Validation Study of Analysis of 1-Phenyl-2-Propanone in Illicit Methamphetamine Samples by Dynamic Headspace Gas Chromatography Mass Spectrometry

A new method based on dynamic headspace sampling (DHS) coupled to GC/MS analysis was developed, optimized and validated for the analysis of 1-phenyl-2-propanone (P2P) in illicit methamphetamine (MAMP) samples. The DHS parameters were investigated to reach the sensitivity suitable for this kind of analysis. The method showed of a good specificity, linearity, accuracy, precision and robustness. The analysis of ten MAMP samples seized by the judicial authority was carried out. P2P was found in all the seizure, confirming that P2P is the starting compound of the synthesis of amphetamines.


Introduction
Amphetamine (AMP) and Methamphetamine (MAMP) are widely abused drugs all over the world [1,2]. In fact, the abuse of amphetaminelike compounds has increased continuously in the last years becoming a global problem in recent years [3]. For this reason, it is important to investigate how they are synthesized and where they are sold and diffused [4], analyzing their chemical impurity profiling. This information is useful for characterizing links between different samples originating from the same seizures, but also for determining the synthetic scheme [5,6], which gives details about the origin of the drug.
There are two major raw materials from which AMP and MAMP are prepared: ephedrine and pseudoephedrine and 1-phenyl-2-propanone (P2P). In order to prevent illicit drug manufacture, law enforcement authorities try to control illicit producers of the main drug precursor P2P (Scheme 1) [7,8].
P2P is colorless or slightly yellowish oil, with a density similar to that of water and a characteristic pleasant flavor [9]. P2P is listed in Table 1 of the United Nations Convention against Illicit Trafficking in Narcotic Drugs and Psychotropic Substances of 1988 and it is strictly controlled all over the world. It is prepared in clandestine laboratories starting from phenyl acetic acid [1]. The determination of P2P therefore is of utmost importance for determining the synthetic route by which MAMP is produced and its origin.
In a previous work we reported a method for determining P2P by static headspace gas chromatography (HS-GC/MS), which allowed us to resolve the case of a particular seizure. This finding was made of cornstarch soaked with P2P and it was sold as drug of abuse ("wet amphetamine") to deceive the consumer [10].
In this frame we were interested in applying the same method for the determination of P2P in seized methamphetamine samples, but we realized that it was not sensitive enough for this kind of analysis. So, we developed, optimized and validated a method based on dynamic headspace sampling [11][12][13][14][15][16], used for the first time in this field, which allowed us to obtain the optimal features for the analysis of impurities in illicit drug samples of MAMP. In this paper we report the results obtained from the analysis of samples containing MAMP seized by the judicial authority and delivered to our laboratory. In all these samples we detected traces of P2P as residual solvent.

Reagents and chemicals
All reagents were of analytical grade and were stored as indicated by the supplier. Propylene carbonate and acetophenone, chosen as internal standard (IS), were purchased from Sigma Aldrich (St. Louis, MO, USA). Water (18.2 MΩ·cm -1 ) was prepared by a Milli-Q System (Millipore, Darmstadt, Germany). Stock solutions of P2P (1 mg/mL) and IS (0.02 mg/mL) were prepared in propylene carbonate.

Samples and sample preparation
Ten samples seized by the judicial authority in 2014 were analyzed ( Figure 2). The samples were characterized by different content of MAMP (Table 1) and different color and crystal shape.
In a 20 mL headspace vial, 25 mg of powder were weighed and added with 1 mL of IS and 2 mL of propylene carbonate. For the preparation of working Standard Samples (WSS) in a 20 mL headspace vial, different volumes of the stock solution of P2P (see 2.1) were made up to 2 mL with propylene carbonate and 1 mL of IS was added.

