Original Article

Development and Validation of an In Vitro Dissolution Method Based on HPLC Analysis for L-Dopa Release From PLGA Nanoparticles


  • Sema ARISOY
  • Özgün SAYINER
  • Tansel ÇOMOĞLU

Received Date: 07.11.2019 Accepted Date: 18.02.2020 Bezmialem Science 2021;9(1):9-19


In the past decade, dissolution testing has emerged as a valauble tool for the characterization of drug product performance in the field of pharmaceuticals. During the development of new formulations, dissolutions tests assist in the evaulation of any changes in the formulation arising during manufacturing process, thereby assuring product quality and performance post manufacturing.


In the present study, a simple high performance liquid chromatography (HPLC) method was developed and validated to quantitate the release of L‐Dopa from poly (D, L‐lactic‐co‐glycolic acid) (PLGA) nanoparticles. The chromatographic separation was performed with a reversed‐phase C18 column, using acetonitrilewater containing 0.05% trifluoroacetic acid (5:95, v/v) as a mobile phase at 280 nm. The developed method was validated for its specificty, linearity, accuracy, and precision according to the ICH guidelines.


The developed method was shown to be linear (r2 ≥ 0.995) in the concentration range of 125-40 μg/mL. The mean % recoveries were found to be 102.59-98.70%, indicating an agreement between the true value and the detected value. Solution stability was guaranteed by the addition of an antioxidant. The analytical method was shown to be suitable for the evaluation of release of L‐Dopa from PLGA nanoparticles. In vitro release of L‐Dopa was studied using sample and separate (SS) and dialysis membrane (DM) methods. To compare SS and DM methods, difference (ƒ1) and similarity (ƒ2) factors were calculated. No significant differences were recorded in the release kinetics of L‐Dopa from nanoparticles using both methods (ƒ1 <15 and ƒ2 >50).


Dissolution test methods were compared and procedure for an analytical method based on HPLC was optimizated and validated for the dissolution of L‐Dopa loaded nanoparticles.

Keywords: Analytical validation, L‐Dopa, PLGA nanoparticles, in vitro drug release, sample and seperation method, dialysis membrane method


Parkinson’s disease (PD) is the second most common neurodegenerative disorder with a global prevalence of 1-3% in the population with age above 65 years. Clinical pathology of PD involves progressive loss of dopaminergic neurons, particularly in nigro‐striatal area and its surrounding pathways. This loss of dopaminergic neurons affects movement and facial expressions in the affected patients (1,2). Conventional treatment for PD involves administration of L‐Dopa, a dopamine precursor. However, L‐Dopa therapy is associated with certain limitations. These include fluctuations in L‐Dopa levels in the plasma owing to erratic gastric emptying and intermittent oral intake (3), poor relative bioavailability (∼5-15%), and availability of <1% of the administered dose in the brain (4-7).

In the past few decades, several polymeric nanoparticles prepared using natural or synthetic polymers have been developed and explored to allow safe and enhanced delivery of drugs like L‐Dopa to the targeted site. Among these, nanoparticles obtained from biodegradable and biocompatible polymers such as  poly‐lactic‐co‐glycolic acid (PLGA) or poly (glycolic acid) have been used most commonly. In general, nanoparticles are preferred for drug administration owing to their high chemical and biological resistance and ability to carry both hydrophilic and lipophilic substances in their active form. In addition, these nanoparticles can be administered in the body via different routes.

Since nanoparticles release small amounts of encapsulated drugs as a function of time, the analytical methods used to study in vitro release of drugs must be highly sensitive to quantify drug concentrations in the dissolution medium. Testing methods used to study in vitro release for nanoparticles based delivery systems can be broadly divided into three categories, membrane diffusion methods [such as dialysis method (DM)], sample and separation methods (SS), and continuous flow methods. Among these, DM is used most commonly to evaluate in vitro release of drugs from nanoparticles, followed by SS. In the present study, DM was used as reference method and SS was used as test method.

Among the currently available analytical methods, HPLC is the most commonly used method employed for the characterization of various pharmaceutical molecules. HPLC‐diode array detector (DAD) offers a quick, sensitive, and accurate method for the separation and identification of drugs in pharmaceutical nanoparticulate formulations (8,9). Although several researchers have investigated in vitro release of drugs from nanoparticles, no HPLC method has been established for the simultaneous determination of L-Dopa. No studies have been reported for the use of DM and SS methods for the quantification of the amount of L‐Dopa released from nanoparticles in dissolution medium. Besides this, in most of the reported studies used L‐Dopa solutions at low pH as standard solution (10-13).

