Bioanalytical Method Development and Validation of RP-HPLC Method For the Estimation of Andrographolide in Plasma, Commercial Formulation and Pharmacokinetic Study

Andrographis paniculata (Burm.f.) Nees, also known as King of Bitters, is a member of the plants in Acanthaceae family, [1] cultivated as medicinal herb in Southeast Asia. It is widely used as a traditional medicine in Taiwan, China, India, and Thailand for the treatment of infections, cold, fever, inflammation, and diarrhoea. Andrographolide (AG), is a diterpenoid lactone as a major constituent of Andrographis paniculata & chemically 3-(2-(decahydro-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenenaphthyl) ethylidene) dihydro-4-hydroxyfuran-2(3H)-one [2].andrographolide have recently attracted considerable interest because of their diverse physiological functions and therapeutic potential, including antioxidant,  anti-inflammatory, antiapoptosis,  antiatherosclerosis , anticancer,  antivirus, and hypoglycaemia effects [3]. The bioanalytical method is employed for the quantitative estimation of drugs and their metabolites in biological media. It plays an important role in the estimation and interpretation of bioequivalence, pharmacokinetic and toxic kinetic studies [4-5]. The sensitivity and selectivity of bioanalytical methods are necessary to conduct preclinical and clinical studies successfully during the drug development process from the initial preclinical phase to the final clinical steps [6-7]. The RP-HPLC chromatographic method is the most used technology for the bioanalysis of small molecules [8-9]. As pointed out by the World Health Organization, there is very limited knowledge about the chemical compositions, pharmacokinetics, pharmacodynamics, and metabolomics of Indian medicinal plants (IMP). the data about authentication, efficacy, and safety of IMP are known and are far from satisfactory to meet the required criteria for worldwide use. Pharmacokinetic profile helps to elaborate the relationship between intensity and time course of pharmacology, the toxicological effects of phytochemicals in the human body, and extends the scope of the use and acceptance by different regulatory bodies.  A few authors have already been reported techniques for the quantification of AG in rat and human plasma by high-performance liquid chromatography (HPLC) or liquid chromatography tandem mass spectrometry (LC-MS/MS) and solid phase extraction for sample preparation which are expensive eventually owing to longer runtime for analysis [1]. Although various analytical methods are available for quantification of the drugs in biological samples, High Pressure liquid chromatography (HPLC) is considered an indispensable analytical method with high specificity and sensitivity for estimation of the drugs. For the drug selected in the present work, the extensive analysis of literature reports shows only a few studies are available on the application of HPLC methods to quantify andrographolide in human plasma [10]. However, no studies are reported on the quantification of commercial herbal formulation containing andrographolide as phytomarker of A.paniculata extract in rat plasma, which is required to estimate of the drug AG in bioanalytical samples during the preclinical studies. Moreover, the HPLC methods reported on human plasma have employed mobile phase mixture involving inorganic buffer species, gradient flow rate and high run time, requiring high plasma volume (100 to 200 ?L). These conditions lead to a complex and time-consuming analytical process for quantifying of the drug.  Several research laboratories were involved in determining the fate of herbal products either by studying the pharmacokinetics of active marker compounds or the herbal products themselves. In the context of the traditional herb A. paniculata, a few papers were published on the pharmacokinetics of the active marker compound andrographolide, as the test material instead of the A. paniculata herb [11-12]. Some studies focused on the preparation of novel drug delivery systems for andrographolide to improve its fate, while others examined the effects of co-administration of andrographolide with standard drugs [13-15]. However, considering the chemical complexity of herbs, these studies had limited scope in understanding their pharmacokinetics and interactions. It is well-documented that phytochemicals have competitive protein binding when taken as herbs or extracts, leading to different pharmacokinetics [16]. The present study was focused on the comparative absorption studies of pure andrographolide with a herbal formulation containing the extract of A. paniculata along with other herbs. The objective of the present work was to develop a simple, accurate, sensitive RP-HPLC method with appropriate validation for determination and quantification of AG and its traditional formulation in rat plasma. The attempts were made to reduce the run time and attain faster retention of the drug and improve the drug’s sensitivity during the chromatographic procedure. Moreover, the developed method was expected to require only plasma micro-quantities for quantification of the drug. These parameters are generally believed to be advantageous as compared with the previously published methods The method was also validated for selectivity, sensitivity, recovery, linearity, accuracy, precision, and stability according to the regulatory guideline. The method was successfully applied for oral pharmacokinetic study to determine AG concentration. The present study describes a simple and reproducible RP-HPLC method using liquid-liquid extraction to detect the plasma concentrations in male Sprague-Dawley rats. The applicability of pharmacokinetics is also demonstrated.

