analysis of underivatized amphetamines and related phenethylamines with high-performance

Journal of Analyt ical Toxicology, Vol. 24, March 2000

Analysis of Underivatized Amphetamines and Related Phenethylamines with High-Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry* Maciej J. Bogusz, Klaus-Dieter KrLiger, and Rolf-Dieter Maier Institute of Forensic Medicine, Aachen University of Technology, 52057 Aachen, Germany

Abstract I

Amphetamine, methamphetamine, illicit designer phenethylamines (MDA, MDEA, MDMA, MBDB, and BDMPEA), and other phenethylamines (benzyl-l-phenylethylamine, cathinone, ephedrine, fenfluramine, norfenfluramine, phentermine, 1-phenylethylamine, phenylpropanolamine, and propylhexedrine) were extracted from serum using a solid-phase extraction procedure. The extracts were examined with high-performance liquid chromatography- atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). The drugs were separated on ODS column in acetonitrile/5OmM ammonium formate buffer (pH 3.0) (25:75) as a mobile phase. Full-scan mass spectra of drugs examined by means of APCI with collision-induced dissociation showed protonated molecular ions and fragments typical for particular drugs. LC-APCI- MS allowed an unequivocal differentiation of all drugs involved. The quantitation was performed using selected ion monitoring of protonated molecular ions and fragments of drugs involved and their deuterated analogues. The limits of detection ranged from 1 to 5 pg/t serum, and the recoveries ranged from 58 to 96%. A linear response was observed for all drugs in the range from 5 to 500 pg/L. The method was applied for routine determination of amphetamine, MDMA, MDA, and MDEA in one run. Solid-phase extraction used assured simultaneous isolation of various groups of basic drugs of forensic interest (opiates, cocaines, phenethylamines, and benzodiazepines) from biofluids.

Introdudion

Selective and sensitive analysis of amphetamine and related compounds such as methamphetamine, methylenedioxyam- phetamines (e.g., "ecstasy"), and other phenethylamines are among the most important tasks of the forensic toxicologist. These drugs particularly ecstasy, were often detected in blood samples taken from impaired drivers, usually together with other drugs such as cannabis or ethyl alcohol (1,2). In the 1990s, several

* Dedicated to Prof.Dr.med. Helmut Althoff on the occasion of his 65th birthday.

reports demonstrating that the intake of amphetamines is associated with high risk, especially when consumed in discos and at rave parties, were published (1,3-7).

Among the analytical procedures applied for the forensic analysis of amphetamines, gas chromatography-mass spectrometry (GC-MS) plays a dominant role. Besides electron impact mode, the chemical ionization mode (positive and negative ions) was advocated in order to avoid the misidentification of some over- the-counter sympathomimetic amines with amphetamine or methamphetamine (8-17).

High-performance liquid chromatography (HPLC) with UV detection is less suitable for blood analysis because of the low absorptivity of amphetamines and is mainly applied in the examination of illicit drug samples (18). HPLC with fluorescence detection showed sufficient sensitivity for detection of amphetamines in biological fluids (19,20), but the selectivity is not comparable with that of MS identification. Among other separation techniques, capillary electrophoresis with diode-array detection (DAD) (21) and thin-layer chromatography with Fourier transform infrared detection (22) have been used for the analysis of forensic samples containing methylenedioxyam- phetamines.

The introduction of HPLC coupled with atmospheric pressure ionization mass spectrometry (LC-API-MS), in either atmospheric pressure chemical ionization (APCI) or electrospray (ESI) mode, created the possibility of taking advantage of the high selectivity of MS detection without derivatization, which is unavoidable in GC-MS procedures. In a previous report (23), we described a procedure for determination of amphetamines and related phenethylamines with LC-APCI-MS or DAD. The drugs were extracted with ether and subjected to derivatization with phenylisothiocyanate in order to enhance the sensitivity of DAD. In the meantime, we have developed a common solid-phase extraction procedure for isolation of basic drugs of abuse, which has been applied for LC-APCI-MS detection of opiate agonists, cocaine and metabolites, flunitrazepam and metabolites, and LSD in biosamples (24-27).

