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Methadone extraction from minimal amounts of biological fluids
43
Forensic Science International, 32 (1986) 43-48 Elsevier Scientific Publishers Ireland Ltd.
METHADONE EXTRACTION FROM MINIMAL AMOUNTS OF BIOLOGICAL FLUIDS
M. CHIARO’ITI,
C. OFFIDANI
and N. DE GIOVANNI
Zstituto di Medicina Legale e delle Assicurazioni, Roma (Italy)
Universita’ Cattolica de1 Sacro Cuore,
(Received March 3, 1986) (Accepted March 25, 1986)
A new solid phase extraction method was studied to extract methadone from small amounts of biological samples, by using little disposable columns prepacked with reverse phase sorbents. Two different commercial kinds of reversed phase columns prepacked with octadecylsilane (ODS) were tested; the results were compared to those obtained by a direct liquid/ liquid extraction method. Gas chromatograms of extracts obtained by means of ODS reversed phase columns showed a good signal/noise ratio resulting in higher sensitivity (20 ng/ml as detection limit), in comparison with the liquid/liquid extraction method. Key words: Methadone; Reversed phase extraction;
Serum analysis
Introduction Methadone monitoring in urine of drug abusers under pharmacological treatment is generally performed by immunoassay techniques [ 1,2] . Nevertheless when accurate quantitative data are required for pharmacokinetics studies, gas chromatographic (GC) analyses are preferred because of their higher specificity [3]. Many GC methods requiring a large sample amount (4-20 ml) are reported in the literature [4], but they are practically unsuitable to the small sample amounts available from newborn babies. In order to monitor serum methadone concentrations in pregnant abusers [5], we developped an analytical method to both extract and concentrate methadone traces by means of ODS reversed phase columns. Our analytical procedure can be carried out easily using 0.5 ml of serum with acceptable sensitivity and good reproducibility. Materials and methods Reagents
All solvents‘ and chemicals used were reagent grade. Methadone hydrochloride was supplied by Supelco Inc. Bellafonte, PA. fl-Diethylaminoethyl0319-0138/86/$03.50
o 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
44
diphenylpropylacetate hydrochloride (SK&F 525A) was a gift of Smith, Kline and French Research Ltd., England. Benzhexol was obtained from commercial pharmaceutical products. Apparatus RP-18 extractions were performed using two kinds of disposable octadecylsilane (00s) packed microcolumns: (a) Sep Pak cartridges by Water Ass. (RP-18 SP); and (b) Baker lo-SPE columns 3 ml LD by J.T. Baker (RP-18 lo-SPE). GC determinations were carried out on a C. Erba Fractovap 4200 equipped with a Flame Ionization Detector. The inlet and detector temperatures were both 225°C. The column used was a 4 mm X 2 m 3% SE 30 on Gaschrom W (100-120 mesh) with nitrogen as carrier gas at 35 ml/min flow rate. Column oven temperature was 200°C. Alternatively GC analyses were made on a 25-m glass capillary column (0.1 pm SE 54 film thickness) employing an HRGC C Erba apparatus, equipped with a cold on-column injector. Injections were done at 40°C the oven was heated in ballistic mode until 120°C and then programmed to 230°C at S”C/min. Hydrogen as carrier gas was at 2.5 ml/min flow rate. Sensitivity, linearity and recovery Different amounts of a standard solution of methadone hydrochloride in water (0.1 mg/ml) were added to drug-free human serum to obtain concentrations ranging between 0.02 and 1.00 pg/ml. A suitable internal standard (I.S.) was added to the samples, which were then extracted as described below. The recovery rates were calculated in comparison with methanol standard solutions of methadone. In ternal standards Various chemical compounds were tested as an I.S. but due to several analytical problems such as the different behaviour of methadone in the extraction procedures, chromatographic mobilities and the presence of coextracted unknown substances, we selected the use of SK&F 525A or benzhexol(O.1 mg/ml) for the extraction procedure. Extraction procedures 1. Direct liquid/liquid extraction. Two drops of n-octyl alcohol containing 20 pi/ml IS. (SK&F or benzhexol) were added to 5 ml of serum and then made alkaline with 0.1 ml of 1 M NaOH. After centrifugation, 4 ml of supernatant were taken and extracted twice with chloroform. The organic layer was removed and evaporated to dryness. Dried extracts were reconstituted with 50 ~1 of methanol and l-p1 aliquots were injected into the gas chromatograph. 2. Reversed phase extraction. The reversed phase extraction can be developed in accordance with two modalities:
