Development and validation of spectrophotometric procedure for quantitative determination of flavonoid content used to control the quality of mixture herbal product.

Species, known as mixture herbal products, are compositions of several types of crushed, sometimes whole, medicinal plant materials with additives; they are a widely used dosage form in the Russian Federation. A large range of species are produced at the pharmaceutical companies. In pharmacopoeial analysis, the most popular and widely used method for the determination of flavonoids, suitable for the standardization of species, is the method of differential spectrophotometry, based on the complexation of flavonoids with aluminum chloride. In accordance with modern requirements for the drugs production, the validation of analytical methods is a prerequisite for the creation of pharmacopoeial monographs projects regulating the quality of pharmaceutical substances of plant origin. Therefore, it is necessary to validate analytical methods for their intended use in evaluating the drug quality. This article discusses/presents the main stages of development and validation (by parameters: accuracy, precision, specificity, linearity) of the methodology for determining total flavonoid content using original species "Fitourol" as a model. Copyright:

INTRODUCTION

Species, mixture herbal products (MHP) is one of the oldest dosage forms, which has retained its value to date due to a number of advantages. These include the presence of active ingredients in the raw material in almost native form, ease of preparation of aqueous extracts that have high affinity to the body due to the nature of the extractant and the natural origin of biologically active compounds (BAS), and low cost.[1] Species under different names (teas, herbal mixtures, MHP, herbal drug mixtures, herbal collections, herb-herb combinations, etc.,) are included in many pharmacopoeias of the world and are still used in almost all traditional medical systems.[23] In folk Russian medicine, MHP have always been one of the main dosage forms. MHP were included in all editions of the domestic pharmacopoeia of Russia.[4]

According to the State Pharmacopoeia of the Russian Federation, IV edition, MHP are mixtures of two or more types of medicinal plant raw materials of various processing methods, possibly with the addition of substances of mineral, synthetic, plant, and animal origin.[5] When standardizing one component herbal drug or multicomponent MHP, the quantitative content of one BAS group (essential oils, tannins, phenolic compounds, and flavonoids) or several BAS groups [Table 1] are assessed.[67] Growing interest in the various biological activities of herbal flavonoids and phenolics outlines the need of determination their content in herbal products.[89]

Table 1

Standardization of mixture herbal products (quantitative content of biologically active substances)

Mixture herbal product name	Composition	Standardization by the content of biologically active substances
Gastrointestinal MHP (Gastrointestinalis species)	Chamomile flowers (Chamomillae recutitae flores) 20%Peppermint leaves (Menthae piperitae folia) 20%Dill fruits (Anethi graveolentis fructus) 20%Sweet flag rhizome (Acori calami rhizomata) 20%Licorice root (Glycyrrhizae radices) 20%	Total flavonoid content in terms of rutin is not <0.9%
Choleretic MHP No 2 (Cholagogae species No 2)	Immortelle flowers (Helichrysi arenarii flores) 40%Common yarrow herb (Achilleae millefolii herba) 20%Peppermint leaves (Menthae piperitae folia) 20%Coriander fruits (Coriandri sativi fructus) 20%	Total flavonoid content in terms of rutin is not <1%
Pectoral MHPNo 3 (Pectorales species No 3)	Marshmallow root (Althaeae radices) 28.8%Licorice root (Glycyrrhizae radices) 28.0%Anise fruits (Anisi vulgaris fructus) 14.4%Pine buds (Pini silvestris gemmae) 14.4%Sage leaves (Salviae officinalis folia) 14.4%	Essential oil content is not <0.4%; Polysaccharides content is not <1.5%
Vitaminic MHP No 2 (Vitaminae species No 2)	Rose fruits (Rosae fructus) 50%Mountain ash fruits (Fructus sorbi) 50%	Ascorbic acid content is not <0.2%
Sedative MHP No 2 (Sedativae species No 2)	motherwort herb (Leonuri herba) 40%hop fruits (Humuli lupuli fructus) 20%peppermint leaves (Menthae piperitae folia) 15%valerian rhizomes with roots (Valerianae officinalis rhizomata cum radicibus) 15%licorice root (Glycyrrhizae radices) 10%	Essential oil content is not <0.2%; total flavonoid content in terms of rutin is not<0.4%
Arfazetin-E MHP(Arphasetinum-E species)	Blueberry shoots (Vaccinii myrtilli cormus) 20%Bean fruit shell (Phaseoli vulgaris fructum valvae) 20%Devil’s bush rhizomes with roots (Eleutherococci senticosi rhizomata et radices) 15%Rose fruits (Rosae fructus) 15%Horsetail herb (Equiseti arvensis herba) 10%Saint-John’s-wort herb (Hyperici herba) 10%Chamomile flowers (Chamomillae recutitae flores) 10%	Total tannin content is not <2%; organic acids content in terms of malic acid is not <3.5%
Urological (diuretic) MHP (Urologicae [diureticae] species)	Bearberry leaves (Uvae ursi folia) 40%Calendula flowers (Calendulae officinalis flores) 20%Dill fruits (Anethi graveolentis fructus) 20%Devil’s bush rhizomes with roots (Eleutherococci senticosi rhizomata et radices) 10%Peppermint leaves (Menthae piperitae folia) 10%	Arbutin content is not <3%/hydroquinone derivatives content in terms of arbutin is not <3%

