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Chromatographic detection of meprobamate and its metabolites in human urine
NOTES
192
Chromatographic
detect&
of meprobamate
and its metabolites
in human
urine is widely Meprobamate (2-methyl-z-propyl-x,3-propanediol dicarbamate) ___ _ _ _ used as one of the tranquilizing agents and the number of meprobamate poisonings rises progressively. In toxicological analysis the main emphasis is on qualitative reliability and on rapidity, while the need for quantitative accuracy is not very high. Thus a chromatographic method is very suitable for this purpose. Paper chromatography of meprobamate has been studied by many authorsl-‘2. MARLGO AND F~ORX have described a thin-layer chromatographic method on silica ge113114,All these methods give satisfactory results, but in most cases of intoxication by an unknown poison, and particularly in combined poisoning, it is necessary to use the chromatographic detection in more than one system to prove the identity of the substance examined. Thus it is clear that the time necessary for analysis is prolonged. We have tried to abolish this disadvantage. When we know the RIP values of a substance and its met,abolites, the discovery, of spots with &7 values which correspond infers the probable identity of the drug. In such a case an analysis in one system can be sufficient for the required proof. Therefore we have studied the chromatographic properties of meprobamate and its metabolites in different systems on paper and thin layers.
E~~traction. Urine was brought to an alkaline pEI with sodium carbonate and meprobamate was extracted by shaking with the same volume of chloroform three times, The organic layers were collected, evaporated on a water-bath and an aliquot was spotted on the starting point of the chromatogram. The remaining urine was then twice extracted with ethyl acetate and the upper phase containing meprobamate metabolites was worked up in the same manner. At a third point, standards of meprobamate and urea were applied (in Fig. I these points are not given). Chronzatogra$lzy, Paper chromatography on Whatman No. I paper and thinlayer chromatography were examined. The adsorbent was prepared by mixing 10.0 g Siloxyde (a preparation of Chema Horni PoEernice, based on Si02), 1.0 g CaSO, l/2 EI,O, and Go.0 g water. The suspension was homogenised, applied to the glass plates, kept for 20 min at room temperature and then I h at 105~. The solvent systems used, the RF values of meprobamate, its metabolites and urea, and the time of development are given in Fig, I. Detection. Two methods were examined. (I) Spraying with a 0.2 % solution of p-dimethylaminobenzaldehyde in hydrochloric acid. The compounds examined yield yellow spots. (2) The paper was placed in a chlorine solution in carbon tetrachloride (as described by MACEI+), dried in air and promptly passed through benzidine-KI solution; this consists of a small amount of benzidine dissolved in 2 ml of acetic acid, the volume being made up to IOO ml with 0.3 y0 KI and filtered-our modification of the RYDON-SMITEI method1 . The thin layers were chlorinated by chlorine gas, and the benzidine-ICI solution was sprayed on the plate. Meprobamate and its metabolites yield intensive blue-violet or blue-gray spots. l
J. Chvomatog.,
19 (wW
1w--194.
193
NOTES
Resuults and discussion
described and confirmed by synthesis two main mepro‘barn&e metabolites in human,urine 17,hydroxy-meprobamate and the N-glucuronide of meprobamate. In our II cases of meprabamate poisoning we have constantly found the spots shown in Fig. I. One of the spots may be hydroxy-meprobamate LUDWIG
et al.
have
,
RF
values
(cross-hatched spots), its metabolites separation of mcprobnmate (blank spots) and urea (hatched spots). C = chloroform extract; E = ethyl acetate extract. (I) ButanolSystems (No. x-4, papcr chromatography, * No. 5-8, thin-layer chromatography): acetic acicl-bvatcr (4 : I :5) ; (2) benzene-butanol-acetic acid-water (3 : I : I : 5) ; (3) benzene-acetic acickvater (2 : 2 : I) ; (4) chloroform-acetic acicl-water ( IOO :2 :5) ; (5) chloroform-acetic acxl (4 : I). In paper (2 : I) ; (8) chloroform-acetone (20: I) ; (6) diethyl ether: (7) benzene-acetone chromatography dcvelopmcnt was by the clesccnding technique, except in the case of system No. 3.
