- Email: [email protected]
Studies in triquinol pharmacokinetics by mass - fragmentography
ZnternationalJournalof~assSpectromefryandZonPhysics, 48 (1983) 105-108 Elsevier Scientific Publishing Company, Amsterdam - Printedin The Netherlands
STUDIES IN TRIQWINOL PHARMACOKINETICS
s.
, P. Pomv’
DEMIREX' , N. MOWVA',
R. G!ZQRGIEvA2,V,
105
BY MASS - FRAGMENTOGRAPMl V.NEZ'JGV'I , A. SIDJIMX?,
N. MAREKOV',
cK2N!QwOvA2
1 Inst. Org. Chemistry, Bulg, Acad. Sci., 1113 Sofia, BULGARIA 2 Chem. Pharmaceutical
Research Institute, 1156 Sofia, BULKZECR
Mass-fragmentography of trimetquinol hydrochloride and its D2-deuterated analogue (an internal standard) is used for the quantitative determination of the triquinol concentration changes in b&xii in dynamics.
INTRODUCTION Trimetaquinol iscquinoline)
(L-l/3,4,5-trimetoxybenzyl/
hydrochloride
- 6,7 - dihydroxy - 1,2,3,4 tetra-
(fig.?) is a bronchdilator
An original synthesis of that drug is developed me TRIQUINOL. radiolaheled
Its pharmacokinetics cwnds
for determining
widely used in clinics.
in Bulgaria under the trade na-
has been investigated till now by the use of
(ref.l,2&3j. The present work describes another method
the dynamics of triquinol concentration
in bleed plasma viz.
mass-fragnientcqraphy.
Fig. I. OOZO-738~J83/0000--0000/$03.00 0
1983 Elsevier Scientific Publishing
Company
*
106
MATERIALS AND MEl!HODS White male rats (Wistar strain, 180-200 g in weight) are treated i-v. with triquinol - 300 mcg/200 g b.weight. Bleed samples are taken in dynamics - 2, 5, IO, 15, 30, 45 and 120 min. after administration. A known quantity of D2 - triquinol hydrochloride
(an internal standard) is added to the hlcod samples. The
protein is precipitated by 40% CC13CoOH
(1 ml/4 ml plasma) and is separated by
centrifugation. The yellow supernatant is saturated with NaCl, extracted twice with CHC13 and alkalized with NH4OH. Triquinol and its eventual metabolites are extracted twice with CHC13, dried over anhydrous Na2S04, evaporated to dryness and sylilated by dissolving in a 100 mcl/? ml plasma of abs. pyridine:hexamethyldisilazane:trimethylchlorosilane
(10:2:1, v/v) and heating the mixture for 1Omin
at 80-90°C. A part of the sample is purified by prep. TLC (silicagel G, chloroform:petr. ether:methanol:acetone
- 40:40:15:15, V/V), the triquinol zone elu-
ted with CH30H, the solvent evaporated and the residue silylated afterwards.
IO
mcl of the substance in the silylating agent is inqected into a quartz crucible and the solvent is evaporated. The smle
is introduced by the direct inlet pro-
be in a "JEOL JMS D-300" mass spectrometer and the probe is heated from 120" to 350°C at a rate lOO'C/min (chamber To-200'C). Peaks at m/z 308 nitored in MID
and 309 are mo-
(mass fragmentography ) mode. The peak area ratio in the corres-
ponding channels is determined with the help of a "JELL JMA 2000" data aoguisition and processing system
and s&ndard
software
(JECL MP7700). The intensity
of the pure sylilated triquinol, its deuterated analcgue and plasma, all treated in the way described above, is also measured and estimated. RESULTS AND DISCUSSION The triquinol mass spectrum shows all characteristic peaks for benzyl - tetra hydroisoquinoline compounds
(fig.l). me
molecular peak is with a low intensity
and the base peak is due to the tetrahydroisoquinoline moiety
(m/z 164 for tri-
quinol and m/z 165, 166 for D2-triquinol). The EI (12 and 70 eV) and the CI (iso C4Hlo) mass fragmentograms at channels 164-166 are not reliable due to the plasma background at the same m/z. The prep. TLC is not enough for leering
the back
ground for a quantitative determination. The silylation of the two hydroxyl groups increases the mass of the tetrahydroisoguinoline
fragment respectively to
m/z 308 and 309 and this is enough for lowering the background. The silylated is volatile but Gc with packed columns lacks sensitivity for quantitative determination. Direct sample introduction is used for that reason. Due to contribution of deuterated triquinol in both channels, for guantitative determination is used the isotope dilution formula (ref.4): (x/y). (pk/E)
+ @k/F) , where:
R= (x/y).(Pl/'B)+ (Ql/F)
107
R
is the ratio of the areas of the respective fragment peaks
-
(determined
experimentally).
