Ultra-performance Liquid Chromatography
5.3 Method Validation
RP-HPLC has been the technique of choice for both pharmaceutical and bioanalytical liquid chromatography–mass spectrometry–mass spectrom- etry (LC-MS-MS) analysis because of the high efficiency of the separations, the compatibility of the mobile phase with biological and lipophilic sam- ples and the easy interfacing with a variety of detectors (Wilson et al., 2005; Rainville et al., 2007).
Separation of the compounds present in the extract is achieved by meticulous assessment, as well as the selection of all separation-relevant parameters such as stationary phase and elution conditions. Preliminary experiments are carried out to optimize the experimental parameters af- fecting both the chromatographic separation of the target compounds in the column selected and their detection by UV or PDA.
After optimization of the HPLC parameters, the developed method is validated with regard to its specificity, linearity, accuracy and precision using the International Conference on Harmonisation (ICH) guidelines. The validity of an analytical method can be verified by establishing sev- eral analytical and statistical parameters. As the major components could be present in several closely related species, therefore, a validated analyt- ical procedure for the separation and quantification of all major activity related compounds is required. Such a method would not only facilitate the standardization of commercial products but also increase the sig- nificance of future pharmacological studies, since the chemically well- defined samples can be utilized (Ganzera et al., 2004). The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH, 2005) guidelines provide details of method validation.
The developed HPLC method is validated in the terms of the fol- lowing parameters: (i) specificity; (ii) selectivity; (iii) linearity; (iv) limit of detection and limit of quantification; and (v) precision expressed as repeatability of retention time, peak area and recovery.
5.3.1 Specificity
The ability of the analytical method to assess the analyte unambiguously in the presence of other components (impurities and degradants) can be demonstrated by evaluating specificity (Dongre et al., 2007).
Specificity requires the method to provide separation of the analyte from process impurities, degradation, excipients and other analytical arte- facts. The specificity of the method can be delineated by the resolution of the integrity of the peak determined by multiple UV wavelength detection;
that is, photodiode array detector or ratio between two wavelengths. How- ever, it may not always be reliable, as many degradants and metabolites may have chromophores similar to those of the analyte. The specificity of the method developed is confirmed by the reliability of the compound peaks corresponding to the standard compounds in the sample.
Further, the robustness of the method is verified by carrying out ex- periments on instruments of different make. The reliability of the method is determined by making small changes in chromatographic conditions, such as the composition of the mobile phase (+5%), pH (+0.1%), etc. (Dey
et al., 2012). 5.3.2 Linearity
The linearity of the method is checked for the standard compounds with their respective calibration curves in the different concentration range, depending on the limit of detection and the limit of quantification. The regression equation and correlation coefficients are obtained with a min- imum of six replicate analyses corresponding to each concentration of a minimum of six concentration levels. The value of the correlation coeffi- cients should be greater than 0.99.
5.3.3 Limit of detection (LOD) and limit of quantification (LOQ)
Limit of detection (LOD) is defined as the lowest amount of sample con- centration that can be detected (signal-to-noise ratio = 3), and the limit of quantification (LOQ) is defined as the lowest amount of sample concen- tration that can be determined quantitatively with suitable precision and accuracy (signal-to-noise ratio = 10). Both LOD and LOQ are also calcu- lated according to the following formula:
LOD = 3.3 ´ (s/s), LOQ = 10 ´ (s/s), (5.6) where s = standard deviation of the blank response and s = the slope of the calibration curve.
5.3.4 Precision and accuracy
The precision of the method is validated by both intraday and interday variations. To determine the intraday variation, six assays are carried out on the same sample at different times during the day. Interday variation is determined by analysing on the next day. Intraday and interday precision of the assay are expressed as relative standard deviation (RSD). RSD is cal- culated as RSD = standard deviation (s)/mean and is taken as a measure
Accuracy of the developed HPLC method is determined by spiking the sample with a known amount of the standards at three concentration levels (low, medium and high) in the calibration range. Spiked samples are analysed under optimized conditions and recovery is obtained.
5.3.5 Lack-of-fit test
The lack-of-fit test is the proper test for linearity. There is always a risk that a regression model is a poor approximation of the true functional relationship between a set of data X and Y. This test is performed to support the adequacy of the regression model and is based on the hy- pothesis that the linear model fits the data (H0) adequately or the linear
model does not fit the data (H1). The test involves the partioning of the
residual sum of squares (SE) into the sum of squares of the lack of fit to the regression model. If the null hypothesis is rejected, the model must be abandoned since no linear relationship exists between the two variables. If the null hypothesis cannot be rejected, then there is no apparent reason to doubt the adequacy of the regression model, so lin- earity is implied.
5.4 Ultra-performance Liquid Chromatography
Using ultra-performance liquid chromatography (UPLC), it is now pos- sible to take full advantage of chromatographic principles to run separ- ations using shorter columns and higher flow rates for increased speed with superior resolution and sensitivity. Faster separations can lead to higher throughput and time savings when running of large samples is re- quired. However, resolution in UPLC separation can reduce method de- velopment time from days, to hours or even minutes.
UPLC operates on the basics of HPLC with a column of submicron particle size (less than 2 μm) and high-pressure flow. Because of smaller particle size, higher back pressure is generated. Therefore, it becomes necessary to have a chromatography system that can operate at a pres- sure of 10,000 psi. As compared to particle size of 5 or 3 μm stationary phase, higher resolution and sensitivity can be achieved with smaller particles (1–2 μm). The increased resolution leads to better separation, increased sensitivity and faster analysis (Wilson et al., 2005; Rainville
et al., 2007). The need to handle elevated pressures in excess of 10,000
psi can be alleviated to some extent by the use of increased column temperature. An increase in temperature leads to a reduction in the viscosity of mobile-phase solvents. An increase in column temperature can result in selectivity and peak elution order. Increasing the column temperature also increases the optimum linear velocity required to run the column effectively.