Sulaf Assi

Handheld Raman spectroscopy offers a rapid and mobile technique for determining drugs nondestructively. The technique offers further advantage for analyzing drug products (DPs) thanks to its ability to measure samples through packaging, thus preserving evidence integrity and continuity. This research evaluated the use of handheld Fourier transform (FT)-Raman spectroscopy for characterization of street DPs (

 = 254) of diverse formulations. Raman spectra of DPs inside their packaging were collected and matched against the instrumental in-built library, and then exported to Matlab 2020a for offline spectral interpretation. Reference analysis was confirmed using gas chromatography–mass spectrometry (GC–MS) and infrared (IR) spectroscopy. The main drugs present were 1,3-trifluoro- methylphenylpiperazine, cocaine, ketamine, 3,4-methylenedioxymethamfetamine, mephedrone, and 1-benzylpiprerazine. The main impurities present in DPs were benzocaine, caffeine, and lactose. The impurities affected the Raman signature (enhanced or inhibited) of DPs, enabling profiling of DPs and visualizing patterns among different products. These latter patterns were featured in the similarities highlighted by the correlation coefficient values between different DPs, and by adjacent clusters of different DPs envisaged in the principal component analysis scores plot.

Crime and violent behaviors have increased because of the widespread use of illicit drugs in contemporary societies. Amsterdam (1027.7), London (701.1), and Copenhagen (780 mg/1000 p/day) had the highest weekend daily mean cocaine usage in 2020 (1). Drug addiction endangers public health, safety, and welfare, as well as the security of the nation. Researchers and authorities require quick and accurate drug detection methods that can be adapted to the ever-increasing spectrum of illicit drugs. Currently, the primary goal of drug analysis is to detect and identify drugs of abuse.

Analytical techniques such as thin layer chromatography, gas chromatography–mass spectrometry (GC–MS), gas chromatography-flame ionization detection (GC-FID), high performance liquid chromatography (HPLC), and liquid chromatography–mass spectrometry (LC– MS) have all been proposed, developed, and used to detect illicit drugs (2). These techniques provide reliable and accurate results in drug analysis, but they are costly, time consuming, and destructive, which limits their practical ability. Vibrational spectroscopic methods, such as Fourier transform infrared (FT-IR) and Raman, address the aforementioned limitations, and they have also been applied for qualitative and quantitative investigations of illicit drugs (3,4).

Raman spectroscopy measures the vibrational modes of a sample. The spectrum is a wavelength distribution of bands that correspond to molecular vibrations in the sample under investigation. The Raman band frequencies and intensities could be used to identify and quantify the measured samples (5). Raman spectroscopy generates molecular-specific spectra with minimum or no sample preparation, permitting non-destructive in situ analysis for different forms of drugs (liquids, tablets, and powders) (6), which is very useful when a substantial number of samples need to be analyzed, and it is essential for avoiding sample contamination, reducing analysis time, and protecting the evidence under investigation. Raman spectroscopy has been used in a variety of applications, including forensics (6), industrial process control (7), biomedical purposes (8), and medical diagnostics (9). When identifying drugs, Raman can distinguish between salts, stereoisomers, and homologs with a sample size of more than 5 mg (10), and it also can reveal a high level of specificity for drugs concealed in alcohol (11). Minor variations in chemical structures can be identified, thus providing a characteristic spectrum for each chemical compound and making the identification of drugs more reliable (12). Raman spectroscopy has recently been used to check drug contents within their packaging (13), evaluate the composition and homogeneity of drug tablets (14), classify street drugs (15), and determine drug authenticity (16). Raman spectra of drug blends are often highly complex because they include spectral features corresponding to all components in the sample. Consequently, chemometric methods must be used to obtain quantitative information from complex spectra (17). This study complements previous studies in evaluating the use of handheld Raman spectroscopy for on-spot identification and characterization of street drugs in their packaging.

 = 254) were measured as received, and included powders, tablets, capsules, liquids, and rocks of different colors. Samples were measured through glass vials using a handheld FT-Raman spectrometer equipped with 490 mW of laser power, a 1064 nm laser wavelength, and cooled InGaAs detector. Spectra were collected over the wavenumber range of 250–2000 cm

. The in-built spectral algorithm was used to search matches of the drug samples against the instrumental libraries. For each sample, the library algorithm gave matches for the drugs or additives that showed spectral features. For offline analysis, the spectra of the products were interpreted considering spectral quality, functional groups, and similarities among products using correlation in wavenumber space (CWS) and principal component analysis (PCA) algorithms. Spectral quality was evaluated for the measured drug products, considering the low spectral resolution in FT-Raman (18). For the CWS method, the momentum product was calculated between the spectra of different products reporting a common drug. A threshold of 0.95 or more was considered a match and vice versa (19). For PCA, drug products’ spectra were explored for patterns among their scores. A false negative was seen when a drug product score was not grouped with product scores of the same drug, whereas false positives were seen when a drug product score was clustered with scores of different drugs. Reference analysis was conducted using GC–MS and attenuated total reflectance-Fourier transform infrared (ATR-FT-IR) spectroscopy. GC–MS and ATR-FT-IR confirmed the identity of the drugs or common impurities present in DPs, respectively (Table I).

Out of the measured DPs, 153 contained drugs or drug mixtures, most of which were not pure. The main drugs found were 1,3-trifluoromethylphenylpiperazine (TFMPP) (

 = 29), 3,4-methylenedioxymethamfetamine (MDMA) (

 = 2), 3,4-methylenedioxypyrovalerone (MDPV) (

 = 2), 1-(3-chlorophenyl)piperazine (mcPP) (

 = 1), and 1-methyl-4-benzylpiperazine (mBZP) (

The main impurities detected were caffeine (

 = 35), which were both Raman active. Lidocaine and procaine were detected less than benzocaine, and they were found in seven and two products, respectively. Benzocaine and lidocaine were mainly found as impurities in cocaine drug products, alongside other impurities such as phenacetin and caffeine. Thus, the pattern of adulterants varied between DPs. For instance, caffeine was also an adulterant in MDMA and piperazine derivatives. On the other hand, lactose was a bulking agent reported in diazepam tablets, and it contributed to the low Raman activity of these products. Likewise, dextrose and sucrose were also found in DPs of tablet- and powder-formulations and contributed to the low Raman activity of these products. Hence, drugs adulterated with the aforementioned sugars were not detected in tablets or powders using Raman spectroscopy where the Raman signal of these tablets was masked by lactose fluorescence.

