INTRODUCTION:

Cholesterol (Fig.1) is a waxy steroid metabolite found in the cell membranes and transported in the blood plasma of all animals. Cholesterol is the principal sterol synthesized by animals¹, but small quantities are synthesized in other eukaryotes, such as plants and fungi. It is almost completely absent among prokaryotes, which include bacteria². Cholesterol has a molecular formula of C₂₇H₄₅OH. This molecule is composed of three regions (shown in the picture): a hydrocarbon tail, a ring structure region with 4 hydrocarbon rings, and a hydroxyl group. The hydroxyl (OH) group is polar, which makes it soluble in water. This small 2-atom structure makes cholesterol an alcohol. The alcohol that we drink, ethanol, is a much smaller alcohol that also has a hydroxyl group (C₂H₅OH). The 4-ring region of cholesterol is the signature of all steroid hormones (such as testosterone and estrogen). All steroids are made from cholesterol. The rings are called "hydrocarbon" rings because each corner of the ring is composed of a carbon atom, with two hydrogen atoms extending off the ring.

The combination of the steroid ring structure and the hydroxyl (alcohol) group classifies cholesterol as a "sterol." Cholesterol is the animal sterol. Plants only make trace amounts of cholesterol, but make other sterols in larger amounts.

The last region is the hydrocarbon tail. Like the steroid ring region, this region is composed of carbon and hydrogen atoms. Both the ring region and tail region are non-polar, which means they dissolve in fatty and oily substances but will not mix with water. Because cholesterol contains both a water-soluble region and a fat-soluble region, it is called amphipathic. Literature survey showed no HPTLC method for the estimation of cholesterol. It was felt that a reliable and rapid method for the estimation of cholesterol was needed. The primary goal was to develop and validate a HPTLC method for the rapid quantization of the drug. The present study illustrates development and validation of a simple, accurate, economical and reproducible procedure for determination of cholesterol by HPTLC.

MATERIALS AND METHODS:

Chemicals:

All other chemicals and reagents used were of analytical grade and were purchased from Merck Chemicals Corporation ltd. Mumbai, India. Deionized and ultra-pure water used in all experiments was obtained from Milli – Q system (Millipore).Silica gel 60F₂₅₄ TLC plates (20×10 cm and 10×10 cm, layer thickness 0.2mm, Merck, Germany) were used as stationary phase.

Fig 1: structures of cholesterol

Equipment:

The instrument used in the present study was Camag HPTLC system comprising Camag Linomat V automatic sample applicator, Hamilton syringe (100µl), Camag TLC scanner III with Wincats software. The HPTLC system consisted of Linomat V auto sprayer connected to a nitrogen cylinder, a twin trough glass chamber (10×10 cm), saturated with filter paper for ten minutes.

HPTLC METHOD AND CHROMATOGRAPHIC CONDITIONS

Selection of solvent:

Cholesterol is freely soluble in acetone, chloroform, ether and ethyl acetate, sparingly soluble in alcohol and insoluble in water. Acetonitrile: 2- propanol (3:1 v/v) was selected.

Fig 2: UV Spectrum of Cholesterol

Selection of wavelength:

The sensitivity of the HPTLC method that uses UV detection depends upon the proper selection of wavelength. An ideal wavelength is one that gives maximum absorbance and good response for the drug to be detected. UV spectrum of Cholesterol on TLC pre-coated plate showed maximum absorption at 215 nm which was selected as the detection wavelength (Fig. 2)

Preparation of Standard cholesterol Solution:

Standard stock solution was prepared by dissolving 62.5 mg of cholesterol in acetonitrile : 2-propanol (3:1) in a 25 ml standard flask and the volume was made up to 25 ml with acetonitrile : 2-propanol (3:1) to obtain a concentration of 2.5 mg/ml. From this 2,4,6,and 8 ml were taken in 10 ml standard flask and volume made up to 10 ml with the same to obtain a concentration of 0.5, 1.0, 1.5, 2.0 µg/ml.

