LSD

Journal of Analytical Toxicology, Vol. 27, January/February2003

A Selectiveand SensitiveMethod for Quantitation of LysergicAcid Diethylamide (LSD) in Whole Blood by Gas Chromatography-IonTrap Tandem Mass Spectrometry Danielle tibong I, St~phane Bouchonnefl,*, and Ivan Ricordel2 1D~partement de Chimie des M~canismes R~actionnels, Ecole Polytechnique, Route de Saclay, 91128 Palaiseau, France and 2Laboratoire de Toxicologie de la Prefecture de Police, 2 Place Mazas, 75012 Paris, France

I Abstract I A gas chromatography-ion trap tandem massspectrometry (GC-ion trap MS-MS) method for detection and quantitation of LSD in whole blood is presented. The sample preparation process, including a solid-phaseextraction step with Bond Elut cartridges, was performed with 2 mt of whole blood. Eight rnicroliters of the purified extract was injected with a cold on-column injection

method. Positivechemical ionization was performed using acetonitrUe as reagent gas; LSD was detected in the MS-MS mode. The chromatogramsobtained from blood extracts showed the great selectivity of the method. GC-MS quantitation was performed using lysergic acid methylpropylamide as the internal standard. The responseof the MS was linear for concentrationsrangingfrom 0.02 rig/m/(detection threshold) to 10.0 ng/mL Several parameters such as the choice of the capillary column, the choice of the internal standardand that of the ionization mode (positive CI vs. El) were rationalized. Decompositionpathwaysunder both ionization modes were studied. Within-day and between-day stability were evaluated.

Introduction Illicit use of lysergic acid diethylamide (LSD) has undergone a significant increase since the early 1990s, leading to a renewal of interest in its analysis in toxicology laboratories. The detection of LSD in urine or blood samples is known to be particularly difficult. Because of its strong hallucinogenic potency, LSD is usually ingested in very small amounts (20-80 1Jg) (1). Moreover, LSD is rapidly metabolized. Its metabolism remains not totally known, but is now assumed to lead to sub9Aulhor Io whom correspondence should be addressed. E-mail address: [email protected].

24

nanogram-per-milliliter concentrations in body fluids, within a few hours after ingestion (2,3). Consequently, the detection of LSD in bio-logicalmatrices remains a verychallenging task. Because of cross-reactivity, immunological techniques (4-9) tend to be progressively replaced by chromatography-mass spectrometry techniques. Tandem mass spectrometric (MS-MS) detection greatly enhances the selectivity and sensitivity of such methods. In most cases, MS--MSis performed using triplequadrupole or ion-trap analyzers. In a very complete gas chromatography- tandem mass spectrometry (GC-MS-MS) study, Nelson and Foltz (10) showed the great efficiency of a triplequadrupole instrument in detecting very small amounts of LSD in urine and blood extracts. The authors pointed out the "strong tendency of LSD to undergo irreversible adsorption during the chromatographic process". LSD was therefore trimethylsilylated prior to chromatographic introduction. The limits of detection thus obtained were about 10 pg/mL for both urine and blood extracts. More recently, LSD detection was performed from urine extracts with a GC-ion trap MS-MS method (11). The limit of detection obtained was 20 pg/mL without derivatization of the analyte. Excellent results were also obtained with a liquid chromatography-mass spectrometry (LC-MS) apparatus. Sauvage and co-workers (12) performed an LC-MS study on blood extracts and determined a limit of quantitation of 20 pg/mL. Another study on the same apparatus led to a detection limit of 25 pg/mL from urine extracts (13). Cougnard and co-workers (14) developed an LC-ion trap MS-MS method allowing the detection of LSD in urine down to 12 pg/mL. LC-MS studies showed the most promising results with very sensitive methods requiring quite simple extraction processes (no need for derivatization of LSD), but GC-MS instruments remain rather less expensive and more widespread in analytical laboratories. Among MS, ion traps allow performing MS-MS experiments at a much lower cost than triplequadrupole instruments. The aim of this study was thus to de-

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Journal of Analytical Toxicology, Vol. 27, January/February2003

velop a selective and sensitive GC-ion trap MS-MS method for quantitation of underivatized LSD in human whole blood.