MHP: Mixture herbal products

The spectrophotometric method is the most popular and widely used quantitative method for the flavonoid determination in pharmacopoeial analysis; it is suitable for MHP standardization. The total flavonoid content (TFC) is calculated in terms of the prevailing component with or without the addition of (in rare cases) the complexing agent, aluminum chloride.[9101112] Together with chromatographic fingerprint analysis, TFC can be helpful for determining the identity, consistency, and stability of herbal drugs containing multiple botanicals.[13] Spectrophotometry method has been widely used in the standardization of herbal substances for determination of various BAS groups; by itself, and also in conjunction with other methods, it allows to achieve acceptable results for standardization.[1415161718]

In accordance with modern requirements for the production of drugs, the validation of analytical methods is a prerequisite for their inclusion in the projects of pharmacopoeial monographs on pharmaceutical substances of plant origin. Therefore, it is necessary to validate analytical methods for their intended use in evaluating the quality of medicines. Evaluation of analytical methods by the validation parameters serves as the basis for the systematic approach formation to the choice of methods for assessing the content of a drug and increasing its standardization level. It cannot but affect the quality of regulatory documentation, and therefore, the quality of pharmaceutical products.[1920]

Previously, we have developed a multicomponent plant collection “Fitourol,” consisting of medicinal plant materials: Cowberry leaves (Vaccinium vitis-idaea [L.]), horsetail herb (Equisetum arvense [L.]), burdock roots (Arctium lappa [L.]), dill fruits (Anethum graveolens [L.]), wormwood herb (Artemisia vulgaris [L.]).[21] In the course of the conducted pharmacological studies, it was established that the water extract of the “Fitourol” collection has a diuretic, saluretic, antiinflammatory, antioxidant, nephroprotective, phosphorus-calcium normalizing metabolism, as well as a pronounced litholytic effect in experimental urolithiasis in laboratory animals caused by intragastric administration of sublethal doses of vitamin D2.[22] The wide range of pharmacological activity of the “Fitourol” collection makes it a promising herbal remedy for use in urological practice.

The aim of this study is to consider the main development and validation stages of methods for determining the content of TFC on the example of the original collection “Fitourol” MHP. For this MHP, a technique was developed for quantifying TFC in terms of luteolin-7-glycoside by differential spectrophotometry with a complexing agent.

MATERIALS AND METHODS

The development of a spectrophotometric method for the quantitative determination of TFC in MHP was carried out by Spectronic Helios Alfa ultraviolet-visible spectrophotometer (Thermo Electron Corporation, England) with 10 mm layer thickness cuvette. A standard sample (SS), luteolin-7-glycoside (CAS No. 5373-11-5, Sigma-Aldrich, USA) was used in the analysis. The MHP was prepared in laboratory conditions; its composition includes: Vaccinii vitis-idaeae folia 30%, Equiseti arvensis herba 30%, Arctii radices 15%, Anethi graveolentis fructus 15%, and A. vulgaris herba 10%.

In the present study, the information-analytical method and the system analysis method were used. In the course of the study, regulatory and technical documentation and validation guidelines for analytical methods were analyzed.

RESULTS AND DISCUSSION

Development of method for quantitative determination of total flavonoid content in the mixture herbal products

The method of differential spectrophotometry, based on the complexation reaction with aluminum chloride (III), is used to determine the flavonoids. As a result, a bathochromic shift of the flavonoid absorption maximum occurs. This method is suitable for the analysis and standardization of MHP, because it allows to analyze the total content of glycosides and aglycones of flavonoids of various groups.

