Bioanalytical RP-HPLC Method Development and Validation for Estimation of Curcumin in Plasma Samples
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The study developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of curcumin in plasma, utilizing hydrochlorothiazide as an internal standard. The method demonstrated strong linearity, precision, and sensitivity, with validation focusing on key parameters such as recovery and stability under various conditions. Overall, the validated RP-HPLC method is suitable for estimating curcumin levels in biological samples.
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Bioanalytical RP-HPLC Method Development and Validation for Estimation of Curcumin in Plasma Samples
- 1. Sagar Kishor Savale. / Asian Journal of Pharmaceutical Analysis and Medicinal Chemistry. 5(2), 2017, 96-101. Available online: www.uptodateresearchpublication.com April – June 96 Research Article CODEN: AJPAD7 ISSN: 2321 - 0923 BIOANALYTICAL RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF CURCUMIN IN PLASMA SAMPLES Sagar Kishor Savale*1 1* Department of Pharmaceutics, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Maharashtra, India. INTRODUCTION Curcumin ((1E, 6E)-1, 7-Bis (4-hydroxy-3- methoxyphenyl)-1, 6 heptadiene-3, 5-dione), a polyphenol known as diferuloylmethane. Molecular formula of CRM C21 H20 O6 and Molecular weight of curcumin (CRM) is 368.39 g/mol. CRM is highly lipophilic drug having a log P value is 1.82 respectively. Dissociation constant of CRM was 8.3±0.04. CRM is a bright yellow-orange powder material. CRM has maximum solubility in methanol and acetonitrile. Melting point of CRM was 185ºc respectively. Reported λmax of CRM is 423 nm. ABSTRACT The present study was aimed at developing a reversed phase high performance liquid chromatography (RP- HPLC) method for determination of curcumin (CRM) in plasma and hydrochlorothiazide was used as an internal standard. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.) coupled with a guard column of silica, mobile phase was consisting of acetonitrile: water with 0.1% formic acid (40:60 v/v). The flow rate was 0.3 ml/min and the drug was detected using PDA detector at the wavelength of 423 nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation and flow rate, were optimised to provide high-resolution and reproducible peaks. The developed method was validated in terms of linearity, recovery, precision, ruggedness, sensitivity (LOD and LOQ) and stability study (short and long-term stabilities, Freeze/thaw stability and post-preparative). KEYWORDS Isocratic, Curcumin, RP-HPLC, Validation and Plasma samples. Author for Correspondence: Sagar Kishor Savale, Department of Pharmaceutics, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Maharashtra, India. Email: [email protected] Asian Journal of Pharmaceutical Analysis and Medicinal Chemistry Journal home page: www.ajpamc.com
- 2. Sagar Kishor Savale. / Asian Journal of Pharmaceutical Analysis and Medicinal Chemistry. 5(2), 2017, 96-101. Available online: www.uptodateresearchpublication.com April – June 97 This inhibits autophosphorylation of EGFR and blocks downstream signalling. There is no bio- analytical method for estimation of CRM in Plasma. Hence this study was aimed at developing a simple, rapid and sensitive method for estimation of analyte (CRM) in tissue samples (plasma) by using RP- HPLC1-3 . MATERIAL AND METHODS Material Curcumin (CRM) supplied as a gift sample from Sunpure Extracts Pvt. Ltd (Delhi, India). All solvents used were of HPLC grade. Formic acid, Methanol and acetonitrile were obtained from MERCK. Chem. Ltd (Mumbai, India). Plasma Samples Plasma samples were obtained from Central Animal House Facility, R.C. Patel Institute of Pharmaceutical education and Research Shirpur. Registration number 651/PO/ReBi/S/02/CPCSEA. The rats were euthanasiazed by using CO2 chamber (carcass disposal: Deep Burying under Soil). The rats were decapitated immediately after blood collection (Figure No.1). Blood samples were anticoagulated with heparin and centrifuged at 6000 rpm for 10 min to obtain plasma. All plasma samples were stored in a deep freezer at −70˚C until HPLC analysis. Instrumentation Analysis were carried out using an Agilent 1200 HPLC system (Agilent technologies, USA). The system was equipped with quaternary pump and photo diode-array detector (PDA). All data were acquired and processed using EZ chrome elite software version 3.3.2. Chromatographic conditions Chromatographic separation was performed by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.) coupled with a guard column. Isocratic elution was performed with acetonitrile: water with 0.1% formic acid (40:60 v/v) at a flow rate of 0.3 mL/min. The mobile phase was selected to give proper resolution of peaks4 . Plasma Samples processing and quality control (QC) samples Certified