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0974-360X (Online)

RESEARCH ARTICLE

Validation of Isocratic RP-HPLC Method and UV Spectrophotometric Method for the Estimation of Loratadine in Pharmaceutical formulations

Samridhi, Sandeep Kumar Singh*

Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi- 835215, Jharkhand, India

*Corresponding Author E-mail: dr.sandeep_pharmaceutics@yahoo.com

ABSTRACT:

The objective of the present work was to develop an accurate, precise and linear Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method and UV-spectrophotometric method and to validate the methods as per ICH guidelines for the quantitative estimation of loratadine. The optimized method employed a C18 ODS column (150×4.6mm, 5µm), a mobile phase of 35:45:20 (v/v) mixture of acetonitrile, methanol and a phosphate buffer solution (0.01M, pH 7.2±0.1), flow rate of 1.0 mL/min and a detection wavelength of 245nm (UV detector). A simple, sensitive and reliable UV-spectrophotometric method has also been developed. The proposed methods of loratadine in methanol were found to be precise with retention time (RT) at 3.59 min (0.295% RSD) and absorption maxima at 247.0 nm (0.072% RSD). The optimized methods of loratadine in 0.1 N HCl (dissolution media) was also found to be precise with RT of 3.60 min and absorption maxima at 280.0 nm. Results of the linearity studies were statistically validated and accuracy was established by drug recovery within the acceptable limits of 98-102%. The limits of detection and quantitation were determined for both the analytical systems. These validated methods were employed for the determination of loratadine release from lipidic formulations and the model independent similarity approach was used to compare the dissolution profiles. The results obtained by either of these methods equally resulted in efficient estimation of drug. However, spectrophotometric method can also present a reliable and reasonably accurate alternative for the determination of loratadine in pharmaceutical formulations.

KEYWORDS: Loratadine, Reverse Phase- High Performance Liquid Chromatography, UV-Spectrophotometry, Method Validation.

INTRODUCTION:

Loratadine (ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6] cyclohepta [1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) is an orally effective, long-acting and non-sedating second generation H1-antihistaminic BCS class II drug being widely prescribed for allergic rhinitis and chronic urticaria. With the global rise in prevalence of allergic diseases, the safer second-generation antihistamines are being preferred owing to their favorable efficacy/safety ratio and having lipophilicity and ionization profiles that make it less able to cross the blood-brain barrier [1-3].

Received on 02.01.2015 Modified on 09.01.2015

Research J. Pharm. and Tech. 8(4): April, 2015; Page 452-461

DOI: 10.5958/0974-360X.2015.00076.1

Loratadine in addition to their antihistaminic properties has also been reported to exert in vitro growth-inhibitory effects on neoplastic mast cells [4]. Thus, in order to achieve desirable efficacy, proper analytical tools must be developed and validated for the estimation of loratadine in the pharmaceutical formulations. Since High Performance Liquid Chromatography (HPLC) is known to have excellent specificity and precision, its optimization for mobile phase composition and validation of the method was carried out for an accurate estimation of loratadine. In this study, we have focused on validating an optimized HPLC method by determining its specificity, precision, linearity, accuracy and limits of detection and quantification. Moreover, for the routine analyses of formulations, a simpler and cheaper UV-Vis Spectrophotometry method was also developed and validated. The analytical methods were developed in methanol as well as the dissolution medium of loratadine i.e. 0.1 N HCl as mentioned in the United States Pharmacopoeia [6]. The validation was performed according to the International Conference on Harmonization (ICH) guidelines for validation of analytical procedures (Q2A and Q2B) provided by the U.S. Department of Health and Human Services, Food and Drugs Administration (FDA) and the parameters were evaluated statistically [6, 7]. The application of the validated methods is hereby exemplified by the estimation of loratadine release from the lipidic formulations by both the methods and comparison of the same by model independent similarity approach given by the US-FDA [8].

MATERIALS AND METHODS:

MATERIALS:

Loratadine pure drug was a generously gifted by Cipla (Mumbai, India). HPLC grade methanol (MeOH), acetonitrile (ACN), orthophosphoric acid, water and potassium dihydrogen orthophosphate were obtained from Sigma Aldrich Ltd. (Mumbai, India). Nylon membrane filters (0.45µm) were purchased from Fischer Scientific. Medium chain caprylic mono and di-glycerides was gifted by Abitec Corporation (Janesville, USA) and polyoxyl 15 hydroxystearate gifted by BASF (Ludwigshafen, Germany). All other chemicals used were of analytical grade.

