AnalyticalMethods

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aDepartamento de Ciencias Farmaceuticas,

Ribeir~ao Preto, Universidade de S~ao Paulo,

fcfrp.usp.br; Fax: +55 16 3602 4178; Tel: +5bDepartamento de Quımica, Faculdade de Fil

Universidade de S~ao Paulo, Ribeir~ao Preto,

Cite this: DOI: 10.1039/c3ay26582j

Received 19th December 2012Accepted 29th July 2013

DOI: 10.1039/c3ay26582j

www.rsc.org/methods

This journal is ª The Royal Society of

Development, validation, and application of a capillaryelectrophoresis method for analysis of cytokineinterferon alpha-2a in pharmaceutical formulations

Fernando Armani Aguiar,a Anderson Rodrigo Moraes de Oliveirab

and Cristiane Masetto de Gaitani*a

A simple CE based method was developed and validated for the determination of interferon alpha-2a in a

pharmaceutical formulation. After optimization, the best results were obtained using 30 mmol L�1

tetraborate buffer at pH 8.50 with 50 mmol L�1 of sodium dodecyl sulfate, and using a diode array

detector at 200 nm. The applied voltage was +25 kV, and the sample injection was performed in the

hydrodynamic mode. All analyses were carried out in a fused-silica uncoated capillary with an i.d. of 75

mm and an effective length of 50.0 cm. Under these conditions, the analysis was achieved in less than 12

min. Linearity was obtained in the range 0.41–1.54 MIU mL�1 (r $ 0.997). The RSD (%) and relative

errors (%) obtained in precision and accuracy studies (intra-day and inter-day) were lower than 5%.

Therefore, this method was found to be appropriate for controlling pharmaceutical formulations

containing interferon alpha-2a.

Introduction

Interferon (IFN) was discovered by Alick Isaacs and Jean Lin-denmann in 1957.1 They observed that a supernatant of chickchorioallantoic membranes incubated with inactivated inu-enza virus was capable of protecting other membranes frominfection by the active virus. This supernatant containedsoluble protein factors that were able to induce a variety ofbiological functions, which are called interferons.1 Nowadays,it is known that IFNs are widely expressed cytokines that havepotent antiviral and growth-inhibitory effects. Thesecytokines are the rst line of defense against viral infectionsand present important roles in immunosurveillance formalignant cells.2,3

There are two major categories of IFNs: type I and type II.IFN-g is the only type II IFN, whereas the type I IFNs consist offour major classes: IFN-a, IFN-b, IFN-s and IFN-u. IFN-acomprises a family of different cytokines.2,4 IFN a-2a has beensuccessfully commercialized as biopharmaceuticals for thetreatment of a variety of diseases (e.g., hepatitis B and C,melanoma, renal cell carcinoma, and leukemia).2,5,6 IFN a-2acontains 165 amino acids with four cysteines and two disuldelinkages. In addition, it has a molecular weight of 19.2 kDa andan isoelectric point (pI) ranging from 5.5 to 7.0.7,8

Faculdade de Ciencias Farmaceuticas de

Ribeir~ao Preto, Brazil. E-mail: crisgai@

5 16 3602 4882

osoa Ciencias e Letras de Ribeir~ao Preto,

Brazil

Chemistry 2013

However, the development of biopharmaceuticals requiresanalytical technology that demonstrates appropriate detectionand quantication. It is necessary to utilize different methods togain a thorough understanding of the product complexity, due tothe inherent heterogeneity present in biopharmaceuticals.9,10 Inthis regard, for ensuring quality and quantity assessment at allresearch and development stages, it becomes generally necessaryanalytical methods, chromatographic and electrophoretic, whichare low time consuming, robust, and precise.11,12

The majority of methods commonly employed for IFNanalysis are based on bioassay,13 immunoassay,14 isoelectricfocusing (IEF),15 and gel electrophoresis techniques16 whichhave certain limitations, including (i) complexity and time-consuming nature of assay development, (ii) relatively largeamounts of sample required to reach a reasonable level ofsensitivity and (iii) high degree of assay variability.17

Liquid chromatography (LC) has been successfully used tomonitor the purity, identity, and chemical stability of biologi-cals obtained through recombinant DNA technology. LC is alsocapable of monitoring small structural variations, which couldlead to signicant changes in the biological activity of thedrug.18–20 Reverse phase liquid chromatography (RP-LC) hasalso been used to analyze IFN, as demonstrated by Silva et al.,21

where the method was developed and validated on a C4 columnwith UV detection and a retention time of 33 min. Additionallythe method described by the European Pharmacopeia uses aC18 column with UV detection and a retention time of 10.50min.22 However, these methods presented drawbacks, such asin the rst case with undesirable long time consumption, andas in both cases with high organic solvent consumption.

