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Rapid detection of some basic drugs by thin-layer chromatography
Note
Rapid detection of basic drugs in illicit preparations may be limited by similarities in their spectral properties. Uttraviolet (UV) spectrophotometry is the most widely available screening method for illicit drugs but lacks specificity. A large number of basic drugs, notzbly phenethylamine derivatives show an UY absorptioR at approximately 257 Rm with low absorptivities (low E values). Thin-layer chromatography (TLC) of basic drrrgs alfows a &her degree of specificity with &creased detection sensitivity. Recently various workers have shown the advantigs of derivative formation in TLC of phenetbylamine derivativesl-(. The use of NBD-Cl (4chloro-7-RitrobeRzo-2, ! ,3-oxzdiazole) has permitted the analyst to screen samples with increased speed and specificitf. Structures of reaction products have been elucidated by mass spectrometry5. This stRdy was RRdertakeR to provide add&lo& TLC dara in reported solvent systemsj*” for phenethylamines aRd other basic drtlgs that exhibit a characteristic absorption at 257 Rm in the U‘J region.
The chromatography &RkS used in this study were giass with approximate dimensions 23 x 12 x 23 cm. The tank tops were sealed with a starch-glycerin paste’; the tmks were lined with %%ztmaR No. 3 chromatography paper. The developing taRks were &lowed to equilibrate for 30 min after solvent iRtroductioR. Analtech prescored silica gel G (250 pm) plates (Mandeg Scientific, Montreal, Canada) were used; she plates were not activated prior to use. The relative humidity throughout data colEection was 23-29”/,. The following developin solvents were used : (1) e+Lhylacet&e-cycIohexaRe (iI) ethyl zcetate-cyclohexane (3:2); and (TEE) dfethyl ether-benzene (::I). Derivative formztion wzs accomplished with 0-i M sodium bicarbonate solution and I T/,(w/v) Nl3D-CI (Aidrich, Milwaukee, Wise., U.S.A.) in methyl isobutyl
(2:3);
ketone. Drug standards were prepared as follows: Solutions of 10 m,o/mI of the salt of each drug were prepared in absolute ethanol or d&i&d water fdepeRdiRg Rpon the solubiiity of each drug).
A!phqxcdize Am&e-&e Aaiietidine Atropine &nzph&zmke Ctiorphetiiamke Ctiorphenterrrine 3,tDimett2oxyzxnphetxni~e Diphesxhydrzmine Eph_e Ethaheptine N-Ethyl-3-p&tidy! kazil2?e Fen&u-amine F-Hydroxyephedrine tIyoscy2mine 3&Me+&yknedioxyuopktimm_* P Mtine N-Mezhyl-$enethy!azzine Pi-Methyi-3-piperidyl klziI2t~ Methy!phenidatt Mem2rnpketine
3-Me’-hoxy-+5methyleriedioxyamphetznine p-Methoxyzmphet2mine Phenethy!zmkle Ph~~~_etiZiII~ Pheny!eptine PhenylpmganotPim~ine Piperidirxe Prccyclidine Propoxyphene Retidoephedrine 2,5-Dimethoxy+ methykmphetam&
4.10 4.60 0.55 0.64 1.45 1-w 3.91
1.58 1.69 0.20 0.23 0.53 0.37 I .$3
0.72 0.38 0.66
0.9f,1.91 .51 3.09 I.@ 2.82
0.33,0-70*s
30 +9,3 5: 7
0.26 0.64 0.30,0.19,0.05~a 0.15
1.09 2.72 1.27,0.82,0.27IS 0-g
l.cQ 0.47,0.3O,O.G9 es 0.23
43 16
0.91 0.34
393 1.45
1.43 0.53
Y
26
0.55
2.36
0.87
Y-Or
II 37
0.23
1.00 3.36 2.82
0.37 1.23 1.03
-
3.?
0.79 0.66
Y-Qr
34
3.72
3.09
2.13
Y
0.95 1.03 0.13 0.15 0.34 023
0.31
0.21,0.45 20
12
42 45 :I 7,4*5
I.13 0.6Q E-03 0.40
Y Or Y
Y Qr,Y Y Y
02
Y Y
Y,Y,Y * 3
-
Y
0.89
0.95 0.66
3.15009’” , .
