methods for selected multi component drugs in their
formulations
Thesis submitted to
The Tamilnadu Dr.M.G.R. Medical University, Chennai, India in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
Submitted by
ARUNADEVI S. BIRAJDAR, M. Pharm.,
JANUARY 2010
J.S.S. COLLEGE OF PHARMACY
OOTACAMUND - 643001
I hereby declare that the thesis entitled “Development and validation of HPLC and Spectrophotometric methods for selected multi component drugs in their formulations”submitted by me for the award of degree ofDoctor of Philosophy of the Tamilnadu Dr.M.G.R. Medical University, Chennai is a record of research work done by me at J.S.S. College of Pharmacy, Ootacamund - 643 001, Tamilnadu, India during the years 2007-2010 under the supervision of Dr.S.N.Meyyanathan and that the thesis had not previously formed the basis for the award of any degree, diploma, associateship, fellowship or other similar title previously.
This is to certify that the thesis entitled “Development and validation of HPLC and Spectrophotometric methods for selected multi component drugs in their formulations” is a record of research work done by Miss. Arunadevi S. Birajdar at J.S.S. College of Pharmacy, Ootacamund - 643 001, Tamilnadu, India during the years 2007-2010 under my supervision and that the thesis had not previously formed the basis for the award of any degree, diploma, associateship, fellowship or other similar title previously. I also certify that the thesis represents independent work done by the candidate.
This is to certify that the thesis entitled “Development and validation of HPLC and Spectrophotometric methods for selected multi component drugs in their formulations” is a record of research work done by Miss. Arunadevi S. Birajdar at J.S.S. College of Pharmacy, Ootacamund - 643 001, Tamilnadu, India during the years 2007-2010 under my supervision as co-guide.
J.S.S. COLLEGE OF PHARMACY, OOTACAMUND
Off campus college of J.S.S. University, Mysore
CERTIFICATE
This is to certify that the thesis entitled “Development and validation of HPLC and Spectrophotometric methods for selected multi component drugs in their formulations” is a record of research work done by Miss. Arunadevi S. Birajdar at J.S.S. College of Pharmacy, Ootacamund - 643 001, Tamilnadu, India during the years 2007-2010 under the supervision of Dr.S.N.Meyyanathan.
Dr.K.Elango
Principal i/cI express my gratitude to Dr. S. N. Meyyanathan, Supervisor for his guidance and constant encouragement throughout the course of this work.
I thank Dr. B. Suresh, Vice Chancellor, J.S.S. University, Mysore for his kind support and facilities provided to me for carrying out this work.
I thank Dr. K. Elango, Principal i/c, J.S.S. College of Pharmacy, Ootacamund for his support and encouragement.
I thank Professor K. Chinnasamy, AICTE and QIP cell for giving me the opportunity to complete this project work.
It is pleasure to express my deep sense of gratitude to Mr. Rajinikant B.Raja Visiting Professor, Mrs Krisnaveni, Mr.S. Muralidaran and Mr.S. Rajan for their constructive suggestion and criticisms.
I thank Dr. M.J.Nanjan, Director, Postgraduate studies & research, Mr. S.Puttarajappa and Mr.J.S.K. Nagrajan for their interest in my work.
I thank Mr. Supe and manufacturers who provided the gift samples for the present project.
I acknowledge the support given by Mrs.Sujatha, Mr.Manju and Mr.Shivaraju helped for my project.
It is my pleasure to thank my beloved Guru Mallinath, my mother Kamala, my (late) father Shantappa, my dear brothers Vaijanath & Naganath and beloved all friends for their constant support and encouragement.
My sincere panamas and respectful regards to our most revered His Holiness Jagadguru Sri Sri Shivarathri Deshikendra Mahaswamigalavaru of Sri Suttur Mutt, Mysore with whose blessings this work has been completed successfully.
Page No
1.0 Introduction
01
2.0 Aims and objectives
22
3.0 Review of literature
24
4.0 Place of research work
62
5.0 Scope and plan of work
63
6.0 Materials and methods
67
7.0 Results
96
8.0 Discussion
101
9.0 Summary and conclusions
102
Bibliography
Annexure I
Drug profiles
Annexure II
Profiles of formulations
Annexure III
1. INTRODUCTION
This thesis deals with the studies carried out by the writer in this laboratory for past three years on the development and validation of HPLC and spectrophotometric methods for selected multi component drugs in their formulations. Before discussing the experimental procedures adopted and the results obtained a brief introduction to the drug analysis, need for multi component drug in formulation analysis by HPLC and spectrophotometric methods and validation would be discussed in detail. The literature on the selected multi component formulations for developing HPLC and spectrophotometric methods would also be reviewed here.
Drug analysis1, namely, identification, characterization and
determination of drugs in dosage forms and biological fluids, play an important role in the development, manufacture and therapeutic use of drug. Drugs are developed and manufactured as dosage form prior to their use by patients. Dosage forms require a variety of tests and standards to assure their therapeutic benefits. Administration of two or more drugs at a time becomes necessary for several therapeutic reasons. There exist a number of drug combinations, referred to as multi component dosage forms, which have proved to be effective due to their combined mode of action in the body. These drug combinations not only offer better therapeutically effective due to additive or synergistic effect, but also administrated as a single dosage, economy in production.
Importance of newer analytical methods
The number of drugs and drug formulations introduced into the market by pharmaceutical industries has been increasing at an alarming rate. These drugs or formulations may be either new entities or partial structural modifications of the existing ones or novel dosage forms (controlled/sustained release formulations) or multi component dosage forms.
Analytical methods for the quantification of the drugs in different combination forms and biological fluids may not be available.
The drug combination may not be official in any pharmacopoeia
A literature search may not reveal any analytical procedure and methods for drug combinations due to the interference caused by excipients
Analytical methods for a drug in combination with other drugs may not be available
Newer methods are also recommended when the existing methods, may require expensive instruments, reagents and solvents used,
may involve cumbersome extraction or separation steps which are time consuming and
may not be simple, rapid, reliable and sensitive. Estimation of drugs in their formulations
Estimation of drugs in their formulations, however, is difficult because of the presence of one or more drug components in addition to additives. In the process of estimation it is important to confirm that one component does not interfere with the estimation of the other. The complexity of these formulations thus poses a challenge to the analytical chemist during the development of dosage form assay methods. Analytical methods2-5 for the
estimation of drugs in their formulations include: 1. Classical separation and analysis
In this process the components of interest are subjected to classical separation techniques like extraction or isolation. A suitable estimation procedure is then selected to quantify the components by gravimetric and volumetric methods.
