FELODIPINE-REVIEW OF ANALYTICAL METHODS DEVELOPED FOR PHARMACEUTICAL DOSAGE FORMS AND BIOLOGICAL FLUIDS

Felodipine (FDP) is a vascular selective L-type calcium channel blocker, in hypertension patients FDP significantly lowers systolic and diastolic blood pressure (BP). It is a lipophilic drug molecule that contains a dihydropyridine ring responsible to show pharmacological activity, it is mainly used to control and prevent essential hypertension. This review article provides a summary of various analytical techniques for determining felodipine in pure form, pharmaceutical formulations, and biological fluids. Various analytical techniques are developed and validated, such as ultraviolet/visible spectrophotometry, high-performance liquid chromatography (HPLC), high-performance thin layer chromatography (HPTLC), and bioanalytical techniques. Estimated validation parameters such as linearity, LOD (Limit of Detection), and LOQ (Limit of Quantification) are discussed for each method. The wavelength of detection ( λmax) , mobile phase, columns, flow rate, retention time (Rt) and sample preparation techniques are all important quality elements for calculating Felodipine via analytical procedures.


INTRODUCTION
Hypertension is one of the main risk factors for atherosclerosis and other life-threatening cardiovascular diseases.Calcium channel blockers are categorised chemically into three groups: benzothiazepines, dihydropyridines, and phenylalkylamines [1].Chemically FDP is ethyl methyl (4RS)-4-(2,3-dichlorophenyl)-2,6dimethyl-1,4-dihydropyridine-3,5 dicarboxylate produces antihypertensive activity due to the presence of dihydropyridine ring [2].Belongs to the class of dihydropyridine and is chemically similar to nifedipine, nimodipine, nisoldipine, nicardipine and nitrendipine.FDP suppresses contractile responses to calcium in potassium-depolarized tissue in cardiac and smooth muscle at therapeutic doses.Contraction of cardiac muscles takes place by binding of calcium to calmodulin protein, which results in the activation of myosin light-chain kinase (MLCK), which causes heart muscle contraction.Myosin light chain is phosphorylated by activated MLCK, causing myosin head attachment to act in, which causes smooth muscle contraction and vasoconstriction.FDP acts by binding to calmodulin protein, hence prevents calcium-calmodulin interaction [3].Dilation of peripheral arterioles is the primary effect of FDP.In vitro researches revealed that selectivity is more for vascular smooth muscle than myocardial muscle when compared to nifedipine or verapamil [4].Felodipine does not cause orthostatic hypotension because it has no effect on venous smooth muscle in clinical doses [5].FDP also have natriuretic and diuretic property as it has direct action on tubular reabsorption and thus prevents retention of salt and water and hence lowers blood pressure and increased cardiac output [6].FDP does not appear to have a significant effect on glomerular filtration rate (GFR), creatinine clearance, glucose tolerance, or plasma lipoprotein concentrations in hypertensive patients [3].
Felodipine (FDP) belongs to the class of dihydropyridine calcium antagonist and it is lipophilic in nature.According to research, oncedaily use of an extended-release (ER) formulation is equivalent to twice-daily administration of conventional tablets in terms of antihypertensive efficacy.At the therapeutic dose, in patients with congestive heart failure (CHF), FDP seemed to have no negative inotropic effect but it might slightly increase myocardial contractility [3,4].FDP gets absorbed in GI tract rapidly and completely when given as an oral solution and reaches peak plasma concentration after 15-90 min (tmax) of administration.It Takes 1h to 2h when administered as plain tablet.It takes 3h to 5h to attain peak plasma concentration when administered as an extended-release tablet.Only about 15% of the drug reaches systemic circulation due to firstpass metabolism.FDP is highly distributed to extravascular tissue.FDP has a volume of distribution of about 10.3L/Kg, which signifies that less than 1% of the drug is concentrated in the blood.Plasma protein binding was found to be 99.64%[7].FDP gets metabolized in the liver by Cytochrome P-450-dependent oxidation to its pyridine analogue [8].Small amount of the drug gets excreted in the urine in its unchanged form.The elimination phase of FDP plasma drug concentration-time curve, which begins 8 to 10 h after administration, reflects the drug's elimination [9].FDP is an orally administered drug, available as extended-release tablets with the strength 2.5 mg 5 mg and 10 mg in the market.FDP can be estimated using a wide range of analytical techniques in formulations and biological samples.

Ultra-violet visible spectrophotometric technique
UV/vis spectrophotometry is a quick, easy, and sensitive approach for detecting and quantifying FDP based on UV absorption and chemical interactions.This method is cost-saving, accurate and precise for the routine analysis of the FDP in tablet dosage form.Table 1 represents the various spectrophotometric methods for determining and estimating FDP in pharmaceuticals, formulations, bulk pharmaceuticals as a whole and in combination with other drugs.