33,382J 11 29 i6 1: 14
0.70,0.51: 2 0.23 0.62 0.34 0.23 0.30
3.0,3.585 1.60 2.a 1.45 I.00 1.27
1.lJ.2:~ 0.37 0.96 0.53 0.37 0.47
lm,Y f 5 Y-Or Or-Rd
46
0.98
4.18
1.53
Or
l IiF x io0 (2vemgz of 3 _nms). ** I?= rehtive to amphetamke (standard). *** R= relative to chforp,hetinimine (standard). 2 R= rehtive to metbmpktzmine (s?adard). EsMr?ltipk iezctiOF3 p?odUCtS. g 5 z Ail spots fiuorescent beHow; 254 nm).
Qi
Y Y
k!!
P
NOTE2
452
Apptoxlnzately 100 pg of each drug were added to a fQ x 75 mm test-t&be, followed by 0.2 m! of ‘&e s~dlum bicarbonate solution. Each &be was &&en and 0.2 d of ths KBD-CI. sot&x~ were added. Tlxe t&es were stoppered and placed in an oven for 30 tin at, 80”. Aliqlsats of 5 ,d of&e upper fmet%yI isobtityi ketone) Izyer were spotted zmci each TLC plate was ekted to a distance of 15 cm. Spots were ob.I . served under vrsxb!e 11&t 83 d ‘t’V G&t (254 nm). Each iiF v&e was recorded. The Rx vahes (relatke to 2 standard mixture, 10 m&ml of chlorphetiramire, methamphe’tine, and am&e&mine) were then cakrrlated. The standard mixture alhed moaitoting chromatographic conditioos and the coliection of Rx values.
TLC data for some basic drags wiL& UV m&ma zt approximately 257 am are tzbulzted in Tab!es I-IIH. Starrdard deviations in al1 RF v&ues are less thzn 0.05.
TLC DA-l-.4 OF NBD-CI
DERIVATIVES
iN DlEXHYL
ETHER-BENZENE
Conqwa3lri
F’-B
VisiiSe coloxr p
Ampheknine J+&g~~ Atropke
69 18
Y at**
_____-
BerzzheL&e Ch!orphe_nkzmine Diphe&ydmmine Ephedrine N-Ethyl-3-pipa-idyl betilate Fenfbzmiize p_Sydroxyepheddne 3,oMet~yvkn~ioxyamphei~mine %&zsaline N-Methy!-phenethylatine N-Methyl-3-pi_peridy1 berrziiate Methylphenkkse
Or Or Or Or-Rd Or,Or,Y”’ Or Y Or
Metham&ietzm%~ . w
15 27 36 63 ‘2 26 57 13,28,36” 63 35 52 27 64 59
j-~~e~ho~y~,5-_~e~hyien~dioxya~zlpheramine
57
Oi
p-Metioxyzmphetzmioe Phenethylaske Phennetrzzine Phenyiepkzke Pher?ylpropanolamine PipeSdine Procyclidioe
63 66 57 17,23”’ 49 55 62 35 35 61
Y Y Or Or,Qr”’ Y Or-Rd Rd Y Or Or
FiCp3X+eZ
P_~?do9pheddTile 2,5-Dim~hoxy~met~yl~~p~et~m~ne
Or
(1 :l)
NOFES
453
The developing solvents I and If were found to be the most suitable for NED-CL derivatives. Tabfe IEEgives data for the first run in each tank only. The second and third runs gave data with standard deviations greater than 0.10 and xere not incEuded. Comparative & values for basic drugs in the tfiree solvent systems permitted tentative idenfikation (see Tzbie IV). The inclusion of UV spectra provided a rapid rretirad for iden&kation of the basic drugs tabulated. For example, chforphetiramine TABLE
IV
Anileridine Aoropine Hyoscyzmize Phesylephrke 3,4-Dimethoxyamphetvnine Chloqhenkzmine Proaoqyphene Pi&inodine
N-Meti+3-piperidyi benrilate N-Ethyl-3-piperidyl b=nzilate Pseudoephedriae p-Hy&oxyephe&ioe Be!Xphetzmi~e hf&ne Pmcyclidine Ephedrine N-MeSxyIphenethytamine Piperidke
Fetifmmine Ethoheptine Methamphetzxnine Phezmnetiae PhenyIpmpaim!amine Dipke;enE?ydrsnine 3-Methoxy~.S-methylenedioxyzmphe’tine hfethylpherddate p-Methoxyanphetzmine Chkqhentermine 3,CMethylen+dicxy~phetiSikiP Xfphapraiine
6 7 7 7,4’ 10,XJ‘ li 11 II
11 12 14 14,S,3’ 16 16 16 1s t6 29 30 31 30 31 33,38’ 31
34 37 42 43
19 25 26 23,16’ 24
18 15 17,23’ -
23 22 35
56 35
26 26 32 30,X,12’ 36 37 31 33 42 43 50 52 49 51 53 53
27 26 35 13,28,36’ 27 37 62 42 57 55 57 -
53 __
57 64 63 -
;: 59
PfKnetAyImine
43 45 45
50 61
&5-Dimethosy-4-methylamphetzmine Amphetamine
46
60
47
64
5s
Or Y-Or,Y’ Y Y
Y
Y-Or
59 -_ s; 53
Y Y Y Or Y Y-C??
