Development of Sensitive Spectrophotometric Method for Analysis of Darifenacin Hydrobromide Liposomes in Rat Plasma

 

N. Raghavendra Babu*, Y. Padmavathi, S. Priyanka, P. Ravi Kumar

Department of Pharmaceutical Analysis and Quality Assurance,

G. Pulla Reddy College of Pharmacy, Osmania University, Mehdipatnam, Hyderabad. Telangana, India.

*Corresponding Author E-mail: nayakaraghavendrababu@gmail.com

 

ABSTRACT:

A new simple and sensitive visible spectrophotometric method for the estimation of darifenacin hydrobromide (DHB) liposomes was developed using diazo coupling reagent, 3-amino phenol in spiked rat plasma. Spectrophotometric method was developed using diazo coupling reaction with the chromogenic reagent, 3-amino phenol for estimation of darifenacin hydrobromide in liposomes. 3-amino phenol forms a yellow colored diazo complex with darifenacin hydrobromide in acidic conditions. The color intensity range was found to be linear in the concentration range of 100-1000ng/mL. Analysis was carried out at its λmax 438nm. The method was validated according to ICH guidelines. The method was optimized and validated in spiked rat plasma according to USFDA guidelines. The developed method was simple and sensitive and the LOQ value was found to be 100ng/mL. All the validation parameters were found to be within the acceptable limits. As the method was sensitive, it was successfully applied to the analysis of darifenacin hydrobromide liposomes in spiked rat plasma.

 

KEYWORDS: 3-amino phenol, darifenacin hydrobromide, ICH guidelines and USFDA guidelines.

 


INTRODUCTION:

Darifenacin hydrobromide1-3 is a medication used to treat urinary incontinence. It works by blocking the M3 muscarinic acetylcholine receptor, which is primarily responsible for urinary bladder muscle contractions. It decreases the urgency to urinate. It should not be used in the people with urinary retention. The chemical name of darifenacin hydrobromide is (S)-2-{1-[2-(2, 3-dihydrobenzofuran-5-yl) ethyl]-3-pyrrolidinyl}-2, 2-diphenylacetamidehydrobromide. Its empirical formula is C28H30N2O2.HBr representing molecular weight of 507.5. It is white to almost white monomorphic crystalline solid, soluble in water (<1mg/mL at 25˚C), chloroform (117mg/mL at 25˚C) and methanol and ethanol (<1mg/mL at 25˚C) and its melting point is 228-230˚C.

 

In the present study, 4-6 when darifenacin hydrobromide reacts with sodium nitrite at 0-5oC, it forms a diazonium salt which is highly reactive and is readily attacked by 3-amino phenol. 3-amino phenol reacts with this highly reactive diazotized compound and leads to the formation of a colored complex which is measured at 438nm.The scheme of reaction between drug and the colorimetric reagent is shown in figure1.

 

MATERIALS AND METHODS:

Chemicals and reagents

Darifenacin hydrobromide7-10 working standard was generous gift from Aurobindo Pharmaceuticals Pvt. Ltd., Hyderabad. 3aminophenol, Sodium nitrite, Hydrochloric acid (Analytical reagent grade) were obtained from Sd fine chemicals limited (SDFCL), Mumbai. Methanol was obtained from Merck specialties private limited, Mumbai. Darilong 7.5mg extended release tablets (Darifenacin Hydrobromide), were manufactured by Sun Pharma laboratories ltd, Jammu, and were purchased from local market.

 

 

Instruments

UV-visible spectrophotometer (Shimadzu 1800), pH meter (Elico LI-127), Digital balance (Shimadzu BL- 220H).Ultrasonic bath sonicator (PCI Analytics 6.5li200H), Refrigerated Centrifuge (EltekRC4100F), Hot Air Oven (Tempo Equipment Private Limited).

 

Experimental animals:                                               

Male Albino Wistar rats (180-200 g) were obtained from National Institute of Nutrition, Hyderabad.

 

Method development:

Preparation of 3-amino phenol (0.5%w/v): Dissolve 125 mg of 3-amino phenol (Sd fine chemicals limited (SDFCL), Mumbai. in 25 mL of distilled water.

 

Preparation of 0.1% sodium nitrite: Dissolve 100 mg of sodium nitrite (Sd fine chemicals limited (SDFCL), Mumbai in 100 mL of distilled water.

