Pragya Nand Badyal; Chetan Sharma; Navdeep Kaur; Ravi Shankar; Abhay Pandey; Ravindra K. Rawal

Review Article

Austin J Anal Pharm Chem. 2015;2(2): 1037.

Analytical Techniques in Simultaneous Estimation: An Overview

Pragya Nand Badyal¹, Chetan Sharma¹, Navdeep Kaur¹, Ravi Shankar², Abhay Pandey¹ and Ravindra K. Rawal¹*

¹Department of Pharmaceutical Analysis, Indo-Soviet Friendship College of Pharmacy (ISFCP), Moga-142001, India

²Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Jammu-180001, India

*Corresponding author: Ravindra K. Rawal, Department of Pharmaceutical Analysis, Indo-Soviet Friendship College of Pharmacy (ISFCP), Moga-142002, India.

Received: March 18, 2015; Accepted: April 16, 2015; Published: April 17, 2015

Abstract

Simultaneous estimation plays a very important role in pharmaceutical world as it is very feasible and time saving. For the multi component analysis various techniques like spectrophotometric techniques (UV-VIS, IR, NMR and MASS spectrometry) and chromatographic techniques (Thin Layer Chromatography, High Performance Liquid Chromatography, Ultra-High Performance Liquid Chromatography, High Pressure Thin Layer Chromatography and Gas Chromatography) is used. These techniques provide high degree of specificity and selectivity and further provide the high degree of assurance that these techniques fit for the simultaneous estimation of the pharmaceutical dosage form. Chromatographic and spectrophotometric techniques together develop new hyphenated techniques which are useful for the simultaneous estimation and impurity profiling. The simultaneous analytical analysis provides specificity and assurance for the identification of the chemical entities in the pharmaceutical formulation. The main objective behind the analytical estimation is to provide the assurance that the particular formulation contains the equal amount of active pharmaceutical ingredient as mentioned in the label.

Keywords: Analytical techniques; Spectrophotometric; HPTLC; Hyphenated techniques; Gas Chromatography

Abbreviations

API's: Active Pharmaceutical Ingredients; UV-VIS: Ultra Violet -Visible; I.R: Infra Red; I.P: Indian Pharmacopoeia; B.P: British Pharmacopoeia; USP: United States Pharmacopoeia; NMR: Nuclear Magnetic Resonance; MALDI: Matrix- Asisted Laser desorption Ionization; GAA: Glacial Acetic Acid; HPLC: High Performance Liquid Chromatography; PAD: Pulsed Amperometric Detection; PDA: Photo Diode Array; NS,CD: Non-suppressed Conductometric Detection; ACN: Acetonitrile; KOH: Potassiun Hydroxide; TLC: Thin layer Chromtography; HPTLC: High Performance Thin Layer Chromatography; UPLC: Ultra Performance Liquid Chromatography; TFA: Triflouro Acetic Acid; GC-MS: Gas Chromatography - Mass Spectrometry; LC-IR: Liquid Chromatography- Infra Red; LCMS: Liquid Chromatography- Mass Spectroscopy; GC-IR: Gas Chromatography-Infra Red; GC-MS-MS: Gas Chromatography- Mass spectroscopy-Mass spectroscopy; LC-MS-MS: Liquid Chromatography-Mass spectroscopy-Mass spectroscopy; GCGC- MS: Gas Chromatography-Gas Chromatography-Mass Spectroscopy; GC-NMR: Gas Chromatography-Nuclear Magnetic Resonance; GC-AES: Gas Chromatography-Atomic Emission Spectroscopy.

Introduction

The development of pharmaceuticals and their analysis has brought the world to the revolutionary extent in health sciences. The process of drug molecule discovery and pharmaceutical investigation or analysis of the formulation provides the safety and therapeutic effect to a high extent. To make API and formulation safe a large number of analytical techniques have been developed. During various stages of preparation of pharmaceutical formulation, the addition of impurities or development of impurities makes the preparation harmful for the administration and may cause other disorders and disease like cancer(mainly from solvents like benzene) [1,2]. In formulation the analytical techniques play the great role in the identification of physical and chemical properties of the formulation. The analytical techniques provide the important description for the evaluation of the toxicity and other impurities [1]. Presently various combinations in dosage forms are present in enormous amount and are increasing rapidly. These multi component formulations provide the increased therapeutic index, multiple actions, less side effects and quicker relief. The analytical process deals with two parts of chemical characterization either it is qualitative or quantitative. The qualitative analytical analysis provides quality and identity of the sample and quantitative analytical analysis provides the amount of chemical identities present in the formulation. The main objective behind the analytical estimation is to provide the assurance that the particular formulation contains the equal amount of active pharmaceutical ingredient as mentioned in the label [3].

For the estimation of multi component in formulation the various instrumental techniques like spectrophotometric and chromatographic techniques are used due to their advantages viz. less time consuming, cheap, specific and accurate which provides result up to high extent [3] (Figure 1).

