ACADEMIA ROMÂNĂ Rev. Roum. Chim., Revue Roumaine de Chimie 2015, 60(9), 891-898 http://web.icf.ro/rrch/ RETENTION BEHAVIOUR OF SOME BENZODIAZEPINES IN SOLID-PHASE EXTRACTION USING MODIFIED SILICA ADSORBENTS HAVING VARIOUS HYDROPHOBICITIES Elena BACALUM,a Mihaela CHEREGIb,* and Victor DAVIDb,* a Research Institute from University of Bucharest – ICUB, 36-46 M. Kogalniceanu Blvd., Bucharest, 050107, Roumania b University of Bucharest, Faculty of Chemistry, Department of Analytical Chemistry, 90 Panduri Ave, Bucharest – 050663, Roumania Received April 6, 2015 The retention properties of six benzodiazepines (alprazolam, bromazepam, diazepam, flunitrazepam, medazepam, and nitrazepam) on four different solid phase extraction silica 1.0 adsorbents with various hydrophobicities (octadecylsilica, octylsilica, phenylsilica, and cyanopropylsilica) were 0.8 H O investigated. The breakthrough curves showed a significant N retention of these compounds on octadecylsilica, octylsilica, 0.6 Br N phenylsilica, excepting alprazolam that has a poor retention on 0 C/C N octadecylsilica. These results can be explained by the 0.4 PHENYL CN hydrophobic character of studied benzodiazepines (octanol- C18 0.2 C8 Bromazepam water partition constant, log Kow, being situated within the interval 1.90-4.45). A poor retention on cyanopropylsilica was 0.0 observed for all studied compounds indicating that π-π and 0 102030405060708090100 Volume (mL) polar intermolecular interactions have a less significant role in their retention on this adsorbent. Generally, the breakthrough curves followed the theoretical shape, with some exceptions due to fluctuations in of the flow-rate of sample loading on the adsorbent bed. INTRODUCTION* imprinted polymers, restricted access materials, and immunoaffinity adsorbents), which allow the Solid-phase extraction (SPE) is a sample development of extraction procedures for specific preparation technique widely used for isolation, applications.1 concentration, clean-up of different samples in Nowadays, SPE is the most popular sample various matrices, based on the same principles of preparation technique for both nonpolar and polar liquid chromatography. Currently there is available analytes due to advantages, such as high an increasing number of adsorbents based on low- selectivity, use of less organic solvents, rare specificity inorganic oxides (chemically bonded emulsion, minimizes time, cleaner extracts, silica) and compound or group-selective materials reproducibility of data, and higher throughput by (such as mixed mode adsorbents, molecularly automatization.2 SPE can be viewed as a simple * Corresponding authors: [email protected]; [email protected] 892 Elena Bacalum et al. chromatographic process with distribution process drophobicity (octadecylsilica, octylsilica, phenyl- performed on a very short column with a low silica, and cyanopropylsilica). These compounds are number of theoretical plate, but involving prescribed drugs worldwide and have been compounds with very different distribution extensively studied from their detection point of coefficients, in which the adsorbent plays the role view, such as by mass-spectrometric techniques,7 or of the stationary phase and the mobile phase is the from the chromatographic behaviour in reversed- solvent of the sample during the adsorption step or phase mechanism.8 This study includes the a selected solvent during the desorption step.2-4 investigations for adsorption/desorption yields The choice of adsorbent which depends measured on four mentioned adsorbents. strongly on the analytes of interest is the key point in SPE because it can control parameters such as selectivity, affinity and capacity. However, it also EXPERIMENTAL depends on the kind of sample matrix and its interactions with both the adsorbent and the Six benzodiazepines (alprazolam, bromazepam, diazepam, 3 flunitrazepam, medazepam, and nitrazepam) were analyte. investigated, which were kindly offered by LaborMed Pharma Also, the knowledge of retention mechanism S.A. Their structures are presented in Fig. 1. Water (resistivity and the behavior of different adsorbent are minimum 18.2 MΩ and TOC maximum 30 ppb) was produced important in solid phase extraction. A specific with a TKA Lab HP 6UV/UF instrument in the laboratory. parameter used to characterize the retention Acetonitrile and methanol (HPLC grade) was purchased from Sigma Aldrich. process in SPE is the breakthrough volume The major molecular parameter indicating the affinity of a established from the breakthrough curve; used to compound towards hydrophobic surface, i.e. octanol-water 9 determine the appropriate adsorbents for isolating partition coefficients (log Kow), for the six studied 5 particular analytes. The breakthrough volume benzodiazepines