Journal of Analytical Toxicology,Vol. 20, March/April 1996
Effectsof Sodium Bicarbonate and Sodium Chloride on
the Elimination of Etorphine in Equine Urine
D.R. Lloyd1, R.J. Rose1,*, A.M. Duffield 2, and C,l. Suann 2
7Departmentof Veterinary Clinical Sciences, University of Sydney, NSW 2006, Australia and 2AustralianJockey Club Laboratory,
Randwick, NSW 2031, Australia
Abstract t
The combination of large doses of sodium bicarbonate and the
potent narcotic, etorphine, has reportedly been given to
racehorses in attempts to improve their performance and also to
"mask" the presence of etorphine in urine samples. The increased
urinary output and pH associated with sodium bicarbonate
(approximately 500 g) administration may reduce the urinary
concentration of etorphine, making it more difficult to detect. Our
experiment was designed to examine the effects of this
combination. Six Thoroughbred horses were used in a latin-square
design with three horse pairs and three treatments consisting of
the following: etorphine (20 pg), etorphine (20 pg) plus sodium
bicarbonate (1.0 g/kg), and etorphine (20 pg) plus sodium chloride
(0.7 g/kg). Sodium chloride was used to distinguish between the
urinary alkalinizing effects of sodium bicarbonate and the diuretic
effects associated with the large electrolyte load. Venous blood
and urine samples were collected prior to and for 24 h posttreatment. Sodium bicarbonate produced a significant metabolic
alkalosis and an increase in urine pH. Both sodium bicarbonate
and sodium chloride produced a profound diuresis. After sodium
bicarbonate and sodium chloride treatments, the urinary
concentration of etorphine, measured by radioimmunoassay (RIA),
was reduced and in some cases could not be detected. Extraction
of the urine samples, prior to RIA analysis, increased the sensitivity
of the assay and in most cases gave a positive result. We conclude
that the coadministration of etorphine and sodium bicarbonate or
sodium chloride can make the detection of etorphine more
difficult because of the dilutional effects associated with the
administration of a large electrolyte load.
Introduction
In recent years, some athletes have resorted to techniques
that make it more difficult to detect drugs in urine samples. An
example of this is the use of diuretics to increase urine volume
and thus reduce the concentration of drugs in urine. This
practice has been recognized by the International Olympic
*Author to whom correspondenceshould be addressed.
Committee's Medical Commission, which has banned the use
of substances and methods that alter the integrity and validity
of urine samples used in doping controls (1). The technique of
diluting urine samples to hinder drug detection by the administration of diuretics has also been recognized as a potential problem in the horse racing industry, particularly in those
racing jurisdictions that permit the use of the diuretic
frusemide (2). Several studies have demonstrated that
frusemide decreases the concentrations of various drugs in
urine (3-7).
A popular routine for racehorse trainers has been the administration of large doses of sodium bicarbonate (approximately 500-600 g), via nasogastric tube, to their horses prior
to racing. Often a number of other ingredients are administered in conjunction with sodium bicarbonate, and together
this concoction has been termed a "milkshake" (8). This practice has now been prohibited in most racing jurisdictions.
Sodium bicarbonate has been administered with the belief
that it may improve the horses' performance by delaying the
onset of fatigue because of an enhanced blood buffering
capacity. We observed that the administration of sodium
bicarbonate (0.5 g/kg) can produce diuresis (9). Consequently,
it is possible that sodium bicarbonate may be used in a similar
way as frusemide to interfere with the detection of other drugs
in urine samples (8,10).
There are also anecdotal reports that sodium bicarbonate has
been used in racehorses to mask the presence of potent opioid
drugs, in particular etorphine. When etorphine (4,5-epoxy-3hydroxy-6-methoxy-oc,17-dimethyl-r
phinan-7-methanol), also known as "elephant juice", and a
sodium bicarbonate based milkshake are administered together, it is known as a "supershake"(11). In the horse, opiate
narcotic analgesics depress pain and, in low doses (approximately 50-100 pg per horse), etorphine has stimulatory or
excitatory locomotor effects (12,13). In addition, etorphine increases the performance time to exhaustion during treadmill
exercise (14), making it a particularly effective drug for stimulating racehorses. The rationale behind the supershake is
that etorphine acts on the central nervous system to stimulate
the horse, and sodium bicarbonate increases the horse's
Reproduction(photocopying)of editorialcontentof thisjournalis prohibitedwithoutpublisher'spermission.
