Hplc and gc analysis


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Hplc and gc analysis

  1. 1. “Quantitative Analysis of Lamotrigine using HPLC & GC analysis and the impact on light on peak value” Authors – Birla, Himanshu Guide - Professor John Nicholson J.W.Nicholson@greenwich.ac.uk 2008 1
  3. 3. 1. The objective of the experiment was to partially validate a high performance liquid chromatography technique for the quantitative analysis of lamotrigine. 2. The drug utilized for the study was lamotrigine taken from Sigma-Aldrich in powder form. The generic lamotrigine came with an amber bottle in 10mg which was then analysed for quantitative analysis. 1.2 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 3
  4. 4. Liquid chromatography is a separation method which is widely used in the chemical, pharmaceutical and biotechnology industry and is of great importance. The principle of liquid chromatography includes the injection of the sample into the system which contains a particulate stationary phase as a column and a moving mobile phase in pushed through the system and hence column. The separation of substances takes place due to differences in the rates of movement through the column and interaction with the stationary phase and movement through the column is facilitated with the help of mobile phase. Chromatography is a general term applied to the separation techniques involving the separation partitioning of the sample in presence of a mobile phase which is generally a liquid or gas and a stationary phase which may be a solid or a liquid. [1] Chromatography is used widely to separate the mixtures and isolate them. It is generally divided into two types, namely Preparative chromatography and Analytical chromatography. Preparative chromatography is used to separate components of the mixture for further use whereas analytical chromatography is done on smaller amount of materials and helps in finding out the proportion of the constituents in the analytical sample [1]. The aim of separation is to obtain the qualitative and quantitative information about the sample. The all important aim of validation of a sample is used to obtain the Quantification of the substance which was observed in this experiment. [1, 2] 1.2.1 HISTORY OF CHROMATOGRAPHY The term “chromatography” was first coined by Tswett after finding a band of colours on the column while separating pigments of green leaves [2]. The thin layer chromatography and gas chromatography came into existence in 1950’s and after that various gel exclusion techniques came in 1960’s. In 1960’s the Journal of Gas Chromatography changed its name to Journal of Chromatography Science and this cerated the interest in liquid chromatography and hence start of a new modern liquid chromatography era. Gas chromatography was a well known and established technique at that time but gas chromatography had a limitation [2]. It was unable to Handle thermally unstable and non-volatile compounds and this limitation drove the interest in favour of liquid chromatography. [2] 4
  5. 5. Liquid chromatography also helped in selective interaction of two chromatographic phases, easy sample recovery and room temperature conditions for the technique to work. Liquid chromatography showed the limitation of inability to show quantification of compounds and resolution between similar compounds. At this stage pressure liquid chromatography showed good separations with the decrease in the separation time with pressurised flow of mobile phase through the column and hence reducing the purification times of compounds. The increase in the pressure to move the mobile phase in liquid chromatography laid the foundation of high performance liquid chromatography (HPLC). [2] High performance liquid chromatography was developed in 1970’s and it was further improved by the use of packed columns and better resolution detectors and now it is the most common techniques used in the analysis and separation of compounds. Even in late 1970’s another technique called reversed phase chromatography helped to improve separation between very similar compounds. [3]. 