GX-274 ASPEC®: AutomatedExtraction of Glucocorticoid andNonsteroidal Anti-Inflammatory
Drugs from Horse Urine
APPLICATION NOTE AN1004

APPLICATION BENEFITSSOLUTIONS

Glucocorticoid and nonsteroidal anti-inflammatorycompounds were isolated from the same sample byautomating multiple elution steps. TRILUTION® LH software
allowed easy automation of manual SPE methods that fit into
existing workflows and provide reproducible and traceable
results. A four-probe GX-274 ASPEC® instrument provides
sufficient throughput to prepare samples for two HPLC/MS/MS systems in parallel, reducing the number of instruments
required in the lab.

Accurate and rapid detection of prohibited substancespresent in complex biological matrices is crucial for testinglaboratories. Robust instrumentation, a flexible softwareinterface that meets the needs of the end user, reproducible
methods and traceability are all needed in regulatedenvironments.

KENSAKU SHIRAI & KENJI KINOSHITA | Laboratory of Racing Chemistry; Utsunomiya, JapanABSTRACT
Three glucocorticoids and three nonsteroidal antiinflammatory drugs were detected simultaneously
from horse urine using the Gilson GX-274 ASPEC®
system with two step extractions and LC/MS/MS
selected reaction monitoring (SRM).

Figure 1

Gilson GX-274 ASPEC® system

INTRODUCTION
Equine drug testing laboratories face the challengeof rapidly and accurately detecting drugs andother foreign substances that are prohibitedin horse racing.1 Automation with the Gilson
GX-274 ASPEC® system provides a reproducible,

Figure 2

The system is equipped with four probes, permitting four samples
to be processed simultaneously with automated solid phaseextraction.

documentable method for sample preparation andfits into workflows for compound detection andquantitation (Figures 1 and 2). With four probes, theGX-274 ASPEC system provides higher throughput
while maintaining reliability and providing a flexible
software interface for development of methods thatmeet the needs of the research or testing lab.
MATERIALS AND METHODS
Solid Phase Extraction
A Gilson GX-274 ASPEC® system was used forthe extraction. 2 The solid phase extraction (SPE)
cartridges used in this study were Presep® RPP-SAX(60 µm particle, 60 mg/3 mL) obtained from Wako
Pure Chemicals. The Presep RPP-SAX cartridges werepre-conditioned with 2 mL of methanol followed by
2 mL of 50 mmol/L ammonium formate.

The sample processing workflow is shown in Figure 3.
Internal standards made up in 1mL of 1 mol/L acetate
buffer (pH 5.0) and 100 µL of beta glucuronidasefrom Pomacea canaliculata solution were added to
1.0 mL of horse urine followed by incubation for30 minutes at 60°C. After cooling the mixture toambient temperature, 4 mL of 0.3 mol/L sodium
dihydrogen phosphate containing 0.01 mol/L
EDTA2Na was added. The mixture was filtered withglass fiber filter paper and a 4.5 mL aliquot wasadded to a pre-conditioned Presep RPP-SAX SPEcolumn. The SPE column was washed with 3 mL ofwater/methanol (95:5) and eluted with 6 mL oft-butyl methyl ether. The resulting eluate was termed
Eluate G and contained the glucocorticoid.

Figure 3

Schematic of the procedure used for two step sample preparation for glucocorticoid and nonsteroidal anti-inflammatory drug detection.

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After the elution, the column was washed with 6 mLof methanol and eluted with 5 mL of 2% (volume)
acetic acid/ethyl acetate. The resulting eluate wastermed Eluate N and contained the nonsteroidal antiinflammatory drug fraction.
Nonsteroidal anti-inflammatory (indomethacin,diclofenac, flunixin) and glucocorticoid(triamcinolone acetonide, betamethasone,methylprednisolone) reference standards werespiked into blank urine as samples and extracted withthe method described.
MS Analysis
To the Eluate G, 2 mL of 1 mol/L sodium hydroxide
was added and mixed for 1 min. The solution wasthen centrifuged for 1 min at 1000xg. The aqueouslayer was removed and the t-butyl methyl ether layer
was evaporated using a nitrogen stream ≤ 40°C. The
residual precipitate, called Residue G, was dissolved

with 500 µL of 0.1 vol% formic acid/acetonitrile (4:1)
mixture. This solution was used as the sample for LC/
MS/MS-1.
Eluate N was dried under the nitrogen streamat ≤ 40°C. The residual precipitate was called
Residue N. The Residue N was dissolved with 1000 µLof methanol. From this, 100 µL of aliquot was taken.
To the aliquot, 900 µL of methanol was added. The
mixture was used as the injection sample for LC/MS/
MS-2 analysis (Table 2).
The injection sample for LC/MS/MS-1 contained
dexamethasone-d4, hydrocortisone-d3 and theinjection sample for LC/MS/MS-2 contained
N-phenylanthranilic acid at 0.2 ng/mL, 10 ng/mL, and10 ng/mL, respectively, as internal standards.
A Prominence HPLC (Shimadzu) in combination witha 4000 QTRAP® MS/MS (AB SCIEX) was used for theLC/MS/MS analysis.

