Available online on 15.09.2022 at ijmspr.com

 International Journal of Medical Sciences and Pharma Research 

Open Access to Medical and Research

Copyright  © 2022 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

LC-ESI-MS/MS techniques for Method Development and Validation for Estimation of Propofol In plasma matrix

Sourabh Mittal¹*, Neetesh Kumar Jain², Sourabh Billore³, Riddhi Bajapai⁴

¹ Department of Quality Assurance, Faculty of Pharmacy, Oriental University Indore-India

² Department of Pharmacology, Faculty of Pharmacy, Oriental University Indore-India

³ Department of Pharmaceutics, Faculty of Pharmacy, Oriental University Indore-India

⁴ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Oriental University Indore-India

Email: sourabhbmittal@gmail.com   drnkjain9781@gmail.com 

To investigate the pharmacokinetic of new drug candidates. To compare pharmacokinetic profiles of different formulations. To monitor drug levels to establish the appropriate dose or frequency of administration. For fast and reliable measurement of the compounds in biological matrices. A bio-analytical method consists of two main components: Sample Preparation. Determination of drug and its metabolites ¹.

Sample preparation is a method used to clean up a sample before analysis and to concentrate a sample to improve its detection. Sample preparation in the Bio-analysis: it is the “last frontier” and starting point for the accurate LC-MS/MS analysis. Processing step includes; Method Development, Validation, Assay Performance and Work Flow ².

1.1 Criteria for Bio-analytical method;

• Simplicity of the developed method
• Short Method Development time and
• Validation failures kept at the minimum
• Develop the most selective sample preparation method.

1.2 How to develop Bioanalytical Method:

Peak Plasma Concentration (C_max): In Cmax, maximum concentration of analyte in biological fluid to be determined from literature survey, which is helps in establishment of target sensitivity of the developing method.

Physicochemical Property:   Physicochemical Properties of an analyte of interest such as solubility, molecular weight, structure, melting point, dissociation constant (pKa) which is helps in selection of the suitable extraction method.

Determination of Lowe r and Upper Limit of Quantification: The lowest concentration of an analyte in a sample that can be quantitatively determined with an acceptable precision and accuracy is usually 1/20^{th of} the Cmax value. After calculating ULOQ and LLOQ value have to prepare standard stock solution from which solution of different concentration are prepared ³.

Selection of drug volume to be spiked: The volume of analyte of interest is depends upon the volume of plasma spiked. Analyte concentration is normally 5% of the spiked plasma volume. For Example; if spiked plasma volume is 500 µL, so the volume of analyte to be added will be 25µL.

Sample preparation: Sample preparation technique is used to the clean up a sample by removing endogenous material as well as to concentrate a sample before analysis to exclude errors in its detection ⁴.

1.3 Application of Bio-Analytical Method:

• The determination of drug concentrations in biological fluids yields the data used to understand the time course of drug action, or pharmacokinetics, in animals and man and is an essential component of the drug discovery and development process.
• Bio-analysis, employed for the quantitative determination of drugs and their metabolites in biological fluids, plays a significant role in the evaluation and interpretation of Bioequivalence ⁵, Pharmacokinetic (PK), and Toxicokinetic studies.
• Selective and sensitive analytical methods for quantitative evaluation of drugs and their metabolites are critical for the successful conduct of pre-clinical and/or bio- pharmaceutics and clinical pharmacology studies.

1.4 Bio-Analytical Method Validation 

• Validation may be defined as documented evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its pre-determined specifications and quality attributes.
• Method validation is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use.

1.5 Drug profile: Propofol

• Propofol is an intravenous anesthetic with a phenolic structure. It is used for both induction and maintenance of anaesthesia.
• Propofol (Diprivan) ⁷ is rapidly acting, has a short recovery time, and possesses antiemetic properties.
• Rapid recovery and its antiemetic properties make Propofol anesthesia very popular as an induction agent for anesthesia. Propofol can also be used to supplement inhalational anaesthesia in longer procedures.
• The recent advent of computer assisted IV drug administration has made more practical the maintenance of anaesthesia with anaesthetic with relatively short half - life. (E.g., Propofol & short acting Phenyl piperidine opioids).
• The technique is called TIVA ⁸ (total intravenous anaesthesia) is done with short acting drug so that rapid recovery occurs even after long infusion.
• MATERIALS AND METHODS:

Table 1: Major Equipment Used

Table 2: Working and Reference Standard Details

Table 3: Materials Used

Drug Stock Solution for Propofol (10.000 mg/ml)

Weigh accurately Propofol standard equivalent to 10.0 mg of Propofol and add appropriate volume of Methanol to make final concentration of Propofol equivalent to 10.000 mg/ml. 

ISTD Stock Solution for Propofol D18 (1.000 mg/ml)

Weigh accurately Propofol D18 standard equivalent to 2.0 mg of Propofol D18 and add appropriate volume of Methanol [9] to make final concentration of Propofol D18 equivalent to 1.000 mg/ml.