The choice of a SS for calculating TFC is based on one condition: The flavonoid standard with aluminum chloride (III) should have a similar curve and a close absorption maximum at the same wavelength, as the absorption maximum of MHP flavonoids with the same complexing agent. Luteolin-7-glycoside was chosen as SS; it has a similar curve and a close maximum absorption in the same area of 402 ± 5 nm with a maximum absorption of flavonoids in “Fitourol” MHP with aluminum chloride (III) [Figure 1].

Figure 1

Absorption spectra of the complexes of “Fitourol” mixture herbal product flavonoids (1) and luteolin-7-glycoside standard sample solution (2) with aluminum chloride

The influence of a number of parameters is studied to select the optimal conditions for the extraction of the total flavonoids from the MHP sample. These include the nature of the extractant, fineness level, the ratio of raw materials and extractant, time and number of extraction [Table 2].

Table 2

Total flavonoid content in of the “Fitourol” mixture herbal products extracts depending on the extraction conditions

Extraction conditions	Total flavonoid content in terms of luteolin-7-glycoside (%)
Ethanol concentration (aqueous solution) (%)
40	0.33±0.01
50	0.41±0.01
60	0.43±0.02
70	0.47±0.02
80	0.46±0.01
96	0.34±0.01
Particle size (fineness level) (mm)
0,5	0.39±0.01
1	0.41±0.01
2	0.46±0.02
3	0.42±0.02
5	0.41±0.01
Raw materials: extractant ratio
1:5	0.42±0.01
1:10	0.48±0.02
1:15	0.45±0.01
1:20	0.43±0.01
Extraction time (min)
15	0.38±0.01
30	0.41±0.01
60	0.47±0.02
75	0.45±0.01
90	0.43±0.01
Number of extractions
1	0.39±0.01
2	0.46±0.01
3	0.44±0.01

Optimal extraction conditions were established experimentally. The results indicate that the optimal conditions for the analysis are: The extractant is 70% ethanol, raw materials: Extractant ratio is 1:10, particle size (fineness level) is 2 mm, double extraction for 60 min. It formed the basis for the quantitative determination method of TFC in terms of luteolin-7-glycoside in the “Fitourol” MHP. Further, the description of the method is given.

Quantitative determination of flavonoid content in terms of luteolin-7-glycoside

A sample of the raw material (analyzed MHP) is crushed to the size of particles passing through a sieve with 2 mm holes. Next, the MHP accurately weighed quantity about 10.0 g is taken, placed in a 200 ml flask, 50 ml of 70% ethanol is added. The flask with the sample in the alcohol-water mixture is heated in a boiling water bath under reflux from the moment of boiling for 1 h. After cooling, resulting extract is filtered through the filter paper into a volumetric flask (100 ml) to separate the raw material particles. The extraction is repeated with 50 ml of 70% ethanol for 1 h. After cooling, the alcohol-water extract is filtered into the same volumetric flask that was filled up to mark with 70% ethanol (solution A). 1.0 ml of solution A is placed in a 25 ml volumetric flask, 2 ml of 2% aluminum chloride (III) solution is added and flask is filled up to mark with 70% ethanol.

Preparation of the reference solution

1 ml of solution A is placed in a 25 ml volumetric flask, 0.1 ml of diluted acetic acid is added, and flask is filled up to mark with 95% ethanol (the solution should be freshly prepared).

The optical density of the obtained solutions was measured by a spectrophotometer after 40 min at a wavelength of 402 ± 5 nm in a cuvette with a layer thickness of 10 mm. The optical density of luteolin-7-glycoside SS solution is measured at the same time at a wavelength of 402 ± 5 nm.

Preparation of the luteolin-7-glycoside standard sample solution

1.0 ml of SS, luteolin-7-glycoside, is placed in a 25 ml flask, 2 ml of a 2% aluminum chloride (III) solution is added, and flask is filled up to mark with 70% ethanol. A solution containing 1.0 ml of SS luteolin-7-glycoside SS, 0.1 ml of diluted acetic acid and 95% ethyl alcohol up to 25 ml is used as a reference solution.

The TFC in the analyzed MHP in terms of luteolin-7-glycoside and absolutely dry raw materials in percent (X) is calculated by the formula:

Where

D* – Optical density of the test solution;

D – Optical density of luteolin-7-glycoside SS solution;

M* – Mass of the MHP sample, g;

M – Mass of CO luteolin-7-glycoside SS, g;

W – Mass loss on drying of the MHP sample, %.

