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    We nanopharmes established in 2000 as a multipurpose company, our proud to introduce ourselves as one of the global supplier companies in Georgia. Nanopharmes markets hundreds of branded product lines and numerous category leaders in the field of Active Pharmaceutical Ingredients, herbals, chemicals, excipients, cement additives, petrochemical additives, polymer additives, packing equipments, polymer and pharmaceutical packs, paper and ...
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    We nanopharmes established in 2000 as a multipurpose company, our proud to introduce ourselves as one of the global supplier companies in Georgia. Nanopharmes markets hundreds of branded product lines and numerous category leaders in the field of Active Pharmaceutical Ingredients, herbals, chemicals, excipients, cement additives, petrochemical additives, polymer additives, packing equipments, polymer and pharmaceutical packs, paper and ...
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Scientific Articles

 Evaluation Of Compatibility Of Tablet Excipients Biology Essay

The present study describes compatibility of anti-HIV drug lamivudine with various selected excipients and a novel synthesized polymer, for the development of its controlled release formulation. Differerntial scannig calorimetry (DSC), Isothermal stability study (ISS) and Fourier transform infrared (FT-IR) spectral analysis were performed to access the compatibility. The compatibility study was performed with various common excipients like spray dried lactose, poly vinyl pyrrolidine K-30, magnesium stearate, talc and a novel synthesized polymer cross-linked sago starch with lamivudine.
In the design of quality drug products, excipients and polymers play an important role. Excipients are the chemical substances which affect the functionality, stability and drug release behavior. Excipients are selected in formulation development on the basis of its compatibility and functionality with the selected active pharmaceutical ingredient.
In recent years, a number of techniques have been introduced for evaluation of drug-excipient compatibility. Differential scanning calorimetry (DSC) is one of the well established techniques in detection of incompatibility in drug/excipient [1-5]. DSC has now become first choice in pharmaceutical industry for compatibility study. Isothermal stability study (ISS) is also became an indirect thermal method for study the compatibility, which involves storage of drug-excipient combinations with or without moisture at high temperature for a specific period of time to accelerate drug ageing and possible interaction. The samples are then visually observed for any type of change in physical appearance, and the drug content determined quantitively [6-8]. Fourier transform infra red (FT-IR) spectroscopy is also used to confirm any type of physical interaction with drug and excipient [9-12].
Lamivudine is an analogue of cytidine. It inhibits both types (1 and 2) of HIV reverse transcriptase and also the reverse transcriptase of hepatitis B. It is phosphorylated to active metabolites that compete for incorporation into viral DNA. They inhibit the HIV reverse transcriptase enzyme competitively and act as a chain terminator of DNA synthesis. The lack of a 3'-OH group in the incorporated nucleoside analogue prevents the formation of the 5' to 3' phosphodiester linkage essential for DNA chain elongation, and therefore, the viral DNA growth is terminated [13].
The purpose of this study is to report the compatibility of lamivudine with pharmaceutical excipients ( spray dried lactose, polyvinyl pyrrolidine K-30, magnesium stearate and talc) and a novel synthesized cross-linked sago starch by DSC, ISS and FT-IR.

MATERIALS AND METHODS
Lamivudine was kindly donated by Ranbaxy Limited, Paonta Sahib, Himachal Pradesh, India. Spray dried lactose (SDL) was kindly gifted from DMV Fonterra excipients, The Netherland. Magnesium stearate (MST) , Talc and Polyvinyl pyrrolidine K-30(PVP) was purchased from Loba Chemie, Mumbai, India. All other chemicals used were of A.R. grade. Double distilled water is used throughout the study.

Determination of drug purity
The drug purity was determined by DSC, HPLC and UV-Vis spectrophotometry. The DSC (Perkin Elmer, USA) of drug lamivudine was done to get the endothermic peak (corresponding to its melting point). The HPLC (Waters, USA) of the drug was done as per the method described elsewhere [14]. The UV-Vis analysis ( Pharmaspec, Shimadzu, Japan) of the drug was done in buffer solution (pH=6.8). The sample was scanned in the range of 200-500nm to confirm its purity.