2. Spectral methods
3. Electro analytical methods
They involve the measurement of current, voltage or resistance as a property of concentration of the drug component. Potentiometry, conductometry and amperometry are some of the important techniques. 4. Chromatographic methods
Chromatography is a method of separation, where the individual components are separated and analyzed. In this technique two or more components are separated by a dynamic differential migrational process, in a system consisting of two phases, one of which moves continuously in a given direction in which the individual components exhibit different mobilities due to the difference in their adsorption or partition or molecular size etc. Most reliable and widely used chromatographic techniques used for the estimation of drugs in their formulations are a. Gas liquid chromatography (GLC)
In this technique a carrier gas is used as the mobile phase which passes over a liquid non-volatile stationary phase, coated on an inert solid support and the separation is effected in accordance with the difference in partition coefficients of the components.
b. High performance thin layer chromatography (HPTLC)
This is a sophisticated, advanced and automated version of the thin layer chromatography. It is the fastest growing technique for the analysis of drugs.
c. High performance liquid chromatography (HPLC)
HPLC is a type of chromatography7-11that employs a liquid mobile
Estimation of multi component dosage forms by HPLC
Most of the drugs in multicomponent dosage forms can be analyzed by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision and ease of automation in this method. HPLC method eliminates tedious extraction and isolation procedures. Some of the advantages are,
Speed (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 substances of low volatility,
Easy sample recovery, handling and maintenance,
Instrumentation lends itself to automation and quantitation (less time and labour),
Precise and reproducible,
Calculations are done by integrator itself and
Suitable for preparative liquid chromatography on a much larger scale.
There are different modes of separation in HPLC. They are normal phase mode, reverse phase mode, reverse phase ion pair chromatography, ion exchange chromatography, affinity chromatography and size exclusion chromatography (gel permeation and gel filtration chromatography).
applications because most of the drug molecules are polar in nature and hence take longer time to elute.
Reverse phase mode is the most popular mode for analytical and preparative separations of compound 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 octa decyl functional group bonded to silica gel and the mobile phase is polar solvent. An aqueous mobile phase allows the use of secondary solute chemical equilibrium (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 longer time. As most of the drugs and pharmaceutical are polar in nature, they are not retained for longer times and hence elute faster. The different columns used are octa decyl silane (ODS) or C18, C8, C4etc (in the order of increasing polarity of the
stationary phase).
In ion exchange chromatography, the stationary phase contains ionic group like NR3+ or SO3-, which interact with ionic groups of the sample
molecules. This is suitable for the separation of charged molecules only. Changing the pH and salt concentration can modulate the retention.
Ion pair chromatography may be used for the separation of ionic compounds and this method can also substitute for ion exchange chromatography. Strong acidic and basic compounds may be separated by reverse phase mode by forming ion pairs (columbic association species formed between two ions of opposite electrical charge) with suitable counter ions. The technique is referred to as reverse phase ion pair chromatography or soap chromatography.
Size exclusion chromatography separates molecules according to their molecular mass. Largest molecules are eluted first and the smallest molecules last. This method is generally used when a mixture contains compounds with a molecular mass difference of at least 10%. This mode can be further subdivided into gel permeation chromatography (with organic solvent) and gel filtration chromatography (with aqueous solvents).
The various components of HPLC are pumps (solvent delivery system), mixing unit, gradient controller and solvent degasser, injector (manual or auto), guard column, analytical columns, detectors, recorders and /or integrators. Recent models are equipped with computers and software for data acquisition and processing.
The choice of the column should be made after a careful consideration of the mode of the chromatographic technique. Three types of columns are available based upon the type of packing and particle size, namely, rigid solids, hard gels porous and pellicular layer beads. The columns of smaller particles (3-10 µ) are always preferred because they offer high efficiency (number of theoretical plates/meter) and speed of analysis.
The different types of detection used in HPLC methods are ultraviolet (UV) detection, fluorescence detection, refractive index detection, mass spectrophotometric detection and electrochemical detection. In most cases, method development in HPLC is carried out with UV detection using a variable wavelength spectrophotometric detector or a diode array detector (DAD).
retention times, peak areas correction factors. With the help of peak area and height values, the peak width can be used for the calculation of number of theoretical plates.
Method development and design of separation method by HPLC
Methods for analyzing drugs in multicomponent dosage forms can be developed, provided one has knowledge about the nature of the sample, namely, its molecular weight, polarity, ionic character and the solubility parameter. An exact recipe for HPLC, however, cannot be provided because method development involves considerable trial and error procedures. The most difficult problem usually is where to start, what type of column is worth trying with what kind of mobile phase. In general one being with reverse phase chromatography when the compounds are hydrophilic in nature with many polar groups and are water soluble.
The organic phase concentration required for the mobile phase can be estimated by gradient elution method. For aqueous sample mixtures, the best way to start is with gradient reverse phase chromatography. Gradient can be started with 5-10% organic phase in the mobile phase and the organic phase concentration (methanol or acetonitrile) can be increased up to 100% within 30-45 min. Separation can be optimized by changing the initial mobile phase composition and slope of the gradient according to the chromatogram obtained from the preliminary run. The initial mobile phase composition can be estimated on the basis of where the compounds of interest were eluted, namely, at what mobile phase composition.
Elution of drug molecules can be altered by changing the polarity of the mobile phase. The elution strength of a mobile phase depends upon its polarity, the stronger the polarity, higher is the elution. Ionic samples (acidic or basic) can be separated, if they are present in undissociated form. Dissociation of ionic samples may be suppressed by the proper selection of pH.
of the organic phase concentration in the mobile phase can be in steps of 5%. If the retention times are too long, an increase of the organic phase concentration is needed.
In UV detection, good analytical results are obtained only when the wavelength is selected carefully. This requires knowledge of the UV spectra of the individual components present in the sample. If analyte standards are available, their UV spectra can be measured prior to HPLC method development.
The molar absorbance at the detection wavelength is also an important parameter. When peaks are not detected in the chromatograms, it is possible that the sample quantity is not enough for the detection. An injection of a volume of 20 µl form a solution of 1 mg/ml concentration normally provides good signals for UV active compounds around 220 nm. Even if the compounds exhibit higher λmax,they absorb strongly at lower
wavelength. It is not always necessary to detect compounds at their maximum absorbance. It is, however, advantageous to avoid the detection at the sloppy part of the spectrum for precise quantization. When acceptable peaks are detected on the chromatogram, the investigation of the peak shapes can help further method development.
The addition of peak modifier to the mobile phase can affect the separation of ionic samples For example; the retention of the basic compounds can be influenced by the addition of small amounts of triethylamine (a peak modifier) to the mobile phase. Similarly for acidic compounds small amount of acetic acid can be used. This can lead to useful changes in selectivity.
at lower pH can be used. For amphoteric solutes or a mixture of acidic and basic compounds, ion-pair chromatography is the method of choice.
The low solubility of the sample in the mobile phase can also cause bad peak shapes. It is always advisable to use the same solvent for preparation of sample solution as the mobile phase to avoid precipitation of the compounds in the column or injector.
Optimization can be started only after a reasonable chromatogram has been obtained. A reasonable chromatogram means that all the compounds are detected by more or less symmetrical peaks on the chromatogram. By a slight change of the mobile phase composition, the shifting of the peaks can be expected. From few experimental measurements, the position of the peak can be predicted within the range of investigated changes. An optimized chromatogram is the one in which all the peaks are symmetrical and are well separated in less run time.
The peak resolution can be increased by using a more efficient column (column with higher theoretical plate number, N) which can be achieved by using a column of smaller particle size, or a longer column. These factors, however, will increase the analysis time. Flow rate doe not influence resolution, but it has a strong effect on the analysis time.