Spectral quality was evaluated considering these parameters: the number of peaks (

); maximum peak position and intensity; wavenumber range; and signal-to-noise ratio (S/N) (19). In this respect, synthetic piperazine, cocaine, and mephedrone products showed strong Raman activity, ketamine products showed medium Raman activity, and MDMA products showed low Raman activity.

Synthetic piperazines’ spectral quality varied depending on the purity of the DPs and the types of impurities present in them (Figure 1). TFMPP samples showed stronger Raman activity than other piperazines, and that was related to their being either pure or cut with caffeine that in turns was Raman active. The ranges of N, maximum intensity, and S/N values for piperazine derivatives were 15–25, 1500–15000 a.u., and 10–20, respectively. Cocaine products demonstrated strong Raman activity, but that depended on the adulterants present in the cocaine sample, which affected the Raman activity (Figure 2). For cocaine, Raman active samples, 

, maximum intensity, and S/N were in the range of 11–27, 1,744–51,242 a.u. and 15–39, respectively. Similarly, mephedrone showed strong Raman activity with ranges of 

, maximum intensity and S/N in the ranges of 25–29, 1500–8000 a.u., and 18–37, respectively (Figure 3). Ketamine products showed medium Raman activity with 

, maximum intensity, and S/N in the ranges of 16–30, 700–4000 a.u., and 5.75–39, respectively (Figure 4). On the other hand, MDMA products exhibited low Raman activity with low S/N < 20), despite the high 

 in most spectra (up to 25) (Figure 5). The maximum intensity seen for MDMA products was in the range of 1500–5000 a.u.

FIGURE 1: Raman spectra of (a) TFMPP and (b) BZP DPs measured using an FT-Raman spectrometer equipped with 1064-nm laser excitation wavelength.

FIGURE 2: Raman spectra of DPs containing (a) cocaine, (b) cocaine and benzocaine, and (c) cocaine and caffeine measured using an FT-Raman spectrometer equipped with 1064-nm laser excitation wavelength.

FIGURE 3: Raman spectra of mephedrone samples in crystal (black) and powder (red) forms measured using an FT-Raman spectrometer equipped with 1064-nm laser excitation wavelength.

FIGURE 4: Raman spectra of ketamine DPs measured using an FT-Raman spectrometer equipped with 1064-nm laser excitation wavelength.

FIGURE 5: Raman spectra of MDMA DPs in the following colors or forms: (a) gray crystal, (b) white crystal, (c) orange powder, and (d) pink tablet measured using an FT-Raman spectrometer equipped with 1064-nm laser excitation wavelength.

Spectral interpretation showed characteristic scattering bands for the major drugs and impurities present in the measured DPs (Table II). Most drugs showed key bands between 500–1700 cm

 (21–25). In this respect, Raman spectra indicated key differences between different drug analogues as well as base or salt forms of the same drug. For drug analogues, piperazine derivatives (TFMPP and BZP) could be differentiated over the ranges 500–1000 and 1300–1700 cm

, respectively (Figure 1). Similarly, cocaine could be discriminated from cocaine HCl mainly in the range between 850–890 cm

, where cocaine base showed three bands around 850, 870, and 890 cm

 corresponding to the C-C stretch of the tropane ring in the cocaine base (20). On the other hand, cocaine HCl only showed two C-C stretching bands corresponding to the tropane ring around 850 and 890 cm

 (20,21). Where benzocaine was present in cocaine DPs, it could be traced by the strong absorption intensity and by the characteristic bands in the range of 850–1700 cm

When CWS was applied to the spectra of the top encountered DPs, variable correlation (

 value) ranges were observed depending on the amount of drug in DPs and on the purity of the specific DP (Figure 6). For instance, TFMPP-related products (DPN213–DPN254) did not show high 

 values against each other, and that they could not be considered a false negative because of the inconsistency in constituents in TFMPP products, indicating the presence of other additives, such as caffeine, dextrose, ephedrine, lactose, phenmetrazine, and sucrose. In this respect, the Raman activity of the constituents impacted the Raman scattering in terms of number of bands and Raman activity of the DP. For instance, caffeine had strong Raman scattering, and that influenced the signature of the TFMPP products containing them (DPN234–249). Nevertheless, the latter products contained impurities such as lactose that was only detected by Raman (and not GC–MS), which in turn influenced their Raman activity. Lactose and other sugars introduced fluorescence in the Raman spectra of TFMPP products (Figure 1). It is important to mention that the physical properties of the DPs did not influence the Raman signature. Hence, TFMPP products of similar chemical makeup and different physical characteristics (formulation, color) showed high 

 values against each other. For example, DPN217 (white tablet formulation) showed an r value of 0.95 against DPN220 (white crystal formulation). Also, DPN228 (pink tablets), DPN235 (white tablets), and DPN246 (blue tablets) showed 

 values of 0.95 against each other. It is worth noting that no false positives were seen for TFMPP-DPs except against a DP containing BZP, and this mismatch could be related to the structural similarity between TFMPP and BZP.

FIGURE 6: (a) Correlation map of the Raman spectra of TFMPP (1:42), cocaine (43:78), ketamine (79:107), MDMA (108:131), caffeine (132:150), mephedrone (151:159), and BZP (160:166) DPs, where 

 values above 0.95 are marked in yellow and those below 0.95 are marked in red. (b) PCA scores plot of the Raman spectra of TFMPP (black circle), cocaine (blue square), ketamine (red star), MDMA (green triangle), mephedrone (cyan triangle) and BZP (black star) DPs.

