Development and Validation of an HPLC Procedure for

Standardization of the Extractives of the Leaves

Nowadays, there is an increased attention in natural products that may counteract the detrimental
effects of environmental or chemical toxins and prevent multiple hepatic disorders in humans. The
abietane diterpene, clerodermic acid, herein isolated from the ethyl acetate/ n-butanol (2:1) fraction
of the 70% ethanol extract (EE) of the defatted leaves of C. inerme has been proven to exert the
highest cytotoxic activity on HEPG-2 liver carcinoma cell lines among other tested isolates. This
compound might therefore be suggested as lead for production of chemo-preventive hepatic
anticancer agent.
Besides upon chromatographic screening (TLC and HPLC), clerodermic acid appeared to be one
of the major constituents of EE. Consequently, an HPLC procedure for reliable standardization of
the leaves of C. inerme was developed based on estimation of its clerodermic acid content. This as
part of the quality assurance process to facilitate the acceptance of the plant as raw material in
herbal formulations. The proposed method was subjected to validation to ensure reliability before
application. The amount of clerodermic acid was estimated in both the parent EE of the leaves and
its active dichloromethane and ethyl acetate/ n-butanol (2:1) fractions.
I. Preparation of the Working Solutions
Solutions of the dried EE of the leaves of C. inerme (5 mg: 2 mL) and its dichloromethane (1 mg: 3 mL)
and ethyl acetate/ n-butanol (2:1) (1 mg: 1 mL) fractions were prepared in methanol and used as working
solutions. Aliquots (20 μL, each) of the working solutions were directly injected into the HPLC system.

Optimization of the Operating Conditions

Several trials were carried out to adjust the HPLC chromatographic conditions; attempts included
the use of various mobile phases with different ratios as eluents.
Satisfactory separation was achieved at room temperature, within 23 minutes, by gradient elution.
The most efficient eluting system consisted of: component A, acetonitrile and component B, 0.3%
phosphoric acid. A stepwise gradient elution program was adopted: 18 - 38% A (0-13 min), 38-
60% A (13-20 min), 60 - 100% A (20-22 min). The system was equilibrated with 100% of A, for
another 1 min, at the end of each run.
The flow rate was adjusted at 1 ml/min and the UV detector set at 325 nm. Aliquots (20 μl, each)
of the working solutions were directly injected into the HPLC system for each determination. All
injections were repeated in triplicates.
These conditions were adjusted as those adopted during HPLC screening and isolation of the
constituents of the ethyl acetate/ n-butanol (2:1) and methanol fractions (page 154). The HPLC
chromatograms of the parent EE, its analyzed fractions and isolated clerodermic acid are
represented in figure 72.
Figure 1: HPLC fingerprints of the EE, its dichloromethane (DCM) and ethyl acetate/ n-
butanol (2:1) (EtOAc) fractions
Standardization of the Extractives
A standard stock solution of clerodermic acid in methanol (1.5 mg/mL) was diluted to yield a
series of seven different concentrations (12, 24, 36, 48, 60, 120 and 300 μg/mL) covering the
concentration range required, based on the level expected in the plant sample. An aliquot (20 μL)
of each prepared dilution was injected in triplicates and corresponding peak areas recorded.
The calibration curve (Figure 73) was then constructed by plotting mean peak areas versus
corresponding concentrations. Results are recorded in table 45.
Table 1: Average peak areas of the different concentrations of clerodermic acid

Concentration (μg/mL) Peak area*

12 101.5
24 236
36 273.5
48 372.5
60 447.5
120 988
300 2401.5
*Average of three determinations

Figure 2: Standard calibration curve of clerodermic acid

The content of clerodermic acid in each extractive was then determined from the pre-established
standard calibration curve and results recorded in table 46.
Table 2: Contents of clerodermic acid in the different extractives of the leaves of C. inerme

Extractive Content of clerodermic acid* (mg %)

EE 9.76
DCM 70.7
EtOAc/BuOH 46.6
*Average of three determinations
EE, ethanol (70%) extract; DCM, dichloromethane fraction; EtOAc/BuOH, ethyl acetate/ n-butanol (2:1) fraction

