Abstract
Corticoids have found their way into the globe of sports, due to their anti‐inflammatory properties, and have often found to be added to dietary supplements for illegally improving the effectiveness of their products. Earlier studies describe the detection of corticoids in several matrices, but this can be an incessant and continuous process as long because the doping practices continue. In this study, we report a technique to verify concurrently 44 of the foremost commonly abused synthetic corticoids (including chiral analogs) in equine plasma supported chiral liquid chromatography‐electrospray ionization mass spectrometry. Polysaccharide i‐cellulose‐5 column was used for chromatographic separation with a gradient mode. The validation studies were also meted out by using equine plasma so as to judge the suitability of the strategy. Detection limits were determined between 0.01 and 0.05 ng/mL and therefore the limit of quantification was between 0.1 and 0.5 ng/mL. Recovery and matrix effect on the analytes was further assessed. Since the developed method was ready to separate the corticoids and to differentiate chiral analogs at very low levels (in picograms), this separation techniques may be employed for the determination (confirmatory analysis) of the corticoids in the forensic and anti‐doping application.
Keywords: anti‐doping applications, corticoids, equine plasma, HRMS, polysaccharide columns
1. INTRODUCTION
Performance‐enhancing substances and methods have become a serious problem in sports competitions. 1 , 2 The rapid developments of analytical methodologies necessitate detection of the increasing number of doping substances and to forestall doping; for the foremost part, synthetic steroids that are unlawfully shaped to be unnoticeable by current drug testing methods. Many of those drugs are made specifically for the competitive sports field and do not have an approved medical use. 3 , 4 , 5 Corticoids (Figure 1) are the steroid hormones that are largely used for the treatment of a variety of diseases like osteoarthritis, rheumatoid, arthritis, infectious disease, gout, aversions, renal and hematological diseases, etc. 6 , 7 , 8 , 9 Spreads of glucocorticoids are developed with varying intensity and duration of their effect. Further, glucocorticoids affect every cell within the body and are often beneficial but now and then potentially negative too. Therefore, it is important to administer these medications with care to have the utmost positive effect and with the smallest amount of side effects. 10 , 11 , 12
FIGURE 1.
Glucocorticoids
Corticosteroid products find their way into the globe of sports and are on the list of drugs banned by different sporting authorities. 3 , 4 , 5 So as to regulate and stop these doping practices, sports authorities have stipulated several guidelines and approved various methods on the idea of reproducibility, sensitivity, adaptability, and pharmacological effect. 13 , 14 , 15 Determinations of corticoids in doping samples pose various difficulties, most of those compounds are thermally labile and their volatility is low. The analysis of this family of compounds is extremely complicated, because of their complex metabolism within the body which provides rise to an outsized number of metabolites, reducing the concentration levels found in urine. There are major advances within the area of spectrum analysis and related detection devices to find small quantities of the drug or their metabolites in body tissues and fluids. 16 , 17 , 18 To support drug metabolism and pharmacokinetic studies of chiral drugs, it is necessary to mix the physical phenomenon of HPLC with the sensitivity of mass spectrometric techniques. Recently, methods using LC‐MS/MS are found to be promising techniques for solving the matter of corticosteroid analysis. 19 , 20 , 21 , 22 , 23 , 24 , 25
Polysaccharide columns offer a large range of chiral separations of racemic components. 26 , 27 , 28 , 29 , 30 This work involves using technique for simultaneous separation and analysis of corticoids by applying chiral reversed‐phase liquid chromatography mass spectrometry. The study concerned 44 corticoids, a number of which are endogenous in humans (hydrocortisone and cortisone) and others are synthetic (triamcinolone; fluocinolone; prednisone; isoflupredone; fluprednisolone‐6α; fludrocortisone; prednisolone; flumetasone; prednisolone hemisuccinate; dexamethasone; diflorasone; betamethasone; paramethasone; fluocinolone acetonide; methylprednisolone; triamcinolone acetonide; beclomethasone; flunisolide; isoflupredone acetate; desonide; fluorometholone; flurandrenolide; prednisolone acetate; deflazacort; meprednisone; paramethasone acetate; ulobetasol; desoximetasone; fluocinolone acetonide acetate; mometasone; dichlorisone; budesonide; flurandrenolone acetonide; halcinonide; fluticasone propionate; amcinonide; triamcinolone hexacetonide; diflucortolone ‐21‐ valerate; norgestrel; medrysone; deoxycorticosterone acetate).