Cocaine DPs featured less false negatives, and that could be attributed to the consistency in the types of impurities detected in the measured cocaine DPs. For instance, samples with a cocaine/benzocaine mixture (DPN48–DPN62) showed 

 values > 0.95 against each other where both constituents in the matrix were Raman active and were strong scatterers. Where more than two constituents were present in a cocaine DP, the 

 value changed, and that also depended on the Raman activity of the constituents. Overall, the constituents were unique to cocaine mixtures where no false positives were observed with cocaine mixtures against other drugs. Similar patterns in 

 values results were seen for ketamine samples that were mainly pure, and showed 

 values < 0.95 against different drugs. Only four ketamine DPs showed low 

 values against other ketamine products (one Raman active and three Raman inactive samples). The remaining DPs (containing MDMA and mephedrone) showed false positives and false negatives related to the high number of impurities present in them, and that had been reported in the literature, especially in mephedrone DPs.

Hence, CWS results were only conclusive when the exact number of impurities were present in the reference and test tablets. Considering the aforementioned challenge in identification, PCA recognized patterns in the DPs scores that corresponded to spectral features contributing to high variances (Figure 6). The first three PCs contributed 88.8% of the variance among the DPs’ spectra, with cocaine DPs’ scores recording the highest variance. This was followed by ketamine DPs’ scores and TFMPP DPs’ scores. Cocaine, ketamine, and TFMPP DPs’ scores showed three distinct clusters with slight overlap in few scores, and that could be related to the caffeine or lactose that were impurities present in DPs of the three drugs. Some MDMA DPs that contained lactose overlapped with products of the same impurity. On the other hand, mephedrone DPs were clearly distinguished from other DPs, and that showed their unique composition.

In summary, handheld FT-Raman spectroscopy complemented GC–MS and IR in characterizing street DPs. It indicated information regarding certain impurities that could not be detected by GC–MS (such as lactose and sucrose). Another advantage encountered over IR was the ability of Raman to characterize Raman active drugs nondestructively regardless of the physical differences between the matrices (tablets, powders, or liquids). However, some limitations were encountered in processing the spectra of the drug products relating to the sensitivity of Raman in detecting drugs that contained impurities, especially fluorescent ones such as lactose. Moreover, the presence of multiple impurities introduced a challenge in the identification and characterization of the drug product. Further understanding of Raman spectra, particularly in samples of multiple impurities, is needed. Therefore, future work will consider investigating drugs in mixtures using a more quantitative approach.

The authors would like to thank Bournemouth University for access to drug products and instrumentation. Special thanks for Amy Williams, Chelsea Bailey, and Angela Buffardi for their help with spectral data collection.

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 (European Monitoring Centre for Drugs and Drug Addiction, Libson, Portugal, September 2020).

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(3) Izake, E.L. Forensic and Homeland Security Applications of Modern Portable Raman Spectroscopy. 

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(5) Yang Y.; Thomas, R. The Benefits of a High-Performance, Handheld Raman Spectrometer for the Rapid Identification of Pharmaceutical Raw Materials. 

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 (John Wiley & Sons, Chichester, United Kingdom, 2005).

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(9) Kong, K.; Kendall, C.; Stone, N.; Notingher, I. Raman Spectroscopy for Medical Diagnostics — From In-Vitro Biofluid Assays to In-Vivo Cancer Detection. 

(10) Lanzarotta, A.; Witowski, M.; Batson, J. Identification of Opioids and Related Substances using Handheld Raman Spectrometers. 

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(15) West, M.J.; Went, M.J. Detection of Drugs of Abuse by Raman Spectroscopy. 

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(17) Müller, J.; Knop, K.; Wirges, M.;  Kleinebudde, P. Validation of Raman Spectroscopic Procedures in Agreement with ICH Guideline Q2 with Considering the Transfer to Real Time Monitoring of an Active Coating Process. 

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(19) Assi, S. Raw Material Identification Using Dual Laser Handheld Raman Spectroscopy. 

(20) Penido, C.A.F.d.O.; Silveira Jr, L.; Pacheco, M.T.T. Quantification of Binary Mixtures of Cocaine and Adulterants Using Dispersive Raman and FT-IR Spectroscopy and Principal Component Regression. 

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 are with the School of Pharmacy and Biomolecular Sciences at John Moores University, in Liverpool in the United Kingdom. 

 is with the Faculty of Science at Lebanese University, in Beirut, Lebanon. 

 is also with the School of Allied Health at Anglia Ruskin University, in Chelmsford in the United Kingdom. Direct correspondence to: 

Articles

Handheld FT-Raman spectroscopy can complement GC–MS and IR in characterizing street drugs.

Characterization of Street Drugs Using Handheld Fourier Transform Raman Spectroscopy

Fingernails can accumulate drugs as a result of chronic exposure. This work employed Raman spectroscopy for detecting cocaine hydrochloride (HCl) and its impurities within fingernails, utilizing orbital raster scanning (ORS) technology, where the laser beam hits multiple positions within the sample. Doing so maintained sensitivity and ensured that more of each sample’s components were represented. Fingernails were spiked with powder and solution forms of cocaine HCl and its impurities, including benzocaine HCl, levamisole HCl, lidocaine HCl, and procaine HCl. The strong Raman scattering observed for these substances indicated a high drug accumulation in the fingernails. Key cocaine HCl bands were seen at 848, 874, and 898 cm

(symmetric stretching-aromatic ring), 1278 cm

 (C=C and C=O stretching). Principal components analysis (PCA) confirmed that 90% (nails spiked with drug powders) and 77.2% (nails spiked with drug solutions) were accounted for in the variance among the data. The findings showed that Raman spectroscopy identified the presence of cocaine HCl and its impurities within fingernails.

 (symmetric stretching-aromatic ring), 1278 cm

The pervasive use of illicit drugs in modern society is a phenomenon that has never occurred in the history of humanity (1). Approximately 275 million individuals have used illicit drugs in 2021, which is an increase of 22% from 2010. It is estimated that projected population growth for 2030 translates into a possible increase of 11% in the percentage of illicit drug users worldwide, with a far higher impact in low-income nations rather than high-income nations. Drug usage has resulted in numerous severe problems, especially addiction (2). Drug addiction has caused young individuals to drop out of school and has made it difficult for parents to care for their children. It undermines ambition, drags individuals into poverty and crime, and wrecks lives. Furthermore, the illicit drug trade hinders social and economic advancement, and it can cause serious danger to both global security and stability in some parts of the world. Within the illegal drug market, cocaine has been one of the most common substances with an estimated 2,000 metric tons produced per year (3). Cocaine is a stimulant that exerts its mechanism of action by producing a build-up of dopamine between synapses. Three main forms of cocaine are available: free base, “crack” rocks, and hydrochloride (HCl) powder (4,5).

