Search Results (87)

Background: Fatty acids (FAs) represent a ubiquitous class of nonpolar alkyl carboxylate metabolites with diverse biological functions. Nutrition, metabolism, and endogenous and exogenous stress influence the overall FA metabolic status and transport via the bloodstream. FAs esterified in lipids are of particular interest, as they represent promising biomarkers of pathological diseases and nutritional status. Methods: Here, we report a validated gas chromatographic-mass spectrometric (GC-MS) method for the quantitative analysis of 32 FAs exclusively bound in esterified lipids. The developed sample preparation protocol comprises three steps using only 5 µL of human serum for Folch extraction, sodium methoxide-catalyzed transesterification in tert-butyl methyl ether, and re-extraction in isooctane prior to a quantitative GC-MS analysis with positive ion chemical ionization (PICI) and selected ion monitoring (SIM). Results: The base-catalyzed transmethylation step was studied for 14 lipid classes and was found to be efficient under mild conditions for all major esterified lipids but not for free FAs, lipid amides, or sphingolipids. To minimize matrix effects and instrument bias, internal fatty acid trideuteromethyl esters (D3-FAME) standards were prepared through isotope-coded derivatization with D3-labeled methylchloroformate/methanol medium mixed with each transmethylated serum extract for the assay. The method was validated according to FDA guidelines and evaluated by analyzing NIST SRM 2378 Serum 1 and sera from three healthy donors. Conclusions: The measured quantitative FA values are consistent with the reference data of SRM 2378, and they demonstrate the application potential of the described method for general FA analysis in esterified lipids as a novel complementary tool for lipidomics, as well as for the analysis of membrane FAs in dry blood spots and red blood cells. Full article

Mass spectrometry (MS) is the only instrumental analytical technology that utilizes unique properties of matter, that is, its mass (m) and electrical charge (z). In the magnetic and/or electric fields of mass spectrometers, electrically charged native or chemically modified (millions) endogenous and (thousands) exogenous substances, the analytes, are separated according to their characteristic mass-to-charge ratio (m/z) values. Mass spectrometers coupled to gas chromatographs (GC) or liquid chromatographs (LC), the so-called hyphenated techniques, i.e., GC-MS and LC-MS, respectively, enable reliable determination of the concentration of analytes in complex biological samples such as plasma, serum, and urine. A particular technology is represented by inductively coupled plasma-mass spectrometry (ICP-MS), which is mainly used for the analysis of metal ions. The highest analytical accuracy is reached by using mass spectrometers with high mass resolution (HR) or by tandem mass spectrometers, as it can be realized with quadrupole-type instruments, such as GC-MS/MS and LC-MS/MS, in combination with stable-isotope labeled analytes that serve as internal standards, like a standard weight in scales. GC-MS belongs to the oldest and most advanced instrumental analytical technology. From the very beginning, GC-MS found broad application in basic and applied research sciences. GC-MS has played important roles in discovering biochemical pathways, exploring underlying mechanisms of disease, and establishing new evidence-based pharmacological therapy. In this article, we make an inventory of the use of instrumental mass spectrometry in the life sciences and attempt to provide a perspective study on the future of analytical mass spectrometry in clinical science, mainly focusing on GC-MS and LC-MS. We used information freely available in the scientific database PubMed (retrieved in August–November 2024). Specific search terms such as GC-MS (103,000 articles), LC-MS (113,000 articles), and ICP-MS (14,000 articles) were used in the Title/Abstract in the “PubMed Advanced Search Builder” including filters such as search period (1970–2024). In total, around 103,000 articles on GC-MS, 113,000 articles on LC-MS (113,000), and 14,000 articles on ICP-MS were found. In the period 1995–2023, the yearly publication rate accounted for 3042 for GC-MS articles and 3908 for LC-MS articles (LC-MS/GC-MS ratio, 1.3:1). Our study reveals that GC-MS/MS, LC-MS/MS, and their high-resolution variants are indispensable instrumentations in clinical science including clinical pharmacology, internal and forensic medicine, and doping control. Long-tradition manufacturers of analytical instruments continue to provide increasingly customer-friendly GC-MS and LC-MS apparatus, enabling fulfillment of current requirements and needs in the life sciences. Quantitative GC-MS and GC-MS/MS methods are expected to be used worldwide hand in hand with LC-MS/MS, with ICP-MS closing the gap left for metal ions. The significance of analytical chemistry in clinical science in academia and industry is essential. Full article

