Characterization of N-acetyltryptophan degradation products in concentrated human serum albumin solutions and development of an automated high performance liquid chromatography–mass spectrometry method for their quantitation

N-acetyltryptophan (NAT) has long been used as a stabilizer in some protein solutions, such as human serum albumin, to prevent oxidative protein degradation. However, the fate of NAT has not been discussed in literature. Two NAT degradation products have been observed in concentrated albumin solutions (20% and 25%) and identified as 1-acetyl-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid and 1-acetyl-3a,8a-dihydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid. To monitor the levels of these two previously unidentified NAT degradation products in concentrated albumin solutions, a fully automated method, incorporating online size exclusion chromatography (SEC) trapping and reversed-phase high performance liquid chromatography–mass spectrometry (HPLC–MS) with multiple reaction monitoring (MRM) analysis, has been developed and validated for their quantitative analysis. The method does not require an internal standard. The only sample manipulation is to obtain an albumin concentration of 4% in all standards and test HPLC samples. A limit of quantitation (LOQ) as low as 20 ng/mL has been achieved for both compounds. This method can readily be adopted for the quantitative determination of other small molecules in concentrated protein solutions.

► Two N-acetyltryptophan degradation products were newly identified.
► A quantitation method incorporating SEC trapping and HPLC–MS was developed.
► The method was validated for their quantitation in human serum albumin solutions.
► Automation minimized sample preparation and increased precision.
► Established an approach for small organic analytes quantitation in protein solutions.