Determination Of Stigmasterol From Polyherbal Formulation Biology Essay

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The extracts were obtained from successive solvent of clerodendrum serratum. 10mg of each extract was weighed and made up to 100ml with solvents such as petroleum ether, chloroform, ethyl acetate and methanol respectively. The concentrations of 1000 μg/ml solutions were prepared and filtered over Whatmann filter paper. 20 μg/spot of each solution was applied on the TLC plate.

10 mg of marker compound was transferred into 10ml standard flask. The volume was made with chloroform to get the concentration of 1000 μg/ml. prepare 50 μg/ml from the above stock solution 300-900ng /spot (3-15 μl/spot) was applied on the HPTLC plate.

Preparation of stock solutions of the extract of Formulation.

The extracts were obtained from the formulation of clerodendrum serratum.10mg of each extract was weighed and made up to 100ml methanol. The concentration of 1000 μg/ml solutions were prepared and filtered over Whatmann filter paper. 20 μg/spot of each solution was applied on the TLC plate.

Fixed Chromatographic Parameters:

Stationary phase

Material: TLC aluminium sheets silica gel 60 F 254, [E.MERCK KGaA]

Plate size: 10Ã-10 cm

Mobile phase:

Solvent: CHCl3:MeOH (95:5 v/v)

Development chamber: Twin Trough Chamber

Chamber saturation time: 20 min.

Development time: 20min.

Drying time of the developed plate: 5 min.

Calibration Parameters:

Calibration mode: Multi level

Evaluation mode: Peak Area

Sample application:

Instrument: CAMAG Linomat 5 "Linomat_100632"

Linomat 5 applicator. WINCAT Software

Spray gas: Inert nitrogen gas.

Dosage speed: 150 ml/s

Sequence

Syringe size: 100 μl

Number of tracks: 8

Application position Y: 10.0 mm

Band length: 6.0 mm.tection - CAMAG TLC Scanner 3

Instrument: CAMAG Scanner 3 "Scanner 3_100904"

Solvent front position: 80.0 mm

Position of first track: 15.0 mm

Slit dimensions: 5.00 Ã- 0.45 mm, Micro

Optimize optical system: Light

Scanning speed: 20 mm/s

Data resolution: 100μm/step.

Measurement

Wavelength: 366nm.

Lamp: D2

Measurement Type: Remission

Measurement Mode: Absorption

Optical Filter: Second order and K400

PM high voltage: 275 V and 313V

OBSERVATION

In Absorption Mode:

The developed HPTLC plates were scanned in absorption mode at 366 nm and the chromatograms of marker compound, all four successive extract and formulations are taken. The concentration and peak Area of the calibration graph, amount of stigmasterol present in successive extracts and formulation are tabulated.

Selection and Prewashing of Plate

A precoated silica gel 60 F254 on aluminium sheet with 100-250 mm thickness was selected for the study. Pre-washing of plate was done with methanol and then it was activated by keeping in an oven at 1150C for 10 minutes.

Selection of solvent

The drug is dissolved in a solvent in which it is soluble and in which the drug shows good stability. Another criteria for selection of solvent is that it has to be volatile, cheap and easily available. stigmasterol is readily soluble in chloroform and shown good stability. Hence chloroform was selected as solvent.

Selection of wavelength

An ideal wavelength is one that give maximum absorbance and good response to the drug to be detected. UV spectrum of drug shows maximum absorbance at 286 nm which was selected as detection wavelength.(Fig1).

Fig1: UV spectrum of standard Stigmasterol on TLC plate

Selection of mobile phase

Solvent system tried

Observation

Chloroform: water

Drug did not move

butyl acetate : chloroform

Drug did not move

n-butanol :butyl acetate

Tailing

Chloroform:Methanol

Compact spot

Optimisation of mobile phase ratio:

Different ratios of Chloroform: Methanol like 5:5, 4:6, 3:7, 2:8 were tried and the ratio of (95:5 v/v) was selected because it gave compact spots with good separation from solvent front and sample application positions and good symmetrical peak.

Fixed Experimental Conditions:

Stationary phase - TLC precoated silica gel 60F254

Mobile phase - Chloroform:Methanol (95:5v/v)

Development chamber - Twin trough glass chamber

Separation technique - Ascending development

Distance developed - 85 mm

Detection wavelength - 366 nm

Rf value - 0.80 ± 0.01

Preparation of standard stock solution

Stock solution was prepared by weighing 10 mg of stigmasterol into a 10ml standard flask and the volume was made upto 10ml with chloroform to get a concentration of 1000µg/ml.

Preparation of Standard Graph

From the stock solution of stigmasterol, 0.5 to 5 µl was spotted on the TLC plate followed by development and scanning of spots. The chromatograms are shown in Fig 3. The peak areas were recorded. Calibration graph was obtained by plotting the peak areas against the corresponding concentration of the standard solutions. The values are tabulated.

