TRANDOLAPRIL ANTIHYPERTENSIVE DRUG

Transcription

1 Chapter-V Trandolapril Introduction TRADLAPRIL ATIYPERTESIVE DRUG 5.01 Drug Profile The IUPAC name of trandolapril is (2S,3aR,7aS)-1-[(2S)-2-[[(2S)-1-ethoxy- 1-oxo-4-phenylbutan 2yl]amino]propanoyl]2,3,3a,4,5,6,7,7a octahydroindole-2- carboxylic acid. Trandolapril is approximately eight times more active as an inhibitor of ACE activity is used to treat high blood pressure. It is believed to exert its antihypertensive effect through the renin-angiotensin-aldosterone system. The effect of trandolapril in hypertension appears to result primarily from the inhibition of circulating and tissue ACE activity thereby reducing angiotensin II formation, decreasing vasoconstriction, decreasing aldosterone secretion, and increasing plasma renin. Decreased aldosterone secretion leads to diuresis, natriuresis, and a small increase of serum potassium. Physical Properties Molecular formula : C Molecular weight : g. Appearance : Colorless and crystalline Solublility : Chloroform, methanol and Dichloromethane 319

2 Chapter-V Trandolapril Introduction Structure Brand names Zetpril (etero C (Genx)) Mavik (Abbott) Tarka (Abbott) Trandolaprilum (alo) : 1 mg, 2 mg, : 1 mg, 2 mg, or 4 mg : 1 mg, 2 mg, or 4 mg : 1mg, 2mg Inactive Ingredients Each tablet contains corn starch, croscarmellose sodium, hypromellose, iron oxide, lactose, povidone; sodium stearyl fumarate. Literature survey Literature survey reveals that some chromatographic methods [ ] are reported. Csermely and his coworkers [298] performed some experiments on determination of some ACE inhibitors by TLC. irogi et al [299] developed a liquid chromatography/tandem mass spectrometry using solid phase extraction to determine the amount of trandolapril and its metabolite trandolaprilat in human plasma. A high performance thin layer chromatography method [300] coupled with densitometric analysis has been developed by Kowalczuk.D, for simultaneous determination of trandolapril and verapamil in two component mixtures and in their combination capsules. A liquid chromatographic tandem mass spectrometric method for the 320

3 Chapter-V Trandolapril Introduction determination of trandolapril in human plasma has been developed and validated [301]. Gumieniczek and opkala [302] have proposed an isocratic PLC procedure for the determination of trandoalpril and verapamil in capsules. LiChrosorb RP18 column with a mobile phase composed of acetonitrile-methanol-phosphate buffer of p= 2.7(2:2:1) and UV detection at 220nm. A rapid and sensitive reverse phase PLC method [303] applied for the qualitative and quantitative assay of trandolapril in pharmaceutical dosage forms. Trandolapril is chromatographed [304] on a reverse phase C 18 column with a mobile phase consisting of methanol: phosphate buffer (p 7.8) in the ratio of 90:10 v/v. The mobile phase is pumped at a flow rate of 1 ml/min. A liquid chromatographic tandem mass spectrometric method [305] for the determination of trandolapril in human plasma has been developed and fully validated. A few spectrophotometric methods [ ] have been reported for the determination of trandolapril in pure and formulations. 321

4 Chapter-V Trandolapril Part-A: PLC-Method Assay of Trandolapril in bulk and formulations by high-performance liquid chromatographic method 5.02 Introduction Chromatography is a simple method of separating a desired component from its impurities or isolating individual components of a mixture. The necessity of isolating the desired compound from these by products to get the drug of the required quality and uniform potency is too obvious. This technique is also used in assay of a component. In the literature it is found that some PLC methods [ ] are reported to determine the trandolapril mostly in human/animal plasma or fluids, in pure, formulations and also in combination with other drugs. The linearity limits, LD and LQ of the reported methods are low, and not applicable in higher concentrations. ence the author has attempted to develop a new reverse phase PLC method for the analysis of pharmaceutical analysis which is applicable in higher concentrations than the concentrations reported earlier Experimental Instrumentation: Shimadzu LC- 20AT Prominance liquid chromatographic system is used for the analysis. Shimadzu LC- 20AT system equipped with binary gradient pump, UV-VIS SPD 20A detector, Column ven and controlled by spinchrom software. Chromatographic conditions: Column : ypersil BDS-C18 (100mm, 4.6mm-ID; 3µm particle size) Detector : UV-VIS SPD 20A Prominance 322

5 Chapter-V Trandolapril Part-A: PLC-Method Wavelength (λ max ) : 215nm Flow Rate : 1.0ml/min. Injection Volume : 20 μl Temperature : 30 o C Run Time : 20 min. Retention Time : 6.06min Method Development (i) Materials and Reagents All the Chemicals and reagents used in the analysis are of PLC grade. Acetonitrile, Methanol, Ammonium dihydrogen orthophosphate and phosphoric acid 85% are purchased from Merck, India and PLC grade water is used to prepare the mobile phase. Stock solutions of trandolapril and sample solutions are prepared in the mobile phase. All solutions are filtered through 0.45 µ membrane filter and degassed using a sonicator. Gift samples of trandolapril and working reference standard from Chandra Lab (an Analytical Testing Laboratory) are used. The formulations are purchased locally. (ii) Preparation of Solutions (a)preparation of buffer solution of p=2.8 (0.02M, Merck; v/w): About 0.544g of ammonium dihydrogen orthophosphate is accurately weighed, transferred into 500ml volumetric flask and dissolved in PLC grade water, made up to the mark by 323