X/Y
- the ratio of the undeuterated to the deuterated prtiuct (initially unknown). the relative intensity of the peaks 308 and 309 m/z for undeuterated triquinol.
Q,,
Q,-
the relative intensity of the peaks 308 and 309 m/z for deuterated triquinol.
E, F
- the mass numbers of the respective
fragments
(I?= 308, F = 309).
The concentration of triquinol (ng/ml plasma) is found from the corresponding R.Ql-Qk ratio x/y (= - E/F). The results of different measurements are averaged Pk-R.P1 and are given in Fig. 2.
.
.
1000
.
900 700
. 4
. 5w
4 4
306 IOP
Fig.
+
+
2:+
- triquinol . - total
+
cont.
4
in plasma
radioactivity
by mass-fragmentography
of plasma
(ref. 3)
- error range 6-C-methyltriquinol tes. The mass
spectrum
and 7-O-methyltriquinol of silylated
are
O-methyltriquinol
the main
triquinol
metaboli-
has a base peak at m/z 250
108
and is monitored parallelly
to m/z 308 and 309. 'Ihe lack of an internal standard
for the metabolite allowed us to determine only only its qualitative It is established that the quantity of O-methyltriquinol
behaviour.
in plasma increases at
the beginning, with a maximum at 10-15 min, followed by an abrupt decrease. These data correspond plasma
to the data of radiolabelled triquinol administered
(Fig. 1). In that case the radioactivity
in
is due to triqufnol and to its
C-methyl metabolite. By deducing the triquinol concentration
from the above me-n-
tioned sum one gets the maximum for the metabolite between IO-30 min.
ACKNCWLEIXGEMENTS:
The authors are grateful to LJNDP/UNESCO project BUL/77/009
for the partial financial support of this study.
REFERENCES
:
1. Meshi T., M. Otsuka, J. Sato, Bicchem. Pharmacol., 19, 2. Kiymoto A., I. Iwasawa, S. Harygaya, Arzn.-Borsch., 20, 3.
Sidgimov A., N. Marekov, R. Georgieva, V. Ognyanova,
4. Pickup J.F., K. McPherson, Anal. Chem., 48,
(1970), 2937. (19701, 46.
in preparation.
(19761, 1885-1890.
STUDIES IN TRIQWINOL PHARMACOKINETICS
s.
, P. Pomv’
DEMIREX' , N. MOWVA',
R. G!ZQRGIEvA2,V,
105
BY MASS - FRAGMENTOGRAPMl V.NEZ'JGV'I , A. SIDJIMX?,
N. MAREKOV',
cK2N!QwOvA2
1 Inst. Org. Chemistry, Bulg, Acad. Sci., 1113 Sofia, BULGARIA 2 Chem. Pharmaceutical
Research Institute, 1156 Sofia, BULKZECR
Mass-fragmentography of trimetquinol hydrochloride and its D2-deuterated analogue (an internal standard) is used for the quantitative determination of the triquinol concentration changes in b&xii in dynamics.
INTRODUCTION Trimetaquinol iscquinoline)
(L-l/3,4,5-trimetoxybenzyl/
hydrochloride
- 6,7 - dihydroxy - 1,2,3,4 tetra-
(fig.?) is a bronchdilator
An original synthesis of that drug is developed me TRIQUINOL. radiolaheled
Its pharmacokinetics cwnds
for determining
widely used in clinics.
in Bulgaria under the trade na-
has been investigated till now by the use of
(ref.l,2&3j. The present work describes another method
the dynamics of triquinol concentration
in bleed plasma viz.
mass-fragnientcqraphy.
Fig. I. OOZO-738~J83/0000--0000/$03.00 0
1983 Elsevier Scientific Publishing
Company
*
106
MATERIALS AND MEl!HODS White male rats (Wistar strain, 180-200 g in weight) are treated i-v. with triquinol - 300 mcg/200 g b.weight. Bleed samples are taken in dynamics - 2, 5, IO, 15, 30, 45 and 120 min. after administration. A known quantity of D2 - triquinol hydrochloride
(an internal standard) is added to the hlcod samples. The
protein is precipitated by 40% CC13CoOH
(1 ml/4 ml plasma) and is separated by
centrifugation. The yellow supernatant is saturated with NaCl, extracted twice with CHC13 and alkalized with NH4OH. Triquinol and its eventual metabolites are extracted twice with CHC13, dried over anhydrous Na2S04, evaporated to dryness and sylilated by dissolving in a 100 mcl/? ml plasma of abs. pyridine:hexamethyldisilazane:trimethylchlorosilane
(10:2:1, v/v) and heating the mixture for 1Omin
at 80-90°C. A part of the sample is purified by prep. TLC (silicagel G, chloroform:petr. ether:methanol:acetone
- 40:40:15:15, V/V), the triquinol zone elu-
ted with CH30H, the solvent evaporated and the residue silylated afterwards.