CHROMATOGRAMS OF STANDARDS

Fig 3: HPTLC chromatogram of Stigmasterol -(300ng/spot)

Fig 4: HPTLC chromatogram of Stigmasterol -(450ng/spot)

Fig 5:HPTLC chromatogram of Stigmasterol -(600ng/spot)

Fig 6: HPTLC chromatogram of Stigmasterol -(750ng/spot)

Fig 7: HPTLC chromatogram of Stigmasterol -(900ng/spot)

Tabel 2: LINEARITY GRAPH FOR STIGMASTEROL

Concentration

Peak area

300ng

2666.65

450ng

3755.30

600ng

4634.20

750ng

5450.18

900ng

6122.25

Fig 8:Calibration graph of STIGMASTEROL

HPTLC FINGER PRINTS OF CLERODENDRUM SERRATUM PLANT EXTRACTS

Fig 9: HPTLC Chromatogram of PEE at 366nm

Fig 10: HPTLC Chromatogram of CE at 366 nm

Fig 11: HPTLC Chromatogram of EAE at 366 nm

Fig 12: HPTLC Chromatogram of ME at 366 nm

Fig 13: FORMULATION - SUPRES

TABLE 3: AMOUNT OF STIGMASTEROL IN EXTRACTS

Extracts

AMOUNT OF STIGMASTEROL(mg)

PETROLEUM ETHER

0.948

CHLOROFORM

0.68

ETHYL ACETATE

0.23

METHANOL

0.10

FORMULATION

0.761

ANALYSIS OF FORMULATION

Preparation of sample solution

10mg of formulation extract (triterpens) each containing 0.761mg of clerodendrum serratum was weighed, Quantity equivalent to 10 mg of standard was weighed, transferred to a 100 ml volumetric flask, extracted and made up to volume with and filtered through a Whatman filter paper and this was used for the study.

The formulation was assayed by spotting 4 µl onto the plate followed by development and scanning. The Chromatograms were recorded which are shown in Fig The amount of stigmasterol was calculated from peak area regression analysis table:4 and the equation is as follows

y = α + βx

α = (Σy) (Σx2) - (Σx) (Σxy)

N Σx2 - (Σx)2

β = NΣxy - (Σx) (Σy)

N Σx2 - (Σx)2

where, x = concentration

y = absorbance

N = Number of pairs of values

Table 4: Analysis of formulation

Drug

Amount 10mg

% label claim +

%RSD

Labeled

Found

Stigmasterol

6.273

1.354

96.93 + 0.3727

VALIDATION OF THE METHOD

The validation of the developed method was carried out in terms of linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), inter and intraday precision, repeatability of sample application, measurement, stability studies and selectivity.

Linearity and Range

Linear regression data showed a good linear relationship over a concentration range of 300 to 900 ng/spot. (Fig: 8) The slope, intercept and correlation co-efficient values were found to be 0.9976.

Accuracy

Recovery studies of the drug was carried out for determining accuracy parameter. It was done by mixing known quantity of standard drug with the pre-analysed sample formulation and the contents were reanalyzed by the proposed method.

Recovery studies carried out at 50 and 100 % levels. The percentage recovery and its %RSD were calculated table 5.

Table 5: Recovery studies

Level

% Recovery

%rsd*

80%

102.98

0.96153

100%

96.5

0.3751

* Mean of three determinations

Precision

Precision of the method was demonstrated by

Intra-day precision

Inter-day precision

Repeatability

Repeatability of measurement

Repeatability of sample application

Intra-day precision

Intra day precision was found out by carrying out the analysis of the standard drug for two different concentrations in the linearity range of drug for three times on the same day and %RSD was calculated, table 6.

Table 6: Intraday precision

Volume applied (µl)

Peak Area

% RSD*

3

2666.6

0.7588

2671.2

2651.4

6

5636.2

0.4784

5636.8

5627.2

* Mean of 3 determinations

Inter-day precision

Inter day precision was found by carrying out the analysis of the standard drug for two different concentrations in the linearity range of drug for two days and %RSD was calculated which is shown in table 7.

Table 7: Inter-day precision

Vol applied (µl)

DAY

PEAK AREA

%RSD*

3

1

2666.6

0.9687

2

2680.3

3

2693.2

6

1

5634.2

0.4550

2

5627.8

3

5647.6

9

1

6122.2

0.8151

2

6145.6

3

6114.S9

* Mean of 3 determinations

Repeatability

Repeatability of sample application

Repeatability of sample application was assessed by spotting 2 and 4µl of drug solution five times on pre-coated TLC plate followed by development of plate and %RSD was calculated, table8.

Table 8: Repeatability of sample application

Volume Applied (µL)

Peak Area

% RSD*

9

6128.0

0.4514

6132.1

6122.7

6121.7

6135.7

6130.9

* Mean of 5 determinations

Repeatability of measurement

Repeatability of measurement of peak area was determined by spotting 2 and 4 µl of formulation on pre-coated TLC plate. After development of the plate, the separated spots were scanned five times without changing position of the plate and %RSD was calculated. (table:9)

Table 9: Repeatability of measurement for formulation

Volume Applied (µl)

Peak Area

% RSD*

9

6184.6

0.6608

6154.1

6158.4

6171.6

6134.5

6118.6

* Mean of 5 determinations

LIMIT OFDETECTION AND LIMIT OF QUANTIFICATION

LOD and LOQ were determined by applying decreasing amount of the drug in triplicate on the plate. The lowest concentration at which the peak is detected is called Limit Of Detection and it was found to be 100ng/spot. (Fig:) The lowest concentration at which the peak is quantified is called Limit Of Quantification and it was found to be 300ng/spot (Fig: 14)

Fig 14 : LOD of stigmasterol (100ng/spot)

Fig15 : LOQ of Stigmasterol (300 ng/spot)

Stability studies

When the developed chromatographic plate is exposed to atmosphere, the analytes are likely to decompose. Hence it is necessary to conduct stability studies.

Stability of the analyte on the plate was studied at different time intervals and peak areas were compared with the peak area of freshly scanned plate.

The developed plate was found to be stable for about 7 hours as the reduction in peak areas was within the limits. The values are shown in table10.

Table 10: Stability of the analyte on plate

Volume Applied (μl)

Time (hours)

Peak Area

6

1

3066.5

2

3115.5

3

3139.6

4

3118.3

5

3143.9

6

3146.8

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