6 Chapter-V Trandolapril Part-A: PLC-Method adjusting the p =2.8 by adding a few milliliters of o-phosphoric acid, filtered and sonicated. (b)preparation of mobile phase(buffer and Acetonitrile; 50:50 v/v): Mobile phase is prepared by mixing accurately measured volumes of 250ml of buffer and 250 ml of acetonitrile in a 1000ml beaker, stirred well, filtered, sonicated and used for the analysis. (c)preparation of drug standards Stock solution (1mg/ml): About 50.0mg of trandolapril is accurately weighed, transferred into a well cleaned, dried 50ml volumetric flask, 30ml of mobile phase is added and sonicated to dissolve the drug. The solution is made up to the mark with mobile phase. Working standard solution (100μg/ml): Working standard solution is prepared daily by accurately measuring 10.0ml of the stock solution into 100ml volumetric flask and made up to the mark with mobile phase. Similarly the working standard solutions of different concentrations 25μg/ml, 50μg/ml, 75μg/ml, 125μg/ml and 150μg/ml are prepared and used for the analysis. Preparation of Sample solutions: Fifty tablets of zetpril are powdered and mixed thoroughly. An amount of the powder equivalent to 50mg of the drug is dissolved in methanol, shaken well and filtered. The filtrate is evaporated to dryness carefully and the residue is dissolved in mobile phase and made up to 100ml. The resulting solution is filtered, sonicated and used for the analysis. 324

7 Chapter-V Trandolapril Part-A: PLC-Method (iii) Procedure: The chromatographic conditions are fixed for the Shimatzu PLC system.the mobile phase is allowed to pump from the mobile phase reservoir into the column ypersil BDS-C18 (100mm, 4.6mm-ID; 3µm particle size) at a flow rate of 1.0 ml/min., keeping the column in thermostat at a temperature of 30 o C. The response of the system is recorded against time at maximum wavelength 215nm. After the elution of the mobile phase through the column, it is found that the base line of the chromatogram is almost parallel to x-axis. ow 20μl of the standard/test solution of trandolapril of is injected into the system, and the chromatogram is recorded Fig.5.01 and Fig.5.02, P: 330. (iv) ptimization of the proposed method: The developed method is optimized and optimum conditions are established by varying the parameters such as concentration of the standard, type of column, temperature of the column, flow rate, injection volume, composition of the mobile phase (polarity), p of the buffer ect one at a time, keeping the others fixed and observing the effect on the retention time, tailing factor and other system suitability parameters Method Validation Proper validation of analytical methods is important for pharmaceutical analysis when ensurance of the continuing efficacy and safety of each batch manufactured relies solely on the determination of quality. The ability to control this quality is dependent upon the ability of the analytical methods, as applied under well- 325

8 Chapter-V Trandolapril Part-A: PLC-Method defined conditions and at an established level of sensitivity, to give a reliable demonstration of all deviation from target criteria. 5.05(i) System Suitability and System Precision: Working standard solution (100μg/ml) of trandolapril is injected six times into the PLC system and the chromatogram is recorded. The system suitability parameters and system precision are evaluated based on the area of the six replicate peaks and found to be within the limits. The results are incorporated in Table-5.01(a) and Table-5.01(b), P: (ii) Linearity of detector response: Linearity between the response of the detector and the different concentration of the standard drug solutions is established by plotting a graph to concentration versus average area of two peaks. The regression parameters are calculated. A series of six working standard solutions of trandolapril are prepared in the concentration range of about 25.0μg/ml to 150μg/ml corresponding to 25% to 150% of target concentration. Each solution is injected into the system and recorded the chromatogram under the standard conditions. A graph is plotted to concentration in µg/ml on X-axis versus response on Y-axis Fig.5.03 (a), P: 331. The detector response is found to be linear with a correlation coefficient of The results are summarized in Table-5.02, P: (iii) Precision of Test method: The precision of the test method is evaluated by assaying six samples of zetpril tablets of 2.0mg and 4.0mg.The average percent of assay of zetpril in tablets is found to be %, % and %RSD is found to be and respectively for 2.0mg and 4.0mg tablets respectively. The results are summarized in Table-5.03, P:

9 Chapter-V Trandolapril Part-A: PLC-Method 5.05(iv) Accuracy of the method: A study of trandolapril from spiked placebo is conducted. Different concentration solutions of the sample are prepared equivalent to about 75%, 100%, and 125% of the target concentration of trandolapril. Sample solutions are prepared in triplicate for each spike level and assayed as per the standard chromatographic conditions. The % recovery is found to be within limits. The results are summarized in Table-5.04, P: 333. (v) Linearity of the test method The percent of recovery of the drug is determined by adding different known amounts of the standard drug to equal amount of test sample. In each concentration the chromatogram is recorded. A linear plot is drawn to average amount of Trandolapril added (mg) versus average of Trandolapril recovered (mg) in accuracy (Fig.5.03 (b), P: 331). The results of the recovery experiments by the developed method are summarized in Table-5.05, P: 334. (vi)limit of Detection and Limit of Quantitation The limit of detection (LD) and the limit of quantitation (LQ) are calculated based on the standard deviation of the response (SD) and the slope of the calibration curve. The results of LD and LQ are summarized in Table-5.02, P: (v) Ruggedness (a)intra Day and Inter Day Precision: This study is conducted using same column on the same PLC system in different days by assaying six separately prepared trandolapril sample solution under similar conditions. The system suitability parameters are evaluated and found to be within limits. The average % assay for the 327

