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FARMACIA, 2009, Vol.LVII, 1
CONTRIBUTIONS TO THE ANALYTICAL
STUDY OF MIDAZOLAM
CAMELIA MALANCIUC, CORINA ARAMĂ, IOANA ŞARAMET,
CRINA-MARIA MONCIU*, MARINELA FLOREA, GEORGE MIHAI
NIŢULESCU
University of Medicine and Pharmacy “Carol Davila” Bucharest,
Faculty of Pharmacy, 6th Traian Vuia str.
*corresponding author: crinamonciu@yahoo.com
Abstract
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.
Rezumat
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 acidobazice în soluţie şi principalele metode de dozare.
Keywords: midazolam; analytical properties; assay methods
Introduction
Midazolam belongs to a relatively new class of benzodiazepine
derivatives which contains a pentaatomic heterocyclic ring condensed in
position 1,2 [1] (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 [2].
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 [3].
FARMACIA, 2009, Vol.LVII, 1
25
Description
Nomenclature
Chemical name: 8-chloride-6-(2-florphenyl)-1-methyl-4H-imidazole
[1,5-a][1,4]benzodiazepine (it is used as maleate and hydrochloride).
International nonproprietary name: midazolamum
Proprietary names: Dormicum®, Dormonid®, Hypnovel®, Ipnovel®,
Midolam®, Versed®.
Structure, chemical formula and molecular mass
C18H13C1FN3=325.8
C18H13C1FN3, C4H4O4(maleate) = 441.9
C18H13C1FN3, HCl (chloride hydrate) = 362.2
Physical properties
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)[5].
It is practically, insoluble in water, easily soluble in acetone and
ethanol, soluble in methanol.
Mass spectrum
The main ions, identified depending on the m/z ratio, are: 310, 312,
311, 163, 325, 75, 39, 297 [6].
IR spectrum
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).
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FARMACIA, 2009, Vol.LVII, 1
Figure 1
IR spectrum of midazolam
Table I
The main bands in the IR absorption spectrum of midazolam
Wave number (cm-1)
Attribution
3435
H2O
3140-3120
υC-H
(from the imidazolic heterocycle)
3027
υCaril-H
(aromatic ring)
2960
υasimCH3
2925
υasimCH2
2847
υsimCH3
1611
υC=N
1580,
C-C)aril
1486
C-C)aril
1453
C-C)aril
1415
υC-Cl
769
υC-F
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).
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27
Figure 2
UV absorption spectrum of midazolam
Thermal analysis
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.
Distribution coefficient
The distribution coefficient n-octanol/water (Koctanol/water) of
midazolam, determined at room temperature, is approximately 2·104 [6].
Figure 3
TG and DTG curves of midazolam
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FARMACIA, 2009, Vol.LVII, 1
Figure 4
The DSC curve of midazolam
Solution properties
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 [6], and the second, Kp =
2·10-2, is due to the nitrogen atom in position 5 in the benzodiazepinic ring [7].
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.
H3 C
H3 C
NH+
N
N
+
N
H / H2 O
NH3 +
HO-
O
Cl
N
Cl
F
F
-OMe
MeOH
tBuOK
H3 C
H3 C
N
N
Cl
N
H
-
N
N
+
H
N
Cl
H
F
Figure 5
The hydrolysis processes of midazolam
F
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FARMACIA, 2009, Vol.LVII, 1
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 [8]. The midazolam photodecomposition products obtained by
irradiation with a mercury lamp, at high pressure, are shown in Fig. 6.
H3 C
H3 C
N
N
N
N
Cl
N
Cl
N
H
O
F
CH3
N
NH2
O
N
N
H
Cl
N
Cl
O
H3 C
H
Cl
N
Cl
O
F
F
F
F
CH3
N
Cl
O
N
O
Figure 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.
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FARMACIA, 2009, Vol.LVII, 1
Table II
The results of the assay of constituting elements
C
H
N
Calculated
66.36
4.02
12.90
Found
66.16
3.92
12.99
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 [9].
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 [10].
Chromatographic determinations
Thin layer chromatographic methods
The European Pharmacopoeia 4th ed. [9] 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.
H3 C
H3 C
N
N
H3 C
N
COOH
N
N
Cl
Cl
N
H
F
N
N
H
H
Cl
F
N
F
Figure 7
Synthesis impurities
The identification of midazolam is made by comparing the
sample’s chromatogram with the chromatogram of the reference substance.
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31
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.
Gas-chromatographic methods
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
1hydroxymethylmidazolam -active metabolite-, 4-hydroxymidazolam and 4hydroxy-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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33
34
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
FARMACIA, 2009, Vol.LVII, 1
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Manuscript received: 20.07.2008