Elsevier

Vibrational Spectroscopy

Volume 110, September 2020, 103112
Vibrational Spectroscopy

Liquid–liquid extraction-assisted SERS-based detection of clonazepam in spiked drinks

https://doi.org/10.1016/j.vibspec.2020.103112Get rights and content

Highlights

  • A simple analytical methodology to detect clonazepam in beverages was developed.

  • SERS and chemometric tools were used to build a classification model.

  • A simple sample pretreatment step was applied.

  • The calculated limit of detection was 1.85 μg mL−1.

Abstract

In this study we aimed to combine the liquid–liquid extraction with low-temperature partition (LLE-LTP) sample preparation method with the SERS analytical technique to detect clonazepam in beverages that are commonly consumed at parties, in bars, and in nightclubs to simulate the analysis of beverages involved in drug-facilitated crimes. Chemometric tools were used to build a classification model to identify the presence of clonazepam in the analyzed drinks. One-class strategy was employed to build classification model. The performance of the SIMCA model was also assessed using sensibility and efficiency for the test set, which was determined to be 0.92 and 0.96, respectively The classification model allowed the appropriate and satisfactorily classification of drinks, even for those drinks that presented a matrix effect during analysis, which were subjected to a quick extraction procedure prior to analyzing them. The methodology proposed here could be used at crime scenes, where portable equipment could be utilized, and presents great potential for application in forensic laboratories, where fast and simple analytical methods must be employed routinely.

Introduction

The production of 1,4-benzodiazepines, a class of anxiolytic drugs that present sedative, relaxing and antidepressant properties, began in the mid-1960s [1]. The chemical structure of 1,4-benzodiazepines comprises a benzene ring fused to a diazepine ring, and different substituent generate distinct benzodiazepine drugs (Fig. 1A).

Benzodiazepines are controlled drugs that are mostly prescribed for the treatment of anxiety and insomnia [2]. Thus, they are readily available for inappropriate use. In Brazil, clonazepam (Fig. 1B), 5-(o-chlorophenyl)-1,3-dihydro-7-nitro-2H-1,4-benzodiazepin-2-one, is the most commonly used benzodiazepine [3]. The inappropriate use of benzodiazepines can occur in three ways: excessive prescription, which could result in high rates of chemical dependency, abuse in cases of self-medication and involuntary administration, and used for sedation for criminal purposes.

Benzodiazepines are mostly used for criminal practices in concert halls and nightclubs, and most victims are female. Drug-facilitated crimes consist of the use of psychoactive substances to incapacitate the victim, and typically include theft and/or non-consensual sexual acts or drug-facilitated sexual assaults. The crime consists of adding central nervous system substances (tablets or drops), such as benzodiazepines (typically clonazepam or flunitrazepam), scopolamine, ketamine and gamma-hydroxybutyrate (GHB, which is known as liquid ecstasy) to alcoholic beverages, which enhance the effect of these substances and impair de victim memory and resistance [4].

The detection of benzodiazepines has been studied mostly in biological samples, such as urine and blood; however, their detection in adulterated drinks has also been investigated [5]. Several analytical techniques have been used to detect and quantify benzodiazepines in beverages for forensic purposes, and the most common ones are high performance liquid chromatography [6,7], gas chromatography coupled with mass spectrometry [8,9], and electrophoresis [10,11]. However, these techniques involve laborious and costly steps as they require large amounts of high-purity solvent and long chromatographic runs, which are often associated with sample pretreatment. More recently, Raman spectroscopy has been used for the forensic study of these compounds [12,13].

Doctor and McCord [12] used the surface-enhanced Raman scattering (SERS) technique combined with solid–liquid extraction (SLE) to analyze 11 benzodiazepines, including clonazepam, diazepam, and flunitrazepam, in urine samples. The limit of detection (LD) of the samples ranged from 5.3–11,000 ng mL−1, and was 890 ng mL−1 for clonazepam. The authors used Au nanoparticles (AuNPs) and magnesium chloride as the SERS substrate and aggregating agent, respectively. The procedure was facile, fast (required less than 20 min), and used a small volume of biological material. Ali and Edwards [13] employed portable equipment to detect the presence of flunitrazepam in Bacardi rum, dark rum, gin, and vodka using the Raman technique, and were able to detect flunitrazepam concentrations as low as 0.01 % w/v. This technique could be used to in situ identify benzodiazepines, and allows to obtain spectra within 10 s, and therefore it is extremely advantageous.

Recently, Paula et al. [14] studied the detection of benzodiazepines in beverages using paper spray ionization mass spectrometry. The authors analyzed samples of beer, soda and vodka, energy drinks and vodka, and light peach juice and vodka mixtures with diazepam, alprazolam, clonazepam, bromazepam, and cloxazolam tablets. A simple and efficient extraction method known as liquid–liquid extraction with low-temperature partition (LLE-LTP) was used to eliminate the matrix effect and detect the presence of all analyzed benzodiazepines in the beverage samples. However, ambient pressure ionization techniques are not yet routinely used in forensic laboratories

The development of methods that could be used to detect analytes (benzodiazepines) quickly, efficiently, and reliably, and that could be used outside of laboratory settings, is crucial for forensic researchers. It is important that the analysis methods take into account the complex matrices of drug-spiked drinks. Therefore, in this study we aimed to combine the LLE-LTP sample preparation method with the SERS analytical technique to detect clonazepam in beverages that are commonly consumed at parties, in bars, and in nightclubs to simulate the analysis of beverages involved in drug-facilitated crimes. Chemometric tools were used to build a classification model to identify the presence of clonazepam in the analyzed drinks.

Section snippets

Chemicals

Analytical grade sodium citrate (Na-cit, 99 % purity) and chloroauric acid (HAuCl4, 98 % purity) were purchased from Sigma-Aldrich and were used as received, without further purification. All aqueous solutions were prepared using Milli-Q deionized water (Ω =18.2 MΩ cm.

The clonazepam (Rivotril® tablet 0.5 mg and oral solution 2.5 mg mL−1) samples used in this study were donated by patients. In addition, pure standard clonazepam powder (20 mg) was donated by the local scientific police. The five

Results and discussion

In this study, we obtained the Raman spectra of tablets and oral solutions of Rivotril®, and the results are presented in Fig. 2A and B. The spectrum of clonazepam, which is the active ingredient of Rivotril®, was also obtained (Fig. 2C).

The Raman spectra of drugs are dominated by the bands of their excipients, which are present in tablets and oral suspensions in greater concentrations than the drugs themselves. The most common excipients found in tablets (Fig. 2 A) are starch, cellulose,

Conclusion

In this study, the efficiency of SERS was investigated for the detection of clonazepam in beverages. The spectra of standard solutions were obtained, and the LD of clonazepam was calculated to be 1.85 μg mL−1.

The results revealed that SERS analysis performed using AuNPs was appropriate for the forensic purpose. This analysis technique did not require sample preparation for distilled beverage samples. However, for more complex drinks, such as beer and energy drinks, a simple extraction step

Declaration of Competing Interest

None

CRediT authorship contribution statement

Isabela M. Alves: Methodology, Investigation, Writing - original draft, Visualization. Nathália O. Melo : Methodology, Investigation. Pablo A. Marinho: Conceptualization, Resources. Mariana R. Almeida : Conceptualization, Formal analysis, Resources, Writing - review & editing, Visualization, Supervision, Funding acquisition.

Acknowledgements

Authors acknowledge the financial support of Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We thank the GRUTAM research group for the use of Raman equipment.

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