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A REVIEW ON DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPLC METHODS FOR ANALYSIS OF ACIDIC DRUGS

High-performance liquid chromatography is one of the fastest, safest and precise technology used for determination and separation of pharmaceutical drugs, impurities and biological samples. High-performance liquid chromatography is versatile and it takes less time for quantification of drugs as compared to old liquid chromatography techniques. This article reviews stability indicating HPLC method developed and validated for acidic drugs and their degradation studies.


INTRODUCTION
High-performance liquid chromatography is also known as highpressure liquid chromatography is an advanced analytical technique that is used for separation, determination, identification, and quantification of active drugs, proteins and impurities [1][2]. HPLC is a type of column chromatography. In column chromatography, the sample is passed through a column with the help of gravity which takes more time for analysis of drugs but in HPLC sample is passed through the column under high pressure up to 400 atmospheres so that fast separation takes place [1][2]. HPLC consist of mobile phase reservoir system, pump, column and detector which records and shows separation result. In HPLC, the pump moves the mobile phase and sample towards the column. Column is the most important part of the HPLC system. In HPLC, column is made up of adsorbent material and most of the columns are made by using silica gel. The elution of the sample depends on the interaction of the sample with the column material and it also depends on the mobile phase. Mobile phase mainly consists of organic liquids such as methanol, acetonitrile and also consists of water. Sometimes buffers are also used as mobile phase. There are two types of elution: (1) HPLC isocratic elution: in which composition of the mobile phase is same during the whole analysis and (2) Gradient elution in which composition is changed during the analysis or separation of sample.

Instrumentation
The instrumentation of HPLC consists of mobile reservoir phase, pump, column, detector and recorder as shown in fig. 1.

Mobile phase reservoir
In HPLC, contents of the mobile phase are stored in a glass container. The mobile phase is a mixture of organic and aqueous liquids in a different proportion which mainly depends on the nature and solubility of drugs [1][2][3][4][5][6][7].

Introduction of acidic drugs
Most of the drugs used in medicines are either weak acids, weak bases or both weak acids and weak bases. pKa value of drugs gives information about the nature of drug whether it is acidic or basic in nature. pKa value gives information about the strength of acids and bases and also indicates the at which pH drug gets 50% ionized. The acidity of a drug depends on the functional group which is present in their structure. Functional groups such as carboxylic acid, amine, and phenolic groups are responsible for the acidic nature of drug. A number of commonly used drugs are carboxylic acid derivatives. These include NSAID-Aspirin, anticancer agent-Methotrexate, antibacterial agent-Amoxicillin, and the diuretic-Furosemide.
Amoxicillin is an antibacterial agent that belongs to the penicillin class. In amoxicillin, the β lactam ring is present and all penicillins are organic acids having various electron donor groups in their structure. In amoxicillin structure, carboxylate (COO-) and amino NH2 groups are electron donor and these groups are responsible for the acidic nature of amoxicillin ( fig. 2) [3].

Fig. 2: Chemical structure of amoxicillin
Methotrexate is an anticancer agent which is a folic acid antagonist and it also contains glutamate tail having a carboxylate group which is an electron donor group and hence methotrexate is acidic in nature ( fig. 3) [3].  Furosemide has a free carboxylic group in its structure due to which furosemide is strong acid than thiazide diuretics ( fig. 5) [3].

Forced degradation study for acyclovir
Acyclovir was subjected to stability indicating a study to check its stability in applied stress conditions. Acid, alkaline, oxidative and thermal hydrolysis stress conditions were applied to acyclovir.
From stability indicating studies, it showed that Acyclovir gets degraded in alkaline and oxidative hydrolysis. 9% drug gets degraded under the alkaline condition and 27% drug gets degraded under oxidative condition ( fig. 8-9) [4].