10 Chapter-V Trandolapril Part-A: PLC-Method two days is found to be 99.79and with a relative standard deviation of 0.562% and 0.393% respectively. The results are summarized in Table-5.06(a), P: 334. Comparison of the results obtained on two different days shows that the assay method is rugged for day to day variability. (b) System to System variability: System to system variability study is conducted on two PLC systems using the same column by assaying six separately prepared trandolapril sample solution under similar conditions. The system suitability parameters are evaluated as per the standard method on both the systems and found to be within limits. The average % assay for the two systems is found to be and with a relative standard deviation of 0.183% and 0.253% respectively. The results are summarized in Table-5.06(b), P: 335. (c) Column to Column variability: The effect of change in column is conducted using two columns on the same PLC system by assaying six separately prepared trandolapril sample solution under similar conditions. The system suitability parameters are evaluated and found to be within limits. The average % assay for the two columns is found to be 99.99and with a relative standard deviation of 0.229% and 0.263% respectively. The results are summarized in Table-5.06(c), P: (vi) Robustness Robustness measures the capacity of an analytical method to remain unaffected by small but deliberate variations in method parameters. It also provides some indication of the reliability of an analytical method during normal usage. A study is conducted to determine the effect of variation in flow rate, change in wavelength, temperature of the 328

11 Chapter-V Trandolapril Part-A: PLC-Method column, composition of the mobile phase and p of the buffer solution on the retention time, asymmetry and area of the peak. Sample solution is prepared in triplicate as per test method and is injected into the PLC system and the instrument response is recorded against in each case. The system suitability parameters are evaluated as per the test method and found to be within limits shown in Table-5.07, P: Results and Discussion: The system suitable parameters such as USP Tailing factor, umber of Theoretical Plates and %RSD are evaluated and found to be within the limits. A linear plot is drawn between concentration and the instrument response (correlation coefficient=0.9999) (Table-5.02, P: 332; Fig.5.03 (a), P: 331). Low %RSD values 0.595,0.244 and and high %recovery 99.95%, % and %corresponding to 75%,100% and 125% spike levels indicate that the method is highly precise and accurate (Table-5.03 and Table-5.04,P: 333). Pharmaceutical formulations are analyzed by the developed method by estimating the amount of drug recovered by standard addition method. A graph is drawn between the amount of drug added and the amount of drug recovered, the plot is linear and regression equation is given by y=1.0038x with r 2 = (Table-5.05, P: 334; Fig.5.03 (b), P: 331). A study is conducted between two analysts, two different systems, two columns and two days and the results are compared. The average % assay and relative standard deviation are within the limits (Table-5.06(a)-Table-5.06(c), P: ). The change in system suitability parameters are evaluated by studying the effect of change in composition of the mobile phase, flow rate, p of the buffer solution, wavelength and column temperature and found to be acceptable (Table-5.07, P: 336). 329

12 Chapter-V Trandolapril Part-A: PLC-Method Fig.5.01Chromatogram of trandolapril (Standard) Fig.5.02Chromatogram of trandolapril (Formulation) 330

13 Weight of the Drug Recovered in µg/ml Mean Peak Area Chapter-V Trandolapril Part-A: PLC-Method Linearity of the Detector Responce for Trandolapril Standard 3.50E E+06 y = 19532x R 2 = E E E E E E Weight of the Trandolapril Standard in (µg/ml) Fig.5.02Linearity of the detector response Linearity of the Test Method y = 1.003x R 2 = Weight of the Drug Added in µg/ml Fig.5.03 (b): Linearity of Test Method 331

14 Chapter-V Trandolapril Part-A: PLC-Method Table-5.01(a): System Suitability System suitability parameters bserved value Acceptance criteria USP tailing factor MT2.0 umber of theoretical plates 4272 LT2000 RSD(six replicate measurements) MT 2.0% Table-5.01(b): System Precision S.o. Concentration (μg/ml) Average Area Statistical parameters Value of the parameters Mean SD %RSD Table-5.02: Linearity of detector response Concentration Average Regression Parameter S.o. μg/ml Area Slope Intercept Correlation Coefficient LD µg/ml LQ µg/ml

15 Chapter-V Trandolapril Part-A: PLC-Method Table-5.03: Precision of the test method Percent of Assay Labeled Amount Sample o. 2.0mg/Tablet 4.0mg/tablet Mean SD %RSD Table-5.04: Accuracy of the method Spike level Sample ID Amount Added(μg/ml) Amount Found(μg/ml) % of Recovery Statistical Analysis 75% Mean SDV %RSD % Mean SDV %RSD % Mean SDV %RSD

16 Chapter-V Trandolapril Part-A: PLC-Method Table-5.05: Linearity of test method Spike level (%) Drug added(mg) Drug Recovered(mg) Regression parameters Slope Intercept Correlation Coefficient Table -5.06(a) Intra Day and Inter Day Precision Percent of assay of Trandolapril Found mg Sample o. Day-1 Day Mean SD %RSD Comparison of precisions with method precision F=1.677,t=0.277 F=3.397,t=0.344 Comparison of precisions between two columns F=2.022 t=

17 Chapter-V Trandolapril Part-A: PLC-Method Table -5.06(b) System to System Variation Percent of assay of Trandolapril Found mg Sample o. Systemt-1 System Mean SD %RSD Comparison of precisions with method precision F=1.532,t=1.470 F=2.935,t=0.386 Comparison of precisions between two systems F=1.926 t=0.662 Table -5.06(c) Column to Column Variation Percent of assay of Trandolapril Found mg Sample o. Column-1 Column Mean SD %RSD Comparison of precisions with method precision F=2.394,t=0.749 F=3.176,t=0.665 Comparison of precisions between two columns F=1.329 t=

18 Chapter-V Trandolapril Part-A: PLC-Method Table 5.07: Study of Robustness Variable Variation USP Plate Count USP Tailing %RSD Standard Flow Rate 0.8ml/min ml/min Wavelength 213 nm nm Column 28 o C Temperature 32 o C Mobile 60 : Phase 40 : Buffer p Acceptance Criteria USP Tailing Factor not more than 2.0, Theoretical Plates not less than Conclusion: The developed procedure for the assay of trandolapril is optimized and validated using the test method and found to be linear, precise, accurate, rugged and robust. The developed method can be applied for the analysis of formulations successfully with excellent percent of recoveries. 336