Preparation of egg cholesterol solution:

Cholesterol is isolated from egg. 25 mg of this cholesterol was weighed and 10 ml of hexane was added. Then it was mixed thoroughly and the hexane layer was decanted into another test tube. Nitrogen gas was then passed through the tube at 35 degree Celsius to evaporate all the hexane. To the residue obtained 10 ml of acetonitrile : 2-propanol (3:1) was added, to obtain a stock solution of concentration 2.5mg/ml. 8ml of the stock was taken in a 10 ml standard flask and the volume was made up with acetonitrile : 2-propanol (3:1) making the concentration 2.0 µg/ml.

Recording the chromatogram:

With the fixed chromatographic conditions all the standard cholesterol solutions of concentration 0.5 to 2.5 µg/ml were applied on the plate, dried, developed, analyzed photo metrically and chromatograms were recorded. The R_fvalue of the standard was found to be 0.55 ± 0.03. Calibration curves were plotted using peak areas of standard cholesterol v/s concentration of the standard solution. This is followed by the application of egg cholesterol solutions of 2.0 to 25 µg/ml (Fig.3). The peak areas of the sample chromatograms were compared and the amount of cholesterol in egg was calculated.

Fig 3: Chromatogram of egg cholesterol

Prewashing of plates:

HPTLC was performed on 10×10cm precoated silica gel 60F₂₅₄ precoated plates from E.Merck. The adsorbent has a very large surface area; it may absorb air and other impurities from atmosphere, particularly volatile impurities, after the pack has been opened. The non-volatile impurities adsorbed by layer can lead to irregular baseline in scanning densitometry. To avoid possible interference from such impurities in quantitative analysis, plates were prewashed with methanol, dried, and activated for 30 minutes at 110 degree centigrade with the plates being placed between two sheets of glass to prevent deformation of the aluminum during heating.

Sample application:

The samples of Cholesterol were spotted on precoated TLC plates in the form of narrow bands of lengths 6mm, with 10mm from the bottom and left margin and with 9mm distance between two bands. Samples were applied under continuous drying stream of nitrogen gas at constant application rate of 150 nLs^-1.

Mobile phase and migration:

Various solvent systems like mixture of a) Chloroform: formic acid: butyl acetate b) Chloroform: methanol: ammonia c) Ethyl acetate: methanol: ammonia: formic acid e) Butyl acetate: acetic acid: hexane f) Toluene: ethyl acetate were tried to separate and resolve spot of Cholesterol from its impurities and other excipients of formulation. The mixture of toluene: acetone: glacial acetic acid (6:1.3:0.1 v/v/v) could resolve with better peak shape. The drug was satisfactorily resolved with R_f value 0.55±0.03. Pre-saturation of TLC chamber with mobile phase was carried out for for 30 minutes.

METHOD VALIDATION_:

Validation of the developed HPTLC method was carried out as per the International Conference on Harmonization (ICH) guidelines^3-6 Q2 (R1) for Linearity, Accuracy, Precision, Limit of Detection, Limit of Quantification, Repeatability and stability studies.

Linearity and calibration curve:

Linearity of the method was evaluated by constructing calibration curves at five concentration levels. 10µl each of standard cholesterol were applied on the plate to obtain concentration in the range of 0.5 to 2.5 mg/ml. The calibration curves were developed by plotting peak areas Vs Concentrations with the help of Win-CATS software. Chromatogram was developed in a twin trough glass chamber; using 20 minutes chamber saturation time. The length of chromatogram run was 80mm. The developed plates were air-dried. Scanning was performed in UV mode at 215nm. The slit dimension was kept at 6×0.45mm at scanning speed of 100nm/s. After completion of scanning peak areas were noted. Peak areas were plotted against corresponding concentrations and least square regression analysis was performed to generate the calibration equation.

Precision:

Intra-day precision was found out by carrying out the analysis of the standard cholesterol at three different concentrations in the range of 5-25µg/spot for three times on the same day. Each concentration was applied in duplicate and %RSD was calculated. The inter-day precision was studied by carrying out the analysis of the standard cholesterol at three different concentrations in the range of 5-25µg/spot for three days and %RSD was calculated. The precision of an analytical method expresses the degree of scatter between a series of measurements obtained from multiple samples of the same homogeneous sample under prescribed conditions.

