17 Alpha ethynyl estradiol (EE) is chemically described as 19-Nor-17α-pregna-1,3,5(10)-trien-20-yne-3,17-diol. It is a derivative of 17β-estradiol. It is first orally bioactive semisynthetic estrogen incorporated in many combined oral contraceptive pills. It is useful in the treatment and management of menopausal symptoms and female hypogonadism (Praveen et al., 2013). However, this was not the longer case. Lately, it was incorporated in combination formulation. It is an estrogen and it acts as estrogen receptor agonist. Eventhough, it is a synthetic derivative of estrogen, its properties are different from estradiol in varied ways. EE is with improved pharmacokinetic properties as compared to the natural estradiol. EE exhibits improved bioavailability when it is orally administered. Moreover, it is more resistant to metabolic pathways and exhibits more effects on specific parts of the body like liver and uterus. These improved pharmacokinetic properties augmented use of this drug in birth control pills in comparison to the estradiol (Panay et al., 2013; Unger, 2015; Kuhl, 2005)
Ethinylestradiol is the English generic name of EE. It can also be spelled as ethynylestradiol, ethynyloestradiol, and ethinyloestradiol. All these names can be pronounced in the same fashion (Morton and Judith, 2012; Elks, 2014).
Initially EE was marketed as individual drug with brand names Esteed, Estinyl, Feminone, Lynoral, Menolyn, Novestrol, Palonyl, Spanestrin, and Ylestrol. However, all these formulations were withdrawn from the market. Later, in large number EE was marketed in combination with progestins. These combinations are being used as oral contraceptives. EE is also present in combination with norelgestromin with brand name Ortho Evra and Xulane which are present as contraceptive patch. It is also present in combination with etonogestrel with brand name NuvaRing which are available as contraceptive vaginal ring.
Its empirical formula is C20H24O2 and molecular weight is 296.403 g/mol. EE is practically insoluble in water, freely soluble in solvents like ethanol and diethyl ether and sparingly soluble in chloroform. It is a synthetic estrane steroid and estradiol derivative. At C17α position, it has ethynyl substitution. It can also be called as 7α-ethynylestradiol or as 17α-ethynylestra-1,3,5(10)-triene-3,17β-diol. There are different prodrugs of EE are available with varied substitutions and derivatives. These prodrugs are known as mestranol (EE 3-methyl ether), quinestrol (EE 3-cyclopentyl ether) and ethinylestradiol sulfonate (EE 3-isopropylsulfonate) (Elks, 2014).
Validation of the analytical procedure is required to confirm the analytical procedure applied and to provide support for identification, and to evaluate quality and purity of the analyte. Analytical method development and validation can have influence quality of the data.
Specificity exercise should be conducted during the validation of the analytical procedures like identification test, impurity profiling and quantitative assay. Procedures required to perform for specificity will be based on the objective of the analysis. It is not possible perform one analytical procedure for specificity hence two procedures need to be performed for specificity.
Linearity should be demonstrated across all the analytical procedures. Linearity can be performed in two ways. In one method, standard stock of drug substance should be diluted and, in another method, separate weighing of the drug substance should be done. Linearity can be performed by visual inspection of the plot of signals with respect to analyte concentration. Linear relationship should be calculated by using suitable statistical methods by calculating regression line using least square method. Regression line can be used as mathematical estimation of degree of linearity. The correlation coefficient, y-intercept, slope of the regression line and residual sum of squares should be calculated and represented. Minimum five concentrations should be used for the estimation of the linearity.
Range should be estimated based on the linearity studies and planed application of the analytical procedure. Analytical procedure should provide adequate degree of linearity, accuracy and precision within or at the extreme range of analytical procedures.
Accuracy should be established for the given analyte throughout the range of analytical procedures and analyte concentrations. Accuracy can be measured by using assays on drug substances and drug products. Accuracy can also be determined by spiking impurities with the known substances. Accuracy should be estimated by using at least 9 estimations with minimum of 3 different concentration levels. These concentration levels should be within specified range and 3 concentrations should be used in replicates. Accuracy can be represented in two ways like 1) percent recovery which estimates known added amount of substance to be analysed to the sample, 2) variance among mean and accepted true value in addition to the confidence intervals.
Precision is required for the validation of analytical tests and quantitative determination of impurities. Precision includes repeatability, intermediate precision and reproducibility. Repeatability can be measured either of the two methods: a) minimum 9 determinations should be preformed comprising of 3 concentrations with three replicates each, or b) minimum three determinations with 100 % concentrations. Use of intended precision should be based on circumstances under which analytical procedure is being carried out. Effects of random events should be established on the analytical procedure. Different variables like days, analysts and equipments should be established. It is not mandatory to study these variables, however these can be studied in matrix experimental setup. Reproducibility can be evaluated by performing inter-laboratory variables on the outcome of the experiment. Data like standard deviation, coefficient of variation and confidence interval should be measured for measuring precision of the method.