Experimental Materialsand samples LSD, LSD-d3 (the methyl group is deuterated), and lysergic acid methylpropylamide (LAMPA)were purchased from Promochem (Molsheim,France) and stored at-18~ Acetonitrile (HPLCgrade) and other chemicals were obtained from Prolabo (Fontenay-sous-Bois, France). Whole blood samples were obtained from the Laboratoire de Toxicologiede la Prefecture de Police (Paris, France). Bond Elut Certify solid-phase extraction (SPE) cartridges (mass: 130 mg, volume: 3 mL) were supplied by Varian-Chrompack (Les Ulis, France).

Samplepreparation The sample preparation process was largely inspired by the procedure describedby Nelson and Foltz (10). Five milliliters of acetonitrile was mixed with 2 mL of whole blood to which LSD had been added. The mixture was agitated for 15 rain before being centrifuged for 15 rain at 3000 rpm. The acetonitrile supematant was transferred to a clean tube and evaporated to dryness. Two milliliters of 0.1M HCI was added. The aqueous solution was then vortex mixed with 5 mL of hexane, and the upper hexane layer was discarded. The pH was adjusted to between 6.0 and 7.0 by the addition of 2.0M NaOH. In accordance with the Bond Elut Certify applications manual (15), the SPE column was conditioned by drawing through it, under vacuum, 3 mL of methanol, then 3 mL of deionized water, followed by I mL of 100mM phosphate buffer (pH 6.0). The LSD aqueous solution previously obtained was vortex mixed with 9.6 mL of 100raM phosphate buffer (pH 6.0). The sample was slowly loaded on the cartridge that was successively rinsed with 3 mL of deionized water, 1 mL of 1.0M acetic acid, and 3 mL of methanol. LSD was slowly eluted with 3 mL of a CH2Cl /isopropanol/NH4OH (78:20:2) mixture. The eluate was dried by evaporation under a N2 stream at 35~ Thirty microliters of acetonitrile was added, and the sample was vortex mixed before injection. LAMPA,the internal standard, was added to 50 pg/l~L of acetonitrite, and the sample was vortex mixed before injection. The concentration factor, from 2 mL of blood to 30 IJL of the final solution, is 66.7. The extraction recovery was determined to be about 60% (see Results and Discussion). It is to be noted that acetonitrile, because of its polarity, is not an ideal solvent for GC analysis. In toxicologylaboratories, however, only small amounts of sample are most of the time available; consequently, acetonitrile is often used to allow LC and GC studies on the same sample. Instrumentation All analysis were performed on a Varian Saturn 2000 apparatus consisting in a GC coupled with an ion-trap MS and fitted with an autosampler. GC The GC was fitted with a temperature-programmed, liquid COs-cooled 1079 injector. A 2.5-m retention gap (internal diameter: 0.53 ram) of deactivated fused silica was

