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A novel method has been established for the rapid separation and determination of free fatty acids from 37 different varieties of raspberry. In this study, a new fluorescent labeling reagent for fatty acids, 2-(4-amino)-phenyl-1-hydrogen-phenanthrene [9, 10-d] imidazole (PIA), has been synthesized and successfully applied to the high-performance liquid chromatography (HPLC) determination of fatty acids in raspberry. The novel method has been optimized by HPLC with fluorescence detection and online mass spectrometry identification (HPLC-FLD-MS/MS). The 22 main fatty acids (FAs) present in raspberry were derivatized by PIA and separated on a reversed-phase Hypersil GOLD column with gradient elution. The main experimental parameters affecting extraction efficiency and derivatization yield were investigated and optimized by response surface methodology (RSM) combined with Box-Behnken design (BBD). Under the optimum conditions, the method was successfully applied for the analysis of 22 fatty acids in 37 different varieties of raspberry. Good linear correlations were observed for all fatty acids with correlation coefficients of > 0.9978. Limits of detection and quantification (LOD and LOQ) were in the range of 0.12 to 0.49 ng/mL and 1.07 to 2.81 ng/mL, respectively. Furthermore, the results indicated that the raspberries were rich in fatty acids, but the contents of the fatty acids varied among the different varieties.
A rapid, sensitive, and selective precolumn derivatization method for the simultaneous determination of eight thiophenols using 3-(2-bromoacetamido)-<i>N</i>-(9-ethyl-9<i>H</i>)-carbazol as a labeling reagent by high-performance liquid chromatography with fluorescence detection has been developed. The labeling reagent reacted with thiophenols at 50°C for 50 min in aqueous acetonitrile in the presence of borate buffer (0.10 mol/L, pH 11.2) to give high yields of thiophenol derivatives. The derivatives were identified by online postcolumn mass spectrometry. The collision-induced dissociation spectra for thiophenol derivatives gave the corresponding specific fragment ions at <i>m/z</i> 251.3, 223.3, 210.9, 195.8, and 181.9. At the same time, derivatives exhibited intense fluorescence with an excitation maximum at λ<sub>ex</sub> = 276 nm and an emission maximum at λ<sub>em</sub> = 385 nm. Excellent linear responses were observed for all analytes over the range of 0.033-6.66 μmol/L with correlation coefficients of more than 0.9997. Detection limits were in the range of 0.94-5.77 μg/L with relative standard deviations of less than 4.54%. The feasibility of derivatization allowed the development of a rapid and highly sensitive method for the quantitative analysis of trace levels of thiophenols from some rubber products. The average recoveries (<i>n</i> = 3) were in the range of 87.21-101.12%.
Graphical abstract Highlights • Five anthocyanidins are identified in Lycium ruthenicum Murray by UPLC-Q-Orbitrap MS. • Five anthocyanins are identified in Lycium ruthenicum Murray by UPLC-Q-Orbitrap MS. • Anthocyanin extracts hanve the activity of anti-gout. • Petunidin-3-glu has the activity of anti-gout. Abstract Lycium ruthenicum Murray (LR) represents an agricultural cash crop found in Northwest China and has been used in traditional folk medicine for a long time. However, detailed qualitative and quantitative analyses of LR anthocyanins, as well as their pharmacological research, remain scarce. In this work, we established a rapid method for the simultaneous identification and quantification of six anthocyanidins and six anthocyanins from LR via UPLC-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap MS) analysis. Finally, five anthocyanidins and five anthocyanins were qualitatively and quantitatively analyzed. Among these, 10 constituents (delphinidin-3-glu, cyanidin-3-glu, petunidin-3-glu, peonidin-3-glu, malvidin-3-glu, delphinidin, cyanidin, petunidin, pelargonidin and malvidin) were detected and petunidin-3-glu proved to be the predominant species in LR. Furthermore, the anti-inflammatory effects of anthocyanin extracts and petunidin-3-glu were investigated using a rat model involving gouty arthritis induced by monosodium urate. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-18 (IL-18), prostaglandin E2 (PE2), cyclooxygenase-1 (COX-1) enzymes in serum, the paw COX-1 mRNA expression and paw volume could be determined to be significantly increased in rats suffering from gouty arthritis induced by monosodium urate. However, these indicators were found to be significantly reduced after treatment with anthocyanin extracts (200 mg/kg b.wt, p.o.) and petunidin-3-glu (40 mg/kg b.wt, p.o.). Taken in concert, our study shows that anthocyanin extracts and petunidin-3-glu may significantly reduce monosodium urate crystal-induced inflammation. Use and administration of these compounds may be potentially valuable for the further development and clinical applicability of the active compounds in this plant.
