Selection of the Optimal conditions for analysis of plant active Pharmaceutical Inulin Ingredients by Thin-layer Chromatography to develop the draft for the National Monograph “Inulin”

 

Nataliia M. Smielova1*, Svitlana M. Gubar2, Olga A. Yevtifieieva1, Elena M. Bezchasnyuk2

1Department of Pharmaceutical Chemistry, National University of Pharmacy, Kharkiv, Ukraine

2State Scientific Research Laboratory for Drug Quality Control, National University of Pharmacy, Kharkiv, Ukraine

*Corresponding Author E-mail: smelova08@gmail.com

 

ABSTRACT:

Inulin is a representative of the class of soluble dietary fibers. One of the stated methods for identification of substances in the monograph “Inulin” of the British Pharmacopoeia 2010 (ВР) is the method of thin-layer chromatography (TLC), in the State Pharmacopoeia of Ukraine (SPhU) the method of TLC for identification of plant active pharmaceutical ingredients (API) of inulin is absent. The Pharmacopoeia provides only quality requirements of such monosaccharides as “Fructose”, “Anhydrous glucose”, “Glucose, monohydrate”, “Anhydrous lactose”, “Lactose monohydrate”. The experimental studies were conducted using nine herbal substances of inulin obtained from plant sources such as chicory, Jerusalem artichoke and agave and two standard substances of inulin with chicory and dahlia. Identification and determination of related impurities of plant API of inulin using TLC were performed according to the method of the monograph “Inulin” of the BP and the method of determination of monosaccharides given in the SPhU. The optimal conditions for the chromatographic determination have been chosen; validation of the method using indicators of specificity, robustness and precision has been conducted. The validated method for analysis of plant API of inulin using TLC has been proposed to be included into the draft for the monograph “Inulin” of the SPhU in the sections “Identification” and “Test for purity”.

 

KEYWORDS: inulin, quality control, standardization, identification, thin-layer chromatography.

 

 


INTRODUCTION:

Inulin is a representative of the class of soluble dietary fibers. It is a rather common polysaccharide on the territory of Ukraine; its largest amount is stored in the roots of elecampane, chicory, in bulbi of Dahlia pinnata, Heliantus tuberosus, etc1,2. Today, inulin is used as a diagnostic agent in determining the glomerular filtration rate in renal disease, as well as a prebiotic, which is able to reach parts of the large intestine almost unchanged and stimulate the growth of the beneficial microflora1,2,3,4. In the literature, there are data on hypoglycemic, hypocholesterolemic, immunomodulatory and other properties of the polysaccharide, which makes it a promising source for development of new drugs5,6,7.

 

 

By its chemical structure, inulin is a mixture of oligo- and polysaccharides with the number of fructose residues from 2 to 60 in D-fructofuranose configuration connected mainly to the β(2→1)-glycoside bonds, and can have one terminal glucose molecule without restorative properties (Fig. 1)1,3,4,8.

 

Fig.1: The chemical structure of inulin

Both the degree of polymerization of the links (the number of fructose residues) and the purity degree of polysaccharide affect the pharmacological activity of inulin. In medical practice the use of high-purity API of inulin is the most reasonable, since the presence of impurities reduces the therapeutic efficacy and affects the safety of the finished product1,4.

 

Currently, there are no national standard documents, which would contain the methods for the quality control of inulin substances. In this regard, standardization issues, including selection of the optimal methods of identification and purity determination of inulin API that would be able to control quality at all stages of production of finished pharmaceutical products and be suitable for detection of a possible adulteration is a topical problem of the study.

 

Thus far, the quality of the inulin substances is controlled by the monographs “Inulin” of the United States Pharmacopoeia 2013 (USP) and BP. However, as noted in works previously published, approaches to standardization in these Pharmacopoeias are somehow different6,8,9,10.