Anal. Methods

Analytical Methods Paper

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Over the past years, capillary electrophoresis (CE) hasadvanced signicantly as a technique for characterization ofbiomolecules, and it has become an important tool in thebiotech and pharmaceutical industries.23–26 CE has become amethod of choice in research and development, and in qualitycontrol for the release of therapeutic biomolecules to themarket.27,28 Many CE methods have been validated in the bio-pharmaceutical industry to meet International Conference onHarmonization (ICH) requirements.29

In this present work, capillary electrophoresis (CE) wasevaluated as a complementary technique for the analysis ofpharmaceutical formulations. CE has been proven to be aneffective technique for protein and peptide separation,30,31

which presents advantages such as simplicity, high efficiency,versatility, rapid analysis, high resolution power, small samplevolume and low operating costs.32,33 Based on this, a simple,rapid, accurate CE method for IFN a-2a determination wasdeveloped. The electrophoretic conditions were optimized andthe method was validated and applied for IFN a-2a determina-tion in commercial formulations.

ExperimentalChemicals and reagents

IFN a-2a was purchased from Sigma Aldrich (St Louis, USA). IFNa-2a 3.0 and 9.0 MIU in 0.5 mL (recombinant human interferonalpha-2a) was purchased from commercial sources in the localmarket. The stock solution (2.03 MIU mL�1) and workingsolutions (0.41, 0.58, 0.78, 1.17, and 1.57 MIUmL�1) of IFN a-2awere prepared in water. The solutions were stored in the fridgeat 4 � 1.0 �C and protected from light. Sodium acetate wasobtained from Merck (Darmstadt, Germany). Acetic acid waspurchased from Labsynth (S~ao Paulo, Brazil). Boric acid andsodium borate 10-hydrate were obtained from J. T. Baker (NewJersey, USA). Sodium dodecyl sulfate (SDS) and lithium chloride(LiCl) were purchased from Sigma Aldrich (St Louis, USA).Sodium hydroxide was purchased from Nuclear (S~ao Paulo,Brazil) and hydrochloric acid was obtained from Zilquımica(S~ao Paulo, Brazil). Water was distilled and puried using aDirect-Q 3 system from Millipore (Massachusetts, USA). Allother chemicals were of analytical grade and of the highestpurity available.

Apparatus and analytical conditions

Analyses were performed on a Beckman Coulter CE system(California, USA)model P/ACEMDQ consisting of an analyzer, anautomatic sampler and a diode array detector operating at200 nm. 32 Karat Soware was used for data acquisition. A fused-silica uncoated capillary obtained from Beckman Coulter(California, USA) of 75 mm i.d., 60.2 cm in total length, and 50.0cm in effective length was used. The electrophoretic separationswere carried out in 30 mmol L�1 sodium tetraborate buffer (pH8.5) containing 50 mmol L�1 of SDS. All experiments were carriedout in the normal mode. Sample injections were performedhydrodynamically at a pressure of 0.5 psi for 10 s. Before the rstuse, the capillary was conditioned by rinsing with 1.0 mol L�1

Anal. Methods

NaOH for 30 min, then with water for 30 min at 25 � 1.0 �C. Atthe beginning of each working day, the capillary was rinsed with0.1mol L�1 NaOH for 15min and thenwith water for 15min. Thecapillary was rinsed with 0.1 mol L�1 NaOH for 3.0 min, water for3.0 min, and running buffer for 4.0 min between consecutiveanalyses. Aer daily use, the capillary was washed with 0.1 molL�1 NaOH for 15 min and then with water for 15 min. Thecapillary was stored in water, when it was not in use.

Method validation

The method was validated for IFN a-2a analysis in standardsolutions. The standard solution stability test and system suit-ability test were performed before starting validation. Thefollowing parameters were evaluated during validation: linearity,precision, accuracy, limit of quantication (LOQ), and robust-ness, following the United States Pharmacopeia (USP) guidelines.

To perform the sample short-term temperature stability test,solutions of 0.41 and 0.90 MIU mL�1 of IFN a-2a prepared inwater (analysis in quadruplicate) were stored in amber vials atambient temperature for 12 h and analyzed. The responses forthe aged solutions were evaluated using a freshly preparedstandard solution.