Many investigating authorities across the world routinely seize cocaine and take judicial actions. Forensic scientists and law enforcement organizations require rapid, accurate, and adaptable techniques of drug detection to combat drugs addiction. Despite the existence of numerous detection techniques, including immunoassays (6), capillary electrophoresis (7), deoxyribonucleic acid (DNA) aptameric sensors (8), and nuclear magnetic resonance (NMR) spectroscopy (9), hyphenated methods are currently the industry standard for analyzing illicit drugs in fingernails and other matrices. Of the hyphenated techniques, gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–MS (LC–MS) are often used in these analyses (10). Both GC–MS and LC–MS are destructive and time-consuming, involving the use of chemicals that can be harmful to both the operator and the environment despite their great sensitivity and molecular specificity. Hence, the use of vibrational techniques, such as Fourier transform infrared (FT-IR) (11) and Raman spectroscopy (12), is considered as a reliable alternative because these methods offer a portable, quick, non-destructive, and cost-effective solution for drug analysis. In particular, Raman spectroscopy is a versatile technique that is applied to analyze different types of forensic samples that cannot be determined by other spectroscopic methods, such as IR spectroscopy (13). Raman spectroscopy is used for both qualitative and quantitative purposes (14,15). Numerous drug classes, including amphetamines, date rape drugs like ketamine, and designer drugs, have been studied using Raman spectroscopy (16). Raman is used because it has better chemical specificity than IR (17). The chemical specificity offers advantages of Raman in detecting physical and chemical differences between isomers and analogues of the same drug (18). Recent developments in data analysis, portable Raman spectrometers, and new excitation wavelengths offer exciting new opportunities for Raman spectroscopy applications in the detection and quantification of drugs of abuse, including investigations carried out immediately at the crime scene. Raman spectroscopy is regarded as a “Category A” technology, offering the maximum level of selectivity through structural information, according to worldwide recommendations given by the Scientific Working Group on Drugs (SWGDRUG) Core Committee (19). An emerging technique for the quick onsite detection of illicit drugs is handheld Raman spectroscopy because it offers additional features over conventional Raman in safety and security where the operator does not need to touch the sample that can be measured inside original packaging (20–23). The aim of this study is to demonstrate the application of rapid, portable methods for the quantitative detection of cocaine HCl and its impurities in human fingernails using handheld Raman spectroscopy in combination with chemometrics.

This work comprised the Raman spectroscopic analysis of 20 sets (11 males and nine females) of fingernails of adults of two ethnicities. Fingernail clippings of 1–2 mm were collected and measured through glass vials. No intervention was made by the researchers during the collection of the fingernail clippings, considering this procedure as an everyday activity. Ethical approval was provided from two institutions: Liverpool John Moores University (PBS/2021/22/04) and the Lebanese University in Lebanon (2022-0104). Five sets of fingernail clippings were then spiked with one of the following drugs or drug impurities: benzocaine (BEN); cocaine HCl (COC); levamisole HCl (LEV); lidocaine HCl (LID); and procaine HCl (PRO). After six weeks of treatment, the spiked nails were measured using a handheld Metrohm Mira XTR Raman spectrometer equipped with a 785-nm laser wavelength, and less than 100 mW of laser output power was used. Raman spectra were collected over a wavenumber range of 400–2300 cm

. The laser beam of the spectrometer used orbital raster scanning (ORS) technology where the beam sampled multiple positions on the sample.

Reference samples of the chosen drugs and impurities were measured through glass vials and included in the instrumental library. The in-built spectral algorithm searched for matches between the reference samples and spiked fingernail clipping samples. Matches were shown for the chosen drug and its impurities and highlighted the key spectral features between the drug and impurities. Offline analysis was also carried out in Matlab 2022b software, whereby the spectra of the drug and impurities were interpreted based on the spectral quality, functional groups, and similarities among the substances identified through the principal component analysis (PCA) algorithm. The spectral quality was evaluated for the measured drug and drug impurities. For PCA, the Raman spectra were investigated for patterns among their PC scores. False negatives were observed when a drug or impurity score was not grouped with the scores of the same drug or impurity and where false positives were seen when a drug or impurity score was clustered with scores of a different drug or impurity.

The measured nails consisted of 20 sets of fingernails obtained from different individuals within the age group of 18–64 years old. The male:female ratio of these individuals was 11:9, and they were of (two) ethnicities: white British and Arab. Each nail set consisted of 7–10 fingernail clippings, and the dimension of each fingernail clipping was 1–2 mm. Of the 20 sets, five were spiked with BEN, COC, LEV, LID, and PRO, respectively. Two types of spiking were made: powder form of the drug (3–5 nails per set) and liquid form (3–5 nails per set) of the drug. A minimum of 20 spectra were collected for each nail set (whether plain or spiked).

Raman spectroscopy was able to detect COC and its impurities in fingernails six weeks post-spiking (Figure 1). In this respect, nail samples showed strong spectral quality with a maximum intensity up to 30,000 a.u.

FIGURE 1: Raman spectra of (a) BEN, (b) COC HCl, (c) LEV HCl, (d) LID HCl, and (e) PRO HCl measured using the handheld Raman spectrometer equipped with a 785-nm laser wavelength; powder (black) and liquid (red) spiked spectra.

Raman activity of the chosen drug and impurities was determined through investigating their Raman spectral quality (Table I). In turn, Raman spectra were evaluated using the following parameters: the number of peaks (

), the maximum peak position and intensity, the wavenumber range, and the signal-to-noise (S/N) ratio. In this respect, the BEN powder, LID powder, and LID solution showed strong Raman activity with N (29–45), maximum intensity (11–74 a.u.), and S/N (33–42). The BEN liquid, COC HCl powder, LEV HCl powder, LEV HCL liquid, PRO HCl powder, and PRO HCl liquid displayed medium Raman activity with N (16–28), maximum intensity (12–66 a.u.), and S/N (20–29). COC HCl liquid showed low Raman activity with N (19), maximum intensity (28 a.u.), and S/N (25).

