INTRODUCTION:

High performance liquid chromatography (HPLC) has proven to one of the major analytical technique used in the qualitative and quantitative analysis of drugs worldwide. The packing material of the column is the basic feature for the growth of this technique which directly responsible for the chromatographic separations. The principle of separation of compounds is given by Van Deemter equation, which is an empirical formula that describes the relationship between linear velocity (flow rate) and plate height (HETP, column efficiency). According to the principle of separation of HPLC, as the particle size of column material decreases, the efficiency of the chromatographic separation, speed and resolution also increases. The HPLC is the most simple, economic, reliable and worldwide used technique in the pharmaceutical analysis.

However certain analytical requirements cannot be fulfilled by HPLC technique. Such as determination of complex samples such as formulation excipients, biological samples, drug metabolites, degradation products, impurities, and drug isomers by HPLC, several problem arises related to determination of analytes at low level (0.1%), speed of analysis and resolution per unit time. For the need of high resolution separation, the researchers have been involved in the designing of sub-2µm particles. The non-porous silica, porous silica and polymeric particles of sub-2µm sizes were developed possesses their own characteristics of separation. Porous packing materials can tolerate higher pressure and give much higher sample capacity than non-porous packings. UHPLC is now becoming an advance and modern technique which gives a new direction for liquid chromatography. UHPLC refers to ultra-high performance liquid chromatography, which enhance mainly in three areas: speed, resolution and sensitivity. UHPLC applicable for particle less than 2μm in diameter to acquire better resolution, speed, and sensitivity compared HPLC. In twenty first centenary pharmaceutical industries are focusing for new ways to in economy and shorten time for development of drugs. The separation and quantification in UHPLC is done under very high pressure (up to 100M Pa). As compare to HPLC, under high pressure it is observed that not any negative influence on analytical column and also other components like time and solvent consumption is less in UHPLC. First time in the year 1999, Waters developed the hybrid particle technology (HPT) column for HPLC. The HPT is the combination of inorganic silica and organic polymeric packings which has high mechanical strength, efficiency, pH stability and peak shape for basic compounds. The second generation hybrid material particle composed with bridged ethylsiloxane/silica hybrid (BEH) structure was developed which provides improved efficiency, strength and pH range. High strength silica (HSS) particle technology has also been used which increases the mechanical stability of silica which provides increased retention time and selectivity of compounds compared to hybrid particles. Charge Surface Hybrid (CSH) Technology was the latest advancement in hybrid materials which contains surface charge within the packing materials to provide enhanced selectivity and better peak shape for entire range of ionic mobile phases [1-17]. From the above development in column packing material and particle size, Waters Company was given the trade name of UHPLC, which was known as UPLC. The objective of the present review paper is to provide the introduction and pharmaceutical applications of UHPLC and examples of some of the most advanced work in the pharmaceutical analysis. The advantages of UHPLC are: Increases selectivity, sensitivity and range of LC analysis, less sample quantity is required, decreases total run time for separation, smaller peak width provides more number of peaks identified, increases resolution performance, less solvent consumption, fast resolution and quantification of related and unrelated compounds, and low cost of analysis. The disadvantages of UHPLC are: Higher pressure required more maintenance, higher pressure reduces the life of the columns, Design and development of sub-2µm stationary phase is complicated, Stationary phase of sub-2 µm are non-regenerable and have limited use, Lack of variety in commercial columns at sub-2 µm particle size, Difficulties in providing higher pressure for solvents. At high operating pressure, the frictional heating of the mobile phase is occurred with HPLC column (3.0 to 4.6 mm) lead to loss of separation efficiency due to non-uniform flow. The frictional heating has been minimized by smaller diameter columns (1 to 2.1mm) which are used in UHPLC. The HPLC was first modified in to UHPLC operated up to 17,500 psi with 1.5 µm particles for the analysis of proteins. The UHPLC was first applied in the separation of various pharmaceutical compounds using UV/TOF detection. The various papers have been reported on UHPLC covering it’s all the features, applications and advantages over HPLC [17-59].