Background: Our study presented a novel LC-MS/MS method for the simultaneous quantification of α-tocopherol (α-TOH) and its phase II metabolites, α-13′-COOH and α-13′-OH, in human serum using deuterium-labeled internal standards (d₆-α-TOH, d₆-α-13′-COOH, d₆-α-13′-OH). Methods: The method addresses the analytical challenge posed by the significantly different concentration ranges of α-TOH (µmol/L) and its metabolites (nmol/L). Previous methods quantified these analytes separately, which caused an increase in workflow complexity. Results: Key features include the synthesis of stable isotope-labeled standards and the use of a pentafluorophenyl-based core-shell chromatography column for baseline separation of both α-TOH and its metabolites. Additionally, solid phase extraction (SPE) with a HybridSPE^® material provides a streamlined sample preparation, enhancing analyte recovery and improving sensitivity. By utilizing deuterium-labeled standards, the method compensates for matrix effects and ion suppression. This new approach achieves precise and accurate measurements with limits of detection (LOD) and quantification (LOQ), similar to previous studies. Calibration, accuracy, and precision parameters align well with the existing literature. Conclusions: Our method offers significant advantages in the simultaneous analysis of tocopherol and its metabolites despite concentration differences spanning up to three orders of magnitude. In contrast to earlier studies, which required separate sample preparations and analytical techniques for tocopherol and its metabolites, our approach streamlines this process. The use of a solid-phase extraction procedure allows for parallel sample preparation. This not only enhances efficiency but also significantly accelerates pre-analytical workflows, making the method highly suitable for large-scale studies. Full article

A new method is presented for the simultaneous determination of 13 multiclass pesticides along with glyphosate. The multiclass pesticides were extracted by creating a soil slurry with 2% ethanol in water (v/v), and then, applying direct-immersion solid-phase microextraction (DI-SPME) with a new type of semi-disposable SPME fiber configuration called LC-Tips. The fibers were then retroextracted to ethanol, and aqueous ammonia was added to the slurry to extract glyphosate. Derivatization of the glyphosate extract was accomplished with a mixture of trifluoroacetic anhydride and trifluoroethanol, after which the reaction mixture was dried and resuspended with the SPME ethanol extract. To this, a mixture of analyte protectants was added, and it was analyzed by GC-MS/MS in multiple-reaction-monitoring mode. All analytes showed a coefficient of determination greater than 0.95 in the 0.1–100 µg/kg calibrated range, and the limits of detection were between 0.1 and 1 µg/kg, except for glyphosate, which was 0.01 µg/kg. The method shows relatively high replicate relative standard deviation (as much as 37% for five extractions at 20 µg/kg), but the isotopically labeled internal standard was effective at mitigating this effect for some analytes. Full article

Τhyreostats (TSs) are veterinary drugs used in livestock farming for fattening. Their administration is banned in the European Union since 1981, and their monitoring for food quality and safety control requires sensitive and confirmatory methods. The present study describes the development and validation of a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of 2-thiouracil (TU), 6-methyl-2-thiouracil (MTU), 6-propyl-2-thiouracil (PTU), 6-phenyl-2-thiouracil (PhTU), tapazole (TAP), and 2-mercaptobenzimidazole (MBI) in bovine muscle tissues. Investigation of the retention mechanism of the six analytes on the selected amide-based stationary phase showed that hydrophilic partition was the dominant interaction. The sample preparation included extraction with ACN/H₂O (80/20), followed by dispersive solid-phase extraction (d-SPE) with C18 sorbent and hexane partitioning. The method was validated according to European guidelines using internal standards, including isotopically labelled ones. The method’s LODs ranged between 2.8 ng g⁻¹ (6-phenyl-2-thiouracil) and 4.1 ng g⁻¹ (2-thiouracil). Application of the proposed method to 48 bovine tissue samples showed non-detectable results. Full article

