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Technical Briefs |
1 Institut für Rechtsmedizin und Verkehrsmedizin der Universität Heidelberg, Vossstrasse 2, D-69115 Heidelberg, Germany;
2
Institut für Rechtsmedizin der Universität Mainz, Am Pulverturm 3, D-55131 Mainz, Germany
aaddress correspondence to this author at: Institute of Legal Medicine, Vossstrasse 2, 69115 Heidelberg, Germany; fax 49-6221-565252, e-mail gisela_skopp{at}med.uni-heidelberg.de
Lysergic acid diethylamide (LSD) is one of the most potent hallucinogenic agents known. Recently, data on emergency department episodes related to the use of drugs commonly thought as "club drugs" have also included LSD (1). Confirmation of LSD use by testing biological fluids is still an analytical challenge because of its extensive, rapid metabolism and its instability (2)(3)(4). After ingestion of a typical street dose (40–120 µg), the concentration of LSD in urine falls to <1 µg/L within a few hours (2)(5)(6). Recently, N-desmethyl-LSD (nor-LSD) and 2-oxo-3-hydroxy-LSD (O-H-LSD) have been identified as LSD metabolites in human urine (7)(8). Measured nor-LSD concentrations were reported to be in the same concentration range as LSD, whereas the measured concentrations of O-H-LSD were severalfold higher (0.02–21.4 µg/L) (7)(9)(10)(11). The application of liquid chromatography–tandem mass spectrometry (LC-MS/MS) improved the detection of LSD use (9)(10)(12)(13)(14)(15); the method is also less susceptible to interferences when used for drug screening (16)(17). In a few studies, O-H-LSD and nor-LSD have also been measured (6)(7)(8)(10)(12)(13)(14)(15). LSD has been shown to rapidly decompose in urine samples exposed to an increased temperature or to sunlight or ultraviolet light (18)(19)(20)(21), but stability data on major LSD metabolites are not yet available.
This study was undertaken to determine the stability of LSD, O-H-LSD, and nor-LSD in urine under different storage conditions by LC-MS/MS. Data on the reaction order type and major influencing factors may be useful in planning transport and storage of urine samples.
Stability was assessed by enriching a drug-free urine sample from a healthy volunteer (author G. Skopp; urine pH 6.5; creatinine, 0.86 g/L) with LSD, O-H-LSD, and nor-LSD (final concentration of each compound, 5 µg/L), using 1.0 or 0.1 g/L stock solutions of each compound in acetonitrile (Cerilliant). Aliquots (2.5 mL; crimp top vials type I glass A with aluminum seals and Teflon liners) with added analytes were stored at -20, 4, and 22 °C up to 7 days protected from light. Two additional series were investigated: samples stored at 40 °C protected from light and at 22 °C exposed to natural light behind window glass for 3 days. Experiments were performed in triplicate, and each sample was measured twice by LC-MS/MS using lysergic acid methylpropylamide (0.1 g/L in acetonitrile; Cerilliant) as the internal standard. Results given are mean values (n = 6).
To establish an efficient method for the simultaneous extraction of the target analytes, in a preliminary experiment we compared a liquid–liquid (n-butyl chloride; Merck), an immunoaffinity (LSD Immunelute; Microgenics), and a solid-phase (Bond Elut Certify, 300 mg; Varian) extraction method after assessment of linearity of the particular calibration curves [calibrators: 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 µg/L and a blank; correlation coefficient (r) >0.99]. Extractions were carried out with room lighting dimmed, and all stock and calibration solutions, which were prepared by diluting the commercial calibrators with acetonitrile, were kept in amber glass vials. We assessed drug stability during sample processing by analyzing three urine samples enriched with each analyte at 5 µg/L. Ion suppression was not be observed by MS-MS down to the lower limit of detection.
Liquid–liquid extraction failed to extract O-H-LSD, and the slope of the calibration curve was lower for immunoaffinity extraction compared with solid-phase extraction (0.018 vs 0.076). The solid-phase extraction method gave the most satisfactory results, and we tested its suitability by analyzing five authentic urine samples from emergency department cases that had tested positive when screened by an immunoassay (LSD CEDIA dau; Microgenics; cutoff value, 0.05 µg/L). Positivity was confirmed, and the concentrations of LSD, O-H-LSD, and nor-LSD were 0–0.81, 0.10 to >10, and 0–1.19 µg/L, respectively, with nor-LSD being present in two of the five specimens.
Briefly, we added 10 µL of lysergic acid methylpropylamide (0.2 mg/L solution) and 2.0 mL of 0.1 mol/L phosphate buffer (pH 6.0) to 1.0 mL of urine. Extraction columns were conditioned with 3 mL of methanol, 3 mL of water, and 3 mL of phosphate buffer (pH 6.0). After the samples were passed through by gravity, the columns were washed with 1 mL of 0.01 mol/L phosphate buffer, pH 6.0. After the addition of 40 µL of acetone, the sorbent was dried for 30 min under reduced pressure (80 mmHg). The analytes were eluted by two 1.0-mL volumes of dichloromethane–isopropanol–ammonium hydroxide (8:20:2 by volume). After evaporation of the eluate under nitrogen at 35 °C and reconstitution of the residue with the mobile phase, 10 µL was injected into the LC-MS/MS system.
