7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50-4000 ng/mg and 30-6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.

Over recent decades, steroidal estrogens have become an emerging and very serious issue as they pose a serious threat to living organisms, soil, plants, and water resources in general. Estrogens have therefore been the subject of considerable scientific attention in order to develop new methodologies for its determination, being able of detecting them at very low concentrations. Those procedures minimize or eliminate the consumption of organic solvents and reagents that may be incompatible with the environment. In this respect, we developed a sensitive, selective method for the simultaneous determination of thirteen natural and synthetic hormones present at the nanogram-per-liter level in various types of water by using continuous solid-phase extraction in combination with gas chromatography and mass spectrometry (GC-MS). The target analytes were preferentially sorbed on an Oasis HLB sorbent column (80 mg) and eluted with acetone (600 µL) for derivatization with a mixture of 70 µL of N,O-bis(trimethylsilyl) trifluoroacetamide and trimethylchlorosilane and 35 µL of petroleum ether in a household microwave oven at 200 W for 4 min. Under optimum conditions, the ensuing method exhibited good linearity (r ≥ 0.998), good precision (RSD ≤ 7%), high recoveries (92-103%), and low detection limits (0.01-0.3 ng L-1). The method outperforms existing alternatives in robustness, sensitivity, throughput, flexibility-it allows both estrogens, progestogens, and androgens to be determined simultaneously-and compliance with the principles of Green Chemistry. It was successfully used to analyze various types of water samples (mineral, tap, well, pond, swimming pool, river, and waste) that were found to contain four estrogens (estrone, 17β-estradiol, 17α-ethinylestradiol, and hexestrol), two progestogens (testosterone, dihydrotestosterone), and one progestogen (progesterone) at concentrations ranging from 3.0 to 110 ng L-1.

A chiral analytical method was proposed based on capillary electrophoresis with laser-induced fluorescence detection coupled with microwave-assisted derivatization for the simultaneous baseline separation and sensitive detection of four stereoisomers of 3-hydroxyaspartate. The derivatization reaction of 3-hydroxyaspartate with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole was greatly accelerated by microwave irradiation. Under the optimized conditions, the derivatization yield was increased by 20% and the derivatization time was shortened by 20 min when compared with those from conventional water bath heating. In addition, the sensitivity was improved by online sample concentration methods. The detection limit of l-threo-3-hydroxyaspartate obtained by large-volume sample stacking with polarity switching was 5.3 nmol/L, which was around 1000-fold lower than that of the capillary electrophoresis/laser-induced fluorescence without stacking. The excellent analytical performance in terms of linearity and precision was also achieved. Furthermore, the developed method was successfully applied to the determination of 3-hydroxyaspartate in the spiked urine, and satisfactory recoveries were obtained ranging from 90.5 to 107.0%.

The metabolic ratios lactate/pyruvate and β-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, β-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency\'s Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and β-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.

Steroid hormones are associated in depth to cellular signaling, inflammatory immune responses, and reproductive functions, and their metabolism alterations incur various diseases. In particular, quantitative profiling of steroids in plasma of patients with gastric cancer can provide a vast information to understand development of gastric cancer, since both sex hormones and glucocorticoids might be correlated with the pathological mechanisms of gastric cancer. Here, we developed a gas chromatography-tandem mass spectrometry-dynamic multiple reaction monitoring (GC-MS/MS-dMRM) method combined with solid-phase extraction (SPE) and microwave-assisted derivatization (MAD) to determine 20 endogenous steroids in human plasma. In this study, MAD conditions were optimized with respect to irradiation power and time. The SPE enabled effective cleanup and extraction for profiling of steroid hormones in human plasma samples. The MAD could improve laborious and time-consuming derivatization procedure, since dielectric heating using microwave directly increase molecular energy of reactants by penetrating through medium. Furthermore, dMRM method provided more sensitive determination of 20 steroids, compared to traditional MRM detection. The limits of quantification of steroids were below 1.125 ng/mL and determination coefficients of calibration curves were higher than 0.9925. Overall precision and accuracy results were below 19.93% and within ±17.04%, respectively. The developed method provided sufficient detection sensitivities and reliable quantification results. The established method was successfully applied to profile steroid metabolism pathways in plasma of patients with chronic superficial gastritis (CSG), intestinal metaplasia (IM), and gastric cancer. Statistical significances of steroid plasma levels between gastric disorder groups were investigated. In conclusion, this method provided comprehensive profiling of 20 steroids in human plasma samples and will be helpful to discover potential biomarkers for the development of gastric cancer and to further understand metabolic syndrome.

A multifunctional visual observation of magnetic ionic liquid (MIL) benzyltrioctylammonium thiocyanatecobalt (II) [N8,8,8,B+]2[Co(SCN)42-] with the long-chain alkyl and benzyl group structures was designed and synthesized as microextraction phase. Designed new structural MIL displays good hydrophobicity, high extraction capacity for both aromatic and aliphatic compounds, and has obvious color markers (blue color) which is easy visual separation from aqueous solution through a magnet. In the present work, a green, efficient and rapid samples pretreatment method based on simultaneous derivatization and extraction of aromatic (tyramine, histamine, phenylethylamine, tryptamine) and aliphatic (spermidine and spermine) biogenic amines (BAs) were performed. Microwave-assisted derivatization coupled with MIL-dispersive liquid-liquid microextraction (DLLME) was established for the determination of six BAs in different food samples via HPLC. The method was successfully applied for the analysis of beer and milk samples, and the recoveries of analytes were 93.0-110.3% and 91.2-111.6%, respectively. The limits of detection (LODs) were 0.51-1.49 μg L-1 for six BAs. These results have demonstrated that the proposed method has offered an effective, accurate, and sensitive methodology for BAs residue detection in food sample, and this method has great potential for the routine analysis of large numbers of samples on measuring different kinds of compounds.

