INTRODUCTION:

Chemically, DAPA (Figure 1) is (1S)-1, 5anhydro-1C {4-chloro-3-[(4-ethoxyphenyl) methyl] phenyl-D-glucitol ¹. Whereas, SAXA (Figure 2), chemically is, (1S,3S,5S)-2-[(2S)-2-Amino-2(3-hydroxyadamantan-1-yl) acetyl]-2-azabicyclo hexane-3-carbonitrile².

Figure 1: Chemical Structure of DAPA

Figure 2: Chemical Structure of SAXA

Literature survey revealed several analytical methods for the estimation for simultaneous estimation of DAPA and SAXA^3-7. The aim of the present work was to develop a simple, accurate, precise and specific RP-HPLC method for the simultaneous estimation of DAPA and SAXA in their combined dosage form and to validate the method with respect to specificity, accuracy, precision, linearity and range.

MATERIALS AND METHODS:

Chemical and Reagents:

Pharmaceutical grade DAPA and SAXA were obtained as a gift samples from Macleods Pharmaceuticals Ltd., Vapi, Gujrat and Cipla Pharmaceuticals Ltd., Mumbai, Maharashtra, respectively. Methanol used in analysis was of HPLC grade and potassium di-hydrogen ortho-phosphate and ortho-phosphoric acid used were of analytical reagent grade. All chemicals were purchased from S D Fine Chemicals, Mumbai, Maharashtra. Tablets containing 10 mg and SAXA 5 mg were prepared in house.

Instrumentation and Chromatographic Conditions:

Binary High-Pressure Gradient HPLC system used in analysis consists of two high pressure gradient pumps (PU-2080 plus) equipped with multichannel UV-Vis detector (UV-2077), purchased from JASCO Corporation, Japan. The sample introduction was performed with 20 mL sample loop injector (Rheodyne 7725i). The data acquisition was done on Borwin software (ver. 1.50).

Other equipment used in analysis consists of digital analytical balance (AUX-220, Shimadzu), Ultrasonic Bath (PCI Analytical Pvt. Ltd., Mumbai, Maharashtra) and pH meter (HANA Instruments, Mumbai, Maharashtra).

All separations were performed on Phenomenex Hyperchlone C18 column (250×4.6 mm, 5m) at ambient temperature using mobile phase consisting of methanol: 20 mM potassium phosphate buffer (pH 3.0) in proportion of (70: 30 %, v/v) in isocratic mode at flow rate of 1 mL/min. The detection was performed at optimum wavelength of 225 nm.

EXPERIMENTAL:

Preparation of Standard Stock Solution:

Standard stock solutions were prepared by dissolving accurately weighed 10 mg of DAPA and 10 mg of SAXA into separate clean and dry 10 mL volumetric flask, in each flask, 8mL of methanol was added, sonicated for 5 min and volume was made up to the 10 mL with the same solvent. The resulting solutions were of 1000µg/mL of DAPA and SAXA, respectively.

Calibration Curve (CC) standards:

From the above prepared stock solutions, six dilutions were prepared and diluted with mobile phase to obtain CC standards with concentrations of 4, 8, 12, 16, 20 and 24 mg/mL of DAPA and 2, 4, 6, 8, 10 and 12 mg/mL of SAXA. Each CC standard was injected in triplicate and the peak area was plotted against the corresponding drug concentration. The straight-line equation was obtained and the regression coefficient (r²) were determined for DAPA and SAXA, respectively.

Analysis of DAPA and SAXA in tablets:

Twenty tablets containing DAPA and SAXA (10mg and 5mg, respectively) were weighed and crushed into fine powder. Powder equivalent to weight of one tablet was weighed and dissolved in 100 mL methanol in a volumetric flask, and sonicated for 15 min. From the resulting solution, suitable aliquot was pipette out and transferred into a 10mL volumetric flask and the volume was made with mobile phase to obtain 20µg/mL of DAPA and 10µg/mL of SAXA, respectively. The prepared dilution was subjected to chromatographic analysis under mentioned conditions in triplicate and the corresponding concentrations were determined using the straight-line equation of DAPA and SAXA obtained in calibration curve experiments.

METHOD VALIDATION:

The developed method was validated as per ICH guidelines Q2(R1)⁸ with respect to specificity, accuracy, precision, linearity and range.

Specificity:

The specificity of the proposed method was established by the complete separation and resolution of DAPA and SAXA from its interfering excipients, if any. Blank tablets were chromatographed. The absence of peaks in the blank runs at the retention time of DAPA and/or SAXA was considered as the indication of specificity.

Accuracy and Precision:

The accuracy and precision were evaluated by fortifying a powder mixture of blank tablets with the amounts of 80, 100 and 120 % of label claimed of DAPA and SAXA. The resulting tablets were analyzed in triplicate for three successive days and the % recovery at each level for each day was determined. The amount found compared with the amount added and the % RSD was considered as an indication of precision. Further, to determine the intermediate precision, results from the intra-day and inter-day studies were subjected to F-test.