Results and Discussion
Applying the conditions previously developed [10], we were able to obtain peaks with a good shape and a good chromatographic response either for P2P or for the IS (acetophenone). On the other hand, we were interested in increasing sensitivity in order to reach a LOD suitable for the analysis of impurities in drugs of abuse. To this end, we studied and optimized the DHS parameters. was added. The organic phase was washed with brine (50 mL) and a saturated solution of sodium sulfite (50 mL) and dried with anhydrous sodium sulfate; and the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography (cyclohexane/ethyl acetate, 97/3, v/v). The yield was 90%. The structure of the synthesized compound was confirmed by 1 H-nuclear magnetic resonance (NMR) spectroscopy. The NMR spectrum was recorded in DMSO-d 6

Optimization of DHS parameters
Incubation time and temperature: In the preliminary analysis [10], the incubation temperature was set at 80°C and the incubation time was 15 min. To favor the transition to the vapor phase of P2P, thus increasing extraction efficiency, we tested an increase of the incubation time (30 min) and an increase of the incubation temperature (100°C). As it is evident from Figure 3, increasing both the temperature and the incubation time led to a marked gain in extraction recovery. When analyzing a real sample of methamphetamine, on the other hand, the peak relative to P2P was not detected, so we decided to further increase the incubation temperature to 150°C. In this way, the chromatographic peak of P2P was evidenced.

Stripping:
The duration of the stripping step is very important to transfer all the analyte to the vapor phase. We tested three different stripping times: 1, 5 and 10 min. Going from 1 to 5 min, there is a significant increase in extraction recovery either for P2P or for the IS, while with a stripping time of 10 min, there is a dramatic increase in the response of acetophenone (IS) but the peak related to P2P remains almost unchanged (Figure 4). So, the ideal stripping time resulted to be 5 min.
Trap adsorption temperature: Generally, the lower the trap's temperature, the greater is the adsorption of the analytes. Three different temperatures were tested: -10, 0 and 10°C. The worst result was obtained at 10°C; between 0 and -10°C there is not a big difference, but, at 0°C, the peaks have a sharper shape and propylene carbonate, the solvent, is less adsorbed ( Figure 5).

Injection:
The injection of the sample is a crucial step. The analyte has to be desorbed in the shortest time as possible, to obtain sharp peaks and increase sensitivity. We evaluated that the best results were obtained with 1 min of injection time and an inlet and transfer line temperatures of 280°C, in order to ensure the immediate volatilization of the analytes and to avoid the formation of condensate inside the instrument.   Specificity: When dealing with seized material synthesized in an illicit way, it is not possible to analyze a "matrix" identical to that of the seizures, which does not contain the analytes. Specificity is assessed if the retention time of standard P2P corresponds perfectly to that of the peak found in unknown samples and the ratio among the four

Validation of the DHS-GC/MS method
The DHS-GC/MS method was validated [17] for the identification and quantification of the analytes meeting the requirements of forensic analysis, analyzing WSS specifically prepared.

Quantitative determination of P2P in MAMP samples seized on the illegal market
The validated DHS-GC method was applied for the determination of P2P in ten methamphetamine samples seized by the judicial authority ( Figure 2). The results are shown in Table 1. P2P was found in all the samples, demonstrating that P2P is at the moment the most frequently used precursor for the synthesis of MAMP [6,7]. In Figure  6, the chromatogram obtained for Sample 1 is shown. The large peak is due to methamphetamine that in the operative conditions passes to the vapor phase but it does not interfere with the analysis of P2P. It is interesting to note that the % of P2P is not correlated with the % of MAMP. There might have been expected that the more MAMP is pure the less is the concentration of P2P, but in Sample 1, which is less pure respect to Sample 10, the % of P2P instead of being higher resulted lower.

Concluding Remarks
A simple, sensitive and efficient dynamic headspace gas chromatography (DHS-GC/MS) method was optimized and validated for the analysis of P2P in MAMP samples seized by the judicial authority; P2P was found in all the seizure, confirming that P2P is the starting compound of the synthesis of amphetamines (Scheme 1). This method could be used to determine the impurity profile of a MAMP sample, thus establishing similarities among different production batches and laboratories in which the synthesis of the drug of abuse was carried out illegally.