1. INTRODUCTION:

Propofol (2, 6-diisopropylphenol) which also known as Diprivan (Marketed Name) is a short-actinggeneral anaesthesia. Being a parenteral emulsion it is injected intravenously. Administered as sedation and also as an add-on regional anaesthesia to the critical conditioned patients. After an antagonistic dose of Propofol or any intravenous infusion, the pharmacokinetic parameters can be explained as a three-compartment open model. In the first compartment model, the distribution and metabolism rate rapidly occurs comparatively than any other anaesthetics. The primary location of forming metabolites is liver, where turned off Propofol and Quinine-alcohol complex can be formed, which is excreted byurine¹. In 1989, FDA (Food and Drug Administration) approved Propofol and is sold as Diprivan by Fresenius Kabi USA.FDA (Food and Drug Administration) classified Propofol as Category B Drug for causing no harm to an unborn or to foetus².

As per the literature review, analytical methods like gas chromatography (GC), high-performance liquid chromatography (HPLC) coupledfluorescence; mass spectroscopy detection method has been applied for the quantification of Propofol^3-5. Hence, the objective of the present study is to establish a reverse phase HPLC-UV method and validation for estimation of Propofol in its pharmaceutical preparation.

Fig 1: Structure of the Propofol

2. MATERIALS AND METHODS:

2.1 Chemicals and Reagents:

Propofol working standard was procured as a gift sample from Ranbaxy, Hyderabad. HPLC grade Methanol was procured from Sigma Aldrich and water of HPLC grade was procured from Milli-Q-RO system.

2.2. Instrumentation and Chromatographic conditions:

Shimadzu Auto-Sampler LC-2010A HT (UV Detector) HPLC System (Tokyo, Japan) was used for chromatographic separations. Inertsil lODS-3V NitrofurantoinC₁₈ (250 x 4.6 mm; 5μ). Column was used for accomplishing Propofol Method Development. Shimadzu UV-Visible Spectrophotometer UV-1700 (Shimadzu Corporation, Japan) was utilized to check the wavelength (λ_max) of the drug. Milli-Q Reverse Osmosis system (Millipore, Bedford, USA) were used for the making of HPLC grade Milli-Pore water. The mobile phases and solutions were degassed by sonicator.

2.3Preparation of Standard and Stock Solutions:

2.3.1 Wavelength Selection:

1.3 mg/ml standard solution was diluted in methanol to produce a concentration of 130µg/ml. From which further diluted were made with methanol to produce a concentration of 13µg/ml. The solution is subjected to UV (Shimadzu 1700) for the scanning of wavelength between 200-400nm. The λ_maxof the drug Propofol was found to be at 270nm.

Fig. 2: UV Spectrum of Propofol

2.3.2. Standard Preparations:

As per the Monograph (IP), Propofol is dissolved in methanol. 14mg of drug was dissolved in methanol in 10ml volumetric flask (1.4mg/ml). From that standard stock solution 140µg/ml were prepared which served as working concentration.

2.3.3Standard preparations for CC:

The calibration standards of 10.0, 30.0, 50.0, 70, 90,110μg/ml of Propofol standard solutions were prepared for 2ml from the working standard solution of Propofol.

3. METHOD VALIDATION:

The RP-HPLC method which had been developed for the quantification of Propofol was validated according to ICH Q2 (R₁A)⁶ guidelines. The Parameters of the validation are specificity, linearity, precision and accuracy, detection limit and quantification limits.

3.1. Specificity:

For the method, validation is specificity, the ability of an analytical method to distinguish the analyte from other chemicals in the sample. The specificity of the method may be assessed by deliberately adding impurities into a sample containing the analyte and testing how well the method can identify the analyte.

3.2. Linearity:

The linearity of an analytical method specifies if the acquired response is linearly proportional to the concentration of the analyte within a definite range. The linearity of the proposed method was assessed over a range of 10-110µg/ml. The working standards were prepared in mobile phase from 1mg/ml standard stock solution and were injected in triplicate under optimized chromatographic conditions and the chromatograms were recorded. The linearity was established based on the correlation co-efficient obtained by plotting a graph with a concentration in mg/ml at the x-axis and peak area of Propofol at the y-axis.