Or-Rd Or Y-Or Y-Or Y -,Y’ Y
Y Y
Y
63 -
Y -
66
Y
61 69
Or Y
NQTES
454
and propoxyphene have identical RF va!ues by the NBD-Cl method; their UV spectra, however, are markedly different. Chlorpheniramine in acid shows an absorption at 265nm, while piopoxyphene in 2&d has an zbsorprion at 257rzm. Slight differences in colours of reaction products asd the presence or absence of a fluorescent reaction product permitted differentiation between most of the drugs tabulated. Multiple rezction products, where detected, are indicated by more than one RF value. The r,ature of these products has not been inv&tigated. Bethanidine, eucatropine, homatropine, hyoscine, mephentermine, methadone, phendimetrazine 2nd propylhexedrine did not react with NBD-Cl, and pethidive gave a weak reaction only. Some of these compounds also show 2n absorption at approximately 357 nm. This fact should be taken into consideration when interpreting analytical data. Typical sensitivities for selected drugs after reaction with NBD-Cl and TLC elution, are listed in Table V. These sensitivities indicate the application of NBD-Cl on small samples, for example, syringe washings. At present the NBD-Cl procedure is being used satisfactorily in forensic drug zr?alysis 2nd drug screens on basic extracts of toxicological material.
TABLE V SENSITIVITIES
OF SELECTED COMPOUNDS Sensitivities were determined after TLC elution.
WITH
NBD-Cl
Compound
Sensitivity (fig)
Amphetamine Ani!eridine Chlorpkniramine Chlorpkntermine Diphenhydrzmine Methylphenidate Methampietamine Phenethylamine Fhenmetrazine 3,4-MethyIenedioxyamphetamine 3-Methoxy4,kneihyienedioxyamphetzmine p-Hydroxyephedrine
0.01 0.03 0.01 0.50 0.01 0.50 0.01 0.01 0.05 0.35 0.35 0.50
ACKNOWLEDGEMENTS
The authors wish to express their appreciation to the Royal Canadian Mounted Police for their assistance, to the Healrh Protection Branch, Health and Welfare, CaRada, for supplying some of the sAand2rd drugs, and to Mrs. Susan Moe for her kind assistance in the preparation of the manuscript_ REFZRENCES I 1. Monforte, R. J. Batii and I. Sunshine, Ck. Ckm., 18 (1977) I3Z9. Z R. S. Eager, C. 5. Kuiinz and E. W. Abnhainson, khd. Bkk4etn., 53 (1973) 290. 3 R. N. Gupte, B. G. Ckiaim and P. M. Deane, J. C?lromctogr. Sci_, 12 (1974) 67. 1 F. van Hoof and A. Xeyn&-ickxcx,Arzl. Chem., $6 (lSi_) 286. 5 J. Reisch. I-I. Alfes, H. J. Kommert. N. Jantos, I-f. Moiiian and D. Ciasing. Pkamratie, 25 (1970) 331. 6 M. A. Shaw and H. FEZ=!,J. C’irrorrzafog~-_, ZC4 (1975) 201.