Figure 1: Different types of analytical techniques used in pharmaceutical world.

    
    
    Figure 1:  Different types of analytical techniques used in pharmaceutical world.

Spectrophotometric Techniques

Spectrophotometric techniques are the important instrumental techniques which are available to pharmaceutical analyst. The basics of spectrophotometric techniques are that they measure the interaction of electromagnetic radiations with sample in quantized form [4]. There are various spectrophotometric techniques which are used in the pharmaceutical world for the analysis of the API's and pharmaceutical ingredients.

UV/VIS Spectroscopy

It is the cheapest and easiest working analytical tool available that is used in the pharmaceutical laboratories and research. The analytical applications of the UV spectroscopy are qualitative and quantitative. As most of the pharmaceutical contains chromophore they can be determined directly. However UV/VIS spectroscopy is not much suitable for the simultaneous estimations with spectral overlapping. The derivative spectroscopy provides the enhancement of specificity and sensitivity in pharmaceutical formulations [5].

The ultraviolet-visible spectrophotometry techniques is one of the most frequently used techniques in the analysis which involves the measurement of amount of ultraviolet and visible radiations absorbed by the pharmaceuticals in a solution. Various techniques has been used in simultaneous estimation by UV/VIS spectroscopy such as: (i) Simultaneous Equation method; (ii) Absorbance ratio method; (iii) Geometric correction method; (iv) Orthogonal polynomial method; (v) Difference spectroscopy; (vi) Derivative spectroscopy [4]. The advantages of the UV/VIS spectroscopy techniques are that they have low time and labor consumption. The precision and accuracy of analyst by using the UV/VIS spectroscopy is up to a high limit [1].

Derivative Spectrophotometry

It is one of the most highly developed spectrophotometric techniques. The origin of derivative spectrophotometry is linked with appearance of spectrophotometers enabling recording of derivative spectra [6]. The derivative spectrophotometry involves the change of normal spectrum to its first, second or higher derivative spectrum [4]. The derivative spectra can be obtained by optical, electronic and mathematical methods. In optical method there is wave length modulation where the wavelength of incident light is quickly modulated over a narrow wavelength range by electromechanical device [6].

If the derivative spectrum is expressed as absorbance (A) as function of wavelength (λ), the derivative spectra are:

Zero order: A = f (λ)

First order: $\frac{d A}{d λ} = f^{'} (λ)$

Second order: $\frac{d^{2} A}{d λ^{2}} = f^{″} (λ)$

The derivative spectra are employed to get the better differences among spectra to resolve the overlapping bands in qualitative analysis and to reduce the outcome of scattering matrix [4].

The strong positive and negative bands with maximum and minimum at same wavelength of an absorption band as inflection point in absorbance band governs the odd (first and second) derivative spectrum whereas the strong positive and negative bands with minimum or maximum at same wavelength as λ _max of absorbance band governs the even (second and fourth) derivative spectrum.

Number of bands = derivative order +1

The merits of derivative spectroscopy are to increase the resolution permitting identification of analyte with close λ_max, to decline the baseline shift arising from instrument or sample handling and diminish the scattering effect thus helpful for analyte present in turbid solution [7] (Table 1,2,3).

Table 1: Simultaneous estimation of drugs in pharmaceutical combination by first-order derivative UV spectrophotometric method.




  
    Drug combination 
    Wavelength�    (nm) 
    References 
  
  
    Adrenaline and Noradrenaline
    394 and 342
    [8] 
  
  
    Amiloride and Furosemide
    241.4 and 343.6
    [9] 
  
  
    Amitryptyline and Chlorpromazine hydrochloride
    254 and 260
    [10] 
  
  
    Amoxicillin and Bromohexine hydrochloride
    278.8 and 326.2
    [11] 
  
  
    Analgin and Adamon
    600 and 310.5
    [12] 
  
  
    Analgin and Hyoscine N-butyl bromide
    291.8 and 219.8
    [13] 
  
  
    Atenolol and Nifedipine
    276 and 340
    [14] 
  
  
    Cephalothin and Cefoxitin
    235 and 236.7
    [15] 
  
  
    Cilazapril and Hydrochlorothiazide
    242.8 and 282.8
    [16]



Table 1:  Simultaneous estimation of drugs in pharmaceutical combination by first-order derivative UV spectrophotometric method.

Table 2: Simultaneous estimation of drugs in pharmaceutical formulation by second-order derivative UV spectrophotometric method.




