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.

Circulating levels of 2-hydroxybutyrate (2HB) are highly related to glycemic status in different metabolomic studies. According to recent evidence, 2HB is an early biomarker of the future development of dysglycemia and type 2 diabetes mellitus and may be causally related to the progression of normal subjects to impaired fasting glucose or insulin resistance. In the present study, we developed and validated a simple, specific and sensitive gas chromatography-mass spectrometry (GC-MS) method specifically intended to quantify serum levels of 2HB. Liquid-liquid extraction with ethyl acetate was followed by 2 min of microwave-assisted derivatization. The method presented acceptable accuracy, precision and recovery, and the limit of quantification was 5 µM. Levels of 2HB were found to be stable in serum after three freeze-thaw cycles, and at ambient temperature and at a temperature of 4 °C for up to 24 h. Extracts derivatized under microwave irradiation were stable for up to 96 h. No differences were found in 2HB concentrations measured in serum or plasma EDTA samples. In summary, the method is useful for a rapid, precise and accurate quantification of 2HB in serum samples assessed for the evaluation of dysglycemia and diabetes mellitus.

BACKGROUND: Smith-Lemli-Opitz syndrome is a birth defect caused by the deficiency of 7-dehydrocholesterol reductase in cholesterol biosynthesis pathway, which leads to accumulation of 7-dehydrocholesterol and reduction of cholesterol in body fluids. To effectively diagnose Smith-Lemli-Opitz syndrome and monitor therapy, a reliable method for simultaneous detection of 7-dehydrocholesterol and cholesterol is needed.
METHODS: In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), 50 μL of human plasma were hydrolyzed at 70℃ for 40 min with 1 M potassium hydroxide in 90% ethanol, and then 7-dehydrocholesterol and cholesterol were extracted by 600 μL of n-hexane for three times. After microwave-assisted derivatization with 70 μL of N,O-bis(trimethylsilyl)trifluoroacetamide at 460 W for 3 min, the analytes were measured by gas chromatography-mass spectrometry.
RESULTS: The limits of detection were 100 ng/mL for 7-dehydrocholesterol and 300 ng/mL for cholesterol. Good linearity was obtained in the range of 1-600 μg/mL for 7-dehydrocholesterol and 10-600 μg/mL for cholesterol, which completely covered the biochemical levels of Smith-Lemli-Opitz syndrome patients that have been reported.
CONCLUSIONS: A time-saving and accurate gas chromatography with mass spectrometry based method was developed for the determination of 7-dehydrocholesterol and cholesterol in human plasma, which also serves as a useful tool for Smith-Lemli-Opitz syndrome diagnosis, treatment, and research.

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.

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.

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.

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.

This paper, for the first time, reported a speedy hyphenated technique of low toxic dual ultrasonic-assisted dispersive liquid-liquid microextraction (dual-UADLLME) coupled with microwave-assisted derivatization (MAD) for the simultaneous determination of 20(S)-protopanaxadiol (PPD) and 20(S)-protopanaxatriol (PPT). The developed method was based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection using multiple-reaction monitoring (MRM) mode. A mass spectrometry sensitizing reagent, 4\'-carboxy-substituted rosamine (CSR) with high reaction activity and ionization efficiency was synthesized and firstly used as derivatization reagent. Parameters of dual-UADLLME, MAD and UHPLC-MS/MS conditions were all optimized in detail. Low toxic brominated solvents were used as extractant instead of traditional chlorinated solvents. Satisfactory linearity, recovery, repeatability, accuracy and precision, absence of matrix effect and extremely low limits of detection (LODs, 0.010 and 0.015ng/mL for PPD and PPT, respectively) were achieved. The main advantages were rapid, sensitive and environmentally friendly, and exhibited high selectivity, accuracy and good matrix effect results. The proposed method was successfully applied to pharmacokinetics of PPD and PPT in rat plasma.