Direct injection LC/ESI‐MS horse urine analysis for the quantification and identification of threshold substances for doping control. I. Determination of hydrocortisone

Abstract

Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for both methods. Matrix effects were evaluated by preparing and analyzing calibration curves in water solutions and different horse urine samples. A great variation of the signal both for hydrocortisone and the internal standard was observed in different matrices. To overcome matrix effects, the unavailability of blank matrix and the excessive cost of the isotopically labeled internal standard, standard additions calibration method was applied. This work is an exploration of the performance of the standard additions approach in a method where neither nonisotopic internal standards nor extensive sample preparation is utilized and no blank matrix is available. The relative standard deviations of intra and interday analysis of hydrocortisone in horse urine were lower than 10.2 and 5.4%, respectively, for the LC/IT-MS method and lower than 8.4 and 4.4%, respectively, for the LC/TOF-MS method. Accuracy (bias percentage) was less than 9.7% for both methods.

Development of an analytical method for the targeted screening and multi-residue quantification of environmental contaminants in urine by liquid chromatography coupled to high resolution mass spectrometry for evaluation of human exposures

“…Moreover, the elimination of the sample preparation step limits the duration of the analytical process. Unlike the more selective techniques, the lack of sample preparation makes the analysis simple and fast [29,30] and this is of particular importance in the context of the Exposome which often requires high throughput methods. However, after a cycle of freezing and thawing, urine becomes cloudier.…”

“…Different researchers showed that the use of diluted urine was preferable to more selective techniques, such as solid phase extraction [29,30]. Furthermore, this generic strategy was used in several studies [28,31] since it is time-saving and allows for the analysis of large cohorts.…”

Direct injection horse‐urine analysis for the quantification and confirmation of threshold substances for doping control. IV. Determination of 3‐methoxytyramine by hydrophilic interaction liquid chromatography/quadrupole time‐of‐flight mass spectrometry

“…[18,19] Horse urine is a complex matrix that contains numerous different compounds, ranging from simple inorganic salts to large proteins. In previous works with direct injection horse urine analysis [11,12] without the use of stable isotope labelled IS, great variability was observed in absolute and relative responses of the analyte and the IS originating from matrix effects, even though a fourfold sample dilution was applied, resulting in quantitative analysis using standard additions approach.…”

“…[7 -10] This work is a continuation of a series of studies [11,12] on the application of direct injection LCMS analysis for the quantification and identification of threshold substances in horse urine. Hydrophilic interaction liquid chromatography was selected as an alternative chromatographic technique using polar stationary phases and low aqueous/high organic mobile phases in order to achieve effective retention of such a polar analyte.…”

“…Additional advantages of HILIC include increased ESI sensitivity due to high organic content in the mobile phase, higher flow rates due to lower column back-pressure and less matrix effect. [7 -10] This work is a continuation of a series of studies [11,12] on the application of direct injection LCMS analysis for the quantification and identification of threshold substances in horse urine.…”