Sedative-hypnotics drugs represent a pharmacologically diverse group of compounds, such as benzodiazepines, barbiturates, and other newest agents (e.g. zaleplon and zopiclone), that are used clinically [1]. Currently, there is no scientifically precise or universally accepted classification scheme for these drugs. Sedative-hypnotics drugs are often prescribed in primary care for insomnia.
Zaleplon and zopiclone are sedative-hypnotic drugs, which are used to induce sleep in the short-term treatment of insomnia. These newest medications are less safe than other benzodiazepines and have a tendency to induce physical dependence [1,2]. Currently, these drugs are the most commonly prescribed sedative-hypnotics agents in the United States of America and the European Union.
Although these drugs are widely prescribed for patients the certain side-effects were identified. It should be noted an adverse cognitive (such as, memory loss) and psychomotor (such as, road traffic crashes) effects, daytime fatigue, addiction, and excess of mortalities with no significant difference from the side-effects of typical benzodiazepines [2-5]. However, with increased use of zaleplon or zopiclone drugs, reports of misuse and possible dependence began to appear in the literature, particularly considering people with a history of drugs and/or narcotics misuse and comorbid psychiatric illness [1,2,4]. For this reason, it is essential to develop a fast and sensitive method for the determination of both analytes in biological matrices.
When developing a fast gas chromatography with negative-ion chemical ionization mass spectrometry (GC/NICI-MS) method matrix effects are a major issue. The effect of co-eluting compounds arising from the matrix (blood, urine) can result in a signal enhancement or suppression [6,7]. During method development, much attention should be paid to diminish matrix effects as much as possible.
The main aim of my study was to develop a new sensitive and specific method based on a fast GC/NICI-MS using solid-phase extraction (SPE) for the quantification of zaleplon and zopiclone at trace level in low-volume blood and urine samples. To the best of my knowledge, this method has been used for the first time for the optimization of sample preparation at different pH values (pH 1.0 - 10.0). Comparison of two SPE sorbents for the determination of both analytes in blood and urine were investigated. The analytes were well retained on Oasis MCX and Oasis HLB sorbents, also sufficient extraction efficiency was achieved at pH 9.0. For further study, a hydrophilic-lipophilic (polymeric) sorbent Oasis HLB was selected due to the polarity of sorbent surface and its large surface area (830 m2 g-1) in order to achieve efficient extraction of the analytes in a single step. The surface area is one of the most important factors for extracting the analytes from blood or urine samples by SPE. Special attention was paid to the selection of washing and eluting solvent in the SPE procedure, resulting in very pure and free from moisture extract, which can successfully be applied for gas chromatography-mass spectrometry. Different solvents or mixtures of solvents for elution of the adsorbed analytes, washing step-eliminating interferences in the sorbent were tested.
The developed method provides significant advantages in comparison with other previously published methods [6,8-10]. It shows higher sensitivity (the limit of detection $\leq 0.60$ ng mL-1 and the limit of quantification $\leq 2.00$ ng mL-1) in blood and urine samples. The mean extraction efficiency was higher than 90.1 % for zaleplon and 82.9 % for zopiclone. The precision for zaleplon and zopiclone was between 3.04 – 10.58 % and 4.08 – 9.52 %, respectively. Whereas the accuracy (Bias value) was in the range from -5.73 to 6.00 % and from -7.00 to 6.32 % for zaleplon and zopiclone, respectively. The results have shown that the developed method is accurate, selective, precise, very fast with excellent recovery, low limits of detection and quantification. Finally, it was demonstrated that this method is applicable for the determination of trace concentrations of zaleplon and zopiclone in whole blood and urine samples. The developed method can be applied in routine toxicological analysis during the investigation of both clinical and forensic cases.