Analytical Techniques in Simultaneous Estimation: An Overview

  

    
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.

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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.

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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, 5µm 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, 5µm
    
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.

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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.

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Table 10: Impurity profiling by TLC

  

    
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] 
  

Table 10:  Impurity profiling by TLC

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Simultaneous identification of drugs: TLC provides the identification of drugs simultaneously in various pharmaceutical dosage forms which is very reliable. Some examples of the drugs which are identified by TLC method are described in Table 11.

Table 11: Simultaneous Identification of Drugs by TLC.

  

    
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] 
  

Table 11:  Simultaneous Identification of Drugs by TLC.

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Determination of the amino acids: Various amino acids can be detected by application of TLC. Various amino acids which are detected by TLC are Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Threonine, Aspartic acid, Aspargine, Glutamic acid, Glutamine, Lysine, Histidine, Arginine, Phenylalanine, Tyrosine, Tryptophan, Cysteine, cystine, Methionine, Proline and Hydroxy Proline [80] and analysis of essential constituents of food by TLC (Table 12)

Table 12: Determination of various important constituents of food used on daily basis.

  

    
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] 
  

Table 12:  Determination of various important constituents of food used on daily basis.

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High Performance Thin Layer Chromatography (HPTLC)

Among the significant modern analytical tools HPTLC is a powerful analytical method equally appropriate for qualitative and quantitative analytical tasks (Table 13). HPTLC is playing important role in today pharmaceutical analytical world, not in competition to HPLC [85]. Separation may result due to adsorption or partition or by both phenomenons's depending upon the nature of adsorbents used on plates and solvents system used for development. Different aspects on HPTLC fundamentals such as qualitative and quantitative analysis have been reported such as phytochemical analysis, biomedical analysis, herbal drug quantification, analytical analysis, finger print analysis and potential for hyphenation (HPTLC-MS, HPTLC- FTIR and HPTLC-Scanning Diode Laser) have been reported [86].

Table 13: Simultaneous estimation of drugs by HPTLC.

  

    
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] 
  

Table 13:  Simultaneous estimation of drugs by HPTLC.

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There are several advantages of using HPTLC for the analysis of compounds as compared to other techniques, like HPLC, spectrophotometry, titrimetry, etc. Some of the advantages of HPTLC are:

• Capability to analyze crude samples containing multicomponents.
• In this process, it is easy to separate colored compounds.
• Several samples can be divided parallel to each other on the same plate resulting in a high output, time saving, and a rapid low-cost analysis.
• Two dimensional separations of the natural as well as pharmaceutical compounds are easy to perform.
• HPTLC can combine and it can be used for different modes of evaluation, allowing identification of compounds having different light absorption characteristics or different colours.
• HPTLC method may help to minimizes exposure risk of toxic organic effluents and significantly reduces its disposal problems as a result reducing environment pollution [87] (Table13).

Gas Chromatography

Gas chromatography is a technique used for separation of mixtures into single entities by a process which lies on the redistribution of components between a stationary phase or supporting material in the form of a liquid-solid or combination of both and gaseous mobile phase. Mechanism of the GC is based on adsorption, mass distribution or size exclusion [97].

Application of GC in quantitative analysis

Assay of pharmaceuticals products: B.P., U.S pharmacopoeia and the European pharmacopoeia are based on the GC as well as on other estimation techniques for the assay of various pharmaceutical products. Some of the pharmaceutical product which are assayed or determination of related substances by the GC are mentioned below:

1. Analysis of methyl testosterone and related substances in API and Tablet.
2. Assay of atropine in eye drops.
3. Quantification of ethanol in a formulation.

Estimation of degradation product in the pharmaceuticals or impurity profiling: GC is the only technique used for the estimation of volatile degradation products in the pharmaceutical preparations and in impurity profiling of the drugs For example

1. Estimation of pivalic acid in dipivefrin.
2. Dimethylanaline in bupivacaine injection.
3. Determination of residual glutaraldehyde in polymeric form [26].
4. Impurity profiling of cocaine [98].