The parameters that are affected by the changes in chromatographic conditions are,
Retention time (Rt) Resolution (Rs), Capacity factor (k’), Selectivity (α),
Column efficiency (N) and Peak asymmetry factor (As) Quantitative analysis in HPLC
i) External standard method
The external method involves the use of single standard or up to three standard solutions. The peak area or the height of the sample and the standard used are compared directly. One can also use the slope of the calibration curve based on standards that contain known concentrations of the compounds of interest.
ii) Internal standard method
A widely used technique of quantitation involves the addition of an internal standard to compensate for various analytical errors. In this approach, a known compound of a fixed concentration is added to the known amount of samples to give separate peaks in the chromatograms to compensate for the losses of the compounds of interest during sample pretreatment steps. Any loss of the component of interest will be accompanied by the loss of an equivalent fraction of internal standard. The accuracy of this approach obviously depends on the structural equivalence of the compounds of interest and the internal standard.
The requirements for an internal standard are,
it must have a completely resolved peak with no interferences, it must elute close to the compound of interest,
it must behave equivalent to the compounds of interest for analysis like pretreatments, derivative formations, etc., it must be added at a concentration that will produce a peak
area or peak height ratio of about unity with the compounds of interest,
it must not be present in the original sample,
it must be stable, unreactive with sample components, column packing & the mobile phase and
The internal standard should be added to the sample prior to sample preparation procedure and homogenized with it. To be able to recalculate the concentration of a sample component in the original sample, one has to determine first the response factor. The response factor (RF) is the ratio of peak areas of sample component (Ax) and the internal standard (AISTD)
obtained by injecting the same quantity. It can be calculated using the formula,
RF = Ax / AISTD
When more than one component is to be analyzed from the same sample, the response factor of each component should be determined.
iii) Standard addition method
In the standard addition method a known amount of the standard compound is added to the sample solution to be estimated. This method is suitable if sufficient amount of the sample is available and is more realistic in the sense that it allows calibration in the presence of excipients or other components.
Estimation of multicomponent dosage forms by UV spectrophotometric methods
Most of drugs in their formulations can be analyzed by spectrophotometric methods. Because it is easy, accurate, fast and economical method for estimation of colorless and colored components. It is very old and basic technique for estimation of known component also guesses functional groups in unknown compounds. Absorption spectra obtained as per functional groups present in the compound. The colorless drug components scanned to 200-400 nm and colored at visible range 400-800 nm. The wavelength fixed at which drug component shows maximum absorbance (λmax). Nowadays spectrophotometric estimation for
Spectroscopic measurement depends on following conditions like, solvents used,
wavelength selected and
selection of spectrophotometric method Selection of solvent
The solute should completely soluble in respective solvent. In UV detection wave length solvent should not interfere for absorbance of drug component and the solution of drugs should be stable for room temperature. Commonly water, mixture of organic solvents and water are used as solvent. Selection of wavelength
The sensitivity UV spectrometric method depends upon the proper selection of the wavelength. An ideal wavelength is one that gives good response for all the components to be detected. This may not, however, be possible in all the cases due to the difference in the nature of the drugs present in the multicomponent dosage forms.
UV spectrums of 10 µg/ml of standard drugs were recorded individually. The spectrums were superimposed to get overlay spectrum for two or three drugs as per combination in formulation. From this overlain spectrum detection wavelength was fixed at which all the drugs show good absorbance.
Selection of spectrophotometric method
The selection of different spectrophotometric methods depends upon λmax obtained for different drug components and overlay spectrum for
i) Analysis of multicomponent drugs by dual wavelength method
Standard stock solution of A and B drugs in the multicomponent formulations were prepared separately in respective solvents to the concentration of 1mg/ml. They were further diluted and the absorbances were measured at the respective wave length of maximum absorption (λmax) for calibration
curve linearity. The sample solution containing both A and B drugs was kept in the sample cell of the double beam spectrophotometer and the absorbance was recorded at A drug λmaxkeeping equivalent strength of standard solution
of B in the reference cell. The absorbance valve was used to calculate concentration of A drug by putting this value in equation of linearity. Similarly, the absorbance was recorded at B drug λmaxkeeping equivalent
strength of standard solution of A in the reference cell. The absorbance value was used to calculate concentration of B drug by putting this value in equation of linearity.
ii) Analysis of multicomponent drugs by simultaneous equation method
If sample contains two absorbing drugs X and Y each of which absorbs at λmax of the other, it may be possible to determine both drugs by a technique of simultaneous equation (Vierodt.s method).
First one should obtain spectra of each pure component and then select two wavelengths where the difference in molar Absorptivity (ε) is maximal.
(ε1/ ε2) λ1and (ε2/ ε1) λ2, neither these wavelengths need necessary coincide
Figure: The individual absorption spectra of substances X and Y, showing the wavelengths for the assay of X and Y in admixture by the method of simultaneous equation
Two simultaneous equations are written as A1= ax1bcx +ay1bcy --- (1)
A2= ax2bcx +ay2bcy --- (2)
A1andA2= Absorbance of mixture at two wavelengthsλ1andλ2
ax1and ax2= Absorptivity of component X atλ1andλ2
ay1and ay2= Absorptivity of component Y atλ1andλ2
These equations are solved for the concentration of each component by rearranging equation 2
Cy= A2-ax2cx /ay2
Substituting for cyin equation 1 and rearranging gives
Cx=A2ay1- A1ay2 /ax2ay1- ax1ay2and
Cy= A1ax2. A2ax1 /ax2ay1- ax1ay2
If sample contains three absorbing drugs X, Y and Z each of which absorbs at λmax of the other, the simultaneous equation can be written as
A1= ax1bcx+ay1bcy+ az1bcz
A2= ax2bcx+ay2bcy+ az2bcz
A3= ax3bcx+ay3bcy+ az3bcz
iii) Analysis of multicomponent drugs by Q-analysis method As the overlain spectrum of drug A and drug B standard solutions recorded. The two wavelengths were selected one at an isoabsorptive point for both the drugs and other wavelength of any one of the drug. The dilutions of the standard and sample solutions were carried out as reported in simultaneous equation method. The absorptivity values for both drugs at the selected wavelength were calculated and employed for Q analysis, the concentration of drugs in sample solution were determined by using the following formula. For drug A in sample
Q0– Q2 A1 Absorbance of sample at fixed wavelength
C1= ---× --- Q0=
---Q1– Q2 a1 Absorbance of sample at isobestic point
For drug B in sample
Q0– Q1 A1 Absorbance of A drug at fixed wavelength
C2=---× --- Q1=
---Q2– Q1 a2 Absorbance of A drug at isobestic point
Absorbance of B drug fixed wavelength Q2=
In equation A1was absorbance of sample at isoabsorptive point and a1and a2
were absorptivities values of A and B respectively at isobestic absorptivity point.
Figure: Wavelength for the assay of absorbance of substances X and Y in admixture by the method of absorbance ratio
iv) Analysis of multicomponent drugs by derivative spectrophotometry
Direct spectrophotometric determination of multicomponent formulation is often complicated by interference from formulation matrix and spectral overlapping; such interferences can be treated in many ways like solving two
simultaneous equations, using Q anlysis, but still may give erroneous results21.