81
Journal of Analytical Toxicology, Vol. 20, March/April 1996
stamina. In addition, because of the diuretic effect of sodium
bicarbonate, etorphine in the urine will be diluted, thereby
making detection more difficult.
Sodium bicarbonate also increases urinary pH (15), which
may influence the rate of excretion of etorphine in the urine.
Basic drugs, such as etorphine, are less likely to transfer into
an alkaline urine and will preferentially remain in a more
acidic solution (2). This effect, combined with the diuresis,
may result in etorphine administration going undetected.
The current study was designed to examine whether the diuretic and urinary alkalinizing effects resulting from sodium
bicarbonate administration (1.0 g/kg) would affect the concentration and detection of etorphine in urine samples over a
24-h period. The effects of a dose of sodium chloride (0.7 g/kg)
were also examined to determine if the concentration of etorphine was affected by a substance that increased urine volume
but did not increase urine pH. In this way it was possible to distinguish between the urinary alkalinizing effects of sodium
bicarbonate and the dilutional effects associated with an increased urine output.
We decided to standardize the times of administration of
etorphine and sodium bicarbonate or sodium chloride by
giving them simultaneously. An alternative would have been to
administer etorphine so that the time of its peak urinary concentration coincided with the peak urine pH or diuretic effect.
However, because each horse is likely to experience peak urine
pH or diuresis at different times, this would introduce another
variable into the experiment that would be difficult to control.
Methods
Pilot study
A pilot study was performed to determine the effects of different dosesof etorphine on the behavior of the horses, as well
as the length of time etorphine could be detected in the urine.
Anecdotal reports indicated that the doses of etorphine likely
to be given are approximately 100 lJg per horse. However, for
practical purposes it was necessary to administer a dose of
etorphine that would not excite the horses and that would
allow urine and blood samples to be collected. Two horses
were used, eachwearing urine collection harnesses,and placed
in specially constructed stocks. One horse was given an intravenous dose of etorphine (Large Animal Immobilon; C-Vet,
Suffolk, England) of 10 IJg, and the second horse was given a
doseof 20 IJg. Urine was collected for 72 h after administration,
and the concentration of etorphine determined on hydrolyzed
urine by radioimmunoassay (RIA) is described. No excitatory
effects were observedwith either dose. Becausethe 20-1Jgdose
did not excite the horses and was closer to the dose of etorphine reportedly used in racehorses, it was considered the
more appropriate dose for the proposed study.
Experimental design
Six horses (three pairs) were used in a latin-square design involving three treatments: etorphine (20 I~g),etorphine (20 IJg)
plus sodium bicarbonate (1.0 g/kg), and etorphine (20 IJg) plus
82
sodium chloride (0.7 g/kg). Water was freely available at all
times.
Each horse wore a urine collection harness and was placed
in a specially constructed stocks 2 h prior to treatment. Immediately prior to treatment a catheter was inserted into the
jugular vein, and venous blood and urine samples were collected to give control samples. The dose of etorphine (20 IJg)
was injected via the catheter, which was then flushed with 20
mL of heparinized saline (4 IU/mL).
Venous blood and urine samples (when freely voided) were
collected every hour up to 15 h postadministration and then at
20, 21, 22, 23, and 24 h postadministration. Blood samples
were collected into 2-mL heparinized syringes and immediately
placed in a crushed ice slurry for blood gas and acid-base
analysis (ABL300;Radiometer, Copenhagen, Denmark) within
1 h of collection. Urine volume and pH (PHM83 Autocal pH
meter; Radiometer) were measured immediately after collection. Portions were stored for specific gravity (Reichert refractometer; Cambridge Instruments Inc., Buffalo, NY) and
osmolality (vapor pressure osmometer 5500; Wescor Inc.,
Logan, UT) measurements. An extra 10 mL was stored at-20~
for etorphine analysis (via RIA), and another 150-200 mL of
urine from each collection time was stored at 4~ for gas chromatographic-mass spectrometric (GC-MS) analysis.
