Propofol Quantification method in Palm Oil Based Nanoemulsion Formula using RP-HPLC

 

Bayu Eko Prasetyo1,2, Norazrina Azmi3, Lia Laila1,2, Ahmad Fuad Shamsuddin4*

1Faculty of Pharmacy, Universitas Sumatera Utara, Medan, 20155, Indonesia.

2Nanomedicine Center of Innovation, Universitas Sumatera Utara, Medan, 20155, Indonesia.

3Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz,

50300 Kuala Lumpur, Malaysia.

4Faculty of Pharmacy and Health Sciences, University Kuala Lumpur Royal College of Medicine Perak

(UniKL RCMP), No. 3 Jalan Greentown 30450 Ipoh, Perak, Malaysia.

*Corresponding Author E-mail: fuad.shamsuddin@unikl.edu.my, bayu@usu.ac.id

 

ABSTRACT:

HPLC is one of the methods that is widely used for the routine determination of drug content in a pharmaceutical dosage form. The objective on this research was to develop a simple method to determine the propofol concentration in a new formulation using a palm oil-based nanoemulsion carrier system (NEMS™ MCT/LCT). The method used was reverse phase high performance liquid chromatography (RP-HPLC) using a C18 column with methanol and water (80:20) as the mobile phase and UV wavelength detection was 276 nm. The retention time obtained for the drug in NEMS MCT/LCT was 6.32 min. This was similar to the retention time of the standard. The correlation coefficient value from the calibration curve was 0.9998. The drug contents were 100.90% and 100.24% for the 1% and 2% NEMS MCT/LCT, respectively. The result indicated that the RP-HPLC method used for the analysis of propofol in the palm oil-based emulsion system was able to accurately determine the concentration of the drug.This method is suitable for routine determination of propofol concentration in palm oil-based emulsion formulations.

 

KEYWORDS: Palm oil, NEMS, LCT, MCT, Propofol, HPLC.

 

 

INTRODUCTION: 

Propofol (Fig. 1) is commonly used in the induction and to maintain anaesthesia in surgical patients1. The drug is also administered in intensive care unit patients2. Some of the advantages of propofol are the rapid onset of hypnosis3,4 and minimal side effects1. Propofol is also shown to be effective in the prevention of emesis 5 and the treatment of pruritus6.

 

Fig. 1: Chemical structure of Propofol

 

The use of palm oil in parenteral pharmaceutical formulation is still new. Palm oil commonly known as cooking oil7,8. Malaysia and Indonesia are the major producers of palm oil9, both are contributing to more than 90% of the world’s palm oil production10..A palm oil-based nanoemulsion system was successfully developed as a carrier system for the parenteral delivery of propofol11. This formulation is a combination of long chain triglycerides (LCT) and medium chain triglycerides (NEMS™ MCT/LCT). This oil combination in propofol development was success reduce the pain intensity when the injection was performed in rats12. Nanoemulsion system is a method that regard as carrier for insoluble drug like propofol and also for drug targeting reason13,14.

 

The RP-HPLC method used in the determination of propofol in NEMS™ MCT/LCT was a modification of the method used by Trapani et al.15. A reverse phase HPLC system is one of the analysis methods of choice due to the easiness, accurateness and suitable for all dosage forms, such as tablets (tilorone dihydrochloride, tenofovir, dexibuprofen)16,17,18, capsules (nilotinib, aprepitant)19,20 or oral solution (risperidone)21. This method also suitable for human plasma samples (clotrimazole22, doravirine, lamavudine and tenofovir disoproxil fumarate23), and also human urine (25-desacetyl rifampicin)24.

 

The study showed a simple and rapid method for routine determination of propofol in palm oil-based nanoemulsion formulation. The developed modified method is successfully  detect and determine propofol in a nanoemulsion system as drug carrier.  In this study, the content of propofol in NEMS™ MCT/LCT stored over six months was also assessed using this method to assess the stability of propofol in the formula.

 

MATERIALS AND METHODS:

Materials:

Palm Oil Olein (local cooking oil, Seri Murni, Malaysia); MCT oil (Enersos, Malaysia). Propofol, glycerol,sodium hydroxide, and sodium oleic were purchased from Sigma-Aldrich (Germany). The used emulsifier was egg lecithin (GmbH, Germany). The solvent for analysis such as methanol was HPLC grade and all other chemicals were of analytical grade (Merck).

