The High Performance Liquid Chromatography Biology Essay

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Analytical chemistry may be derived as the science and art of determining the composition of material in terms of the elements of compounds contained. By means of analytical techniques both qualitative analysis (the presence or absence of one or more elements) and quantitative analysis (how much amount is present) can be done.

The qualitative and quantitative analysis can be done by various analytical methods: various analytical techniques can be revised and some of them give accurate result,


Chromatography: Any of various techniques used for analyzing or separating a sample mixture of gases, liquids, or dissolved substances, based on the differential competition for molecules of the sample between a mobile phase sample and a stationary phase sample

Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.

The analyte is the substance to be separated during chromatography.

Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample.

A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.

A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.


Gas chromatography; Gel permeation chromatography; Liquid chromatography; Supercritical fluid chromatography; Column Chromatography; Liquid Chromatography; Ion-Exchange Chromatography; Thin-Layer and Paper Chromatography; Electrophoresis; HPLC


HPLC was introduced commercially in 1969 and since then it has undergone extensive modifications and innovation which lead to its emergence as the foremost analytical tool for quantitative analysis. HPLC is a type of liquid chromatography that employs a liquid mobile phase and a very finely divided stationary phase. In order to obtain a satisfactory flow rate liquid must be pressurized to a few thousands of pounds per square inch.

The rate of distribution of drugs between stationary and mobile phase is controlled by diffusion process. If diffusion is minimized, a faster and effective separation can be achieved. The technique of high performance liquid chromatography is so called because of its improved performance when compared to classical column chromatography. Advances in column technology, high pressure pumping system and sensitive detectors have transformed liquid column chromatography into high speed, efficient, accurate and highly resolved method of separation.

For the present study, the drug Nevirapine was selected for their estimation. The HPLC method was considered the choice of estimation, since this method is the most powerful of all chromatographic and other separative methods. The HPLC method has enabled analytical chemist to attain great success in solving his analytical problems.

The HPLC is the method of choice in the field of analytical chemistry, since this method is precise, accurate and linear and also it offers the following advantages.

Speed (many analysis can be accomplished in 20 minutes or less).

Greater sensitivity (various detectors can be employed).

Improved resolution (wide variety of stationary phases)

Reusable columns (expensive columns but can be used for many analysis).

Ideal for the substances of low volatility.

Easy sample recovery, handling and maintenance.

Instrumentation leads itself to automation and quantitation (less time and less labour)

Precise and reproducible.

Calculations are done by integrator itself.

Suitable for preparative liquid chromatography on a much large scale.

Types of HPLC

The type of HPLC methods include:

Normal phase chromatography

Reversed phase chromatography.

Normal Phase Chromatography

The term normal phase refers to a system where a stationary phase is polar and mobile phase is a relatively non polar liquid (hexane, benzene, CHCL€³, etc). In this mode most probably used stationary phase is silica gel. The silica structure is saturated with silanol groups at the end and 'OH' groups attached to silicon atoms are the active binding sites. Best separating compounds include plasticizer, dyes, steroids, amines, alkaloids, alcohols, phenols, aromatic and metal complexes.

Reversed Phase Chromatography

A most popular mode for analytical and preparative method for separation of compounds of interest in chemical, biological, pharmaceutical, food and biomedical sciences. In this mode the stationary phase is a non polar hydrophobic packing with octyl or octadecyl functional group bonded to the silica gel and mobile phase is a polar solvent. An aqueous mobile phase allows the use of secondary solute chemical equilibria (such as ionization control, ion suppression, ion pairing and complexation) to control retention and selectivity.

The polar compound gets eluted first in this mode and non-polar compounds are retained for a larger time. Since most of the drugs and pharmaceuticals are polar in nature, they are not retained for longer time and eluted faster. The different columns used are octadecyl silane (ODS) or C18, C8, C4 etc (In order to increase the polarity of the stationary phase). Hence by varying the organic moiety in the silianization reagent (dimethyl chlorosilane derivatives) different stationary phase of polarities can be realized.