Applications of UHPLC in Pharmaceutical Analysis:

1. Method Development and Validation:

According to FDA, validation is defined as establishing documented evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes. Method development and validation is a time-consuming and complicated process: labs need to evaluate combinations of mobile phase, pH, temperature, column chemistries, and gradient profiles to get a robust, reliable separation for every activity. Using UPLC, analysis times become as short as one minute, methods can be optimized in just one or two hours, significantly reducing the time required for method development and validation [60].

2. Manufacturing and QA/QC:

Identity, purity, quality, safety and efficacy are the important factors to be considered while manufacturing a drug product. The successful production of quality pharmaceutical products requires that raw materials and finished pharmaceutical products meet purity specifications. Continued monitoring of material stability is also a component of quality assurance and control. UPLC is used for the highly regulated, quantitative analyses performed in QA/QC laboratories. The supply of consistent and high quality products plays an important role in a registered analytical method [61].

3. Determination of FDC Products:

A fixed dose combination (FDC) product is a formulation of two or more drugs combined in a single dosage form available in certain fixed doses. Method development for two or more drugs becomes very complex if the solubility varies greatly. Hence they need to be analyzed by more sophisticated techniques such as UPLC-PDA, UPLC-QTOF/MS. A large number of samples per day can be analyzed due to the short analysis times [62].

4. Stability testing/Stress Testing:

According to ICHQ1A (R2), forced degradation studies or forced degradation studies of the drug substance are carried out for identification of degradation products and to determine the intrinsic stability of the molecule and to develop the stability-indicating power of the analytical methods. The analytical technique mostly used for forced degradation studies is HPLC-UV and/or HPLC-MS but the main drawback is that these techniques are time consuming and not provide adequate information regarding resolution of all the impurities and degradation products. The UHPLC/Q-TOF-MS system provide changeable collision energy values allows the generation of mass information with acceptable accuracy and precision, which is ultimately helpful in structure elucidation, identification of fragmentation patterns of the drugs, identification of degradation products and establishment of degradation mechanisms [63-69]. The authors have been successfully applied the UPLC technique for determination of aceclofenac and paracetamol and their degradation products in tablets [70].

5. Detection and Identification of Impurities:

Impurity profiling is mandatory requirement by various regulatory agencies and is directly related with the quality, safety and efficacy of a drug product. Impurities are the related substances of drugs which develop during formulation, or upon storage of drugs. Mass spectrometry is one of the best techniques to detect impurities. Impurities present can be detected by the additional peaks, highest value of mass peaks than compound itself and from the fragmentation pattern. The rapidly change of collision energy generates the molecular fragments, which allows fast identification of the compounds along with their related impurities. On the basis of various fragments of the compounds, one can easily propose its structure [71-76].

6. Dissolution Testing:

For quality control and release in drug manufacturing, dissolution testing is essential in the formulation, development and production process. The dissolution profile is used to demonstrate reliability and batch-to-batch uniformity of the active ingredient. For quality control and release in drug manufacturing, dissolution testing is essential in the formulation, development and production process. In sustained-release dosage formulations, testing higher potency drugs is particularly important where dissolution can be the rate-limiting step in medicine delivery. The dissolution profile is used to demonstrate reliability and batch-to-batch uniformity of the active ingredient. Additionally, newer and more potent formulations require increased analytical sensitivity. UPLC provides precise and reliable automated online sample acquisition. It automates dissolution testing, from pill drop to test start, through data acquisition and analysis of sample aliquots, to the management of test result publication and distribution [77].

7. Pharmacokinetic and Bioequivalence Studies:

For pharmacokinetic, toxicity, and bioequivalence studies, quantitation of a drug in biological samples is an important part of development programs. The sensitivity and selectivity of UPLC-PDA at low detection levels generates accurate and reliable data that can be used for a variety of different purposes, including statistical pharmacokinetics (PK) analysis. The drugs are generally of low molecular weight and are tested during both preclinical and clinical studies [78-82].

