Pharmaceutical Analysis may be defined as the application of analytical processs used to find the pureness, safety and quality of drugs and chemicals. The term “ Pharmaceutical analysis ” is otherwise called quantitative pharmaceutical chemical science. Pharmaceutical analysis includes both qualitative and quantitative analysis of drugs and pharmaceutical substances starts from majority drugs to the finished dose signifiers. In the modern pattern of medical specialty, the analytical methods are used in the analysis of chemical components found in human organic structure whose altered concentrations during disease provinces serve as diagnostic AIDSs and besides used to analyse the medical agents and their metabolites found in biological system.
Qualitative inorganic analysis seeks to set up the presence of given component or inorganic compound in a sample.
Qualitative organic analysis seeks to set up the presence of a given functional group or organic compound in a sample.
Quantitative analysis seeks to set up the sum of a given component or compound in a sample.
The term “ quality ” as applied to a drug merchandise has been defined as the amount of all factors, which contribute straight or indirectly to the safety, effectivity and dependability of the merchandise. These belongingss are built into drug merchandises through research and during procedure by processs jointly referred to as “ Quality control ” . Quality control warrants with in sensible bounds that a drug merchandises
Is free of drosss.
Is physically and chemically stable
Contains the sum of active ingredients as stated on the label and
Provides optimum release of active ingredients when the merchandise is administered.
Most modern analytical chemical science is categorized by two different attacks such as analytical marks or analytical methods.
By Analytical Targets
By Analytical Methods
Bioanalytical chemical science
1.1 INTRODUCTION FOR CHROMATOGRAPHY2-5:
High public presentation liquid chromatography is the procedure, which seperates mixture incorporating two or more constituents under high force per unit area. In this the stationary stage is packed in column one terminal of which is attached to a beginning of pressurized liquid nomadic stage.
High public presentation liquid chromatography is the fasted turning analytical technique for the analysis of drug. Its simpleness, high specificity and broad scope of sensitiveness makes its ideal for the analysis of many drugs in both dosage signifier and biologic fluids.
HPLC is besides known every bit High public presentation liquid chromatography. It is indispensable signifier column chromatography in which the stationary stage is consists of a little atoms ( 3-5oAµm ) wadding contained in a column with a little dullard ( 2-5mm ) , one terminal of which is attached to beginning of pressurized liquid eluent ( nomadic stage ) .
Different Types of Principles:
Harmonizing to the stages involved, HPLC can be classified into several types, which are as follows:
Normal Phase Chromatography ( NPC )
Reverse – Phase Chromatography ( RPC )
Liquid – Solid Chromatography or surface assimilation HPLC
Liquid – Liquid Chromatography or Partition HPLC
Ion exchange Chromatography or Ion exchange HPLC
Size exclusion or gel pervasion or steric exclusion HPLC
Normal Phase Chromatography ( NPC ) :
In normal stage chromatography, the stationary stage is more polar so the nomadic stage, and the nomadic stage is a mixture of organic dissolvers with out added H2O ( e.g. isopropane with hexane ) and the column wadding is either an inorganic adsorbent ( silicon oxide ) are a polar bonded stage ( cyanno, glycol, amino ) on a silica support. Sample keeping in normal stage chromatography additions with the mutual opposition of nomadic stage lessenings. They are eluted in the order of increasing mutual oppositions.
Reverse – Phase Chromatography ( RPC ) :
In reverse-phase chromatography, the stationary stage is less polar than the nomadic stage and the nomadic stage is a mixture of organic and aqueous stage. Reverse-phase chromatography is typically more convenient and rugged than the other signifiers of liquid chromatography and is more likely to ensue in a satisfactory concluding separation. High public presentation RPC columns are efficient, stable and consistent. In this, the solutes are eluted in the order of their decreasing mutual oppositions. These are prepared by handling the surface silanol group of site with an organic chloro silane reagent.