Nanoparticles were specially designed to ensure delivery of L‐Dopa to brain by endocytosis when administered nasally. To represent dissolution profile of L‐Dopa released from the nanoparticles, two buffered solutions mimicking pH conditions present in the endolysosomal compartment (pH 4.5) and brain (pH 7.4) were used (14). Several previous studies have reported evidences for the stability of L‐Dopa in acidic conditions (10,12,13,15). The present study aimed to develop and optimize an analytical method to determine the levels of L‐Dopa in different media having a higher pH as compared to the acidic media reported earlier for L‐Dopa.

In the present study, a double emulsion‐solvent evaporation method was used to prepare PLGA nanoparticles, wherein methylene chloride, polyvinyl alcohol, and PLGA were used as organic solvent, surfactant, and polymer, respectively. In order to determine the release profile of L‐Dopa from PLGA nanoparticles, SS and DM methods were compared. In general, it is believed that in vitro release method reflects the changes in the manufacturing procedure that in turn affects the performance of the drug entrapped in the nanoparticles. Thus, in vitro dissolution test methods used in the study were compared and HPLC method was further validated to ensure the robustness of the developed analytical method. The study also included stability tests for the L-Dopa released from the PLGA nanoparticles.



For HPLC studies, Agilent 1100 series integrated HPLC system with DAD, pump, auto‐sampler, and degasser unit was used. Reversed phase HPLC analysis was carried out using a 250x4.6 mm, 5 µm reversed‐phase C18 HPLC column obtained from Macherey‐Nagel. For in vitro drug release studies, orbital shaker and ultra centrifuge (Sigma® 30KS) purchased from Sigma were used.

Reagents and Materials

L-Dopa was a kind gift from ILKO Pharmaceuticals (Ankara, Turkey). Deionised water used in the study was obtained from a Millipore water supplier. Trifluoroacetic acid (TFA), NaCl, Na2HPO4, KH2PO4, acentonitrile (HPLC grade), and Tween 80 were procured from Merck. For HPLC analysis, the mobile phase was filtered through a 0.45 µm membrane filter (Millipore, Barcelona) and degassed using an ultrasonic bath, prior to use. Cellulose dialysis tubing (14,000 MWCO), polypropylene co‐polymer centrifuge tubes, and polyvinyl alcohol (PVA) were purchased from Sigma‐Aldrich.

Chromatographic System and Conditions

First, the wavelength for absorption maxima for the drug was selected based on its UV spectrum. L‐Dopa was characterized by a absroption maxima at 280 nm which was further used for HPLC analysis. Mobile phase for HPLC analysis comprised of TFA solution (0.05% v/v, pH 3) and acetonitrile at 95:5 (v/v). For chromatographic separation, 250x4.6 mm, 5 µm C18 reversed‐phase HPLC column was used. A flow rate of 1 mL/min and run time of 7 minutes with 10 µL injection volume were used for HPLC analysis (1). The method used in the present study was developed and validated as per the considerations of the ICH guideline Q2 (R1) that involved several parameters including specificity, linearity, detection and quantification limits, repeatability and intermediate precision, accuracy, and stability (16).

Preparation of Reagents

Suitable analytical procedures should be used to define the amount of L‐Dopa used during dissolution test. Two stock solutions of L‐Dopa at concentration of 100 µg/mL were prepared by dissolving a suitable amount of L‐Dopa in buffered solutions at pH 4.5 and pH 7.4. Prior to use, all solutions were sonicated for 30 min. For each stock solution, 6 diluted samples in the concentration range of 1.25-40 µg/mL (ppm) (1.25, 2.5, 5, 10, 20, and 40 µg/mL) were prepared from the stock solutions. The stability of L‐Dopa in the diluents (phosphate buffer solution at pH 4.5 and pH 7.4) was investigated for a period of 48 hours at different tempratures (4 ºC, 25 ºC, and 37 ºC) in both the presence and absence of ascorbic acid.