 

Preparation of Propofol-loaded NEMS™ MCT/LCT:

The propofol nanoemulsion formulation were produced based on Prasetyo, et al.11. The concentrations of propofol (1% and 2% propofol in NEMS™ MCT/LCT) were prepared in 10ml vial dosage form and kept at storage temperature (2-8 oC) until analysis.

 

Chromatographic Condition:

The HPLC system (Waters, USA) used consisted of a HPLC pump (Waters 600), a line degasser AF, an auto sampler (Waters 2707), a PDA Detector (Waters 2998) and an Empower computer system for analysis. A reversed phase ODS Hypersilcolumn, C18 (25 cm×4.6mm; 5µm) was used as the stationary phase. The flow rate was set to 1mL /min and detection was set at 276 nm. Methanol and distilled water (80:20) were used as the mobile phase. The mobile phase was filtered through a 0.45µm membrane filter and sonicated for 20 minutes in an ultrasonic bath (Branson, USA).

 

Preparation of Standard Solution:

Standard stock solution was prepared by dissolving 50 mg of propofol standard in methanol to obtain a solution with final concentration of 1000µg/mL. The standard stock solution was then diluted in methanol to prepare working solutions in a concentration range of 100 - 500 µg/mL.

 

Method Validation:

The parameters of the validation assayed were linearity, repeatability, accuracy, precision and system suitability according to ICH Q2 (A) guidelines25.

 

Linearity:

The linearity was determined from five replicate injections of each concentration to obtain a calibration curve for analysis of propofol standard solutions. The calibration curves were obtained by plotting peak area versus concentration.

 

Repeatability:

Six replicate injections at a concentration of 300µg/mL were done to analyse the injection repeatability and the Relative Standard Deviation (RSD) value was calculated to determine the variation.

 

Accuracy:

The standard addition method was carried out to check the accuracy of the method.  Three different levels (50%, 100% and 150%) of sample solutions were tested. All of these solutions were injected directly into the HPLC system for analysis.

 

Precision:

Precision was assessed by intra-day and inter-day variability at five different concentrations (100, 200, 300, 400 and 500µg/mL). The intra-day precision was expressed as RSD of results with 3 replicates during the same day. The inter-day precision was calculated by comparing the result on three different days during the same week.

 

Sensitivity:

Limit of detection (LOD) and limit of quantification (LOQ) were assessed from the signal-to-noise ratio. LOD is the lowest concentration level resulting in a peak height of three times the baseline noises. LOQ is the lowest concentration level resulting in a peak height higher than 10 times.

 

Specificity:

The specificity of the HPLC method was determined to provide a quantitative system suitability test report. The parameters like retention time (Rt), theoretical plates (N), tailing factor (T) and capacity factor were calculated.

 

Propofol determination of sample solution:

Determination of total concentration of propofol-loaded NEMS™ MCT/LCT 1% and 2% were carried out by dissolving 100µl of the nanoemulsion with methanol to a 5mL in a volumetric flask. The mixture was vortexed for 1min to ensure complete solubilisation.  The solution was then filtered through a 0.45µm membrane filter and 10µL was injected for analysis.

 

Stability test for propofol-loaded NEMS™ MCT/LCT:

Determination of propofol concentration in propofol-loaded NEMS™ MCT/LCT was also conducted during the storage at 4±1oC and 16±1oC for 6 months. Propofol concentrations were determined after 1, 3 and 6 months of storage with the same method to assess the stability of propofol in the formula.

 

RESULTS AND DISCUSSION:

The propofol determination in the NEMS™ MCT/LCT was successfully conducted with this RP-HPLC. The chromatograms of blank, propofol standard and propofol sample are shown in Fig.1 a, b and c, respectively. The retention time of the peak was 6.32 min.

 

 

Fig.1: Chromatograms of blank (a), propofol standard (b) and propofol in the sample (c)

 

Linearity:

The calibration curve of propofol standard solution over the range of 100 to 500µg/mL is shown in Fig.2. The curve of the peak area vs. concentration was proven to be linear, with a correlation coefficient of 0.9998 and the regression equation was y = 4400 x + 18700.

 

Fig.2: The calibration curve of propofol standard in methanol

 

Repeatability:

The repeatability was expressed as relative standard deviation (RSD). The results for injection repeatability showed that RSD value of retention times and peak areas of propofol analysis were well below 2% Table1.

