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UV-DerivativeSpectrophotometricDetermination of RitonavirCapsules and Comparison withLC MethodCarolina Lupi Dias a , Ana Maria Bergold a & PedroEduardo Fröehlich aa Programa de Pós-Graduação em CiênciasFarmacêuticas, Faculdade de Farmácia, UFRGS ,Porto Alegre, BrazilPublished online: 10 Aug 2009.

To cite this article: Carolina Lupi Dias , Ana Maria Bergold & Pedro EduardoFröehlich (2009) UV-Derivative Spectrophotometric Determination of RitonavirCapsules and Comparison with LC Method, Analytical Letters, 42:12, 1900-1910, DOI:10.1080/00032710903060701

To link to this article: http://dx.doi.org/10.1080/00032710903060701

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SPECTROPHOTOMETRY

UV-Derivative SpectrophotometricDetermination of Ritonavir Capsulesand Comparison with LC Method

Carolina Lupi Dias, Ana Maria Bergold,

and Pedro Eduardo FroehlichPrograma de P�oos-Graduacao em Ciencias Farmaceuticas, Faculdade de

Farm�aacia, UFRGS, Porto Alegre, Brazil

Abstract: An ultraviolet-derivative spectrophotometric method (UV-D) has beenproposed as an alternative to a previously described liquid chromatographic (LC)method for the quantitative determination of ritonavir in soft gelatin capsules.The spectrophotometric method is based on recording the second-derivative spec-tra for ritonavir at 222.3 nm of its solutions in methanol. The linear dynamicrange was 10.0–30.0 mg �mL�1 with a correlation coefficient of 0.9995. Meanrecoveries were between 99.2% and 100.2% for the tested capsules samples. Meanintra- and interassay relative standard deviations (RSDs) were less than 2.0%. Thestatistic analysis showed that LC and UV-D methods were equivalent to assayritonavir capsules.

Keywords: Quality control, ritonavir in soft gelatin capsule, UV-derivativespectrophotometry

Received 9 January 2009; accepted 30 April 2009.Address correspondence to Carolina Lupi Dias, Programa de P�oos-Graduacao

em Ciencias Farmaceuticas, Faculdade de Farm�aacia, UFRGS, Avenida Ipiranga,2752=704, Porto Alegre CEP 90610-000, RS, Brazil. E-mail: carolina.lupi@.ufrgs.br

Analytical Letters, 42: 1900–1910, 2009Copyright # Taylor & Francis Group, LLCISSN: 0003-2719 print=1532-236X onlineDOI: 10.1080/00032710903060701

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INTRODUCTION

Human immunodeficiency virus (HIV) is the etiologic agent of acquiredimmunodeficiency syndrome (AIDS). Ritonavir (RTV) (Fig. 1) is apeptidomimetic inhibitor of both the HIV-1 and HIV-2 proteases,enzymes that are essential for viral growth (Tavares 1996; Raffanti andHaas 2001; Sethi 2002; Chen et al. 2003; Korolkovas 2004; Abbott2006). Protease inhibitors (PIs) have been responsible for marked reduc-tions in morbidity and mortality in patients with advanced HIV infection(Murphy 2003) through the production of noninfectious viruses. Cellsincubated in the presence of HIV protease inhibitors produce viral parti-cles that are immature (Raffanti and Haas 2001; Chen et al. 2003; Abbott2006). Thus, inhibition of HIV protease becomes one of the most impor-tant approaches for the therapeutic intervention of HIV infection (Chenet al. 2003).

Ritonavir (RTV), chemically designated as 10-hydroxy-2-methyl-5-(1-methylethyl)-1-(2-(1-methylethyl)-4-thiazolyl)-3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid, 5-thiazolylmethylester, (5S-(5R�,8R�,10R�,11R�)), was approved by the U.S. Food andDrug Administration (FDA) in March 1996 and is available as soft gela-tin capsules and oral solution (Norvir1) (Budavari et al. 2001; FDA 2004;Abbott 2006). The inactive ingredients of RTV soft gelatin capsules(100mg) are butylated hydroxytoluene, ethanol, gelatin, iron oxide, oleicacid, polyoxyl 35 castor oil, and titanium dioxide (Abbott 2006).

