Phosphoric acid

Petroleum ether, methanol, and phosphoric acid were analytical pure and purchased from Beijing Chemical Factory (Beijing, China). HPLC-grade acetonitrile and HPLC-grade methanol were purchased from Fisher (Fisher Scientific, Fair Lawn, NJ, USA). The ultrapure water was prepared from a gradient water purification system (Water Purifier, Sichuan, China). Eicosapentaenoic acid (EPA, Lot No: 32510020), docosahexaenoic acid (DHA, Lot No: 0501808), and arachidonic acid (ARA, Lot No: 2491801) were purchased from TanMo Quality Testing Technology Co., Ltd. (Beijing, China). α-Linolenic acid (ALA, Lot No: U-62A-J25-C), linoleic acid (LA, Lot No: U-52A-JA3-C), and oleic acid (OA, Lot No: U-46A-MA6-C) were purchased from ANPEL Laboratory Technologies (Shanghai) Inc. (Shanghai, China). The purity of each standard compound was higher than 99%, as determined by HPLC. Six kinds of PUFAs standards were formulated to different concentrations using HPLC-grade methanol and filtrated with a 0.22 μm filter membrane prior HPLC injection.
Jilin Province Rana Industry Association provided a total of 27 samples of Oviductus Ranae, which were collected from the Changbai Mountains area in Jilin Province, China. Collection sources covered the major production areas of Changbai Mountains. The specific collection information is shown in Table 1, and Figure 2 shows the geographical distribution of the samples.

Polysuccinimide (PSI, M_w ≈ 100,000) was prepared by Luoyang Kang’en Chemical Company. EGDGE was obtained from Shanghai RuFa Chemical Technology Corporation. Sodium hydroxide, phosphoric acid, and ethanol were of analytical grade and purchased from Beijing Chemical Reagent Company. The water used for the experiments was previously deionized. Corynebacterium glutamicum (No. 10,109) and Bacillus Subtilis (No. 20,643) were used, and the strain was deposited in the China Centre for Industrial Culture Collection. The glutamic acid fermentation medium contained glucose (80 g/L), Na₂HPO₄·12H₂O (3 g/L), MgSO₄·7H₂O (1.8 g/L), KCl (1.7 g/L), methionine (2 g/L), MnSO₄·H₂O (2.5 mg/L), FeSO₄·7H₂O (2.5 mg/L), Vitamin B₁ (0.5 mg/L), molasses (1 g/L), corn steep liquor (4 mL/L), and soybean cake meal hydrolysate (20 mL/L). The γ-PGA fermentation medium contained glutamic acid fermentation broth (30 g/L), glucose (20 g/L), citric acid (20 g/L), (NH₄)₂SO₄ (5 g/L), K₂HPO₄·3H₂O (2.5 g/L), MgSO₄ (0.15 g/L), and MnSO₄ (0.04 g/L). The pH was controlled to within 7.3 ± 0.1.

All reagents used in this study were analytically pure and used as received without further purification. FP (Grade 40, radius 55 mm) was purchased from Whatman. Chemical reagents included ferric chloride hexahydrate (FeCl₃·6H₂O), anhydrous ethanol (C₂H₆O), sodium borohydride (NaBH₄), diphenylcarbonyl dihydrazide (C₁₃H₁₄N₄O), acetone (CH₃COCH₃), potassium dichromate (K₂Cr₂O₇), o-phenanthroline (C₁₂H₈N₂), and anhydrous sodium acetate (CH₃COONa). Above chemical reagents were produced by Tianjin Fuchen Chemical Reagent Factory. Sodium hydroxide (NaOH), hydrochloric acid (HCl), sulfuric acid (H₂SO₄), and phosphoric acid (H₃PO₄) were produced by Beijing Chemical Factory.

Three hundred and seventy seven batches of amoxicillin and potassium clavulanate ODFs produced by 26 manufacturers were collected in post-marketing surveillance in 2012 and 2014. There were 74 batches of tablets, 78 batches of dispersible tablets, 10 batches of chewable tablets, 96 batches of granules, and 120 batches of oral suspensions; 211 samples of amoxicillin capsule were from 100 batches provided by ZhuHai United Laboratories. The amoxicillin capsules included mixed intermediate granules of amoxicillin capsules as well as filled capsules and/or packaged capsules of the same batch. A reference standard of amoxicillin trihydrate (lot number: 130409-201011; content: 85.8%) and potassium clavulanate (lot number: 130429-201307; content: 95.0%) were provided by the US National Institutes for Food and Drug Control.
Methanol was purchased from Fisher Scientific (Pittsburgh, PA, USA). Phosphoric acid was obtained from Beijing Chemical Works (Beijing, China). Sodium dihydrogen phosphate dihydrate was purchased from Sinopharm Chemical Reagents (Beijing, China).