 

Preparation of stock solutions: Standard darifenacin hydrobromide, 100mg was weighed and transferred to 100mL volumetric flask and dissolved in 0.1N HCl. The flask was shaken and was made up to the mark with 100mL 0.1N HCl to give a solution containing 1000µg/mL solution. From this stock solution 1mL was pipette out into another 100mL volumetric flask and the volume was made up to 100mL to give 100µg/mL.

 

The method was developed based on diazotization coupling reaction between Darifenacin hydro bromide and 3 amino phenol.

 


 

The step by step reaction scheme is as follows:

 

Figure1 Proposed reaction pathway between Darifenacin hydrobromide and 3-aminophenol

 


 

 

Plasma sample preparation:

The blood samples were collected from retro-orbital puncture into disodium EDTA vials (20mg disodium EDTA in 1mL water, 1mL of blood requires 50µL of disodium EDTA). Plasma was separated from blood samples by centrifugation at 1400 rpm for 7 minutes. After centrifugation, plasma layer gets separated and it is collected and stored at -200C for further use.

 

Extraction procedure:

·      A pool of blank rat plasma was obtained.

·      100μL of plasma sample in was spiked with appropriate volume of the stock solution and 400μL of methanol was added.

·      The mixture was vortexed for 1 minute.

·      Centrifuged at 3000 rpm for 12minutes at 40C.

·      The organic layer was separated & filtered through a 0.2μm cellulose acetate filter.

·      The organic layer was evaporated on a Savant vacuum evaporator at about 60°C. The residue was reconstituted in 10mL of solvent and used for the measurement of the absorbance at the absorption maxima of three drugs.

 

Determination of Absorption maximum:

0.2 ml 0f drug solution was taken in a 10mL volumetric flask to which 1mL of sodium nitrite (0.1%w/v) was added. This reaction was carried out by keeping the flask in an ice tray. To this reaction mixture 1mL of 3-amino phenol (0.5%) was added and the volume was made up to 10mL with distilled water to give a solution of 2µg/mL solution. The absorbance of resulting coloured solution was measured against respective blank solution in visible region i.e., 400-800 nm which shows a maximum absorbance at 438nm and the absorption spectrum is shown in figure 2.

 

Figure 2  Spectrum of Darifenacin hydrobromide and 3-aminophenol reagent.

 

Construction of Calibration curve in rat plasma:

5 µg/mL darefenacin hydrobromide solution was prepared by transferring 0.5mL of 100 ug/mL stock solution into a 10mL volumetric flask followed by the addition of 1mL of 0.1% sodium nitrite and 1mL of 0.5% 3-amino phenol. From this, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mL were transferred to ependroff tubes containing  100 µL of plasma and 400 µL of methanol to get 100, 200, 400, 600, 800, 1000ng/mL and these were centrifuged at 3000 rpm for 12min. The  supernatant organic layer was collected and made up to the volume to 5 mL with distilled water and the absorbance of these standard solutions was measured at 438nm  in a UV-Visible spectrophotometer against blank plasma treated in the same fashion as standard solutions. The calibration curve was constructed between concentration of drug and absorbance and shown in  figure 3 and calibration curve data is given in table1.

                             

Table 1 Calibration curve data

S.No

Concentration (ng/mL)

Absorbance*

1

100

0.088

2

200

0.181

3

400

0.379

4

600

0.561

5

800

0.770

6

1000

0.958

*Average of three determinations


Figure 3 Calibration curve of darifenacin hydrobromide with 3-amino phenol Reagent in rat plasma

 

Liposomes preparation:

Liposomes are prepared by conventional thin film method. Lipid (soya lecithin) and drug solution (Darifenacin hydrobromide 1 mg/mL) are dissolved in methanol: chloroform (1:2 v/v). This mixture is dried to a thin film by slowly reducing the pressure from 500 to 1 mbar at 40˚C and 80 rpm using rotary flash evaporator. The film is kept under vacuum (1 mbar) for overnight at room temperature and subsequently flushed with nitrogen. Then, the film deposited is hydrated with PBS (pH 7.4) by rotation for 2 hrs at room temperature. The obtained vesicles are ultrasonicated for size reduction using sonicator for 3 mins. Blank or placebo is prepared in the same manner omitting the drug solution.