45
(A) RP-18 SP cartridges were activated as suggested by Water Ass. and equilibrated with 2 ml of phosphate buffer l/15 M (pH 7.0). Samples (0.5-l ml) added with 20 &ml I.S. (SK&F 525A is necessary) were pumped through the cartridges. They were then washed with phosphate buffer (2 ml) and with 2 ml of ethyl acetate. Methadone elution was obtained with 1.5 ml of methanol. Methanolic eluate was evaporated to dryness and then reconstituted with 25 ~1 of methanol. One microliter was injected into the gas chromatograph. (B) RP-18 10.SPE were used and the extraction procedure was almost like the previous one, only a few variations were made: columns activation was made with 6 ml of methanol and 6 ml of phosphate buffer (pH 7.0). After pumping of the serum, 0.5 ml with 20 pi/ml IS. (SK&F 525A or benzhexol), the column was washed with 3 ml of phosphate buffer and 1 ml of ethyl acetate to flush the small amount of residual water from the column. This procedure was carried out by the Baker lo-SPE Extraction Vacuum Manifold. At any step of the reversed phase extraction we checked the possible presence of methadone in the fluids filtered through the column. The drug was found only at the elution step and 1.5 ml of methanol were sufficient to elute the methadone in both kinds of microcolumns. Results and discussion Reversed phase extraction provides cleaner extracts and higher recovery in comparison with conventional liquid/liquid extraction procedure. In Fig. la typical pattern resulting from direct extraction is reported, while in Figs. 2 and 3 chromatographic responses after RP-18 extractions are shown.
methadone internal
standard
(benz’~c:o’)
_’
Fig. 1. Gas chromatographic pattern obtained after direct liquid/liquid extraction dure No. 1) on a packed column.
(proce-
46
0.4
ornat
10.6 .-.m._._ ._.-.-.-
16.1
Fig. 2. Gas chromatographic Sep Pak Cartridges (procedure
Internal
rtrrdatd
standard
(S6hf
pattern obtained after ODS reversed No. 2-A) on a packed column.
(benzhex@t) 6’26 A)
phase extraction
using
Because of the clean extracts obtained with RP-18 columns, very low amounts of methadone can be quantified from small amounts of biological samples. With the RP-18 extractions we observed a detection limit of 20 ng/ ml, while by direct liquid/liquid extraction the smallest detectable methadone amount was 60 ng/ml.
J
-
8.4 10.8 f- -._.__ _,_ _
16.1 -
_
._ _.-.I_L-
-.-._.‘_‘--_
internal
standard
(6Mf
626 1)
f
Fig. 3. Gas chromatographic pattern obtained after ODS reversed Baker lo-SPE columns (procedure No. 2-B) on a packed column.
phase extraction
using
The direct extraction method involves an increase in analytical time, higher sample handling and lower absolute recovery (approx. 68%). These characteristics make the direct extraction procedure undesirable when small samples have to be analyzed in a short time and high sensitivity is required, as in toxicological studies during delivery and on newborn babies. On the contrary, RP-18 extraction involves easier sample handling, allows a faster analysis (only 5 min) with higher absolute recovery (better than 95%). Before extraction-concentration on the RP-18 columns, serum buffering was performed at alkaline, neutral and acid pH. A basic pH (greater than 8) destroys the column packing while no real differences were observed employing neutral or acid pH (approx. 4). Hence, we preferred a column conditioning at the same pH of serum avoiding sample buffering before the analysis. After sample addition to the microcolumns, we tested various column , METHADONE
CALIBRATION
(obtained
I
with
I
2-B
I
0.1
methadone
CURVE method)
I
I
0.5
concent
ration
I 1
( pg / ml 1
Fig. 4. Methadone calibration curve obtained Baker lo-SPE columns (procedure No. 2-B).