Preparation of luteolin-7-glycoside standard sample solution

A accurately weighed quantity of CO luteolin-7-glycoside SS (0.03 g) is placed in a 100 ml volumetric flask, dissolved in 50 ml of 70% ethanol and flask is filled up to mark with 70% ethanol, stirred.

Validation of methods for quantitative determination total flavonoid content in mixture herbal products

Validation assessment of the developed methodology is estimated by the criteria: Trueness, precision (repeatability and reproducibility), specificity, and linearity.

The trueness of the method is established by checking the method with model SS solutions. Model solutions are prepared in three concentrations with a SS content in % to the initial concentration of 80%, 100%, and 120%. The determination is carried out in triplicate for each concentration. The usability criterion is the average % recovery when checking solutions of 80%, 100%, 120% concentrations, that are corrected for 100% and its average value should be in the range of 98.0%–102.0%. Luteolin-7-glycoside SS solutions were used as model solutions. The initial concentration of luteolin-7-glycoside was 0.4 mg in 25 ml. According to our results, the % recovery ranged from 99.49% to 101.99%, and its average value was 99.93% [Table 3].

Table 3

The results of validating the method by trueness

n	The real value of the measured quantity in the model mixture (mg)	Experimentally measured quantity
		Absolute value (mg)	Recovery (%)
1	0.32
1.1		0.323	100.9
1.2		0.319	99.57
1.3		0.316	98.77
Average value			99.75
2	0.4
2.1		0.394	98.49
2.2		0.402	100.43
2.3		0.408	101.99
Average value			100.3
3	0.48
3.1		0.485	101.02
3.2		0.475	98.9
3.3		0.401	100.29
Average value			99.74
The average recovery percentage for three concentrations in triplicate			99.93

The testing of method repeatability is carried out in different days using a spectrophotometer by the same specialist on one MHP sample in six replications with 100% concentration of the active substance in the test solution. The assessment of repeatability is carried out by coefficient of variation (CV). The acceptance criterion is performed when the variation coefficient for six measurements is not more than 2%. The TFC in terms of luteolin-7-glycoside in “Fitourol” MHP was 0.43%. According to our results, CV is in the range from 1.22% to 1.26% [Table 4].

Table 4

The results of method repeatability testing

Date of experiment	“Fitourol” MHP with total flavonoids content in terms of luteolin-7-glycoside of 0.43%
	№	Results of determination	Standard deviation (S)	The coefficient of variation (CV), %
1st day	1	0.42
	2	0.42
	3	0.43
	4	0.42
	5	0.42
	6	0.43
Average value		0.425	0.00516	1.22
2nd day	1	0.43
	2	0.43
	3	0.43
	4	0.43
	5	0.42
	6	0.42
Average value		0.43	0.00516	1.26

CV: Coefficient of variation, MHP: Mixture herbal products

The reproducibility is established according to the results of analysis of one collection sample in two laboratories in six replications with 100% concentration of the active substance in the test solution. The acceptance criterion is the value of the variation coefficient for six measurements, which should be no more than 2%. The TFC in terms of luteolin-7-glycoside in the collection “Fitourol” was 0.43%. The variation coefficient value for six measurements was 1.35% [Table 5].

Table 5

Results of reproducibility method testing

Laboratory	“Fitourol” MHP with total flavonoids content in terms of luteolin-7-glycoside of 0.43%
	n	Results of determination	Standard deviation (S)	The coefficient of variation CV (%)
1	1	0.42
	2	0.42
	3	0.43
	4	0.43
	5	0.43
	6	0.42
Average value		0.425	0.00548	1.35
2
	1	0.42
	2	0.43
	3	0.43
	4	0.43
	5	0.43
	6	0.42
Average value		0.43	0.00516	1.27

MHP: Mixture herbal products, CV: Coefficient of variation

The determination of linearity is carried out at five different dilutions levels of the test solution in the range of at least 80%–100% of the analyte concentration in the test solution. The criterion for acceptability of linearity was the correlation coefficient value, it should be no <0.98, and the data set can be described by a straight line. Determination of linearity was performed at five concentrations levels of luteolin-7-glycoside SS. For the analysis, an accurately weighed quantity 0.02 g of luteolin-7-glycoside SS was weighed, placed in a 25 ml volumetric flask, 20 ml of 70% ethanol was added and flask was filled up to mark with 70% ethanol (solution A). Next, 1 ml; 2 ml; 3 ml; 4 ml; 5 ml of solution A were placed in five 25 ml volumetric flasks. To the solutions 2 ml of a 2% aluminum chloride (III) solution was added and flasks were filled up to mark with 70% ethanol, stirred. The optical density of each obtained solutions was measured by a spectrophotometer at a wavelength of 402 ± 5 nm in 10 mm layer thickness cuvette. In the experiment, the linearity of the method is observed in the concentration range of 0.32–1.6 mg%; it is confirmed by a high correlation coefficient (r = 0.999) [Table 6 and Figure 2].