Lycium ruthenicum Murr. (LR) is a perennial shrub commonly used as a nutritional food and medicine. Herein, we identified 12 anthocyanins from LR, with petunidin derivatives constituting approximately 97% of the total anthocyanin content. Furthermore, the potential mechanism of anthocyanins exerting neuroprotective effects in d-galactose (d-gal)-treated rats was explored. Behavioral results showed that anthocyanins relieved d-gal-induced memory disorder. Additionally, anthocyanins reduced receptor for advanced glycation end products (RAGE) and suppressed oxidative stress caused by d-gal. Anthocyanins suppressed microgliosis and astrocytosis and reduced the overexpression of nuclear factor kappa B (NF-κB), interleukin-1-β (IL-1β), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α). Moreover, anthocyanins lowered C-jun N-terminal kinase ( p-JNK), caspase-3 levels, and the B-cell lymphoma 2-associated X protein/B-cell lymphoma 2 (Bax/Bcl-2) ratio. Thus, anthocyanins from LR attenuated memory disfunction, neuroinflammation, and neurodegeneration caused by d-gal, possibly through the RAGE/NF-κB/JNK pathway, representing a promising, safe candidate for prevention and therapy of neurodegenerative diseases.
Abstract This presented study describes a method based on high performance liquid chromatography combined with fluorescence detection (HPLC-FLD) using N-(2-iodoacetyl)-1-pyrenemethylamine (NIPA) as a novel fluorescence labeling reagent for the determination of thyreostats in bovine milk. Five thyreostats, belonging to the group of imidazole and thiouracil, were investigated in this work: tapazole (TAP), thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU) and phenylthiouracial (PhTU). Thyreostats were specifically purified by a silver ion solid phase extraction (Ag-SPE) cartridge and then labeled using NIPA. The labeled derivatives showed excellent fluorescence property with maximum excitation and emission wavelengths of 330 nm and 375 nm, respectively. The labeled derivatives were separated on a reversed-phase Eclipse SB-C18 column within 12 min. Excellent linearity (R2 > 0.995) of all thyreostats was achieved with the limits of detection (LODs) and the limits of quantitation (LOQs) in the low micrograms per liter range of 0.21–0.30 μg/L and 0.70–1.00 μg/L, respectively. Satisfactory recoveries in the range of 93.5–98.0% were obtained for all thyreostats. The developed method has been successfully applied to analyze thyreostats in bovine milk with good applicability. Thirty bovine milk samples have been investigated, and varying levels of thiouracil were detected in thirteen of these samples. The highest level in the raw milk reached a value of 4.5 μg/L. To our best knowledge, this study is the first to report the presence of naturally occurring thiouracil in milk by HPLC-FLD analysis. Highlights • A pre-column derivatization HPLC-FLD method was developed for the determination of thyreostats in milk samples. • LOD was in the low micrograms per liter range of 0.21–0.30 μg·L−1. • The proposed method was successfully applied to the determination of thyreostats in milk sample. • This study is the first to report the presence of naturally occurring thiouracil in milk by HPLC-FLD analysis.
In this study, a simple analytical method for the determination of γ-aminobutyric acid, gabapentin, and baclofen by using high-performance liquid chromatography with fluorescence detection was developed. An amidogen-reactive fluorescence labeling reagent, 4-(carbazole-9-yl)-benzyl chloroformate was first used to sensitively label these analytes. The completed labeling of these analytes can be finished rapidly only within 5 min at the room temperature (25°C) to form 4-(carbazole-9-yl)-benzyl chloroformate labeled fluorescence derivatives. These labeled derivatives expressed strong fluorescence property with the maximum excitation and emission wavelengths of 280 and 380 nm, respectively. The labeled derivatives were analyzed using a reversed-phase Eclipse SB-C18 column within 10 min with satisfactory shapes. Excellent linearity (R² > 0.995) for all analytes was achieved with the limits of detection and the limits of quantitation in the range of 0.25−0.35 and 0.70−1.10 μg/L, respectively. The proposed method was used for the simultaneous determination of γ-aminobutyric acid and its analogs in human serum with satisfactory recoveries in the range of 94.5-97.5%.