reference standards of CRM was weighed accurately and transferred 100 mg of CRM as working standard into 100 ml of volumetric flask, add about 100 ml of methanol and sonicated (1000 µg/mL solution). The working standard solutions was 100-400 ng/mL solution5 . Preparation of standard solutions of internal standard (IS) Internal standard such as hydrochlorothiazide, add 100 mg of IS in 100 ml of methanolic working solution (1000 µg/ml). The working standard solutions was 30 µg/mL solution Preparation of Plasma samples The whole procedure was carried out at room temperature. To 100 µl of CRM standard solution 100 µl of blank plasma sample, 100 µl of IS hydrochlorothiazide (30 µg/ml) were spiked and added extraction solvent 2 mL of acetonitrile was added and vortexed mixture for 20 min. This sample was ultracentrifuged at 10,000 rpm for 10 min. The supernatant layer was collected and 20 µl was analyzed by HPLC system6-8 . Method development Methods development was important to judge the quality, reliability and consistency of analytical results. It is the process for proving that analytical method is acceptable for use to measure the concentration of drugs7 . Method validation Validation of an analytical method is the process which is established by laboratory studies to evaluate the performance uniqueness of the procedure meet the requirements for its intended use. The validation process for analytical procedures begins with planned and systematic collection by the applicant of the validation data to support analytical procedures. The following are typical analytical performance characteristics which may be tested during methods validation: linearity, recovery, precision, sensitivity (LOD and LOQ) and stability study (short and long-term stabilities, Freeze/thaw stability and post-preparative). The linearity of a bioanalytical method is its ability to elicit test results that are directly, or by a
- 3. Sagar Kishor Savale. / Asian Journal of Pharmaceutical Analysis and Medicinal Chemistry. 5(2), 2017, 96-101. Available online: www.uptodateresearchpublication.com April – June 98 well‐defined mathematical transformation, proportional to the concentration of analyte in sample within a given range (100-400 ng/mL). Percent recovery of the proposed method was determined on the basis of standard addition method. The percent recovery as well as average percent recovery was calculated. Recovery should be assessed using minimum 9 determinations over minimum 3 concentrations level covering specified range. Recovery study was performed three different level 80 %, 100 % and 120 %. The precision is the measure of either the degree of reproducibility or repeatability of analytical method. It provides an indication of random error. Intra-day precision was determined by analysing, the three different concentrations 200 ng/ml, 300 ng/ml and 400 ng/ml for plasma samples analysis, for three times in the same day and Inter-day variability was assessed using above mentioned three concentrations of plasma samples were analysed by three different days, over a period of one week. Sensitivity refers to the smallest quantity that can be accurately measured. It also indicates the capacity of the method to measure small variations in concentration. Sensitivity of the proposed method were estimated in terms of Limit of Detection (LOD) and Limit of Quantitation (LOQ). For plasma sample analysis 100-400 ng/ml. The linear regression equation of the calibration curve was used to determine the LOD and LOQ. The stability of CRM in plasma samples was assessed under different storage conditions. Stability was expressed as the concentration ratio of analytes in sample under each storage condition against those in the freshly prepared sample. All stability assessments were assayed at three concentrations. Three samples were determined for short-term stability by putting them on the bench top at room temperature for 12 h and 24 h, respectively, prior to extraction. To evaluate freeze/thaw stability, three samples were subject to three freeze-thaw cycles with each cycle stepping from defrosting at room temperature to freezing at -20°C for 12 h. To determine the post- preparative stability, the extracted samples were stored in the sampler for 24 h. The long-term stability was performed by processing and analysing samples of plasma and brain kept at -20°C for 40 days8-12 . RESULTS AND DISCUSSION Method development Mobile phase consisting of acetonitrile: water with 0.1% formic acid (40:60 v/v) was tried and drug was resolved properly. This method showed the best peak shape and ideal detection response. Furthermore, strong organic solvent in the reversed- phase chromatography can reduce static retention and shorten analysis time. Acetonitrile: water with 0.1% formic acid (40:60 v/v) mobile phase was optimized to give proper resolution of