The object of the present study is a comprehensive characterization of midazolam, from the analytical and physico-chemical point of view, based on the data in the literature and on our own research. The study presents physical constants, mass spectra, IR and UV absorption spectra, thermal analysis, acid-base properties in solution and the main methods used for the assay of midazolam.
Lucrarea prezintă, pe baza datelor din literatură şi a cercetărilor proprii, o caracterizare cuprinzătoare a midazolamului, din punct de vedere fizico-chimic şi analitic. Se prezintă constante fizice, spectrele de masă, IR, UV, analiza termică, proprietăţile acido-bazice în soluţie şi principalele metode de dozare.
Midazolam belongs to a relatively new class of benzodiazepine derivatives which contains a pentaatomic heterocyclic ring condensed in position 1,2  (the first pharmacologically active benzodiazepine, chlordiazepoxid, was synthesized in 1957 by L.H. Sternbach and L.O. Randall).
Midazolam contains an imidazolic ring condensed in position 1,2 and was synthesized by Hoffmann-La Roche Laboratories in 1976. This ring induces its specific properties, such as: strong basic character due to the nitrogen atom in the cycle, resulting in water soluble salts formation (it is used as maleate and hydrochloride), increased stability to hydrolytic degradation and a much faster metabolic inactivation compared to other classic benzodiazepines, mainly by the oxidation, both at the methyl group, and the imidazolic ring level .
Midazolam has hypnotic, sedative and anxiolitic action, as well as serious amnesic properties. It is widely used as an anesthetic due to its short half time (1.5 – 3.5 hours) and in artificially ventilated patients sedation in intensive care units. Midazolam has also anticonvulsivant properties .
Chemical name: 8-chloride-6-(2-florphenyl)-1-methyl-4H-imidazole [1,5-a][1,4]benzodiazepine (it is used as maleate and hydrochloride).
Midazolam is a crystalline, white or pale yellow powder, with a weak specific odour, melting point 161-164°C (colorless crystals obtained from ether-dichloromethane and hexane melt at 158-160°C).
It is practically, insoluble in water, easily soluble in acetone and ethanol, soluble in methanol.
The main ions, identified depending on the m/z ratio, are: 310, 312, 311, 163, 325, 75, 39, 297 .
The IR absorption spectrum of midazolam was recorded in a KBr disc (1mg substance in 400 mg KBr), using a Bruker spectrometer (Fig. 1).
The main absorption bands were attributed (Table I).
IR spectrum of midazolam
The main bands in the IR absorption spectrum of midazolam
Wave number (cm-1)
(from the imidazolic heterocycle)
The UV absorption spectrum
The UV absorption spectrum of the midazolam was registered using a UV-Vis Kary 100 bio spectrophotometer (Varian Inc.) and an ethanolic sample solution with 4.08·10-5 g midazolam/mL (Fig. 2).
UV absorption spectrum of midazolam
Thermogravimetric (TG), differential thermogravimetric (DTG) and differential scanning calorimetry (DSC) curves were recorded using a Nicolette equipment, in an aluminum crucible (4.94 mg sample). The temperature range was 20-700°C and 60-200°C, respectively, with a 10°C/min heating rate.
The TG and DTG curves (Fig. 3) indicate the thermal stability of the substance up to 196°C.
The DSC curve (Fig. 4) indicates, through the endothermic peak at 164°C, the melting, with a 78.55 J/g thermal effect.
The thermal behavior (TG, DTG, DSC curves) indicates the thermal stability of midazolam.
The distribution coefficient n-octanol/water (Koctanol/water) of midazolam, determined at room temperature, is approximately 2·104 .
TG and DTG curves of midazolam
The DSC curve of midazolam
In aqueous solution, midazolam ionises in steps. The first ionization step, due to the nitrogen atom in position 2 on the imidazolic ring, is characterized by a protolysis constant Kp = 6,31·10-7 , and the second, Kp = 2·10-2, is due to the nitrogen atom in position 5 in the benzodiazepinic ring .
