Simultaneous Estimation of Tramadol HCl, Paracetamol and Domperidone in Pharmaceutical Formulation by Thin-Layer Chromatographic-Densitometric method

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Preparation of standard solution
A Combined standard stock solution containing 100µg/mLDMP, 3250 µg/m PCM and 375 µg/ml TMD was prepared in methanol. Calibration was done by applying mix standard solution ranging from 1-6  µl by micro liter syringe with the help of automatic sample applicator Linomat V on TLC Plate, which gives concentration of  100-600 ng/spot of DMP , 3250-19500 ng/spot of  PCM  and 375-2250 ng/spot of TMD. Each concentration was spotted six times on the TLC plates. The plate was developed on previously described mobile phase. The peak areas plotted against the corresponding concentrations to obtain the calibration graphs.

2.4 Method Validation
The developed method was validated for linearity and range, specificity, accuracy, precision, Limit of detection, Limit of quantitation, robustness and solution stability as per ICH guidelines. 

2.4.1 Linearity and Range
Aliquots of standards 1-6µl TMD, PCM & DMP solutions were loaded in the 10 x 10 Silica gel 60F254TLC plate using 100μl Hamilton syringe and Camag – Linomat -5 instrument.

2.4.2 Specificity
The specificity of the method was ascertained by analyzing PCM, DMP and TMD in presence of excipients commonly used for tablet formulations. The bands of PCM, DMP and TMD were confirmed by comparing RF values and respective spectra of sample with those of standards. The peak purity of PCM, DMP and TMD was assured by comparing the spectra at three different levels, that is, peak start, peak apex and peak end positions, as listed in Table 1.

2.4.3 Accuracy (% Recovery)
The recovery study was car