Preparing HPLC To Attain Central Place In Diagnostic Labs

Backed by advances in the field of liquid chromatography, diagnostic services are now able to offer faster turnaround times and measure analytes in patient types and disease states that were previously problematic.

Chromatography is the backbone of analytical laboratories all over the globe with high performance liquid chromatography (HPLC) representing the largest portion. HPLC is generally seen as the most versatile chromatographic methodology, with applications in chemistry, bio-analytical sciences, and the pharmaceutical industry. The versatility of HPLC has stemmed from its unique, universal applicability to a very diverse range of analytes; from small molecules and ions to polymers and large biomolecules. The technology is constantly evolving, driven, in the main part, by a wide variety of available stationary phases and the implementation of more sophisticated technologies such as more sensitive detectors, allowing for robust and reproducible separation conditions. Adding to the inherent advantages in analytical analyses, HPLC is nondestructive and therefore, well-suited for preparative applications.

Moreover, rather than simply being a way to identify which patients have a specific disease, diagnostics are now used to support clinical development of drugs, predict disease before symptoms begin, forecast the progress of a disorder, and identify patients who are most likely to respond/not respond to specific treatment. Many forms of chromatography have been used over the years in the clinical laboratory for the separation and quantification of a variety of clinically relevant analytes. The last decade has seen a series of advances in the field of liquid chromatography that have resulted in improvements for many clinical diagnostic services. As a result, diagnostic services are able to offer faster turnaround times and measure analytes in patient types and disease states that were previously problematic. Considering all these applications, HPLC can be considered as a multi-tool of chromatographic techniques. 

Although having matured as a technique for more than 50 years, classical HPLC has been challenged – or complemented – in the last decade by the availability of ultrahigh performance liquid chromatography (UHPLC), capable of using smaller size particles that create elevated pressures exceeding 400 bars. With the increasing need to reduce the ecological footprint in the lab, UHPLC technology has the potential to help lab managers meet this challenge; the lower flow rates, smaller column diameters, reduced equilibration times, and lowered overall system volumes all combine to directly reduce the amount of organic solvents used during analysis. Ultimately this leads to a single advantage: the reduction in overall operating costs!

Indian market dynamics
The Indian HPLC market in 2017 is estimated at Rs 1241 crore, with 6045 units. Across all segments, there was a uniform 7 percent increase over 2016, in value and quantity terms, since prices remained constant over the year. This is very different from 2016, when prices had increased by about 15–20 percent in rupee terms over 2015.

Basic HPLC systems (5000-8000 psi; 350-600 bar) continue to hold sway with a 75 percent share in quantity and a 66 percent share in value terms in 2017. Modular systems are increasingly being preferred over their integrated counterparts. Complex application demand is increasing, especially in the pharma and biopharma industries, traditional HPLC is not adequate to fulfill the requirement, and UHPLC with specialized detection system is being explored. Fast HPLC (9800-20,000 psi; 900-1400 bar) is where the same (or better) separation is obtained, but with a shorter runtime. This is obtained simply by exploiting certain parameters (the column particle size, internal diameter, length, and particle morphology) to increase the efficiency of the separation. If the column and conditions are more efficient, the same job is done in less time. Specialty systems, comprising gel permeation chromatography instruments, supercritical fluid chromatography instruments, bio-LC, and ion exchange chromatography instruments cater to niche demand.

Customers are looking for single vendor support for multi-vendor products. The pharmaceutical industry continues to drive this segment. Food, specialty chemicals, and agro led the non-pharma sector over the last couple of years. The Indian buyer is continuously giving weightage to operating cost, and the growth and increased spend will be determined by how much the vendors are able to decrease the operating cost. Leading pharma companies continue to face serious compliance issues with regulatory authorities, particularly for FDA approvals, albeit today, India has the 2nd largest number of USFDA-approved manufacturing plants outside the US. They are continually investing in remedial measures, making regular procurement of equipment a low priority. The smaller companies, which export to non-US markets as Middle East, Russia, and other developing nations, are better placed.

Technological advances
With many second-generation instruments debuted by all major manufacturers in prior years, now the market is seeing mostly line extensions, application-specific systems, modules (particularly new MS or MS add-ons), and software products.

