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13DOI: 10.5281/zenodo.3958549 rv i?=S~7f)?
UDC: 543.544.5:547.794.3 Open £ Access
Development and validation of the high-pressure liquid chromatographic method for the quantitative determination of propylthiohinothiadiazole
Andrei Uncu
Scientific Center for Drug Research, Nicolae Testemitanu State University of Medicine and Pharmacy
Chisinau, the Republic of Moldova
Author's ORCID iD, academic degrees and contributions are available at the end of the article
"Corresponding author: [email protected] Manuscript received July 28, 2020; revised manuscript August 14, 2020; published online August 26, 2020
Abstract
Background: Propylthiohinothiadiazole is a thiadiazole derivative with pronounced antimycobacterial action. Substance is researched in order to develop methods of analysis and standardization, analytical documents regulating quality for use as an active principle in the development of pharmaceutical forms. Material and methods: 3 series of propylthiohinothiadiazole, internal reference standard of 2-(propylthio)-5H-[1,3,4]-thiadiazole[2,3-b]-quinazolin-5-one substance with concentration 99.98%, chromatographic system Shimadzu LC-20AD high-pressure liquid chromatography (HPLC) with UV-VIS detector, acetonitrile and dimethylsulfoxide of purity grade "pro HPLC analysis" (Sigma Aldrih and Merck), bidistilled purified water. Results: Linearity is proved for concentrations of 5-30 ^g/ml, the linear regression equation is y=36472x-44580, R2=0.9999. The limit of detection is 0.729 ^g/ml and the limit of quantification is 2.210 ^g/ml. It was established that the method is accurate (mean recovery values at 80%, 100% and 120% concentration levels were close to 100%). The accuracy of the method was expressed by repeatability and intermediate accuracy. The variation of the chromatographic conditions established that the method is robust. For all validation parameters, relative standard deviation was less than 1. Conclusions: The validation results show that the developed HPLC method is simple, fast, accurate and reproducible. Key words: high-pressure liquid chromatography, propylthiohinothiadiazole, dosing, validation.
Cite this article
Uncu A. Development and validation of the high-pressure liquid chromatographic method for the quantitative determination of propylthiohinothiadiazole. Mold Med. J. 2020;63(3):32-37. doi: 10.5281/zenodo.3958549.
Introduction
Tuberculosis remains one of the most common infectious diseases worldwide. According to the World Health Organization (WHO), there is an incidence of 10 million cases, which would be equivalent to 133 cases per 100.000 people [1]. The Republic of Moldova is among the 18 countries in the European Region where tuberculosis control is a priority and among the 30 countries in the world with a high burden of multidrug-resistant tuberculosis (MDR TB) [2]. Currently, the WHO recommends 11 common international drug names for the treatment of tuberculosis, of which 4 are first-line (isoniazid, rifampicin, pyrazinamide, ethambutol) and 7 are second-line (aminoglycosides, qui-nolones, thioamides, etc.) [3, 4 ].The evolution of pharmaceutical technologies and clinical trials have reduced the duration of treatment for tuberculosis from 24 months to 6-8 months of intensive treatment, the basic method being the administration of therapeutic combinations, with preparations from the first and second line simultaneously [5]. However, the latest studies show a rate of resistance to treatment in about 73% of patients, 35% of whom have multi-drug resistance since the first weeks of treatment [3]. There are several causes: non-compliance with treatment; stopping or abandoning treatment at the first reduction of symptoms, then resuming it after a while; late diagnosis;
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mycobacterial mutations over time and lack of alternative medication [3, 4, 6].
Thus, finding and obtaining new compounds with an-timycobacterial potential is an important and very current research topic. A promising direction is the development of new substances with a high activity against MDR myco-bacteria by valuing thiadiazole (quinazolinone) derivatives with structures similar to Triptantrine alkaloid [7, 8].
Propylthiohinothiadiazole (2-(propylthio)5H-[1,3,4] thiadiazolo-[2,3-b]quinasolin-5-one) is a thiadiazole derivative, synthesized in the Laboratory of Organic Synthesis and Biopharmaceuticals of the Institute of Chemistry, Chisinau,being one of the most promising substances in the series of about 80 derivatives obtained. This compound has a pronounced antimycobacterial activity (p99%) and low toxicity [9, 10, 11]. The substance is being researched in order to develop methods of analysis and standardization, of the Analytical Documents of Quality Standardization (DAN) for use as an active principle in the development of antimycobacterial pharmaceutical forms.