19 Table 5.08(a) ptimum conditions established for methods M2(a) and M2(b) for TDP Parameter ptimum range Conditions in procedure Remarks max (nm) M2(a) M2(b) Effect of buffer on color development Volume of buffer required for maximum intensity of color (ml) Effect of volume of dye MB (M2(a))) SF (M2(b)) Choice of organic solvent for extraction of colored complex p-9.8 Variations of the p less than 6.0 and greater than11.0 resulted in low absorbance values ptimum volume of 1.0ml of buffer is sufficient for maximum color development 1.0ml of MB (for M2(a))and 0.5 ml of SF (for M2(b)) dye is necessary for covering the broad range of beer s law limits Chloroform Chloroform Water immiscible solvents tested for the extraction of the colored complex into organic phase include chlorobenzene, dichloro methane, CCl 4, C 6 6 and butanol. CCl 3 is preferred for its selective extraction of the colored drug-dye complex from the aqueous phase. Effect of the ratio of organic to aqueous phase on extraction 1:1 1:1 The extraction of the colored species in to Chloroform layer is in complete when the ratio of chloroform to aqueous phase is more than the specified ratio in each case Effect of shaking time (min) Constant absorbance values are obtained for the shaking period of 1-5 Effect of temperature on the colored species (C 0 ) Stability of the colored species Laboratory - Temperature (28 5) Immediate to 60 min Laboratory- Temperature (28 5) min. At low temperature (<20 0 C) and at high temperature (>35 0 C) the extraction of the colored species is found to be improper and the stability of the colored species is found to be very less. 10 min The colored species after separation from organic phase is stable for 60 min, after wards the absorbance gradually decreases. 346

20 Table 5.08(b) ptimum conditions established in method M11for TDP Parameter ptimum range Conditions in procedure Remarks max (nm) Volume of AMV(1.80 x 10-1 M) ml 5.0ml 5.0ml of AMV(1.80 x 10-1 M) is necessary for covering broad range of Beer s law limits Effect of vol. f con 2 S 4 on color development ml 3.0ml Less than 3.0ml of con 2 S 4 resulted in low absorbance values and grater than 5.0ml resulted in instability of the colored product. Effect of the order of addition reagents on color development. TDP, AMV, Con. 2 S 4 TDP, AMV, Con. 2 S 4 If the order of addition is changed, low absorbance values resulted. Effect of temperature and time C 10-20min 75 0 C 15min. The stability of the colored species is found to be less, if the temperature exceeds 80 0 C. Stability period after final dilution Immediate - 30min 5min - 347

21 Chapter - V Trandolapril SPECTRPTMETRIC ASSAY F TRADLAPRIL- ATIYPERTESIVE DRUG 5.08 Introduction Analytically useful functional groups present in Trandolapril have not been fully exploited for designing suitable visible spectrophotometric methods [ ] and therefore still offer a scope to develop more number of new visible spectrophotometric methods with better sensitivity, selectivity, precision and accuracy. The author has made some attempts in this direction and succeeded in developing a few spectrophotometric methods. 5.09(i) Experimental UV-Visible Spectrophotometer: Elico SL159 model, 2nm high resolution, double beam, 1cm length quartz coated optics and wavelength range nm instrument is used for all the spectral measurements. 5.09(ii) Preparation of solutions (a) Standard Solution of Trandolapril Trandolapril stock solution (0.1%) is freshly prepared by transferring accurately weighed 100mg of trandolapril into 100ml volumetric flask, dissolved in methanol, and then made up to the mark with double distilled water. Test solution is prepared by taking accurately weighed portion of powdered tablets equivalent to 100mg of the drug, dissolved in 20ml of methanol, shaken well and filtered. The filtrate is diluted to 100ml with methanol to get 1mg/ml of drug. 337

22 Chapter - V Trandolapril (b) Preparation of reagents All the solutions are prepared by using analytical grade chemicals, reagents and double distilled water. Method M1(a), M1(b), M1(c) and M1(d) Aqueous solutions of ARS ( 0.2%, 5.49 x 10-3 M), BTB( 0.1%, 1.60 x 10-3 M), M( 0.2%, 6.11 x 10-3 M), TPooo( 0.2%, 5.71 x 10-3 M) and Cl solution(0.1m) are prepared in the same way as described under TT in Chapter II P: Method M2(a) and M2(b) MB solution (Fluka; 0.2%, w/v 6.25x10-3 M): Prepared by dissolving 200mg of MB in 100ml of distilled water and subsequently washed with chloroform to remove chloroform soluble impurities SF- solution (Fluka; 0.2%, w/v x 10-3M): Prepared by dissolving 200 mg of Safranin in 100ml of distilled water and subsequently washed with chloroform to remove chloroform soluble impurities. Buffer solution p Cl: 7.0gms of 4 Cl and 6.8ml of liquid ammonia solution are mixed and diluted to 100ml with distilled water and p is adjusted to 9.8. Method M5 (a) Solutions of MBT (0.2%, 8.56 x 10-3 M) and Ce (IV) (1%, 9.35 x 10-3 M) are prepared in the same way as described under TT in Chapter II, P:

23 Chapter - V Trandolapril Method M11 AMV solution (Loba; 0.3%, 1.80x10-1 M): Prepared by dissolving 300mg of ammonium metavanadate in 100ml of distilled water Method M15 Aqueous solutions of p-ca (Sd-fine; 0.1%, 4.785x10-3 M) is prepared in the same way as described under TT in Chapter II, P: 74. Method M17 Aqueous solutions of PA (0.1%, 4.36 x 10-3 M) and Buffer p 9.8 are prepared in the same way as described under TT in Chapter II P: (iii) Procedures proposed for the assay of Trandolapril After a systematic and detailed study of the various parameters involved the following procedures are proposed for the assay of trandolapril in bulk and pharmaceutical formulations. Method M1(a), M1(b), M1(c) and M1(d) Into a series of 125ml separating funnels containing aliquots of standard TDP solution ( μg/ml, μg/ml, μg/ml, μg/ml for methods M1(a), M1(b), M1(c) and M1(d) respectively), 6.0ml of 0.1M CI solution and 1.0ml of 0.2%ARSdye solution M1(a), 1.0ml BTB dye solution M1(b), 2.0ml of 0.2% M dye solution M1(c), 2.0ml 0.2%TPooo dye solution M1(d), are added successively. The total volume of aqueous phase in each separating funnel is adjusted to 15ml with distilled water. To each separating funnel 10ml of chloroform is added and the contents are shaken 339