3.3. Precision and Accuracy studies:

The precision of the developed method can be assessed by intra-day and inter-day studies.6 independent injections of 3 different concentrations i.e. 10, 50, 110µg/ml (LQC, MQC&HQC level) were used to study the precision of the proposed method. Intra-day precision, as well as repeatability, was examined by injecting the samples on the same day and the inter-day precision study was carried out by injecting the same samples on 2 different days.The mean values and the values of %RSD were calculated.

3.3. Limit of detection and limit of quantification:

The sensitivity of a method determines how capable is the method of detecting the lowest possible concentration of analyte without any noise. This is assessed by the parameter of LOD and LOQ. Limit of Detection is the smallest concentration of the analyte that can be detected by the developed method which evokes a computable response (signal-to-noise ratio 3) where Limit of Quantification is the smallest concentration of the analyte which generates a response that can be precisely quantified. (Signal to noise ratio 10).

LOD and LOQ can be calculated by the formula:

LOD = 3.3 σ/S

LOQ = 10 σ/S;

Where σ = Standard deviation of the response; S = Slope of the deviation curve.

3.5. Robustness and Ruggedness:

The ruggedness and robustness of the developed were studied by bringing about slight changes in the experimental conditions (Analyte, reagent source and columns of various bands) and chromatographic conditions (pH, mobile phase composition and mobile phase ratio and flow rate).

3.6. System Suitability:

The system suitability testing is used to verify that an analytical method was suitable for its intended purpose the day analysis was done. It is an essential parameter to ensure the quality of the method for correct measurements. Chromatographic parameters viz; the number of Theoretical Plates (N), Retention time (R_t), Resolution (R_s) and Peak Asymmetric factor (A) were scanned on injecting 6 replicates of the standard Propofol at a concentration of140µg/ml.

4. RESULTS AND DISCUSSIONS:

4.1. Method development:

The proposed method was designed by optimizing the chromatographic conditions by pertaining to various trial runs altering the mobile phase composition, the ratio of the mobile phase, pH, column, column length to attain symmetrical analyte peak at a sufficiently short run time. Methanol was used as an organic modifier in the mobile phase. Initially, various ratios of Methanol and water employed as the mobile phase for separations, exhibiting peak asymmetry.

Finally, a symmetrical analyte peak with an acceptable short run time was achieved employing Methanol and water in a ratio of 85:15 v/v at a flow rate of 1ml/min, with an Inertsil ODS 3VNitrofurantoin column (250mm x 4.6mm, 5µ) being utilized as the stationary phase and monitored at a wavelength of 270nm.Propofol was eluted at 7.20 mins. The mobile phase was prepared by filtering through a 0.45µ PTFE (Poly Tetra fluoro ethylene) membrane filter before incorporating in the HPLC system. The chromatograms were recorded and processed on a class VP Data station.

Fig. 3: Chromatogram of Propofol

4.2. Accuracy and Precision:

The Accuracy method was expressed as % mean recovery for three different concentration levels (10, 30, 50µg/ml) by standard addition method. Triplicate analyses were performed at each level. Percent mean recovery was calculated. The % RSD was found to be in the range of 1.21 to 3.45%. The developed method was also used for the assay of the marketed Propofolinjec table formulation. The results obtained were comparable to that of the label claim and is tabulated in table 1. The intra-day and inter-day precision studies carried out showed a 0.999% RSD of respective indication the precision of the method.

Table 1: Precision Studies:

Sl. No	Concentration Added(µg/ml)	Intra-day (µg/ml)	Inter-day (μg/ml)
Sl. No	Concentration Added(µg/ml)	Amount found ±RSD (n=6)	Amount found ±RSD (n=6)
1.	10	9.41 ±3.01	9.10 ± 3.45
2.	50	49.2 ± 2.41	48.9 ± 2.64
3.	110	111.7 ± 1.37	110.1 ± 1.21

*SD: Standard Deviation, RSD: Relative Standard Deviation.

4.3. Linearity:

A calibration curve was plotted for 6 different concentrations of drug vs. The corresponding peak. The graph showed excellent co-relation between the concentrations and peak area when observed within the range of 10µg/ml to 110µg/ml for the drug. The correlation co-efficient for Propofol was 0.999.

