Difference Spectroscopic Methods Research Articles

UV Spectrophotometric Methods to Quantify Alogliptin Benzoate and Pioglitazone Hydrochloride

Three new, precise, accurate and sensitive UV-Spectrophotometric methods namely Ratio Difference Spectroscopic Method (RDSM), First Derivative of Ratio Spectra Method (DR1) and Area Under Curve Method (AUC) were developed and validated for simultaneous assessment of alogliptin benzoate (ALO) and pioglitazone hydrochloride (PIO) in tablet dosage form. In RDSM, ratio spectra of both the drugs were recorded by dividing the mixtures using interfering drug as divisor. Then the difference between the amplitudes of obtained ratio spectra was measured at 288 and 291 nm for ALO and 236 and 245 nm for PIO. The second method DR1, where the first derivative of ratio spectra of both the drugs were recorded and the first derivative signal was measured at 290 nm for ALO and 276.8 nm for PIO. The scaling factor was fixed as 1 and wavelength interval (Δλ) as 2 for recording the first derivative of ratio spectra. In the third method (AUC), peak area of recorded zero order spectra was measured at 276 ± 10 nm for ALO and 267.8 ± 10 nm for PIO. All three proposed methods were validated according to “International Conference on Harmonization” (ICH) guidelines parameters. For all three methods, ALO and PIO obeyed Beer’s law in the range of 0.5-5 & 1.8-18 µg/ml, respectively. The % RSD of repeatability of measurement, intra-day and inter-day precision were found to be less than 2 for all three methods. Limit of detection (LOD) and Limit of quantification (LOQ) of the drugs were calculated which proved the sensitivity of the methods. The accuracy ranged between 98-101% for all three methods. No interference from pharmaceutical excipients present in the formulation was observed. These proposed methods were found to be simple, sensitive, accurate and precise and can be applied to the simultaneous estimation of ALO and PIO in combined tablet formulation and also appropriate for routine quality control analysis.

Development and Validation of Difference Spectrophotometric Method for the Quantitative Estimation of Mesalamine in Bulk Drug and Dosage forms.

A sensitive, precise, economic spectrophotometric method i.e. a difference spectroscopic method was developed for the estimation of mesalamine in bulk and dosage (tablet and suppository) forms. Difference spectroscopic method was developed by measuring the difference absorbance of the drug in 0.1N HCl and in 0.1 N NaOH showing maximum absorbance at 330nm. Beer's law was obeyed in the concentration range of 5–25 μg/ml. The LOD and LOQ were found to be 0.3 μg/ml and 0.9 μg/ml respectively. Correlation coefficient was observed as 0.999. The method was validated as per ICH guideline Q2R1 and the results were within the official limits. The developed difference spectroscopic method was applied for quantitative analysis of the drug in bulk and pharmaceutical dosage forms.

The effect of allyl isothiocyanate on RNA

Allyl isothiocyanate (AITC), finds in cruciferous vegetables. AITC shows bactericidal, fungicidal activities and anticancer activity by inhibition of cell proliferation, cell cycle arrest and apoptosis induction and tumor growth inhibition. In this study, the interaction of Allylisothiocyanate with yeast RNA in aqueous solution at physiological conditions using constant RNA concentration and various molar ratios of AITC/RNA complexes are examine. Fourier transform infrared (FTIR) and uv-visible difference spectroscopic methods are used to determine the AITC binding mode, binding constant and stability of RNA in AITC/RNA complexes in aqueous solution. Spectroscopic evidence shows that AITC interacts with RNA. The external binding of AITC to RNA with overall binding constant of K(AITC-RNA)=7.207×10 6 M -1 are also presented. RNA remains in A conformation upon AITC complexation. Keywords : Allyl isothiocyanate; RNA; FTIR; UV-vis; binding constant; conformation.

Development of Difference Spectroscopic Method for the Estimation of Lincomycin Hydrochloride in Bulk and Pharmaceutical Solid Dosage forms

A simple, precise and accurate difference spectroscopic method has been developed for the estimation of lincomycin hydrochloride (LMH) in bulk and pharmaceutical dosage form. The proposed method is based on the measurement of absorbance of Lincomycin hydrochloride at maxima 209 nm and minima 220 nm. The measurement value is the amplitude of maxima and minima between two equimolar solutions of the analyte in different chemical forms, which exhibits different spectral characteristics. Since the drug was freely soluble in distilled water, a stock solution (1mg/ml) was prepared with distilled water. Further dilution was made by using 0.1N sodium hydroxide and phosphate buffer of pH6.8 separately. The maxima and minima in the difference spectra of lincomycin hydrochloride were at 209 nm and 220 nm, respectively. Difference in absorbance between these maxima and minima was calculated to find out the amplitude. This amplitude was plotted against concentrations. The drug obeyed the beer's law in the concentration range 5-25μg/ml and showed good correlation. The result of analysis was validated by recovery studies.