24 Chapter - V Trandolapril for 2min. The two phases are allowed to separate and the absorbance of the separated chloroform layer is measured at max (435nm for ARS; 420nm for BTB; 420nm for M; and 510nm for TPooo) against a similar reagent blank. The amount of TDP is deduced from the calibration curves (Fig , P: ). Method M2(a) and M2(b) Aliquots of standard drug solution (TDP) μg/ml for method M2(a) or M2(b) ( g/ml) and 1.0ml of p 9.8 buffer solution are placed separately in a series of 125ml separating funnels. A volume of 0.5ml of MB for method M2(a) (1.0ml of Safranin- for method M2(b)) are added respectively. The total volume of aqueous phase in each funnel is adjusted to 10.0ml with distilled water. Then 10.0mlof chloroform is added in each separating funnel and the contents are shaken for 2min and allowed to separate. The organic layer is collected through cotton plug and the absorbance is measured immediately at 640nm (for method M2(a)) and at500 nm (for method M2(b)) against a reagent blank. All the colored species are stable for 2 hours. The amount of drug (TDP) in a sample solution is obtained from the Beer s Lambert plot (Fig , P: 351). Method M5(a) Aliquots of standard TDP solution ( g/ml) are transferred into a series of 25ml calibrated tubes. Then 0.5ml (8.56 x 10-3 M) of MBT solution is added and kept aside for 5min. After that 2.0ml (1.58 x 10-2 M) of cerric ammonium sulphate is added and kept aside for 10min. The volume is made up to the mark with distilled water. The absorbance is measured at 630nm against a similar reagent blank. The amount of TDP is computed from its calibration graph. (Fig5.20, P: 352). 340

25 Chapter - V Trandolapril Method M11 Aliquots of standard TDP drug solution ( g/ml)) are delivered in to a series of 20ml calibrated tube. To each tube 5.0ml of AMV (1.80x 10-1 M) reagent and 0.5ml of 18 M 2 S 4 are added to each tube and the contents are heated for 20min in boiling water bath. After cooling the volume is made up to 20ml with distilled water. The resulting absorbance of the green color is measured at 760nm against a reagent blank. The amount of drug is computed from to appropriate calibration graph (Fig5.21, P: 352). Method M15 Into a series of 10ml calibrated tubes containing aliquots of standard TDP solution ( g/ml), 2.0ml of chloranilic acid (4.785 x 10-3 M) is added and kept aside for 30 min at lab temperature. The volume in each tube is made up to the mark with chloroform. The absorbance of the colored species is measured at 540nm against a reagent blank. The amount of the drug is calculated from Beer s law plot (Fig.5.22, P: 352). Method M17 Into a series of 50 ml separating funnels containing aliquots of drug ( μg/ml) solutions, 2 ml of p 9.8 buffer and 2.0 ml of 0.1% picric acid solutions are added successively. The total volume of aqueous phase in each separating funnel is adjusted to 15 ml with distilled water. To each separating funnel 10 ml of chloroform is added and the contents are shaken for 2 min. The two phases are allowed to separate and the absorbance of the separated chloroform layer is measured at 410 nm 341

26 Chapter - V Trandolapril against a reagent blank prepared under similar conditions. The amount of drug is deduced from the calibration graph (Fig.5.23, P: 352) 5.09(iv) ptimum Conditions The optimum conditions for the maximum color development and maximum stability of the proposed methods are established by varying the parameters one at a time, keeping the others fixed and observing the effect produced on the absorbance of the colored species. The optimum conditions thus established are incorporated in recommended procedures. Method - M1(a), M1(b), M1(c) and M1(d) The procedure to establish optimum conditions for these methods are as same as described in Chapter II of TT (Table2.08(a); P:97 ). Method - M2(a) and M2(b) The optimum conditions in these methods are fixed based on the study of the effects of various parameters such as type of acid for buffer, concentration of acid, concentration. of dye MB (M2(a)), SF- (M2(b)) or choice of organic solvent, ratio of organic phase to aqueous phase, shaking time, temperature, intensity and stability of the colored species in organic phase. The author performed controlled in pediments by measuring absorbance at max 655nm (M2(a) or 530nm (M2(b) of a series of solutions varying one and fixing the other parameter and the results are recorded in Table5.08 (a), P:

27 Chapter - V Trandolapril Method M5(a) The procedure to establish optimum conditions for the method are as same as described in Chapter II of TT (Table2.08(c) P: 99). Method M11 The method involves the reaction of TDP with AMV in acid medium. The effect of various parameters, such as concentration and volume of AMV, nature and strength of acid, order of addition of reagents, solvent for final dilution are studied and the optimum conditions developed and recorded in Table 5.08(b), P: 347. Method M15 The procedure to establish optimum conditions for this method are as same as described in Chapter II of TT (Table 2.08(k), P: 107). Method M17 The procedure to establish optimum conditions for this method are as same as described in Chapter II of TT (Table 2.08(m), P:109) Validation of the methods 5.10 (i) Linearity Studies Linearity plots are drawn for the proposed methods by taking concentration on x-axis and absorbance on y- axis and found to be passing linearly through the origin. Regression parameters such as slope, intercept and correlation coefficient, standard deviation on slope, standard deviation on intercept, limit of detection and limit of 343