Derivative spectrophotometry is a useful means of resolving two overlapping spectra and eliminating matrix interferences or interferences due to an
indistinct shoulder on side of an absorption band24. Derivative
spectrophotometry involves the conversion of a normal spectrum to its first, second or higher derivative spectrum. In the context of derivative spectrophotometry, the normal absorption spectrum is referred to as the fundamental, zero order or D spectrum. The absorbance of a sample is differentiated with respect to wavelength λ to generate first, second or higher order derivative
[A] = f (λ ): zero order
[d2A/d λ2] = f (λ ): second order
The first derivative spectrum of an absorption band is characterized by a maximum, a minimum, and a cross-over point at the λmax of the absorption band. The second derivative spectrum is characterized by two satellite maxima and an inverted band of which the minimum corresponds to the λmax of the fundamental band.
The important features of derivative technique include enhanced information content, discrimination against back ground noise and greater selectivity in quantitative analysis. It can be used for detection and determination of impurities in drugs, chemicals and also in food additives and industrial wastes.
Figure: (b) First, (c) Second, (d) Third and (e) fourth derivative Spectrum of (a) Gaussian peak.
Validation of analytical method
Validation is a process that confirmation or establishment by laboratory studies that a method developed is accurate, precise and rugged 12-13. In simple terms, validation of an analytical procedure is to demonstrate
The various validation performance parameters are, accuracy,
precision (repeatability and reproducibility), specificity,
linearity and range,
limit of detection (LOD)/limit of quantitation (LOQ), selectivity/specificity,
ruggedness/robustness, stability and
system suitability. Accuracy
The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method should be established across its range. Accuracy is calculated as the percentage of recovery by the assay of the known added amount of analyte in the sample, or as the difference between the mean and the accepted true value, together with confidence intervals.
Accuracy is calculated from the test results as the percentage of analyte recovered by the assay. Dosage form assays commonly provide accuracy within 3-5% of the true value.
Precision
of single determination and are commonly in the range of 0.3 to 3% for dosage form assays.
Specificity
The International Conference of Harmonization (ICH) documents define specificity as the ability to assess unequivocally the analyte into the presence of components that may be expected to be present, such as impurities, degradation products and matrix components.
In case of assay, demonstration of specificity requires that the procedure is unaffected by the presence of impurities or excipients. In practice, it can be done by spiking the substance or product with appropriate levels of impurity or recipients and demonstrating that the assay results are unaffected by the presence of this extraneous material. If impurity or degradation product standards are unavailable specificity may be demonstrated by comparing test results of samples containing impurities or degradation product to a well characterized procedure. This comparison should include sample stored under relevant stress conditions e.g., light, heat, humidity, acid/base hydrolysis and oxidation.
Selectivity
Linearity and Range
The linearity of an analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in sample within a given range. It should be established across the range of the analytical procedure. Linearity is usually expressed in terms of the variance around the slope of the regression line calculated according to an established mathematical relationship from test results obtained by the analysis of samples with varying concentrations of analyte.
The range of an analytical method is the interval between the upper and lower levels of analytic (including these level) that have been demonstrated to be determined with a suitable level of precision, accuracy, and, linearity using the method as written. The range is normally expressed in the same unit as test result (e.g. percent / ppm).
Limit of Detection
Limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitative, under the stated experimental conditions. The detection limits is usually expressed as the concentration of analyte (e.g., percentage ppb) in the sample.
Limit of Quantitation
Limit of quantitation (LOQ) is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. It is expressed as the concentration of analyte (e.g., percentage, ppb) in the sample.
Ruggedness
Robustness
The robustness of analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. A good practice is to vary important parameters in method systematically and measure their effect on separation. Such parameters include mobile phase composition and pH, mobile phase additives, column temperature, flow rate etc.
Stability
Stability of sample, standard and reagents used in HPLC and UV method is required for a reasonable time to generate reproducible and reliable results. For example 24 hour stability is desired for solutions and reagents that need to be prepared for each analysis. Long term column stability is critical for method ruggedness since even a best HPLC column will eventually degrade and loose its initial performance.
System suitability tests
2. AIM AND OBJECTIVES
There are numbers of formulations containing combination two or three drug components have been proved more effective, sustained action in combination form. The drug combination not only gives better therapeutic effect due to additive or synergistic effect, but also easy for administration as a single dose, economy in production, distribution and treatment costs. Although several countries have banned the use of some irrational combinations, several multicomponent dosage forms continue to exist and still introducing new formulations in market with two and three drugs combinations.
Analysis of dosage forms containing single drug is easier than compared to analysis of multicomponent mixtures containing two or three drug components either by UV spectrophotometry or HPLC method. In such formulations interference from excipients can occur. The proper sample preparation methods should be adopted. Analysis of multicomponent dosage form by the extraction of individual drugs is time consuming tedious and always errors in results due to incomplete extraction. Therefore it is very essential to develop simple, rapid and accurate instrumental methods like UV spectrophotometric and more sensitive HPLC method for multicomponent formulations. Most of the drugs in multicomponent dosage forms can be analyzed by the UV spectrophotometric and HPLC methods because of many advantages like rapidity, specificity, accuracy, precision and easy of automation of HPLC method eliminate tedious extraction or isolation procedures.
3. REVIEW OF LITERATURE
The detailed literature survey was carried out for the following multi component drug formulations about the development and validation of UV and HPLC methods.
1) Ambroxol Hcl in combination with Cetirizine and Ambroxol Hcl in combination with Levo cetirizine di hydrochloride of different formulations like tablets, capsules and syrup.
2) Ambroxol Hcl in combination with Loratadine of tablets.
3) Nebivolol in combination with Valsartan of capsule dosage form and Nebivolol in combination with Hydrochlorothiazide of tablets.
4) Olmesartan medoxomil in combination with Hydrochlorothiazide of tablets.
5) Rabeprazole in combination with domperidone of sustained release capsules.
6) Mosapride in combination with Pantoprazole of enteric coated capsules. 7) Diclofenac sodium in combination with Rabeprazole of capsules.
8) Tramadol in combination with Paracetamol of tablets.
9) Tramadol in combination with Paracetamol and Aceclofenac of tablets. 10) Tramadol in combination with Paracetamol and Domperidone of tablets. 11) Atrovastatin in combination with Fenofibrate of tablets.
12) Pseudoephedrine in combination with Levo cetirizine and Ambroxol Hcl of tablets.
