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High Performance HPLC
Jayata Sharma presents an overview of HPLC technique
that has revolutionised diabetes monitoring and thalassemia detection in India.
High Performance Liquid Chromatography (HPLC) is a diagnostic
technique used for identifying and purifying proteins and many other molecules
in the blood. It is employed for diagnosing various haemoglobin disorders in
the blood like thalassemia major or minor or estimation of HbA1c for diabetes.
Of the three main types of chromatography—gas (GC), liquid (LC) and supercritical
fluid (SFC), HPLC falls in the SFC section.
Advantage HPLC
 "HPLC
helps estimate analytes, which are in very low concentration in blood. This
was not possible with earlier techniques"
- Dr Nilesh Shah,
Senior Vice President, Metropolis Technologies Mumbai
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Earlier, various methodologies such as the NESTROFT test,
sickling tests, HbA2 columns, capillary electrophoresis, Iso electric focusing,
and electrophoresis were employed to detect haemoglobin abnormality. However,
HPLC made haemoglobin abnormality detection much more accurate, faster and automated.
"Earlier techniques could not match HPLC and failed to provide total and
accurate information of a patient's haemoglobin condition," says Dr Arvind
Lal, Chairman and Managing Director, Dr Lal PathLabs, New Delhi. Dr Lal PathLabs
was the first Indian pathology provider to introduce HPLC in the medical laboratory
in 1993.
 "Earlier
techniques could not match HPLC and failed to provide total and accurate
information of a patient's haemoglobin"
- Dr Arvind Lal,
Chairman and Managing Director, Dr Lal PathLabs New Delhi
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In an age where diagnostic techniques are constantly replaced
with more advanced ones, HPLC stood the test of time for more than three decades
through modifications. Introduced in the '70s in a crude form, HPLC was not
really used by any lab. In fact, it was available only in the R&D labs in
the US and the UK. Subsequently, features like column packing material, online
detectors and reverse phase were added. By the '80s, it was ready to be used
for separation of chemical compounds/analytes.
HPLC Helps
 "HPLC
has enabled identification of
couples at risk of giving birth to
homozygotes for b-thal"
- Dhiren Wagle,
Country Manager
Bio-Rad Laboratories, NCR
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HPLC is defined as an analytical technique for separation
and determination of organic and inorganic solutes in any sample, especially
biological, pharmaceutical, food, environmental and industrial. One can say
that HPLC is similar to finding out how much caffeine there is in a cup of coffee
(or tea, or cola).
HPLC is also widely used for the estimation of HbA1c. "In HbA1c, the haemoglobin
fractions are separated based on the dynamic interaction between the protein
molecule (sample), stationary phase (cartridge), and the mobile phase (reagents),"
explains Dhiren Wagle, Country Manager, Bio-Rad Laboratories, NCR. The results
are then reported as chromatograms with the relative percentage of various haemoglobin
fractions.
With today's advanced instrumentation and chemistry supplies in the field of
pathology, capabilities have increased —earning the name 'high performance'
for HPLC.
"HPLC is also used in analysis of cardiac risk markers
like homocysteine, scatecholamine, separation of peptides and proteins, and
for the analysis of fungal toxins like ochratoxin and afflotoxin," says
Dr Harish Ahuja, HOD-Pathology, Jaslok Hospital, Mumbai. In addition, HPLC identifies
individuals with clinically-silent variants like Hb QIndia, Hb J, Hb DIran and
new variants like Hb C.
How It Works
 The
process of HPLC starts with a liquid chromatographic process. The liquid (blood
sample in this case) permeates through a porous solid stationary phase and elutes
the solutes into a flow-through detector. HPLC works when the analyte is forced
through a column—usually a small tube packed with small round particles
with a certain surface chemistry (stationary phase) by pumping a liquid (mobile
phase) at high pressure of 20-200 kg/cm² through the column.
The stationary phase is usually in the form of small-diameter
(5-10 mm) uniform particles, packed into a cylindrical column. The sample to
be analysed is introduced in a small volume to the stream of mobile phase. The
amount of retardation depends on the nature of the analyte, stationary phase
and mobile composition.
The typical column is constructed from a rigid material (such
as stainless steel or plastic) and is generally 5-30 cm long with an internal
diameter in the range of 1-9 mm. HPLC is measured on three scales of chromatography.
They are analytical—just data (high sensitivity), semi-preparative—data
and a small amount of purified analyte (gram) and preparative—larger quantities
of purified analytes (kilograms)-high capacity. The tests performed by HPLC
techniques cost from Rs 600 to Rs 3,000 based on the type of analyte to be tested.
However, it can go up to Rs 7,500 for urinary metanephrines for detection of
pheochromocytoma.
It is reported that HPLC is 99 per cent accurate. HPLC is preferred due to its
high speed, better resolution, sensitivity, reproducibility, accuracy and automation.
"Screening by HPLC has enabled identification of couples at risk of giving
birth to homozygotes for b-thal," informs Wagle.
However, the flip sides of HPLC are its high cost, complexity, low sensitivity
of some compounds, irreversibly absorbed compounds not being detected and difficulty
in detection of coelution. Moreover, it is tedious to perform and needs skilled
labour.
Future Improvements
Currently, Dr Lal PathLabs is conducting research on improving the accuracy
of HPLC analysis by enhancing the software capabilities. "Recent advancement
in manufacturing technology has helped in better detection of haemoglobin variants
by incorporating minute differences in the retention time," informs Dr
Lal. HPLC has always developed and grown with the times. In recent times, 'miniaturisation'
has introduced numerous testing strategies in chemical pathology.
Miniaturisation has reduced the amount of patient's sample used and has evolved
ways through which we can now detect molecules of interest, which has significant
implications for various therapies.
The detection of inborn errors of metabolism (IEM) from a blood sample by HPLC
and use of mass spectrometry for the detection of lead and other heavy metal
contamination and thereby disease, is a striking example of miniaturisation.
Experts believe, in future improved HPLC methods will replace old photometric
assays and immunoassays. "HPLC shall remain the mainstay methodology in
purification and separation of proteins, peptides and nucleotides," affirms
Dr Lal.
In addition, research is being done for analysis of various diagnostic markers
using HPLC. Industry experts consider that once the markers are standardised
and affordable, the use of HPLC will definitely increase.
healthcare@expressindia.com
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