a fast ce method for the achiral separation of methadone and its major metabolites,...

Tamsin Kelly*Philip DobleMichael Dawson

Centre for Forensic Science,Faculty of Science,University of Technology,Sydney, NSW, Australia

Received February 9, 2007Revised June 28, 2007Accepted July 6, 2007

Research Article

A fast CE method for the achiral separationof methadone and its major metabolites,2-ethylidene-1,5-dimethyl-3,3-diphenylpyr-rolidine and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline

The utilization of dynamic doubly coated capillaries for a fast separation of methadone andits two major metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP) was investigated. The coated capillarieswere prepared using a polycation of poly(diallyldimethylammonium chloride) and a poly-anion of dextran sulfate. A fast achiral separation was developed using the coated capillarieswith a BGE of 100 mM phosphate buffer at pH 2.6. Complete achiral separation of metha-done, EDDP and EMDP was achieved, with migration times of approximately 4 min. Themethod offers considerable advantages with respect to BGE simplicity and analysis timecompared to previously published CE methods for methadone and its related analytes.

Keywords:

Dynamic doubly coated capillaries / 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyr-rolidine / 2-Ethyl-5-methyl-3,3-diphenyl-1-pyrroline/ Methadone / Poly(diallyl-dimethylammonium chloride) DOI 10.1002/elps.200700090

3566 Electrophoresis 2007, 28, 3566–3569

1 Introduction

When using bare silica capillaries, the EOF is highly de-pendent on the pH of the BGE. Considerable EOF exists atelevated pH conditions, while under acidic conditions theEOF is dramatically reduced, but still present [1]. This effectarises due to differences in the extent of silanol dissociationon the surface of the capillary under different pH conditions[2]. Small variations in the EOF give rise to the limitedmigration time reproducibility commonly associated withsome CE applications. The EOF not only influences themigration time of the analyte, but it may also influence theapparent selectivity factor.

The coating of capillaries in CE is often used to stabilizeor alter the speed and/or direction of the EOF, as well as toassist in the elimination of peak tailing associated with theadsorption of cationic analytes on the negatively charged

silanol groups present on the capillary wall [2]. Both perma-nently and dynamically coated capillaries have been devel-oped. Permanently coated capillaries usually involve thecovalent bonding of functional groups to the capillary sur-face, whereas dynamically coated capillaries are generatedusing the BGE additives. It has been proposed that dynami-cally coated capillaries offer greater stability due to theirpotential for continuous regeneration [3].

Recent developments in the preparation of dynamicallycoated capillaries that result in pH-independent EOF allowfor the possibility of very fast cationic separations. Therefore,even under low pH conditions, the provision of a consider-able EOF enhances the apparent mobility of charged basicdrugs, reducing the analysis time. The coating procedureinvolves an initial coating of a polycation, followed by apolyanion. Various types of polycations and polyanions havebeen previously utilized, including poly(diallyldimethyl-ammonium chloride) (PDDAC) and dextran sulfate (DS) [4,5], and CElixir reagents (A and B) [3, 6, 7].

Zakaria et al. [4] utilized a doubly coated fused-silica cap-illary with b-cyclodextrin (bCD) as a pseudostationary phasefor the modeling and optimization of the separation of 14aromatic bases by electrokinetic chromatography, with ananalysis time of approximately 2 min. The authors con-

Correspondence: Dr. Philip Doble, Centre for Forensic Science,Faculty of Science, University of Technology, Sydney, PO Box123, Broadway, NSW Australia 2007E-mail: [email protected]: 161-2-9514-1460

Abbreviations: �CD, b-cyclodextrin; DS, dextran sulfate; EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EMDP, 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline; MET, methadone; PDDAC,poly(diallyldimethylammonium chloride)

* Present address: Curtin University of Technology, Perth, W.A.,Australia

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2007, 28, 3566–3569 CE and CEC 3567

cluded that the PDDAC/DS dynamically coated capillariesproduced stable EOF values over the pH range of 3.5–7.5(BGE: 20 mM citrate buffer with bCD concentrations of 0–10 mM). Zakaria et al. [5] also utilized PDDAC/DS capillariesin the separation of six related opiate alkaloids, includingmorphine, codeine and thebaine, using sulfated bCD as acation exchange pseudostationary phase.