Experimental

MATERIALS AND METHODS

Andrographolide and the internal standard (IS) propyl paraben were kindly provided by Natural Remedies Ltd., Bengaluru India and SD Fine chemicals, Mumbai, India respectively. Acetonitrile and Methanol (HPLC grade) and analytical grade of ethylene diamine tetra acetic acid disodium (Na EDTA), glacial acetic acid, formic acid (98–100%), perchloric acid (70%) were purchased from Merck India Ltd. Ultrapure water generated in house using a MiliQ Plus System (Millipore, MA, USA) was used throughout the analysis.

Instrumentation and Chromatographic Conditions

HPLC system (Agilent 1260 infinity series) coupled with UV detector, auto sampler and column oven, was used to study the concentration of andrographolide in rat plasma. A reversed-phase HPLC system was used that involves polar mobile phase and non-polar, hydrophobic stationary phase. Samples were separated using ZORBAX Eclipse Plus C18 4.6 mm x 25cm column with a pore size of 5µm (Agilent Technologies) as a stationary phase, and the column temperature was ambient. The mobile phase consisted of Acetonitrile: Water (30:70 v/v) For all samples, the flow rate of the mobile phase was 1mL/min with a fixed total run time of 14 minutes and the injection volume of the sample was 10µL. The mobile phase was filtered using 0.45µm membrane filter paper and subjected to degassing in an ultrasonic cleaner before running the HPLC analysis. The detection was done by UV detector with measuring wavelength at ?_max (231nm), determined earlier by UV spectrophotometric method. The chromatograms data was obtained by using Lab Solution software (Agilent Corporation).

Collection of Blood and Separation of Plasma

Study protocols (Protocol No. IAEC/RES/19/16 and IAEC/ RES/23/07) was approved by the Institutional Animal Ethics Committee (IAEC), RCPIPER prior to commencement of the work. Healthy male Wistar rats were obtained from Central Animal Facility, RCPIPER (Maharashtra, India). The animals were kept in standard plastic cages maintained under controlled conditions (23 ± 2⁰ C, 60 ± 5% RH and 12 hr dark -light cycle) and provided standard laboratory pellet food with water ad libitum. Blood samples were collected from the retro orbital sinus of the rats into 2 mL polypropylene centrifuge tubes containing 100 mL of 10% w/v solution of Na-EDTA. The blood-EDTA mix was centrifuged at 8000 rpm for 30 min at 4⁰C. The clear supernatant plasma was pooled into fresh tubes and stored at -20⁰ C till further use [10].

Preparation of Standard Stock and Working Solution

The standard stock solution of andrographolide and Internal standard (IS) Propyl paraben was sonicated for about 10 minutes to produce stock concentrations 200 µg/mL with acetonitrile each. The standard stock solution of andrographolide was further diluted with mobile phase to get working solutions of different concentrations like 2, 4, 6, 8, 10,12 ?g/mL. Each drug solution was filtered through 0.45?m membrane filter before use and 20?L of each concentration was injected into the HPLC column.

Preparation of Calibration Standard and Quality Control (QC)Samples

Sample preparation for calibration of plasma spiked solutions and QC sample were prepared using aliquots of 500µl of rat plasma were added in working standard solutions of mixture of AG (100 µl) and IS (10 µl). Then the mixture of each concentration was extracted with 2ml of acetonitrile. Then resulting mixture was shaken mechanically for 5-10 min. after centrifugation at 5000 rpm for 15 min, the supernatant was transferred to clean test tube and evaporated to dryness on water bath. The residue was reconstituted with 100µl of mobile phase and vortex for 60 s and the centrifugation procedure was repeated from that solution 20µl aliquot of the supernatant was injected into optimized HPLC system. Quality Control samples (low 4 µg/ml, mid 6 µg/ml, high 8 µg/ml) were prepared by considering the manner from which calibration working standard solution and sample preparation is treated as above same. The amount of AG in both calibration curves were determined by their respective linear equations. Y=mx+c