Forced degradation study for levodopa
Marketed drug formulation consisting of Levodopa, Carbidopa and Entacapone were subjected to stress conditions of acidic, alkaline, thermal and peroxide degradation. In acidic stress condition, Levodopa gets degraded ( fig. 12-14) [5]. A new, simple, rapid stability indicating RP-HPLC analytical method was developed for the simultaneous estimation of Flucloxacillin and Amoxicillin in bulk and pharmaceutical dosage form. The chromatographic separation for the two drugs was attained on a Thermosil C18 (4.6 mm x 250 mm, 5 µm) column as the stationary phase at ambient temperature. The mobile phase composed of potassium dihydrogen phosphate buffer (adjusted to pH 3 by using orthophosphoric acid) and Methanol (70:30%, v/v) in isocratic mode at a flow of 1 ml/min, and UV detection at 225 nm. The run time was 8 min ( fig. 15-16) [6].

Forced degradation study for folic acid
Marketed formulation of methotrexate and folic acid was subjected to stress conditions such as acidic, alkaline, oxidative, photolytic, reduction, thermal and hydrolysis conditions. Methotrexate and Folic acid are stable under applied stress conditions like thermal, acidic, photolytic stress conditions. No degradants were observed under stability indicating the study of Methotrexate and Folic acid [7].

Salicylic acid
New RP-HPLC stability indicating method was developed and validated for the determination of Salicylic acid in a pharmaceutical dosage form. Shimadzu Prominance L20 AD HPLC equipped with SPD 20A UV-Vis detector was used for the study of the analysis of salicylic acid in a pharmaceutical dosage form. RESTEX allure C18 column (4.6 mm x 12 mm, 3 µm) was used as the stationary phase. For isocratic elution, a mixture of water, methanol, and glacial acetic acid in a ratio of 65:35:1,v/v as mobile phase at a wavelength of 254 nm. The flow rate was 1 ml/min. The retention time was 7.65 min ( fig. 20-21) [8].

Forced degradation study for salicylic acid
Salicylic acid was subjected to stress conditions like alkaline, acidic, neutral and photolytic stress conditions. No degradation was found in alkaline, acidic, photolytic and neutral stress conditions [8].  . 22) [9].

Forced degradation study for furosemide
The marketed formulation of Spironolactone and Furosemide was subjected to acid, alkaline, thermal, oxidation and photolytic degradation. In acidic condition, 8.72% drug degraded ( fig. 23) [9].

Forced degradation study for Ibuprofen
The standard mixture of Paracetamol and Ibuprofen was subjected to stability indicating studies such as acidic, basic, reduction and oxidative stress conditions. During stability studies, it was observed that Ibuprofen was stable in acidic condition but it showed degradation in strong basic condition within the limit ( fig. 25) [10].

Forced degradation study for methotrexate
Marketed drug formulation of Methotrexate and Folic acid were subjected to stress conditions such as acidic, alkaline, oxidative, photolytic, reduction, thermal and hydrolysis conditions. The methotrexate and folic acid were stable under applied stress conditions like thermal, acidic, photolytic stress conditions. No degradants were observed under stability indicating studied of Methotrexate and Folic acid [11].  27) [12].

Forced degradation study for phenobarbitone
The working standard of Phenobarbitone was subjected to various stress conditions such as acid, base, oxidation, thermal, dry heat and photolytic stress conditions. From forced degradation studies it was found that Phenobarbitone gets degraded in acid, base, oxidative and thermal stress conditions ( fig. 28-31) [12].

Phenytoin
A simple, new and stability indicating method was developed and validated for the estimation of Phenytoin in a pharmaceutical dosage form. The separation was achieved by using Zorbax C18 (4.6 mm x 250 mm, 5 µm) column at ambient temperature and mobile phase contained acetonitrile and water in a ratio of 50:50%, v/v in isocratic mode. The detection was done at 200 nm and the flow rate was 1 ml/min ( fig. 32) [13].

Forced degradation study for phenytoin
Phenytoin subjected to stress conditions such as acid, base, oxidative and photolytic condition and from forced degradation studies it showed that Phenytoin gets degraded in oxidative and basic stress conditions ( fig. 33) [13].

CONCLUSION
High-Performance Liquid Chromatography has a wide variety of uses in many fields such as analysis, separation, and identification of pharmaceutical drug, impurity and biological samples. This article gives an overview of analysis and stability indicating method development and validation of acidic drugs.