pressfit into an on-column injector glass insert, up to the internal reduction. The insert was set so that the reduction end is in the upper part of the injector, allowing the syringe needle to enter the retention gap when injecting. The other end of the retention gap was pressfit to a 30-m analytical column (internal diameter: 0.25 ram, film thickness: 0.25 IJm); three columns were evaluated: a DB5-MS from J&W Scientific and a CP WAX52CB and a CP-Sil 8CB-MSfrom Chrompack. Such an assembly permits injecting large volumes of sample for trace analysis; all experiments were performed automatically injecting 8 pL of sample at a rate of 1 IJL/s. Helium was used as the carrier gas, and the flow was held constant at 1.4 mL/min with an electronic flow controller. Considering the boiling point of acetonitrile (81.6~ the injector was held at 60~ for 0.25 rain after the beginning of the injection and then raised to 300~ at 200~ where it was held for 18.55 rain. Rapid heating lead to sample concentration at the analytical column entrance which was held at 200~ during injection. The comparison with chromatograms that we had previously recorded in splitless mode shows that temperature-programmed cooled injection allows significant reducing in peak tailing for both LSD and LAMPAand greatly improves sensitivity and repeatability. The column was ramped from an initial temperature of 200~ held for 1.50 rain, increased to 220~ at 30~ then increased to 280~ at 20~ and finally increased to 300~ at 2~ where it was held for 4.83 min. The total duration of the method was 20 rain. The transfer line was maintained at 300~ to avoid condensation of LSD and LAMPAbefore entering the ion trap. MS. In the Saturn 2000 MS, molecules are ionized between the ion trap electrodes, just as they are eluted from the capillary column. The ion trap electrodes and manifold temperatures are 250~ and 70~ respectively. Tuning of the MS was automatically performed, using the ions resulting from electron ionization (EI) of perfluorotributylamine. These ions also permit to optimize the axial modulation value (3.5 V in this case). In MS-MS experiments, the electron multiplier voltage was set 300 V above the value automatically optimized for a gain of 10s. In our experiments, positive chemical ionization (CI) was performed using acetonitrile (ACN) as reagent gas. The proton affinity of acetonitrile (188.4 kcal/mol), slightly below that of isobutane (195.9 kcal/moi), allows efficient protonation of LSD and of I~,MPA by proton exchange with ACNH§ Performing chemical ionization with acetonitrile is low cost and more convenient than with usual CI gas (CH4, isobutane, and NH3); it does not require gas tank and gauge. The vapor pressure of ACN is sufficient to provide a constant flow that is adjusted with a manual needle valve. In the EI mode, the m/z 323.4 (M§ parent ions of LSD and LAMPAwere isolated within a 3-unit mass window;m/z 326.4 was isolated in the case of LSD-d3.Ions dissociated under resonant excitation; the collision-induced dissociation (CID) voltage was optimized at 0.65 V for an excitation time of 30 ms. In the CI mode, m/z 324.4 (MH§ parent ions (m/z 327.4 for LSD-d3)were also isolated within a 3-unit mass window;the CID voltage was optimized at 0.75 V for an excitation time of 30 ms. In both EI and CI modes, daughter ions were collected in the m/z 170 to m/z 290 range, with a scan rate of 0.8 s/scan. 25

Journal of Analytical Toxicology, Vol. 27, January/February 2003

Results and Discussion Capillary column selection The first step of the method development was devoted to the choice of the separative column. Three columns were tested: a polar stationary phase CP WAX52CB and two low-polarity stationary phase DB5-MS and CP-Sii 8CB-MS columns. Although LSD presents a polar structure, the CP WAX52CB column was rapidly discarded because of the intense bleeding of the stationary phase at high temperatures. Such a bleeding complicates tuning of the MS and cannot allow low thresholds for LSD detection. The choice between both low polarity columns was more difficult. As a matter of fact, at temperatures such as 300~ the DB5-MScolumn bleeding is significantly lower than that of the CP-Sil 8CB-MS column, leading, in full scan recording, to mass spectra which are less polluted by stationary phase ions. In counterpart, the CP-Sil 8CB-MS column allows a better separation of both peaks of LSD and LAMP&resulting in better accuracy when performing quantitation with LAMPA as the internal standard (especially for concentrations of LSD above 2 ng/mL in blood). Figure 1 compares the m/z 223 selected ion profiles obtained from both columns in the CIMS-MS mode. In MS--MSexperiments, the problem of column bleeding is solved since the resulting ions are not trapped in the MS. Because it best separates LSD and LAMPA,the CP-Sil 8CBMS column was retained for LSD quantitation. It is to be noted that the "ZB-5" capillary column, used by Sklerov and coworkers (11) in their method to quantitate LSD from urine extracts, seems to better separate LSD and LAMPAthan our method.

Internal standardselection

using multiple reaction monitoring (MRM)instead of MS--MS. In the MRMprocess, parent ions of LSD and LSD-d3are isolated and fragmented in turn. Daughter ions of each one are recorded in separated channels so that LSDand LSD-d3can be separately visualized and integrated. We performed MRM experiments in both EI and CI modes. In both cases, the results showed that the MRM process reduced the sensitivity by about 30% compared with MS-MS experiments. Because it allows the quantitation of LSD by MS-MS rather than by MRM, LAMPAwas chosen as the internal standard.

