Spectrophotometric determination of lamuvidine using acidic ... - NOPR

Indian Journal of Chemical Technology Vol. 18, November 2011, pp. 431-438

Spectrophotometric determination of lamuvidine using acidic dye and coupling reagent M B Rahul Reddy, B M Gurupadayya * & T Anil Kumar Department of Pharmaceutical Analysis, JSS College of Pharmacy, JSS University, Mysore 570 015, India Received 5 October 2010 ; accepted 15 November 2011 Two simple, extraction-free spectrophotometric methods (Method-I and Method-II) for the quantitative estimation of lamuvidine (LMV) in bulk drug and pharmaceutical formulations (tablets) have been developed. First method is based on the interaction of LMV with 0.1% methanolic solution of bromophenol blue to form a stable, red-colored, ion-pair complex showing the peak at 595 nm. Second method is based on the oxidation followed by coupling of 3-methyl-2-benzothiazolinone hydrazone with LMV in the presence of ferric chloride to form green-colored chromogen exhibiting absorption maximum at 659 nm. Both the methods follow Beer’s law in the concentration range 1-8 µg mL-1. Linear relationships with good correlation coefficients (0.991-0.9967) are found between absorbance and corresponding concentrations of the drug. The reliability and performance of the proposed methods are validated statistically, the recovery ranges from 99.4 ± 0.1% to 99.9 ± 0.3%. The results of analysis for the two methods have been validated statistically and by recovery studies. Keywords: 3-methyl-2-benzothiazolinone , Acidic dye, Bromophenol blue, Lamivudine, hydrazone, Spectrophotometric study

Lamivudine (LMV), chemically (2R-cis)-4-amino-1(2-hydroxymethyl)-1,3-oxathiolan-5-yl-2-(1H)-pyrithiacytidine, is the active ingredient of many pharmaceutical preparations concerned with the treatment of human immune deficiency virus that causes AIDS1-3. It is also active against hepatitis B virus. LMV is official in Martindale, the Extra pharmacopoeia. Literature survey revealed that few methods are available for the assay of LMV and most of the methods found in the literature are applicable to the determination of the drug in body fluid. High performance liquids chromatography is the most widely used technique and has been applied for the determination of LMV in human plasma4-10, rabbit plasma11, cerebrospinal fluid12, human serum13, blood cells14 and also for the determination of drug metabolites in urine15,16. LMV in human serum has also been determined by capillary electrophoresis17,18. There are also reports on the development of methods using chloramines T19, para dimethyl cinnamlehyde20, para dimethyl benzaldehyde21, potassium permanganate22, N-bromosuccinamide23, phloroglucinol24, potassium bromated and bromide mixture25 reagents for the estimation of LMV in pharmaceuticals. Some of the reported methods suffer from the use of unstable —————— * Corresponding author. E-mail: [email protected]

reagents, expensive chemicals and liquid liquid extraction22,23(Tables 1 and 2). Some combination methods developed for drug in combination with stavudine have been assayed by HPTLC26 and with abacavir27, zidovudine28 by reverse phase HPLC in tablet dosage forms. In addition, LMV is also simultaneously estimated along with stavudine and nevirapine by reverse phase HPLC method, developed in tablet dosage forms29. The aim of the present investigation was to develop a simple and sensitive visible spectrophotometric method for the quantitation of LMV in pure drug and in pharmaceutical formulations. The method uses the well-known oxidation followed by coupling of 3-methyl-2benzothiazolinone hydrazone (MBTH) with LMV in the presence of ferric chloride to form green-colored chromogen exhibiting absorption maximum at 659 nm and the interaction of LMV with 0.1% methanolic solution of bromophenol blue (BPB) to form a stable, red-colored, ion-pair complex peaking at 595 nm. The structure of lamivudine is represented in Fig. 1. Experimental Procedure Apparatus

A UV/VIS spectrophotometer (1800, Pharmaspec, Shimadzu, Japan) with 1.0 cm matched quartz cells was used for absorbance measurements.