Instrumentation:

High Performance Liquid Chomatography (HPLC)

High Performance Liquid Chromatography (HPLC) analyses were performed using a quaternary gradient HPLC Knauer (Germany) with Smartline manager-5000 degasser and Smartline pump-1000 equipped with a UV detector. A Rheodyne injector with sample loop of 20µL, attached to the HPLC system was manually used for sample injections. A digital weighing balance (Mettler Toledo, Japan), a digital pH meter (Systronics, India), a bath sonicator (Bandelin, Germany) and a solvent filtration apparatus (Millipore, India) were employed in this study.

UV- Visible Spectrophotometer:

A double beam UV-Visible Spectrophotometer-1800 (Shimadzu, Japan) equipped with UV probe software (version 2.41) was employed for recording the absorption spectra. The absorption of the ultraviolet radiation by the molecule leading to transitions among the electronic energy levels of the molecule, is being expressed by absorbance (A) which is itself recorded as the spectrophotometer by the ratio between the reference beam and sample beam intensities [9]. The matched sample and reference cells made of quartz having path lengths of 1 cm each were employed in the study. The samples were scanned over the range of 200 nm to 400 nm to obtain the absorption maxima of the solute (λ_max). The absorbance of the test samples having varied concentrations were obtained at the wavelength of absorption maximum.

METHOD:

Chromatographic Conditions:

HPLC was performed on reverse phase Thermo Scientific® ODS C18 column (150×4.6mm, 5 µm) in isocratic mode, at 30 ºC. The liquid-phase chromatography of loratadine was carried out using methanol as solvent and also in dissolution medium. The optimized degassed mobile phase consisted of 35:45:20 (v/v) mixture of acetonitrile, methanol and a phosphate buffer solution (0.01M, pH 7.2±0.1, adjusted with dilute orthophosphoric acid). The flow rate was set to be 1.0 mL/min with an injection volume of 20µL.

Preparation of Mobile Phase:

The buffer solution was prepared by dissolving 1.3609 g of anhydrous potassium dihydrogen orthophosphate and 34.7 mL of 0.2M of sodium hydroxide, in 1.0 L Milli-Q water and the pH was adjusted to 7.2±0.1 using orthophosphoric acid. HPLC grade ACN and MeOH were mixed with the buffer solution in a ratio of 35:45:20 (v/v) and the mixture was bath sonicated (Bandelin, Germany) for about 15 min for proper mixing. The mobile phase solution was then filtered through 0.45 µm nylon membrane filter in a solvent filtration apparatus, followed by bath sonication for 15 min for proper mixing and for removal of the air bubbles, if any.

Preparation of Stock and Working Standard Solution:

Accurately weighed 10 mg of loratadine was dissolved in 10 mL of the solvent and sonicated for 5 min for proper mixing. This was considered as a stock solution of 1 mg/mL. One mL of the stock solution was pipette out and diluted upto 10 mL with the solvent to obtain a concentration of 100.0 mcg/mL. This solution was suitably diluted to achieve working standard solution for HPLC analysis (12.0 mcg/mL) and spectrophotometric analysis (10.0 mcg/mL). The various concentrations for construction of calibration curve and its contemplation, were made by suitable dilutions of the stock solution of the drug.

RESULTS AND DISCUSSIONS:

Method Validation:

Method validation demonstrates and establishes the suitability of the performance characteristics of the proposed analytical method for the determination of loratadine. The optimized RP-HPLC method and the developed UV-spectrophotometric method were validated for its selectivity, accuracy, precision, linearity, range, determination limit and quantitation limit according to the ICH guidelines provided by the U.S. FDA.