IO
mcl of the substance in the silylating agent is inqected into a quartz crucible and the solvent is evaporated. The smle
is introduced by the direct inlet pro-
be in a "JEOL JMS D-300" mass spectrometer and the probe is heated from 120" to 350°C at a rate lOO'C/min (chamber To-200'C). Peaks at m/z 308 nitored in MID
and 309 are mo-
(mass fragmentography ) mode. The peak area ratio in the corres-
ponding channels is determined with the help of a "JELL JMA 2000" data aoguisition and processing system
and s&ndard
software
(JECL MP7700). The intensity
of the pure sylilated triquinol, its deuterated analcgue and plasma, all treated in the way described above, is also measured and estimated. RESULTS AND DISCUSSION The triquinol mass spectrum shows all characteristic peaks for benzyl - tetra hydroisoquinoline compounds
(fig.l). me
molecular peak is with a low intensity
and the base peak is due to the tetrahydroisoquinoline moiety
(m/z 164 for tri-
quinol and m/z 165, 166 for D2-triquinol). The EI (12 and 70 eV) and the CI (iso C4Hlo) mass fragmentograms at channels 164-166 are not reliable due to the plasma background at the same m/z. The prep. TLC is not enough for leering
the back
ground for a quantitative determination. The silylation of the two hydroxyl groups increases the mass of the tetrahydroisoguinoline
fragment respectively to
m/z 308 and 309 and this is enough for lowering the background. The silylated is volatile but Gc with packed columns lacks sensitivity for quantitative determination. Direct sample introduction is used for that reason. Due to contribution of deuterated triquinol in both channels, for guantitative determination is used the isotope dilution formula (ref.4): (x/y). (pk/E)
+ @k/F) , where:
R= (x/y).(Pl/'B)+ (Ql/F)
107
R
is the ratio of the areas of the respective fragment peaks
-
(determined
experimentally).
X/Y
- the ratio of the undeuterated to the deuterated prtiuct (initially unknown). the relative intensity of the peaks 308 and 309 m/z for undeuterated triquinol.
Q,,
Q,-
the relative intensity of the peaks 308 and 309 m/z for deuterated triquinol.
E, F
- the mass numbers of the respective
fragments
(I?= 308, F = 309).
The concentration of triquinol (ng/ml plasma) is found from the corresponding R.Ql-Qk ratio x/y (= - E/F). The results of different measurements are averaged Pk-R.P1 and are given in Fig. 2.
.
.
1000
.
900 700
. 4
. 5w
4 4
306 IOP
Fig.
+
+
2:+
- triquinol . - total
+
cont.
4
in plasma
radioactivity
by mass-fragmentography
of plasma
(ref. 3)
- error range 6-C-methyltriquinol tes. The mass
spectrum
and 7-O-methyltriquinol of silylated
are
O-methyltriquinol
the main
triquinol
metaboli-
has a base peak at m/z 250
108
and is monitored parallelly
to m/z 308 and 309. 'Ihe lack of an internal standard
for the metabolite allowed us to determine only only its qualitative It is established that the quantity of O-methyltriquinol
behaviour.
in plasma increases at
the beginning, with a maximum at 10-15 min, followed by an abrupt decrease. These data correspond plasma
to the data of radiolabelled triquinol administered
(Fig. 1). In that case the radioactivity
in
is due to triqufnol and to its
C-methyl metabolite. By deducing the triquinol concentration
from the above me-n-
tioned sum one gets the maximum for the metabolite between IO-30 min.
ACKNCWLEIXGEMENTS:
The authors are grateful to LJNDP/UNESCO project BUL/77/009
for the partial financial support of this study.
REFERENCES
:
1. Meshi T., M. Otsuka, J. Sato, Bicchem. Pharmacol., 19, 2. Kiymoto A., I. Iwasawa, S. Harygaya, Arzn.-Borsch., 20, 3.
Sidgimov A., N. Marekov, R. Georgieva, V. Ognyanova,
4. Pickup J.F., K. McPherson, Anal. Chem., 48,
(1970), 2937. (19701, 46.
in preparation.
(19761, 1885-1890.