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Área de trabajo: Farmacéutica.

Título: Determinación de Proteínas y Carbohidratos por HPLC 2D con detección por Aerosol Cargado.

Título original: Determination of Proteins and Carbohydrates by 2D HPLC (RPLC and HILIC) with Charged Aerosol and Ultraviolet Detection.

Autor: Marc Plante, Bruce Bailey, Qi Zhang, David Thomas Thermo Fisher Scientifi c, Chelmsford, MA.

Introduction

Many of today’s pharmaceutical formulations use proteins and other biotechnological APIs. Along with these newer formulations come different excipients, namely surfactants, amino acids, salts, and carbohydrates. Carbohydrates are used to assist in the stabilization of the proteins in solution, and are used in different amounts, depending on the protein and the product requirements. Many carbohydrates are found in these formulations, including the sugars glucose, sucrose, lactose, maltose, and trehalose and the sugar alcohols sorbitol and mannitol. The quantitation of these carbohydrates in bioformulations can be complicated, since the carbohydrates are typically separated using HILIC where the protein API would irreversibly retain, but when using reverse phase, they have no retention. A method was developed that separates the carbohydrate excipient from the protein, and quantifies both simultaneously. The method injects the sample on to the reverse phase column, where the protein is retained, and the carbohydrates are not. These are captured into a 500 µL sample loop while being mixed with acetonitrile in preparation for the HILIC analysis. Once the carbohydrates are captured, the gradient elution of the protein is conducted, as well as the analysis of the carbohydrates by HILIC simultaneously.

 

Methods

Sample Preparations Standards for BSA and each of the carbohydrates were dissolved in deionized water at 10 mg/mL and then diluted to 1000 µg/mL for the protein with each of the carbohydrate combinations (sorbitol and sucrose; mannitol and lactose; and glucose, maltose, and trehalose) also at the same concentration. Standards were serially diluted by 50% each down to a lowest concentration of 3.9 µg/mL

 

Results

Calibration The HILIC method is designed to provide resolution between the seven, different carbohydrates. There is partial overlap of the sorbitol, mannitol, and glucose analytes, but they are rarely found together in a single formulation. The other carbohydrates are well resolved. Standards were prepared from 3.9 to 1000 µg/mL in water and injected in triplicate.

 

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