DEVELOPMENT AND VALIDATION OF DIFFERENCE SPECTROSCOPIC METHOD FOR THE ESTIMATION OF TOLPERISONE HCL IN BULK AND PHARMACEUTICAL DOSAGE FORM

Difference spectroscopic method is based on the principle that tolperisone hydrochloride can exhibit two different absorption spectra in basic and acidic medium. A stock solution (1 mg/ml) was prepared with distilled water. Further dilution was made by using 0.1 N sodium hydroxide and 0.1 N hydrochloric acid separately. The maxima and minima in the difference spectra of tolperisone hydrochloride were at 231nm and 263nm, respectively. Difference in absorbance between these maxima and minima was calculated to find out the amplitude. This amplitude was plotted against concentration. Linearity was found in the concentration range of 10-50µg/ml. The LOD and LOQ of tolperisone hydrochloride were found to be 0.064µg/mL and 0.196µg/mL respectively. The percentage recovery study of the drug for the proposed method was found in the range of 99.31 - 99.94% for tolperisone hydrochloride. The proposed method is recommended for routine analysis since it is rapid, simple, precise and accurate.

Development of difference spectrophotometric method for the estimation of leflunomide in tablet dosage form

A new simple, accurate, precise, highly sensitive and reproducible difference spectrophotometric method for the determination of leflunomide in bulk and pharmaceutical dosage form is described. Difference spectroscopic method is based on the principle that leflunomide exhibit two different forms; in acidic and basic medium which differs in their absorption spectra. The difference spectra were obtained by reading the absorbance of leflunomide in 0.1N HCl in the reference cell and the absorbance of leflunomide in 0.1N NaOH in the sample cell and vice versa; in the difference spectrum maxima and minima were seen at 293.5nm and at 261.5nm respectively. The amplitude values were calculated, which was plotted against concentration. The method is found to be linear in the concentration range of 2-12 ?g/ml. The percentage recovery was found to be between the ranges from 98.92 % to 99.08 %. The proposed method was statistically validated and successfully applied for analysis of tablet dosage forms.

Simultaneous Determination of Norfloxacin and Tinidazole Binary Mixture by Difference Spectroscopy

A simple and rapid difference spectroscopic method was developed for the simultaneous determination of binary mixture of norfloxacin (NF) and tinidazole (TZ) without prior separation. The proposed method depends upon measuring the absorbance of NF at 291.6 nm which is the zero crossing point on the difference spectra of TZ in 0.1 N NaOH vs. 0.1 N HCl. Similarly, the absorbance of TZ was measured at 344.4 nm which is the zero crossing point on the difference spectra of NF. Beer's law was obeyed in the concentration range of 2-20 and 5-50 µg/mL for NF and TZ, respectively. The lower limits of detection (LOD) of NF and TZ are 0.23 and 0.36 µg/mL, respectively, while the lower limits of quantification (LOQ) of NF and TZ were 0.70 and 1.08 µg/mL, respectively. The precision of the method was satisfactory; the maximum value of relative standard deviations did not exceed 1.5% (n=10). The accuracy, expressed as recovery is between 98.25 and 101.8% with relative error of 0.29 and 0.23 for NF and TZ, respectively. The proposed method was successfully applied for the determination of both drugs in bulk powder, laboratory prepared mixture and commercial dosage forms such as tablets without interference from the commonly encountered excipients and additives. The results obtained are in good agreement with those obtained by the reference methods.