SCHEMATIC DIAGRAM OF HPLC
Pumps are required to present a changeless flow of nomadic stage at force per unit areas runing from 1 – 550 saloon pumps capable of force per unit area up to 6000 pounds per square inch provide a broad scope of flow rates of nomadic stage, typically from 0.01-10ml min-1. Low flow rates ( 10-100i?lmin-1 ) are used with micro dullard columns, intermediate flow rates ( 0.5-2ml min-1 ) are used with conventional analytical HPLC columns, and fast flow rates are used for preparatory or semi preparatory columns and for slurry packing techniques.
Mechanical pumps of the reciprocating Piston type position a throbing supply of nomadic stage. A damping device is at that place forward required to smooth out the pulsations so that inordinate noise at high degrees of sensitiveness or low force per unit area does non take away from sensing of little measures of sample. This type of pump is largely used.
Dual – Piston reciprocating pumps produce an about pulse free flow because the two Pistons are carefully faced so that as one is make fulling the other is pumping. These pumps are more expensive than individual Piston pumps but are of benefit when utilizing a flow sensitive sensor such as UV or refractile index sensor.
B. Injection Systems:
Injection ports are of two basic types, ( A ) those in which the sample with injected straight into the column and ( B ) those in which the sample is deposited before the column recess and so brush by a valving action into the column by the nomadic stage.
HPLC columns are made of high quality chromium steel steel, Polish internally to a mirror coating. Standard analytical columns are 4-5 mm internal diameter and 10-30 centimeter in length. Shorted columns ( 3-6 centimeter ) incorporating a smaller atoms size packing stuff ( 3 or 5 i?m ) produce similar or better efficiencies, in footings of the figure of theoretical home bases ( about 7000 ) , that those of 20 centimeters columns incorporating 10 i?m irregular atoms and are used an short analysis clip and highest throughput of samples are required. Micro bore columns of 1-2 millimeter internal diameter and 10-25 centimeter in length have certain advantages of lower sensing bounds and lower ingestion of dissolver, the latter being of import if expensive HPLC – class dissolvers are used. HPLC are besides being carried out on the semi preparatory graduated tables by utilizing columns of 7-10 millimeter or 20-40 millimeter internal diameter severally.
The most widely used sensors for liquid chromatography are
Any with chromophores
UV-grade non UV absorbing dissolvers
Has grade of selectivity and utile for many HPLC applications
UV-grade non UV absorbing dissolvers
Highly selective and sensitive, frequently used to analyse derivitized compounds
Compounds with different RI than nomadic stage
Can non run nomadic stage gradients
Charged or polar compounds
Mobile stage must be carry oning
Excellent for ion exchange compounds
Readily oxidized or reduced compounds, specially biological samples
Mobile stage must be carry oning
Very selective and sensitive
Theoretical rules of HPLC:
The clip is required between the injection point and the peak upper limit is called the keeping clip. It is denoted as the Rt. It is chiefly utile for the qualitative analysis for the designation of compound.
It represents the molar ratio of the compound in the stationary stage and the nomadic stage. It is independent of column length and nomadic stage flow rate. It is denoted as the “ K ” . It should be kept 1-10. If “ K ” values are excessively low it is likely that the solutes may be adequately resolved and for high ‘k ‘ values the analysis clip is excessively long. It can be calculated by
tr – t0
K = — — — — — — — —
tr = Retention clip, t0 = Dead clip.
Closer survey of a chromatographic show that the Gaussian signifiers is normally non wholly symmetrical. The graph spread out to a greater or lesser extent, organizing a tail. It reduces the column home base figure which intern influences the declaration. Tailing is chiefly due to deteriorated column, overloading column, excess column-volumes, and mutual exclusiveness of sample with standard and/or nomadic stage. Practically it can be calculated or determined at 10 % of the entire extremum tallness. It must non be greater than 2.0
The grade of separation of one constituent from another is described by the declaration. It is by and large denoted by ‘Rs ‘ . It is measured as the difference in keeping clip and the arithmetic mean of the two peak breadths.
tr2 – tr1
Rs = — — — — — — — — — — –
0.5 ( w1 + w2 )
tr2 = Retention clip of first extremum w1 = breadth of first extremum
tr1 = Retention clip of 2nd extremum w2 = breadth of 2nd extremum
Theoretical home bases:
It is of import belongings of the column. It reflects its quality of separation and its ability to bring forth crisp, narrow extremum and accomplishing good declaration of extremum. ‘N ‘ denotes it.
