The metrological parameters of diosmin quantitation in pharmaceutical dosage forms

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Introduction

Several drugs based on the pharmaceutical substance diosmin (3ʹ,5,7-trihydroxy-4ʹ-methoxy flavone-7-rutinoside) have been registered in the Russian Federation [1]. The corresponding pharmacopoeial monographs propose a quantitative determination of the main active ingredient and specific impurities using high-performance liquid chromatography to control their quality.

An urgent issue is the analytical method development using simple methods of analysis that are available in routine practice, with acceptable metrological characteristics [2]. Spectrophotometry is used as an alternative variant of the quantitative analysis of diosmin [3].

This study aimed to determine the metrological characteristics of the method for the quantitative determination of diosmin by spectrophotometry in some drugs.

Materials and methods

The objects of the study were drugs based on the pharmaceutical substance diosmin, namely Venarus, Detralex (tablets and suspension), and Phlebopha.

A pharmacopoeial standard sample of diosmin (Hyderabad, India) was used as a comparison sample.

Sample preparation of the analyzed drugs was performed by extracting sodium hydroxide solution at a concentration of 0.02 mol/L. Ultraviolet spectrophotometry was used for the quantitative determination of diosmin. The values of specific absorption indices at wavelengths of 268 and 370 nm were determined based on the corresponding equation parameters of the calibration dependences.

Statistical processing of analytical data was performed using the methods of variation statistics, correlation, one-way analysis of variance using computer programs ChemMetr 1.0, ChemMetr Evaluation 1.0 [4, 5], and Statistica 6.0 (Statsoft Inc., USA) [6].

The theoretical value of the convergence limit RSD_r of the investigated quantitative determination methods was calculated according to the Horwitz equation [7]:

RSD_r = 0.67 · RSD_R; RSD_R = 2^{1 – 0.5 · log C}; С = w/100;

where w is the concentration of the analyte in the sample, % (wt.); RSD_R is the reproducibility limit, %; and C is the concentration factor.

The theoretical value calculation of the standard deviation S_av and the relative error of the mean ε_av, based on the convergence limit RSD_r of the method was performed according to the equations:

S_av = (RSD_r · x_av) · 100; ∆x = S_av · t (P, f); ε_av = (∆x/x_av) · 100,

where x_av is the average value of the analyte content in the sample; t (P, f) is the Student’s t-test; and Δx is the half-width of the confidence interval of the analyte content mean value.

The calculation of the relative error ε_av of the method for quantitative analysis of diosmin was performed by the equation:

$\epsilon =\sqrt{{\left(\frac{{\sigma }_{x1}}{x_1}\right)}^2+{\left(\frac{{\sigma }_{x2}}{x_2}\right)}^2+...+{\left(\frac{{\sigma }_{xn}}{x_n}\right)}^2},$

where σ_x1, σ_x2, and σ_xn are the values of the absolute measurement errors of the individual stages of the quantitative determination procedure; x₁, x₂, and x_n are the values of the measured quantities during the implementation of the quantitative determination method.

Results and discussion

The choice of the extractant for sample preparation of the analyzed drugs was determined by the solubility of diosmin. Diosmin is practically insoluble in water and ethyl alcohol. The solubility of diosmin in sodium hydroxide solutions is due to the ionization of phenolic hydroxyl groups (formation of phenolates). Therefore, a sodium hydroxide solution at a concentration of 0.02 mol/L was used for the preparation of standard sample solutions of diosmin and its extraction from the analyzed drugs.

The technique working range with the linear nature of the optical density depending on the diosmin concentration was 0.0001%–0.001% and 0.0002%–0.002% for analytical wavelengths of 268 and 370 nm, respectively.

The specific absorption indices values of diosmin at wavelengths of 268 and 370 nm in a sodium hydroxide solution at a concentration of 0.02 mol/L were 463.0 ± 24.6 and 259.0 ± 9.9, respectively.

The relative errors of the above absorption indices, which were determined in five series of parallel determinations and affected the overall error of the determination results, were 5.3% and 3.8%.

The metrological characteristics of the method for the quantitative determination of diosmin by spectrophotometry are presented in Table 1.

