Analysis of Amphetamines and Synthetic Cathinones in Hair Samples Using LC–Tandem Mass Spectrometry and Solid-Phase Extraction

In this study, the procedure for analyzing amphetamines and synthetic cathinones (also known as "bath salts") in hair samples using a mixed-mode solid-phase extraction (SPE) is described. Samples of hair were digested with a dilute solution of base (containing internal standards), neutralized, and diluted with an aqueous phosphate buffer (pH 6). Each sample was applied to a conditioned SPE column, after which the sorbent was rinsed with deionized water, acetic acid, and methanol. After drying, the analytes were eluted and collected from the SPE column with 3 mL of an elution solvent consisting of methylene chloride–isopropanol–ammonium hydroxide. To the eluate, 200 µL of mobile phase was added and the samples were evaporated to the mobile phase for analysis by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Chromatography was performed in gradient mode using a C18 column and a mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. The total run time for each analysis was under 5 min.

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Amphetamine (α-methylphen-ethylamine) (Figure 1) belongs to a class of compounds known as sympathiomimetic amines (1). This class of drugs includes the illicit drugs methamphetamine, methylenedioxyamphetamine (MDA), and methylenedioxymethamphetamine (MDMA), as well as ephedrine and pseudoephedrine, which can be found in over-the-counter medications. Amphetamine is administered as a prescription medication (for example, Adderall [Shire Pharmaceuticals]) for treating medical issues such as narcolepsy, obesity, or hypotension (2), whereas methamphetamine, MDA, and MDMA are considered controlled substances — that is, pharmaceuticals with little or no medical use.

Figure 1: Structure of amphetamine.

Synthetic cathinones are derived structurally from the parent compound (Figure 2) and have become noticeable in the scientific literature in recent times because of the fatalities arising from administration (3–6). The drugs are commonly referred to as "bath salts" because they were originally packaged with names such as "Ivory Wave" and marketed as "not for human consumption" or "research chemicals". These drugs are now scheduled in the same way as other controlled substances.

Figure 2: Structure of cathinone.

The popularity of amphetamines is because of their euphoria effect and ease of synthesis. Their use or abuse is generally verified by the analysis of biological samples, such as urine, blood, oral fluid, or hair. Of these samples, hair is a biological matrix that has been used as an alternative to urine or blood for drug testing because it allows noninvasive sampling and can document the use of the drugs over a longer period of time than blood or urine (7). In this study, amphetamine in the form of Adderall was determined in the hair of a subject along with several other amphetamines and a range of synthetic cathinones. Amphetamine is deactivated during metabolism in the human system, undergoing deamination to form phenylacetone, which is converted to benzoic acid and excreted in a conjugated form (2). A small amount of the parent is oxidized to norephedrine, which is also metabolized to the parahydroxylated forms of this compound, all of which is pharmacologically active and are thought to contribute to the effects of the drug (8,9). The therapeutic, toxic, and fatal concentrations of amphetamines in samples such as blood and urine are well documented (2,10), but not so much for the synthetic cathinones because of the recent nature of their abuse. Most of the published studies on hair analysis have been performed in the area of work place drug testing and drugs or driving cases (11,12), not postmortem studies.

Gas chromatography coupled to mass spectrometry (GC–MS) has been reported as a technique for quantifying amphetamines in hair (13). GC–MS analysis of amphetamines requires derivatization using compounds such as heptafluorobutyric anhydride (HFAA) or pentafluoropropionic anhydride (PFAA) (14,15). Because amphetamine exists as a d–l isomeric pair, some laboratories have used chiral modification to separate the isomers in samples such as hair (16). The ratio of the isomeric forms may indicate whether or not the amphetamine has been taken legally. GC–MS analysis of the cathinones used similar fluoroacyl derivatives (3). Liquid chromatography coupled to tandem mass spectrometry (LC–MS–MS) is gaining popularity for analysis in this matrix (17,18). The use of solid-phase extraction (SPE) described in this article uses the LC mobile phase as a keeper solvent for amphetamine, reducing its volatility. In previous methods the addition of methanolic hydrochloric acid or a solvent such as dimethylformamide has been reported, and the solvent was evaporated to dryness (19). The addition of the mobile phase presents the LC–MS–MS with a more amenable analytical solvent. SPE has been reported in the analysis of hair samples previously (20–22), but not using this type of keeper solution format.

