Miniaturization trend in modern high-performance liquid chromatography (HPLC) instrumentation is reflected by micro, nano, capillary or ultra-high-performance liquid chromatography. The latter was the great revolution in liquid chromatography that occurred, with significant advances in instrumentation and column technology, with outstanding improvement in resolution, speed, and sensitivity. Speed, sample throughput, and selectivity are the driving forces in the selection of the ideal analytical tool in an analytical laboratory. HPLC offers a variety of versions and thus it is the separation technique of choice with a wide range of applications. Therefore, it is a powerful tool fully adapted by most analytical chemists and is currently the state-of-the art in separation science. Reasonably, the advances in hardware manufacture have turned HPLC from a workhorse into a racehorse in the lab.

Of course no matter how sophisticated hardware is used in the analytical method, sample preparation will always be the bottleneck as it is the most determinative step, at least until a universal and ideal technique is discovered. Automation is now more mandatory than ever. Trends in sample preparation techniques are represented by micro-extraction techniques including online solid-phase extraction (SPE), microwave-assisted solvent extraction (MASE), solid-phase microextraction (SPME), supercritical fluid extraction (SFE), single-drop microextraction, (SDME), fabric-phase sorptive extraction (FPSE), stir-bar sorptive extraction (SBSE), and others, all promising in reducing solvent consumption. New materials of nanostructures, such as graphene, carbon nanotubes, biomaterials, and immunoaffinity materials are also introduced in the sample pre-treatment step either for clean-up or for isolation. All these techniques comply with green analytical chemistry demands and ensure environmental protection and public safety. Applied prior to HPLC offer, the necessary selectivity, sensitivity, and lower detection are required to meet the legislation criteria and give a solution to any analytical problem.

Global Market

The global chromatography instruments market is expected to reach USD 9.223 billion by 2020 from USD 7.062 billion in 2015, at a CAGR of 5.5 percent. The liquid chromatography instruments market has witnessed various technological advancements to meet the needs of biotechnology and biopharmaceuticals. These advancements have led to the development of miniaturized, automated, and computerized devices. These technological advancements have increased the convenience and ease of use of chromatography devices. Improved methods, advent of U-HPLC, efficient column techniques along with a rise in lab-on-a-chip or nano-HPLC, are the contributing factors driving the market.

New international certification for pharmaceutical excipients, increased importance of chromatography tests in drug approvals, and incessant new product launches are major factors driving the chromatography market. In addition, the chromatography instruments market has witnessed various technological advancements to meet the needs of biotechnology and biopharmaceutical companies. Moreover, emerging market across India and China, increasing usage of chromatography in the purification of monoclonal antibodies, growing use of chromatography in proteomics, and green chromatography are likely to create opportunities for market growth. The high growth in HPLC market can be attributed to government investments in biomedical industry and strategic expansions of chromatography players globally. However, high cost of chromatography equipment is likely to pose as a restraint for the growth of the market.

Technology Trends

Next-generation column-packing technology. Column-packing technology played a major role in the progress of HPLC. Without the proper choice of column and appropriate operating conditions, method development and optimization of HPLC separations can be both a frustrating and unrewarding experience. The technology to make and pack nanobore and capillary columns is being optimized in gen-next laboratories; for instance, the recent successes in proteomics are resulting in the usage of capillary and nano-LC columns, often in two-dimensional configurations. These columns excel in sample-limited, high-sensitivity applications. The current widespread use of LC-MS has encouraged developments in shorter and narrower-bore columns that have flow rate requirements within the optimum range of MS ionization techniques. The importance of high-throughput LC columns based on conventional packings and recently on monolith technology is a direct result of the need for increased productivity.

A unique feature of silica-based monolithic HPLC columns over packed-particle columns is the ability to independently control the macro- and meso-pore sizes as well as the silica skeleton size. This control is achieved through the sol-gel preparation process; as a result, this process permits the design of HPLC columns that show simultaneous high-separation efficiency and high permeability, not entirely possible with most packed-particle columns. In the latter case, the particle size determines the separation efficiency and permeability but in a reciprocal manner, that is, big particles lead to high permeability and low efficiencies whereas small particles lead to the opposite relation. The SPP columns do provide some of the advantages of high efficiency, but with lower permeability than the silica monoliths.

En-route to miniaturization. Nano HPLC or lab-on-a-chip concept is today an established tool for research laboratories. The main advantage of nano-LC is enhanced sensitivity, as compounds enter the MS in more concentrated bands. This is particularly relevant for determining low abundant compounds in limited samples. Nano-LC columns can produce peak capacities of 1000 or more, and very narrow columns can be used to perform proteomics of 1000 cells or less. Also, nano-LC can be coupled with online add-ons such as selective trap columns or enzymatic reactors, for faster and more automated analysis. With a smaller internal-diameter column, nano-HPLC keeps the sample more concentrated, rather than it getting diluted across a relatively wider column. Nonetheless, the smaller scale creates complexities in connecting the parts. Microfluidic chip-based technology for high-sensitivity nano-LC/MS seamlessly integrates sample preparation, sample enrichment, and separates nano-columns, tubing, and connections. This system can also be adapted to specific applications by using different chips. A wide variety of chips with specific chemistries enables a broad spectrum of applications, including intact monoclonal antibody characterization, monoclonal antibody sequence confirmation, and in-depth characterization of the myriad post-translational modifications of these complex molecules.