Selectivity and Specificity:

A reverse phase HPLC method was optimized for the analysis of loratadine and the mobile phase composition was selected for efficient elution and good resolution. Out of the various compositions explored, a ratio of 35:45:20 (v/v) of ACN, MeOH and buffer solution was found to be optimum for efficient elution of the analyte at a wavelength of 245.0 nm (UV detector) with a flow rate of 1 mL/min at room temperature. The optimized retention time was obtained at 3.59 min with a run time of 5 min. Specificity was determined to unequivocally assess the analyte in presence of expected components in order to ensure the identity of the analyte. This was achieved by recording the chromatograms for blank (control, methanol) and the standard solution (12 mcg/mL loratadine in methanol) in triplicate (Figure 1a and 1b). Solvent chromatogram revealed absence of any peak in and around the retention time of loratadine in methanol (3.59 min). An insignificant small peak at 1.78 min observed in the chromatogram of standard drug solution was also found to be present in the blank solvent chromatogram indicating the presence of solvent peak. However, no interactions between the blank and sample peaks were observed upon comparison of the chromatograms. This indicated that the method was selective and specific to the detection of loratadine, with no interference with the drug’s peak in methanol at the retention time of 3.59 min in the proposed mobile phase composition and chromatographic conditions. Moreover, the standard solution of the drug in 0.1N HCl reported the retention time of 3.60 min. Also, absence of any peak in blank (0.1N HCl) chromatogram adds to the specificity of the peak (Figure 1c and 1d). The absorption maxima of loratadine (10.0 mcg/mL) in distilled methanol and in 0.1N HCl was found to be 247.0 nm and 280.0 nm respectively. However, a broader peak obtained around 280.0 nm in 0.1N HCl and the solvent shift towards a longer wavelength is probably due to protonation where all molecular orbitals shift to lower energies, as also reported by other researchers [6, 10]. The precision, linearity, accuracy, detection and quantitation limits were further obtained for validation of these analytical methods.

Precision:

The precision of the system was investigated by determination of repeatability or intra-day. Repeatability was assessed by six determinations of standard drug concentration from the same homogeneous sample and under similar conditions over a short period of time. The precision was expressed statistically by the standard deviation (SD) and per cent relative standard deviation (% RSD). The parameters taken for the RP-HPLC method were retention time and the peak area while that for UV-spectrophotometric method were absorption maxima (λ_max) and absorbance as shown in Table 1. The analytical methods were found to be precise in both methanol and 0.1 N HCl as indicated by the SD falling well-within ± 5% of the mean and % RSD explicitly lying well below 2 % (Table 1).

Table 1: Results of system precision data for the RP-HPLC method and UV-spectrophotometric method developed for loratadine in methanol and dissolution media (0.1 N HCl).

System Precision
*RP-HPLC method*
	Methanol		0.1 N HCl
S. No	Retention Time (min)	Peak Area *(mAUmin)**	Retention Time (min)	*Peak Area (mAUmin)**
1	3.591	11.75351	3.595	17.91200
2	3.611	11.80114	3.595	18.36215
3	3.595	11.85235	3.594	18.28496
4	3.595	12.06998	3.595	17.81086
5	3.578	11.67823	3.611	17.98548
6	3.591	11.65399	3.611	17.85812
Mean	3.593	11.80153	3.600	18.03560
SD	0.011	0.151	0.008	0.232
% RSD	0.295	1.279	0.213	1.285
*UV-spectrophotometric method*
	Methanol		0.1 N HCl
S. No	Absorption maxima (nm)	Absorbance	Absorption maxima (nm)	Absorbance
1	246.8	0.378	280.2	0.236
2	247.2	0.376	281.0	0.234
3	247.0	0.381	280.8	0.234
4	246.8	0.377	278.8	0.235
5	247.2	0.378	279.6	0.237
6	247.0	0.378	280.0	0.238
Mean	247.0	0.378	280.06	0.236
SD	0.179	0.002	0.807	0.002
% RSD	0.072	0.443	0.288	0.693

(b)

(c)

(d)

(f)

(e)

(a)

Figure 1: Chromatograms of (a) methanol (blank), (b) loratadine in methanol, (c) 0.1 N HCl (blank), (d) loratadine in 0.1 N HCl. Figure 1(e) and 1(f) shows UV spectrum of loratadine in methanol and loratadine in 0.1 N HCl respectively.

Table 2: Linearity studies: Calibration data and results of regression analysis of loratadine in methanol and 0.1 N HCl by the RP-HPLC method.