Based on the application of non-instrumental or instrumental methods, detection limit can be determined by using different methods like based on visual evaluation, based on signal-to-noise ratio and based on the standard deviation of the response and the slope. Based on the standard deviation of the response and the slope can be estimated by using methods based on the standard deviation of the blank and based on the calibration curve. Detection limit value and method used for estimating detection limit should be represented in the results. Respective chromatograms can be considered as acceptable, if visual evaluation or signal to noise ration used as method for estimating detection limit.
Based on the application of non-instrumental or instrumental methods, quantitation limit can be determined by using different methods like based on Visual evaluation, based on signal-to-noise ratio and based on the Standard Deviation of the Response and the Slope. Based on the Standard Deviation of the Response and the Slope can be estimated by using methods based on the Standard Deviation of the Blank and based on the Calibration Curve. Quantitation limit value and method used for estimating detection limit should be represented in the results. Quantitation limit can be validated by analysing different samples with concentration near quantitation limit.
Robustness evaluation of the analytical method should be considered during the development phase and it should decided based on the procedure implemented in the study. It should demonstrate reliability of the method. If in case, parameters to be analysed are susceptible to variation, then analytical procedures should be controlled and related statement should be mentioned in the analytical procedure. System suitability parameters can be established in the robustness evaluation. It makes sure that validity of the analytical method can be maintained all the time. In case of HPLC, following are the examples of variations in analytical procedure: PH of the mobile phase, mobile phase composition, solvents and chemicals of varied lots or suppliers, temperature and flow rate.
System suitability testing is the integral component of the analytical method development. System suitability testing is based on the concept that equipment, electronics, analytical operations and analytes are the integral components of the analytical procedures and these should be evaluated as such. System suitability parameters to be established in the particular analytical method depends on the particular procedure being validated. System suitability parameters comprises of capacity factor, repeatability, relative retention, resolution, tailing factor and theoretical plates.
In RP-HPLC, pump needs to pass solvent with pressure through the column which comprises of filled solid adsorbent material. Solvent should contain mixture of samples from which required analyte gets separated. Components of the sample mixture interact with the adsorbent with different affinities which results in the separation of required analyte as solvent flow out of the column. Characteristics of RP-HPLC includes speed, greater sensitivity, improved resolution, re-usable columns, easy sample recovery, handling and maintenance, instrumentation tends itself to automation and quantitation, precise and reproducible results, calculations are done by integrator itself. Reverse-phase high-performance liquid chromatography (RP-HPLC) performs separation mainly based on the hydrophobicity. In RP-HPLC, solute in the mobile phases binds hydrophobically to the immobilised hydrophobic ligands present on the stationary phase which is also called as sorbent. Solute can be put on the sorbent in the presence of aqueous buffer and it can be eluted by using organic solvents. In RP-HPLC, elution can be categorised in gradient and isocratic elution. In gradient elution, quantity of organic solvent goes on increasing with time and in isocratic elution quantity of organic solvent remains constant. Solutes can be eluted with increasing power of hydrophobicity of the solute molecules. Most commonly used packing material for RP-HPLC is microparticulate porous silica. This material helps in achieving higher velocity and it can be useful in rapid separation. Modification need to be done in silica by using derivatized silane containing an n-alkyl hydrophobic ligand. This modification leads to increasing hydrophobicity of the sorbent. Most commonly used ligand is C18. C8 and C4 ligands can also be used in RP-HPLC. Retention time of analyte can be raised by adding more amount water to the mobile phase because affinity of the hydrophobic analyte to the hydrophobic stationary phase can be improved as compared to the affinity towards hydrophilic mobile phase. On the other hand, retention time of analyte can be decreased by adding more amount of organic solvent to the mobile phase. Chemical characteristics of the solute molecules also plays important role in the separation. Analytes with the hydrophobic characteristics retained for the longer duration because of its less interaction with the hydrophilic mobile phase. Analytes with hydrophobic characteristics contain groups such as C-H, C-C and S-S. Solutes with more hydrophilic characteristics retained less because of their more interaction with hydrophilic mobile phase. Analytes with hydrophilic characteristics contain groups such as -OH, NH2, COO– and NH3. pH of mobile phase is also important factor because it can alter hydrophobic characteristics of analyte. Hence, buffering agent like sodium phosphate can be used to change pH. There are less chances of damage of RP-HPLC column as compared to the normal phase chromatography. However, RP-HPLC aqueous bases should not be used in the RP-HPLC columns. Aqueous acids can be used, however, it should not expose to the columns for the longer duration. Immediately after the use RP-HPLC columns should be flushed with suitable solvent to remove buffers. Required efficiency and amount of sample loading decides the dimension of column to be selected for analysis. Resolution can be increased by increasing column length. Internal diameter of the column can be selected based on the sample capacity and detection sensitivity. Operating temperature can also influence resolution because temperature can affect viscosity of the mobile phase. Most of the regulatory agencies are making compulsion to analyse the drug with RP-HPLC before its release in the market.