The purpose of the present paper was to use the same isolation

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Journal of Analytical Toxicology, Vol. 24, March 2000

procedure for the determination of amphetamines and related phenethylamines with LC-APCI-MS without any derivatization. The use of a standardized extraction procedure for simultaneous isolation of potentially all basic drugs of abuse may save labora- tory time and (usually limited) sample. It must be added that in April 1998, German Parliament issued a law stating that the mere presence of any defined drug of abuse in the blood of a driver is equivalent to impairment. The actual list of drugs included tetrahydrocannabinol (THC), morphine, benzoylecgonine, amphetamine, methylenedioxymethamphetamine (MDMA), and methylenedioxyethylamphetamine (MDEA). The following concentrations of drugs were suggested as unequivocal evidence for their presence in blood: THC, 2 IJg/L; morphine, 20 IJg/L; amphetamine, MDMA, and MDEA, 50 IJg/L; and benzoylecgonine, 150 IJg/L (28). In the available literature only some studies were found, which concerned examination of amphetamines with LC-MS in biological fluids. Verweij and Lipman (29) compared the sensitivity of detection of MDMA, methylenedioxyamphetamine (MDA), and MDA (pure drugs) using thermospray-MS, LC-APCI-MS, and LC-ESI-MS. Goff et al. (30) determined MDMA and MDA with LC-ESI-MS in blood and liver taken from rabbits after administration of MDMA, and in maggots fed the rabbit tissues. To our knowledge, no systematic study on all forensically relevant phenethylamines with LC-API-MS has been performed.

Experimental

Reagents Drug standards. Amphetamine, methamphetamine, MDMA,

MDA, MDEA, ephedrine, 1-phenylethylamine, phenylpropanolamine, phentermine, and propylhexedrine were obtained from Sigma-Aldrich (St. Louis, MO). Fenfluramine and norfenflu-

Table I. Measured Ions and Retention Times of Substances Involved*

Flow rate Substance Ions measured IS (mL/min)

Amphetamine 147, 136, 119, 91 A-d11 f 0.3 MDMA 199, 194, 163, 133 MDMA-ds 0.3 MDA 180, 163,147 A-du 0.3 MDEA 215, 208, 163 MDEA-d7 0.3 MBDB 215, 208, 177, 147 MDEA-d7 0.3 BDMPEA 260, 262,245, 215 MDEA-d7 0.4 Methamphetamine 160, 150, 119, 91 MA-dl0 0.3 Phentermine 160, 150, 133, 91 MA-dl0 0.3 Cathinone 150, 147, 132 A-d~ 0.3 Phenylethylamine 147,122, 105 A-du 0.3 Propylhexedrine 204, 156, 157 NF 0.8 Ephedrine 160, 166, 148 MA-dm 0.3 Phenylpropanolamine 152, 147, 134 A-du 0.3 Fenfluramine 232, 212, 159 BEA 0.8 Noffenfluramine 21Z 204, 187, 159 BEA 0.8 BEA 212, 204, 108, 105 NF 0.8

* Protonated molecular ions are given in bold characters, the ions of internal standards in italics. t Abbreviations: A, amphetamine, MA; methamphetamine; NF, noffenfluramine; BEA,

benzyl-1 -phenylethylamine.

ramine were supplied by Servier (Orleans, France). 2S-Cathinone by Radian (Austin, TX) and N-benzyl-l-phenylethylamine (BEA) were supplied by Fluka (Buchs, Switzerland). N-Methyl-l-(3,4- methylenedioxyphenyl)-2-butanamine (MBDB) and 4-bromo-2,5- dimethoxyphenethylamine (BDMPEA) were purified from street drug samples.

Deuterated internal drug standards. Amphetamine-dn, methamphetamine-dl0, MDEA-ds, and MDMA-d7 were obtained from High Standards Products (Inglewood, CA).

Serum validation standards. Serum samples were obtained from local blood bank were prescreened for absence of drugs with immunoassay (CEDIATM). Samples showing no reaction (zero) to amphetamines were spiked individually with the listed drugs to the concentrations of 5, 50, 100, and 500 IJg/L. The concentration of appropriate internal standard was always 50 1Jg/L. The control serum BTMF S-plus from Medichem (Steinenbronn, Germany), containing amphetamine (56 1Jg/L), MDMA (121 IJg/L), and MDEA (81 IJg/L) was used for precision and accuracy studies. This serum was also spiked with MDA to the concentration of 50 IJg/L.

