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Detection of 1,4-Benzodiazepine by Using Different Analytical Methods

Anupama P*

Department of Pharmaceutical analysis and Quality Assurance, Vignan Pharmacy College, Jawaharlal Nehru Technological University, Guntur, India

*Corresponding Author:
P. Anupama
Department of Pharmaceutical analysis and Quality Assurance
Vignan Pharmacy College, Guntur, India
E-mail: [email protected]

Received date: 5/12/2016; Accepted date: 29/12/2016; Revised date: 20/12/2016

Visit for more related articles at Research & Reviews: Journal of Pharmaceutical Analysis


Benzodiazepines (BDZs) are the chemicals having the flexible restorative qualities. These are the kind of psychotropic medication, that is, they concern the brain and can revise attitude. Benzodiazepines upgrade the impact of the neurotransmitter gamma-aminobutyric corrosive (GABA) and are along these lines utilized restoratively as anxiolytics, sedatives, hypnotics, anticonvulsants in epilepsy and midway acting muscle relaxants. Benzodiazepines (BDZs) are for the most part regularly utilized as anxiolytic or potentially trancelike medications as a ligand of the GABA-benzodiazepine receptor. Besides, some of benzodiazepines are generally utilized as a hostile to depressive and narcotic medications, furthermore as against epileptic medications and now and again can be valuable as an assistant treatment in headstrong epilepsies or hostile to alcoholic treatment. The across the board utilization of this class of medications has sometimes raised worry about recreational benzodiazepine mishandle and has prompted to the wrong impression that benzodiazepines have a generally high manhandle risk among recreational medication clients. The mishandle or abuse of benzodiazepines is universally boundless which implies that any scientific research facility may experience a scope of these mixes. As a rule, benzodiazepines experienced in the unlawful market are redirected from legitimated sources.


Benzodiazepines, epilepsy, neurotransmitter


As of late an expansive number of investigative and pharmacological reviews on 1,4-benzodiazepines and their metabolites in organic specimens have been depicted [1]. The predominant measure strategies incorporate fluid chromatography (LC/HPLC) [2], gas chromatography (GC) [3], micellar fluid chromatography (MLC)[4], micellar electrokinetic chromatography (MEKC)[5], potentiometry [6], spectrophotometry[7], fluorimetry [8], slim electrophoresis [9] and immunoassay [10]. Methodology for evaluation of medications investigated by various creators [11-13] included benzodiazepines in the bio-tests blood, plasma, serum, or oral liquid (salivation, and so forth.) utilizing fluid chromatography combined with single-stage or couple mass spectrometry (LC–MS, LC– MS–MS) [14]. Writing dedicated to medication testing in hair by HPLC/GC [15] has been checked on. The utilization of fine electrophoresis to the location and assurance of 1,4-benzodiazepine sedatives in plans and body materials has been basic assessed [16]. A survey tended to the utilization of elite fluid chromatography (HPLC) and narrow electrophoresis (CE) as proclivity partition techniques to describe medications or potential drugs–biopolymer connections [17]. Another arrangements with the diverse strategies for investigation of specific sedatives; phenothiazines, thioxanthenes, and benzodiazepine subordinates in organic liquids of pharmaceutical premium [18]. Just a single expound report secured chromatographic techniques, HPLC, LC–MS, GC, GC–MS distributed throughout the years 1992 to 1997 for the estimation of benzodiazepines in natural specimens, which included examples readiness incompletely [19]. Writers assessed diverse parts of HPLC [20] and GC [21] chromatographic techniques in beforehand distributed articles. Brief exchange on HPLC and GC chromatographic techniques has been incorporated in this. This audit incorporated all conceivable systematic techniques dedicated for the assurance of benzodiazepines in pharmaceutical details and natural specimens.

Properties and physiological activity

Various benzodiazepines have allegedly been integrated and more than fifty of these are showcased for clinical use all through the world. The established benzodiazepines depend on a 5-aryl-1,4-benzodiazepine structure. The seven-membered amino-ring was basic for its partiality towards the BDZs-restricting site and the atomic lipophilic properties of various BDZs assumed a huge part in their comparing receptor fondness [22-30].

Benzodiazepines are feeble fundamental medications and as free bases are lipidsoluble and waterinsoluble. Stock arrangements of 1, 4-benzodiazepines in methanol, ethanol or acetonitrile are steady for 3-6 months, when they were kept at - 4ºC oblivious. Benzodiazepines are steady in organic media when put away at - 20°C for a few weeks or months. Considers demonstrated that some of benzodiazepines are photolabile, and the photoinstability of alprazolam increments as the pH diminishes.

