Targeted quantitation using HPLC IR and UV acquisition

This tutorial walks you through the workflow for analyzing targeted HPLC data starting from input file generation, to processing the data in SmartPeak, to reviewing the data in SmartPeak, to reporting the results.

Objectives

1. Obtaining the SOP for the workflow.

2. Choosing a data set for demonstrating the workflow.

3. Creating an optimized SmartPeak input templates for running the workflow.

The Workflows include

1. Calculating the calibration curves to generate quantitation methods for each component using Standard samples

2. Processing Unknown samples using the quantitation methods

Notes

Due to the non-standard formats used by HPLC and GC vendors, customized raw data file parsing routines are needed. Currently, Thermo HPLC text file inputs are supported. Additional vendor input files can be supported upon request.

Steps

The tutorial includes the following steps :

1. Setting up the input files

The data set used can be found in HPLC_UV_Standards and HPLC_UV_Unknowns for the HPLC UV Standards and HPLC UV Unknowns respectively.

1. Defining the workflow in SmartPeak

For HPLC UV Standards analysis, the following steps are saved into the workflow.csv file. Alternatively, steps can be replaced, added or deleted direclty from SmartPeakGUI. A detailed explanation of each command step can be found in Workflow Commands.

workflow_HPLC_UV_Standards.csv

workflow_step
LOAD_RAW_DATA
MAP_CHROMATOGRAMS
EXTRACT_CHROMATOGRAM_WINDOWS
ZERO_CHROMATOGRAM_BASELINE
PICK_MRM_FEATURES
CHECK_FEATURES
SELECT_FEATURES
CALCULATE_CALIBRATION
STORE_QUANTITATION_METHODS
QUANTIFY_FEATURES
STORE_FEATURES

The calibration curve for each transition’s quantitation method can be inspected after all workflow steps have been run, to do so please click on view and then “Calibrators”. From the transition tab select Antranilicacid and Indole as transition_group to plot their concentration curves within the given concentration range as shown below:

To inspect the features for the selected transition groups, select “Features (line)” from the view menu then open the features tab (can be opened from the view menu as well) to select the “asymetry_factors” and “logSN” in the plot column. The line plot illistrates the value for each transition group and feature as shown below:

The features can also be plotted as a heatmap, under “view” select “Features (heatmap)” then select the “left_width” feature to display transition groups as a heatmap and compare the values from the same injection as shown below:

The workflow step STORE_QUANTITATION_METHODS writes the calibration model for each transition, an excerpt can be seen below:

Generated sequence1_quantitationMethods.csv

IS_name	component_name	feature_name	concentration_units	llod	ulod	lloq	uloq	correlation_coefficient	n_points	transformation_model	transformation_model_param_y_weight	transformation_model_param_y_datum_min	transformation_model_param_y_datum_max	transformation_model_param_x_weight	transformation_model_param_x_datum_min	transformation_model_param_x_datum_max	transformation_model_param_symmetric_regression	transformation_model_param_slope	transformation_model_param_intercept
_	Antranilicacid	intensity	ug/mL	0.0	0.0	0.5	2500	0.998679668124795	7	linear	ln(y)	-1.0e15	1.0e15	ln(x)	-1.0e15	1.0e15	FALSE	1.353587567241049	0.369814545757549
_	Indole	intensity	ug/mL	0.0	0.0	0.5	50.0	0.998763546720702	6	linear	ln(y)	-1.0e15	1.0e15	ln(x)	-1.0e15	1.0e15	FALSE	0.995574540930201	3.242340261658038

This file is used to apply the predefined calibration model to each transition by running the QUANTIFY_FEATURES workflow step.

The workflow steps for HPLC UV Unknowns are :

workflow_HPLC_UV_Unknowns.csv

workflow_step
LOAD_RAW_DATA
MAP_CHROMATOGRAMS
EXTRACT_CHROMATOGRAM_WINDOWS
ZERO_CHROMATOGRAM_BASELINE
PICK_MRM_FEATURES
QUANTIFY_FEATURES
CHECK_FEATURES
SELECT_FEATURES
STORE_FEATURES

To inspect the features for the selected transition groups, select “Features (line)” from the view menu then open the features tab (can be opened from the view menu as well) to select the “asymetry_factors” and “logSN” in the plot column. The line plot illistrates the value for each transition group and feature as shown below:

The features can also be plotted as a heatmap, under “view” select “Features (heatmap)” then select the “asymetry_factors” feature to display transition groups as a heatmap and compare the values from the same injection as shown below:

To plot the intensities over time for given injections and transitions, view the “chromatogram” from the “view” menu then select the injections and transitions to plot from their respective tabs on the left. The following shows the chromatogram for two injections using Antranilicacid and 5-HTP2 transitions and their intensity differences over time.

1. Running the workflow in SmartPeak

   To run the analysis, please follow the steps for Using SmartPeak GUI or Using SmartPeak CLI to execute the workflow steps, review the results, and report the results.

2. Reporting the results

   To export the results, select “Report” from the “Actions” which will show the “Create Report” window:

   

   Based in the data you wish to export, select the desired “Sample types” from the left pane and select the “Metadata” from the right pane then click on of the buttons below to create the report with the selected items in the csv format. More details on exporting the results can be found in Export report.