Development of Difference Spectroscopic Method for the Estimation of Balsalazide in Bulk and in Formulation

A simple, precise and accurate difference spectroscopic method has been developed for the estimation of balsalazide in bulk and in pharmaceutical dosage form. The proposed method is based on the principle that balsalazide can exhibit two different chemical forms in basic and acidic medium that differ in the absorption spectra in basic and acidic medium. Since the drug was freely soluble in distilled water, a stock solution (1 mg/mL) was prepared with distilled water. Further dilution was made by using 0.1 M sodium hydroxide and 0.1 M hydrochloric acid separately. The maxima and minima in the difference spectra of balsalazide were at 460 nm and 354 nm, respectively. Difference in absorbance between these maxima and minima was calculated to find out the amplitude. This amplitude was plotted against concentration. Beer’s law is valid in the concentration range of 2‐20 μg/mL. The results of analysis have been validated statistically and by recovery studies.

Interaction of tRNA with Safranal, Crocetin, and Dimethylcrocetin

Saffron is the red dried stigmas of Crocus sativus L. flowers and used both as a spice and as a drug in traditional therapeutic. The biological activity of saffron in modern medicine is in development. Its numerous applications as an anti-oxidant and anti-cancer agent are due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). The aim of this study was to examine the interaction of transfer RNA with safranal, crocetin, and dimethylcrocetin in aqueous solution at physiological conditions. Constant tRNA concentration (6.25 mM) and various drug/tRNA (phosphate) molar ratios of 1/48 to 1/8 were used. FT-IR and UV-Visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants and the effects of drug complexation on the stability and conformation of tRNA duplex. External binding mode was observed for safranal crocetin and dimethylcrocetin, with overall binding constants Ksafranal = 6.8 (± 0.34) × 103 M−1, KCRT = 1.4 (± 0.31) × 104 M−1, and KDMCRT = 3.4 (± 0.30) × 104 M−1. Transfer RNA remains in the A-family structure, upon safranal, crocetin and dimethylcrocetin complexation.

DNA Interaction with Saffron's Secondary Metabolites Safranal, Crocetin, and Dimethylcrocetin

Saffron comes from the dried red stigmas of the Crocus sativus L. flower. Except for its use in cooking and in traditional medicine, it has numerous applications as an antitoxic, antioxidant, and anticancer agent due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). However, there has been no information on the interactions of these secondary metabolites with individual DNA at molecular level. This study was designed to examine the interaction of safranal, crocetin (CRT), and dimethylcrocetin (DMCRT) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.25 mM) and various drug/DNA(phosphate) molar ratios from 1/48 to 1/2. FTIR and UV-visible difference spectroscopic methods are used to determine the drug binding sites, the binding constants, and the effects of carotenoids and safranal complexation on the stability and conformation of DNA duplex. Both intercalative and external binding modes were observed, with overall binding constants K(safranal) = 1.24 x 10(3) M(-1), K(CRT) = 6.2 x 10(3) M(-1) and K(DMCRT) = 1.85 x 10(5) M(-1) A partial B- to A-DNA transition occurs at high carotenoids and safranal concentrations.

Spectrophotometric, Difference Spectroscopic, and High-Performance Liquid Chromatographic Methods for the Determination of Cefixime in Pharmaceutical Formulations

Three simple and sensitive spectrophotometric, difference spectroscopic, and liquid chromatographic (LC) methods are described for the determination of cefixime. The first method is based on the oxidative coupling reaction of cefixime with 3-methyl-2-benzothiazolinon hydrazone HCI in presence of ferric chloride. The absorbance of reaction product was measured at the maximum absorbance wavelength (wavelength(max)), 630 nm. The difference spectroscopic method is based on the measurement of absorbance of cefixime at the absorbance maximum, 268 nm, and minimum, 237 nm. The measured value was the amplitude of maxima and minima between 2 equimolar solutions of the analyte in different chemical forms, which exhibited different spectral characteristics. The conditions were optimized, and Beer's law was obeyed for cefixime at 1 to 16 microg/mL and 10 to 50 microg/mL, respectively. The third method, high-performance LC, was developed for the determination of cefixime using 50 mM potassium dihydrogen phosphate (pH 3.0)-methanol (78 + 22, v/v) as the mobile phase and measuring the response at wavelength(max) 286 nm. The analysis was performed on a Lichrospher RPC18 column. The calibration curve was obtained for cefixime at 5 to 250 microg/mL, and the mean recovery was 99.71 +/- 0.01%. The methods were validated according to the guidelines of the U.S. Pharmacopoeia and also assessed by applying the standard addition technique. The results obtained in the analysis of dosage forms agreed well with the contents stated on the labels.