Preparation of Nanoparticles

L‐Dopa loaded nanoparticles were prepared using a double emulsion solvent evaporation method with PVA as a stabilizer. Briefly, L‐Dopa and PLGA were first dissolved in dichloromethane (DCM). Further, distilled water was added to the resulting solution and mixed using an ultrasonic homogenizer to form a primary water‐in‐oil (W/O) emulsion. Following this, the primary emulsion was emulsified in PVA solution with homogenization to form a double water‐in‐oil‐in‐water (W1/O/W2) emulsion. The resulting double emulsion was stirred using a magnetic stirrer at a constant rate at room temperature (25 °C) to evaporate the organic solvent. The resulting nanoparticle suspension was further incubated at 4 °C overnight to ensure hardening of the PLGA matrix by allowing DCM to fully partition to the external aqueous phase. The nanoparticles were recovered by ultracentrifugation. The supernatant was carefully removed and pellet containing nanoparticles was washed twice with distilled water to remove free drug and excess surfactant. The sample was further subjected to lyophilization.

Dissolution Test Development

Dissolution Medium

The term “sink condition” is generally defined as the ability of the dissolution medium to dissolve at least three times the amount of drug present in the dosage form. Percentage of drugs released from the nanoparticles should be detected using the developed analytical method. Selection of the most suitable media conditions is based upon the stability of the analyte in the test medium and its application in terms of in vivo performance. Since the nanoparticles were specifically designed to deliver L-Dopa to brain via endocytosis using a nasal route of administration, the in vitro release of L‐Dopa from PLGA nanoparticles was studied at pH 4.5 to mimic the endolysosomal pH and at pH 7.4 to mimic the brain pH (17). Phosphate buffer solution at pH 4.5 was prepared by dissolving 6.8 g potasium dihydrogen phosphate in 1 L distilled water containing 2 % Tween 80 (v/v) and 0.1 % (w/v) ascorbic acid (aa). Phosphate buffer solution at pH 7.4 was prepared by dissolving 2.38 g disodium hydrogen phosphate, 0.19 g potassium dihydrogen phosphate, 8 g sodium chloride, 2% Tween 80, and 0.1% aa in 1 L distilled water. In the present study, 2% (w/v) Tween 80 was used to enhance the solubility of L‐Dopa in aqueous solution and 0.1% (w/v) aa was added to protect L‐Dopa from oxidation during the assay (18).

Method devolopment

In Vitro Drug Release (SS Method)

L‐Dopa loaded nanoparticles weighed to contain 45 µg L‐Dopa were suspended in 5 mL of buffered solution. The nanoparticle suspensions were transferred to tubes and incubated in an orbital shaker bath at 37±0.5 °C with rotation at 100 rpm. The tubes were removed from the water bath at 30, 60, 120, and 240 minutes, and centrifuged at 2000 rpm for 20 min. The supernatant was carefully removed and used for further analyis. The nanoparticle pellets were resuspended in 5 mL of fresh buffer (pH 4.5) and placed back in the shaker bath. The supernatant was used to determine the concentration of L‐Dopa. All experiments were perfomed in triplicates and results were expressed as mean ± variation.

In Vitro Drug Release (DM Method)

L-Dopa loaded nanoparticles containing 45 µg L‐Dopa were suspended in 0.5 mL of buffered solution and inserted in a dialysis bag. The dialysis bag was placed in 4.5 mL (total 5 mL) of buffered solution at pH 4.5. This was further incubated at 37±0.5 °C in an orbital shaker bath at 100 rpm. To evaluate drug release as a function of time, 1 mL sample aliquots were collected at 30, 60, 120 and 240 minutes and replaced with 1 mL fresh buffer (pH 4.5). Drug concentrations in the aliquots were determined using the above mentioned analytical method. All experiments were performed in triplicates and results were expressed as mean ± variation.

Statistical Calculations

All results are reported as mean ± standard deviation of replicates. For drug release (%) at different time intervals, two tailed t‐test was performed using Prism Software Version 6.0, where differences were considered to be significant with p<0.05. Microsoft Office Excel® was used to calculate ƒ1 and ƒ2 factors.


The present study involved development of a HPLC based analytical method to determine time dependent release of L‐Dopa from PLGA nanoparticles synthesized using a double emulsion solvent evaporation method. For reversed‐phase HPLC analyis, mobile phase comprised of TFA solution (0.05%  v/v) at pH 3 and acetonitrile at ratio of 95:5 (v/v). The chromatographic separation was carried out using a 250x4.6 mm, 5 µm C18 HPLC column. The flow rate for HPLC analysis was kept at 1 mL/min with run time of 7 minutes and 10 µL injection volume. No interfence was observed after injection of L‐Dopa solution, placebo, and nanoparticles.