 

Table 1. The metrological parameters of diosmin quantitation in pharmaceutical dosage form by means of UV-spectrophotometry / Таблица 1. Метрологические характеристики методики количественного определения диосмина методом спектрофотометрии в некоторых лекарственных препаратах

Drug/Wavelength	μ, mg%	f	xav, mg%	S2	S	P	t (P, f)	Dx	ε, %	εav, %	δ, %
Detralex susp./268 nm	0.9	9	0.92	0.021	0.15	0.95	2.26	0.33	36.06	11.40	1.79
Detralex susp./370 nm	1.8	9	1.69	0.022	0.15	0.95	2.26	0.34	19.82	6.27	5.82
Venarus/268 nm	0.9	9	0.86	0.01	0.098	0.95	2.26	0.22	25.80	8.16	4.48
Venarus/370 nm	0.9	9	0.89	0.011	0.10	0.95	2.26	0.24	26.38	8.34	1.10
Detralex tabl./268 nm	0.9	9	0.92	0.015	0.12	0.95	2.26	0.28	30.06	9.51	2.09
Detralex tabl./370 nm	0.9	9	0.86	0.006	0.076	0.95	2.26	0.17	20.09	6.35	5.04
Phlebopha/268 nm	0.6	9	0.60	0.011	0.10	0.95	2.26	0.24	38.99	12.33	0.29
Phlebopha/370 nm	1.2	9	1.20	0.02	0.14	0.95	2.26	0.32	26.31	8.32	0.32

Note. μ: true (accepted reference) level of diosmin concentration in the pharmaceutical dosage form; x_av: average diosmin concentration in pharmaceutical dosage form (experimental value); f: degrees of freedom; S²: dispersion; S: standard deviation; P: confidence level; Δx: confidence interval half-width of the average diosmin concentration; ε: relative single measurement error, %; ε_av: relative average value measurement error; δ: relative mean value bias of diosmin concentration in pharmaceutical dosage form from the true value of the concentration.

Примечание. μ — истинное значение содержания диосмина в лекарственном препарате (принятое опорное значение); x_сред — среднее значение содержания диосмина в лекарственном препарате, определенное экспериментальным путем; f — число степеней свободы; S² — дисперсия; S — стандартное отклонение; P — уровень доверительной вероятности; ∆x — полуширина доверительного интервала среднего значения; ε — относительная ошибка (погрешность) единичного определения; ε_сред — относительная ошибка (погрешность) среднего значения; δ — относительное отклонение среднего значения содержания диосмина в лекарственном препарате от истинного значения содержания (систематическая ошибка).

 

A significant difference was noted in the relative error of the mean value at two analytical wavelengths for all analyzed drugs, so that the value of the determination error at a wavelength of 268 nm was statistically significantly greater than that of 370 nm, except for the Venarus.

The relative error in determining the average value of the diosmin concentration was within 8%–12% for the analytical wavelength of 268 nm and 6%–8% for 370 nm.

The results of one-way analysis of variance confirm the presence of statistically significant differences for the parameter “relative error of the mean value of the diosmin concentration in the pharmaceutical dosage form,” and the significance level of the F-criterion was <0.05. The bias of determinations did not significantly differ at analytical wavelengths of 268 nm and 370 nm (Table 2).

 

Table 2. Findings of variance analysis for comparison of the relative mean error and bias to quantitate diosmin in the drugs by UV-spectrophotometry / Таблица 2. Результаты дисперсионного анализа для сравнения относительной ошибки среднего значения и систематической ошибки при определении диосмина в лекарственных препаратах методом спектрофотометрии

Parameters	Total sample variance SStot	Effect variance MSef	Intergroup variance SS	Error variance MSerr	F-criterion	Significance level p
εav 268 nm / εav 370 nm	18.36	18.36	14.60	2.43	7.54	0.033
δ268 nm / δ370 nm	1.65	1.65	31.91	5.32	0.31	0.60

Note. ε_av: relative error of average value; δ: relative deviation (bias) of diosmin average concentration in pharmaceutical dosage form from the true value of the concentration.