Ambroxol Hydrochloride
Z. Dincer and coworkers14 have reported quantitative determination of
ambroxol in tablets by derivative UV spectrophotometric method and HPLC. Determination of ambroxol in tablets was conducted by using first-order derivative UV spectrophotometric method at 255 nm (n = 5). Standards for the calibration graph ranging from 5.0 to 35.0 µg/ml were prepared from stock solution. The proposed method was accurate with 98.6 ± 0.4% recovery value and precise with coefficient of variation of 1.22. These results were compared with those obtained by reference methods, zero-order UV spectrophotometric method and reversed-phase high-performance liquid chromatography (HPLC) method. A reversed-phase C18 column with aqueous phosphate (0.01 M) –
acetonitrile - glacial acetic acid (59:40:1, v/v/v) (pH 3.12) mobile phase was used and UV detector was set to 252 nm. Calibration solutions used in HPLC were ranging from 5.0 to 20.0 µg/ml. Results obtained by derivative UV spectrophotometric method was comparable to those obtained by reference methods, zero-order UV spectrophotometric method and HPLC, as far as ANOVA test, F(calculated) = 0.762 and F(theoretical) = 3.89, was concerned.
H. Basan and coworkers15 have reported derivative UV
J.E.Koundourellis and coworkers16 have reported high performance
liquid chromatographic determination of ambroxol in the presence of different preservatives in pharmaceutical formulations. The method separates ambroxol from methyl- ethyl-, propyl and butyl paraben and from other multi-component mixtures. The retention behaviour of ambroxol and parabens as a function of both pH and mobile phase composition was investigated. The eluents were monitored with a UV detector at 247 nm. Linear relationships between the amount of pharmaceutical compounds and peak heights were confirmed at the concentrations of 0.74 - 14.08 µg/ml. The high recovery (no extraction of the samples is required) and the low % RSD confirm the suitability of the proposed method for the determination of ambroxol in different pharmaceutical preparations.
M. Heinänen and coworker17 have reported validation of an HPLC
method for the quantification of ambroxol hydrochloride and benzoic acid in a syrup as pharmaceutical form stress test for stability evaluation. A method is described for ambroxol, trans-4-(2-amino-3,5-dibromobenzylamino) cyclohexanol hydrochloride, and benzoic acid separation by HPLC with UV detection at 247 nm in a syrup as pharmaceutical presentation. Optimal conditions were: column symmetry shield RPC8, 5 µ 250 x 4.6 mm, and
methanol/(H3PO4 8.5 mM/triethylamine pH=2.8) 40:60 v/v. Validation was
performed using standards and the pharmaceutical preparation which contains the compounds described above. Results from both standards and samples show suitable validation parameters. The pharmaceutical grade substances were tested by factors that could influence the chemical stability. These reaction mixtures were analyzed to evaluate the capability of the method to separate degradation products. Degradation products did not interfere with the determination of the substances tested by the assay.
Krupa M. Kothekar and coworkers18 have reported quantitative
C18 column (25cm X 4.6mm, 5µm). The mobile phase constituted of buffer:
acetonitrile: methanol (650:250:100) with triethylamine and pH adjusted to 5.2 with dilute orthophosphoric acid was delivered at the flow rate 1.0 ml/min. Detection was performed at 220 nm. Separation was completed within 10 min. Calibration curves were linear with coefficient correlation between 0.99 to 1.0 over a concentration range of 7 to 22 mg/ml of levofloxacin and 50 to 150 mg/ml for ambroxol hydrochloride respectively. The relative standard deviation (R.S.D) was found <2.0%.
Meiling Qi and coworkers19 have reported simultaneous determination
of roxithromycin and ambroxol hydrochloride in a new tablet formulation by liquid chromatography. Chromatographic separation of the two drugs was achieved on a diamonsil™ C18 column (200 mm×4.6 mm, 5 μm). The mobile
phase consisting of a mixture of acetonitrile, methanol and 0.5% ammonium acetate (39:11:50 (v/v), pH 5.5) was delivered at a flow rate of 1.0 ml/min. Detection was performed at 220 nm. Linearity, accuracy and precision were found to be acceptable over the concentration range of 201.2 – 2012.0 μg/ml for roxithromycin and 42.7– 427.0 μg/ml for ambroxol hydrochloride, respectively. Separation was complete in less than 10 min. The proposed method can be used for the quality control of formulation products.
Cetirizine hydrochloride
Sevgi Karakus and coworkers20 have reported development and
validation of a rapid RP-HPLC method for the determination of cetirizine (CET) or fexofenadine (FEX) with pseudoephedrine (PSE) in binary pharmaceutical dosage forms. The chromatographic separation of PSE, FEX and CET was achieved on a zorbax C8(150mm×4.6 mm; 5µm particle size) column using UV
mixtures were 10 – 80 and 5 – 40 µg/ml with LOD values of 0.75 and 0.27 µg/ml for PSE and FEX, respectively. Correlation coefficients (r) of the regression equations were greater than 0.999 in all cases. The precision of the method was demonstrated using intra- and inter-day assay R.S.D. values which were less than 1% in all instances. No interference from any components of pharmaceutical dosage forms or degradation products was observed. According to the validation results, the proposed method was found to be specific, accurate, precise and could be applied to the quantitative analysis of these drugs in capsules containing PSE – CET or extended-release tablets containing PSE – FEX binary mixtures.
Sayeed Arayne and coworkers21 reported for determination and
quantification of cetirizine Hcl in dosage formulations by RP-HPLC. The chromatographic system consisted of shimadzu LC-10 AT VP pump, SPD-10 AV VP with UV/visible detector and a CBM-102 bus module integrator. Separation was achieved on the U bondapak 125 Å C18 10 µm column at room
temperature. The samples were introduced through an injector valve with a 10 µl sample loop. Acetonitrile-water (1:1 v/v) was used as mobile phase, with flow rate 2 ml/min. pH was adjusted to 2.9 with phosphoric acid. UV detection was performed at 205 nm. The results obtained showed a good agreement with the declared content. Recovery values for cetirizine hydrochloride were 99.19 -100.82 %. The proposed method is reliable rapid, precise, selective and may be used for the quantitative analysis of cetirizine Hcl, in presence of hyoscine butyl bromide as internal standard. The method was valid used for the determination in raw materials, bulk drug and formulations. The limit of quantification was 5 -30 ng, while the limit of detection was 0.4 ng.
A.F.M. El Walily and coworkers22 have developed spectrophotometric
determined by the measurement of its first (1D) and second (2D) derivative amplitudes at 239 (peak) and 243–233 nm (peak-to-trough), respectively. The aqueous solutions obeyed beer’s law in the concentration ranges of 1.2–10.0 and 0.8–10.0 mg/ml for 1D and 2D measurements, respectively. The colorimetric procedure was based on measuring the absorbency of the coloured chromogen resulted from the reaction between cetirizine sodium salt in polar solvent (DMF) and chloranil at 556 nm. The relation with concentrations was linear over 120–250 mg/ml. Optimization of the reaction conditions was studied. At the same time, investigation of the complex formed was made with respect to its composition and the associated constant. A simple liquid chromatographic assay has been developed for the determination of cetirizine dihydrochloride in the presence of one of its synthesis precursor (hydroxyzine hydrochloride). A bondapak-C18 column was used with a mobile phase consisting of acetonitrile :
0.01 M ammonium dihydrogen phosphate (32:68, v/v) containing 0.1% w/v tetra butyl ammonium hydrogen sulphate adjusted to pH 3 with phosphoric acid at a flow rate of 2 ml/min. With salicylic acid as internal standard, quantitation was achieved with UV detection at 230 nm based on the peak height ratios. Beer’s law was obeyed in a concentration range of 3–35 mg/ml and the regression line equation was derived with a correlation coefficient of 0.9999. The validity of the methods was further confirmed using the standard addition method. The proposed procedures were successfully applied to the determination of cetirizine in bulk and tablet form, with high percentage of recovery, good accuracy and precision.