Synthesis of novel polymer
Cross-linking of starch was done with POCl3 in alkali containing sodium hydroxide as described by Zheng et al [15]. The sago starch (50g, dry basis) was dispersed in distilled water (200ml), and then starch slurry was adjusted to pH 9.0 with 0.5 N NaOH solutions. The cross-linking reagent POCl3 was added drop wise in different concentrations (0.5-2.5%w/v). The starch dispersion was stirred for 1 h and stored for 12 h at room temperature for completion of the reaction. The starch suspension was adjusted to pH 6.5, by adding 1 N HCl which leads to termination of the reaction. Extensive washing was done to ensure the removal of un-reacted salt. After drying overnight at 400C in a vacuum oven, the cross-linked starch was grounded and sieved (60 meshes).
(1)

Compatibility study by Differential scanning calorimtery
A differential scanning calorimetry ( JADE DSC, Perkin Elmer, USA) was used to study the thermal analysis of drug-excipient compatibility. Firstly, binary mixtures of lamivudine and excipients (in 1:1 mass/mass ratio). The drug-excipient mixture was scanned in the temperature range of 50-2200C under an atmosphere of nitrogen. The heating rate was 200C/min and the obtained curves were observed for any type of interaction.

Fourier-transform infrared (FT-IR) study
FT-IR spectra was recorded on a Bruker spectrophotometer (Model no 220, Germany) using KBr discs in the range of 4000-450 cm-1. FT-IR analysis has been performed using sample of lamivudine with various excipients ( SDL, PVP K-30, MST, Talc and CLSS) at 1:1 mass/mass ratio.

Isothermal stability study[16,17]
In isothermal stability study (ISS), samples of drug and different excipients (Table-1) were weighed directly in 5 ml glass vials (n=3). After mixing on a cyclomixer for 3 min, 10% (w/w) water was added in each of the vial. The glass vials, after teflon sealing, were stored at 500C in hot air oven. Drug- excipient blends without added water and stored in refrigerator served as controls. The drug-excipient blends were periodically examined for any change in physical appearance. Samples were quantitively analyzed using UV-Vis spectrophotometer (Pharmaspec 1700, Shimadzu, Japan) after 4 weeks of storage at above conditions.

Analysis of samples in Isothermal stability study:
The stored samples were quantitively analyzed using UV-Vis spectrophotometer. The drug–excipients samples were diluted in phosphate buffer solution (pH =6.8).. The samples were centrifuged, filtered and analyzed at 270 nm in UV-Vis spectral analysis.

RESULTS AND DISCUSSION
The purity of drug was assessed by HPLC and the purity was confirmed by getting chromatogram with same retention time as specified in reference (shown in Fig ). The UV-Vis spectrophotometer analysis of drug lamivudine showed maximum absorption at (λmax) 270 nm which confirms its purity (Fig. 3).
The DSC curve of drug lamivudine showed a sharp endothermic peak at 182.730C (ΔH= 74.54J/g ) with a melting temperature (Tonset = 177.390C). In the DSC curves of binary mixtures (Fig.5), they exhibited neither the shifting nor the disappearance of peaks. The retention of original peak suggested that the drug was physically stable in combinations with all the selected excipients. The novel synthesized cross-linked sago starch was also shown none type of interaction with the drug (Fig. 6).
In the isothermal stability studies, drug-excipient binary mixtures showed no change in physical appearance at ambient temperature.The blends remain physically stable and no discoloration,liquefaction or gas formation was observed during storage.There is also no significant drug degradation was observed with any type of excipients. Table-1 showed % drug remaining at the end of the study at 500C.
Pure lamivudine showed the characteristic band peaks at 1651.12 cm-1 which corresponds to cystedine nucleus. A characteristic bands peak at 3407.58 cm-1 and 3198.77 cm-1 owing to amino and hydroxy group present in lamivudine. Peaks present at 1287.37 cm-1 and 1160.32 cm-1 owing to asymmetrical and symmetrical stretching of C-O-C group present in oxathiolane ring of lamivudine.All the binary mixture of drug and excipient (Fig.7) showed none type of physical interaction except with magnesium stearate. In the FT-IR spectral diagram of drug-magnesium stearate, there is introduction of absorption bands at 2955.18 cm-1 and 2850.32 cm-1, which might be a type of physical interaction, but in thermal analysis (DSC and IST) there is no confirmation for the same.

CONCLUSION
Compatibility study in preformulation stage of formalationa development is now become an essential step. The thermoanalysis provides information about the thermal stability and decompositon of drug and used excipients. The results demonstrated the suitability of drug lamivudine with various excipients like spray dried lactose, PVP K-30, magnesium stearate, talc and novel synthesized cross-linked sago starch. The DSC and ISS showed none type of interaction in all drug-excipient combinations, while FT-IR showed only one interaction with magnesium stearate. But this interaction was not reconfirmed with DSC and ISS, so magnesium stearate is compatible with lamivudine.




