Calibration curves and limit of detection .1 Methods .1 Methods

Stock solutions (2 mg/ml) of all standards were prepared in DMSO in methanol (0.1% DMSO): the flavanones - hesperidin, narirutin, hesperetin, naringenin and the isoflavones - daidzin, genistin, daidzein, genistein, and apigenin, taxifolin and rutin used as internal standards. Stock solutions were sonicated for 2 min until fully dissolved and stored at –20 °C until analysed. A serial dilution was prepared for each compound from 5 – 100 µg /ml. Samples were filtered (PTFE 0.2 µm pore size) prior to HPLC analysis.

Calibration curves were constructed for each standard at six concentrations in duplicate. Regression analysis was assessed to determine the linearity of calibration curves. The result showed that the peak area was linearly correlated with on-column amounts over the range of (20 – 393 µM), (18.5 – 370 µM), (16.5 – 331 µM), (18.3 – 367 µM) and (18.5 -370 µM) for daidzein, genistein, hesperetin, naringenin and apigenin, respectively.

There are several definitions which are used to define limit of detection (LOD) and limit of quantification (LOQ). In general, LOD is taken as the lowest concentration of an analyte in sample that can be detected, but not quantified. The LOQ is the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated condition of the test (Shrivastava and Gupta, 2011).

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The method used to estimate the detection and quantification limit is based on calculation from the calibration line at low concentration using the equation below:

LOD = F*SD/b LOQ = F*SD/b Where

F: factor of 3.3 and 10 for LOD and LOQ, respectively SD: Standard deviation of intercept,

b: Slope of the regression line

The LOD and LOQ for each standard were established at their particular maximum UV wavelength when the signal to noise ratio of peak height was at three and ten respectively. Recovery, accuracy and selectivity of the standards were evaluated intra-day by performing triplicate injections for each concentration. The inter-day reproducibility test was carried out by analysing two injections on two different days. Each aliquot of these solutions were injected on to the HPLC system and calibration curves were done by plotting concentration against peak area of each compounds and then regression equation was calculated for each curve for each flavanone and isoflavone. Accuracy of HPLC methods was evaluated by analysing blank urine samples spiked and non-spiked by appropriate concentration of the target compounds. The recovery was calculated by comparing the detemined amounts of extracted urine with the known amounts added. Moreover, the areas of peaks and retention times were identified in human urine with and without enzymatic analysis to identify evidence of inference from the matrix or other analytes. Control samples were prepared for each urine sample in duplicate, replacing the enzyme-enriched buffer with non-enriched buffer.

2.5.2 Results

Calibration curves were performed for each standard at 6 concentrations in duplicate. The R² was greater than 0.99 for each standard using the chromatographic conditions mentioned in the methods previously (section

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2.3.3.1) and (section 2.3.3.2) for glucoside and aglycone form, respectively. The slopes of peak area aginst the concentration of the standard (µg/ml) were as follows: daidzein 46.2, genistein 43.1, hesperetin 25.2, naringenin 26.1, daidzin 10.2, genistin 6.2, narirutin 13.3, hesperdin 13.3, apigenin 16.7, taxifolin 8.46 µg/ml. The limits of detection and quantification for these methods for isoflavones and flavanones standards are presented in Table 2.1. The precision of the methods were tested by both intra-day and inter-day and coefficient of variation was below 3% and 4.5%, respectively. The accuarcy of intra-day and inter-day accuracy were demonstrated across the concentration range with relative error of less than 6%. Standard curve of flavanones and isoflavones are shown in figure 2.4 and 2.5.

Table 2.1: Limit of detection and quantification of isoflavones and flavanones compounds.

Limit of detection (LOD) (µg/ml)

Limit of quantification (LOQ) (µg/ml)

daidzein 0.19 0.66

genistein 0.34 1.12

naringenin 0.35 0.74

hesperetin 0.22 0.74

Daidzin 0.91 3.01

Genistin 1.04 3.4

narirutin 0.68 2.27

hesperidin 2.5 8.4

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Figure 2.4 Standard curves: (A) daidzein, (B) genistein at 260 nm (C) hesperetin and (D) naringenin, 10 µl injected in the column at 280 nm, by using HPLC-DAD.

0

Absorbance at 260 nm (mAU)

Concentration (µg/ml)

Absorbance at 260 nm (mAU)

Concentration (µg/ml)

Absorbance at 280 nm (mAU)

Concentration (µg/ml)

Absorbance at 280 nm (mAU)

Concentration (µg/ml)

D

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Figure 2.5 Standard curves of (A) Narirutin (B) Hesperidin at 280 nm and (C) daidzin (D) genistin, 5 µl injected in the column at 260 nm, by using HPLC-DAD.

The identification of flavanoids in the food and biological samples were based on comparison of retention times to authentic standards, and the UV absorption spectral characteristics, as shown in figure 2.6.

0

Absorbance at 280 nm (mAU)

Concentration (µg/ml)

Absorbance at 280 nm (mAU)

Concentration (µg/ml)

Absorbance at 260 nm (mAU)

Concentration (µg/ml)

Absorbance at 260 nm (mAU)

Concentration (µg/ml)

D

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Figure 2.6: UV absorption spectra of flavanone and isoflavone standards daidzein (A), genistein (B) at 270 nm, hesperetin (C), naringenin (D) at 280 nm.

An HPLC chromatogram showing a mixture of flavanone and isoflavone aglycone standards is shown in Figure 2.7. The peaks of the flavanones and isoflavones were well separated at 6.8, 7.8, 8.4, 8.7 and 10 min for daidzein, naringenin, genistein, hesperetin and apigenin, respectively. An HPLC chromatogram of a mixture of flavanone and isoflavone glycosides standards are shown in Figure 2.8 The peaks are well separated at 11.1, 14.0, 15.4, 17.4 and 23.1 min for daidzin, genistin, narirutin, hesperidin and apigenin, respectively.

Daidzein (A) Genistin (B)

Hesperetin (C) Naringenin (D)

Wavelength (nm) Wavelength (nm)

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Figure 2.7: Reversed –phase HPLC chromatogram of a mixture of aglycone standards. Peak identity: P1- daidzein, P2- naringenin, P3- genistein, P4- hesperetin, P5- apigenin.

Figure 2.8: Reversed –phase HPLC chromatogram of a mixture of glycoside standards. Peak identity: P1- daidzin, P2- genistin, P3- narirutin, P4- hesperidin, P5- apigenin.

Absorbance at 280 nm (mAU)

Retention time / min

Absorbance at 280 nm (mAU)

Retention time /min

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2.5.3 Verification and identification of flavanone and isoflavone peaks

The identification of eluted flavonoids was determined based on the following methods:

a) UV Absorption spectra. Most flavonoids have a distinct UV spectrum, which is very useful for determining compounds expected in samples.

However it is difficult to identify the aglycone or conjugate, or very similar flavonoid subclasses using spectral characteristics.

b) Retention times. Hydrophobic aglycone flavonoids will elute later in reserved-phased HPLC, and hydrophilic glycosides or conjugate metabolites will elute earlier. Therefore comparing the retention time of the reference standard compound with the eluted unknown, especially in combination with the UV spectrum can help identify the flavonoid.

c) Spiking. The sample was spiked with a known amount of flavonoid standard to verify the change in peak area of the target flavonoids; the peak area was compared before and after spike.

d) Mass. For some samples (hesperetin conjugate metabolites), LC-MS was used to identify the mass of the parent compound. Along with the retention time and the UV spectrum this will add to the evidence to identify the right flavonoid. see chapter 7for more details