Development and Validation of an Analytical Method for Kaempferol Using UV-Vis Spectrophotometry

Kaempferol contain naturally occurring polyphenolic flavonoids which are commonly included in herbal and nutraceutical formulations ^[1-3]. Kaempferol has been extensively studied for its strong antioxidant, anti-inflammatory, and anticancer properties ^[4-7]. The molecular structure of the kaempferol is shown in Figure 1 ^[5]. Kaempferol has gained significantly attention in herbal, pharmaceutical, and nutraceutical products due to their notable therapeutic effects. Despite their pharmacological significance, few studies have validated a UV-visible spectrophotometric method for their quantification ^[6-8]. Although often combined in dietary supplements, a straightforward, cost-effective, and reproducible technique for their simultaneous analysis remains unavailable, especially in quality control labs. While methods like HPLC and LC-MS provide accuracy, they are costly, time-consuming, and require advanced equipment ^[9-13]. This research aims to develop a simple, reliable, and validated UV spectrophotometric method for measuring kaempferol in bulk ^[8]. Therefore, UV-visible spectrophotometry offers a rapid, economical, and user-friendly alternative for the simultaneous quantification of kaempferol in bulk drugs ^[13]. This study aims to fill this analytical gap and ensure that the proposed method meets international standards for accuracy, precision, linearity, and robustness, thereby strengthening quality assurance in herbal product manufacturing ^[14-15].

Fig. 1: Molecular Structure of Kaempferol

1. MATERIALS AND METHODS:

2.1 Instrumentation:

A pre-calibrated double-beam UV-Visible spectrophotometer (UV-530, Jasco), employed with Spectra Manager software, was used for method development. Quartz cuvettes with a 1 cm path and 3 cm length were used for spectral analysis. The analytical balance (Essae, Vibra HT) with an internal calibration system was used for weighing. An ultrasonic bath (PCI Analytics, India; 6.5 L capacity) was used for assisting the complete solubilisation of kaempferol.

2.2 Chemicals and Reagents:

Kaempferol was purchased from TCI Chemicals (India) Pvt. Ltd., Chennai. Acetonitrile was purchased from Merck. Deionized water was obtained from a water purification system (Lablink Xtrapure). For method development, all chemicals employed in the study were of at least analytical grade.

2.3 Preparation of Standard Solutions:

Accurately weighed 100 mg of kaempferol was transferred into a 100 mL volumetric flask and dissolved in 100 ml acetonitrile (Mother stock). Further, dilutions were prepared using co-solvent system comprising of acetonitrile and water in a 40:60 (v/v) ratio to achieve working standard solution of 100 μg/mL (Stock I). Stock-I was suitably diluted to achieve the kaempferol solution of 15 μg/mL strength (Stock II).

2.4 Determination of wavelength of maximum absorbance (λ_max)

The wavelength of maximum absorbance (λ_max) of Kaempferol was determined using a UV-Visible spectrophotometer.  The spectrophotometer was set in the spectrum management mode. A working stock II solution (15 μg/mL) of kaempferol was scanned over the wavelength of 200 to 800 nm with medium scanning speed. The wavelength of maximum absorbance (λ_max) was identified using the help of software settings. The scanning and λ_max identification process was repeated three times to achieve the reproducible results.

2.5 Preparation of calibration curve:

Freshly prepared stock-II solution of kaempferol was diluted with co-solvent to achieve six calibration standards viz., CAL-STD-1 (1 μg/mL), CAL-STD-2 (2 μg/mL), CAL-STD-3 (4 μg/mL), CAL-STD-4 (8 μg/mL), CAL-STD-5 (12 μg/mL) and CAL-STD-6 (14 μg/mL). The spectrophotometer was set in Fixed Wavelength Measurement mode, and the absorbance of each calibration standard was measured at pre-identified wavelength of maximum absorption of 360 nm. The procedure was repeated thrice to ensure consistency of the results which were reported in terms of mean ± SD.

2.6 Validation of the Method:

The developed UV-visible spectrophotometric method for kaempferol was validated using the ICH Q2 (R1) guidelines ^[16]. The developed method was assessed for linearity, accuracy, precision, robustness, limit of detection (LOD), and limit of quantitation (LOQ).

