This Information Applies To: Agilent MassHunter Quantitative Analysis "Quant-My-Way" user interface (version B.09.00 or later)
Issue
To process the data files in the batch and obtain quantitative results, you must either create a new quantitative method or use a previous quantitative method and then apply it to the batch.
Steps to follow
1. Create a new batch and add the applicable data files (Add Samples) to begin the quantitative analysis then initially save the batch. Refer to How to Create a New Batch in Agilent MassHunter Quantitative Analysis to create a new batch.
2. To define calibration standards in the batch table, set the Type to Cal and set the Levels (Level ID) in the batch table (see Figure 1).
The Level (Level ID) if set as a number will also set the numeric value for the Conc. field for all the compounds in that Level in the Calibration Table if the Level contains text the numeric value for the Conc. field for all the compounds in that Level will be set to 1.00.
3. While still in the Batch view, select a representative calibration standard, see Figure 1. As an example the selected calibration standard data file is STD3. Common practice is to select a midrange calibration standard.
Select Method > New > New Method Using Manual Setup in the main Quantitative Analysis screen (see Figure 2) to switch to the Method Edit view.
Figure 2. Create new method option
4. Use the Sample Information window in the Method Edit view to manually add new compounds to the method.
a. Zoom (right-click and drag) the chromatogram for the first compound of interest.
b. Left-double-click the peak apex to obtain the apex retention time and spectrum.
c. Zoom (right-click and drag) the spectrum around the quantifier ion, left-click directly above the quantifier ion to select the ion as the quantifier. The ion will be labeled with a Blue triangle.
d. Right-click the Blue triangle and select New Compound.
e. Enter the Compound Name in the Compound Setup table.
f. Zoom (right-click and drag) the spectrum around the qualifier ion, left-click directly above the qualifier ion to select the ion as the qualifier. The ion will be labeled with a Blue triangle.
g. Right-click the Blue triangle and select New Qualifier – repeat step f. and g. for the remaining qualifiers.
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Note: If the data was acquired within Scan Mode and the Sample Information window contains the Total Ion Chromatogram (TIC), after step 4b. you can right-click the spectrum and select Search Library to library search against either a large commercial library (e.g. NIST17.L) or an application-specific user created library to assist in confirming the peak’s identity.
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Repeat the steps above to add all the compounds in this calibration standard (click the Sample Information region and click the double-headed arrow icon to zoom out the window, then continue to create another compound), or see the following video to create new compounds (see Figure 3)
Figure 3. Video - Create new compound in Method Edit view
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Note: For highly complex chromatograms selecting a chromatographic peak apex may generate a spectrum from coeluting or overlapping compounds with ions from more than one compound, which if library searched may provide ambiguous results (Scan mode data). Try a left-click and drag from several peak widths before to several peak widths after the chromatographic peak to select a time range (blue highlight) and then right-click that range and select Deconvolution.
If there is more than one compound present with several unique ions in the selected time range multiple Retention Time and Deconvoluted Spectra should be generated. Each of those spectra can be then library searched to assist in confirming those deconvoluted chromatographic peaks identities. Also the ions present in each of the deconvoluted spectra are relatively unique to that particular deconvoluted compound retention time and may provide quantifier and qualifier ion choices with less overlapping compound interference. |
Figure 4 demonstrates how to create a new compound for a highly complex chromatogram using deconvolution:
Use the MassHunter\Data\QuantExample\MS\VOA\CAL_L07.D data file for the TIC peak / Scan at 10.337 min for the following example.
When mixed spectra of Tetrachloromethane and Benzene are Library Searched against NIST17, mixed matches at a Match Factor of ~63 to 65 are returned for each of these two compounds.
After Deconvolution you should get two compounds.
Figure 4. Video Co-eluting / Overlapping compound Deconvolution
5. Compound Setup – all parameters have been created from Step 4. (see Figure 5).
Review that the default setting for Criteria of Close RT is applicable to your application or laboratory protocol.
Criteria (Peak Selection Criterion) represents the ranking logic to select the peak in the integrated quantifier signal as the primary hit (top ranked).
Greatest Response ranks the peaks for the integrated quantifier signal by the greatest response, which determines the primary hit (top, ranked), irrespective of the presence, quality, and fidelity of integrated qualifier signal ratios.
Close RT ranks the peaks for the integrated quantifier signal by closest to expected retention time, which determines the primary hit (top, ranked), irrespective of the presence, quality, and fidelity of integrated qualifier signal ratios.