Isolation procedure The method, already applied for several other basic drugs

(24-27), was used with minor modification. Solid-phase extraction (SPE) cartridges (200-rag Bond Elut C18) were supplied by Varian Analytichem (Harbor City, CA). The cartridges were rinsed with 1 mL methanol, 1 mL H20, and 2 mL of 0.01M ammonium carbonate buffer (pH 9.3) before use. Spiked serum samples or forensic blood samples were centrifuged 5 rain at 14,000 xg, and 0.2-mL to 1-mL volumes of supernatant were vortex mixed with 2 mL of 0.01M ammonium carbonate buffer (pH 9.3) and with appropriate internal standards (listed in Table I). After 10 min centrifugation at 5000 x g, 2 mL of clear supernatant was applied on the SPE cartridge and slowly passed through (approximately 5 rain). The SPE cartridge was rinsed with 2 mL of 0.01M ammo-

nium carbonate buffer (pH 9.3) and vacuum dried for 5 rain. The retained drugs were eluted with 0.5 mL methanol/0.5M acetic acid (9:1) under gravity

Rt force to standard, capped Eppendorf 1-mL (min:s) polypropylene tubes. After addition of 10 1JL of 1

mmol HCI, the eluates were dried under nitrogen, reconstituted in 100 1JL of HPLC mobile phase, and centrifuged 4 rain at 14,000 x g. Clear supernatant (5-20 IJL) was injected into LC-MS. The only modification in this procedure was that the amount of HC1 in the drying phase was doubled; during the pilot experiments it was stated that the use of 10 tJL rather than 5 IJL HC1 increased the recovery by about 20%.

3:39 4:20 3:50 5:16 5:52 6:50 4:13 4:25 2:58 3:07 3:52 3:06 2:46 7:02 4:22 4:33

HPLC The separations were performed in isocratic

conditions on Superspher 100 RP 18 column (125 mm x 3-ram i.d., 4-tJm particle size, E. Merck, Darmstadt, Germany). The mixture of acetonitrile and 50raM pH 3.0 ammonium formate buffer (25:75) was used as a mobile phase. The flow rates and the retention times observed for particular compounds are listed in Table I.

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Figure 1. Mass spectra and proposed fragmentation patterns of amphetamine (A), methamphetamine (B), MDMA (C), MDA (D), MDEA (E), BDMPEA (F), MBDB (G), and cathinone (H).

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APCI-MS A SSQ 7000 single quadrupole instrument (Finnigan MAT, San

Jose, CA) equipped with an APCI source was used in positive ionization mode. The following APCI inlet conditions were applied: sheath gas (nitrogen) pressure 70 psi, auxiliary gas (nitrogen) 20 mL/min, heated vaporizer temperature 450~ heated capillary temperature 190~ corona current 5 t~A. In order to establish the appropriate selected ion monitoring (SIM) conditions, the full- scan mass spectra of substances involved were taken in the range of 50 to 300 amu at octapole offset values of 10 V. For this examination, the loop injections of pure drugs dissolved in the mobile phase (50 ng/5 I~L), without HPLC separation, were applied.

On the basis of observed mass spectra, the procedures were written for SIM detection of particular drugs. Table I shows the ions monitored in SIM procedures for particular drugs and for respective internal standards.

Validation The validation was performed in duplicate on three different

days, using serum standards spiked to the concentrations of 5, 50,

100, and 500 IJg/L of a particular drug and stored at -20~ until extraction. Additional accuracy and precision standards, which contained amphetamine, MDMA, MDA, and MDEA, were examined three times on different days. Analytical recovery for amphetamine, MDMA, MDA, and MDEA was tested on three different days at the concentration levels of 5, 50, and 100 Vg/L and was defined as percent peak areas of corresponding amounts of a given drug spiked postextraction to blank serum extract. The recovery of other drugs was determined in the same manner on two different days. The stability of extracted drugs was tested with three samples of extracted BTMF S-plus serum. Reconstituted extracts were frozen again after analysis, stored at -20~ and reexamined 2-3 more times within 8 weeks. These extracts were stored, as usual, in capped Eppendorf tubes.

Results and Discussion

APCI mass spectra

Table II. Validation Data for Examined Drugs

LOD* Recovery f Drug (pg/L) tinearity r (%)