INDIAN J. CHEM. TECHNOL., NOVEMBER 2011

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Table 1—Comparison of reported HPLC methods with present method Column

Mobile phase

Linear range, µg mL-1

Remarks

Ref.

Phenyl -

-

10-5000ng/mL -

7 8

Octylsilane

Phosphate buffer-CH3CN

0.6-17.6

C-18

Methanol-Water

406.10-4020.05 ng mL-1

Hypersil BDS C-18

Triethyl amine buffer

25–2000 ng mL-1

BDS Hypersil C-18

MeOH-CH3COONH4

0.5-100

Applicable to human plasma Applicable to seminal human plasma, tandem mass spectrometric detection Applicable to human plasma less accurate (RE, 0.1-11%) Applicable to human plasma; involves protein precipitation prior to separation Applicable to rabbit plasma and run time was 15 min Applicable to metabolite in human urine; less precise (RSD 4.7-22.4%)

9 10 11 16

Table 2—Comparison of reported spectrophotometric methods with present method Linear range, µg mL-1 Remarks (E, l mol-1 cm-1)

Reagent

λmax nm

Chloramine-T, Methyl orange Chloramine-T, Indigo carmine P-Dimethylamino Cinnamaldehyde (PDCA) P-Dimethylamino Benzaldehyde (PDAB) Vanillin KMnO4- fast green FCF

520 610 496 476 474 640

NaIO4-MBTH

620

Iron (III)- Ferricyanide

740

NBS-Celestine Blue

540

Cobalt-thiocyanate

610

Ammonium Molybdate

700

NaNO2-Phloroglucinol

445

0.1-1.0 0.25-3.5 2-10 2-10 2-10 1-8 (1.28 × 104) 0.6-6.0 (2.02 × 104) 9.0-75.0 (1.24 × 103) 0.7-6 (1.6 × 104) 1.5 - 15 (7.7 × 103) 11-150 (1.0 × 103) 2-10

Folin-ciocalteu reagent Iron (III)-phenanthroline

684 472

2-10 3-15

-

2.5-7.5 mg

Titrimetric method and less accurate

BrO-3- Br-1 Methyl Orange

520 610

Method is tedious and time consuming Method is tedious and time consuming

25

BrO-3- Br-1 Indigocarmine

0.125-1.5 (7.4 × 104) 1.0-8.0 (1.7 × 104)

Bromophenol Blue

595

1-8 (6.75 ×103)

No heating/extraction; use no expensive chemicals; highly sensitive method

Present method

MBTH

659

1-8 (2.86 × 103)

No heating/extraction; use no expensive chemicals; highly Sensnsitive method

Present method

Titration with potassium bromate and Bromide

Ref.

Extraction procedure Extraction procedure Less sensitive Narrow absorption range Narrow absorption range Uses an oxidant, which is unstable in solution -

19 19 20 21 21 22

Uses an expensive chemical

22

Uses an unstable solution

23

Involves an extraction step with organic solvent Requires heating ; least sensitive Absorbance measured at shorter wavelength Less sensitive Less sensitive

22

23 23 24 24 24 25

25

REDDY et al.: SPECTROPHOTOMETRIC DETERMINATION OF LAMUVIDINE

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series of 10 mL calibrated flasks by means of a micro burette. To all the calibrated flasks, 1 mL of 0.1% methanolic solution of BPB was added. The solutions were swirled and allowed to stand for 5 min and the volume was diluted to the mark with methanol and mixed well. The absorbance was measured at 595 nm against the corresponding reagent blank and calibration graph was constructed.