A.M.Y. Jaber and coworkers23 have reported determination of cetirizine
dihydrochloride (CZ), related impurities and preservatives in oral solution and tablet dosage forms using HPLC. The chromatographic system used was equipped with a hypersil BDS C18, 5 μm column (4.6 × 250 mm) and a detector
set at 230 nm in conjunction with a mobile phase of 0.05M dihydrogen phosphate:acetonitrile:methanol:tetrahydrofuran (12:5:2:1, v/v/v/v) at a pH of 5.5 and a flow rate of 1ml/min. The calibration curves were linear within the target concentration ranges studied, namely, 2×102 – 8×102 μg/ml and 1–4 μg/ml for CZ, 20–100 μg/ml for preservatives and 1–4 μg/ml for CZ related impurities. The limits of detection (LOD) and quantitation (LOQ) for CZ were, respectively, 0.10 and 0.34 μg/ml and for CZ related impurities were in the ranges of 0.08–0.26 μg/ml and 0.28–0.86 μg/ml, respectively. The method proved to be specific, stability indicating, accurate, precise, robust and could be used as an alternative to the European pharmacopoeial method set for CZ and its related impurities.
B.Paw and coworkers24 have reported development and validation of a
HPLC method for the determination of cetirizine in pharmaceutical dosage forms. Methanol was found to be a suitable extraction solvent for tablets and for preparing solutions from drops and oral liquids. The samples were chromatographed on a nova-Pak C18column and UV detected at 227 nm. The
elution was achieved isocratically with a mobile phase of 0.067 M phosphate buffer pH 3.40/acetonitrile (1:1, v/v). Ketotifen was applied as an internal standard. The method was validated for linearity, precision, accuracy and limit of detection. The recovery (mean ± SD) for tablets was 100.88% ± 0.8967, for drops 100.35% ± 0.4062 and for solutions 101.20% ± 1.1698.
Levo cetirizine
M.R. Morita and coworkers25 have reported determination of levo
cetirizine (I) in human plasma by liquid chromatography–electrospray tandem mass spectrometry: Application to a bioequivalence study. Sample preparation was made using a fexofenadine (II) addition as internal standard (IS), liquid– liquid extraction using cold dichloromethane, and dissolving the final extract in acetonitrile. I and II (IS) were injected in a C18 column and the mobile phase
and product ion combinations ofm/z389 > 201 for I andm/z502 > 467 for II. The limit of quantification and the dynamic range achieved were 0.5 ng/ml and 0.5– 500.0 ng/ml. Validation results on linearity, specificity, accuracy, precision and stability, as well as its application to the analysis of plasma samples taken up to 48 h after oral administration of 5 mg of levo cetirizine dichloride in healthy volunteers demonstrate its applicability to bioavailability studies.
Nebivolol Hcl
H.Y.Aboul-Enein and co worker26 have reported HPLC enantiomeric
resolution of nebivolol on normal and reversed amylose based chiral phases. Racemic nebivolol, a adrenergic blocker showing very promising beta-adrenergic antagonist properties in comparison to other beta-beta-adrenergic blockers has been resolved by HPLC under normal and reversed phase modes. The columns used were Chiralpak AD and Chiralpak AD-RH containing amylose tris (3, 5-dimethyl phenyl carbamate) as the chiral selector. The mobile phases used were pure ethanol and 1-propanol. The flow rates used were 0.5, 1.0 and 1.5 ml/min. The best resolution was achieved at 0.5 ml/min. flow rate with ethanol and 1-propanol on both Chiralpak AD and Chiralpak AD-RH stationary phases. The values of infinity for both alcohols on Chiralpak AD were 1.38 while on Chiralpak AD-RH these values were 1.41 and 1.38 respectively. The values of Rs for ethanol and 1-propanol were 2.63 and 1.71 on Chiralpak AD and 1.73 and 1.76 on Chiralpak AD-RH respectively.
N.V.Ramakrishna and coworkers27 have reported rapid quantification of
nebivolol in human plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The method involved a simple single-step liquid-liquid extraction with diethyl ether/dichloromethane (70/30). The analyte was chromatographed on waters symmetry C18
and the weighted (1/x2) calibration curves were linear over the range 50 - 10,000
pg/ml. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in human plasma were 10 and 50 pg/ml, respectively. The within- and between-batch accuracy and precision were found to be well within acceptable limits (<10%). The analyte was stable after three freeze-thaw cycles (deviation <10%). The average absolute recoveries of nebivolol and tamsulosin, used as an internal standard, from spiked plasma samples were 73.4 ± 3.7 and 72.1 ± 2.0%, respectively. The assay method described here was applied to study the pharmacokinetics of nebivolol.
Valsartan
S. Tatar and coworker28 have reported comparison of UV- and second
derivative-spectrophotometric and LC methods for the determination of valsartan in pharmaceutical formulation. For the first method, UV-spectrophotometry, standard solutions were measured at 205.6 nm. The linearity changes were found to be 2.0 - 10.0 µg/ml in ethanol and the regression equation was A = 0.105 C+0.04264 (r=0.9997). For the second method, the distances between two extreme values (peak-to-peak amplitudes), 221.6 and 231.2 nm were measured in the second order derivative-spectra of standard solutions. Calibration curves were constructed by plotting dλ A/dλ2 values
against concentrations, 0.5 - 4.0 µg/ml of valsartan standards in ethanol. Regression equation of linear calibration graph was calculated as D = 0.029 C – 0.00237 (r=0.9996). The third method was based on high-performance liquid chromatography on C18column using acetonitrile, phosphate buffer as a mobile
B.R.Kadam and coworker29 have developed quantitative analysis of
valsartan and hydrochlorothiazide in tablets by high performance thin-layer chromatography with ultraviolet absorption densitometry. Standard and sample solutions of valsartan and hydrochlorothiazide were applied to precoated silica gel G 60 F254HPTLC plates and the plates were developed with chloroform–ethyl acetate–acetic acid, 5 : 5 : 0.2 (v/v), as mobile phase. UV detection was performed densitometrically at 248 nm. The retention factors of valsartan and hydrochlorothiazide were 0.27 and 0.56, respectively. The linear range was 800–5600 ng per spot for valsartan and 125–875 ng per spot for hydrochlorothiazide; the correlation coefficients, r, were 0.9998 and 0.9988, respectively. The method was validated in accordance with the requirements of ICH guidelines and was shown to be suitable for purpose. The method was successfully used for determination of the drugs in tablets. Tablet excipients did not interfere with the chromatography.
