Development and application of a sensitive liquid chromatography-tandem mass spectrometry method for the quantitative analysis of 11 free fatty acids in human serum using a derivatisation strategy.

A stable isotope dilution-liquid chromatography-tandem mass spectrometry method based on a derivatisation strategy involving an N,N'-carbonylimidazole solution (CDI) with 4-(dimethylamino)-benzenemethanamine was developed for the determination of 11 free fatty acids (FFAs) in human blood samples. Serum samples were subjected to liquid‒liquid extraction and centrifuged, and the supernatant was collected for a two-step derivatisation reaction with a CDI and 4-(dimethylamino)-aniline acetonitrile solution. The derivatised solution was separated on a ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) column with a mobile phase consisting of water-acetonitrile in gradient elution and then detected by tandem mass spectrometry using electrospray ionisation (ESI) and multiple reaction monitoring (MRM) in positive ion mode and quantified using the isotope internal standard method. The effects of the derivatisation reaction time, temperature and concentration of derivatisation reagents on the response values of the analytes were investigated. The optimal conditions were as follows: 1.0 mg mL-1 CDI acetonitrile solution at 25 °C for 25 min, followed by a reaction with a 1.0 mg mL-1 4-(dimethylamino)-benzenemethanamine acetonitrile solution at 70 °C for 30 min. Under the optimal conditions, the limits of detection (LODs) of the 11 FFAs were in the range of 3.0-14.0 ng mL-1; the limits of quantification (LOQs) were in the range of 8.0-45.0 ng mL-1; and the mean recoveries ranged from 83.4 to 112.8%, with intraday and interday precisions ranging from 0.7 to 9.1% and 3.7-9.5%, respectively. The experimental method is simple in terms of the pretreatment operation, accurate and reliable, and can be applied to the sensitive determination of FFAs in human blood samples.

Determination of six volatile fatty acids in human serum, urine and faeces by low temperature derivatisation combined with HPLC-MS/MS.

A stable isotope dilution-liquid chromatography tandem mass spectrometry method based on a low-temperature derivatization strategy with 3-nitrophenylhydrazine (3-NPH) was developed for the determination of six volatile fatty acids (VFAs) in serum, urine, and feces. Ice acetonitrile was used to precipitate proteins and extract the target analytes. The extract was derivatized with 3-NPH methanol solution at 4 °C. BEH C8 (1.7 µm, 2.1 × 100 mm) column was used for chromatographic separation, and acetonitrile-water (both containing 0.01 % formic acid) were used as the mobile phase with a gradient elution of 10 min. Electrospray ionization source (ESI) in negative ion multiple reaction monitoring (MRM) mode were used for analyte detection. The regression coefficients R2 of the calibration curves for the six VFAs were in the range of 0.9963-0.9994, and the LOQs were in the range of 0.02-0.5 µg mL-1, with the recoveries in the range of 85.3-104.3 %, and the intra- and inter-day precision in the range of 1.8-9.1 %. The method is simple, accurate and reliable, and has been applied in the sensitive determination of VFAs in complex biological samples.

Yu Linzhu alleviates primary ovarian insufficiency in a rat model by improving proliferation and energy metabolism of granulosa cells through hif1α/cx43 pathway.

