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 »  Abstract
 » Introduction
 »  Materials and Me...
 » Results
 » Discussion
 » Conclusion
 »  References
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SHORT COMMUNICATIONS
Year : 2021  |  Volume : 53  |  Issue : 6  |  Page : 480-483
 

A preliminary study on urinary excretion patterns of methylprednisolone after oral and intra-articular administration and effect on endogenous glucocorticosteroids profile


1 National Dope Testing Laboratory, Ministry of Youth Affairs and Sports, J. N. Stadium, New Delhi, India
2 Anti-Doping Lab Qatar (ADLQ), Doha, Qatar
3 School of Studies in Zoology, Jiwaji University, Gwalior, Madhya Pradesh, India

Date of Submission26-Sep-2020
Date of Decision23-May-2021
Date of Acceptance11-Nov-2021
Date of Web Publication30-Dec-2021

Correspondence Address:
Alka Beotra
House No. 278, Sector 37, Near DAV School, Faridabad, Haryana
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijp.ijp_946_20

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 » Abstract 


INTRODUCTION: The use of glucocorticosteroids (GCs) through oral, intravenous, intramuscular, or rectal routes is prohibited in sports. Its use is permitted through inhalation, topical and intra-articular route of administration. Methylprednisolone (MP) is available for use by different routes for anti-inflammatory and immunosuppressive purposes. To discriminate its intake by permitted & forbidden routes, a reporting level of 30 ng/ml is set by World Anti-Doping Agency. The aim of this study was to compare MP's excretion profile following oral & intra-articular administration & to evaluate its effect on endogenous GCs profile.
MATERIALS & METHODS: The MP was administered through oral and intra-articular route to different patients & urine samples were collected up to 100 h. The urine samples were hydrolyzed, extracted, and analyzed on Liquid chromatography-mass spectrometry/MS.
RESULTS: MP levels in urine exceeded the reporting limit of 30 ng/ml after oral (8 mg) and intra-articular administration (80 mg) routes. After oral intake (8 mg), MP levels exceeded the reporting level up to 24 h. However, after intra-articular injection (80 mg), the MP could be detected above the reporting level up to 80 h.
CONCLUSION: The findings reveal that the MP can exceed the reporting level in urine even after administration by permitted route (i.a.). Further analysis of four endogenous GCs (Cortisol, Cortisone, TH Cortisone, and 11-deoxycortisol) showed a decreased excretion following administration of MP by oral & intra-articular routes.


Keywords: Doping, endogenous glucocorticosteroid, intra-articular, Liquid chromatography-mass spectrometry, methylprednisolone, Oral


How to cite this article:
Upadhyay AK, Dubey S, Ahi S, Beotra A, Bhardwaj A, Shukla S, Jain S. A preliminary study on urinary excretion patterns of methylprednisolone after oral and intra-articular administration and effect on endogenous glucocorticosteroids profile. Indian J Pharmacol 2021;53:480-3

How to cite this URL:
Upadhyay AK, Dubey S, Ahi S, Beotra A, Bhardwaj A, Shukla S, Jain S. A preliminary study on urinary excretion patterns of methylprednisolone after oral and intra-articular administration and effect on endogenous glucocorticosteroids profile. Indian J Pharmacol [serial online] 2021 [cited 2022 Jan 29];53:480-3. Available from: https://www.ijp-online.com/text.asp?2021/53/6/480/334361





 » Introduction Top


The use of oral, rectal, intravenous, and intramuscular glucocorticosteroids (GCs) is prohibited in sports by World Anti-Doping Agency (WADA), whereas dermatological, inhaler, and intraarticular routes of administration are permitted justifying therapeutic use of glucocorticoids.[1] In addition, WADA has put a reporting limit of 30 ng/ml for GCs which means that positive findings (adverse analytical findings) would only be reported if the concentration of GC is found >30 ng/ml. Methylprednisolone (MP) is a potent synthetic glucocorticosteroid mainly used to treat various health conditions such as arthritis, severe allergic reactions, certain cancers, eye, and skin conditions, and immune system disorders. The administration of GCs through prohibited routes requires a valid therapeutic use exemption (TUE) granted by the TUE committee duly appointed by the testing authority to a sports person, before its intake.[2] However, a reporting limit of 30 ng/ml can be exceeded by the permissible routes of administration and needs to be investigated further to avoid such false-positive results.[3] Several Studies are published for the detection of MP and its metabolite in urine and other matrices using gas chromatography-mass spectrometric (GC-MS) and high-performance liquid chromatographic (HPLC).[4],[5] However, due to ionization compatibility and sensitivity, liquid chromatography-MS/MS is a first choice for the detection of MP and its metabolites.[6],[7],[8],[9] Studies have been conducted for the detection of MP metabolite through different routes of administration in rats and humans.[10],[11],[12],[13] However, no comparative study is performed to evaluate MP excretion in urine after oral and intra-articular administration and its effect on endogenous GCs profile. Hence, the goal of this work was to compare MP's excretion profile following two different routes of administration and to evaluate its impact on the endogenous GCs profile as it may function as a tool for differentiating the routes of administration of MP.


