ANALYTICAL METHODS FOR THE QUANTITATION OF AMLODIPINE BESYLATE AND ATORVASTATIN CALCIUM IN PHARMACEUTICAL DOSAGE FORM AND BIOLOGICAL FLUIDS

Amlodipine is the best-prescribed medication for cardiovascular disease major risk factor for hypertension and atorvastatin well known for diabetic. First discussed low cost ultraviolet-visible technique for the determination and quantitation of drugs in pharmaceuticals and biological fluids. Chromatographic techniques have an application with respect to trace analysis. Different types of chromatography such as high-performance liquid chromatography, high performance thin layer chromatography have most frequent applications in the field of pharmaceutical as well as biomedical analyses. Chromatography combined with mass spectrophotometry has the ability to collect molecular ion, followed to prepare a spectrum to assess molecular weight as well as structure. High-performance liquid chromatography coupled with mass spectrophotometry is a reliable and dynamic technique for the analysis of small and large drugs molecule. The advantages and disadvantages of all techniques are compared with each other with respect to sensitivity, reproducibility and other important parameters. The investigation also focused for the quantitation on both drugs in pharmaceutical preparations and plasma samples with the help of all available analytical techniques.


INTRODUCTION
Amlodipine besylate ( fig. 1a) is scientifically known as (RS)-3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro -6-methyl-3,5-pyridinedicarboxylate benzene sulfonate. It was first introduced and prescribed for coronary artery disease. It was also helpful for angina and peripheral artery disease [1]. Now days mostly targeted for the patient having hypertension. The European Society of Cardiology was conducted survey until the year 2000 and their report based on statistical analysis showed nine hundred seventy-two million people were in this category. The number will increase with time and expected by the year 2025, approximately 1.56 billion [2]. This drug is under the umbrella of calcium channel blocker. The mechanism of such blockers to control the transportation of calcium to coronary (mainly smooth muscle) and arteries, that reflects on muscles became relaxes, reduces peripheral resistance and ultimately lowering the blood pressure [3]. Atorvastatin ( fig. 1b) is chemically known as (βR, δR)-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-lH-pyrrole-1heptanoic acid. The synthetic drug, atorvastatin is generally under the class known statins. These reductase inhibitors are familiar as 3-hydroxyl-3methylglutaryl-coenzyme A (HMG-CoA). All drugs are prescribed for cardiovascular disease and reduction of heart attack, as well as for clinical demand [4][5][6][7][8][9][10]. Active pharmaceutical ingredients (APIs) such as amlodipine are commonly manufactured as their acid addition salts to promote solubility and improve both stability and bioavailability. The objective of this review article was to research more about ultraviolet-visible and chromatographic technique's applications, specifically for the quantification of amlodipine and atorvastatin in bulk, pharmaceutical formulations, and biological fluids. Compared the result between all the developed method for amlodipine and atorvastatin about the limit of detection and quantitation value. Discussed briefly the importance of all analytical techniques with respect to cost, time of analysis, sensitivity and limitations.

UV-visible spectrophotometer
Spectroscopic methods are mostly used for the determination of drugs in bulk as well as with pharmaceutical formulations. Ultraviolet (UV) and visible spectrophotometry are important and common techniques for the quantitative analysis of drugs. Because these are low-cost techniques, simple and no requirement of pretreatment as well as any elaborate preparatory step prior to assay. The visible spectrophotometer is depending on the redox and complex formation reaction. However, some deficiencies are also with the techniques with respect to the 6 presence of two or more drugs have similar UV characteristic. The detailed literature survey studies elaborate on the advantages and disadvantages among all developed method for both drugs using UVvisible spectrophotometer  are presented in table 1.