The regression equations for the calibration curve were found to be y=5.7424x +2.1421 (Figure 3) and y=7.4159x +9.5333 (Figure 4) for phosphate buffer solution at pH 4.5 and phospate buffer saline solution at pH 7.4, respectively. The regression coefficient r2 was calculated to be 0.9998 and 0.995 for buffered solutions at pH 4.5 and pH 7.4, respectively. Linearity data calculated using Prism Software Version 6.0 are summarized in Table 1. Repeatability analysis for buffered solutions at pH 4.5 and pH 7.4 were characterized by relative standard deviation (RSD) % of 3.07-0.03% and 0.83-0.01%, respectively. For inter day assays, buffer at pH 4.5 was found to have precision with RSD % of 1.44-0.01% while buffer at pH 7.4 showed precision of RSD % 1.00% and 0.01%. The mean % recoveries for buffered solutions at pH 4.5 and pH 7.4 were calculated to be in the range of 102.59-98.70 and 101.92-100.00, respectively. LD and LQ values were found to be 0.0408 and 0.1235 µg/mL, respectively, for buffer at pH 4.5. For buffer at pH 7.4, LD and LQ values were 0.0636 and 0.1928 µg/mL, respectively. When the analysis was performed as function of temperature over a period of 48 hours, the amount of remaining L‐Dopa was found to be 97.46% at 4 ºC, 95.54% at 25 ºC, and 89.63% at 37 ºC for buffer at pH 4.5 in presence of ascorbic acid. In comparison to this, the remaining L‐Dopa in buffered solution at pH 7.4 in the presence of aa over a period of 48 hours was calculated to be 103.21% at 4 ºC, 88.21% at 25 ºC, and 75.85% at 37 ºC.

The differences in release rate were evaluated for SS and DM method. ƒ1 was found to be 6.02 and ƒ2 was 95.38. No differences were observed in the test and reference methods for all the time intervals for which drug release (%) was studied (p=0.853; p<0.05).


Optimization of the Chromatographic Method

The HPLC method developed in the present study to determine the release of drugs provided a reliable quality control analysis. The wavelength selection for HPLC analysis was done on the basis of absorption maxima obtained for three different concentrations of L‐Dopa solutions according to the acquired ultraviole (UV) spectra. A wavelength of 280 nm was selected for HPLC analysis because it provided high sensitivity, required for the quantitation of significantly low concentations of the drug present in the dissolution samples.

The detection of L‐Dopa required an adequate mobile phase comprising of a suitable ratio of polar to non‐polar solvent. For an acceptable chromatographic separation, several parameters including pH of the mobile phase and percentage of organic modifier were tested. HPLC analysis was conducted at a flow rate of 1 mL/min with a run time of 7 minutes and 10 µL injection volume. L-Dopa at a concentration of 10 ppm was injected into the system. The chromatograms obtained for the HPLC analysis showed that the use of acidic mobile phase with reversed‐phase C18 column provided high solubility for L‐Dopa resulting in symmetric and sharp peaks. As shown in Figure 1, TFA solution was determined as acidic buffer solution for HPLC analysis.

For the mobile phase used in HPLC analysis, when the ratio of acetonitrile to 0.05 % (v/v) TFA solution in water was changed from 10:90 (v/v) to 5:95 (v/v) a sharp peak for the active substance was obtained. Following this, a ratio of 2.5:97.5 (v/v) for acetonitrile to 0.05% (v/v) TFA solution was also tested for mobile phase to allow for better separation. However, peak of L‐Dopa couldn’t be determined at this ratio in contrast to our previous experiments. In order to protect the column’s integrity, pH for 0.05% (v/v) TFA solution was adjusted to pH 3 using 0.1 N HCl solution and thus the resulting peak tailing was acceptable (Figure 1).

For the sample peak, symmetry factor was found to be 1.058 which was within the limit of <1.5 as established for various pharmacopoeias (Figure 1). Therefore, all the HPLC analysis performed in the study were carried out using a 250x4.6 mm, 5 µm C18 HPLC column with a flow rate of 1 mL/min, run time of 7 minutes, 10 µL injection volume, 25 °C column temperature, and mobile phase comprising of acetonitrile and 0.05% (v/v) TFA solution at 5:95 (v/v) (pH 3) (1).