Importance of Polarity in HPLC

The relative distribution of solute between two phases is determined by the interactions of the solute species with each phase. In both normal phase and reversed phase HPLC, the eluting power or solvent strength of the mobile phase is mainly determined by its polarity. The relative strengths of these interactions are determined by the polarity of the sample and the mobile and stationary phase.

Reverse Phase Mobile Phases

The power of HPLC in terms of being able to resolve many compounds is mainly due to the diversity of mobile phases or mobile solvents available. The mobile phase in HPLC, however, has a great influence on the retention of the solutes and the separation of component mixtures.

The primary constituent of reversed phase-mobile phase is water. Water miscible solvents such as methanol, ethanol, acetonitrile, dioxane, tetrahydrofuran and dimethyl formamide are added to adjust the polarity of the mobile phase. They should be of high quality, either distilled or demineralised. The most widely used organic modifiers are methanol, acetonitrile and tetrahydrofuran. Methanol and acetonitrile have comparable polarities but the latter is an aprotic solvent. This factor may be important if hydrogen bonding plays a significant role in the separation. When inorganic salts and ionic surfactants are used, the mobile phase should be filtered before use since these additives frequently contain a significant amount of water-insoluble contaminants that may damage the column. Degassing is quite important with reverse-phase mobile phases.

Polarity is a term that is used in chromatography as an index of the ability of compounds to interact with one another. It is applied very freely to solute, stationary and mobile phase.

If the polarities of stationary phase and the mobile phase are similar, it is likely that the interactions of solute with each phase may also be similar, resulting in poor separation. Retention of solutes is usually altered by changing the polarity of the mobile phase. Successful chromatographic separation requires a proper balance of intermolecular force among three participants in separation process i.e Analyte, Mobile phase and Stationary phase.

Methods of Quantitative Analysis in HPLC

The sample or solute is analysed quantitatively in HPLC by either peak height or peak area measurements. Peak areas are proportional to the amount of constant rate. Peak heights are proportional to the amount of material only when peak width are constant and are strongly affected by the sample injection techniques. Once the peak height or the peak areas are measured, there are five principle evaluation methods for quantifying the solute3.

a) Calibration by Standards

Calibration curves for each component are prepared from pure standards, using identical injection volumes of operating conditions for standards and samples. The concentration of solute is read from its curve if the curve is linear.

X = K x area

Where, X = Concentration of solute

K = Proportionality constant (slope of the curve)

In this evaluation method only the area of the peaks of interest is measured. Relative response factors must be considered when converting areas to volume and when the response of a given detector differs for each molecular type of compounds.

b) Internal Standard Method

In this technique a known quantity of the internal standard is chromatographic and area vs. concentration is ascertained. Then a quantity of the internal standard is added to the raw sample prior to any sample pretreatment or separation operations. The peak area of the standard in the sample run is compared with the peak area when the standard is run separately. This ratio serves as a correction factor for variation in sample size, for losses in any preliminary pretreatment operations, or for incomplete elution of the sample. The material selected for the internal standard must be completely resolved from adjacent sample components, must not interfere with the sample components and must never be present in samples.

Area of sample

Area ratio = -----------------------------------

Area of internal standard

Area ratio of sample

Sample concentration = -----------------------------

Area ratio of standard

c) Area Normalization

The technique is often used for the sample having identical components. It is used to evaluate the absolute purity of the sample. The procedure is to total up the areas under all peaks and then calculate the percentage of the total area that is contributed by the compound of interest. For this method the entire sample must be eluted, all components must be separated and each peak must be completely resolved.

d) Standard Addition Method

If only few samples are to be chromatographed, it is possible to employ the method of standard addition(s). The chromatogram of the unknown is recorded, then a known amount of analyte(s) is added and the chromatogram is repeated using same reagents, instruments and other conditions. From the increase in the peak area (or peak height), the original concentration can be computed by interpolation.

The detector response must be a linear function of analyte concentration and yield no signal at zero concentration of the analyte. Sufficient time must elapse between addition of the standard and actual analysis to allow equilibrium of added standard with any matrix interferant.