Effect of Physical Form on Raman Activity

Key variations were seen in the Raman activity between drugs in solid versus liquid forms (Figure 2). The degree of variation depended on the type of drug being analyzed because some drugs showed no difference in Raman activity in their solid forms compared to their liquid forms, which was the case for LEV and PRO; both showed medium Raman activity in their solid and liquid forms. LID also showed strong Raman activity in both its solid and liquid form. Some drugs, such as BEN and COC, showed differences in Raman activity between their solid and liquid forms. In this respect, BEN and COC showed stronger Raman activity in their solid form compared to their liquid forms (that was medium). Variations between drugs and impurities in their solid and liquid forms suggested that physical properties of the substance influenced their Raman activity.

FIGURE 2: Raman spectra of fingernails spiked with BEN powder (black) and BEN liquid (red) forms measured using the handheld Raman spectrometer equipped with 785-nm laser wavelength.

Previous work has reported the Raman activity of key endogenous compounds such as carbohydrates, lipids, and proteins (24). Figure 3 shows the Raman spectra of endogenous compounds. Carbohydrates have been associated with the bands 1471 (CH

 (C-C stretching) (25). The amino acids cystine and cysteine have been linked to C-S stretching vibrations that showed Raman scattering at 623 and 643 cm

 (26). These amino acids make up the disulfide bridges related to the microstructure of fingernails. Disulfide cross-linking is determinant for physical and mechanical properties of a fingernail’s keratin, and it can often be used to identify disease or nail degradation such as diabetes (27). For example, bands at 830 and 850 cm

 corresponded to the presence of denatured tyrosine (amino acid) in keratin (26), which is advantageous for establishing patterns between healthy, diseased, or aged fingernails. The strong C-C stretching band at 935 cm

 was associated with the α-helix of the fingernail’s nail plate (28). Proteins have also corresponded to S-S stretching bands at 509–514 cm

 were representative of lipids and proteins, respectively (24).

FIGURE 3: Raman spectra of a fingernail clipping using the handheld Raman spectrometer equipped with 785-nm laser wavelength.

The Raman activity of fingernails was also evaluated using the aforementioned parameters. The tested fingernails (

 = 20) showed strong Raman activity with 38 peaks and a S/N ratio of 56. Fingernails also demonstrated a maximum peak position of 1457 cm

The Raman activity of fingernails impacted the level of Raman scattering, and this was apparent in terms of the number of bands and the Raman activity of the fingernail clippings. It is important to mention the incorporation of ORS technology and how this improved the representation of the sample’s identify and composition. Traditional Raman methods incorporated a small interrogation area, whereas ORS technology ensured a larger interrogation area. The ORS technique included a tightly focused laser beam scan over a large sample area, which provided high intensity spectra and resolution, resolving previous limitations of conventional Raman spectroscopy (29). Thus, highly complex structures could be analyzed. The ORS technique ensured that all components within the fingernail clipping were collected, and a high spectral resolution was maintained. Hence, Raman spectra of fingernails were highly representative of the heterogeneous components found within fingernails, such as BEN, COC HCl, LEV HCl, LID HCl, and PRO HCl (Figure 4).

FIGURE 4: Raman spectra of (a) fingernail clipping spiked with cocaine powder, specifically cocaine HCl powder, (b) fingernail clipping spiked with cocaine HCl powder, and (c) unspiked fingernail clipping measured using the handheld Raman spectrometer.

Raman Scattering of Drugs in Spiked Nails

In the case of COC, the physical properties of the drug influenced its Raman scattering. For example, COC powder showed medium Raman scattering, whereas the COC solution showed weak Raman scattering. However, some drugs, such as LID, were unaffected by physical properties and demonstrated strong Raman scattering in both the powder and liquid forms. BEN was also a strong scattering regardless of the physical state. LEV and PRO showed medium scattering in both their powder and liquid forms.

Spectral interpretation showed characteristic scattering bands for the chosen drug and impurities, which suggested that Raman spectra indicated a key difference between the sets of spiked fingernails. The tested drug and impurity showed key bands between 400–1700 cm

. In this respect, Raman spectra showed key differences between the spiked fingernail sets. Fingernails spiked with COC HCl could be differentiated from BEN over the ranges of 1600–1712 cm

(30). PRO could also be discriminated from BEN and COC over a range of 1600–1712 cm

. For example, PRO showed key bands at 1605 (C=C stretching), 1640 (NH

 (30). Similarly, LEV could be differentiated from COC over a range
of 460–1004 cm

, where COC showed key bands at 848, 878, and 898 cm

, which have been associated with the C-C stretching of the tropane ring. COC also showed bands at 1278 (C-N stretching), 1453 (asymmetric CH3 deformation), 1605 (C=C stretching), and 1712 cm

 (C=O stretching) (Figure 4) (30). On the other hand, LEV showed key bands at 469 (C-S stretching), 627 (C-C bending of the benzene ring), 969 (In-plane bending related to the imidazole ring) and 1000 cm

 (stretching of the mono-substituted aromatic ring) (31,32). Furthermore, LID showed characteristic bands at 954 and 991 (C-C stretching), 1044 (C-C stretching of the aromatic ring), 1094 (C-N stretching), and 1275 cm

 (C-C and C-N stretching of the aromatic ring) (30) (Table II).