Amphetamine 2.0 y = 10.117x- 2.308 1.000 86 • 6 MDMA 1.O y = 4,079x + 18,931 0,9986 90 • 4 MDA 2.0 y = 26.305x + 0.775 0.9990 96 • 5 MDEA 1,0 y= 34.029x- 14.688 1.0000 87 • 5 MBDB 1,0 y = 5.367x + 1.294 1.0000 87 • 6 BDMPEA 2.0 y = 2.249x + 3.243 0.9999 86 • 7 Methamphetamine 1.0 y = 5.252x- 3.628 0,9999 82 • 2 Phentermine 1.0 y = 35.751x + 1.642 1.0000 96 + 3 Cathinone 5.0 y = 0.1095x + 8.24 0.999] 88 • 5 Phenethylamine 2.0 y = 51.648x + 1.680 0.9999 58 _* 6 Propylhexedrine 2,0 y = 59,861x + 0.401 1.0000 86 • 3 Ephedrine 1.0 y = 1t ,976x- 3.205 1,0000 58 • 6 Phenylpropanolamine 1.0 y = 10.126x- 2.969 1.0000 63 • 7 Fenfluramine 1.0 y = 8.037x + 4.881 0.9995 96 • 2 Norfenfluramine 1.0 y = 3,756x + 6.129 0.9987 90 • 3 [3EA 2.0 y = 0.11 l x - 0.663 1.0000 79 • 1

* Defined as signal-to-noise ratio of 3. * Defined as percent peak area of corresponding amounts of nonextracted drugs added to blank serum extract

and injected into LC-MS. Determined in duplicate at the concentration levels of 5, 50, and 100 pg/L on three different days (for amphetamine, MDMA, MDA, and MDEA) or on two different days (for other drugs).

Figures 1 and 2 show the mass spectra of 16 examined phenethylamines. In applied conditions, several fragments were observed beside the protonated molecular ion for all drugs but propylhexedrine. Observed fragmentation patterns allowed an unequivocal differentiation between drugs with identical molecular mass, such as MDEA and MBDB or methamphetamine, phentermine, and cathinone. Also, sympathomimetic phenethylamines used in over-the-counter prepa- rations showed quite different mass spectra than amphetamine or metharnphetamine. Figure 3 shows the mass spectra of deuterated internal standards: amphetamine-du, rnethamphetamine-dl0, MDMA-ds, and MDEA-dT.

LC-APCI-MS results Applied chromatographic conditions ensured

fast elution of examined drugs and chromatographic peaks of acceptable symmetry. The analysis time in each case did not exceed 8 min. For most of the drugs, a basic separation was achieved. Two pairs of drugs were not completely resolved: amphetamine/MDA and methamphetamine/phentermine. In the case of amphetamine and MDA, a

Table III. Validation Data for Amphetamine, MDMA, MDA, and MDEA

Amphetamine MDMA MDA MDEA Accuracy Precision Accuracy Precision

Recovery (%) (%CV) Recovery (%) (%CV)

Accuracy Precision Accuracy Precision Recovery (%) (%CV) Recovery (%) (%CV)

BTMF serum 100 4 110 3 112 4 112 9 5pg/L 87+4 102 11 86+4 103 10 97• 106 12 89_+6 96 6 50 pg/L 84+5 98 4 94• 101 3 98+7 100 5 84• 103 5 100 pg/L 86 • 7 96 6 90 • 4 97 4 92 • 2 95 7 88 + 2 95 5

* Recovery (mean • SD from three determinations); BTMF = amphetamine 56 pg/L, MDMA ]21 pg/L, MDA 50 pg/L, MDEA 81 pg/L.

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lournal of Analytical Toxicology, Vol. 24, March 2000

140

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simultaneous quantitation of both drugs is possible because these drugs have completely different mass spectra. The mass spectra of methamphetamine and phentermine show differences only in one fragment. Therefore, for simultaneous quantitation, these drugs have to be chromatographically separated. This is possible using a mobile phase containing 15% acetonitrile and 85% ammonium formate buffer. The validation results are given in the Tables II and III. For the majority of drugs, the absolute recovery of some 80-90% was noted, and the limits of detection ranged from 1.0 to 5.0 pg/L. Only in the cases of ephedrine, phenylpropanolamine, and 1-phenethylamine, the recovery was around 60%. All drugs showed linear response in the measured concentration range of 5 to 500 pg/L serum. Extracted drugs, stored in a refrigerator, were stable over at least 8 weeks (Table IV).