3500 X L ( centimeter )
Theoretical home bases = — — — — — — — — — — —
displaced person ( Aµm )
L = length of the column – in centimeter, displaced person = diameter of the atom ( Aµm )
It follows that if the exchange is fast and efficient, the theoretical home base will be little in size and there will be big figure of home bases in the column.
Height equivalent to theoretical home base ( HETP ) :
Number of home bases straight relative to the column length ( L ) and reciprocally relative to the diameter of the atoms ( displaced person ) . The value of H is a standard for the quality of a column. Lower the HETP, higher is the efficiency of the column. ‘ Its value depends upon atom size, flow rate, viscousness of nomadic stage.
H = L/N
L = Length of column, N = No. of theoretical home base
1.3 HPLC method development6-8:
The broad assortment of equipment, columns, eluent and operational parametric quantities involved makes high public presentation liquid chromatography ( HPLC ) method development seem complex. The chief aim of method development is to obtain a good separation with minimal clip and attempt. Based on the end of separation, the method development is preceded. The stairss involved are
Information on sample, define separation ends
Need for particular HPLC process, sample pretreatment, etc.
Choose sensor and sensor scenes
Choose LC method, preliminary tally ;
Estimate best separation conditions
Optimize separation conditions
Check for jobs or demand for particular process
Validation for release to routine research lab
The undermentioned must be considered when developing an HPLC method:
Keep it simple
Try the most common columns and stationary stages foremost
Thoroughly investigate binary Mobile stages before traveling on to tertiary
Think of the factors that are likely to be important in accomplishing the coveted declaration.
Mobile stage composing, for illustration, is the most powerful manner of optimising selectivity whereas temperature has a minor consequence and would merely accomplish little selectivity alterations. pH will merely significantly affect the keeping of weak acids and bases.
VALIDATION OF ANALYTICAL METHOD IN PHARMACEUTICAL ANALYSIS:
Validation is documented grounds, which is completed to guarantee that an analytical method is accurate, consistent and robust over the specific scope. The quality of the analytical information is a cardinal factor in the success of a drug development plan. The procedure of method development and proof has a direct impact on the quality of these informations.
Method proof is the procedure to corroborate that analytical process employed for a specific trial is suited for its intended usage. Method needs to be validated or revalidated
Before their debut into everyday usage
Whenever the conditions alterations for which the method has been validated, e.g. , instrument with different features
Whenever the method is changed, and the alteration is outside the original range of the method.
Depending on the usage of the check, different parametric quantities will hold to be measured during the assay proof. ICH and several regulative organic structures and Pharmacopoeia have published information on the proof of analytical processs
1.4 METHOD VALIDATION PARAMETERS 9-10:
The end of the proof procedure is to dispute the method and find the bound of allowed variableness for the conditions needed to run the method. The undermentioned statistical parametric quantities are to be determined to formalize the developed method.
Correlation coefficient ( R ) :
When the alterations in one variable are associated or followed by alterations in the other, it is called correlativity. The numerical step of correlativity is called the coefficient of correlativity and is defined by the relation.
i?“ ( ten – ten ‘ ) ( y -y ‘ )
R = — — — — — — — — — — — — — — — — — — — — — — — — — –
a?s i?“ ( x -x ‘ ) 2 i?“ ( y -y ‘
Regression equation= I + aC
Y2 – Y1
a = incline = — — — — — — — –
X2 – X1
I = Intercept = arrested development – a C
As a per centum of average optical density.
S = a?s i?“ ( X- Ten! ) 2/N – 1
Where, X = observed values
Ten! = Arithmetical mean = i?“X/N
N = Number of divergences
For practical reading it is more convenient to show ‘S ‘ in footings of per centum of the approximative norm of the scope of analysis is used in the computation of ‘S ‘ . This is called co-efficient of fluctuation ( C.V ) or percent comparative criterion divergence ( % RSD ) .
C.V OR % RSD = 100* S/ Ten!
