By: Kianshar D Research Team

ABSTRACT

High performance liquid chromatography (HPLC) has been one of the most important methods for the separation and analysis of pharmaceutical raw materials and drugs since many years ago. HPLC is an adoptive technique for numerous applications in the pharmaceutical industries, research , analytical and clinical laboratories, clinical laboratories due to its flexibility and performance. HPLC is the commonly used technique in case of qualitative assessment and quantitative analysis of the compounds of interest in pharmaceutical products like Diazepam , Ciprofloxacin , Dopamine , Fluoxetine , Levamisole , etc. It is the used for analyzing of many natural products such as ginseng, plant extracts and herbal medicines . In the recent years HPLC has been approved to be a useful method in clinical laboratories for determination of Hb1Ac and vitamin D .

This short article exhibits an overview of analysis of large number of key HPLC applications which can be useful for the people who are in charge with the analysis of pharmaceutical compounds in the industries , research companies or colleges.

Keywords: HPLC application, Pharmaceutical, Pharmaceutical quality control , purification , isolation , preparative HPLC , analytical HPLC

INTRODUCTION

High Performance Liquid Chromatography ( HPLC ) is a modern technique which enables separation and determination of the chemical compounds in the sample.

According to the nature of stationary and mobile phase there are 2 types of HPLC ; Reversed Phase HPLC ( RP-HPLC ) and Normal Phase HPLC ( NP-HPLC ).

NP-HPLC uses polar stationary phase , usually silica and non-polar mobile phase like hexane, methylene chloride, chloroform, diethyl ether, or mixtures of them. In NP-HPLC Polar samples are retained on the polar surface of the column packing longer than less polar materials.

In Reverse Phase HPLC the nature of stationary phase is nonpolar (hydrophobic) while the mobile phase is a polar liquid, such as mixtures of water, methanol, acetonitrile or mixtures of them. It works on the principle of hydrophobic interactions thus the more nonpolar the material is, the longer it will be retained.

Reversed-phase HPLC (RP-HPLC) is one of the most popular methods due to its speed, column stability, and capacity to separate a wide range of compounds.Almost 90% of all analyses of low molecular weight samples are carried out using RP- HPLC.

In the attached table a great number of HPLC applications has been listed. The table includes some important information such as the name of the medicines ( from A to Z ) , type of analytical column , mobile phase composition , flow rate and detector.*

HPLC Applications in pharmaceutical industry

The Process of creating a new drug can be divided into three main stages: 1. Drug discovery 2. Drug development 3. Drug manufacturing.

Some important HPLC applications have been defined within all the steps of the process of creating a new drug These applications include separation and quantification of different analytes by analytical HPLC or isolation and purification of the compounds and extracts by preparative HPLC.

From variety of HPLC applications in pharmaceutical and some other related industries we can name the below topics :

Pharmaceutical applications

1. Determination of Shelf-life

2. Identification of active ingredients.

3. Pharmaceutical quality control.

4. Tablet dissolution of pharmaceutical dosage forms

Food and Flavor analysis

1. Analysis of components and quality in water and nonalcoholic drinks.

2. Sugar analysis in fruit juices.

3. Analysis of polycyclic compounds in vegetables.

4. Analysis of preservatives

5. Analysis of residual pesticides in food samples

Environmental applications

1. Bio-monitoring of pollutant.

2. Trace analysis of phenol compounds in drinking water.

3. Identification of diphenhydramine in sedimented samples.

4. Separation and identification of carbonyl compounds

5. Determination of 3-mercaptopropionic acid by HPLC

Forensics applications

1- Investigation of doping in athletes

2. Identification of anabolic steroids in serum, urine, sweat & hair.

3. Forensic analysis of textile dyes.

4. Determination of narcotics in blood and urine.

Clinical applications

1- Determination of Vitamin D in blood.

2- HbA1c analysis in blood of the diabetics.

3- Analysis of antibiotics in blood plasma.

4-. Determination of bilirubin & biliviridin in plasma in hepatic disorders.

5-Analysis of the precursors and metabolites of catecholamines like epinephrine and dopamine in order to provide diagnosis of diseases such as Parkinson and muscular dystrophy.

6. Detection of endogenous neuropeptides in brain extracellular fluids

New applications

HPLC analysis method has been developing to identify, quantitation or isolating and purifying the compounds of interest. During recent years the HPLC technique has been adopted to be used in many different new studies :

1- HPLC is used in stability studies of pharmaceutical products such as antibiotics and biotechnology-based drugs like insulin, streptokinase, etc.

2- HPLC is used in inorganic chemistry for separating anions & cations.

3-. HPLC is widely used in forensic science for the separation and identification of morphine and its metabolites from blood plasma, and for the detection of different poisons or intoxicants such as carbon monoxide, alcohol , cholinesterase inhibitors, heavy metals, hypnotics, etc.

4-. HPLC is used in environmental studies for analyzing the pesticide content in foods and drinking water.

5- HPLC is the main technique for determination of soluble and fat soluble vitamins in supplements and food products.

6- HPLC is used for determination of antioxidants and preservatives in different products.

7- Quality control of various cosmetics like lipsticks, creams and ointments is satisfactorily done by HPLC method.

8- Analysis the extracts of different pharmaceutical herbs such as licorice cinchona, digitalisand ergot is done by HPLC. The technique of preparative HPLC has important role in purification of the components of interest containing in pharmaceutical herbs.

9- HPLC is used for the separation of herbicides in the agrichemical industry.

10- HPLC is the main tool for separation and analysis of amino acids, carbohydrates, proteins, lipids and steroidal hormones.

11- HPLC is used for separation and identification of Psychotropic drugs and antibiotics such as butyrophenones, neuroleptics, phenothiazines , antidepressants , benzodiazepines , chloramphenicol, Cotrimoxazole, Penicillins, and sulphonamides.

12- HPLC can be used for controlling microbiological processes engaged in the production of some antibiotics like tetracyclines, chloramphenicol and streptomycins.

13- To monitor the steps of an organic synthesis and isolating products in the reaction the HPLC is a necessary instrument.

14- In nucleic acids research HPLC instrument has vital importance for some purposes like

a. studying the regulatory effects of cyclic nucleotides.

b. determination of the composition of hydrolysates of nucleic acids

c. metabolite profiling of normal and diseased subjects

d. separation and purification of nucleic acids.

CONCLUSION

High-performance liquid chromatography ( HPLC ) is one of the most valuable techniques for analysis the various compounds in the field of pharmaceutical research and production. By ability of determination of trace compounds and isolating the compounds of interest HPLC has been more and more getting vital instrument in analytical chemistry, pharmaceutical and drug science, clinical diagnosis, food and beverage technology, combinatorial chemistry, polymer chemistry, and environmental chemistry. HPLC has an important role in the pharmaceutical industry ; in finding and isolating the active ingredients , in quality control of raw materials, in formulation , in quality control of finished products and determination of shelf life.

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* Most of the analysis have been practically done by DanChrom HPLC model A2 in the Chromatography lab of Kianshar D company.

 

 

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