Determination of Estradiol Chromatographic Purity Using an Ultra High Performance Convergence Chromatography System

purpose

Using Waters ACQUITY UPC ² â„¢ system estradiol impurity analysis can be obtained, and the United States Pharmacopeia (USP) method comparable or better results.

background

Currently, the United States Pharmacopoeia (USP) uses estradiol (esradiol) chromatographic purity using a 4.6 x 250 mm silica gel column with 2,2,4-trimethylpentane, n-butyl chloride, methanol 45:4: The mobile phase of 1 has a flow rate of 2 mL/min. Since many laboratories want to limit the use of aliphatic hydrocarbons and chloride solvents, alternative chromatographic methods such as supercritical fluid chromatography (SFC) must be evaluated. The Waters ACQUITY UPC ² System was used to develop methods for determining the purity of estradiol chromatography. Results ^{Ultra Performance Convergence Chromatography â„¢ (UPC 2} â„¢) and Comparative obtained directly from the current U.S. Pharmacopeia method of detecting impurities in estradiol. The results of the two methods were similar, and the sensitivity of the UPC ² method for detecting estradiol impurities was higher than that of the normal phase HPLC method used in the US Pharmacopoeia. In addition, with UPC ² , the run time of the sample is greatly reduced, and the total cost per analysis is also significantly reduced (based on solvent usage and waste disposal costs).

The chromatographic purity of estradiol was measured using the UPC2 method at a rate three times that of the current normal phase HPLC method , while the cost of a single analysis was reduced by more than 100 times.

solution

Estradiol was prepared and analyzed using current US Pharmacopoeia methods, as shown in Figure 1. The results of the HPLC analysis were compared with the results of the ACQUITY UPC ² system analysis (using the same sample preparation method) as shown in FIG.

Column: ACQUITY UPC ² BEH, 2.1 x 150 mm, 1.7 micron

Mobile phase: A = CO ₂ B = 1:1 methanol / isopropanol

Back pressure: 130 bar / 1880 psi

Column temperature: 45 °C

Detection: UV / PDA, 280 nm

The results obtained by the two test methods are compared in Table 1. Both the normal phase HPLC method and UPC ² detected at least 5 impurities in an amount of less than 0.1% by area. In both methods, the signal-to-noise ratio of the peak in the range of 0.01% is about 3:1, and the value obtained by the UPC ² result is slightly higher. The maximum impurity (about 0.05% by area) measured by the UPC ² method has a signal-to-noise ratio of 16:1 and the normal phase HPLC method measures 9:1. These experimental results clearly show that the ACQUITY UPC ² system can be successfully used to analyze trace impurities in estradiol. The run time of the UPC ² method was significantly shorter than the time used for the normal phase HPLC method (20 min vs. 60 min), thereby increasing laboratory productivity. Cost analysis for each run showed that the solvent cost for normal phase HPLC was $5.89, while the cost for each run was only $0.05 with UPC ² . The mixed chloride waste liquid to be treated by the normal phase HPLC method was 108 Ml 2 , 2,4-trimethylpentane, 9.6 mL of n-butyl chloride and 2.4 mL of methanol. The waste liquid to be treated produced by the UPC ² method was 0.60 mL each of methanol and isopropanol. The CO ₂ used in the separation is discharged through a laboratory exhaust pipe. Using the UPC ² method, waste disposal costs are reduced by as much as 150 times. 2,2,4-Trimethylpentane, 9.6 mL of n-butyl chloride and 2.4 mL of methanol. The waste liquid to be treated produced by the UPC ² method was 0.60 mL each of methanol and isopropanol. The CO ₂ used in the separation is discharged through a laboratory exhaust pipe. Using the UPC ² method, waste disposal costs are reduced by as much as 150 times.