Identify Quantify Pharmaceutical Ingredient Enalapril Maleate Products Countries Biology Essay

 


Quantification of API in commercial tablets from the UK market by NIR spectroscopy using diffuse reflectance and imaging techniques.
Working principally with intact tablets.

Abstract
The aim of this project was to identify and quantify the active pharmaceutical ingredient Enalapril Maleate in given different tablet products from different countries by Near Infrared Spectroscopic method. There were number of steps involved in the development of experimental method for Qualitative and Quantitative analysis of pharmaceutical products which are based on the comparison of the NIR spectrum of a sample with the spectra of authentic drug. There were three types of sample preparation done for collecting spectra which included intact tablets, powdered tablets and KBR discs made with tablet powder. There were two type of reference used in the experiment for better results and select the better one for the model preparation. This analysis was done by powerful chemo metric methods such as multiple linear regression method, Partial least square regression method. In the beginning the spectra were processed and multivariate model constructed for data acquisition. The acquired data then processed from different models, which gave equation for prediction of the active constituent in the sample. The constructed model equations then tested with external validation which shown the best regression model for quantification. The results show NIR spectroscopic method is rapid, non-destructive and reliable method for qualitative and quantitative analysis of pharmaceutical products and could be helpful to identify the counterfeit drugs among products.

Introduction
Identification of counterfeit medicines
Currently the pharmaceutical industry is facing biggest threat from counterfeit products. The definition of the counterfeit drug was given by WHO in 1982 is as follows: “A counterfeit medicine is one which is deliberately and fraudulently mislabeled with respect to identity and/or source. Counterfeiting can apply to both branded and generic products and counterfeit products may include products with correct ingredients or with the wrong ingredients, without active ingredients, with insufficient active ingredients or with fake packing”. The definition was changed in the FIT conference in Sydney 2003 as “Counterfeiting in relation to medical products means the deliberate and fraudulent mislabeling with respect to the identity, composition and/or source of a finished medicinal product or ingredient for the preparation of medicinal product” . This also impacts on patient health because they receive improper dose or even without proper ingredients, resulting in increase of their disease condition and potential disability and death. The industry is threatened by damage of its reputation resulting from the counterfeit products from the market places of its brand name.
Nowadays numbers of fake products are coming in the market under the famous brand names which have already become well-known in the pharmaceutical industry. There are different tricks to copy and mimic the original genuine products which are available in the market. The counterfeit medicines are prepared in terms of different ways. The marked active ingredient may not present in the packed product or active ingredient is not present within labeled range. Sometimes the costlier active substance is substituted by cheaper or less active substances .
The counterfeiting is not only problem of the developing country but it is also a notable offence in developed countries like USA, UK etc. The possibilities of counterfeit medicines in wealthier countries are in the area of new expensive lifestyle medicines, such as hormones, steroids and anti-histamines while in developing countries like India, China, Brazil the most counterfeited medicines are those used to treat life threatening conditions such as Malaria, tuberculosis and HIV. Hence, the problem of counterfeited medicines is more serious in developing countries.
The conventional methods for identification of counterfeit medicines are titration, mass spectroscopy and high performance liquid chromatography (HPLC). The HPLC method is very accurate method but needed high cost of experiment with highly skilled personnel to perform the test. Hence it is difficult for developing countries to carry out any identification of counterfeited medicines because of lack of fund for proper laboratory .
Near Infrared Spectroscopy is a spectroscopic analytical method. The near Infrared region is situated between the red band of the visible light and the mid-infrared region . The spectral regions span the wavelength 750-2500 nm. It is widely used techniques for the analytical purposes in pharmaceutical and food industries due to its speed and precision .
Its main applications are in medical diagnostics, pharmaceutical industries because of several advantages of NIR spectroscopy like low cost per sample analyzed no need of sample preparation, wide range of products and parameters . The NIR spectroscopy is nondestructive and highly reproducible method for analyses. NIR spectroscopy can be used in identification of drug substances, excipient in short time of period. Direct reading of spectra is possible in Near Infrared technique because many packing materials allow penetrating Infrared radiation to the product. The obtained spectra used to convert into electronic library for comparison of authentic and false product among the given sample products .
The NIR region was discovered by Herschel in 1800. Although, the actual practice of NIR experiment was started in early 1920s when the potential of this analytical technique was incorporated in newfangled manner by U.S. department of agriculture .
Figure 1 Wavelength range of NIR region
Recently, NIR has achieved significant importance in pharmaceutical industries for quality control testing and process monitoring mainly for raw materials.
Although this technique has many advantages in the pharmaceutical industries but some drawbacks also limits its use in pharmaceutical industry as it lacks the ability of mid infrared spectroscopy to identify samples by simple inspection of spectra. Furthermore, quantitative analyses needs complicated micro computing and chemo metrics like mathematical techniques for calibrations which recently developed in advent of computer science knowledge .
However, there was initial ignorance at the NIR spectroscopy but now it is gaining popularity as a results of improvement in instruments, software and chemo metrics. There are sophisticated soft wares available for the interpretation of the spectra and chemo metric calculations .
In this experiment tablets are measured intact with no sample preparation. The NIR spectra were recorded using a FOSS NIR System equipped with a Rapid Content Analyzer. The data are recorded and processed using Vision software version 2.51. Chemo metric method like Partial least square regression was used for data treatment using software. The method of taking spectra was simple and fast. The general idea of the authenticity of the spectra was obtained by processing the spectra.