Several liquid chromatographic (LC) analytical methods have beendescribed to quantify RTV alone and combined with other anti-HIVagents in plasma. According to the final text for inclusion in the interna-tional pharmacopoeia (WHO 2005) and first supplement of the Americanpharmacopeia (USP 2006), a nonaqueous titrimetric method with poten-tiometric end point and an LC method, respectively, are recommendedfor assay of RTV bulk substance. However, only one LC method hasbeen reported to assay RTV capsules (Dias et al. 2005). The aim of this

Figure 1. Molecular structure of ritonavir.

Determination of Ritonavir Capsules 1901

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work is to propose an alternative spectrophotometric method, equivalentto the reported LC method, to determine RTV in this dosage form.

Ultraviolet (UV) spectrophotometry is a method to assay bulk sub-stance, but it can suffer from background interference by excipients ofpharmaceutical formulation. Ultraviolet-derivative (UV-D) spectropho-tometry (zero-crossing method) has been described as an analyticaltechnique of great utility to determine substances under the influenceof increased background absorption and overlapping, without prelimin-ary separation (Talsky, Mayring, and Kreuzer 1978; O’Haver and Begley1981; Rojas, Ojeda, and Cano Pavon 1988; Hackmann, Beneton, andSantoro 1991; Paschoal et al. 2003; Rocha and Teixeira 2004). SeveralUV-derivative spectrophotometric methods have been described as tech-niques to quantify drugs alone or in association when there is interferencefrom other compounds (associated drugs, excipients, or potential degra-dation products) (Toral et al. 2002; Mabrouk et al. 2003; Morelli 2003;Busaranon, Suntornsuk, and Suntornsuk 2005; Karpinska and Szostak2005; Afkhami and Bahram 2005). We report the development and fullvalidation of a simple UV-D spectrophotometric assay for the quantita-tive determination of RTV in soft gelatin capsules according to theofficial codes (FDA 1994; ICH 2005; USP 2006).

MATERIALS AND METHODS

Chemicals

The RTV chemical reference substance (CRS) was kindly given byCrist�aalia Produtos Quımicos Farmaceuticos Ltda (Sao Paulo, Brazil).Norvir capsules (Abbott) were purchased in the market. All chemicalsand solvents used were of analytical grade. Methanol was from Merck(Darmstadt, Germany). Butylated hydroxytoluene (BHT) was from ViaFarma (Brazil), polyoxyl 35 castor oil was from Alfa Quımica (Brazil),and oleic acid was from SP Farma (Brazil).

Apparatus

A double-beam UV-visible spectrophotometer (Shimadzu, Japan), modelUV-1601 PC, was used for spectrophotometric measurements. The soft-ware employed was UVPC personal spectroscopy software, version 3.9.For all the tested solutions, the second derivative spectra (D2) wasrecorded over the range 250–220 nm against solvent in a 1-cm quartzcells, fixing Dk at 2 nm and scaling factor 30. The amplitude values ofD2 were measured at 222.3 nm for RTV (zero crossing of excipients).

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Standard and Sample Solutions

The RTVCRS was accurately weighted (50mg) and dissolved in a 100-mLvolumetric flask with methanol to generate a stock standard solution con-taining 500 mg �mL�1 analyte. Working standard solutions were preparedby diluting the stock standard solution to appropriate concentrations.

Twenty units of Norvir capsules (100mg of RTV) were separatelyweighted. Their contents were washed with methanol, collected in a100-mL volumetric flask, diluted to volume, mixed, and filtered to yielda concentration of 10mg �mL�1. This solution was appropriately dilutedwith methanol to 20 mg �mL�1 before D2 measurements.