Fig. 1—Chemical structure of lamivudine Reagents and materials

LMV chemical references (gift sample) used for this study were obtained from Strides Arcolab Laboratories Ltd., Bangalore, India; it was reported to be 99.8% pure and was used as received. All reagents and solvents used were of analytical grade. The formulations selected for this study were obtained from pharmaceutical stores in Mysore and are LMV brands: Lamivir HBV (Cipla LTD., Sikkim, India), Hepitec (GSK, India) and Lamidac (Zydus Cadila, India). Standard solutions of pure reference LMV (1000 µg mL–1) were prepared in methanol for Method-I and LMV (1000 µg mL–1) were prepared in water for Method-II. A 0.1% bromophenol blue solution (Merck Specialities Private Limited) was prepared in 95% pure methanol (Merck Specialities Private Limited), diluting it in a calibrated flask and filtering using glass wool. A 1% FeCl3 solution was prepared in water and filtered using glass wool. A 0.5% MBTH reagent (Loba Chemie Private Ltd) was prepared in water and filtered using glass wool. Standard solutions for Method – I and Method– II A stock solution of LMV (1 mg mL–1) in methanol was prepared (Method–I); it was later diluted to 1-8 µg mL–1 using methanol. A stock solution of LMV (1 mg mL–1) in distilled water was prepared (Method – II); it was later diluted to 1-8 µg mL–1 using distilled water. Analytical procedures

Method I (BPB method) Different aliquots (0.1–0.8 mL) of the standard 100 µg mL–1 LMV solution were transferred into a

Method II (MBTH method) Varying aliquots (0.1–0.8 mL) of the standard 100 µg mL–1 LMV solutions were transferred into a series of 10 mL calibrated flasks by means of a micro burette. To all the calibrated 10 mL flasks, 1 mL of 0.5% MBTH reagent was added. The solutions were swirled and allowed to stand for 5 min. A 1 mL of 1% FeCl3 solution is added to all the flasks. The solutions were swirled and allowed to stand for 5 min, the volume was diluted to the mark with distilled water and finally mixed well. The absorbance was measured at 659 nm against the corresponding reagent blank and calibration graph was constructed. Procedure for tablet

The content of ten tablets (depending on the content per tablet) was powdered and mixed thoroughly. An amount of the powder equivalent to 100 mg of active component was weighed into a 100 mL volumetric flask; about 60 mL of methanol (Method–I), water (Method–II) was added and shaken thoroughly for about 20 min. The volume was made up to the mark with respective solvent, shaken and filtered using filter paper. The filtrate was diluted sequentially to get 0.1 mg mL–1 of the drug. Results and Discussion Method development

The methods are based on (Method-I) the application of acidic dyes for the spectrophotometric determination of LMV. The structural formula of LMV feature amine group suggests the use of acidic dyes (BPB) as chromogenic reagents. The acid dye technique is a general procedure for the quantitative analysis of a variety of pharmaceutical amines. In practice, an aqueous solution containing the amine and a suitable indicator dye is shaken with an organic solvent. The concentration of the resulting ion-pair is then determined spectrophotometrically. Few studies have reported the analysis of pharmaceutical compounds through formation of ion-pair, without extraction, followed by spectrophotometric

434


estimation30. The reaction path way is represented in Fig. 2. When added in increasing concentrations of LMV to a fixed concentration of BPB there is a proportional increase in absorbance at the respective λmax. Preliminary experiments were performed to fix the upper concentrations of the dye that could be determined spectrophotometrically. Method–II is based on the oxidation followed by coupling of 3-methyl-2-benzothiazolinone hydrazone with LMV in the presence of ferric chloride to form a green colored chromogen31. Actually, this is an iron catalyzed oxidative coupling reaction of MBTH with the drug. Under reaction conditions, on oxidation, MBTH loses two electrons and one proton forming an electrophilic intermediate, which is the active coupling agent32,33. This intermediate undergoes electrophilic substitution with the drug to form the colored product. The reaction pathway is represented in Fig. 3.