Principles of NIRS:-
NIRS is based on the absorption of the light due to overtones and combination of fundamental vibrational bands. The NIR region lies between the visible and MIR regions of the electromagnetic spectrum and is defined by the American Society for Testing and Materials (ASTM) as the spectral region spanning the wavelength range 780-2526 nm. There is a condition of the radiation to be absorbed is the frequency of the light should be exactly the same as a fundamental vibration frequency for a specific molecule and a molecule should go under a change in its dipole moment by virtue of its fundamental vibration .
The vibrational frequency for a diatomic molecule can be determined on the assumption of the harmonic oscillation model, where an atom shifts from its equilibrium position with strength proportional to the shift (Hook’s law) .
Where is the speed of the light, k the bonding force constant and m is the reduced mass.
Polyatomic molecules possess several fundamental frequencies so they may exhibit simultaneous changes in the energies of two or more vibrational mode; the frequency observed will be the sum of or difference between the individual fundamental energies. This results in very weak bands that are called combination and subtractions bands. Thus vibrations can also be observed at approximately the multiples, e.g. twice, 3 times, 4 times etc. of the fundamental vibrational frequencies. These vibrations are called overtones. These overtones and frequencies combinations are observed in the near infrared region of the electromagnetic spectrum and are characterized by a large number of band overlap due to broad bands. This gives a fingerprint characteristic to the NIRS which makes it very useful for identification purpose .
Figure 2 Finger print region of the important functional group in NIR region
Resonance overtones of C-H, N-H, O-H, and S-H and combination of the fundamental vibrational modes have important information when they processed with related chemometric algorithms. When fundamental vibration compare with NIR absorption band in corresponding to mid infrared region later one has been found weaker in frequency. Near Infrared Radiation has low molar absorption therefore it could penetrate up to several millimeters into solid material. Hence, the NIR region is less sensitive and it permits operation in the reflectance mode and recording the spectra of the solid samples.
‘Reflectance spectroscopy measures the light reflected by the sample surface which contains a specular component and a diffuse component’. Specular reflectance described by Frensel’s law, contain little information about composition; consequently its contribution to measurements is minimized by adjusting the detector’s position relative to the sample. On the other hand diffuse reflectance, which is described by the Kubelka-Munk theory, is the basis for the measurements by this technique .
One widely used practical alternative is a relationship between concentration and relative reflectance similar to Beer’s law, namely: .
A= log 1/R = a´c
Where A is apparent absorbance, R relative reflectance, c concentration and a´ a proportionality constant. However, this relationship is different from Kubelka-Munk equation in terms of theoretical basis but it provides satisfactory results under typical conditions used in many diffuse reflectance spectroscopic applications.

 