It was not possible to perform statistical analysis on the
urine data between treatment groups as the horses urinated infrequently when etorphine only was given, providing inadequate data for statistical comparisons to be made. The correlations between urinary specific gravity and etorphine
concentration and also urinary specific gravity and urinary
osmolality for the three treatment groups were determined
using least-squares regression. The venous blood pH results
were analyzed by analysis of variance with time as the repeated
measures factor. Where the F values were significant a post-hoc
Tukey test was used. All results are reported as the mean plus
or minus the standard error of the mean.
Detection of etorphine in the urine
RIA detection. The concentration of etorphine was determined using the method described in the Etorphine [l~Sl]
Double Antibody Radioimmunoassay kit (Diagnostic Products
Corp., Los Angeles, CA). All samples, calibrators, and controls
were analyzed in duplicate, and background values for etorphine equivalentswere obtained from the control urine samples.
Extraction of etorphine for RIA analysis. The screening of
urine samples for etorphine by the method outlined above
revealed that there was a remarkable decrease in the urinary
etorphine concentrations when either sodium bicarbonate or
sodium chloride was coadministered with etorphine (Figure 1).
A basic back extraction procedure, rather than direct analysis
of urine, was used to measure the etorphine concentrations in
17 of these urine samples and four preadministration urine
samples.
Urine samples were prepared for RIA screening by the following method: Urine samples (5 mL) were adjusted to pH 5
and hydrolyzed with I drop per milliliter of concentrated glucuronidase-aryl sulfatase (Boehringer Mannheim, Mannheim,
Germany) overnight at 37~ Each urine sample was adjusted
Journal of Analytical Toxicology, Vol. 20, March/April 1996
to pH 9.5 + 0.5, dichloromethane (5 mL) was added, and the
capped tubes were rotoracked for 10 rain and centrifuged at
2000 rpm for 10 min. The aqueous layer was aspirated to waste,
and the dichloromethane was vortex mixed with 1 mL 0.2M
hydrochloric acid. After centrifugation at 2000 rpm for 10
min, the acidic layer was transferred to another tube, and the
pH was adjusted to 9.5 • 0.5 with 10% ammonium hydroxide.
Hexane (3 mL) was added, and the tube was vortex mixed (30
s). The organic phase was dried (anhydrous sodium sulfate),
transferred to a Kimble tube, and evaporated at 50~ under N2
gas. The residue was reconstituted in 50 pL of buffered saline
(pH 7) and transferred to polypropylene tubes for RIA screening
as previously outlined.
Preparation of urine for GC-MS analysis
Extraction
Enzymatically hydrolyzed urine (10 mL; pH 9.5 • 0.5) was
extracted with hexane (three times with 2-mL portions)
by rotoracking for 15 rain, and then it was centrifuged at
2000 rpm for 10 rain. The combined hexane extracts were
vortex mixed (30 s) twice with 2 mL 0.25M sulfuric acid and
centrifuged at 2000 rpm for 10 min. The aqueous phase was
vortex mixed (30 s) twice with hexane (2 mL) and centrifuged
at 2000 rpm for 10 rain, and the hexane was discarded. The pH
of the aqueous fraction was adjusted to 9.5 • 0.5 and extracted
with hexane (four times with 2-mL portions) and centrifuged
at 2000 rpm for 10 min. The combined hexane extracts were
dried (anhydrous sodium sulfate), and the organic phase was
dried under N2 gas at 50~
and a 2-pL aliquot was used for positive ion chemical ionization
(PCI) GC-MS analysis.
Pentafluorobenzoylation. Hydrolyzed urine was extracted
and base back extracted as previously described, except that
diisopropyl ether was used as the organic solvent. The dry
residue was reacted with 2,3,4,5,6-pentafluorobenzoyl chloride (Sigma Chemical Co., St Louis, MO) (10 drops of a 10%
solution in ethyl acetate) at 50~ for 30 rain. The reaction
mixture was evaporated to dryness under N2 gas at 50~ the
residue was reconstituted in dry ethyl acetate (50 tJL), and a
2-pL aliquot was used for negative ion chemical ionization
(NCI) GC-MS analysis.
