GB/T 33412-2016 in English

GB/T 33412—2016 Standard Overview

National Standard of the People's Republic of China GB/T 33412—2016 "High Performance Liquid Chromatography for the Determination of Hydroxycitric Acid in Biological Products" specifies the method for the determination of hydroxycitric acid in health (functional) biological products using high performance liquid chromatography (HPLC). This standard is applicable to the detection of hydroxycitric acid in biological products such as oral liquids, capsules, and granules.

Background of Standard Formulation

Hydroxycitric acid is an important biologically active substance, widely used in medicine, food and other fields. In order to standardize its determination method in biological products and ensure the accuracy and consistency of the test results, the National Standards Administration organized the drafting of this standard. This standard is based on GB/T1.1—2009 "Guidelines for Standardization Work Part 1: Structure and Writing Rules of Standards", and is scientific, practical and operational.

Technology Evolution Analysis

With the development of high performance liquid chromatography technology, the detection method has gradually shifted from traditional spectrophotometry to the more accurate and efficient HPLC method. This standard uses the external standard method for quantification, combined with a UV detector, to ensure high sensitivity and accuracy of the determination. Compared with previous standards, this standard is more refined in terms of reagent preparation, instrument requirements and data processing.

Comparison of standard frameworks

Reagent and instrument requirements

According to clauses 4.1 to 5.7 of the standard, the reagents and instruments required for the test include:

• High performance liquid chromatograph (with UV detector)
• Analytical balance (sensitivity 0.01g and 0.001g)
• High-speed centrifuge (10000g)
• Ultrasonic cleaner
• Methanol (chromatographic grade), phosphoric acid, hydroxycitric acid standards (CAS No.: 6205-14-7), etc.

Determination steps interpretation

According to clauses 7.1 to 7.3 of the standard, the determination steps are as follows:

1. Liquid chromatography conditions: Use Atlantis T3C18 chromatographic column (150mm×4.6mm, 5μm), the mobile phase is 0.1% phosphoric acid aqueous solution + methanol (volume ratio 99:1), the flow rate is 0.6mL/min, the column temperature is room temperature, and the detection wavelength is 210nm.
2. Standard curve drawing: Pipette hydroxycitric acid standard solutions of different concentrations, filter and inject, draw the standard curve with peak area as the ordinate and concentration as the abscissa, and the correlation coefficient is not less than 0.999.
3. Sample analysis: Prepare the sample solution, filter and measure under the same conditions, and calculate the content using the standard curve.

Result calculation and method characteristics

According to clauses 8.1 to 9.3 of the standard, the calculation formula for the determination result is as follows:

• Solid sample: ρ = (A - α)/b; w = ρ × V / m
• Liquid sample: ρi = ρ × V / Vi

Determination range: Inject 10μL, under the premise of correlation coefficient ≥0.999, the linear measurement concentration response range of hydroxycitric acid is 0.1 mg/mL～1.000 mg/mL.

Implementation suggestions

In order to ensure the effective implementation of the standard, it is recommended that:

• The laboratory should be equipped with instruments and equipment that meet the requirements and calibrate them regularly.
• The operator needs to be professionally trained and familiar with the operation of HPLC and UV detectors.
• Prepare reagents strictly according to standards to ensure data accuracy.
• Establish a quality control system and regularly verify the recovery and precision of the method.