Rapid detection of basic drugs in illicit preparations may be limited by similarities in their spectral properties. Uttraviolet (UV) spectrophotometry is the most widely available screening method for illicit drugs but lacks specificity. A large number of basic drugs, notzbly phenethylamine derivatives show an UY absorptioR at approximately 257 Rm with low absorptivities (low E values). Thin-layer chromatography (TLC) of basic drrrgs alfows a &her degree of specificity with &creased detection sensitivity. Recently various workers have shown the advantigs of derivative formation in TLC of phenetbylamine derivativesl-(. The use of NBD-Cl (4chloro-7-RitrobeRzo-2, ! ,3-oxzdiazole) has permitted the analyst to screen samples with increased speed and specificitf. Structures of reaction products have been elucidated by mass spectrometry5. This stRdy was RRdertakeR to provide add&lo& TLC dara in reported solvent systemsj*” for phenethylamines aRd other basic drtlgs that exhibit a characteristic absorption at 257 Rm in the U‘J region.
The chromatography &RkS used in this study were giass with approximate dimensions 23 x 12 x 23 cm. The tank tops were sealed with a starch-glycerin paste’; the tmks were lined with %%ztmaR No. 3 chromatography paper. The developing taRks were &lowed to equilibrate for 30 min after solvent iRtroductioR. Analtech prescored silica gel G (250 pm) plates (Mandeg Scientific, Montreal, Canada) were used; she plates were not activated prior to use. The relative humidity throughout data colEection was 23-29”/,. The following developin solvents were used : (1) e+Lhylacet&e-cycIohexaRe (iI) ethyl zcetate-cyclohexane (3:2); and (TEE) dfethyl ether-benzene (::I). Derivative formztion wzs accomplished with 0-i M sodium bicarbonate solution and I T/,(w/v) Nl3D-CI (Aidrich, Milwaukee, Wise., U.S.A.) in methyl isobutyl
(2:3);
ketone. Drug standards were prepared as follows: Solutions of 10 m,o/mI of the salt of each drug were prepared in absolute ethanol or d&i&d water fdepeRdiRg Rpon the solubiiity of each drug).
A!phqxcdize Am&e-&e Aaiietidine Atropine &nzph&zmke Ctiorphetiiamke Ctiorphenterrrine 3,tDimett2oxyzxnphetxni~e Diphesxhydrzmine Eph_e Ethaheptine N-Ethyl-3-p&tidy! kazil2?e Fen&u-amine F-Hydroxyephedrine tIyoscy2mine 3&Me+&yknedioxyuopktimm_* P Mtine N-Mezhyl-$enethy!azzine Pi-Methyi-3-piperidyl klziI2t~ Methy!phenidatt Mem2rnpketine
3-Me’-hoxy-+5methyleriedioxyamphetznine p-Methoxyzmphet2mine Phenethy!zmkle Ph~~~_etiZiII~ Pheny!eptine PhenylpmganotPim~ine Piperidirxe Prccyclidine Propoxyphene Retidoephedrine 2,5-Dimethoxy+ methykmphetam&
4.10 4.60 0.55 0.64 1.45 1-w 3.91
1.58 1.69 0.20 0.23 0.53 0.37 I .$3
0.72 0.38 0.66
0.9f,1.91 .51 3.09 I.@ 2.82
0.33,0-70*s
30 +9,3 5: 7
0.26 0.64 0.30,0.19,0.05~a 0.15
1.09 2.72 1.27,0.82,0.27IS 0-g
l.cQ 0.47,0.3O,O.G9 es 0.23
43 16
0.91 0.34
393 1.45
1.43 0.53
Y
26
0.55
2.36
0.87
Y-Or
II 37
0.23
1.00 3.36 2.82
0.37 1.23 1.03
-
3.?
0.79 0.66
Y-Qr
34
3.72
3.09
2.13
Y
0.95 1.03 0.13 0.15 0.34 023
0.31
0.21,0.45 20
12
42 45 :I 7,4*5
I.13 0.6Q E-03 0.40
Y Or Y
Y Qr,Y Y Y
02
Y Y
Y,Y,Y * 3
-
Y
0.89
0.95 0.66
3.15009’” , .