High performance liquid chromatography
High-performance liquid chromatography (HPLC) technique is more accurate and based on the properties of the analyte with the existing mobile phase and stationary phase. Depending on the stationary and mobile phase, different types of chromatography techniques are developed. Recently high-performance liquid chromatography has achieved lots of attention in the field of pharmaceutical analysis in dosage forms and biological fluids because of its simplicity, sensitivity and high specificity. The conventional rule for the analysis of polar compounds by utilizing a non-polar stationary phase with the polar mobile phase and vice versa for the non-polar compounds. Stationary phase binding the analyte is directly proportional with the surface area of the nonpolar segment of the analyte associate the ligand as well as with aqueous eluent. One of the main parameters found as evidence about the quality and efficacy of drug products and formulations is stability testing. The products nature is changing with humidity, temperature, light, retesting time, storage conditions, shelf life, so it is necessary to control the environmental factors. To develop method different types of the column (ODS C 18 , BDS C8, ODS C8, BDS C18 and Discovery HS C18 The ultra-performance liquid chromatography (UPLC) is a new and modern technique for liquid chromatography. Worldwide HPLC was a predominant technique for the last 30 to 40 y for the drug analysis. Speed, sensitivity, and resolution are the main keywords for the drugs analysis with UPLC compare to HPLC. The particle size can play a significant role in this type of chromatography that governed by the well-known Van Deemter equation. For UPLC, the preferred size diameter is less than 2μm to get more sensitivity, short analysis time and improved resolution. The quality of the product analyzed by UPLC will give better with less time. However, the main disadvantage is related to the column life. The analysis requiring high pressure (100 M Pa) that damage the column efficiency. The novel and selective methods were developed for the determination of both drugs with a marketed formulation as single or combined dosage form [84][85][86][87][88][89][90][91][92][93][94][95][96][97][98] (table 3).

Thin layer chromatography
) with a combination of different mobile phases containing buffers, organic modifier (acetonitrile, methanol) can be used. Amlodipine and atorvastatin were quantified using HPLC with ultraviolet in bulk and pharmaceutical dosage form   (table 2).

Ultra pressure liquid chromatography
Thin-layer chromatography (TLC) is a simple separation technique typically for the mixture of nonvolatile compounds. The adsorbent material, specifically cellulose, silica gel, aluminum oxide is covered on plastic or glass sheet, known as the stationary phase. Aleppo bentonite with modified phenyl support can also be applied as the stationary phase, has the ability to separate amlodipine and atorvastatin with a mobile phase consisting of sodium phosphate buffer and acetonitrile (50:45, v/v) in pharmaceutical dosage form [99]. Methanol, toluene triethylamine combination and silica gel adsorbent distinct atorvastatin in pharmaceutical formulation with high resolution [100]. However, triethylamine can be replaced with chloroform and acetic acid in tablet dosage form on the aluminum plate [101]. However, propanol and water system (70:30, v/v) has the capability to separate amlodipine with a detection limit of 0.4 µg [102].

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Bulk drug and tablets [83] High-performance thin layer chromatography High-performance thin layer chromatography (HPTLC) is an accelerated separation technique and flexible enough to determine the drug sample. The advantages associated with HPTLC are a short analysis time for the complex sample without pretreatment, independent construction of chromatogram with multiple samples, easy to transfer samples that will increase the confidence and reliability of the technique. It can be employed for qualitative and quantitative purposes. Several combinations of mobile phases with different size of plates have been successfully studied by HPTLC in pharmaceutical preparations [103][104][105][106][107][108][109][110][111][112][113][114][115] (table 4).