Fig. I, Chromatographic
the structure of the other compounds is still unknown to us. Since we, extract from an alkaline medium the glucuronide is not extracted. Ilowever, for toxicological proof it is not necessary to know the structure of the metabolites, if the substances found are really metabolites of the drug investigated. In choosing a system which gives good. separations and saves,time, the thin-layer chromatography system No. 8, chloroform-acetone (4: I) is suggested, while the second detection method seems to be the more sensitive. An important factor is the size of the aliquots used. Too small an amount is not detected, while too large an amount is not well separated. For this reason we use J. Chvomatog.,
xg (1965)
xg2-rg4
‘&(&g,
.I’94
two extractions. Chloroform removes the greater part of meprobamate which interferes with the,separation of the meprobamate metabolites. Thus it ,is recommended that two different aliquots df both the extracts, corresponding to 0.2 and 0.6 ml of’ urine, are used. An approximate estimate of the period of time from the_onset of poisoning can be deduced from the size of the’spots of meprobamate and its metabolites. Laboratory for TOASGOJO~~ and FOYMZS~G Chtwnistry, Charles Urtiversidy, ,Prugzce (Czechoslovakia)
IV0
HYNIE
JIND~X K~NIG I
I H. ARAMAKI, &&.?&dU TO .%Sa, 12 (1959) 44, J. I?.DOUGLAS AND A. SCHLOS~ER,J. CltromuZog., G (1961) 540. 3 A. DRESSLER, Dead. Z. Ges. Gericltll. Med., 50 (IQGO) 457. 4 J. L. EMERSON, T. S. MIYA AND G. K. W. YzM.J. P?zarmacoZ._?ZxfilZ. Therap., Izg (1960) 89. 5 I, HYNIE, I<.I
Received December zznd, 1964 J. Glwomatog., x9 (xgG5) Igz--I94
Iodine
quenched
fluorescence,
detectioti of organic
eompounds
a sensitive,
non-dtistructive
method
for
the
on chrdmatoplatss
In order to detect compounds
on thin layer plates of alumina or silica gel it is commonly necessary to alte? or ,destroy the spots in order to make coloured derivatives of the. compound under investigation. The introduction of a fluorescent dye’ or phosphor into the thin layer material, thereby.making the spots visible by virtue of their U.V. absorption or fluorescence, constituted a notable advance. Sensitive, non-destructive methods of detection are of especial value when quantity recovery of the separated bands is required ‘from ,preparative thin-layer chromatoplates. Iodine ,applie,d either,’ as ‘vapour or as, a. spray in methanol can also, be used for this because it forms ti loose complex with most organic compounds1 ‘yet reacts with few?. During a test ‘of detection methods suitable. for aliphatic acids it was found ‘that the, combinatioti lof fluorescent dye and, iodine treatment resulted in a considerable irni provement in sensitivity over. either m,ethod alone. ‘. ‘, J.. Chvokaiog.,
Ig (1965)
IQ&197
‘,
:, :
’
192
Chromatographic
detect&
of meprobamate
and its metabolites
in human
urine is widely Meprobamate (2-methyl-z-propyl-x,3-propanediol dicarbamate) ___ _ _ _ used as one of the tranquilizing agents and the number of meprobamate poisonings rises progressively. In toxicological analysis the main emphasis is on qualitative reliability and on rapidity, while the need for quantitative accuracy is not very high. Thus a chromatographic method is very suitable for this purpose. Paper chromatography of meprobamate has been studied by many authorsl-‘2. MARLGO AND F~ORX have described a thin-layer chromatographic method on silica ge113114,All these methods give satisfactory results, but in most cases of intoxication by an unknown poison, and particularly in combined poisoning, it is necessary to use the chromatographic detection in more than one system to prove the identity of the substance examined. Thus it is clear that the time necessary for analysis is prolonged. We have tried to abolish this disadvantage. When we know the RIP values of a substance and its met,abolites, the discovery, of spots with &7 values which correspond infers the probable identity of the drug. In such a case an analysis in one system can be sufficient for the required proof. Therefore we have studied the chromatographic properties of meprobamate and its metabolites in different systems on paper and thin layers.
E~~traction. Urine was brought to an alkaline pEI with sodium carbonate and meprobamate was extracted by shaking with the same volume of chloroform three times, The organic layers were collected, evaporated on a water-bath and an aliquot was spotted on the starting point of the chromatogram. The remaining urine was then twice extracted with ethyl acetate and the upper phase containing meprobamate metabolites was worked up in the same manner. At a third point, standards of meprobamate and urea were applied (in Fig. I these points are not given). Chronzatogra$lzy, Paper chromatography on Whatman No. I paper and thinlayer chromatography were examined. The adsorbent was prepared by mixing 10.0 g Siloxyde (a preparation of Chema Horni PoEernice, based on Si02), 1.0 g CaSO, l/2 EI,O, and Go.0 g water. The suspension was homogenised, applied to the glass plates, kept for 20 min at room temperature and then I h at 105~. The solvent systems used, the RF values of meprobamate, its metabolites and urea, and the time of development are given in Fig, I. Detection. Two methods were examined. (I) Spraying with a 0.2 % solution of p-dimethylaminobenzaldehyde in hydrochloric acid. The compounds examined yield yellow spots. (2) The paper was placed in a chlorine solution in carbon tetrachloride (as described by MACEI+), dried in air and promptly passed through benzidine-KI solution; this consists of a small amount of benzidine dissolved in 2 ml of acetic acid, the volume being made up to IOO ml with 0.3 y0 KI and filtered-our modification of the RYDON-SMITEI method1 . The thin layers were chlorinated by chlorine gas, and the benzidine-ICI solution was sprayed on the plate. Meprobamate and its metabolites yield intensive blue-violet or blue-gray spots. l
J. Chvomatog.,
19 (wW
1w--194.