1.2.2 REVERSED PHASE CHROMATOGRAPHY: - [2] Reversed Phase Chromatography is a technique slightly different from the normal chromatography. The difference in reversed phase chromatography is the stationary phase. In the reversed phase chromatography the stationary phase is non-polar and hence it is more advantageous as compared to normal chromatography. As the stationery phase is non-polar, water can be used as mobile phase and the strength of the mobile phase can be increased with the help of organic solvents. The aqueous mobile phase provides transparent mobile phases at low cost and they have high compatibility with biological samples and electrochemical detectors. The interactions of the solute with the stationary phase are weak, which provides mass transfer with less possibility of irreversible adsorption and provides rapid equilibrium with solvent changes and hence helpful in gradient elution methods. HPLC gained importance because of its reliable technique that is high pressure application on the mobile phase to push its movement through the densely packed 5
  6. 6. column. This technique hence enables faster separations and increased resolution as compared to liquid chromatography. Until 1980’s HPLC was commonly used to separate the chemical compounds but later with further improvements in the technique the quantification, purification and identification of the compounds also started to immerge with the use of HPLC. The development of computerised methods also added in improved results and reproducibility and hence helping the HPLC as the most common and widely used technique for the analysis and separation of compounds.[3] 1.2.3 THEORY OF OPERATION [2] Chromatography is a separation technique which employs of two phases, one stationery phase and one mobile phase. The mixture is injected into the system with the help of the injection through an injector and is carried by the mobile phase into contact with the stationary phase. Separation takes place as the components of the mixture move and partition differently between the stationery and mobile phases. The theory of operation basically works on two parameters which include: - [2] 1) Thermodynamic considerations 2) Kinetic considerations Thermodynamic considerations involve the basic interaction between the mobile phases containing the compound with that of the stationary phase. Hence thermodynamic considerations define the retention times of the sample. [2] The time required for the sample to elute from the system is equal to the time the sample spends in the moving mobile phase plus the time spent in the stationary phase. If there is no interaction takes place with the stationary phase then the retention times becomes the hold up time or dead time and it is the time required for the mobile phase molecules to cross the column. [2] Kinetic considerations in an HPLC experiment refers to the moving speed of the mobile phase and hence kinetics of the system is responsible for peak maxima, peak 6
  7. 7. width and band broadening and hence thermodynamic and kinetic factors of an HPLC system defines the peak and hence separation of the compound. [2] 1.2.4 INSTRUMENTATION OF HPLC [3] The HPLC technique is carried out with the help of different experimental parts, but the most important part is the column as the column is responsible for separation and hence the analysis of the sample. The instrumentation of HPLC contains following parts which are discussed in details later. The instrumentation includes:1) Column Efficiency 2) Mobile Phase 3) Stationary Phase 4) Injectors 5) Pumps 6) Columns and Column packing 7) Detectors Column Efficiency Column efficiency basically defines the capability of the mobile phase to distinguish and separate the analytical compounds. The column efficiency basically depends on the number of theoretical plates and the efficiency of the column to separate; it defines column’s packing and kinetics of the mobile phase through the column. The change in the column efficiency can be seen with the help of peak shapes [3]. If the peaks show “Tailing” then it implies that there is some problem in the column and hence the change in the packing is causing the peak shape to change. The column efficiency is calculated by the following equation:- N = A (TR/W2) Where, “N” is the number of theoretical plates. “A” is the constant dependent upon the height where peak width measured. 7