2. EXPERIMENTAL SECTION
2.1. Experimental materials
HPLC grade methanol and methyl t‐ butyl ether were procured from Fisher chemicals (Germany). LC‐MS grade acetonitrile was obtained from Merck KGaA (Germany); while formic acid, acetic acid, trifluoroacetic acid, and ammonium formate were procured from Sigma (United States). Hydrocortisone‐d4, triamcinolone, fluocinolone, fluprednisolone, fludrocortisone, prednisolone hemisuccinate, prednisolone, diflorasone, fluocinolone acetonide, dexamethasone, flunisolide, fluorometholone, deflazacort, halobetasol, fluocortalone, fluocinolone acetonide acetate, dichlorisone, and halcinonide were procured from Toronto Research Chemicals (Canada). Isoflupredone, betamethasone, paramethasone acetate, paramethasone, beclomethasone, cortisone, isoflupredone acetate, prednisolone acetate, difluocortolone valerate, levonorgestrel, dextronorgestrel, medrysone, deoxycorticosterone acetate, and mometasone obtained from Steraloids Inc (United States). Flumethasone, hydrocortisone, prednisone, and methylprednisolone were procured from B‐Dent Global (New Zealand). Triamcinolone acetonide, desonide, meprednisone, desoximetasone, budesonide, amcinonide, fluticasone propionate were obtained from Sigma (United States). The Columns (Lux cellulose‐1, Lux cellulose‐2, Lux i‐cellulose‐5, Lux amylose‐1, Lux i‐amylose‐1, and Lux amylose‐2) used for the separation were bought from Phenomenex (United States), and the Zorbax from Agilent (United States).
2.2. Sample preparation
2.2.1. Preparation of non‐extracted sample (NES) mixture
Stock standard solutions of every drug molecule were prepared in methanol, acetonitrile, tetrahydrofuran, or water. Mixtures of drug molecules were prepared by mixing equal volumes of the stock solutions of the individual drugs and the resulting mixture was further diluted with methanol or acetonitrile. All preparations were stored in dark at 2‐5°C.
2.2.2. Plasma
Horse plasma samples were extracted by using a liquid‐liquid extraction procedure. The plasma sample (1.0 mL), internal standard (d4‐hydrocortisone; 10 µL; 1.0 µg/mL) and 4% phosphoric acid (1 mL) to displace the corticoids from plasma proteins were added to a clean Kimble tube. The sample was vortexed and extracted with methyl t‐butyl ether (2 × 5 mL). The organic layer was dried under nitrogen (at 60°C) and reconstituted with 50 µL of methanol: water mixture (1:1), then transferred to an HPLC autosampler vial.
2.2.3. Mobile phases
The mobile phase B consisted of acetonitrile or methanol with/ without additives. An aqueous solution of additives was used as mobile phase A. The acidic buffer [0.01‐0.1% (v/v)] was prepared by dissolving formic acid, ammonium formate, or trifluoroacetic acid in deionized water. While the basic or neutral buffer [0.01‐0.1% (v/v)] was prepared by dissolving ammonium bicarbonate, ammonium acetate, or ammonia. The pH was adjusted to the right value with acetic acid; in some instances, triethylamine was added before adjusting the pH. All solutions were filtered through 0.45 m nylon membranes (Teknokroma) and degassed with helium before use.
3. INSTRUMENTATION
3.1. Liquid chromatography
The screening for corticoids was performed by using Zorbax C18 column (4.6 × 150 mm, 3.5 µm). The sample was eluted with 0.02% HCOOH in water (mobile phase A) and ACN (mobile phase B). The chiral chromatographic separation was performed using various cellulose and amylose chiral HPLC columns (4.6 × 250 mm, 5 µm) with the same chromatographic conditions. Racemic mixture separations were attempted in both isocratic and gradient elution mode, at the numerous temperature ranges of 25‐50˚C, flow rates of 0.6‐1.0 mL/min, and with mobile phases of different eluting strength.
3.2. Mass spectrometry
The mass spectrometric analysis was performed on Thermo Scientific Q Exactive high‐resolution accurate mass spectrometer in addition to Dionex UltiMate 3000 UHPLC+ operated in positive ion mode and therefore the S‐lens value was set to 50. The MS data was acquired at 70000 resolutions over the mass range m/z 50‐750. The capillary temperature was 320˚C and capillary voltage was set to 4 kV (sheath gas 45 units and auxiliary gas 10 units). The precursor ion detection was carried out employing a full‐scan MS experiment while the product ion detection was achieved employing a data‐independent acquisition experiment at collision energy 10 eV.