PCA scores recognized patterns in the spiked fingernail scores, which corresponded to the spectral features and contributed to a high variance (Figure 5). The first three PCs contributed 70.4% of the variance among the spiked fingernail spectra. PC scores indicated that Raman spectroscopy and chemometrics demonstrated the ability to differentiate fingernails spiked with COC from other drug impurities. PC1 demonstrated the highest variance among the data (42.8%) and showed scores relating to BEN, COC, LID, and PRO. PC2 showed the second highest variance (20.8%) and accounted for BEN, LEV, and LID, and PC3 showed the third highest variance (6.8%) with scores relating to BEN. The combination of Raman spectroscopy and PCA analysis was successful in identifying and classifying the different sets of spiked fingernails where each set of spiked fingernails showed a distinct cluster (Figure 4). In this respect, no false positive was observed but false negatives were seen for the scores of BEN, LEV, and PRO spectra. This observation could be related to two main reasons—the first being the inconsistency of distribution of drugs within fingernails, and the second being related to the nature of ORS in hitting multiple spots within the sample. To address this limitation in future, Raman microscopy could be used where larger areas of the sample could be measured and studied in further detail.

FIGURE 5: PCA scores plot of the Raman spectra of fingernails spiked with BEN (blue), COC HCl (red), LEV HCl (green), LID HCl (magenta), and PRO HCl (cyan).

Raman spectroscopy offered a rapid and specific detection method for fingernails’ endogenous constituents, COC HCl, and its impurities. The results showed ORS technology resolved previous issues of conventional Raman spectroscopy, such as low spectrum intensity and resolution, and enhanced the Raman signal of fingernails spiked with drugs and impurities. This was ensured by a greater interrogation area and therefore advancing the overall sample’s identity. Further research is needed into looking at the influence of spiking over a longer duration of time and identifying the differences between acute and chronic drug exposure.

The authors would like to thank Paul Bonser and Ian Jackson for their support of the project and for the Metrohm Mira XTR spectrometer.

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(2) United Nations Office on Drugs and Crime, 

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 (Centre for Addiction and Mental Health, Toronto, Ontario, Canada, 3rd ed., 1998).

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Goodman & Gilman’s Pharmacological Basis of Therapeutics

 (McGraw-Hill Medical, New York, NY, 2011).

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(11) H. Schulz, M. Baranska, R. Quilitzsch, and W. Schütze, 

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 (John Wiley & Sons, New York, NY, 2005).

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 (Cengage Learning, Boston, MA, 6th ed., 2006).

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Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) Recommendations, version 8.0

(21) H. Muhamadali, A. Watt, Y. Xu, M. Chisanga, C. Jones, D. Ellis, et al., 

(22) J. Omar, B. Slowikowski, C. Guillou, F. Reniero, M. Holland, and A. Boix, 

(24) M. Gniadecka, O. Nielsen, D. Christensen, and H. Wulf, 

(25) E. Wiercigroch, E. Szafraniec, K. Czamara, M. Pacia, K. Majzner, and K. Kochan, 

(26) A. Chiriac, D. Azoicai, A. Coroaba, F. Doroftei, D. Timpu, and A. Chiriac, 

(28) B. Barry, H. Edwards, and A. Williams. 

(29) A. Geravand, and S. Hashemi Ne-zhad, 

(30) I. Abdulazeez, S. Popoola, T. Saleh, and A. Al-Saadi, 

(31) R. Kranenburg, J. Verduin, Y. Weesepoel, M. Alewijn, M. Heerschop, and G. Koomen, 

(32) S. Shi, F. Yang, W. Yao, and Y. Xie, 

are with the School of Pharmacy and Biomolecular Sciences at Liverpool John Moores University, in Liverpool, United Kingdom. 

 is with the School of Engineering and Technology at Liverpool John Moores University, in Liverpool, United Kingdom. 

 is with the Faculty of Science at Lebanese University, in Beirut, Lebanon. Direct correspondence to: 

The results of this study showed that orbital raster scanning (ORS) technology, which ensured a greater interrogation area, also resolved issues of conventional Raman spectroscopy, such as low spectrum intensity and resolution.

Using Handheld Raman Spectroscopy Equipped with Orbital Raster Technology for Field Detection of Cocaine and its Impurities in Fingernails

The abuse of novel psychoactive substances (NPS) has been increasing over the last few years especially as these products have become so readily available through the Internet. These products are emerging mostly as analogues or precursors of well-known drugs of abuse, such as amphetamine. However, a major problem that is more complicated than their abusive potential is that these products may not contain what is on the label. In fact, they have been shown to contain a mixture of drugs and excipients that may be toxic or even lethal. The number of deaths caused by NPS products has been increasing over the last year. This increase stimulates the need for sensitive techniques to identify these substances and detect any organic impurity in their products. Hyphenated mass spectrometric techniques, such as gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) offer some advantages. These techniques can both separate the constituents as well as confirm the chemical identity of the individual constituents in the product. 

Novel psychoactive substances (NPS), also called "legal highs," represent a major threat to society, particularly to the younger generation. The threat caused by these drugs ranges from chronic psychological or physical illnesses to death. These drugs emerge as analogues or precursors of well-known drugs of abuse to bypass regulations, and the popularity of abusing them is increasing worldwide (1–4). The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) witnessed the emergence of 41 NPS in 2010, which was high compared to 24 substances in 2009 and 13 substances in 2008 (1). This was the largest number of substances reportedly introduced in a single year and included the following derivatives: 15 cathinones, 11 cannabinoids, a synthetic derivative of the plant based substance arecoline, five phenethylamines, one tryptamine, one piperazine, one cocaine, one ketamine, one phencyclidine, one aminoindan, one benzofuran, one aliphatic amine, and a substance that was described as designer medicine (1). The largest threat of these drugs is "amphetamine type" stimulants whose use, according to the United Nations report, has exceeded that of heroin and cocaine (3). 

Several factors contribute to the emergence of these new drug derivatives, such as the drop in the purity of ecstasy, heroin, and cocaine over the last few years (3); the change in the sociological and behavioral context (5); the change in the drug phenomena from life-threatening treatment to lifestyle drugs; and the emergence of unregulated Internet websites. These drugs are marketed in an attractive way and can be found under the names "Legal," "Speed," "Research Chemical," "Plant Food," "Bath Salts," and "Not for human consumption" (6–8). They also are sold at a cheap price. A survey by Schmidt and colleagues showed that an average price of a designer drug in the UK was 9.69 GBP (9). In addition, a survey conducted by Carhart-Harris and colleagues showed that the popularity of common NPS was related to their legal status (2,10). The issue associated with the emergence of NPS is not simply related to increased use, but in the chemical diversity of the products available (1). The EMCDDA warns that the situation could become even more complex if the future witnesses a growing trend in the synthesis of new illicit drugs based on pharmaceutical products (1).  