The system suitability test was also performed to evaluate thereproducibility of the system for the analysis to be performed,using eight replicate injections of a standard solution contain-ing 0.90 MIU mL�1 of IFN a-2a. The migration time, tailingfactor and plate number were determined.

The linearity of the method was determined by constructingthree calibration graphs using six concentration levels coveringthe range from 0.41 to 1.57 MIU mL�1. Three replicate injec-tions of the standard solutions were made and the peak heightof the electropherogram was plotted against the concentrationsof IFN a-2a to obtain the respective calibration graph. The datawere then subjected to regression analysis by the least-squaresmethod to calculate the calibration equation and correlationcoefficient (r).

The limit of quantication (LOQ) is dened as the lowestconcentration that could be determined with accuracy andprecision below 5% over six analytical runs under the experi-mental conditions established. The LOQ was determined byusing standard solution with 0.41 MIU mL�1 of IFN a-2a (thelowest point included in the calibration curve).

The precision and accuracy of themethod were determined byintra- and inter-day studies. Intra-day precision was determinedby analyzing samples at three different concentrations of IFNa-2a (0.41, 0.78 and 1.57 MIU mL�1) on the same day and underthe same experimental conditions. Inter-day precision wasassessed by performing the analysis on two different days. Theaccuracy was calculated by the ratio between the experimentallydetermined average concentration and the corresponding theo-retical concentration. The results obtained for precision andaccuracy were expressed in terms of relative standard deviation(RSD, %) and relative error (RE, %), respectively.

Robustness, i.e. the measure of the method's capacity toremain unaffected by small but deliberate changes in electro-phoretic conditions, was studied for IFN a-2a standard

This journal is ª The Royal Society of Chemistry 2013

Table 1 Variables selected as factors and values chosen as levels to evaluate therobustness of the method

Factors Optimal Low level High level

pH buffer 8.5 8.3 8.7SDS concentration (mM) 50 45 55Voltage (kV) 25 23 27Capillary temperature 20.0 15.0 25.0

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solutions by analyzing them under conditions slightly changedfrom the dened method. The robustness test was evaluated byusing an experimental design procedure. The procedureselected was a 2-level 24–1 (eight experiments) fractional facto-rial design performed by the selection of four factors: pH,concentration of SDS, temperature and voltage studied at twolevels: high and low. The factors and their levels evaluated inthis study are presented in Table 1. The responses evaluatedwere peak height and migration time. The obtained responseswere processed by Minitab 15 statistical soware (State College,PA, USA) to evaluate the signicance of the effects.

Analysis of IFN a-2a in pharmaceutical formulations

For the quantication of IFN a-2a in the pharmaceutical formu-lations (samples containing 3 and 9 MIU of IFN a-2a per 0.5 mL),these solutions were ltered and concentrated by use of acentrifugal lter device with a membrane of 10 kDa nominal cut-off (Amicon Ultra-4, Millipore, Massachusetts, USA). This ltra-tion procedure was performed to remove the excess saltremaining aer formulation, which is composed of 3.60 mg ofsodium chloride, 0.10 mg of polysorbate 80, 5.00 mg of benzylalcohol as a preservative and 0.38 mg of ammonium acetate.Aer that, the concentrated solution of IFN a-2a was diluted to anappropriate concentration with water (0.90 MIU mL�1). Fivesamples of each formulation were prepared individually andinjected. This procedure was performed for two consecutive days.

Results and discussionMethod optimization

In capillary electromigration techniques, during the optimiza-tion phase of the method, the correct choice of the buffer is veryimportant. It should be able tomaintain constant the pH duringall time of analysis. In addition, the buffer may affect the elec-troosmotic ow (EOF) and electrophoretic mobility which arevery sensitive to pH variation.34,35

Based on that, the effects of the sodium tetraborate(100 mmol L�1) and sodium phosphate buffer (100 mmol L�1)at different pH values were investigated. Initially, the back-ground electrolyte (BGE) consisting of sodium phosphate at pHranging from 4.5 to 8.5 was tested. Besides employing sodiumphosphate buffer solution, no peak was observed beforereaching 20 min. Probably, it occurred due to factors such asprotein adsorption on the capillary inner wall, or lowmobility ofthe protein at pH near its pI (data no shown). Next, sodiumtetraborate buffer at pH values from 8.0 to 9.5 was tested. When