 

Quantitative analysis of liposomes of darifenacin hydrobromide

Assay of liposomes of darifenacin hydrobromide was carried out. A sample solution was prepared in triplicate. From the 5µg/mL solution of liposomes, 0.8mL was pipette into 10 mL graduated tube followed by the addition of each 1mL of sodium nitrite (0.1%) and 3-amino phenol (0.5%) and volume was made up to the mark with distilled water. This gives 400ng/mL sample solution. Absorbance of this concentration was measured against blank omitting the drug solution and % purity was calculated and reported in table 2.

 

Table 2 Assay data of darifenacin hydrobromide liposomes

S. No

Concentration (ng/mL)

Absorbance

Assay (%)

Mean*

1

400

0.374

98.2

 

98.6

2

400

0.382

98.7

3

400

0.377

99.1

*Average of three determinations

 

Sample preparation in spiked rat plasma.

The separated organic layer was taken from processed blank plasma and after filtration its absorbance was measured directly without spiking it with either sample or solvent. Standard solution of darifenacin hydrobromide liposomes 400ng/mL spiked in plasma was prepared and absorbance was measured. Blank plasma absorbance was also recorded. The spectrum of blank plasma and sample spiked in plasma are presented in the fig 4 and fig 5.

 

Figure 4 Absorption spectrum of blank plasma

 

Figure 5 Absorption spectrum of darifenacin liposomal solution

 

Fig 6 Overlay spectrum of darifenacin hydrobromide liposomal solution spiked in rat plasma (100-1000ng/mL)

 

Bio analytical method validation11-14

All analytical procedures were validated according to USFDA guidelines 'Bioanalytical Method Validation'.

 

Sensitivity (LLOQ)

It is the lower limit of quantification (LLOQ).The lowest concentration of the calibration curve where the analyte response was more than five times the blank response was selected as LLOQ. At this concentration absorbance was reproducible with defined accuracy and precision.

LLOQ is the least concentration in the calibration curve which is 100ng/mL.

 

Acceptance criteria

The analyte response at the LLOQ was more than five times the blank response.

 

Linearity                                                   

Preparation of Quality control (QC) standards in plasma

Calibration samples were prepared by spiking with appropriate amounts of sample & organic solvent (methanol) into 100μL of control plasma. From each concentration 10μL of sample and 400μl of methanol were taken and spiked into 100μL control plasma to get 100, 200,400, 600, 800, 1000ng/mL.

 

Linear regression analysis was performed for checking the linearity of the data obtained. The response of the drug was found to be linear in the concentration range 100ng-1000ng/mL.

 

The linear regression equation for darifenacin hydrobromide was y=0.00102x-0.020 with r2 0.993.

The calibration curve data is shown in table 3 and calibration curve is shown in fig. 7.

Table 3 Calibration curve data

S. No

Concentration (ng/ml)

Absorbance *

1

100

0.070

2

200

0.217

3

400

0.414

4

600

0.517

6

800

0.743

6

1200

0.906

 

 

Fig 7 Calibration graph of darifenacin HBr liposomal solution with plasma

Acceptance criteria

Coefficient of determination should not be less than 0.999.

 

Accuracy

Accuracy was determined by spiking with appropriate amounts of sample into 100μL of control plasma. From 5 μg/mL stock solution, 320, 400 and 480μL samples were taken. To these, 400μL of methanol was added and these solutions are spiked into 100μL control plasma to get 320, 400 and 4800ng/mL. 320ng/mL- LQC, 400ng/mL –MQC and 480ng/mL-HQC The three standard solutions each five times was measured spectrophotometrically. Recovery determined should be within +15%.

 

Accuracy of the method was determined at three different concentration levels. Mean and % RSD values were calculated and are shown in Table 4. The results indicate that the method was accurate.

 

Acceptance criteria

% RSD for analyte concentration in five replicate standard samples should not be more

than 15.0.

 

Recovery

Recovery was determined at LQC (320ng/mL), MQC (400ng/mL) and HQC (480ng/mL) levels. Concentrations were prepared by spiking 320, 400 and 480μL of darifenacin hydrobromide liposomal solution into 100μL of control plasma and 400μL of organic solvent. The obtained results were reported in table 5.