Spectrofluorimetric methods
The spectrofluorimetric methods are also used to estimate FDP in tablet dosage forms, in addition to the UV-visible spectrophotometric techniques.The spectrofluorimetric techniques are used because they are highly selective, sensitive, simple to operate, and cost-effective.Mohamed AM and his colleagues developed micelle-enhanced spectrofluorimetric techniques to determine FDP and Nimodipine in formulations and human plasma, and the sample was treated with 2% Tween-80 solution.Using tween-80, fluorescence intensity was measured at 423 nm after getting excited at 385 nm.In the range of 0.05-4.0g/ml, the standard fluorescence-concentration curve was found to be linear.For the reported linearity range LOD and LOQ were found to be 2-0.02µg/mland 2-0.05µg/ml respectively [10].Table 1 represents the spectrofluorimetric methods for determining and estimating FDP in pharmaceuticals, formulations and in biological matrix.

HPTLC
For the quantitative determination of felodipine in solid dosage form and in bulk, simple, precise, and sensitive HPTLC and RP-HPTLC methods have been developed and were validated as per ICH.These techniques can be used to analyse Felodipine in bulk and pharmaceutical preparations on a regular basis [21,22].Table 2 represents the HPTLC methods for determining and estimating FDP in pharmaceuticals, formulations.FDP: Felodipine, NA: Not available, RP: Reverse phase, NP: Normal phase

High-performance liquid chromatography
HPLC is a simple and sensitive method for estimating and measuring FDP in the presence of impurities, employing a simple mobile phase and minimal amounts of samples, and it has been validated in terms of accuracy, precision, stability, sensitivity, specificity, and robustness.

Ultra-performance liquid chromatography
Ultra-high-performance liquid chromatography (UPLC) is a more efficient and effective method for determining FDP.To identify and quantify FDP, its impurities, and degradation products, simple and accurate RP-UPLC methods are developed.Table 3 represents the HPLC/UPLC methods for the determination and estimation FDP in pharmaceutical, formulations.

Biological matrices
For the determination and quantification of FDP in biological matrices, various bioanalytical methods have been developed.Bioanalytical methods are useful for identifying and quantifying drugs and their metabolites in biological matrices, which helps in drug evaluation of bioequivalence, pharmacokinetics, and pharmacodynamic studies [23].Various analytical approaches have been developed, including hyphenated techniques for estimating FDP as a single entity and in combination, which requires less time.Table 4 represents various analytical methods for determining and estimating FDP in biological matrix (serum and plasma).

DISCUSSION
FDP is calcium channel antagonist [54,55] which belongs to the class of dihydropyridine anti-hypertensive agents, and it was approved by FDA in 1991.In vitro studies have revealed that FDP is highly vascular selective, and it does not cause orthostatic hypotension as they don't have any effect on venous smooth muscles in clinical dose.FDP is a lipophilic molecule that is soluble in methanol and is used as a solvent for UV-Visible spectroscopy to determine FDP concentrations in bulk and tablets.Spectrofluorometric method is highly selective, sensitive, simple to operate, cost-effective and doesn't require any derivatization.The developed HPTLC method is easy and inexpensive, and it can be used for routine analysis.The HPLC methods were used to determine FDP, and the results with the UV detector showed greater sensitivity, specificity, and accuracy.The separation is accomplished using a UPLC approach that does not require the use of an ion-pair reagent in the mobile phase.Gas chromatography methods for the determination of FDP is also developed where helium was used as carrier gas.To determine FDP and evaluate pharmacokinetic parameters and toxicological properties, several bioanalytical procedures like HPLC, UPLC, LC-MS/MS, LC-ESI-MS/MS, LC-Tandem MS have been developed.Biological matrices like plasma and serum as a single drug alone and also in association with other drugs have been used for the studies.All these methods utilized derivatization, protein precipitation, Liquid-Liquid extraction, and solid-phase extraction methods that are used for sample preparation in biological matrices.
Future developments in hyphenated methods may widen the path for analysing FDP in biological fluids and finished products, which could be more useful for FDP therapeutic monitoring.HPLC is the most extensively used analytical technique since it is cost-effective, has good sensitivity, and accurate and robust results are obtained hence it is used in routine analysis.

CONCLUSION
The present review examines the several analytical methodologies for estimating FDP that are available.For the estimation of FDP various analytical methods such as spectrophotometry, spectrofluorimetric, HPTLC, HPLC, UPLC are employed.HPLC, UPLC, LC-MS/MS, LC-ESI-MS/MS, LC-Tandem-MS, GCMS are the various bioanalytical techniques developed for the estimation of FDP in biological fluids.Since it is relatively easy, economical, and sensitive, HPLC is the most extensively used technique for the determination and quantification of FDP in bulk, formulations, and biological fluids.LC-MS/MS, LC-ESI-MS/MS, LC-Tandem-MS, GCMS are the hyphenated techniques for estimation of FDP.Analysts and skilled formulators should work together in the foreseeable future to develop more environmentally safe techniques for estimating FDP that use less toxic solvents.More HPLC approaches can help with FDP evaluation in biological fluids and bulk formulations.Further developments in UV spectrophotometric methods might help to estimate FDP in the future, as they are reliable and can be employed on regular basis.

Fig. 3 :
Fig. 3: An outlook of various analytical methods proposed for estimation of FDP

Table 1 : Spectrofluorimetric and spectrofluorometric methods for determining and estimating FDP in pharmaceuticals, formulations and in biological matrix
FDP: Felodipine, NA: Not available, MeOH: Methanol, ACN: Acetonitrile, HCl: Hydrochloric acid