 

One of the stated methods for identification of substances in the monograph of BP is the method of TLC based on determination of fructan by its structural monosaccharides – D-fructose and D-glucose formed after hydrolysis of the main substance10. In the SPhU the TLC method for identification of inulin API is absent. The Pharmacopoeia provides only quality requirements of such monosaccharides as “Fructose”, “Anhydrous glucose”, “Glucose, monohydrate”, “Anhydrous lactose”, “Lactose monohydrate”11. The comparative characteristic of the methods for determination of inulin in accordance with BP and monosaccharides according to the SPhU is given in Fig. 2.


 

Fig.2: The comparative characteristic of the TLC methods for inulin analysis according to BP and monosaccharides according to the SPhU


When developing the national monograph “Inulin”, it is important not only to identify the main substance, but to make a conclusion about the purity of the substance analyzed. It is the TLC method that is the most versatile, sensitive and economical from this point of view12,13,14.

 

However, to introduce the method in the sections Identification” and “Test for purity”, it is necessary to select the most optimal conditions of chromatography, adapt the method to the level of equipment of national laboratories and validate the method of analysis, which guarantees accuracy of the results obtained.

 

The aim of this work is to determine the optimal conditions for analysis of plant inulin API by the TLC method and validation characteristics (specificity, robustness and precision) of the method in order to develop the draft for the national monograph “Inulin” in the sections Identification” and “Test for purity”.

 

MATERIAL AND METHODS:

The experimental studies were conducted using nine herbal substances of inulin obtained from plant sources such as chicory (samples 01, 04, 05, 06, 07, 09), Jerusalem artichoke (sample 03) and agave (samples 02, 08). Chromatography was carried out in accordance with the requirements of the TLC methods in the monographs “Inulin” of the BP and monographs “Fructose”, “Anhydrous glucose”, “Glucose monohydrate”, “Anhydrous lactose”, “Lactose monohydrate” of the SPhU (Fig. 2)6,10,11.

 

TLC plates:

The following plates were used: TLC Silica gel 60 (Merck) on the aluminum substrate; TLC Silica gel 60 (Merck) on the glass substrate; Silica gel on TLC (Supelco) on the aluminum substrate.

 

Plates were tested for the chromatographic separation ability in accordance with the requirements of the SPhU 2.2.2711: the solution for determination of TLC plates R suitability representing a mixture of bromocresol green, methyl orange, methyl red and Sudan red G was applied, then it was chromatographed in the solvent system of methanol Rtoluene R (20:80). The TLC plate is considered to be suitable if it contains four clearly separated areas where Ris in the tolerance range.

 

Mobile phase:

For the analysis two separate mobile phases were chosen:

·      Mobile phase 1: Glacial acetic acid R – chloroform R – water R (70:60:10) – given in the BP, monograph “Inulin”10.

·      Mobile phase 2: Water R – methanol R – glacial acetic acid R – ethylene chloride R (10:15:25:50) – given in the SPhU, monographs “Fructose”, “Anhydrous glucose”, “Glucose monohydrate”, “Anhydrous lactose”, “Lactose monohydrate”12.

 

Test solutions (TS):

·      TS-1 – solution of inulin (for samples 1-9): dissolve 0.20 g of the inulin substance in hot water R, allow to cool, dilute to 20.0 mL with water R and mix.

·      TS-2 – solution of inulin after hydrolysis (for samples 1-9): dissolve 0.20 g of the inulin substance and 0.8 g of oxalic acid R in hot water R, boiling for 10 minutes, allow to cool, dilute to 20.0 mL with water R and mix.

 

Reference standard substances (markers):

Due to the fact that it was planned to apply the TLC method not only to identify inulin and determine the purity of the substance in addition to the standard substances – D-fructose and Dglucose specified in the BP other markers that were frequent admixtures when obtaining substances of inulin (lactose, sucrose) or used in adulteration of fructan (maltodextrin, Sigma-Aldrich, cat. No. 419672, MFCD00146679) were also selected. The results were also assessed compared to standard substances of inulin with chicory (Sigma-Aldrich, cat. No. I2255, SLBQ7169V) and with dahlia (Sigma-Aldrich, cat. No. I3754, SLBN1201V).

