Tropic acid

A urine volume containing 0.5 mmol of creatinine was analyzed, in order to normalize metabolites differences affected by hydratation. Urine pH was adjusted to 14 by adding 30% NaOH.
Urine metabolites were oxidized with 0.5 mL of hydroxylamine hydrochloride (2.5 mg/mL in water) and heated at 60 °C for 60 min. After cooling, the samples were acidified by using several drops of 2.5 M H₂SO₄ until pH 1 was reached. Internal standards, dimethylmalonic acid (10 mg/mL in 1:1 of H₂O/CH₃CH₂OH (v:v)), pentadecanoic acid (10 mg/mL in CH₃CH₂OH), and tropic acid (10 mg/mL in H₂O) (Sigma-Aldrich, St. Louis, MO, USA) were prepared and then added to the sample where they had the final concentrations of 10 μM. Samples were extracted three times with 2 mL of ethyl acetate and centrifuged at 3000 rpm for 5 min. At the end of centrifugation, the organic phase was collected and saturated with approximately 1 g of Na₂SO₄ (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at RT to remove water. After centrifugation (3000 rpm for 10 min), the organic phase was collected and evaporated at 40 °C under a gentle nitrogen flow.
Finally, the sample was derivatized in 50 µL of N,O-bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich, St. Louis, MO, USA) at 60 °C for 30 min. The mixture was cooled at RT and transferred into 250 µL conical glass inserts in order to carry out the subsequent GC-MS analysis.

Progesterone injection was purchased from Xianju Pharma (Taizhou, China). Pentobarbital was obtained from Xiya Reagent (Chengdu, China). Ultrapure water was produced by a Milli-Q plus water purification system (Milford, USA). N,O-bis(trimethylsilyl)-trifluoroacetamide-trimethylchlorosilane (BSTFA-TMCS) (99 : 1, v/v), urease, margaric acid, tropic acid, methoxylamine hydrochloride, pyridine, and metabolite standards were purchased from Sigma-Aldrich (St. Louis, USA). Absorbable gelatin sponge was purchased from Jinling Pharmaceutical Co. (Nanjing, China).
PSF was purchased from Tianxiang Co. (Yueyang, China) and identified by Professor Zhuxin Wang (Hunan university of Chinese medicine, Changsha, China). A voucher specimen (number HHBJC20140913) is deposited in the Key Laboratory of Hunan Province for Traditional Chinese Medicine in Obstetrics & Gynecology Research (the Maternal and Child Health Hospital of Hunan Province, Changsha, China). Referring to previous method [25 (link)], a 500 g of PSF was two times extracted with 5 L of hot ethanol for 1 hour and then the two extracts were filtrated. The filtrate was dried in vacuo to yield 63.9 g of the PSF extract.