Evolutionary development of analytical methods provides researchers a wide range of modern, advantageous and cost-effective techniques. High-pressure liquid chromatography (HPLC) is one of them, being based on the separation of components between two phases under pressure,
allows the identification and assay of individual substances in pharmaceutical forms, but also the possible impurities from synthesis or degradation. Quantitative determination of medicinal substances is one of the most important stages of the analysis, being also the main quality parameter. The requirements assigned to a quantitative method in the process of pharmaceutical analysis are stipulated in various guidelines, which regulate the analytical procedures. The validation of analytical methods, in accordance with the guidelines of the International Conference on Harmonization (ICH), aims to "demonstrate that the method is appropriate to the proposed purpose" [12].
Propylthiohinothiadiazole, being a newly-synthesized compound, has no formalized methods of analysis. Thus, this study proposes the development for the first time of the HPLC method for propylthiohinothiadiazole assay and its validation in accordance with the provisions of the ICH guidelines.
Material and methods
The experimental researches were performed within the Laboratory of analysis, standardization and control of drugs (LASCD) of Nicolae Testemitanu State University of Medicine and Pharmacy.
The elaboration of the propylthiohinothiadiazole assay method was performed based on the requirements of the ICH guideline "Q2R1: For analytical and validation procedures" [12].
The study used the drug substance propylthiohinothia-diazole, synthesized in the Laboratory of Organic Synthesis and Biopharmaceuticals of the Institute of Chemistry, synthesis series: MF1-001, MF1-002, MF1-003; internal reference standard for 2-(propylthio)5H-[1,3,4]thiadiasolo-[2,3-b] quinasolin-5-one substance, purified by recrystallization from LASCM (concentration 99.98%).
Apparatus: Shimadzu LC-20AD HPLC chromatographic system, Zorbax Eclipse Plus C18 analytical column, 5 mm, 4.6 x 250 mm was used; UV-VIS detector, wavelength -300 nm.
Chemicals: Acetonitrile (ACN) purity grade "pro HPLC analysis" (Sigma Aldrich), dimethylsulfoxide (DMSO) purity grade "pro analysis" (Merck), bidistilled purified water.
Chromatography conditions: The mobile phase was prepared by mixing ACN with purified water in proportions of 80:20 by volume, filtering the solution under vacuum through a Millipore XF 5423050 capron filter (0.2-0.45^m), and degassing in the DONAU-LAB SONIC DLS 660 T/H. The temperature of the chromatographic column was 30°C; injection volume 20 ^l; mobile phase flow - 1 ml/min. The retention time was 4.5 min.
Preparation of the standard solution: Approximately 0.001 g (exact mass) of the internal reference standard is placed in a 25.0 ml volumetric flask, dissolved in 5 ml of DMSO, stirred until dissolved, 10 ml of mobile phase is added and mixed, then made up with the same solvent to the quota and mixed (stock standard solution). Place 5 ml
of standard stock solution in a 10 ml volumetric flask and make up to the mark with the same solvent. The solution is used freshly prepared.
Chromatographic system utility control: Before performing the analysis, the chromatographic column is stabilized with mobile phase for 10 min, then chromatographed 20 ^l standard solution until an established retention time (4.5 min) is obtained after two consecutive injections; at least 3 chromatograms are obtained. The chromatographic system is considered useful when: the performance of the column, calculated after the propylthiohinothiadiazole peak, is at least 2000 theoretical plates; the asymmetry coefficient of the peak, calculated at the level ^ of the peak height does not exceed 1.5; the relative standard deviation of the peak area does not exceed 2.0%.
Preparation of the sample solution: Approximately 0.001 g (exact mass) of propylthiohinothiadiazole was accurately weighed and transferred into a 25.0 ml volumetric flask, 5 ml of DMSO was added and stirred until the substance dissolved. 10.0 ml of mobile phase was added, then it was mixed and made up to the level with the same solvent. 5.0 ml of obtained solution was placed into a 10 ml volumetric flask and made up to the mark with the same solvent.
Quantitative determination: Each 20 ^l of sample solution and standard solution were chromatographed, obtaining at least 5 chromatograms of each solution under the same conditions indicated above. Then it was determined the mean value of the propylthiohinothiadiazole peak area from the chromatograms of sample solution (San) and standard solution (Sst). The chromatograms of the standard and sample solutions with a concentration of 20 ^g/ml are shown in fig.1.