3.3. Data Analysis
The data was acquired and analyzed with Thermo Xcalibur software. The precursor ion exact mass with the mass tolerance of 5 ppm within the full scan is taken for identification, while a minimum of three fragment ions in data‐independent acquisition mode was considered for confirmation.
4. RESULT AND DISCUSSION
Initially, the plan was to develop a screening method, using the LC‐MS behavior of individual corticosteroid molecules and their major fragment ions. The goal was achieved with the Zorbax C18 column. Mobile phase A consisted of 0.02% HCOOH in water and acetonitrile was used as mobile phase B (Table 1). Using the developed method forty‐four (44) corticoids were successfully identified based on fragmentation pattern and retention time. The major fragments with theoretical values within 5 ppm tolerance are shown in Table 2.
TABLE 1.
The elution gradient used for screening method
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
|---|---|---|
| 00.00 | 98 | 02 |
| 08.00 | 05 | 95 |
| 12.00 | 05 | 95 |
| 12.01 | 98 | 02 |
| 14.00 | 98 | 02 |
Column: Zorbax C18 (4.6 × 150 mm, 3.5 µm); Method: gradient; Mobile phase A: 0.02% formic acid in water, Mobile phase B: acetonitrile; Run time: 15 min; Flow Rate: 0.6 mL/min.
TABLE 2.
LC‐MS behavior of individual corticosteroid molecules using a Zorbax C18
| Major Fragments (m/z) [Calculated/Observed] | ||||||||
|---|---|---|---|---|---|---|---|---|
| No | Corticoids | Structure | RT (min) | Exact Mass(m/z) | Precursor ion Mass (M+H) [Calculated/Observed] | 1 | 2 | 3 |
| 1 | Triamcinolone |
|
7.19 | 394.1792 |
395.18644/ 395.18672 |
375.18046/ 375.18029 |
357.16900/ 357.16966 |
339.15882/ 339.15913 |
| 2 | Fluocinolone |
|
7.71 | 412.1697 |
413.17702/ 413.17630 |
393.17079/ 393.17007 |
373.16456/ 373.16372 |
253.12230/ 253.12182 |
| 3 | Prednisone |
|
8.30 | 358.178 |
359.18530/ 359.18329 |
341.17470/ 341.17470 |
323.16417/ 323.16419 |
295.16926/ 295.16942 |
| 4 | Isoflupredone |
|
8.25 | 378.1843 |
379.19153/ 379.19140 |
341.17419/ 341.17459 |
359.18530/ 359.18510 |
323.16287/ 323.16378 |
| 5 | Fluprednisolone‐6α |
|
8.25 | 378.18425 |
379.19153/ 379.19152 |
341.17470/ 341.17474 |
323.16417/ 323.16417 |
359.18530/ 359.18447 |
| 6 | Fludrocortisone |
|
8.37 | 380.1999 |
381.20720/ 381.20641 |
325.17894/ 325.17934 |
265.15980/ 265.15864 |
239.14310/ 239.14293 |
| 7 | Prednisolone |
|
8.26 | 360.1937 |
361.20095/ 361.19987 |
343.19003/ 343.19016 |
325.17933/ 325.17928 |
307.16878/ 307.16892 |
| 8 | Flumetasone |
|
8.80 | 410.1905 |
411.19776/ 411.19610 |
391.19150/ 391.19083 |
371.18530/ 371.18488 |
253.12231/ 235.12224 |
| 9 |
Prednisolone hemisuccinate |
|
8.71 | 460.2097 |
461.21633/ 461.21633 |
443.20590/ 443.20590 |
325.17917/ 325.17917 |
307.16864/ 307.16864 |
| 10 | d4‐hydrocortisone |
|
8.37 | 366.2344 |
367.24170/ 367.24200 |
331.22030/ 331.22037 |
313.21006/ 313.21006 |
273.21513/ 273.21513 |
| 11 | Hydrocortisone |
|
8.37 | 362.2093 |
363.21660/ 363.21448 |
327.19550/ 327.19559 |
309.18490/ 309.18501 |
121.06480/ 121.06516 |
| 12 | Dexamethasone |
|
8.84 | 392.1999 |
393.20718/ 393.20548 |
355.19040/ 355.19038 |
