Ž .
The LCrMS system consisted of a Hewlett-Packard Palo Alto, CA Model 1050 LC Ž
. pump and a Finnigan San Jose, CA Model TSQ-7000 triple-quadrupole mass spec-
Ž .
trometer with a standard Finnigan electrospray ESI ion source. All chromatography Ž
. Ž
was performed on a YMC Wilmington, NC J-sphere ODS-M80 LC column 2 = 250 .
mm . The mobile phase flow rate was 0.2 mlrmin and all other buffers and gradients were the same as those used to prepare the metabolite fractions. Nitrogen was used as a
nebulizing gas and the ion source capillary temperature was 2308C. For full scan MS experiments, the instrument was scanned over the range of 150–700 amu at 1 srscan.
For MSrMS experiments, the instrument was scanned from 15–380 amu at 1 srscan. Argon was used as the collision gas and collision energy was 40 V.
2.6.3. Liquid scintillation counting LSC Total
14
C contents in tissues, fluids, and HPLC effluent fractions were determined by Ž
. LSC, as previously described Plakas et al., 1998 . Radioactivity in tissues or fluids was
Ž .
expressed in units of concentration mg equiv. of FLUrg or ml or as a percentage of the administered dose. With no practical means of solubilizing bone for LSC, caudal
Ž .
vertebrae specimens ; 0.5 g from each fish were extracted twice with 4 ml acidified methanol by shaking overnight at room temperature in a mechanical shaker. Methanolic
extracts were combined and aliquots were analyzed by LSC.
2.6.4. Plasma protein binding Ž
Plasma protein binding of parent FLU was determined by ultrafiltration Centrifree .
14
Micropartition System, Amicon, Beverly, MA . C-FLU was added to control plasma at
Ž .
concentrations ranging from 0.125 to 8 mgrml. Duplicate aliquots 0.4 ml of spiked Ž
. plasma were added to the ultrafilters and centrifuged at 1000 = g for 1 h 258C . Entire
ultrafiltrates were analyzed for
14
C-FLU contents by LSC, representing unbound drug. Values were corrected for non-specific absorption of unbound drug to the ultrafiltration
Ž .
apparatus ; 6 . Non-specific absorption was determined by the corresponding analy- sis of control plasma ultrafiltrates spiked with
14
C-FLU. Additionally, plasma specimens
14
Ž .
collected from fish dosed orally with C-FLU as described above were analyzed for
protein binding of total radioactive residues. 2.6.5. Pharmacokinetics
Ž .
The computer program WinNonlin Scientific Consulting, Cary, NC was used to derive pharmacokinetic values for parent FLU concentrations in plasma. Oral bioavail-
ability was calculated from the areas under the plasma concentration-time curves, normalized for dose.
3. Results
3.1. Pharmacokinetics Parent FLU levels in plasma declined slowly after intravascular dosing and were
Ž .
Ž .
measurable G 0.01 mgrml for up to 168 h Fig. 1 . Elimination half-life was 25 h
Ž .
Fig. 1. Mean parent FLU concentrations in plasma after intravascular dosing at 1 mgrkg B or oral dosing at Ž .
Ž .
5 mgrkg Ø in channel catfish ns 5 .
Ž .
Ž .
Table 1 . Other pharmacokinetic parameters Table 1 indicate moderate distribution of Ž
. Ž
FLU outside of the vascular system V , 527 mlrkg , and slow clearance Cl , 14.9
ss b
. mlrhrkg .
Ž .
After oral dosing, parent FLU was measurable in plasma within 15 min Fig. 1 . Peak plasma concentration was 3.1 mgrml at 14 h. Absorption and elimination half-lives
Ž .
were 4.9 and 22 h, respectively Table 1 . The bioavailability of FLU was 44. FLU levels were not quantifiable beyond 168 h.
3.2. Plasma protein binding Plasma protein binding of
14
C-FLU in spiked plasma was extensive, but saturable, Ž
. declining from 88 at 0.125 mgrml to 55 at 8 mgrml Fig. 2 . Similar values were
Table 1 Pharmacokinetic values for parent FLU in channel catfish
Ž .
Ž .
Intravascular 1 mgrkg Oral 5 mgrkg
Parameter Unit
Value Parameter
Unit Value
t el
h 24.6
t abs
h 4.94
1r 2 1r2
AUC mgPhrml
67.3 t
el h
21.8
1r 2
Cl mlrhrkg
14.9 AUC
mgPhrml 149
b
V mlrkg
527 C
mgrml 3.06
ss max
MRT h
35.5 t
h 13.7
ma x
F 44.3
Pharmacokinetic parameter abbreviations: t el, elimination half-life; AUC, area under the plasma concentra-
1r 2
tion-time curve; Cl , total body clearance; V , apparent volume of distribution at steady-state; MRT, mean
b ss
residence time; t abs, absorption half-life; C
, maximum plasma concentration; t , time to C
; F,
1r 2 max
max max
fraction of the oral dose available systemically.