  8. 8. “TR” is the retention time. “W” is the peak width. [2] [Fig.1 taken from ref. no. 3] Mobile Phase:The mobile phase is another important part of the HPLC. The mobile phase is a continuous solvent which runs across the HPLC system throughout the experiment. The mobile phase is basically a carrier for the compound which is subjected to analysis. The mobile phase carries the sample into the column where the separation takes place. The interaction of the mobile phase with the sample and the column collectively defines the extent of separation and movement of the sample through the system [2]. Methanol is a common mobile phase used in the HPLC, though other polar organic compounds, such as acetonitrile, are also widely used. The interaction of the sample with that of the mobile phase determines its movement and separation. If the sample is having a high affinity with that of the solvent then the sample will elute from the system faster and the retention time of the sample at the column and the system will decrease and the elution will be fast hence it may lead to less separation and vice-versa is also true. The mobile phase is subjected to a number of alterations to get the best suited mobile phase for the sample in order to get maximum separation. [2, 3] Stationary Phase: The stationary phase is the base at which the separation takes place. The solid support containing the column forms the stationary phase and mobile phase continuously runs over the stationery phase during the experiment. The sample comes into the system from the injector and hence joins the mobile phase from the injector and runs over the stationary phase (column) where the separation takes place. When the sample solution moves to the stationary phase along with the mobile phase then the interactions between the sample solution and the column helps the separation. The amount of time 8
  9. 9. sample solution spends at the stationary phase or the column interface before finally eluting out is called the retention time of the sample. Samples having higher interaction towards the mobile phase as compared to the column will elute faster. [3] The samples having good interaction towards the column will elute out from the system slower hence the samples having higher affinity towards the column or stationary phase show greater retention times as compared to the samples which have higher affinity to the mobile phase. There are different stationary phases used according to the need of the experiment and the sample and detailed discussions is not included in this part of introduction. The different columns include: - [3] 1) Solid-Liquid columns (Adsorption) 2) Liquid-Liquid 3) Size Exclusion 4) Normal Phase 5) Reversed Phase 6) Ion Exchange 7) Affinity Injector:The injector is the source from where the sample enters the HPLC system and hence meets the mobile phase and move towards the column. The injection technique plays a very important role in the experiment, as the injection technique for sample injection is responsible for total peak width and hence the injection of the sample should be done with care. The injection of the sample in the HPLC system is a bit difficult because of the high pressure on the mobile phase exerted by the pumps to push the mobile phase through the column. The difficult injection technique is responsible for improper precision and reproducibility and is termed as “syringe error”. The problem of syringe error is now been sorted out with the help of auto samplers. Until now the most common method of sample injection is through the Rotary Valve injectors. These injectors contain ports which allow the sample to be injected into the external 9