The method was applied for the routine determination of amphetamine, MDMA, MDA, and MDEA in forensic drug samples in one chromatographic run. For this purpose, the SIM proce-

Table IV. Concentrations of Drugs in Serum Extract Stored at-20~ and Analyzed Several Times*

Day 10 Day 22 Day 55

A (56 pg/L) 97 __. 8 - 99 -+ 7 MDMA (121 pg/L) 101 +7 9 7 + 8 100+6 MDA (50 pg/L) 98 _+ 3 91 _+ 4 101 + 5 MDEA (81 i~g/L) 104 • 4 98 _+ 2 102 _ 3

* The results are presented as percent of initial value (mean _ SD from three samples).

dure was written to monitor relevant ions (119 and 136 for amphetamine, 147 for amphetamine-du, 163 for MDA, MDMA, and MDEA, 180 for MDA, 194 for MDMA, 199 for MDMA-dg, 208 for MDEA, and 215 for MDEA-d?). Figure 4 shows the mass chromatograms of blank serum extract, serum spiked with drug standards, and casework blood serum containing amphetamine, MDMA, and MDA, respectively. In the first phase of method imple- mentation, over 40 forensic samples were examined in parallel with the present method and with the LC-APCI-MS method used previously (23). Virtually identical results were obtained with both procedures.

Hence, the LC-APCI-MS method enabled the selective and sensitive determination of all forensically relevant phenethylamines without any defivatization procedure. The "in source" fragmentation, provided by a single quadrupole instrument, was sufficient for the positive identification of all drugs examined. It seems that the single quadrupole LC-MS is a real alternative to much more expen- sive triple quadrupole LC-MS--MS instruments. The solid-phase extraction procedure allowed the isolation of all relevant amphetamines, as well all other basic drugs of abuse, in one run. This is of practical importance, particularly in Germany. The exten- sion of road traffic law regulation in this country confronted the toxicologists working in this country with the need for a rapid and selective method for the drug mentioned (i.e., morphine, benzoylecgonine, amphetamine, MDMA, MDEA, and THC). Among German authors coping with this problem, Weinmann et al. (31) developed a common solid-phase extraction method with consecutive simultaneous GC-MS quantitation of amphetamine, benzoylecgonine, morphine, and codeine in serum samples. The limit

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D

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of quantitation for amphetamine as a pentafluoropropionyl deriva- tive was about 2 pg/L. Kderstein and Sticht (32) also used GC-MS with perfluorobutyryl derivatization for confirmative determination of opiates, cocaine, benzoylecgonine, cannabinoids, and

amphetamines (amphetamine, MDMA, MDA, MDEA) in the blood of suspected drivers. Separate extraction procedures were used for particular groups of drugs. The present report shows an alternative option for the simultaneous isolation of all basic drugs mentioned

in the legal regulation with consecutive LC-APCI- MS assay without derivatization. It must be added

i" A that the extracts of drugs not subjected to defivatization were very stable when frozen, and practically

r no changes were observed in the concentrations of r drugs involved over eight weeks of storage. The

same was observed previously for other drugs of abuse (24-27).

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Conclusions

Solid-phase extraction procedure may be applied for simultaneous isolation of psychoactive phenethylamines, as well as for other alkaline drugs of abuse of forensic importance.

LC-APCI-MS (single quadrupole) allowed the differentiation of underivatized amphetamines and other forensically relevant phenethylamines with specificity and sensitivity sufficient for forensic blood examination.

References

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2. M.R. Moeller and M. Hartung. Ecstasy and related substances--serum levels in impaired drivers. J. Anal ToxicoL 21:591 (1997).

3. J.A. Henry, K.J. Jeffreys, and S. Dawling. Toxicity and deaths from 3,4-methylenedioxymethamphetamine ("ecstasy"). Lancet 340:384-387 (1992).

4. A.R.W. Forrest, J.H. Galloway, I.D. Marsh, G.A. Strachan, and J.C. Clark. A fatal overdose with 3,4- methylenedioxyamphetamine derivatives. Forensic 5ci. Int. 64:57-59 (1994).

5. C.M. Milroy, J.C. Clark, and A.R.W. Forrest. Pathology of deaths associated with "ecstasy" and "eve" misuse. J. Clin. PathoL 49:149-153 (1996).

6. V. Fineschi and A. Masti. Fatal poisoning by MDMA (ecstasy) and MDEA: a case report. Int. J. Leg. Med. 108:272-275 (1996)�9

7. W. Weinmann and M. Bohnert. Lethal monointox- ication by overdosage of MDEA. Forensic 5ci. Int. 91:90-101 (1998).

8. R.W. Taylor, S.D. Le, S. Philip, and N.C. Jain. Simultaneous identification of amphetamine, methamphetamine using solid-phase extraction and gas chromatography/nitrogen phosphorus detection or gas chromatography/mass spectrometry. J. AnaL Toxicol. 13:293-295 (1989).

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Manuscript received March 4, 1999; revision received April 28, 1999.

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