28 Chapter - V Trandolapril quantification are evaluated and presented in Table 5.10(a)-Table 5.10(d) P: (ii) Precision The precision of each proposal methods is ascertained from the absorbance values obtained by actual determination of six replicates of a fixed amount of TDP in total solution. The percent relative standard deviation and percent range of error (at 0.05 confidence limits) are calculated for the proposed methods and are given in Table 5.11(a) -Table 5.11(c), P: (iii) Accuracy To determine the accuracy of each proposed method, different amounts of bulk samples of TDP within the Beer s law limits are analyzed by the proposed method. The percent of recovery and percent of relative standard deviation are evaluated. The results are incorporated in Table 5.12(a) -Table 5.12(j), P: (v) Limit of Detection and Limit of Quantification The LD and LQ are calculated from the standard deviation of intercept the slope of the linearity plot Table 5.10(a) -Table 5.10(d), P: Assay of pharmaceutical formulations Commercial formulations containing trandolapril are successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for the formulations are compared statistically with F-test and t-test and found to be not different significantly. Present of recoveries are determined by adding standard 344

29 Chapter - V Trandolapril drug to preanalysed formulations. The results of the recovery experiments by the proposed methods are also listed in Table5.13 (a) Table 5.13(e), P: Results and Discussions 5.12 (i) Spectral Characteristics In order to ascertain the optimum wavelength of maximum absorption ( max ) of the colored species formed, specified amount of TDP is taken and color is developed by following the above mentioned procedures individually. The absorption spectrum is scanned on a spectrophotometer in the wavelength region of 370nm to 900nm against similar reagent blank. The absorption curves (Fig Fig.5.13, P: ) of the colored species show characteristics absorption maxima (ii) ptical Characteristics In order to test whether the colored species formed in the above methods, adhere to Beer s law or not, the absorbances at appropriate wavelength of a set of solutions containing varying amounts of TDP and specified amounts of reagents (as given in the recommended procedures for each method) are recorded against the corresponding reagent blank for each method. The Beer s law plots of these systems are recorded against the corresponding reagent blanks and are represented graphically (Fig Fig5.23 P: ). Beer s law limits, molar absorptivity, Sandell s sensitivity and optimum photometric range are calculated and presented in Table 5.09(a)-Table 5.09(d), P:

30 Absorbance Absorbance Absorbance Absorbance Method M1(a):ARS+CCl 3 Method M1(b):BTB+CCl [TDP]=3.10x10-5 M [ARS]=3.66x10-4 M,[Cl]=4.0x10-2 M Blank Test [TDP]=6.19x10-5 M [BTB]=1.07x10-4 M Buffer P =3.5 Blank Test Wavelength nm Fig Absorption Spectrum of TDP with ARS methodm1(a) Wavelength nm Fig Absorption Spectrum of TDP with BTB methodm1(b) MethodM1(c):M+CCl 3 Method M1(d):TPooo+CCl [TDP]=7.74x10-5 M [M]=8.15x10-4 M,[Cl]=4.0x10-2 M [TDP]=1.55x10-5 M [TPooo]=3.8x10-4 M,[Cl]=4x10-2 M Blank Test Blank Test Wavelength nm Fig Absorption Spectrum of TDP with M methodm1(c) Wavelength nm Fig Absorption Spectrum of TDP with TPooo methodm1(d) 348

31 Absorbance Absorbance Absorbance Absorbance Method M2(a):MB+CCl3 Method M2(b):SF-+CCl [TDP]=2.67x10-5 M [MB]=4.17x10-4 M Buffer P =9.8 Blank Test [TDP]=1.55x10-5 M [SF-]=5.714x10-3 M Blank Test Wavelength nm Wavelength nm Fig Absorption Spectrum of TDP with MB method M2(a) Fig Absorption Spectrum of TDP with SAF- method M2(b) Method M5(a):MBT+Ce(IV) [TDP]=1.86x10-5 M [MBT]=1.71x10-4 M [Ce(IV)]=1.27x10-3 M Blank Test Method M11:AMV+ 2 S 4 [TDP]=1.16x10-5 M [AMV]=4.49x10-2 M [ 2 S 4 ]=5.42M Blank Test Wavelength nm Fig Absorption Spectrum of TDP with MBT+Ce(IV) method M5(a) Wavelength nm Fig Absorption Spectrum of TDP with AMV method M11 349

32 Absorbance Absorbance Absorbance Absorbance Method M15:p-CA+CCl 3 Method M17 :PA+CCl [TDP]=9.29x10-5 M [p-ca]=3.83x10-4 M Blank Test [TDP]=3.10x10-5 M [PA]=5.82x10-4 M,[Cl]=4.0x10-2 M Blank Test Wavelength nm Fig Absorption Spectrum of TDP with p-ca method M Wavelength nm Fig Absorption Spectrum of TDP with PA method M Method M1(a): ARS+CCl 3 [TDP]=1.55x10-5 M x10-5 M [ARS]=3.66x10-4 M [Cl]=4.0x10-2 M Method M1(b):BTB+CCl 3 [TDP]=3.10x10-5 M -1.86x10-4 M [BTB]=1.07x10-4 M Buffer P = Weight of the Drug in μg/ml Weight of the Drug in μg/ml Fig.5.14 Beer s Law Plot of TDP with ARS method M1(a) Fig.5.15 Beer s Law Plot of TDP with BTB method M1(b) 350