  10. 10. loop. The activation of ports helps the sample to get incorporated into the mobile phase flow and the sample is delivered into the system. Since the introduction of the auto samplers the precision and reproducibility has been greatly improved. The most common injection loops volumes are from 5 to 20uL. [3] Pumps:The pumps play an important role in the HPLC system as they are responsible for the constant flow of mobile phase through the system. Pumps are responsible for the constant flow and pressure of the mobile phase and hence help in getting proper precision during the experiment. There are different types of pumps applied in the HPLC. Some commonly used pumps include: - [3] Reciprocating Piston Pumps- These pumps consists of a motor driven piston which moves back and forth in a hydraulic chamber. These pumps help in moving the mobile phase from the reservoir and then push the mobile phase into the system. The flow rate can be changed by changing the settings of such pumps. [3] Syringe Type Pumps- These pumps are responsible for small columns as they deliver a definite amount of volume and then they need to get refilled. These pumps generally have the volume ranging from 250-500uL. The pump operates with the help of a motor and delivers mobile phase at constant rate. [3] Constant Pressure Pump- These types of pumps provide a constant pressure to mobile phase but the mobile phase is pushed through the system with pressure from gas cylinder. A low pressure gas source is needed for this pump. These pumps provide continues mobile phase flow rates. [3] Detectors: - [3] The detectors are the instruments which take the response from the HPLC system and show them as a peak in the Chromatogram. Detectors work at the elution of the sample and define the separation or sample change as a peak in the chromatogram. Detectors work just after the stationary phase and detect the sample as soon as they elute out from the stationary phase. The bandwidth and the peak areas can be adjusted with the help of these detectors as they can be subjected to a change easily and are 10
  11. 11. easily adjustable. The most common detector used in the HPLC is the UV spectrometer. The UV spectrometer works on the principle of absorbance and shows that with the increase in the concentration of the sample the peak area increases. There are many detectors used in the HPLC instrumentation and some of them are listed below: - [3] 1) UV Detectors 2) Fluorescent Detectors 3) Electrochemical Detectors 4) Mass Spectroscopy Detectors 5) NMR Detectors 6) Light Scattering Detectors 7) Near Infrared Detectors. 1.3 VALIDATION When a method has been developed it is important to validate it to confirm that it is suitable for its intended use. The validation of the system tells how good the methods are and how good they are specifically for the intended application [1]. Validation is a technique which establishes the documented evidence that produces high degree of assurance about the product or technique and its accuracy and precision. Validation of an analytical technique is a procedure which is carried out to obtain justified evidence of the ability of the technique to provide accurate and précised results [4]. In the pharmaceutical industry validation and validation methods have a great importance. All analytical techniques used for the development of pharmaceutical products and for the determination of their quality, validation are done [4]. Partial validation is the process in which the validation is carried out on the analytical sample and system but in partial validation all the components of validation are not included. Partial validation is carried out on the following components: - 11
  12. 12. 1) Accuracy 2) Precision 3) Repeatability 4) Reproducibility 5) Linearity and Range 6) Robustness 7) Limit of Detection 8) Limit of Quantification 9) Recovery. …………………………………….[1] 1.3.1 Accuracy and Precision: The precision of an analytical technique is the closeness of the series of individual measurements of a sample when the analytical procedure is applied repeatedly to multiple experiments of the same sample. The precision in a technique is calculated by the coefficient of variation between the techniques and hence defines the standard deviation from the results found earlier. This is also called as relative standard deviation (RSD) and is further divided into repeatability (Intra-day precision) and (Inter-day precision) and reproducibility (between-laboratory precision). [1] 1.3.2 Linearity and Range:The linearity of an analytical method is the ability of the technique to produce the results which are directly proportional to the concentration of the sample. The results are then transformed into a mathematical expression and then plotted in a graph mode to give the linearity in the results. Linearity is calculated by the equation of regression. The results of the experiment are plotted on the graph. Generally 6-8 points are taken on the graph to find out the linearity of the results. The linearity of the sample defines that the change in the sample is directly proportional to the results obtained that is with the increase of the sample concentration the peak area in the 12