Advantages of RP-HPLC include : 1 ) High level of resolution can be attained using varied chromatographic conditions, 2) structurally similar and distinct chemical compounds can be efficiently separated, 3) high recovery and high productivity can be achieved, and 4 ) high level of reproducibility can be achieved mainly due to stability of sorbent.
Various components of RP-HPLC include a solvent delivery system, including pump, sample injection system, a chromatographic column, a detector, a strip chart recorder, data handling device and microprocessor control. Solvent delivery system is used to pump mobile phase under pressure. Mobile phase flow through the pump single or more reservoirs and with the uniform flow rate. Based on the eluting power of mobile phase, RP-HPLC separation can be classified in to normal phase and reverse phase. With increase in the polarity of the mobile, eluting power increase in the normal phase. With the increase in the polarity of the mobile phase, eluting power decreases in the reversed phase chromatography. Pumps are the main components of the solvent delivery system in the RP-HPLC. Pump efficiency can have impact on retention time, reproducibility and detector sensitivity. Displacement pump, Reciprocating pump and Pneumatic or constant pressure pump are the three different types of pumps which can be used in RP-HPLC. Sample injection system can be used to introduce sample into the injection port. Loop injection, Valve injection and On column injection are the three types of sample injection system which can be used in the RP-HPLC. Material used in the manufacture of chromatographic columns in the RP-HPLC are heavy glass or stainless-steel tubing. These materials can be useful to withstand high pressure. Generally, these columns are 10-30 cm in length and with 4-10 mm inside diameter. Stationary phase filled in these columns is usually with particle diameter 25 µm or less. It is evident that columns with internal diameter of approximately 5 mm give good results. Column packing material used in the RP-HPLC is usually of small and rigid particles. Main function of detectors used in the RP-HPLC is to monitor mobile phase. Universal detectors and selective detectors are the two types of detectors can be used in the RP-HPLC. Universal detectors are also known as the bulk property detectors and these detectors measure physical property of the mobile phase either with solute or without solute. Refractive index, dielectric constant or density can be measure using bulk property detectors. Selective detectors are also known as solute property detectors and these detectors can measure physical properties of the detectors which cannot displayed by mobile phase. UV absorbance, fluorescence or diffusion current are the examples of the solute specific detectors. These detectors are approximately 1000 times more sensitive as compared to the bulk property detectors and can detect even nanogram quantity of the sample. UV-Vis absorbance detector is the most commonly used detector in the HPLC system (Mendham et al., 2006; Skoog et al., 1980; Beckett and Stenlake, 1997).
EE and mestranol are the two most widely used synthetic contraceptive estrogens available. Mestranol contains one extra methyl group attached to C-3. Mestranol should be converted to EE in the liver to exhibit its pharmacological effect. It is evident that mestranol is 50 % less active as compared to EE. EE is present in the dose range of 20 – 50 µg in oral dosage forms. EE is an oral contraceptive agent. There are numerous contraceptive formulations available in the market containing EE along with other drugs. Thorough literature survey revealed that various analytical methods are existing for the estimation of EE in bulk drugs and combination formulations. New formulations were developed for the EE with low dose levels to maintain balance between safety and efficacy. Due to presence of low level of doses and requirement of long duration treatment, quality of EE containing formulations need to be maintained. Quality of drugs and its formulations can be evaluated by applying analytical methods including HPLC. Literature survey revealed that few analytical methods are available for the quantitation for EE in the solid dosage forms (Strusiak et al., 1982). However, these methods are associated with disadvantages like complex method of sample preparation and solvent system preparation. A colorimetric method is available for the quantification of the EE in the solid dosage forms. This method is based on the formation of azo dye due to condensation of the diazotized 5?chloro?2, 4?dinitroaniline with ethinyl estradiol (Mohamed et al., 1975). Hence, this method is having less sensitivity as compared to the HPLC method. Most of the methods available for the quantitation of EE were developed for simultaneous quantification along with another drug. EE is available in combination with several drugs. These drugs include drospirenone, levonorgestrel, gestodene, norgostimate, norethisterone and levonorgestrelin. Several methods are available for the simultaneous quantification of these drugs with EE. HPTLC and RP-HPLC methods are available for the simultaneous quantification of EE and drospirenone. HPLC, RP-HPLC and spectroscopic methods are available for the simultaneous quantification of EE with levonorgestrel. Derivative spectroscopy, RP-HPLC and UPLC-MS/MS methods are available for the simultaneous quantification of EE with levonorgestrel (Prabhakar and Deshpande, 1999; Fakhari et al., 2006; Durga et al., 2004; Matejícek and Kubán, 2007; Van et al., 1987; Sarat and Rambabu, 2012; Parmar et al., 2012; Berzas et a., 1997). LC-MS/MS method is available for the estimation of EE in human plasma using tandem mass spectroscopy (Shou et al., 2004). Other methods are also available for the estimation of EE in biological samples like plasma, hepatocytes and breast milk. Methods developed for estimation of EE from biological samples are more advanced comprising of photoionization/mass spectroscopy detection system and integrating LC-MS/MS following derivatisation. Complexity of these methods include complex extraction method for sample preparation and complex mobile phases preparation including buffer preparation. Since, HPLC conditions for these analytical methods for the simultaneous quantification are complex in nature. Several methods are available for the analysis of EE from the biological samples like human plasma, serum, cerebrospinal fluid, rat plasma, human cell line culture medium, human urine and milk. Most of these methods are LC–MS/MS with detection systems comprising of APPI, ESI, Electrochemical detection, DAD and GC-MS. Most of the analytical methods available for the determination of EE in the biological samples can not be applied for solid dosage forms because of complexity of the available methods. Most of the available methods for EE determination in the biological samples are based on the gas and liquid chromatography with mass detection. Hence, it is necessary to develop and validate method for the estimation of EE in contraceptive pills. As a result, this method can be widely used for estimation of EE due to its ease of procedure and cost effectiveness.