Linearity studies: Calibration data by RP-HPLC method
*Methanol*
S. No.	Concentration (mcg/mL)	*Peak Area (mAUmin)**			*Mean Peak Area (mAUmin) (± SD)**	Regression Analysis
S. No.	Concentration (mcg/mL)	I	II	III	*Mean Peak Area (mAUmin) (± SD)**	Regression Analysis
1	4	4.74404	3.78262	4.70056	4.409073 (0.54296)	Best-fit values Slope : 1.0064 ± 0.005986 Intercept : 0.3281 ± 0.09325
2	8	9.37227	8.32868	7.45472	8.385223 (0.960025)
3	12	12.9708	11.83086	11.95351	12.25172 (0.625751)	95% Confidence Intervals Slope : 0.9897 to 1.023 Intercept : 0.06927 to 0.5870
4	16	17.66563	15.85278	16.0743	16.5309 (0.988924)
5	20	21.28635	19.97881	19.96124	20.4088 (0.760031)	Goodness of fit r² : 0.9999 p-value : < 0.0001
6	24	26.88692	23.69797	22.96641	24.5171 (2.084665)	Goodness of fit r² : 0.9999 p-value : < 0.0001
*0.1 N HCl*
S. No.	Concentration (mcg/mL)	*Peak Area (mAUmin)**			*Mean Peak Area (mAUmin) (± SD)**	Regression Analysis
S. No.	Concentration (mcg/mL)	I	II	III	*Mean Peak Area (mAUmin) (± SD)**	Regression Analysis
1	4	7.70300	6.52800	6.09305	6.77468 (0.832841)	Best-fit values Slope : 1.5503 ± 0.02072 Intercept : 0.02747 ± 0.3227
2	8	12.49899	12.28589	12.00289	12.26259 (0.248869)
3	12	17.91200	17.62300	18.22900	17.92133 (0.303108)	95% Confidence Intervals Slope : 1.506 to 1.594 Intercept: -0.6567 to 0.7117
4	16	24.28087	25.01106	24.81814	24.70336 (0.378386)
5	20	31.38799	31.62889	31.08808	31.36832 (0.270941)	Goodness of fit r² : 0.9972 p-value : < 0.0001
6	24	36.71896	37.47825	37.89775	37.36499 (0.597501)	Goodness of fit r² : 0.9972 p-value : < 0.0001

(a)

(b)

(c)

(d)

Figure 2: Linearity and contemplation plot by RP-HPLC method: Plots of standard curve of loratadine in (a) methanol, (b) 0.1 N HCl. Plots of actual concentration versus observed concentration in (c) methanol and (d) 0.1 N HCl.

Table 3: Linearity studies: Calibration data and results of regression analysis of loratadine in methanol and 0.1 N HCl by the UV-spectrophotometric method.

Linearity studies: Calibration data by UV-Spectrophotometric method
*Methanol*
S. No.	Concentration (mcg/mL)	Absorbance			Mean Absorbance (± SD)	Regression Analysis
S. No.	Concentration (mcg/mL)	I	II	III	Mean Absorbance (± SD)	Regression Analysis
1	0	0.000	0.000	0.000	0.000 (0.000)	Best-fit values Slope : 0.0368 ± 0.0002707 Intercept :0.00782 ± 0.004880
2	5	0.203	0.208	0.202	0.204 (0.003)
3	10	0.386	0.376	0.38	0.381(0.005)	95% Confidence Intervals Slope : 0.03625 to 0.03739 Intercept:-0.002392 to 0.01804
4	15	0.569	0.552	0.561	0.561 (0.009)
5	20	0.733	0.725	0.722	0.727 (0.006)
6	25	0.946	0.925	0.914	0.928 (0.016)	Goodness of fit r² : 0.9990 p-value : < 0.0001
7	30	1.133	1.119	1.109	1.120 (0.012)	Goodness of fit r² : 0.9990 p-value : < 0.0001
*0.1 N HCl*
S. No.	Concentration (mcg/mL)	Absorbance			Mean Absorbance (± SD)	Regression Analysis
S. No.	Concentration (mcg/mL)	I	II	III	Mean Absorbance (± SD)	Regression Analysis
1	0	0	0	0	0.000 (0.000)	Best-fit values Slope : 0.02374 ± 0.0001707 Intercept: -0.002405 ± 0.006154
2	10	0.238	0.237	0.237	0.237 (0.001)
3	20	0.473	0.47	0.474	0.472 (0.002)	95% Confidence Intervals Slope : 0.02338 to 0.02410 Intercept: -0.01529 to 0.01048
4	30	0.699	0.683	0.704	0.695 (0.011)
5	40	0.957	0.945	0.937	0.946 (0.010)
6	50	1.172	1.203	1.237	1.204 (0.033)	Goodness of fit r² : 0.9990 p-value : < 0.0001
7	60	1.418	1.407	1.415	1.413 (0.006)	Goodness of fit r² : 0.9990 p-value : < 0.0001