Nanoparticles used in the study were specifically designed to be transported to brain via nasal route by means of endocytosis. To evaluate the dissolution profile of L‐Dopa from PLGA nanoparticles, two buffered solutions at pH 4.5 and pH 7.4 were used to mimic pH of endolysosomal compartment and brain, respectively (14). As mentioned earlier, several studies have previously established the stability of L‐Dopa in acidic conditions (10,12,13,15). Therefore, present study focussed on developing and optimizing a HPLC based analytical method to determine the amount of released L‐Dopa in different media conditions having pH values higher than those reported in previous studies. In addition to this, buffered solutions at pH 4.5 and pH 7.4 with and without L‐Dopa were also injected and analyzed using the same method to check for any interference in the peaks. As shown in Figure 2, no interference was observed (Figure 2).

Method Validation

In the present study, the analytical method was developed and validated according to the ICH guideline Q2 (R1) (16). The developed method was validated for various characteristics including linearity, accuracy, precision, specificity, stability, and detection limit and quantification limit. The method thus established was further utilised to quantitate the amount of the drug released from L‐Dopa loaded nanoparticles.


For the evaluation of linearity, standard solutions at different concentrations of L‐Dopa were used. ICH guidelines suggest use of a minimum of five concentration levels for linearity studies. In the present study, linearity for L‐Dopa was determined over a range of 1.25-40 µg/mL at six different concentrations. All the analysis were performed in triplicates (n=3) (16). For each concentration, samples were analysed six times, and the resulting peak areas were documented and analysed. The results for the regression analysis are shown in Figures 3 and 4. A good linear relationship (r2 ≥0.995) was observed between the concentrations and their respective peak areas as provided by the detector (Figures 3 and 4). The regression coefficient r2 values  ≥0.995 are considered to be acceptable according to the ICH guidelines (16). Generally, correlation intercept is calculated to evaluate the acceptability of the linearity of the data. In fact, for better analysis of the data, slope of line should also be calculated (Table 1) (8). All these results showed that all calibration curves were characterized by suitable linearity according to ICH guidlines (16).


The values for the absolute differences between the mean assay results for the developed method were obtained using repeatability and intermediate precision tests. All these values met the acceptance criteria of RSD %, RSD <2.0. In general, repeatability establishes the precision of multiple sampling under the same operating conditions over a short interval of time. In comparison to this, precision establishes variations arising from the same laboratory under variable operating conditions, for example different days, analysts or equipment. Thus, all these results suggest that the developed method was reproducible and precise (19).


Repeatability proves precision of the method under same operational conditions over a short period of time. Repeatability also refers to intra‐assay sensitivity. In the present study, low (1.25 µg/mL), medium (5 µg/mL), and high (20 µg/mL) concentrations of L‐Dopa used in the calibration curve were analyzed to determine mean, standard deviation (SD), and RSD (n = 6). As per the standard guidelines, RSD % for standard peak must be < 2.0 (20). As shown in Tables 2 and 3, the developed method met the standart requirements of this analytical validation parameter for medium (5 µg/mL) and high (20 µg/mL) concentrations of the drug (20). In case of L‐Dopa concentration of 1.25 µg/mL, which represents the lower concentration, % RSD was >2.0. However, there are several studies that extend the limits for % RSD to 5-6% (8). Since the concentration of 1.25 µg/mL represents the lowest point of the calibration curve, so extension of limits might be suitable for this method.

Intermediate Precision

The variations in terms of analyst for the developed method were determined using replicate injections of the above mentioned concentrations and analyzed using different analysts on the same day (Tables 4 and 5). Intermediate precision was performed using RSD % of six repeated assays on samples at three concentration levels. RSD % values were found to be in the range of 1.44-0.1% for all concentration levels and pHs studied. For intermediate precision, RSD % value should not exceed 2.0% The results summarized in Tables 4 and 5 show that the developed method met the requirements set for this analytical validation parameter (16).


