Amphetamine and Methamphetamine in Urine by GC/MS Method

RESEARCH ARTlCLES / B1LiMSELARAŞTIRMALAR

Procedure for Determination of

Amphetamine and Methamphetamine in Urine by GC/MS Method

Yalçın DUYDU*

Procedure for Determination of Amphetamine and Methamphetamine in Uri11e by GC/MS Method Summary : in the present study a procedure for the determination of amphetamine and methamphetamine in urine by Gas Chromoıography - Mass Spectrometry (GC/MS) was presented. Amphetamine and nıethamphetamine were extracted by solid-plıase extraction (SPE) and trifluoroacetic anhydride (TF A) was used as the derivatization reagent. The derivatized extracts were analyzed by GCIMS which operated in the selected ion monitoring (SiM) mode. Deuterated analogs of the analytes were used as internal standards.

Recovery rates of 93.6% and 94.lo/o for amphetamine and methamphetamine respetcively were obtained.

Key words: Amphetamine, Methamphetamine, GC/MS

Received Revised Accepted

method 26.12.1997 26.2.1998 26.2.1998

INTRODUCTION

lllicit arnphetarnine and rnelharnphetarnine are largely derived by synthesis in clandestine la- boratories. There is a rnarked variation in !he oc- currence of arnphetarnine and ınethaınphetarnine in different parts of the world. Arnphetarnine is rnore prevalent in Europe whereas ınethaınphetamine is the more common drug in !he USA, Japan and South-East Asia. in connection wilh lheir never- ending popularity and extensive use, the analytical procedures for determining amphetamine and me- lhamphetamine in biological fluids have become rnore irnportant in recent yearsl.

Arnphetarnine is most frequently laken orally or in-

İdrarda GC/MS metodu ile amfetamin ve metamfetamin tayini

Özet : Bu çalışmada GC/MS metodu ile idrarda amfetamin ve metanıfetamin için bir tayin yöntemi sunulmuştur. Am- fetamin ve metamfetamin katı faz ekstraksiyonu (SPE) ile

ekstrakte edildikten sonra trifluoroasetik anhidrid (TFA) ile derivatize edilmiş ve derivatize edilen extract GC/MS de se- çilmiş ion tarama (SlM) kon~munda analiz edilmiştir. Bu yöntemde amfetamin için %93.6, metamfetamin için %94.1 Verim değerleri hesaplanmıştır.

Anahtar kelinıeler: Amfetamin, metamfetamin, GC/MS metodu

tranasally as the sulfate or phosphate salt, while me- thamphetarnine is most frequently prepared for in- jection or for smoking although is also available in tablet form (2,3). Following 2.5-15 rng, oral doses of

Amphetaınine peak plasrna levels of 30-170 mg/l are reached in 2 hours and plasrna elimination half lives range from 8 to 12 hours. B!ood concentrations in fatalities are normally above 500 mg/]2.3.

Arnphetamine and methamphetamine begin to ap- pear in the urine within 20 rninutes of administra- tion and both are excreted unchanged Typically 20- 30% of the adrninistrated dose of amphetarnine is excreted unchanged. The recommended target anal- ytes are, therefore, !he unchanged drugs. Benzoic acid, hippuric acid and hydroxylated rnetabolites

*

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cou!d be evalualed as !he major metabolites of am- phetamine2,4,5,6. Methamphetamine is excreted alsa as the unchanged drug over 44% of the administrat- ed dose. Amphetamine and 4-0H- methamphelamine appear as the major metabolites of metamphetamine2. The metabolic pathways of amphetamine and methamphetamine are summar- ized in Figure 1.

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Figure 1. Metabolic pathways of amphetamine and methamphetamine.

Many studies have been carried out on the de- termination of amphetamine and me- thamphetamine in recent years⁷,s,9 _ The Gas chro- matographic method (GC) is one of the most used procedures for analyzing amphetamine in biological fluids. A Gas Chromatographic method for de- termination of amphetamine in urine had been car- ried aut in our department also in 1983 and am- phetamine was detected by its N-acetyl derivatives by using FID detectorlO. in the present study we used TFA as a derivatization agent and the pro- cedures were performed by using GC/MS in com- bination with solid-phase extraction. This combina- tion provided extremely high recovery and reproducibility values.

MATERIALS AND METHODS

Chemicals and Reagents : Amphetamine, me- thamphetamine and their analog deuterated intemal standards were supplied by Radian Corporation, Austin, TX. Amphetamine-D₅ and me- thamphetamine - D₅ were used as intemal stan- dards in GC/MS. Derivatization was carried out with Trifluoroacetic Anhydride (TFA) which was supplied by Pierce. Amphetamine and me- thamphelamine were extracted using Bond Elut Cer- tify solid-phase extraction columns (Varian Sample Preparalion Products, Harbor City, CA). The other chemiça]s were supplied by Merek.