Systematic approach for the project starteded with the well-defined objective and emphasis was given to product and process understanding. Process control was implemented through understanding of the science behind analytical method development and validation. Ultimate goal of the project was to entrench quality of the pharmaceutical products for the patient safety. Strategy of project included method development, method validation and application of validated for the estimation of EE from the tablet. Method development included: titration of different mobile phase for resolution of EE from other excipients, selection of flow rate of mobile phase, column and detection system. Method validation included preparation of calibration curve and measurement of validation parameters like precision, accuracy and limit of detection. Ultimate objective of this project was to develop robust method for estimation of EE in contraceptive pills. It was helpful in reducing and controlling variability. Knowledge management is the important aspect in method development and validation. All the information required for method development gathered at the start of the project and it was utilized at each of the respective steps. Changes and improvements in the method were implemented based on the information collected in the knowledge repository. Knowledge repository stored all the information which was implemented for different stages of the method lifecycle. Potential variables and their respective acceptable ranges were decided during procedure design. Analytical method development lifecycle categorised into three stages: 1. Procedure design, 2. Procedure performance and 3. Procedure performance verification. Routine monitoring of the process included special reason of variation, unacceptable reason variation and continual improvement.
At the beginning of the project, physicochemical characteristics of the analyte were collected from the literature. Column screening was performed either through literature or through performing required experiments. Data collected from the literature also can be verified by performing experiments. Chemistry of the analyte was thoroughly studied because selection of most of the HPLC parameters mostly depend upon the chemistry of the molecule. Knowledge about the separation science was gathered to implement it at each stage of the method development and validation. Knowledge about separation science is very important before initiating quality by design experiment. Method was developed which can be applicable for multiple projects and products. Efforts were taken to develop method which can be performed with ease and it would be cost effective because requirements of the modern methods are more as compared to the simple methods. For the quantitation of the analyte, sample response was calculated against the external standard response. Standard solution was prepared using solution which generally would not contain sample matrix. Following factors were considered during method development for EE in contraceptive pills : chemical and analytical structure, pka, solubility of analyte in different polarity solvents, dilution effect, selection of detector, sample preparation, solution stability, science behind selection of stationary phase, selection of mobile phase PH and buffers, type of separation either isocratic or gradient. Items or materials required for method development were selected on following criteria: short run time, exhibit reproducibility on all types of HPLC brands, long life for the column, impurities separation at the baseline level, controlled column temperature and each peak integration and quantitation. Items or materials required for method development were selected which were not fulfilled following criteria : requirement of QC scientist to alter experimental conditions to meet system suitability, filtration after sample preparation and mobile phase preparation, instrument specificity and vendor specificity. Each step of the method development like mobile phase, flow rate and column selection were finalised after performing preliminary experiments. It would be helpful in critically evaluating performance of each step and streamlining final method optimization. Critical parameters were identified during the validation phase and acceptance limits were established for method system suitability.
Steps followed during the validation of the protocol:
Weighted one pill of their packs and grinded them, put in 10 ml volumetric flask and added methanol. The content of the volumetric flask was filtered through Whatman filter paper and diluted up to mark with methanol.
For the optimization of the mobile phase, several compositions of mobile phases were tried. Mobile phases compositions like 60% Acetonitrile 40% Water, 70% Acetonitrile 30% Water, 50% Acetonitrile 50% Water and 40% Acetonitrile 60% Water were tried. For mobile phase optimisation, stock solution of EE with concentration with middle range (30µg/ml) was selected.
Stock solution of EE standard solutions (0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 ml) were transferred into series of 10 ml volumetric flasks. Volume of the volumetric flasks were adjusted to mark with methanol to achieve concentrations of 10, 20, 30, 40 and 50 μg/ml of EE respectively. Respective peak area against concentration in μg/ml of EE was plotted to obtain calibration curve and regression equation was obtained. Calibration curve experiment was repeated three times.