Optical and EPR spectroscopic studies of demetallation of hemin by L-chain apoferritins

Earlier crystallographic and spectroscopic studies had shown that horse spleen apoferritin was capable of removing the metal ion from hemin (Fe(III)-protoporphyrin IX) [G. Précigoux, J. Yariv, B. Gallois, A. Dautant, C. Courseille, B. Langlois d’Estaintot, Acta Cryst. D50 (1994) 739–743; R.R. Crichton, J.A. Soruco, F. Roland, M.A. Michaux, B. Gallois, G. Précigoux, J.-P. Mahy, D. Mansuy, Biochemistry 36 (1997) 15049–15054]. We have carried out a detailed re-analysis of this phenomenon using both horse spleen and recombinant horse L-chain apoferritins, by electron paramagnetic resonance spectroscopy (EPR) to unequivocally distinguish between heme and non-heme iron. On the basis of site-directed mutagenesis and chemical modification of carboxyl residues, our results show that the UV–visible difference spectroscopic method that was used to establish the mechanism of demetallation is not representative of hemin demetallation. EPR spectroscopy does establish, as in the initial crystallographic investigation, that hemin demetallation occurs, but it is much slower. The signal at g = 4.3 corresponding to high spin non-heme-iron (III) increases while the signal at g = 6 corresponding to heme-iron decreases. Demetallation by the mutant protein, while slower than the wild-type, still occurs, suggesting that the mechanism of demetallation does not only involve the cluster of four glutamate residues (Glu 53, 56, 57, 60), proposed in earlier studies. However, the mutant protein had lost its capacity to incorporate iron, as had the native protein in which the four Glu residues had been chemically modified. Interestingly, a signal at g = 1.94 is also observed. This signal most likely corresponds to a mixed-valence Fe(II)–Fe(III) cluster suggesting that a redox reaction may also be involved in the mechanism of demetallation.

Interaction of Antioxidant Flavonoids with tRNA: Intercalation or External Binding and Comparison with Flavonoid-DNA Adducts

Antioxidants are essential to good health. Flavonoids are powerful antioxidants, and prevent DNA damage. The antioxidative protections are related to their binding modes to a DNA duplex and complexation with free radicals in vivo. Recently we reported the interaction of flavonoids with DNA in vitro (Kanakis et al., J. Biomol. Struct. Dyn. 22, 719-724, 2005), where polyphenol different binding modes were discussed. The aim of this study was to examine the interaction of transfer RNA with quercetin (que), kaempferol (kae), and delphinidin (del) in aqueous solution at physiological conditions and to make a comparison with the corresponding pigment-DNA adducts. Constant tRNA concentration (6.25 mM) and various drug/RNA(phosphate) molar ratios of 1/48 to 1/8 were used. FTIR and UV-visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants, and the effects of drug complexation on the stability and conformation of tRNA duplex. Both intercalative and external binding modes were observed. Structural analysis showed que, kae, and a del intercalate tRNA duplex with minor external binding to the major or minor groove and the backbone phosphate group with overall binding constants K (que) = 4.80 x 10(4) M(1), K (kae) = 4.65 x 10(4) M(1), and K (del) = 9.47 x 10(4) M(1). The stability of adduct formation is in the order of del > que > kae. A comparison with flavonoids-DNA adducts showed both intercalation and external bindings with the stability order K (que) = 7.25 x 10(4) M(1), K (kae) = 3.60 x 10(4) M(1), and K (del) = 1.66 x 10(4) M(1). Low flavonoid concentration induces helical stabilization, whereas high pigment content causes helix opening. A partial Bto A-DNA transition occurs at high drug concentration, while tRNA remains in the A-family structure.

Difference spectroscopic and reverse phase HPLC methods for the estimation of cefdinir in pharmaceutical dosage forms

Two simple efficient and reproducible difference spectroscopic and reverse phase high performance liquid chromatographic methods have been developed for the estimation of cefdinir in pharmaceutical dosage forms. Difference spectroscopic method is based on the measurement of absorbance of cefdinir at maxima 265 nm and minima 230 nm. The measured value is the amplitude of maxima and minima between two eqimolar solutions of the analyte in different chemical forms, which exhibit different spectral characteristics. Beer's law was obeyed in the concentration range of 10 to 35 mg/ml. The second method, a High Performance Liquid Chromatography, was developed for the estimation of cefdinir, using 50 mM ammonium acetate (pH 3.0±0.1 adjusted with 10% phosphoric acid) and methanol (80:20% v/v) as the mobile phase with flow rate of 1 ml/min, and measuring the response at 285 nm. An external standard calibration method was employed for quantitation. Beer's law was obeyed in the concentration range of 15 to 125 mg/ml. The results obtained in the analysis of dosage forms agree well with the labelled contents.