  13. 13. result increase and hence defines the peak area increases proportionally with the increase in sample concentration. [1] 1.3.3 Limit of Detection is defined as the concentration of the sample that results in a peak height three times the noise when injected into the chromatographic system. LOD is the lowest quantity of the substance which can be distinguished from the absence of that substance. It is estimated from the mean of blank and the standard deviation of the blank. [1] 1.3.4 Limit of Quantification is defined as the lowest concentration of the sample that can be quantified with acceptable accuracy and precision. 1.3.5. Recovery of the sample is the desirable result of sample preparation and it is an important part of extraction experiments. The absolute recovery is the ratio of response measured for a spiked sample treated according to the whole analytical procedure and with the same quantity the sample substance and directly injected into the HPLC system. The recovery is referred as the ratio of the responses between extracted spiked samples and extracted pure samples. The relative recovery can be used to calculate the absolute recovery and hence can be useful in the detection of sample losses during the process. [1] 1.3.6 Robustness is the test which is used to examine the effects that can change the results of the experiment. In robustness the experimental parameters are altered to a small extent and then the results are taken. Robustness is the technique which is not approved by FDA guidelines and is not considered most appropriate for validation. The parameters of the experiment are subjected to a small change to find out the changes in the result. The parameters which can be altered include pH of the buffer, mobile phase, and temperature and detection wavelength. [1] 13
  15. 15. Fig 2………………….. [7]. 1.4 LAMOTRIGINE :- (LAMICTAL) 15
  16. 16. Lamotrigine is a new antiepileptic agent drug which is chemically not similar to other anticonvulsants and generally used as an add-on therapy in patients with epilepsy. Lamotrigine is effective against partial and secondary generalised tonic clonic seizures as monotherapy or as an add-on. [5]. Lamotrigine or Lemictal is an anticonvulsant drug used in the treatment of epilepsy and bipolar disorder. Lamotrigine is commonly used in epilepsy to treat partial seizure, primary and secondary tonic clonic and Lennox Gas taut syndrome. Lamotrigine is found to work in the treatment of mood disorders as well, which includes rapid cycling and mixed bipolar state. The main advantages of lamotrigine over other antiepileptica agents like valproate is that it has fewer side effects and does not require blood monitoring. [6]. The CNS and nerves are made up of many nerve cells called neurons which communicate among themselves through electrical signals. These signals are carefully regulated for the brain and nerves to function properly. Sometimes there is an abnormality in the brain leads to rapid and repetitive electrical signals firing at the nerve endings, the brain becomes over-stimulated and normal function is disturbed. This results in fits or seizures. [7] 1.4.1 MECHANISM OF ACTION: Lamotrigine is a sodium channel blocker which prevents epileptic fits by preventing excessive electrical activity in the brain. Lamotrigine prevents sodium from entering nerve cells and stops the conduction of the electrical signals and block use dependesnt sodium channels. Sodium entry into the nerve cells is necessary for the electrical signal to be passed on to other nerve cells. Lamotrigine prevents the sodium entry and hence helps stabilise the electrical activity in the brain. [7] Lamotrigine prevents the conduction of electrical impulse as well as it prevents the release of a neurotransmitter called glutamate from the nerve cells in the brain. Neurotransmitters are chemicals that are stored in nerve cells and are involved in conducting messages between the nerve cells. Glutamate is a neurotransmitter that acts as a natural 'nerve-exciting' agent. It is released when electrical signals 16