If an instrumental reading (area/height) 'Rx' is obtained, from a sample of unknown 'x' and a reading 'Rt' is obtained from the sample to which a known concentration 'a' of analyte has been added, then 'x' can be calculated from:

X Rx

-------- = ----------

x+a Rt

A correction for dilution must be made if the amount of standard added changes the total sample volume significantly. It is always advisable to check the result by adding at least one other standard.

e) External Standard Method

It employs a separate injection of a fixed volume of sample and standard solution. The peaks are integrated and concentration is calculated.

Peak area of sample

Sample concentration = ------------------------------- x Conc. Of Standard

Peak area of standard

The selection of suitable chromatographic (HPLC) system for a given mixtures of solutes cannot be made with certainty and must be confirmed by experiment. If the chemical nature of the sample components is known, then the phase system can be selected from the literature references. If nothing is known about the chemical nature of sample, then the sample solubility will give some indication as to which chromatographic method to employ. The essential parts of high performance liquid chromatographic system are solvent reservoir, pump, injection port, column, detector and recorders.

Solvent reservoir

Stainless steel and glass are used for making solvent reservoir. They should be inert to a variety of aqueous and non-aqueous mobile phases. Stainless steel should be avoided for solvents containing halide ions. If the reservoir is to be pressurized, glass is to be avoided. The capacity of the reservoir should be greater than 500ml. The aqueous and organic solvents are degassed prior to use in order to prevent the formation of gas bubbles in the detector. Degassing is done by following methods.

By stirring the mobile phase in vacuum

Purging with helium gas and


Finally the solvent is filtered through Millipore filter before introducing into the reservoir.


the pumps must be constructed from materials that are inert to all mobile phases. Materials used are stainless steel, glass and Teflon. They should generate a pressure up to 800 psi at a flow rate of up to 3ml/minute and should provide pulse less solvent flow. The solvent should be dampened to remove pulses, since the presence of pulses in solvent flow may cause spurious results with some detectors. The pump should produce reproducible and constant flow rate HPLC pumps can be classified into two groups according to the manner in which they operate. They are constant flow rate pumps and constant pressure pump. The two principle types of constant flow rate pump are reciprocating piston pump and syringe dive pump. Reciprocating pump has filling and pumping cycle. During the filling cycle a piston is withdrawn from a syringe type chamber. Two check-valves are connected to this chamber such that during the piston withdrawal, solvent flows from the reservoir to the pump outlet. The volume of solvent discharged from the pump in unit time can be changed by altering the distance that the piston travels (or) the number of cycles. The advantage of this pump is unlimited volume of the solvent reservoir since it is external to the pump.

Syringe drive pump is a single stroke displacement pump in which all of the mobile phase is contained within the pump. The piston inside the chamber is actuated by a screw feed drive connected to a stepping motor. The volume displaced by the pump per unit time is controlled by the voltage applied. This pump produces a pulse less flow and requires no check valves.

Constant pressure pump can deliver a constant flow rate if the pump operates against a constant column back pressure and if the viscosity of the mobile phase remaining constant. Constant pressure pump may be simple gas displacement pump (or) pneumatic amplifier pump simple gas displacement pump is a reservoir such as coil of tubing to which pressure is applied from a gas cylinder. The disadvantage of this pump is limited solvent capacity. Pneumatic amplifier pump is a modification of simple gas displacement pump. The gas pressure is applied to a large piston, which is connected to a small diameter piston, which is in contact with mobile phase when all the mobile phase is used up, the piston returns quickly by pneumatic means, thus refilling the chamber. The advantage of this pump is pulse less flow and unlimited solvent capacity.

Sample injection system

The injection of sample on to the column presents some unique problems because of high pressure involved in HPLC. The volume of sample used ranges from 0 to 500 μl. The various injection methods are

Syringe Injection

The sample is injected by two method using micro syringes, which are designed to with stand pressure up to 1500 psi. The injection is done through a self-sealing elastomeric septum.

Stop Flow Injection

The type of syringe injection can be made as high pressure but not through the septum. Here the flow of the solvent is momentarily stopped and the sample is directly injected on to the head of the column. This method is simple and convenient.