337.17980/ 337.17986 |
237.12739/ 237.12751 |
| 13 | Diflorasone |
|
8.80 | 410.1905 |
411.19764/ 411.19764 |
391.19152/ 391.19090 |
371.18530/ 371.18479 |
353.17473/ 353.17341 |
| 14 | Betamethasone |
|
8.84 | 392.1999 |
393.20718/ 393.20718 |
355.19040 355.19040 |
337.17980/ 337.17980 |
237.12739/ 237.12739 |
| 15 | Paramethasone |
|
8.84 | 392.1999 |
393.20718/ 393.20718 |
375.19600/ 375.19603 |
355.19040/ 355.18978 |
337.17980/ 337.17927 |
| 16 |
Fluocinolone acetonide |
|
9.24 | 452.2011 |
453.20788/ 453.20788 |
433.20197/ 433.20197 |
413.19575/ 413.19575 |
337.14268/ 337.14268 |
| 17 |
Methyl prednisolone |
|
8.75 | 374.2093 |
375.21660/ 375.21634 |
357.20600/ 357.20558 |
339.19550/ 339.19512 |
321.18491/ 231.18442 |
| 18 |
Triamcinolone acetonide |
|
9.20 | 434.2105 |
435.21774/ 435.21785 |
415.21150/ 415.21155 |
397.20100/ 397.20098 |
357.16970/ 357.16955 |
| 19 | Beclomethasone |
|
9.00 | 408.1704 |
409.17762/ 409.17807 |
391.16710/ 391.16682 |
337.17980/ 337.17969 |
319.16926/ 319.16912 |
| 20 | Cortisone |
|
8.41 | 360.1937 |
361.20095/ 361.20072 |
343.19040/ 343.19056 |
267.17434/ 267.17439 |
163.11174/ 163.77796 |
| 21 | Flunisolide |
|
9.20 | 434.2105 |
435.21685/ 435.21685 |
397.19999/ 397.19999 |
339.15817/ 339.15817 |
321.14797/ 321.14797 |
| 22 |
Isoflupredone acetate |
|
9.28 | 420.1948 |
421.20209/ 421.20197 |
383.18491/ 383.18491 |
341.17419/ 341.17416 |
295.16913/ 295.16913 |
| 23 | Desonide |
|
9.32 | 416.2199 |
417.22716/ 417.22690 |
399.21660/ 399.21633 |
341.17470/ 341.17483 |
323.16417/ 323.16414 |
| 24 | Fluorometholone |
|
9.44 | 376.205 |
377.21202/ 377.21202 |
339.19516/ 339.19516 |
321.18460/ 321.18460 |
279.17396/ 279.17396 |
| 25 | Flurandrenolide |
|
9.38 | 436.22612 |
437.23339/ 437.23339 |
285.16451/ 285.16455 |
295.16861/ 295.16859 |
361.18012/ 361.18021 |
| 26 |
Prednisolone acetate |
|
9.36 | 402.2042 |
403.21151/ 403.21111 |
385.20081/ 385.20081 |
307.16903/ 307.16903 |
325.17952/ 325.17952 |
| 27 | Deflazacort |
|
9.64 | 441.2151 |
442.22233/ 442.22233 |
400.21184/ 400.21164 |
312.19580/ 312.19525 |
225.12739/ 225.12713 |
| 28 | Meprednisone |
|
8.90 | 372.1937 |
373.20095/ 373.20027 |
355.19040/ 355.18893 |
337.17980/ 337.17930 |
309.18491/ 309.18471 |
| 29 |
Paramethasone acetate |
|
9.86 | 434.2105 |
435.21774/ 435.21786 |
319.16876/ 319.16879 |
337.17911/ 337.17911 |
379.18995/ 379.18995 |
| 30 | Halobetasol |
|
9.96 | 428.1566 |
429.16367/ 429.16367 |
409.15764/ 409.15691 |
389.15141/ 389.15086 |
253.12230/ 253.12190 |
| 31 | Desoximetasone |
|
9.44 | 376.205 |
377.21226/ 377.21225 |
357.20575/ 357.20589 |
339.19550/ 339.19546 |
303.17550/ 303.17439 |
| 32 | Fluocinolone acetonide acetate |
|
10.42 | 494.2116 |
495.21823/ 495.21823 |
475.21265/ 475.21253 |
455.20643/ 455.20619 |
337.14343/ 337.14276 |
| 33 | Mometasone |
|
10.34 | 426.1365 |
427.14297/ 427.14397 |
409.13271/ 409.13271 |
373.15544/ 373.15544 |
237.12705/ 237.12705 |
| 34 | Dichlorisone |
|
9.30 | 412.1208 |
413.12687/ 413.12687 |
377.15141/ 377.15036 |
341.17473/ 341.17390 |
237.12739/ 237.12698 |
| 35 |
Budesonide (R & S) |
|
11.18 | 430.2355 |
431.24281/ 431.24253 |
