Development Of Calibration Curve Biology Essay

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A stock solution of Atorvastatin calcium1mg/ml was prepared using acetonitrile and water as solvent. From this stock solution 10, 20, 30, 40 and 50 μg/ml concentrations were prepared. The λmax of the drug was determined by scanning one of the dilutions between 400 and 200 nm using a UV-visible spectrophotometer. At this wavelength, the absorbance of all the other solutions was measured against a blank (acetonitrile and water (1:1) without drug). Standard curve between concentration and absorbance was plotted. Results were shown in table.

Solubility studies of API (LS 4)

The solubility of the Atorvastatin calcium was determined in four different non-volatile solvents i.e. Propylene glycol, Polyethylene glycol 400, Tween 80 and Polyethylene glycol 200 by adding an excess amount of drug to 5 ml of selected non-volatile solvents in glass vials. The vials were kept at 25± 0.5o C in isothermal shaker for 72 hours to reach equilibrium. After equilibriation has achieved the supernatant was taken and filtered through a 0.22μ membrane filter and concentration of Atorvastatin calcium was determined in the non-volatile solvents after proper dilutions using UV-Visible spectrophotometer at 246nm against blank (blank sample having the same dilutions as that of drug but without drug). Results were shown in table.

Compatibility study:

FTIR spectral matching technique was used for detection of any possible chemical interferences between the drug and carrier neusilin US2 which is an adsorbent. A combination of drug and carrier was prepared and mixed with suitable quantity of KBr to obtain about 100 mg of mixture which was then compressed to form a transparent pellet using a KBr pellet press at 9 tons pressure. It was scanned from 4000 to 400 cm-1 in Schimadzu FTIR spectrophotometer. The IR spectrum of the physical mixture was compared with that of pure drug and neusilin US2 and spectral matching was done to detect any appearance and disappearance of peaks.

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Optimization of gelling agent: HPC(LF) and HPMC K4M are proposed as gelling agents for the present study. Different amounts of 5, 8, 12, 16mg of HPC(LF) was added to 0.06ml(76mg) of non-volatile solvent (propylene glycol) containing drug by continuous stirring until a clear viscous gel was obtained. Similarly Different amounts of i.e. 5, 10, 16, 18mg of HPMC K4M was added to 0.06ml(76mg) of non-volatile solvent (propylene glycol) containing drug by continuous stirring until a clear viscous gel was obtained.

Optimization of adsorbent quantity

Different amount of adsorbent 20, 40, 60, 80,100,120mg of neusilin was added to 0.06ml(76mg) of non-volatile solvent i.e. propylene glycol containing drug by continuous mixing until a free flowing blend was obtained.

Precompression studies of the prepared liquisolid powder systems

Differential scanning calorimetry:

Thermograms of the samples were recorded on DSC Q-200 (TA Instruments, USA). Samples were placed in T-zero aluminum pans and the lids were crimped using T- Zero press. Thermal behavior of the samples was investigated under at scanning rate of 10oC/min, covering a temperature range of 50-200oC. The instrument was calibrated with indium standard.

X-ray Powder Diffractometer (XRPD):

X-Ray Powder Diffractometry is one of the most powerful and established technique for material structural analysis, capable of providing information about the structure of a material at the atomic level. Low and High temperature measurement facilities available. The physical forms of drug in ideal batches were characterized by means of X-ray powder diffraction. The tablets were crushed to fine powder with the help of pestle and mortar. The fine powder was packed into sample holder and the sample was scanned using the instrument parameters listed below.  Instrument used: Powder X-ray diffractometer  Make, Model: PANalytical, X'Pert PRO  Goniometer: Theta/Theta vertical  Measuring circle: 480 mm  Radiation: Cu K α (wavelength=1.5418 Ao)  Detector: X'Celerator***  Voltage current: 45 kV, 40 mA  Scan range: 3o - 44o 2θ  Step size: 0.02o 2θ  Time per step: 200 sec

 Scan mode: Continuous  Divergent slit: Automatic 10 mm  Anti scattering slit: Automatic 10 mm  Specimen length: 10 mm  Synchronous rotation: On

Flow properties:

The flow properties of the prepared powders were determined by angle of repose, bulk density, compressibility, hausner's ratio. The angle of repose was measured using fixed funnel apparatus and remaining bulk density, compressibility, hausner's ratio were measured using bulk density apparatus.