Accuracy of the validated method was estimated by addition of standard EE at three different levels. These three different levels were comprised of lower, middle and upper level. In accuracy estimation, known amount of EE were added to the previously analysed samples and percentage recovery was estimated.
Quantitative assay for EE was performed at three different levels. It includes between the tablets, within the tablets and comparing the tablets.
For estimating EE between the tablets, three different brands of EE like RIGEVIDON, MICROGYNON and CILEST were used. Each of these tablets were grinded in the fine powder and added into the 10 ml volumetric flask followed by addition of methanol. Each of the EE sample solution was filtered and made volume upto 10 ml. Each of the sample solution was put into the autosampler for injection into the HPLC.
For estimating EE within the tablets, solution prepared during estimation between the tablets was used. Along with this, standard stock solution of EE at 8 different concentrations was prepared. Sample solution in the instrument was doubled and made it from 25 to 50.
For comparing the tablets, another tablet was taken from the pack and same procedure was repeated like estimation between the tablets.
For the optimization of the HPLC parameters, different mobile phases were titrated. These mobile were 60% Acetonitrile 40% Water, 70% Acetonitrile 30% Water, 50% Acetonitrile 50% Water and 40% Acetonitrile 60% Water. At the start, 60% acetonitrile and 40% mobile phase was injected into the HPLC instrument. With this mobile phase, peak was observed at 3.71 min. With change in mobile phase to 70% acetonitrile 30% water, peak appeared at 2.54 min. With the increase in the water concentration like 50 and 60 %, retention time was obtained at higher value. With decrease in water concentration upto 30 %, retention time changed from 3.42 to 2.542 min. Hence, 60% Acetonitrile 40% Water was selected as the mobile phase for the estimation of EE.
Table 1 : Optimization of mobile phase
|
Mobile Phase |
Concentration (µg/mL) |
Retention time (min) |
Peak area |
|
60% Acetonitrile 40%Water |
30 |
3.71 |
236170 |
|
70% Acetonitrile 30%Water |
30 |
2.452 |
192080 |
|
50% Acetonitrile 50%Water |
30 |
5.59 |
211075 |
|
40% Acetonitrile 60%Water |
30 |
12.61 |
210145 |
Different flow rates were tried for the optimization of the HPLC parameters. These flow rates were comprised of 0.5, 0.75, 1.25, 1.5 and 1.75 ml/min. Middle concentration of EE which was 30 µg/ml was used for the optimization of the flow rate. Thin and long peak was observed for the flow rate 0.5, sharp and thin peak was observed for flow rate 0.75, sharp and long peaks were observed for flow rate 1.25 and 1.5 and sharp and too long peak was observed for flow rate 1.75. In HPLC analysis, generally sharp and long peak used to be considered as the ideal peak. Since, flow rate 1.25 ml/min exhibited sharp and long peak and optimum retention time and peak area, it was selected as the flow rate for method development and validation of HPLC method for EE estimation. It was observed that flow rate above 1.75 ml/min damaged the column.
Table 2 : Optimization of flow rate
|
Flow Rate (ml/min) |
Concentration (µg/ml) |
Retention time (min) |
Peak area |
Shape of peak |
|
0.5 |
30 |
8.06 |
527136 |
Thin and long |
|
0.75 |
30 |
4.84 |
347779 |
Sharp and thin |
|
1.25 |
30 |
2.64 |
187619 |
Sharp and long |
|
1.5 |
30 |
2.16 |
158858 |
Sharp and long |
|
1.75 |
30 |
1.73 |
135085 |
Sharp and too long |
Different wavelengths like 214, 234, 254, 274 and 294 were tried for the optimization HPLC parameters. Middle concentration (30 µg/ml) of the stock solution was used for the optimization of the wavelength. Optimum wavelength for the quantitation of the compound can be selected based on its maximum wavelength, peak area and nature of the peak. Generally, in HPLC methods more peak area and sharp and long peak considered as the optimum wavelength for the quantitation of the compounds. Wavelengths at 214, 234, 254 and 274 nm exhibited very short and broad peak, short and broad peak, so long and thin peak and too long with sharp and thin peak respectively. All these types of peak didn’t demonstrate optimum resolution of EE. Wavelength at 294nm exhibited long and sharp peak. This peak demonstrated optimum resolution of EE. Moreover, this wavelength gave more peak area. Hence, 294 nm was considered as the optimum wavelength for the quantitation of the EE using proposed HPLC method.