 

Table1: Injection Repeatability for Propofol Analysis

Injection number

Retention time (min)

Peak area

1

6.334

1336250

2

6.331

1333441

3

6.325

1340097

4

6.331

1343441

5

6.324

1345751

Mean±SD

6.329 ± 0.0043

1339796 ± 5044.77

RSD (%)

0.0680

0.38

 

Accuracy:

Accuracy value was calculated as the percentage of recovery from 3 known quantities samples. The result for recovery was found to be 98.46 - 101.22% for propofol analysis when sample was spiked in the level of 50%, 100% and 150%. The recovery and RSD value for each concentration are given in Table 2.

 

Table2: Accuracy of Propofol Analysis

Concentration (µg/mL)

Mean ± SD

RSD (%)

Recovery (%)

100

98.46±0.16

0.16

98.46

200

202.45±0.36

0.18

101.22

300

300.21±1.15

0.38

100.07

 

Precision:

The precision of the analysis in the intra and inter-day variations in the peak areas of propofol solution were considered in terms of RSD value and the results are represented in Table 3.As shown in Table3, RSD value for intra-day and inter-day precision data at five concentrations were all below 2%.

 

Table3: The precision of Propofol Analysis

Concentration (µg/mL)

Precision

Intra-day

Inter-day

Mean±SD

RSD (%)

Mean±SD

RSD (%)

100

98.54±0.15

0.15

98.19±0.44

0.45

200

202.25±0.30

0.15

201.60±0.29

0.14

300

300.24±0.82

0.27

299.73±1.91

0.64

400

399.14±0.86

0.22

400.28±3.40

0.85

500

500.29±0.12

0.02

499.70±1.76

0.35

 

The results of all system suitability parameters, LOD and LOQ values of HPLC method are shown in Table 4. This method showed a respectable sensitivity and selectivity for propofol analysis with the theoretical plate value more than 2000 and tailing factor below than 2.

 

Table4: System Suitability Parameters

Parameters

Observations

Retention time (Rt)

6.32

Theoretical plates (N)

6933.92

Tailing factor (T)

1.29

Capacity factor (k)

2.58 

LOD (µg/mL)

0.1

LOQ (µg/mL)

0.25

 

The propofol contents in the NEMS™ MCT/LCT formulations were determined from the regression equation of the calibration curve. The results are shown in Table5. The concentration of propofol in NEMS™ MCT/LCT fulfilled the requirement of the United States Pharmacopoeia (USP)26, which stated that concentrations determined should be in the range of 98 to 102%. The propofol-loaded NEMS™ MCT/LCT formulations showed good stability. No changes in propofol concentrations were observed after 6 months storage at 4±1oC and 16±1oC (Table6).

 

Table5: The Result of Propofol Concentration of Propofol in NEMS™

Formulation

Concentration of propofol (mg/mL)

% recovery

Propofol 1% in NEMS™ MCT/LCT

10.09±0.037 mg/mL

100.90

Propofol 2% in NEMS™ MCT/LCT

20.05±0.154 mg/mL

100.24

 

 

Table6: Propofol Contenta in Propofol in NEMS™ at Various Storage Temperatures

Formulation

Temperatur

(oC)

Percentage Recovery of Propofol

Storage time (Months)

0

1

3

6

Propofol 1% in NEMS™ MCT/LCT

4±1

100.90±0.04

100.60±0.06

100.80±0.13

100.10±0.04

16±1

100.90±0.04

100.90±0.04

100.10±0.10

100.00±0.03

Propofol 2% in NEMS™ MCT/LCT

4±1

100.24±0.15

100.60±0.17

100.55±0.15

101.25±0.09

16±1

100.24±0.15

100.20±0.06

100.10±0.11

100.75±0.12

a Data were expressed as % recovery, as mean± SD, n= 6.

 

This method was applicated to evaluate propofol concentration in nanoemulsion with palm oil as the oil phase for the developed formula. The similar study had been done by Mukherjee (2021) for propofol determination in commercial product with retention time obtained was 7.1 minutes27. However, this study used different type of instrument, mobile phase ratio and UV wavelength condition, and the result showed shorter retention time which was 6.32 minutes.

 

Conclusion:

The method developed was found to be simple, rapid and sensitive for the determination of propofol concentrations in a nanoemulsion system. Propofol-loaded NEMS™ MCT/LCT showed an excellent recovery and good stability during 6 months of storage at 4±1oC and 16±1oC.

Acknowledgement:

The authors thank to the Malaysian Technology Development Corporation for supporting this project under the UKM-MTDC-BF 0003-2008 grant.

 

CONFLICT OF INTEREST:

Authors have declared no conflict of interest to declare

 

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Received on 28.08.2022            Modified on 12.12.2022

Accepted on 17.02.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(6):2622-2626.

DOI: 10.52711/0974-360X.2023.00430