The propylthiohinothiadiazole content (X%) in the substance is determined according to the formula:
X% =
S *mt * W *P*[100-U] *100
/in cf tltl fl L J
S *m
st a
* a *Wf * 100
x st
in which:
San - the mean value of the peak area, calculated from the chromatograms of the sample solution;
Sst- the average value of the peak area, calculated from the chromatograms of the standard solution; mp - mass of the substance, g; Us - humidity of the standard substance, %; m - mass of standard substance, g; P0 - active substance content, %;
W „ and W - the volumes of solutions for the standard
st an
and the analyte.
Validation of the method. The method was validated according to the ICH guide, being determined by the parameters: linearity, accuracy, precision, sensitivity (LOQ and LOD) and robustness, solution stability [12].
Linearity. The linearity of the results of an analysis procedure represents its ability to obtain results directly proportional to the analyte concentration in the sample. The linearity of the HPLC method of propylthiohinothiadiazole assay on the concentration ranges 5-30 ^g/ml was investigated. Thus, the standard stock solutions of propylthiohi-
nothiadiazole 40 ^g/ml were initially prepared. From the stock standard solution, 5 samples with concentrations 5, 10, 15, 20 and 30 ^g/ml were prepared by dilution, using mobile phase as solvent and then were injected into the chromatograph, obtaining chromatograms and peak areas. The determinations were performed in triplicate, being constructed by the calibration curve (fig. 2). Linear regression analysis was used to evaluate the linearity of the calibration curve using the least squares method.
Accuracy. Accuracy is defined as a characteristic of the approximation of analytical results to true value and is a measure of the deviation of the mean value found by analysis from true value. It is evaluated by applying the method to samples with known concentrations [13]. To determine the accuracy of the HPLC propylthiohinothiadiazole assay method, the standard addition (sample enrichment) method was used by analyzing in triplicate solutions with concentrations of 80%, 100% and 120% (8.0 ^g/ml, 10 ^g/ml and 12.0 ^g/ml) and the percentage recovery of the amount of substance was calculated, the relative standard deviation (RSD) value being evaluated for each concentration level (tab. 3).
Precision. Determination of the precision of the method was performed by evaluating repeatability and intermediate accuracy [14]. Repeatability was determined for 6 samples, at the concentration level of the drug substance of 100%, on the same day, respecting the same conditions (tab. 4). The intermediate accuracy was determined using the same procedure for identical samples, in the same laboratory, by different operators, using different equipment and in a specific time interval. The intermediate accuracy was investigated in 2 different days, under the same conditions, performing 6 determinations (tab. 5).
Sensibility (LOQ and LOD). The limit of detection (LOD) and limit of quantification (LOQ) of propylthiohi-nothiadiazole were determined by analyzing the substance solutions and measuring the signal-to-noise ratio. The limit of detection (LOD) is the concentration, which is due to the signal/noise ratio of about 3:1, while the limit of quantification (LOQ) is the concentration that gives a signal/noise ratio of about 10:1 with RSD values (n = 3) less than 10%.
Robustness. The robustness of an analytical method is demonstrated by assessing the ability of the method to remain unaffected by small, deliberate variations in parameters. It was determined by varying the following chromato-graphic conditions: flow rate of the mobile phase by ± 0.1 ml/min, amount of acetonitrile in the mobile phase by ± 2% and column temperature by ± 5°C [12] (tab. 6).
Stability of the solution. The stability of standard and sample analytical solutions was determined by analyzing them immediately after preparation and after 24 hours of refrigeration and room temperature (25°C). Three determinations were performed, the peak areas were evaluated, the analyte concentration in the sample (relative to a freshly prepared reference solution) and the RSD were calculated (tab. 7).
Statistical analysis. Statistical analysis was carried out by using the Statistical Package for the Social Sciences (IBM SPSS Statistics) 10.5 software.
Results and discussion
Method Development and Optimization. Preliminary study of the physicochemical properties of propylthiohi-nothiadiazole allowed the development of the HPLC analytical method by preliminary selection of chromatographic conditions, including detection wavelength, mobile phase, stationary phase, and sample preparation procedure. For this purpose, a series of tests were performed varying the ratio between acetonitrile and water, optimizing the chromato-graphic conditions on the Zorbax Eclipse Plus C18 column, 5 |im, 4.6 x 250 mm. The results of the method optimization are summarized in tab. 1. The mobile phase consists of acetonitrile and water in a ratio of 80:20 v/v with a flow rate of 1 ml/minute, injection volume 20 ^l, running time 6 minutes and column temperature 300C at wavelength (X) 300 nm. It was determined that these are the optimal conditions under which propylthiohinothiadiazole was eluted forming symmetrical peaks, resolution, and adequate analysis time, with retention time around 4.5 minutes (fig. 1.A and fig. 1.B).