  
    Drug combination 
    Wavelength (nm) 
    References 
  
  
    Acrivastine and Pseudoephedrine    hydrochloride
    288 and 270.2
    [17] 
  
  
    Cefatoxime sodium and Cefadroxil    monohydrate
    257 and 279
    [18] 
  
  
    Telmisartan� and Metoprolol
    224 and 299.5
    [19] 
  
  
    Prasugrel and Asprin
    252.4 and 267.6
    [20] 
  
  
    Ibuprofen and Famotidine
    272.8 and 290
    [21] 
  
  
    Citicoline and Piracetam
    274.6 and 206.8
    [22] 
  
  
    Diclofenac sodium and    Thiocolchicoside
    249 and 246
    [23]



Table 2:  Simultaneous estimation of drugs in pharmaceutical formulation by second-order derivative UV spectrophotometric method.

Table 3: Simultaneous estimation of drugs in pharmaceutical estimation by thirdorder derivative UV spectrophotometric method.




  
    Drug combination 
    Wavelength (nm) 
    References 
  
  
    Atenolol and Amlodipine
    264 and 308
    [24] 
  
  
    Amitryptyline and    Chlorpromazine hydrochloride
    254 and 260
    [10]



Table 3:  Simultaneous estimation of drugs in pharmaceutical estimation by thirdorder derivative UV spectrophotometric method.

IR Spectroscopy

Pharmaceutical quality control and quality assurance depends on monitoring the composition and uniformity of the drug substance during processing and in the pharmaceutical product. Various tests have been used to determine identity, quality and strength. Vibrational spectroscopic techniques, including Mid-infrared, Near-infrared and Raman, have been proposed as other alternative approaches [25]. Infra-red is an important technique which gives sufficient information about the structure and its functional groups of a compound. This technique provides a spectrum containing large number of absorption bands i.e. functional groups can be derived from which the structures of organic compound can be studied or demonstrated. The absorption of infra-red radiations (quantized) causes the various bands in a molecule to stretch and bend with respect to one another [26]. The most important region for the organic chemist is 2.5μ to 15μ in which molecular vibrations can be detected. The ordinary infra-red region extends from 2.5μ to 15μ. The region from 0.8μ to 2.5μ is called Near IR region and that from 15μ to 200μ is called Far IR region. When the electromagnetic radiations are passed through sample which are absorbed by the bonds of the molecules in the sample causing them to stretch or bend. The wavelength of the radiation absorbed is characteristic of the bond absorbing it (Table 4).

Table 4: Determination of pharmaceutical drugs by IR spectroscopy.




  
    Drug name 
    Near IR 
    Mid IR 
    Far IR 
    Property Description 
    References 
  
  
    Indomethacin-saccharin 
    
       
    
    
       
    
     
    Characterization of cocrystals 
    [27] 
  
  
    Enalapril
     
    
       
    
     
    Characterization of six salt forms
    [27] 
  
  
    Celecoxib
     
    
       
    
     
    Characterization of polymorphs
    [27] 
  
  
    Omperazole sodium
     
    
       
    
     
    Characterization of API in salt form
    [27] 
  
  
    Acyclovir and    lactose 
     
    
       
    
     
    Drug excepient compatibility
    [27] 
  
  
    Bicifadine HCl 
     
    
       
    
     
    Characterization of polymorphs
    [27] 
  
  
    Piroxicam    monohydrate 
    
       
    
     
     
    Quantification during
      isothermal dehydration 
    [27] 
  
  
    Troglitazone
    
       
    
     
     
    Drug (crystalline versus amorphous) distribution in
      solid dispersion
    [27] 
  
  
    Theophylline    and caffeine
    
       
    
    
       
    
     
    Characterization of hydrate formation during wet Granulation
    [27] 
  
  
    Sulfathiazole
    
       
    
     
     
    Polymorph screening and processing-induced
      transformation (PIT)
      screening
    [27] 
  
  
    Prediction of    DNA
     
     
    
       
    
    Oligonucleotide of DNA
    [28]



Table 4:  Determination of pharmaceutical drugs by IR spectroscopy.

Shortcomings and limitations of infrared spectroscopy

Infra-red spectroscopy has proved to be one of the most important and needy methods for characterization of both quantitatively and qualitatively of large number of inorganic compounds encountered in research as well as in industry yet it suffers some shortcomings which are mentioned below:

It is impossible to determine the unknown substance from the individual IR spectrum. Example a mixture of paraffin and alcohol will give the same IR spectrum as by the higher molecular weight alcohols.
In case of Near IR region technique extensive method development is required before the technique can be used for analysis of pharmaceuticals. Development of method requires a highly trained professional analyst with computing knowledge [29].

Application of IR spectroscopy

Various pharmacopoeias like IP, BP and USP are used to identify API's and to check the purity at short interval of the time which indirectly increases the productivity eg: Amylobarbitone, Betamethasone, Dexamethasone, Cyclophosphamide, Sulphalene. Assay of pharmaceutical dosage form: A few remarkable examples are codeine phosphate in tablet, aspirin-phenacetein-caffeine tablet, meprobamate in tablet have been reported [30].

Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance is a division of analytical chemistry and spectroscopy which deals with the radio frequency waves which produces transitions among magnetic energy levels of nuclei of a molecule. The magnetic energy levels are created by keeping the nuclei in magnetic field. The first observation of NMR was observed and studied by Purcell and Bloch at 1945. Ethyl Alcohol was the first compound which was studied and demonstrated by this technique in 1951 [31].

When the energy in the form of radiofrequency is applied, the applied energy is equal to the precessional frequency, the adsorption of energy takes place and NMR spectrum is obtained. On increasing the strength of magnetic field it will cause raise in precessional frequency but it does not cause the transition from ground state to excited state. Without the magnetic field and radiofrequency there will be no reason to cause the formation of NMR spectrum. NMR is used for the quantitative and qualitative analysis to determine the impurities of the drug, to elucidation of natural drug products and to determination metabolites of drugs in body fluids. The enhancement or power of NMR can be increased by following ways:

Hyper-polarization
Two dimensional(2D)
Three dimensional(3D) and higher dimension multifrequency techniques
Distortionless enhancement by polarization transfer (DEPT)
Correlation spectroscopy (COSY)
Nuclear overhouser enhancement spectroscopy (NOESY)

Incredible natural abundance double quantum transfer experiment (INADEQUATE), and with the addition of chromatographic techniques ie hyphenated techniques [32].

Application of NMR

Food Chemistry: It is used for the authentication of the wine aging and identification of the fatty oil's constituents in food and other beverages.
Clinical application: It is used for the identification and studies of metabolites in biological fluids in vivo or in vitro and used for diagnosis and helpful in treatment of diseases [33].
Study of the Hydrogen Bonding: Hydrogen bonds in the metal chelates as well as in organic compounds can be determined by this technique. Hydrogen bonding results in the decrease in the electron shield protons and signal is shifted towards low field.
Impurity profiling of the pharmaceuticals: NMR play has an important role in the impurity profiling of pharmaceuticals (Table 5).

Table 5: Simultaneous estimation of drugs in presence of impurity by NMR spectroscopy.




  
    Drugs 
    Impurity 
    References 
  
  
    Heparin
    Galactosamine
    [34] 
  
  
    Fluticasone propionate
    Monomeric or dimeric impurity
    [35] 
  
  
    Ropivacaine hydrochloride
    Drug associated impurity
    [36] 
  
  
    Anastrozole
    Degradation associated impurity
    [37]



Table 5:  Simultaneous estimation of drugs in presence of impurity by NMR spectroscopy.

Mass Spectrometry

In mass spectroscopy the compound or a complex molecule is ionized, the ions are separated on the basis of their mass/charge ratio, and the number of ions generated during the ionization represents mass/charge unit which is recorded as the spectrum. Commonly electron impact mode is applied for bombarding the molecules in vapour phase with high energy electron beam and records as a spectrum of positive ions, which have been separated on the basis of mass/charge [38]. Some important features of mass spectroscopy are such as (i) small quantity of sample is used in this process which is ionized by the ion source which generally produces cations; (ii) the mass analyzers used in the mass spectrometer separate ions according to their mass to charge ratio; (iii) the ions which are formed are detected by the detector and displayed, which is called mass spectrum.

Advantages of mass spectrometry

It is highly sensitive and accurate technique.
Small amount of sample is required from nanogram to microgram can be analyzed.
Resolution time is up to high extent.
Highly specific due to fragmentation and helps in study of structure.

Disadvantages of mass spectrometry

Sample recovery cannot be achieved due to destructive nature of the process.
Very costly and required high maintenance.

Introduction of sample is difficult due to small sample size [29].

Application of mass spectrometry

Determination of molecular mass of the compounds: peak having highest m/e ratio shows the molecular mass of the compound
Characterization of polymers: Determination of polymers and elucidation of polymer structure can be done by this technique. The structure of the polymer can be distinguished on the basis of arrangement of atoms.
Impurity profiling in pharmaceuticals by Mass spectrometers: Mass spectrometry is the finest and latest technique used for the detection of impurities in the complex compounds. If the molecular weights of the impurities are larger than major components their detection is easier due to their higher mass peaks which are free from major constituents. For example: i) Determination of impurity (toxic elements) in Hydroxyapatite [39]; ii) Determination of impurity (Anhydro-simvastatin, Simvastatin dimer) in Simvastatin [40].
Mass spectrometry is also used for the analysis of simple or complex proteomes using quantitative mass spectrometry [41].
Analytical biological quantitative estimation: Quantitative analysis of antiretroviral drugs (Ritonavir. Saquinavir, Amprenavir, Indinavir, Nelfinavir, Tipranavir, Carbamazepine. As internal standard, carbamazepine for nevirapine, indinavir for amprenavir, lopinavir for tipranavir, indinavir for nelfinavir, saquinavir for ritonavir, nelfinavir for indinavir, and tipranavir for lopinavir were used) in lysates of peripheral blood mononuclear cells using MALDI-triple quadrupole mass spectrometry have been reported [42].