Capillary electrophoresis
Capillary electrophoresis (CE) is based on charge and size under electric field separate molecules. The capillary tube is made of glass loaded with an electrolyte solution. Electrophoretic mobility is an important parameter for the separation, chemical constituents as well as solvent viscosity. However, the limitation with gel electrophoresis with regards to applied voltage due to ohmic heating damage the gels and restrict the separation. Need to require large voltage often 10-20 thousand for experiments with CE. Different types of capillary electrophoresis are capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic capillary chromatography (MEKC), capillary electro chromatography (CEC), capillary isoelectric focusing (CIEF) and capillary isotachophoresis (CITP).
Phosphate buffer (pH 6.5) and methanol mixture combined (80:20, v/v) used as background electrolyte with capillary fused silica column under 15 KV voltage at room temperature for the determination amlodipine and atorvastatin [116]. In tablet formulation no interference from present common excipients [117]. Phosphate buffer can separate both drugs in 5 min with high precision but in the presence of acidic products need to involve borate buffer [118][119]. It is likewise possible to quantify within 3 min in combined dosage form with high efficiency and resolution [120]. CZE method validated for solid dosage form in the presence of electrolyte as a methanol borate buffer [121]. MEKC succeeded by controlling surfactant, sodium dodecyl sulfate (SDS) concentration and acquired within 2 min [122] compared to CZE [117] 13 min migration time having comparable resolution. High and ultra-pressure liquid chromatography combined with mass spectrophotometry High pressure and ultra-performance liquid chromatography with the mass spectrometry (LC-MS, UPLC-MS) are analytical techniques that combine liquid chromatography with mass spectrophotometer. The aim to develop a fast and reliable analytical method for the determination of atorvastatin and amlodipine together with the presence of metabolites using the above techniques. The MS combined with LC has high selectivity and sensitivity. The techniques are commonly applied for bioavailability and pharmacokinetics investigation. Mostly quantification of parent drug, active and inactive metabolites are of interest for various studies with biological fluids. It is also required to verify the interaction between drug to drug and side effects as well as the toxicity of different metabolites after metabolism in the human body. The LCMS [123][124][125][126][127][128][129][130][131][132][133][134][135][136] (table 5) and UPLC-MS [137][138][139][140][141][142] (table 6) were used for the determination of amlodipine, atorvastatin and their metabolites in pharmaceutical dosage forms and biological fluids.

DISCUSSION
The UV-visible spectrophotometer does not require any pH adjustment and very close to the pharmacopeia method for both drugs. Recently HPTLC can be applied as an alternative option for traditional TLC method. It is easy to handle with software, which is not possible with TLC. HPTLC enhanced the capability to determine impurity with the help of the hydrophilic phase combined silica gel, an important parameter in all pharmacopoeias. One single run is enough to achieve two parameters such quantity and its impurity by HPLC, main pharmacopeias method to quantify the assay percentage. The setting of parameters for the reaction is not easy as well as for characterization with CE that is why maybe not recommended in official method. UPLC is a high-cost instrument compare with other chromatographic methods, ability to study pharmacokinetics such as adsorption, metabolism. The limit of detection (LOD) and limit of quantitation (LOQ) value is very less with chromatographic method combined with mass spectro-photometry (table 7). Amlodipine and atorvastatin concentration in pharmaceutical formulations and biological fluids are based on different parameters and the calibration curve of the analyte (table  8). During method development and validation, accurately quantified the analyte as well provide brief information related to impurity profiling, a key equipment for the formulation process of the drug.     Simple operation procedure, fast, cost effective, accurate in readings and used widely

CONCLUSION
The main objective of this review is to provide sufficient information for the researcher, academic, scientist about the analysis of important calcium channel and statins receptor. After going through with the proposed review, people can understand the common method for the analysis of drugs. These medications are very significant due to its growing demands in our daily life that is why it was one of the fastest growing products in the pharmaceutical industry. The ultraviolet and visible spectrophotometer are low-cost instrument as well as easy to handle and available everywhere. It is more suitable for pure pharmaceutical formulations. The biological fluids and trace analysis will go for highly sensitive instrument combined with the mass spectrophotometer. Therefore, the present review will give an idea for determination and quantification of both drugs using UV-visible, HPLC, HPTLC, UPLC, LCMS and UPLC-MS in bulk, pharmaceutical formulations, and biological fluids.

AUTHORS CONTRIBUTIONS
All the author have contributed equally

CONFLICT OF INTERESTS
The authors report no conflicts of interest