HPLC-UV method for simultaneous determination of irbesartan, candesartan, gliquidone and pioglitazone in formulations and in human serum      
Agha Zeeshan Mirza*   
Science and Technology Unit, Umm Al-Qura University, Makah 21955, KSA        

Abstract: In this study, we reported and validated a novel and sensitive reversed-phase liquid chromatographic method for the simultaneous determination of irbesartan, candesartan, gliquidone and pioglitazone. Separation was performed at 230 nm using a mobile phase consisting of methanol–water (90:10, v/v) with a flow rate of 1 mL/min. pH was adjusted to 3.5 with phosphoric acid. The concentration-response relationship was found linear over a concentration range of 5–25 μg/mL for all of the analytes tested. The limits of detection and quantification were 0.83 and 2.78 for irbesartan, 0.30 and 1.01 for candesartan, 1.11 and 3.93 for gliquidone, and 0.41 and 1.41 μg/mL for pioglitazone, respectively. Described method permitted the successful determination of these drugs in human serum. The developed method was simple, rapid, and it did not require extensive sample purification.                   
Keywords: Candesartan; Gliquidone; Irbesartan; Pioglitazone; RP-HPLC; Method development
CLC number: R917                Document code: A                 Article ID: 10031057(2018)427308

1. Introduction
According to the WHO, there are 347 million people worldwide, who have been diagnosed with diabetes[1], and more than 80% of diabetes-related deaths occur in developing countries[2]. In diabetic patients, a strong association has been found between hypertension and the adverse effects of diabetes, which is responsible for an increased risk of coronary activities. It has been observed that patients with both diabetes and hypertension are twice as likely to develop cardiovascular disease compared with non-diabetic people with hypertension[3].
In hypertensive patients with cardiovascular complications, angiotensin II receptor inhibitors are more effective[4] and preferred drugs to use in combination with anti-diabetic therapy[5]. The irbesartan (Fig. 1), a known angiotensin II type 1 receptor antagonist, prevents the effects of angiotensin II and improves metabolic parameters, and it is primarily recommended for treatment of hypertensive patients with type 2 diabetesmellitus[6]. Candesartan (Fig. 1), a benzimidazole, is an antihypertensive drug and used in the treatment of angina. It is selective to the angiotensin II receptor, significantly binds to the receptor[7] and lowers blood pressure levels[8].

Figure 1. Structures of irbesartan (A), candesartan (B), gliquidone (C) and pioglitazone (D).
Gliquidone (Fig. 1) is commonly used to treat type 2 diabetes mellitus[9]. It works on the sulfonylurea receptors by stimulating the release of insulin, and at pharmacological concentrations, gliquidone stimulates PPAR-γ activating properties and appears to be potent as pioglitazone[10].
Pioglitazone (Fig. 1), a PPAR-γ, is also used to treat type 2 diabetes mellitus. It lowers glucose levels and has potential cardiovascular benefits[11]. It is a commonly prescribed drug and also available in combination with sulfonylurea or metformin[12].
These are different classes of drugs that can be co-administrated. In the present study, we aimed to develop a simultaneous HPLC method for the determination of irbesartan, candesartan, gliquidone and pioglitazone in pharmaceuticals and in human serum utilizing simple, sensitive, and least time-consuming approach. Several HPLC methods have already been reported for the determination of these drugs using different detectors. Analysis of irbesartan has been conducted by using a fluorescence detector[13]; irbesartan and hydrochlorothiazide in plasma are identified using UV detector[14]; different sartans (losartan, irbesartan, valsartan, candesartan cilexetil) in the presence of candesartan metabolite are examined using fluorescence detection[15]; and irbesartan in human plasma and urine samples has been identified using fluorescence detector[16]. Recently, a stability-indicating HPLC-DAD method of irbesartan with simultaneous determination of atorvastatin and amlodipine has also been reported [17].
To the best of our knowledge, no simultaneous method for determination of gliquidone and pioglitazonewith irbesartan and candesartan in human serum has been reported. However, we have reported a method for the simultaneous determination of gliquidone and pioglitazone by UV spectrophotometer[18] and HPLC[19,2025]. Methods of simultaneous determination of pioglitazone with olmesartan[26], felodipine[27], metformin, glimepiride[28]and metformin, or glibenclamide and glimepride[29] have also been reported. In the present work, we validated the established method according to ICH[30] and USFDA guidelines[31]. The proposed method was successfully applied for the determination of these drugs.
2. Experimental
2.1. Materials
Purospher® column with a dimension of (5 µm, 25 cm×0.46cm) RP-18 end-capped was utilized. The Shimadzu HPLC (Japan) LC-10 AT VP pump and SPD-10 A VP UV/vis (Japan) detector were used in the experiment.
2.2. Reagents and chemicals
Irbesartan (Irecon® 75 mg) and candesartan (Treatan®4 mg) from Barrett Hodgson Pakistan (Private) Limited were purchased from the local pharmacy. Gliquidone and pioglitazone hydrochloride reference standard were kindly gifted by Pharmatec (Private) Limited and AGP (Private) Limited Karachi, respectively. Glurenor® and Poze® tablets were obtained from the local pharmacy. Analytical-grade orthophosphoric acid and HPLC-grade methanol were provided by Merck (Germany).
2.3. Preparation of standard solutions
Stock standard solutions of irbesartan, candesartan, gliquidone and pioglitazone (100 µg/mL each) were prepared by accurately weighing 10 mg of each of irbesartan, candesartan, gliquidone and pioglitazone. These chemicals were transferred to 100-mL measuring flasks and dissolved with methanol. These stock solutions were further diluted with methanol to obtain the working concentrations (525 μg/mL).
2.4. Procedure for commercial tablets
Concentration solution (100 µg/mL) of 20 tablets of each drug was prepared by dissolving the appropriate amount of each powder in methanol. All the sample solutions were sonicated, filtered and then chromatographed.
2.5. Procedure for human serum
Reported methods for drug serum analysis[20-22] and USFDA guidelines[31] were utilized. Three mL of blood samples of healthy volunteers were used and processed to separate plasma, and acetonitrile was then added. After vortexed for 1 min, the samples were processed and filtered using 0.45-micron membrane filter. The serum samples were mixed with irbesartan, candesartan, gliquidone and pioglitazone at desired concentrations as described above.
2.6. Chromatographic conditions
The mobile phase of methanolwater (90:10, v/v) was chosen, and phosphoric acid was used to adjust pH 3.5. At a flow rate of 1.0 mL/min, all analytes were detected at a wavelength of 230 nm (Fig. 2).