Standards for Validation of the Method
Recovery ( 98-102 % )
RSD & lt ; 2 %
Correlation Coefficient ( R ) & gt ; 0.99
& gt ; 24h or & gt ; 12h
3. LITERATURE REVIEW
Sasmitha Kumar et al22 ; has been developed UV spectroscopic method for appraisal of Rizatriptan benzoate.The drug shows maximal soaking up at 277 nanometers and 281 nanometers and obeys beer-lamberts jurisprudence in the concentration of 0.5-20 Aµg/ml at 277 nanometers and 0.5-80 Aµg/ml at 281 nm severally. The per centum recovery was found to be 97-100 % .
Madhukar et al23 ; was developed rearward stage high public presentation liquid chromatographic method for finding of Rizatriptan benzoate. The proposed method utilised column L1 inertsil ODS-3v, 250 nmx4.6 millimeter holding atom size, 5Aµm. The nomadic stages were comprised of A, B of Acetonitrile and buffer pH 6.5 at UV sensing 225 nm.The method shows recovery 96.64-97.71
Sachin jagthap et al24 ; has been developed stableness bespeaking reversed stage high public presentation liquid chromatographic method for the finding of Rizatriptan benzoate in majority pulverization and in pharmaceutical preparations. The method utilizes c18 column holding dimension 250mmx4.6 millimeter holding atom size,5.0 Aµm utilizing a nomadic stage 0.01M Na dihydrogen phosphate buffer: Methanol, at a flow rate 1ml/min at ambient temperature and detected at 225 nm.and the method was validated harmonizing to ICH guidelines
Quizi zhang et al25 ; has been developed, a high public presentation liquid chromatographic method for the finding of Rizatriptan benzoate in human plasma.using asingle measure liqid liqid extraction with metyl third butyl quintessence, the analytes separated usig amobile stage dwelling of 0.05 % v/v triehylamine in H2O seting pHs 2.75 with 85 % phosphorous acid and acetonitrile.fluroscence sensing was performed at an excitement wavelength of 225 nanometers and an emanation wavelength of 360 nm.The one-dimensionality for rizatriptan was within the concentration scope of 0.5-50ng/ml.
Rajendra Kumar et al26 ; was developed and validated stableness a stableness bespeaking high public presentation liquid chromatographic method for Rizatriptan benzoate.The force debasement surveies were performed on bulk sample of Rizatriptan benzoate. The method utilizes a zorbax SB-CN column with dimension of 250 mmx4.6 millimeter, 5um column. The nomadic stage consists of a mixture of aqueous K dihydrogen ortho phosphate ( ph3.4 ) , acetonitrile and methyl alcohol.
Rauza bagh et al27 ; has been developed a spectroscopic method for analysis of Rizatriptan benzoate in majority and tablet dose signifier. The Rizatriptan benzoate shows maximal optical density at 225 nanometer. Beer ‘s jurisprudence was obeyed in the concentration scope of 1-10Aµg/ml.
2. DRUG PROFILE 20-21:
2.1 RIZATRIPTAN Benzoate:
Chemical name: N, N diethyl -5- ( 1H-1,2,4-triazol-1-1-ylmetyl ) -1H
Indole-3 Ethanamine monobenzoate
Molecular Formula: C15H19N5.C6H5COOH
Molecular weight: 391.47
Description: White crystalline pulverization
Melting point: 178-1800C
Solubility: Meagerly soluble in H2O and methyl alcohol
Storage: Air tight container protect from visible radiation.
Drug Class: Anti migraine drug
2.2 THERAPEUTIC RATIONAL:
Mechanism of action:
Rizatriptan binds with high affinity to human 5-HTIB and 5-HTID receptors taking to cranial blood vas bottleneck.
Wholly absorbed from GI piece of land, absolute bioavailability is 45 % plasma peak concentration attained with in 1-1.5 hours ( conventional tablet ) or 1.6-2.5 hours ( orally disintegrating tablet ) after unwritten disposal.
Crosses placenta and is distributed in to milk in animate being, no surveies in pregnant or nursing adult females.
Metabolized chiefly via oxidative deaminization by Mao-A to an inactive indole acetic acid metabolite
Excreted chiefly in piss ( 14 % of dosage as unchanged drug and 51 % a indole acetic acid metabolite.
