Journal of Chemistry

Research Article

A Simple RP-HPLC Method to Simultaneously Assay the Contents of Lamivudine, Tenofovir, and Nevirapine in Fixed Dose Combined Oral Antiviral Medicines

Table 2

Results from validation carried out on the developed method.


Validation parameter		3TC	NVP	TDF	Remarks

Specificity/selectivity (S2)	Retention time (mean ± SD)	3.269 ± 0.0074 min (N = 5)	5.423 ± 0.0015 min (N = 5)	7.555 ± 0.0024 min (N = 5)	Retention times are significantly different from each other (F_2,12 = 1.102e + 006; ). Method selectively and specifically identifies the three analytes.

Accuracy (S3)	% recovery (mean ± SD)	36 µg/ml = 99.30% ± 0.34 (N = 3)	16.8 µg/ml = 102.99% ± 0.45 (N = 3)	24 µg/ml = 101.75% ± 0.28 (N = 3)	Developed method accurately estimates the content of the analytes. Acceptance criteria: [98.00%–102.00%].
		45 µg/ml = 100.80% ± 0.32 (N = 3)	21 µg/ml = 99.71% ± 0.72 (N = 3)	30 µg/ml = 98.58% ± 0.71 (N = 3)
		54 µg/ml = 97.90% ± 0.46 (N = 3)	25.2 µg/ml = 98.22% ± 0.51 (N = 3)	36 µg/ml = 98.18% ± 0.26 (N = 3)

Linearity and range	Regression equation	Y = 14487x + 38521	Y = 9363x + 11417	Y = 5283x + 8162	Acceptance criteria: [R² > 0.9900] linearity of responses established for the specified analytes concentration ranges.
	R²	0.9959	0.9948	0.9971
	Sy.x	25214	8543	5062
	F-value, value	3900,	3090,	5429,
	Range	15 µg/ml–90 µg/ml	7 µg/ml–42 µg/ml	10 µg/ml–59 µg/ml

LOD		5.5032 µg/ml	3.1496 µg/ml	3.9267 µg/ml

LOQ		16.6762 µg/ml	9.5443 µg/ml	10.0433 µg/ml

Precision (S4)	Repeatability (3 conc terms)	RSD = 0.4000% ± 0.2291, N = 3	RSD = 0.7967% ± 0.342, N = 3	RSD = 0.9467% ± 0.6704, N = 3	Acceptance criteria: [RSD <2.0%]. Two-way ANOVA showed that the differences in the responses produced from the different concentrations on different days not were significant (). Developed method responses were precise.
Precision (S4)	Intermediate precision (3 analysts for 3 days)	RSD = 0.52%–1.70%, N = 9; F_(4,18) = 1.786,	RSD = 0.41%–1.94%, N = 9; F_(4,18) = 2.890, .	RSD = 0.29%–1.63%, N = 9; F_(4,18) = 0.08900,

Robustness (S5)		(i) Retention time = 3.416 ± 0.019 min and 3.131 ± 0.131 min at 0.90 ml/min and 1.1 mil/min, respectively. (ii) % deviation of peak area < ±5%	(i) Retention time = 8.375 ± 0.033 min and 3.417 ± 0.022 min at 0.90 ml/min and 1.1 mil/min, respectively. (ii) % deviation of peak area < ±5%	(i) Retention time = 9.573 ± 0.011 min and 6.783 ± 0.036 min at 0.90 ml/min and 1.1 mil/min, respectively. (ii) % deviation of peak area < ±5%	Deliberate changes in flow rate resulted in retention times of the analytes maintaining their distinction from each other. Hence, peaks maintained resolutions at 0.90 mil/min [F_(2,6) = 60322; ] and 1.10 mil/min [F_(2,6) = 1949; ]. Deliberate changes in the wavelength detection did not show significant differences in peak areas (RSD <2%; % deviation < 5%). In addition, proportionate detection was maintained (). Method is fairly robust.

Minimal deviations of results from acceptance criteria.