by ODS reversed phase extraction
using
48
wash solvents and chose ethyl acetate, because it allows a good eluate cleaning and at the same time has a low affinity to methadone and the IS. Column washes using diethyl ether or chloroform, resulted in clean extracts but loss of methadone occurred during this step. On the contrary, the USCof less polar solvents (like n-heptane) resulted in a dirtier final methanolic eluate. The response curve for methadone was found to be linear in the range 0.02-1.00 pg/ml (Fig. 4). The correlation coefficients calculated for the regression line were respectively 0.9752 using benzhexol as IS. and 0.9886 using SK&F 525A. Five subsequent analyses performed on standard serum containing 0.5 pg/ ml of methadone, showed a standard deviation of about 2% for all the methods. Benzhexol was the first choice as an I.S. because of the shorter retention time with our GC conditions. However, with the RP-18 SP, an unknown peak with a retention time close to that of benzhexol is eluted from the column (Fig. 2). In this case the use of SK&F 525A is necessary. The outlined method demonstrated that ODS sample extraction provides rapidity, accuracy, sensitivity and high recovery, as required in routine toxicological analyses and pharmacokinetic studies. References 1 T. Manning, J.M. Bidanset, S. Cohen and L. Lukash, Evaluation of the Abuscreen for methadone. J. Forensic Sci., 21 (1976) 112-121. 2 S.D. Ferrara, L. Tedeschi, F. Castagna and M. Marigo, Comparison of GLC-EMIT analysis for the assay of methadone and its major metabolite in urine. Forensic Sci., 11 (1978) 181-188. 3 R.K. Lynn, R.M. Leger, W.P. Gordon, G.D. Olsen and N. Gerber, New gas chromatographic assay for the quantification of methadone. Application in human and animal studies. J. Chromatogr., 131 (1977) 329-340. 4 C.E. Inturrisi and K. Verebely, A gas-liquid chromatographic method for the quantitative determination of methadone in human plasma and urine. J. Chromatogr., 65 (1972) 361-369. 5 C. Offidani, M. Chiarotti and N. De Giovanni, Methadone in pregnancy: clinicaitoxicological aspects. J. Z'oxicol.Clin. Toxicol., 24 (1986) (in press).
Forensic Science International, 32 (1986) 43-48 Elsevier Scientific Publishers Ireland Ltd.
METHADONE EXTRACTION FROM MINIMAL AMOUNTS OF BIOLOGICAL FLUIDS
M. CHIARO’ITI,
C. OFFIDANI
and N. DE GIOVANNI
Zstituto di Medicina Legale e delle Assicurazioni, Roma (Italy)
Universita’ Cattolica de1 Sacro Cuore,
(Received March 3, 1986) (Accepted March 25, 1986)
A new solid phase extraction method was studied to extract methadone from small amounts of biological samples, by using little disposable columns prepacked with reverse phase sorbents. Two different commercial kinds of reversed phase columns prepacked with octadecylsilane (ODS) were tested; the results were compared to those obtained by a direct liquid/ liquid extraction method. Gas chromatograms of extracts obtained by means of ODS reversed phase columns showed a good signal/noise ratio resulting in higher sensitivity (20 ng/ml as detection limit), in comparison with the liquid/liquid extraction method. Key words: Methadone; Reversed phase extraction;
Serum analysis
Introduction Methadone monitoring in urine of drug abusers under pharmacological treatment is generally performed by immunoassay techniques [ 1,2] . Nevertheless when accurate quantitative data are required for pharmacokinetics studies, gas chromatographic (GC) analyses are preferred because of their higher specificity [3]. Many GC methods requiring a large sample amount (4-20 ml) are reported in the literature [4], but they are practically unsuitable to the small sample amounts available from newborn babies. In order to monitor serum methadone concentrations in pregnant abusers [5], we developped an analytical method to both extract and concentrate methadone traces by means of ODS reversed phase columns. Our analytical procedure can be carried out easily using 0.5 ml of serum with acceptable sensitivity and good reproducibility. Materials and methods Reagents
All solvents‘ and chemicals used were reagent grade. Methadone hydrochloride was supplied by Supelco Inc. Bellafonte, PA. fl-Diethylaminoethyl0319-0138/86/$03.50