Method Validation

A large number of preliminary tests using analyte solutions at constantconcentration and variable concentration of excipient formulation weremade to select the most convenient derivative order, analytical wave-length (k), Dk, and scaling factor.

The D2 spectrophotometric method was validated according to pub-lished guidelines (ICH 2005; USP 2006) and compared with the reportedLC method (Dias et al. 2005). The specificity of the analytical methodwas evaluated with a solution of inactive ingredients: BHT, ethanol, oleicacid, and polyoxyl 35 castor oil. Their concentrations in pharmaceuticalformulation were based on the literature (Kibbe, Wade, and Weller 2000)and calculated for the average weight of content (�939.1mg) of thecapsules. Linearity was tested by analyzing five different RTV concentra-tions covering the range of 10.0–30.0mg �mL�1 (n¼ 3). Assay performancewas determined from replicate analysis of samples at 20.0 mg �mL�1

(n¼ 6), evaluated by repeatability and intermediate precision andexpressed in terms of RSDs from mean intra- and interday assay.Accuracy (recovery test) of the method was evaluated by spiking knownamounts of standard (5.0, 10.0, and 15.0 mg �mL�1) in three sample solu-tions with 10.0 mg �mL�1 to provide final concentrations of 15.0, 20.0,and 25.0 mg �mL�1, respectively. The accuracy was expressed in termsof recovery percentage values (%R) of the theoretical concentration.

RESULTS AND DISCUSSION

Specificity

The best solubility for RTV and inactive ingredients of formulationwas obtained using methanol. The parameters were selected through

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elimination of background interferences and overlaps. Solutions ofstandard, sample, and excipient formulation were evaluated over therange 400–200 nm in the zero (D0), first (D1), and second (D2) derivativeorders. Polyoxyl 35 castor oil and BHT were the excipients that showedgreatest absorption in the region studied and therefore largest interfer-ence with original RTV spectra. Results showed that D2 measurementof RTV is possible at 222.3 nm because at this wavelength the influenceof excipient was not observed.

The original and D2 derivative spectra for standard, sample, andexcipient (isolated and mixed) solutions are showed in Figs. 2 and 3.The results of the preliminary specificity test are presented in Table 1,where the absence of interference upon D2 measurement for RTV, dueto increasing proportion of inactive ingredients, can be observed.

Linearity

Standard solutions prepared at five concentrations (10.0, 15.0, 20.0, 25.0,and 30.0 mg �mL�1) were measured in triplicate every day for three

Figure 2. (I) Zero-order spectra of excipient solution (BHT, ethanol, oleic acid,and polyoxyl 35 castor oil) in methanol. (II) Zero-order spectra of RTV standardsolution (20 mg �mL�1). (III) Zero-order spectra of RTV standard solution (A),BHT solution (B), and polyoxyl 35 castor oil solution (C) in methanol. (IV) Theircorresponding D2 spectra, respectively.

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consecutive days. The average correlation coefficient (r) obtained wasgreater than 0.9995. The average equation for three calibration curveswas y¼ 0.0008þ 0.0148 x, in which y¼ absorbance and x¼RTV concen-tration (mg �mL�1). Figure 4 contains the overlay of D0 and D2 derivativespectra of RTV standard solutions. The linearity data were evaluatedusing analysis of variance (ANOVA), and this showed that the regressionequation was linear (Fcalculated¼ 7605.30>Fcritical¼ 4.96; P¼ 0.05) withno deviation from linearity (Fcalculated¼ 2.17<Fcritical¼ 3.71; P¼ 0.05).

Figure 3. (a) UV zero-order spectra of RTV standard and sample (capsule) solu-tions (20mg �mL�1) in methanol. (b) Their corresponding D2 spectra, respectively.

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Thus, the results showed that the D2 absorbance was proportional toRTV concentration.