Sample Preparation and Extraction Procedure

Sample preparation technique used for the study plays a significant role in bioanalytical samples. it helps to remove the matrix of interfering biological compounds. It is essential to reduce the effect of the matrix formed due to biological components. The protein precipitation extraction procedure was used to extract andrographolide from plasma using ethanol as the extracting solvent. Samples were prepared by adding 10 µl of each working solution of andrographolide to 500 µl of blank plasma and vortexed (SPINIX, Mumbai) for 3 minutes. Then 10 µl of internal standard solution (IS) of propyl paraben (10 µg/ml in ACN) equivalent to 5 µg/ml in plasma was added to the plasma and vortexed for 1 minute. Then 500?L of ACN was added to precipitate proteins and vortexed for 5 min and centrifuged at 5000 rpm in a micro centrifuge (REMI Scientifics, India) for 15 min. Supernatant was taken and dried in vacuum oven at 40?C. Dried samples were then redispersed in 100?L ACN and vortexed. The supernatant was transferred into a microcentrifuge tube and from this 20?L was injected for HPLC analysis.

Bioanalytical Method Validation

The developed HPLC conditions were validated as per the ICH guideline for bioanalytical method development and validation for andrographolide. The developed method was validated for its linearity, accuracy and precision, specificity and selectivity, the limit of detection (LOD) and limit of quantitation (LOQ) [17].

Calibration of Andrographolide Solution

The six points of calibration curve were constructed by plotting peak area ratio of andrographolide to the internal standard propyl paraben spiking (500 µl) with and without rat plasma. A solution containing 10µg/ml of IS was also added in each concentration. The linearity of the method was determined by the estimation of the regression coefficient (R²) value. The desirable value of R² should not be less than 0.999. [Table 1]

Quantification of Andrographolide in formulation

From the standard stock solution of andrographolide, 6 µg/ml sample was prepared and analyzed them at 5 replicates. Similarly, from the standard formulation (stimuli), equivalent to 10mg of andrographolide paniculata extract (APE) was prepared. From this solution 6 µg/ml solution of formulation was prepared. Concentration and % recovery were calculated by using linearity equation [Table 1-2].

Accuracy and Precision

Precision and accuracy were determined by analyzing three replicates at three different concentration levels. The three quality control samples were LQC, MQC and HQC. The low QC (LQC) was 4µg/ml, mid QC (MQC) was 6µg/ml, and the high QC (HQC) was 8 µg/ml. The samples for all quality control levels were prepared by the same method used for preparing the samples for linearity. All concentrations were spiked into plasma with internal standard and subjected to all the steps mentioned above of sample preparation. The concentration of quality control samples was calculated by using the linearity equation. The precision was expressed as % RSD between the accuracy values at each QC concentration levels. Accuracy was calculated using the following equation.

measured concentration.

Accuracy (% recovery) = –––––––––––––––––––× 100?dedconcentration.             

Repeatability was measured by multiple injections of a homogenous sample of 6µg/ml. The precision was calculated by %RSD.

Specificity and Selectivity

The selectivity of this method investigated by analyzing pooled blank rat plasma. Blank plasma checked by interference using proposed Protein precipitation method and HPLC condition compared with spiked sample concentration of andrographolide and LLOQ of andrographolide in plasma.

Sensitivity

Sensitivity is measured using Lower Limit of Quantification (LLOQ) is the lowest concentration of the standard curve that can be measured with acceptable accuracy and precision. The LLOQ should be established using at least five samples independent of standards and determining the co-efficient of variation and appropriate confidence interval. Measured by using LLOQ (2µg/ml) by analyzing 5 replicates. The accuracy and precision was determined by calculating % recovery, RSD and SD.

Recovery Study

The accuracy of the method was determined from recovery studies by adding known amount of standards at 80, 100, and 120% level to the placebo followed by replicate quantitative analyses by the proposed method. Recovery is calculated by considering both peak area of extracted sample and unextracted samples of andrographolide. The results are noted in Table 3.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The quantitation limit is a parameter of quantitative assay for low levels of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products. The limit of detection (LOD) and limit of quantitation (LOQ) were determined using the following formulae. The results are mentioned in Table 2

LOD =3.3*SDS

LOQ=10*SDS

Where SD the standard deviation between area ratios of six replications of low concentration standard and S is the slope of the regression line. The obtained LOD and LOQ values were then prepared in plasma and injected into the system for six replicate measurements. The relative standard deviation (% RSD) for LOD and LOQ were calculated by the following equation.