o 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
44
diphenylpropylacetate hydrochloride (SK&F 525A) was a gift of Smith, Kline and French Research Ltd., England. Benzhexol was obtained from commercial pharmaceutical products. Apparatus RP-18 extractions were performed using two kinds of disposable octadecylsilane (00s) packed microcolumns: (a) Sep Pak cartridges by Water Ass. (RP-18 SP); and (b) Baker lo-SPE columns 3 ml LD by J.T. Baker (RP-18 lo-SPE). GC determinations were carried out on a C. Erba Fractovap 4200 equipped with a Flame Ionization Detector. The inlet and detector temperatures were both 225°C. The column used was a 4 mm X 2 m 3% SE 30 on Gaschrom W (100-120 mesh) with nitrogen as carrier gas at 35 ml/min flow rate. Column oven temperature was 200°C. Alternatively GC analyses were made on a 25-m glass capillary column (0.1 pm SE 54 film thickness) employing an HRGC C Erba apparatus, equipped with a cold on-column injector. Injections were done at 40°C the oven was heated in ballistic mode until 120°C and then programmed to 230°C at S”C/min. Hydrogen as carrier gas was at 2.5 ml/min flow rate. Sensitivity, linearity and recovery Different amounts of a standard solution of methadone hydrochloride in water (0.1 mg/ml) were added to drug-free human serum to obtain concentrations ranging between 0.02 and 1.00 pg/ml. A suitable internal standard (I.S.) was added to the samples, which were then extracted as described below. The recovery rates were calculated in comparison with methanol standard solutions of methadone. In ternal standards Various chemical compounds were tested as an I.S. but due to several analytical problems such as the different behaviour of methadone in the extraction procedures, chromatographic mobilities and the presence of coextracted unknown substances, we selected the use of SK&F 525A or benzhexol(O.1 mg/ml) for the extraction procedure. Extraction procedures 1. Direct liquid/liquid extraction. Two drops of n-octyl alcohol containing 20 pi/ml IS. (SK&F or benzhexol) were added to 5 ml of serum and then made alkaline with 0.1 ml of 1 M NaOH. After centrifugation, 4 ml of supernatant were taken and extracted twice with chloroform. The organic layer was removed and evaporated to dryness. Dried extracts were reconstituted with 50 ~1 of methanol and l-p1 aliquots were injected into the gas chromatograph. 2. Reversed phase extraction. The reversed phase extraction can be developed in accordance with two modalities:
45
(A) RP-18 SP cartridges were activated as suggested by Water Ass. and equilibrated with 2 ml of phosphate buffer l/15 M (pH 7.0). Samples (0.5-l ml) added with 20 &ml I.S. (SK&F 525A is necessary) were pumped through the cartridges. They were then washed with phosphate buffer (2 ml) and with 2 ml of ethyl acetate. Methadone elution was obtained with 1.5 ml of methanol. Methanolic eluate was evaporated to dryness and then reconstituted with 25 ~1 of methanol. One microliter was injected into the gas chromatograph. (B) RP-18 10.SPE were used and the extraction procedure was almost like the previous one, only a few variations were made: columns activation was made with 6 ml of methanol and 6 ml of phosphate buffer (pH 7.0). After pumping of the serum, 0.5 ml with 20 pi/ml IS. (SK&F 525A or benzhexol), the column was washed with 3 ml of phosphate buffer and 1 ml of ethyl acetate to flush the small amount of residual water from the column. This procedure was carried out by the Baker lo-SPE Extraction Vacuum Manifold. At any step of the reversed phase extraction we checked the possible presence of methadone in the fluids filtered through the column. The drug was found only at the elution step and 1.5 ml of methanol were sufficient to elute the methadone in both kinds of microcolumns. Results and discussion Reversed phase extraction provides cleaner extracts and higher recovery in comparison with conventional liquid/liquid extraction procedure. In Fig. la typical pattern resulting from direct extraction is reported, while in Figs. 2 and 3 chromatographic responses after RP-18 extractions are shown.
methadone internal
standard
(benz’~c:o’)
_’
Fig. 1. Gas chromatographic pattern obtained after direct liquid/liquid extraction dure No. 1) on a packed column.
