|Darasanapalli Meenakshi1*, Maste Meenaxi1
|Corresponding Author:Darasanapalli Meenakshi Email: [email protected]|
|Received:01 March 2013 Accepted: 07 March 2013|
|Citation: Darasanapalli Meenakshi*1 , Maste Meenaxi1 “A Review on Ultra Performance Liquid Chromatography” Int. J. Drug Dev. & Res., April- June 2013, 5(2): 29-34.|
|Copyright: © 2013 IJDDR, Darasanapalli Meenakshi et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.|
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UPLC is a rising chromatographic separation technique whose packing materials have smaller particle size lesser than 2.5μm which improves the speed, resolution and sensitivity of analysis. When many scientists experienced separation barriers with conventional HPLC, UPLC extended and expanded the utility of chromatography. The main advantage is a reduction of analysis time which also reduces solvent consumption. The analysis time, solvent consumption and analysis cost are very important factor in many analytical laboratories. The time spent for optimizing new methods can also be greatly reduced. This results in many analysis in a day and quick results which is of very importance to the industries and research laboratories. UPLC principle is same as that of HPLC that is based on Van Deemter equation but decrease in particle size has increased efficiency at increased flow rates.
|Ultra performance liquid chromatography (UPLC).|
|The conventional separation method HPLC (High Performance Liquid Chromatography) has many advantages like robustness, ease of use, good selectivity and adjustable sensitivity but main limitation is lack of efficiency due to low diffusion coefficients in liquid phase and slow diffusion of analytes in the stationary phase. [1,2] The Van Deemter equation shows that efficiency increases with use of smaller particle size but this leads to a rapid increase in back pressure, while most of the HPLC system can operate only up to 400 bar. Thus columns filled with particles less than 2μm are used with these systems, to accelerate the analysis without loss of efficiency, while maintaining an acceptable loss of load. Efficiency of HPLC separations can be improved by elevating temperature which lowers the viscosity of mobile phase and also allows for high flow rates thus significantly column backpressure is reduced.[3, 4] Use of monolithic columns provides lower flow resistances than conventional particle packed columns as these are polymerized porous support structure .[5, 6, 7]|
|The principle of UPLC is based on Van Deemter equation which describes the relationship between flow rate and HETP or column efficiency.The equation is as follows :|
|H = A + B/B + CB|
|Where A = Eddy diffusion|
|B = Longitudinal diffusion|
|C = Equilibrium mass transfer|
|B = flow rate|
|The eddy diffusion A , is smallest when the packed column particles are small and uniform. The B term representing longitudinal diffusion or the natural diffusion tendency of molecules diminishes at high flow rates and so this term is divided by B . The C term equilibrium mass transfer is due to kinetic resistance to equilibrium in the separation process and this kinetic resistance is the time lag involved in moving from the gas phase to the packing stationary phase and back again. The greater the flow of gas, more a molecule on the packing tends to lag behind molecules in the mobile phase. Thus this term is proportional to B.|
|Hence it is possible to increase throughput, and thus the speed of analysis without affecting the chromatographic performance. The advent of UPLC has demanded the development of a new instrumental system for liquid chromatography, which can take advantage of the separation performance (by reducing dead volumes) and consistent with the pressures (about 8000 to 15,000 PSI, compared with 2500 to 5000 psi in HPLC). The efficiency is proportional to column length and inversely proportional to the particle size. Thus the column can be shortened by the same factor as the particle size without loss of resolution and this resulted in application of UPLC in the detection of additional drug metabolites, superior separation and improved spectral quality [10, 11].|
|Decreases run time and increases sensitivity|
|Reducing analysis time so that more product can be produced with existing resources|
|Provides the selectivity, sensitivity, and dynamic range of LC analysis|
|Maintains resolution performance.|
|Expands scope for multi residue methods|
|Fast resolving power quickly quantifies related and unrelated compounds|
|Operation cost is reduced|
|Less solvent consumption|
|Increases sample throughput and enables manufacturers to produce more material that consistently meet or exceeds the product specifications, potentially eliminating variability, failed batches, or the need to re-work material [12 , 13]|
|Due to increased pressure more maintenance is required and it reduces the life of the columns of this type but so far performance similar or even higher have been demonstrated by using stationary phases of size around 2μm without the adverse effects of high pressure.|
|In addition, the stationary phases of less than 2μm are generally non- regenerable and thus have limited use.[14 , 15]|
Method Development / Validation
|Method development and validation is a timeconsuming and complicated process. The labs need to evaluate multiple combinations of mobile phase, pH, temperature, column chemistries and gradient profiles to arrive at a robust and reliable method. UPLC helps in critical laboratory function by increasing efficiency, reducing costs, and improving opportunities for business success.|
|UPLC reduces the analysis times as short as one minute and methods can be optimized in just one or two hours, thus significantly reducing the time required to develop and validate a method.|