Linearity and Range:

The mean amounts of drugs found during accuracy and precision studies were plotted against the amounts of the drugs added. The data pairs were subjected to regression analysis and the corresponding slopes and intercepts were determined for DAPA and SAXA, respectively.

RESULTS AND DISCUSSION:

Different mobile phases were tried to achieve the separation and resolution of DAPA and SAXA. Initially, when methanol and acetonitrile were tried with water as an aqueous phase, inadequate separation with unaccepted peak shape were obtained for DAPA and SAXA which suggests replacing the water with buffer of appropriate strength as an aqueous phase. When mobile phase comprising of methanol: 20 mM potassium phosphate buffer (pH 3.0) was tried with C18 column, at flow rate of 1 mL/min, adequate retention of DAPA and SAXA (6.458 min and 4.708 min, respectively) at 225 nm. Good peak shape with acceptable system suitability parameters (theoretical plates: DAPA: 9659, SAXA: 7769; asymmetry: DAPA: 1.18, SAXA: 1.16) were obtained. The chromatogram of DAPA and SAXA is presented in Figure 3.

Figure 3: Representative chromatogram of SAXA and DAPA

In calibration curve experiments, DAPA was found linear in the range of 4–24 mg/mL and SAXA 2–12 mg/mL, respectively. The straight-line equations and regression coefficients obtained for DAPA was y=30662x-4982, 0.9995 and for SAXA y=22070x +27035, 0.9989, respectively.

When tablets prepared on lab scale were analyzed, the results obtained were in good agreement with the nominal amounts of the drugs. DAPA was found 100.30 % and SAXA was found 99.81 %, respectively.

When the blank tablets and tablets containing the DAPA and SAXA were subjected to chromatographic analysis, no interfering peaks were observed at the retention times of the DAPA and SAXA suggesting the specificity of the developed method.

The results obtained for accuracy and precision studies of DAPA and SAXA are presented in Table 1 and Table 2, respectively. The mean values of amount found was close to the amount added and the low % RSD values indicates the acceptable accuracy and precision of the developed method. To determine the intermediate precision, when the data of accuracy and precision studied obtained at each QC level were subjected to F-test, it was observed that F (calculated) were found less than the F (tabulated), which concludes no significant difference between intra- and inter-day precision and good intermediate precision.

When the data obtained from accuracy and precision studies of amount found was plotted against the amount added, a straight-line equation was obtained with y=0.971x+0.285 with r²=0.999 for DAPA and y=0.937x-0.003 with r²=0.999 for SAXA, respectively. It was observed at 95 % confidence interval, slope encompassing 1 and intercept encompassing 0, suggest the linearity of the developed method in the range of 80 to 120 % of the label claimed.

Table 1: Accuracy and precision studies for DAPA

Amount added	Amount Found			Within mean square (WMS)	Between Mean Square (BMS)	F value
10+8=18	17.96	18.09	17.80
	17.85	17.88	17.78	0.0193	0.0412	2.1
	17.83	17.77	17.51
Mean	17.88	17.91	17.69
±SD	0.06954	0.1635	0.1624
%RSD	0.3888	0.9127	0.9179
10+10=20	19.21	19.26	19.46
	19.68	19.25	19.16	0.0302	0.03	1.2
	19.35	19.07	19.29
Mean	19.41	19.19	19.30
±SD	0.2399	0.1075	0.1463
%RSD	1.235	0.5601	0.7579
10+12=22	21.82	22.10	22.01
	21.91	21.71	21.76	0.0219	0.0025	0.11
	21.94	21.74	21.95
Mean	21.89	21.85	21.91
±SD	0.0585	0.2144	0.1281
%RSD	0.2673	0.9811	0.5847

Table 2: Accuracy and precision studies for SAXA

Amount added	Amount Found			Within mean square (WMS)	Between Mean Square (BMS)	F value
5+4=9	9.08	9.01	8.89
	9.06	9.07	9.04	0.0050	0.0027	0.54
	8.93	8.96	8.97
Mean	9.02	9.01	8.97
±SD	0.0812	0.0563	0.0713
%RSD	0.900	0.624	0.795
5+5=10	9.91	9.80	9.80
	9.92	10.01	9.92	0.0057	0.0027	0.47
	9.88	9.98	9.88
Mean	9.91	9.93	9.87
±SD	0.0201	0.1122	0.063
%RSD	0.2028	1.1305	0.638
5+6=11	10.59	10.68	10.47
	10.60	10.51	10.49	0.0079	0.0051	0.64
	10.56	10.69	10.68
Mean	10.59	10.63	10.55
±SD	0.020	0.099	0.115
%RSD	1.898	0.9387	1.090