33,382J 11 29 i6 1: 14
0.70,0.51: 2 0.23 0.62 0.34 0.23 0.30
3.0,3.585 1.60 2.a 1.45 I.00 1.27
1.lJ.2:~ 0.37 0.96 0.53 0.37 0.47
lm,Y f 5 Y-Or Or-Rd
46
0.98
4.18
1.53
Or
l IiF x io0 (2vemgz of 3 _nms). ** I?= rehtive to amphetamke (standard). *** R= relative to chforp,hetinimine (standard). 2 R= rehtive to metbmpktzmine (s?adard). EsMr?ltipk iezctiOF3 p?odUCtS. g 5 z Ail spots fiuorescent beHow; 254 nm).
Qi
Y Y
k!!
P
NOTE2
452
Apptoxlnzately 100 pg of each drug were added to a fQ x 75 mm test-t&be, followed by 0.2 m! of ‘&e s~dlum bicarbonate solution. Each &be was &&en and 0.2 d of ths KBD-CI. sot&x~ were added. Tlxe t&es were stoppered and placed in an oven for 30 tin at, 80”. Aliqlsats of 5 ,d of&e upper fmet%yI isobtityi ketone) Izyer were spotted zmci each TLC plate was ekted to a distance of 15 cm. Spots were ob.I . served under vrsxb!e 11&t 83 d ‘t’V G&t (254 nm). Each iiF v&e was recorded. The Rx vahes (relatke to 2 standard mixture, 10 m&ml of chlorphetiramire, methamphe’tine, and am&e&mine) were then cakrrlated. The standard mixture alhed moaitoting chromatographic conditioos and the coliection of Rx values.
TLC data for some basic drags wiL& UV m&ma zt approximately 257 am are tzbulzted in Tab!es I-IIH. Starrdard deviations in al1 RF v&ues are less thzn 0.05.
TLC DA-l-.4 OF NBD-CI
DERIVATIVES
iN DlEXHYL
ETHER-BENZENE
Conqwa3lri
F’-B
VisiiSe coloxr p
Ampheknine J+&g~~ Atropke
69 18
Y at**
_____-
BerzzheL&e Ch!orphe_nkzmine Diphe&ydmmine Ephedrine N-Ethyl-3-pipa-idyl betilate Fenfbzmiize p_Sydroxyepheddne 3,oMet~yvkn~ioxyamphei~mine %&zsaline N-Methy!-phenethylatine N-Methyl-3-pi_peridy1 berrziiate Methylphenkkse
Or Or Or Or-Rd Or,Or,Y”’ Or Y Or
Metham&ietzm%~ . w
15 27 36 63 ‘2 26 57 13,28,36” 63 35 52 27 64 59
j-~~e~ho~y~,5-_~e~hyien~dioxya~zlpheramine
57
Oi
p-Metioxyzmphetzmioe Phenethylaske Phennetrzzine Phenyiepkzke Pher?ylpropanolamine PipeSdine Procyclidioe
63 66 57 17,23”’ 49 55 62 35 35 61
Y Y Or Or,Qr”’ Y Or-Rd Rd Y Or Or
FiCp3X+eZ
P_~?do9pheddTile 2,5-Dim~hoxy~met~yl~~p~et~m~ne
Or
(1 :l)
NOFES
453
The developing solvents I and If were found to be the most suitable for NED-CL derivatives. Tabfe IEEgives data for the first run in each tank only. The second and third runs gave data with standard deviations greater than 0.10 and xere not incEuded. Comparative & values for basic drugs in the tfiree solvent systems permitted tentative idenfikation (see Tzbie IV). The inclusion of UV spectra provided a rapid rretirad for iden&kation of the basic drugs tabulated. For example, chforphetiramine TABLE
IV
Anileridine Aoropine Hyoscyzmize Phesylephrke 3,4-Dimethoxyamphetvnine Chloqhenkzmine Proaoqyphene Pi&inodine
N-Meti+3-piperidyi benrilate N-Ethyl-3-piperidyl b=nzilate Pseudoephedriae p-Hy&oxyephe&ioe Be!Xphetzmi~e hf&ne Pmcyclidine Ephedrine N-MeSxyIphenethytamine Piperidke
Fetifmmine Ethoheptine Methamphetzxnine Phezmnetiae PhenyIpmpaim!amine Dipke;enE?ydrsnine 3-Methoxy~.S-methylenedioxyzmphe’tine hfethylpherddate p-Methoxyanphetzmine Chkqhentermine 3,CMethylen+dicxy~phetiSikiP Xfphapraiine
6 7 7 7,4’ 10,XJ‘ li 11 II
11 12 14 14,S,3’ 16 16 16 1s t6 29 30 31 30 31 33,38’ 31
34 37 42 43
19 25 26 23,16’ 24
18 15 17,23’ -
23 22 35
56 35
26 26 32 30,X,12’ 36 37 31 33 42 43 50 52 49 51 53 53
27 26 35 13,28,36’ 27 37 62 42 57 55 57 -
53 __
57 64 63 -
;: 59
PfKnetAyImine
43 45 45
50 61
&5-Dimethosy-4-methylamphetzmine Amphetamine
46
60
47
64
5s
Or Y-Or,Y’ Y Y
Y
Y-Or
59 -_ s; 53
Y Y Y Or Y Y-C??