Monolithic columns. Since late 1970s, particulate columns (5 µm) have been widely used in the field of chromatography. The decrease in the particle size gives better column efficiency but results in a higher back pressure. With the advent of monolithic columns, higher column efficiency can be provided with minimum back pressure. The recent invention and development of monolithic columns is a major technological change in column technology; indeed the first original breakthrough to have occurred in this area since Tswett invented chromatography, a century ago. The monolithic column is made up of continuous porous material, sealed against the wall of a tube, instead of beads. The decrease in chemicals and samples usage, improved sensitivity, reusable frit-less columns, and lower back pressure make them more efficient and user-friendly columns in the present world. Currently, they are available in three different types, that is, silica-based, polymer-based, and hybrid monolithic columns.

Micro-pillar array column. Another new column format that has been introduced to increase the separation performance is the micro-pillar array column. The perfect order of these pillars significantly reduces the band broadening as a result of heterogeneous flow paths in the column, and hence the overall peak dispersion, while the inter-pillar distance can be tuned to decrease the column back pressure, allowing the use of very long columns. The distinctive properties of these columns
can be used to address challenging separation problems, as encountered in lipidomics.

Core–shell technology. In contrast to the typical silica-based LC particles, core–shell particles consist of a solid, impermeable inner silica core that is surrounded by an outer shell of conventional fully porous silica. Due to this unique core–shell morphology, columns packed with core–shell particles deliver significantly greater efficiency than columns packed with fully porous media of equivalent particle size. Greater efficiency will translate into improved resolution and increased peak height (sensitivity). With that improved resolution, one can afford to go to shorter column lengths and/or increased flow rates (within allowable adjustment ranges) to dramatically reduce analysis times compared to the original method while still meeting initial system suitability requirements.

Fast HPLC. HPLC using short columns (3–10 cm) packed with small particles (<3 µm) and high flow rates has recently become a good strategy to save time and solvent consumption in HPLC. Many laboratory budgets do not allow the purchase of new UHPLC systems, and workload is constantly growing. Fast  HPLC incorporates the use of faster mobile phase flow rates and smaller particles to obtain compound separations in a lower time and with an equivalent resolution to a traditional HPLC system. Fast HPLC allows to run more samples in the same time frame by developing fast methods with the existing HPLC systems in the laboratory.

Integrated solution. Manufactures are now launching HPLC systems build off by incorporating new modules (i.e. dual gradient pump and a unique dual auto-sampler) to offer three workflows that increase productivity in a single system – dual LC, tandem LC or LC-MS, and inverse gradients. Dual LC allows to run two samples in parallel or two different applications in parallel, doubling throughput or obtaining more information from a single sample, respectively. Tandem LC or LC-MS uses two individual pumps, for example, a dual gradient pump and two columns. Whilst one is performing an analytical run on one column using one pump, the other column can be re-conditioned on the second pump. One can then simply switch the column used for the next sample. In this way, the detector does not sit idle waiting for the column to be re-conditioned before the next run begins. This not only saves time, but also maximizes the utilization and ROI of the detector. Inverse gradient maximizes productivity. By using a dual-gradient pump it is possible to perform an inverse gradient on the second pump to match the analytical gradient. The two gradients then combine before the CAD so that it receives a constant solvent composition which greatly improves uniformity of response for quantitation.

Outlook
The main advantages of HPLC method over other techniques are its high selectivity, sensitivity, reliability, and versatility with low cost of operations. Its use in the clinical laboratory has steadily increased over the past decades as its unmatched analytical performance and versatility allows for testing of many different types of clinically relevant analytes. With increasing automation in the overall processing of HPLC with minimal human intervention, integration of HPLC
with mass spectrometry (MS) and possibility of sequential testing, it has become more user-friendly and allows routine laboratories to provide better diagnostic services with a shorter turnaround time.

Now is a great time for life science organizations to invest in HPLC technology. As the demand for HPLC systems increases in the life science industry, manufacturers are looking to develop systems that are tailored to the needs of life science research and manufacturing organizations. Together with increasing health awareness amongst the Indian population, government initiatives and strict regulations, testing labs are forced to go far more selective; precise and automated diagnostic tools like HPLC would find a central place in any diagnostic lab.