.319 µg L⁻¹ and B_max (1) = 0.240 μg g⁻¹, and K_d (2) = 34.6 µg L⁻¹ and B_max (2) = 5.82 μg g⁻¹. The elemental composition of magnetic sorbent was as follows: 17.55, 37.33, 9.14, 34.94 wt% for Si, C, Fe and O, respectively. The extraction protocol was optimized, and the obtained results were explained using theoretical analysis. Finally, the analytical method was validated prior to application to pharmacokinetic study in which the ANT was administrated intravenously to three healthy volunteers. The results prove that the novel sorbent could be useful in extraction of ANT and ANT-OH from human plasma and that the analytical strategy could be a versatile tool to explain a potential and pharmacological activity of ANT and ANT-OH.     

Stir Bar Sorptive Extraction: A New Quantitative and Comprehensive Sampling Technique for Determination of Chemical Signal Profiles from Biological Media

Various applications of a novel sampling procedure in chemical ecology are outlined. The stir bar extraction method features the analytical reproducibility needed in recording the analytical profiles of volatile and semivolatile components of biological mixtures. This methodology has been demonstrated here through the examples of small volume urine samples, glandular tissue volatiles, and the air blown through animal cages. Its analytical merits are compared with those of the previously established purge-and-trap (dynamic headspace) technique.     

Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.     

Determination of Polycyclic Aromatic Hydrocarbons in F-15J,C-130H,and F-4EJ Aircraft Exhaust

There are occupational health concerns at Japan Air Self-Defense Force bases in regard to the exposure of military flightline personnel to carcinogens in aircraft emissions, such as polycyclic aromatic hydrocarbons (PAHs). To characterize the PAHs in military aircraft emissions from different types of engines, aerosol and gas samples were separately collected downwind from aircraft with a turboprop engine (C-130H), turbojet (F-4EJ), and turbofan (F-15J). The gas-phase PAHs were determined by gas chromatography coupled to mass spectrometry and the aerosol-phase PAHs were determined by high-performance liquid chromatography with fluorescence detection. The F-4EJ engine was a source of naphthalene vapor and aerosol PAHs, including carcinogens such as chrysene, benzo (b) fluoranthene, benzo (k) fluoranthene, benzo (a) pyrene, dibenzo (a,h) anthracene, benzo (ghi) perylene, and indeno (1,2,3-cd) pyrene. These heavier (five and six-ring) PAHs were also included in the emissions from the F-15J with its newer, high-temperature F-100 turbofan engine, but the concentrations were approximately one-tenth of those in the F-4EJ. In contrast to these fighter aircraft, the C-130H was found to be a significant aerosol source of the lighter, three-ring (phenanthrene and anthracene) and four-ring (fluoranthene and pyrene) PAHs, but not the heavier ones. These results demonstrate that various aircraft are sources of PAHs in the military flightline environment.     

Adenylate Kinase 4—A Key Regulator of Proliferation and Metabolic Shift in Human Pulmonary Arterial Smooth Muscle Cells via Akt and HIF-1α Signaling Pathways

Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.     

Structures,practical sources (gymnosperm seeds), gas‐liquid chromatographic data (equivalent chain lengths), and mass spectrometric characteristics of all‐cis Δ5‐olefinic acids

In view of the increasing interest of lipid researchers in the biological effects of all‐cis Δ5‐unsaturated polymethylene‐interrupted fatty acids (Δ5‐UPIFA) from vegetable origin, this paper is concerned with their occurrence in practical sources (gymnosperm seeds), structures, and identification by gas‐liquid chromatography (GLC) and mass spectrometry (MS). The use of equivalent chain lengths (ECL) determined by calculations based on available standards, in close agreement with data determined with easily available commercial gymnosperm seeds specific for each Δ5‐UPIFA, allows identification of all Δ5‐UPIFA. These tentative identifications are supported by GLC‐MS of the appropriate (4, 4‐dimethyloxazoline and picolinyl ester) derivatives. ECL are particularly useful to identify Δ5‐UPIFA in tissue lipids from animals experimentally fed oils containing these acids, with no interference with polyunsaturated fatty acids of endogenous origin.     

Determination of Ni and Zn in an Oil Matrix using Schiff Base‐Assisted Extraction Followed by a Flame Atomic Absorption Spectrometer: A Simple Strategy to Determine Ni and Zn

In the present work, a new and simple Schiff base‐assisted extraction strategy for Ni and Zn from an edible oil matrix with subsequent determination using a flame atomic absorption spectrometer was suggested. According to the green approach, laborious sample‐pretreatment procedures were eliminated via complexation of the analytes with N,N′‐bis(4‐methoxysalycylidene)‐2‐hydroxy‐1,3‐propanediamine (4MSHP) and transferred from the oil phase to the aqueous phase. The complexation properties of 4MSHP, Ni, and Zn were investigated using UV–vis spectrophotometry. The experimental conditions that affect the extraction efficiency were optimized using central composite design. The optimum conditions for the extraction of Ni and Zn were as follows: a volume to oil mass ratio of 0.83 to 1.31 mL g^?1 of 4MSHP solution; 62.3‐ and 50.6‐min, stirring time; 27.3 and 31.1 °C, temperature, respectively. The detection limits (3s_bm^?1) were 0.41 μg g^?1 for Ni and 0.16 μg g^?1 for Zn. Validation of the suggested work was performed by the analysis of organometallic standard‐doped n‐hexane solutions as certified reference materials under the optimum experimental conditions. The recovery percentages were warranted the accuracy and found as 98.2 ± 1.8% for Ni and 99.8 ± 1.2% for Zn. In addition, relative SD values were below 5% for both the analytes. The Student's t‐test showed that there was no significant difference between the found and doped amount of analytes at 95% confidence level. The features such as the detection technique, cheapness, eco‐friendly solvent usage, and practicality were better compared to the literature.     

Comparative Investigation of the Volatile Urinary Profiles in Different <Emphasis Type="Italic">Phodopus</Emphasis> Hamster Species

Stir bar sorptive extraction method was used for investigation of the urinary volatile profiles in male and female Phodopus campbelli and Phodopus sungorus hamsters. Additionally, female Phodopus roborowsky urinary profiles were characterized. A quantitative analytical approach allowed comparisons of 17 selected compounds in urine. Results showed that campbelli and sungorus species show similar urinary volatile profiles for males and females. Differences appeared only in concentrations. Several unique compounds, such as pyrazine derivatives, were found to be gender- and age-specific. P. roborowsky females exhibited a completely different urinary volatile profile from campbelli and sungorus females, featuring a unique set of substituted quinoxalines.