Experimental

Chemicals and Reagents: Amphetamine, amphetamine-d₅, methamphetamine, methamphetamine-d₅, MDA, MDA-d₅, MDMA, MDMA-d₅, butylone, ethylone, flephedrone, mephedrone, mephedrone-d₅, methylone, methedrone, methcathinone (4-MEC), methylenedioxypyravalerone (MDPV), and pyravalerone were obtained from Lipomed as 1-mg/mL methanolic solutions. Acetonitrile, acetic acid (glacial), concentrated ammonium hydroxide solution (32% by volume), formic acid, isopropanol, methanol, and methylene chloride were obtained from Fisher Scientific. The SPE columns (CSDAU206) were obtained from UCT Inc. Deionized (DI) water was laboratory grade, and it was generated in the Massachusetts State Police Crime Laboratory (MSPCL). All chemicals were of ACS grade.

Acetic acid was prepared as a 0.1 M solution by diluting glacial acetic acid (5.8 mL to 500 mL) and then increasing the volume to 1 L by adding DI water and mixing well. Formic acid was prepared as a 0.1% (v/v) solution by adding 1 mL of the acid to 900 mL of DI water and diluting it to 1 L (mobile-phase A). Acetonitrile containing 0.1% formic acid (v/v) was prepared by adding 1 mL of formic acid to 900 mL of acetonitrile and diluting to 1 L (mobile-phase B). Aqueous sodium hydroxide was prepared as a 0.1 M solution by adding 4 g of the solid to 500 mL of DI water and dissolving before diluting to 1 L. Phosphate buffer (pH 6, 0.1 M) was purchased from Fisher Scientific as a ready-to-use solution.

Chromatographic Analysis: Analysis was performed using an API 3200 Q-Trap instrument supplied by Applied Biosystems. The chromatographic system consisted of a Shimadzu CBM 20 A controller, two Shimadzu LC 20 AD pumps (including a degasser), a Shimadzu SIL 20 AC autosampler, and a Shimadzu CTO AC oven (set at 10 °C). The instrument was fitted with a 50 mm × 2 mm, 5-µm d_p Cadenza-C18 column from Imtakt USA (formerly Silvertone Sciences) that was attached to a 5 mm × 2 mm Cadenza-C18 guard column obtained from the same supplier. The LC column oven was maintained at 40 °C throughout the analyses. The injection volume was 10 µL. The mobile phase was delivered at a flow rate of 0.5 mL/min. The mobile-phase gradient program started at 5% mobile-phase B, rose to 90% B in 4.0 min, and then returned to 5.0% B. The instrument was readied for reinjection after 5.1 min.

Mass spectrometry was performed using positive multiple reaction monitoring (MRM). The mass spectrometer conditions for each of the amphetamines and synthetic cathinones are shown in Table 1. Tandem MS was performed using the following conditions: curtain gas setting, 15; collision gas setting, medium; ion spray voltage setting, 5000 V; temperature setting, 650 °C; ion source gas 1 setting, 50; ion source gas 2 setting, 50. The tandem mass spectrometer conditions are shown in Table 1. The analytical data were collected using Analyst software version 1.5.2 supplied by Applied Biosystems.

Table 1: Tandem mass spectrometry conditions.

Sample Preparation for Analysis:

Calibrators and Controls: A solution of amphetamines and synthetic cathinones (amphetamine, methamphetamine, MDA, MDMA, butylone, ethylone, flephedrone, mephedrone, methylone, methedrone, methcathinone [4-MEC], methylenedioxypyravalerone [MDPV], and pyravalerone) was prepared at a concentration of 1 µg/mL by the dilution of 10 µL of stock solution with acetonitrile to 10 mL in a volumetric flask. A 1-µg/mL solution of the internal standards (amphetamine-d₅, methamphetamine-d₅, MDMA-d₅, MDA-d₅, and mephedrone-d₃) was prepared by diluting 100 µL of the stock solution (100 µg/mL) to 10 mL with acetonitrile in a volumetric flask. The choice of internal standard was based on the fact that deuterated analogues of amphetamines and synthetic cathinones would not only chromatograph in a very similar mode to the drugs themselves, but would also be extracted via SPE as efficiently as amphetamine and the synthetic cathinones and would not likely be observed in the samples under analysis.

