For accomplishing optimal separation of all analytes in a short analysis time, UHPLC conditions such as the column, mobile phase, gradient elution program, flow rate, injection volume and column temperature were optimized. An Acquity UPLC BEH Cl8 column (50mm x 2.1 mm id, 1.7pm) was ultimately chosen for good separation efficiency and better peak shapes. Further optimization results showed that mobile phase system composed of 0.1% formic acid in water and 0.1% formic acid in acetonitrile achieved good separation along with better ionization at a flow rate of 0.3 mL/min and 50°C column temperature within 13 min.

The mass spectrometric conditions were optimized by direct infusion of 50ng/mL solution of each analyte into mass spectrometer at a flow rate of lOpL/min using a Harvard syringe pump (Harvard Apparatus, South Natick, MA, USA). MS spectra were recorded in both positive and negative ionization modes. Finally, negative ionization mode was selected due to the high signal sensitivity of all target analytes. Further to achieve most abundant, specific and stable MRM transition for each analyte, the compound dependent MRM parameters (DR ER CE and CXP) and source parameters (curtain gas, GS1, GS2 and ion source temperature) were optimized. The optimized UHPLC-MS/MS method in MRM acquisition mode was applied to quantify fifteen bioactive constituents in the six Ocimum species using andrographolide as an IS.

Analytical Method Validation For Quantitative Analysis of 15 Analytes in Six Ocimum Species

This UHPLC-QqQLn-MS/MS method for quantitation was validated according to the guidelines of International Conference on Harmonization (ICH, Q2R1, 2005) by determining linearity, LOD, LOQ, precision, accuracy and solution stability.

Linearity, LOD and LOQ

The stock solution was diluted with LC-MS grade acetonitrile to provide a series of concentrations in the range of 0.5-500 ng/mL for the construction of calibration curves. The linearity of calibration was determined using the ana-lytes-to-IS peak area ratios versus the nominal concentration, and the calibration curves were constructed with a weight (I Z.v2) factor by least-squares linear regression. The LOD and LOQ were determined based on calibration curve method by the following equations: LOD = (3.3 x Sxy)/Sa and LOQ = (10 x Sn.)/ Sa, where Sn, is the residual standard deviation of the regression line, and Sa is the slope of a calibration curve. All the calibration curves indicated good linearity with correlation coefficients (r2) from 0.9989 to 1.0000 within the test ranges. The LOD for 15 analytes varied from 0.041 to 0.357 ng/mL and LOQ from 0.124 to 1.082 ng/mL.

Precision, Stability and Recovery

The intra- and inter-day variations were checked to determine the precision of the developed method. It was completed by determining fifteen analytes with IS in six replicates during a single day and by duplicating the experiments on three consecutive days. Variations of the peak area were taken as the measures of precision and expressed as % RSD. The overall intra- and inter-day precisions were not more than 1.95%. Stability of sample solutions stored at room temperature was examined by replicate injections of the sample solution at 0,2, 4, 8. 12 and 24h. The RSD values of stability of the 15 analytes were <2.91%.

A recovery test was carried out to evaluate the accuracy of this method. The test was performed by adding known amounts of the 15 analytical standards at low, medium and high levels into samples. The spiked samples were analyzed at each level by the proposed method in triplicate, and average recoveries were determined. The analytical method developed had good accuracy with overall recovery in the range from 95.10% to 103.04% (RSD < 1.68%).

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