 

Reference solutions (RS):

·      RS-1: dissolve 0.1 g each of fructose CRS, glucose CRS, lactose CRS and sucrose CRS in water R and dilute to 20 mL with water R and mix.

·      RS-2: dissolve 0.20 g of the inulin with chicory in hot water R, allow to cool, dilute to 20.0 mL with water R and mix.

·      RS-3: dissolve 0.20 g of the inulin with dahlia in hot water R, allow to cool, dilute to 20.0 mL with water R and mix.

·      RS-4: dissolve 0.20 g of the inulin with chicory and 8 g of oxalic acid R in hot water R, boiling for 10 minutes, allow to cool, dilute to 20.0 mL with water R and mix.

·      RS-5: dissolve 0.20 g of the inulin with dahlia and 8 g of oxalic acid R in hot water R, boiling for 10 minutes, allow to cool, dilute to 20.0 mL with water R and mix.

·      RS-6: dissolve 0.10 g of the maltodextrin in water R, allow to cool, dilute to 20.0 mL with water R and mix.

 

Chromatography:

The chromatographic procedure was performed according to the requirements of the SPhU “2.2.27. Thin-Layer Chromatography”12.

 

 

Drying, derivatization:

After the process of elution and drying in a flow of warm air, derivatization of the analyzed compounds was carried out using the following reagents:

·      The mixture of aniline R, diphenylamine R and orthophosphoric acid R – according to the method of the BP10;

·      The solution of thymol R in the mixture of sulfuric acid R96 % ethanol – according to the method of SPhU11.

 

Drying of the plates after derivatization was conducted in a drying cabinet at a temperature of 130 ± 10oC for 5-15 min. The result was assessed in the daylight. The stability of the chromatographic results was studied for 1 hour.

 

The validation characteristics (specificity, robustness and precision) were studied in accordance with the requirements of the general monograph of the SPhU 5.3.N.2 “Validation of analytical methods and tests”12.

 

RESULTS AND DISCUSSION:

Test for separation of the stationary phase:

The primary objective of the study was to evaluate the TLC plate separation12. It has been proven that the components of the solution for determining the suitability of TLC plates R are clearly separated in all plates analyzed. Therefore, all plates proposed can be used for further analysis.

 

Substantiation for the mobile phase selection:

The optimal mobile phase for the analysis of inulin API and its hydrolysis products is the mixture of glacial acetic acid R – chloroform R – water R (mobile phase 1) in the ratio of 70:60:10, it provides complete separation on all TLC plates proposed. The eluent used in the SPhU method for the analysis of monosaccharides appeared to be unsuitable for separation of the mixture components in the solution of inulin after hydrolysis (TS-2). Therefore, mobile phase 1 was used for further studies.

 

Specificity:

Validation of the method for analysis was started with determination of specificity13, which was performed on one plate compared to the standard substances for test solutions of inulin before and after hydrolysis.

 

In the mobile phase of glacial acetic acid – chloroform – water (70:60:10) there were chromatographic areas for all the samples of inulin after hydrolysis (TS-2) and for inulin from chicory (RS-4) and dahlia (RS-5) after hydrolysis at the level of areas of the standard substances of fructose and glucose (RS-1). Thus, it confirms the structural composition of the polysaccharide (Fig. 3).

 

Fig. 3: The chromatogram of the tested API of inulin after hydrolysis, TS-2 (1-9); inulin from chicory after hydrolysis, RS‑4 (10); inulin from dahlia after hydrolysis, RS-5 (11), solution of the standard substances of fructose, glucose, sucrose and lactose, RS-1 (12); solution of the standard substance of maltodextrin, RS-6 (13) on a Silica gel 60 TLC plate (Merck)

 

The specificity in comparison with the possible impurities of inulin and compounds used in adulteration of fructan was determined. Simultaneously, solutions of inulin API (1-9) prior to hydrolysis (TS-1), solution of markers of fructose, glucose, sucrose and lactose (RS-1); solutions of inulin from chicory (RS-2) and dahlia (RS-3) and solution of maltodextrin marker (RS-6) were applied on one TLC plate. The results of the specificity determination are presented in Fig. 4.