(proce-
46
0.4
ornat
10.6 .-.m._._ ._.-.-.-
16.1
Fig. 2. Gas chromatographic Sep Pak Cartridges (procedure
Internal
rtrrdatd
standard
(S6hf
pattern obtained after ODS reversed No. 2-A) on a packed column.
(benzhex@t) 6’26 A)
phase extraction
using
Because of the clean extracts obtained with RP-18 columns, very low amounts of methadone can be quantified from small amounts of biological samples. With the RP-18 extractions we observed a detection limit of 20 ng/ ml, while by direct liquid/liquid extraction the smallest detectable methadone amount was 60 ng/ml.
J
-
8.4 10.8 f- -._.__ _,_ _
16.1 -
_
._ _.-.I_L-
-.-._.‘_‘--_
internal
standard
(6Mf
626 1)
f
Fig. 3. Gas chromatographic pattern obtained after ODS reversed Baker lo-SPE columns (procedure No. 2-B) on a packed column.
phase extraction
using
The direct extraction method involves an increase in analytical time, higher sample handling and lower absolute recovery (approx. 68%). These characteristics make the direct extraction procedure undesirable when small samples have to be analyzed in a short time and high sensitivity is required, as in toxicological studies during delivery and on newborn babies. On the contrary, RP-18 extraction involves easier sample handling, allows a faster analysis (only 5 min) with higher absolute recovery (better than 95%). Before extraction-concentration on the RP-18 columns, serum buffering was performed at alkaline, neutral and acid pH. A basic pH (greater than 8) destroys the column packing while no real differences were observed employing neutral or acid pH (approx. 4). Hence, we preferred a column conditioning at the same pH of serum avoiding sample buffering before the analysis. After sample addition to the microcolumns, we tested various column , METHADONE
CALIBRATION
(obtained
I
with
I
2-B
I
0.1
methadone
CURVE method)
I
I
0.5
concent
ration
I 1
( pg / ml 1
Fig. 4. Methadone calibration curve obtained Baker lo-SPE columns (procedure No. 2-B).
by ODS reversed phase extraction
using
48
wash solvents and chose ethyl acetate, because it allows a good eluate cleaning and at the same time has a low affinity to methadone and the IS. Column washes using diethyl ether or chloroform, resulted in clean extracts but loss of methadone occurred during this step. On the contrary, the USCof less polar solvents (like n-heptane) resulted in a dirtier final methanolic eluate. The response curve for methadone was found to be linear in the range 0.02-1.00 pg/ml (Fig. 4). The correlation coefficients calculated for the regression line were respectively 0.9752 using benzhexol as IS. and 0.9886 using SK&F 525A. Five subsequent analyses performed on standard serum containing 0.5 pg/ ml of methadone, showed a standard deviation of about 2% for all the methods. Benzhexol was the first choice as an I.S. because of the shorter retention time with our GC conditions. However, with the RP-18 SP, an unknown peak with a retention time close to that of benzhexol is eluted from the column (Fig. 2). In this case the use of SK&F 525A is necessary. The outlined method demonstrated that ODS sample extraction provides rapidity, accuracy, sensitivity and high recovery, as required in routine toxicological analyses and pharmacokinetic studies. References 1 T. Manning, J.M. Bidanset, S. Cohen and L. Lukash, Evaluation of the Abuscreen for methadone. J. Forensic Sci., 21 (1976) 112-121. 2 S.D. Ferrara, L. Tedeschi, F. Castagna and M. Marigo, Comparison of GLC-EMIT analysis for the assay of methadone and its major metabolite in urine. Forensic Sci., 11 (1978) 181-188. 3 R.K. Lynn, R.M. Leger, W.P. Gordon, G.D. Olsen and N. Gerber, New gas chromatographic assay for the quantification of methadone. Application in human and animal studies. J. Chromatogr., 131 (1977) 329-340. 4 C.E. Inturrisi and K. Verebely, A gas-liquid chromatographic method for the quantitative determination of methadone in human plasma and urine. J. Chromatogr., 65 (1972) 361-369. 5 C. Offidani, M. Chiarotti and N. De Giovanni, Methadone in pregnancy: clinicaitoxicological aspects. J. Z'oxicol.Clin. Toxicol., 24 (1986) (in press).