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METHADONE ISOLATION FROM HUMAN
PLASMA BY LIQUID-LIQUID EXTRACTION
AND SOLID-PHASE EXTRACTION.
COMPARATIVE STUDY.
DANIELA-SAVETA POPA, BELA KISS, LAURIAN VLASE, DANA
MUNTEAN, FELICIA LOGHIN
Faculty of Pharmacy, University of Medicine and Pharmacy “Iuliu
Haţieganu” Cluj-Napoca
*
corresponding author: dpopa@umfcluj.ro
Abstract
Optimal experimental conditions for the methadone isolation from human
plasma were established for ulterior quantification of the drug by performant analytical
methods. The methadone isolation efficiency was determined by liquid-liquid extraction
(LLE) and solid-phase extraction (SPE) depending on the pH. Different organic solvents
(ethyl ether, ethyl acetate and hexane) or solvent mixtures and different SPE cartridges
were tested. The methadone was quantified by an original LC-MS-MS method. Maximum
recovery for the extraction was obtained with solvents as ethyl ether (recovery 99.05%, n =
3) or hexane (recovery 100.75%, n = 3) without pH adjustment, and with Sep C18
cartridges (recovery 98.24%, n = 3) at pH 6.85. LLE showed the same efficiency as SPE for
the methadone isolation from plasma, depending on the experimental conditions: solvent or
solid phase nature, and the pH of the extraction medium. The best extraction methods can
successfully be applied in clinical and forensic toxicology for the efficient isolation of
methadone from human plasma samples.
Rezumat
S-au studiat condiţiile experimentale optime pentru izolarea metadonei din
plasma umană, în vederea cuantificării ei ulterioare prin metode analitice performante. S-a
determinat eficacitatea izolării metadonei atât prin extracţie lichid-lichid (ELL), cât şi prin
extracţie pe fază solidă (EFS), dependente de pH. S-au testat diferiţi solvenţi organici (eter
etilic, acetat de etil, hexan) sau amestecuri de solvenţi, respectiv diferite cartuşe de extracţie
pe fază solidă. Determinarea cantitativă a metadonei s-a realizat printr-o metodă LC-MSMS originală. Metadona se extrage din plasma umană cu randament maxim, utilizând
solvenţi precum eterul etilic (regăsire 99,05%, n = 3) sau hexanul (regăsire 100,75%, n = 3)
fără ajustarea pH-ului, respectiv cartuşe de extracţie Sep C18 (regăsire 98,24%, n = 3) la
pH 6,85. ELL s-a dovedit a fi la fel de eficientă cu EFS pentru izolarea metadonei din
plasmă, dependent de condiţiile experimentale aplicate: natura solventului, respectiv a fazei
staţionare, şi pH-ul mediului de extracţie. Cele mai bune metode de extracţie se pot aplica
cu succes în toxicología clinică şi medico-legală în vederea izolării eficiente a metadonei
din probe de plasmă umană.


methadone
plasma


solid phase extraction
liquid-liquid extraction
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INTRODUCTION
Methadone is an opioid widely used in the treatment of severe pain
and in the maintenance treatment of opioid addicts [1]. As methadone
pharmacokinetics has a large inter-individual variability, the monitoring of
methadone levels in blood samples during the methadone maintenance
treatment is important in order to achieve optimum treatment [2].
Several HPLC [3-7] or GC-MS [8-10] chromatographic methods
are available for methadone quantification in plasma samples after liquid–
liquid extraction (LLE) [3, 5] or solid-phase extraction (SPE) [4-6, 10].
The aim of the present study was to compare the efficiency of
several LLE and SPE methods for methadone isolation from plasma and to
mark out optimal experimental conditions that can later be applied for the
quantification of the drug by highput analytical methods.
MATERIALS AND METHODS
Reagents and materials
Methadone was standard reference from Lipomed AG (Arlesheim,
Switzerland). All chemicals were of analytical-reagent grade. HPLC-grade
acetonitrile, HPLC-grade methanol, ethyl ether, 98% formic acid and sodium
hydroxide were purchased from Merck (Darmstadt, Germany), concentrated
ammonia from Fluka (Buchs, Switzerland), ethyl acetate, hexane,
dichloromethane, disodium phosphate and monopotassium phosphate from
Sigma-Aldrich (Steinheim, Germany). Bidistilled, deionised water pro
injectiones was purchased from Infusion Solution Laboratory of University of
Medicine and Pharmacy Cluj-Napoca, Romania. The human blank plasma
was supplied by the Blood Donation Centre Cluj-Napoca, Romania.