413.23230/ 413.23213 |
341.17470/ 341.17471 |
323.16417/ 323.12438 |
| 36 |
Flurandrenolide acetate |
|
10.71 |
478.23663/ 478.23668 |
479.24311/ 479.24304 |
343.16994/ 343.16990 |
361.18042/ 361.18047 |
323.16360/ 323.16366 |
| 37 | Halcinonide |
|
11.05 | 454.1922 |
455.19932/ 455.19932 |
377.15143/ 377.15143 |
359.14097/ 359.14097 |
227.14268/ 227.14268 |
| 38 | Amcinonide |
|
11.15 | 502.2367 |
503.24395/ 503.24405 |
483.23849/ 483.23754 |
399.18076/ 399.17988 |
339.15939/ 339.15903 |
| 39 |
Fluticasone propionate |
|
10.99 | 500.1844 |
501.19164/ 501.19164 |
481.18602/ 481.18602 |
313.15996/ 313.15996 |
293.15348/ 293.15348 |
| 40 |
Triamcinolone hexacetonide |
|
12.21 | 532.28363 |
533.29091/ 533.29090 |
415.20625/ 415.20628 |
397.19778/ 397.19774 |
399.15645/ 399.15640 |
| 41 |
Diflucortolone‐21‐ valerate |
|
11.49 | 478.2531 |
479.26042/ 479.26042 |
459.25370/ 459.25370 |
439.24773/ 439.24773 |
375.19602/ 375.19602 |
| 42 |
Levonorgestrel & Dextronorgestrel |
|
10.82 | 312.2089 |
313.21621/ 313.21614 |
245.18999/ 245.18998 |
295.20564/ 295.20577 |
277.19508/ 277.19544 |
| 43 | Medrysone |
|
10.89 | 344.2351 |
345.24212/ 345.24212 |
327.23153/ 327.23153 |
309.22068/ 309.22068 |
135.08044/ 135.08044 |
| 44 |
Deoxycorticosterone acetate |
|
11.25 | 372.2301 |
373.23734/ 373.23754 |
331.22677/ 331.22666 |
295.20564/ 295.20566 |
109.06479/ 109.06519 |
Column: Zorbax C18 (4.6 × 150 mm, 3.5 µm); Method: gradient; Mobile phase A: 0.02% formic acid in water, Mobile phase B: acetonitrile; Run time: 15 min; Flow Rate: 0.6 mL/min; RT‐Retention time.
The next focus was to develop a separation method, the separation of corticoids and their isomers if they were doped together. A range of columns and buffers were used to evaluate peak separation. Individual, as well as mixture of analytes, were injected with each method; however, no conclusions could be made with reverse‐phase C18 chromatographic results.
The polysaccharide‐based chiral columns provided good separation with reversed‐phase techniques during our previous studies 18 , 22 , 26 , 28 ; thus, the focus was shifted to chiral columns. Parameters like gradient, buffer, flow rate, and temperature were studied to get good resolution. An optimal separation was obtained via i‐cellulose‐5 column (Table 3 detail the optimized chromatographic conditions). Within the developed method, all 44 corticoids were found to elute at different retention times with realistic resolution (Figures 2, 3 & 4). A gradient method was applied (flow rate of 0.6 mL/min and column temperature of 50˚C), using 0.1% HCOOH in water (mobile phase A) and 0.1% HCOOH in acetonitrile (mobile phase B). By the optimized method, spiked samples of assorted concentrations (1‐1000 ng) were analyzed (Table 4).
TABLE 3.
Optimized chromatographic condition
| Method | Mobile Phase | Gradient(time vs. % of mobile phase B) | Column oven temp. (˚C) | Flow rate(mL/min) |
|---|---|---|---|---|
| Gradient | A: 0.1% HCOOH in water | 0‐05, 15‐40, 35‐95, 40‐95, 42‐05, 45‐05 | 50 | 0.6 |
| B: 0.1% HCOOH in acetonitrile |
Column: Zorbax C18 (4.6 × 150 mm, 3.5 µm).
FIGURE 2.