Whilst effective for pain relief, methadone is an opioidthat is primarily used therapeutically in the management ofwithdrawal symptoms in heroin-dependent users duringmaintenance therapy. The main metabolic pathways ofmethadone involve sequential N-demethylation followed byspontaneous cyclization. The two main metabolites ofmethadone are 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrro-lidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline(EMDP); methadone, EDDP and EMDP are all chiral com-pounds. Methods for the analysis of methadone, EDDP andEMDP in biological matrices, such as urine, plasma, serumand saliva, for therapeutic drug monitoring purposes can beconsidered in two broad categories. Achiral methods focuson the determination of the total methadone, EDDP and/orEMDP concentrations, while chiral methods determine theconcentration of each analyte stereoselectively. Such meth-ods have been utilized to investigate correlations betweendose of methadone administered and the resulting con-centration of the parent drug and/or its major metabolites invarious biological specimens, and whether the concentra-tions relate to effective treatment outcomes. Several chiralCE methods based on bCD BGE additives have been report-ed for the separation of methadone and/or EDDP enantio-mers [8–15]. Kelly et al. [16] detailed the simultaneous chiralseparation of methadone, EDDP and EMDP using CZE.However, limited CE methods have been published for theachiral separation of methadone and EDDP [10, 17], ormethadone and both its major metabolites (EDDP andEMDP) [18].

The objective of this study was to employ a dynamicdoubly coated capillary in a simple and fast CE method forthe achiral separation of methadone, EDDP and EMDP.

2 Materials and methods

All buffers were prepared in Milli-Q grade (18 MOcm–1)water by dilution of stock solutions of phosphoric acid(H3PO4), sodium dihydrogen phosphate (NaH2PO4) and/orsodium hydrogen phosphate (Na2HPO4?2H2O) (Sigma-Aldrich, Sydney, Australia). Sodium hydroxide (1M NaOH,HPCE grade) was obtained from Agilent Technologies (Syd-ney, Australia). PDDAC (20 wt% in water) and DS were pur-chased from Sigma-Aldrich. All BGE were degassed by soni-cation under vacuum and filtered using 0.45 mm Nylon sy-ringe filters (Bonnett Equipment, Sydney, Australia) prior touse. (R,S)-Methadone hydrochloride (MET) was kindlydonated by Clinical Pharmacology, St Vincent’s Hospital(Darlinghurst, New South Wales, Australia). (R,S)-EDDP

perchlorate (EDDP, 1 mg/mL MeOH) and (R,S)-EMDP(EMDP, 1 mg/mL ACN) standards were purchased fromDiagnostic Consultants (Sydney, New South Wales, Aus-tralia), LGC Promochem (Hertfordshire, England) orLipomed (Arlesheim, Switzerland). Aqueous standards of(R,S)-methadone, (R,S)-EDDP, (R,S)-EMDP were preparedin Milli-Q water to give a concentration of 20 mg/mL.

Experiments were conducted on an Agilent ChemStationCapillary Electrophoresis System (Agilent Technologies,USA), equipped with a diode array detector. Uncoated 50-mmid fused-silica capillaries of total length of 65 cm and effec-tive length of 56.5 cm were obtained from Polymicro Tech-nologies (Phoenix, USA). A detection window was preparedin each capillary by burning the outer polyimide coating.

The coated capillaries were prepared by a method pre-viously described by Zakaria et al. [4]. In summary, the capillarywas flushed with 1M NaOH for 30 min, then with Milli-Qwater for 15 min before standing for 30 min. A solution of 1wt% PDDAC was then flushed through the capillary for15 min, after which the capillary was allowed to stand for15 min before a 2-min flush with Milli-Q water. Finally, 1.5%w/v DS solution was flushed through the capillary for 15 min.After 30 min standing, the capillary was flushed with Milli-Qwater for 5 min.

Unless stated otherwise, all separations were performedat 257C, with an applied voltage of 30 kV (positive polarity)and detection was performed at 195 nm. Hydrodynamicinjections were used at 50 mbar for 2 or 5 s. Replicate (n = 5or 8) injections of each standard solution were performed foreach BGE investigated. At the start of the working day, thecoated capillary was flushed with the running buffer for15 min. Between different BGE, the capillary was flushed for15 min with the new BGE, and then allowed to stand for afurther 15 min. The migration of the EOF was verified foreach BGE by injection of a neutral marker (10% acetone in10% buffer solution). Furthermore, a flush of the runningbuffer was performed for 3 min before each injection.