The accuracy was determined in terms of recovery of known amounts of L‐Dopa. For testing the accuracy of analytical methods, three concentrations (1.25, 5, and 20 µg/mL representing low, medium, and high concentrations, respectively) of the drug, covering the linear range of analytes, were prepared by diluting the stock solutions. The mean % recoveries were found to be in the range of 101.92-98.70%, thus implying an agreement between the true value and the found value (Tables 6 and 7). All these results indicate that the developed method met the requirements for method verification according to ICH guidelines (16).


Specificity of an analytical method can be defined as the ability to assess an analyte unequivocally in the presence of expected components. This definition has a descriptive effect on the identity of an analyte. In our study, the specificity tests were performed using three different polymers for nanoparticle preparation and L‐Dopa in two different media conditions. It was decided that the HPLC method was specific for the determination of L‐Dopa according to the data summarized in Table 8. L‐Dopa samples were injected five times and similar retention times were observed in all cases. All the nanoparticles prepared using different polymers were injected with or without L‐Dopa. No interference was observed between the peaks of nanoparticles and L‐Dopa. Thus, the described HPLC method was specific for buffered solution at pH 7.4 buffer and selective for buffered solution at pH 4.5.


The stability of L-Dopa in aqueous solution was evaluated to verify any spontaneous degradation of the samples during preparation (21). The aqueous solutions of L‐Dopa were found to be unstable. However, aqueous solutions have been previously shown to be stable in the presence of high concentrations of acidic substances. Stability of L-Dopa remained uneffected in the presence of light. The storage condition for the active substance was specified to be at 2-8 °C. In addition to this, the oxidation of L‐Dopa can be avoided in the presence of antioxidants. Therefore, information was obtained for the stability of L‐Dopa in the presence of aa in the body and during the validation and formulation studies (3,18,22). Stock solution of L-Dopa at 0.4 mg/mL concentration was prepared using buffered solutions at pH 4.5 and pH 7.4. The solutions were mixed and vortexed until solid particles disappeared. The samples were further divided into aliquots of 3 mL. The stability of the solutions containing ascorbic acid was extended up to 48 hours. No oxidation was observed in the solutions containing aa at 4 °C. Thus, all these results suggest that aa should be added as a preservative in the dissolution medium during dissolution studies. No interference was recorded between the peaks of L-Dopa and ascorbic acid. The results of stability studies are summarized in Tables 9 and 10.

Limit of Detection (LD) and Limit of Quantification (LQ)

Limit of detection (LD) represents the lowest concentration level in a peak area where signal level is atleast three fold of the baseline‐to‐noise. Limit of quantification (LQ) represents the lowest concentration level that can be precisely provided by a peak area with a given signal‐to‐noise. Calibration curve was calculated using L‐Dopa concentrations in the range of 1.25-40 µg/mL. The following equations were used:

Detection limit (LD) =3.3 a/s;

Quantification limit (LQ)  =10 a/s;

where “a” is the standard deviation of y‐intercept of regression lines and “s” is the slope of the calibration curve. For LD and LQ, no defined limit is available in the literature and these are specific for each method. As shown in Table 12, the results met the requirements for in vitro dissolution tests (23).

In Vitro Drug Release

In order to design a suitable dissolution medium for a poorly soluble drug, the first method involves increasing the volume of aqueous sink conditions or decreasing the amount of dissolved drug. The second approach is based upon the addition of anionic or non‐ionic surfactans and solubilization of the drug by co‐solvents up to 40%. In another approach, pH is altered to obtain a better sink condition. The last two approaches are less cumbersome and have been employed more widely in dissolution tests in the pharmaceutical industry. To enhance the solubility of L‐Dopa 2% Tween 80 was added to the dissolution medium. In addition to this, ascorbic acid was also used in the mediums to protect L‐Dopa against oxidation during in vitro release.

The cumulative amount of L‐Dopa released from polymeric material was plotted as a function of time. As shown in Figure 4, only 5-6% of the drug content was released from the nanoparticles as evaluated for both methods. In order to achieve sink conditions, L-Dopa concentrations shouldn’t exceed 20% of its saturation solubility in dissolution medium which was maintained in the the present study. Thus, inhibited release of L‐Dopa was not contributed by insufficient sink conditions. In addition, it did not arise owing to inadequate loading of nanoparticles either (amount of released drug as a function of the encapsulated drug substance). Interactions between L‐Dopa and PLGA prevented complete dissolution. Moreover, 5-6% of the drug content was suitable enough for the dicussion of the two methods. In vitro release profiles of the test formulations were similar to the reference formulation. In literature, it is mentioned that release rate of the encapsulated drug is progressively higher in SS method as compared to DM (Figure 5) (24). This might be contributed by the differences in the hydrodynamics of the system as the nanoparticles are present in the dialysis bag in one methode while in the other formulation is dispersed in a flask (25).