Weigh accurately Propofol D18 standard equivalent to 2 mg of Propofol D₁₈ and add appropriate volume of Methanol to make final concentration of Propofol D₁₈ equivalent to mg/mL Correct the final concentration for Propofol D18 accounting for its potency and the actual amount weighed. Provide the batch number and store in refrigerator at 5±3°C [10]. Use this solution within 7 days from the date of preparation.

Pipette out 200µL of ISTD stock solution (1.000 mg/mL) and dilute up to 100.0 mL with methanol. Provide the batch number and store in refrigerator at 5 ± 3°C. Use this solution within 2 days from the date of preparation.

Weigh accurately Propofol standard equivalent to 10 mg of Propofol and add appropriate volume of Methanol to make final concentration of Propofol equivalent to 10.000 mg/mL Correct the final concentration for Propofol accounting for its potency and the actual amount weighed. Store in refrigerator at 5±3°C [11]. Use this solution within 7 days from the date of preparation.

Take 0.500mL of Drug Stock Solution, 10.000 mg/mL in polypropylene tube. Make up the volume to 10.0mL with Methanol. Store in refrigerator at 5 ± 3°C. Use this solution within 7 days from the date of preparation.

Prepare the working solution (SS) for DISS [12, 13] in Methanol using Drug Stock solution, 10.000/mL, as described in the table below.

Prepare the CC standards by spiking the respective CC spiking solutions in screened K₃EDTA human Plasma [14] as described in the table below:

1. For storage into deep freezer, aliquot either 0.200 mL or appropriate required volume of each sample into separate pre-labeled tubes cap them and store in deep freezer at - 20±5°C and at -78±8°C.
2. After aliquoting CC Standards, discard the leftover quantity of samples if any.

Prepare the QC samples by spiking the respective QC spiking solutions [15] in screened K3EDTA human Plasma as described in the table below:

Aliquot 0.100mL of each standard into separate pre-labeled tubes for processing. Note:

1. For storage into deep freezer, aliquot either 0.200mL or appropriate required volume of each sample into separate pre-labeled tubes cap them and store in deep freezer at - 20±5°C and at -78±8°C.
2. After aliquoting QC Samples, discard the leftover quantity of samples if any.

   Preparation of Mobile Phase (Acetonitrile: Mobile Phase Buffer: 70:30v/v,):

Separately measure 700mL of Acetonitrile and 300mL of Mobile Phase Buffer in measuring cylinder. Transfer both the contents into a reagent bottle and mix the contents thoroughly. Store at ambient temperature [16]. Use this solution within 3 days from the date of preparation.

Table 4: Trials for Optimization of Chromatographic Condition

Table 5: Trials for Optimization of Column

Step 1: Aliquot 0.100 ml of sample into pre-labelled tubes.

Step 2: Add 50 µl of ISTD dilution (2000.000ng/ml) to all the samples except STD BL and vortex for about 30 seconds. Add 50 µl Methanol to STD BL sample.

Step 3: Add 500 µl of Formic Acid in water, 2 % v/v to all samples and vortex to mix.

Step 4: Arrange the required number of pre- labelled Strata-X 33µm Polymeric Reversed Phase 30mg/1ml extraction cartridges [17, 18] on EZYPRESS 48-48 Position Positive Pressure Processor. Condition the cartridges with 1.0ml Methanol followed by 1.0 ml Water.

Step 5: Load the prepared samples on conditioned cartridges carefully.

Step 6: Wash the cartridges with 1.0 ml of Water, followed by Methanol in water, 10

% v/v and dry the cartridges for about 5 minutes by applying positive pressure at maximum flow rate or by applying full vacuum.

Step 7: Elute the contents from the cartridges with 0.300 ml Acetonitrile into pre- labelled tubes.

Step 8: Transfer appropriate volume of each sample into separate pre- labelled Autosampler vials, arrange them in the Autosampler and inject by using LC- ESI-MS/MS.

3.1 Identification of Drug and ISTD

Identification of drug and internal standard was done by using Mass spectrometer. Parent ion and product ion of drug and ISTD.

Table 6: Q1 and Q3 Mass for Drug and ISTD

Table 7: Optimized Chromatographic Condition

3.2 Precision and Accuracy:

Table 8: Result Table for Intra - run Precision & Accuracy I

Table 8: Result Table for Recovery

3.3 Selectivity or Specificity:

Table 9: Result Table for Specificity

3.4 Ruggedness:

Table 10: Result Table for Ruggedness (Different Equipment)

