GLC Identification and Determination of 3,4-Methylenedioxyamphetamine(MDA) from Plasma and Urine K. K. MIDHA", I. J. McGILVERAY, S. P. BHATNAGAR, and J. K. COOPER
Abstract 0 A GLC method for qualitative and quantitative determination of 3,4-methylenedioxyamphetaminein biological fluids is described. After extraction of the drug and the internal standard, 4-methoxyamphetamine, from plasma and urine, the solvent is evaporated and the residue is mixed with 20 11 of freshly distilled ether. Aliquots (1-2 pl) then are injected into the gas chromatograph. Both the drug and the internal standard give well-separated symmetrical peaks. Flame-ionization detection allows concentrations of 0.125 fig of 3,4-methylenedioxyamphetaminefrom plasma to be determined with a precision of 3.16%. The method is applicable to the estimation of 4-methoxyamphetamine, employing 3,4methylenedioxyamphetamine as the internal standard. For identification purposes, various derivatives such as Schiff bases, isothiocyanates, trifluoroacetates, and heptafluorobutyrates of both compounds were formed following extraction from urine and their GLC behavior was investigated. Derivative formation was confirmed by GLC-mass spectrometry. Mass spectral data for these derivatives are presented. Keyphrases 0 3,4-Methylenedioxyamphetamine-GLC analysis, mass spectrum, plasma and urine, human, dog 4-Methoxyamphetamine-GLC analysis, mass spectrum, plasma and urine, human, dog GLC-analysis, 3,4-methylenedioxyamphetamine and 4-methoxyamphetamine, plasma and urine, human, dog
3,4-Methylenedioxyamphetamine (MDA) (I) is chemically related to both mescaline and amphetamine. Some investigators found that the drug possesses stimulant and minor sympathomimetic activity; I inhibited monoamine oxidase (1) and increased muscular rigidity in a patient with Parkinson's disease (2). I t was evaluated as an adjunct to psychotherapy (3), and another study (4)compared the subjective effects of I with those of amphetamine. Because of the widespread abuse of I on the street (under such names as Mellow Drug of America, Love Drug, Love Pill, or MDA), it was felt that there was a need for its toxicological identification and quantitative estimation from biological fluids such as urine and plasma. A literature review on the analysis of this drug showed only one published method (5), which lacks details for quantitation from biological Table I-Recovery of I and I1 from Plasma Determined by GLC Assaya Micrograms Ad d ed to 1 ml of Plasma
I
1.04 4.00 Mean 100.29t 1.15% 11 __
1.0 4.0
Mean Mean Micrograms Percent Recovered Recovery
1.00 4.04
of I Added to Plasma by GLC
Pg
n
Mean Peak - ~ . Height Rat i o
0.125
6 6 4 4 4 4
0.053 0.107 0.206 0.406 0.820 1.663
I Added,
0.250 0.50
1 .o 2.0 4.0
UMean
.
~
~
SD
cv, %a
0.002 0.003 0.006 0.002 0.012 0.046
3.16
C V = 2.22%,y = t n x , where Fn = 0.415
2
2.54
2.83 0.53
1.47 2.16
0.004, r z = 1.
fluids. The present paper describes a GLC procedure for the positive identification and quantitation of I from plasma and urine. EXPERIMENTAL Reagents-Acetone', ethyl acetate', cnrbon disulfide*, and ether3 were distilled in glass prior to use. Heptafluorobutyric anhydride4 and trifluoroacetic anhydride4 were refluxed on phosphorus pentoxide for 3 hr before glass distillation. The 15, 115, dl-amphetamine5, methamphetamine5, and n-pr~pylamphetamine~ were donated. Pyridine4 was silylation grade, and all other chemicals employed were analytical grade. Stock solutions of I (100 pg/ml) and the internal standard, 4methoxyamphetamine (11) (100 ug/ml), were prepared fresh weekly by dissolving their hydrochloride salts in water. Appropriate dilutions of each were made fresh daily as required. Plasma Level Study-3,4-Methylenedioxyamphetamine hydrochloride, 10 mg/kg, was administered to a male dog weighing 8.38 kg. Samples of blood (10 ml) were withdrawn from the femoral vein by means of heparinized tubes6 a t 12 appropriate time intervals. The blood samples were centrifuged, and the plasma was transferred to another tube before storing a t -loo. General P r o ced u r e f o r Extraction of I-To the sample [human or canine plasma (2ml) or urine (5 ml) spiked with I or biological fluids from dosed animals], placed in ~ cr ew - ca ppe d~ cen-
I
CH
2.19 0.96 Caledon Laboratories Ltd., Geor etown, Ontario, Canada.