Fig. 1.A. Chromatograms of propylthiohinothiadiazole solutions with concentration 20 ^g/ml: A - standard solution.
B S ft ? i -, Oj fif . ti« , « , « . / s Dtt.A Ch1 «0 *
Fig. 1.B. Chromatograms of propylthiohinothiadiazole solutions with concentration 20 ^g/ml: B - sample solution.
Linearity. By evaluating the averages of the peak areas obtained in the HPLC determinations, represented in relation to the corresponding concentrations, the calibration graph was obtained. The results of the linearity study (tab. 2, fig. 2) showed a linear relationship over the concentration range of 5-30 ^g/ml for propylthiohinothiadiazole.
From the regression analysis, a linear equation was obtained: y=36472x-44580, and the correlation coefficient (r2) was 0.9999, which indicates a linear relationship between the analyte concentration and the area of the chromato-graphic peak.
Table 1
Results of the optimization of the HPLC method of propylthiohinothiadiazole dosing
Used Column Mobile phase Flow rate Wavelength Observation Result
Nucleosil 100, C-18, 15 |im., 4 x 150 mm Acetonitrile: water 80:20 1.0 ml/min 23S nm Poor resolution 1.3 Method rejected
Zorbax Eclipse Plus C18, 5 mm, 4.6 x 250 mm Acetonitrile: water 80:20 1.0 ml/min 300 nm Good resolution 2.4 Method accepted
Zorbax Eclipse Plus C18, 5 mm, 4.6 x 250 mm Acetonitrile: water 6S:3S 1.0 ml/min 300 nm Poor resolution 1.8 Method rejected
Table 2
Calibration data for propylthiohinothiadiazole
Solution concentration, ng/ml Retention time, min Peak area
S 4.483 13S014.0
10 4.487 2706S7.0
1S 4.471 40S1S8.0
20 4.477 S41687.0
30 4.479 823649.0
0 5 10 15 20 25 30
Concentration, [ig'inl
Accuracy. Determining the accuracy of the analytical quantitative procedure, the selectivity of the results obtained in this method was found to achieve the true value. As in-
Fig. 2. Calibration curve of standard propylthiohinothiadiazole solution.
Table 3
Results of accuracy of developed method of assay of propylthiohinothiadiazole
Concentration levels, % Number of determinations Theoretical concentration of spiked sample, pg/ml Peak area Concentration in spiked sample, ng/ml % Recovery Average, % RSD
80 1 8 6746764.SS 8.04S 100.S6 100.S30
2 8 6747213.28 8.046 100.S8
3 8 6739989.94 8.037 100.46 0.061
100 1 10 84334SS.69 10.04S 100.4S 100.710
2 10 846027S.02 10.077 100.77
3 10 8472697.44 10.091 100.91 0.240
120 1 12 10110S46.81 12.033 100.28 100.260
2 12 10108824.S7 12.031 100.26
3 12 1010S489.14 12.027 100.23 0.031
Note: RSD - relative standard deviation
dicated in table 3, RSD values are between 0.03-0.24. The triplicate analysis of solutions with concentrations of 80%, 100% and 120% (8.0 ^g/ml, 10 ^g/ml and 12.0 ^g/ml) demonstrated a percentage recovery of the substance of at least 100.23% and maximum 100.91%, values that were within the accepted limits from 98.0% to 102.0%, which indicates the applicability of the method for quantitative analysis.
Precision. The precision expresses the narrowness of the agreement (degree of dispersion, coefficient of variation) between a series of measurements that come from several series of the same homogeneous sample (independent results) under identical working conditions. Precision provides data on random errors and has no relation to the true value. Because all measurements contain random errors, the result of a single measurement cannot be accepted as true. An estimate of this error is required to predict the range in which the true value is found, which is done by repeating
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the measurement several times. From this process two important parameters are obtained, namely the average value and the variability of the measurements [12]. The results of the determinations showed that the method is accurate within acceptable limits. RSD was calculated for retention time, peak area and concentration, all values being less than 1% (tab. 4 and tab. 5).
Sensibility: detection limit and quantification limit (LOD and LOQ). The limit of detection (LOD) is the smallest amount of analyte in a sample that can be detected, but not necessarily quantified, while the limit of quantity (LOQ) is the smallest amount of analyte in a sample that can be determined quantitatively with adequate accuracy [15]. The results of the determinations showed LOD of 0.729 ^g/ ml and LOQ of 2.210 ^g/ml respectively.