E. Satana and coworkers30have reported simultaneous determination of
Hydrochlorothiazide
F. Liu and coworkers31 have reported determination of
hydrochlorothiazide in human plasma by liquid chromatography/tandem mass spectrometry. The analyte and irbesartan, used as the internal standard, were precipitated and extracted from plasma using methanol. Analysis was performed on a phenomenex kromasil C8 column with water and methanol
(27:73, v/v) as the mobile phase. Linearity was assessed from 0.78 to 200 ng/ml in plasma. The analytical method proved to be applicable in a pharmacokinetic study after oral administration of 12 mg hydrochlorothiazide tablets to 20 healthy volunteers.
S.R.Sathe and coworker32 have reported simultaneous analysis of
losartan potassium, atenolol, and hydrochlorothiazide in bulk and in tablets by high-performance thin-layer chromatography with UV absorption densitometry. After extraction with methanol, sample and standard solutions were applied to prewashed silica gel plates and developed with toluene–methanol– triethylamine 6.5:4:0.5 (v/v) as mobile phase. Zones were scanned densitometrically at 274 nm. The Rf values of losartan potassium, atenolol, and hydrochlorothiazide were 0.60, 0.43, and 0.29 respectively. Calibration plots were linear in the ranges 1000–5000 ng per band for losartan potassium and atenolol and 250–1250 ng per band for hydrochlorothiazide; the correlation coefficients, r, were 0.9994, 0.9993, and 0.9994, respectively. The suitability of this method for quantitative determination of these compounds was proved by validation in accordance with the requirements of pharmaceutical regulatory standards. The method was used for routine analysis of these drugs in bulk and in a formulation.
F.Al-momani33 has developed determination of hydrochlorothiazide and
v/v) adjusted to pH 4.1 by glacial acetic acid. The detection was at 220 nm. The method was tested for linearity, accuracy, recovery, and specificity.
G. Carlucci and coworkers34have developed simultaneous determination
of losartan and hydrochlorothiazide in tablets by high-performance liquid chromatography. The procedure, based on the use of reversed phase high performance liquid chromatography, is linear in the concentration range 3.0 -7.0 µg/ml for losartan and 0.5 - 2.0 µg/ml for hydrochlorothiazide, is simple and rapid and allows accurate and precise results. The limit of detection was 0.08 µg/ml for losartan and 0.05 µg/ml for hydrochlorothiazide.
E.Dinc and coworker35 have reported spectrophotometric quantitative
determination of cilazapril and hydrochlorothiazide in tablets by chemometric methods. Four chemometric methods were applied to simultaneous determination of cilazapril and hydrochlorothiazide in tablets. Classical least-square (CLS), inverse least-least-square (ILS), principal component regression (PCR) and partial least-squares (PLS) methods do not need any priori graphical treatment of the overlapping spectra of two drugs in a mixture. For all chemometric calibrations a concentration set of the random mixture consisting of the two drugs in 0.1 M Hcl and methanol (1:1) was prepared. The absorbance data in the UV-vis spectra were measured for the 15 wavelength points (from 222 to 276 nm) in the spectral region 210 - 290 nm considering the intervals of Δλ = 4 nm. The calibration of the investigated methods involves only absorbance and concentration data matrices. The developed calibrations were tested for the synthetic mixtures consisting of two drugs and using the Maple V software the chemometric calculations were performed. The results of the methods were compared each other as well as with HPLC method and a good agreement was found.
Serap Saglik and coworkers36have reported simultaneous determination
derivative spectrophotometric method. Calibration curves were constructed by plotting d4A/dλ4 values at selected wavelengths against concentrations. HPLC
analyses were carried out on C18 column with gradient elution by using 10 mM
H3PO4and CH3CN as mobile phase. Benazepril was used as internal standard
and the substances were detected at 215 nm. Commercially available tablets containing 20 mg fosinopril and 12.5 mg hydrochlorothiazide were analyzed by fourth derivative spectrophotometric and HPLC methods. The results were compared statistically at 95% confidence level with each other. There was no significant difference between the mean percentage recoveries and precision of the two methods.
Olmesartan medoxomil
M.Celebier and coworker37 have reported determination of olmesartan
medoxomil in tablets by UV-vis spectrophotometry. The solutions of standard, tablet and synthetic tablet were prepared in acetonitrile and in NaOH - Water. 258 nm and 250 nm were chosen for acetonitrile and for NaOH - Water solutions respectively. The developed method was validated with respect to stability, linearity, sensitivity, specificity, precision, accuracy, robustness and ruggedness. The linearity range of the method was 1.0 - 70.0 µg/ml for acetonitrile solutions and 1.0 - 75.0 µg/ml for NaOH - Water solutions. The developed and validated method was applied for the determination of olmesartan medoxomil in pharmaceutical dosage forms.
T. Murakami and coworkers38 have reported identification of a
degradation product in stressed tablets of olmesartan medoxomil by the complementary use of HPLC hyphenated techniques. An unknown degradation product (DP-1) increased in olmesartan medoxomil (OLM) tablets stored at 40ºC/75% RH, reaching 0.72% after 6 months. The molecular weight and fragment information obtained by LC-MS suggested that DP-1 was a dehydrated dimer of olmesartan (OL) and the presence of ester carbonyl group was indicated by solvent-elimination LC-IR analysis. LC 1H NMR confirmed
use of HPLC hyphenated techniques without complicated isolation or purification processes.
C.Mustafa and coworkers39have reported development of a CZE method
for the determination of olmesartan medoxomil (OLMD) in tablets. The influences of pH, buffer concentration, applied voltage and capillary temperature on the migration time of OLMD were investigated. About 50 mM pH 6.5 phosphate buffer was used as background electrolyte. The optimum instrument parameters were found to be 30°C temperature with 30 kV applied voltage and diode array detection was carried out at 210 nm. OLMD was hydro dynamically injected (Pinj = 50 mbar, tinj = 3 s) and an internal standard,
diflunisal (IS), was used to improve the precision and repeatability. Under these conditions, the migration time of OLMD was 2.32 min and the total analysis time was shorter than 5 min. Linearity range for the developed method was found to be 2.0 – 50.0 μg/ml and the limit of detection was 0.5 μg/ml. The developed method was applied for the analysis of OLMD in pharmaceutical tablet formulations.
Rabeprazole sodium
G.M. Reddy and coworkers40 have reported identification and
2-[[[4-(3-methoxymethyl]pyridin-2-yl] methane sulfinyl] – 1 -[[4 - (3-methoxy propoxy)-3-methyl]pyridin-2-ylmethyl]-1H-benzimidazole (impurity IV); 2-[[[4-methoxy-3-methyl-2-pyridinyl] methyl] sulfinyl]-1H-benzimidazole (impurity V); 2-[[[4-(3-methoxypropoxy)-3-methyl-2-pyridine-1-oxide] methyl] sulfinyl]-1H-benzimidazole (impurity VI).
S. S. Sabnis and coworkers41 have reported the spectrophotometric
simultaneous determination of rabeprazole sodium and itopride hydrochloride in capsule dosage form. The method is based on ratio spectra derivative spectrophotometry. The amplitudes in the first derivative of the corresponding ratio spectra at 231nm (minima) and 260nm were selected to determine rabeprazole sodium and itopride hydrochloride, respectively. The method was validated with respect to linearity, precision and accuracy.