  17. 17. conduction takes place in the nerve cells and due to its nature it excites more nerve cells. It is believed that glutamate plays a key role in causing epileptic seizures. [3] Reducing the release of glutamate from the nerve cells in the brain also helps in stabilising the electrical conductance in the brain and hence helps in preventing epilepsy. [7] Lamotrigine is also used as a mood stabiliser for treating people with the psychiatric illness, bipolar affective disorder. Lamotrigine proved to be effective in people with bipolar disorder who have not responded to the traditional mood stabilisers (lithium, carbamazepine, valproate). Lamotrigine is used for treating episodes of high or low mood and for helping to prevent episodes of ill health in these people. It is not fully understood how lamotrigine works in this illness, but is thought to be to do with the reduction of glutamate in the brain. [6] 1.4.2 Properties of Lamotrigine: - [6] Chemical Formula C9 H7 Cl2 N5 6-(2, 3-dichlorophenyl)-1, 2, 4-triazine-3, 5-diamine Figure 1 (Taken from google image) Mol. Mass- 256.091 g/mol Pharmacokinetic data Bioavailability- 98% Protein binding -55% 17
  18. 18. Metabolism -Hepatic Half life -24-34 hours (healthy adults) Excretion- Renal Storage- 2-8o C [8] Insoluble in water [8] 1.4.3 Market Preparations of Lamotrigine: - Lamictal and Lamictin from GSK is the main market preparation of lamotrigine. Lamotrigine is available as an oral medication intended for oral use and it is available in tablet form with the dosage of 25 mg, 100mg, 150mg and 200mg. The tablet of the lamotrigine is given in the figure: - [6] Tablet of Lamotrigine Fig.2 (taken from Wikipedia) 1.4.4 INDICATIONS: - [6] Labelled indications: - Epilepsy and Bipolar Disorders I 1. Lennox-Gastaut syndrome 2. Partial Seizures 3. Secondarily Generalised Tonic Clonic seizures 4. Bipolar Mania I 5. Mixed and Rapid Cycling Mania Off-Labelled Indications:1. Peripheral Neuropathy 2. Trigeminal Neuralgia 3. Neuropathy Pain 18
  20. 20. 2.1 Materials Chemical Name Supplier Orthophosphoric acid Fisher scientific UK Ltd. HPLC grade methanol Fisher scientific UK Ltd. Lamotrigine powder Sigma Aldrich UK Ltd. Acetonitrile Fisher scientific UK Ltd. De-ionized water Made in the HPLC lab of University of Greenwich 2.2 Equipment 1. HPLC pump: (I) Model :- 6000 A solvent delivery system manufactured by WATER ASSOCIATES, serial no. 18105. (II) AGILANT 1200 Systems 1200 Series isocratic pump 41310 A Serial No. DE 62956516 2. Detector :- (I) PHILIPS Pye Unicham PU 4025 UV Detector. (II) AGILANT Systems 1200 Series VWD 41314 B. Serial No. DE 71359377 3. Integrator: - Model number SP4270, Manufactured by: Spectra Physics. 4. Column: - C 18 Bondapack column from WATER ASSOCIATES Length 25 cm , Serial No. 27324. 20
  21. 21. 5. Injector: - RHEODYNE 7725i 25uL injector made in USA. 6. Syringe: - 50 uL Syringe manufactured by; - SGE Ltd. Australia. 7. Membrane Filters: - Manufactured by Fisher UK limited. 8. Weighing Balance: - Model AC 88, Manufactured by: Metter instrument. 2.3 Chromatographic conditions:1. The Flow rate was 1ml/min. 2. UV Detection was done at 210nm. 3. A Rheodyne 20-µl loop injector was used. 4. C 18 Bondapack column from WATER ASSOCIATES 25 cm long. 5. 50 µL Syringe for injection. 2.4 Mobile Phase:1. The Mobile phase was prepared using Acetonitrile and Water ( 49.9:49.9 v/v) with 0.2 % Orthophosphoric acid. 2. The Mobile phase was prepared and the Degassed using membrane filters of 0.45 µm thickness under vaccum. 2.5 Chromatographic system and conditions:HPLC method was performed using an Agilant systems HPLC pump with a Rheodyne 7725 I injector. The absorbance was taken with AGILANT Systems 1200 Series VWD 41314 B. The separation was done on C-18 µBondapack column. The mobile phase was prepared with acetonitrile and 21