Dry oral cavity
Pain tightness/pressure in neck/throat/jaw.
Dose and disposal:
The dose scope of Rizatriptan benzoate is 10-30mg orally one time daily.Rizatriptan benzoate can be administer orally disintegrating tablet with out repasts.
New RP-HPLC METHOD FOR THE ESTIMATION OF RIZATRIPTAN
Benzoate IN TABLET DOSAGE FORM
A simple contrary stage HPLC methods was developed for the finding of Rizatriptan benzoate in tablet dose signifier. Zorbax Eclipse XBD C18 ( 250 centimeter A- 4.6 millimeter ) column in isocratic manner with nomadic stage Buffer ph 5.0: Methanol ( 80:20 ) was used and pH-5 adjusted with tri ethylamine. The flow rate was 1.0 ml/min and UV sensing at 225nm. The keeping clip 3.0 min. The proposed method was besides validated.
Shimadzu LC-10A HPLC
Vacuum pump – Gelmon scientific discipline
Elico SL-164 dual beam UV-Visible spectrophotometer
Ultra sonicator 3.5L 100 ( pci )
Water HPLC class
Methanol HPLC class ( Merck )
Potassium dihydrogen inorganic phosphate ( AR Grade )
Triethylamine ( AR Grade )
1. Choice of wavelength:
After solubility survey for the drug dissolver was selected and appropriate concentration of Rizatriptan benzoate criterions with dissolver were prepared. The solution were so scanned by utilizing doubl beam UV-Visible spectrophotometer the scope between 200-400nm.The overlain spectra for the both drug were observed and maximal wavelength was eventually selected.
2. Choice of nomadic stage:
To develop a precised and robust HPLC method for finding of Rizatriptan benzoate, its standard solution were injected in the HPLC system. After literature study and solubility informations different composing of nomadic stage of different flow rates were employed in order to find the best status for effectual separation of drugs.
3. Choice of column:
Initially different C8 and C18 columns were tried for selected composing of nomadic stage and quality of extremums were observed for the drugs. Finally the column was fixed upon the satisfactory consequences of assorted system suitableness parametric quantities such as column efficiency, keeping clip, chasing factor / extremum dissymmetry of the extremums.
Other parametric quantities such as flow rate, column temperature etc. were selected by changing its value up to certain degrees and consequences were observed. The value at satisfactory consequences were obtained has been selected for the method. The concluding choice of chromatographic conditions as follows
Optimized chromatographic conditions
Stationary stage ( column )
Zorbax Eclipse XBD C18 ( 250cm x4.6 millimeter ) , 5Aµm
Buffer: Methanol ( 80:20 )
Volume of injection cringle
Preparation of Buffer ph 5.0:
Dissove 2.76 gram of K dihydrogen inorganic phosphate in 1000ml of HPLC H2O plus 5.0 mlof Triethylamine. Mix and adjust PH 5.0 with orthophosporic acid. Filter with 0.45u nylon filter.
Preparation of nomadic stage:
The nomadic stage was prepared by blending Buffer: Methanol ( 80:20 ) . the solution was so filtered through 0.45I?m membrane filter and sonicated.
Preparation of standard stock solution:
Standard solution of the pure drug was prepared by fade outing 73.0 milligram of Rizatriptan benzoate in 100ml volumetric flask. The drugs were dissolved by utilizing nomadic stage as a dilutant. Add about 50ml of dilutant and sonicate to fade out. Make up the volume with dilutant. Mix good. Further dilute 5.0ml of the above solution to 250ml with dilutant, mix good.
Preparation of sample solution:
Weighted and crushed 20 integral tablet. and taken accurately 1000 milligram of Rizatripatan benzoate in into a100ml volumetric flask. Add about 50ml of dilutant and sonicate for 15 min and do up the volume with dilutant. Mix good, filter through 25 millimeters 0.45 u nylon, discard 4ml filtrate. Further dilute 5ml of the solution to 250 milliliter with dilutant and mix good.