Use of GC in bio analysis: Use of GC in the bio analysis of the drugs in the plasma and its metabolism increase the efficacy of this type of chromatography.

1. Determination of Valproic acid in the plasma can be determined by this method.
2. Quantification of bupivacaine in plasma [26].
3. Measurement of isoprene solubility in water, human blood and plasma [99].
4. Simultaneous determination of 3,4-Dihyroxyphenylglycol, Catecholamines and 3,4-Dihydroxyphenylalanine in plasma [100].

Environment analysis: Use of GC-GC played important role in investigation of Oil-Sand Napthenic acid which are waste products formed during the the Oil-sand digestion and extraction processes [101].

Simultaneous estimation by GC: Simultaneous estimation of the pharmaceutical formulations can be done by this technique which provides the use of less time and increase in the sensitivity. For example:

Simultaneous estimation Miconazole Nitrate and Metronidazole in Different Pharmaceutical Dosage Forms [102].

Simultaneous estimation of the amino acids in the food suppliments [103].

Simultaneous estimation of Turpentine oil, Chlorbutol, Para- Dichlorobenezene and Benzocaine [104].

Ultra-High Performance Liquid Chromatography

Ultra-high performance liquid chromatography (UPLC) has marked a radical and outstanding change through opening new doors intended for analyst to fetch rapid analytical separation techniques without sacrificing high-quality results obtain former from high performance liquid chromatography (HPLC). The principle is based on the principal of use of stationary phase consisting of particles less than 2μm while HPLC columns are typically filled with particles of 3 to 5μm. The underlying principles of this evaluation are governed by the Van Deemter equation, which is an empirical formula that describes the relationship between linear velocities (flow rate) and plate height (HETP or column efficiency) [105].

The potential of UPLC is to get better analysis of the samples that are encountered during pharmaceutical development and manufacturing. UPLC generated higher separating efficiencies through the make use of smaller diameter particle packing and higher operating pressures. A commercial system competent of generating much higher pressures than used in standard HPLC has been evaluated to determine its potential in routine analysis. UPLC has been shown to generate high peak capacities in short times and this is found to be quite beneficial in analyzing the complex mixtures that comprise metabolism samples. The application of UPLC resulted in the detection of additional drug metabolites, improved the spectrum quality and separation efficiency [106]. UPLC is one of the advantageous analytical techniques in less run time, high sensitivity, high resolution; faster analysis due to fine particle size, cost effective, less solvent consuming and real time analysis is possible with the manufacturing processes in pharmaceutical industries as well as in research programs. But it has some restrictions also such as increased pressure requires for high maintenance, less column life [107] and non regenerable phases of size 1.7μm [108] (Table 14).

Table 14: Application of UPLC in simultaneous estimation.

  

    
Drugs 
    
Mobile phase 
    
Detection 
    
Reference 
  
  

    
Abacavir Sulphate and Lamivivudine
    
Triethylamine phosphate buffer (pH 2.5) and MeOH (50:50%) v/v
    
UV detection at 230 nm
    
[109] 
  
  

    
Aspirin and Dipyidamole
    
Triethylamine phosphate buffer (pH 2.5) and MeOH (50:50) v/v
    
UV detection at 230 nm
    
[110] 
  
  

    
Losartan potassium, Atenolol and Hydrochlorothiazide
    
Water : ACN : Triethyl amine: Ortho phosphoric Acid (60:40:0.1:0.1)    v/v/v/v
    
UV detection at 225 nm
    
[111] 
  
  

    
Ceftriaxone and sulbactum injection
    
0.05M Sodium di-hydrogen ortho-phosphate dehydrate : ACN (86:14) v/v
    
PDA detection at 254 nm and 195 nm
    
[112] 
  
  

    
Bambuterol Hydrochloride and Montelukast
    
0.025 % TFA in water as aqueous and 0.025% TFA in ACN as organic    solvent
    
PDA detection at 210 nm
    
[113] 
  
  

    
Impurities in Telmisartan and Chlorthalidone
    
The mobile phase A consists pH 4.5 buffer & ACN in the ratio 90:10    (v/v). Mobile phase B consists pH 4.5 buffers & ACN in the ratio 20:80    v/v
    
Detection at 290 nm
    
[114] 
  
  

    
Metformin and pioglitazone
    
0.2% Triethylamine in water: ACN (70:30) v/v ph adjusted with    orthophosphoric acid .
    