Chromatography

Chromatography is moderately a new technique which was developed by M. Tswett, a botanist in 1906 in Warsaw. In that year he was successful in elucidating chlorophyll, xanthophylls and several other colored substances from vegetable extracts through a column of calcium carbonate. The calcium carbonate act as an adsorbent and other various dissimilar substances get adsorbed to different extent and give rise to colored bands at different positions on the column. Tswett named this system of colored bands as the Chromatogram and the method as Chromatography after the Greek words chroma and graphos meaning "color" and "writing" respectively. In 1930's chromatography in the form of TLC and Ion Exchange chromatography was introduced as separation techniques. In 1941, Martin and Synge introduced Partition and Paper chromatography. They introduce Gas chromatography in 1952.

Chromatography is a non-destructive method from which multi component can be derived and separated chromatography is the most important single analytical technique used today and will most expected continue to be so far for the predictable future. It is the foundation stone of molecular and pharmaceutical analytical chemistry and recently it is coupled with atomic absorption and Mass spectroscopy which has extended its application in the world pharmaceutical analysis.. Various chromatographic techniques have been used for simultaneous estimation of API's or pharmaceutical dosage forms such as (i) Paper chromatography; (ii) High performance liquid chromatography (HPLC); (iii) Thin layer chromatography (TLC); (iv) High performance liquid chromatography (HPTLC); (v) Gas chromatography (GC ); (vi) Ultra-high performance liquid chromatography (UPLC); (vii) Column chromatography.

High Performance Liquid Chromatography (HPLC)

Chromatography is the most commonly used analytical technique in pharmaceutical analysis. The technique is used by the chemists to separate and determine species in variety of organic, inorganic, and biological materials [26]. HPLC has been around for about 35years and it is the major separation technique use. HPLC is one of the best method of choice for analyzing various varieties of natural and synthetic compounds [43]. Various types of HPLC's have been used for simultaneous estimations e.g. normal phase HPLC, reversed phase HPLC, size exclusion HPLC, ion-exchange HPLC, bio-affinity HPLC.

In case of normal phase HPLC, the stationary phase is polar and mobile phase is non polar. Adsorption extends with increase in polarity, and the interaction with polar analyte and polar stationary phase increases the elution time [44]. Few drugs enlisted in Table 6 which is simultaneously estimated by normal phase HPLC.

Table 6: Simultaneous estimation of drugs by normal phase HPLC.




  
    Drugs 
    Mobile phase 
    References 
  
  
    Drotaverine hydrochloride and Omperazole
    n-heptane: Dichloromethane: Methanolic Ammonia (5%) : MeOH(50:25:1:4)    v/v/v
    [45] 
  
  
    Benzoyl peroxide and Benzoic Acid
    MeOH : water (65:35) v/v
    [46] 
  
  
    Tocopherols and Tocotrienols
    Hexane : 1,4-dioxane (95.5:4.5) v/v
    [47]



Table 6:  Simultaneous estimation of drugs by normal phase HPLC.

Furthermore, RP-HPLC is opposite to normal phase HPLC. The polarities of the mobile and stationary phase are reversed. It is the most popular mode of liquid chromatography. Almost 90% of all analysis of low molecular weight sample is carried out using RPHPLC [48] (Table 7).

Table 7: List of few drugs estimated simultaneously by RP-HPLC.




  
    Drugs 
    Mobile phase 
    References 
  
  
    Aceclofenac and Paracetamol
    MeOH and water (70:30) v/v
    [49] 
  
  
    Losartan potassium and Amlodipine besylate
    0.02% Triethylamine in water: ACN (60:40)v/v
    [50] 
  
  
    Naproxen and Esomeprazole
    Part A: Mixing buffer, ACN and MeOH in the ratio of (70:20:10) v/v/v
      Part B: Mixing buffer, ACN in the ratio of (20:80) v/v
      Buffer is prepared by dissolving 0.71 g (0.005M) of sodium perchlorate    in 1000 mL of water, added with 5 mL of N-butyl amine. The pH of the buffer    is adjusted to pH to 8.7 using diluted solution of Perchloric acid.
    [51] 
  
  
    Paracetamol and Etroricoxib
    MeOH : ACN: Phosphate buffer (40:20:40)v/v
    [52] 
  
  
    Cefixime and Cloxacilin
    Phosphate buffer : ACN: MeOH (80:17:3)v/v
    [53] 
  
  
    Metoprolol and Hydrochlorothiazide
    Di-sodium hydrogen phosphate: MeOH: ACN (525:225:250) v/v
    [54] 
  
  
    Chlorpheniramine Maleate and Phenylephrine
    ACN: Phosphate buffer (55:45) v/v
    [55] 
  
  
    Lansoprazole and Domperidone
    ACN and MeOH (81:19) v/v
    [56] 
  
  
    Ambroxal hydrochloride and Loratidine
    ACN and Ammonium acetate (50:50) v/v
    [57]



Table 7:  List of few drugs estimated simultaneously by RP-HPLC.