Or-Rd Or Y-Or Y-Or Y -,Y’ Y
Y Y
Y
63 -
Y -
66
Y
61 69
Or Y
NQTES
454
and propoxyphene have identical RF va!ues by the NBD-Cl method; their UV spectra, however, are markedly different. Chlorpheniramine in acid shows an absorption at 265nm, while piopoxyphene in 2&d has an zbsorprion at 257rzm. Slight differences in colours of reaction products asd the presence or absence of a fluorescent reaction product permitted differentiation between most of the drugs tabulated. Multiple rezction products, where detected, are indicated by more than one RF value. The r,ature of these products has not been inv&tigated. Bethanidine, eucatropine, homatropine, hyoscine, mephentermine, methadone, phendimetrazine 2nd propylhexedrine did not react with NBD-Cl, and pethidive gave a weak reaction only. Some of these compounds also show 2n absorption at approximately 357 nm. This fact should be taken into consideration when interpreting analytical data. Typical sensitivities for selected drugs after reaction with NBD-Cl and TLC elution, are listed in Table V. These sensitivities indicate the application of NBD-Cl on small samples, for example, syringe washings. At present the NBD-Cl procedure is being used satisfactorily in forensic drug zr?alysis 2nd drug screens on basic extracts of toxicological material.
TABLE V SENSITIVITIES
OF SELECTED COMPOUNDS Sensitivities were determined after TLC elution.
WITH
NBD-Cl
Compound
Sensitivity (fig)
Amphetamine Ani!eridine Chlorpkniramine Chlorpkntermine Diphenhydrzmine Methylphenidate Methampietamine Phenethylamine Fhenmetrazine 3,4-MethyIenedioxyamphetamine 3-Methoxy4,kneihyienedioxyamphetzmine p-Hydroxyephedrine
0.01 0.03 0.01 0.50 0.01 0.50 0.01 0.01 0.05 0.35 0.35 0.50
ACKNOWLEDGEMENTS
The authors wish to express their appreciation to the Royal Canadian Mounted Police for their assistance, to the Healrh Protection Branch, Health and Welfare, CaRada, for supplying some of the sAand2rd drugs, and to Mrs. Susan Moe for her kind assistance in the preparation of the manuscript_ REFZRENCES I 1. Monforte, R. J. Batii and I. Sunshine, Ck. Ckm., 18 (1977) I3Z9. Z R. S. Eager, C. 5. Kuiinz and E. W. Abnhainson, khd. Bkk4etn., 53 (1973) 290. 3 R. N. Gupte, B. G. Ckiaim and P. M. Deane, J. C?lromctogr. Sci_, 12 (1974) 67. 1 F. van Hoof and A. Xeyn&-ickxcx,Arzl. Chem., $6 (lSi_) 286. 5 J. Reisch. I-I. Alfes, H. J. Kommert. N. Jantos, I-f. Moiiian and D. Ciasing. Pkamratie, 25 (1970) 331. 6 M. A. Shaw and H. FEZ=!,J. C’irrorrzafog~-_, ZC4 (1975) 201.