Calibrators were prepared by the addition of 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 ng of amphetamines and synthetic cathinones into 10-mg samples of drug-free hair samples. The hair samples had been previously decontaminated by washing two times with methylene chloride (10 mL) and two times with DI water (10 mL) before being air dried for 24 h. Each wash step was analyzed for the presence of drugs and found to be negative. Next, 50 ng of the internal standard was added to these samples. Control samples were prepared by the addition of 2 ng and 8 ng of amphetamine and synthetic cathinones to drug-free hair samples in addition to 50 ng of the internal standard. All determinations were performed in duplicate. A negative control sample was prepared by the addition of only the internal standard (50 ng) to a sample of drug-free hair samples (10 mg). Calibrators, control samples, and test samples were treated in an identical mode with regard to sample extraction.

To assess the performance of the procedure, calibration curves were constructed twice daily over five consecutive days using the spiked controls, and from these data intraday and interday values were obtained.

Sample Extraction: A 1-mL volume of 0.1 M aqueous sodium hydroxide solution was added to each sample (calibrator, control, and test) in a clean glass tube (75 mm × 12 mm) that was then capped. The tubes and contents were digested for 30 min at room temperature. Then, 4 mL of 0.1 M phosphate buffer (pH 6) was added to the solution, which was mixed and centrifuged at 3000 rpm for 10 min before it was applied to the SPE column.

Solid-Phase Extraction: SPE columns were conditioned by the sequential addition of 3 mL of methanol, 3 mL of DI water, and 1 mL of 0.1 M phosphate buffer (pH 6). Each liquid was allowed to percolate through the sorbent using gravity without allowing the sorbent to dry out between steps.

Following the passage of the methanol, DI water, and 0.1 M phosphate buffer (pH 6) through the SPE columns, each diluted sample (that is, calibrator, control, and case item) was loaded on to an individually marked SPE tube and allowed to pass through the sorbent using gravitational flow. The columns were then washed with sequential additions of 3 mL of DI water, 1 mL of 0.1 M acetic acid, and 3 mL of methanol. The SPE columns were then dried by applying a vacuum to the SPE manifold at 15 in. of mercury pressure using an electric vacuum pump.

The analytes were eluted from the SPE columns by the addition of 3 mL of a 78:20:2 methylene chloride–isopropanol–ammonium hydroxide solution. This solution was prepared daily by adding 2 mL of concentrated ammonium hydroxide solution to 20 mL of isopropanol and mixing well. Then, 78 mL of methylene chloride was added to this solution and the resultant solution was transferred to a clean screw-top bottle for use. A screw-top bottle ensures that the basicity of the solution remains high by eliminating any loss of ammonia from the bottle. The elution solvent was allowed to flow through the SPE sorbent with the aid of gravity and collected in separate glass tubes (75 mm × 12 mm). Glass tubes were chosen because they are standard laboratory materials within this toxicology laboratory.

Following elution, 200 µL of a solution containing 95% of mobile-phase A and 5% of mobile-phase B was added to each sample tube. The tubes were vortex mixed for approximately 1 min before the eluates were evaporated to the mobile phase using a gentle stream of nitrogen at 35 °C. After further vortex mixing, the samples were transferred to an autosampler vial (2 mL) containing a low-volume insert (250 µL) and the vial was capped for analysis.

