Isocratic HPLC Method for Simultaneous Determination of Amlodipine and Xipamide in Human Plasma

In this analytical study, two antihypertensive drugs are subjected to such analysis using chromatographic chemistry. One of them is amlodipine (AML), which is chemically, 3 -ethyl 5-methyl (4RS)-2-[(2-aminoethoxy) methyl]-4 -(2-chlorophenyl)-6-methyl1,4-dihydropyridine (Figure 1). Amlodipine is a long-acting calcium channel blocker (dihydropyridine derivative) and it is mainly used as an anti-hypertensive and anti-anginal drug [2]. The other drug is xipamide (XIP) which is a sulfonamide diuretic drug. Chemically, It is 4 -chloro5 -sulphamoylsalicylo2′,6′ -xylidide; (Figure 1) and it is used for the treatment of high blood pressure and edema of cardiac, hepatic, or renal origin. It is a nonthiazide diuretic with a greater natriuretic effect Abstract


Introduction
The approach of separation chemistry science is signifi cantly growing and is representing a major challenge for scientists in general, and chemists in particular, to fi nd a compromise between developing or analyzing new products and achieving economical and environmental objectives due to an increased recognition of environmental safety and continuous industrial ecology all over the world [1].
In this analytical study, two antihypertensive drugs are subjected to such analysis using chromatographic chemistry.
It is a nonthiazide diuretic with a greater natriuretic effect Abstract An HPLC method had been developed and validated for rapid simultaneous separation and determination of the two antihypertensive drugs, amlodipine and xipamide in human plasma within 5 minutes. Drugs are extracted from plasma using methanol, the environmentally benign solvent, for protein precipitation technique. Separation was carried out on a Thermo Scientifi c ® BDS Hypersil C 8 column (5μm, 2.50x4.60 mm) using a mobile phase of methanol: 0.025M KH 2 PO 4 (70: 30, v/v) adjusted to pH 3.49 with ortho -phosphoric acid at ambient temperature. The fl ow rate was 1 ml/min and maximum absorption was measured using DAD detector at 235nm. The retention times of amlodipine and xipamide were recorded to be 4.51 and 3.38 minutes, respectively, indicating a shorter analysis time. Limits of detection were reported to be 0.17 and 0.25 μg/ml for amlodipine and xipamide, respectively, showing a high degree of the method sensitivity. The method was then validated according to FDA guidelines for the determination of the drugs clinically in human plasma specially regarding pharmacokinetic and bioequivalence studies. than the thiazides with a less abrupt onset and longer duration of action [3]. Combination of both drugs is synergistic for hypertensive drugs and many medical prescriptions include combinations of both drugs in addition to other -blockers and ACE inhibitors specially for congestive heart failure (CHF) patients.
To the best of our knowledge and comprehensive survey,

Materials and reagents
All solvents and reagents were of an HPLC analytical grade The human plasma was kindly provided by Zagazig University Hospital and was tested to be drug and disease free. Plasma was kept frozen before use, and was then stored either at -4º C between uses or at -20º C for freeze-thaw cycle stability studies.

Optimization of Chromatographic Conditions
All chromatographic conditions are illustrated in Table 1.
Spectroscopic analysis of the drugs in the range of 200-400 nm showed that amlodipine and xipamide have UV absorbance maxima ( max ) at 237 nm and 231 nm, respectively, as depicted in Figure 2. Therefore, the chromatographic detection was performed at 235 nm as the appropriate wavelength using a DAD detector. The method was performed on a Thermo Scientifi c ® BDS Hypersil C 8 column (5μm, 2.50×4.60 mm) Table 1.
Furthermore, under several trials of mobile phase

Method Validation
The method was validated according to food and drug administration guidelines for bioanalytical methods validation [27,28].    Table 1. these peaks were not obtained for the blank solution. Also, the specifi city studies revealed that the presence of human plasma didn't show any kind of interference with the sharp and wellresolved peaks of amlodipine and xipamide (Figure 4).

Robustness:
The robustness of the methods was evaluated by making deliberate subtle changes (± 0.05) in the fl ow rate, pH of mobile phase and mobile phase composition ratio keeping the other chromatographic conditions constant. The changes effect was studied on the basis of percent recovery and standard deviation of both drugs. Table 4 shows that the changes had negligible infl uences on the results as revealed by mean recoveries in the acceptable range (99.5-101.38) and small SD values (≤1.92) for both drugs Table 4.

Limits of detection and limits of quantifi cation:
For determining the limits of detection and quantitation, the method based on signal-to-noise ratio (3:1 for LOD & 10:1 for LOQ) was adopted. Limits of detection were reported to be 0.17 and 0.25 μg/ml, while limits of quantifi cation were calculated to be 0.58 and 0.82 for amlodipine and xipamide, respectively (Table 3) showing that the proposed method is highly sensitive and applicable for pharmacokinetic and bioequivalence studies where detection of small concentrations in plasma is required.

Analysis of human plasma:
The proposed method was also applied for determination of amlodipine and xipamide in human plasma samples using protein preciptation procedure.
Retention times of amlodipine and xipamide in plasma samples and the other system suitability parameters were also pretty similar to those in pure forms. The calibration curves in the spiked plasma were also found to be linear over the clinical range of 2.50-15μg/ml for amlodipine and 0.50-5μg/ml for xipamide (Table 5). According to Clark´s Analysis book [29],   Table 1.    (Table 6) showing that the method is highly precise in plasma samples. Also, Stability studies were conducted by applying plasma freeze-thaw cycles at -20 0 C (over three days) using the same validation QC samples in plasma and results are summarized in Table 6.
These results show that the proposed method has an adequate precision and stability for simultaneous determination of both drugs in plasma Tables 5, 6.

Conclusion
The developed analytical method represents a kind of chromatographic chemistry for rapid and simultaneous estimation of amlodipine and xipamide within 5 minutes.
The results obtained indicate that the proposed method is rapid, sensitive, accurate, selective, robust and reproducible.
Protein precipitation was carried out for drug extraction from plasma using methanol instead of liquid liquid extraction.