Analysis was performed on an API 365 mass spectrometer (Applied Biosystems) with a TurboIon ionization source operated in the positive-ion mode. The device was interfaced to a quaternary HPLC pump equipped with an autosampler (series 200; Perkin-Elmer). The samples were eluted from a Zorbax Eclipse XDB C8 column (2.1 x 150 mm; particle size, 5 µm; Agilent) with acetonitrile–methanol–20 mmol/L ammonium acetate buffer, pH 6.0 (33:33:34 by volume), as the mobile phase at a flow rate of 250 µL/min.
LC-MS/MS data were recorded in the multiple-reaction monitoring mode (LSD, m/z 324
223; O-H-LSD, m/z 356237; nor-LSD, m/z 310209; lysergic acid methylpropylamide, m/z 324223) with an Apple Macintosh G3 Power PC with Masschrom 1.1.1 software (Applied Biosystems). The limits of detection and quantification were determined according to DIN 32645 at a probability of 90% (22). The absolute recovery was established by comparing the peak areas of extracted calibrators (n = 4) with the peak areas of calibrators injected directly into the LC-MS/MS system. Accuracy was tested by repeated extractions (n = 5) at three concentrations (0.5, 1.0, and 5.0 µg/L) of each analyte in urine. Precision was assessed by analyzing enriched urine samples containing each analyte at 0.5, 1.0, or 5.0 µg/L (n = 5) on 5 different days. The evaluation data are summarized in Table 1
.
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From the interday precision data, we chose a mean CV value of 6% to establish the stability of the analytes. Values were not considered different as long as the difference between the initial concentration and the currently measured concentration did not exceed the critical difference (d): d = 2 · 
· SD = 17% (23). All analytes were stable in samples stored at -20 °C during the whole observation period, whereas at a higher temperature, the concentrations of LSD, O-H-LSD, and nor-LSD steadily decreased. LSD was stable in urine samples stored at 4 and 22 °C for 3 days protected from light. This finding is in accordance with results reported in the literature (3)(4)(9)(20).
The reaction kinetics were investigated by graphic representation, and apparent first-order reaction kinetics could be established for all samples stored protected from light (Table 1
). A plot of the natural logarithm of the particular rate constants determined at different temperatures against the reciprocal of the temperature in degrees Kelvin gave an Arrhenius relationship (24), which clearly demonstrated the temperature dependence of LSD, O-H-LSD, and nor-LSD degradation. The mechanism of the thermal degradation of LSD is not yet fully understood (8).
In samples exposed to natural sunlight behind window glass, the concentrations decreased very rapidly. A linear correlation relating the natural logarithm of the drug concentration to its time of storage could not be obtained for these samples, unlike the specimens protected from light. Already after 1 day of storage, the concentration of LSD decreased to 3% of its initial concentration, whereas the concentrations of nor-LSD and O-H-LSD were 21% and 69%, respectively, of their starting concentrations. In samples stored for 3 days, LSD was no longer detectable, and the concentrations of nor-LSD and O-H-LSD had decreased to 3% and 6% of their initial value. When irradiated under ultraviolet light, LSD undergoes rapid catalytic hydration at the C-9,10 double bond (25). The most interesting finding was that both metabolites were less susceptible to photodegradation than LSD itself. O-H-LSD was far more stable than nor-LSD, suggesting further interaction at the C-1,2 double bond.
As the most important result, these data clearly revealed the higher stability of LSD metabolites compared with the parent drug under the same storage conditions. Considering in addition that the concentration of O-H-LSD was always higher compared with those of LSD and nor-LSD and that the half-life of nor-LSD has been reported to be 
10 h, in contrast to 3.6 h for LSD (2)(15), simultaneous analysis of LSD and its metabolites offers a longer time period for detecting LSD use than does analysis of LSD as the sole analyte; simultaneous analysis may also improve the detection of LSD use even if thermal or light-induced degradation of LSD has occurred.
Generally, the simultaneous determination of a parent drug and major metabolites offers advanced aspects of interpretation in drug analysis and improves analytical plausibility. This implies the need to establish appropriate analytical methods and stability data. The higher concentration of O-H-LSD as well as the increased detection time and the higher stability of both metabolites compared with the parent drug as a traditional marker strongly suggests that O-H-LSD and nor-LSD be included in future confirmation protocols. The present investigation also highlights the importance of providing customers with information about the need to protect clinical and forensic urine samples to be analyzed for LSD from light and to transport them rapidly.
References
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