The similarity factor (ƒ2) is a measurement of the similarity in the percent (%) dissolution between the two profiles (26). The difference factor (ƒ1) is proportional to the average difference between the two profiles. FDA guidelines recommend that ƒ1 <15 and ƒ2  >50 are indicative of equivalence in dissolution profiles (27).

ƒ2 = 50 x log [1 + (1/n) Σnt=1(Rt - Tt)2] -0.5x100)

ƒ1 = [Σnt=1 (Rt - Tt ) / Σnt=1Rt] x100

As shown in Table 12, no signicant differences were obtained in the release kinetics of L‐Dopa as studied using both methods (ƒ1 <15 and ƒ2  >50) (24,28). A two tailed t‐test was used to compare the dissolution profiles. No differences were observed in the drug release (%) for the test and reference methods at all time intervals (p=0.853; p<0.05).


The analytical methods developed and validated in the present study were found to be simple, sensitive, accurate, and precise. The results of the study indicated that the developed methods are suitable for dissolution studies as well as routine quality control analysis of L‐Dopa present in nanoparticulate formulations. DM and SS methods were characterized by similar in vitro release profiles, with acceptable precision (<10% SD). SS method showed faster release from formulations as compared to those observed using dialysis bag (Table 12). No significant differences were reported between DM and SS methods used for release kinetics study (ƒ1 <15 and ƒ2 >50). For further studies, the optimal method with in vivo relevance needs to established keeping into consideration in vivo to in vitro corelation.


Ethics Committee Approval: In vitro study.

Informed Consent: In vitro study.

Peer-review: Internally and externally peer reviewed.

Authorship Contributions

Data Collection or Processing: S.A., Ö.S., Analysis or Interpretation: T.Ç., Literature Search: S.A., Ö.S., Writing: S.A., T.Ç.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.

