High-Throughput RPLC-MS/MS Quantification of Short- and Medium-Chain Fatty Acids.

Short- and medium-chain fatty acids (SMCFA) are monocarboxylic acids with a carbon chain length of 1-12 carbon atoms. They are mainly produced in humans by the gut microbiota, play crucial metabolic roles, are vital for intestinal health, and have multifaceted impact on immune and neurological functions. Accurate detection and quantification of SMCFA in different human biofluids is achieved using 3-nitro phenylhydrazine (3-NPH) derivatization of the free fatty acids followed by reverse phase liquid chromatography (RPLC) separation and detection by tandem mass spectrometry (MS/MS). Here, we describe the simultaneous measurement of 14 SMCFA and lactate in detail. All 3-NPH-SMCFA-hydrazones are separated in less than 5 min with an 8-min total run time (injection-to-injection). Linear dynamic range over 0.1-500 µM is achieved for most SCFAs, while it is 0.05-100 µM for MCFAs. Validation of the procedure depicts good linearity (R2 > 0.98) and repeatability (CV ≤ 20%). The lower limit of detection (LLOD) is 10-30 nM. The lower limit of quantification (LLOQ) is 50-100 nM for most analytes, while it is 0.5 µM for acetate. In conclusion, the method offers several benefits compared to alternative methods regarding throughput, selectivity, sensitivity, and robustness.

Analysis of methamphetamine in urine by GC/MS coupled with solid-phase dispersive extraction and solid-phase derivatization.

In this study, we investigated a solid-phase dispersive extraction (SPDE) method and solid-phase derivatization method using the same solid-phase gel to extract methamphetamine (MA) from urine samples more efficiently and perform trifluoroacetic acid (TFA) derivatization for MA analysis by gas chromatography/mass spectrometry (GC/MS). N-Methylbenzylamine (NMe-BA) was added to the urine sample as a surrogate, and MA was extracted by SPDE using Oasis® HLB gel as a solid-phase agent. After drying the solid-phase gel of the SPDE, anhydrous TFA was added to the MA-absorbed HLB gel in order to derivatize MA with TFA on the solid-phase, followed by elution of the TFA derivative from this gel using ethyl acetate. As a validation of the analytical method, the limit of detection (S/N = 3) and the limit of quantification (S/N > 10) of MA were 0.002 µg/mL and 0.01 µg/mL, respectively. And the average recovery rate was 97.6-100.1%, repeatability was 5.6-10.7%, and intermediate precision was 10.1-11.4% for low (0.02 µg/mL), intermediate (0.1 µg/mL), and high (1 µg/mL) concentrations of MA added in urine. The developed pretreatment method enabled continuous extraction, cleanup, and TFA derivatization of urinary MA on the same solid-phase. Thus, urinary MA can be analyzed easily, rapidly, and with high sensitivity and accuracy.

Advances and challenges in liquid chromatography-spectrometry (LC-MS) methodology for quantifying androgens and estrogens in human serum and plasma.

Accurate quantification of androgens and estrogens is critical for elucidating their roles in endocrine disorders and advancing research on their functions in human biology and pathophysiology. This review highlights recent advances and ongoing challenges in liquid chromatography- mass spectrometry (LC- MS) methodology for quantifying androgens and estrogens in human serum and plasma. We summarized current approaches for analyzing the different forms of androgens and estrogens, along with their reported levels in publications from 2010 to the present. These published levels pointed out the inconsistencies in reference intervals across studies. To address these issues, advances in derivatization methods and chromatographic separation techniques are reviewed. Future perspectives for improving the accuracy and consistency of hormone quantification in clinical and research settings were also proposed.

Determination of 4-n-butylresorcinol by fluorescence derivatization based on dopamine.

4-n-butylresorcinol (4nBR) is a frequently utilized as whitening ingredients in skincare cosmetics. Compared with other whitening ingredients, it can effectively inhibit tyrosinase with lower toxicity and superior inhibition efficacy. Under alkaline conditions, an induced oxidative coupling reaction can occur between 4nBR and dopamine (DA) to generate strong fluorescent substance azamonardine with an intense emission band centering at 476 nm when excited at 440 nm. This phenomenon can be used to establish a fluorescence analysis method for 4nBR. The results indicated that the linear range of the method was 1.0-24.0 nmol L-1, and the detection limit was as low as 0.25 nmol L-1. The method showed high sensitivity, good selectivity, mild experimental conditions and low cost. The proposed method was successfully used to detect 4nBR in cosmetics, and the results were consistent with those of HPLC. The spiking recoveries were between 98.2% and 108 %.

Comprehensive profiling and evaluation of free/conjugated Phytosterols in crops using chemical derivatization coupled with UHPLC-ESI-QTOF-MS/MS.