Close RT with Qualifiers ranks the peaks for the integrated quantifier signal by closest retention time with expected integrated qualifier signal peak ratio limits met which determines the primary hit (top ranked). If there are no integrated quantifier signal peaks with expected qualifier peak ratio limits met the rank will be equivalent to Close RT.
Greatest Q-Value ranks the peaks for the integrated quantifier signal by the best match of the expected integrated qualifier signal peak ratios from their QVALUE. The QVALUE is a measure of the presence, quality, and fidelity of the integrated qualifier signal ratios.
6. Qualifier Setup - all parameters have been created from Step 4. (see Figure 6).
Uncertainty type - Percentage Uncertainty, set as Relative or Absolute Uncertainty.
For example, 80% Relative Response, 20% Uncertainty.
Absolute = 80% +/- 20% = Range of 60 to 100 %.
Relative = 80% +/- ((80*20)/100)% = range of 64 to 96%.
Rel. Resp. - Expected Qualifier Ion response as Percentage of Quant Ion.
Uncertainty - value—+/- % limit range.
7. Retention Time Setup – all parameters have been created from Step 4, except for the Left / Right RT Delta, which is set to the default, +/- 1.000 Min (see Figure 7).
RT – Expected Retention Time for the Compound
Left RT Delta – Signal baseline before the Expected RT of the Compound
Right RT Delta – Signal baseline after the Expected RT of the Compound
RT Delta Units
Minutes – Minutes (e.g. for compound AA at 7.56 minutes and a +/- of 1.00 minutes the extracted signal will be from 6.56 to 8.56 min.)
Percent – Percentage (e.g. for compound AA at 7.56 minutes and a +/- of 10 percent the extracted signal will be from 6.804 to 8.316 min.)
Minutes would be the usual setting for chromatograms with similar chromatographic peak widths across the chromatogram such as in temperature programmed gas chromatography separations.
Percent would be the usual setting for chromatograms with increasing chromatographic peak widths across the chromatogram such as in isothermal gas chromatography separations.
The compound’s RT Window is defined by the Left / Right RT Delta, and needs to be wide enough to allow the integrator to identify chromatographic baseline either side of the chromatographic peak for that compound. The integrator must always be able to accurately determine the peak start, peak apex, and peak stop. Do not overly reduce the RT window in an attempt to increase compound selectivity. Generally the RT window should never be less than 3X the peak width at baseline (e.g. for a 6 second / 0.1 minute wide peak at baseline an 18 second / 0.3 minute RT Window would be the practical minimum). To improve compound identification selectivity, adjust the Globals > Non-Reference Window (for non-ISTD compounds) and Globals Reference Window (for ISTD compounds). This will improve rejection of close eluting peaks and if the RTs are stable can be set to less than the chromatographic peak width (e.g. for a 6 second / 0.1 minute wide peak at baseline with stable RTs 6 seconds / 0.1 minutes would significantly improve rejection of close eluting peaks).
Figure 7. Retention time setup
8. ISTD Setup – For compounds requiring ISTD quantitation, each compound must be assigned an ISTD (see Figure 8).
For each ISTD Compound set the Type to ISTD and check the ISTD Flag plus enter the ISTD Conc. and optionally check the Time Reference Flag.
For each Compound requiring ISTD quantitation set the ISTD (ISTD Compound Name).
In Figure 8, Compounds CC and DD have been set as ISTD’s and Compound AA set to use Compound CC as its ISTD and Compound BB set to use Compound DD as its ISTD.
9. Concentration Setup – Sets the concentration of each compound for the calibration standards that will be processed.
Where the Batch Table contains calibration standards that have their Sample Type set to Cal. and the Compound Method Expected Concentration (Exp. Conc.) entered, then this information can be used to easily create a populated calibration table.
Click Method > Calibration Curve > Create Levels from Calibration Samples (see Figure 9). The Level IDs are populated from the Cal. Sample Type rows in the batch table. If Level ID is a numeric value it will also populate the concentration field.
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Note: For compounds that are being ISTD quantitated, MassHunter Quantitative Analysis requires exact matching of all calibration Level IDs between those non-ISTD compounds and their corresponding ISTD compound.
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If a compound has the Enable box unchecked, then that calibration level will not be included in the calibration curve calculations.
Figure 9. Create levels from calibration samples option
If some or all compound concentrations are the same, then they can be copied to other compounds by right-clicking the Calibration Table and selecting Copy Calibration Levels To… (see Figure 10).
Figure 10. Copy calibration levels to...option
In the Copy Calibration Levels To dialog box you can select one (click) or more compounds (shift + click) or Select All and click Ok complete the copying of those levels to the selected compounds(see Figure 11).