33 Absorbance Absorbance Absorbance Absorbance Method M1(c):M+CCl3 [TDP]=3.87x10-5 M x10-4 M [M]=8.15x10-4 M,[Cl]=4.0x10-2 M Method M1(d):TPooo+CCl 3 [TDP]=7.74x10-6 M x10-5 M [TPooo]=3.81x10-4 M [Cl]=4.0x10-2 M Weight of the drug in μg/ml Weight of the Drug in μg/ml Fig.5.16 Beer s Law Plot of TDP with M method M1(c) Fig.5.17 Beer s Law Plot of TDP with TPooo method M1(d) Method M2(a):MB+CCl 3 [TDP]=1.55x10-5 M x10-5 M [MB]=4.17x10-4 M Buffer of p = Method M2(b):SF -+CCl 3 [TDP]=7.74x10-6 M -4.65x10-5 M [SF-]=5.714x10-4 M [Cl]=4.0x10-2 M Weight of the Drug in μg/ml Weight of the Drug in μg/ml Fig.5.18 Beer s Law Plot of TDP with MB method M2(a) Fig.5.19 Beer s Law Plot of TDP with SF- method M 2(b) 351

34 Absorbance Absorbance Absorbance Absorbance Method M5(a):MBT+Ce(IV) [TDP]=9.29x10-6 M x10-5 M [MBT]=1.71x10-4 M [Ce(IV)]=1.27x10-3 M Method M11:AMV+ 2 S 4 [TDP]=5.81x10-6 M x10-5 M [AMV]=6.73x10-4 M,[ 2 S 4 ]=5.42M Weight of the Drug in μg/ml Fig.5.20 Beer s Law Plot of TDP with MBT method M5(a) Weight of the Drug in μg/ml Fig.5.21 Beer s Law Plot of TDP with AMV method M11 Method M15:p-CA+CCl 3 Method M17:PA+CCl [TDP]=1.55x10-5 M x10-5 M [p-ca]=9.57x10-4 M [TDP]=1.55x10-5 M-9.29x10-5 M [PA]=5.82x10-4 M,[Cl]=4.0x10-4 M Weight of the Drug in μg/ml Weight of the Drug in μg/ml Fig.5.22 Beer s Law Plot of TDP with p- CA method M15 Fig.5.23 Beer s Law Plot of TDP with PA method M17 352

35 % of Transmitance % of Transmitance % of Transmitance % of Transmitance 0 Method M1(a):ARS+CCl 3 0 Method M1(b):BTB+CCl LG(Concentration in μg/ml) Fig.5.24 Ringbom Plot of TDP with ARS method M1(a) LG(Concentration inμg/ml) Fig.5.25 Ringbom Plot of TDP with BTB method M1(b) 0 Method M1(c):M+CCl 3 0 MethodM1(d):TPooo+CCl LG(Concentration in μg/ml) Fig.5.26 Ringbom Plot of TDP with M method M1(c) LG(Concentration in μg/ml) Fig.5.27 Ringbom Plot of TDP with TPooo method M1(d) 353

36 %of Transmitance % of Transmitance % of Transmitance % of Transmitance 25 Method M2(a):MB+CCl 3 0 MethodM2(b):SF-+CCl LG(Concentration in μg/ml ) Fig.5.28 Ringbom Plot of TDP with MB method M2(a) LG(Concentration in μg/ml) Fig.5.29 Ringbom Plot of TDP with SF- method M2(b) 0 Method M5(a):MBT+Ce(IV) 0 Method M11:AMV+ 2 S LG(Concentration in μg/ml) Fig.5.30 Ringbom Plot of TDP with MBT+Ce(IV) method M5(a) LG(Concentration in μg/ml) Fig.5.31 Ringbom Plot of TDP with AMV method M11 354

37 % of Transmitance %of Transmitance 0 MethodM15:p-CA+CCl LG(Concentration in μg/ml) Fig.5.32 Ringbom Plot of TDP with p-ca method M15 0 Method M17:PA+CCl LG(Concentration in μg/ml) Fig.5.33 Ringbom Plot of TDP with PA method M17 355

38 Table 5.09(a): ptical characteristics of the proposed methods for TDP S.o ame of the Parameter M1(a) M1(b) M1(c) 1 Maximum Wavelength λ max 435 nm 420 nm 420 nm 2 Beer's Law Limits µg/ml ptimum Photometric Range µg/ml Sandell's Sensitivity(µg/cm 2 / Absorbance) 7.84E E E-01 5 Molar Absorptivity lt/mole/cm 5.95E E E+03 Table 5.09(b): ptical characteristics of the proposed methods for TDP S.o ame of the Parameter M1(d) M2(a) M2(b) 1 Maximum Wavelength λ max 510 nm 650 nm 500 nm 2 Beer's Law Limits µg/ml ptimum Photometric Range µg/ml Sandell's Sensitivity(µg/cm 2 / Absorbance) 3.55E E E-02 5 Molar Absorptivity lt/mole/cm 1.08E E E+04 Table 5.09(c): ptical characteristics of the proposed methods for TDP S.o ame of the Parameter M5(a) M11 M15 1 Maximum Wavelength λ max 630 nm 760nm 540 nm 2 Beer's Law Limits µg/ml ptimum Photometric Range µg/ml Sandell's Sensitivity(µg/cm 2 / Absorbance) 6.06E E E-02 5 Molar Absorptivity lt/mole/cm 7.63E E E+03 Table5.09 (d): ptical characteristics of the proposed methods for TDP S.o ame of the Parameter M17 1 Maximum Wavelength λ max 410 nm 2 Beer's Law Limits µg/ml ptimum Photometric Range µg/ml Sandell's Sensitivity(µg/cm 2 / Absorbance) 5.56E-02 5 Molar Absorptivity lt/mole/cm 7.83E