Size Exclusion HPLC

Size exclusion or Gel chromatography is one of the latest types of the liquid chromatographic procedures. It is the most potent technique that is particularly applicable to high molecular-weight species [58]. It separates biomolecules on the basis of true size difference; small molecules of analyte can enter the pores of gel without difficulty and therefore spend more time in these pores escalating their retention time. On the other hand, large analyte spend little time in the pores and elute quickly [59].

Ion-exchange HPLC

In ion exchange chromatography, the stationary phase is ion exchange resin and ions of the opposite charge are electro statically attached to the surface of the resin. When the mobile phase is passed through resin, the electro statically bound ions are free as other ions which are bounded preferentially. This technique involves the exchange equilibria between ions in solution and ions of like sign on the resin [60] (Table 8).

Table 8: Pharmaceutical compounds estimated by Ion-exchange chromatography.




  
    Analyte 
    Column 
    Eluent 
    Detection 
    Method 
    References 
  
  
    Amikacin
    Anion Exchanger
    0.115 N NaOH
    PAD
    Assay
    [61] 
  
  
    Bethinicol chloride injection
    Weak cation Exchanger
    20 mM������ CH3SO3H
    NS,CD
    Assay
    [61] 
  
  
    Erythromycin
    Cation Exchanger
    Mixture of ACN, NaOH and water
    PAD
    Assay
    [61] 
  
  
    Kanamycin sulfate
    Anion Exchanger
    0.115 N NaOH
    PAD
    Assay
    [61] 
  
  
    Fenoldopam
    Anion Exchanger
    2.8mM NaHCO3 + 2.2mM Na2CO3+ 0.8mM 4-cyanophenol+2% ACN
    NS,CD
    Assay
    [61] 
  
  
    Paracetamol
    Waters IC-PAK A HR
    5 mM LiOH in 5% ACN at 1mL/min
    UV at 300nm
    Quantitation in solid dosage form
    [62] 
  
  
    Oxytetracycline,
      tetracycline,
      chlortetracycline,
      doxycycline.
    Dionex OmniPac PCX 100 (250 � 4mm)
    0.2M HCL in ~28% ACN at 1 mL/min
    Direct UV at 300 nm
    Method developed for primarily residual testing
    [63] 
  
  
    Caffeine, Theobromine,��    Theophylline
    DionexHPLC-CS3(cationic coloumn)
      Dionex OmniPac PAX-100(anionic)
       
    100mM HCL at1mL/min(cation)
      15mM KOH in 1% ACN at 1mL/min
    Direct UV at 274nm
    Quantitation in injections and tablets .
       
    [64] 
  
  
    Flucloxacillin and Amoxicillin
    Zorbax 300-SCX (Agilent)
      250�4.6 mm, 5�m particles
    0.025M ammonium dihydrogen phosphate (pH 2.6)-ACN (95/5) at 1.5mL/min
    UV at 225nm
    Used in QC test in pharmaceutical injection products.
    [65] 
  
  
    Methenamine, Methenamine mandelate, Methenamine hippurate
    Zorbax SCX-300(Agilent), 150�4.6 mm, 5�m
    ACN-sodium perchlorate monohydrate (0.1M, pH05.8), 1mL/min
    UV at 212nm
    Assay of pharmaceutical tablets
    [66] 
  
  
    Simultaneous determination of chloride, bromide and iodide in food    stuff .
    Anion-Exchange resin column (0.6 cm � 13 cm)
    4.0 mM Na2CO3 with flow rate of 0.70mL/min
       
    Optical detector
    Simultaneous Estimation
    [67]



Table 8:  Pharmaceutical compounds estimated by Ion-exchange chromatography.

Bio Affinity HPLC

Affinity chromatography is a precious tool in areas such as biochemistry, pharmaceutical science, clinical chemistry, and environmental testing, where it has been used for both the purification and analysis of compounds in complex sample mixtures. Affinity chromatography is a liquid chromatographic technique that uses a "biologically related" agent as a stationary phase for the purification or analysis of pharmaceutical components. The method of analysis is usually based on the same types of specific, reversible interactions and reactions that are initiated in biological system, such as the binding of an enzyme with a substrate or an antibody with an antigen. These interactions are exploited in affinity chromatography by immobilizing (or adsorbing) one of a pair of molecules on to a solid support which is called as a stationary phase. This immobilized molecule provides the site for binding to particular compounds in a sample [68].