Enalapril Maleate:-
Enalapril Maleate is the maleate salt of enalapril, an ethyl ester of a long-acting angiotensin converting enzyme inhibitor, enalaprilat. The chemical name of enalapril maleate is (s)-1-[N-[1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl]-L-proline, (Z)-2-butenedioate salt (1:1). Its empirical formula is C20H28N2O5*C4H4O4 and its structural formula is:
Figure 3 Structural formula of Enalapril Maleate
Enalapril Maleate is available as off white crystalline powder. Its molecular weight is 492.53. It is sparingly soluble in water, soluble in ethanol, and freely soluble in methanol.
Enalapril is a prodrug which is activated by hydrolysis of the ethyl ester to enalaprilat, which is active angiotensin converting enzyme inhibitor .
It is available as tablet dosage form in different range of 2.5mg, 5 mg, 10 mg and 20 mg tablets for oral administration. The tablet also contains following excipients: anhydrous lactose and zinc stearate and iron oxide.
Clinical Pharmacology: Mechanism of Action:-
EM mainly inhibits angiotensin converting enzyme in human subjects and animals. ACE is a peptidyl dipeptidase that catalyzes the conversion of angiotensin 1 to the vasoconstrictor substance, angiotensin 2. Furthermore angiotensin 2 stimulates secretion of aldosterone from adrenal cortex. This inhibition of Renin Angiotensin aldosterone system is the beneficial pharmacological effect of the enalapril .
Pharmacokinetic and Metabolism:-
The maximum plasma concentration of enalapril maleate reaches within one hour of administration. It has maximum plasma concentration about 60%. Food administration does not affect its oral absorption. The only metabolite of the enalapril is enalaprilat observed in urine. The half-life of the drug is 11 hours.
Pharmacodynamics effect:-
After several clinical studies it was found that the oral administration of enalapril maleate causes reduction in both supine and standing blood pressure usually with no orthostatic component. The antihypertensive effect of EM is seen after 1 hour of oral administration. In the treatment of heart failure EM shows good results like decrease in the vascular resistance, blood pressure, pulmonary capillary wedge pressure and heart size, and increase in cardiac output and exercise tolerance .
Indication and Usage:-
Enalapril maleate tablets are indicated to use in the treatment of Hypertension effectively alone or in combination with other anti-hypertensive agents such as thiazide type diuretics. It is also used in the treatment of Asymptomatic Left ventricular dysfunction and symptomatic congestive heart failure.
Contraindication:-
The use of Enalapril Maleate is restricted in hypersensitive patient to this product having history of angioedema related to previous treatment with an angiotensin converting enzyme inhibitor and in patients with heredity or idiopathic angioedema.
Chemometrics:-
Chemometrics is the science of extracting information from chemical systems by data driven means. The spectra obtained are needed to be processed to achieve clear results of the collected data. This statistical treatment of the spectral data, modeling and classification were performed by Vision software. This package provides multiple linear regression, partial least square regression, correlation in wavelength space algorithms.
The obtained absorption bonds are severely overlapping and difficult to interpret because they are the result of combinations and overtones of the fundamental mid-IR bonds. The spectra are complicated due to overlapping by other peaks. There are different parameter which affects the spectra like particle size, crystalline forms and variation in optical path length. Therefore the well-defined knowledge of interpretation of the spectral data by chemometrics required for better results . The use of chemometrics in NIRS is essential in number the steps of the process. From the experimental design to quantification of the regression model it plays crucial role in the method in each step. It can be used for development of predictive models, mathematical pretreatments of the spectra and sample grouping .
The processed spectra are both advantageous and dis advantageous for the original spectra. Though use of chemometrics clears the spectra and enhances resolution and facilitates computational treatment but it causes the worsening of signal to noise ratio and increases the complexity of the spectra.

Standard Normal Variate (SNV)
SNV and SNV D2 (Second derivative of SNV) are light scattering correction method used to normalize spectra whereas mean centering remove offset from the data. The difference in the spectra was because of granular and powdery nature of the sample which effects on its particle size. The correction in the factors is done in different way .

Correlation in wavelength space (CWS)
Correlation method identifies the similarity between a reference spectrum and a sample spectrum of library. It is a cosine of an angle between the two spectra. The two spectra are indicated as identical when their correlation coefficient is equal to 1 but it was set to 0.975.
Mathematically the equation of the correlation coefficient between two spectra X and Y is defined as:
r = Σixiyii  2498= wavelength 1110
√ Σxi Σiyi

Partial Least Square Regression (PLSR)
It is a calibration set up method first introduced by the Swedish statistician Herman Wold. It is used for complex matrices or analytes variables for which Multiple Linear Regression is not suitable. This technique can construct a linear model by projecting the lab data vs computed data in the regression equation. These data X and Y are projected in the new spaces.
In this method, the whole infra-red region of electromagnetic spectrum is needed to scan because a single peak could not account the matrices and analytes variables. However, there must be large number of samples used for a precise calibration. This method is more suitable when the matrix of predictors has more variables than observation .
Experimental Method:-

Materials
Total 15 product of Enalapril Maleate tablet were used for constructing libraries and models for analysis purposes. These products came from different manufacturers, different batches and different countries. The tablets have two dose strengths one was 5 mg and the other was 20 mg. All of the tablets got over their expiry dates but all were stored in good packing condition so there was no chance of contamination. The active ingredient of the all tablet is Enalapril Maleate. The tablet also contain various excipients mainly lactose. The list of different sample products is given below with batch number, manufacturer name and dose strengths.