 

Table 4. Accuracy data of darifenacin hydrobromide liposomal solution in plasma

S.No

Accuracy level

Concentration

(ng/mL)

Absorbance

Mean*±SD

(n = 3)

%RSD

1

80%

320

0.371

0.335± 0.004

0.9

0.376

0.374

2

100%

400

0.462

0.398± 0.00360

1.1

0.459

0.460

3

120%

480

0.560

0.495± 0.0060

1.2

0.556

0.554

*Average of three determinations

 

Table 5 Recovery data of darifenacin hydrobromide tablets and liposomes

Formulation

Sample

(ng/mL)

Pure

(ng/mL)

Amt of plasma added (µL)

Amt of methanol added (µL)

Total amount added(ng/mL)

Total amount recovered (ng/mL)

Recovered pure*+ SD

Darilong 7.5mg

100

100

100

400

200

192.15

96.07+

0.0026

 

100

400

100

400

500

492.15

98.43+

0.0035

 

100

800

100

400

900

878.43

96.60+

0.701

Darifenacin HBr liposomes

100

100

100

400

200

186.74

 

92.86+

0.0024

 

100

400

100

400

500

461

89.43+

0.0027

 

100

800

100

400

900

778.4

85.6+

0.508

*Average of five determinations

 

 


Acceptance criteria

% Recovery should be between 85% to 105%.

 

Precision

Samples were prepared by spiking with appropriate amounts of sample into 100μL of control plasma. From 5 μg/mL stock solution, 600μL, 800μL and 1000μL samples were taken. To these, 400μL of methanol was added and these solutions are spiked into 100μL control plasma to get 600, 800 and 1000ng/mL. 600ng/mL- LQC, 800ng/mL –MQC and 1000ng/mL-HQC.

 

Variations of results within the same day (intraday) and variation of results between days (inter day) were analyzed.

 

Repeatability

Repeatability assessment of an analytical method is performed by analyzing six replicates of single concentration that is 600ng/mL. Absorbances of samples were recorded at 438nm. The % relative standard deviation (RSD) was calculated. The results obtained were shown in table 6.

 

Table 6 Repeatability data

S. No

Concentration  (ng/mL)

Parameter

Absorbance *

% RSD

1

600

0.517

 

 

1.2

2

600

0.521

3

600

0.524

4

600

0.518

5

600

0.513

6

600

0.521

*Average of six determinations

 

Intraday precision

The intra-assay precision of the proposed method was determined on samples of drug solutions at varying concentration levels (LQC, MQC and HQC) by analyzing five replicates of each sample as a batch in a single assay run on the same day. The results obtained were shown in table 7.

 

 

Inter day precision

The Inter-assay precision was determined by analyzing the same samples on the next two, days and %RSD was calculated. The results obtained were shown in table 8.

 

 Table 7 Intra-day precision data

S. No

Concentration (ng/mL)

Mean absorbance*

Mean± Standard deviation*

%RSD

Morning

Afternoon

1

LLOQ (100)

0.078

0.092

0.00121±0.0018

1.8

2

LQC (600)

0.565

0.577

0.5651±0.0011

1.1

3

MQC (800)

0.774

0.785

0.7663±0.00026

1.2

4

HQC (1000)

0.963

0.976

0.971±0.0025

1.6

*Average of six determinations

 

 

Table 8 Inter-day precision data

S. No

Concentration

(ng/mL)

Mean absorbance*

Mean± Standard deviation*

%RSD

Day 1

Day 2

1

LLOQ (100)

0.070

0.074

0.0721±0.001

1.2

2

LQC (600)

0.562

0.568

0.5657±0.0013

1.11

3

MQC (800)

0.780

0.774

0.7801±0.0038

1.83

4

HQC (1000)

0.964

0.957

0.964±0.0025

1.48

*Average of five determinations

 


Acceptance criteria

The % RSD for sample absorbances should not be more than 15%.

 

Quantitative analysis of liposomes of darifenacin hydrobromide in rat plasma

From the 1000 µg/mL solution of liposomes, 100 µg/mL solution was prepared. From this, 5 µg/mL solution was prepared followed by the addition of 0.1% sodium nitrite and 0.5% 3-amino phenol. From this, 0.8mL was transferred to ependroff tube to which 100 µL of plasma and 400 µL were added and centrifuged at 3000 rpm for 12min. The organic layer was collected and made up to the volume to 5 mL with suitable solvent and the absorbance of this concentration was measured spectrophotometrically. The obtained results were reported in table 9.