Validation of the Proposed Standardization Method

The proposed method was subjected to elementary validation studies according to the guidelines
of the International Conference of Harmonization (ICH, 2005) and United States Pharmacopoeia
(USP, 2009). The following validation characteristics were examined: specificity, linearity,
precision, accuracy, limit of detection, limit of quantification, and sample stability testing. The
isolated clerodermic acid was used as standard.
A. Specificity
The specificity is the ability to detect the analyte peak in the presence of other components or
impurities. Specificity was determined by performing three injections: the first with the sample
(working solution), the second with the standard (isolated clerodermic acid) and the third with the
solvent (blank). All experiments were performed in triplicates. No interference was observed for
the main peak with other peaks detected in the chromatogram.
B. Linearity
Linearity is the ability (within a given range) to obtain a response or signal with a magnitude (y =
peak height or peak area) directly proportional to the amount (C = concentration) of the analyte in
the sample (ICH, 2005).
Linearity was assessed by linear regression analysis, which was calculated by the least square
method. As shown previously in figure 73, page 186, the Correlation Coefficient (R2) for the
standard calibration curve was 0.999. This indicates linearity of the peak area of clerodermic acid
in the range of 12-300 μg/mL.

Correlation coefficient (R2) = 0.999; Regression equation: y = 8.0025x + 2.7162

C. Accuracy
Accuracy expresses the closeness or agreement between the accepted value, either as a
conventional true value or an accepted reference value and the test results (ICH, 2005); this is
determined by comparing the results obtained for the analyzed samples with the calculated
amounts. Recovery experiments were thus performed to evaluate the accuracy of the method. The
bias of the analytical procedure was investigated during the method validation study using the
recovery data of the tested extract spiked with the standard.
Three individual samples of the ethanol (70%) extract (EE, working solution, 2.5 mg/mL) were
spiked with three concentration levels of clerodermic acid (24, 48 and 120 μg/mL). The spiked
samples were analyzed in triplicates. Accuracy was determined by calculating the difference
between the average value found during analysis and the theoretical amount. Accuracy was
expressed as mean percent recovery at the three concentration levels examined. The % RSD was
calculated and results, presented in table 47 and figure 74, indicate that the data obtained were
within acceptance criteria (98-102%).

Figure 3: HPLC chromatograms of the EE of the leaves of C. inerme, isolated Clerodermic

acid & EE spiked with Clerodermic acid
Table 3: Accuracy study of the proposed standardization method

Clerodermic Added *Theoretical Detected

acid in clerodermic clerodermic clerodermic Recovery
%RSD
extract acid acid acid %
(μg/mL) (μg/mL) (μg/mL) (μg/mL)
244 24 134 136 3.8 101.49
244 84 146 145 2.3 99.5
244 120 182 179 1.4 98.4
*Mean of clerodermic acid concentration in the extract + added pure clerodermic acid concentration

D. Precision
Precision expresses the closeness of agreement (degree of scatter) between series of measurements
obtained from multiple sampling of the same homogeneous sample under the prescribed conditions
(ICH, 2005). Precision of the assay is determined by repeatability (intra-day variability) and
intermediate precision (inter-day variability).
1. Repeatability (intra-day variability)
Repeatability was estimated by analyzing two different concentrations (36 and 60 μg/mL) of
clerodermic acid six times in the same day. Repeatability was expressed in terms of relative
standard deviation (% RSD) of the peak area and results are recorded in table 48.
Table 4: Repeatability for retention time and peak areas expressed as % RSD

C3 (36 μg/mL) C5 (60 μg/mL)

Run Peak Area Peak Area
Rt (min) Rt (min)
(mAU) (mAU)
1 22.31 284 21.95 442
2 22.1 281 22 458
3 22.28 276 22.07 462
4 22.27 282 22.11 466
5 22.23 272 22.16 454
6 22.19 284 22.31 465
Mean ± SD 22.23 ± 0.076 279.83 ± 4.833 22.1 ± 0.127 457.83 ± 8.95
% RSD 0.343 1.727 0.577 1.956
SD= standard deviation; % RSD= relative standard deviation
C3, C5= Clerodermic acid concentrations (36, 60 μg/mL respectively)

2. Intermediate precision (inter-day variability or ruggedness)

Intermediate precision was determined by analyzing, in triplicates, two concentrations of
clerodermic acid (36 and 60 μg/mL) on three different days. Intermediate precision was expressed
in terms of relative standard deviation (%RSD) of peak area, and results recorded in table 49.
Table 5: Intermediate precision for peak areas of two concentrations of clerodermic acid
over three days

Peak Area* (mAU)