Table 3 : Optimization of wavelength
|
Wave length (nm) |
Concentration (µg/ml) |
Retention time (min) |
Peak area |
Shape of peak |
|
214 |
30 |
3.42 |
127174 |
Very short and broad |
|
234 |
30 |
3.42 |
241007 |
Short and broad |
|
254 |
30 |
3.42 |
170764 |
So long, sharp and thin |
|
274 |
30 |
3.42 |
265451 |
Too long, Sharp and thin |
|
294 |
30 |
3.42 |
288376 |
Long and sharp |
Different columns were tried for the development of HPLC method for EE. All the used columns were from Phemomenex. Hypersil and Luna columns were 5 C18 columns were used with size 150×4.60mm. These columns were of serial number 115799, 97002 and 220652. Out of these three columns, serial number 115799 column was used for method development because out of other two columns, one column was broken and another column didn’t worked.
Table 4 : Optimisation of column
|
Selected column |
Broken column |
Column didn’t produce desired results |
|
|
Brand |
Phenomenex |
Phenomenex |
Phenomenex |
|
Type |
HYPERSIL 5 C18 |
LUNA 5u C18 |
|
|
Size |
150×4.60mm |
150×4.60mm |
150×4.60mm |
|
Serial Number |
115799 |
97002 |
220652 |
The developed method was validated according to the International Conference on Harmonization (ICH) guidelines for the quantitation of EE in contraceptive pills.
Linear correlation was obtained in the concentration range of 10 – 50 µg/ml for EE. Regression analysis data is mentioned table no 5 and calibration curve is given in the figure no 2. Obtained correlation coefficient value is high in the concentration range 10 – 50 µg/ml. It indicates that developed method is linear in the concentration range 10 – 50 µg/ml. Range is the interval between upper and lower concentration of analyte which was observed over a range of 10 – 50 µg/ml for EE.
Table 5: Regression analysis data for EE
|
Parameters |
Data |
|
Concentration range (µg/ml) |
10 – 50 |
|
Slope |
9122.5 |
|
Intercept |
15288 |
Table 6 : Linearity range for EE
|
Concentration (µg/ml) |
Retention time (min) Mean for 3 Injections |
Peak area Mean from 3 Injections |
|
10 |
3.72 |
88134 |
|
20 |
3.72 |
150362 |
|
30 |
3.72 |
261600 |
|
40 |
3.72 |
344804 |
|
50 |
3.72 |
447039 |
In figures 3 – 7, chromatograms of EE were presented at 264 nm. These chromatograms represent increasing concentrations of EE like 10, 20, 30, 40 and 50 µg. It has been observed that there is increase in the peak area with increase in the concentration of EE. Mobile phase comprising of 60% Acetonitrile 40% Water was used for the linearity analysis of EE. These chromatograms were helpful in the selection of mobile phase and flow rate. Peak parameters of the chromatograms like height, asymmetry and tailing were considered.
Accuracy of the developed method was computed by recovery experiment which was performed by standard addition method. Accuracy and precision were estimated by using different concentrations like lower, middle and higher. Different concentrations used were 10, 30 and 50 µg/ml. Experiment for each of the concentration was repeated three times. Accuracy precision values obtained for 10, 30 and 50 µg/ml were 9, 8 and 9 respectively. Accuracy precision values obtained for 10, 30 and 50 µg/ml were 2, 2 and 2 respectively.
Table 7 : Accuracy and precision data
|
Accuracy |
Precision |
Concentration (µg/ml) |
Repeat |
|
9 |
2 |
10 |
3 |
|
8 |
2 |
30 |
3 |
|
9 |
2 |
50 |
3 |
For the estimation of LOD, 0.5, .025 and 0.125 µg/ml concentrations were used. For the LOQ, 1, 1.5 and 2.5 µg/ml concentrations were used. For the estimation of LOD and LOQ, standard solution with lower concentration 10µg/ml was used and it was diluted with the methanol to achieve desired concentrations for the estimation of LOD and LOQ. Diluted samples were injected into the autosampler for the estimation of LOD and LOQ values. Peak areas obtained for the estimation of LOQ were as follows: peak area of 19249, 13899, and 10702 were obtained for the concentrations of 2.5, 1.5 and 1 µg/ml respectively. As 2.5 µg/ml exhibited good response, it was considered as the LOQ for the developed method for the estimation EE in the contraceptive pills. Peak areas obtained for the estimation of LOD were as follows : peak area of 7580, 2133, and 847 were obtained for the concentrations of 0.5, 0.25 and 0.125 µg/ml respectively. 0.125 µg/ml was considered as the LOD for the developed method for the estimation of EE in the contraceptive pills.
