Simultaneous steroids measurement in dogs with hyperadrenocorticism using a column-switching liquid chromatography-tandem mass spectrometry method

Abstract

We developed an analytical method using an on-line column-switching liquid chromatography with triple quadrupole mass spectrometry (LC/MS/MS) for quantifying multiple steroids in serum. Using the developed method, we evaluated the serum concentration of nine steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 21-deoxycortisol, deoxycorticosterone, progesterone, 17α-OH-progesterone and aldosterone) in dogs with hyperadrenocorticism (HAC). Serum was mixed with stable isotope internal standards and thereafter purified by the automated column-switching system. The limit of detection ranged 2-16 pg/ml for nine steroids. In the baseline samples, five steroids (cortisol, corticosterone, cortisone, 11deoxycortisol, and 17α-OH-progesterone) were detected in all dogs. The concentrations of cortisone, 11deoxycortisol, and 17α-OH-progesterone in dogs with HAC (n = 19) were significantly higher those in dogs without HAC (n = 15, p < 0.02). After the adrenocorticotropic hormone stimulation test, six steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 17α-OH-progesterone, and deoxycorticosterone) were above the limit of quantification in all dogs. Cortisol, corticosterone, cortisone, and deoxycorticosterone concentrations of dogs with HAC were significantly higher than those of dogs without HAC (p < 0.02). In addition, 11-deoxycortisol and 17α-OH-progesterone concentration was higher in dogs with HAC than in dogs without HAC (p = 0.044 and p = 0.048, respectively). The on-line column-switching LC/MS/MS would be feasible for measuring multiple steroids in dog serum. The results suggest that cortisone, 11-deoxycortisol, and 17α-OH-progesterone would be related to HAC. Further studies are warranted to assess the clinical feasibility of steroid profile in dogs with HAC.

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

“…The achieved LOD was 0.002 ng/mL. The lowest LOQs of all the reviewed gestagen protocols were reported as 0.005 ng/mL for 17α-OH-progesterone and 0.007 ng/mL for progesterone [ 66 ]. The analysis of the serum samples for the combination of gestagens with estrogens was described in Section 4.2 .…”

“…Throughout the three subgroups, C18 columns were the most prevalently chosen stationary phase in LC-MS and were used in 91% of all the methods. Only two methods utilized π–π interactions for the chromatographic separation using Kinetex biphenyl or XBD phenyl columns [ 61 , 66 ]. Pre-columns [ 67 , 68 ] and trap-columns [ 66 ] were only utilized in the gestagen methods.…”

“…Only two methods utilized π–π interactions for the chromatographic separation using Kinetex biphenyl or XBD phenyl columns [ 61 , 66 ]. Pre-columns [ 67 , 68 ] and trap-columns [ 66 ] were only utilized in the gestagen methods. The trap column is used due to its superiority in separating hydrophilic compounds.…”

Serum steroid profiling of hepatocellular carcinoma associated with hyperadrenocorticism in dogs: A preliminary study

“…Cortisol is the main and potent glucocorticoid in dogs with HAC. Meanwhile, other adrenal steroids, such as 17α-OH progesterone, corticosterone, or 11-deoxycortisol, have been considered to cause HAC ( 19 – 21 ). Therefore, it may be helpful to measure multiple adrenal steroids, so-called steroid profile, to understand the link between HAC and HCC in dogs.…”

“…The chemical derivative reagent is an additional sample pretreatment method that reacts to the functional groups of target analytes (amine, hydroxyl, or carbonyl groups) to enhance better ionization ( 29 ). In our previous LC/MS/MS method, it was feasible to measure the serum levels of nine steroid hormones; however, nine steroids may not be enough for the steroid profile ( 19 ). Thus, we combined the conventional non-derivatization and keto-derivatization methods for measuring multiple steroid hormones and investigated the relationship between HAC and HCC in dogs using serum steroid profiling.…”