(a)

(b)

(c)

(d)

Figure 3: Linearity and contemplation plot by UV-spectrophotmetric method: Plots of standard curve of loratadine in (a) methanol, (b) 0.1 N HCl. Plots of actual concentration versus observed concentration in (c) methanol and (d) 0.1 N HCl.

The calibration curve was construed by plotting concentration (mcg/mL) versus mean absorbance. The linear relationship were statistically evaluated by regression analysis (method of least squares) to obtain best-fit values, goodness-of-fit and 95% confidence intervals with the help of GraphPad Prism software (version 5.01) (Keymaker-ZWT). The results reveal high degree of linearity in the RP-HPLC method in methanol with regression coefficient (r²) of 0.9999, which indicates that the concentration of the solute is well correlated to the peak area. A relatively lower yet significant linearity (r² 0.9972, p <0.0001) was also achieved in dissolution medium (0.1 N HCl) (Figure 2a and 2b). The linearity obtained in the UV-spectrophotometric method was sufficiently good, evident by the r² value of 0.9990, which being more than 0.995 fulfils the criteria for acceptability (Figure 3a and 3b). Moreover, the significance of the linear model which is dependent on the p value was also found to be < 0.05 in both the methods in both media, indicating a significant linear relationship (p value = <0.0001). This linearity indisputably determines the ability of the analytical procedure to obtain test results in the specified range.

Accuracy:

Accuracy of the optimized analytical methods were determined by the recovery experiments wherein % mean recovery is obtained by analyzing a known concentration of the analyte at a minimum of five concentration levels lying within the specified range [7]. Accuracy is affected by systematic errors and random errors and thus, three determinations being performed at each level to check reproducibility of the results. The observed concentration was calculated by utilizing the regression equation obtained by calibration plot. This reflection of the validation of calibration plot therefore may also be termed as contemplation of the method. The mean % recovery obtained by the RP-HPLC method was in the range of 99.46% to 101.35% and 98.9 % to 100.89% in methanol and 0.1 N HCl respectively with nearly 100% sample recovery being observed (Table 4). UV-spectrophotometric method reported mean recovery of 98.18% to 101.59% and 98.68 % to 101.6 % in methanol and 0.1 N HCl respectively, which also were found to be within the acceptable recovery limits of 98 % to 102% (Table 5).

Figure 4: Graphical representation of per cent loratadine release from lipidic formulations (F1 and F2) by the validated RP-HPLC method and UV-spectrophotometric method.

Table 4: Accuracy studies: Contemplation of the calibration curve in methanol and 0.1 N HCl by RP-HPLC method.

Accuracy studies: Contemplation of the method by RP-HPLC method
*Methanol*
S.No.	Actual Concentration (mcg/mL)	Peak Area *(mAUmin)**			Mean Observed Concentration (mcg/mL)	% Recovery
S.No.	Actual Concentration (mcg/mL)	I	II	III	Mean Observed Concentration (mcg/mL)	Mean	SD	% RSD
1	6.0	6.29447	6.40981	6.29654	6.333607	99.46	1.093	1.099
2	10.0	10.47427	10.4399	10.2737	10.39596	100.04	1.066	1.065
3	14.0	14.45163	14.2286	14.45635	14.37886	99.73	0.924	0.926
4	18.0	18.56502	18.3314	18.6101	18.50217	100.33	0.826	0.823
5	22.0	22.59827	23.0057	22.70136	22.76844	101.35	0.957	0.944
*0.1 N HCl*
S.No.	Actual Concentration (mcg/mL)	Peak Area *(mAUmin)**			Mean Observed Concentration (mcg/mL)	% Recovery
S.No.	Actual Concentration (mcg/mL)	I	II	III	Mean Observed Concentration (mcg/mL)	Mean	SD	% RSD
1	6.0	9.09814	9.32773	9.43211	5.972066	99.53	1.837	1.845
2	9.0	13.7896	13.64788	14.04275	8.901015	98.90	1.434	1.449
3	12.0	18.36215	18.8548	18.2496	11.90824	99.23	1.730	1.744
4	15.0	23.78766	22.95585	23.42638	15.06964	100.46	1.794	1.785
5	18.0	28.30381	27.97662	28.11452	18.1282	100.71	0.589	0.584
6	22.0	34.78581	33.50864	35.02163	22.19647	100.89	2.387	2.366

