Mediterranean Journal of
Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online
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SHORT COMMUINCATION article
A comparative study of intravenous midazolam marketed in Libya
Amal K. Belaid 1 * , Ruwida M. Kamour 1 , Zaema A. Elbaroudi 1
1
Walid EL-Nifati EL-Fetori , and Rashad A. Ghrew 2
1
Department of Medicinal and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Tripoli, Tripoli, Libya
2
Chemical unit, Drug Administration, Food and Drug Control Center, Tripoli, Libya
*
Author to whom correspondence should be addressed
Received: 12-09-2024, Revised: 28-10-2024, Accepted: 06-11-2024, Published: 31-12-2024
Copyright© 2024. This open-access article is distributed under the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
HOW TO CITE THIS
Belaid et al. (2024) A comparative study of intravenous midazolam marketed in Libya.
Mediterr J Pharm Pharm Sci. 4 (4): 9-14. [Article number: 175]. https://doi.org/10.5281/zenodo.14042921
Keywords: Assay, benzodiazepine, chromatographic, Infrared, Pharmacopeia
Abstract: Midazolam, a benzodiazepine medication, is used for sedation during diagnostic and therapeutic
medical procedures. Insufficient doses of sedatives, including midazolam, can result in patient anxiety and
awareness during the procedure. Several brands of midazolam are available in the Libyan market. This study aims
to identify and estimate the medication content of intravenous midazolam in various marketed products using
different analytical methods. Product identity was confirmed using Infrared methods (IR) and retention times of
High-Performance Liquid Chromatographic methods (HPLC). Quantification was performed using a rapid
reverse-phase HPLC method. Chromatographic analysis was conducted on a C18 column (250 mm×3.3 mm I.D.,
5.0 μm particle size) with a mobile phase comprising acetonitrile, methanol, and 0.065 M ammonium acetate
buffer (50: 20: 30, v/v/v), adjusted to a pH of 5.5±0.02 with orthophosphoric acid, at a flow rate of 1.0 ml/min.
Ultraviolet (UV) detection was set at 220 nm. The identification results met British Pharmacopeia (BP) standards.
However, the midazolam content in the Tunisian brand was shallow compared to the products from Germany and
Switzerland. Thus, post-marketing testing is essential to assess the quality of critical drugs like midazolam, and
further investigations, including clinical evaluations and regulatory follow-up, are necessary.
Introduction
Benzodiazepines exert their effects on the central nervous system (CNS) by interacting with GABAA receptors, which are
distributed across various regions of the brain. Midazolam (MDZ) a hydrophilic benzodiazepine, is highly soluble in water,
contributing to its rapid onset and short duration of action. As part of the benzodiazepine class (Figure 1), MDZ induces a
range of effects, including anxiety relief, sedation, muscle relaxation, hypnosis, and anticonvulsant activity. Compared to
barbiturates, which can lead to coma when administered in high doses, benzodiazepines are considered safer [1, 2]. MDZ’s
binding to GABAA receptors in the CNS produces an inhibitory effect, leading to its sedative and amnesic properties. With
a short duration of action (1.5-3.5 hrs), it is widely used as an anesthetic agent, particularly in intensive care units [3]. As
noted earlier, MDZ (Figure 2) is a member of the benzodiazepine family, known for its favorable efficacy and safety profile.
This highlights the importance of chemical, pharmacokinetic, and clinical studies of this class of compounds [4, 5]. MDZ
was first synthesized by Hoffmann-La Roche Laboratories in 1976, exhibiting modified properties compared to
Belaid et al. (2024) Mediterr J Pharm Pharm Sci. 4 (4): 9-14. 9
Mediterranean Journal of
Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online
www.medjpps.com ISSN: 2958-3101 print
classical benzodiazepines [6]. Incorporating a fused imidazole ring endows MDZ with increased basicity and
hydrophilicity, along with enhanced stability against metabolic degradation. The nitrogen atom in the imidazole
ring is primarily responsible for the basicity of MDZ, with a pKa of 6.15, enabling the formation of water-soluble
chloride and maleate salts. Consequently, MDZ can be formulated for parenteral administration, allowing for
intramuscular or intravenous delivery [6, 7].