GC-MS analysis
A Varian Instruments 3400 gas chromatograph interfaced to
a triple stage quadruple mass spectrometer (model 700;
Finnigan MAT,San Jose, CA) was used for GC-MS analysis. The
GC injector temperature and MS transfer line were both maintained at 275~ and the MS ion source was set at 180~ Samples were injected at an initial GC oven temperature of 100~
(3 min), and the temperature was programmed to 300~
(3 min) at 20~
For NCI, the final temperature was main-
A
4
O
E
-5
>
t"E
Derivatization of urine extract
Acetylation. The dry residue was acetylated with acetic an-
3
2
1
0
hydride (3 drops) in pyridine (3 drops) at 75~ for 45 rain, and
the reaction mixture was evaporated to dryness under N2 gas at
50~ The residue was reconstituted in ethyl acetate (50 pL),
I
I
I
I
I
I
I
I
I
1.05
I
I
I
I
)
I
I
i
i
i
B
1.04
>
1.03
5000
O
1.02
r
O
ffl
4000
E
3500
1.01
1.00
I
I
I
I
I
I
I
I
I
I
3000
e 1200
2500
E
0
9
C
O
2000
g 1000
1500
8OO
1000
(D
500
.m
600
~
4
6
8 10 12 14 16 18 20 22 24
Time postadministration (h)
Figure 1. Changes (mean plus or minus standard error of the mean) in urinary etorphine concentrations after the administration of etorphine (20 pg)
( I - t ) , etorphine (20 pg) plus sodium bicarbonate (1.0 g/kg) (O-O), and
etorphine (20 IJg) plus sodium chloride (0.7 g/kg) (A-A) (n = 6). Etorphine
concentrations were measured by RIA analysis.
I-L
4OO
i
I
pre 2
-
i
i
i
i
pre 2
4
6
8 10 12 14 16 18 20 22 24
i
i
i
i
i
Time postadministration (h)
Figure2. Changes(meanplusor minusstandarderrorofthe mean)in (A)
urine volume, (g) specificgravity,and (C) urine osmolalityafter the
administrationof etorphine(20 pg)(O-e),etorphine(20 pg) plus sodium
bicarbonate(1.0g/kg)(O--O),and etorphine(20 pg) plussodiumchloride
(O.7~g)(A-A)(n = 6).
83
Journal of Analytical Toxicology, Vol. 20, March/April 1996
tained at 300~ for 10 rain. The GC column was an Ultraphase
1 (Hewlett-Packard,Palo Alto, CA),and helium was the carrier
gas (flow rate, 1 mL/min). For PCI, the spectrometer was
scanned from m/z 60 to 550 in 0.5 s, and for NCI, the spectrometer was scanned from rn/z 100 to 620 in 0.5 s. Methane
was used as the chemical ionization reagent gas (0.12 kPa).
Results
Effects of sodium bicarbonate and sodium chloride on fluid
and acid-base balance
The administration of sodium bicarbonate and sodium chloride produced an increase in the urine volume (Figure 2),
which was maintained for approximately 20 h postadministration. The diuretic effectwas pronounced after sodium chloride administration, and there was a peak urine volume of
3.3 • 0.9 L/h at 5 h postadministration. Sodium bicarbonate
administration resulted in a lower average urine output than
sodium chloride, but was higher than the horses that received
only etorphine; at 5 h postadministration, the average value
A
7.4-8
was 1.3 • 0.2 L/h compared with 0.5 • 0.2 L/h for the etorphine-treated horses. There was a marked drop in urine specific
gravity and osmolality (Figure 2) after both sodium bicarbonate and sodium chloride administration, but this was more
pronounced with sodium chloride, indicating the dilute nature
of the urine associated with the excretion of large electrolyte
loads. The values returned to normal levelsby 22 h. In the control horses and horses given sodium chloride, there was a drop
in urine pH of approximately 0.7-1 pH units over the first
16 h postadministration (Figure 3). Urinary pH then increased
and returned to the resting level after 20 h. The administration
of sodium bicarbonate caused a rise of approximately 0.6 pH
units, which was sustained for approximately 12 h and returned to resting levels by 24 h.
