SOUJANYA SATHI
REGISTRATION NO: 099051482
DRUG DICOVERY AND DEVELOPMENT
UNIVERSITY OF SUNDERLAND
INTRODUCTION
USES IN PHARMACEUTICAL INDUSTRY
HPLC has a considerable range of applications inboth clinical research and routine clinical analysis. The variety of molecules iscommonly analyzed by this method.
Methods based on chroma- tography from small cation exchange columns as wellas various electrophoresis methods have been usedsuccessfully to resolve glycated from non-glycated
Hemoglobin’s.
Cationexchange HPLC, however, can resolve all the subtypes of glycated haemoglobin both from each other andfrom the F, S, and C forms. This form of assay show excellent precision with rapid separation, and manymanufacturers market HPLC systems dedicatedentirely to this purpose.
The ability of HPLC to resolve closely relatedmolecules makes it the method of choice for detailedinvestigation of many congenital metabolic disordersor diseases.
For instance, though amino acids inplasma or urine, or both, can be investigated withpaper chromatography, thin layer chromatography,or high voltage electrophoresis, these methods give
relatively poor separation and results are difficult toquantify.
Ion exchange HPLC methods have beenmuch more successful in separating, identifying, and
quantifying the main amino acid species in plasma andin urine.
A high resolutiontechnique like HPLC can differentiate between thesestates with the same basic method.
Other general types of molecule can also be separatedinto individual smaller molecules by HPLC to givemetabolic profile.
With ion exchange HPLC techniquesthe biogenic amines can be analyzed.
Simmonds et al recently described an anion exchangeHPLC procedure for the separation of the majornucleotides and their corresponding deoxyderivatives.
Reverse phase HPLC has been used successfullyfor identifying and quantifying individual urinaryporphyrins.
Many HPLC methods have been developed for thestudy of vitamins and their metabolites.
One of the main areas in which HPLC is used is intherapeutic drug monitoring like
Whenthe therapeutic dose is close to the toxic dose, whensigns of toxicity are difficult to detect clinically, whenthe rate of metabolism varies widely between patients,or when drug metabolism is impaired owing to organdysfunction or altered by other drugs.
Monitoringwhen rates of metabolism might vary is especiallyimportant if the drug metabolite is the therapeuticallyactive form or the toxic form.
Drugs that are commonly monitored by HPLCinclude antiasthmatic drugs (theophylline and its
active metabolities, including caffeine), anticonvulsantssuch as carbamazepine, phenobarbitone,
phenytoin, ethosuximide, primidone and valproate,tricyclic antidepressants, and cardioactive drugs suchas procainamide and propranolol. In many cases thedrug may be monitored by radioimmunoassay, butwhen no antibody to the drug (or metabolite) exists, orif simultaneous measurement of a mixture of drugs ormetabolites is required then HPLC is more effective.[4]
Adsorption (normal phase) HPLC-separation of molecules on the basis of theirsolubility in water
Chiral molecules-those containing a carbon atom to which is bound four differentgroups.
Ion exchange HPLC-separation of molecules on the basis of their net charge
Liquid phase-the solvent passing through the HPLC column
Reverse phase HPLC-separation of molecules on the basis of their solubility inorganic solvents
Size exclusion HPLC-separation of molecules on the basis of their size.Solid phase-the surface of the solid particles (in an HPLC column) with which
molecules can interact(2)
HPLCIMS holds great promise for measuring drug concentrations
LC-MS
Liquid chromatography-mass spectrometry (LC-MS, or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high sensitivity and selectivity. Generally its application is oriented towards the specific detection and potential identification of chemicals in the presence of other chemicals. [17]
Pharmacokinetics: LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals and is thus the most frequently used technique in the field of bioanalysis. These studies give information about how quickly a drug will be cleared from the hepatic blood flow, and organs of the body. MS is used for this due to high sensitivity and exceptional specificity compared to UV (as long as the analyte can be suitably ionised), and short analysis time.
The major advantage MS has is the use of tandem MS-MS. The detector may be programmed to select certain ions to fragment. The process is essentially a selection technique, but is in fact more complex. The measured quantity is the sum of molecule fragments chosen by the operator. As long as there are no interferences or ion suppression, the LC separation can be quite quick. It is common now to have analysis times of 1 minute or less by MS-MS detection, compared to over 10 mins with UV detection.[3][4][5]
Proteomics / metabolomics: LC-MS is also used in the study of proteomics where again components of a complex mixture must be detected and identified in some manner. The bottom-up proteomics LC-MS approach to proteomics generally involves protease digestion and denaturation (usually trypsin as a protease, urea to denature tertiary structure and iodoacetamide to cap cysteine residues) followed by LC-MS with peptide mass fingerprinting or LC-MS/MS (tandem MS) to derive sequence of individual peptides.[6]
LC-MS/MS is most commonly used for proteomic analysis of complex samples where peptide masses may overlap even with a high-resolution mass spectrometer. Samples of complex biological fluids like human serum may be run in a modern LC-MS/MS system and result in over 1000 proteins being identified, provided that the sample was first separated on an SDS-PAGE gel or HPLC-SCX.