 

Fig. 4: The chromatogram of the tested inulin API prior to hydrolysis, TS-1 (1-9); inulin from chicory prior to hydrolysis, RS‑2 (10); inulin from dahlia prior to hydrolysis, RS-3 (11); solution of the standard substances of fructose, glucose, sucrose and lactose, RS-1 (12); solution of the standard substance of maltodextrin, RS-6 (13)  on a Silica gel 60 TLC plate (Merck)

 

As can be seen from Fig. 4, on the chromatogram of inulin substances without hydrolysis there were chromatographic areas at the level of the standard substances of fructose, glucose, sucrose and lactose, as well as other chromatographic areas. The areas of marker substances and test solutions were completely separated, coincided by their coloring and location, indicating the separation specificity of the components of the mixtures.

 

According to the results of the chromatograms (Fig. 3, 4), this method can be used not only for identification (by the products of inulin hydrolysis), but also for determination of the purity of the inulin substances analyzed (by solutions of inulin prior to hydrolysis).

 

Robustness:

The next step was to determine robustness; for this purpose the effect of chromatographic conditions (the effect of the stationary phase type, saturation of the chamber, the volume of application, the distance from the starting line to the finish line, elution frequency, duration of hydrolysis, the effect of the developer, stability of solutions for application) on the final result was assessed12,14,15.

 

Stability of solutions before chromatography:

The stability of the solutions studied was assessed in parallel on the same TLC plate. The solution of inulin API before hydrolysis (TS-1) and after hydrolysis (TS-2) was applied 3 hours before (Fig. 5, areas – 1, 3) and immediately prior to chromatography (Fig. 5, areas – 2, 4). The resulting individual areas did not differ against each other by location, color, intensity and shape, indicating the stability of the solutions analyzed within a specified period of time.

 

Fig. 5: Stability of test solutions of inulin API prior to hydrolysis, TS-1 (1, 2); after hydrolysis, TS-2 (3, 4); solution of the standard substances of fructose, glucose, sucrose and lactose, RS-1 (5): 1, 3 – samples applied 3 h prior to chromatography; 2, 4 – samples applied immediately before chromatography on a Silica gel 60 TLC plate (Merck)

 

The effect of the substrate type:

For analysis TLC plates of different manufacturers (“Merck”, “Supelco”), coated with silica gel on the aluminum and glass substrates, were used. The effect of the substrate type on the result of the separation ability of the components of the mixture and variation of Rf values for the corresponding areas was assessed. The separation of the mixture components was typical for all the plates (Fig. 6). From the standpoint of economy and convenience, it was advantageous to use TLC Silica gel 60 (Merck) and Silica gel on TLC (Supelco) plates with the aluminum substrate.

 

Fig. 6: The effect of the substrate type: А – TLC Silica gel 60 (Merck) on the glass substrate; В - TLC Silica gel 60 (Merck) on the aluminum substrate; С – Silica gel on TLC (Supelco) on the aluminum substrate; 1 – TS-1; 2 – TS-2; 3 – RS-1 on a Silica gel 60 TLC plate (Merck)

 

The effect of the volume of the solution applied:

The study compared the results obtained when applying test solutions and reference solutions, the volumes of their samples were 5 μl, 6 μl, 7 μl and 8 μl. The samples were applied in strips using a Hamilton microsyringe, their size was 10 mm × 2 mm (Fig. 7).

 

Fig. 7: The effect of the volume of the solution applied: 1-4 – solution of inulin API prior to hydrolysis, TS-1; 5-8 – solution of inulin API after hydrolysis, TS-2; 9-12 – solution of the standard substances of fructose, glucose, sucrose and lactose, RS-1; 13-16 – solution of the standard substances of maltodextrin: 1, 5, 9, 13 – 5 μl; 2, 6, 10, 14 – 6 μl; 3, 7, 11,14 – 7 μl; 4, 8, 12, 16 – 8 μl on a Silica gel 60 TLC plate (Merck)

 

As we can see, at low values of the substance quantity, applied on the plate, the areas poorly develop, and with large volumes there is blurring of the chromatographic areas and the change of their shape. Therefore, the optimal volumes for application were 5.0 μl for TS-1 (50 µg), TS-2 (50 µg), RS-2 (50 µg), RS-3 (50 µg), RS-4 (50 µg), RS-5 (50 µg), RS-1 (25 µg each of the marker of fructose, glucose, sucrose and lactose), RS-6 (25 µg). The width of the chromatographic area of samples allowing to obtain reproducible results was 10 mm × 2 mm.