Interaction of Arsenic Trioxide As2O3with DNA and RNA

Arsenic salts have been used for centuries to treat a variety of medical conditions ranging from infectious disease to cancer. More recently, trivalent arsenic trioxide was found to exhibit high antitumor activity towards hematological malignancies. Even though much is known about antitumor activity and DNA damage by As2O3, there has been no report on the interaction of arsenic trioxide with isolated DNA or RNA. Therefore, it was of interest to examine the interaction of As2O3 with DNA and RNA in aqueous solution at physiological pH. FTIR and UV-visible difference spectroscopic methods were used to characterize the nature of drug-DNA and drug-RNA interactions and to determine the As binding site, the binding constant, the sequence selectivity, the helix stability, and the biopolymer secondary structure in the As2O3-polynucleotide complexes in vitro. The FTIR spectroscopic studies were conducted with As2O3-polynucleotide (phosphate) ratios of 1/40, 1/20, 1/10, and 1/5, with a final DNA (P) or RNA (P) concentration of 6.25 mmol/l. Spectroscopic results showed As2O3 binds to DNA and RNA at G-C, A-T, and A-U bases, and no interaction with the backbone PO2 group. As2O3-DNA and -RNA adducts showed one type of binding with overall binding constant of K(As2O3-DNA) = 1.24 x 10(5) M(-1) and K(As2O3-RNA) = 2.60 x 10(5) M(-1). The As2O3-polynucleotide complexation is associated with a partial biopolymer aggregation and no major alterations of B-DNA or A-RNA structure.

A Comparative Study of Fe(II) and Fe(III) Interactions with DNA Duplex: Major and Minor Grooves Bindings

The involvement of the Fe cations in autoxidation in cells and tissues is well documented. DNA is a major target in such reaction, and can chelate Fe cation in many ways. The present study was designed to examine the interaction of calf-thymus DNA with Fe(II) and Fe(III), in aqueous solution at pH 6.5 with cation/DNA (P) (P = phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the cation binding site, the binding constant, helix stability and DNA conformation in Fe-DNA complexes. Structural analysis showed that at low cation concentration (r = 1/80 and 1/40), Fe(II) binds DNA through guanine N-7 and the backbone PO(2) group with specific binding constants of K(G) = 5.40 x 10(4) M(1) and K(P) = 2.40 x 10(4) M(1). At higher cation content, Fe(II) bindings to adenine N-7 and thymine O-2 are included. The Fe(III) cation shows stronger interaction with DNA bases and the backbone phosphate group. At low cation concentration (r = 1:80), Fe(III) binds mainly to the backbone phosphate group, while at higher metal ion content, cation binding to both guanine N-7 atom and the backbone phosphate group is prevailing with specific binding constants of K(G) = 1.36 x 10(5) M(-1) and K(P) = 5.50 x 10(4) M(-1). At r = 1:10, Fe(II) binding causes a minor helix destabilization, whereas Fe(III) induces DNA condensation. No major DNA conformational changes occurred upon iron complexation and DNA remains in the B-family structure.

DNA Interaction with Naturally Occurring Antioxidant Flavonoids Quercetin, Kaempferol, and Delphinidin

Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. However, there has been no information on the interactions of these antioxidants with individual DNA at molecular level. This study was designed to examine the interaction of quercetin (que), kaempferol (kae), and delphinidin (del) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.5 mmol) and various drug/DNA(phosphate) ratios of 1/65 to 1. FTIR and UV-Visible difference spectroscopic methods are used to determine the drug binding sites, the binding constants and the effects of drug complexation on the stability and conformation of DNA duplex. Structural analysis showed quercetin, kaempferol, and delphinidin bind weakly to adenine, guanine (major groove), and thymine (minor groove) bases, as well as to the backbone phosphate group with overall binding constants Kque = 7.25 × 104M−1, Kkae = 3.60 × 104M−1, and Kdel = 1.66 × 104M−1. The stability of adduct formation is in the order of que>kae>del. Delphinidin with a positive charge induces more stabilizing effect on DNA duplex than quercetin and kaempferol. A partial B to A-DNA transition occurs at high drug concentrations.