Thin Layer Chromatography

In 1958, Stahl demonstrated purpose and use of TLC in analysis, a method based on adsorption chromatography. Presently TLC is an important analytical tool for the qualitative and quantitative analysis, of number of natural as well as synthetic products. The TLC technique is very important tool in analysis of alkaloids, glycosides, isoprenoids, lipid components, sugars and derivatives and practically all bio constituents.. In TLC sample is spotted on the plate with micropipette and the chromatogram is developed by placing the bottom of the plate or strip. The solvent or the mobile phase is drawn up by the phenomenon of capillary action, and the sample components move up the plate at different rates, depending on their solubility and their degree of affinity towards mobile phase. The spots will generally move at certain fraction of the rate at which the solvent moves, and they are characterized by the Rf value: Rf = distance solute moves /distance solvent from moves [69] TLC plays important role in the analysis of different groups of drugs. The last few years shows that interest in TLC application in pharmaceutical analysis has increased with improvements in TLC instrumentation such as TLC combined with densitometry or with MS and IR. As TLC has some advantages as comparison to HPLC and GC methods are absence of UV activity or when absence of volatility in compounds, TLC is cheap equipment and easy to work in comparison to HPLC and GC and allows parallel separation and quantitative determination of many samples at the same time [70]. The application of TLC has been limited due to low sensitivity and does not appropriate for volatile compounds. Mainly, TLC is frequently used by BP monographs as part of number of identity tests performed on pure substances or used to identify the marker compounds. There are some examples of TLC based identity tests described in pharmacopoeial monographs as shown in Table 9. Various impurities present in the pharmaceutical API's can be detected by TLC. Some drugs with impurities which can be detected by TLC can be detected (Table 10).

Table 9: List of some drugs estimated by TLC.




  
    Drug examined 
    Stationary phase 
    Mobile phase 
    Visualization reagent 
    Reference 
  
  
    Polymyxin B, Framycetin, and Dexamethasone
    Silica gel 60 and F254 silica gel 60 plates
    MeOH and MeOH-n-butanol-Ammonia (25%)-Chloroform    (14:4:9:12) v/v/v/v
    0.3% Ninhydrin solution
    [71] 
  
  
    Oxo-steroids
    Silicagel GF254
    Chloroform: MeOH(97:3) v/v/
    Dansylhydrazine was used as a prelabeling    reagent.
    [72] 
  
  
    Aceclofenac, Paracetamol, and Chlorzoxazone
    Silica gel 60 F 254
    Toluene:2-Propanol: Ammonia (4:4:0.4 )v/v/v
    Detection at 274nm
    [73] 
  
  
    Levamisole
    Silica gel 60F254
    MeOH:Toluene:Chloroform (14:36:50 )v/v/v
    UV light 223nm
    [74]



Table 9:  List of some drugs estimated by TLC.

Drug combination	Wavelength (nm)	References
*Atenolol and Amlodipine*	264 and 308	[24]
*Amitryptyline and Chlorpromazine hydrochloride*	254 and 260	[10]

Drugs	Impurity	Reference
Ethambutol	2-amino butanol	[75]
Framycetin sulphate	Neamine	[75]
Norgestrel	3,17a-diethinyl-13-ethyl-3,5-gonadiene-17-ol	[75]
Clopidogrel	(+)-(S)-(o-chlorophenyl)-6,7-dihydrothieno3,2-c.pyridine-5(4H)-acetic acid	[76]

Drug	Dosage Form	Stationary phase	Mobile phase	Identification	References
Clotrimazole, Miconazole, and Ketoconazole	Creams and Ointments	Silica gel F254	n- hexane: Chloroform: MeOH: Diethylamine� (50:40:10:1) v/v	By UV at 220nm	[77]
Neomycin sulfate, Polymixin B sulfate, Zinc bacytracin and Methyl and Propyl hydroxybenzoates	Opthalmic Ointment	Silica gel	MeOH:n-butanol: Ammonia 25%-Chloroform (14:4:9:12)v/v/v/v for determination of antibiotics and n-pentane:Glacial Acetic Acid (66:9)v/v for methyl and propyl hydroxybenzoates.	Antibiotic were identified by using ninhydrin ethanol solution, while densitometric measurements were made at ?=550nm. Hydroxy benzoates were identified by UV measurements at ?=260 nm	[78]
Atorvastatin calcium and Fenofibrate	Pharmaceutical Dosage form	Silica gel 60 F254	Toluene:MeOH:Triethylamine (7:3:0.2 v/v	densitometrically at 258 nm	[79]

Food sample	Analyte determined	Stationary phase	Mobile phase	Determination	Refernces
Green salad	Ascorbic Acid	Silica Gel F254	Ethanol:1.0% acetic Acid(9:1) v/v	By UV at 254 nm	[81]
Potatoes	Vitamin C	Kiesel gel-G	Oxalic acid, MeOH, Chloroform (2gm:20:60)	UV radiation	[82]
Fish Liver	Vitamin A	Alumina	Cyclohexane	Molybdophosphoric acid	[82]
Vegetable oils	VitaminE	Kiesel gel-G	Dichloromethane, trichloroethylene	sbCl2 2,2-bipyridyl Fecl3	[82]
Coca butter	Triglyceride	Silica impergnated by silver nitrate	Carbon tetrachloride: Chloroform: Acetic Acid and small volumes of ethanol (60:40:0.5) v/v/v	0.2% Ethanolic solution of dibromo-R-fluorescein	[83]
Ziziphusmauritiana	Sugar	Silica gel	Butanol: Water : Acetic Acid (55:30:15) v/v/v	Visulaized under UV the sprayed with 80% Folin -C phenyl reagent	[84]