Chromatogram of corticoids (1) Triamcinolone; (2) Fluocinolone; (3) Prednisone; (4) Isoflupredone; (5) Fluprednisolone‐6α; (6) Fludrocortisone; (7) Prednisolone; (8) Flumetasone; (9) Prednisolone hemisuccinate; (10) d4‐hydrocortisone; (11) Hydrocortisone; (12) Dexamethasone; (13) Diflorasone; (14) Betamethasone by developed chiral separation method
FIGURE 3.
Chromatogram of corticoids (15) Paramethasone; (16) Fluocinolone acetonide; (17) Methyl prednisolone; (18) Triamcinolone acetonide; (19) Beclomethasone; (20) Cortisone; (21) Flunisolide; (22) Isoflupredone acetate; (23) Desonide; (24) Fluorometholone; (25) Flurandrenolide; (26) Prednisolone acetate; (27) Deflazacort; (28) Meprednisone by developed chiral separation method
FIGURE 4.
Chromatogram of corticoids (29) Paramethasone acetate; (30) Ulobetasol; (31) Desoximetasone; (32) Fluocinolone acetonide acetate; (33) Mometasone; (34) Dichlorisone; (35) Budesonide; (36) Flurandrenolone Acetonide; (37) Halcinonide; (38) Fluticasone propionate; (39) Amcinonide; (40) Triamcinolone hexacetonide; (41) Diflucortolone ‐21‐ valerate; (42) Norgestrel; (43) Medrysone; (44) Deoxycorticosterone acetate by developed chiral separation method
TABLE 4.
Validation details of studied corticoids by the developed method
| No | Corticoids | RT | RRT | Recovery (%) | LOD (ng/ml) | LOQ (ng/ml) | Accuracy (%) | Intra‐dayprecision RSD (%) | Inter‐dayprecision RSD (%) | Correlation coefficient | Matrix effect (%) |
|---|---|---|---|---|---|---|---|---|---|---|---|
| d4‐hydrocortisone | 18.23 | Ref. Std. | |||||||||
| 1 | Triamcinolone | 12.82 | 0.703 | 89 | 0.01 | 0.1 | 98.2 | 2.8 | 3.1 | 0.999 | ‐6.0 |
| 2 | Fluocinolone | 13.84 | 0.760 | 87 | 0.01 | 0.1 | 96.8 | 1.6 | 2.2 | 0.998 | ‐2.5 |
| 3 | Prednisone | 16.40 | 0.900 | 91 | 0.05 | 0.5 | 100.5 | 2.2 | 2.5 | 0.999 | ‐8.1 |
| 4 | Isoflupredone | 16.61 | 0.911 | 95 | 0.02 | 0.2 | 102.2 | 2.8 | 3.3 | 0.999 | +12.0 |
| 5 | Fluprednisolone‐6α | 17.35 | 0.952 | 88 | 0.01 | 0.1 | 97.7 | 4.0 | 4.8 | 0.999 | ‐6.7 |
| 6 | Fludrocortisone | 17.60 | 0.966 | 94 | 0.02 | 0.2 | 99.1 | 1.9 | 2.1 | 0.998 | ‐7.0 |
| 7 | Prednisolone | 17.97 | 0.987 | 96 | 0.02 | 0.2 | 98.3 | 4.5 | 4.1 | 0.998 | ‐5.8 |
| 8 | Flumetasone | 18.04 | 0.990 | 78 | 0.02 | 0.2 | 101.2 | 3.3 | 3.5 | 0.998 | +1.8 |
| 9 | Prednisolone hemisuccinate | 18.13 | 0.993 | 93 | 0.05 | 0.5 | 99.2 | 2.9 | 2.8 | 0.998 | +4.5 |
| 10 | d4‐hydrocortisone | 18.20 | 1.000 | 88 | 0.05 | 0.5 | 100.5 | 4.1 | 3.8 | 1.000 | ‐3.4 |
| 11 | Hydrocortisone | 18.30 | 1.004 | Not validated | |||||||
| 12 | Dexamethasone | 18.60 | 1.021 | 95 | 0.01 | 0.1 | 100.2 | 2.7 | 3.5 | 0.999 | +3.1 |
| 13 | Diflorasone | 18.75 | 1.027 | 87 | 0.01 | 0.1 | 98.9 | 0.9 | 1.2 | 0.999 | ‐6.1 |
| 14 | Betamethasone | 19.35 | 1.061 | 94 | 0.01 | 0.1 | 101.5 | 3.9 | 3.2 | 0.999 | ‐2.7 |
| 15 | Paramethasone | 19.59 | 1.075 | 92 | 0.01 | 0.1 | 97.9 | 2.7 | 3.8 | 0.998 | ‐4.2 |
| 16 | Fluocinolone acetonide | 19.75 | 1.084 | 83 | 0.01 | 0.1 | 99.0 | 3.5 | 4.7 | 0.998 | +6.5 |
| 17 | Methyl prednisolone | 20.04 | 1.098 | 90 | 0.02 | 0.2 | 101.3 | 2.1 | 3.4 | 0.999 | ‐1.2 |