Figure 1: General structure of benzodiazepines Figure 2: Chemical structure of midazolam
Unlike other benzodiazepines, MDZ demonstrates high stability against hydrolysis. Its rapid onset of action can
be attributed to its fast metabolic inactivation, facilitated by the methyl group at position one on the fused
imidazole ring [8]. Because of its precision, accuracy, and sensitivity; liquid chromatography, and especially
HPLC, is widely applicable for MDZ quantification and its metabolites in biological matrices of humans, rats,
and rabbits' plasma [9-11], as well as methods to evaluate the quality of pharmaceutical formulations, compiled
in the review [12]. This study involves the identification of MDZ using the IR scanning method, along with
quantification via HPLC. The drugs tested are listed in Table 1.
Table 1: Origin of the three drugs tested
Samples Origin
A Switzerland
B Germany
C Tunisia
Material and methods
Infrared (IR) spectroscopy: Liquid samples were extracted to obtain clearer IR spectra, and the spectra were
recorded using an IR spectrometer (Shimadzu, Japan). The extraction procedure was as follows:
1. 15.0 mg of samples B and C were weighed into separate beakers.
2. A few milliliters of 5.0 M ammonia were added to each sample.
3. The mixture was transferred to a separatory funnel.
4. For each sample, 7.5 mL of dichloromethane was added, and the mixture was shaken and allowed to
separate.
5. 7.5 mL of dichloromethane was also added, shaken again, and separated.
6. Sodium sulfate was used to dry the extract.
7. The samples were filtered using a Buchner funnel.
8. The filtrate was dried in an oven at 50°C.
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The IR absorption spectrum for MDZ was recorded using a KBr disc (1.0 mg of the substance mixed with 200
mg KBr) on an IR spectrometer (Bruker, USA). Infrared spectroscopy data revealed that IR spectra for samples
A, B, and C were recorded over the range of 4000 to 500 cm⁻¹. The findings are shown in Figures 3, 4, and 5.
Figure 3: IR spectra of sample A
Figure 4: IR spectra of sample B
Figure 5: IR spectra of sample C
The IR spectra revealed characteristic peaks corresponding to functional groups, as summarized in Table 2.
Differences in spectra suggested variability in the excipient composition across the brands, potentially
contributing to inconsistencies in drug content.
Table 2: Location of some functional groups of midazolam on IR spectra
Peak assignment Wave number cm-1
C=O 1090
C=N 1611
C-H 3200-3500
from the imidazolic heterocycle
Belaid et al. (2024) Mediterr J Pharm Pharm Sci. 4 (4): 9-14. 11
Mediterranean Journal of
Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online
www.medjpps.com ISSN: 2958-3101 print
High-Performance Liquid Chromatographic Methods (HPLC analysis): In this study, HPLC analysis was
performed using a UFLC system (Shimadzu, Japan). The chemicals used, including orthophosphoric acid and
methanol, were supplied by Carlo Erba Reagents (France), and triethylamine was also utilized. These reagents
were critical in ensuring the precise measurement of MDZ concentrations across different products [9].
Chromatographic conditions: The method for MDZ assay was adapted from BP 2008 guidelines using HPLC
(UFLC, Shimadzu, Japan). The analysis utilized an SPD-20AV detector, with a mobile phase consisting of 77:28
(v/v) methanol and an equal volume mixture of 0.1 M orthophosphoric acid and 0.03 M trimethylamine.