Detection at 243 nm
    
[115] 
  
  

    
Thiocolchicoside and Aceclofenac
    
5% ammonium acetate buffer and MeOH in the ratio of (40:60) v/v
    
PDA detection at 276 nm
    
[116] 
  
  

    
Vitamins, caffeine and
      
 
      
Preservatives
    
The mobile phase consisting of A : buffer (0.1% Trifluoro acetic acid    in water) and B : a mixture of 50% ACN and 50% MeOH with a timed gradient    programme

    
Detection at 200, 254, and 290 nm
    
[117] 
  

Table 14:  Application of UPLC in simultaneous estimation.

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Hyphenated Techniques

A hyphenated technique is combination or coupling of two different analytical techniques with the help of proper or various interfaces. The word Hyphenated technique comes from the combination of separation-separation, separation-identification and identification-identification techniques. The term "hyphenation" was first adapted by Hirschfield in 1980 to describe a possible combination of two or more instrumental analytical techniques in a single run. The main objective of coupling of different analytical techniques to obtain an information-rich detection for both identification and quantification compared to that with a single analytical technique [118].

Hyphenated technique is usually comprised of separation and detection technique which are coupled to give a new technique. The use of hyphenated technique provides the high degree of assurance for the pharmaceutical estimation and impurity profiling (Figure 2).

Figure 2: Diagram representing coupling of two techniques using different interfaces.

    

    

    Figure 2:  Diagram representing coupling of two techniques using different interfaces.
    
    

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GC-MS

This hyphenated technique developed from the coupling of GC and MS, was the first of its type to become functional for research and development purposes. Mass spectra obtained from this hyphenated technique presents more structural information based on the interpretation of fragmentations. Compounds that can be easily volatile, small, and stable in high temperature in GC conditions can be easily analyzed by GC-MS. The most common derivatization technique is the conversion of the analyte to its trimethylsilyl derivative which provides the accurate and precise results.

LC-IR

The hyphenated technique is a coupling of an LC and IR or FTIR and is known as LC-IR or HPLC-IR. While HPLC is one of the most dominant separation techniques available today, the IR or FTIR is a useful spectroscopic technique for the identification of organic compounds, because the mid-IR region provides maximum possibilities of functional groups e.g., -OH, -COOH, and so on. However, combination of HPLC and IR is difficult and the evolution in this hyphenated technique is extremely slow because of large number of bands of the mobile phases thus it is difficult to understand the small signal generated by the sample components [119].

LC-MS

Hyphenated techniques LC-MS or HPLC-MS is composed of an LC (Liquid chromatography) with a mass spectrometer (MS). The information obtained from a single LC-MS run, on the structure of the compound is limited in use because the ionization techniques used in this technique are soft ionization techniques which present the molecular ion species with only a few fragment ion. Hyphenated techniques such as HPLC coupled with UV and Mass spectrometry (LC-UV-MS) has been proved to be more useful in biological screening for rapid survey of natural products [120].

LC-NMR

The on-line coupling of high-performance liquid chromatography (HPLC) principles to high-resolution NMR spectrometers offers a powerful tool for analyzing and characterizing complex chemical mixtures without the need of chemical separation. LC-NMR promises to be of great value in the analysis of complex compounds of all types, particularly the analysis of natural products and drugrelated metabolites in biofluids. The major advancement have been made in LC-NMR technology, a strong case can be made that HPLC purification of metabolites followed by conventional tube NMR is equally useful and may provide results in very less time and show very high sensitivity [121]. Hyphenated techniques provide the high degree of word for the compounds purity and impurity (Table 15).