Drugs	Stationary phase	Mobile phase	Detection	Reference
Valsartan and Hydrochlorothiazide	Silica gel 60F254	Chloroform : MeOH : Toluene : Glacial acetic acid (6:2:1:0.1) v/v/v/v	By UV at 260 nm.	[88]
Emtricitabine and Tenofovir	Silica gel 60F254	Chloroform: MeOH (9:1) v/v	By UV at 265nm.	[89]
Telmisartan and Ramipril	Silica gel 60F254	Acetone: Benzene : Ethyl acetate : Glacial acetic acid in the proportion of (5:3:2:0.03) v/v/v/v	Densitometrically using a UV detector at 210 & 296 nm.	[90]
Diosgenin and Levodopa	Silica gel 60F254	Toluene : Ethyl acetate: Formic acid : GAA in the ratio (2:1:1: 0.75 ) v/v	194 nm for Diosgenin and 280 nm for Levodopa using absorbance reflectance mode.	[91]
Diclofenac Sodium and Misoprostol	Silica gel 60F254	Toluene : Ethyl Acetate : Ethanol : Glacial Acetic acid (8:2:1:0.1) v/v/v/v	Densitometric evaluation of the separated zones was performed at 220 nm	[92]
Pseudoephedrine and Cetirizine	Silica gel 60F254	Ethyl Acetate : MeOH : Ammonia (7:1.5:1) v/v/v	Spectrodensitometric scanning at a wavelength of 240nm	[93]
Telmisartan and Amlodipine	Silica gel 60F254	Ethyl acetate : 1, 4 Dioxane : MeOH : 25% Ammonia (15:1.5:3:1.5) v/v/v/v	By UV detection at 323 nm.	[94]
Perindopril Erbumine and Indapamide	Silica gel 60F254	Dichloromethane : MeOH : Glacial acetic acid (9.5:0.5:0.1) v/v/v	By UV detection at 215 nm.	[95]
Atorvastatin calcium and Ezetimibe	Silica gel 60F254	Toluene: MeOH (8:2) v/v	Densitometric evaluation was performed at 240 nm	[96]

Analytical Techniques in Simultaneous Estimation: An Overview

Abstract

Abbreviations

Introduction

Spectrophotometric Techniques

UV/VIS Spectroscopy

Derivative Spectrophotometry

IR Spectroscopy

Shortcomings and limitations of infrared spectroscopy

Application of IR spectroscopy

Nuclear Magnetic Resonance Spectroscopy

Application of NMR

Mass Spectrometry

Advantages of mass spectrometry

Disadvantages of mass spectrometry

Application of mass spectrometry

Chromatography

High Performance Liquid Chromatography (HPLC)

Size Exclusion HPLC

Ion-exchange HPLC

Bio Affinity HPLC

Thin Layer Chromatography

High Performance Thin Layer Chromatography (HPTLC)

Gas Chromatography

Application of GC in quantitative analysis

Ultra-High Performance Liquid Chromatography

Hyphenated Techniques

GC-MS

LC-IR

LC-MS

LC-NMR

Simultaneous Estimation by Hyphenated Techniques

Simultaneous Bioanalytical Techniques used in the Medical Diagnosis

Conclusion

Acknowledgements

References

Drug	Impurity	Techniques	Reference
Quetiapine	Degredation impurities	LC-MS/MS	[122]
Lopinavir	Methanesulfonate and Ethyl Methanesulfonate	LC-MS/MS	[123]
Acyclovir	Drug associated impurity	LC-MS	[124]
Irbesartan	Degredation impurity	LC-NMR	[125]
N-acetylcysteine	Cysteine, cystine, N,N-diacytylcysteine	LC-UV-MS	[126]
Triton	1,4-dioxane	GC-MS	[127]
Selenium	Elemental impurities	ICP-MS	[128]

Medical conditions	Analytical Techniques	Reference
Coronory heart diseases	MRI	[140]
Overdose of Barbiturate Amphetamine,Cocaine Heroine and Cannabis.	LC-MS, EIA, GC-MS,� LC-MS/MS,LC-NMR, ELISA	[141-145]
Detection of cadmium, cobalt, chromium, iron, molybdenum, nickel, selenium, titanium, vanadium and zinc in blood	Neutron activation Analysis	[146]
Asphyxia	Gas chromatography-thermal conductivity	[147]