  22. 22. water and the Flow rate was kept at 1ml/min. The experiment was done at room temperature and 20 µl of samples was injected into the HPLC system. 2.6 Standard Stock Solution of Lamotrigine:The Lamotrigine powder was taken from Sigma-Aldrich ltd in 10 mg amber coloured bottle. The lamotrigine standard solution was prepared with help of methanol. Accurate amount of lamotrigine was weighed and transferred to a 10 ml volumetric flask and made it up to 10 ml. This gave lamotrigine standard solution as 10mg of lamotrigine in 10 ml methanol. 2.7 Working Standard solution:The working standard solution contained 25µg/ml of lamotrigine. 250 uL of the standard solution was taken and transferred to 100ml volumetric flask and the solution was made up to 100ml with methanol. . 2.7 Procedure:Preparation of Lamotrigine Stock solution 10 mg of Lamotrigine was taken and dissolved in Methanol and made upto 10 ml in volumetric flask. Then further dilutions to get desired test solutions were done from this stock solution using the calculations:- 22
  23. 23. The equation used for making the dilutions for test solutions: C1V1= C2V2 Where, C1 = Concentration of the Stock solution V1= Unknown volume to be calculated C2 = Concentration of dilution to be prepared V2= Volume of the volumetric flask in which the dilution is made 2.8 DILUTIONS MADE FOR THE EXPERIMENT:Dilutions made from working standard solution:Lamotrigine 0.5 ml 1.0 ml 1.5 ml 2.0 ml 2.5 ml Methanol (vol. make up ) 100 ml 100 ml 100 ml 100 ml 100 ml Conc. in PPM 5 10 15 20 25 These different dilutions were injected in the HPLC system to get the results. CHAPTER 3 PARAMETERS FOR PARTIAL VALIDATION 3.1-Determination of Standard Deviation:S. No. Concentration Peak Area (AUC) 1 10 µg/ml 2.70 2 10 µg/ml 2.68 23
  24. 24. 3 10 µg/ml 2.74 4 10 µg/ml 2.74 5 10 µg/ml 2.75 6 10 µg/ml 2.73 The Mean of the above readings comes to be:Mean = 2.73 Median = 2.74 The Standard Deviation of the above readings was calculated from the site http://www.physics.csbsju.edu/stats/descriptive2.html. The Standard Deviation of the above readings came as:Standard Deviation (S.D) = 0.02495 From the Standard Deviation we can calculate the Relative Standard Deviation. Relative Standard Deviation (RSD) = (SD/Mean) x 100 i.e. RSD = (0.02495/ 2.73) x 100 = 0.91% 3.2 Linearity:Linearity of the test sample was calculated by plotting the graph of different concentrations against their respective peak areas. The graph when plotted showed a straight line with the Regression Coefficient r2 value 0.9994. From the graph, the value of slope was taken for further calculation of LOD and LOQ. Readings for the Concentration and respective AUCs of different concentrations Concentration Mean Peak area (AUC) 24
  25. 25. 1 5 10 15 20 25 400 1204 2293 3543 4649 5746 Chart Title 7000 y = 225.52x + 115.91 R2 = 0.9994 6000 5000 4000 peak area AUC 3000 Linear (peak area AUC) 2000 1000 0 0 10 20 30 From the above graph we get the equation:- y= 225.52x + 115.91 Hence we get, Slope=S= 225.52 Table for Peak areas of different concentrations with their Standard Deviation:Concentration Peak Peak S.No µg/ml Area (1) Area (2) Peak Area (3) Mean S.D 25
  26. 26. 1 1 µg/ml 362 409 430 400 0.34 2 5 µg/ml 1213 1198 1201 1204 0.79 3 10 µg/ml 2280 2298 2301 2293 0.114 4 15 µg/ml 3547 3539 3544 3543 0.040 5 20 µg/ml 4658 4619 4671 4649 0.27 6 25 µg/ml 5790 5772 5675 5746 0.61 Total S.D = 2.164 From the above table we get the Total Standard Deviation S.D = 2.164 3.3 Limit of Detection (LOD) = 3 x S.D/s Where s = slope = 225.52 Hence LOD = 3 x 2.164/225.52 = 0.028 µg/ml 3.4 Limit of Quantification (LOQ) = 10 x S.D/s Hence LOQ = 10 x 2.164/225.52 = 0.096 µg/ml. The limit of detection and limit of quantification showed the results that the concentration of the sample was very less and hence the system was very sensitive to even smaller amounts of samples as well. 3.4 Quantification:Readings were taken from equal injections of a test solution and standard solution and compared and then the content of lamotrigine in solution was calculated by Cs (At/As) At As 26
  27. 27. 3695 5545 3620 5571 3672 5574 Where Cs is the concentration of lamotrigine in the working standard solution which is 25 µg/ml. Hence Quantified value of the test solution = 25(3662/5563) = 16.45µg/ml 3.6 Precision and Repeatability:The Precision of HPLC method was done by Repeatability which includes Intra Day and Inter Day precision. The results were taken by injecting the concentrations of 5, 10 and 20 and the peak areas were taken for precision by calculating the standard deviation. 3.6.1 INTRA-DAY PRECISION ----- TABLE 1. Date 13/12/2007 Concentration S.No. (PPM) Peak Area AUC 1173 S.D R.S.D 27
