4.2.1 Extraction

Extraction of LSD from blotter paper, the most common form found in the illicit market, is a relatively simple process. Commonly, 100 μg of LSD salt are found on examination of blotter paper. Virtually all illicit preparations contain LSD and its isomer iso-LSD (See Fig. 4.2). The proportion of LSD to iso-LSD allows for the determination of source of the drug since the amount of each of these drugs can vary dramatically between batches (10–70% iso-LSD) depending on the source (illicit laboratory). Iso-LSD has no pharmacological activity, therefore sources with high proportions of iso-LSD have less pharmacological activity than those with proportionately higher LSD. In addition to blotter paper, LSD is also sold on gelatin cubes, sugar cubes, microdots (small tablets) and in liquid form in small dropper bottles. Isolation and identification of LSD from sugar cubes and the liquid form has been described by Kilmer [7] using GC-MS and high-performance liquid chromatography (HPLC). Veress described an extensive evaluation of parameters involved with the isolation of LSD from blotter paper. Solvent, time, temperature and extraction method's effects were characterized and analysis using HPLC with UV detection was evaluated [8].

Extraction of LSD from biological matrices is a far more challenging task due to the inherent low concentrations of the drug. Even with the sensitivity associated with selection ion monitoring MS, analysis of LSD is difficult and commonly accomplished using elaborate extraction schemes. Extraction of the drug from blood, plasma and tissue homogenates requires some different handling than that used for urine in order to afford the elimination of cells and proteins is important in these cases. Numerous approaches have been used for this problem over the years, and any of the currently available methods can be employed successfully with LSD. Plasma often only requires dilution with a buffer to prevent plugging of solid-phase extraction cartridges. Methods of cold precipitation, salting out of proteins and addition of solvents such as acetonitrile have also been used as a preliminary step in the extraction of drugs, including LSD, from these sample matrices. Once this is accomplished, the extracts are typically handled the same as urine.

The concentrations found in routine analysis of LSD pose a challenge for benchtop GC-MS systems. As a result, extraction becomes more critical to ensure high recovery along with a very high level of selectivity to the analyte. One method for the extraction of LSD from urine involves a combination of liquid- and solid-phase extractions [9]. Urine samples were made basic with ammonium hydroxide, saturated with sodium chloride and extracted into 1-chlorobutane. Following evaporation, the extract was dissolved in isooctane:methylene chloride:triethylamine (50:50:0.1) and poured through solid-phase extraction columns and eluted with methanol:methylene chloride:triethylamine (0.2:10:0.01). The extract from the column was then dried and reconstituted in 1-chlorobutane and the alkaloids extracted with 3 mL of phosphate buffer. This extract was then washed with 1-chlorobutane followed by making the solution basic with ammonium hydroxide and saturating with sodium chloride. The drugs of interest were then extracted using 1-chlorobutane. This extract was evaporated then reconstituted in ethanol containing triethylamine. The authors felt triethylamine helped to recover the LSD from the glass tube by displacing the LSD that had bound to glass surfaces. The extract was then derivatized with BSTFA and analyzed by GC-MS. This extraction procedure is extensive and laborious. It was the author's belief that to reliably conduct routine analysis of LSD using standard benchtop GC-MS systems required this degree of clean-up to ensure the drugs were efficiently recovered (69%) and potential interferences eliminated from the sample extract.

This method also included an additional step to convert iso-LSD, a common contaminant of illicit LSD, into LSD using ethanolic sodium hydroxide and heat (50°C for 10 min). This process converted iso-LSD into LSD with approximately 98% efficiency, thus effectively combining both of the compounds into one single peak. This allowed assessment of the total amount of LSD, in either isomeric form, in the sample since neither of these drugs is naturally occurring nor have any legitimate source. Although at its surface a reasonable procedure, it has not been adopted by the forensic community.