Separation was carried out on an Ascentis C18 column with dimensions of 250 mm length and 4.6 mm internal
diameter. The flow rate of the mobile phase was maintained at 1.0 ml/min, with the oven set to 40°C, and detection
was performed at a wavelength of 220 nm. The injection volume was 3.0 μl. Due to the absence of a reference
standard, product A was utilized as the reference for comparison with the other two products [9].
Sample preparation: Each ampoule contained 5.0 mg/ml of MDZ. For preparation, the content of three ampoules
was mixed (15 mg in total), and a volume equal to 10 mg of each product was transferred into a 50 ml volumetric
flask, and the mobile phase was added to reach a final volume of 50 ml. This will prepare a solution containing
0.01%w/v of MDZ. The resulting solutions were filtered using a Pall 0.2 μm GHP Acrodisc 13 mm filter and
injected into the HPLC apparatus for analysis [9].
uV(x100,000)
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 6: HPLC data (midazolam chromatogram); Red: A; Black: B; Blue: C.
Figure 6 shows the HPLC chromatograms for the three MDZ products, identified by the following color codes:
Red: Product A, Black: Product B and Blue: Product C. In all chromatograms, a peak at approximately 5.8 min
(retention time) corresponds to MDZ, confirming its identity in products B, and C, relative to the reference product
A. The peak areas were used to calculate the percentage recovery of MDZ in the injections. The content of MDZ
in product B was found to be 86.6%, while product C had a significantly lower content of 46.5% when compared
to product A, Table 3.
Belaid et al. (2024) Mediterr J Pharm Pharm Sci. 4 (4): 9-14. 12
Mediterranean Journal of
Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online
www.medjpps.com ISSN: 2958-3101 print
Table 3: Chromatographic data for A, B and C products
Product Retention time (min) Area % recovery
A 5.8 2181439 Reference
B 5.7 1889937 86.6%
C 5.8 1015042 46.5%
Discussion
As product A was sourced from a reliable manufacturer, the results for products B and C were compared to it.
The identification results for all three products were comparable, with similar IR spectra, and retention times,
suggesting that the chemical identities of the drugs were alike [11, 12]. However, more investigations for
possibilities of degradation or the presence of impurities are needed especially with the appearance of the small
peak at 1.5 min observed with product C, Figure 6, and BP [12]. These impurities may have risen from
substandard manufacturing processes or inappropriate storage or handling and therefore need to be studied
thoroughly according to ICH guidelines [13, 14]. The chromatographic data showed similarities in the chemical
structure of the main compound in the three products. However, the variation in peak areas and heights indicated
that product C had significantly lower MDZ content. These stark differences highlight the need for testing
multiple batches from the same manufacturer to ensure consistent quality. Appropriate actions, such as stricter
regulation or potential withdrawal of certain batches, should be considered if further testing shows substandard
content.
Conclusion: Midazolam (Product C) is widely used in Libyan clinics, despite the significant discrepancy in drug
content. Considering these results, further investigations into different batches are essential along with different
chemical methods of analysis and clinical studies. Moreover, feedback reports must be provided to regulatory
authorities to ensure quality control management, and subsequently, stricter measures may be necessary for
pharmaceutical products that do not meet acceptable standards, including potential withdrawal from the market.
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Author contribution: AKB &WEE conceived and designed the study. AKB, ZAE, WEE & RAG collected and analyzed data.
AKB, RMK & ZAE performed data analysis and interpretation. AKB & RMK drafted and revised the manuscript. All authors
approved the final version of the manuscript and agreed to be accountable for its contents.
Conflict of interest: The authors declare the absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Ethical issues: Including plagiarism, informed consent, data fabrication or falsification, and double publication or submission
were completely observed by the authors.
Data availability statement: The raw data that support the findings of this article are available from the corresponding author
upon reasonable request.
Author declarations: The authors confirm that all relevant ethical guidelines have been followed and any necessary IRB and/or
ethics committee approvals have been obtained.
Belaid et al. (2024) Mediterr J Pharm Pharm Sci. 4 (4): 9-14. 14