|With UPLC, separation speed and efficiency allows rapid development of methodologies|
|The following parts of UPLC are important :|
|UPLC columns whose high stability allows a wide range of column temperatures and pH to be explored. UPLC Column Manager which easily evaluate column temperatures from 10°C below room temperature to 90°C and enables to use HPLC methods on the UPLC before scaling to UPLC.|
|UPLC Calculator puts information at fingertips.|
Forced Degradation Studies
|One of the most important factor that impacts the quality and safety of pharmaceuticals is chemical stability. The stability of product defines storage conditions and shelf life as well as the selection of proper formulations and protective packaging for it and also it is required for regulatory documentation.|
|Forced degradation or stress testing is carried out under harsher conditions than used for accelerated stability testing. Generally it is performed early in the drug development process.|
|The laboratories cause the potential drug to degrade under a variety of conditions like peroxide oxidation, acid and base hydrolysis, photo stability and temperature to understand resulting byproducts and pathways that are necessary to develop stability indicating methods. However the FDA and ICH require stability testing data to understand how with time the quality of an API or a drug product changes under the influence of environmental factors such as heat, light, pressure and humidity.|
|The most commonly used technique for doing forced degradation experiments is HPLC with UV and / MS detection for peak purity, mass balance and identification of degradation products but these HPLC-based methodologies are time-consuming and provide medium resolution to ensure that all of the degradation products are accurately detected. Combining the UPLC separations with the UPLCspecific photodiode array and MS detection helps to identify degradation products and as well as shortens the time required for developing an stabilityindicating methods.|
|Impurity profiling should be capable of reproducibly separating and detecting all of the known and unknown impurities of the active compound. The detection and quantification of drug substances and their impurities in raw materials and final product testing is an essential part for drug development and formulation process. It is important to accurately measure low-level impurities with the presence of active pharmaceutical component thus it requires high resolution as profiling becomes complicated in the presence of excipients in the sample and often resulting in long HPLC analysis times to achieve sufficient resolution.|
|UPLC PDA Detector involves two analytical flow cells for maximum flexibility according to application requirements, one for maximum chromatographic resolution and a second for high sensitivity.|
|UPLC also involves the latest peak detection algorithms and custom calculations to optimize data processing and reporting which confidently detect impurities in compounds even at trace levels.|
|To characterize impurities MS and MS/MS data is necessary. The sensitivity and flexibility of exact mass time-of-flight mass spectrometry combined with the high resolving power of the UPLC system allows rapid profiling and identification of impurities.|
|UPLC combined with MS has been successfully employed for the identification of drug and endogenous metabolites.|
|For formulation development and production process drug release in manufacturing product is essential to know so dissolution testing is important. The dissolution profile is used to demonstrate reliability and batch-to-batch uniformity of the active ingredient. In sustained-release dosage formulations, testing of higher potency drugs is particularly important as their dissolution can be the ratelimiting step in medicine delivery. Additionally, newer and more potent formulations requires increased analytical sensitivity. UPLC provides precise and reliable automated online sample acquisition. It automates dissolution testing from tablet dropping to test starting through data acquisition and analysis of sample aliquots and also to the management of test result publication and distribution.|
Bioequivalence / Bio-analysis Studies
|For pharmacokinetic, toxicity and bioequivalence studies quantitation of a drug in biological samples is an important part of development programs. Several biological matrices are used for quantitative bioanalysis, the most commonly used are blood, plasma, and urine.The primary technique used for quantitative bioanalysis is LC-MS/MS.|
|The sensitivity and selectivity of UPLC-MS/MS at low detection levels generates accurate and reliable data that can be used for different purposes including statistical pharmacokinetics (PK) analysis.|
|Quantitative bioanalysis is also an integral part of bioequivalence studies which includes to determine if new formulations of existing drugs allows the compound to reach the bloodstream at the similar rate and exposure level as the original formulation. UPLC-MS/MS solutions are proven to increase efficiency, productivity and profitability for bioequivalence laboratories.|
|UPLC Sample Organizer increases efficiency by accommodating large numbers of samples in a temperature- controlled environment, ensuring maximum throughput also increase the sensitivity of analysis, quality of data including lower limits of quantitation (LLOQ) and productivity of laboratory by coupling the UPLC System with fast acquisition rates of tandem quadrupole MS systems.|
|Easy acquisition, quantification and reporting full system data using a security-based data collection software ensures the highest quality of results.|