Figure 11. Select compound to copy calibration levels
10. Calibration Curve Setup – There are many combinations of Curve Fit (CF) type, Curve Fit Origin (CF Origin), and Curve Fit Weighting (CF Weight) available. The default settings are CF Linear, CF Origin Ignore, and CF Weight None (see Figure 12).
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Note: If your application has only a single calibration level, then CF Origin must be set to Force to display a calibration line for any Curve Fit.
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Figure 12. Calibration curve setup
11. Globals Setup – Review the default settings as applicable to your application or laboratory protocol in particular in relation to the following quantifier and qualifier integrated peak recognition settings (see Figure 13).
Correlation Window determines if peaks from integration of the quantifier and qualifier signals are due to the same chromatographic elution (i.e., set the time range limit to be a match for retention time). The units are minutes with a default of 2 minutes. Typically when optimized this is set to the chromatographic peak width at baseline or less based on your application or laboratory protocol.
Non Reference Window determines if peaks from integration of the quantifier and qualifier signals are within an acceptable retention time limit (window) to the expected retention time or adjusted expected retention time (if using Time Reference ISTD for that compound). The units are in percent or minutes and the default is 200 percent. Typically when optimized this is set to a small multiple of the chromatographic peak width at baseline or less based on the application or laboratory protocol.
Reference Window determines if ISTD peaks with the Time Reference Flag set from integration of the ISTD quantifier and ISTD qualifier signals are within an acceptable retention time limit (window) to the expected retention time. The units are in percent or minutes and the default is 80 percent. Typically when optimized this is set to a small multiple of the chromatographic peak width at baseline (often larger than the Non Reference Window) or less based on the application or laboratory protocol.
12. Click Method Tasks > Save / Exit > Validate– this will check the method to make sure that all necessary fields are correctly populated. The Method Error List window is displayed at the bottom of the screen which will display errors and warnings. All errors must be fixed before the method can be applied to the batch. For those errors listed, double-click each of the lines in the Method Error List window. The field in the Method Table window which contains the error is displayed (see Figure 14). After fixing all the errors, click Validate once again to ensure that no errors or problematic warnings are remaining.
13. Method Tasks > Save / Exit > Exit – Exit the Method Editor and select whether to Apply Method to the batch and Additional batch processing after applying the method options.
Select Analyze and click Yes to apply the method to the batch and return to Batch View (see Figure 15).
If the batch contains Calibration (Cal), Quality Control (QC) or Continuing Calibration (CC) Sample types then Analyze batch applies these to the Calibration Table. If they already exist in the calibration table from the same data file, then the previous response is replaced (e.g. when changing integrator types or integrator settings that change the response values).
Yes – Apply to batch and return to Batch View.
No – Discard any changes, don’t apply method to batch.
Cancel – return to Method Editor.
Analyze - fully rebuild the calibration curve (replace responses and curve fits) if the Batch contains Cal, QC or CC sample types and then recalculates compound concentrations.
Quantitate partially rebuild the calibration curve (does not replace responses) but calculates compound concentrations based on the existing calibration curve responses, enabled levels, and the edited method curve fit type, curve fit origin and curve fit weighting.
Integrate simply determines the responses for compounds in the batch. It does not apply the calibration curve fit to obtain calculated concentrations.
If None is selected the batch will need to be either Analyzed or Quantitated once back in Batch View.
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Note: In MassHunter Quant B.07.00 or later, if the batch contains any calibration samples (Cal only not CC or QC) Analyze Batch will Clear Calibration for all the existing batch method calibration levels (clear all the calibration responses but the previous calibration levels will still be present in the calibration table).
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The Calibration Curve is recalculated based on those calibration samples contained in the batch at the time of the Analyze Batch occurring. A single calibration sample (Cal. sample type) in the batch will change the batch method to a single point calibration.
To preserve the previous calibration responses and calibration curves use Quantitate Batch or if a calibration sample is being used to verify the current calibration and you need the responses to be added to the calibration table then change the type to CC or QC prior to Analyze Batch. The calibration curve equation only uses the Cal sample types in the calibration table not the CC and QC sample types, even though they are present in the calibration table from Analyze Batch.
14. The compounds Calculated Concentration (Calc. Conc.} are calculated from the current calibration curve settings and displayed in the batch table (see Figure 16).
If required you can also calculate and display the Final Concentration (Final Conc.), to account for sample preparation procedures with use of Dilution (Dil.), Sample Amount (Amt.) and Total Sample Amount (Tot. Amt.) according to How to Calculate the Final Concentration in MassHunter Quantitative Analysis.
Figure 16. The quantitative result
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