Applications of High Performance Liquid Chromatography (HPLC) in Pharmaceutical Analysis

APPLICATIONS OF HPLC IN PHARMACEUTICAL ANALYSIS

Modern HPLC finds its abundant applications not only confined to isolation of natural pharmaceutically active compounds, control of microbiological processes but also assay of pure drugs and their dosage forms. A few typical examples will be discussed below :

1. ISOLATION OF NATURAL PHARMACEUTICALLY ACTIVE COMPOUNDS

Some plant alkaloids and glycosides can be isolated as stated below :

Chromatographic Conditions :

Column              :  Size-25 cm × 4.6 mm ID ;

Adsorbent           :  Lichrosorb RP-8 ;

Mobile-phase      :  Water/Acetonitrile-Gradient Elution ;

Detector      :         UV 254 nm

2. CONTROL OF MICROBIOLOGICAL PROCESSES

Various microbiological processes are used in the production of a number of antibiotics, for instance :

penicillins, tetracyclines, chloramphenicol and streptomycins. The major areas of such operations being :

·              kinetics of the microbiological process,

·              monitoring of the on-going process,

·              isolation and purification of active ingredients,

·              purity control of active constituents, and

·              monitoring derivatization reactions of these compounds.

HPLC-controlled analysis of a microbiological process during Penicillin Production : Chromatographic conditions are as follows :

Column              : Size-25 cm × 4.6 mm ID ;

Adsorbent          : Lichrosorb-NH₂^(R) (10 μ m) ;

Mobile-phase :  0.005 M H₂SO₄ buffer (pH 4.4))/acetonitrile (50 : 50) ; Flow rate : 3 ml min^–1 ;

Detector      :         UV-220 nm ;

Microbial cleavage of Penicillin-G into 6-AMP and phenylacetate is as shown below :

Figure : 11 (a) and (b) shows the HPLC chromatograms of penicillins in ‘production sample’ and ‘standard solution’ respectively :

3. ASSAY OF CEPHALOSPORINS

Several commercially available cephalosporin antibiotics have been adequately separated by HPLC methods under the following experimental parameters

Column              :  ODS-SIL-X-II,

Mobile-phase       :         0.95 M Ammonium Carbonate/Methanol (95 : 5) ;

Detector      :  UV-220 nm ;

The loss of absorption with cleavage of the β-lactam was used by Marrelli* to analyze the concentra-tions of cephalosporin C in the presence of other UV absorbing species.

4. ASSAY OF FRUSEMIDE**

Theory : HPLC analysis of frusemide and its decomposition products is carried out by using simultane-ous fluorescence and UV detection.

It is noteworthy that fluorescence detection is a very specific technique, especially when excitation and emission wavelengths can be selected. In addition to this, sensitivity for compounds with photoluminescence properties can be higher by factors of 100 to 1000 when compared with that of other detectors.

The chromatographic conditions for Frusemide determination are as stated below :

Column                : Size : 250 × 4.6 mm ID ;

Adsorbent            : Lichrosorb(R) RP-8, 10 μ m ;

Mobile-phase       : Gradient elution-2 minutes, from 20% B to 37% B in 15 minutes, where, A = Water (pH 2.7) and B = Acetonitrile ;

Detector                : (i) Fluorescence : Excitation : 275 nm ; Emission : above 405 nm ;

Figure : 12 (a) and (b) depict the HPLC chromatograms of frusemide by fluorescence and UV detectors respectively.

5. ASSAY OF THEOPHYLLINE

Theophylline invariably contains other related substances as impurities, namely : theobromine, caffeine and β-hydroxypropyltheophylline.

The chromatographic conditions for HPLC are as stated below :

Sample size :                  10 µL ;       

Column       :   size – 250 × 4.6 mm ID ;

Adsorbent   :   Lichrosorb (R) RP-8, 10 µm ;

Mobile-phase       :   0.02 M KH2PO4 Buffer (pH 3.5)/Acetonitrile (95 : 5) ;

Detector      :                  UV-254 nm ;

Figure 30.13, illustrates the HPLC chromatogram of theophylline assay with four distinct peaks.

6. ASSAY OF CORTICOSTEROIDS

            mixture of six corticosteroids, namely : deoxycortisone, hydrocortisone acetate, cortisone, prednisone, hydrocortisone and prednisolone can be assayed by HPLC method. The chromatogrpahic param-eters for the assay are as follows :

Sample size          : 10 μ L

Column               : size-250 × 4.6 mm ID ;

Adsorbent           : Lichrosorb^(R) DIOL : 10 μ m ;

Mobile-phase      :  Gradient elution of A (n-Hexane) and B (Isopropanol) ;

Detector               :  UV-254 nm ;

Figure 30.14, clearly shows the six well-elaborated and distinct peaks of all the constituents stated earlier under the above HPLC parameters.

7. ASSAY OF DICHLORPHENAMIDE*

Materials Required : Dichlorphenamide sample : 100 mg ; dichlorphenamide RS : 100 mg ; Mobile-phase [solution containing 0.02 M NaH₂PO₄ and 0.2 M Na₂HPO₄ in a mixture of equal volumes of acetonitrile and water] : 50 ml ;

Procedure : The chromatographic procedure may be performed using μ Bondapack C18 column as the stationary phase and the above mentioned mobile-phase with a flow rate of 1.0 ml per minute and a detection wavelength of 280 nm. Carry out the HPLC analysis using solutions in the mobile-phase containing (1) 0.05% w/v of dichlorphenamide RS and (2) 0.05% w/v of dichlorphenamide sample.

Calculations : Calculate the content of C₆H₆Cl₂N₂O₄S₂ using the declared content of the same in dichlorphenamide RS.

8. ASSAY OF HUMAN INSULIN*

Materials Required : Solution (1) : dissolve 40 mg of Human Insulin in sufficient of 0.01 M HCl (0.3648 g of HCl in one litre DW) to produce 10 ml ; solution (2) : Mix thoroughly 900 μ L of 0.01 M HCl to 100 μ L of solution (1) ; Solution (3) : dissolve 40 mg of Human Insulin EPCRS in sufficient 0.01 M HCl to produce 10 ml ; Solution (4) : mix 1 ml of solution (3) with 1 ml of a solution containing 4 mg of porcine insulin RS : Mobile-phase ‘A’ : Dissolve 28.4 g of anhydrous Na₂SO₄ in sufficient water to produce 1000 ml, add 2.7 ml of orthophosphoric acid, adjust of pH 2.3, if necessary, with ethanolamine, filter and degas by passing He through the solution ; Mobile-phase ‘B’ : Mix 500 ml of mobile-phase ‘A’ with 500 ml of acetonitrile, filter and degas by passing He through the solution.

Procedure : The HPLC is carried out using (a) a Vydac C18 column, for proteins and peptides, maintained at 40 °C, (b) as the mobile phase at a flow rate of 1 ml per minute, a mixture of 48 volumes of mobile phase ‘A’ and 52 volumes of mobile phase ‘B’ prepared and maintained at a temperature of not less than 20 °C, and (c) a detection wavelength of 214 nm.

Adopt the following steps sequentially :

Calculations : Calculate the content of human insulin, C₂₅₇H₃₈₃N₆₅O₇₇S₆, from the peak areas and using the declared content of C₂₅₇H₃₈₃N₆₅O₇₇S₆ in human insulin EPCRS.

9. COGNATE ASSAYS

A number of pharmaceutical substances can be assayed by HPLC method as detailed in Table 30.1.