Repeatability:

Repeatability of measurement of peak area was determined by spotting 10µl of standard cholesterol solution (2µg/ml) on TLC plate and developing the plate. After developing the plate developed spot was scanned six times without changing the position of the plate and %RSD was calculated. Repeatability of sample application was assessed by spotting 10µl of standard drug solution six times on a TLC plate by automatic spotter, followed by development of plate and recording to the peak areas for six spots.

Accuracy⁷:

Recovery studies of the drugs were carried out for determining accuracy parameter. To study the accuracy parameter the recovery studies of the drugs were carried out at two levels for cholesterol. Quantity equivalent of 25 mg of egg cholesterol was taken and to this the standard cholesterol was added at 50% and 100% levels. The contents were analyzed by the proposed method and chromatographs obtained. The percentage recovery and %RSD were calculated.

%Recovery =

Limit of Detection and Limit of Quantification:

The detection limit (LOD) of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. LOD was calculated using the following formula,

3.3× Standard Deviation of the Y- intercept

LOD = ----------------------------------------------------------

Slope of the calibration curve

The quantification limit (LOQ) of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

LOQ was calculated using the following formula,

10 × Standard Deviation of the Y- intercept

LOQ = ----------------------------------------------------------

Slope of the calibration curve

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

RESULTS AND DISCUSSIONS:

Linearity:

The linear regression data showed a good linear relationship over a concentration range of 5-25 mg/ml. The slope, intercept and correlation co-efficient values were found to be 133.57, 485.36 and 0.9986 respectively (Fig. 4). It showed that good correlation between regression coefficient and concentration of the drug (Table 1).

Fig 4: calibration curve

Table 1: Peak area obtained for pure cholesterol (5-25 µg/spot)

Concentration (µg/spot)	Peak area
5	1109.1
10	1851.2
15	2510.4
20	3220.3
25	3756.0

Table 2: Intraday precision

Concentration (µg/spot)	Peak Area		% RSD*
5	919.2	910.0	0.5853
	921.0	924.0
	916.0	914.0
10	1718.5	1695.4	0.1709
	1711.0	1705.0
	1689.2	1713.0
15	2130.6	2080.1	0.6928
	2094.0	2120.0
	2112.3	2107.8

* RSD of six observations

Precision:

The intra-day and inter-day relative standard deviations found in the range 0.57 to 0.86% and 0.58 to 0.90% respectively. The smaller values of intra-day and inter-day variation in the analysis indicate that the method is precise (Table 2-3).

Table 3: Inter day precision

Volume applied (µg/spot)	Day	Peak Area		% RSD*
5	1	919.2	909.0	0.58
	2	922.0	918.0
	3	909.4	913.0
10	1	1710.5	1708.0	0.61
	2	1703.0	1689.0
	3	1675.5	1716.0
15	1	2157.4	2163.9	0.90
	2	2174.0	2184.0
	3	2198.3	2146.5

* RSD of six observations

Repeatability:

The RSD for measurement of peak area was calculated and was found to be 1.37%. In repeatability of sample application the %RSD for the peak area values were calculated and found to be 1.58%. The RSD values for measurement of peak area and sample application were both below the instrumental specifications (i.e.1%); ensuring proper functioning of HPTLC system (Table 4-5).

Table 4: Repeatability of measurement

Volume applied (µg/spot)	Peak Area	% RSD*
20	3398.0	1.37
	3518.6
	3514.5
	3516.2
	3514.7
	3510.1

* RSD of six observations

Table 5: Repeatability of sample application

Volume applied (µg/spot)	Peak Area	% RSD*
20	3390.5	1.58
	3410.0
	3319.5
	3443.1
	3479.0
	3398.0

* RSD of six observations

Accuracy:

The % recovery was found to be 99.01; 96.05 (at 100 and 50% levels respectively), which is satisfactory. The results of recovery study indicate that the proposed method is accurate for estimation of drug in tablet dosage form (Table 6).