 » Materials and Methods Top


Drug administration and sample collection

Drug Preparation of MP (8 mg, Elpred-M, Lancer) was orally taken by three male patients (age 25–35 years) and urine samples were collected up to 100 h. Urinary samples after intra-articular injection (depo-medrol; 80 and 40 mg, Pfizer) of MP were collected from two female patients who were under medical treatment. Before sample collection written consent was obtained from both patients. The study was duly approved by the institutional ethics committee of the National Dope Testing Laboratory (NDTL), India, though it did not involve any drug administration at NDTL. The administration of the drug was as per the doctor's prescription for the treatment and consent of the patient to give urine samples for the analysis was taken.

Reagents and reference standards

The reference standard of MP was purchased from Sigma Aldrich, USA, and cortisol, cortisone, tetrahydrocortisone, 11-deoxycortisol, and d4-cortisol (internal standard) were purchased from Toronto Research Chemicals, Canada. All the reagents and chemicals used in the study were of analytical or mass spectrometric grade. The deionized water was obtained from Mili-Q (Millipore, Milford, USA). Standard stock solutions were prepared at a concentration of 1 mg/ml in methanol and further diluted at required levels to prepare calibrators and quality controls.

Sample analysis

The urine samples were prepared using an existing protocol in the laboratory.[8] In brief, 2 ml of the sample was hydrolyzed after adding β-glucuronidase enzyme ( Escherichia More Details coli) and phosphate buffer. The hydrolysate was extracted in alkaline (7% K2CO3, pH 9–10) and acidic medium (6N Hcl, pH 2–3) with tertiary methyl ethyl ether and ethyl acetate, respectively. The extracts were combined and dried under nitrogen evaporation and dissolved in the 50:50 Mobile phase.

The extracts were analyzed using Sciex API 3200 QTrap Mass spectrometer coupled with Agilent ultra-HPLC 1290 system in positive ionization electrospray ionization, multiple reaction monitoring modes. The instrument conditions were optimized to get optimum sensitivity. Separation of analyte was achieved on inertsil ODS (C-18, 3 μ, 4.6 mm × 50 mm) column using 1% formic acid and acetonitrile (ACN) in gradient program 15% ACN to 60% ACN in 5 min, held for 1 min then 100% ACN in 7 min followed by 15% ACN till 11 min, at a flow rate of 0.7 ml/min.


 » Results Top


For method specificity, the chromatogram of the spiked samples with MP and four endogenous corticosteroids was compared with that of the blank urine sample. The method was found fit for the purpose and we did not observe any urine matrix interference at the retention times of the targeted ion transitions.

The analysis of excretion urine revealed increased MP levels (>30 ng/ml) in all the volunteers (n = 3) till 24 h after oral administration. The MP levels remained elevated (3–4 times of reporting limit of 30 ng/ml) till 9 h postoral administration [Figure 1]a. The results of intra-articular (40 mg) administration showed an increased level of MP (>30 ng/ml) up to 3 h only. However, in case of an elevated dose of intra-articular (80 mg), all the urine samples crossed the reporting limit till 70 h and decreasing the trend (<30 ng/ml) till 100 h [Figure 1]b. The urinary excretory profile of four endogenous GCs (cortisol, cortisone, tetrahydro cortisone, and 11-deoxycortisol) showed suppression of excretion after intake of MP through both oral and intra-articular routes.
Figure 1: Methylprednisolone profiles after administration of (a) of oral dose 8 mg to 3 volunteers (b) of intra articular dose of 40 mg & 80 mg

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 » Discussion Top


Our work supports previous study performed on MP oral and topical application were both routes crosses the prohibited concentration of 30 ng/ml. Based on the finding, it is apparent that, although intra-articular is a permitted route for GCs, it may produce adverse analytical findings for MP. The comparison of endogenous GCs profiles did not provide any conclusive findings to discriminate the routes of intake of MP [Figure 2] and [Figure 3]. The suppression of endogenous GCs profiles was similar for oral and intra-articular routes especially for the tetrahydrocorticone and Cortisone, which substantiates the systemic effect of the intra-articular route. However, a detailed excretion study with more number of volunteers is required to substantiate the finding of this preliminary study. Profiling of endogenous GCs after administration of MP by both the administration routes on more number of subjects is underway. The comparative analysis of the decrease in endogenous corticosteroid profiles between oral and intra-articular routes may act as a marker to differentiate the exact route of administration of MP in sports doping analysis.
Figure 2: Urinary excretion profile of endogenous Glucocorticosteroids (oral dose 8 mg); (a) Voluenteer-1 (b) Voluenteer-2 (c) Voluenteer-3

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Figure 3: Urinary excretion profile of endogenous Glucocorticosteroids; (a) volunteer-1, Intra-articular Dose 40mg (b) volunteer-2, Intra-articular Dose 80mg.

