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As published in CLI April 2006

Applications of HPLC in clinical diagnostics


HPLC has been widely used for years as an analytical method for sample separation in pharmaceutical quality control and life science research. Due to the power and flexibility of this technique, numerous applications have been adopted for routine use in the clinical laboratory. A few of these applications are discussed in this article.
The analysis of catecholamines, mainly in plasma and urine samples, is challenging because of their presence at low concentrations, their susceptibility to oxidation and the fact that they are unstable at basic pH. HPLC provides distinct advantages, since the method can analyse such samples with high resolution. Rapid analysis without prior derivatisation is rapidly becoming the method of choice compared to earlier techniques such as column chromatography. BioRad kits for analysis of urinary catecholamines and plasma catecholamines (UCATS and PCATS) are used in the identification and quantitation of urine and plasma catecholamines respectively. Plasma free metanephrines There remains a lack of agreement about the most efficient and cost-effective method of diagnosing pheochromocytomas, which are rare neuroendocrine tumours. In a recent study the relationship between tumour size, location and catecholamine content with plasma and urinary metanephrines or catecholamines was analysed in 275 patients with pheochromocytoma. Plasma samples were assayed by HPLC, and 24h urinary outputs were measured by HPLC or by liquid chromatography with tandem mass spectroscopy. Using these methods it was concluded from the study that measurement of plasma free metanephrines (normetanephrine and metanephrine) provides a useful test both for the presence of these tumours and their size and location [1, 2]. Alcohol abuse The high prevalence of chronic alcohol abuse in many countries has increased the need for the monitoring of alcohol consumption in these patients. Carbohydrate-deficient transferrin (CDT) is widely utilised as a specific marker of excessive alcohol use. Individuals who have been drinking excessively over many weeks typically show increased relative amounts of glycoforms. The main advantage of CDT compared with the conventional biochemical indicators of chronic alcohol abuse (e.g. liver enzymes) is its high specificity for alcohol abuse with a resulting low rate of false-positive identifications. Several analytical methods for CDT have been developed. Transferrin glycoforms were originally separated, identified, and quantified by isoelectric focusing (IEF), and those glycoforms that were isoelectric at pH 5.7 or above, after

Any analytical technique must satisfy the need to provide sensitive, timely and reliable results. HPLC meets these needs for clinical chemistry. The method is able to cope with polar, thermally labile or relatively non-volatile compounds. HPLC is a separation technique in which the mobile phase is an aqueous solution or an organic solvent that is miscible with water, and the compounds of interest are dissolved in water or an organic solvent such as methanol. The major advantage of the technique 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; about 14% 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 the different components of the mixture migrate through the column at different rates. The apparatus of a typical liquid chromatograph system includes an eluent system, a pump, an automatic sampler, the analytical column, a detector and a recorder. The most essential component of this apparatus is the analytical column that is used to separate the components of interest in biological samples. The most widely used detector for HPLC is the UV-Visible absorbance detector. This is a sensitive detector for organic molecules that absorb light within the UV or visible wavelength spectrum, which is widely applicable to many compounds. The difficulty often faced in analysing clinical samples is resolving the compounds of interest from interfering substances, as well as the need to detect trace quantities of analytes. In addition to analysing compounds which are the

result of metabolism in both healthy and diseased states, HPLC can also be used to measure therapeutic drug levels as as well as the presence of drugs of abuse. Good sample preparation is essential for the successful analysis of specimens, which include biological samples such as blood and urine. Blood in particular needs to be processed prior to analysis to remove any interfering compounds that would cause either contamination or blockage of the column, giving inaccurate results. These problems can be prevented with the use of a guard column with larger particle size packing material. Sample preparation also involves the typical methods of solvent extraction, ultrafiltration or solid-phase extraction. With total automation, time spent on sample extraction and clean-up procedures is minimised, while the sensitivity and simplicity of the technique are maintained. This has proven extremely useful in the analysis of catecholamines and in determination of amino acid metabolism. Catecholamines Catecholamines are biologically active amines that perform a number of important neuronal and hormonal functions in humans. HPLC techniques have the advantage of being able to simultaneously detect several analytes and metabolites at low cost compared to other techniques such as LC/MS and RIA. Determination of the levels of the parent amines and their metabolites have been widely used as diagnostic tools in a number of diseases, i.e. pheochromocytoma, hypertension, heart disease, muscular dystrophy and Parkinson's disease. Bio-Rad offers HPLC-based products that are used for the quantitative determination of vanillylmandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5HIAA) in urine for diagnosis of pheochromocytoma, neuroblastoma and carcinoid syndrome