Matrix Effects: Studies of the matrix effects were performed according to a previously published procedure (23). In this part of the study, aliquots of amphetamine and synthetic cathinones (covering the linear range) were introduced into 200 µL of a solution containing 95% of mobile-phase A and 5% mobile-phase B. Each elution solution was evaporated to remove the organic component until only the mobile phase remained and then they were analyzed by LC–MS–MS (analysis A). Concurrently, a set of hair samples were subjected to the SPE process; after elution of the analytes from the SPE columns, the elution solvent was spiked with amphetamine, and 200 µL of mobile phase (95:5 A–B) was added, and then the solution was evaporated to the mobile phase (analysis B). A second set of hair samples was spiked with amphetamine and synthetic cathinones and processed via the SPE method. After elution, 200 µL of mobile phase was added and after vortex mixing, the solution was evaporated to the mobile phase (analysis C). The data (peak areas) for analyses A, B, and C were collected by the data analysis software. By comparing the peak areas of analysis B with those of A, an assessment of matrix effects was made. The comparison of peak areas for C with B provided data for the recoveries.

Amphetamine and synthetic cathinone solution (concentration: 50 ng/mg) was infused into the tandem mass spectrometer using the on-board syringe pump (controlled by the data analysis software) via a Hamilton syringe (model 1001TLL, 1 mL volume, supplied by Fisher Scientific) at a flow rate of 5 µL/min. At the same time as the amphetamine solution was flowing into the mass spectrometer, a 10-µL aliquot of the SPE-extracted hair matrix (samples of hair confirmed to contain no drug material) was injected using the autosampler syringe on the Shimadzu liquid chromatograph. The liquid chromatograph and mass spectrometer were arranged so that samples from the liquid chromatograph were mixed into the flow of amphetamine and synthetic cathinones via a three-port tee section before the total flow entered the mass spectrometer. Any suppression effects on the amphetamine could be monitored at the MRM step for the noted drugs.

Selectivity: When analyzing samples of hair extracts via SPE and LC–MS–MS it is essential to ensure that the interfering effects of other drug compounds can be eliminated. In this procedure, samples of hair extracts were spiked with 49 drugs at a concentration equivalent to 100 ng/mg of hair sample: (bupropion, lidocaine, methadone, amitriptyline, nortriptyline, thioridazine, trazodone, mesoridazine, meperidine, diphenhydramine, phenyltoloxamine, imipramine, desipramine, benztropine, trimethoprim, diltiazem, haloperidol, strychnine, morphine, codeine, 6-acetylmorphine, oxycodone, oxymorphone, hydrocodone, noroxycodone, hydromorphone, diazepam, nordiazepam, oxazepam, temazepam, alprazolam, α-hydroxyalprazolam, lorazepam, triazolam, α-hydroxytriazolam, flunitrazepam, 7-amino-flunitrazepam, chlordiazepoxide, midazolam, α-hydroxymidazolam, flurazepam, desalkyl-flurazepam, cocaine, ecgonine methyl ester, ecgonine ethyl ester, benzoylecgonine, cocaethylene, clonazepam, and 7-amino-clonazepam) and extracted according to the SPE method. It was observed that the interfering effect of these compounds was not found to be significant.

Results and Discussion

Recovery: The mean recovery of amphetamine and synthetic cathinones from drug-free hair samples was determined to be 95% (±2%). This is an excellent indicator for the efficiency of the extraction procedure of amphetamines and synthetic cathinones using hair as a matrix. This procedure was performed twice daily over a period of five days.

Imprecision of Analysis: The results of the analysis of the spiked control samples of hair (2 ng/mg and 8 ng/mg, respectively) are shown in Table 2. Analysis of the control samples was performed at the same time as the calibration curves were constructed — that is, over a period of five days. Control samples were prepared by adding the amphetamine and synthetic cathinone solution to the hair sample (10 mg) in the digestion mixture and treating as per the test samples.

Table 2: Precision results for control hair samples shown as percent recovery.

Intraday and interday variation for the analysis of amphetamines and synthetic cathinones was found to be less than 7% and less than 10%, respectively. Ion suppression studies revealed that suppression of the monitored ions was less than 5%. This method was found to be linear (r² > 0.995) over the dynamic range 0.1–10 ng/mg.