Depending on the pH value, midazolam hydrolysis in aqueous solution is due to the azomethinic group from the benzodiazepine ring (Fig. 5). The cycle opens in acidic solution, and at pH>4 the benzodiazepinic structure recovers.
The hydrolysis processes of midazolam
It has been studied the photochemical decomposition of midazolam, in aqueous solution irradiated with a mercury lamp at high pressure. The decomposition products were isolated and identified. The pH and the irradiation source influence over the photo decomposition process were investigated . The midazolam photodecomposition products obtained by irradiation with a mercury lamp, at high pressure, are shown in Fig. 6.
Midazolam photodecomposition products
Methods of analysis
Assay of constituting elements
The quantitative determination of carbon, nitrogen and hydrogen in the structure of midazolam was made with a Perkin Elmer 2400 series II CHNS/O elemental analyser. The obtained values, compared to the theoretical ones, are presented in Table II.
The results of the assay of constituting elements
Protometric titration in anhydrous medium
Midazolam can be titrated as a diprotic base, using a mixture of anhydrous acetic acid and acetic anhydride as titration medium, in the presence of perchloric acid. The equivalence point is determined potentiometerically .
Spectrometric determinations in visible
Literature quotes indirect procedures in order to determine midazolam, through derivation, in the presence of different reagents, by visible spectrometric methods. Thus, from the reaction of midazolam with 2,3-dichloro-5,6-dicyano-p-benzoquinone results a red-orange charge transfer complex, with an absorption maximum at 462,5 nm; this complex allows the determination of the substance in bulk and in pharmaceutical dosage forms .
The European Pharmacopoeia 4th ed.  indicates the identification of midazolam and of related substances through thin layer chromatography (TLC). From the synthesis of midazolam, the main related substances are shown in Fig. 7.
The identification of midazolam is made by comparing the sample’s chromatogram with the chromatogram of the reference substance. As adsorbent, silica gel GF254 is used and for development, the solvent mixture glacial acetic acid/water/methanol/ethyl acetate (2/15/20/80, v/v). Plates are examined in UV light at 254 nm.
Most of the methods in literature investigate midazolam, the synthesis impurities and/or some metabolites, through gas-chromatography and HPLC.
The gas-chromatographic methods in literature are focused upon the determination in biologic fluids (plasma, urine), using the electron capture detection or mass spectrometric detection [11–13; for other references, please contact the authors].
High performance liquid-chromatographic methods
In liquid chromatographic methods quoted in literature, most frequently reversed phase chromatographic systems, the detection is made, either through mass spectrometry or through UV spectrometry [14-16, for other references, please contact the authors].
The HPLC methods with mass spectrometric detection were used in the separation, the detection and quantification of midazolam and some of its metabolites from different biologic liquids (in the human body the midazolam is transformed through hydroxylation into 1-hydroxymethylmidazolam -active metabolite-, 4-hydroxymidazolam and 4-hydroxy-1-hydroxymethylmidazolam), as well as in pharmacokinetic studies.
Thus, midazolam was determined from pig plasma, using detomidine as internal standard; from human plasma, together with its active metabolite, 1-hydroxymidazolam, after solvent extraction using diethyl ether, with an acetonitril/ammonium acetate (pH 4,7) mixture as mobile phase, and prazepam as internal standard (detection limit: 0,65 ng/mL) or in human hair, using a mixture of acetonitril/phosphate tampon 10-1 M; with the solvent system water/acetonitril with 1% acetic acid added. Also, it was determined, in a mixture with other anesthetics, in dog plasma, using as eluent a mixture of methanol/ ammonium acetate 0,1M (3:2).
In Table III the types of samples and the characteristics of the chromatographic systems which use the UV detection, quoted in the specific literature for the determination
of midazolam are presented.
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