Table 5: Accuracy studies: Contemplation of the calibration curve in methanol and 0.1 N HCl by UV-spectrophotometric method.

Accuracy studies: Contemplation of the method by UV- Spectrophotometric method
*Methanol*
S.No.	Actual Concentration (mcg/mL)	Absorbance			Mean Observed Concentration (mcg/mL)	% Recovery
S.No.	Actual Concentration (mcg/mL)	I	II	III	Mean Observed Concentration (mcg/mL)	Mean	SD	% RSD
1	0.0	0.000	0.000	0.000	0.000	100.00	0.000	0.000
2	2.5	0.099	0.097	0.100	2.45438	98.18	1.656	1.687
3	7.5	0.279	0.289	0.281	7.44986	99.33	1.912	1.925
4	12.5	0.472	0.478	0.480	12.6983	101.59	0.903	0.889
5	17.5	0.646	0.635	0.650	17.224	98.42	1.203	1.222
6	22.5	0.815	0.843	0.816	22.1292	98.35	1.913	1.945
7	27.5	1.025	1.049	1.038	27.8925	101.42	1.184	1.167
*0.1 N HCl*
S.No.	Actual Concentration (mcg/mL)	Absorbance			Mean Observed Concentration (mcg/mL)	% Recovery
S.No.	Actual Concentration (mcg/mL)	I	II	III	Mean Observed Concentration (mcg/mL)	Mean	SD	% RSD
1	0	0.000	0.000	0.000	0.000	100.00	0.000	0.000
2	5	0.120	0.116	0.118	5.080	101.60	1.688	1.661
3	15	0.356	0.37	0.366	15.459	102.06	2.028	1.968
4	25	0.582	0.586	0.598	24.939	99.76	1.405	1.409
5	35	0.822	0.838	0.842	35.291	100.83	1.276	1.265
6	45	1.042	1.048	1.06	44.405	98.68	0.859	0.871
7	55	1.326	1.314	1.338	56.050	101.91	0.921	0.903

Detection Limit:

The detection limit as defined by the ICH guidelines is the lowest amount of analyte in the sample which can be detected but not necessarily quantitated. An approach towards the estimation of same, is done by determination of signal-to-noise ratio expressed by 3.3 times the ratio of standard deviation of response to the slope of calibration plot [7]. This detection limit, often known as limit of detection (LOD) is found to be 0.306 mcg/mL in methanol and 0.687 mcg/mL in 0.1 N HCl, for the optimized HPLC method in the specified linearity range. LOD was found to be 0.437 mcg/mL in methanol and 0.855 mcg/mL in 0.1 N HCl, for the UV-spectrophotometric method in the linearity range.

Quantitation Limit:

The lowest amount of loratadine concentration which can be quantitatively, precisely and accurately determined by the proposed RP-HPLC method in the specified linearity range was found to be 0.927 mcg/mL in methanol and 2.082 mcg/mL in 0.1 N HCl,. This was calculated based on signal-to-noise ratio of 10:1, expressed by 10 times the ratio of standard deviation of response to the slope of calibration plot [7]. The LOQ in the spectrophotometric was found to be 1.325 mcg/mL in methanol and 2.592 mcg/mL in 0.1 N HCl. This quantitation limit, also referred to as limit of quantitation (LOQ) is mostly used in determination of impurities and/ or degradation products.