Evaluation of the prepared gel based liquisolid and DC tablets:

All the prepared LS and DC tablets were evaluated for the average weight, friability, hardness, disintegration, drug content and in vitro drug release adopting the following procedures.

Weight variation:

The weight variation test was performed as per USP [14] and results for all the batches of LC and DC tablets were shown in Table ...

Hardness

Monsanto hardness tester was used to determine the hardness of the tablets. It consisted of a barrel containing a compressible spring held between two plungers. The tablet was placed in contact with lower plunger and zero reading was noted. The plunger was then forced against a spring by turning the thread bolt until the tablet fractured. The force required to fracture the tablet was recorded.

Friability Testing:

Friability of the tablets was determined by using Roche friabilator. Ten tablets from each batch were placed in the friabilator and rotated at 25 rpm for a period of 4 minutes. The friability was determined using the following formula..

Disintegration test

The disintegration test was carried out using disintegration test apparatus as specified in the Indian Pharmacopoeia [15] and results for all the batches of LC and DC tablets were shown in Table ...

Drug Content:

The drug content of tablets was determined by UV Spectrophotometer. Five tablets were powdered and a quantity equivalent to 100 mg of the drug was transferred into a beaker containing phosphate buffer pH 6.8. The solution was stirred for 1 hour, filtered and the absorbance was measured at 246 nm against blank solution. The drug content was calculated using the formula

Formulation of Gel liquisolid compacts: Several gel based liquisolid compacts as LS1 to LS10 were prepared using cadmach 16 station tablet compression machine. Atorvastatin calcium was dispersed in propylene glycol and then solubilized by gently warming followed by sonicating the solution. This solution was converted in to gel by HPC &HPMC K-4M which are having interconnected pore morphology which helps in the high and quick solvent absorption. To this calculated amount of adsorbent neusilin US2 was added in different concentration (80mg, 100mg and 120mg) to form a freely flowing powder. To above powder blend, aerosil (coating material) and cross povidone (super disintegrant) were added. Finally, MCC pH 102 (diluents) was added. Then, to this powder blend 0.5% PVP in IPA was added drop wise until wet mass was formed. Finally, the wet mass was passed through sieve.no.16 to obtain granules. The granules were dried at room temperature for 30 minutes. The dried granules were passed through sieve no.22 and evaluated for flow properties and then compressed using 12mm and 13mm dies.

Formulation of directly compressible tablets:

Two batches of DC tablets (LS 11, LS 12) were prepared using cadmach 16 station tablet compression machine. The known quantity of drug and the calculated amount of adsorbent, gelling agent and other excepients without non-volatile solvent i.e. propylene glycol were triturated to form a free flowing powder. To this powder blend 0.5% PVP in IPA was added drop wise until wet mass was formed. Finally wet mass was passed through sieve no 16 to obtain granules. The granules were dried at room temperature for 30 mins. The dried granules were passed through sieve number 22 and evaluated for flow properties and then compreesed using 10mm die.

Invitro dissolution studies:

The Invitro dissolution studies of prepared LS 1to LS 12 formulations were carried out using USP type 2 (The paddle method) dissolution apparatus. Phosphate buffer 6.8 was selected as dissolution media which was maintained at 37±0.5OC. The dissolution process was carried out using 900 ml of Phosphate buffer 6.8 with a rotation speed of 75 RPM and temperature of 37±0.5OC. Prior to dissolution the phosphate buffer was deaerated by sonication. The formulation LS 1 to LS 12 were placed in to the dissolution media and then an aliquot of 5 ml sample was withdrawn at 0, 5, 10, 15, 30, 45, and 60 minutes of time intervals respectively. After each sampling an equal quantity of buffer was replaced to maintain the skin conditions. The UV absorbance of filtered samples was measured at 246nm and the cumulative drug release in percentage at various time intervals were calculated.

BIOANALYTICAL METHOD FOR ATORVASTATIN CALCIUM

Selection of the wavelength

10 μg/ml Atorvastatin calcium was weighed accurately in the into the 10 ml volumetric flask and 5ml of ACN:Water (50:50) was added and kept for sonicator to dissolve. Then the volume is made up with the same solution. This solution was scanned and uv spectrum was recorded. 254nm was selected for both drug and internal standard which shows good response.