Quantification of API in commercial tablets from the UK market by NIR spectroscopy using diffuse reflectance and imaging techniques.
Working principally with intact tablets.
Abstract
The aim of this project was to identify and quantify the active pharmaceutical ingredient Enalapril Maleate in given different tablet products from different countries by Near Infrared Spectroscopic method. There were number of steps involved in the development of experimental method for Qualitative and Quantitative analysis of pharmaceutical products which are based on the comparison of the NIR spectrum of a sample with the spectra of authentic drug. There were three types of sample preparation done for collecting spectra which included intact tablets, powdered tablets and KBR discs made with tablet powder. There were two type of reference used in the experiment for better results and select the better one for the model preparation. This analysis was done by powerful chemo metric methods such as multiple linear regression method, Partial least square regression method. In the beginning the spectra were processed and multivariate model constructed for data acquisition. The acquired data then processed from different models, which gave equation for prediction of the active constituent in the sample. The constructed model equations then tested with external validation which shown the best regression model for quantification. The results show NIR spectroscopic method is rapid, non-destructive and reliable method for qualitative and quantitative analysis of pharmaceutical products and could be helpful to identify the counterfeit drugs among products.
Introduction
Identification of counterfeit medicines
Currently the pharmaceutical industry is facing biggest threat from counterfeit products. The definition of the counterfeit drug was given by WHO in 1982 is as follows: “A counterfeit medicine is one which is deliberately and fraudulently mislabeled with respect to identity and/or source. Counterfeiting can apply to both branded and generic products and counterfeit products may include products with correct ingredients or with the wrong ingredients, without active ingredients, with insufficient active ingredients or with fake packing”. The definition was changed in the FIT conference in Sydney 2003 as “Counterfeiting in relation to medical products means the deliberate and fraudulent mislabeling with respect to the identity, composition and/or source of a finished medicinal product or ingredient for the preparation of medicinal product” . This also impacts on patient health because they receive improper dose or even without proper ingredients, resulting in increase of their disease condition and potential disability and death. The industry is threatened by damage of its reputation resulting from the counterfeit products from the market places of its brand name.
Nowadays numbers of fake products are coming in the market under the famous brand names which have already become well-known in the pharmaceutical industry. There are different tricks to copy and mimic the original genuine products which are available in the market. The counterfeit medicines are prepared in terms of different ways. The marked active ingredient may not present in the packed product or active ingredient is not present within labeled range. Sometimes the costlier active substance is substituted by cheaper or less active substances .
The counterfeiting is not only problem of the developing country but it is also a notable offence in developed countries like USA, UK etc. The possibilities of counterfeit medicines in wealthier countries are in the area of new expensive lifestyle medicines, such as hormones, steroids and anti-histamines while in developing countries like India, China, Brazil the most counterfeited medicines are those used to treat life threatening conditions such as Malaria, tuberculosis and HIV. Hence, the problem of counterfeited medicines is more serious in developing countries.
The conventional methods for identification of counterfeit medicines are titration, mass spectroscopy and high performance liquid chromatography (HPLC). The HPLC method is very accurate method but needed high cost of experiment with highly skilled personnel to perform the test. Hence it is difficult for developing countries to carry out any identification of counterfeited medicines because of lack of fund for proper laboratory .
Near Infrared Spectroscopy is a spectroscopic analytical method. The near Infrared region is situated between the red band of the visible light and the mid-infrared region . The spectral regions span the wavelength 750-2500 nm. It is widely used techniques for the analytical purposes in pharmaceutical and food industries due to its speed and precision .
Its main applications are in medical diagnostics, pharmaceutical industries because of several advantages of NIR spectroscopy like low cost per sample analyzed no need of sample preparation, wide range of products and parameters . The NIR spectroscopy is nondestructive and highly reproducible method for analyses. NIR spectroscopy can be used in identification of drug substances, excipient in short time of period. Direct reading of spectra is possible in Near Infrared technique because many packing materials allow penetrating Infrared radiation to the product. The obtained spectra used to convert into electronic library for comparison of authentic and false product among the given sample products .
The NIR region was discovered by Herschel in 1800. Although, the actual practice of NIR experiment was started in early 1920s when the potential of this analytical technique was incorporated in newfangled manner by U.S. department of agriculture .