This journal is ª The Royal Society of Chemistry 2013

sodium tetraborate buffer solution was employed, an irregularand wide base peak was observed. These results can beexplained by the reduced protein interaction with the capillarywall, as well as by the increased net negative charge of theprotein at pH higher than its pI.36,37

Aer these results, the electrolyte 50 mmol L�1 sodium tet-raborate buffer pH 8.5 was supplemented with different addi-tives, such as LiCl and SDS. The use of additives is a commonpractice and it is used tomodify themobility of the solute,modifythe EOF, or solubilize compounds in the sample matrix andreduce the interaction of solutes with the capillary wall.11,38,39

Positively charged moieties on the basic amino acid residuesof the protein may interact with the acidic silanol groups evenwith the protein at the pI as a result of the distribution of thecharge density on the protein molecule. This way, the use ofhigh concentrations of ionic salt in BGE solutions has been onestrategy for minimizing undesirable interactions betweenproteins and the surface of fused-silica capillaries.38,40,41 On theother hand, this strategy must be carefully taken because thismight lead to excessive Joule heating and/or denaturation ofproteins.42

The effect of the running buffer additive LiCl at 25 mmol L�1

on the IFN alpha-2a analysis was investigated. LiCl is a neutralsalt that is able to stabilize tertiary structures of some proteins insolution, so it can prevent adsorption of the protein to thecapillary wall.43 However, no peak was observed during theanalysis time in this work. The obtained results might be sup-ported by the fact that Li+ showed to be little effective in blockingsilanol groups in the silica capillary. It could be weakly bound tothe double layer yielding the analyte solubility adversely.38,43,44

Another strategy was adding the surfactant SDS to the BGE.Surfactants may bind to proteins either to the same extent,resulting in protein–surfactant complexes of approximatelyconstant charge-to-mass ratio (e.g. protein–SDS complexes), orto a different degree which can enlarge differences in the elec-trophoretic mobilities between proteins.38,45 In the presence ofSDS, an improvement in the analysis of IFN a-2a was observed.Thus, sodium tetraborate buffer 50 mmol L�1 pH 8.5 withaddition of 50mmol L�1 of SDS was selected as the start point tooptimize the method of analysis.

At a pH value of 8.5, the inuence of SDS in the range of10–50 mmol L�1 in the analysis of IFN a-2a was witnessedby performing experiments in sodium tetraborate buffer(50 mmol L�1). These experiments showed an increase of themigration time when the surfactant was increased, in agree-ment with the literature (Fig. 1A). This fact can be explained as apossible increase in the negative net charge because of SDSattachment.46 Additionally, it should also be considered that theincrease of SDS concentration reduces the mobility due to anincrease of the BGE viscosity.38,47,48 Even with the increase inmigration time, the concentration of 50 mmol L�1 SDS wasselected. On the other hand, the SDS appears to be involved inthe peak sharpening (Fig. 1B).45,49 Therefore, 50 mmol L�1 SDSwas selected for subsequent analysis. During analyses with SDS,a range of pH values (8.0–9.5) was evaluated. However, at pH 8.5peak IFN a-2a showed better shape and efficiency (data notshown).

Anal. Methods

Fig. 1 Effects of (A) SDS concentration and (C) sodium tetraborate buffer concentration on the migration time; effects of (B) SDS concentration and (D) sodiumtetraborate buffer concentration on the plate numbers. Experimental conditions: capillary, 50 cm effective length and 75 mm i.d.; applied voltage, 20 kV; temperature,20 �C; sample, 0.5 psi for 10 s injection. **p < 0.01 compared with the SDS 50 mmol L�1 group. ***p < 0.001 compared with the buffer 30 mmol L�1 group.

Fig. 2 Representative electropherograms of the analysis of (A) peak 1 – IFNalpha-2a standard (0.90 MIU mL�1) and (B) peak 1 – formulation preservative andpeak 2 – IFN alpha-2a (0.90MIUmL�1) in pharmaceutical formulations.Optimizedconditions: sodium tetraborate buffer 30 mmol L�1 with SDS 50mmol L�1, pH 8.5,25 kV, 20 �C. A capillary of 50 cm effective length and 75 mm i.d. was used.