Both sodium chloride and sodium bicarbonate administration caused considerable alterations to the horses' acid-base
status, as shown by the changes in blood pH (Figure 3).
Sodium bicarbonate administration caused an alkalosis, resulting in a significant increase in blood pH from a resting
value of 7.382 • 0.006 to a peak value of 7A54 • 0.006 at 7 and
10 h postadministration, whereas sodium chloride caused a
mild acidosiswith blood pH decreasing from a resting value of
7.361 • 0.01 to 7.312 • 0.006 at 2 h after treatment. Blood pH
had returned to resting levelsby 7 and 20 h in sodium chloride
and sodium bicarbonate treated horses, respectively.
7.46
7.4-4-
Table I. Urinary Etorphine (Et) Concentrations (and
Etorphine Equivalents) Derived from RIA Analysis
Directly on Urine and on the Same Urine Samples after
Solvent Extraction and Purification from Treated and
Untreated Horses
7.#2
-r 7.4,0
r.~
7.38
7.36
Urinaryetorphine
concentration(pg/mL)
7.3#
7.32
Sample
(treatment)
7.30
7.28
i
i
[Dre 2
i
i,
i
i
i
i
i
i
1
i
8.8
,,
8.2
8.0
o
6.6
6.4
pre
l
l
J
~
I
I
I
I
~
~
I
2
4 6 8 10 12 14 16 18 20 22 24
(h)
Time postadministration
I
Figure 3. Changes (mean plus or minus standard error of the mean) in (A)
venous blood and (8) urine pH after the administration of etorphine (20 IJg)
( I - I ) , etorphine (20 iJg) plus sodium bicarbonate (1.0 g/kg) (O-O), and
etorphine (20 pg) plus sodium chloride (0.7 g/kg) (A-A) (n = 6).
84
extractionof
etorphine
I
4. 6 8 10 12 1# 16 18 20 22 24.
Time postadministration (h)
8.#
directanalysis
on urine
Preadministration
Preadministration
Preadministration
Preadministration
Et + NaHCO3 (4 h)
Et + NaHCO3 (4 h)
Et + NaHCO3 (9 h)
Et + NaHCO3 (9 h)
Et + NaHCO3 (10 h)
Et + NaHCO3 (11 h)
Et + NaHCO3 (11 h)
Et + NaHCO 3 (14 h)
Et + NaHCO 3 (20 h)
Et + NaHCO3 (22 h)
Et + NaC] (3 h)
Et + NaCI (4 h)
Et + NaCI (10 h)
Et + NaCI (11 h)
Et + NaCI (20 h)
Et + NaCI (20 h)
Et + NaCI (20 h)
429.78
215.21
162.53
133.82
594.28
405.25
77.71
105.10
141.47
108.89
77.23
119.26
30.35
75.63
576.64
622.01
63.06
51.45
104.71
96.01
38.03
373.92
196.67
308.20
115.87
81,003
64,643
1603.1
377.50
2284.0
19.49
2448.1
2779.1
1335.7
2886.0
34,431
30,057
482.16
2072.7
2482.9
1950.2
904.44
Journal of Analytical Toxicology, Vol. 20, March/April 1996
RIA detection of etorphine
The variations in urinary concentration of etorphine
(20 pg) with time are shown in Figure 1. It should be noted
that not all the horses urinated at each of the collection times,
and therefore, the points on the graphs only indicate the values
that were obtained at each time. This was especially the case
when the horses were given etorphine only, which may account for the larger standard error bars. In horses that received etorphine only as well as etorphine plus sodium bicarbonate, the peak urinary concentration of etorphine occurred
at 2 h postadministration. The peak concentrations that were
found at 2 h were 3896 • 779 and 3530 • 513 pg/mL for horses
that received etorphine only (n = 3) and etorphine plus sodium
bicarbonate (n = 4), respectively. This was followed by a profound decrease in urinary etorphine concentrations in horses
that received sodium bicarbonate; the mean concentration
was 405 ___86 pg/mL at 5 h. In the etorphine-only treated
group, the concentration had decreased to 1312 • 228 pg/mL
by 6 h postadministration and slowly declined to a mean concentration of 733 • 141 pg/mL by 24 h. The administration of
sodium chloride with etorphine produced an increase in urinary etorphine concentration at 2 h postadministration that
was less than that for etorphine plus sodium bicarbonate and
the etorphine-only treated groups; the mean concentration
was 1730 • 432 pg/mL at 2 h (n = 4). The urine etorphine concentration remained at this level for the next 2 h and then fell
to a concentration of 399 • 145 pg/mL at 4 h. By 6 h postadministration the urinary etorphine concentration in the horses
that received sodium bicarbonate and sodium chloride was
between 20 and 260 pg/mL, and it remained at this level until
IE+086
436
[MH-H20]
~176
1
1133
13:14
m/z454
21 h when it rose by approximately 300 pg/mL.