Figure 1 Wavelength range of NIR region
Recently, NIR has achieved significant importance in pharmaceutical industries for quality control testing and process monitoring mainly for raw materials.
Although this technique has many advantages in the pharmaceutical industries but some drawbacks also limits its use in pharmaceutical industry as it lacks the ability of mid infrared spectroscopy to identify samples by simple inspection of spectra. Furthermore, quantitative analyses needs complicated micro computing and chemo metrics like mathematical techniques for calibrations which recently developed in advent of computer science knowledge .
However, there was initial ignorance at the NIR spectroscopy but now it is gaining popularity as a results of improvement in instruments, software and chemo metrics. There are sophisticated soft wares available for the interpretation of the spectra and chemo metric calculations .
In this experiment tablets are measured intact with no sample preparation. The NIR spectra were recorded using a FOSS NIR System equipped with a Rapid Content Analyzer. The data are recorded and processed using Vision software version 2.51. Chemo metric method like Partial least square regression was used for data treatment using software. The method of taking spectra was simple and fast. The general idea of the authenticity of the spectra was obtained by processing the spectra.
Principles of NIRS:-
NIRS is based on the absorption of the light due to overtones and combination of fundamental vibrational bands. The NIR region lies between the visible and MIR regions of the electromagnetic spectrum and is defined by the American Society for Testing and Materials (ASTM) as the spectral region spanning the wavelength range 780-2526 nm. There is a condition of the radiation to be absorbed is the frequency of the light should be exactly the same as a fundamental vibration frequency for a specific molecule and a molecule should go under a change in its dipole moment by virtue of its fundamental vibration .
The vibrational frequency for a diatomic molecule can be determined on the assumption of the harmonic oscillation model, where an atom shifts from its equilibrium position with strength proportional to the shift (Hook’s law) .
Where is the speed of the light, k the bonding force constant and m is the reduced mass.
Polyatomic molecules possess several fundamental frequencies so they may exhibit simultaneous changes in the energies of two or more vibrational mode; the frequency observed will be the sum of or difference between the individual fundamental energies. This results in very weak bands that are called combination and subtractions bands. Thus vibrations can also be observed at approximately the multiples, e.g. twice, 3 times, 4 times etc. of the fundamental vibrational frequencies. These vibrations are called overtones. These overtones and frequencies combinations are observed in the near infrared region of the electromagnetic spectrum and are characterized by a large number of band overlap due to broad bands. This gives a fingerprint characteristic to the NIRS which makes it very useful for identification purpose .
Figure 2 Finger print region of the important functional group in NIR region
Resonance overtones of C-H, N-H, O-H, and S-H and combination of the fundamental vibrational modes have important information when they processed with related chemometric algorithms. When fundamental vibration compare with NIR absorption band in corresponding to mid infrared region later one has been found weaker in frequency. Near Infrared Radiation has low molar absorption therefore it could penetrate up to several millimeters into solid material. Hence, the NIR region is less sensitive and it permits operation in the reflectance mode and recording the spectra of the solid samples.
‘Reflectance spectroscopy measures the light reflected by the sample surface which contains a specular component and a diffuse component’. Specular reflectance described by Frensel’s law, contain little information about composition; consequently its contribution to measurements is minimized by adjusting the detector’s position relative to the sample. On the other hand diffuse reflectance, which is described by the Kubelka-Munk theory, is the basis for the measurements by this technique .
One widely used practical alternative is a relationship between concentration and relative reflectance similar to Beer’s law, namely: .
A= log 1/R = a´c
Where A is apparent absorbance, R relative reflectance, c concentration and a´ a proportionality constant. However, this relationship is different from Kubelka-Munk equation in terms of theoretical basis but it provides satisfactory results under typical conditions used in many diffuse reflectance spectroscopic applications.
Enalapril Maleate:-
Enalapril Maleate is the maleate salt of enalapril, an ethyl ester of a long-acting angiotensin converting enzyme inhibitor, enalaprilat. The chemical name of enalapril maleate is (s)-1-[N-[1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl]-L-proline, (Z)-2-butenedioate salt (1:1). Its empirical formula is C20H28N2O5*C4H4O4 and its structural formula is:
Figure 3 Structural formula of Enalapril Maleate
Enalapril Maleate is available as off white crystalline powder. Its molecular weight is 492.53. It is sparingly soluble in water, soluble in ethanol, and freely soluble in methanol.
Enalapril is a prodrug which is activated by hydrolysis of the ethyl ester to enalaprilat, which is active angiotensin converting enzyme inhibitor .
It is available as tablet dosage form in different range of 2.5mg, 5 mg, 10 mg and 20 mg tablets for oral administration. The tablet also contains following excipients: anhydrous lactose and zinc stearate and iron oxide.