Table 15: Impurity profiling of some compounds by hyphenated techniques.

  

    
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] 
  

Table 15:  Impurity profiling of some compounds by hyphenated techniques.

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Simultaneous Estimation by Hyphenated Techniques

Use of hyphenated techniques in the pharmaceuticals, environmental sciences and study of pharmacokinetic parameters by simultaneous estimation played an important role. Few examples of the various hyphenated techniques which are reported and their role in different fields are enlisted below.

Simultaneous determination of opiates, cocaine and major metabolites of cocaine in human hair by gas chromatography/mass spectrometry (GC/MS) [129].

Differentiation of the regioisomeric 2-, 3-, and 4-trifluoromethyl phenylpiperazines (TFMPP) by GC-IRD and GC-MS [130].

Simultaneous determination of melamine, ammelide, ammeline and cyanuric acid in milk and milk products by gas chromatographytandem mass spectrometry [131].

Rapid sensitive speciation analysis of butyl- and phenyltin compounds in water by capillary gas chromatography atomic emission spectrometry (GC-AES) after in-situ ethylation and in-liner preconcentration [132].

Investigation of the composition of Pinus peuce needle oil by GCMS and GC-GC-MS [133].

Simultaneous determination of rosuvastatin and fenofibric acid in human plasma by LC-MS/MS with electrospray ionization [134].

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS [135].

Simultaneous determination of pioglitazone and its two active metabolites in human plasma by LC/MS/MS [136].

Simultaneous Bioanalytical Techniques used in the Medical Diagnosis

The application of analytical tools in the clinical as well as in diagnostic fields is up to the mark. The bioanalytical techniques like GC-MS, HS-GC/MS, Gas chromatography-thermal conductivity are used in the diagnosis of the diseases. All these technique are noninvasive techniques and have a great potential in medical as well as in the development of medical biomedical analytical method. The various approaches of bioanalytical tools like chromatography, spectroscopy, and immunoassay have advantages in the field of narcotics and toxicology. Diseases like AIDS, Hepatitis, Acute respiratory disorders can be diagnosed by the use of analytical tools [137-139]. In future these hybrid techniques like LC-NMR, LC-MS/MS, GC-MS/MS etc can be used in field of medical and forensic science (Table 16).

Table 16: Analytical tools used in toxicology and medical diagnosis.

  

    
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] 
  

Table 16:  Analytical tools used in toxicology and medical diagnosis.

Close

Conclusion

From the above studies, it has been concluded that analytical techniques can be used successfully for the simultaneous estimation of many drug combinations as medicines which are given to the patients should be free from impurity and other interferences which can affect the therapeutic index and may show harmful effects to the patients. Above techniques are also used to elucidate and determine the structure of natural compounds. The advantages of the simultaneous estimation are fast, simple, less time consuming, accurate and sensitive for research purpose where no new method of estimation and analysis has been reported yet. This review also represents the advancement of chromatographic and spectroscopic techniques to hyphenated techniques. Hence, the simultaneous estimation of chemical entities using various analytical techniques are very much valuable for the future needs in pharmaceutical as well as other fields of investigation. Nevertheless, more expansion and advancement is needed in these techniques which will be beneficial to the analytical and bioanalytical researchers in developing strategies for new analytical method and high output results in the laboratories.

Acknowledgements

The authors express their sincere thanks to Mr. Parveen Garg (Chairman), I.S.F. College of Pharmacy, Ferozepur Road, Moga- 142001, India for providing the necessary support and motivation for this study.

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Citation: Badyal PN, Sharma C, Kaur N, Shankar R, Pandey A and Rawal RK. Analytical Techniques in Simultaneous Estimation: An Overview. Austin J Anal Pharm Chem. 2015;2(2): 1037. ISSN:2381-8913

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