Development of open tubular columns for application in nano liquid chromatography coupled to tandem mass spectrometry

Author(s)

Natalia Gabrielly Pereira dos Santos

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Química de São Carlos (IQSC/BT)
• Defense date: 2023-11-07

Examining board members

Fernando Mauro Lanças; Deyber Arley Vargas Medina

• Advisor: Fernando Mauro Lanças

Abstract

The development of this doctoral thesis consisted of one of the main works written by the student during the postgraduate period, which can be considered as innovative themes within the field of chromatography, such as the application of open tubular columns in chromatography liquid, and the coupling of the technique with mass spectrometry with electron impact ionization. In addition, the last chapter brings the development of an analytical methodology for the analysis of N-nitrosamines in drugs from the sartan class, an impurity with carcinogenic potential which has been the subject of discussion and control in the pharmaceutical market. Therefore, this work aimed to contribute to the development of WCOT-type open tubular columns for nanoLC-ESI-MS/MS. At first, a methodology for the preparation of these columns was proposed, adapting the method proposed by Kurt Grob to the laboratory\'s experimental conditions. Then, a screening of stationary phases for coating the WCOT column was performed. The phases tested were: OV-73, OV-17, OV-210, OV-225 and OV-275, purchased from Ohio Valley. For column evaluation, atrazine, clomazone and metolachlor standards were used as analytical standards. The stationary phase that presented the best results for nanoLC- ESI-MS/MS, among those evaluated, was OV-210 (50% trifluoropropyl 50% dimethyl poly siloxane), which went on to the next stage of the work: WCOT column optimization. In this optimization step, parameters such as column length, internal diameter, mass of stationary phase for column coating, injection volume and kinetic parameters such as optimal linear velocity and performance-velocity evaluation, using Van Deemter and Poppe graphs, were evaluated. respectively. Finally, the optimized WCOT column was coupled to a packed trapping column, which enabled better chromatographic separation results. The other experimental work consisted in the development of an analytical methodology for the analysis of N-nitrosamines in medicines, in this case, in Losartan Potassium, widely consumed in the treatment of hypertension. The methodology consisted of automating MEPS-type sample preparation (packaged sorbent microextraction) in an arduino-controlled robotic system. The analytical method was developed and validated in UHPLC-APCI-MS/MS, and showed good results for linearity, intra-day and inter-day precision, among other figures of merit. What stands out most about this methodology is the high-throughput capacity of the robotic system, which allows the preparation of six simultaneous samples in less than 20 minutes, which is a great advance for the monitoring of these impurities in routine analyses. (AU)

• FAPESP's process: 19/26263-4 - Development of open tubular capillary columns for micro and nano liquid chromatography coupled to sequential mass spectroscopy
• Grantee: Natalia Gabrielly Pereira dos Santos
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)