  1. Zhou YZ, Alany RG, Chuang V, Wen J. Optimization of PLGA nanoparticles formulation containing L-DOPA by applying the central composite design. Drug Dev Ind Pharm 2013;39:321-30.
  2. Sharma S, Lohan S, Murthy R. Formulation and characterization of intranasal mucoadhesive nanoparticulates and thermo-reversible gel of levodopa for brain delivery. Drug Dev Ind Pharm 2014;40:869-78.
  3. Luinstra M, Grasmeijer F, Hagedoorn P, Moes JR, Frijlink HW, de Boer AH. A levodopa dry powder inhaler for the treatment of Parkinson’s disease patients in off periods. Eur J Pharm Biopharm 2015;97(Pt A):22-9.
  4. Vasa DM, Buckner IS, Cavanaugh JE, Wildfong PL. Improved Flux of Levodopa via Direct Deposition of Solid Microparticles on Nasal Tissue. AAPS Pharm Sci Tech 2017;18:904-12.
  5. Pahuja R, Seth K, Shukla A, Shukla RK, Bhatnagar P, Chauhan LKS, et al. Trans-blood brain barrier delivery of dopamine-loaded nanoparticles reverses functional deficits in parkinsonian rats. ACS Nano 2015;9:4850-71.
  6. Nedorubov A, Pavlov A, Pyatigorskaya N, Brkich G, Aladysheva Z. HPLC-MS/MS Method Application for the Determination of Pharmacokinetic Parameters of Intranasal Delivered L-DOPA in Rats. J Pharm Sci Res 2018;10:2489-92.
  7. Gambaryan P, Kondrasheva I, Severin E, Guseva A, Kamensky A. Increasing the Efficiency of Parkinson’s Disease Treatment Using a poly (lactic-co-glycolic acid)(PLGA) Based L-DOPA Delivery System. Exp Neurobiol 2014;23:246-52.
  8. Souza PRdSe, Carvalho JMd, Albert ALM, Moreira JC, Leandro KC. Development and validation of a method for the determination of valproic acid in pharmaceutical formulations by high performance liquid chromatography with diode array detection (HPLC-DAD). Vigilância Sanitária em Debate 2013;1.
  9. Fuster J, Negro S, Salama A, Fernández-Carballido A, Marcianes P, Boeva L, et al. HPLC-UV method development and validation for the quantification of ropinirole in new PLGA multiparticulate systems: Microspheres and nanoparticles. Int J Pharm2015;491:310-7.
  10. Raut PP, Charde SY. Simultaneous estimation of levodopa and carbidopa by RP-HPLC using a fluorescence detector: its application to a pharmaceutical dosage form. Luminescence 2014;29:762-71.
  12. Dhawan R, Ravi S, Subburaju T. Compatibility and stability studies of levadopa, carbidopa, entacapone and natural bioenhancer mixture. IJPAR 2014;3:474-81.
  13. Reddy BJC, Sarada NC. A Simple Validated Stability Indicating RP-HPLC Method for the Determination of Three Antiparkinsonism Compounds in Oral Contraceptive Tablet Formulations. Int Jornal ChemTech Research 2017;10:636-46.
  14. Gao X, Tao W, Lu W, Zhang Q, Zhang Y, Jiang X, et al. Lectin-conjugated PEG–PLA nanoparticles: preparation and brain delivery after intranasal administration. Biomaterials 2006;27:3482-90.
  15. Junnotula V, Licea-Perez H. Development and validation of a simple and sensitive method for quantification of levodopa and carbidopa in rat and monkey plasma using derivatization and UPLC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 2013;926:47-53.
  16. ICH. Q2(R1) Validation Of Analytical Procedures: Text And Methodology. 2005.
  17. Piazza J, Hoare T, Molinaro L, Terpstra K, Bhandari J, Selvaganapathy PR, et al. Haloperidol-loaded intranasally administered lectin functionalized poly (ethylene glycol)–block-poly (D, L)-lactic-co-glycolic acid (PEG–PLGA) nanoparticles for the treatment of schizophrenia. Eur J Pharm Biopharm 2014;87:30-9. 
  18. Pappert EJ, Buhrfiend C, Lipton JW, Carvey PM, Stebbins GT, Goetz CG. Levodopa Stability in Solution: Time Course, Enviromental Effects, and Practical Recomendations for Cl,n,cal Use. Mov Disord 1996;11:24-6.
  19. Bhatnagar P, Vyas D. Stability Indicating HPLC Method for Simultaneous Estimation of Entacapone, Levodopa and Carbidopa in Pharmaceutical Formulation. J Chromatogr Sep Tech 2015;6:7.
  20. Ermer J, Miller JHM. Method validation in pharmaceutical analysis: A guide to best practice: John Wiley & Sons; 2006.
  21. Mendez AS, Steppe M, Schapoval EE. Validation of HPLC and UV spectrophotometric methods for the determination of meropenem in pharmaceutical dosage form. J Pharm Biomed Anal 2003;3:947-54.
  22. Ravani L, Sarpietro MG, Esposito E, Di Stefano A, Sozio P, Calcagno M, et al. Lipid nanocarriers containing a levodopa prodrug with potential antiparkinsonian activity. Mater Sci Eng C Mater Biol Appl 2015;48:294-300.
  23. Mehta J, Patidar K, Patel P, Kshatri N, Vyas N. Development & validation of an in vitro dissolution method with HPLC analysis for misoprostol in formulated dosage form. Anal Methods 2010;2:72-5.
  24. D’Souza SS, DeLuca PP. Methods to assess in vitro drug release from injectable polymeric particulate systems. Pharm Res 2006;23:460-74.
  25. D’Souza SS, DeLuca PP. Development of a dialysis in vitro release method for biodegradable microspheres. AAPS PharmSciTech 2005;6:E323-E8.
  26. Gonjari DI, Karmarkar AB, Hosmani AH. Evaluation of in vitro dissolution profile comparison methods of sustained release tramadol hydrochloride liquisolid compact formulations with marketed sustained release tablets. Digest Journal of Nanomaterials and Biostructures 2009;4(4):651-61. Epub 2009.
  27. FDA. Dissolution Testing of Immediate Release Solid Oral Dosage Forms, 1997.
  28. Shazly G, Nawroth T, Langguth P. Comparison of Dialysis and Dispersion Methods for In Vitro Release Determination of Drugs from Multilamellar Liposomes. Dissolut Technol 2008.