8. Identification of Metabolites:

After the development stage of any chemical compound, its metabolite identification is a mandatory process. For complete knowledge of therapeutic effectiveness of the compound, it is essential to identify all of related metabolites. It has been used successfully in metabolite study using selected MS/MS transitions of molecular ion and product ions for quantification in sample matrix. The identification of the human metabolites of paracetamol was carried out by HPLC monolithic column and sub-2 μm particle UPLC columns along with TOF-MS. The UHPLC system produced the sensitivity and detected metabolites by three times more than the HPLC system. The metabolite profiling has been investigated in various biological samples by applying UHPLC/Q-TOF coupled with MetaboLynx™ software. Biotransformation of new chemical entities (NCE) is necessary for drug discovery. When a compound reaches the development stage, metabolite identification becomes a regulated process [83-96].

9. Metabonomic Studies:

The biochemical changes in the drug product when it exposed to human system is known as metabonomics. Metabonomic studies are helpful in the discovery new metabolites of drug. By the identification of metabolites, its therapeutic and toxic aspects can be determined. It has been employed in pharmaceutical development particularly in the fast in-vitro and in-vivo studies of drug products. Metabonomic studies are carried out in labs to accelerate the development of new medicines. The chemical structures of various constituents in red and white ginseng used in Chinese formulations were determined by this technique. Metabonomics provides a rapid and robust method for detecting these changes, improves understanding of potential toxicity, and allows monitoring the efficacy. The correct implementation of metabonomic and metabolomic information helps similar discovery, development, and manufacturing processes in the biotechnology and chemical industry companies. UPLC used for accurate, reliable and reproducible analysis of amino acids in the areas of protein characterizations, cell culture monitoring and the nutritional analysis of foods [97-101].

10. Peptide Mapping:

The identification of chemical structure of the Peptide connected within the protein is known as peptide mapping. This technique provides authentic results in the when employed in the analysis complex molecules such as proteins. The structural identification of proteins can become easier by applying the time-of-flight mass detection [102].

11. Analysis of Natural Products and Herbal Medicine:

Complex samples from natural products and traditional herbal medicines have been determined. For example, separation of Asian ginseng and adulterated American ginseng preparations by qualitative and quantitative analysis through this technique. The method is helpful in identification and structure elucidation of various isomeric compounds such as ginsenoside and pseudoginsenoside. The chemical constituents of a famous Chinese herbal formulation used for the treatment of intestinal and stomach injure was studied by fingerprinting [103]. UPLC provides high-quality separations and detection capabilities to identify active compounds in highly complex samples that results from natural products and traditional herbal medicines [104].

12. Screening of Antibiotics in Surface and Wastewater:

This technique has been applied in the detection of antibiotics from surface water, influent and effluent wastewaters. These compounds are carried by washing of machineries and transferred in to the wastewater during the manufacturing process. Several antibiotics such as ofloxacin, ciprofloxacin along with other were determined and confirm the presence of the residues of such substances in wastewater in the pharmaceutical industries. The identification and quantitation of various pharmaceutical compounds belonging to different therapeutic classes such as analgesics and anti-inflammatory, cholesterol lowering agents, psychiatric drugs, anti-ulcer agents, antibiotics and beta-blockers were carried out by multi-residue method. The separation time for all the compounds was 10 min. The mass measurement of the compounds was performed by collision induced dissociation (CID) method by selecting the various values of collision energy for fragmentation of compounds [105].

13. Therapeutic Drug Monitoring:

The monitoring of β-lactam antibiotic concentration in plasma of patients with different pharmacokinetics. UPLC-MS/MS method was applied for the simultaneous estimation of two β -lactamase inhibitors and seven β- lactam antibiotics in human plasma. The main benefit of the technique is the faster speed of analysis compared to other approaches used for this type of multiple analytes. This technique is now widely applied for the toxicological analysis of biological specimens. These toxicants may be created during biotransformation of drugs, by-products, intermediates, isomers, impurities, degradation products, excipients of drug products. These materials are present in blood; plasma and urine at very low level were easily identified and quantified [106-108].