Drug development:LC-MS is frequently used in drug development at many different stages including Peptide Mapping, Glycoprotein Mapping, Natural Products Dereplication, Bioaffinity Screening, In Vivo Drug Screening, Metabolic Stability Screening, Metabolite Identification, Impurity Identification, Degradant Identification, Quantitative Bioanalysis, and Quality Control.[7]
Hplc/Ms holds a promise for measuring drug concentration in body fluids, particularly because many drugs are large or labile molecules that do not lend themselves to analysis by GC/MS.HPLC/MS has two distinct areas of application in drug analysis: Qualitative analysis of a drug or quantification of a drug. [8]
LC-NMR
Hyphenated analytical techniques such as LC-MS, which combines liquid chromatography and mass spectrometry, are well-developed laboratory tools now widely used in the pharmaceutical industry. However, in most cases MS alone is insufficient for complete structural elucidation of unknown compounds. Traditionally nuclear magnetic resonance (NMR) experiments are performed on more or less pure samples, in which the signals of a single component dominate. Therefore, the structural analysis of individual components of complex mixtures is normally time-consuming and less economic. The combination of chromatographic separation techniques with NMR spectroscopy offers advantages for the on-line separation and structural elucidation of unknown compounds. Mixtures such as crude reaction mixtures in drug discovery can be analyzed without prior separation. Experiments in combining an HPLC with NMR on the study of mixtures were introduced to the scientific community in the early 1980s. However, LC-NMR was not widely practical due to its low sensitivity, approximately six orders in magnitude inferior to that of MS. Another challenge comes from the measurement of proton signals in mobile phase. However, recent developments in higher magnetic field strength and electronics that improve the sensitivity of probes, together with advanced solvent suppression techniques, have made LC-NMR measurement practical.[16]
During the last decade, LC-NMR has been fully commercialized and its application in both academia and industry has been growing rapidly. The emphasis is therefore on describing the experimental design, the practical applications, and the recent developments in technology. With all the applications to date, LC-NMR spectroscopy is still a relatively insensitive technique due to the poor mass sensitivity of the NMR detection system. To this end, several other hyphenated NMR techniques have been developed to enhance the sensitivity of this technique. LC-SPE-NMR dramatically increases the sensitivity up to a factor of four by utilizing a solid phase extraction device after the LC column. Capillary LC-NMR also significantly lowers the detection limit to a low nanogram range through integration of capillary LC with NMR detection. Other breakthroughs such as cryo-LC-probe technology combine the advantages of sample flow and the enhanced sensitivity from a cryogenically cooled NMR probe.
DISPLACEMENT CHROMATOGRAPHY
The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.[15]. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations.[1]
HILLIC
Partition chromatography was the first kind of chromatography that chemists developed. Partition chromatography uses a retained solvent, on the surface or within the grains or fibres of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of somecoulombic and/or hydrogen donor interaction with the solid support. Molecules equilibrate (partition) between a liquid stationary phase and the eluent. Known as Hydrophilic Interaction Chromatography (HILIC) in HPLC, this method separates analytes based on polar differences. HILIC most often uses a bonded polar stationary phase and a non-polar, water miscible, mobile phase. Partition HPLC has been used historically on unbounded silica or alumina supports. Each works effectively for separating analytes by relative polar differences, however, HILIC has the advantage of separating acidic, basic and neutral solutes in a single chromatogram.[1]
If you are trying to increase retention of hydrophilic molecules by RPC, there is a versatile, effective alternative to consider: hydrophilic interaction chromatography (HILIC). A rival technique to RPC for separating polar peptides, HILIC is easy to use and works best where RPC works worst: with polar solutes which aren't retained well on RPC. HILIC has been used successfully with phosphopeptides, crude extracts, peptide digests, membrane proteins, carbohydrates, histones, oligonucleotides and their antisense analogs, polar lipids and in preparative applications where changing the order of elution affects isolation yields.[20]
NORMAL PHASE CHROMATOGRAPHY
Also known as normal-phase HPLC (NP-HPLC), or adsorption chromatography, this method separates analytes based on adsorption to a stationary surface chemistry and by polarity. It was one of the first kinds of HPLC that chemists developed. NP-HPLC uses a polar stationary phase and a non-polar, non-aqueous mobile phase, and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction strength depends not only on the functional groups in the analyte molecule, but also on steric factors. The effect of sterics on interaction strength allows this method to resolve (separate)structural isomers.