Limit of Detection and Limit of Quantification: The limit of detection (LOD) of a particular method can be defined as the level at which the signal-to-noise ratio for the particular analyte is greater than or equal to 3:1. The limit of quantification (LOQ) for the method is the level at which the signal-to-noise ratio for a particular analyte is greater than or equal to 10:1. In this study, LOD values were determined empirically by analyzing extracted samples of drug-free hair fortified with amphetamines and synthetic cathinones by LC–MS–MS according to the SPE method. This was performed until the lowest level at which each of the respective analytes just failed the signal-to-noise ratio of 3:1. This was observed as 0.05 ng/mg. In terms of LOQ, samples of drug-free hair samples were spiked with amphetamines and synthetic cathinones at concentrations below 10 ng/mg and extracted according to the SPE procedure until the analytes just failed a signal-to-noise ratio of 10:1; this value was found to be 0.1 ng/mg. This is better than the recommendations by the Society of Hair Testing (SoHT) of 0.2 ng/mg (24). Representative chromatograms at LOQ and genuine hair sample are shown in Figures 3 and 4.

Figure 3: Chromatogram of hair analysis containing amphetamines and synthetic cathinones at the LOQ (0.1 ng/mg). See Table 1 for compound identification.

Solid-Phase Extraction: Because hair is a solid matrix, the sample requires digestion to produce a liquid that is able to flow through the SPE column. As the digest is basic, buffering to pH 6 permits both efficient flow and optimal sorbing of the drugs onto the SPE sorbent. In using a mixed-mode (C8 and strong cation-exchange chemistries), the sample can be cleaned up via aqueous acid and methanol washes, which leaves the drugs in a much cleaner state than when they were originally applied to the SPE column. This effect is noted in the low matrix effects and ion suppression values.

Figure 4: Chromatogram of genuine hair demonstrating the presence of amphetamine only (upper trace). Lower trace indicates internal standards. See Table 1 for compound identification.

In this new method, the SPE eluates are not evaporated to dryness as in typical SPE procedures, but are evaporated to a solution of mobile phase. Without the addition of methanolic hydrochloric acid to reduce their volatility, these drugs are known to be lost during this evaporation step. Using the mobile phase as a keeper solvent permits the volatile amphetamines and synthetic cathinones to be kept in solution and results in high recovery values by eliminating the loss during the evaporation.

Tandem MS: In this method, LC–MS–MS has been successfully applied to the extraction and analysis of amphetamine and synthetic cathinones rather than GC–MS in which a multistep derivatization procedure (that is, reaction with a fluoroacyl reagent such as heptafluorobutyric anhydride), evaporation, and reconstitution in a volatile solvent is required not only to quantify, but also to confirm the identity of the amphetamine or synthetic cathinone. By using LC–MS–MS with specific MRM values, amphetamine and synthetic cathinones can be targeted, confirmed, and quantified in hair samples without the use of derivatization. Coupling this procedure with a quick LC method offers analysts the ability to determine concentrations of the drug within a short turnaround time.

Concentrations of Amphetamines and Synthetic Cathinones in Genuine Hair Sample: It was observed that the donor hair sample contained only amphetamine (confirmed by a prescription of Adderall). The concentration was found to be 1.2 ng/mg. The synthetic cathinones (butylone, ethylone, flephedrone, mephedrone,methylone, methedrone, methcathinone [4-MEC], methylenedioxypyravalerone [MDPV], and pyravalerone) were not observed in the sample and neither were methamphetamine, MDA, or MDMA. Although studies were not performed on different coloured hair samples, the recoveries of the drugs on donated hair samples (grey) were notably high.

Conclusion

The use of hair samples is recognized as an alternative specimen of choice for the forensic and clinical analysis of drugs. Analysts around the world are going to be asked to test it on a routine basis. With this in mind, this SPE and LC–MS–MS procedure will offer these facilities the ability to perform a quick and efficient analysis of amphetamine and synthetic cathinones in small samples of hair. The novel use of the mobile phase as the keeper solvent serves to improve efficiency and maintain high recovery values.

Albert A. Elian and Kerrie T. Donovan are with the Massachusetts State Police Crime Laboratory in Sudbury, Massachusetts, USA.

Jeffery Hackett is with UCT Inc., in Bristol, Pennsylvania, USA.

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