Tris(hydroxymethyl)aminomethane (TRIS), also known as tromethamine, is a weak base commonly used as a biological buffer system in the production of biological products. It can cause hypoglycemia, hypotension, nausea, vomiting, excessive intake or renal insufficiency can cause alkalemia, also inhibit breathing, and even stop breathing. Therefore, it is necessary to test the TRIS content in biological products. Then how to detect it?

At present, the quantitative detection of TRIS mainly adopts the method of acid-base titration, but its limit of quantitative is high, and it is susceptible to interference of various acid base groups. The biological products contain a large amount of carboxyl groups, amino groups and anions and cations, so TRIS in the product cannot be quantitatively detected by titration.

In order to solve the above problems, the researchers [1] invented a method for detecting TRIS Buffer in biological products by ion chromatography, and the specific steps are as follows:

(1) Water for injection are added to the biological freeze-dried product to dissolve and filter, or the biological aqueous solution is directly filtered, and the filtrate is taken as the test solution; TRIS is added to the water for injection to dissolve, and the solution of different concentrations is obtained as a reference solution after the gradient dilution;

(2) A pulse amperometric detector and the method of post-column replenishment are used. The column was a CS16 cation exchange column with a mobile phase of 40 mmol/L methanesulfonic acid solution at a flow rate of 1 mL/min. The post-column replenishment was an alkaline solution of sodium hydroxide or potassium hydroxide at a concentration of 500 mmol/L with a flow rate of 0.3mL/min;

(3) Quantitative analysis used the external standard method, the prepared various concentrations of the reference solution were tested, and the concentration-peak area standard curve of TRIS was drawn to obtain the regression equation of the standard curve; The sample solution was tested under the same conditions, and the content of TRIS in the test sample was calculated by the above regression equation.

The detection limit is 3.14×10-5mmol/L, the minimum limit of quantification is 1.05×10-4mmol/L, the recovery rate is 93.8%~100.1%, and the reproducibility range is 0.10%~0.52%. (RSD, n=5).

Compared with the traditional acid-base titration method, the method has lower detection limit and lower limit of quantitation, and is not interfered by the acid base group carried on the protein, and is not interfered with the anion and cation in the sample, and does not require derivatization and complex sample preparation. It can detect the content of TRIS (CAS 77-86-1) in different biological products, has good selectivity, and has no other peak interference in the TRIS retention time region. It has high detection sensitivity and fast analysis speed, and can be applied to the content detection and quality evaluation of TRIS in biological products of related industries.

Reference

[1] Yu Xiaoping, Yu Jin. A method for the determination of TRIS in biological products by ion chromatography. 2018, CN107782829A.