Passive smoking from environmental tobacco smoke not only increases the risk of lung cancer and cardiovascular disease but may also be a stressor triggering neuropsychiatric and other disorders. To prevent these diseases, understanding the relationship between passive smoking and stress is vital. In this study, we developed a simple and sensitive method to simultaneously measure nicotine (Nic) and cotinine (Cot) as tobacco smoke exposure biomarkers, and cortisol (CRT), serotonin (5-HT), melatonin (MEL), dopamine (DA), and oxytocin (OXT) as stress-related biomarkers. These were extracted and concentrated from saliva by in-tube solid-phase microextraction (IT-SPME) using a Supel-Q PLOT capillary as the extraction device, then separated and detected within 6 min by liquid chromatography–tandem mass spectrometry (LC−MS/MS) using a Kinetex Biphenyl column (Phenomenex Inc., Torrance, CA, USA). Limits of detection (S/N = 3) for Nic, Cot, CRT, 5-HT, MEL, DA, and OXT were 0.22, 0.12, 0.78, 0.39, 0.45, 1.4, and 3.7 pg mL⁻¹, respectively, with linearity of calibration curves in the range of 0.01–25 ng mL⁻¹ using stable isotope-labeled internal standards. Intra- and inter-day reproducibilities were under 7.9% and 14.6% (n = 5) relative standard deviations, and compound recoveries in spiked saliva samples ranged from 82.1 to 106.6%. In thirty nonsmokers, Nic contents positively correlated with CRT contents (R² = 0.5264, n = 30), while no significant correlation was found with other biomarkers. The standard deviation of intervals between normal beats as the standard measure of heart rate variability analysis negatively correlated with CRT contents (R² = 0.5041, n = 30). After passive smoke exposure, Nic levels transiently increased, Cot and CRT levels rose over time, and 5-HT, DA, and OXT levels decreased. These results indicate tobacco smoke exposure acts as a stressor in nonsmokers. Full article

Mass spectrometry (MS) is a widely used analytical technique including medical diagnostics, forensic toxicology, food and water analysis. The gold standard for quantifying compounds involves using stable isotope-labeled internal standards (SIL-IS). However, when these standards are not commercially available, are prohibitively expensive, or are extremely difficult to synthesize, alternative external quantification techniques are employed. We hereby present a novel, convenient and cheap quantification approach—quantification via post column infusion (PCI). As a proof of concept, we demonstrated PCI quantification for the immunosuppressant tacrolimus in whole blood using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The validation results met the criteria according to the guideline on bioanalytical method validation of the European Medicine Agency (EMA), achieving imprecisions and inaccuracies with coefficient of variation and relative bias below 15%. Anonymized and leftover whole blood samples from immunosuppressed patients receiving tacrolimus were used for method comparison (PCI quantification vs. conventional internal standard (IS) quantification). Both methods showed strong agreement with a Pearson correlation coefficient of r = 0.9532. This novel PCI quantification technique (using the target analyte itself) expands the quantification options available in MS, providing reliable results, particularly when internal standards are unavailable or unaffordable. With the current paper, we aim to demonstrate that our innovative PCI technique has great potential to overcome practical issues in quantification and to provide guidance on how to incorporate PCI in existing or new LC-MS methods. Moreover, this study demonstrated a more convenient method for correcting matrix effects in comparison to alternative PCI techniques. Full article

The abuse and irrational use of tetracyclines (TCs) in human medicine and animal husbandry has become a serious concern, affecting the ecological environment and human health. The aim of this study was to develop a sensitive and selective method using fully automatic solid-phase extraction coupled with ultra-performance liquid chromatography–tandem mass spectrometry for the determination of twelve TCs in water. Four isotope-labeled internal standards for TCs were used to correct matrix effects. Several parameters affecting extraction efficiency were systematically optimized, and the optimum experimental conditions found were 1.0 L water sample with 0.5 g/L Na₂EDTA (pH 3.0) extracted and enriched by CNW HLB cartridge and eluted by 4 mL of acetone:methanol (v/v, 1:1). The enrichment factors were up to 798−1059 but only requiring about 60 min per six samples. Under the optimized conditions, the linearity of the method ranged from 0.2 to 100 μg/L for 12 TCs, the detection limits were as low as 0.01−0.15 ng/L, and the recoveries were in the range of 70%–118%, with relative standard deviations less than 15%. The developed method can be successfully utilized for the determination of 12 TCs in pure water, tap water, river water, and mariculture seawater. In summary, three and six TCs were detected in river water and mariculture seawater, respectively, with total concentrations of 0.074–0.520 ng/L (mean 0.248 ng/L) and 0.792–58.369 ng/L (12.629 ng/L), respectively. Tetracycline (TC) and oxytetracycline (OTC) were the dominant TCs in river water, while doxytetracycline (DXC) and OTC were dominant in mariculture seawater. Full article