For SPE the following cartridge types were used: Oasis® MCX, 30
mg (Waters Corporation, Massachusetts, Ireland), Isolute® HCX, 130 mg
(International Sorbent Technology, Mid Glamorgan, UK) and SNAP
Cartridge – Sep C18, 300 mg (Lida Manufacturing Corporation, Kenosha,
WI, USA).
Apparatus
The following apparatus were used in this study: 204 Sigma
Centrifuge (Osterode am Harz, Germany); Analytical Plus Balance (MettlerToledo, Switzerland) and Discovery Balance (Ohaus, Pine Brook, NJ,
SUA); Vortex Genie 2 (Scientific Industries, New York, USA); ultrasonic
bath Elma Transsonic 700/H (Singen, Germany); SPE automated extractor
Chromabond® Vacuum manifolds (Macherey-Nagel, Düren, Germany);
evaporator with nitrogen stream (Cole-Parmer, USA).
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117
The HPLC system was a 1100 series model (Agilent Technologies,
Darmstadt, Germany) consisting of a G1312A binary pump, an in-line
G1379A degasser, a G1329A autosampler, a G1316A column thermostat
and an Agilent Ion Trap Detector (1100 VL; Agilent Technologies).
Chromatographic separation was performed at 45ºC on a Zorbax
SB-C18 (100 mm x 3.0 mm, 3.5 m I.D) column (Agilent Technologies)
protected by an in-line filter under isocratic conditions. The mobile phase
was a 45:55 (v/v) mixture of acetonitrile and 0.2% (v/v) formic acid in
water. The flow rate of mobile phase was 1 mL/min.
Chromatograms were processed using Quant Analysis software.
The detection of methadone was in the multiple-reaction
monitoring (MRM) mode, using the ion trap mass spectrometer equipped
with an atmospheric pressure electrospray ionisation ion source. The
electrospray ion source parameters were as follows – capillary 4000 V,
nebulizer 70 psi (nitrogen), dry gas nitrogen at 12 L/min, dry gas
temperature 350ºC. The ion transition monitored was m/z 310→ m/z 265.
Standard solutions
A stock solution of methadone with a concentration of 0.5 mg/mL
was prepared by dissolving an appropriate quantity of reference substance in
5 mL methanol. The marked plasma sample with a concentration of 50
ng/mL was obtained by diluting specific volumes of methadone stock
solution with blank plasma.
Sample preparation
All analysis were performed in triplicate.
For LLE, spiked plasma samples (0.5 mL), with or without
alcalinisation using 1M sodium hydroxyde (100 μL), were mixed with 5 mL
organic solvent (Table I) at vortex-mixer for 5 minutes. After centrifugation,
the organic phase was separated and evaporated to dryness at 45ºC under a
nitrogen stream. The dry extract was reconstituted in 500 μL of
water:acetonitrile 80:20 (v/v) and 5 μL of aliquot were injected into the
chromatograph.
For SPE, to 0.5 mL spiked plasma sample, 2.5 mL 50 mM
phosphate buffer (pH 6.85) was added. The tested cartridges were
successively conditioned with methanol, distillated water and 50 mM
phosphate buffer (pH 6.85). After sample addition, the cartridges were
successively washed with 0.5 mM phosphate buffer (pH 6.85) and a solution
of formic acid 2%. The volumes of the solutions used for conditioning and
washing were 3 mL for Oasis MCX and Isolute HCX, and 5 mL for Sep
C18, respectively. The elution was performed in two consecutive phases,
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with: a) methanol (1 mL for Oasis MCX and Isolute HCX, and 2 mL for
Sep C18, respectively) and b) methanol:concentrated ammonia (19:1, v/v)
(1 mL for Oasis MCX and Isolute HCX, and 2 mL for Sep C18,
respectively). Each eluate was evaporated to dryness at 45ºC under a
nitrogen stream. The dry extract was reconstituted in 500 μL
water:acetonitrile 80:20 (v/v) and a sample of 5 μL aliquot was injected into
the chromatograph.