Application of the Validated Methods for Estimation of Drug Release from Pharmaceutical Formulations:

The quantity of drug present in the pharmaceutical formulations can be determined by both chromatographic and spectrophotometric methods. Lipid based loratadine nanoparticulate formulations were prepared by incorporating medium chain caprylic mono and di-glycerides as oil phase and polyoxyl 15 hydroxystearate as the surfactant phase in similar manner as reported by us previously [11]. Varying proportions of excipients were explored to obtain a stable formulation. After various preliminary trials, two lipidic formulations (F1 and F2) were prepared, each containing 10 mg of loratadine. The oil and the surfactant phases taken in formulation F1 were 104.4 mg and 181.63 mg respectively. Formulation F2 was loaded with 126.58 mg of the oil and 197.98 mg of the surfactant. Each formulation was filled in hard gelatin capsules and immersed in 900 mL of 0.1 N HCl with the help of sinkers and rotated at 50 rpm at 37± 0.5ºC [6]. Samples were withdrawn at timed intervals of 5, 10, 15, 20, 30, 45, 60, 90 and 120 minutes and analyzed by both the in-house validated chromatographic and spectrophotometric method. The dissolution experiments were performed in triplicate, and data were expressed as mean value ± S.D. The percent drug released as a function of time was plotted using OriginPro software (version 9.0) (Microcal Software Inc., Northampton, MA) and is presented in Figure 4. The comparison of the dissolution profiles of the formulations by the RP-HPLC and UV-spectrophotometric method were done based on model independent approach using difference factor (f1) and similarity factor (f2), calculated as per the US-FDA guidelines for dissolution testing of immediate release solid oral dosage forms [8]. The difference factor (f1) which measures the relative error between the two curves and is calculated by the percent (%) difference between the two curves at each time point, was found to be 4.314 % and 6.55 % in case of F1 and F2 formulations respectively. The similarity factor (f2) representing the measure of similarity in percent dissolution between two curves was found to be 73.594 % and 65.299 % for F1 and F2 formulations respectively. Since f1 lies within 0-15 % and f2 being greater than 50% in both the formulations, the sameness or equivalence of the two curves was ensured (as per the guidelines). Thus, the dissolution profiles of loratadine release obtained from the validated RP-HPLC method and UV-spectrophotometric method were found to be similar, for both the formulations. However, more consistent and reproducible results were obtained when analyzed by RP-HPLC method as compared to the spectrophotometric results.

CONCLUSION:

A simple isocratic reverse phase high performance liquid chromatography (RP-HPLC) method was optimized for the determination of loratadine in methanol and in 0.1 N HCl (dissolution medium) and was validated according to ICH guidelines as exemplified by specificity, high degree of linearity, accuracy, precision, LOD and LOQ. The validation was also carried out for the developed UV-spectrophotometric method for estimation of loratadine in both media. The low % RSD of retention time, peak area, absorption maxima and absorbance revealed the precision of both the instrumental methods. Excellent linear relationship (r² of 0.9999, p value <0.0001) observed in the concentration range of 4mcg/mL to 24mcg/mL was also acknowledged by per cent mean recovery of 99.46% to 101.35% in methanol and 98.9 % to 100.89% in 0.1 N HCl, thereby demonstrating the accuracy of the RP-HPLC method.

The UV-spectrophotometric method developed for loratadine followed the Beer’s law in the range of 5mcg/mL to 30mcg/mL with the % mean recovery in the range of 98.18% to 101.59%. Conclusively, both the analytical methods were well validated for the determination of loratadine. The sensitivity of the methods was evident from the low detection and quantitation limits. The application of the validated methods for analysis of drug release revealed good correlation and similarity in the drug release profiles between the validated chromatographic and spectroscopic tools. Thus, although the RP-HPLC method is more accurate and reproducible, the developed and validated UV-spectrophotometric method can be used as a cheap and reliable alternative with good precision for the quantification of loratadine in the formulations.

ACKNOWLEDGEMENT:

The authors thank Vice-Chancellor, Birla Institute of Technology for providing the facilities. One of the authors (Samridhi) gratefully acknowledges the financial support in the form of INSPIRE-JRF (IF120784) provided by the Department of Science and Technology, Government of India (Ref. No. DST/INSPIRE fellowship/2012 dated 25 February 2013).

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