A validated HPLC-UV method and optimization of sample preparation technique for norepinephrine and serotonin in mouse brain

Figures & data

Figure 1. (A) Chemical structure of norepinephrine. (B) Chemical structure of serotonin.

Figure 2. (A) Chromatogram showing standard graphs of norepinephrine and serotonin. (B) Chromatogram showing the norepinephrine and serotonin in mice brain homogenate.

Figure 3. Recovery values given by different sample preparation methods when a fixed amount of standards (norepinephrine, 200 ng mL⁻¹ and serotonin, 750 ng mL⁻¹) were spiked into normal brain samples.

Table 1. Analytical parameters for norepinephrine and serotonin.

Regression equation parameter	Norepinephrine	Serotonin
Range (ng mL^−1)^a	50–2000	31.25–4000
Slope	27.053	24.44
y-Intercept	830.33	1955.6
Correlation coefficient	0.994	0.996
Retention time (min)	2.17 ± 0.004	3.7 ± 0.005
LOD (ng mL^−1)	25	15.63.
LOQ (ng mL^−1)	50	31.25

^an = 6. LOD, limit of detection; LOQ, limit of quantification.

Table 2. Accuracy test results (n = 6).

Compound	Concentration ofhomogenate (ng mL^−1 ± SD)	Added standard conc. (ng mL^−1)	% of drug recovered
Norepinephrine	220.45 ± 5.64	55.11	88.88
		110.23	86.04
		220.45	88.52
		330.68	89.01
Serotonin	752.76 ± 10.76	188.19	87.92
		376.38	89.61
		752.76	86.87
		1129.14	86.43

Table 3. Repeatability and intra- and inter-day precision (n = 6) for the standard solution of norepinephrine and serotonin.

	Repeatability	Intra-day precision		Inter-day precision
Conc. (ng mL^−1)	%RSD	Conc. (ng mL^−1) ± SD (n = 6)	%RSD	Conc. (ng mL^−1)  ± SD (n = 6)	%RSD
Norepinephrine
100	2.59	99.87 ± 1.78	1.78	99.65 ± 1.92	1.93
500	2.33	495.76 ± 9.42	1.9	496.87 ± 10.58	2.13
1000	2.289	982.87 ± 15.72	1.60	985.15 ± 22.95	2.33
Serotonin
250	2.92	246.76 ± 6.96	2.82	245.43 ± 7.02	2.86
500	2.04	493.95 ± 8.05	1.63	492.12 ± 8.56	1.74
1000	3.06	991.76 ± 18.15	1.83	993.81 ± 22.24	2.23

Table 4. Repeatability and intra- and inter-day precisions of sample.

	Repeatability	Intra-day precision		Inter-day precision
Compounds	%RSD	Conc. (ng mL^−1)  ± SD (n = 6)	%RSD	Conc. (ng mL^−1)  ± SD (n = 6)	%RSD
Norepinephrine	10.63	218.87 ± 20.55	9.39	222.79 ± 22.52	10.11
Serotonin	9.96	758.82 ± 64.12	8.45	756.33 ± 71.39	9.44

Table 5. Robustness data of the HPLC method using control samples.

Actual	Used	Level	Mean conc. (ng mL^−1) ± SD (n = 6)	%RSD
Norepinephrine
0.05% formic acid:acetonitrile (90:10)	95:5	+5	221.54 ± 24.08	10.87
	90:10	0	220.87 ± 21.79	9.87
	85:15	−5	222.33 ± 22.27	10.02
280 nm	282 nm	+5	217.70 ± 21.53	9.89
	280 nm	0	220.72 ± 21.54	9.76
	278 nm	−5	221.54 ± 22.44	10.13
Serotonin
0.05% formic acid:acetonitrile (90:10)	95:5	+5	756.74 ± 70.67	9.34
	90:10	0	758.81 ± 70.87	9.63
	85:15	−5	755.43 ± 74.63	9.88
280 nm	282 nm	+2	754.98 ± 79.80	10.57
	280 nm	0	758.98 ± 72.94	9.61
	278 nm	−2	756.24 ± 74.26	9.82