SD% RSD= ––– ×100%                                                     

Where SD is the standard deviation and M is the mean value of six replicate measurements. The acceptance criteria for LOD and LOQ is % RSD less than 33% and 10%, respectively.

Pharmacokinetic Study

Male wistar albino rats weighing about 200–250g were selected for the oral bioavailability & pharmacokinetic studies. Animals were housed in well-ventilated cages and maintained under standard conditions [at 21 ± 2°C, 60-80% Relative humidity, 12:12 hr L: D (Light and Dark) cycle] in the departmental animal house. The animals were fed with standard pelletized feed (Amrut Rat Feed, Pune) and water was provided ad libitum. The study was approved by Institutional Animal Ethical Committee (IAEC)registered with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India (Registration No: 651/PO/ReBi/S/02/CPCSEA & 19/07/2014).

Study Design

In vivo oral bioavailability study was carried out using wistar albino rats. These rats were fasted overnight with free access to water before drug administration. Three groups of wistar albino rat’s each with six animals was used for the study. Group A is named as control, Group B as standard and Group C as test sample. The control group was served with distilled water. Standard animal group received andrographolide at the dose of 50 mg/kg b.w [18] in aqueous suspension (0.5% w/v sodium-carboxy methyl cellulose solution in water) by gastric gavages. The test group received syrup at the dose of 5 ml. After administration, 0.5 ml blood sample was withdrawn by puncturing the retro-orbital plexus in heparinized eppendorff’s tubes at different time interval (0, 30, 60, 120, 180, 300, 360, 720 min.). Plasma was separated from blood by centrifugation for 15 min. at 5000 × g rpm. An aliquot of 50 ????L of plasma was mixed with 100 ????L of acetonitrile and then centrifuged at 10,000 ×g for 15min at 4?C. The supernatant was used to determine the concentration of andrographolide HPLC. Pharmacokinetic parameters of AG were calculated using non-compartmental methods. The maximum concentration (C_max) and the time to reach the maximum concentration (T_max) were obtained directly by visual inspection of the data from the plasma concentration versus time plot. Areas under the plasma-concentration-time curve from time zero to the last measurable AG sample time and to infinity (AUC_0-t and AUC_0-inf) were calculated by trapezoid rule. The elimination rate constant (K_el) was derived from the slope of the log plasma concentration versus time plot, and elimination half-life (T_1/2) was calculated by formula T_1/2 = 0.693/K_el [1] for the period of 0 to 6 h. Determination of C_max, T_max, AUC, is done by using PK SOLVER 0.2 software.

RESULTS AND DISCUSSION

Pharmaceutical analysis requires very precise and accurate assay methods to quantify drugs either in Pharmaceutical or biological samples. Analytical methods are used for product research, product development and quality control purposes. The assay methods must be sensitive, selective, precise, and reproducible. Such methods play a significant role in the evaluation and interpretation of pharmacokinetic data. The main analytical phases comprise method development, method validation and sample analysis. The method for the analysis of andrographolide in test solutions and biological samples was developed and validated in accordance with ICH guidelines, and all parameters were within acceptable ranges. The obtained results were comparable to previous similar studies that focused on the analysis of andrographolide from herbs and biological samples [11-17]. The validated method offered advantages, including the use of simple and cost-effective solvents, low retention time, and significant peak purity of the analyzed samples. The research conducted was one-of-a-kind, comparing the absorption of andrographolide (pure phytomarker) with a polyherbal formulation containing A. paniculata.

Bioanalytical Method validation:

HPLC Method and Extraction of Drug from Plasma

Bioanalytical method development and validation of AG by using Agilent Eclipse plus C18 (100 × 4.6, 5?m) as stationary phases. The developed RP-HPLC method was very simple, involving an equal volume ratio of acetonitrile and water (pH adjusted 7.5 by triethanolamine; 30:70, v/v). Total chromatographic run time was 14 min. Plasma extraction method developed was a simple liquid-liquid extraction. AG analysis in plasma sample was developed using liquid-liquid extraction involving acetonitrile as extracting organic solvent. A rapid and sensitive RP-HPLC bioanalytical method for the estimation of AG was developed and validated according to ICH guidelines Figure.1.

