Ammonium formate

An analytical standard of dimethoate with a purity of >99.0% (Restek, Bellefonte, PA, USA) was used for the sorption–desorption experiments. Its physicochemical properties are listed in Table 1 [62 ]. To prepare the standard solution, dimethoate was dissolved in MS-grade methanol (Honeywell, Charlotte, NC, USA) and further diluted with methanol or the corresponding mobile phase. For the soil sorption experiment, a 1000 mg/L stock solution of dimethoate was prepared by dissolving 250 μL Chromgor^® 40 in acetonitrile ultra-gradient-grade (J.T. Baker, Deventer, The Netherlands). Working solutions with dimethoate concentrations ranging from 5.0 to 100.0 mg/L were then prepared by dilution with a 0.01 M calcium chloride solution (CaCl₂, Acros Organics, Morris Plains, NJ, USA).
All solvents and chemicals were of analytical grade. These included: ammonium formate (Sigma-Aldrich, St. Louis, MO, USA), sodium acetate, sodium hydroxide, sodium pyrophosphate, potassium dichromate, sulfuric acid and phenolphthalein (Kemika d.d., Zagreb, Croatia). In addition, glucose (Merck, Darmstadt, Germany) and a certified EDTA standard (41.06 wt% C, 5.51 wt% H and 9.56 wt% N) were used (LECO Corporation, Saint Joseph, MI, USA). Deionized water was purified using a Siemens Ultra Clear system (Munich, Germany) to ensure high quality experimental conditions.

The following chemicals were used throughout the lipid extraction and analysis, water (Hypergrade for LC-MS, LiChrosolv®, MerckKGaA), acetonitrile (MSsuprasolve®, Sigma Aldrich), methanol (HPLC grade ≥99.9%, Sigma Aldrich), chloroform (stabilsed with amylenes, ≥99.5%, Sigma Aldrich), isopropanol (Optima™LC/MS Grade, Fisher Scientific), ammonium formate (99.995%, Sigma Aldrich).