Table 1:

LC/MS/MS conditions for glucocorticoid analysis of Residue G
HPLC: PROMINENCE LC-20 (SHIMADZU)
ColumnAtlantis T3(Waters), L: 50 mm, ID: 2.1mm, particle size: 3µmColumn temperature

40°CMobile phase

A = 0.1vol% formic acid, B=acetonitrile

Gradient profile

Time (min)

00.5

6.0

6.01

7.5

7.51

7.6

9.5

A(%)

8080

502280

80

80

B(%)

20205098

98

202020Flowrate (mL/min)

0.2

0.2

0.2

0.2

0.2

0.2

0.4

0.4

Ionization accelerator

Acetonitrile with 10mM glycerol was added to column eluate at 0.1mL/min.

Injection volume

5 µL

MASS SPECTROMETER: 4000 Q TRAP (AB SCIEX)
Ionization method:

Electrospray ionization

Ion polarity:

Negative polarity mode

Ionization temperature:

600°C

Ionization spray voltage:-4500V

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Table 2:

LC/MS/MS conditions for nonsteroidal anti-inflammatory drug analysis of Residue N
HPLC: PROMINENCE LC-20 (SHIMADZU)
ColumnAtlantis T3(Waters), L: 50 mm, ID: 2.1mm, particle size: 3µm

Column temperature

40°C

Mobile phase

A = 0.1vol% formic acid, B=acetonitrile

Gradient profile

Time (min)

00.5

5.5

7.5

7.51

7.6

9.0A(%)

80

80

2280

80

80

B(%)

20209898

202020Flowrate (mL/min)

0.2

0.2

0.2

0.2

0.2

0.4

0.4Ionization accelerator

Acetonitrile with 10mM glycerol was added to column eluate at 0.1mL/min

Injection volume5 µL

MASS SPECTROMETER: 4000 Q TRAP (AB SCIEX)
Ionization method:

Electrospray ionization

Ion polarity:

Negative polarity mode

Ionization temperature:

600°C

Ionization spray voltage:-4500VRESULTS AND DISCUSSION
Examples of the drug detection using this system areshown in Figure 4. For all drugs detected, spiked
urine gave drug peaks’ relative retention times within± 2% of those of the peaks of reference
standards-only injections. There were nocontaminant peaks which hindered the detection of
the desired peaks from the analytes.
The detection sensitivity was evaluated by testing
the recovery of reference standards (n=5) (Table 3).
Glucocorticoids demonstrated a 67%–73% recovery,
while the nonsteroidal anti-inflammatory drugs wererecovered at 81%–97%.

Figure 4

Examples of glucocorticoid Betamethasone (BTM) detection (left)
and nonsteroidal anti‑inflammatory drug Flunixin (FNX) detection(right).
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Table 3:

Detected drugs and selected internal standard ions with recovery rates
PRECURSORION (m/z)

SELECTEDION (m/z)

SPIKED CONC.
(ng/mL)%RECOVERY%RSD(N=5)

Indomethacin

356.0

312.0

100

812.2

Diclofenac

294.0

214.0

50974.8

Flunixin

295.0

234.0

10096

3.4

N- phenylanthranilic acid

212.0

168.0

GLUCOCORTICOIDS

PRECURSORION (m/z)

SELECTEDION (m/z)

SPIKED CONC.
(ng/mL)%RECOVERY

%RSD
(N=5)

Triamcinolone acetonide

479.3

337.1

573

5.9

Betamethasone

437.2

361.0

0.2

683.3

Methylprednisolone

419.2

343.1

1067

3.0

Dexamethasone-d4

441.1

362.9Hydrocortisone-d3

410.2

127.9

NONSTEROIDAL ANTI-INFLAMMATORY DRUGS
Detected Drugs

Internal Standard

Detected Drugs

Internal Standard

CONCLUSIONS AND BENEFITS
Automating multiple elution steps into different
collection tubes allows parallel extractions ofglucocorticoid and nonsteroidal anti-inflammatorycompounds from the same sample tube.
With four probes, just one GX-274 ASPEC®
instrument provides sufficient throughput to prepare
samples for two HPLC/MS/MS systems in parallel.
This reduces the number of instruments required inthe lab, saving money and bench space.
The flexible TRILUTION® LH software allows easytranslation of manual SPE methods to fit into yourpersonalized workflows for compound detection andquantitation.

REFERENCES
1. Association of Racing Commissioners
International, Inc. Uniform Classification
Guidelines for Foreign Substances. Version
9.00 revised April 2015. http://arcicom.
businesscatalyst.com/
2.

Gilson Guide to SPE Automation. 2015. http://
www.gilson.com/Resources/Gilson_Guide%20
to_SPE_Automation_2015.pdf

3.

Wako Pure Chemicals Industry, Ltd. Page
accessed 11/6/15. http://www.wako-chem.co.jp/
english/labchem/product/analytical/presep_
rpp_sax/index.htm

The washable probes offer an economical alternative
to disposable tips and are proven to avoid crosscontamination.

ACKNOWLEDGMENTS
Special thanks to Takeshi Yokono and Naohiro
Imamura of M&S Instruments Inc.

Automated SPE provides verifiable results that canbe tracked and trusted.

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