2.preparation of standard solution

a. Preparation of standard stock solution for Atorvastatin calcium

1mg/ml of Atorvastatin calcium stock solution was prepared by dissolving 10 mg of drug in the 10ml of ACN: Water (50:50). This solution was the stored in refrigerator at -200 C ± 20C until analysis.

b. Preparation of standard stock solution for IS

1mg/ml of Atorvastatin calcium stock solution was prepared by dissolving 10 mg of drug in the 10ml of ACN: Water (50:50). This solution was the stored in refrigerator at -200 C ± 20C until analysis.

c. preparation of calibration curve

From the standard stock solution control rat plasma were spiked with10,30,60,90,110,140,160 and 180ng/ml of Atorvastatin calcium and 1mcg of IS. This solution was vortex and extracted with SPE cartridge by solid phase extraction method. This quality control sample were prepared by bulk.Quality control were lower limit of quantification was (LLOQ QC), 30.0ng /ml, middle limit of quantification was(MQC) 160ng /ml and higher limit of quantification was (HQC) 180 ng /ml.

These samples were stored below -50°C until use.

3. Optimization of chromatographic conditions for Atorvastatin calcium and Olanzapine (IS).

Stationary phase used wasHibarC8 column of specification of(250 x 4.6 mm i.d., 5μ) with the mobile Phase ratio of Acetonitrile and20 Mm Phosphate buffer at the pH range of 6.5 (40:60). Flow rate was 1 ml/min with the sample volume of 20 μl using Rheodyne 7725i injector. The detection was carried out with 254 nm. Data station used was LC-20AD of shimazu model

Preparation of Mobile Phase

25Mm of potassiumdihydrogen ortho phosphate was prepared for 500ml with Millipore water and the pH was adjusted to 6.5 by using Triethyl amine. This solution wasfilter through 0.45μ filter paper and the solution was degassed in sonicator.

IN VIVO ORAL BIOAVAILABILITY STUDIES

200-250g of Male Albino wistar rats was used for the oral bio-availability studies. Animal Ethical clearance were approved by Institutional Animal Ethical Committee, J.S.S. College of Pharmacy, Ooty (Proposal no. JSSCP/IAEC/M.PHARM/Ph.ceutics/01/2011-2012). All the animals were treated as per the study protocol. This study was carried out by giving free access to water.

GROUPINGS :

12 animals were grouped in to two groups in such a way that each group contains six animals. Group one animals received test formulation (), group two animals received standard formulation (drug suspension). All the formulations were administered using oral needle at a dose of 0.8 mg which is equivalent to human dose 10mg. conversion factor = 0.07 (10*0.08= 0.8mg).

Blood sample were collected at the interval of (0.25, 0.5, 0.75,2,4,6,8,12,24 hrs) in an Eppendorf tubes containing 0.3ml anticoagulant (Sodium citrate) and centrifuged immediately. After centrifugation, the plasma obtained was collected in the Eppendorf tube and stored at -20°C until further analysis.

Preparation of blank plasma Blank rat plasma (0.5 ml) was transferred into 2.0 ml Eppendorf tube and 0.1 ml of mobile phase was added. The resulting solution was vortexed for 5 minutes. The plasma was extracted using SPE cartridge with ACN- WATER (50:50) and analyzed.

Preparation of plasma samples CollectedPlasma samples (0.5 ml) from study subjects was transferred into 2.0 ml Eppendorf tube and 0.5 ml (1mcg/mL) of Internal Standard was added. This solution was vortexed for 2-3 minutes. This resulting plasma was extracted using SPE C-18cartridge with ACN: WATER (50:50) as the extraction fluid and analyzed in RP-HPLC.

Method of analysis

The above extracted plasma sample were injected into the above mention optimized chromatographic conditions and the chromatograms were recorded. Recorded sample response factor was calculated from the calibration curve and the Pharmacokinetic parameters like concentration maximum (Cmax),area under the curve (AUC) , area under the total plasma concentration-time curve AUC, elimination rate constant K and half life(t1/2).

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