Clinical parameters monitored in the study included uterine artery flow rate (mean pulsatility index), body mass index (BMI), mean arterial pressure (MAP), and weight gain. Pregnant individuals underwent uterine artery flow rate assessment via pulse color Doppler throughout the entire pregnancy. The BMI and MAP were calculated using established formulas as detailed in our previous work (18 (link), 19 ).
Serum lipid parameters, including total cholesterol, HDL cholesterol, LDL cholesterol (calculated using Friedewald’s equation), and apoA-I, were determined using commercially available kits and methods as already explained in details (17 (link), 18 (link)). ApoM mass concentrations were quantified using the Enzyme-Linked Immunosorbent Assay (ELISA) technique (FineTest Biotech Inc., Wuhan, China).
High-performance liquid chromatography (HPLC) grade analytical standards were used for quantification of sphingosine (SPH), sphinganine (SAP), S1P, ceramide C16:0 (Cer C16:0), ceramide C24:0 (Cer C24:0) (Avanti Polar Lipids, Birmingham, USA), sphinganine-1-phosphate (SAP1P), and sphingomyelin C16:0 (SM C16:0) (Cayman Chemical, Ann Arbor, USA). SPH d17, S1P d17, and ceramide C17:0 (Cer C17:0), obtained from Avanti Polar Lipids (Birmingham, USA), and sphingomyelin C17:0 (SM C17:0) purchased from Cayman Chemical (Ann Arbor, USA) were used as internal standards (IS). Methanol (HPLC grade) was purchased from Fisher (Pittsburgh, USA), chloroform, and ammonium formate from Sigma Aldrich (St. Louis, USA), trifluoroacetic and formic acid from Thermo Fisher Scientific (Waltham, USA).
Sphingolipid quantification employed liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), beginning with plasma lipid extraction via liquid-liquid extraction. A 50 µL plasma aliquot was introduced into the IS-coated (SPH d17, S1P d17, Cer C17:0, and SM C17:0 mixture) glass vials. Subsequently, 2 mL of an extraction mixture (methanol:chloroform blend in a 2:1 ratio with 0.1% trifluoroacetic acid) was added to each vial and vigorously vortexed for 30 seconds. In the ensuing step, 0.67 mL of chloroform was introduced, and the test tubes were once again thoroughly mixed for 30 seconds. Following this, 1.15 mL of HPLC-grade water was added to establish a 1:1:0.9 ratio for methanol, chloroform, and water. After vortexing for 30 seconds and centrifugation for 20 minutes at 2000xg, a protein layer could become distinguishable in the interlayer. The lower chloroform layer was transferred to a new clean tube, dried to solid, and reconstituted in 30 µL of HPLC-grade methanol. It underwent further 30 seconds vortexing and centrifugation at 1500xg for 10 minutes prior to the injection into the column.
Sphingolipids underwent chromatographic separation using a Zorbax Eclipse Plus C8 column (4.6 x 150 mm, 5 µm) (Agilent Technologies, Santa Clara, USA) with a gradient elution of solvent A (1 mM ammonium formate in methanol with 0.2% formic acid) and solvent B (2 mM ammonium formate in water with 0.2% formic acid). The mobile phase initially consisted of 80% A and 20% B, with subsequent linear gradients to 90% A at 10.6 minutes, which was held for 6 minutes. Subsequently, a linear gradient to 99% A was applied up to 66 minutes, and back to 80% A at 68 minutes. The final condition was sustained for further 7 minutes until the end of run. Flow rate was 0.5 mL/min with a 15 µL sample injection volume.
Analyte m/z transitions were provided in Table 1. Quantification was done using multiple-reaction-monitoring (MRM) on triple quad mass spectrometer Agilent 6420 equipped with an electrospray ionization (ESI) ion source (Agilent Technologies, Santa Clara, USA). Source conditions included a gas temperature of 340 °C, vaporizer temperature of 250 °C, gas flow rate of 12 L/min, and nebulizer pressure at 20 psi. Positive and negative capillary voltages were set at 4500 V and 4000 V, respectively.
Intra-run variabilities ranged from 3.8% to 18.8%. Inter-run variabilities varied from 4.1% to 19.4%. Calibration ranges were: SPH (3.91-1001.7 nmol/L), SAP (3.50-223.7 nmol/L), S1P (10.3-1317.6 nmol/L), SAP1P (1.02-524.2 nmol/L), Cer C16:0 (0.055-14.1 µmol/L), Cer C24:0 (0.083-17.8 µmol/L), and SM C16:0 (7.89-1010.0 µmol/L).

Erjingtiao dried chili pepper (Capsicum annuum L. ‘Erjingtiao’) was purchased from the Longquanyi district vegetable wholesale market, Chengdu, Sichuan Province. Third-grade rapeseed oil (Yihai Kerry, Chongqing, China) was purchased from supermarkets in the Longquanyi district, Chengdu, Sichuan Province.
MS-grade methanol, MS-grade acetonitrile, and HPLC-grade 2-propanol were purchased from Thermo Fisher Scientific, Inc. (Cleveland, OH, USA), HPLC-grade formic acid and ammonium formate were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 2-methyl-3-heptanone was purchased from Beijing Manhag Biotechnology Co., Ltd. (Beijing, China).

This experiment utilized a variety of solutions and reagents. Liquid chromatography-mass-spectrometry-grade methanol and acetonitrile were both purchased from Thermo (Waltham, MA, USA). We sourced 2-chlorophenylalanine from Aladdin (Carlsbad, CA, USA). High-performance liquid-chromatography-grade formic acid was obtained from TCI (Tokyo, Japan). Liquid-chromatography-grade ammonium formate was purchased from Sigma (Darmstadt, Germany). The experimental water was ultrapure water with a resistivity of 18.2 MΩ/cm, and it was purified using a Millipore ultrapure water system (Burlington, NJ, USA). Additionally, ACS-grade chloroform was sourced from Wokai (Beijing, China). These solutions and reagents provided a solid guarantee for the accurate conduct of the experiment.