 

The effect of the plate activation:

The analysis was carried out by both the preliminary activation of plates with 0.3 % solution of sodium acetate, followed by drying in the flow of warm air and without immersion into the appropriate solution. The conclusion was made that the most effective and complete separation of the compounds analyzed was achieved on the plates preliminary impregnated with sodium acetate solution. This is due to the ability of carbohydrates to form complexes with inorganic components and the best fixation to the stationary phase.

 

Before using, the plates were also activated by heating for 10-15 min at the temperature of 100-105 ºС to remove the residual moisture, which could reduce the activity of the sorbent16.

 

The effect of the chamber saturation:

The study was conducted in the chromatographic chamber saturated and unsaturated with the eluent. It was found that due to the chamber saturation the process of elution was faster, i.e. the time of chromatography reduced.

 

The effect of the distance that the mobile phase should pass:

Such distances of the solvent front as 10 cm, 12 cm and 15 cm were compared (Fig. 8). The optimal value is 10 cm, since the increase in the distance that the mobile phase should pass does not significantly affect the separation ability, however, it increases the duration and cost of the analysis.

 

Fig. 8: The effect of the distance that the mobile phase should pass: А – 8 cm; В – 10 cm; С – 12 cm; D – 15 cm; 1 – solution of inulin API prior to hydrolysis , TS-1; 2 – solution of inulin API after hydrolysis, TS-2; 3 – solution of the standard substances of fructose, glucose, sucrose and lactose, RS-1 on a Silica gel 60 TLC plate (Merck)

 

Elution frequency:

The chromatographic profile of separation of the substance areas depending on the elution frequency on different plates was compared. It was found that when using the eluent twice and drying the plate in a flow of warm air between each immersion into the mixture of the mobile phase the optimum and complete separation of the components of the test solutions and reference solutions was achieved. This is confirmed by the corresponding Rf values for solutions of the markers (Tab. 1).

 

Table 1: The dependence of Rf values for standard substances of fructose, glucose, sucrose and lactose on the plates of various types depending on the elution frequency.

The area analyzed

Elution frequency

once

twice

The area corresponding to fructose

0.347 ± 0.05

0.438 ± 0.05

The area corresponding to glucose

0.303 ± 0.05

0.381 ± 0.05

The area corresponding to sucrose

0.213 ± 0.03

0.274 ± 0.03

The area corresponding to lactose

0.135 ± 0.03

0.165 ± 0.02

 

Determination of the optimal time for hydrolysis:

To confirm the structural composition of inulin the necessary condition is hydrolysis of the substance to the constituents of monosaccharides. The best time for destruction of inulin with the solution of oxalic acid was determined by comparing chromatographic profiles in 5, 10, 15, 20, 25, 30, 60, 90, 120, 150 min after heating on a water bath under reflux (Fig. 9).

 

Fig. 9: The chromatogram of the solution of inulin API after hydrolysis (TS-2) on a water bath heating under reflux for 5 min (1), 10 min (2), 15 min (3), 20 min (4), 25 min (5), 30 min (6), 60 min (7), 90 min (8), 120 min (9), 150 min (10); solution of the standard substances of fructose, glucose, sucrose and lactose, RS-1 (11) on a Silica gel 60 TLC plate (Merck)

 

As can be seen from Fig. 9, the optimal time for hydrolysis is 10 min, further heating leads to appearance of side products, their number increases in proportion with the increase of the hydrolysis duration.

