Simplified Liquid Chromatography–Mass Spectrometry Methods for Gestagen Analysis in Animal Fat and Liver

Abstract

Gestagens, a class of veterinary drugs also called progestogens, are synthetic hormones used to increase feed efficiency and rate of gain in heifers. The Canadian Food Inspection Agency analyzes progestogens melengestrol acetate (MGA), megestrol acetate, and chlormadinone acetate using liquid chromatography−mass spectrometry (LC−MS). Our conventional gestagen method for kidney fat has many time-consuming steps, including solid-phase extraction. A sample preparation procedure having fewer cleanup steps was developed for routine diagnostic analysis of kidney fat and provided similar results faster, and at lower cost. A confirmatory liver method for gestagens, developed using salt-assisted extraction, employed minimal clean-up steps that resulted in high chemical background at the desired lower limit of quantification (LLOQ). Differential ion mobility spectrometry, specifically high-field asymmetric waveform ion mobility spectrometry (FAIMS), was used to filter chemical background in the gas phase. The effect of the ionization probe position on FAIMS parameters, including sensitivity, is described. With LC-FAIMS-MS, chemical background for each gestagen was virtually eliminated, resulting in a quantitative liver method having the desired 0.6 ng/g LLOQ and estimated limits of detection (LODs) up to 140 times lower than LC-MS. Incurred MGA samples, analyzed using kidney fat and liver methods from the same animal, show levels within the quantitative ranges of both methods.

Sample Preparation Techniques for Growth-Promoting Agents in Various Mammalian Specimen Preceding MS-Analytics

“…Then, several salts (NaCl; Na 2 SO 4 and MgSO 4 ) were subsequently added, with shaking in between. After centrifugation, the supernatant was evaporated to dryness, and the residue was reconstituted in ACN/0.1% FA in water (7/3, v/v ) [ 71 ]. While the longer extraction method using SPE has shown good results, it can be replaced by the shortened method to safe time.…”

“… Chromatography Ionization Detection Monitoring Mode Column Temperature [°C] Flow Rate [mL/min] Mobile Phase A Mobile Phase B Reference LC ESI + TQ MRM Biphenyl (100 mm × 2.1 mm, 1.7 µm) and trap column Unison UK-C1 HT (10 mm × 4.6 mm, 3 µm) n.a. 0.4, 0.8, 0.4 Pump A MeOH/water Pump B, 50% ammonium fluoride in water/50% ammonium fluoride in MeOH [ 66 ] LC HESI + TQ SRM C18 (50 mm × 2.1 mm, 1.7 µm) 45 0.4 0.1% FA in water MeOH [ 71 ] LC ESI + QExactive tSIM HSS T3 (100 mm × 2.1 mm, 1.8 µm) and HSS T3 VanGuard pre-column (5 mm × 2.1 mm, 1.8 µm) n.a. 0.45 0.1% FA in water 0.1% FA in ACN [ 67 ] LC ESI + …”