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Under the optimal pH condition (pH ¼ 8.5) with a SDSconcentration of 50 mmol L�1, the buffer concentration rangingfrom 30 to 150 mmol L�1 was evaluated. The gradual increase inconcentration caused an increase in the migration time(Fig. 1C), which can be explained by the increased viscosity,35

which provides a reduction in the mobility of analytes.50,51

Another explanation may be the decrease of zeta potentialwhich produces a decrease in EOF.35,52 Furthermore, the exces-sive Joules effect caused by higher running buffer concentra-tions may lead to bubbles and may cause an unstable baseline,or even interrupt the separation process.50 So, a short migrationtime and a better efficiency were achieved with sodium tetra-borate buffer 30 mmol L�1 (Fig. 1D). Therefore, 30 mmol L�1

sodium tetraborate buffer was chosen.The inuence of temperature and applied voltage was

investigated, and a temperature of 20 �C and a voltage of 25 kVwere selected. Fig. 2A shows the electropherogram of IFN a-2aunder the selected conditions.

Method validation

In the development of any quantitative method, the procedureof method validation is a key step. An analytical methodproducing valid measurements for an intended applicationneeds to be “t for purpose”. It is possible to discover whetherthe developed method is suitable for the desired applicationduring method validation.53

A system suitability test of the electrophoretic system wasperformed before each validation run. Eight-fold injections ofstandard solutions were made and the migration time, tailingfactor and plate number were determined. Since there is no

Anal. Methods

specic guidance for system suitability tests in electrophoreticmethods, the guide for validation of chromatographic methodswas used as a reference.54 For all system suitability tests theevaluated parameters were in accordance with the guide, asshown in Table 2. Next, these parameters showed that theequipment was able to perform the analysis.

The short-term stability of aqueous solution of IFN a-2a wasdetermined by keeping the solution at room temperature (21 �2.0 �C) for 12 h. The short-term stability shows that the samplesdid not present any signicant degradation in the concentra-tions studied (0.90 and 0.41 MIUmL�1). Fcalc (0.148 and 0.053) <Fcrit (0.246); p values (0.89 and 0.85) > 0.05. The level of

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Table 2 Results of the system suitability test for IFN a-2a analysis

ParametersUSPrecommendation Day

Averagevalue RSDa (%)

IFN a-2a

Migrationtime

RSDb (%) < 2 1 11.9 1.62 11.0 0.9

Tailing factor TF < 2 1 0.83 —2 0.86 —

Plate numbers Values > 2000 1 11 544 —2 10 283 —

a Values from eight replicate analyses. b RSD (%), relative standarddeviation expressed as a percentage.

Table 4 Precision and accuracy of the method for IFN a-2a analysis

Parameters IFN a-2a

Nominal concentration (MIU mL�1) 0.41 0.78 1.54

Within-daya (n ¼ 3)Analyzed concentration (MIU mL�1) 0.39 0.80 1.50Precisionb (RSD, %) 4.8 3.1 2.9Accuracyc (RE, %) �3.9 2.6 �2.3

Between-dayd (n ¼ 2)Analyzed concentration (MIU mL�1) 0.39 0.81 1.49Precisionb (RSD, %) 2.6 2.3 4.7Accuracyc (RE, %) �4.6 3.1 �2.9

a n ¼ number of determinations. b Precision expressed as relativestandard deviation percentage (RSD, %). c Accuracy expressed asrelative error percentage (RE, %). d n ¼ number of days.

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signicance was set at 95%. The results showed that IFN a-2awas stable for at least 12 h. The IFN a-2a standard is not stablein aqueous solution for a long time, as mentioned by themanufacturer. Owing to these reasons, long-term stability wasnot carried out.

The calibration plots were evaluated for linearity by the lack-of-t test. The calibration plot for the method was appropriateover the concentration range of 0.41–1.54 MIU mL�1 for IFNa-2a. A correlation coefficient (r) higher than 0.99 was obtained.The linear model for the relationship between the concentra-tion and response was considered to be appropriate as nosignicant lack of t was observed. The limit of quanticationwas the lowest concentration of the calibration curve. Theprecision and accuracy at this level over six analytical runs werewithin adequate limits, with RSD (%) and RE (%) less than 5%.The results are shown in Table 3.