Optimization of reaction conditions

Effect of MBTH concentration The study on MBTH concentrations reveals that the reaction is dependent on MBTH reagent (Fig. 4). The absorbance of the reaction solution increases as the MBTH concentration increases, and the highest absorption intensity is attained at MBTH concentration of 0.5% (w/v). A higher MBTH concentration up to 1.25% has no effect on the absorption values. Further experiments are carried out using 0.5% MBTH. Effect of FeCl3 concentration The study on FeCl3 concentrations reveals that the reaction is dependent on FeCl3 reagent (Fig. 4). The absorbance of the reaction solution increases as the FeCl3 concentration increases, and the highest absorption intensity is attained at FeCl3 concentration of 1% (w/v). A higher FeCl3 concentration up to

Fig. 2—Proposal of the reaction pathway between lamuvidine and bromophenol blue


435

Fig. 3—Proposal of reaction pathway between lamuvidine and MBTH

Fig. 4— Effect of MBTH and FeCl3 on the reaction with LMV

1.6% has no effect on the absorption values. Further experiments are carried out using 1% FeCl3. Effect of BPB concentration For the first method various concentrations of BPB are tried and the absorbance maximum is found with 0.1% of BPB. A higher BPB concentration up to 1% has no effect on the absorption values. Further experiments are carried out using 0.1% BPB. The color intensity increases with time. The maximum

Fig. 5— Effect of time on reaction of BPB with LMV

absorbance is seen only after 5 min and this is found to be stable after 5 min as shown in the Fig. 5. All the experimental procedures were carried out in room temperature. Reaction products

The absorbance spectra of the red colored product (LMV-BPB) with λmax of 595 nm and that of the green colored product (LMV-MBTH) with λmax 659 nm are shown in Fig. 6. The above mentioned blanks have practically negligible absorption in both systems.


436 Analytical data

Method validation A linear correlation is found between absorbance at λmax and LMV concentration. They show negligible intercept as shown below: A= 0.050 ± 0.0023 γ; R = 0.9913, n=6 (Method – I) A= 0.045 ± 0.0013 γ; R = 0.9967, n=7 (Method – II) where A is the absorbance; γ, the concentration in µg mL-1; R, the regression coefficient; and n, the number of concentration levels. Linearity To establish linearity of the proposed methods, a separate series of solutions of LMV (1-8 µg) has been prepared from the stock solutions and analyzed. Least square regression analysis is performed on the obtained data. Accuracy The accuracy of the method is the closeness of the measured value to the true value for the sample. To determine the accuracy of the proposed method,

Fig. 6—Absorbance spectra of LMV-BPB and LMV-MB reaction products (initial conc. of LMV 10 µg mL-1)

different levels of drug concentrations – lower concentration (LC, 80%), intermediate concentration (IC, 100%) and higher concentration (HC, 120%) have been prepared from independent stock solutions and analyzed. Accuracy is assessed as the percentage relative error and mean % recovery (Table 3). To provide an additional support to the accuracy of the developed assay method, a standard addition method is employed, which involves the addition of different concentrations of pure drug to a known pre analyzed formulation sample and the total concentration is determined using the proposed methods. The % recovery of the added pure drug is calculated as % recovery = [(Ct–Cs)/Ca] × 100, where Ct is the total drug concentration measured after standard addition; Cs, the drug concentration in the formulation sample; and Ca, the drug concentration added to formulation. Precision Repeatability is determined by using different levels of drug concentrations (same concentration levels taken in accuracy study), prepared from independent stock solutions and analyzed. Inter-day, intra-day and inter-instrument variations are studied to determine intermediate precision of the proposed analytical methods (Table 4). Different levels of drug concentrations (6 times) are prepared three different times in a day and studied for intra-day variation. The same procedure is followed for three different days to study inter-day variation. One set of different levels of the concentrations is re-analyzed using the Shimadzu 1800 UV–VIS spectrophotometer connected to computer with UV-PC software has been used to study inter-instrument variation. The percentage relative standard deviation (% R.S.D.) of the predicted concentrations from the regression equation is taken as precision. Precision studies are also carried out using the real samples of LMV in a similar way to standard solution to prove the usefulness of the method.