are shown in Table 1, together with their pKa depends on kinetic parameters of adsorbent and on values. Bromazepam and flunitrazepam have similar log Kow 6 values. the retention parameters. Stock standard solutions of 2 mM of benzodiazepines The aim of this study was to investigate the were prepared in methanol. Working solutions of 20 µM were retention behaviour of six 1,4-benzodiazepines prepared by further dilution in water. Stock standard solutions (namely, alprazolam, bromazepam, diazepam, of 400 ppm benzodiazepines in methanol were prepared that were used to obtain working solutions of 20 ppm by dilution flunitrazepam, medazepam, and nitrazepam) on four in water. SPE adsorbents with different polarity and hy- H3C N CH 3 N H O O N N N N Cl N Br N Cl N Alprazolam Bromazepam Diazepam CH 3 CH O 3 O N H N N N O2N N N Cl O2N F Flunitrazepam Medazepam Nitrazepam Fig. 1 – Chemical structures of studied benzodiazepines. Retention behaviour of benzodiazepines 893 Table 1 10 11-13 The values of log Kow for benzodiazepines studied, and pKa Molecular Benzodiazepines CAS Number log K pK weight ow a Alprazolam 28981-97-7 308.77 3.87 2.4 Bromazepam 1812-30-2 316.16 1.93 2.9 Diazepam 439-14-5 284.74 2.70 3.3 Flunitrazepam 1622-62-4 313.29 1.91 1.8 Medazepam 2898-12-6 270.76 4.43 6.2 Nitrazepam 146-22-5 281.27 2.45 3.2 Table 2 Characteristics of silica based adsorbents used in this study Adsorbent Surface Particle Average Carbon Retention Type of Cartridge type mass area size pore size loading mechanism interaction mg m2/g µm Å % SampliQ C18 200 541 45 60 25.7 Reversed phase Hydrophobic SampliQ C8 200 571 45 60 15.6 Reversed phase Hydrophobic SampliQ Hydrophobic 200 562 45 60 12.6 Reversed phase Phenyl and π-π Reversed phase π-π, dipole- Finisterre 200 n.a* 50 60 4 and normal dipole, and Cyano phase hydrophobic * – not available An off-line automated solid-phase extraction SPE HT400E cartridges, hydrophilic modified reversed-phase system was used. The SPE off-line procedure was described in 14 (Oasis HLB, a hydrophilic–lipophilic balanced detail elsewhere. First the adsorbent was conditioned with 5 23 mL methanol and then accommodating with 5 mL water. The copolymer) cartridge and adsorbents containing a volume of the individual solution of benzodiazepines added cationic-exchange group or anionic-exchange was 2.5 mL. Fractions of 2.5 mL were collected until the loss functionalities were investigated for the solid- of analyte could be observed, indicating saturation of the phase extraction of some benzodiazepines in serum adsorbent bed. The adsorbent was never allowed to dry during and urine.24,25 Recently, molecularly imprinted either the conditioning or sample loading steps. All SPE 26,27 experiments were performed at room temperature. The flow solid-phase extraction (MISPE), on-line rate was kept constant during the experiments at 1 mL/min. enrichment and clean-up on a restricted access Four types of adsorbents containing octadecyl (C18), octyl extraction column,28,29 and multiwalled carbon (C8), phenyl and cyanopropyl silica have been used; with the nanotubes (MWCNTs) adsorbents30 for physico-chemical properties presented in Table 2. Chemically bonded silicas, usually with C8 and C18 organic groups, are benzodiazepines extraction were also used. Parallel by far the most commonly used SPE materials. Octadecylsilica extraction of benzodiazepines was also adsorbent is considered one of the most non-selective accomplished on a 96-well disk solid phase adsorbents. extraction technique.31 Although benzodiazepines The absorption spectra of benzodiazepines were recorded with a Jasco V-530 double beam spectrometer, in 1 cm quartz have structural similarities, they have various cells, and used to choice of the maximum sensitivity of UV behaviors towards adsorbents used in SPE because 32 detection of these benzodiazepines. The absorbance were of differences in polarity. measured at λmax = 220 nm for alprazolam, at λmax = 226 nm This paper is focused on the investigation of for bromazepam and diazepam, and at λmax = 254 nm for breakthrough curve of six benzodiazepines on four flunitrazepam, medazepam, and nitrazepam. different types of SPE silica adsorbents (octadecyl, octyl-, phenyl- and cyanopropylsilica). As presented RESULTS AND DISCUSSION in Table 2, the four used adsorbents are involved in the retention mechanisms including: hydrophobic, Many SPE procedures for extraction of π-π and dipole-dipole. Among them, π-π benzodiazepines in various sample matrixes are interactions may have a great contribution to the reported in the literature.15,16 These procedures retention on both cyano and phenyl adsorbents. include uses of C2, C8, C18,17-20 mixed-mode Dipole-dipole interactions can be dominant for columns21 and polar cyano