Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 A new robust technique for testing of glucocorticosteroids in dogs and horses Terry E. Webster Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Veterinary Toxicology and Pharmacology Commons Recommended Citation Webster, Terry E., "A new robust technique for testing of glucocorticosteroids in dogs and horses" (2007). Retrospective Theses and Dissertations. 15029. https://lib.dr.iastate.edu/rtd/15029 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. A new robust technique for testing of glucocorticosteroids in dogs and horses by Terry E. Webster A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Toxicology Program o f Study Committee: Walter G. Hyde, Major Professor Steve Ensley Thomas Isenhart Iowa State University Ames, Iowa 2007 Copyright © Terry Edward Webster, 2007. All rights reserved UMI Number: 1446027 Copyright 2007 by Webster, Terry E. All rights reserved. UMI Microform 1446027 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ii DEDICATION I want to dedicate this project to my wife, Jackie, and my children, Shauna, Luke and Jake for their patience and understanding without which this project would not have been possible. iii TABLE OF CONTENTS LIST OF FIGURES iv LIST OF TABLES vi CHAPTER 1. INTRODUCTI ON 1 CHAPTER 2. LITERATURE REVIEW 2 CHAPTER 3. METHOD S AND MATERIALS 19 CHAPTER 4. RESULTS 26 CHAPTER 5. SUMMARY AND INTERPRETATION 53 CHAPTER 6. CONCLUSIONS 55 APPENDIX : COMPOUND STRUCTURES 56 REFERENCES CITED 61 ACKNOWLEDGEMENTS 67 iv LIST OF FIGURES Figure 1. Chemical structure of cortisol with steroid numbering system 2 Figure 2. Route from cholesterol to cortisol 4 Figure 3. Dexamethasone and b etameth asone with major metabolites 6 Figure 4. Illustration of electron impact for molecule ABC 12 Figure 5. Diagram of chemical ionization reactions 13 Figure 6. Total Ion chromatograph of 100 ng/ml standard in cani ne urine 32 Figure 7. Ion c hromatograph using filters for c ortisone and hydrocortisone 33 Figure 8. Ion chromatograph and mass spectra of cortisone and hydrocortisone 34 Figure 9. Chromatograph and mass spectra of dexamethasone and betamethasone 35 Figure 10 . pH vs. drug recovery for first set of analytes (100 ng/ml) in equine plasma. 39 Figure 11 . pH vs. drug recovery for second set of analytes (100 ng/ml) in equine plasma. 39 Figure 12. pH vs. drug reco very for third set of analytes (100 ng/ml) in equine plasma. 40 Figure 13. Extracted dexamethasone calibration curve in canine urine. 40 Figure 14. Dexamethasone elimination study area counts vs. hours post dose 43 Figure 15. De xamethasone elimination study results last 48 hours on smaller scale 43 Figure 16. Dexamethasone elimination study concen trations vs. hours post dose 44 Figure 17. Hydrocortisone concentration in equine dexamethasone study 46 Figure 18. Cortisone concentration in dexamethasone equine urine study 47 Figure 1 9. Dexamethasone concentration in dexamethasone equine urine study 47 Figure 20 . Comparison of dexamethasone concentration to cortisone and hydrocortisone 48 Figure 21 . Chromatograph and mass spectra of fludrocortisone metabolite 50 Figure 2 2. Chromatographs of fludrocortisone and metabolite 51 Figure 23. Chemical structure of fludrocortisone 51 Figure 24. P roposed c hemical stru cture of fludrocortisone metabolite 52 v Figure 25. Structures of amcinonide through cortisone 56 Figure 26. S tructures of deoxycorticosterone to flumethasone 57 Figure 27. S tructures of fluocinolone acetamide to medrysone 58 Figure 28. S tructure s of methyprednisolone to prednisone 59 Figure 29. S tructures of triamicinolone and triamicinolone acetomide 60 vi LIST OF TABLES Table 1. List of various endogenous and exogenous glucocorticoids 8 Table 2. HPLC gradient with m obile phase composition gradient 24 Table 3. Ions and retention times (RT) using LC/MSn 26 Table 4. Mass spectrometer tune parameters 28 Table 5. Extraction recoveries of select analytes in canine urine at various pH levels 35 Table 6. Average recoveries and R 2 of standards 0.5 - 200ng/ml in stripped canine urine 36 Table 7. Extraction recoveries of select analytes in stripped equine plasma at varying pH levels at 100 ng/m l 37 Table 8. Canine urine volume and pH value in dexam ethasone study 41 Table 9. Dexamethasone in canine urine results 42 Table 10 . Hydrocortisone in equine urine dexamethasone study 44 Table 11. Cortisone in equine urine dexamethasone study 45 Table 12. Dexamethasone in equine urine study 45 Table 1 3. Calibration curve in fludrocortisone canine urine study 48 Table 1 4. Fludrocortisone for run s 1 -3 in canine urine 49 Table 1 5. Hydrocortisone in