The background concentration of etorphine equivalents in
the preadministration urine samples varied between 133 and
429 pg/mL. Therefore, when sodium bicarbonate or sodium
chloride was administered with etorphine, a negative result
could be returned between 6 and 20 h postadministration,
whereas positive results would be obtained for the entire 24-h
period in horses that received only etorphine. In some instances during the 6-20-h period, the values were lower than
those obtained before etorphine was administered, suggesting
that the background was diluted to a lower level in addition to
decreasing the etorphine concentration. The increase in the
etorphine concentration at 21-24 h in the horses given sodium
bicarbonate and sodium chloride coincided with an increase in
the urine specific gravity (Figure 2), indicating that there was
a return by this time to the normal urine concentrating capacity. Although this seems a likely explanation for these
changes, it is not known how the background levels change
over time in an untreated horse. However, the urinary specific
gravity and etorphine concentration were poorly correlated,
and there were correlation coefficient (r) values of 0.351, 0.542,
and 0.536 for control, sodium bicarbonate, and sodium chloride treated horses, respectively. There was a good correlation
between the urinary specific gravity and the urinary osmolality for all three treatment groups, and the r value was 0.951.
Solvent extraction of etorphine from urine samples was used
to increase the etorphine concentration prior to RIA analysis,
3J
§
E+05
' 8.286
100
80
50
60
+
E
RIC
475
7:24
~" 100
E+08
1.299
40
454
IMHI+
20
50
366
258
I
482
164
....
87 121 a.,78 .~ 286:+
+ + ._s 2.a~. LL..~. . . .
,
. . . . .
1O0
....
,+-+-,
200
,
400
,
9
i+---,.
,
.......
, . . . . . .
600
800 1000
Scan number
,
......
1200
,.,
1400
Figure 4. PCI (methane) GC-MS analysisof an extract from equine urine
1 h postadministrationof etorphine (20 pg). Etorphineacetate eluted at a
retention time of 13 min and 14 s (scan 1133).
250
- , - - ,
. . . . .
200
- - - - i
. . . .
300
m/z
tl!
'
9
- . - i - - -
400
9
- - -
494
r
5 2 1
r
. . . . .
500
Figure 5. The PCI (methane) mass spectrum of etorphine acetate from a
sample identified in Figure 4 at 1 h postadministration of etorphine (20 lag).
The protonated molecular ion occurred at m/z454 with the elimination of
water providing the base peak at m/z 436.
85
Journal of Analytical Toxicology, Vol. 20, March/April 1996
reducing the background interference. Table I shows the urinary etorphine concentrations derived from RIA analysis
directly on urine and on the same urine samples after solvent
extraction and purification. The samples chosen for this study
were preadministration urine specimens, as well as urine samples that produced negative results or were considered to be
borderline between a positive result and background on RIA
analysis of urine. A positive screen for etorphine would be indicated if the concentration was greater than 500 pg/mL after
extraction. It is evident that in most cases the extraction procedure resulted in the RIA screening of etorphine, although in
several instances a negative result was still obtained (Table I).
