INTRODUCTION:

Pioglitazone is a member of the class of thiazolidenediones that is 1,3-thiazolidine-2,4-dione substituted by a benzyl group at position 5 which in turn is substituted by a 2-(5-ethylpyridin-2-yl) ethoxy group at position 4 of the phenyl ring. It shows hypoglycemic activity. It has a role as an insulin-sensitizing drug, an EC 2.7.1.33 (pantothenate kinase) inhibitor and a xenobiotic. It is a member of thiazolidenediones, an aromatic ether and a member of pyridines¹.

Pioglitazone triggers the nuclear peroxisome proliferator activated receptor-γ (PPAR-γ), which assist to the increased transcription of various proteins regulating glucose and lipid metabolism^2-3. These proteins escalates the post-receptor actions of insulin in the liver and peripheral tissues, which assist to improved glycaemic control with no increase in the endogenous secretion of insulin^4-5.

The drug has been well tolerated by adult patients of all ages in clinical studies. Oedema has been reported with mono therapy, and pooled data have shown hypoglycaemia in 2 to 15% of patients after the addition of pioglitazone to sulphonylurea or insulin treatment. There have been no reports of hepatotoxicity⁶.

Pioglitazone is an orally administered insulin sensitizing thiazolidinedione agent that has been developed for the treatment of type 2 diabetes mellitus⁷.

Figure 1 Pioglitazone chemical structure

In multivariate technique it shows the change of solitary species analysis out-of one reliant changeable to a self-reliant changeable, since the wavelengths are concurrently comprise in the calibration model. Therefore estimation of species is feasible in the analytical system⁸.

In optimization state, the mathematical technique applied gives a significant resolution, sensitivity, rapidity and minimum cost for the quality analysis for concerning of mixtures. Statistical perspective are elaborated below.

Assuming that analyte (X) absorbance was calculated at five wavelengths stand (λ = 214, 216, 218, 220 and 222nm), subsequent equations are noted as follows to all wavelengths.

Aλ214 = f × C_x + k1…………………………………..(1)

Aλ216 = g × C_x + k2………………………………….(2)

Aλ218 = h × C_x + k3………………………………….(3)

Aλ220 = i × C_x + k4…………………………………..(4)

Aλ222 = j × C_x + k5…………………………………..(5)

Where, Aλ appear for climax zone of the analyte; f, g, h, i and j are slopes functions of linear regression of the analyte; k1, k2, k3, k4 and k5 are intercepts functions of linear regression at five chosen wavelengths and C_x appears for concentration of analyte. Equations shown above 1 to 5 can be noted as follows:

AT = f × C_x+ g × C_x+ h × C_x+ i × C_x+ j × C_x+ KT which can be simplified as……………………………(6)

AT = C_x (f + g + h + i + j) + KT……………………...(7)

Where, AT and KT appears for total climax zone gained and total of intercepts of regression equations at five-wavelengths stand individually. The concentration of analyte (X) in the solution of unspecified concentration can be measured using equation below:

C_x = ……………………………………….(8)

In case such as multivariate technique it contains the application of regression equations which is based on two variables i.e absorbance against concentration where it’s restored for the forecast of an unspecified concentration of analyte against the absorbance⁹.

So far in the literature for the estimation of Pioglitazone in biological samples and pharmaceutical formulations techniques like HPLC^9-13, HPTLC^14-19, RP-HPLC^20-29UV^30-45 spectroscopy, potentiometric^46-49and LC-MS/MS⁵⁰was useful for analysis, from which HPLC method have been mostly used. Multivariate method using UV spectrophotometer have been useful for determination of Pioglitazone in pharmaceutical dosage form. The main objective of this method was to evolve fast, simple, sensitive and reliable, multivariate technique. This method is used for quality analysis of Pioglitazone in pharmaceutical dosage form.

MATERIAL AND METHODS:

Chemicals and reagents

1. Sodium Hydroxide 0.1M (NaOH):

Solubility:

1. Sodium Hydroxide 0.1M: Freely Soluble

2. Water: practically insoluble

Instrumentation:

1. Perkin-Elmer UV-Visible spectrophotometer

2. Electrical balance

3. Sonicator

METHOD DEVELOPMENTS:

Selection of solvent:

The suitable solvent of Pioglitazone was found to be 0.1M NaOH. Therefore solvent 0.1M NaOH was chosen to solubilize the sample.