Table 3—Assay of drug in pharmaceutical formulations (tablets) Formulation

Lamivir Lamidac Hepitec **

Label claim mg per tablet

150 100 150

Percentage found**± SD

Student t-value

f-value

BPB

MBTH

Reference method

BPB

MBTH

BPB

MBTH

99.8±0.55 99.6±0.45 99.4±0.24

99.9±0.54 99.9±0.43 99.9±0.22

101.5±0.92 98.42±1.18 102.6±1.54

0.04 0.06 0.02

0.15 0.23 0.44

1.87 1.44 2.33

2.33 1.99 3.01

Mean value of five determinations. Tabulated t-value at 95% confidence level is 2.77; tabulated f-value at 95% confidence level is 6.39.


437

Table 4—Intra-day precision and intra-day error of the methods LMV, µg mL-1

Method

er a, %

RSD, %

CLb, µg mL-1

Taken 1

Found 0.9

0.6

0.4

0.9 ± 0.002

BPB

2 3 4

1.92 2.89 3.91

0.13 0.1 0.15

0.2 0.2 0.1

1.98 ± 0.002 2.98 ± 0.004 3.99 ± 0.001

MBTH

5 6 7

4.96 5.88 7.97

0.11 0.25 0.25

0.1 0.1 0.1

4.99 ± 0.003 5.99 ± 0.002 6.99 ± 0.002

a

er – Relative error. CL – Confidence limits at 95% confidence level for seven degrees of freedom. a Mean value of seven determinations. b

Table 5—Analytical and validation parameters for the assay of LMV Parameter

Method _ I

Method _ II

Color λmax, nm Beer’s law range, µg mL–1 Molar absorptivity, L mol-1 cm-1 Limit of detection, µg mL–1 Limit of quantification, µg mL–1 Sandell’s sensitivity, µg/cm2 Intercept a ± Sa Slope b ± Sb (γ, LMV µg mL–1) Correlation coefficient (R)

Red 595 1-8 0.6075 × 103 0.1 0.3 0.0038 0.050

Green 659 1-8 0.286 × 103 0.3 0.9 0.0094 0.045

0.0023

0.0013

0.9913

0.9967

Limit of detection (LOD) and limit of quantitation (LOQ)

The LOD and LOQ for Acyclovir, Valacyclovir and Ganciclovir by the proposed method are determined using calibration standards. LOD and LOQ are calculated as 3.3 σ/S and 10 σ/S respectively, where S is the slope of the calibration curve and σ is the standard deviation of y-intercept of regression equation. The linearity, slope and the intercepts are calculated using the regression equation. The optical and validation parameters are listed in Table 5. Precision and accuracy of the proposed methods are tested by carrying out the determination of three replicates of pure and commercial samples of the drug, whose concentration is within Beer’s law range. Values of the standard deviation (SD) and relative standard deviation (RSD) are calculated. The two methods have been applied to various pharmaceutical formulations and recovery studies have been made by the standard-addition method (Table 3). The results were in agreement with the labeled amounts. For comparison, a conventional UV spectrophotometric

method34, is used for parallel comparison. The limit of detection (LOD) and limit of quantification (LOQ) are calculated according to the current ICH guidelines35. Conclusion Two useful methods for the determination of LMV have been developed and validated as per ICH guidelines. The above two methods can be used to monitor the content uniformity of tablets and purity of raw material. The proposed methods do not take more than 15-20 min. The methods could be considered for the determination of LMV in quality control laboratories. Acknowledgement The authors are thankful to Strides Archo Labs, Bangalore, India for providing LMV chemical reference (gift sample) for this study. References 1 2 3 4

5

6 7 8 9

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