Interaction of antitumor drug Sn(CH3)2Cl2 with DNA and RNA

Sn(CH3)2Cl2 exerts its antitumor activity in a specific way. Unlike anticancer cis-Pt(NH3)2Cl2 drug which binds strongly to the nitrogen atoms of DNA bases, Sn(CH3)2Cl2 shows no major affinity towards base binding. Thus, the mechanism of action by which tinorganometallic compounds exert antitumor activity would be different from that of the cisplatin drug. The aim of this study was to examine the binding of Sn(CH3)2Cl2 with calf thymus DNA and yeast RNA in aqueous solutions at pH 7.1–6.6 with constant concentrations of DNA and RNA and various molar ratios of Sn(CH3)2Cl2/DNA (phosphate) and Sn(CH3)2Cl2/RNA of 1/40, 1/20, 1/10, 1/5. Fourier transform infrared (FTIR) and UV–visible difference spectroscopic methods were used to determine the Sn(CH3)2Cl2 binding mode, binding constant, sequence selectivity and structural variations of Sn(CH3)2Cl2/DNA and Sn(CH3)2Cl2/RNA complexes in aqueous solution. Sn(CH3)2Cl2 hydrolyzes in water to give Sn(CH3)2(OH)2 and [Sn(CH3)2(OH)(H2O)n]+ species. Spectroscopic evidence showed that interaction occurred mainly through (CH3)2Sn(IV) hydroxide and polynucleotide backbone phosphate group with overall binding constant of K(Sn(CH3)2Cl2–DNA)=1.47×105M−1 and K(Sn(CH3)2Cl2–RNA)=7.33×105M−1. Sn(CH3)2Cl2 induced no biopolymer conformational changes with DNA remaining in the B-family structure and RNA in A-conformation upon drug complexation.

Taxol Anticancer Activity and DNA Binding

The interaction of taxol with DNA has major biological importance since it is shown the presence of higher concentration of taxol in the nucleus, than in the human lung tumor cell. Therefore, in this report we examine the interaction of taxol with calf-thymus DNA in aqueous solution at physiological pH, using constant DNA concentration (25 or 1.25 mM phosphate) and various taxol/DNA (phosphate) ratios 1/200 to 1/2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods are used to characterize the nature of drug-DNA interaction and to determine the taxol binding site, the binding constant, sequence selectivity, helix stability and biopolymer secondary structure in the taxol-DNA complexes in vitro. Structural analysis showed that taxol is an external DNA binder with no affinity towards DNA intercalation. The major target of taxol is A-T, G-C bases and the backbone PO(2) groups. Two bindings were observed for taxol-DNA complexes with K(1)= 1.4 x 10(4) M(-1) and K(2)=3.5 X 10(3) M(-1). The taxol-DNA interaction is associated with a partial helix stabilization and no major alterations of B-DNA structures.

AZT Binds RNA at Multiple Sites

Azidothymidine (AZT) is a widely used inhibitor of type I human immunodeficiency virus (HIV) reverse transcriptase that act as a DNA chain terminator. Studies have shown primer unblocking and rescue of DNA synthesis AZT-resistant HIV-1 reverse transcriptase on DNA and RNA templates. Our recent study showed AZT bindings to the G-C, A-T base pairs and the backbone phosphate group of DNA duplex resulting in partial DNA conformational changes. This study was designed to examine the interaction of AZT with RNA in aqueous solution at physiological condition, using different drug/RNA (phosphate) molar ratios of 1/800 to 1/2 and constant RNA concentration of 1.25 or 12.5 mM (phosphate). Capillary electrophoresis, FTIR, and UV-visible difference spectroscopic methods and molecular modeling were used to determine the drug binding sites, binding constants, and the effects of AZT complexation on RNA conformation. Structural analysis showed that AZT binds RNA through G-C and A-U bases with two binding constants of K1=7.3 x 10(5) M(-1) and K2=1.90 x 10(5) M(-1). The drug distributions were 54% with G-C, 36% A-U, and 10% with the backbone phosphate group. RNA remains in A-family structure and drug sugar pucker in the C2'-endo/anti conformation in the AZT-RNA complexes. Molecular modeling studies show hydrogen bondings between RNA and AZT donor groups.