Phytosterols are naturally existed in crops but their detection is constrained by sensitivity and accuracy due to the inefficient analytical approaches. This study hypothesizes that an untargeted analytical method combining chemical derivatization with ultrahigh performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry can identify the various composition and contents of phytosterols in different crops. The results showed that chemical derivatization significantly enhanced intensity of phytosterols compared with non-derivatized samples. Using precursor ion scanning (PIS) of m/z 252.0690, dansyl chloride-labeled phytosterols were identified, demonstrating that rapeseeds had the highest content of total phytosterol (3981.2 ± 95.3 mg/kg), followed by sunflower seeds, flaxseeds, corn and rice, respectively. Principal component analysis revealed significant variations in phytosterol distribution among 15 crop samples, suggesting the applicability of phytosterol profile as a marker for phytosterols-contained crops. Hence, the proposed analytic approach proves high efficiency and accuracy in determining phytosterols and advances the study for phytosterol-enriched crops.

Simultaneous determination of glyphosate, glufosinate and their metabolites in soybeans using solid-phase analytical derivatization and LC-MS/MS determination.

Glyphosate and glufosinate are the most widely used herbicides worldwide. We developed a simple and rapid analytical method for detecting glyphosate, glufosinate, and their metabolites (N-acetyl glyphosate: Gly-A, N-acetyl glufosinate: Glu-A, and 3-(hydroxymethylphosphinyl)propanoic acid: MPPA) in soybeans. The method involved extraction with water, trapping in a mini-column containing polymer-based resin with strong anion exchange groups, dehydration with acetonitrile, and solid-phase analytical derivatization at ambient temperature for 1 min using N-(tert-butyldimethylsilyl)-N-methyl trifluoroacetamide (MTBSTFA), followed by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination. This method offers a straightforward and rapid analysis, using on-solid phase dehydration and rapid derivatization at an ambient temperature with MTBSTFA, yielding reliable results for glyphosate, glufosinate, and their metabolites. The method was applied to both domestic and imported soybean samples. Glyphosate, glufosinate, and Glu-A were detected in imported feed soybeans and processed soybean meal for feed use, reflecting the current conditions of GM soybean cultivation.

Pipette-tip kapok fiber-based solid-phase extraction/in-situ derivatization for the rapid and green analysis of furfural compounds.

Furfural compounds, including 5-hydroxymethylfurfural, furfural, and 5-methylfurfural, are common in foods and pose health risks. This study presents a pipette-tip solid-phase extraction with in-situ derivatization (PT-KF-SPE/ISD) method for rapid analysis of furfural compounds in various food matrices. Utilizing natural kapok fiber as an efficient adsorbent, this method integrates extraction and derivatization into a single step via a simple pull-push operation. Derivatization with 2,4-dinitrophenylhydrazine increases the hydrophobicity and ultraviolet absorption of furfural compounds, enabling sensitive liquid chromatography-ultraviolet detection. The method shows good linearity, sensitivity, and reproducibility, with limits of detection in ranges of 3.9-6.0 ng/mL. Real sample analysis confirms its applicability in detecting furfural compounds in beverages and herbal products, offering a reliable and eco-friendly solution for food safety and quality control. Five greenness assessment metrics demonstrate the method's excellent environmental friendliness. This approach highlights the advantages of combining natural adsorbents with in-situ derivatization for efficient food analysis.

Analysis of glycerol bound ω-oxo-fatty acids as ω-dioxane-FAME-derivatives.

ω-oxo-fatty acids, also known as aldehydic fatty acids, are major products of fatty acid oxidation and pose potential health risks. When bound to glycerol, ω-oxo-fatty acids (core aldehydes) can be ingested with food. Challenges in GC-MS quantification include the absence of an appropriate internal standard. Additionally, substantial analyte losses during sample preparation, caused by the high volatility of short-chain compounds, alter their pattern based on molecular weight. In this study, among various tested derivatization methods, the formation of ω-dioxane derivatives demonstrated improved recovery rates after three evaporation cycles. For methyl 7-oxo-heptanoate, recovery increased from 43 % to 88 %, while recovery rates for different chain lengths and a novel synthesized internal standard improved from a range of 43 %-76 % to 87 %-92 %. Additionally, ω-dioxane derivatives displayed favorable GC-MS behavior, enabling clear identification and increased sensitivity. Finally, ω-oxo-fatty acids were quantified as their ω-dioxane-derivatives in thermally treated sunflower and rapeseed oil.

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.

Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis coupled with High Performance Liquid Chromatography (HPLC) for subsequent detection. This method can be performed in anaerobic glove bag settings. It requires readily available HPLC instrumentation for ligand quantitation and is adaptable to meet the needs of a variety of substrate affinity measurements.

Design, Synthesis, and Acaricidal Activity of 2,5-Diphenyl-1,3-oxazoline Compounds.

By using a scaffold hopping/ring equivalent and intermediate derivatization strategies, a series of compounds of 2,5-diphenyl-1,3-oxazoline with substituent changes at the 5-phenyl position were prepared, and their acaricidal activity was studied. However, the synthesized 2,5-diphenyl-1,3-oxazolines showed lower activity against mite eggs and larvae compared to the 2,4-diphenyl-1,3-oxazolines with the same substituents. We speculate that there is a significant difference in the spatial extension direction of the substituents between the two skeletons of compounds, resulting in differences in their ability to bind to the potential target chitin synthase 1. This work is helpful in inferring the internal structure of chitin synthase binding pockets.