Standard solution preparation:

Standard solution has been formulated in which 100mg of Pioglitazone drug powder is dissolved into 100ml of 0.1MNaOH to get a concentration of 1mg/1mL.

Sample solution preparation:

Ten tablets of Pioglitazone was grinded using pastle with motar and weight equivalent to 100mg of the drug was transferred into 100ml of 0.1M NaOH to get concentration of (1mg/1mL). The solution was further processed for working concentration ranging from 8-16 µg/mL and scanned in UV spectrophotometry, where chart with absorbance against concentration was plotted to get linearity chart.

Estimation of λ_max: Standard solution of Pioglitazone was dissolved into 0.1M (NaOH) to make concentration of (10µg/mL) which have been examined in the region from 200-400nm using UV.

Figure. 2 UV Spectrum of Pioglitazone at 218 nm.

METHOD VALIDATION:

The technique was approved as per ICH Q2B guidelines for linearity, precision, accuracy and reproducible.

Linearity:

Standard solution of Pioglitazone was dissolved into 0.1M NaOH to make concentration in the range of 8-16µg/mL. Where absorbance was recorded over a range surrounding 218nm i.e., 214, 216, 218, 220 and 222nm, in orderly to enhance good correlation and reduce instrumental variations. A graph was constructed showing the absorbance of five separate concentration was noted at five separate wavelengths and curves were shown below.

Figure 3 UV Spectrum of Pioglitazone showing linearity at 218 nm.

The responsiveness of multivariate calibration was estimated in calculation of LOQ and LOD using the following formula.

LOQ = 10 S/σ and

LOD = 3.3 S/σ

Where, S represent concentration of standard deviation while

σ appears to be standard curve of the slope.

Precision:

10ml of standard solution of drug was arranged and dissolved into a conical flask containing 100ml of 0.1M NaOH as solvent. To these mixture, 1.2mL was taken using pipette and transferred into 10mL standard flasks, the volume is filled up with sodium hydroxide to make concentrations of 12µg/mL. The aliquots were scanned six times a day for intraday precision and six days at same time for interday precision at five wavelengths using UV spectroscopy.

Figure 4: UV Spectrum of Pioglitazone showing intra-day precision

Figure 5: UV Spectrum of Pioglitazone showing intraday precision

Accuracy:

The accuracy of multivariate technique have being estimated using standard addition method. An already arranged stock solutions of standard and sample, 10mL of the solutions was taken using pipette and dissolved into 100mL standard flask with solvent to make a concentration of 100μg/mL. 1mL of the standard solution were pipetted into three 10mL standard flasks and 0.8, 1.2, 1.6mL of the sample solution was poured to the above standard flasks and mixed. Standard flasks was filled with solvent. The aliquots were scanned using UV spectroscopy.

Figure 6: UV spectrum of pioglitazone showing accuracy

RESULTS AND DISCUSSION:

Pioglitazone λmax was found to be at 218nm with solvent 0.1M NaOH. The curves were found to be linear for the concentration ranging from 8-16μg/mL. Therefore the analytical data of linear regression for the curves shows great linear relations with R² = 0.9969. Equation of linear regression has shown Y = 0.0486x + 0.1856 while LOD as well as LOQ was originated in the range of 1.2765-3.8683 μg/mL and 2.490-3.045μg/mL. Finally the %RSD for the precisions was found to be 0.734-0.954, at all the five wavelengths, where according to ICH guidelines it is within the acceptance limits.

Linearity:

Five separate concentrations 8-16 μg/mL was measured at five different wavelengths, 214, 216, 218, 220 and 222nm, for linearity studies. The table was tabulated and shown below. Figure 7-16 shows calibration graphs and residual plots respectively.

TABLE I: UV calibration of five different wavelengths:

Conc. In (µg/ml)	(nm)
Conc. In (µg/ml)	214	216	218	220	222
8	0.336	0.398	0.434	0.587	0.539
10	0.515	0.609	0.684	0.735	0.814
12	0.564	0.684	0.768	0.867	0.958
14	0.496	0.626	0.742	0.867	0.972
16	0.439	0.612	0.790	0.868	0.986

Minimum value of standard deviation for five wavelengths shows the technique have being accurate, moreover calculations of LOD and LOQ was shown in table II below.