Table 8 : LOD and LOQ data
|
LOD |
LOQ |
Concertation (µg.ml) |
Peak area |
|
LOQ |
2.5 |
19249 |
|
|
1.5 |
13899 |
||
|
1 |
10702 |
||
|
LOD |
0.5 |
7580 |
|
|
0.25 |
2133 |
||
|
0.125 |
874 |
Quantitation of the EE in the contraceptive pills were estimated in three different types of samples. These comprised of between the samples, within the samples and comparing the tables. For the quantitation of EE in contraceptive pills, three different tablets with brand names like RIGEVIDON, MICROGYNON and CILEST were used. Labelled claim for each of these tablets were 30 µg. Weight of RIGEVIDON, MICROGYNON and CILEST tablets were 0.0874, 0.0863 and 0.1032 g respectively. Obtained peak areas for EE for RIGEVIDON, MICROGYNON and CILEST were 11199, 9596 and 13550 respectively. Estimated concentration of EE in RIGEVIDON, MICROGYNON and CILEST were 2.90, 2.77 and 3.16 µg respectively. EE retention time obtained for each these tablets were 3.21
Y = 9122.5 x – 15288 formula was used for the calculation of EE concentration in respective tablets.
Table 9 : Estimation of EE in Rigevidon, Microgynon and Cilest
|
Tablet |
Weight (g) |
Declared amount (µg) |
Peak area |
Retention time (min) |
% RSD |
% Recovery |
Concentration quantified (µg) |
|
RIGEVIDON |
0.0874 |
30 |
11199 |
3.21 |
11.322 |
84.5 |
2.90 |
|
MICROGYNON |
0.0863 |
30 |
9596 |
3.21 |
5.944 |
82.5 |
2.77 |
|
CILEST |
0.1032 |
30 |
13550 |
3.21 |
6.375 |
95 |
3.16 |
Variability within the tablet was estimated by using sample solutions prepared during the estimation of EE between the tablets. Volume injected in this method was changed from 25 to 50. Along with these samples, samples solutions of 8 different concentrations of EE were prepared and all these samples were run together in HPLC. 8 different prepared concentrations were 0.5, 1, 5, 10, 20, 30, 40 and 50 µg/ml and peak areas obtained for these concentrations were 3567, 13497, 38851, 139667, 263829, 428672, 601204 and 762043 respectively. Obtained peak areas for EE for RIGEVIDON, MICROGYNON and CILEST were 19233, 33525 and 19233 respectively. EE retention time obtained for each these tablets were 3.08 min. Concentration of EE in each tablet was estimated by using linear regression equation.
Y = 9122.5 x – 15288 formula was used for the calculation of EE concentration in respective tablets.
Table 10: Estimation of EE in Rigevidon, Microgynon and Cilest within the tablets
|
Compound run with tablet |
Concentration (µg/mL) |
Retention time (min) mean from 3 times |
Peak area Mean from 3 times |
Concentration quantified (µg) |
|
Ethinyl estradiol |
10 |
3.08 |
139667 |
20.31 |
|
Ethinyl estradiol |
20 |
3.08 |
263829 |
30.59 |
|
Ethinyl estradiol |
30 |
3.08 |
428672 |
48.66 |
|
ethinyl estradiol |
40 |
3.08 |
601204 |
65.96 |
|
ethinyl estradiol |
50 |
3.09 |
762043 |
85.21 |
|
ethinyl estradiol |
5 |
3.05 |
38851 |
5.39 |
|
ethinyl estradiol |
1 |
3.09 |
13497 |
3.15 |
|
ethinyl estradiol |
0.5 |
3.04 |
3567 |
2.06 |
|
RIGEVIDON |
3.08 |
19233 |
3.78 |
|
|
MICROGYNON |
3.08 |
33525 |
5.35 |
|
|
CILEST |
3.08 |
19233 |
3.78 |
For comparison of the EE estimation among different tablets, another tablet were taken from the each pack and quantitation of EE was performed. RIGEVIDON, MICROGYNON and CILEST of these tablets were 0.095, 0.095 and 0.102 respectively. Obtained peak areas for EE for RIGEVIDON, MICROGYNON and CILEST were 11260, 21558 and 16103 respectively. Estimated concentration of EE for RIGEVIDON, MICROGYNON and CILEST were 2.91, 4.03 and 3.44 µg respectively. EE retention time obtained for RIGEVIDON and MICROGYNON were 3.45 min. EE retention time obtained for CILEST was 3.29 min. Concentration of EE in each tablet was estimated by using linear regression equation.
Y = 9122.5 x – 15288 formula was used for the calculation of EE concentration in respective tablets.