Clinical Pharmacology: Mechanism of Action:-
EM mainly inhibits angiotensin converting enzyme in human subjects and animals. ACE is a peptidyl dipeptidase that catalyzes the conversion of angiotensin 1 to the vasoconstrictor substance, angiotensin 2. Furthermore angiotensin 2 stimulates secretion of aldosterone from adrenal cortex. This inhibition of Renin Angiotensin aldosterone system is the beneficial pharmacological effect of the enalapril .
Pharmacokinetic and Metabolism:-
The maximum plasma concentration of enalapril maleate reaches within one hour of administration. It has maximum plasma concentration about 60%. Food administration does not affect its oral absorption. The only metabolite of the enalapril is enalaprilat observed in urine. The half-life of the drug is 11 hours.
Pharmacodynamics effect:-
After several clinical studies it was found that the oral administration of enalapril maleate causes reduction in both supine and standing blood pressure usually with no orthostatic component. The antihypertensive effect of EM is seen after 1 hour of oral administration. In the treatment of heart failure EM shows good results like decrease in the vascular resistance, blood pressure, pulmonary capillary wedge pressure and heart size, and increase in cardiac output and exercise tolerance .
Indication and Usage:-
Enalapril maleate tablets are indicated to use in the treatment of Hypertension effectively alone or in combination with other anti-hypertensive agents such as thiazide type diuretics. It is also used in the treatment of Asymptomatic Left ventricular dysfunction and symptomatic congestive heart failure.
Contraindication:-
The use of Enalapril Maleate is restricted in hypersensitive patient to this product having history of angioedema related to previous treatment with an angiotensin converting enzyme inhibitor and in patients with heredity or idiopathic angioedema.
Chemometrics:-
Chemometrics is the science of extracting information from chemical systems by data driven means. The spectra obtained are needed to be processed to achieve clear results of the collected data. This statistical treatment of the spectral data, modeling and classification were performed by Vision software. This package provides multiple linear regression, partial least square regression, correlation in wavelength space algorithms.
The obtained absorption bonds are severely overlapping and difficult to interpret because they are the result of combinations and overtones of the fundamental mid-IR bonds. The spectra are complicated due to overlapping by other peaks. There are different parameter which affects the spectra like particle size, crystalline forms and variation in optical path length. Therefore the well-defined knowledge of interpretation of the spectral data by chemometrics required for better results . The use of chemometrics in NIRS is essential in number the steps of the process. From the experimental design to quantification of the regression model it plays crucial role in the method in each step. It can be used for development of predictive models, mathematical pretreatments of the spectra and sample grouping .
The processed spectra are both advantageous and dis advantageous for the original spectra. Though use of chemometrics clears the spectra and enhances resolution and facilitates computational treatment but it causes the worsening of signal to noise ratio and increases the complexity of the spectra.
Standard Normal Variate (SNV)
SNV and SNV D2 (Second derivative of SNV) are light scattering correction method used to normalize spectra whereas mean centering remove offset from the data. The difference in the spectra was because of granular and powdery nature of the sample which effects on its particle size. The correction in the factors is done in different way .
Correlation in wavelength space (CWS)
Correlation method identifies the similarity between a reference spectrum and a sample spectrum of library. It is a cosine of an angle between the two spectra. The two spectra are indicated as identical when their correlation coefficient is equal to 1 but it was set to 0.975.
Mathematically the equation of the correlation coefficient between two spectra X and Y is defined as:
r = Σixiyii  2498= wavelength 1110
√ Σxi Σiyi
Partial Least Square Regression (PLSR)
It is a calibration set up method first introduced by the Swedish statistician Herman Wold. It is used for complex matrices or analytes variables for which Multiple Linear Regression is not suitable. This technique can construct a linear model by projecting the lab data vs computed data in the regression equation. These data X and Y are projected in the new spaces.
In this method, the whole infra-red region of electromagnetic spectrum is needed to scan because a single peak could not account the matrices and analytes variables. However, there must be large number of samples used for a precise calibration. This method is more suitable when the matrix of predictors has more variables than observation .
Experimental Method:-
Materials
Total 15 product of Enalapril Maleate tablet were used for constructing libraries and models for analysis purposes. These products came from different manufacturers, different batches and different countries. The tablets have two dose strengths one was 5 mg and the other was 20 mg. All of the tablets got over their expiry dates but all were stored in good packing condition so there was no chance of contamination. The active ingredient of the all tablet is Enalapril Maleate. The tablet also contain various excipients mainly lactose. The list of different sample products is given below with batch number, manufacturer name and dose strengths.