Fig. 2. Plasma protein binding of FLU as determined in vitro.
found for residual
14
C in plasma of orally dosed animals, depending on concentration or Ž
. time after dosing data not shown . For example, mean binding values were 67 at 24 h
Ž
14
. peak
C levels and 89 at 168 h. 3.3. Tissue distribution and elimination
After oral dosing with
14
C-FLU, total residue concentrations in the tissues were Ž
. Ž
. highest at 12–24 h Table 2 . At 24 h, liver had the highest concentrations 6.2 mgrg
Ž .
and muscle had the lowest 1.8 mgrg of any tissues analyzed. Concentrations in plasma, trunk kidney, head kidney, and spleen were similar, ranging from 3.3 to 4.4
mgrg at 24 h. Rates of elimination of radioactive residues were also similar between individual tissues. In muscle,
14
C concentrations declined below the limit of determina-
Table 2 Ž
.
a
Concentration of radioactive residues mg equiv.rg or ml in the bile and tissues of catfish after oral
14
Ž .
administration of C-FLU 5 mgrkg
Ž . Tissue
Time after dosing h 3
6 12
24 72
168 336
Bile 0.480.24
6.307.52 12.26.18
33.915.3 11971.1
91.034.9 82.427.7
Liver 2.152.12
4.622.91 5.462.01
6.151.24 1.770.74
0.340.20 0.080.04
Plasma 1.710.46
3.160.96 4.001.97
4.100.89 3.001.20
0.490.44 0.020.02
Trunk kidney 0.950.24
2.921.35 3.781.54
4.411.16 1.540.52
0.250.17 0.050.02
Head kidney 0.750.27
2.421.12 3.801.75
3.370.82 1.070.38
0.220.16 0.070.03
Spleen 0.700.27
2.421.16 3.351.26
3.340.78 1.070.46
0.240.18 0.110.06
Skin 0.420.12
1.320.74 1.840.73
2.180.72 0.840.20
0.260.16 0.090.05
b
Muscle 0.280.12
1.030.68 1.800.91
1.840.47 0.420.19
0.070.06 –
a
Values are the means.d. of five animals.
b
Ž .
Below the limit of determination - 0.005 mg equiv.rg .
Fig. 3. Parent FLU concentrations in muscle after oral dosing at 5 mgrkg in channel catfish. Values are the means.d. of five animals.
Ž .
tion by LSC - 0.005 mgrg at 336 h. In skin, concentrations were initially similar to those in muscle, but were eliminated more slowly. Residue levels in bone, as estimated
by solvent extraction, were 0.11 mgrg at 3 h, 0.66 mgrg at 24 h, and 0.03 mgrg at 336 Ž
. h data not shown .
Ž .
Parent FLU concentrations in muscle, as determined by HPLC Fig. 3 , were nearly Ž
. identical to total residue concentrations, by LSC Table 2 . Parent FLU levels were 1.8
Ž .
Ž mgrg at 24 h and 0.07 at 168 h Fig. 3 . Parent FLU was not quantifiable - 0.01
. mgrg beyond 168 h. Estimated half-life for elimination of FLU in muscle was 26 h.
Ž .
14
Fig. 4. Renal excretion of FLU and its metabolites total residues after oral dosing with C-FLU at 5 mgrkg
Ž .
in channel catfish: B, concentration mg equiv.rml ; Ø , cumulative dose.
3.4. Excretion Residues were highly concentrated in bile, relative to the tissues. Total residue
concentrations increased rapidly from 0.48 mgrml at 3 h to 119 mgrml at 72 h, and Ž
. Ž
. then declined slowly to 82 mgrml at 336 h Table 2 . At peak levels 72 h , the amounts
of
14
C contained in the gall bladder represented 3–4 of the administered oral dose. Ž
. In urine, total residue concentrations were highest 0.88 mgrml at the 12–24 h
Ž .
14
sampling interval Fig. 4 . By 168 h, C levels in urine were 0.04 mgrml and at 336 h,
- 0.01 mgrml. Cumulatively, 4.9 of the oral dose was excreted in urine over the
2-week sampling period. 3.5. Metabolism
Only parent FLU was found in muscle and plasma after oral dosing with
14
C-FLU, under our conditions of analysis. In bile, parent FLU was a minor component at the
Fig. 5. Radiochromatograms of bile and urine collected from channel catfish after oral dosing with
14
C-FLU at 5 mgrkg.
Ž .
earlier sampling times e.g., 12 of total residues at 24 h , but was not detectable beyond 72 h. Residues in bile were mostly composed of a single, polar metabolite with a
Ž .
retention time of ; 13 min Fig. 5 . The molecular weight of this metabolite was 368, Ž
by LCrMS. This finding, along with a significant fragment ion at mrz 80 80 relative .
abundance in the negative ion MSrMS spectrum, is consistent with the metabolite being a taurine conjugate of FLU.
Ž .
In urine, four radioactive peaks were found Fig. 5 . Parent FLU comprised 72 of total radioactive residues at 3 h, 55 at 24 h, and 38 at 168 h. The taurine conjugate
of FLU also was identified, comprising 11 of total residues at 3 h, 32 at 24 h, and Ž
59 at 168 h. Two minor metabolites, which eluted as a doublet peak retention time, .
9.5–10 min by HPLC, comprised - 10 of total radioactivity. These minor metabo- lites were both identified as hydroxyl-FLU, and were probably structural isomers. Liver
Ž .
and kidney contained mostly 80 parent FLU at 24 h.
4. Discussion