fludrocortisone canine urine study 49 Table 1 6. Cortiso ne in fludrocortisone canine urine st udy 49 Table 1 7. Fludrocortisone metabolite areas in canine urine 50 1 CHAPTER 1. INTRODUCTION Gluc oco rticosteroids are a class of steroids that have been used to obtain beneficial and detrimental resul ts in the horse and dog racing industry. Gluc oco rticosteroids , also known as glucocorticoids, are used to treat inflammation in horses and dogs that have been stressed or injured to allow the animals to return to the racing circuit more quickly than natur al ly . While these treatments, when used correctly , wou ld bene fit th e animal just as anti -inflamma tories benefit humans, they can also cause detrimental effects to the health of the animal. Glucocorticoids are a key ingredient in maintaining homeostasi s in the body . This homeostasis is affected by t rauma, exercise and cold that increas e production of the corticotrop in releasing factor in the hypothalamus (Wilke et al 1982) . The corticotropin releasing factor increases the production of glucocorticoids (Wilke et al 1982 ). There are two reasons for interest in the ability to detect glu cocorticoids in racing animals. First, glucocorticoid residuals in the animal are undesirable in the racing industry as the residuals can be evidence of dosing the anima l. Since these chemicals decreas e the amount of pain the animal experien ces, the animal may increase trauma due to lack of feedback. The decreased pain may be a result of reduced pressure on the nerves due to less inflammation but the exact reason is un clear (Martindale, 2005). The second reason is that the treatment with gluco co rticoids can affect performance that could affect the outcome of the pari -mutuel races. Par i-mutuel racing, dogs and horses, generated over $4 billion in revenues and receipts in the United States in 2002 (U.S. Census Bureau 2002 ). This paper presents research to characterize and quantify glucocorticoi ds in equine urine and plasma and in canine urine. This paper also summarizes an elimination study of an under -examined glucocor ticoid. The research incorporates a liquid \liquid extraction from a small aliquot of sample. This aliquot is analyzed for many glucocorticoids in a single liquid chromatography mass spectrometry run. 2 CHAPTER 2. LITERATURE REVIEW Cortisol (hydrocortison e) is the principal glucocorticoid in horse and dog blood and also the most potent naturally produced corticosteroid ( Gower , 1984) . Cortisol is produced by the adrenal gland, decreases the body’s response to stress, decreases inflammation, and can cause e uphoria. One study states the no rmal range in horses to be 50 -3500 nmol/L (Irvine et al 1988) . Cortisol maintains homeostasis in the body . I ntroduction of compounds that mimic cortisol decrease s cortisol production (Toutain et al , 1984) . Synthesis : “The adrenal cortex produces corticosteroids which are a group of C 21 steroids” (Gallciano , 1985) . “This group known as 4 -en -3-ones contains a double bond at C-4 and an oxo group at C -3. These compounds also have a side chain at C -17 and may or may not co ntain a hydroxyl group. The 17 - hydroxylated glucocorticoids are more powerful than the non -hydroxylated count erparts. There is hydroxyl at C -21 and an oxo group at C -20. ” “There also needs to be an oxygen function, hydroxyl or oxo group which may or ma y not be at C -11” (Gower , 1984) . Th e steroid numbering system is illustrated using cortisol in figure 1. Figure 1 . Chemical structure of c ortisol with steroid numbering system (Wilke , 1982) 3 Effects: Gower (1984) stated corticosteroids increase blood glucose level by promoting the hepatic conversion of a mino acids to glucose. These compounds also oppose insulin in allowing glucose into adipose cells, which in effect mobilizes fat and releases free fatty acids to the liver. Corticosteroids cause a net reduction in protein synthesis in muscle and lymphatic tissue , which reduces antibody synthesis. This net protein loss is due to increased protein catabolism (Martindale, 2005). Deluca (1984) stated g lucocorticoids also suppress intestinal calcium absor ption therefore lowering plasma calcium levels. Lower plasma calcium levels can l ead to bone decalcification. Corticosteroids counteract the histamine and serotonin effects of increased capillary permeability and dilation which are part of the inflammato ry response. This increase d capillary resistance reduces the movement of leucocytes to the site of infection. Therefore, phagocytosis decreases (Gower , 1984) . These chemicals can also cause adrenal gland atrophy (Ralston and Stenhouse, 1990).