In this study, an ecofriendly analytical method was developed for determining glutathione (GSH) levels in biomatrix samples. 9-(bromomethyl)acridine was used for the first time as a derivatization reagent in GSH analysis. Microwave-assisted derivatization reduced the reaction time to 1 min. After derivatization, coacervative extraction was employed to extract GSH derivative from the complex biomatrix and to increase sensitivity. Because the negatively charged group of the GSH derivative was neutralized by the extracting agent Aliquat 336, aggregates formed without any coacervating agents. Furthermore, capillary liquid chromatography coupled with ultraviolet detection was applied to decrease waste generation and increase selectivity. This method successfully quantified GSH levels in various biomatrices, including erythrocytes, HaCaT cells, BALB/3T3 cells, and 3T3-L1 fibroblasts. This method only required a low sample volume (≤10 μL). A standard addition method was utilized to spike the biomatrix samples with 0-4.8 nmol GSH to construct calibration curves. The proposed method performed well, with a determination coefficient of 0.999 and relative standard deviations of less than 6.59% for the slope and the intercept, as determined by linear regression analysis. The limit of detection of GSH in the standard solution was 800 nM or 0.4 pmol. Compared to non-derivatized GSH, the proposed method for detecting derivatized GSH provides 750-fold greater sensitivity.

Hydroxyl-containing cholesterol and metabolites (HCMs) are potential biomarkers for Alzheimer\'s disease. Therefore, quantitative analysis of HCMs can serve as an indicator of clinical diagnosis and drug treatment. In this work, we developed an accurate, sensitive and rapid method for the determination of HCMs in rat blood microdialysates by microwave-assisted stable isotope labeling derivatization (MA-SILD) magnetic dispersive solid phase extraction (MDSPE) coupled with ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) in the multiple reaction monitoring mode (MRM). In this respect, a pair of new SILD reagents, d0-/d3-3-N-methyl-2\'-carboxyl Rhodamine 6G (d0-/d3-MCR6G), were designed, synthesized and used to label HCMs. Rhodamine 6G with permanently positive charge was introduced into HCMs, improving ionization efficiency and enhancing detection sensitivity. In addition, the d3-MCR6G labeled standards were served as internal standards, reducing the matrix effect and guaranteeing accurate quantification. Various factors affecting MA-SILD and MDSPE were optimized. Furthermore, good linearity was obtained with R2 > 0.994 over the concentration range of 2-3000 pg/mL. The limits of detection (LODs) and quantitation (LOQs) were in the range of 0.24-0.31 and 1.5-1.8 pg/mL, respectively. Acceptable precision (1.6-12.6%), accuracy (87.9-105.2%), matrix effect (85.4-111.2%) and derivatization efficiency (>98%) were achieved. On the whole, this method was validated and applied to accurate, sensitive and simple quantitation of HCMs in rat blood microdialysates. This work would provide some technical support in the diagnosis and treatment of AD or other neurological diseases.

In this work, a GC-MS/MS method was developed for the determination of anabolic-agent residues in bovine urine. The optimized sample preparation was as follows: enzymatic hydrolysis by β-glucuronidase-sulfatase enzyme from Helix pomatia for 16 h at 37.5 °C, liquid-liquid extraction with diethyl ether, solid-phase extraction with HLB and aminopropylsilane cartridges, and microwave-assisted derivatization using 25 μL of MSTFA/NH4I/ethanethiol and full microwave power for 2 min. The method was validated according to Decision 657/2002/EC, Codex Alimentarius, and Manual da Garantia da Qualidade Analítica guidelines. The acceptability criteria for quantitative analysis were met for α-ethinylestradiol, α-nandrolone, β-estradiol, β-zearalanol, β-zearalenol, drostanolone, ethisterone, dienestrol, diethylstilbestrol, hexestrol, megestrol, methyltestosterone, and zearalenone. The analytes α-zearalenol, α-zearalanol, and norethandrolone were validated for qualitative analysis.

Simultaneous detection of oleanolic acid and ursolic acid in rat blood by in vivo microdialysis can provide important pharmacokinetics information. Microwave-assisted derivatization coupled with magnetic dispersive solid phase extraction was established for the determination of oleanolic acid and ursolic acid by liquid chromatography tandem mass spectrometry. 2\'-Carbonyl-piperazine rhodamine B was first designed and synthesized as the derivatization reagent, which was easily adsorbed onto the surface of Fe3O4/graphene oxide. Simultaneous derivatization and extraction of oleanolic acid and ursolic acid were performed on Fe3O4/graphene oxide. The permanent positive charge of the derivatization reagent significantly improved the ionization efficiencies. The limits of detection were 0.025 and 0.020 ng/mL for oleanolic acid and ursolic acid, respectively. The validated method was shown to be promising for sensitive, accurate, and simultaneous determination of oleanolic acid and ursolic acid. It was used for their pharmacokinetics study in rat blood after oral administration of Arctiumlappa L. root extract.