Day
C3 (36 μg/mL) C5 (60 μg/mL)
1 273 447.5
2 285 444
3 274 450
Mean ± SD 277.33 ± 6.658 447.17 ± 3.014
% RSD 2.401 0.674
*Average of three determinations
C3, C5= Clerodermic acid concentrations (36, 60 μg/mL respectively)
SD = standard deviation; % RSD = relative standard deviation
The relative standard deviation values of the repeatability and intermediate precision were less
than 2.00 and 3.00 %, respectively and demonstrate that the HPLC method is precise.
E. Suitability range
The range of suitability of a given analytical procedure is the interval between the minimum and
maximum concentrations of the compound to be determined in which the linearity is observed
(ICH, 2005), the characteristics of repeatability limits and the accuracy is maintained at a
sufficiently high level. The range of purity of the method has been set at concentration range of
12-300 μg/mL of the standard compound, since the method has been shown to be precise, accurate
and linear within this range.
F. Limit of Detection (LOD) and Limit of Quantification (LOQ)
The limit of detection (LOD) is the lowest concentration of an analyte that the analytical process
can reliably differentiate from the background level, at a signal-to-noise (S/N) ratio of 3:1. The
limit of quantification (LOQ) is the minimum concentration of the analyte that can be determined
at an acceptable precision and accuracy under rated conditions of analysis. It is usually regarded
as the amount for which the signal-to-noise (S/N) ratio should not be less than 10:1 (ICH, 2005).
LOD and LOQ were estimated based on the standard deviation (SD) of the response and the slope
of the linearity curve.

Limit of Detection (LOD) = SD × 3/S

Limit of Quantitation (LOQ) = SD × 10/S
Where:
SD = standard deviation of the y intercepts of regression lines
S = slope of the linearity curve obtained by regression analysis
The LOD and LOQ of clerodermic acid were found to be 1.12 and 3.39 μg/mL, respectively.
G. Robustness
The robustness of an analytical procedure is the characteristic of its stability with respect to small
variations of the different system parameters (main system factors e.g. flow rate, pH,
temperature…, etc.) possible under real conditions (ICH, 2005).
Preliminary testing of the robustness of the proposed method was established by making deliberate
minor variation in the flow rate employed.
Flow rate was changed from 1 mL/min to 0.9 and 1.1 mL/min and %RSD was calculated for one
clerodermic acid concentration (36 μg/mL). The chromatographic elution pattern remained
unaffected and the low values of RSD established the robustness of the method. Results are
presented in table 50 and figure 75.
Table 6: Results of robustness study for the proposed standardization method for analysis of
clerodermic acid based on minor variation in flow rate

Flow rate Mean Peak Mean Rt* ± SD

% RSD % RSD
(mL/min) Area* ± SD (min)
 1 280.67 ± 4.163 1.483 22.29 ± 0.021 0.093
0.9 281.33 ± 2.082 0.739 22.32 ± 0.006 0.026
1.1 275.33 ± 3.055 1.109 22.3 ± 0.017 0.078
*Average of three determinations
SD= standard deviation; % RSD= relative standard deviation

Figure 4: HPLC chromatograms of isolated Clerodermic acid at different flow rates (1, 0.9
& 1.1 mL/min)
Criteria for validation of the quantitative RP-HPLC-UV method proposed for standardization of
the EE of the leaves of C. inerme, using the isolated clerodermic acid as standard, are collectively
recorded in table 51.
Table 7: Criteria for validation of the HPLC-based analytical procedure proposed for
standardization of the extractives of the leaves of C. inerme

No. Parameter Acceptance criteria Results

No interference with other

1 Specificity Passes
peaks
2 Linearity R2 = 0.995 to 1 0.999
3 Accuracy Recovery = 98-102 % 98.4 to 101.49
Precision
4 Repeatability RSD < 2% 0.343 to 1.956
Intermediate Precision RSD < 3% 2.401 and 0.674
5 Suitability Range ------ 12-300 μg/mL
6 LOD ------ 1.12 μg/mL
7 LOQ ------ 3.39 μg/mL

8 Robustness RSD < 3% 0.026 to 1.483

Conclusion
Analysis was performed on a LiChrospher 100 RP-C18 column by stepwise gradient elution using
appropriate acetonitrile: 0.3% phosphoric acid mixtures with the UV detector set at 325 nm. The
conditions of the developed RP-HPLC/UV standardization method were optimized using the
isolated clerodermic acid as standard.
The method was validated and all parameters were found within acceptance limits. The method
was found to be simple, precise, linear, accurate, reproducible, selective, robust, stable and time
saving and thus reliable for identification and quantitation of clerodermic acid in the ethanol extract
(EE) of the leaves of Clerodendrum inerme.
The contents of clerodermic acid in the parent EE, and its dichloromethane and ethyl acetate/ n-
butanol (2:1) fractions, evaluated by the proposed method, were 9.76, 70.7 and 46.6 mg% (w/w
relative to the dry weight of the individual extractives), respectively.