Fructose sodium diphosphate injection is a drug used to treat hypophosphatemia and is commonly used as an adjuvant therapy for myocardial ischemia in clinical practice. Hypophosphatemia can occur in acute situations such as blood transfusion, surgery under extracorporeal circulation, and parenteral nutrition, and is also associated with chronic diseases such as chronic alcoholism, long-term malnutrition, and carbonate depletion in chronic respiratory failure. Fructose sodium diphosphate injection is widely used in clinical practice, but due to its structural characteristics, the determination of fructose sodium diphosphate is often carried out using derivative UV spectrophotometry; At the same time, impurities may be introduced in the production process of fructose diphosphate sodium, including related substances such as fructose phosphate, glucose phosphate, and fructose. These impurities are strong polar compounds with low content and cannot be detected in ordinary reverse phase chromatography and differential detectors. There are literature reports on the use of paper electrophoresis for separating and detecting phenylenediamine after color development, as well as thin-layer chromatography for detection [2-4]. There are also literature reports on the use of solid composite enzyme method to determine the content of fructose sodium diphosphate [5]. These methods are not only cumbersome to operate, but also have issues with repeatability and sensitivity.

This experiment uses ion chromatography pulse amperometry to separate and determine sodium fructose diphosphate and three related substances, with good separation accuracy, high sensitivity, and easy operation. An effective method for detecting fructose sodium diphosphate and its related substances has been established.

Test condition instrument: ICS-5000 chromatograph (ThermoFisher company), equipped with quaternary gradient pump, ED amperometric detector, AS-AP automatic sampler chromatography column: CarboPac PA 20 protection column 6 0 µ m, 50 × 3 mm, (P/N: 060144) CarboPac PA 20 separation column 6 0 µ m, 150 × 3 mm, (P/N: 060142) column temperature: 30 ℃; Rinsing solution: NaOH/NaOAc gradient rinsing flow rate: 0.4 mL/min; Quantitative loop: 20 µ L; detection method: pulse integral ampere detection (four potential waveform)

After diluting the sample by an appropriate multiple, it was filtered through a 0.22 µ m nylon filter membrane and the filtrate was directly analyzed on the machine.

The actual sample analysis of commercially available fructose sodium diphosphate injection was carried out. After dilution, it was filtered through a 0.22 µ m filter membrane and determined according to the selected chromatographic conditions. The external standard method was used for quantification. The spectrum is shown in Figure 2, and the analysis results are shown in Table 2.

Results and Discussion: The selection of chromatographic columns and optimization of eluent gradient conditions. The structure of fructose diphosphate sodium contains two phosphate groups, which can be separated by anion exchange chromatography. At the same time, its related substances are fructose phosphate, glucose phosphate, and fructose, all of which contain hydroxyl groups and can have signals on an amperometric detector. To ensure the separation of chromatographic peaks and the rationality of retention time, we chose the CarboPac PA 20 sugar column and NaOH/NaOAc gradient elution method. The chromatograms of several substances under the above chromatographic conditions are shown in the following figure (Figure 1)

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