Ionization and fragmentations Figure 2 shows the MS-MS spectra resulting from CID on ionized LSD (Figure 2A) and on protonated LSD (Figure 2B) Spectra were recorded with LSD injected at 250 pg/mL. CID spectra of LAMPAare not displayedbecause they are almost the same as those of I,SD in both ionization modes. With the aim of interpreting decomposition pathways, MS-MS was performed on all the major daughter ions: rn/z 280, 265, 222, 221, 207, 196, and 181 in El and m/z 281,251, 223, 208, and 197 in CI. Parent ions were isolated within a I mass-to-charge ratio window and submitted to resonant excitation under several CID voltage values in the range from 0.1 to 1.0 V. MS-MS was also performed on the molecular ions of LSD-d3 in both ionization modes. The EI-MS-MS spectrum of LSD is nearly the same as the one reported by Sklerov and co-workers (11) with the same apparatus. There is only one slight difference between both EI spectra: the relative intensity of the m/z 196 ion is slightly greater in our spectrum. The m/z 223 ion is not interpreted as a daughter ion; it is assumed to result: (i) from the 13Cisotopic contribution of the m/z 222 structure and (ii) from fragmen-

Two internal standards were evaluated for LSD quantitation: LSD-d3 and LAMPA.LSD-d3cannot be separated from LSD by GC; coe]ution implies to perform detection of both compounds

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Figure 1. The m/z 223 selected ion profiles of a blood extract of LSD (2 ng/mL before extraction); LAMPA was added, after extraction, to 50 pg/pL. The selected ion profiles were recorded in the CI-MS-MS mode on the m/z 324.4 parent ion. The chromatographic separation was performed on DB5-MS capillary column (A). The chromatographic separation was performed on a CP-Sil 8CB-MS capillary column (B).

26

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ration of auto-protonated LSD which have been isolated with the molecular ion (MS-MS spectra of Figure 2 were acquired with a 3 mass-to-charge ratio isolation window). CID on daughter ions showed the following transitions: m/z 280 dissociates into m/z 265, m/z 207, and m/z 181 and m/z 222 dissociates into m/z 221 and m/z 207. Other selected ions do not dissociate into ions of the reference spectrum in Figure 2A. Structures for the main daughter ions are proposed in Figure 3. The base peak of the spectrum, m/z 222, is assumed to result from loss of diethylformarnide from the molecular ion; it fragments in turn followingeliminations of the H. and "CH3radical species to provide m/z 221 and 207, respectively.Another fragmentation pathway consists in the loss of CH3NCH2 through a retro-Diels Alder mechanism leading to m/z 280. The latter leads to m/z 265 by 'CH3 loss or to ra/z 181 by [CON(Et)CH2CH2]. loss. The m/z 196 ion results from direct elimination of diethylpropeneamide from the molecular ion, presumably via a concerted mechanism analogous to the retroDiels-Alder one. The comparison of LSD and LSD-d3 spectra shows that m/z 265 and 181 ions are not mass-shifted, whereas m/z 222, 221, and 196 ions are shifted to 225, 224, and 199, respectively. This is in good agreement with the proposed structures. The m/z 280 and 207 ions are partially mass-shifted of 3 mass units. This partial mass shift shows evidence that several competitive mechanisms are implied in the formation of these ions: those proposed in Figure 3 and at least another one implying structures for the m/z 280 and 207 ions that include the methyl group. CID of the m/z 283 ion of LSD-d3 showed the