We previously found that Ratanasampil (RNSP), a traditional Tibetan medicine, improves the cognitive function of mild-to-moderate AD patients living at high altitude, as well as learning and memory in an AD mouse model (Tg2576); however, mechanism underlying the effects of RNSP is unknown. In the present study, we investigated the effects and molecular mechanisms of RNSP on oxidative stress-induced neuronal toxicity using human neuroblastoma SH-SY5Y cells. Pretreatment with RNSP significantly ameliorated the hydrogen peroxide- (H2O2-) induced cytotoxicity of SH-SY5Y cells in a dose-dependent manner (up to 60 μg/mL). Furthermore, RNSP significantly reduced the H2O2-induced upregulation of 8-oxo-2′-deoxyguanosine (8-oxo-dG, the oxidative DNA damage marker) but significantly reversed the expression of repressor element-1 silencing transcription factor (REST) from H2O2 associated (100 μM) downregulation. Moreover, RNSP significantly attenuated the H2O2-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 (ERK 1/2) in SH-SY5Y cells. These observations strongly suggest that RNSP may protect the oxidative stress-induced neuronal damage that occurs through the properties of various antioxidants and inhibit the activation of MAPKs. We thus provide the principle molecular mechanisms of the effects of RNSP and indicate its role in the prevention and clinical management of AD.
Amino acids are indispensable components of living organisms. The high amino acid content in Nitraria tangutorum Bobr. fruit distinguishes it from other berry plants and is of great significance to its nutritional value. Herein, using 10-ethyl-acridine-3-sulfonyl chloride as a fluorescent pre-column labeling reagent, a method for the efficient and rapid determination of amino acid content in N. tangutorum by pre-column fluorescence derivatization and on-line mass spectrometry was established and further validated. The limits of detection (signal-to-noise ratio = 3) were between 0.13 and 1.13 nmol/L, with a linear coefficient greater than 0.997 and a relative standard deviation between 1.37% and 2.64%. In addition, the method required a short analysis time, separating 19 amino acids within 20 min. Subsequently, the method was used to analyze the amino acid content of Nitraria tangutorum Bobr. from tissues retrieved from seven regions of the Qinghai-Tibet Plateau. Nitraria tangutorum Bobr. was shown to contain a large amount of amino acids, with the total content and main amino acid varying between the different tissues. This research supports the nutritional evaluation, quality control, and development and utilization of Nitraria tangutorum Bobr.
• Simultaneously identified and quantified 18 phenolic compounds from LR fruit by UPLC-Q-Orbitrap MS. • Catechin, naringenin and 9 phenolic acids are the first time to conduct qualitative and quantitative analysis in LR. • Total phenolics content and total anthocyanin content were determined. • The antioxidant activities in vitro of the LR were also evaluated.<br><b>Lycium ruthenicum</b> Murray (LR) is a functional food, and it has long been used in traditional folk medicine. However, detailed qualitative and quantitative analyses related to its phenolic compounds remains scarce. This work reports, for the first time, the establishment of a rapid method for simultaneous identification and quantification of 25 phenolic compounds by UPLC-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap MS). This method was validated by LODs, LOQs, precision, repeatability, stability, mean recovery, recovery range and RSD. The confirmed method was applied to the analysis of phenolic compounds in LR. Finally, 18 phenolic compounds in LR were qualitatively and quantitatively analyzed. Among them, 11 constituents were detected for the first time, which included two flavonoids (catechin and naringenin) and seven phenolic acids (gallic acid, vanillic acid, 2,4-dihydroxybenzoic acid, veratronic acid, benzoic acid, ellagic acid and salicylic acid). Moreover, Phloretin and protocatechuate, belonging to the dihydrochalcone flavonoid and protocatechuic acid respectively, were also identified and quantified. The total phenolics content (20.17 ± 2.82 mg/g) and the total anthocyanin content (147.43 ± 1.81 mg/g) were determined. In addition, the antioxidant activities of the LR extract were evaluated through 2,2-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging activity, ferric reducing antioxidant power (FRAP) and total antioxidant activity (T-AOC) assays.