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) gaining traction. Liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) has recently become a popular alternative to traditional ligand-binding assays for quantitative determination of biopharmaceuticals. LC-MS-MS offers several advantages such as improved accuracy and precision, better selectivity, and generic applicability without the need for raising analyte-directed antibodies. Another benefit of LC-MS-MS is that it gives clinical laboratories the ability to multiplex, identifying and quantifying several analytes of interest simultaneously. Multiplexing lowers the cost per test. LC-MS-MS offers other cost savings and increased throughput via simplified or minimal sample preparation for some applications, such as dilute-and-shoot or protein crash, compared to more time-consuming and expensive sample-preparation methods like solid-phase extraction or derivatization. Clinical laboratories have also experienced situations in which manufacturers unexpectedly withdraw immunoassays from the market, leaving labs searching frantically for alternate methods to get test results back to the ordering physician. These experiences have given labs another reason to turn to LC-MS-MS. MS-MS also exhibits flexibility and versatility in enabling laboratories to offer novel laboratory-developed tests (LDTs) for biomarkers or for newly approved medications before FDA-approved kits or immunoassays to measure them come on the market. In addition, the sensitivity of LC-MS-MS may allow lower limits of detection for some analytes, such as steroids, compared to immunoassays and other methods.

Compared to ligand-binding assays, LC-MS-MS has a number of analytical advantages such as a larger linear dynamic range; higher accuracy and precision because of the possibility to apply internal standards; the ability to quantify multiple analytes simultaneously; and the fact that it does not necessarily require immunological reagents.

UV detection for HPLC. The application of microprocessors in HPLC systems has simplified instrument manipulation, and enabled the development of fully automated systems. Additional information about the structure of the compounds being eluted can be obtained with the widely used variable-wavelength UV detectors. HPLC with UV is the technique of choice for determination of impurities of analgesic, antibiotic, anti-viral, anti-hypertensive, anti-depressant, gastro-intestinal, and anti-neoplastic agents. The standard UV detector for HPLC measures the absorbance of monochromatic light of fixed wavelength in the UV or visible wavelength range (typically between 190 nm [UV] and 400 nm [blue light]) against a reference beam and relates the magnitude of the absorbance to the concentration of analyte in the eluent passing through a flow cell contained within the instrument.

Road Ahead

Continued investment in column technology is bound to continue since the HPLC and UHPLC columns market has been very strong. Columns are considered a consumable. An average instrument uses seven columns per year; so as the number of instruments grows, so does the columns market. The overall market for columns such as analytical, preparative, capillary-nano, packings, and column accessories will foresee an overall growth of 3.5 percent, but a higher growth in the UHPLC segment. Tremendous strides have been made in particle and stationary-phase technology, but user demands in industry for productivity and sensitivity improvements continue to push further developments in columns that are more efficient, faster, and more inert. These needs stretch from the research laboratory and through all phases of development up to manufacturing and quality control.

Even with all of the current improvements in column-particle technology, work is continuing on further improvements in superficially porous particles. Current techniques for making SPPs are based on a multilayer process or a coacervation process, and most commercial SPPs use one of these two processes. There are alternatives being investigated that result in improved TPP and SPP materials by allowing the formation of more uniform meso-pores compared to present packings.

The recent trends in HPLC column technology with shorter columns packed with smaller particles or superficially porous particles used at a higher flow rate are a direct result of the need for increasing sample throughput. When higher throughput is required, analysts do not want to have to spend time redoing or revalidating the method but want to get the same elution order and resolution achieved with the original column but faster. To maintain the integrity of the separations, the experimental conditions must be adjusted so that everything looks the same, except that the analysis time is shorter.

The future of LC-MS-MS is bright as manufacturers continue to improve, automate, and simplify the technology and make this highly complex instrumentation more like automated, FDA-approved chemistry analyzers. Eventually, more simplified, automated LC-MS-MS instruments will enable additional operators. The release of more ready-to-use, FDA-approved reagent kits for LC-MS-MS will also minimize the effort needed for method development and extensive validation, making LC-MS-MS accessible to more labs. Further improvements in sensitivity, specificity, and throughput will also facilitate more clinical applications.

Dr Meenakshi Sharma, Consultant Hematopathologist, Manipal Hospital, Jaipur
Second Opinion
Applications of HPLC in Clinical Diagnostics

High-performance liquid chromatography (HPLC) has been widely used for years as an analytical method for sample separation in pharmaceutical quality control and life science research. The major advantage of HPLC is the flexibility it allows in choosing the stationary and mobile phases that best separate the particular sample components. More than half of the types of columns used in HPLC are reverse-phase; around a quarter are normal-phase; and about 14 percent are ion-exchange columns. Obtaining high resolution at high flow rates is the key requirement of any HPLC system. Separation of the different components of a mixture is achieved by allowing the sample to migrate through the column at selected experimental conditions. Discrete bands are formed because different components of the mixture migrate through the column at different rates. {mosimage}

A few of the applications of HPLC in clinical laboratory are:

Diagnosis of hemoglobinopathies. HPLC is capable of separating more than 45 commonly encountered hemoglobin variants within 12 minutes. The simplicity of sample preparation and superior resolution of the method with complete automation makes it an ideal methodology for routine diagnosis of hemoglobin disorders.

Catecholamines. Various assay permits the quantitative determination of vanillylmandelic acid (VMA), homovanillic acid (HVA), and hydroxyindoleacetic acid (5-HIAA) only in urine by HPLC for diagnosis of pheochromocytoma, neuroblastoma, and carcinoid syndrome.

Alcohol abuse. HPLC is the proven method of choice because it allows reproducible separation and visible detection of different transferrin glycoforms.

Diabetes monitoring. It is a highly sensitive technique that allows quantitation of glycated hemoglobins and more specifically hemoglobin A1c, which is an excellent measure of long-term blood glucose control.

Vitamins. Levels of most vitamins - vitamin A, vitamin B, vitamin B2, 25 OH vitamin D3, vitamin C, vitamin E, and vitamin K can be measured using HPLC.

Dr Meenakshi Sharma
Consultant Hematopathologist,
Manipal Hospital, Jaipur

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