14. Screening of Synthetic Compounds:

The technique has become critical tools for use in high-throughput quality control screening of synthetic medicinal compounds. The use of more conventional techniques such as NMR cannot address these high throughput analytical needs due to relatively poor sensitivity, high sample purity requirement, necessity of operator expertise and the use of costly solvents. To simplify the analytical procedures, automation in combination with application of software for exact mass measurement is demonstrated [69, 70].

15. Identification of Unknown Pesticides:

The method is used in the identification of pesticides in the vegetables and fruits. Pesticides having the complicated chemical structure of organic and inorganic compounds. The identification of these toxic materials from vegetables is essential to public health, which can be determined by this sensitive method. The pesticide residue present at very low concentration level collected from packaging materials used for packaging of fruits were analyzed by technique [109].

16. Analysis of Doping Agents:

Method has been applied for doping control and analyses of doping agents from various classes such as β-blockers, stimulants, diuretics, and narcotics. However the developed methods have been used for screening screening of the doping agents, then finally applied for quantification. The method was found reliable for the determination of these restricted drugs by the World Anti-Doping Agency (WADA). The applied method was validated as per the guidelines described in the World Anti-Doping Code [110].

17. Screening of Organic Pollutants in Water:

It is applied for determination of organic contaminants in natural and waste water. The samples were obtained from solid-phase extraction procedure. The analysis was carried out by standard addition technique. Organic pollutants from various sources were mixed with water samples in the range concentration levels and analyzed by this technique. The developed method was applied for detection and identification of several organic contaminants, antibiotics, anti-inflammatory and analgesic drugs [111-114].

18. High-throughput Screening (HTS):

Systematic approach of screening and assaying the organic, synthetic and pharmaceutical compounds are known as high-throughput screening (HTS). The HTS technique has been used widely in drug design and discovery for identification, quantification, characterization of chemical compounds. By applying the HTS methods, the therapeutic, metabolic, pharmacokinetic, toxicological, analytical and degradation data of new drugs can be obtained. It reduces the cost, time, materials and solvent consumption during drug development.^[1-7] The design and discovery of new drug is depends on complete information regarding its therapeutically useful metabolites and derivatives. The therapeutic effectiveness of the compound depends on formation of its all metabolites during biotransformation process and it is essential to identify all these metabolites. The metabolism of drug and formation of metabolites and its derivatives during drug metabolism is known as metabonomics. In the era of developed and modified chromatographic techniques, the UHPLC/Q-TOF-MS technique is relatively new technique and has been used worldwide in drug discovery and development [115-118].

19. Iodinated Byproducts in Drinking Water (IBP):

With the help of UPLC coupled to electrospray ionization-triple quadrupole mass spectrometer (ESI-tqMS), several iodinated byproducts in water (IDBPs) treated with chlorine and chlorine-ammonia have been analyzed quantitatively and their structures were proposed [119].

20. Toxicity Studies:

During the drug development process, toxicity issue causes a fall out of drug candidates and this causes monetary loss to the organization. It is a complicated task to estimate candidate drugs for possible inhibition or initiation of metabolizing enzymes, toxicity or drug-drug interactions in the body. UPLC allows precise detection due to its high resolution. Further, its sensitivity also allows the detection of the peaks at low concentrations. These factors lessen the time for analysis and decrease failure of sample analysis [120].

CONCLUSION:

UHPLC technique has a wide range of its applications in chromatographic analysis. It has been successfully applied for identification and determination of compounds in almost every area of chromatographic and pharmaceutical analysis. It provides the fast, better chromatographic separation and shorter chromatographic run time. From the above discussion, it can be concluded that this technique should be applied to obtain the better results as compared to conventional HPLC technique.

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Received on 21.03.2017 Accepted on 20.05.2017

Asian J. Pharm. Ana. 2017; 7(2): 124-131.

DOI: 10.5958/2231-5675.2017.00020.5