C.V. Garcia and coworkers42 have reported development and validation
of a dissolution test for rabeprazole sodium in coated tablets using a reverse-phase liquid chromatographic method. After test sink conditions, dissolution medium and stability of the drug, the best conditions were: paddle at 75 rotations per minute (rpm) stirring speed, Hcl 0.1 M and borate buffer pH 9.0 as dissolution medium for acidic and basic steps, respectively, volume of 900 ml for both. The quantitation method was also adapted and validated. Less than 10% of the label amount was released in the acid step, while more than 95% was achieved over 30 min in the basic one. The dissolution profile for tablets was considered satisfactory. The dissolution test developed was adequate for its purpose and could be applied for quality control of rabeprazole tablets, since there is no official monograph.
A. El-Gindy and coworkers43 have reported spectrophotometric and
was based on HPTLC separation followed by densitometric measurement of the spots at 284 nm. The separation was carried out on merck HPTLC sheets of silica gel 60 F254, using acetone-toluene-methanol (9:9:0.6 v/v) as mobile phase.
The third method depends on first derivative of the ratio spectra (1DD) by measurement of the amplitudes at 310.2 nm. Moreover, the proposed HPLC method was utilized to investigate the kinetics of the oxidative and photo degradation processes. The pH-rate profile of degradation of RA in britton-robinson buffer solutions within the pH range 3-11 was studied. In addition, the activation energy of RA degradation was calculated in britton-robinson buffer solution pH 7.
Shan Ren and coworkers44 have reported effect of pharmaceutical
excipients on aqueous stability of rabeprazole sodium. The chemical stability of a proton-pump inhibitor, Rabeprazole sodium, was evaluated in simulated intestinal fluid (pH 6.8) containing various ‘Generally Recognized As Safe (GRAS)’-listed excipients, including brij® 58, poleaxes 188, cremophor RH40,
gelucire 44/14 and PEG 6000. After incubation at 37 and 60 °C, the amounts of rabeprazole and its degradation product, thioether-rabeprazole, were quantitated by HPLC analysis. The main degradation product was separated and characterized by LC/MS. The degradation of rabeprazole followed first-order kinetics. In the absence of any excipients, the rate constants (k) obtained at 37 and 60 °C were 0.75 and 2.78 /h, respectively. In contrast, the addition of excipients improved its stability. Among several excipients tested in this study, brij® 58 displayed the greatest stabilizing effect. For instance, at 37 and 60 °C,
brij® 58 reduced the k values to 0.22 and 0.53 /h, respectively. The stabilizing
mechanisms of these hydrophilic polymeric excipients with optimal HLB values could be partially explained in terms of their solubilizing efficiency and micellar formation for thioether-rabeprazole. In conclusion, Rabeprazole formulations that contain suitable excipients would improve its stability in the intestinal tract, thereby maximizing bioavailability.
N.V. Ramakrishna and coworkers45 have reported high-performance
plasma using solid-phase extraction. Following solid-phase extraction using waters oasis trade mark SPE cartridges, the analyte and internal standard (pantoprazole) were separated using an isocratic mobile phase of 5 mM ammonium acetate buffer (pH adjusted to 7.4 with sodium hydroxide solution)/acetonitrile/methanol (45/20/35, v/v) on reverse phase waters symmetry C18 column. The lower limit of quantitation was 20 ng/ml, with a
relative standard deviation of less than 8%. A linear range of 20-1000 ng/ml was established. This HPLC method was validated with between- and batch precision of 2.4-7.2% and 2.2-7.3%, respectively. The between- and within-batch bias was -1.7 to 2.6% and -2.6 to 2.1%, respectively. Frequently co administered drugs did not interfere with the described methodology. Stability of rabeprazole in plasma was excellent, with no evidence of degradation during sample processing (auto sampler) and 3 months storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.
Diclofenac sodium
J. Ghasemi and coworkers46 have reported simultaneous
spectrophotometric determination of benzyl alcohol and diclofenac in pharmaceuticals using methods based on the first derivative of the optical density ratio.The first method makes use of a derivative of the double-divisor-ratio spectrum of optical density. In this case, the linear determination ranges
are 30 – 97 µg/ml for benzyl alcohol and 12 – 45 µg/ml for diclofenac. In the
second method, the analytical signals are measured at wavelengths corresponding to either maxima or minima for both drugs in the spectra of the first derivative of the ratio of optical densities of the sample and the standard
solution of one of the drugs. Two sensitive and accurate spectrophotometric
signals are measured at wavelengths corresponding to either maxima or minima for both drugs in the spectra of the first derivative of the ratio of optical densities of the sample and the standard solution of one of the drugs. In this case, the linear determination ranges are 32–95 µg/ml for benzyl alcohol and 14–45 µg/ml for diclofenac. Both analytical procedures do not require any separation of the components. The proposed methods were satisfactorily applied to the rapid simultaneous determination of benzyl alcohol and diclofenac in commercial pharmaceutical preparations and semi synthetic pharmaceuticals containing these drugs.
J. Krzek and coworker47 have reported densitometric determination of
diclofenac, 1-(2,6-dichlorophenyl)indolin-2-one and indolin-2-one in pharmaceutical preparations and model solutions. The effect of pH, temperature and ultra violet (UV) radiation on diclofenac concentration was investigated. Chromatographic separation was performed on TLC silica gel coated plates with the mobile phase: cyclohexane-chloroform-methanol (12:6:1, v/v/v). Densitometric detection was carried out in UV at λ 248 nm. The conditions for good separation and the detection limit were established. The recovery for diclofenac was 99.20%, for 1-(2,6-dichlorophenyl)indolin-2-one--92.34% and for indolin-2-one--95.85%. The method was used for quality assessment of diclofenac in pharmaceutical preparations. Reliable results comparable to those determined by high performance liquid chromatography (HPLC) were obtained.
Domperidone
Maurizio Cignitti and coworkers48have reported UV spectroscopic study
and conformational analysis of domperidone. The UV absorption spectra of domperidone, 2-(3H)-benzimidazolone and 5-Cl-2-(3H)-benzimidazolone in CH3CN have been studied both in the absence and in the presence of hard acids.
conformational space of domperidone. The results show a large number of conformers lying with 3 kcal/mol with respect to the lowest energy structure.
I. Ali and coworkers49 have developed screening of domperidone in
wastewater by high performance liquid chromatography and solid phase extraction methods. Domperidone is a dopamine D2receptor antagonist, which
has been used as antiemetic agent in human beings. It has been found in wastewater released by some pharmaceutical industries leading to the contamination of surface and ground water. The column used was waters symmetry C18 (15cmx0.46mm, 5µm). The mobile phase used was phosphate
buffer (50mM, pH 3.5) acetonitrile (80:20, v/v) at the flow rate 2.0ml/min. The detection was achieved by using UV mode at 230nm. The retention, separation and resolution factors were 2.63, 3.00 and 3.20, respectively. The percentage recovery of domperidone from wastewater was 95.0%. Celiprolol was used as the internal standard to access the percentage extraction of domperidone from wastewater.
T. Sivakumar and coworkers50 have reported development and