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 » Conclusion Top


The finding suggests that the permitted route (i.e., intra-articular) may also produce positive findings for MP.

Therefore, our study suggests that the stakeholders involved in the annual review of WADA the prohibited list need to be vigilant about considering removing the intraarticular route of administration from the permissible route. Athletes and health experts, however, be cautious when considering an intra-articular MP administration during a competition period and obtain prior administration permission and exemption for MP use.

Acknowledgment

Financial assistance from the Ministry of Youth Affairs and Sports is acknowledged.

Financial support and sponsorship

Ministry of Youth Affairs and Sports.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

1.
World Anti-Doping Agency, The World Anti-doping Code. Available from: https://www.wada-ama.org/sites/default/files/wada_2020_english_prohibited_list_0.pdf. [Last accessed on 2020 Aug 25; Last updated on 2020 Jan 01].  Back to cited text no. 1
    
2.
World Anti-Doping Agency, The World Anti Doping Code – International Standard for Therapeutic Use Exemptions (ISTUE); 2019. Available from: https://www.wada-ama.org/en/resources/therapeutic-use-exemption-tue/international-standard-for-therapeutic-use-exemptions-istue/. [Last accessed on 2020 Aug 25; Last updated on 2020 Jan 01].  Back to cited text no. 2
    
3.
Panusa A, Regazzoni L, Aldini G, Orioli M, Giombini A, Minghetti P, et al. Urinary profile of methylprednisolone acetate metabolites in patients following intra-articular and intramuscular administration. Anal Bioanal Chem 2011;400:255-67.  Back to cited text no. 3
    
4.
Rodchenkov GM, Uralets VP, Semenov VA. Determination of methylprednisolone metabolites in human urine by gas chromatography-mass spectrometry. J Chromatogr 1987;423:15-22.  Back to cited text no. 4
    
5.
Vree TB, Lagerwerf AJ, Verwey-van Wissen CP, Jongen PJ. High-performance liquid chromatography analysis, preliminary pharmacokinetics, metabolism and renal excretion of methylprednisolone with its C6 and C20 hydroxy metabolites in multiple sclerosis patients receiving high-dose pulse therapy. J Chromatogr B Biomed Sci Appl 1999;732:337-48.  Back to cited text no. 5
    
6.
Reddy IM, Beotra A, Jain S, Ahi S. A simple and rapid ESI-LC-MS/MS method for simultaneous screening of doping agents in urine samples. Indian J Pharmacol 2009;41:80-6.  Back to cited text no. 6
[PUBMED]  [Full text]  
7.
Pozo OJ, Marcos J, Matabosch X, Ventura R, Segura J. Using complementary mass spectrometric approaches for the determination of methylprednisolone metabolites in human urine. Rapid Commun Mass Spectrom 2012;26:541-53.  Back to cited text no. 7
    
8.
Ahi.S, Dubey S, Upadhyay A, Yadav SR, Priyadarshi R, Beotra A, et al. Identification of prednisolone, methylprednisolone and their metabolite in human urine using HPLC (+) ESI-MS/MS and detection of possible adulteration in Indian herbal drug preparations. Ibnosina J Med BS 2012;4:44-52.  Back to cited text no. 8
    
9.
Sridhar SR, Mercy LL, Prasanth TS. A novel LC–MS/MS assay for methylprednisolone in human plasma and its pharmacokinetic application. Asian J Pharm Sci 2016;3:459-68.  Back to cited text no. 9
    
10.
Simoes SM, Calcada M, Horta L, De La Torre X. Methylprednisolone detection in urine following local and oral administrations. Recent advances in doping analysis (13). Sport und Buch Strauβ, Koln 2005. p. 411-414.  Back to cited text no. 10
    
11.
Panusa A, Aldini G, Orioli M, Vistoli G, Rossoni G, Carini M. A sensitive and specific precursor ion scanning approach in liquid chromatography/electrospray ionization tandem mass spectrometry to detect methylprednisolone acetate and its metabolites in rat urine. Rapid Commun Mass Spectrom 2010;24:1583-94.  Back to cited text no. 11
    
12.
Panusa A, Orioli M, Aldini G, Carini M. A rapid and sensitive LC-ESI-MS/MS method for detection and quantitation of methylprednisolone and methylprednisolone acetate in rat plasma after intra-articular administration. J Pharm Biomed Anal 2010;51:691-7.  Back to cited text no. 12
    
13.
Matabosch X, Pozo OJ, Monfort N, Pérez-Mañá C, Farré M, Marcos J, et al. Urinary profile of methylprednisolone and its metabolites after oral and topical administrations. J Steroid Biochem Mol Biol 2013;138:214-21.  Back to cited text no. 13
    


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  [Figure 1], [Figure 2], [Figure 3]



 

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