Примечание. ε_сред — относительная ошибка (погрешность) среднего значения; δ — относительное отклонение среднего значения содержания диосмина в лекарственном препарате от истинного значения содержания (систематическая ошибка).

 

The analysis of the correlation dependences “relative error of the mean” and “systematic error” in the sample of results combined for two analytical wavelengths show the presence of a high relationship, as the value of the correlation coefficient r is −0.73.

The above dependencies, calculated for analytical wavelengths of 268 nm and 370 nm, are characterized by a very high relationship, with the values of the correlation coefficients r of −0.94 and −0.98, respectively. Therefore, an increased relative error of the diosmin content mean value in drugs is associated with an increased contribution of random errors, including errors in sample preparation (varying the degree of diosmin extraction). The magnitude of the systematic determination error does not depend on the wavelength used by the analyst.

A predictive calculation of the relative error of diosmin extraction degree (sample preparation) was performed using the example of the analysis of Detralex tablets, which was based on the following principles:

• considering the minimum levels of errors at each stage of the analysis, except for the extraction of diosmin from a weighed sample of tablets;
• availability of experimental data on the value of the total relative error in determining diosmin;
• square additivity of the relative errors of individual stages of the analysis (Table 3).

 

Table 3. The error evaluation of diosmin quantitation in Detralex^® tablets using UV-spectrophotometry / Таблица 3. Метрологическая оценка ошибки методики количественного определения диосмина в таблетках «Детралекс» методом спектрофотометрии

No.	Analysis stage	Absolute error σX	Relative error (σX/x) · 100, %
1	Taking an accurate sample of 1.0 g	0.0002 g	0.020
2	Measuring the volume of 250 ml (volumetric flask)	0.3 ml	0.12
3	Dispensing a 1 ml aliquot (pipette)	0.01 ml	1.00
4	Measuring the volume of 500 ml (volumetric flask)	0.5 ml	0.10
5	Measurement of optical density at 268 nm (spectrophotometer SF-2000)	0.004	0.93
6	Error of the specific absorption index value, 268 nm	–	5.30
7	Calculated diosmin recovery: ChemMetr Evaluation 1.0 program/Horwitz equation	–	7.95/16.60
Calculated relative error εav, % (excluding the sample preparation relative error, ChemMetr Evaluation 1.0 program)			5.2
Calculated convergence limit RSD_r, % (Horwitz equation)			7.7
Calculated relative error εav, % (Horwitz equation)			17.4
Relative error εav, %, determined during the experiment			9.5

 

The relative sample preparation stage error, namely the degree of extraction of diosmin from Detralex tablets was 8%. The variant of calculating the total relative error of analysis according to the algorithm based on the Horwitz equation reached 17%.

Regardless of the used prognostic calculation variant, the stage of sample preparation makes the greatest contribution to the error of the method of spectrophotometric determination of diosmin in Detralex tablets.

Conclusion

Therefore, as a research result, the metrological characteristics of the method for the quantitative determination of diosmin by spectrophotometry in a number of drugs were determined. The contribution values to the relative error of the specific absorption technique indices of diosmin at analytical wavelengths of 268 and 370 nm, as well as the extraction degree of diosmin from the analyzed dosage forms (for example, Detralex tablets analysis), have been established. The proposed computational algorithms can be implemented for the theoretical assessment of the sample preparation error for other multi-component objects of analysis in the quality control of medicines.

The authors declare no conflict of interest.

About the authors

Alexander V. Voronin

Samara State Medical University

Author for correspondence.
Email: dimmu2000@mail.ru

Candidate of Pharmaceuticals Sciences, Associate Professor, Head of the Department of Chemistry of Pharmaceutical Faculty

Russian Federation, Samara

Maksim N. Kachalkin

Samara State Medical University

Email: maxroso@yandex.ru

Assistant of the Department of Chemistry of Pharmaceutical Faculty

Russian Federation, Samara

Alexander V. Karpov

Samara State Medical University

Email: karpov.sasha111@gmail.com

Student of Pharmaceutical Faculty

Russian Federation, Samara

References

Supplementary files