Lorazepam was appeared to be immediately photodegradated by direct sunlight based radiation, with a halflife time lower than one summer sunny day. Actually, oxazepam, diazepam and alprazolam appeared to be exceedingly impervious to photodegradation with half-life times of 4, 7 and 228 summer sunny days, individually [31- 40]. months, when they were kept at - 4ºC oblivious. Benzodiazepines are steady in organic media when put away at - 20°C for a few weeks or months. Considers demonstrated that some of benzodiazepines are photolabile, and the photoinstability of alprazolam increments as the pH diminishes.

Other nitrogen molecules, as in the 7-amino subordinates, can likewise be protonated. The hydroxyl amass in the 3-hydroxy subordinates can be deprotonated at high pH values, while the N-oxide gather in 4-Noxidederivatives is protonated at low pH values. Major metabolic pathway of 1,4-benzodiazepines includes hepatic hydroxylation by means of cytochrome P450 in this way shaping hydroxy-metabolites. As of late, flubromazepam (7- bromo-5-(2-fluorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one), a moment fashioner benzodiazepine, was advertised. It seems to have a to a great degree long disposal half-existence of more than 100 h. One monohydroxylated compound and the debrominated compound could be recognized as the transcendent metabolites.

These medications vary in their pharmacokinetic and metabolic properties. Specifically, they are biotransformed by various cytochrome P450 isoforms furthermore by various UDP-glucuronosyltransferase subtypes. The most imperative reviews on the metabolic attributes of a few 1,4-benzodiazepines, completed from 1998 onwards, are accounted for and quickly talked about. Benzodiazepines tie with particular receptors in the sensory system that are the piece of GABA neurotransmitter framework. GABA (gamma-amino-butyric corrosive) is the real neurotransmitter for the support of chloride channel which controls the anxiolytic movement [41-50]. GABA receptor, creatively called the benzodiazepine site, which is unique in relation to the GABA restricting site. Authoritative of benzodiazepine subsidiaries to that specific site, improve the impact of GABA to close down cerebrum movement all the more successfully.

Bio-Test Planning and Separation of Benzodiazepines

Entire blood, serum, plasma, pee, spit, liver tissues are the natural specimens typically broke down for benzodiazepines. Tests gathered in a tube containing an anticoagulant (e.g. heparin, EDTA, citrate or oxalate) can be kept for 6 h at room temperature, or for l-2 days at 4°C. For longer stockpiling period tests ought to be solidified at - 20°C. Pee tests must be put away by solidifying at - 20°C or by the expansion of an additive specialist, for example, toluene, boric corrosive or concentrated hydrochloric corrosive [51-60]. 1,4-Benzodiazepines are generally present at follow levels (μg mL-1 or ng mL-1) in a complex natural lattice and the possibly meddling mixes should be expelled before examination. Along these lines test pretreatment including protein expulsion took after by extraction ought to be fit for concentrating the specimen and decreasing the measure of meddling substances. By and large, a solitary extraction or pre-fixation system is sufficient to lessen the example unpredictability or to enhance the LODs of the techniques. Blood, plasma and serum are frequently being deproteinized and hair needs brooding, while pee may require hydrolysis preceding the seclusion methodology. Extraction of hair was performed by overnight hatching in a blend of methanol:acetonitrile:formiate support pH 3 (10:10:80). Hair tests were extricated with a blend of water: acetonitrile: 1 M trifluoroacetic corrosive (80:10:10, v/v) utilizing a 5 min concurrent pounding/extraction step. Spit needs no deproteination as it contains protein insignificant. Enzymatic processing [hydrolysis] of plasma, pee, hair, tissue tests of benzodiazepines before extraction to free the conjugated part of the medication is prescribed. β-Glucuronidase or Gluculase [β-Glucuronidase + Sulphatase] has been utilized to discharge benzodiazepines from a conjugate. Normally plasma or serum protein precipitation comprises of blending one volume of plasma or serum with three volumes of corrosive or natural dissolvable took after by vortex-blending and centrifugation, which discharges the 1,4-benzodiazepines from protein-restricting locales evacuating 99% of the proteins. The expansion of sodium octylsulphate or sodium dodecylsulphate that disturb the structure of proteins is an option approach to discharge 1,4-benzodiazepines from proteins without precipitation. Different techniques, for example, ultramicro-filtration and harmony dialysis expel proteins from blood tests. Hair tests are washed before hatching ideally with dichloromethane or 0.1% sodium dodecylsulfate. Blend of β-glucuronidase and arylsulfatase at pH 4, or 8 M urea–0.2 M thioglycolate arrangement at pH 3 are accounted for hair brooding. An outline included expounded test planning and separation innovations for the chromatographic assurance of 1,4-benzodiazepines in natural lattices. Dried blood spots (DBS) examining has picked up prominence in the bioanalytical group as a contrasting option to traditional plasma testing, as it gives various advantages as far as test accumulation and coordinations. DBS examination is basic bringing down the expenses and ecological effect [61-70].