* Fisher Scientific Co., MontrBal, duBbec, Canada.
1.01 4.02
Mean 100.30 t 0.62% at1
99.72 100.86
SD of Percent Recovery
Table 11-Estimation
= 4.
188 / J o u r n a l of P h a r m a c e u t i c a l Sciences
100.24 100.42
1.23 1.36
Mallinckrodt Chemical Works Ltd., MontrBal, QuBbec, Canada Pierce Chemical Co., Rockford, Ill. Dr. Keith Bailey, Pharmaceutical Chemistry Ilivision, Drug Research Laboratories, Health Protection Branch, Ottawa, Canada. Vacutainers, Becton Dickinson and Co., Mississauga, Ontario, Canada. Lined with Teflon (du Pont).
It
m/c 136
+.I t
m m/e
1
2
8
4
0
1
MINUTES
2
8
4
135
I
0
MINUTES
Figure 1-Gas-liquid chromatogram of human plasma. Key: A, control plasma; B, plasma spiked with I (0.5 pglml) and I1 (3.0 pglml); peak I , endogenous material; peak II, 11;and peak I l l , I. m/e
trifuge tubes (20 ml), are added 1ml of I1 (3.0 pglml), 5 ml of 0.2 M phosphate buffer (pH 7.2), 1 ml of 1N HCl, and 5 ml of ether. The sample is extracted by mixing for 5 min a t 40 rpm, followed by centrifugation a t 3000 rpm for 5 min. The ethereal layer is removed by aspiration, and the extraction is repeated with another 5 ml of ether. Following centrifugation, the ethereal layer is discarded and an aliquot of the aqueous layer is transferred (8 ml for plasma and 11 ml for urine) into another similar centrifuge tube containing 2 ml of 1 N NaOH. The sample is extracted with 6 ml of ether by mixing as before for 10 min a t 40 rpm, followed by centrifugation a t
105
- [COI
m / e 77
m/e
51
Scheme I-Postulated fragmentation of I U
w
c
a:
u
0
a:
uz
0
I
I
I
1
I
10
20
30
40
50
HOURS
3000 rpm for 5 min. Five milliliters of the ethereal layer is transferred into a custom-made evaporating tube (6) containing a small antibumping granuleQ. The extraction is repeated as before with another 6 ml of ether. Six milliliters of the ethereal layer is removed and combined with the first extract. The combined extracts are Concentrated'O under a stream of dry nitrogen at 40' to a volume of approximately 20-30 pl. Aliquots (1-2 pl) then are injected into the gas chromatograph" equipped with a flame-ionization detector. Derivatization-Acetone Schiff Bases and Isothiocyanate Derioatiues-To the concentrated ethereal extracts from plasma or urine is added 0.5 ml of acetone (for Schiff base) or carbon disulfide (for isothiocyanate). The tubes are swirled on a mixerI2 for 30 sec and then concentrated to 50 p1 a t 60' under a stream of dry ni-
Figure 2-Plasma concentration of I in a dog weighing 8.38 kg following a single oral dose of I-HCl (10 mglkg).
British Drug House, Toronto, Ontario, Canada. Dri-Bath, Fisher Scientific Co., MontrBal, QuBbec, Cana-
lo Thermolyne
da. l1 8 Rob-Rack,
Fisher Scientific Co., Montreal, Quebec, Canada.
Model F A l , Perkin-Elmer, Montreal, QuBbec, Canada. Vortex Genie, Fisher Scientific Co., Montreal, Quebec, Canada.