Table 6

The results of testing the method linearity

Optical density (D)	The concentration of luteolin-7-glycoside SS (mg %)	Correlation coefficient	Y=b × x + a
			b	a
0.130	0.32	0.999	0.397	0.006
0.265	0.64
0.385	0.96
0.515	1.28
0.640	1.60

SS: Standard sample

Figure 2

Graph of optical density versus CO concentrations of luteolin-7-glycoside. OY - optical density, OX - concentration, mg%

The specificity of the method is confirmed by MHP flavonoids' light-absorbance at a certain wavelength compared with SS. The acceptance criterion is the identity of the SS absorption spectrum of with aluminum chloride with the MHP absorption spectrum with the same complexing agent. According to the developed method of TFC quantitative determination, the colored complexes of “Fitourol” MHP and CO luteolin-7-glycoside SS were obtained. The absorption spectra of the obtained complexes were recorded by a spectrophotometer at the wavelength range from 350 to 460 nm. The presented absorption spectra [Figure 1] confirm the identity. Therefore, specificity is satisfactory. The method specificity is also proved by the method with additives. The test is carried out on one sample of collection in 3 concentrations with SS additives. The acceptance criterion is the value of the relative experimental error with additives, which must be within the random error of the method. The test was carried out on one sample of the “Fitourol” MHP in three concentrations with the addition of luteolin-7-glycoside SS. The relative error value of experiments with additives is within the random error of the method, which indicates the absence of a systematic error in TFC determination and the method specificity [Tables 7 and 8].

Table 7

The results of testing the method specificity

Number	TFC in the “Fitourol” MHP (mg)	Added luteolin-7-glycoside SS (mg)	The expected content of flavonoids (mg)	The resulting content of flavonoids (mg), average of three definitions	Error
					Absolute (mg)	Relative (%)
1	0.75	0.03	0.78	0.79	+0.01	+1.28
2	0.75	0.04	0.79	0.81	+0.02	+2.5
3	0.75	0.05	0.80	0.79	−0.01	−1.25

MHP: Mixture herbal products, SS: Standard sample

Table 8

The results of testing the method specificity

n	f	Р (%)	t (Р, f)	x̄ (%)	S2	S	Δx̄	Ē (%)
5	4	95	2.78	0.47	0.00013	0.0114	0.0141	3.04

The quantitative determination method of the TFC in the “Fitourol” MHP in terms of luteolin-7-glycoside can be positively evaluated by parameters: Trueness, precision (repeatability and reproducibility), specificity, and linearity [Table 9].

Table 9

The validation results of the quantitative determination method of total flavonoid content in the “Fitourol” mixture herbal products in terms of luteolin-7-glycoside

Number	Validation parameters	Acceptance criteria	Experimental data
1	Trueness	Recovery 98.0%-102%	99.93%
2	Repeatability	The variation coefficient is not >2%	1st day CV=1.22% 2nd day CV=1.26%
3	Reproducibility	The variation coefficient is not >2%	1st laboratory: CV=1.35 2nd laboratory: CV=1.27
4	Specificity	1. The proximity of the absorption spectra2. The relative error of experiments with additives should be within the random error of the method, which indicates the absence of a systematic error	1. The proximity of the absorption spectra of the colored complexes of the water-alcohol extraction of the studied collection and luteolin-7-glycoside SS has been proved2. The relative error of experiments with additives is within the random error of the method, which indicates the absence of a systematic error
5	Linearity and range	The correlation coefficient must be at least 0.98	0.999 (0.32-1.6) mg %

SS: Standard sample, CV: Coefficient of variation

CONCLUSION

Thus, the main stages of the development and validation of methods for the quantitative determination of the TFC in terms of luteolin-7-glycoside in the “Fitourol” MHP are considered. This algorithm can be used in the development of regulatory documentation for MHP, the main biologically active substances of which are flavonoids. The method, developed and validated according to this algorithm, can be used in setting standards and developing projects of Pharmacopoeia monographs to include the State pharmacopoeia of the Russian Federation of a subsequent edition.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.