The effect of the developer:

Under the action of the solution for detection by the method of the SPhU (thymol in the mixture of sulfuric acid R96 % ethanol) all areas of monosaccharides are colored in a pink and pink-brown color. While according to the method of BP the diphenylamine-aniline-phosphate reagent has a response with different monosaccharides and disaccharides, and gives a wide range of shades for compounds differing by the type of the glycosidic bond. This allows differentiating structurally different sugars with close Rf values. However, since a more intense coloration of the chromatographic areas was achieved, it was proposed to use the ratio of the mixture components according to the method: 2 ml of aniline R and 2 g of diphenylamine R were dissolved in 100 ml of methanol R, 15 ml of orthophosphoric acid (85 %) R was added and mixed13,16.

 

Stability of the derivatization results:

Fig. 10 shows the study results of the effect of the conditions for heating the plate in the temperature range of 130 ± 10 ºС for 5-15 min. As we can see, the color of chromatographic areas and their intensity weakened at a temperature less than 130 ºС. It was also found that heating the plate for 10 min was sufficient for the process of derivatization.

 

 

 

 

Fig. 10: Heating the plate for 10 min at 120 ºС (А), at 130 ºС (В), at 140 ºС (С); the heating time – 5 min (D), 10 min (E), 15 min (F); 1 – solution of inulin API prior to hydrolysis, TS-1; 2 – solution of inulin API after hydrolysis, TS-2; 3 - solution of the standard substances of fructose, glucose, sucrose and lactose, RS-1 on a Silica gel 60 TLC plate (Merck)

 

Stability of chromatographic results:

The stability of the results was assessed in 5, 15, 30 and 60 min after chromatography. The difference between the areas studied was not detected within the time specified.

 

Precision:

When studying the precision of the method, the Rf values for the areas of markers of fructose, glucose, sucrose and lactose (RS-1) were calculated on one TLC plate, on different plates and on different days, by different analysts. The metrological characteristics of the Rf values for the areas of fructose, glucose, sucrose and lactose are presented in Tab. 2.

 


Table 2: Precision of Rf values for the areas corresponding to fructose, glucose, sucrose and lactose.