Precision

The intraday and interday precisions were estimated from the triplicatemeasurements of six different sample solutions prepared at 20.0mg �mL�1

of RTV and analyzed for 4 different days. Means and RSDs were obtainedfrom calculated RTV concentrations in capsules over all 4 days (Table 2).The variability was low with RSD values ranging from 0.50% to 1.74%;thus RSD< 2.0%. These results indicated that this assay is reproducible.

Table 1. Results of preliminary specificity test of the D2 measure-ment of RTV in the presence of excipients (n¼ 3)

Excipients(mg �mL�1)a,b,c RSDd (%)

Concentration ofRTV found (mg �mL�1)

Relativeerror (%)

0.00 0.2 22.2410.0 0.6 22.18 �0.315.0 1.6 22.38 0.620.0 1.4 22.15 �0.425.0 1.7 22.20 �0.230.0 1.5 22.29 0.2

aConcentration of excipients proportional to the sample solutionscontaining 0.0–30.0mg �mL�1 of RTV.

bTheoretical concentration of RTV CRS added for each propor-tion of excipients¼ 20.0 mg �mL�1.

cTotal mean absorbance¼ 0.3374 (RSD¼ 0.4%).dRelative standard deviations.

Figure 4. Overlay of UV zero-order spectra (a) and D2 spectra (b) for RTVstandard solutions (10.0–30.0 mg �mL�1) in methanol.

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Accuracy

Accuracy was calculated as the percentage recovery of known addedamounts of RTV CRS in the sample solutions using three concentrationlevels covering the specified range and three replicates of each concentra-tion. The mean accuracy of the method ranged from 99.2% to 100.2%,indicating that this assay is reliable (Table 3).

Comparison of D2 Spectrophotometric and LC Methods

The D2 spectrophotometric assay was compared with a previouslydescribed LC method (Dias et al. 2005). The t-test was used (t-table value

Table 2. Assay results of capsules containing 100mg of RTV by the proposed D2

spectrophotometric method and the reported LC method

D2 spectrophotometric method LC method

Consecutivedays

Intraday assay(%)� SEMa,b RSD (%)a,c

Intraday assay(%)� SEMa,b RSD (%)a,c

I 100.7� 0.21 0.50 100.4� 0.26 0.64II 100.4� 0.70 1.72 100.3� 0.19 0.45III 100.6� 0.71 1.74 100.3� 0.32 0.80IV 101.5� 0.53 1.29 100.3� 0.18 0.45

aMean of six determinations.bStandard error of the mean.cRelative standard deviations.Note. Interday assay (RSD, %): 100.8% (1.38) for D2 spectrophotometric

method and 100.3% (0.57) for LC method.

Table 3. Recovery of RTV in capsules solution

D2 spectrophotometric method LC method

Added(mg �mL�1)a

Found(mg �mL�1)a

Recovery(%)a

Added(mg �mL�1)a

Found(mg �mL�1)a

Recovery(%)a

5.0 5.32 100.2 20.0 21.3 101.110.0 10.59 99.2 40.0 41.9 99.415.0 16.07 100.1 60.0 63.0 99.8

aMean of three determinations.Note. Mean absolute recovery, % (RSD, %): 99.8 (0.55) for D2 and 100.1 (0.89)

for LC.

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2.013 for 46 degrees of freedom) to compare both methods. The assaydata obtained from the two methods showed nonsignificant differenceat the confidence level of 95% (P¼ 0.05) with the statistic t (1.490) beingsmaller than the critical value.

CONCLUSIONS

The importance of RTV in the clinical treatment of AIDS justifies theneed of analytical procedures assuring the quality of its pharmaceuticaldosage form. The proposed D2 spectrophotometric method for thequantitative determination of RTV in capsules offers selectivity, withno interference from the excipients, when compared with the conven-tional UV-visible spectrophotometric method. It presents simplicity,accuracy, and precision and is an alternative to the LC method: the sta-tistical analysis by Student’s t-test suggests that the two methods areequivalent.

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