| 18 | Triamcinolone acetonide | 20.22 | 1.107 | 87 | 0.05 | 0.5 | 98.0 | 2.7 | 4.0 | 0.998 | ‐4.0 |
| 19 | Beclomethasone | 20.32 | 1.114 | 92 | 0.01 | 0.1 | 100.9 | 4.9 | 4.3 | 0.999 | ‐4.4 |
| 20 | Cortisone | 20.60 | 1.129 | 86 | 0.01 | 0.1 | 98.6 | 4.9 | 4.1 | 0.998 | ‐3.8 |
| 21 | Flunisolide | 21.31 | 1.169 | 90 | 0.02 | 0.2 | 99.1 | 2.7 | 3.6 | 0.998 | +10.1 |
| 22 | Isoflupredone acetate | 21.45 | 1.176 | 79 | 0.05 | 0.5 | 99.8 | 3.2 | 2.9 | 0.999 | +3.5 |
| 23 | Desonide | 22.04 | 1.210 | 84 | 0.05 | 0.5 | 102.0 | 1.8 | 2.0 | 1.000 | ‐12.6 |
| 24 | Fluorometholone | 22.20 | 1.217 | 88 | 0.02 | 0.2 | 100.8 | 3.5 | 4.8 | 0.999 | ‐6.2 |
| 25 | Flurandrenolide | 22.33 | 1.225 | 83 | 0.05 | 0.5 | 100.1 | 3.8 | 4.0 | 0.998 | ‐4.9 |
| 26 | Prednisolone acetate | 22.56 | 1.238 | 87 | 0.05 | 0.5 | 99.0 | 3.2 | 3.9 | 0.999 | ‐3.7 |
| 27 | Deflazacort | 22.90 | 1.256 | Not validated | |||||||
| 28 | Meprednisone | 23.08 | 1.266 | Not validated | |||||||
| 29 | Paramethasone acetate | 23.50 | 1.288 | 89 | 0.05 | 0.5 | 99.8 | 2.9 | 4.6 | 0.999 | +1.2 |
| 30 | Ulobetasol | 23.85 | 1.309 | 92 | 0.05 | 0.5 | 98.5 | 3.1 | 4.0 | 0.998 | ‐8.9 |
| 31 | Desoximetasone | 24.00 | 1.317 | 83 | 0.05 | 0.5 | 97.8 | 3.7 | 5.2 | 0.999 | ‐3.1 |
| 32 | Fluocinolone acetonide acetate | 24.18 | 1.326 | 88 | 0.05 | 0.5 | 103.1 | 5.1 | 4.8 | 0.998 | +2.8 |
| 33 | Mometasone | 24.85 | 1.364 | 93 | 0.05 | 0.5 | 102.8 | 2.9 | 4.1 | 0.998 | ‐11.0 |
| 34 | Dichlorisone | 25.05 | 1.375 | 92 | 0.05 | 0.5 | 101.2 | 4.8 | 5.3 | 0.999 | ‐8.9 |
| 35 | Budesonide R or S | 25.70 | 1.411 | Not validated | |||||||
| 36 | Budesonide S or R | 26.20 | 1.437 | Not validated | |||||||
| 37 | Flurandrenolide acetate | 26.30 | 1.442 | 85 | 0.05 | 0.5 | 97.9 | 3.9 | 6.1 | 0.999 | ‐6.5 |
| 38 | Halcinonide | 26.95 | 1.481 | 91 | 0.05 | 0.5 | 98.8 | 4.7 | 7.0 | 0.998 | +5.2 |
| 39 | Fluticasone propionate | 27.17 | 1.490 | 94 | 0.01 | 0.1 | 103.0 | 6.2 | 5.5 | 0.998 | +3.2 |
| 40 | Amcinonide | 27.30 | 1.498 | 86 | 0.01 | 0.1 | 100.7 | 3.2 | 4.8 | 0.999 | ‐4.1 |
| 41 | Triamcinolone hexacetonide | 28.70 | 1.570 | 92 | 0.02 | 0.2 | 97.7 | 5.5 | 6.8 | 1.000 | ‐3.7 |
| 42 | Diflucortolone ‐21‐ valerate | 29.00 | 1.591 | 92 | 0.05 | 0.5 | 102.3 | 2.7 | 4.3 | 0.999 | ‐4.9 |
| 43 | Levonorgestrel or dextronorgestrel | 30.00 | 1.644 | Not validated | |||||||
| 44 | Dextronorgestrel or levonorgestrel | 30.40 | 1.666 | Not validated | |||||||
| 45 | Medrysone | 31.50 | 1.730 | 90 | 0.01 | 0.1 | 101.0 | 6.1 | 7.2 | 0.998 | +3.1 |
| 46 | Deoxycorticosterone acetate | 34.70 | 1.904 | 88 | 0.02 | 0.2 | 99.3 | 3.5 | 4.0 | 0.999 | ‐1.9 |
The method was found to be an accompaniment to separate the R/S enantiomers of budesonide, norgestrel, and epimeric dexamethasone, betamethasone & paramethasone (Figure 5). The developed method was also very much applicable for the routine confirmation analysis of structural isomers of corticoids like triamcinolone acetonide/ paramethasone acetate/ flunisolide, diflorasone/ flumetasone, fluorometholone/ desoximetasone, prednisolone/ cortisone, and isoflupredone/ 6α ‐fluoroprednisolone (Figure 5).