Loop Injection (or) Sampling Valve

This system is the most widely used injection system, which provides precise injection volumes against high back pressures. In this method a sample loop of fixed capacity is connected to a high-pressure valve and the sample is filled onto the sample loop through a syringe.


HPLC column are made up of stainless steel or glass, which differs in length and inside diameter depending on the application. The two types of columns are analytical column and preparative column. Standard analytical columns are 4-5mm in internal diameter and 10-30 cm in length. The particle size used ranges from 5-10 micrometers. Preparative columns are 20-50 mm in internal diameter and 20-100 cm in length. The particle size used ranges from 37-50 micrometers.


All detectors used in HPLC may be of selective type and non-selective type in which the former detectors only a part of the components and the later detects almost all the components. The choice of the detector depends on the mobile phase, nature of the analyte and the required sensitivity. The detectors used are Refractive index detector, U.V. absorption detector, fluorescence detector, electrochemical detector, mass detector and radioactive detector.


The signals from the detector are recorded as deviation from a baseline. Two open recorders are used with instruments having two detectors. The peak position along the curve relative to the starting point, denotes the particular component, with proper calibration the height or area of peak is a measure of the amount of the component present in the sample.

Best column, best mobile phase, best detection wavelength, efforts in their selection can make a world of difference while development HPLC method for routine analysis. Determining the ideal combination of these factors assures faster delivery of desired results a validated method for separation.


The word "validation" means assessment of validity or action of validity or action of providing effectiveness.


FDA defines validation as establish the documented evidence which provides a high of assurance that a specific process will consistently produces a product of predetermined specifications and quantity attributes .

EUMGP define validation as "action of proving in accordance with the principle of Good manufacturing practice"(GMP),That any material, activity or system actually lead to expected result


The action of proving that any material, process, activity, procedure, system, equipment or mechanism and intended result.


Method validation is the documented successful evolution of an analytical method that provides a high level assurance that such method will consistently yield result that are accurate with in previously established specifications.


The primary objective of validation is to form a basis for written procedures for production of process which are designed to assure that the drug product have the identity, quality and purity. They are represented to possess

Assurance of quantity

Govern met regulation

Analytical method validation:

Method validation is the process for establishing that performance characteristics of the analytical method are suitable for the intended application.

Chromatographic methods need to be validation before first routine use. To obtain the most accurate results, all of the variables of the method should be considered, including sampling procedure, sampling preparation, chromatographic separation, detection and data evaluation using the same matrix as that of the intended sample. The validity of an analytical method can only be verified by laboratory studies. All validation experiments us to make claims or conclusions about validity of the method should be documented in report.


Identification test for impurities

Quantitative test for impurities

Limit test control of impurities

Quantitative test for the active moiety in samples of drug substance or drug product or other selected components in the drug product.


REGULATORY compliance

Minimize rejection and reworking

Minimize utility cost

Minimize complaints

Reduce testing requirements

More rapid and reliable start-up new equipment

Easier scale-up from development

Easier maintainace of equipment

More rapid automation


The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) is a project that brings together the regulatory authorities of Europe, Japan and the United States and experts from the pharmaceutical industry in the three regions to discuss scientific and technical aspects of pharmaceutical product registration. The purpose of ICH is to reduce or obviate the need to duplicate the testing carried out during the research and development of new medicines




(d)Limit of detection

(e)Limit of quantitaion



(a)ACCURACY: The accuracy of a measurement system is the degree of closeness of measurements of a quantity to its actual (true) value.

(b)PRECISON: The precision of a measurement system, also called reproducibility or repeatability, is the degree to which repeated measurements under unchanged conditions show the same results















(d)LIMIT OF DETECTION: The detection limit, lower limit of detection, or LOD (limit of detection), is the lowest quantity of a substance that can be distinguished from the absence of that substance (a blank value) within a stated confidence limit (generally 1%).

(f)ROBUSTNESS: It is the method of capacity of an assay to remain unaffected by small but deliberated variation in method parameters and provide an indication of its reliability in normal usage degradation and variation in chromatography columns, mobile phase and inadequate method development are common cause of lack of robustness.