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blood sugar control is necessary to prevent the complications of diabetes. The technique of HPLC is invaluable for monitoring glycaemic control. It is a highly sensitive technique that allows quantitation of glycated haemoglobins and, more specifically, Haemoglobin A1c, which is an excellent measure of long term blood glucose control. The main advantages of using the HPLC method are better resolution and reproducibility. High accuracy and reproducibility are two key requirements in order to monitor a patient's glycaemic control. Automated HPLC methods display excellent within- and between-run precision [3]. Many diabetes monitoring platforms are dedicated walk-away systems, necessary because of the volume of samples that need to be processed per day. Rapid separation (~ 90 seconds) is needed without compromising the level of resolution of the HbA1c peak from potential interfering compounds, i.e, labile HbA 1c and carbamylated HbA1c. The advantages of utilising an HPLC system for quantitating HbA1c are exemplified in the Bio-Rad VARIANT II and VARIANT II TURBO, which are ideal for test volumes of from 500 to more than 2000 samples per month. These systems have excellent within- and between-run precision, and have the added capability of detecting haemoglobin variants. Furthermore, the D-10 Rack-Loader provides a solution for test volumes ranging from 10-50 samples a day. In addition these platforms are also extremely useful in the screening for thalassaemia. Vitamins Vitamins are an important component of the daily diet. Some of the many vitamins of clinical interest include Vitamin A (retinol), vitamin B1 (thiamine), vitamin B2 (riboflavin), 25-OH vitamin D3, vitamin C, vitamin E (alpha-tocopherol) and vitamin K. Levels of most vitamins can be measured using common techniques such as UV absorption, spectrophotometric procedures, TLC, GC and HPLC. Vitamin B1, found in human blood, milk and cerebrospinal fluid, is measured by reversephase HPLC after appropriate sample preparation. Though standard techniques exist for the measurement of vitamins, problems are frequently encountered due to the complexity and lability of these compounds, which are either fat or water soluble. HPLC is a proven method for isolating analytes of interest from other interfering compounds as well as yielding accurate and reproducible results.

Figure 1. The Bio-Rad VARIANT II HPLC system.

complete iron saturation, were collectively named CDT. For routine determination of CDT, several commercial and non-commercial methods based on ion-exchange chromatographic separation on minicolumns followed by immunoassay are available, such as the Bio-Rad % CDT-TIA, HPLC, and capillary electrophoresis (CE). HPLC is the proven method of choice because it allows reproducible separation and visible detection of the different transferrin glycoforms. Genetic transferrin variants and glycoform types that may cause incorrect determination of CDT with the minicolumn immunoassays are readily identified from a unique pattern of peaks in the chromatogram. Diabetes monitoring Diabetes is a chronic but treatable condition, which is caused by insulin resistance or an inability to produce insulin. For such individuals, tight

References
1. Eisenhofer G et al. Pheochromocytoma catecholamine phenotypes and prediction of tumour size and location by use of plasma free metanephrines. Clinical Chemistry 2005; 51: 735 - 744. 2. Lenders JW et al. Biochemical diagnosis of pheochromocytoma: which test is best? JAMA 2002; 287: 1427 -34 3. www.ngsp.org 4. HPLC in Clinical Chemistry by I.N. Papadoyannis.

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