Calculation: % Assay Rizatriptan
spl country venereal disease wt 5 100 250 269.4 Avg wt
= — — — — — — x — — — — -x — — — x — — — — — x — — — — x — — — — — x — — — — — — -x P
std country 100 250 spl wt 5 391.4 LC
6. RESULT AND DISSCUSION
1 ) Specificity:
Specificity of an analytical method is its ability to mensurate accurately and specifically the analyte of involvement without interventions from clean and placebo.
There should non be any intervention extremums due to blank or placebo solution with the chief extremum.
1 ) There is no intervention from clean and placebo with the chief extremum.
2 ) The peak pureness base on ballss for the chief extremum in the standard readying, sample.
readying and placebo.
2 ) One-dimensionality:
Linearity was determined at five degrees over the scope of 80 % to120 % of the trial solution.
A standard stock solution was prepared and further diluted to achieve concentration about 80 % , 90 % , 100 % , 110 % , 120 % . Each standard readying was injected in triplicate. The average country at each degree was calculated and graph of average country versus concentration was plotted. The correlativity co-efficient R, Y intercept, incline regration line, were calculated. and recorded in Table-1
The correlativity coefficient value should non be less than 0.99.
Table-1: Drumhead study of Rizatriptan:
Conc. In Aµg/ml
Correlation co-efficient R
Slope of arrested development line
Graph 1: One-dimensionality secret plan of Rizatriptan
The correlativity value is 0.9998. The countries obtained are straight relative to the concentration of analyte in the sample. Threefore, method is additive in the scope.
3 ) Accuracy:
The truth was performed at three degrees viz, 80 % , 100 % , 120 % with regard to the trial concentration. Known sum of Rizatriptan benzoate working criterion was added to the placebo, analysed as per trial process and milligram of Rizatriptan benzoate found in the trial solution was calculated.
Each sample was prepared in the triplicate at each degree and injected. The consequences are recorded in Table-2.
Recovery at each degree and average recovery should be between 95.0 % to 105 % with RSD at each degree should non be more than 2.0 % .
Table-2: Drumhead study of Rizatriptan:
Conc. ( % )
Aµg of added
Aµg of found
% Average recovery
The per centum at each degree, average recovery, and RSD meets the established credence standards. Hence, method is accurate.
4 ) Preciseness:
a ) System preciseness:
Rizatriptan benzoate criterion was prepared and injected in six replicate, RSD was calculated and recorded in Table-3.
The RSD for six replicate injections of standard readying should non be more than 2.0 %
Table-3: Drumhead study of Rizatriptan:
The RSD of six replicate injections is within bound, hence system is precise.
B ) Method preciseness:
Method preciseness was established by assaying six readyings under the same status. Six replicate of sample were prepared at trial concentrationby one analyst and injected on the same equipment and the same day.and consequence recorded in Table-4
RSD for six replicate analysis should non be more than 2.0 % .
Table-4: Drumhead study of Rizatriptan:
Weight of sample
The consequence obtained lies good within credence standards. Hence method is precise.
5 ) Robustness:
The hardiness of the method was established by doing calculated minor fluctuation in the undermentioned parametric quantity.
A ) Change flow rate: ( 0.2 )
Low flow rate ( LF ) : 0.8ml/min
High flow rate ( HF ) : 1.2ml/min
The consequence of alterations made delibrately in the chromatogram status was obsereved on the system suitableness trial and recorded in Table-5
Robustness: Change in flow rate
Table-5: Drumhead study of Rizatriptan:
Area of Rizatriptan benzoate
L.F: 0.8 ml/min.
Hafnium: 1.2 ml/min.
6 ) Stability OF THE ANALYTICAL SOLUTION:
The sample solution was prepared at working concentration and initial check determined. Solution was stored up to 24 hour room temperature, and analyzed against newly prepared criterion. The assay value obtained at 24hrs was compared with initial check value. The consequence are recorded in Table-6.
The difference in the check values should non be more than 2.0 % from the initial value.
Table-6: Drumhead study of Rizatriptan:
After 24 hours at room temperature
Consequence: The consequences obtained are good within the credence standards. Therefore, the sample solution and standard solution are stable upto