(g)ROGUDNESS: Ruggedness is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of test conditions such a different laboratories, analysis, instrument, reagent, assay times, temperature, days etc. It can be expressed as lack influence of the operation and environmental variable on the test results of the analytical method


Although various methods have been developed for the estimation of Nevirapine by HPLC method, as per literature review of the methods like UV-spectrophotometer, and HPLC for the estimation these drug individually and in combination with other drugs. But I am trying to develop a new estimation method of this drug by RP- HPLC method individually.


1)Lokamatha KM et al.,: Nevirapine (NVP) is an antiretroviral drug, classified as Biopharmaceutics Classification System (BCS) Class II drug, has a drawback of variable dissolution rates with resultant decrease in oral bioavailability. In the present study, attempts were made to improve the aqueous solubility and dissolution rate of NVP via complexation with β-cyclodextrin (β-CD). The complexation of NVP with β-CD was investigated by phase solubility studies in pH 1.2 and pH 6.8 as the NVP exhibited pH dependent solubility. Solid binary complexes (1:1M) were made by kneading, solvent evaporation and microwave method. All solid complexes were characterized by performing dissolution studies in 0.1 N HCl and pH 6.8 and by analytical techniques such as DSC, FT-IR, P-XRD and SEM. The phase solubility profiles were classified as AL-type, indicating the formation of 1:1 inclusion complex. Stability constants (K1:1) calculated from the phase solubility diagrams were found to be pH dependent. Analytical studies confirmed the formation of inclusion complexes with β-CD. All binary systems exhibited higher dissolution rates in 0.1 N HCl and pH 6.8 than their corresponding mixtures and pure drug. The statistical analysis showed that binary system prepared by microwave method was much superior to the others (P < 0.05). The release of drug from the preparations is followed predominately first order kinetics compared to Hixson-Crowell's cube root law. The prepared solid complexes reflect the vital role of β-CD to improve the solubility and dissolution rate of NVP, both in gastric and intestinal pH via complexation process, which could minimize the variable dissolution rates with increase in the oral bioavailability.]

2)PURNIMA D. et al.,: A simple, accurate and precise HPTLC method has been developed and validated for the estimation of nevirapine from bulk drug and tablet formulations. The separation was achieved on TLC plates using appropriate solvent system. The spots so developed were densometrically scanned at 283 nm. The linearity of the method was found to be within the concentration range of 2.50μg/ml to 62.50μg/ml. The validation parameters, tested in accordance with the requirements of ICH guidelines, prove the suitability of this method. The method was successfully applied for determination of drug in tablets, wherein no interference from tablet excipients was observed, indicating the specificity of the developed method. Thus the proposed method can be used successfully for routine analysis of nevirapine from capsule and tablet formulations.

3)Prasada Rao CH et al.,: A reverse phase high performance liquid chromatography [RP-HPLC] method has been developed for the estimation of Nevirapine in bulk drug and pharmaceutical dosage forms. The quantification was carried out on Octa Decyl Silane column in isocratic mode, with mobile phase consisting of methanol and acetate buffer in the ratio of 60:40[v/v].The mobile phase was pumped at a rate of 1.0 ml/min and the detection was carried out at 280 nm and the linearity was found to be in the range of 25 to 100 μg/ml. The regression equation was found to be Y=18984x +6262.9 with correlation coefficient [r2] of 0.9998. The % recovery values were found to be in the range of 100.18-101.08%. The proposed method was validated for accuracy and precision. Statistical analysis proves that the method was found to be simple, precise, accurate, rapid and reproducible and can be used for the routine determination of Nevirapine in bulk drug and in pharmaceutical formulations