LOD and LOQ:

The limit of detection was found to be 1µg/spot (Fig.5), while the limit of quantification was found to be 3µg/spot (Fig. 6).

Fig 5: LOD of cholesterol

Table 7: Stability of plate

Time (hrs)	Conc. (µg/spot)	Peak area
0	20	3535.0
0	25	4449.2
1	20	3348.2
1	25	4386.9
2	20	3781.2
2	25	4599.4
4	20	3535.3
4	25	4385.0
6	20	3512.7
6	25	4425.6
10	20	3335.0
10	25	4213.5
24	20	3757.3
24	25	4607.0
48	20	3980.7
48	25	4964.1

Fig 6: LOQ of cholesterol

Stability studies:

The developed plate was found to be stable for 24 hrs as the reduction in peak areas was within the limits. The drug solution was found to be stable for two days at refrigeration conditions and 24hrs at room temperature (Table7- 8).

Table 8: Stability of solution

Time in hrs	Conc. (µg/spot)	Peak area
Time in hrs	Conc. (µg/spot)	Room temperature	Refrigerator
0	20	3535.0	-
0	25	444.2	-
1	20	3725.0	3523.0
1	25	4392.4	4447.5
2	20	3678.9	3486.5
2	25	4104.9	4361.5
4	20	3818.7	3510.1
4	25	4446.3	4349.0
6	20	3710.0	3765.7
6	25	4249.5	4471.3
10	20	3710.0	3798.0
10	25	4249.5	4551.6
24	20	3741.0	3681.9
24	25	4119.0	4629.0
48	20	2735.0	3701.3
48	25	3011.3	4721.6

CONCLUSION:

A simple, rapid, precise and accurate HPTLC method was developed for the estimation of cholesterol. Acetonitrile: 2-propanol (3:1) was employed as the solvent. The wavelength for the estimation was selected as 215 nm. The mobile phase fixed after optimization was toulene: acetone: glacial acetic acid (6:1.3:0.1 v/v/v). The R_f value was found to be 0.55 ±0.03. The method was validated as per ICH guidelines in planar chromatography, when analytes are absorbed on highly active polar surfaces of silica sorbent layer in the presence of air, substances can decompose more easily. Therefore cholesterol was tested for its stability on chromatographic plates for different time periods. The developed plates were found to be stable for 24 hrs. Linearity experiment was performed and it was found to be linear in the range of 5 to 25µg/spot. The correlation co-efficient value for cholesterol was found to be 0.998. The precision study of the method was determined by running replicate samples. Under this intraday and interday precision and repeatability studies were performed. The low percentage RSD values indicate that the proposed method had good precision. LOD and LOQ for cholesterol were found to be 1 and 3 µg/spot. In this method accuracy was determined by calculation of percentage recovery and it was found to be within the limits.

REFERENCES:

1. Emma Leah (May 2009). "Cholesterol" Lipidomics Gateway.

2. "Phylogenetic and biochemical evidence for sterol synthesis in the bacterium Gemmata obscuriglobus". Proc. Natl. Acad. Sci. U.S.A. 100 (26): 15352–7.

3. ICH Harmonised Tripartite Guideline, Validation of Analytical Preocedures: Text and Methodology Q2 (R1), Nov 2005.

4. ICH. Q2B Validation of analytical procedure: Methodology. International Conference on Harmonization, Geneva; 1996 March.

5. Validation of Analytical procedure: Methodology (Q2B), ICH Harmonised Tripartite Guideline.

6. ICH Q2B Text on validation of analytical procedures. (2005)

7. P.D. Seth, Qualitative analysis of drugs and formulations, 4^thedition, 1996, Pp. No. 1-19.

Received on 23.06.2010 Modified on 05.07.2010

Research J. Pharm. and Tech. 4 (1): January 2011; Page 155-159

S. No	% Level	Amount Added(mg)	Amount Found(mg)	%Recovery	% RSD*
1	50	12.5	12.00	96.05	0.12
2	100	25.0	24.80	99.01	0.15