Determination of tolbutmide by Ultra High Performance Convergence Chromatography (ACQUITY UPC2TM) System

purpose

Using Waters (Waters®) ACQUITY UPC ² â„¢ system, successfully measuring tolbutamide drug content USP normal phase HPLC method is converted into a supercritical fluid chromatography method.

background

Supercritical liquid chromatography (SFC) is a normal phase chromatographic separation technique that uses CO2 as the primary mobile phase, typically using a polar solvent such as MeOH as a modifier. Since the principle of SFC is similar to the principle of HPLC, the current method should be able to be converted to the SFC method, thereby reducing the amount of solvent used and treatment, reducing the cost per analysis, while enhancing environmental protection. Chromatographic methods converted to SFC must maintain data quality and must obtain experimental results consistent with current normal phase chromatography methods. Currently, the United States Pharmacopoeia (USP) specifies a normal phase HPLC method containing a drug containing carbendazim (benzene sulfonamide, CAS # 64-77-7). Isocratic separation using a 4.0 x 300 mm silica gel column (L3), flow rate 1.5 mL/min, mobile phase 475:475:20:15:9 n-hexane: water-saturated n-hexane solution: tetrahydrofuran: glacial acetic acid The solution was mixed and the run time was approximately 20 minutes. As in most Pharmacopoeia methods, this method is proven and reliable. However, the analysis process uses

A complex mobile phase mixed solvent containing n-hexane and tetrahydrofuran. Many laboratories want to eliminate the use of these solvents for environmental and cost reasons.

The new ultra-high performance convergent chromatography (UPC ² TM) method yields data comparable to current HPLC methods , even better, at 10 times faster than current HPLC methods and consumes less solvent.

solution

The invertase was mixed with the internal standard, methylglycine, and samples were prepared and analyzed using the current USP method. The results of the analysis were compared with the results obtained using the ACQUITY UPC2 method. The conditions of the UPC2 method are as follows:

Column: ACQUITY UPC2 BEH, 3.0 x 100 mm, 1.7 microns

Temperature: 50 °C

Mobile phase: 95% CO2: 5% methanol/isopropanol (1:1) with 0.2% TFA

Flow rate: 2.5 mL/min

Back pressure: 120 Bar/1740 psi

Detector: UV / PDA, 254 nm

The current normal phase HPLC method yields an acceptable chromatographic separation (see Figure 1), although the internal standard chromatographic peak tailing is severe (tailing factor 1.65). Since the listed adaptation criteria have been passed (the relative standard deviation of repeated injections does not exceed 2.0%; the resolution R of tolazamide and methylprednisolone is not less than 2.0), no further improvement has been made.

The results obtained by the newly developed UPC ² method also met the requirements of the US Pharmacopoeia (the retention time RSD values ​​of methyl sulphonate and tolazamide were 1.2% and 0.9%, respectively, and the area RSD values ​​of the two compounds were less than 0.90). %, n=6), while maintaining the separation between the two target compounds (R = ~15), the running time is greatly shortened. The tailing of the internal standard, tolazamide, was greatly improved (tailing factor 1.2). It should be noted that the use of UPC2 to separate and detect many small peaks from the mixture indicates that the method has high separation efficiency. In this example, approximately 29 mL of n-hexane and less than 1 mL of each of tetrahydrofuran and ethanol were used each time for normal phase HPLC analysis. In contrast, approximately 0.25 mL of methanol and isopropanol were used per injection in the UPC ² method. This shows that by converting the normal phase HPLC method to the UPC ² method, the use of the organic solution can be greatly reduced. Based on current solvent prices, the cost of each normal phase HPLC analysis is approximately $1.40, and the cost per UPC ² analysis is approximately $0.01, indicating that the cost can be greatly reduced by converting the normal phase HPLC method to the UPC ² method.

to sum up

The AC Pharmacy HPLC method can be successfully converted to the UPC ² method using ACQUITY UPC ² . The data from this new UPC ² method is comparable to current HPLC methods, even better, at 10 times faster than current HPLC methods, and consumes less solvent. We get high-quality analytical data faster, increasing laboratory productivity and reducing the cost of analysis per sample. The ACQUITY UPC ² system is an ideal solution for laboratories wishing to convert the current normal phase HPLC method into a more efficient and cost effective method, while also enhancing health, safety and environmental protection.