Analytical Method Validation

The proposed UPLC-MRM method for quantitative analysis was validated according to the guidelines of International Conference on Harmonization (ICH, Q2R1) by determining linearity, lower LOD, lower LOQ, precision, solution stability and recovery. MRM extracted ion chromatogram of analytes and IS are shown in Figure 3.3.

Linearity, LOD and LOQ

The IS method, wherein a known amount of a reference compound is added to all samples, was employed to calculate the contents of 13 analytes in Piper species. The stock solution was diluted with methanol to different working

TABLE 3.1 List of Compound-Dependent MRM Parameters, DP and Collision Energy (ce) for Each Analyte and IS

s.

NO.

RT

(MIN)

ANALYTE

Q1

MASS

(DA)

Q3

MASS

(DA)

DP

(V)

CE

(EV)

POLARITY

1

1.41

Protocatechu ic acid

153

109

-64

-22

~

2

2.06

Caffeic acid

179

135

-48

-21

-

3

2.81

Ferulic acid

193

134

-58

-23

-

4

3.33

Rosmarinic acid

359

161

-65

-22

-

5

3.48

Vanillic acid

167

108

-55

-22

-

6

3.93

Luteolin

285

133

-139

-38

-

7

3.96

Quercetin

301

151

-107

-31

-

8

4.12

Palmatine (IS)

352

336

80

26

+

9

4.45

Kaempferol

285

239

-95

-35

-

10

4.45

Apigenin

269

117

-71

-45

-

11

4.97

Piperlongumine

318

221

40

14

*

12

5.3

Piperlonguminine

21A

201

60

25

*

13

5.33

Curcumin (IS)

367

217

-60

-10

-

14

5.41

Piperine

286

201

45

30

*

15

7.27

Ursolic acid

455.1

455

-75

-9

+ = [м+Hh - = [m-h]-

Source: Reproduced from Ref. Chandra et al. 2015 with permission from Elsevier.

concentrations for the construction of calibration curves. The linearity of calibration was performed by the analytes to IS peak area ratios vs. the nominal concentration, and the calibration curves were constructed with a weight factor (1/x2) by least-squares linear regression. The applied calibration model for all curves was у = ax + b, where у = peak area ratio (analyte/IS), x = concentration of the analyte, a = slope of the curve and b = intercept. The LODs and LOQs were measured with S/N of 3 and 10, respectively, as criteria. The results are listed in Table 3.2. All the calibration curves indicated good linearity with correlation coefficients (r2) from 0.9986 to 0.9999 within the test ranges. The LODs for each analyte varied from 0.02 to 1.34ng/mL and LOQs from 0.06 to 3.88ng/mL. As previously reported (Kozukue et al. 2007; Friedman et al. 2008), LOD for piperine was 15-30 ng/mL, but in our study, LODs for all analytes are less than 1.34 ng/mL. Thus, the developed UPLC-ESI-MS/MS method is more sensitive than the previously reported methods.

UPLC-MRM extracted ion chromatogram of analytes

FIGURE 3.3 UPLC-MRM extracted ion chromatogram of analytes: (a) pro- tocatechuic acid, (b) caffeic acid, (c) ferulic acid, (d) rosmarinic acid, (e) vanillic acid, (f) luteolin, (g) quercetin, (h) kaempferol, (i) apigenin, (j) piperlongumine, (k) piperlonguminine, (I) piperine, (m) ursolic acid and IS, i.e., (n) curcumin and (o) palmatine. (Reproduced from Ref. Chandra et al. 2015 with permission from Elsevier.)

 
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