|Year : 2021 | Volume
| 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
Awanish Kumar Upadhyay1, Sachin Dubey1, Shobha Ahi1, Alka Beotra2, Akhilesh Bhardwaj1, Sangeeta Shukla3, Shila Jain1
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 Submission||26-Sep-2020|
|Date of Decision||23-May-2021|
|Date of Acceptance||11-Nov-2021|
|Date of Web Publication||30-Dec-2021|
House No. 278, Sector 37, Near DAV School, Faridabad, Haryana
Source of Support: None, Conflict of Interest: None
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 Sep 28];53:480-3. Available from: https://www.ijp-online.com/text.asp?2021/53/6/480/334361
| » Introduction|| |
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. 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. 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. 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)., However, due to ionization compatibility and sensitivity, liquid chromatography-MS/MS is a first choice for the detection of MP and its metabolites.,,, Studies have been conducted for the detection of MP metabolite through different routes of administration in rats and humans.,,, 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|| |
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.
The urine samples were prepared using an existing protocol in the laboratory. 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|| |
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|| |
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|| |
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.
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.
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