The resolution (Rs) of adjacent peaks was determinedbased on the migration times and baseline peak widths ofeach peak.

3 Results and discussion

(R,S)-Methadone, (R,S)-EDDP and (R,S)-EMDP standardswere initially injected with a BGE consisting of 100 mMphosphate buffer, at pH 2.6 on an uncoated capillary. As all ofthe basic analytes were protonated at this pH, all of the ana-lytes migrated toward the detector before the EOF peak, andas expected, resolution was not observed for the enantio-meric pairs. The migration times of (R,S)-MET, (R,S)-EDDPand (R,S)-EMDP were 7.9, 7.3 and 7.6 min, respectively.This achiral separation had an analysis time of approximate-ly 9 min. An example electropherogram of the achiralseparation of methadone, EDDP and EMDP using anuncoated capillary is illustrated in Fig. 1.


3568 T. Kelly et al. Electrophoresis 2007, 28, 3566–3569

Figure 1. Example electropherogram for the achiral separation ofmethadone, EDDP and EMDP using an uncoated capillary. Con-ditions: 50 mm id665 cm uncoated capillary, 100 mM phosphatepH 2.6, applied voltage 30 kV, temperature 257C, injection 5 s at50 mbar, concentration 20 mg/mL of (R,S)-methadone, (R,S)-EDDP and (R,S)-EMDP in water, detection l 195 nm;migration order: 1. EDDP (tm 7.245), 2. EMDP (tm 7.583), 3. MET (tm

7.923).

A dynamic double-coated capillary was then investigatedto speed up the separation of methadone, EDDP andEMDP. The buffers consisted of 100 mM phosphate at pH2.6 and 3.1, with an applied voltage of 30 kV. Both BGEwere found to be suitable for the achiral separation of thethree analytes. The average effective mobilities and the res-olution values obtained for the three analytes at pH 2.6 and3.1 (100 mM phosphate) are given in Table 1. Similar effec-tive mobilities and complete achiral resolution (Rs .4.7) ofeach analyte was observed at pH 2.6 and 3.1. 100 mMphosphate at pH 2.6 was selected as the optimal BGE interms of the shortest analysis time (6 min including EOF).The migration times of (R,S)-MET, (R,S)-EDDP and (R,S)-EMDP were 3.6, 3.4 and 3.5 min, respectively. An exampleelectropherogram of the achiral separation of methadone,EDDP and EMDP using a PDDAC/DS-coated capillary isillustrated in Fig. 2.

The repeatability of the uncoated versus coated capil-laries for the achiral separation of methadone, EDDP andEMDP was evaluated over 56 injections. Using the uncoatedcapillary, the mean (6 SD, n = 56) migration time observedfor (R,S)-MET, (R,S)-EDDP and (R,S)-EMDP was 8.176(6 0.052), 7.386 (6 0.047), 7.815 (6 0.049), respectively.This corresponds to a coefficient of variation (% CV) of0.6 % for each analyte. Using the coated capillary, the mean(6 SD, n = 56) migration time observed for (R,S)-MET,(R,S)-EDDP and (R,S)-EMDP was 3.467 (6 0.038), 3.313(6 0.035), 3.399 (6 0.037), respectively. The reduced migra-tion time using the coated capillary resulted in a slightlyhigher % CV of 1.1 % for each analyte. Therefore, the

Figure 2. Example electropherogram for the achiral separation ofmethadone, EDDP and EMDP using a PDDAC/DS-coated capil-lary. Conditions: 50 mm id665 cm PDDAC/DS-coated capillary,100 mM phosphate pH 2.6, applied voltage 30 kV, temperature257C, injection 2 s at 50 mbar, concentration 20 mg/mL of (R,S)-methadone, (R,S)-EDDP and (R,S)-EMDP in water, detection l195 nm; N.B. Rs (1,2) refers to resolution value between peaks 1and 2 etc.; migration order: 1. EDDP (tm 3.401), 2. EMDP (tm 3.485),3. MET (tm 3.554).