Partition and NP-HPLC fell out of favor in the 1970s with the development ofreversed-phaseHPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica oraluminachromatographic media. Recently it has become useful again with the development ofHILICbonded phases which improve reproducibility. [1]
REVERSE PHASE HPLC (RPC)
RPC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RPC to qualify drugs before their release.
HPLC can readily be used to identify drug metabolitesthat may be important in drug toxicity.
A rolefor HPLC in both research and routine clinical analysis.
Reversed phase chromatography has proven itself to be an indispensable technique in the purification of biomolecules. In recent years, with the advent of high performance media and instrumentation, reversed phase chromatography has been applied to the purification of biomolecules such as peptides, proteins and oligonucleotides. Reversed phase chromatography has proven so successful for biomolecule purification in the research laboratory that it is now routinely applied for process scale purification of synthetic peptides, and recombinant peptides and proteins.[19]
Ion Pair Reverse-Phase Chromatography:
A Versatile Platform for the Analysis of RNA.
Ion pair reverse-phase chromatography (IP RP HPLC) has been widely applied for the study of nucleic acids, in particular DNA [9-10]. More recently, a number of studies have utilised IP RP HPLC for the purification and analysis of RNA, demonstrating its versatility in a variety of different applications; from the routine purification of synthetic oligoribonucleotides, through to the analysis of complex RNA:RNA interactions. This research demonstrates that IP RP HPLC is a versatile platform for the analysis of RNA. Careful selection of the chromatography conditions including the ion pair reagent, temperature and additives to the mobile phase, facilitates the operation of the IP RP HPLC under different modes, enabling the study of a wide of range RNAs and biological systems.[18]
SIZE-EXCLUSION CHROMATOGRAPHY
Size-exclusion chromatography (SEC), also known asgel permeation chromatographyorgel filtration chromatography separates particles on the basis of size. It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of purification. It is also useful for determining thetertiary structureandquaternary structureof purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.
This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) for the molecular weight comparison of different commercially available low-molecular weightheparins.
The main analytical application is to determine molecular weight of single macromolecules and for determination of molecular weight distributions of polydispersed polymers.This method is less used for quantitative determinations of specific macro molecules and in that case reverse phase or ion exchange is preferred.[9]
ION-EXCHANGE CHROMATOGRAPHY
In ion-exchange chromatography, retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the same charge are excluded. Types of ion exchangers include:
- Polystyrene resins
- Cellulose anddextranion exchangers (gels)
- Controlled-pore glass or porous silica
This form of chromatography is widely used in the following applications: water purification, preconcentration of trace components, ligand-exchange chromatography, ion-exchange chromatographyof proteins, high-pH anion-exchange chromatography of carbohydrates and oligosaccharides, and others.
And it’s a method of choice for analysis of inorganic ions and preferable to reverse phase for analysis of small organic ions. [9]
Rapid ion exchange chromatography of plasma amino acids has been achieved on polystyrene-based cation exchange resins.[10]
AQUEOUS NORMAL-PHASE CHROMATOGRAPHY
Aqueous normal-phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reverse-phase solve.
UPLC
This technique may therefore enhance the sensitivity and expand the scope of analysis to smaller cell populations and possibly to single mammalian cells.
The “ultra” HPLC approach offers a wide range of applications, and is highly attractive for those situations which require ultra-high sensitivity such as those done with limited sample quantities (Lindon et al., 2003). The limitations of this approach are that it requires special high pressure pumping equipment and that its peak capacity is still too low to adequately resolve complex mixtures often encountered in proteomics.
The HPLC separation takes in excess of 12min while the UPLC accomplishes the same separation in under 30seconds. UPLC can also be used to significantly improve the success of the drug discovery process. By the mid-1990s, high performance liquid chromatography (HPLC) directly coupled to mass spectrometry (MS) was in routine use in drug metabolism laboratories for these types of studies (Mutton et al., 1998). Enhanced selectivity and sensitivity, and rapid, generic gradients made LC–MS the predominate technology for both quantitative and qualitative analyses.
At a time when many scientists have reached separation barriers with conventional HPLC, UPLC presents the possibility to extend and expand the utility of chromatography.UPLC fulfills the promise of increased resolution, speed, and sensitivity predicted for liquid chromatography. It might become the gold standard for quantification in clinical and forensic toxicology and doping control if the cost of the equipment can be markedly reduced, if current disadvantages, for example irreproducibility of fragmentation, reduction of ionization by matrix, etc., can be overcome, and, finally, if one of the increasing number of quite different techniques can become the apparatus standard.[11]
LC-IR
The hyphenated technique developed from the coupling of an LC and detection method IR or FT-IR is known as HPLC-IR.And it is a useful technique for identification of organic compounds because in the mid IR region the structure of organic compounds has many absorption bands that are characteristics of particular functionalities.Eg: -OH,-COOH.However the combination is difficult and the hyphenated technique is slow and it is sensitive.