4)Kuo YC, Chung JF et al., : Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) coated with human serum albumin (HSA) were fabricated for formulating nevirapine (NVP). Here, NLCs contained low-melting-point oleic acid (OA) in the internal lipid phase. The results revealed that the two nanoparticles were uniformly distributed with the average diameter ranging from 145 to 180 nm. The surface HSA neutralized the positive charge of dimethyldioctadecyl ammonium bromide (DODAB) on SLNs and NLCs and reduced their zeta potential. In a fixed ratio of solid lipids, SLNs entrapped more NVP than NLCs. The incorporation of OA also reduc ed the thermal resistance of NLCs and accelerated the release of NVP from the nanocarriers. When incubated with DODAB-stabilized SLNs, the viability of human brain-microvascular endothelial cells (HBMECs) reduced. However, the surface HSA increased the viability of HBMECs about 10% when the concentration of SLNs was higher than 0.8 mg/mL. HSA-grafted SLNs and NLCs can be effective formulations in the delivery of NVP for viral therapy.

5)Zhou XJ, Sheiner LB, et al: The population pharmacokinetics of nevirapine (NVP), zidovudine (ZDV), and didanosine (ddI) were evaluated in a total of 175 patients infected with human immunodeficiency virus randomized to receive either a double combination of ZDV plus ddI or a triple combination of NVP plus ZDV plus ddI as a substudy of the AIDS Clinical Trials Group Protocol 241. Levels (approximating 3.5 determinations/patient) of the three drugs in plasma were measured during 44 of a total 48 weeks of study treatment, and a set of potential covariates was available for nonlinear mixed-effect modeling analysis. A one-compartment model with zero-order input and first-order elimination was fitted to the NVP data. Individual oral clearance (CL) and volume of distribution (V) averaged 0.0533 liters/h/kg of body weight and 1.17 liters/kg, respectively. Gender was the only covariate which significantly correlated with the CL of NVP. ZDV and ddI data were described by a two-compartment model with zero-order input and first-order elimination. Individual mean oral CL, VSS (volume of distribution at steady state), and V of ZDV were 1.84 liters/h/kg and 6.68 and 2.67 liters/kg, respectively, with body weight and age as correlates of CL and body weight as a correlate of VSS. The average individual oral CL, VSS, and V of ddI were 1.64 liters/h/kg and 3.56 and 2.74 liters/kg, respectively, with body weight as a significant correlate of both CL and VSS. The relative bioavailability (F) of ZDV and ddI in the triple combination compared to that in the double combination was also evaluated. No significant effects of the combination regimens on the F of ddI were detected (FTRIPLE = 1.05 and FDOUBLE = 1 by definition), but the F of ZDV was markedly reduced by the triple combination, being only 67.7% of that of the double combination. Large (>50%) intraindividual variability was associated with both ZDV and ddI pharmacokinetics. Individual cumulative area under the plasma drug level-time curve of the three drugs was calculated for the entire study period as a measure of drug exposure based on the individual data and the final-model estimates of structural and statistical par

6) Renu chada et al.,: The study is aimed at exploring the utility of thermoanalytical methods in the solid-state characterization of various crystalline forms of nevirapine. The different forms obtained by recrystallization of nevirapine from various solvents were identified using differential scanning calorimetry and thermogravimetric analysis (TGA). The appearance of desolvation peak accompanied by weight loss in TGA indicated the formation of solvates: hemi-ethanolate (Form I), hemi-acetonitrilate (Form II), hemi-chloroformate (Form III), hemi-THF solvate (Form IV), mixed hemi-ethanolate hemi-hydrate (Form V), and hemi-toluenate (Form VI). The higher desolvation temperatures of all the solvates except toluenate than their respective boiling point indicate tighter binding of solvent. Emphasis has been laid on the determination of heat capacity and heat of solution utilizing microreaction calorimeter to further distinguish the various forms. The enthalpy of solution (ΔH sol), an indirect measure of the lattice energy of a solid, was well correlated with the crystallinity of all the solid forms obtained. The magnitude of ΔH sol was found to be −14.14 kJ/mol for Form I and −2.83 kJ/mol for Form V in phosphate buffer of pH 2, exhibiting maximum ease of molecular release from the lattice in Form I. The heat capacity for solvation (ΔC p) was found to be positive, providing information about the state of solvent molecules in the host lattice. The solubility and dissolution rate of the forms were also found to be in agreement with their enthalpy of solution. Form (I), being the most exothermic, was found to be the most soluble of all the forms.