|A significant number of candidate drugs falls out of the development process due to toxicity and this results in great loss to the manufacturers. It is difficult to evaluate candidate drugs for possible toxicity, drug-drug interactions, inhibition, and/or induction of metabolizing enzymes in the body. Failure to properly identify these potential toxic events causes a compound to be withdrawn from the market. The high resolution of UPLC enables accurate detection and integration of peaks in complex matrices and extra sensitivity allows peak detection for samples generated by lower concentration incubations and sample pooling. These are important for automated generic methods as they reduce failed sample analysis and saves time.|
|UPLC-MS/MS provides following advantages:|
|• It doubles the throughput with no loss in method robustness.|
|• Tandem quadrupole MS provides sensitivity and selectivity for samples in matrix using multiple reaction monitoring (MRM) for detection and automated compound optimization.|
|• Increase in throughput and sensitivity allows rapid measurement of potential p450 inhibition, induction and drug-drug interactions.|
|Thus this UPLC-based approach helps labs to determine candidate toxicity and drug-drug interactions which enables organizations to be more confident in the viability of candidate drug that will progress to late- stage clinical trials.|
Identification of Metabolite
|Biotransformation of new chemical entities (NCE) is necessary for drug discovery. When a compound reaches the development stage, metabolite identification becomes a regulated process. It is of the utmost importance for labs to successfully detect and identify all possible metabolites of a candidate drug. Key for analysts in metabolite identification is maintaining high sample throughput and providing results to medicinal chemists as soon as they are available. UPLC-MS/MS addresses the complex analytical requirements of biomarker discovery by offering sensitivity, resolution, dynamic range and mass accuracy.|
Manufacturing / QA / QC
|Identity, purity, quality, safety and efficacy are important factors to be considered while manufacturing a drug product. Continues monitoring of material stability is also a component of quality assurance and control.|
|UPLC is used for the highly regulated, quantitative analysis performed in QA/QC laboratories. The supply of consistent, high quality consumable products plays an important role in a registered analytical method.|
|Maximum efficiency is essential for analytical laboratories that are constantly challenged to increase throughput and deliver results to research chemists in pharmaceutical discovery. UPLC and UPLC-MS systems and software enable versatile and open operation for medicinal chemistry laboratories with easy-to-use instruments, a user-friendly software interface, and fast, robust analysis using UV/ MS for nominal and exact mass measurements. UPLC increases productivity in both chemistry and instrumentation by providing more information per unit of work as it gives increased resolution, speed and sensitivity for liquid chromatography. When many scientists experience separation barriers with conventional HPLC, UPLC extends and expands the utility of chromatography. The main advantage is a reduction of analysis time which also meant reduced solvent consumption. Analysis time, solvent consumption, and analysis cost are very important in many analytical laboratories. The time spent for optimizing new methods can also be greatly reduced. The time needed for column equilibration while using gradient elution and during method validation is much shorter. Sensitivity can be compared by studying the peak width at half height. It was found that the sensitivity of UPLC was much higher than that of conventional HPLC. Tailing factors and resolution were similar for both techniques. Peak area repeatability (RSD) and peak retention time repeatability (RSD) were also similar for both techniques. A negative aspect of UPLC could be the higher backpressure than in conventional HPLC. This backpressure can be reduced by increasing the column temperature. Overall, it seems that UPLC offers significant improvements in speed, sensitivity and resolution as compared to conventional HPLC.|
Tables at a glance
|1) Zhang Y.H., Gong X.Y., Zhang H.M., Larock R.C., and Yeung E.S., J.Comb. Chem. 2, 450-452 (2000).
2) Zhou C., Jin Y., Kenseth J.R., Stella M., Wehmeyer K.R. and Heineman W.R, J. Pharmac. Sci. 94, 576- 589 (2005).
3) Zhu J., Goodall D.M., and Wren S.A.C., LCGC 23(1), 54-72 (2005).
4) Greibrokk T. and Andersen T., J. Chromatogr, A1000, 743-755 (2003).
5) Gerber F., Krummen M., Potgeter H., Roth A., Siffrin C., and Spoendlin C., J. Chromatogr., A1036, 127-133 (2004).
6) Tanaka N., Kobayashi H., Nakanishi K., Minakuchi H., and Ishizuka N., Anal. Chem. 73, 420-429A (2001).
7) Wu N. , Dempsey J. , Yehl P.M., Dovletoglu A., Ellison A., and Wyvratt J., Anal. Chem. Acta 523, 149-156 (2004).
8) Swartz M.E. Ultra Performance Liquid Chromatography (UPLC): An Introduction, Separation Science Re-Defined, LCGC Supplement, p. 8(2005).
9) Swartz M.E., Ultra Performance Liquid Chromatography(UPLC): An Introduction, Separation Science Re-Defined, LCGC Supplement, p. 10(2005).
10) MacNair J.E., Patel K.D. and Jorgenson J.W., Anal.Chem. 71, 700-708 (1999).
11) Wu N., Lippert J.A. and Lee M.L., J. Chromatogr., A 911, 1-12 (2001) .
12) Jerkovitch A.D., Mellors J.S., and Jorgenson J.W., LCGC 21(7),2003.
13) Wu N., Lippert J.A., and Lee M.L., J. Chromotogr., 911(1),2001.
14) Swartz M., LCGC 23(1), 46-53 (2005).
15) Broske A.D., et al., Agilent Technologies application note 5988-9251EN (2004).
16) Goodwin L, White SA, Spooner N. Evaluation of ultra- performance liquid chromatography in the bioanalysis of small molecule drug candidates in plasma. J.Chromatogr. Sci 2007; 45(6): 298-304.