BACKGROUND: Yu Linzhu (YLZ) is a classical Chinese traditional formula, which has been used for more than 600 years to regulate menstruation to help pregnancy. However, the mechanism of modern scientific action of YLZ needs to be further studied. METHODS: Thirty SD female rats were divided into three groups to prepare the blank serum and drug-containing serum, and then using UHPLC-QE-MS to identify the ingredients of YLZ and its drug-containing serum. Twenty-four SD female rats were divided into four groups, except the control group, 4-vinylcyclohexene dicycloxide (VCD) was intraperitoneally injected to establish a primary ovarian insufficiency (POI) model of all groups. Using vaginal smear to show that the estrous cycle of rats was disturbed after modeling, indicates that the POI model was successfully established. The ELISA test was used to measure the follicle-stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH) levels in the serum of rats. HE stain was used to assess the morphology of ovarian tissue. The localization and relative expression levels of CX43 protein were detected by tissue immunofluorescence. Primary ovarian granulosa cells (GCs) were identified by cellular immunofluorescence. CCK8 was used to screen time and concentration of drug-containing serum and evaluate the proliferation effect of YLZ on VCD-induced GCs. ATP kit and Seahorse XFe24 were used to detect energy production and real-time glycolytic metabolism rate of GCs. mRNA and protein expression levels of HIF1α, CX43, PEK, LDH, HK1 were detected by RT-PCR and WB. RESULTS: UHPLC-QE-MS found 1702 ingredients of YLZ and 80 constituents migrating to blood. YLZ reduced the FSH while increasing the AMH and E2 levels. In ovarian tissues, YLZ improved ovarian morphology, follicle development, and the relative expression of CX43. In vitro studies, we found that YLZ increased the proliferative activity of GCs, ATP levels, glycolytic metabolic rate, HIF1α, CX43, PEK, HK1, LDH mRNA, and protein levels. CONCLUSIONS: The study indicated that YLZ increased the proliferation and glycolytic energy metabolism of GCs to improve follicular development further alleviating ovarian function.

Effects of Yu Linzhu on ovarian function and oocyte mitochondria in natural aging mice.

OBJECTIVE: To study the effect of Yu Linzhu on ovarian function and mitochondria in natural aging mice. METHODS: Female BALB/c mice were selected as normal group at 7-8 weeks and natural aging group at 9 months. The natural aging group was divided into Yu Linzhu intervention group and non-intervention group by intragastric administration once a day for 6 weeks. The morphology and blood flow of ovary were observed by ultrasound. Ovarian morphology and follicle were observed by HE staining. Hormone levels were analyzed by ELISA. Serum oxidative stress were detected by radioimmunoassay. The distribution of mitochondria in oocytes was observed by fluorescence staining. The ultrastructure of oocytes and the morphology of mitochondria were observed under electron microscope. The mitochondrial membrane potential was detected by JC-1. RESULTS: Two groups of aging mice had serious disturbance of estrus cycle. The ovarian area of the mice in the aging non-intervention group was smaller than that in the normal group, and the ovarian area of the mice in the aging intervention group recovered. The ovarian blood flow was weak or even disappear in the aging non-intervention group, and the blood flow in the intervention group was improved. The ovarian volume of mice in the non-intervention group was smaller than that in the normal group. Some ovarian tissues were adhered to the surrounding tissues. While in the intervention group, the ovarian volume increased, the degree of adhesion decreased, the infiltration of ovarian interstitial lymphocytes decreased, and the zona pellucida recovered. Granular cell arrangement returned neatly, egg cell shape recover regular and the number also increased. In the non-intervention group, E2 (Estrogen), AMH (Anti-Mullerian hormone) decreased (P = 0.0092 and P = 0.0334, respectively), FSH (Follicle stimulating hormone) increased (P < 0.0001). In the intervention group, FSH decreased (P = 0.0002), LH (luteinizing hormone) decreased and E2, AMH increased. In the non-intervention group, GSH-Px (Glutathione peroxidase) decreased (P = 0.0129), SOD (Superoxide dismutase) decreased, ROS (reactive oxidative species), MDA (Malondialdehyde) increased. In the aging intervention group, ROS, MDA decreased and GSH-Px increased. In the non-intervention group, mitochondrial expression was scattered at the concentrated distribution point, the length of mitochondria was mostly long and the average volume increased, the density decreased, the number decreased and some mitochondria fused, and lesions such as swelling, vacuolar degeneration and inclusion body formation, membrane potential decreased (P = 0.0002). In the aging intervention group, mitochondria were evenly distributed, the mitochondria were basically round, the distribution density was moderate, the inner ridge was clear, and the membrane potential of the aging intervention group increased. CONCLUSION: Yu Linzhu can improve the ovarian function of natural aging mice by improving the mitochondrial function of oocytes.