The deuterated internal standards were obtained as solutions at concentrations of 100 µg/mL Stock deu- tered internal standard solutions were prepared by diluting to give a final concentration of 20 µg/mL Amphetamine and methamphetamine standards were also obtained as solutions at concentrations of 1 mg/ml and were also diluted to give a concentra- tion of 10 µg/ml. These final solutions were used as stock standard solutions.

0.1 M Phosphate Buffer, pH 6: 13.61 g of KH2P0⁴ was added to 900 mL deionized water and dis- solved. The pH was adjusted to 6.0 with 1.0 M KOH and !he total volume was brought up to 1.0 L with deionized water.

2% Ammonium Hydroxide in Ethyl Acetate: 2 mL of concentrated ammonium hydroxide was added to 98 mL of ethylacetate and shaken vigorously far 5 min.

Instrumentaton : Analyses were performed on a Hewlett Packard Model 5890 series il Gas Chro- matograph and 5971 MSD with a 7673 autosamp!er and a 12 m x 0.2 mm x 0.33 µm film HP Ultra-1 cap- illary column. The other parameters were given be- low;

Colurnn pressure: 8.5 psi, Septum purge flow: 0.8 mL/min, Split went flow: 20 mL/min, Colurnn flow:

1.04 ml/min, Column temp. 1: 60°C (time: 0.5 rnin, in- crement: 20°C/min) Colurnn temp. 2: 220°C (time: O, increment: 40°C/ınin), Colurnn temp. 3: 275°C (time:

2 min), Injector temp.: 250°C, interface temp.: 280°C, injection mode: splitless, Dwell Time (DT): 100 rns.

Ions monitored were: amphetamine-TFA m/z 140, 118, 91; methamphetamine-TFA m/z 154, 118, 110;

amphetamine-D₅-TFA m/z 144, 123, 58; me- thamphetamine-D5-TFA m/z 158, 113. For quan- tification the ion ratios were m/z 140/144 far am- phetamine and m/z 154/158 far methamphetamine (The bold ion numbers were used far quantification).

Preparation of standards: 0.5 µg/ml, 1.0 µg/mL, 1.5 µg/ml of amphetamine and methamphetamine standards were prepared in 2 ml of drug free urine.

Ali the standards were extracted and derivatized as described in the axtraction and derivatization pro- cedure below.

Extraction procedure : 2 mi of urine (prepared above) and 2 mi 0.1 M phosphate buffer (pH: 6.0) were added to a suitable test tube. The solution was vortexed and poured on an SPE column, previously activated by !he sequential addition and elution of 2 mL of methanol and 2 mL of 0.1 M phosphate buffer (pH:6.0). The buffered urine was eluted with vacu- um. The column was washed by the addition and elution of 1 mL 1.0 M acetic acid and dried under maximum vacuum for 2 min. The analytes were eluted into a 5 mi screw capped tube by adding 2 mL of freshly prepared 2% aınmonium hydroxide in ethylacetale. The eluent was fully evaporated at 30-40°C under a slow flow of nitrogen after 20 µ! of methanol with 1 % of HCI had been added to the el- uent to prevent evaporation of the am- phetamines2·H

Derivatization: The residue of the evaporated solution was reconstituted with 200 µl of TFA. The tube was capped, vortexed and placed in a heating block at 70'C far 30 min. After cooling, 1 µ! of the derivatized solution was injected into !he GC/

Ms2.12.

Linearity : 100, 200 and 300 µ! of each analyte were added to the drug free urine (as described in prep- aration of standards) and the linearity of the ex- tracted amphetamine and methamphetamine de- termined. These urine samples were extracted and derivatized in the described manner. The ion ratios for quantitating each analyte were plotted against their respective concentrations. The plots were sub- jected to linear regression analyses (Fig 4).

Recovery Stud.ies : Drug-free urine was used to pre- pare standard solutions of 1 µg/ml of amphetamine and methamphetamine. These standard solutions were extracted in the previously described manner, with the exception !hat no internal standards were added to the urine prior to extraction. After extrac- tion and prior to derivatization, 20 µl of deuterated internal standards were added to the extracts. The derivatized extracts were analyzed under the de- scribed conditions. The area ratio of the extracted drugs to their respective internal standards was compared to the corresponding area ratio of un- extracted 1 µg amphetamine and methamphetamine standards. The recovery results are !he mean ol three determinations (Table 1).

Reproducibility studies : Reproducibilities of the analyses were determined by calculating coefficients of variation. Both amphetamines (0.1 / 0.3 µg/mL) and methamphetamine (0.1/0.3 µg/mL) containing urine standards was analyzed in 10 separate runs Table 2).

RESULTS AND DISCUSSION

in !his study !he solid-phase extraction (SPE) and the TFA derivatization lechniques were used si- multaneously in combination with !he GC/MS method. This combination is very rapid and pro- vided an excellent linearity, recovery and re- producibility far routine laboratory use. The limits of detection for amphetamine and me- thamphetamine were calculated as 15 ng/mL at our working conditions13.

