Development of normal phase HPLC based method for the determination of retinyl palmitate in fortified edible oils

Figures & data

Table 1. System suitability and validation parameters.

Parameters	Retinyl palmitate standard (21.6 µg/mL)
*Tailing factor (USP)	0.98 ± 0
*Resolution (USP)	3.28 ± 0.15
Theoretical plates/meter (USP)	88,666
*Retention time (min)	8.13 ± 0.28
Relative standard deviation (%)	0.35

USP: United States Pharmacopoeia; *Values are Mean ± SD of three replications

Table 2. Linearity of response for vitamin A standard (retinyl palmitate).

Standard concentration (µg/mL)	Area (y)	Mean Area (y) (n = 3)	Intercept (-c)	Slope (m)	Correlation coefficient (R^2)
3.93	320,231	319,744	18,322	81,934	0.9998
	319,669
	319,333
7.87	640,635	641,483
	642,183
	641,631
15.75	1,267,904	1,268,347
	1,267,915
	1,269,224
31.5	2,513,031	2,511,962
	2,513,376
	2,509,479
63	5,165,965	5,166,941
	5,168,278
	5,166,582

Table 3. LOD and LOQ of retinyl palmitate standard.

Retinyl palmitate standard
Concentration (µg/mL)	Area (y)	Mean area (y)	SD	LOD (µg/mL)	LOQ (µg/mL)
7.87	640,635	641,483	784.54	0.029	0.096
	642,183
	641,631

Table 4. Recovery data for retinyl palmitate standard.

Concentration (µg/mL)	Mean Area (y)	Mean found concentration (µg/mL)	Recovery (%)	Mean recovery (%)	RSD (%)	Mean RSD (%)
3.93	319,744	4.13	104.99	102.34	0.14	0.10
7.87	641,483	8.05	102.32		0.12
15.75	1,268,348	15.70	99.71		0.06

Table 5. Recovery data for spiked sample.

	Sample conc. (µg/mL)	Added conc. (µg/mL)	Expected conc. (µg/mL)	Found conc. (µg/mL)	Recovery (%)	Mean recovery (%)	RSD (%)	Mean RSD (%)
Rec-1	0.049	7.87	7.92	8.02	101.55	97.13	0.67	0.44
Rec-2	0.049	15.75	15.80	15.30	96.81		0.57
Rec-3	0.049	31.5	31.5	29.31	93.04		0.07

Rec: Recovery sample

Table 6. Solutions stability data of the proposed HPLC method.

	Time interval	Area (y)	Mean area (y)	Rt (min)	Mean recovered (µg/mL)	Mean recovery (%)	RSD (%)
Standard (63 µg/mL)	0 h	5,165,965	5,166,941	8.38	63.29	100.45	0.02
		5,168,278		7.86
		5,166,582		7.88
	After 24 h at Light	5,064,818	5,052,960	8.32	61.89	98.25	0.26
		5,039,196		7.02
		5,054,867		6.95
	After 24 h at Dark	5,112,238	5,115,779	7.93	62.66	99.46	0.16
		5,125,287		7.95
		5,109,813		7.96
	After 24 h at Refrigerator	5,044,580	5,024,745	8.01	61.55	97.7	0.40
		5,024,958		8.01
		5,004,699		8.00
Sample (0.04 µg/ml)	0 h	7,707	7,959	8.03	-	-	3.33
		7,935		8.05
		8,236		8.06
	After 24 h at Light	6,324	6,052	8.09	0.029	72.5	4.27
		5,809		8.12
		6,025		8.12
	After 24 h at Dark	6,689	6,667	7.72	0.041	102.5	7.0
		6,190		7.44
		7,122		7.65
	After 24 h at Refrigerator	5,239	5,151	8.15	0.033	82.5	1.88
		5,047		8.18
		5,168		8.17

Table 7. Precision data of retinyl palmitate standard (21.6 µg/mL).

Time Interval	Area (y)	Mean area (y)	Rt (min)	Tail factor	Peak resolution	Mean recovered (µg/mL)	Mean recovery (%)	RSD (%)
Intra-day precision
0 h	1,783,384	1,778,782	7.80	0.98	3.10	21.93	101.54	0.35
	1,781,296		8.27	0.98	3.38
	1,771,667		8.31	0.98	3.35
3 h	1,767,380	1,760,184	8.35	0.98	3.38	21.71	100.49	0.37
	1,754,814		7.94	0.98	3.07
	1,758,359		9.19	0.96	3.63
6 h	1,650,501	1,646,810	8.25	0.98	3.22	20.29	93.92	0.28
	1,641,571		9.41	0.95	3.50
	1,648,359		9.09	0.96	3.42
Inter-day precision
Day 1	1,783,384	1,778,782	7.80	0.98	3.10	21.93	101.54	0.35
	1,781,296		8.27	0.98	3.38
	1,771,667		8.31	0.98	3.35
Day 2	1,595,419	1,576,749	8.42	0.98	3.37	19.47	90.13	1.04
	1,564,785		8.87	0.97	3.47
	1,570,045		9.14	0.97	3.42
Day 3	1,493,188	1,473,784	8.98	0.98	3.40	18.21	84.31	1.28
	1,455,572		8.86	0.98	3.33
	1,472,593		8.52	0.97	3.83

Table 8. Robustness data of the proposed HPLC method (28 µg/mL of retinyl palmitate standard).

Parameters		Mean area (y)	Mean RT (min)	Mean recovery (%)	Mean RSD (%)
Mobile phase composition	80% (A pump), 20% (B pump)	2,125,518	6.33	93.30	0.53
	75% (A pump), 25% (B pump)	2,126,374	6.48	93.49	0.36
	85% (A pump), 15% (B pump)	2,074,871	8.15	91.24	1.03
Wavelength	323 nm	2,106,144	6.018	92.60	0.06
	326 nm	2,126,374	6.48	93.49	0.36
	329 nm	2,104,384	6.023	92.53	0.2
Flow rate	1.2 mL/min	1,780,449	5.07	78.41	0.14
	1 mL/min	2,126,374	6.48	93.49	0.36
	0.8 mL/min	2,041,274	7.56	89.78	0.42

Figure 1. Specificity of analytical solutions; chromatogram of (a) blank solution, (b) standard solution, and (c) retinyl palmitate in fortified edible oil.

Figure 2. Calibration curve of vitamin A standard (retinyl palmitate).

Table 9. Vitamin A content in different fortified edible oils.

Types of fortified oil	Sample concentration (mg/mL)	Mean area (y)	Mean Rt (min)	Vit A (mg/100 g)	RSD (%)	Fortification standard (mg/100 g)
Soybean	1.13	4,588.5 ± 4.95	7.43 ± 0.01	1.01 ± 0.008	0.11	1.5–3.0
Sunflower	1.06	5,132 ± 11.31	9.005 ± 0.46	1.15 ± 0.03	0.22
Palm	1.19	4,554 ± 32.53	8.36 ± 0.00	0.98 ± 0.002	0.71

Values are Mean ± SD of three replications.