2.6.1 Linearity & Range:

The linearity of the proposed UV Spectrophotometric method of kaempferol was evaluated using predefined six calibration standards, as mentioned in section 2.5. By using calibration curves of absorbance vs. concentration, the linear least squares regression analysis was carried out. The linearity of the proposed method was established by evaluating the correlation coefficient (r²), which states a linear relationship between absorbance and concentration. The range of the UV spectrophotometric method of kaempferol was defined by the lower and upper concentration limits over which acceptable linearity was consistently observed.

2.6.2 Accuracy:

The accuracy of the proposed UV-visible spectrophotometric method was established using quality control standards (QC-STD). Three distinct quality control standards of kaempferol, viz. QC-STD-1, QC-STD-2, and QC-STD-3 having nominal concentrations of 1.5 μg/mL, 7 μg/mL, and 13 μg/mL respectively, were prepared in triplicate and used for the proposed study. The QC-STD were analysed for its kaempferol content using the proposed UV-visible spectrophotometric method at three different time intervals in a day for three consecutive days. The intra-day and inter-day accuracy of the proposed method was established in terms of % difference, calculated using the following formula.

% Difference=Mean Measured Concentration- Nominal ConcentrationNominal Concentration×100

2.6.3 Precision:

Precision of the method was conducted to assess the reliability of the anticipated analysis process. The QC-STD as mentioned in section 2.6.2 were employed to determine precision of proposed method in terms of % relative standard deviation (RSD). The intra-day and inter-day precision was established by analysing the QC-STDs at three different time intervals during the day & the same procedure was repeated on three consecutive days. The precision of the method was established in terms of % RSD, calculated by following formula,

%RSD=StandardDeviation SDMean ×100

2.6.4 Robustness:

The robustness of the proposed UV method for Kaempferol was assessed by making a small deliberate change in the co-solvent mixture of acetonitrile and water. The acetonitrile: water ratio was adjusted to 38:62 v/v and 42:58 v/v to check whether such alterations affect the results. The three QC-STDs were analysed with above mentioned co-solvent ratio, and the mean standard deviation (SD) and percent relative standard deviation (%RSD) was calculated. The method was robust in case the % RSD values were within the acceptable limit of ≤ 2%.

2.6.5 Limit of Detection and Limit of Quantification:

According to ICH guidelines, the limits of detection (LOD) and quantification (LOQ) for kaempferol was calculated based on standard deviation of the regression line and slope of the calibration curve, using the following formula.

LOD= 3.3 x SDS

LOQ=10 x SDS

Where,

SD = Standard deviation of Y-intercept

S = Slope of calibration curve

2.7 Estimation of Kaempferol in Marketed Formulation:

The proposed UV-visible spectrophotometric method was employed to quantify and estimate Kaempferol in marketed formulations to ensure the methods reliability for routine quality assessment of Kaempferol capsules. An amount equivalent to 50 mg of kaempferol was transferred in a 10 mL volumetric flask and dissolved in 10 ml of acetonitrile. The solution was subjected to ultrasonication for 10 minutes. Further, it was diluted at a 1:4 v/v ratio of cosolvent to achieve the desired theoretical concentration of 12 µg/mL. The kaempferol content was analysed using the pre-validated method and the percentage of the kaempferol in capsules was shown in terms of mean ± SD.

2. RESULTS AND DISCUSSION

3.1 Determination of the wavelength:

For accurate quantitative analysis of Kaempferol by UV spectrophotometry, initially, the λ_max of Kaempferol was established. When a 15 µg/mL solution of Kaempferol repeatedly scanned over the range of 200 to 800 nm showed the maximum absorbance at 360 nm which is evident in the figure 2.

 

360 nm

 

Fig. 2: UV-Visible Spectra of Kaempferol

 

 

Fig. 3: (A) Calibration curve of kaempferol (Replicate 1)

 

 

Fig. 3: (B) Calibration curve of kaempferol (Replicate 2)

 

 

Fig. 3 (C) Calibration curve of kaempferol (Replicate 3)

CONCLUSION

A precise and sensitive UV-Visible spectrophotometric method for kaempferol was developed and validated. The proposed UV-Visible spectrophotometric method showed excellent linearity, accuracy, precision, and robustness in accordance with ICH guidelines. The low LOD & LOQ values confirmed its sensitivity. Results from testing commercial capsules closely matched with label claims, making the proposed UV-Visible spectrophotometric method suitable for routine quality control testing.

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