Ms grade methanol

MS-grade methanol (047192), MS-grade acetonitrile (51101), HPLC-grade 2-propanol (022906) were purchased from ThermoFisher (USA). HPLC-grade formic acid (5438040250) and HPLC-grade ammonium formate (714690-4X4L) were purchased from Sigma (Germany).

MS-grade methanol, MS-grade acetonitrile, HPLC-grade 2-propanol were purchased from Thermo Fisher. HPLC-grade formic acid and HPLC-grade ammonium formate were purchased from Sigma. CSS is composed of seven herbs, including Citrus reticulata Blanco, Radix Bupleuri, Ligusticum chuanxiong Hort, CyperusrotundusL., Citrus aurantium L., Paeonia lactiflora Pall. and Glycyrrhiza uralensis Fisch., and the ratio is 4:4:3:3:3:3:1. The herbs were purchased from the pharmacy of Jiangsu Provincial Hospital of Traditional Chinese Medicine, Nanjing University of Chinese Medicine. All herbs were authenticated by the herbal medicine botanist Professor Yunan, Zhao, Nanjing University of Chinese Medicine.

MS-grade methanol, MS-grade acetonitrile and HPLC-grade isopropanol were purchased from ThermoFisher (Thermo Fisher Scientific Co., Waltham, Massachusetts, USA). HPLC-grade formic acid, HPLC-grade ammonium formate and MTBE were purchased from Sigma-Aldrich (Sigma Chemical Co., St. Louis, Missouri, USA).

Sigma Aldrich provided Ammonium acetate (NH₄AC), Merck provided Acetonitrile, and Fisher provided ammonium hydroxide (NH₄OH) and methanol. Thermo Fisher provided MS-grade methanol, MS-grade Acetonitrile, and HPLC-grade 2-propanol. Sigma provided HPLC-grade formic acid and HPLC-grade ammonium formate. All chemicals used in this study were of high purity and quality, ensuring the reliability and reproducibility of the results.

For each sample, approximately 20 mg of clamp frozen tissue was weighed out on dry ice into a pre-cooled Precellys tube (Hard tissue homogenising CK28-R - 2 mL; Bertin Instruments, France). After weighing, 25 μL/mg of pre-cooled extraction buffer (50% (v/v) mass spectrometry (MS)-grade methanol (Thermo Fisher Scientific, UK), 30% (v/v) MS-grade acetonitrile (Romil, UK), 20% MS-grade water (Thermo Fisher Scientific, UK) and 100 ng/mL HEPES free acid) was added to each sample along with 20 μL internal standard containing 1 nmol ¹³C₄-succinate and 1 nmol ¹³C₃-malonate. Samples were homogenised using a Precellys 24 tissue homogeniser (6500 rpm, 2 × 15 s, Bertin Instruments, France) and transferred back onto dry ice. Following homogenisation, samples were centrifuged (17,000 g, 10 min, 4 °C) and the supernatant transferred to a pre-cooled microcentrifuge tube on wet ice. Samples were centrifuged again (17,000 g, 10 min, 4 °C) and the supernatant transferred to a pre-cooled MS vial. Vials were stored at −70 °C until analysis.

Naoxintong Capsule (NXT) (lot No. 170589) was provided by Shanxi Buchang Pharmaceutical Co., Ltd. (Xianyang, China). Myristic acid-D27 (98%) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, USA). MS grade formic acid, streptozotocin (STZ) and glucose were purchased from Sigma-Aldrich Co. (St. Louis, USA). Insulin was obtained from Novo (China) Pharmaceutical Co., Ltd. (Beijing, China). Rodent standardized diet was provided by Guangdong Medical Laboratory Animal Center (Foshan, China). Rodent high-fat diet (HFD) with 60 kcal% fat was purchased from Research Diets Inc. (D12492, New Brunswick, USA). MS grade methanol and acetonitrile were purchased from Thermo Fisher Scientific Inc. (Fair Lawn, USA). Deionized water was purified by the Milli-Q system (Millipore, Merck KGaA, Darmstadt, Germany) and filtered through 0.22 mm membrane filter prior to use.