Inter - run precision for all high, middle and low quality control samples was found to be within acceptance limit of 15.00 %.The method is considered valid for extraction and analysis of Propofol in K₃EDTA human samples within investigated concentration range of 5.000 - 5000.000 ng/ml using 0.1 ml processing volume.The validation criteria used in present study is instrument stability, sample preparation strategy, calibration, precision and accuracy, specificity and selectivity of drugs. Upon injection of spiked plasma after a valid extraction procedure into LC-MS/MS instrument at LLOQ, specificity and selectivity shows acceptable results which means that instrument at this lowest concentration is capable to give a reproducible result. There was no significant peak observed from endogenous compounds at retention time of analyte and internal standard which means chromatographic conditions were quite perfect for satisfactory separation within chromatographic run time (2 minutes).The overall matrix effect value: % CV obtained from all 10 plasma lots were < 15 % for both analytes including their ISTD and did not show any peak area differences which means matrix effect does not affect this test procedure and sample preparation strategy used.Further, good results were obtained in plas ma calibration curves. The results obtained from their intra and inter-run precision and accuracy was determined using six sample replicates at four different concentration QC levels (LLOQ QC, LQC, MQC and HQC) that reveals acceptability of data included in accuracy within ± 15.0 % deviation from nominal values and precision of < 15% relative standard deviation(RSD) % CV , except for LLOQ, where it should not exceed < 20 % of deviation which shows that method validated to meet acceptance criteria of industrial guidance for bio-analytical method validation.Hence, it can be concluded that simple, rapid and sensitive isocratic reverse phase liquid chromatographic-tandem mass spectrometric method was devised to quantify Propofol in human plasma and can be successfully applied for bioequivalence studies in human subjects.

REFERNCES:

1. Ahuja S and Rasmussen HT, HPLC Method Development for Pharmaceuticals, Academic Press, 2007, pp. 485-489.

2. Sharma BK, Industrial methods of chemical analysis, 20th edition, GOEL publishing house, 2005, 68-80, 114-165, pp. 286-320.

3. Basics of LC/MS, Primer, Agilent Technologies, Innovating the HP Way, pp. 1- 36.

4. Buick AR, Doig MV, Jeal SC, Land GS and McDowall RD, "Method Validation in the Bio-analytical Laboratory", Journal of Pharmaceutical and Biomedical Analysis, 1990; 8: pp 629-637. https://doi.org/10.1016/0731-7085(90)80093-5

5. Shah VP, Midha KK, Dighe SV, Analytical Method Validation: Bioavailability, Bioequivalence and Pharmacokinetic studies. Pharmaceutical Research 1992; 9:588-592. https://doi.org/10.1023/A:1015829422034

6. Dabhi MJ, Bio-analytical Method Development and its Validation, 2009.

7. Indian Pharmacopoeia, Government of India, Ministry of Health & Family Welfare, Indian Pharmacopoeia commission, Ghaziabad, 2014, vol.-III, 2576 - 2577.

8. British Pharmacopoeia-2016, British pharmacopoeia commission, Volume II Ph Eur monograph 1558(for Propofol) 691.

9. USP-34/NF29 2011, volume II, Asian Ed., United state Pharmacopoeial Convention, Inc., Rockville, MD, 4046 - 4049.

10. Vaiano F., Serpelloni G., Focardi M., Fioravanti A., Mari F., Bertol E., "LC- MS/MS and GC-MS methods in propofol detection: Evaluation of the two analytical procedures", Journal of Pharmaceutical and Biomedical Analysis 39, 2005, 1-6 https://doi.org/10.1016/j.forsciint.2015.07.013

11. Leea S.Y., Parka N., Jeonga E., Wia J., Kimb C., Kim J.Y., Kyo Inc M., Honga J.; "Comparison of GC/MS and LC/MS methods for the analysis of propofol and its metabolites in urine", Journal of Chromatography B 2012; 900:1- 10 https://doi.org/10.1016/j.jchromb.2012.05.011

12. Alvarez J.C., Abe E., Etting I., GuenM.L., Devillier P., Grassin-Delyle S., "Quantification of remifentanil and propofol in human plasma: a LC-MS/MS assay validated according to the EMA guideline", Bioanalysis, 2015, 1675-168 https://doi.org/10.4155/bio.15.89

13. Hamd M., Wada M., Ikeda R., Kawakami S., Nakashima K.; "Validation of an LC-MS/MS Method for the Determination of Propofol, Midazolam, and Carbamazepine in Rat Plasma: Application to Monitor Their Concentrations Following Co-administration", The Pharmaceutical Society of Japan, 2015; 30(8):1250-1253 https://doi.org/10.1248/bpb.b15-00191

14. Pyo J.S.; "Selective and Accurate Determination Method of Propofol in Human Plasma by Mixed-Mode Cation Exchange Cartridge and GC-MS", Journal of analytical methods in chemistry, 2016, 1-6. https://doi.org/10.1155/2016/9531769

15. Ranshang zhang. Propofol Composition and methods of treating chronic pain by administering Propofol derivative United states patent, US8546453 B2, 2013

16. Wilson, Louis j., Dale B., James N. Apparatus and method for modelling and predicting sedative effect of drug such as Propofol on patients, International Patent, US2014/059829, 2014

17. Diaone N. Thompson, Gerold L. Mosher. Formulations containing Propofol and a sulfoalkyl ether cyclodextrin, United states patent, US7034013B2, 2006

18. Bosteels, Arnaud. Combination of Remifentanyl and Propofol, International Patent, PCT/EP2015/080788, 2016