transition m/z 283 -~ m/z 210 that is not in agreement with the structures proposed and confirms the hypothesis of a third mechanism. It is to be noted that Nelson and Foltz (10) reported similar conclusions in their CI-MS-MS study on decompositions of LSD. Whatever the ionization mode, none of the usual MS mechanisms allow to rationalize this fragmentation pathway. The CI-MS-MS spectrum of LSD provides six daughter ions that are sufficientlyabundant to be retained for identification of the compound: m/z 281,251,223, 222, 208, and 197. These ions are the protonated analogues of those displayed in the EI mode except m/z 251 that results from diethylamine elimination from the pseudomolecular ion. CID on daughter ions showed the following transitions: m/z 281 dissociates into m/z 208 and m/z 251 dissociates into m/z 223 that leads to m/z 222 and m/z 208. Other daughter ions do not fragment into ions of the reference spectrum in Figure 2B. Structures for the main daughter ions are proposed in Figure 4. It is to be noticed that two structures are proposed for the m/z 208 ion. Structure a in Figure 4 results from methyl elimination from the m/z 223 ion, and structure b in Figure 4 results from diethylamine elimination from m/z 281. The fact that the m/z 180 ion is of very low abundance in the spectrum and that no CO elimination is observed from m/z 208 is not in favor of structure b in Figure 4 because CO eliminations are assumed to be low energy-costing fragmentations. CID experiments on LSD-d3 showed that m/z 251, 223, 222, and 197 are totally mass-shifted, whereas m/z 281 and 208 ions are only partially mass shifted. These results are in good agreeO

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Figure 4. Decomposition pathways from collision-induced dissociation of protonated LSD.

27


ment with the proposed fragmentation pathways, but, as previously reported for EI-MS-MS, they show evidence that at least another mechanism is implied in the formation ofrn/z 281 and 208 ions, in competition with those proposed. This non-elucidated mechanism necessarily implies rn/z 281 and m/z 208 structures including the methyl group of LSD. The comparison of our results with those obtained by Nelson and Foltz (10), who performed CID on protonated LSD with a triple-quadrupole MS is very informative. The ion trap MS provides less fragment ions than the triple-quadrupole instrument, especially for the lower mass-to-charge ratio ions. In our CI-MS-MS spectrum, relative abundances ofm/z 192, 182, and 180 ions are less than 6%; they are 12, 17, and 54%, respectively,in the CID spectrum recorded on a triple-quadrupole apparatus. This phenomenon can be explained by two factors: (i) collisions with helium atoms are much less efficientthan those with argon atoms, presumably leading to a lower increase in internal energy of parent ions and (ii) triple-quadrupole analyzers favors secondary fragmentations because daughter ions may undergo energetic collisions during the CID process, which is not the case with ion trap MS where, in the resonant mode, daughter ions are thermalized by the cooling effect of helium (16). Both ionization modes provide MS-MS spectra that display at least five characteristic daughter ions for identification of LSD. In each case, one fragment ion is particularly abundant (m/z 222 in EI, rn/z 223 in CI) and will be thus obviously selected to quantize the analyte. It can be noticed, however, than the ElMS-MS mode provides more fragment ions with high relative abundances and is thus more "informative" than the C]-MS--MS mode. In counter part, the CI-MS-MS mode provides spectra in which the base peak (m/z 223) is more largelypredominant; this can be particularly interesting to perform sensible and accurate quantitation. Quantitation The sensitivity of the GC-MS-MS method was first evaluated apart from the sample preparation process. Ten solutions of neat LSD in acetonitrile, ranging from 1.0 to 10.0 pg/IJL,were injected in both EI-MS-MS and CI-MS-MS modes. We compared the rn/z 222 and m/z 223 selected ion profiles resulting from EI-MS/MS and CI-MS-MS experiments, respectively. At 1.0 pg/pL, the 223 selected ion profile displays a LSD peak with a signal-to-noise ratio (SIN) of 6. The major characteristic ions of LSD in CI-MS-MSare significantly above ions from noise. In EI-MS-MS, a peak with a SIN ratio of 5 can only be achieved for a concentration of 7 pg/laL. In addition, the EI-MS-MS spectra of LSD and LAMPAare significantly more contaminated than those resulting from CI-MS-MS. The CI ionization mode was consequently retained for the further steps of the method development. Six qualifying ions were retained for LSD (and LAMPA)identification:rn/z 281,251,223, 222 208, and 197; the major ion, rn/z 223, was retained for quantitation. The extraction recoveryof the sample preparation step was estimated as follows. Eight samples of whole blood spiked with neat LSD were extracted according to the described process; four replicates were spiked to 0.25 ng/mL and four to 0.50 ng/mL. According to the concentration factor, calibration was performed by injecting 10 solutions of neat LSD in acetonitrile 28