The area analyzed

Rf average

RSD, %

Rf maximum - Rf minimum

Precision on one type of plate, one day

TLC Silica gel 60 (Merck) on the aluminum substrate

The area corresponding to fructose

0.39

0.93

0.010

The area corresponding to glucose

0.34

0.98

0.010

The area corresponding to sucrose

0.25

0.89

0.005

The area corresponding to lactose

0.15

1.11

0.005

TLC Silica gel 60 (Merck) on the glass substrate

The area corresponding to fructose

0.49

0.90

0.010

The area corresponding to glucose

0.43

0.77

0.010

The area corresponding to sucrose

0.30

1.11

0.010

The area corresponding to lactose

0.19

0.87

0.005

Silica gel on TLC (Supelco) on the aluminum substrate

The area corresponding to fructose

0.43

0.91

0.010

The area corresponding to glucose

0.37

0.98

0.010

The area corresponding to sucrose

0.27

0.92

0.005

The area corresponding to lactose

0.15

1.08

0.005

Precision on 3 plates of the same type, one day

TLC Silica gel 60 (Merck) on the aluminum substrate

The area corresponding to fructose

0.41

2.27

0.025

The area corresponding to glucose

0.36

2.84

0.025

The area corresponding to sucrose

0.26

2.06

0.015

The area corresponding to lactose

0.16

2.79

0.010

TLC Silica gel 60 (Merck) on the glass substrate

The area corresponding to fructose

0.50

1.47

0.020

The area corresponding to glucose

0.44

2.11

0.020

The area corresponding to sucrose

0.30

1.98

0.015

The area corresponding to lactose

0.19

2.29

0.010

Silica gel on TLC (Supelco) on the aluminum substrate

The area corresponding to fructose

0.43

1.96

0.020

The area corresponding to glucose

0.37

2.36

0.020

The area corresponding to sucrose

0.28

1.92

0.015

The area corresponding to lactose

0.16

1.93

0.010

Intermediate precision

TLC Silica gel 60 (Merck) on the aluminum substrate

The area corresponding to fructose

0.41

4.69

0.040

The area corresponding to glucose

0.37

4.92

0.040

The area corresponding to sucrose

0.26

4.63

0.030

The area corresponding to lactose

0.16

4.84

0.015

TLC Silica gel 60 (Merck) on the glass substrate

The area corresponding to fructose

0.49

2.64

0.040

The area corresponding to glucose

0.45

3.12

0.035

The area corresponding to sucrose

0.31

3.06

0.025

The area corresponding to lactose

0.20

3.36

0.015

Silica gel on TLC (Supelco) on the aluminum substrate

The area corresponding to fructose

0.44

3.62

0.035

The area corresponding to glucose

0.38

2.52

0.025

The area corresponding to sucrose

0.16

3.39

0.015

The area corresponding to lactose

0.16

3.39

0.015

The validated TLC method for analysis of inulin API proposed for the draft for the monograph “Inulin” of the SPhU is given below.

 


Method:

Plate: TLC silica gel plate R. Place the plate in 0.3 % solution of sodium acetate, dry in the flow of warm air. Activate in a drying cabinet at the temperature of 100-105 ºС for 10-15 min.

 

Test solution (a). Dissolve 0.20 g of the inulin substance in hot water R, allow to cool, dilute to 20.0 mL with water R and mix.

 

Test solution (b). Dissolve 0.20 g of the inulin substance and 0.8 g of oxalic acid R in hot water R, boiling for 10 minutes, allow to cool, dilute to 20.0 mL with water R and mix.

 

Reference solution (a). Dissolve 0.1 g each of fructose CRS, glucose CRS, lactose CRS and sucrose CRS in water R and dilute to 20 mL with water R and mix.

 

Reference solution (b). Dissolve 0.1 g of maltodextrin CRS in water R and dilute to 20 mL with water R and mix.

 

Mobile phase: glacial acetic acid R, chloroform R, water R (70:60:10 V/V/V); accurately measure the volumes of the components of the mixture.

 

Application: 5 μl for test solutions (а, b) and reference solutions (a, b) as bands of 10 mm.

Development A: over a path of 10 cm.

Drying A: in a current of warm air.

Development B: immediately, over a path of 10 cm using the same mobile phase.

Drying B: in a current of warm air.

Mixture for detection: dissolve 2 ml of aniline R and 2 g of diphenylamine R in 100 ml of methanol R, add 15 ml of orthophosphoric acid (85 %) R, and mix.

Detection: spray with mixture for detection and heat at 130 °C for about 10 min; examine in daylight.

System suitability: reference solution (a):

– the chromatogram shows 4 clearly separated spots.

 

Results: the two principal zones of fructose and glucose in the chromatogram obtained with the test solution (b) is similar in position and colour to the principal zones of fructose and glucose in the chromatogram obtained with reference solution (a). The zones in the chromatogram obtained with test solution (a) remains on the line of application, there should not be zones at levels of the reference solutions (a, b). Fig. 11 shows the sequence of zones on the chromatogram of reference solutions (a, b) and test solutions (а, b).

 

Fig. 11: The graphic presentation of the location of the areas in chromatography

 

CONCLUSION:

1    When studying nine plants with inulin API obtained from chicory, Jerusalem artichoke and agave, and two standard samples of inulin from chicory and dahlia, the optimal conditions for the TLC analysis have been determined to identify the purity of substances.

 

2    It has been found that the TLC method presented meets the requirements of the SPhU by the main validation characteristics (specificity, robustness and precision).

3    The method for analysis of inulin API using TLC has been proposed to be included into the draft for the national monograph “Inulin” of the SPhUin the sections Identification” (by solution of inulin after hydrolysis) and “Test for purity” (by solutions of inulin prior to hydrolysis).

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 06.01.2019          Modified on 10.02.2019

Accepted on 01.03.2019        © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(6): 2862 – 2870.

DOI: 10.5958/0974-360X.2019.00482.7