peaks (Figure No.2). METHODS VALIDATION Linearity For plasma sample analysis linearity concentration in the range was100-400 ng/mL. The correlation coefficients (R2 ) of CRM in plasma was found to be 0.9996. Recovery, Precision and Sensitivity study Recovery studies of plasma samples for the proposed method were carried out, respective data is obtained and mentioned in (Table No.1) Recovery study was determined at three levels 80 %, 100 %, 120 % at each level three determinations were performed. Intra-day and Inter-day precision of plasma sample analysis was reported in (Table No.1). The % RSD for CRM was less than 2.0 %. The results are showing that the proposed method was precise. Sensitivity of the proposed method were estimated in terms of Limit of Detection (LOD) and Limit of Quantitation (LOQ). The linear regression equation of the calibration curve was used to determine the LOD and LOQ. Limit of detection, limit of quantitation of plasma sample analysis were reported in (Table No.1) respectively13-14 . Stability study The results demonstrated that CRM were stable in plasma sample at room temperature for 12 h, in the sampling for 24 h and after three freeze-thaw cycles. All analytes were stable after stored at room temperature for 24 h. Even when stored in a long-
- 4. Sagar Kishor Savale. / Asian Journal of Pharmaceutical Analysis and Medicinal Chemistry. 5(2), 2017, 96-101. Available online: www.uptodateresearchpublication.com April – June 99 term freezer set at -20°C for 40 days, all analytes remained stable. Stability data for CRM shown in (Table No.2) and the results suggested that the tissue sample containing CRM can be stored under common laboratory conditions without any significant degradation of all analytes. Stability of CRM was investigated using different concentrations of QC plasma samples. Excellent recoveries of CRM were observed at different storage conditions and no significant loss of CRM in either plasma was observed. Table No.1: Recovery, Precision and Sensitivity study Recovery S.No Analysis Drug Initial amount (ng/ml) Added Amount (ng/ml) % Recovery % RSD (n = 3) 1 Plasma CRM 200 188 99.57 0.66 2 200 200 96.19 0.25 3 200 202 100.37 0.16 Precision Analysis Drug Con. (ng/ml) Mean ± SD % RSD (n = 3) Mean ± SD 4 Plasma CRM 200 200.25 ± 0.27 0.11 200.22 ± 0.22 5 300 299.29 ± 0.74 0.16 292.74 ± 0.50 6 400 400.11 ± .056 0.35 391.11 ± 0.43 Sensitivity Analysis Drug LOD LOQ 7 Plasma CRM 52.66 ± 0.11 195.22 ± 0.28 Table No.2: Stability study (Plasma analysis) S.No Nominal (ng/ml) 3 freeze-thaw cycles short-term room temperature post-preparative stability (24 h) long-term room temperature (40 d)(12 h) (24 h) 1 200 99.56 ± 1.41 98.16 ± 3.38 100.13 ± 5.19 102.13 ± 2.99 98.33 ± 6.63 2 300 100.08 ± 0.29 100.09 ± 1.36 100.52 ± 3.16 103.32 ± 0.35 103.86 ± 14.26 3 400 100.65 ± 1.63 99.56 ± 2.87 99.24 ± 6.62 100.54 ± 0.49 99.89 ± 8.58 Figure No.1: Plasma sample collection and analysis steps
- 5. Sagar Kishor Savale. / Asian Journal of Pharmaceutical Analysis and Medicinal Chemistry. 5(2), 2017, 96-101. Available online: www.uptodateresearchpublication.com April – June 100 Figure No.2: Typical chromatogram of CRM in Plasma Samples CONCLUSION In this study, we developed and validated a highly sensitive and specific RP-HPLC method for the quantitative analysis of CRM in plasma samples. Validation of analytical method for estimation for CRM was determined by evaluating linearity, precision, recovery, sensitivity (LOD-LOQ) and stability (short and long-term stabilities, Freeze/thaw stability and post-preparative) in order to establish the suitability of analytical method. The method was validated in compliance with ICH guidelines is suitable for estimation of analytes with excellent recovery, precision, linearity and stability. ACKNOWLEDGEMENT I wish to acknowledge all those who are involved directly or indirectly for compilation of this article. It has been a great honour to work with such a professional. CONFLICT OF INTEREST We declare that we have no conflict of interest. BIBLIOGRAPHY 1. Savale S. Simultaneous Determination of Curcumin and Gefitinib in Pure Form by Using UV Spectrophotometric Method, Hygeia: journal for drugs and medicines, 9(1), 2017, 1-8. 2. Savale S K. UV Spectrophotometric Method Development and Validation for Quantitative Estimation of Halcinonide, Asian Journal of Biomaterial Research, 3(3), 2017, 22-25. 3. Savale S K. UV Spectrophotometric Method Development and Validation for Quantitative Estimation of Curcumin, Asian Journal of Biomaterial Research, 3(4), 2017, 14-18. 4. Savale S K. UV Spectrophotometric Method Development and Validation for Quantitative Estimation of Paracetamol, Asian Journal of Biomaterial Research, 3(4), 2017, 33-37. 5. Savale S K. UV Spectrophotometric Method Development and Validation for
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