in the range from 5 pg/1JLto 1 ng/1JL.The peaks were integrated on the m/z 223 ion profile. The response was linear over the range; the correlation coefficient was 0.999. Considering the eight samples, the percent recoverywas determined between 53 and 67%. Twelve solutions of LSD-spiked whole blood were prepared with concentrations ranging from 0.02 ng/mL to 10.0 ng/mL. They were extracted following the sample preparation process described in the experimental section. LAMPAwas added to 50 pg/IJL, between extraction and injection, as the internal standard for GC-MS quantitation. LSD and LAMPApeaks were integrated on the m/z 223 ion profile The response for LSD was linear over the concentration range; the correlation coefficient was 0.995 when performing automated integration and 0.998 with manual integration. At the quantitation threshold of 0.02 ng/mL, the SIN ratio of the LSD peak is 4:1 on the m/z 223 selected ion profile. Figure 5 displays the LSD spectrum at the quantitation threshold, for comparison with the "reference" spectrum of Figure 2B (neat LSD at 250 pg/mL in acetonitrile). The spectrum at the quantitation threshold displays the six qualifying ions with relative abundances close to those of the "reference" spectrum, except for the rn/z 281 ion whose relative abundance is about three times that of the reference spectrum. The spectrum, however, allows unambiguous identification of the analyte. The protocol of within-day and between-day stability determination was inspired from that reported by Sklerov and coworkers (11). Within-day stability was evaluated by 10 successive injections of an extracted 250-pg/mL standard. The average concentration was determined at 240 • 26 pg/mL with a coefficient of variation of 10.8%. Between-day stability was evaluated by four series of five injections over a three-week period using two extracted standards at 250 pg/mL and 1.0 ng/mL, respectively.For the f~rstone, the average concentration was determined at 243 _+37 pg/mL with a coefficient of variation of 15.2%. For the second one, the average concentration was determined at 987 9 80 pg/mL with a coefficient of variation of 8.1%. The relative importance of the coefficientsof variation for both within-day and between-day stabilities is imputed to two

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Figure 5. @MS-MS spectrumof LSDat the quantitationthreshold;LSD was extracted from a whole blood sample spiked with LSD at 0.02 nglmL.

Journal of Analytical Toxicology,Vol. 27, January/February2003

factors: (i) adsorption of LSD on the capillary column and on the electrodes of the MS and (ii) the relative complexityof the sample preparation process. The first point has been reported and commented in previous studies; it is clear that LSD analysis implies regular conditioning of the column and cleaning of the MS, especiallywhen large amounts are injected (calibration of the quantitation method). The relative complexity of the sample preparation process poses the problem of its reproducibility as well as the problem of its duration (severalhours). The SPE and solvent evaporation steps are especiallytime-consuming. A simpler sample preparation process such as the one described by Sauvage and co-workers (12) in their LC-MS method should be tested. As a matter of fact, the low selectivity of a liquid-liquid extraction process without purification step could be compensated by the great selectivityof the CI-MS--MS method.

Conclusions The combination of GC and ion-trap MS-MS provides a very sensitive method for detection and quantitation of LSD. The very low quantitation threshold is rationalized by two factors. First, performing liquid injection allows injection of a large volume of sample: 8 IJL of sample is injected versus only 1 I~L in a recent study, performed on the same apparatus, with an injection temperature above the boiling point of the solvent (11). Compared with splitless injection, it is to be noticed that cold on-column injection also reduces peak tailing and the "tendency for LSD to undergo irreversible adsorption during the chromatographic process" (10). The great sensitivity of the method is also due to the combination of positive chemical ionization with MS-MS analysis. Because of its selectivity, chemical ionization leads to chromatograms that are much less contaminated than those obtained by electron ionization (interference originates from the matrix and from bleeding of the column). The MS-MS procedure eliminates the last polluting ions; it permits to increase the capacity of storage of parent ions and thus to significantlyincrease signal to noise ratios for peaks of interest.