7) Rosamaria Mangiacasale et al.,: Endogenous, nontelomeric reverse transcriptase (RT) is encoded by two classes of repeated elements: retrotransposons and endogenous retroviruses. Expression of RT-coding genes is generally repressed in differentiated nonpathological tissues, yet is active in the mammalian germ line, embryonic tissues and tumor cells. Nevirapine is a non-nucleoside RT inhibitor with a well-characterized inhibitory activity on RT enzymes of retroviral origin. Here, we show that nevirapine is also an effective inhibitor of the endogenous RT in murine and human cell lines. In addition, progenitor and transformed cells undergo a significant reduction in the rate of cell growth upon exposure to nevirapine. This is accompanied by the onset of differentiation, as depicted in F9 and C2C7 progenitor cells cultures in which nevirapine triggers the expression of differentiation-specific markers. Consistent with this, an extensive reprogramming of cell cycle gene expression was depicted in nevirapine-treated F9 cultures. Furthermore, nevirapine exposure rescued the differentiation block present in acute myeloid leukemia (AML) cell lines and primary blasts from two AML patients, as indicated by morphological, functional and immunophenotypic assays. The finding that an RT inhibitor can modulate cell proliferation and differentiation suggests that RT may represent a novel target in the development of therapeutical approaches to neoplasia.


Chemical structure

Chemical name : 11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido [3,2-b:2',3'-e][l,4] diazepin-6-one.

Molecular formula : C15H14N4O

Molecular weight : 266.30

Description : a white to off-white crystalline powder

Solubility : soluble in Dimethyl Sulphoxide (DMSO) and Sparingly soluble in

Dichloro methane(MDC) and in dimethyl formamide(DMF).

Melting point : 247 to 249 C

Sulfated ash : Not more than 1.0 mg/g.

Loss on drying : 0.5 % max

Category : Antiretroviral (Non-Nucleoside Reverse Transcriptase Inhibitor)

Storage :Store tablets and oral suspension at 25 C (77F), with excursions permitted

between 15 C to 30 C (59 F to 86 F).

Mechanism of action:

Nevirapine falls in the non-nucleoside reverse transcriptase inhibitor (NNRTI) class of antiretrovirals. Both nucleoside and non-nucleoside RTIs inhibit the same target, the reverse transcriptase enzyme, an essential viral enzyme which transcribes viral RNA into DNA. Unlike nucleoside RTIs, which bind at the enzyme's active site, NNRTIs bind allosterically at a distinct site away from the active site termed the NNRTI pocket.

Nevirapine is not effective against HIV-2, as the pocket of the HIV-2 reverse transcriptase has a different structure, which confers intrinsic resistance to the NNRTI class.

Resistance to nevirapine develops rapidly if viral replication is not completely suppressed. The most common mutations observed after nevirapine treatment are Y181C and K103N, which are also observed with other NNRTIs. As all NNRTIs bind within the same pocket, viral strains which are resistant to nevirapine are usually also resistant to the other NNRTIs, efavirenz and delavirdine.


Tiredness, nausea, vomiting, or diarrhea may occur. Drowsiness may rarely occur.


Nevirapine is readily absorbed ( > 90%) after oral administration in healthy volunteers and in adults with HIV-1 infection. Absolute bioavailability in 12 healthy adults following single-dose administration was 93 &pluamn; 9% (mean &pluamn; SD) for a 50 mg tablet and 91 &pluamn; 8% for an oral solution. Peak plasma nevirapine concentrations of 2 &pluamn; 0.4 ?g/mL (7.5 ?M) were attained by 4 hours following a single 200 mg dose. Following multiple doses, nevirapine peak concentrations appear to increase linearly in the dose range of 200 to 400 mg/day. Steady-state trough nevirapine concentrations of 4.5 &pluamn; 1.9 ?g/mL (17 &pluamn; 7 ?M), (n=242) were attained at 400 mg/day. Nevirapine tablets and suspension have been shown to be comparably bioavailable and interchangeable at doses up to 200 mg. When VIRAMUNE (200 mg) was administered to 24 healthy adults (12 female, 12 male), with either a high-fat breakfast (857 kcal, 50 g fat, 53% of calories from fat) or antacid (Maalox? 30 mL), the extent of nevirapine absorption (AUC) was comparable to that observed under fasting conditions. In a separate study in HIV-1 infected patients (n=6), nevirapine steady-state systemic exposure (AUC?;) was not significantly altered by didanosine, which is formulated with an alkaline buffering agent. VIRAMUNE may be administered with or without food, antacid or didanosine.


