Differential diagnosis of gallstones by using hypericin as a fluorescent optical imaging agent

Table 1 Calibration data of the chromatographic methods

Cholesterol			Hypericin
Conc.	PA ± SD	RT ± SD (min)	Conc.	PA ± SD	RT ± SD (min)
(×10-3 M)	(n = 4)		(×10-6 M)	(n = 4)
2.6	32715275 ± 532288	19.60 ± 0.19	0.6	961398 ± 14312	9.85 ± 0.14
5.2	64995700 ± 622439	19.83 ± 0.26	1.3	3120453 ± 33528	9.63 ± 0.03
7.8	91376450 ± 1113357	19.79 ± 0.14	2.5	5782945 ± 90738	9.41 ± 0.13
10.3	124007250 ± 2116420	19.97± 0.11	5.0	10563681 ± 134692	9.75 ± 0.11
12.9	151245250 ± 2660042	20.03 ± 0.12	10.0	20814150 ± 226368	9.61 ± 0.07

Conc.: Concentration; PA: Peak area; RT: Retention time; SD: Standard deviation.

Table 2 System suitability

System suitability parameters	Cholesterol	Hypericin
Theoretical plates (N)	7700	5500
Asymmetry factor (As)	1.02	0.75
Tailing factor (TF)	1.00	0.61

Table 3 Results of the intra-day precision studies

Cholesterol			Hypericin
Conc.	RSD-PA	RSD-RT	Conc.	RSD-PA	RSD-RT
(×10-3 M)			(×10-6 M)
2.6	1.6%	0.9%	0.6%	1.5%	1.4%
5.2	0.9%	1.3%	1.3%	1.1%	0.3%
7.8	1.2%	0.7%	2.5%	1.6%	1.4%
10.3	1.7%	0.5%	5.0%	1.3%	1.1%
12.9	1.7%	0.6%	10.0%	1.1%	0.7%

Conc.: Concentration; PA: Peak area; RSD: Relative standard deviation; RT: Retention time.

Table 4 Results of the recovery studies

Compound	Labelled amount	Added amount	Found amount	Recovery	RSD
Cholesterol	1.0 mg	0.80 mg	0.79 mg	99.8%	1.6%
		1.00 mg	1.00 mg	100.0%	1.1%
		1.20 mg	1.19 mg	99.6%	1.2%
Hypericin	0.0025 μg	0.0020 μg	0.0029 μg	108.8%	0.4%
		0.0025 μg	0.0026 μg	101.7%	0.1%
		0.0030 μg	0.0032 μg	103.6%	1.6%

RSD: Relative standard deviation; SD: Standard deviation.

Table 5 Linearity of the high performance liquid chromatography methods for cholesterol and hypericin

Compound	LR	Slope	SD (LR)	R2	LOD (M)	LOQ (M)
Cholesterol	y = 1 × 107 x + 3 × 106	11712479	2055881	0.998	0.58 × 10-3	1.76 × 10-3
Hypericin	y = 2 × 106x + 2 × 105	2066297	433480	0.998	0.69 × 10-6	2.1 × 10-6

LR: Linear regression; SD (LR): Standard deviation of regression line; R²: Coefficient of correlation; y: Peak area; x: Compound concentration; LOD: Limit of detection; LOQ: Limit of quantification.

Table 6 Results of the analysis of cholesterol, mixed and pigment stones

Type of stones	Cholesterol		Hypericin	P value
	Conc. (× 10-3 M)	Percentage	Conc. (× 10-6 M)
	IPA/DMSO extracts	in stone	IPA/DMSO extracts
Cholesterol	7.68 ± 0.25	83%-86%	2.01 ± 0.38
Mixed	6.65 ± 0.12	71%-75%	0.50 ± 0.19	0.011
Pigment	0.08 ± 0.02	1%-3%	0.23 ± 0.06

Conc.: Concentration; DMSO: Dimethyl sulfoxide; IPA: 2-propanol.