Table 11 : Comparison of Estimation of EE in Rigevidon, Microgynon and Cilest
|
Tablet |
Weight (g) |
Declared amount (µg) |
Peak area |
Retention time (min) |
% RSD |
% Recovery |
Concentration quantified (µg) |
|
RIGEVIDON |
0.095 |
30 |
11260 |
3.45 |
11.5 |
85 |
2.91 |
|
MICROGYNON |
0.095 |
30 |
21558 |
3.45 |
6.5 |
86 |
4.03 |
|
CILEST |
0.102 |
30 |
16103 |
3.29 |
7.1 |
96 |
3.44 |
Effective analytical method development can result in meeting objective of each step. Certain regulatory agencies require method validation which demonstrates suitability of use of method for the intended use. EE solubility was studied initially, which helped in selecting suitable mobile phase and column for EE method development and validation using HPLC system. Three important critical components of HPLC method development include : sample preparation (% each each aqueous and organic solvents, pH and sample size), analysis conditions (% of each aqueous and organic solvents, pH, flow rate, temperature, wavelength and type and age of column) and standardization (wavelength, peak integration and standard concentration). In this EE method development and validation, all these components like mobile phase optimization, flow rate selection, wavelength selection and column selection were finalised after performing preliminary experiments. Analytical method validation can be used to demonstrate scientific accuracy of analysis. Validation of the method is necessary during the regulatory submissions. Validation of the method can demonstrate developed analytical method measure correct substance, can measure substance in the correct amount and can measure in the appropriate range. Validation of the method can be helpful in identifying behaviour of the method and establishing performance limits of the method (Rozeta et al., 2013; Bouabidia et al., 2010).
For the development of HPLC method, different combinations of solvent systems were tried and these solvent systems comprised of water and acetonitrile. Final decision on the selection of combination of solvent system was based on various factors like sensitivity of the system, preparation ease, compatibility with the drug, time and cost. Combination of mobile phase and flow rate were selected based on the peak parameters like height, asymmetry and tailing. Stability of the column and run time were also another factor responsible for the selection of run time.
Type of analyte, concentration range of analytes, details of the equipment and procedure were mentioned in the project. Range usually doesn’t reflect higher and lower level of analyte, however it mainly depends on the intended application of the analytical method. A study need to be carried out for the substances which can interfere with the estimation of the EE and these interfering substances need to be used as excipients. It would be helpful as guide for the selection guide for the formulation of pills containing EE. Developed method is effective in estimating EE in different pharmaceutical formulations. Analyte extraction and clean up are the important steps in the method development and validation of the method. In the developed method for EE, extraction method for EE was kept very simple. Since, extraction method is simple, there is no requirement of clean up in this developed method. Validity of the proposed method was established by performing recovery studies. These recovery studies were performed by standard addition method. Known quantity of the drug was spiked with the pill formulations and quantity of the EE was estimated by the developed method. Each estimation was repeated for three times. In this proposed method, no internal standard was used because there was no extraction step involved in the quantitation of EE from the oral contraceptive pills. As accuracy of the developed method was established, it was not required to use internal standard. This method for the quantitation of EE was proved to be superior to the previously reported methods in literature and official methods because this method exhibited lower LOD and LOQ values. Moreover, preparation mobile phase for this method is simple as compared to the earlier methods. Hence, this method is proved to be economical as compared to the previous methods (Rambla-Alegre et al., 2012).
Previously developed method for the quantification EE from the solid dosage form used fluorescence detection system for the quantification of EE. However, in this analytical method UV detection system was used for the detection of EE. As compared to the fluorescence detection, UV detection is readily available for the detection of compounds in the HPLC analysis. Hence, developed method can be widely used for the quantification of the EE in the solid dosage forms (Strusiak et al., 1982). Colorimetric method is also available for the quantification of EE in the solid dosage forms (Mohamed et al., 1975). However, colorimetric method is associated with the disadvantage of interference with the excipients. Hence, developed HPLC method with UV detection system can be used instead of colorimetric method for quantification of EE in the solid dosage form. EE containing formulations might be available along with vitamins and minerals, hence there might be interference in its quantification using colorimetric method.
This project demonstrates a simple, accurate and reliable validated analytical method for quantification of EE in the contraceptive pills using HPLC. It was evident that, developed method has wide applicability and scope. This method can be applied in the routine analysis of the EE containing contraceptive pills.
Patient consuming pharmaceutical dosage forms expect safe and efficacious dosage forms. As large number of pharmaceutical drugs are present in the market in varied dosage forms, it is necessary to quantitate these drugs. Most of the pharmaceutical regulatory agencies present around the world, mandate to maintain quality, purity and potency of the product during its commercial lifecycle. Hence, there is always need for development of validated analytical method which is precise, accurate, selective and sensitive. Developed method should be used for the routine analysis of the drugs and it formulations. In the present work, validated analytical method was developed as per the ICH guidelines for the quantitation of EE in tablets. A HPLC method was developed for the quantification of EE in contraceptive pills. Developed method was utilized for different brands of EE and it was also utilized for both coated and uncoated tablets. Form the obtained results, it was evident that developed method can be applied to different brands and different types of tablets. System suitability parameters for the developed method were within the limit. Advantages of the developed method as compared to the existing method include simple method of sample preparation and detection system. Hence, this method is less time consuming and cost effective as compared to the earlier developed methods. Developed analytical method is simple, precise and sensitive for the quantification of the EE in the contraceptive pills. Moreover, applicability of the developed method in different formulations was proved. In conclusion, this method can be applicable for the estimation of EE in different types of single drug containing formulations. However, for estimation of EE in formulation containing combination drugs, modifications in the sample preparation need to be carried out.
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