Figure 2.
Representative chromatogram of pioglitazone (1), irbesartan (2), candesartan (3) and gliquidone (4). 
3. Results and discussion
Hypertensive patients with diabetes are normally prescribed with antihypertensive drugs due to combination therapy. There is a requirement for the simultaneous analysis of these drugs in human serum using HPLC. The method validation characteristics of present methodwere determined according to ICH guidelines[30]. Linearity, accuracy, precision, limit of detection and quantitation, robustness and specificity were tested.
3.1. Calibration curves
Linear relationships were established by plotting graph between concentrations and area below the curve of a peak for irbesartan, candesartan, gliquidone and pioglitazone. The concentration ranges were found to be 525 μg/mL (n = 3) for each drug. The linearity of the method was performed using calibration curves (Fig. 3).The coefficient of determination obtained for irbesartan, candesartan, gliquidone and pioglitazone was 0.9965, 0.9973, 0.9966 and 0.9976, respectively (Table 1).

Figure 3. Linearity diagram of proposed method.

Table 1. Linear regression functions and their statistical parameters.
3.2. Precision and accuracy
Precision was evaluated through a repeatability test by calculating the relative standard deviation (% RSD). The % RSD values (Table 2) was determined by spiking 8, 10 and 12 μg/mL (n = 3) of every analyte. 

Table 2. Accuracy, precision and recovery of method.

. Limit of detection and quantification
The limits of detection and quantification were calculated according to ICH Q2B recommendations[30] and are summarized in Table 1. Using following equation, LOD and LOQ were determined.
Here σ represented standard deviation and S is the slope of the calibration curve.
3.4. Robustness
Robustness was studied by planned modifications in flow rate, wavelength and column temperature. The flow rate was altered by 0.1 units, from 0.9 to 1.1 mL/min, while a change of wavelength to (230±1) nm had no significant effect on results. Similarly, column temperature on the resolution was studied at 25 and 32 °C, and these changes had no impact on chromatographic performance (Table 3). 

Table 3. Robustness of the proposed method.

3.5. Specificity
In specificity studies, it was observed that common excipients (lactose, magnesium stearate and starch) did not affect the analytes (Table 2). The chromatogram (Fig. 2) indicated well-resolved peaks of irbesartan, candesartan, gliquidone and pioglitazone.
3.6. Assay of drugs in commercial tablets
The present method was successfully applied for irbesartan, candesartan, gliquidone, and pioglitazone in their tablets. The results (Table 4) indicated the suitability of the method for routine analysis of irbesartan, candesartan, gliquidone and pioglitazone from their drug products.

Table 4. Recovery of irbesartan, candesartan, gliquidone and pioglitazone incommercial tablets.

3.7. Application of the method
The study was conducted in accordance with guidelines of the US Food and Drug Administration (USFDA)[31]. The current method had an advantage over previously developed HPLC procedures using liquid-liquid extractionfrom plasma samples for the determination of irbesartan,  candesartan, gliquidone and pioglitazone, showing efficient recovery values (Table 5, Fig. 4). 
Table 5. Recovery of irbesartan, candesartan, gliquidone and pioglitazone in human serum.

Figure 4. Representative chromatogram of pioglitazone (1), irbesartan (2), candesartan (3) and gliquidone (4) in human serum.
4. Conclusions
An efficient method for the determination of irbesartan,candesartan, gliquidone and pioglitazone in formulations and human serum was established in the present study, and no interference of excipients was found. No additional extraction procedures were required, and results obtained were in accordance with the labeled claim of formulations. The proposed methods could be used for determination of irbesartan, candesartan, gliquidone and pioglitazone in clinical samples.
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Received: 2018-01-05, Revised: 2018-02-23, Accepted: 2018-03-11.
*Corresponding author. Tel.: +92-334-367-1246; Fax: +92-9713131, E-mail:dr.zeeshan80@gmail.com     
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