Acknowledgments We greatly acknowledgeVarianS.A. (France) for its technical support, especially Christophe Chamard and Xavier Witz for their helpful advice about the chromatographic part, Michel Lesieur for all the precious hints about ion trap MS.

References 1. D.G. Upshall and D.G. Wailling. The determination of LSD in human plasma following oral administration. Clin. Chim. Acta 36:67-73 (1972). 2. H.K. Lira, D. Andrenyak, R Francom, R.L. Foltz, and R.T. )ones. Quantification of LSD and N-demethyI-LSD in urine by gas chromatography/resonance capture ionization massspectrometry. Anal Chem. 60:1420-1425 (1988). 3. D.I. Papac and R.L. Foltz. Measurement of lysergic acid diethylamide (LSD) in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry. J. Anal. Toxicol. 14: 189-190 (1990). 4. J.T.Cody and S. Valtier. Immunoassay analysis of lysergic acid diethylamide. J. Anal. ToxicoL 21: 459-464 (1997). 5. A.H.B. Wu, X.Y. Feng, A. Pajor, T.G. Gomet, S.S.Wong, E. Forte, and J. Brown. Detection and interpretation of lysergic acid diethylamide results by immunoassay screening of urine in various testing groups. J. Anal. ToxicoL 21:181-184 (1997). 6. N.P. Cassels and D.H. Craston. The effects of commonly used adulterants on the detection of spiked LSD by an enzyme immunoassay. 5ci. Justice 38(2): 109-117 (1998). 7. A.J. McNally, I(. Goc-Szkutnicka, Z. Li, I. Pilcher, S. Polakowski, and S.J.Salamone. An online immunoassay for LSD: comparison with GC-MS and the Abuscreen | RIA. J. Anal. Toxicol. 20: 404~,08 (1996). 8. Z. Li, K. Goc-Szkutnicka, A.J. McNally, I. Pilcher, S. Polakowski, S. Vitone, R.S. Wu, and S.J. Salamone. New synthesis and characterization of (+)-Iysergic acid diethylamide (LSD) derivatives and the development of a microparticle-based immunoassay for the detection of LSD and its metabolites. Bioconjugate Chem. 8: 896-905 (1997). 9. S. Kerrigan and D.E. Brooks. Immunochemical extraction and detection of LSD in whole blood. J. Immunol. Methods 224:11-18 (1999). 10. C.C. Nelson and R.L. Foltz. Determination of lysergic acid diethylarnide (LSD), iso-LSD and N-demethyI-LSD in body fluids by gas chromatography/tandem mass spectrometry. Anal Chem. 64:1578-1585 (1992). 1t. ].H. Sklerov, K.S. Kalasinsky, and C.A. Ehom. Detection of tysergic acid diethy]amide (LSD) in urine by gas chromatography-ion trap tandem mass spectrometry. J. Anal ToxicoL 23:474-478 (1999). 12. M.E Sauvage, R Marquet, S. Ragot, E Lachatre, J.L. Dupuy, and G. Lachatre. Determination of LSD and three of its metabolites or isomers in serum and blood samples by LC-ES-MS. Toxicorama 10(2): 73-79 (1998). 13. H. Hoja, E Marquet, P. Verneuil, H. Lotfi, J.L. Dupuy, and G. Lachatre. Determination of LSD and N-demethyI-LSD in urine by liquid chromatography coupled to electrospray ionization mass spectrometry. J. Chromatogr. B 692(2): 329-335 (1997). 14. T. Cougnard, C. Charlier, and G. Plomteux. Ultra-rapid determination of urinary LSD and nor-LSD by liquid chromatography-mass spectrometry. Toxicorama 11 (2): 99-102 (1999). 15. Bond Elut Certify Applications Book. Varian S. A., Les Ulis, France. 16. R.E. March. An introduction to quadrupole ion trap mass spectrometry. J. Mass 5pectrom. 32:351-369 (1997). Manuscript received April 5, 2001 ; revision received July 26, 2001.

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