Fig. 4: Linearity of Propofol

Table 2: Recovery studies in Formulation:

Drug	Label claim	Amount taken for assay	Amount obtained (μg/ml) ±RSD (n=6)	Percentage Recovery (%)
Propofol	200mg	10	9.5 ± 2.67	95.00
		50	49.8 ± 1.89	99.60
		110	112 ± 1.21	101.81

4.4. Detection Limit and Quantification Limit:

The Detection and Quantification limit represents the sensitivity of the proposed method. The LOD and LOQ were found to be 10ng/ml and 100ng/ml, respectively indicating the sensitivity of the method.

4.5. Specificity:

The specificity test demonstrates that the used excipients did not interfere with the peak of the main compound. No peaks were eluted along with the retention time of Propofol (Fig.3).

Hence, the results showed that the developed method was selective for the determination of Propofol in the formulation.

4.6. System Suitability:

Replicates of the standard at working concentrate were injected to observe changes in separation, retention time and asymmetry of the peaks and validate the system suitability parameters. The system suitability found to be within limits and is summarized in Table 3.

Table 3: System Suitability Studies:

Sl. No	Parameters	Propofol
1.	Retention Time (min)	7.1
2.	Theoretical Plates (N)	3333
3.	Tailing Factor	0.5
4.	Asymmetric Factor	1
5.	Linearity Range (μg/ml)	10-110
6.	Slope	88048
7.	Correlation Coefficient	0.999
8.	LOD (ng/ml)	10
9.	LOQ (ng/ml)	100

Assay of the marketed formulation:

The Assay of the formulation was performed as per the Indian Pharmacopeia. According to Pharmacopeia, the test solution was prepared by the diluting the 80mg of Propofol to 100ml with the propan-2-ol. Two reference solutions were prepared for the assay of the marketed formulation of the drug. Reference solution a: 0.08 percent w/v solution of Propofol RS in Propan-2-ol containing 6.8 percent v/v of water, Reference solution b: A solution of 0.00008 percent w/v of Propofol RS and 0.00008 percent w/v of Propofol impurity RS in Propan-2-ol containing 6.8 percent v/v of water.

Methanol and water were used as the mobile phase in the ratio of 85:15 v/v and 270nm wavelengths for the chromatographic conditions. Inertsil ODS-3V NitrofurantoinC₁₈column was used as the stationary phase.

The recovery percentage of the marketed formulation of Propofol is 101.81%.

Fig. 2: Typical Chromatogram of Propofol Formulation

5. CONCLUSION:

A rapid, simple, sensitive, precise, accurate RP-HPLC method was developed for Propofol and the developed method was validated as per ICH Q2 (R₁A) guidelines. The %RSD of less than 3.45% indicated the method to be precise. The drug showed good linearity over concentrations ranging from 10-110µg/ml. Recovery studies that were carried out expressed the accuracy of the method. The mean recovery of the validated from ranged between 95.21 to 101.81%. Thus it may be concluded that an accurate precise and rapid RP-HPLC method have been developed and validated for routine quantification of Propofol in bulk and pharmaceutical formulations.

6. CONFLICT OF INTEREST STATEMENT:

The authors declare that there are no conflicts of interest.

7. ACKNOWLEDGEMENT:

The authors are grateful to Ranbaxy, Hyderabad for providing the Propofol standard as a gift sample.

REFERENCES:

1. Jason Yarbrough, Ralph Harvey: Determination of Propofol Using High Performance Liquid Chromatography in Whole Blood with Fluorescence Detection. Journal of Chromatographic Science 2012; 50:162–166.

2. URL:https://www.rxlist.com/diprivan-drug.html

3. H. Zhang, P. Wang: HPLC determination of cisatracuriumbesylate and Propofol mixtures with LC-MS identification of degradation products. Journal of Pharmaceutical and Biomedical Analysis 16 (1998) 1241–1249.

4. Karthick Vishwanathan, James T. Stewart: HPLC determination of a Propofol and Remifantanil mixture. J. LIQ. CHROM. & REL. TECHNOL., 22(6), 923–931 (1999).

5. T.B. Vree, A.J. Lagerwerf, C.P. Bleeker: Direct high-performance liquid chromatography determination of Propofol and its metabolite quinol with their Glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man. Journal of Chromatography B, 721 (1999) 217–228.

6. ICH Guidelines Validation of analytical procedures: Text and methodology Q2 (R1).

Received on 27.03.2019 Modified on 13.02.2020

Research J. Pharm. and Tech. 2021; 14(6):3139-3142.

DOI: 10.52711/0974-360X.2021.00547