All RIA screening of etorphine in equine urine must be confirmed by GC-MS before etorphine is declared to be present in
that urine specimen.
GC-MS detection of etorphine
Two urine samples from horses given etorphine were analyzed using GC-MS to confirm that the positive results derived
from the RIA analysis were due to the presence of etorphine.
One sample was acetylated using acetic anhydride in pyridine
and analyzedusing PCI (methane) GC-MS analysis.Etorphine
acetate eluted at a retention time of 13 min and 14 s (scan
1133) (Figure 4), and its PCI mass spectrum is shown in Figure
5. The protonated molecular ion [MH § occurred at mlz 454,
and the elimination of water provided the basepeakat mlz 436.
The second urine sample was derivatized using 2,3,4,5,6pentafluorobenzoylchloride and analyzedusing NCI (methane)
GC-MS analysis. Etorphine 2,3,4,5,6-pentafluorobenzoate
eluted at 15 rain and 59 s (scan 1431) (Figure 6), and its mass
100
1159
13:33
m/z 605
1431
15:59
spectrum is reproduced in Figure 7; the molecular anion [M-]
occurred at m/z 605.
Discussion
The administration of sodium bicarbonate to racehorses has
been a controversialpractice in the horse racing industry in recent years. It is apparent that sodium bicarbonate has been
widely administered in the Standardbred racing industry
(8,15), and authorities in several countries have now prohibited
its use, alone or in the form of a milkshake. Most countries
have now instigated testing procedures focusing on the measurement of bicarbonate or total carbon dioxide concentrations in venous blood. The administration of sodium bicarbonate (1.0 g/kg) or an equimolar dose of sodium chloride
produced changes in the fluid and acid-base balance that affected the concentration of etorphine in equine urine. Sodium
chloride administration resulted in a slight metabolic acidosis,
whereas sodium bicarbonate treatment resulted in a profound
metabolic alkalosis. The decrease in urinary etorphine concentration can be attributed to the diuresis caused by the administration of the large electrolyte loads. Although it has
been suggested that the administration of sodium bicarbonate
may be able to reduce the rate of etorphine excretion into the
urine by increasing the urine pH (8), it seems that the decrease in urinary etorphine concentration is more likelydue to
a dilutional effect.Sodium bicarbonate produced an increase in
the urinary pH, whereas sodium chloride produced a decrease
100 "
605 E+06
[M-]
E+06
1.150
1,03
80"
50
60
E
100
RIC
E+08
"1.203
167
9
148
n.- 40 9
541
196
523
50
20 .
I
498 '
203
130
I
I
I-
'
.
5OO
. .
, . . . .
1000
Scan number
, . . . .
283
07
4r~
T9
I
3~ 1. s
I
I,
, ....
15oo
Figure 6. NCI (methane) GC-MS analysis of an extract from equine urine
I h postadministration of etorphine (20 pg). Etorphine 2,3,4,5,6-pentafluorobenzoate e]uted at 15 min and 59 s (scan 1431).
86
393
238
100
200
300
400
500
600
m/z
Figure 7. NCI (methane) mass spectrum of etorphine 2,3,4,5,6-pentafluorobenzoate from a sample identified in Figure 6 at I h postadminis-
trationofetorphine(20pg).Themolecularanion[M-]occurredat m/z605.
Journal of Analytical Toxicology,Vol. 20, March/April 1996
in urinary pH; yet there was a greater reduction in the urinary
etorphine concentration associated with sodium chloride. In
addition, there was a greater urine output when the horses received sodium chloride compared with sodium bicarbonate.
This finding was surprising, as equimolar doses of sodium
chloride and sodium bicarbonate were administered. It seems
likely that the increase in urinary volume was responsible for
the lower etorphine concentration rather than the effects of a
change in urine and blood pH.