193
NOTES
Resuults and discussion
described and confirmed by synthesis two main mepro‘barn&e metabolites in human,urine 17,hydroxy-meprobamate and the N-glucuronide of meprobamate. In our II cases of meprabamate poisoning we have constantly found the spots shown in Fig. I. One of the spots may be hydroxy-meprobamate LUDWIG
et al.
have
,
RF
values
(cross-hatched spots), its metabolites separation of mcprobnmate (blank spots) and urea (hatched spots). C = chloroform extract; E = ethyl acetate extract. (I) ButanolSystems (No. x-4, papcr chromatography, * No. 5-8, thin-layer chromatography): acetic acicl-bvatcr (4 : I :5) ; (2) benzene-butanol-acetic acid-water (3 : I : I : 5) ; (3) benzene-acetic acickvater (2 : 2 : I) ; (4) chloroform-acetic acicl-water ( IOO :2 :5) ; (5) chloroform-acetic acxl (4 : I). In paper (2 : I) ; (8) chloroform-acetone (20: I) ; (6) diethyl ether: (7) benzene-acetone chromatography dcvelopmcnt was by the clesccnding technique, except in the case of system No. 3.
Fig. I, Chromatographic
the structure of the other compounds is still unknown to us. Since we, extract from an alkaline medium the glucuronide is not extracted. Ilowever, for toxicological proof it is not necessary to know the structure of the metabolites, if the substances found are really metabolites of the drug investigated. In choosing a system which gives good. separations and saves,time, the thin-layer chromatography system No. 8, chloroform-acetone (4: I) is suggested, while the second detection method seems to be the more sensitive. An important factor is the size of the aliquots used. Too small an amount is not detected, while too large an amount is not well separated. For this reason we use J. Chvomatog.,
xg (1965)
xg2-rg4
‘&(&g,
.I’94
two extractions. Chloroform removes the greater part of meprobamate which interferes with the,separation of the meprobamate metabolites. Thus it ,is recommended that two different aliquots df both the extracts, corresponding to 0.2 and 0.6 ml of’ urine, are used. An approximate estimate of the period of time from the_onset of poisoning can be deduced from the size of the’spots of meprobamate and its metabolites. Laboratory for TOASGOJO~~ and FOYMZS~G Chtwnistry, Charles Urtiversidy, ,Prugzce (Czechoslovakia)
IV0
HYNIE
JIND~X K~NIG I
I H. ARAMAKI, &&.?&dU TO .%Sa, 12 (1959) 44, J. I?.DOUGLAS AND A. SCHLOS~ER,J. CltromuZog., G (1961) 540. 3 A. DRESSLER, Dead. Z. Ges. Gericltll. Med., 50 (IQGO) 457. 4 J. L. EMERSON, T. S. MIYA AND G. K. W. YzM.J. P?zarmacoZ._?ZxfilZ. Therap., Izg (1960) 89. 5 I, HYNIE, I<.I
Received December zznd, 1964 J. Glwomatog., x9 (xgG5) Igz--I94
Iodine
quenched
fluorescence,
detectioti of organic
eompounds
a sensitive,
non-dtistructive
method
for
the
on chrdmatoplatss
In order to detect compounds
on thin layer plates of alumina or silica gel it is commonly necessary to alte? or ,destroy the spots in order to make coloured derivatives of the. compound under investigation. The introduction of a fluorescent dye’ or phosphor into the thin layer material, thereby.making the spots visible by virtue of their U.V. absorption or fluorescence, constituted a notable advance. Sensitive, non-destructive methods of detection are of especial value when quantity recovery of the separated bands is required ‘from ,preparative thin-layer chromatoplates. Iodine ,applie,d either,’ as ‘vapour or as, a. spray in methanol can also, be used for this because it forms ti loose complex with most organic compounds1 ‘yet reacts with few?. During a test ‘of detection methods suitable. for aliphatic acids it was found ‘that the, combinatioti lof fluorescent dye and, iodine treatment resulted in a considerable irni provement in sensitivity over. either m,ethod alone. ‘. ‘, J.. Chvokaiog.,
Ig (1965)
IQ&197
‘,
:, :
’