RESULTS AND DISCUSSION
The results are presented in Tables I and II. The LC-MS-MS
method applied for the quantification of methadone is an original method,
previously developped in our laboratory and validated for the determination
of methadone in human plasma [11].
Ethyl ether and hexane can quantitatively extract methadone from
human plasma without pH adjustment. Due to methadone basic character
(pKa = 8.3) [12], one would expect that extraction in alkaline medium to be
superior to that at pH 7. At pH 7, not only the extraction recovery was
superior, but also the reproductibility was better (reflected by the coeficient
of variation).
Methadone quantification mode LC-MS-MS can influence the
obtained results. The endogenous interferents from alkaline extracts can
generate ion supression and consequently, the recovery at alkaline pH is
smaller.
Methadone was extracted from plasma by SPE at pH 6.85. Pure
methanol has no ability to eluate methadone from cartridges. In exchange, a
quantitative elution was obtained with a mixture of methanol:concentrated
ammonia (19:1, v/v), with the recovery values depending on the type of
solid phase cartridge.
The best results were obtained in case of Sep C18 cartridges, with a
recovery of 98.24% and a very good reproductibility (CV % = 2.77).
Mercolini and colab. have obtained similar results in a study in which
several solid-phases were tested, with cationic exchange/lipophilic retention
(Bond Elut Certify), hydrophilic/lipophilic retention (Oasis HLB) or
reverse-phase sorbents (cyclohexyl-, CH, and octyl-, C8). The best
methadone extraction was obtained with C8 cartridges (recovery between
96-99%, HPLC-DAD quantification method) [9]. Therefore, in the case of
methadone, the use of extraction cartridges with double retention (reverse
stage and ionic exchange) such as those tested in the present study (Oasis ®
MCX and Isolute® HCX) is not justified.
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Solvent
Table I
Recovery of methadone from human plasma
by LLE using different solvents and different pH conditions
Individual
Mean
pH
CV Recovery
concentration concentration SD
plasma
(%)
(%)
(ng/ml)
(ng/ml)
40.59
Ethylic ether
11
39.06
32.67
12.42 38.00 65.34
37.38
4.62 12.36 74.76
29.42
2.87
9.75
58.84
49.24
3.40
6.90
98.48
28.77
2.71
9.43
57.54
34.54
5.58 16.15 69.09
49.52
0.90
1.82
50.38
2.46
4.88 100.75
18.36
40.06
Hexane
11
32.05
40.03
26.73
Ethyl acetate
11
32.44
29.10
Ethylic
ether:hexane
50/50
Ethylic ether:
ethyl acetate
50/50
Ethylic ether:
dichloromethane
75/25
53.17
11
47.31
47.25
29.91
11
30.73
25.67
38.24
11
28.13
37.26
50.51
Ethylic ether
7*
49.31
99.05
48.75
48.66
Hexane
7*
49.27
53.19
* plasma pH
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FARMACIA, 2008, Vol.LVI, 2
Table II
Recovery of methadone from human plasma
by LLE using different cartridges at pH 6.85
Eluate
Cartridge
Methanol
Oasis®
MCX
Isolute®
HCX
Methanol:
conc.ammonia
(19:1, v/v)
Oasis®
MCX
Isolute®
HCX
Sep C18
Individual
concentration
(ng/ml)
0.29
0.15
0.13
0.13
0.00
0.15
38.66
35.86
31.85
39.17
37.10
22.56
48.07
48.64
50.66
Mean
concentration
(ng/ml)
SD
CV
(%)
Extraction
output
(%)
0.19
0.09
47.48
0.38
0.09
0.08
86.89
0.19
35.46
3.42
9.66
70.91
32.94
9.06
27.49
65.89
49.12
1.36
2.77
98.24
CONCLUSION
LLE proved the same efficiency as SPE in the isolation of
methadone from human plasma, depending on the experimental conditions:
solvent and solid-phase nature, as well as pH. The best extraction methods
can successfully be applied in clinical and forensic toxicology for the
efficient isolation of methadone from human plasma samples.
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