To overcome the dilution effect of large electrolyte loads
upon the excretion of etorphine into urine, an extraction step
was used prior to RIAanalysis. This preconcentrated etorphine
and effectively increased the sensitivity of the RIA assay, also
reducing the levels of background substances. The presence of
background levels of substances that register as etorphine
equivalents has also been recognized as a problem in immunoassay tests on equine urine (16,17). In all urine samples
that had borderline amounts of etorphine (concentrations in
the range of 400-600 pg/mL), the extraction procedure gave
positive results. In those samples that had low etorphine concentrations and would have been regarded as negative, extraction resulted in positive results using RIA in nearly all
instances. There were several cases where the administration
of the electrolyte load reduced the urinary concentration of
etorphine and etorphine equivalents to levels less than those
found in urine samples prior to etorphine administration.
Other researchers (17), using RIA analysis, confirmed the
results found in the current experiment: hydrolysis and
extraction increase the sensitivity of the assay and therefore
increase the length of time a single dose of etorphine can be
detected in equine urine samples.
A previous study that examined the relationship between specific gravity and background etorphine equivalents concluded
that there was no correlation between these measurements
(18). This study only examined the relationship between specific
gravity and the level of background etorphine equivalents in
postrace horse urine samples where etorphine had not been
administered. It was thought that a relationship between specific
gravity and etorphine concentration may be evident after a dose
of etorphine and with the production of a very dilute urine.
However, in the current study, urinary specificgravity and etorphine concentration were poorly correlated in control, sodium
bicarbonate, and sodium chloride treated horses. Despite this
poor correlation, it can be observed in Figure 2 that the decrease
in specific gravity when sodium bicarbonate and sodium chloride are administered relates to the time in which a decrease in
urinary etorphine concentration occurs (Figure 1). The increase
in etorphine concentration, which occurs after 21 h, in sodium
bicarbonate and sodium chloride treated horses is likely to be
due to a return of the renal concentrating capacity after the
diuretic effectsof the electrolyte load have passed. Similar trends
have also been noted after the diuretic effects of a dose of
frusemide have passed with the urinary concentrations of
fentanyl, phenylbutazone, and morphine (5,6).
Although the coadministration of etorphine and sodium
bicarbonate or sodium chloride resulted in a decreased urinary
etorphine concentration, other dosing schedules may produce
similar or greater decreases in urinary drug concentrations. It
is possible that sodium bicarbonate and sodium chloride could
produce a more profound decrease in the urinary etorphine
concentrations if the administration of etorphine and the electrolyte solution were timed so that the peak diuresis coincided
with the time of peak etorphine excretion, between 1 and 3 h
postadministration. A similar approach was used by other
researchers (7) when investigating the excretion of several
drugs in conjunction with frusemide administration.
Another factor to consider is that in the current study the
horses were allowed free access to water at all times. If water
was withheld for a period of time after the administration of
etorphine and sodium bicarbonate or sodium chloride, the
reduction in urinary etorphine concentration may be less
marked because of less dilution, resulting from a decreased
urine output. However, it should be noted that after the
administration of sodium bicarbonate (0.5 g/kg) there is still an
increase in the volume of urine produced if water is withheld
for a period of 6 h posttreatment (9).
The current studies were performed in resting horses. Urine
samples for detection of etorphine were collected following
racing, which could affect urine volume and pH. This may
have compounding effects on urinary drug excretion patterns.
Further studies investigating the effects of strenuous exercise
may be of value.
In conclusion, we have demonstrated that the administration
of the so-called supershake, consisting of sodium bicarbonate
and etorphine, was capable of reducing the urinary concentrations of etorphine. Although in most cases it was still possible to detect the presence of etorphine by solvent extraction
of urine prior to RIAanalysis, it seems likely that there is an increased risk of some cases of etorphine administration going
undetected when urine is used for screening. However, the
dose of etorphine administered to the horses in the present
study was lower than that reportedly illegally given prior to
racing, and it is possible that the supershake would not be as
effective masking the presence of larger doses.
Acknowledgments
We would like to thank Claire Ward, Sally Martyr, Pamela
Manning, Michael Eaton, and Shirley Ray for their valuable
technical assistance. This study was supported by the New
South Wales Racing Research Fund, and laboratory facilities
were provided by the Racecourse Development Fund.
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Manuscript received December 16, 1994;
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