Vol. 65, No. 2, February 1976 / 189
I
cn,.
cn-Nn-
c - C F CF~CF, ~
cn,o-
Q-
m / r 239
m/r 191
+ C Fa CFz CFz
4
8
0
MINUTES
4
i
l+
m/o 1 4 8
1-
m h 133
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CHz-$H-NH-C-CFzCF~CF3
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Abstract 0 A GLC method for qualitative and quantitative determination of 3,4-methylenedioxyamphetaminein biological fluids is described. After extraction of the drug and the internal standard, 4-methoxyamphetamine, from plasma and urine, the solvent is evaporated and the residue is mixed with 20 11 of freshly distilled ether. Aliquots (1-2 pl) then are injected into the gas chromatograph. Both the drug and the internal standard give well-separated symmetrical peaks. Flame-ionization detection allows concentrations of 0.125 fig of 3,4-methylenedioxyamphetaminefrom plasma to be determined with a precision of 3.16%. The method is applicable to the estimation of 4-methoxyamphetamine, employing 3,4methylenedioxyamphetamine as the internal standard. For identification purposes, various derivatives such as Schiff bases, isothiocyanates, trifluoroacetates, and heptafluorobutyrates of both compounds were formed following extraction from urine and their GLC behavior was investigated. Derivative formation was confirmed by GLC-mass spectrometry. Mass spectral data for these derivatives are presented. Keyphrases 0 3,4-Methylenedioxyamphetamine-GLC analysis, mass spectrum, plasma and urine, human, dog 4-Methoxyamphetamine-GLC analysis, mass spectrum, plasma and urine, human, dog GLC-analysis, 3,4-methylenedioxyamphetamine and 4-methoxyamphetamine, plasma and urine, human, dog
3,4-Methylenedioxyamphetamine (MDA) (I) is chemically related to both mescaline and amphetamine. Some investigators found that the drug possesses stimulant and minor sympathomimetic activity; I inhibited monoamine oxidase (1) and increased muscular rigidity in a patient with Parkinson's disease (2). I t was evaluated as an adjunct to psychotherapy (3), and another study (4)compared the subjective effects of I with those of amphetamine. Because of the widespread abuse of I on the street (under such names as Mellow Drug of America, Love Drug, Love Pill, or MDA), it was felt that there was a need for its toxicological identification and quantitative estimation from biological fluids such as urine and plasma. A literature review on the analysis of this drug showed only one published method (5), which lacks details for quantitation from biological Table I-Recovery of I and I1 from Plasma Determined by GLC Assaya Micrograms Ad d ed to 1 ml of Plasma
I
1.04 4.00 Mean 100.29t 1.15% 11 __
1.0 4.0
Mean Mean Micrograms Percent Recovered Recovery
1.00 4.04
of I Added to Plasma by GLC
Pg
n
Mean Peak - ~ . Height Rat i o
0.125
6 6 4 4 4 4
0.053 0.107 0.206 0.406 0.820 1.663
I Added,
0.250 0.50
1 .o 2.0 4.0
UMean
.
~
~
SD
cv, %a
0.002 0.003 0.006 0.002 0.012 0.046
3.16
C V = 2.22%,y = t n x , where Fn = 0.415
2
2.54
2.83 0.53
1.47 2.16
0.004, r z = 1.
fluids. The present paper describes a GLC procedure for the positive identification and quantitation of I from plasma and urine. EXPERIMENTAL Reagents-Acetone', ethyl acetate', cnrbon disulfide*, and ether3 were distilled in glass prior to use. Heptafluorobutyric anhydride4 and trifluoroacetic anhydride4 were refluxed on phosphorus pentoxide for 3 hr before glass distillation. The 15, 115, dl-amphetamine5, methamphetamine5, and n-pr~pylamphetamine~ were donated. Pyridine4 was silylation grade, and all other chemicals employed were analytical grade. Stock solutions of I (100 pg/ml) and the internal standard, 4methoxyamphetamine (11) (100 ug/ml), were prepared fresh weekly by dissolving their hydrochloride salts in water. Appropriate dilutions of each were made fresh daily as required. Plasma Level Study-3,4-Methylenedioxyamphetamine hydrochloride, 10 mg/kg, was administered to a male dog weighing 8.38 kg. Samples of blood (10 ml) were withdrawn from the femoral vein by means of heparinized tubes6 a t 12 appropriate time intervals. The blood samples were centrifuged, and the plasma was transferred to another tube before storing a t -loo. General P r o ced u r e f o r Extraction of I-To the sample [human or canine plasma (2ml) or urine (5 ml) spiked with I or biological fluids from dosed animals], placed in ~ cr ew - ca ppe d~ cen-
I
CH
2.19 0.96 Caledon Laboratories Ltd., Geor etown, Ontario, Canada.