Electroanalytical Methods


The particle particular terminal strategy has the benefits of selectivity, effortlessness and velocity over the strategies having weaknesses, for example, absence of selectivity, need of a costly instrument or being tedious. The anodes depended on poly (vinylchloride) (PVC) or poly(esterurethane) films, doped with medication tetraphenyl borate (TPB) or medication phosphotungstic corrosive (PTA) particle match edifices as electroactive materialsThe particle specific terminal system has been ended up being touchy, solid, and can be utilized with great precision and high rate recuperation without pretreatment methodology. Cyclodextrinbased potentiometric sensors are actualized for midazolam and diazepam in pharmaceuticals. The strong contact terminals for midazolam and diazepam depend on polymeric films consolidating separately beta-cyclodextrin and (2-hydroxiproyl)- gammacyclodextrin as ionophores, 2-fluorophenyl 2-nitrophenyl ether as plasticizer and potassium tetrakis (p-chlorophenyl) borate as ionic added substance. Chlordiazepoxide particle specific anodes in view of particle partners, chlordiazepoxidium-phosphomolybdate (I) and chlordiazepoxidium-phosphotungstate (II), demonstrated a decent selectivity for the potentiometric assurance of the chlordiazepoxide in pharmaceutical readiness [71-80].


Since the 1,4-benzodiazepines are effortlessly lessened at a mercury anode through the two-electron diminishment of the 4,5-azomethine useful gathering, decrease of metals (copper, gold, platinum, and palladium), and in addition that of the ligands. Much of the time adsorption wonders happened and their impact on voltammetric signals must be deliberately investigated. The voltammetric conduct was then deciphered regarding complex arrangement. Coordinate square-wave voltammetry (SWV) and square-wave cathodic stripping voltammetry (SWCSV) at hanging mercury drop terminals have been created for assurance of the psychoactive 1,4- benzodiazepines; clonazepam, bromazepam, midazolam, diazepam, medazepam, and flurazepam. The arrangement of radical species from two benzodiazepines, loprazolam and flunitrazpam, in various media (protic, aprotic and blended media) has been analyzed utilizing cyclic voltammetry. Electron paramagnetic reverberation (EPR) spectroscopy demonstrated that both drags were lessened electrochemically to their relating nitro radical anions. Diazepam in plasma and oxazepam in pee were resolved at the ideal conditions in the voltammetric estimations utilizing adjusted carbon-glue terminals. The augmentations weakening strong state electrochemical strategy, in view of the voltammetry of microparticles approach, grants measure, by means of standard increments technique, 1,4-benzodiazepine in plans with no need of test disintegration utilizing weakening with a reference electroactive compound. In light of diminishment conduct, an immediate differential heartbeat voltammetric technique has been produced and approved for the assurance of midazolam in parenteral measurement. a logical technique in view of adsorptive cathodic stripping voltammetry (AdCSV) for the concurrent assurance of 1,4- benzodiazepines was produced utilizing a hanging mercury drop anode as a working terminal and Ringer cradle (pH 10.0) as a supporting electrolyte. Examination was performed with better accuracy, bring down location points of confinement and a great deal more quickly, and the strategies were connected to the investigation of monetarily accessible tablets with least example control [81-90].


The 1,4-benzodiazepines; medazepam, diazepam, flurazepam, nitrazepam, and clonazepam, were explored utilizing differential heartbeat polarography (DPP) and cyclic voltammetry (CV) at a mercury cathode in 0.10 M KNO3 and pH 7.0-0.1 by method for copper complexation. A computerized differential heartbeat polarography was utilized for the approval of CZE connected to the division and assurance of four benzodiazepines; chlorodiazepoxide, diazepam, flurazepam, nitrazepam alongside omeprazole and metronidazole in pharmaceutical plans.

Chromatographic Techniques

With regards to this survey accentuate has been given to TLC, micellar fluid chromatography (MLC) and micellar (electrokinetic) narrow chromatography (MECC). Expound discourse on chiral fluid chromatography (Chiral LC) has been endeavored instead of HPLC, GC strategy as later two strategies are independently surveyed. Chromatographic strategies can either be utilized to screen natural specimens for the nearness of at least one benzodiazepines, or affirm the nearness of at least one benzodiazepines taking after an underlying immunoassay or other screening test.