The linearity of an analytical method is its ability to elicit that test results are proportional to the concentration of analyte in samples within a given range [20]. Linearity was plotted over 6-points concentration ranges: 2, 4, 6, 8, 10,12 ?g/mL for AG in plasma. The Linear calibration curve (Table 1) with a correlation coefficient (R²) 0.9986 was obtained. The R² indicated an excellent linear correlation between peak area ratios (AG to IS) and concentration of Andrographolide. The linearity equation was y = 50376x + 17496.

Precision and Accuracy

The accuracy of an analytical method describes the closeness of test results to the actual concentration of analytes. In contrast, precision is a measure of the degree of repeatability and reproducibility of the analytical method. In this research, only intra-day precision was evaluated as part of the partial method validation study. The accuracy and precision of the method were assessed by analyzing the three Quality control (QC) samples (4 µg/ml LQC, 6 µg/ml MQC, and 8 µg/ml HQC). The method employed in this research bears a simple and rapid liquid-liquid extraction with excellent recovery, sharp peak shapes, and consistent performance of the HPLC system. Therefore, the accuracy and precision of the method evaluated at three quality control levels met the acceptance criteria. This bioanalytical method was used in the pharmacokinetic studies to quantify the andrographolide in the plasma of rats (Table 2 and 3).

Table 2. The validation values for determination of andrographolide in rat plasma

Table 3. Accuracy study of Andrographolide in Commercial Hepatoprotective Formulation spiked with rat plasma

Selectivity and Specificity

Specificity and selectivity were studied for the examination of the presence of interfering components. The developed HPLC method was found to be selective and specific as it was able to differentiate and quantify AG in the presence of other plasma component and the IS. IS was selected as internal standard because of its adequate resolution with AG, satisfactory peak shapes, stability, and consistency in area count during the analysis. During the validation process, no peaks were found at AG and IS retention times in mobile phase and plasma as illustrated in Fig.1. The chromatogram of the same showed good separation with low background noise. The retention time for IS and AG were about 4.2 min and 8.3 min, respectively. The total chromatographic run time was 15.0 min (Table 2,4).

Table 4. Amount of andrographolide in Commercial Hepatoprotective Formulation *

CHF*= Commercial Hepatoprotective Formulation

LOD and LOQ

The limit of detection (LOD) is the smallest concentration of the analyte that gives the measurable response while the limit of quantification (LOQ) is the smallest concentration of the analyte, which gives response that can be accurately quantified¹⁶. The minimum detectable concentration of AG (LOD) was found to be 0.2129µg/ml, whereas the quantitative limit (LOQ) was 0.6452 µg/ml. The highly sensitive method is suitable enough to quantify low drug concentration in a pharmacokinetic study.

Pharmacokinetic Parameters

The established HPLC method was sensitive enough to assess the pharmacokinetic parameters following a single dose of andrographolide in experimental rats. The mean plasma drug concentration-time profile of AG is shown in Figure 3. The pharmacokinetic parameters values such as maximum plasma concentration (C_max), time required for maximum plasma concentration (T_max), area under the curve (plasma concentration) from the initial time to 6 h (AUC _0-t), area under the curve (plasma concentration) from time zero to infinity (AUC_0-?), the elimination rate constant (K_el), and terminal half-life (t_1/2) have been summarized in Table 5. Andrographolide was quickly and almost completely absorbed into blood following the oral administration of Syrup containing A. paniculata extract at a dose of 100mg/kg body weight in rats. Following the oral administration of syrup (a single therapeutic dose, equal to 10 mg of andrographolide) to rats, maximum plasma levels of approximately 17.89 ng/mL were reached after 3 hours, as quantified by UV diode-array detection method. It was found that the pharmacokinetics of andrographolide in rats were highly variable among individuals Figure 2.

Table 5. Pharmacokinetic parameters of andrographolide in arts after oral administration of standard AG (20 mg/kg) and Commercial Hepatoprotective Formulation (250 mg/kg CHF)

*AG=Andrographolide; CHF*= Commercial Hepatoprotective Formulation; Values are in (mean±SD, n=6)

       
           
       
Figure 3. Typical HPLC chromatograms of (A) a rat plasma sample spiked with IS 30 min after oral administration of pure AG (20 mg/kg) and 30 min after receiving oral doses of 250 mg/kg CHF (B)