* Fisher Scientific Co., MontrBal, duBbec, Canada.
1.01 4.02
Mean 100.30 t 0.62% at1
99.72 100.86
SD of Percent Recovery
Table 11-Estimation
= 4.
188 / J o u r n a l of P h a r m a c e u t i c a l Sciences
100.24 100.42
1.23 1.36
Mallinckrodt Chemical Works Ltd., MontrBal, QuBbec, Canada Pierce Chemical Co., Rockford, Ill. Dr. Keith Bailey, Pharmaceutical Chemistry Ilivision, Drug Research Laboratories, Health Protection Branch, Ottawa, Canada. Vacutainers, Becton Dickinson and Co., Mississauga, Ontario, Canada. Lined with Teflon (du Pont).
It
m/c 136
+.I t
m m/e
1
2
8
4
0
1
MINUTES
2
8
4
135
I
0
MINUTES
Figure 1-Gas-liquid chromatogram of human plasma. Key: A, control plasma; B, plasma spiked with I (0.5 pglml) and I1 (3.0 pglml); peak I , endogenous material; peak II, 11;and peak I l l , I. m/e
trifuge tubes (20 ml), are added 1ml of I1 (3.0 pglml), 5 ml of 0.2 M phosphate buffer (pH 7.2), 1 ml of 1N HCl, and 5 ml of ether. The sample is extracted by mixing for 5 min a t 40 rpm, followed by centrifugation a t 3000 rpm for 5 min. The ethereal layer is removed by aspiration, and the extraction is repeated with another 5 ml of ether. Following centrifugation, the ethereal layer is discarded and an aliquot of the aqueous layer is transferred (8 ml for plasma and 11 ml for urine) into another similar centrifuge tube containing 2 ml of 1 N NaOH. The sample is extracted with 6 ml of ether by mixing as before for 10 min a t 40 rpm, followed by centrifugation a t
105
- [COI
m / e 77
m/e
51
Scheme I-Postulated fragmentation of I U
w
c
a:
u
0
a:
uz
0
I
I
I
1
I
10
20
30
40
50
HOURS
3000 rpm for 5 min. Five milliliters of the ethereal layer is transferred into a custom-made evaporating tube (6) containing a small antibumping granuleQ. The extraction is repeated as before with another 6 ml of ether. Six milliliters of the ethereal layer is removed and combined with the first extract. The combined extracts are Concentrated'O under a stream of dry nitrogen at 40' to a volume of approximately 20-30 pl. Aliquots (1-2 pl) then are injected into the gas chromatograph" equipped with a flame-ionization detector. Derivatization-Acetone Schiff Bases and Isothiocyanate Derioatiues-To the concentrated ethereal extracts from plasma or urine is added 0.5 ml of acetone (for Schiff base) or carbon disulfide (for isothiocyanate). The tubes are swirled on a mixerI2 for 30 sec and then concentrated to 50 p1 a t 60' under a stream of dry ni-
Figure 2-Plasma concentration of I in a dog weighing 8.38 kg following a single oral dose of I-HCl (10 mglkg).
British Drug House, Toronto, Ontario, Canada. Dri-Bath, Fisher Scientific Co., MontrBal, QuBbec, Cana-
lo Thermolyne
da. l1 8 Rob-Rack,
Fisher Scientific Co., Montreal, Quebec, Canada.
Model F A l , Perkin-Elmer, Montreal, QuBbec, Canada. Vortex Genie, Fisher Scientific Co., Montreal, Quebec, Canada.
Vol. 65, No. 2, February 1976 / 189
I
cn,.
cn-Nn-
c - C F CF~CF, ~
cn,o-
Q-
m / r 239
m/r 191
+ C Fa CFz CFz
4
8
0
MINUTES
4
i
l+
m/o 1 4 8
1-
m h 133
(.>
CF: m/e 69
mle77
!
CHz-$H-NH-C-CFzCF~CF3
l+
CHI
m / e 375
8
MINUTES
cn3
CH mcn-cn,
CH30-Q-
m l r I69
n
I* cn,-