Superior Fluid Chromatography

There was no undeniable inclination for decision of section, with the exception of turned around stage C18 or C8, which was more predominant than the option stages. Solid sections, comprising of ceaseless beds with through pores of natural or inorganic grid, have discovered expanding applications for HPLC partitions. In any case, other pressing materials, expansion of natural modifiers or other chromatographic instruments could be considered for decreasing pinnacle following. Another HPLC polymer stationary stage, MSpak GF segment, comprising of an exceptionally crosslinked hard gel of polyvinyl liquor, set up fast and basic chromatographic strategies for breaking down benzodiazepines by direct infusion of tests (plasma, pee) without the extraction strategy or without the section exchanging method. Father identification gives an enhanced intends to bar co-eluting substances. Some encouraging outcomes have been acquired by utilizing electrochemical identifiers, however additionally work is required to perceive how these indicators contrast and the entrenched bright locators, as far as general execution. Specifically, the mix of HPLC with MS seems to offer an imperative contrasting option to traditional GC–MS for the recognizable proof new metabolites of the medications. With the presentation of more delicate identifiers (most extreme affectability 0.001 AUFS), novel section frameworks and computerization of the gear, the utilization of HPLC in benzodiazepine examination is probably going to increment. In fine HPLC, a technique for expanding significance for these objects is being utilized with bright, fluorescence or electrochemical location and even with MS applying delicate ionization interfaces like thermospray ionization (TSP), electrospray ionization (ESI), sonic splash ionization (SSI), air weight synthetic ionization (APCI) or photoionization (APPI). The utilization of HPLC-MSMS for subjective and quantitative investigation of benzodiazepines has been portrayed all the more as of late. MS modes; electrospray ionization (ESI), different response checking (MRM), data subordinate securing (IDA), improved item particle examine (EPI) were analyzed for recognition and measurement of 21 benzodiazepines in human hair tests. Electrospray ionization was observed to be more effective than environmental weight substance ionization. The utilization of a portable period of high pH brought about higher maintenance and higher electrospray ionization signals than the customary low pH versatile stages. Considering the advantages of a high pH portable stage on both chromatography and mass spectrometry, its utilization ought to be energized [91-95]. The explanation behind utilizing couple mass spectrometry is to expand furthest reaches of recognition without the requirement for concoction derivatization. Online extraction LC–MS/MS strategy has been produced for the concurrent quantitative affirmation of benzodiazepines and metabolites in pee and in hair. An examination demonstrates that the comparing LOD and LOQ qualities are around three requests of size lower in HPLC than in CZE. Ultra-execution fluid chromatography– tandem mass spectrometry (UPLC-MS/MS or TOF-MS) test strategy was created and approved for concurrent assurance of benzodiazepines in human pee, serum, plasma entire blood, hair.

Chiral fluid chromatography (HPLC) is a settled zone of bioanalytical science and is regularly utilized amid the procedures of medication disclosure and improvement. Synchronous enantioseparation of two benzodiazepines, (±)-oxazepam and ±)- lorazepam was done utilizing polymeric surfactants, poly(sodium N-undecenoxy carbonyl- L,Lleucyl-valinate) (poly-L,L-SUCLV) and dictated by Chiral micellar EKC (CMEKC) coupled to ESI-MS [96-100].


Chromatography can be kept away from by the utilization of compound duplicated immunoassay systems, however these are not particular for every medication. A scope of immunoassay techniques utilizing EIA, ELISA, FPIA, agglutination or motor cooperation of microparticles, or RIA strategies are presently accessible. Cross reactivities to benzodiazepines are variable with the end goal that nobody unit will perceive all benzodiazepines and their pertinent metabolites at fixations prone to be experienced amid helpful utilize. Earlier hydrolysis of pee to change over glucuronide metabolites to immunoreactive substances enhances location limits for some benzodiazepines. Be that as it may, it is generally non-particular, tedious and gives just semi-quantitative information.


Benzodiazepines are as of now among the most every now and again recommended medicates everywhere throughout the world. They go about as anxiolytics, tranquilizers, hypnotics, amnesics, antiepileptics and muscle relaxants. Benzodiazepines are regularly utilized for the treatment of epilepsy, shakings, and numerous psychiatric issues. The across the board utilization of this class of medications has every so often raised worry about recreational benzodiazepine mishandle and has prompted to the incorrect impression that benzodiazepines have a generally high manhandle risk among recreational medication clients. Along these lines, the division and recognizable proof of these mixes is of incredible intrigue. Techniques reported in this paper took into consideration a basic, precise, fast and reproducible evaluation of BDZs in pharmaceutical plans and additionally natural examples. The depicted methodologies may be extremely helpful for pharmaceutical lab and explores in which broad benzodiazepines examinations are performed. All in all, the division of benzodiazepines is performed utilizing highperformance fluid chromatography (HPLC).