The toxicity and death case reports caused by these drugs have been increasing over the last few years (1,2,11). According to the United States Poison Control Center, 2237 calls were received from January to May 2011 relating to "bath salts" from 47 states; whereas, the number of calls in 2010 relating to these drugs was 303 (3,12). The cases from the last few years include paranoia and psychotic illnesses (13), acute liver failure (14,15), myocardial inflammation (16), acute sympathomimetic toxicity, hallucinations (1,17–24), and even death (1,20,25–29). The toxic effects associated with these products are not only related to the NPS pharmacological effect and dose, but also to the impurities and adulterants found in them. Often, these products do not only contain what is stated on the product label, or "label claim," but also contain a mixture of drugs and impurities. These additional substances are both deliberate (for example, to counteract a side effect) and accidental (such as, resulting from a poor quality synthesis). They range from organic to inorganic materials and include illicit drugs, pharmaceutical active ingredients, chemical precursors, and common excipients (30). For instance, analysis of products from patients admitted to the emergency department showed that out of 33 products analyzed 19% of nonliquid samples did not contain any drug and 23% contained a pharmaceutical active ingredient (31). The remaining samples contained both classic and novel recreational drugs, including methamphetamine, ketamine, chlorophenylpiperazine, and 1-benzylpiperazine. This highlights a significant issue of confirming the identity of these substances and ultimately the quantity in which they are present in a product.  

Hyphenated mass spectrometric techniques, such as gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS), offer the advantage of selectivity and sensitivity for the analysis of NPS. These techniques can separate and potentially identify NPS of similar chemical structure, in contrast to rapid colorimetric tests that are often unable to discriminate between chemical analogues and isomers. The objective of this review is to highlight the MS methods used in the literature to date for the analysis of NPS. The review focuses on NPS products purchased from the Internet as well as product seizures. Most of the references in the literature cited the use of GC–MS; whereas only one reference utilized the application of LC–MS. 

GC–MS has been the mass spectrometric method of choice for NPS analysis because GC can often separate structural isomers and MS provides fragmentation that is essential in identifying different analogues of a substance. Also, data collected by electron ionization (EI) is easily checked against fragmentation libraries. This is beneficial when analyzing NPS products that are often contaminated with additional analogues, isomers, synthesis precursors, and other active ingredients or organic impurities. 

 Figure 1:  Chemical structures of (a) amphetamine and (b) cathinone.			

GC–MS has been used to identify drug analogues in NPS products seized (32,33) and purchased from the Internet (34). For example, the cathinone "mephedrone" was identified in two products initially believed to be "amphetamine" (Figure 1) (32). Italian police seized these products in the form of an off-white powder and a white tablet, labeled with a dolphin logo (Table I), which initially tested positive for amphetamine using a standard colorimetric test. The products were further analyzed using GC–MS, yet the data did not confirm the presence of amphetamine. Examination of the mass spectrum showed the presence of mephedrone (MW 177) and its major fragment at 

 58, which corresponded to the immonium ion (Table II). The drug had retention times of 6.03 min and 11.06 min before and after derivatization with 2,2,2-trichloroethyl chloroformate. Derivatization of mephedrone increased the major fragment from 

 232, 234, and 236, showing the three chlorine atoms with their clustering pattern. Another case was observed for two samples obtained during a large product seizure submitted by the police department in Australia (33). The GC–MS analysis showed that the samples had very similar mass spectra with the same major ion peak of 

 58 corresponding to cathinone fragmentation, yet the chromatograms showed peaks at different retention times, 5.54 and 6.96 min. The two drugs were identified as the 3-fluoromethcathinone analogues 3,5-difluoromethcathinone and 3,5-dichloromethcathinone, respectively. Another case identified a mixture of cathinone analogues in capsules purchased from the Internet that claimed to contain only the active ingredient mephedrone (34). The products purchased were Spirit, Sub Coca 2, Neo Dove, and Sub Coca. The products all were different colors. However, the results showed that only the first product (Spirit) contained mephedrone (retention time = 4.36 min), the second product (Sub Coca 2) contained a mixture of α-phthalimidopropiophenone (retention time = 6.45 min) and 2-fluoroamphetamine (retention time = 2.56 min), and the third (Neo Dove) and fourth (Sub Coca) contained a mixture of caffeine, mephedrone, 

-ethylcathinone, and α-phthalimidopropiophenone (Table I). The MS spectra of the four substances showed the same fragmentation pattern as the previous cathinones. 

 Table I: Details of the substances found in novel psychoactive substances products			

Because the synthesis of NPS is often done in uncertified laboratories, there is little effort made to control or purify isomeric forms. The incidence of analogues present as isomers, yet still different to that on the label claim of an NPS product, was much more prominent in the literature. These included isomers of fluoromethcathinone (35), methylmethcathinone (36), 

-ethylethcathinone (37), methylenedioxycathinone (38), isocathinone (39), and naphyrone (40).  

A number of studies developed methods using isomer standards to identify a single isomer present in a sample. A method to distinguish the 2-, 3-, and 4-isomers of fluoromethcathinone was developed using GC–EI-MS (35). The chromatogram of the 4′-isomer (9.28 min) was clearly distinguished from the 2′-isomer (9.15 min) and the 3′-isomer (9.23 min). However, the three isomers were not stable so acetylation was needed to improve their stability. In this respect, the retention time of the acetylated derivatives of 4′-, 3′-, and 2′-isomer increased to 11.44, 11.46, and 11.66 min, respectively. The fragmentation of the three isomers was the same as the cathinone derivatives with a major ion peak corresponding to the immonium ion at 

 58. The presence of the 4′-isomer of fluoromethcathinone was then confirmed in three capsule products labeled as Sub Coca Dragon, High Spirit, and Neo Dove 2. However, in this case GC–MS could not detect caffeine and methylamine salt, which were present in the capsules and were confirmed using nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. A method to distinguish 2-, 3-, and 4-isomers of methylmethcathinone in NPS products also was developed (36–38). All isomers showed the same MS fragmentation pattern with the major ion peak at 

 58 (36), yet they were successfully separated 2<3<4 by GC between 6 and 9 min, respectively. The seized product analyzed was found to contain the 4- methylmethcathinone (mephedrone) isomer as well as benzocaine and showed GC retention times of 8 and 12 min, respectively. 