Derivatization is one of the most commonly used technique for increasing !he volatility and thermal stability of the molecuJe12. The character of the molecule is changed from active to iner!. Thus the interactions between the column support and the derivatized form of the analyte are reduced to a minimum and the chromatograms exhibit well- resolved Gaussian-shaped peaks with no observable interferencesl2,14. Selected !on Monitoring (SIM) chromatograms and mass spectra of the tri- fluoroacethyl derivatives (TFA) of standards and in- ternal standards are shown in Fig. 2,3.

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The analytes were derivatised to improve their chro- matographic properties and to provide higher mass ions for measurement than underivatized analytes give (interference is lower at higher masses). The commonly used derivatives are pen- tafluoropropionyl, heptafluorobutyryl (HFB), N- trifluoroacetyl-1-prolyl chloride (TPC) and tri- fluoroacetyl (TFA). Although some publications were made in the pası concerning the lack of stabil- ity of trifluoroacetyl derivatives, TFA has been used successfully in a number of studies in recent years as a derivatization agentlZ,14. in our study we ob- served that with use of deuterated internal stan- dards, TFA derivatives were quantitatively stable

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for at least one day. Fuller et al. (1992) performed a similar study on codeine and morphine by using TFA as the derivatization agent and found that the trifluoroacetyl derivatives were stable over a period of 2 to 3 days. A siınilar study was performed by El- lerbe et al. (1993) and the stability of trifluoroacetyl derivatives of amphetamine and methamphetamine was also found to be 2 days. Our observations were in agreement with both of the studies.

Linearity : As shown in Figure 4, the "R2" values of amphetamine and methamphetamine were cal- culated as 0.9999 an 0.9974 respectively. The y- intercepts were -0.0148 for amphetamine and - 0.0804 for methamphetamine. These results pro- vided us with and adequate quantification in our working range (Fig 4).

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Recovery : The recovery rates for ampehatimine and methamphetaine were shown in Table 1. Jons- son et al! (1996) and Tatsuno et all (1996) reported 83% and 88% recovery rates respectively in their studies. Our extraction procedure provides rel- atively higher recovery rates than these studies (15,16).

Table 1. The results of the recovery studies Recovery (%)

Mean' Range

Ampehatamine 93.6 92.9 - 94.2

Methamphetamine 94. l 93.2 - 95.7

* The results are the mean of three determinations (1 µg/ml)

Reproducibility : An adequate reproducibility was provided by this method. The coefficient of vari- ation (CV) far amphetamine and methampehetaine can be seen in Table 2.

Table 2. The results of the reproducibility studies

Reproducibility

Drugs Actual ^Found~ S.D. C.V.

µg/ml µg/ml µg/ml

Amph. 0.1 0.3 0.103 0.305 0.005 0.007 4.9 2.4 (0.096-0.115) (0.291-0.316)

Methamph 0.1 0.3 0.101 0.303 0.005 0.0011 5.1 3.6 (0.094-0.116) (0285-0.319)

. • The concetrations are !he mean of ten determinations

Solid-phase extraction, which is emerging as a very important sample preparation technique, was pre- ferred to other traditional extraction procedures, such as liquid-liquid extraction (LLE). Higher re- coveries of the analytes, higher selectivity and high- ly purified extracts, reduction in organic solvent consumption and time were observed as the ad- vantages of SPE procedure over liquid/liquid ex-

traction17,ıs. Bound Elute Certify®, which we used in this study, represents a copolymeric bonded silica column. Depending on the elution fluid and pH con- dition these copolimeric bonded silica sorbents, have three different types of interactions: hy- drophobic, polar and ionic. These interactions pro- vide the selectivity, resulting in extremely clean ex- tracts leading to an improved precision of analysis17,18.

In !his method an extraction (SPE method) and de- rivatization procedure (TFA) of amphetamine and methamphetamine in urine was described. This combination was used several times to detennine urinary amphetamine and methamphetamine in many earlier studies. However some modifications, especially in eluting and derivatization procedures, were made in our study .. These modifications pro- vided us with betler recovery rates when we com- pared these with the earlier studies14,15. Con- sequently when the . linearity, recovery and reproducibility values that we obtained from our study were laken into consideration, this method can be recommended for routine laboratory use.

ACKNOWLEDGMENTS

Thls study was performed in CBFT laboratory (ltaly) and therefore 1 would like to thank S.D. Ferrara and L. Tedeschi far their technical and scientific support.

I anı also grateful to N. Vural far discussions and far critically reading the manuscript.

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marı urine by liquid chromatography - mass spec- trometry, J Ana/. Toxicol., 20, 281-286, 1996.

17. Chen X., Wijsbeek )., Van Veen )., Franke j.P., de Zeeuw R.A., Solid - Phase Extraction for the screen- ing of acidic, neutral and basic drugs in plasma using a single-column procedure on Bound Elute Certify, J,

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18. Lillsunde P., Korte T., Comprehensive drug scree!1-- ing in urine using Solid-Phase Extraction and com- bined TLC and GC/MS identification, J Anal. Tox- icol., 15, 71-81, 1991.