One procedure for the analysis of LSD in serum involved liquid–liquid extraction. After making the sample basic, 1-chlorobutane was used to extract the LSD. The solvent was then transferred to a silanized glass vial and evaporated under nitrogen. The dried extract was dissolved in methanol then injected into GC using flame ionization detection. Some samples showed interference when using this simple extraction procedure. In such cases, the extraction scheme was expanded to further clean up the extract. This was accomplished by taking the dried extract and redissolving in phosphate buffer and washing with 1-chlorobutane and cyclohexane (1:1). The LSD was then extracted from the buffer using 1-chlorobutane. Following extraction, the extract was derivatized with MSTFA with pyridine and analyzed by electron ionization GC-MS. The extraction efficiency of this method was reported to be 76% for the single-step extraction and 66% for the more extensive procedure. This method was reported to be linear from 100 pg/mL to 10 ng/mL [10].

An automated procedure for the extraction of LSD and nor-LSD from blood, serum, plasma and urine has also been described [11]. This method resulted in recovery of LSD at 95% or above at concentrations of 0.1–5 ng/mL. Within-run precision of the assay was less than 3% and between-run was less than 10%. The reproducibility of nor-LSD by this method was not as good as for LSD and the method was therefore only used for qualitative identification of nor-LSD. Detection limits using liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) following this extraction procedure were 25 pg/mL for both LSD and nor-LSD and quantitative limits were 50 pg/mL for both drugs.

The extraction of LSD and nor-LSD from hair has been reported by Nakahara et al.[12]. The hair was first washed using sodium dodecyl sulfate (SDS) and dried in a desiccator. A solution (2 mL) of methanol:5 M HCl (20:1) was added to samples (20 mg) of hair that were placed into an ultrasonicator for 1 h then stored at room temperature for 14 h. The samples were filtered, neutralized with ammonium hydroxide and evaporated. Purification was accomplished by extracting the drug from 0.1 M sodium hydroxide using dichloromethane. The extract was then derivatized and analyzed by GC-MS using both deuterated LSD and lysergic acid methyl propylamide (LAMPA) as internal standards.

Immunoaffinity extraction of compounds found in very low concentrations in biological matrices is an appealing method for several reasons. The specificity of the antibody to the compound of interest allows for isolation of only those compounds that bind to the antibody. The extraction specificity is thus directly related to the specificity of the antibody to the drug of interest. Unlike other methods that extract compounds that share similar physical and chemical properties, this process will extract only those compounds that bind to the antibody. Several reports of using immunoaffinity extraction have been published. Francis and Craston [13] developed a method for immunoaffinity extraction of LSD. Analysis of the extract by HPLC and LC-MS using electrospray ionization gave detection limits of approximately 500 pg/mL. Although a viable technique, the detection limit for this method was higher than would be considered desirable in most cases. (The previous US Department of Defense cutoff for LSD was 200 pg/mL which required a control at not more than 100 pg/mL.) A similar immunoaffinity extraction procedure has been described [14] that employed electrospray ionization LC-MS. The detection limit reported for that method was also 500 pg/mL. Immunoaffinity extraction was also described by Henion and coworkers [15,16]. The process identified LSD and a number of metabolites in a unique on-line extraction process. This method offered a significant advantage since the extraction was on-line but the distinct disadvantage of the antibody not being covalently bonded to the support material. Therefore, when the drug was eluted from the column, so too was the antibody necessitating generation of a new column to analyze each sample. This method was useful for the isolation of the drug of interest and several metabolites from a relatively large volume of urine. It also allowed isolation of metabolites from the samples in sufficient amounts to allow characterization of the metabolic products. An immunochemical method for the isolation of LSD has also been reported by Kerrigan and Brooks [17]. Recoveries of the LSD were reported to be greater than 80%. The method worked well with both blood and urine samples. Use of this method with blood samples gave excellent results. No special preparative procedures were required with blood samples, they were simply diluted with buffer and applied to the resin.

A commercially available immunoaffinity resin is available that has been used for the analysis of LSD in urine samples (LSD ImmunElute, Microgenics, Pleasanton, CA, USA). This allows for the analysis of LSD and several metabolites without requiring investigators to prepare their own immunoaffinity resin for the purpose of extraction.

As seen from the examples above, the extraction of LSD from biological matrices is a complex process and in many ways dependent on the analytical method being used.