39 Table 5.10(a): Regression characteristics of the proposed methods for TDP S.o ame of the Parameter M1(a) M1(b) M1(c) 1 Slope (b) 1.38E E E-03 2 Intercept(a) 3.13E E E-03 3 Standard Deviation on Slope(S b ) 2.12E E E-05 4 Standard Deviation on Intercept(S a ) 5.50E E E-03 5 Correlation Coefficient ( r ) Limit of Detection (LD) µg/ml Limit of Quantification (LQ) µg/ml Table 5.10(b): Regression characteristics of the proposed methods for TDP S.o ame of the Parameter M1(d) M2(a) M2(b) 1 Slope (b) 2.51E E E-02 2 Intercept(a) 1.35E E E-03 3 Standard Deviation on Slope(S b ) 4.36E E E-04 4 Standard Deviation on Intercept(S a ) 5.66E E E-03 5 Correlation Coefficient ( r ) Limit of Detection (LD) µg/ml Limit of Quantification (LQ) µg/ml Table 5.10(c): Regression characteristics of the proposed methods for TDP S.o ame of the Parameter M5(a) M11 M15 1 Slope (b) 1.77E E E-02 2 Intercept(a) -2.67E E E-02 3 Standard Deviation on Slope(S b ) 1.11E E E-04 4 Standard Deviation on Intercept(S a ) 1.73E E E-03 5 Correlation Coefficient ( r ) Limit of Detection (LD) µg/ml Limit of Quantification (LQ) µg/ml

40 Table 5.10(d): Regression characteristics of the proposed methods for TDP S.o ame of the Parameter M17 1 Slope (b) 1.82E-02 2 Intercept(a) -2.33E-03 3 Standard Deviation on Slope(S b ) 1.98E-04 4 Standard Deviation on Intercept(S a ) 5.14E-03 5 Correlation Coefficient ( r ) Limit of Detection (LD) µg/ml Limit of Quantification (LQ) µg/ml Table 5.11(a): Precision of the proposed methods for TDP S.o. ame of the Parameter M1(a) M1(b) M1(c) 1 Amount Taken(μg/ml) Amount Found (μg/ml) Standard Deviation (S) %Relative Standard Deviation %Recovery Table 5.11 (b): Precision of the proposed methods for TDP S.o. ame of the Parameter M1(d) M2(a) M2(b) 1 Amount Taken(μg/ml) Amount Found (μg/ml) Standard Deviation (S) %Relative Standard Deviation %Recovery Table 5.11 (c): Precision of the proposed methods for TDP S.o. ame of the parameter M5(a) M11 M15 M17 1 Amount Taken(μg/ml) Amount Found (μg/ml) Standard Deviation (S) %Relative Standard Deviation %Recovery

41 Table-5.12(a): Accuracy of the proposed Method M1(a) Sample ID Amount Taken µg/ml Amount Found µg/ml Percent of Recovery Statistical analysis Mean SD %RSD Mean SD %RSD Mean SD %RSD Table-5.12(b): Accuracy of the proposed Method M1(b) Sample ID Amount Taken µg/ml Amount Found µg/ml Percent of Recovery Statistical analysis Mean SD %RSD Mean SD %RSD Mean SD %RSD

42 Table-5.12(c): Accuracy of the proposed Method M1(c) Sample ID Amount Taken µg/ml Amount Found µg/ml Percent of Recovery Statistical analysis Mean SD %RSD Mean SD %RSD Mean SD %RSD Table-5.12(d): Accuracy of the proposed Method M1(d) Sample ID Amount Taken µg/ml Amount Found µg/ml Percent of Recovery Statistical analysis Mean SD %RSD Mean SD %RSD Mean SD %RSD

43 Table-5.12(e): Accuracy of the proposed Method M2(a) Sample ID Amount Taken µg/ml Amount Found µg/ml Percent of Recovery Statistical analysis Mean SD %RSD Mean SD %RSD Mean SD %RSD Table-5.12(f): Accuracy of the proposed Method M2(b) Sample ID Amount Taken µg/ml Amount Found µg/ml Percent of Recovery Statistical analysis Mean SD %RSD Mean SD %RSD Mean SD %RSD

44 Table-5.12(g): Accuracy of the proposed Method M5(a) Sample ID Amount Taken µg/ml Amount Found µg/ml Percent of Recovery Statistical analysis Mean SD %RSD Mean SD %RSD Mean SD %RSD Table-5.12(h): Accuracy of the proposed Method M10 Sample ID Amount Taken µg/ml Amount Found µg/ml Percent of Recovery Statistical analysis Mean SD %RSD Mean SD %RSD Mean SD %RSD

45 Table-5.12(i): Accuracy of the proposed Method M15 Sample ID Amount Taken µg/ml Amount Found µg/ml Percent of Recovery Statistical analysis Mean SD %RSD Mean SD %RSD Mean SD %RSD Table-5.12(j): Accuracy of the proposed Method M17 Sample ID Amount Taken µg/ml Amount Found µg/ml Percent of Recovery Statistical analysis Mean SD %RSD Mean SD %RSD Mean SD %RSD

46 Table5.13 (a): Assay of Formulations of Trandolapril Reference Method [303] Amount Amount found in Percent of Recovery Sample Taken proposed methods* Ref.Method Proposed methods** (mg/tablet) M1(a) M1(b) M1(a) M1(b) Zetpril 2 Mean %REC SD ±0.014 ±0.028 %RSD ±1.69 ±0.680 ±1.419 F-test t-test Mavik 2 Mean %REC SD ±0.052 ±0.042 %RSD ±1.20 ±2.608 ±2.587 F-test t-test Mavik 4 Mean %REC SD ±0.047 ±0.035 %RSD ±1.69 ±1.182 ±0.883 F-test t-test Tarka 4 Mean %REC SD ±0.036 ±0.029 %RSD ±1.20 ±1.151 ±0.731 F-test t-test *Average of six determinations are considered, AVG=Average, SD=Standard deviation, F=F-test value, t=t-test value; Theoretical values at 0.05 level of confidence limit F=5.05, t= **%REC=% of Recovery, %RSD=%of Relative standard deviation; Recovery studies, 2.0 and 4.0mg added to the preanalyzed formulations (Average of six determinations) 364