Accurate measurement of steroid hormones is crucial to elucidate new mechanisms of action and diagnose steroid metabolism-related diseases. This study presents a simple, sensitive, and automated analytical method for nine representative steroid hormones. The method involves on-line coupling of in-tube solid-phase microextraction (IT-SPME) with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The steroid hormones were extracted and enriched on a Supel-Q PLOT capillary column using IT-SPME. Subsequently, they were separated and detected within 6 min using a Discovery HS F5-3 column and positive ion mode multiple reaction monitoring system via LC–MS/MS. Calibration curves of these compounds using each stable isotope-labeled internal standard (IS) showed linearity with correlation coefficients greater than 0.9990 in the range of 0.01–40 ng/mL, with limits of detection (S/N = 3) of 0.7–21 pg/mL. Moreover, intra- and inter-day variations were lower than 8.1 and 15% (n = 6), respectively. The recoveries of these compounds from saliva samples were in the range of 82–114%. The developed IT-SPME/LC–MS/MS method of steroid hormones is a highly sensitive, specific, and non-invasive analytical method that allows extraction and enrichment with no organic solvents, and enables direct automated online analysis by simply ultrafiltrating a small sample of saliva. Full article

Sulfite, a widely used food additive, is subject to regulated labeling. The extraction of sulfite as the stable hydroxymethylsulfonate (HMS) form and its quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been recognized for their good sensitivity, selectivity, and versatility across various food materials. This study aimed to develop a cost-effective and simpler method for sulfite quantitation, while maintaining the superior sensitivity and selectivity of mass spectrometry (MS). To achieve this, we introduced paper spray ionization (PSI), an ambient desorption ionization technique that could achieve the direct measurement of analytes without employing separation. We also employed a novel internal standard (IS) structurally similar to the analyte, replacing the more expensive isotopically labeled IS. Although the PSI-MS/MS method developed in this study exhibited slightly lower analytical performance compared to the conventional LC-MS/MS, it remained effective for sulfite analysis in dried fruits. Full article

Ergot alkaloids (EAs) formed by Claviceps fungi are one of the most common food contaminants worldwide, affecting cereals such as rye, wheat, and barley. To accurately determine the level of contamination and to monitor EAs maximum levels set by the European Union, the six most common EAs (so-called priority EAs) and their corresponding epimers are quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The quantification of EAs in complex food matrices without appropriate internal standards is challenging but currently carried out in the standard method EN 17425:2021 due to their commercial unavailability. To address the need for isotopically labeled EAs, we focus on two semi-synthetic approaches for the synthesis of these reference standards. Therefore, we investigate the feasibility of the N⁶-demethylation of native ergotamine to yield norergotamine, which can subsequently be remethylated with an isotopically labeled methylating reagent, such as iodomethane (¹³CD₃-I), to yield isotopically labeled ergotamine and its C⁸-epimer ergotaminine. Testing the isotopically labeled ergotamine/-inine against native ergotamine/-inine with HPLC coupled to high-resolution HR-MS/MS proved the structure of ergotamine-¹³CD₃ and ergotaminine-¹³CD₃. Thus, for the first time, we can describe their synthesis from unlabeled, native ergotamine. Furthermore, this approach is promising as a universal way to synthesize other isotopically labeled EAs. Full article