 

Table 9 Assay data of darifenacin hydrobromide liposomes in spiked rat plasma

S. No

Concentration (ng/mL)

Absorbance

Assay (%)

Mean*

1

800

0.749

99.8

 

99.16

2

800

0.755

99.2

3

800

0.751

98.5

*Average of three determinations

 

CONCLUSION:

A new simple, sensitive visible spectrophotometric method has been developed for the estimation of darifenacin hydrobromide liposomes using diazo coupling reagent, 3-amino phenol in spiked rat plasma. The method was developed using diazo coupling reaction with the chromogenic reagent, 3-amino phenol for estimation of darifenacin hydrobromide in liposomes. Darifenacin after reaction with sodium nitrite under acidic conditions formed a highly reactive intermediate which upon reaction with 3-amino phenol forms a yellow colored diazo complex in acidic conditions. The color intensity range was found to be linear in the concentration range of 100-1000ng/mL. Analysis was carried out at its λmax 438nm. Bioanalytical method validation for darifenacin hydrobromide liposomes in spiked rat plasma has been performed according to USFDA guidelines

 

Method was successfully applied to the analysis of darifenacin hydrobromide liposomes in spiked rat plasma. Visible spectrophotometric methods are commonly used for analysis of drugs in pure form and formulations. Sensitivity of visible spectrophotometry is high when compared to UV and chromatographic methods. The method in the present study was developed using simple chromogenic reagent 3-amino phenol in spiked rat plasma. As the sensitivity of method was high, this method can be applied for pharmacokinetic studies of newly developed formulations of darifenacin hydrobromide.

 

REFERENCES:

1.     H P. Rang, M M Dale, J M. Ritter, R. J. Flower, G Henderson. Rang and Dale’s Pharmacolology. 7th edition. Spain: Elsevier; 2012.

2.     Material Safety Data Sheet of darifenacin hydrobromide.p.1-9. Available from: Datasheets.scbt.com/sc-204708.pdf.

3.     Product block of darifenacin hydrobromide. Available from: www.scbt.jp/datasheet-204708-darifenacin-hydrobromide.html

4.     The Merck Index. 14th edition. USA: Merck Research Laboratories; 2006. 477p.

5.     Remington The science and Practice of Pharmacy. Volume-II. 21st edition. New Delhi: Wolters Kluwer India Pvt ltd; 2006.

6.     Burgess C, Knowles A, Practical Absorption Spectrometry Techniques in visible and Ultra Violet Spectrometry. Volume-3. London: Chapman and Hall;1984.

7.     Micheal E, Schartz IS Krull. Analytical method development and validation Japan: Elsevier Sciences; 2001.

8.     ICH, Q2 (R1) Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH harmonized Tripartite Guideline, Nov 2005.

9.     International Conference on Harmonization, “Q2A: Text on Validation of Analytical Procedures”, Federal Register 60(40), 11260-11262 (1995).

10.   International Conference on Harmonization, “Q2B: Validation of Analytical procedures: Methodology, 1997; 62 (96), 27463-27467.

11.   Bioanalytical Method Validation, Guidance for Industry, United states Food and Drug Administration, May 2001.

12.   Sathish NK, Pradeep V, UV Spectrophotometric determination of darifenacin hydrobromide in bulk and pharmaceutical dosage forms. Der Pharmacia Sinica. 2011; 2(2): 169-76.

13.   Sai Praveen P, Jagathi V, Devala Rao G, Sudhakar G. Visible Spectrometric method for the determination of darifenacin. Research J Pharm Bio Chem Sci. 2010; 1(2): 254-56.

14.   Sridharan D, Umarani L, Thenmozhi A, Pavan kumar, Aswani Dutt C, Venkata Ramanaiah M, Phanikishore Y, Development and Validation of UV spectrophotometric method of darifenacin hydrobromide in bulk and tablet dosage form. Asian J Pharm Ana. 2011; 1(3): 43-45.

 

 

 

Received on 13.01.2017       Accepted on 13.02.2017     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2017; 7(1): 41-47.

DOI:  10.5958/2231-5675.2017.00008.4