Table 1. Average effective mobilities (meff610–5, cm2s–1V–1) andresolution values obtained for the three analytes at pH2.6 and 3.1

pH EDDPmeff

EMDPmeff

METmeff

Rs EDDPEMDP

Rs EMDPMET

2.6 (n = 8) 22.15 20.70 19.55 5.73 4.713.1 (n = 5) 21.72 20.23 19.14 6.30 4.98

Conditions: 50 mm id665 cm PDDAC/DS-coated capillary,100 mM phosphate pH 2.6 or 3.1, applied voltage 30 kV, temper-ature 257C, injection 2 s at 50 mbar, concentration 20 mg/mL of(R,S)-methadone, (R,S)-EDDP and (R,S)-EMDP in water, detectionl 195 nm.

repeatability of the achiral separation using the coated capil-lary has been clearly demonstrated, without the need forregenerating the dynamic coating.

This separation of methadone, EDDP and EMDP repre-sents a simple and fast method for the analysis of the threeanalytes in less than 6 min. Although previous methods haveachieved complete achiral separation of all three analytes(methadone, EDDP and EMDP) using MEKC [18], the anal-ysis time was approximately 60 min.

4 Concluding remarks

The application of dynamic doubly coated capillaries allowedfor a simple, fast and robust achiral separation of metha-


Electrophoresis 2007, 28, 3566–3569 CE and CEC 3569

done, EDDP and EMDP using a BGE of 100 mM phosphate,pH 2.6. The coated capillaries were prepared using a polyca-tion of PDDAC and a polyanion of DS. Complete separationof the three analytes was obtained with a total analysis timeof 6 min.

5 References

[1] Frazier, R. A., Ames, J. M., Nursten, H. E., Capillary Electro-phoresis for Food Analysis: Method Development, RoyalSociety of Chemistry, London 2000.

[2] Altria, K. D., J. Pharm. Biomed. Anal. 2003, 31, 447–453.

[3] Lurie, I. S., Bethea, J., McKibben, T. D., Hays, P. A. et al., J.Forensic Sci. 2001, 46, 1025–1032.

[4] Zakaria, P., Macka, M., Haddad, P. R., Electrophoresis 2002,23, 1844–1852.

[5] Zakaria, P., Macka, M., Haddad, P. R., J. Chromatogr. A 2003,985, 493–501.

[6] Lurie, I. S., Panicker, S., Hays, P. A., Garcia, A. D. et al., J.Chromatogr. A 2003, 984, 109–120.

[7] Boone, C. M., Jonkers, E. Z., Franke, J. P., de Zeeuw, R. A. etal., J. Chromatogr. A 2001, 927, 203–210.

[8] Trkulja, S., Kovar, K. A., J. Sep. Sci. 2004, 27, 557–559.

[9] Esteban, J., Pellin, M. d. l. C., Gimeno, C., Barril, J. et al.,Toxicol. Lett. 2004, 151, 243–249.

[10] Lanz, M., Thormann, W., Electrophoresis 1996, 17, 1945–1949.

[11] Frost, M., Köhler, H., Blaschke, G., Electrophoresis 1997, 18,1026–1034.

[12] Ramseier, A., Caslavska, J., Thormann, W., Electrophoresis1999, 20, 2726–2738.

[13] Cherkaoui, S., Rudaz, S., Varesio, E., Veuthey, J.-L., Electro-phoresis 2001, 22, 3308–3315.

[14] Rudaz, S., Cherkaoui, S., Gauvrit, J.-Y., Lantéri, P. et al.,Electrophoresis 2001, 22, 3316–3326.

[15] Thormann, W., Caslavska, J., Ramseier, A., Siethoff, C., J.Microcol. Sep. 2000, 12, 13–24.

[16] Kelly, T., Doble, P., Dawson, M., Electrophoresis 2003, 24,2106–2110.

[17] Thormann, W., Lanz, M., Caslavska, J., Siegenthaler, P. et al.,Electrophoresis 1998, 19, 57–65.

[18] Molteni, S., Caslavska, J., Alleman, D., Thormann, W., J.Chromatogr. B 1994, 658, 355–367.


a fast ce method for the achiral separation of methadone and its major metabolites,...

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