The amount of macrolide (MAL) residues in aquatic products, including oleandomycin (OLD), erythromycin (ERM), clarithromycin (CLA), azithromycin (AZI), kitasamycin (KIT), josamycin (JOS), spiramycin (SPI), tilmicosin (TIL), tylosin (TYL), and roxithromycin (ROX), was determined using solid-phase extraction and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The residues were extracted with 1% ammonia acetonitrile solution and purified by neutral alumina adsorption. Chromatographic separation was completed on an ACQUITY UPLC BEH C₁₈ column with acetonitrile–0.1% formic acid aqueous solution as the mobile phase, and mass spectrometry detection was performed by multiple reaction monitoring scanning with the positive mode in an electrospray ion source (ESI⁺). Five isotopically labeled compounds were used as internal standards for quality control purposes. The findings indicated that across the mass concentration span of 1.0–100 μg/L, there was a strong linear correlation (R² > 0.99) between the concentration and instrumental response for the 10 MALs. The limit of detection of UPLC-MS/MS was 0.25–0.50 μg/kg, and the limit of quantitation was 0.5–1.0 μg/kg. The added recovery of blank matrix samples at standard gradient levels (1.0, 5.0, and 50.0 μg/kg) was 83.1–116.6%, and the intra-day precision and inter-day precisions were 3.7 and 13.8%, respectively. The method is simple and fast, with high accuracy and good repeatability, in line with the requirements for accurate qualitative and quantitative analysis of the residues for 10 MALs in aquatic products. Full article

Adenosine is a multifunctional nucleoside with several roles across various levels in organisms. Beyond its intracellular involvement in cellular metabolism, extracellular adenosine potently influences both physiological and pathological processes. In relation to its blood level, adenosine impacts the cardiovascular system, such as heart beat rate and vasodilation. To exploit the adenosine levels in the blood, we employed the liquid chromatography method coupled with mass spectrometry (LC–MS). Immediately after collection, a blood sample mixed with acetonitrile solution that is either enriched with ¹³C-labeled adenosine or a newly generated mixture is transferred into the tubes containing the defined amount of ¹³C-labeled adenosine. The ¹³C-enriched isotopic adenosine is used as an internal standard, allowing for more accurate quantification of adenosine. This novel protocol for LC–MS-based estimation of adenosine delivers a rapid, highly sensitive, and reproducible means for quantitative estimation of total adenosine in blood. The method also allows for quantification of a few catabolites of adenosine, i.e., inosine, hypoxanthine, and xanthine. Our current setup did not allow for the detection or quantifying of uric acid, which is the final product of adenosine catabolism. This advancement provides an analytical tool that has the potential to enhance our understanding of adenosine’s systemic impact and pave the way for further investigations into its intricate regulatory mechanisms. Full article

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware. Full article

Accurate determination of the concentration of alcohols and their metabolites is important in forensics and in several life science areas. A new headspace gas chromatography–mass spectrometry method has been developed to quantify alcohols and their oxidative products using isotope-labeled internal standards. The limit of detection (LOD) of the analytes in the developed method was 0.211 µg/mL for methanol, 0.158 µg/mL for ethanol, 0.157 µg/mL for isopropanol, 0.010 µg/mL for n-propanol, 0.157 µg/mL for acetone, and 0.209 µg/mL for acetaldehyde. The precision and accuracy of the method were evaluated, and the relative standard deviation percentages were found to be less than 3%. This work demonstrates the application of this method, specifically in quantifying the concentration of oxidative products of alcohol and other minor alcohols found in hand sanitizers, which have become an essential household item since the COVID-19 pandemic. Apart from the major components, the minor alcohols found in hand sanitizers include methanol, isopropanol, and n-propanol. The concentration range of these minor alcohols found in ethanol-based hand sanitizer samples was as follows: methanol, 0.000921–0.0151 mg/mL; isopropanol, 0.454–13.8 mg/mL; and n-propanol, 0.00474–0.152 mg/mL. In ethanol-based hand sanitizers, a significant amount of acetaldehyde (0.00623–0.231 mg/mL) was observed as an oxidation product, while in the isopropanol-based hand sanitizer, acetone (0.697 mg/mL) was observed as an oxidation product. The concentration of acetaldehyde in ethanol-based hand sanitizers significantly increased with storage time and temperature, whereas no such increase in acetone concentration was observed in isopropanol-based hand sanitizers with storage time and temperature. In two of the selected hand sanitizers, the acetaldehyde levels increased by almost 200% within a week when stored at room temperature. Additionally, exposing the hand sanitizers to a temperature of 45 °C for 24 h resulted in a 100% increase in acetaldehyde concentration. On the contrary, the acetone level remained constant upon the change in storage time and temperature. Full article