Robustness. The robustness of the analytical method was tested to assess the lack of influence of minor changes
Table 4
Results of determinations of repeatability of the HPLC method for propylthiohinothiadiazole assay
No Retention time, min Peak area Assay, %
1 4.483 84334SS.69 100.4S
2 4.487 846027S.02 100.77
3 4.471 8472697.44 100.91
4 4.477 8439984.0S 100.S3
5 4.479 84334SS.69 100.4S
6 4.484 8439984.0S 100.S3
Average 4.480 8446641.98 100.60
RSD, % 0.128 0.191 0.188
Table 5
Results of intermediate precision determinations in the validation of the propylthiohinothiadiazole HPLC method
Day 1 Day 2
No. Retention time, min Peak area Assay, % Retention time, min Peak area Assay, %
1 4.483 84334SS.69 100.4S 4.49S 9275724.35 101.73
2 4.487 846027S.02 100.77 4.937 9303352.96 102.03
3 4.471 8472697.44 100.91 4.922 9189046.32 100.78
4 4.477 8439984.0S 100.S3 4.9S6 9388533.41 102.96
5 4.479 84334SS.69 100.4S 4.988 9307879.46 102.08
6 4.484 8439984.0S 100.S3 4.943 9268835.80 101.6S
Average 4.480 8446641.98 100.60 4.949 9288895.38 101.87
RSD, % 0.128 0.191 0.188 0.4S0 0.698 0.698
Table 6
Results of the determination of the robustness of the HPLC method for dosing propylthiohinothiadiazole
Variable parameters Retention time, min Peak area Assay, %
Mobile phase flow 0.9 ml/min 4.482 8434755.24 99.72
0,5 4.472 8461005.87 100.03
Acetonitrile: purified water 80:20 4.486 8473097.57 100.17
Acetonitrile: purified water 81.6:18.4 4.479 8439784.07 99.78
Column temperature 300 C 4.481 8432915.21 99.70
Column temperature 350 C 4.482 8438584.13 99.77
The average value 4.480 8.45E+06 99.86
Standard deviation S2 2.19E-0S 2.70E+08 3.77E-02
S 0.00S 16418.795 0.194
Relative standard deviation RSD 0.104 0.194 0.194
Table 7
Results of solution stability testing for HPLC propylthiohinothiadiazole assay method
Determined parameters Storage period Retention time, min Peak area Assay, % RSD for the peak area
Standard solution 0 h 4.483 84S7894.12 - 0.03
24 h at 2S0C 4.479 84S7387.08 100.09 0.17
24 h at 40C 4.482 84S7614.21 100.10 0.07
Sample solution 0 h 4.484 84347SS.24 0.0S
24 h at 2S0C 4.484 843291S.21 99.70 0.21
24 h at 40C 4.479 8433089.S7 99.71 0.11
in the conditions of chromatography using for obtaining the results of the analysis. The test results showed that at a minor change in the method conditions, such as the composition and flow rate of the mobile phase, the temperature of the column, the method is robust. A good separation has been achieved, and the RSD values are within the limits and do not exceed 2.0% (tab. б).
Stability of the solution. After testing the stability of the solution, the concentration of propylthiohinothiadiazole in the solutions varied from 99.7% to 100.1% and the RSD was not higher than 1.0%, which indicates a high stability of the sample and standard solutions during 24 hours both by refrigeration and under normal conditions. The results of the solution stability test are presented in Table 7.
Conclusions
The HPLC method of assay of propylthiohinothia-diazole was developed and validated, which proved to be simple, fast, accurate and precise, sensitive and robust. The selected chromatographic conditions and mobile phase provide a good resolution for the test substance. The retention time does not exceed 5 min. During the experiments, the rigors of the ICH guidelines regarding the validation of the analysis methods were taken into account. The developed and validated HPLC method can be included in the Quality Standardization Analytical Documents for the propylthio-hinothiadiazole quantitative determination.
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Author's ORCID iD and academic degrees
Andrei Uncu, PharmD, PhD Applicant - https://orcid.org/0000-0002-6419-2596. Author's contribution
AU designed the study, conducted the laboratory work and performed the analytical part of the laboratory work, interpreted the data, drafted, revised and approved the manuscript.
Funding
This study was supported by Nicolae Testemitanu State University of Medicine and Pharmacy. The trial was the author's initiative. The author is
independent and takes responsibility for the integrity of the data and accuracy of the data analysis.
Ethics approval and consent to participate
No approval was required for this study.
Conflict of Interests
No competing interests were disclosed.
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