FIGURE 5.
Chromatogram of corticosteroid isomers by developed chiral separation method
In furtherance, the method was applied for the confirmatory analysis of authentic administration samples (positive sample of horse racing) to verify the Sex hormone binding globulin (SHBG) effects (Figure 6). The results further confirmed the developed method is ready to detect and separate the corticoids, and to differentiate chiral analogs at very low levels (even in picograms per millilitre).
FIGURE 6.
Chromatogram of Flumethasone (approx. 20 ng/mL), Dexamethasone (approx. 20 ng/mL), Methyl prednisolone (approx. 5 ng/mL) and Triamcinolone acetonide (approx. 15 ng/mL) by developed chiral separation method (in post‐administration plasma samples)
The exact life time of the used i‐cellulose‐5 chiral column is yet unknown, but the chromatographic performance was acceptable even after 2000 injections. While a slight loss of column performance was observed especially after repeated use of additives or aggressive mobile phase conditions, and those effects were often mitigated by utilizing a regeneration procedure. 31
5. METHOD VALIDATION
The suitability of the developed method was assessed by validation parameters like retention time (absolute retention time changes from run to run, thus, relative retention times RRT were used), sensitivity (LOD & LOQ of corticoids in horse plasma were determined by signal to noise ratio technique), linearity (1, 5, 10, 20, 40, 50 ng/mL), recovery (assessed by spiking a series of samples i.e. 1.0, 5.0, 10, and 50 ng/mL in horse plasma, performed twice) intra‐day and inter‐day imprecision (a group of 18 spiked samples i.e.1.0 ng/mL were evaluated for 3 validation days) and matrix effects (Table 4). Ion suppression and enhancement were determined by comparing the common peak intensities of every target analyte obtained from the analyses of reference standards and urine samples (n = 6) spiked at 10 ng/mL.
6. CONCLUSION
The present work shows that 44 corticoids can be identified in a single LC/MS/MS run and proved reproducible results even at very low concentrations. The accuracy and precision were monitored through the analysis of control equine plasma spikes and were shown to be excellent with accuracies within the acceptance criteria. Detection limits were determined as 0.01‐0.05 ng/ mL, and also the obtained limits of quantification are in between 0.1‐0.5 ng/mL. Matrix effect and recovery on the analytes were additionally evaluated. Hence, the developed method is useful for confirmatory analysis and will accelerate the analysis of corticoids or related compounds in LC/MS/MS‐based routine doping control procedures.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
ETHICAL APPROVAL
This article doesn't contain any studies with humans/animals.
ACKNOWLEDGMENTS
The authors are thankful to Dr. Ali Ridha, Director General, (Central Veterinary Research Laboratory) for the priceless support and suggestions. The authors are acknowledging the support & assistance of Dr. U. Wernery (Scientific Director, Central Veterinary Research Laboratory), and Equine Forensic Unit staff for this project.
Karakka Kal AK, Karatt TK, Nalakath J, et al. Simultaneous analysis of 44 frequently abused corticosteroid drugs using polysaccharide‐based chiral column‐HRMS approach. Anal Sci Adv. 2021;2:427–439. 10.1002/ansa.202000166
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