Table II: Showing linearity of LOD and LOQ for all wavelengths.

(Abs)	Equation of regression	R²	Standard deviation	LOD (µg/mL)	LOQ % (µg/mL) RSD
214	Y = 0.0257x + 0.0266	0.9985	0.0128	1.6436	4.9805	2.55
216	Y = 0.0371x + 0.2292	0.9939	0.0226	2.0102	6.0916	4.52
218	Y = 0.0486x + 0.1856	0.9969	0.0188	1.2765	3.8683	3.77
220	Y = 0.0597x + 0.0248	0.9982	0.0208	1.1497	3.4841	4.16
222	Y = 0.0654x + 0.0248	0.9989	0.0231	1.6559	3.5321	4.63

Precision:

The low standard deviation value for all wavelengths shows that the method was precise. The % RSD value for the intraday and interday precision was in the range of 0.734-0.954, therefore it is within the acceptance criteria of less than 3% for all wavelengths. Minimum value of the %RSD shows that the technique is precise.

Table III: Showing Intra-day precision:

*Day*	*(nm*)
	214	216	218	220	222
1	0.557	0.561	0.617	0.608	0.572
2	0.565	0.562	0.625	0.597	0.571
3	0.551	0.563	0.619	0.603	0.583
4	0.559	0.553	0.612	0.607	0.574
5	0.552	0.559	0.615	0.607	0.576
6	0.553	0.567	0.619	0.613	0.576
SD	0.0053	0.0047	0.0044	0.0054	0.0042
%RSD	0.9543	0.8319	0.7123	0.8883	0.7343

Table IV: Showing Inter-day precision:

Days	(nm)
	2% RSD 14	216	218	220	222
1	0.558	0.543	0.643	0.603	0.583
2	0.554	0.533	0.632	0.601	0.582
3	0.547	0.544	0.632	0.612	0.576
4	0.555	0.538	0.637	0.605	0.586
5	0.561	0.545	0.631	0.607	0.574
6	0.551	0.548	0.635	0.614	0.585
SD	0.0079	0.0077	0.0083	0.0084	0.0081
%RSD	0.9014	0.7989	0.7113	0.8410	0.8374

Table V: Showing study recovery:

(Abs)	Quantity available in (µg/ml)	Quantity added (µg/ml)	Absorbance	Quantity recovered (µg/ml)	% Recovery
214	8	0	0.329	7.671	95.89
		4	0.561	11.973	99.78
		8	0.939	15.761	98.51
216	8	0	0.386	7.614	95.18
		4	0.609	11.144	92.87
		8	0.984	15.641	97.76
218	8	0	0.434	7.565	94.56
		4	0.684	11.535	96.13
		8	0.968	15.577	97.36
220	8	0	0.487	7.548	94.35
		4	0.735	11.532	96.10
		8	0.967	15.547	97.17
222	8	0	0.439	7.571	94.35
		4	0.714	11.533	96.11
		8	0.958	15.581	97.38

Recovery:

Recovery percentage of the drug from synthetic compound have been in the range of 92.87 – 99.78 %w/w, it is within the acceptance limit 94-104%w/w according to ICH guidelines.

CONCLUSION:

The proposed UV spectrophotometric using multivariate technique was approved by assessing diverse approved parameters. Moreover the developed method does not require any complicated solvents and it is simple, sensitive, precise, accurate and reproducible for determination of Pioglitazone in pharmaceutical dosage form. The multivariate technique can be strongly suggested for quality analysis of Pioglitazone in pharmaceutical formulation.

ACKNOWLEDGEMENT:

Appreciations towards SRM Collage of Pharmacy and SRM Institute of Science and Technology in general for providing various sources and helping us to complete the research work by authors.

CONFLICTS OF INTEREST:

There is NO conflict of interest on the study reported by authors.

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Received on 10.02.2020 Modified on 19.04.2020

Research J. Pharm. and Tech. 2021; 14(1):14-20.

DOI: 10.5958/0974-360X.2021.00004.4