The precision and accuracy of the CE method were evaluatedby calculating RSD (%) and RE (%), respectively, for threedeterminations of IFN a-2a at three different levels of concen-trations (0.41, 0.78, and 1.54 MIUmL�1) in two consecutive daysunder the same experimental conditions (Table 4). The RSD and

Table 3 Linearity and LOQ of the methoda

Parameters Day IFN a-2a

ANOVA lack oftb/bFtab

F value p value

Linear equation 1 y ¼ 101.08x � 83.968 1.66 0.222 y ¼ 69.877x � 70.039 1.36 0.29

Correlationcoefficient (r)

1 0.9972 0.997

Range (MIU mL�1) 1 0.41–1.542 0.41–1.54 RSD (%) RE (%)

LOQ (MIU mL�1) 1 0.41 4.8 �3.92 0.41 2.6 �4.6

a Calibration graphs were prepared in triplicate (n ¼ 3) forconcentrations of 0.41, 0.58, 0.78, 1.17 and 1.57 MIU mL�1 of IFN a-2a; y ¼ Ax + B, where y is the peak height of the analyte, A is theslope, B is the intercept, and x is the concentration of the measuredsolution in MIU mL�1. b Ftab (0.05) ¼ 2.95. RSD – relative standarddeviation; RE – relative error.

This journal is ª The Royal Society of Chemistry 2013

relative error were less than 5% in both experiments (within-dayand between-day). This performance suggests that the methodis completely suitable for quantitative determination of thestudied drug.

In the determination of the method robustness, which is themeasure of the method's capacity to remain unaffected by smallbut deliberate changes in electrophoretic conditions, thesignicance of the effects was evaluated by a Pareto chart. Asshown in Fig. 3, little changes in voltage and temperature wereable to affect the migration time with a signicant difference.This way, these parameters should be controlled duringanalyses.

Application of the method in parenteral formulations of IFNa-2a

The applicability of the proposed method was evaluated bydetermination of IFN a-2a in parenteral formulationscommercially available.

To demonstrate the performance of the ltration procedure,the recovery was assessed by the application of an analyticalprocedure to synthetic mixtures of the pharmaceutical formu-lation spiked with standards of IFN (0.90 MIU mL�1). Then,these recoveries were analyzed and compared against thereference standard solution. During the ltration stage, lossesare liable to occur, but no statistically signicant loss of analyteswas observed. A recovery higher than 99% was observed with agood precision (RSD < 8%).

A typical electropherogram of IFN a-2a in pharmaceuticalformulation is given in Fig. 2B. There were no signicantinterfering peaks that could interfere with the analyte, and astable baseline was maintained throughout. Quantication wasachieved in terms of relative peak heights and the results wereexpressed as RSD (n ¼ 5) of the obtained recoveries for theanalyte. The range of recovery for IFN a-2a was 101.4–104.9%with RSD less than 4.9%. Results are shown in Table 5. Theresults indicate that the method obtains good accuracy and issuitable for the pharmaceutical formulation analysis. The IFNa-2a (3 and 9 MIU 0.5 mL�1) analyzed is consistent with itsspecications.

Anal. Methods

Fig. 3 Pareto charts representing the effects of the variables and their interactions on the migration time (A) and height (B) of the IFN a-2a peak.

Table 5 Determination of IFN alpha-2a in pharmaceutical form on 2 consecutive days

IFN alpha-2aa (3 MIU per syringe) IFN alpha-2aa (9 MIU per syringe)

1st day 2nd day 1st day 2nd day

% foundb RSDc (%) % foundb RSDc (%) % foundb RSDc (%) % foundb RSDc (%)104.3 4.9 101.4 3.9 102.3 2.4 103.8 2.1

a Manufacturer's specications. b Mean of ve replicate analyses. c Relative standard deviation.

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Conclusions

In the present work a new, simple, and inexpensive analyticalmethod was developed and validated for analysis of IFN a-2a. Itwas based on CE using SDS as an additive and a simple sodiumtetraborate buffer. The method has the advantage of allowingthe analysis of IFN a-2a in less than 12 min with high efficiency.The method has been successfully used for the analysis ofcommercial pharmaceutical formulations. Compared withprevious methods described in the literature, the method pre-sented in this study has the following advantages: (i) low timeconsumption and (ii) no consumption of organic solvents.Thus, this method can be routinely applied in the pharmaceu-tical industry and it may be extended to other applications, e.g.it is a valuable alternative to analyses that employ HPLC.

Acknowledgements

The authors are grateful to Fundaç~ao de Amparo a Pesquisa doEstado de S~ao Paulo (FAPESP), Conselho Nacional de Desen-volvimento Cientıco e Tecnologico (CNPq), and to Coor-denaç~ao de Aperfeiçoamento de Pessoal de Nıvel Superior(CAPES) for nancial support and for the grant of researchfellowships.

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Anal. Methods

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