  28. 28. 1. 5 1169 0.050 0.42 0.076 0.32 0.226 0.48 1179 2306 2. 10 2311 2321 4661 3. 20 4674 4705 TOTAL RSD = 1.22 3.6.2 INTRA-DAY PRECISION ------TABLE 2 Concentration S.No. 1. Peak Area (PPM) AUC 1174 S.D R.S.D 5 1179 .00251 0.021 0.151 0.64 0.66 1.40 1177 2337 2. 10 2366 2344 4723 3. 20 4624 4749 3.6.3 INTER-DAY PRECISION AND REPEATEBILITY Concentration S.No. 1. Date 14/12/2007 Peak Area (PPM) AUC 1127 S.D R.S.D 5 1131 .00208 0.018 0.214 0.91 0.110 0.22 1128 2349 2. 10 2359 2318 4853 3. 20 4869 4874 TOTAL RSD = 1.14 28
  29. 29. The results obtained for Repeatability and Precision taking the interday and intra Day results which shows that all the readings show RSD below 2.0%. The results were taken from inter and intraday precision and repeatability and the RSD for the results were taken and compared. All the results showed the RSD under the limits of acceptance and the method came out to be linear and precise. CHAPTER 4 RESULTS AND CONCLUSION 4.1 RESULT AND CONCLUSION:The Partial Validation of Quantitative Analysis of Lamotrigine was done using HPLC and the parameters taken to partially validate the technique include Linearity, Quantification, Specificity, Precision and Repeatability. The results obtained in the technique along with their use in validation are detailed below:4.2- LINEARITY: - The linearity was evaluated with the help of linear regression analysis and linearity gave the values of LOQ and LOD. 29
  30. 30. 4.3- Regression coefficient r2 was 0.9994 which shows great degree of linearity. 4.4- LOQ and LOD: - From the linearity taken the LOD and LOQ were calculated with the help of slope from the regression equation. The calculated LOQ = 0.096 µg/ml LOD = 0.028 µg/ml# 4.5- PRECISION AND REPEATIBILITY:The precision and repeatability was taken by Intra Day and Inter Day analysis and the results were taken to calculate the Relative Standard Deviation (RSD). The RSD for both Intra day and Inter day came under the desired value of 2 % and hence the results comply within the limits. RSD for Inter day came to be 1.14 % RSD for Intra day came to be 1.22 % 4.6 -CONCLUSION:The HPLC validation technique for the Quantitative analysis of Lamotrigine gives results under the limits of acceptance and the method used is linear and precise. CHAPTER 5 DISCUSSION 5.1 DISSCUSION:The method used in this experiment for the HPLC of lamotrigine was slightly different from the method taken as reference. The changes in the mobile phase were done and a simpler mobile phase was used containing acetonirile and water. The method showed linearity with regression coefficient 0.9994 and precision with RSD less than 2% for both Inter and Intra day precision. According to the USP the value of RSD (which is the measure of the spread of data in comparison to the mean of the data should not be more than 2%) [9] And hence the results came under the limits of acceptance and the method was found to be linear and precise. 30
  31. 31. REFERENCES 1. Johan Lind Holm, Development methods,Comprehensive and Validation of HPLC Summaries of Uppsala Dissertations from Faculty of Science and Technology 995, Acta Universities Upsaleinsis, pp 87 Sweden 2004. 2. Thomas H Stout, Handbook of Pharmaceutical Analysis, chapter 3. Published in 3. A Acid Free paper 2002, United States of America. Guide to HPLC, http://www.pharm.uky.edu/ASRG/HPLC/hplcmytry.html 4. N.A Epshtein, Validation of HPLC Techniques for Pharmaceutical Analysis, Pharmaceutical Chemistry Journal vol. 38, no.4, 2004. 31
  32. 32. 5. J. Emami, N Ghassami, Development and Validation of HPLC method for Determination of Lamotrigine, Journal of Pharmaceutical and Biomedical Analysis 40 (2006) 999-1005. 6. Wikipedia, Lamotrigine last modified 03:27, 13 December 2007. 7. netdoctor.co.uk/medicines/100001450.html last updated 15/3/2007. 8. Sigma-Aldrich sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/L37911000014 50.html last updated 15/3/2007. 9. J.C Miller, Statistics for Analytical chemistry, III rd edition. 1993. 32
  33. 33. 33