Table II: Ions observed from the analysis of novel psychoactive substances using chemical ionization (CI) and electron ionization (EI) mass spectrometry 			

In some cases, samples contained a substance and an isomer of the substance, demonstrating the typically poor quality of the product obtained. Isomers of mephedrone and ethcathinone were detected in two seized products using GC–EI-MS (37). The isomers were detected at different retention times than mephedrone (8.2 min) and ethcathinone (6.5 min) and were seen at 6.4 min for isomephedrone and 5.4 min for isoethcathinone. Both isomers had a major ion peak at 

 134 corresponding to isocathinone instead of 

 58, which corresponds to cathinone (Table II). Benzocaine also was found in these products. Methods to distinguish 3,4-methylenedioxymethcathinone and butylone isomers found in two seized products using GC–EI-MS also was developed (38). The chromatogram of the first sample showed the separation of 3,4-methylone and 3,4-MDPV from two other impurities, benzocaine and phenacetin, and were between 12 and 16 min (Table I). On the other hand, the second sample lacked these two impurities and the GC data showed the separated isobutylone and 3,4-butylone between 13 and 15 min. The mass spectra showed base peaks for methylone, butylone, and MDPV at 

 58, 72, and 126, respectively (Table II). Isomers of pentedrone and pentylone, were seen in two NPS product seizures: white powder seized from the customs department at the airport in Austria and green capsules brought to the police from a head shop in Germany (39). For the white powder, the mass spectrum in chemical ionization (CI) mode for both isomers showed the same [M+H]

192, but the mass spectrum in EI mode showed a difference in the major ion peak of the characteristic immonium ion that was 

 120 for isopentedrone (Figure 2 and Table I). Similarly, the capsules showed to contain pentylone and iso-pentylone had similar results with [M+H]

 236 in CI mode and showed different major ion peaks in EI mode of the characteristic immonium ion that was at 

 86 and 164 for pentylone and isopentylone (Figure 2), respectively. A recent study using GC–ion trap-EI-CI-MS to distinguish α- and β-isomers of naphyrone also was done (40). In this case, both isomers showed similar mass spectra with a major ion peak at 

 126 corresponding to the formation of the iminium ion in EI mode, and [M+H]

 282 in CI mode. However, both isomers had different retention times at 12.41 and 12.86 min corresponding to α and β-naphyrone respectively. The β-isomer was detected in one powder sample from the Internet, whereas both isomers were found in a second powder sample (Table I). 

Figure 2:  Chemical structures of (a) pentedrone, (b) isopentedrone, (c) pentylone, and (d) isopentylone. 			

In many cases, NPS products contain mixtures of isomers, analogues, and organic and inorganic impurities. This was observed with the analysis of 24 NPS products purchased from the Internet (41). Most of these products were labeled as naphyrone (NRG-1) but contained one or a binary mixture of other cathinone analogues or isomers, an anesthetic, a stimulant, or other substances with no psychoactive effect. The cathinones found included 4-methyl-N-ethylcathinone, butylone, flephedrone, MDPV, mephedrone, and naphyrone (Table I). The substances with no psychoactive effects included benzocaine, caffeine, lidocaine, procaine, and inorganic materials (Table I). A GC–MS method was developed to separate all substances found in the purchased products. Thus, the retention times for the cathinone derivatives were 7.97, 9.54, 6.73, 11.53, 7.64, and 12.41 min, corresponding to 4-methyl-

-ethylcathinone, butylone, flephedrone, MDPV, mephedrone, and naphyrone, respectively. The mass spectra for these cathinones showed major fragmentations corresponding to the iminium ion at 

 58, 58, 126, 72, 126, and 72, respectively (Table II). On the other hand, benzocaine, caffeine, lidocaine, and procaine showed retention times of 8.36, 9.9, 10.05, and 10.92 min, respectively. Whereas all these products contained a single substance or binary mixtures of two substances, further binary mixtures and more complicated quaternary mixtures of cathinones were detected in an additional three products. Two of the products were labeled as mephedrone and one was labeled as naphyrone (42). Five different cathinones were identified in these samples (Table I) and had retention times of 6.73, 9.95, 11.12, 11.53, and 9.39 min, corresponding to 4-fluoromethcathinone, pentylone, MDPBP, MDPV, and MPPP, respectively. The mass spectra in EI mode showed a major ion peak corresponding to these cathinones at 

 58, 86, 112, 126, 98, and 164, respectively (Table II). In addition, the [M+H]

  values detected for these cathinones in CI mode were observed at 

 182, 236, 262, 276, and 218, respectively. 

A number of studies used GC–MS to identify substances chemically unrelated to the label claim. In some cases, NPS products contained neither an isomer nor an analogue of their label claim, but instead contained a completely different substance. This was shown with seven products purchased from the Internet and labeled as MDAI (three products), Benzofury, 5-IAI, NRG-3, and E2. Apart from one MDAI sample, the GC–MS analysis of the products showed that they contained completely different ingredients to their label claim (43). These products contained a mixture of BZP, 3-TFMPP, and caffeine, except E2, which contained only caffeine (Table I). The chromatography method was able to separate the compounds present that appeared at 5, 5.3, 5.7, and 7 min corresponding to BZP, 3-TFMPP, MDAI, and caffeine, respectively. The major ion peaks observed in the mass spectra were 

Gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) techniques offer advantages in separating and confirming the identity of constituents in novel psychoactive substances.

Sulaf Assi

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Using Handheld Raman Spectroscopy Equipped with Orbital Raster Technology for Field Detection of Cocaine and its Impurities in Fingernails

Identification of Novel Psychoactive Substances Using Hyphenated Mass Spectrometric Techniques