Nevirapine is highly lipophilic and is essentially nonionized at physiologic pH. Following intravenous administration to healthy adults, the apparent volume of distribution (Vdss) of nevirapine was 1.21 &pluamn; 0.09 L/kg, suggesting that nevirapine is widely distributed in humans. Nevirapine readily crosses the placenta and is also found in breast milk [see Use In Specific Populations]. Nevirapine is about 60% bound to plasma proteins in the plasma concentration range of 1-10 ?g/mL. Nevirapine concentrations in human cerebrospinal fluid (n=6) were 45% (&pluamn;5%) of the concentrations in plasma; this ratio is approximately equal to the fraction not bound to plasma protein. Metabolism/Elimination



Shimadzu HPLC, 10 AT detector, Rheodyne injector with 20µl loop was used, mode of operation - Isocratic and Temperature ambient

HPLC columns -Inertsil ODS C18 Column 150 X 4.6 mm , 5 µ

Electronic Balance

Ultra Sonicator

Thermal Oven

pH analyzer

Triple Quartz Distillation Unit

HPLC Injecting Syringe (25 ml) (HAMILTON)


Acetonitrile HPLC grade

Methanol HPLC grade

Water-HPLC grade

Phosphoric acid HPLC grade



Drug is dissolved in suitable solvents and the resulting solution was filtered through millipore filter paper then the solution was injected in HPLC and the chromatogram was recorded from the chromatogram we detected retention time, peak area height, purity of the drug.


INSTRUMENT : shimadzu

COLUMN : BDS Hypersil C18 150 x 4.6 mm, 5µm



FLOW RATE :1ml/minute


MOBILE PHASE : Acetonitrile : Buffer (30:70)

RUN TIME : 4.083 min


From the optical characteristics of these proposed methods, it was found that trail method II for Nevirapine obey Accuracy, Precision and Linearity within the concentration respectively.

From the results, it was found that the percentage recovery values of pure drugs from the pre analyzed solution of formulation were 99.31% for Nevirapine, which indicates that the proposed method is accurate and also reveals that the commonly used excipients and additives in the pharmaceutical formulations were not interfering in the proposed method.






















Standard deviation


Retention time





















Area obtained

Average area

Actual result




































The Robustness studies were performed by changing the wavelength and flow rate and reported in table 3.4

Table 3.4


Robustness study



Peak area

Assay (mg)

Assay %


Wave length







Flow rate

1 ml/min

1.1 ml/min





All parameters including flow rate, temperature, detection, wave length, and sensitivity were maintained constant throughout the procedure except for robustness study.

Table 3.5



Date of analysis




Satish teki





Basha khan





Satish teki





Basha khan




A HPLC method was developed for estimation of Nevirapine in tablet dosage form using HPLC chromatography.

Shimadzu HPLC was used with column BDS Hypersil C18 150 x 4.6 mm, 5µm at wave length 243nm at ambient temperature. Flow rate was set at 1ml/min and injection volume was 1ml. Acetonitrile : sodium phosphate(30 : 70) and run time was found to be 4.083 min.

The developed method was validated for various parameters like accuracy , precision, linearity, robustness, rugedness as per ICH guidelines.

The proposed HPLC method for determination of nevirapine tablet formulation was found to be satisfactory and could be used for the routine analysis of nevirapine .

The proposed method were proved to be superior to most of the reported method and this can be used as alternative method for the routine determination of selected drugs under the study in bulk and pharmaceutical dosage forms.


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Viramune (nevirapine) tablets; Viramune (nevirapine) oral suspension prescribing information