Agilent MassHunter Quantitative Data Analysis - Batch Table, Compound Information Setup, Calibration Curve and Globals Settings
Souhrn
Význam tématu
Quantitativní hmotnostní spektrometrie je základním nástrojem pro přesné stanovení koncentrací analytů v různých oblastech, od farmacie a environmentální analýzy po potravinářský a klinický výzkum. Správná kalibrace, identifikace a integrace signálů umožňuje dosahovat vysoce reprodukovatelných a validních výsledků, což je klíčové pro rozhodování v laboratorní praxi a regulaci kvality.
Cíle a přehled studie
Cílem prezentovaného webináře a přidruženého materiálu je představit komplexní prostředí softwaru Agilent MassHunter Quantitative Analysis a ukázat postupy optimalizace kvantitativní metody. Autoři popisují hlavní zobrazení Batch At a Glance, Method Editor a Compounds At a Glance, včetně nástrojů pro manuální integraci, tvorbu kalibračních křivek a globálních nastavení metod.
Použitá metodika a instrumentace
Softwarový balíček Agilent MassHunter Quantitative Analysis slouží pro zpracování dat z hmotnostních spektrometrů triple quadrupole (QQQ), time-of-flight (TOF) a kombinačních QTOF systémů. Hlavní kroky pracovního postupu:
- Batch At a Glance: přehled dávky vzorků, zobrazení výsledků koncentrací, výstrahy a odlehlých hodnot.
- Method Editor: úprava metodických úloh, nastavení extrakčních oken pro přesnou hmotnost (MZ), konfigurace prekurzorů a produktových iontů, definice vnitřních standardů, segmentace měření.
- Compound Information: detailní zobrazení chromatogramů analytů, automatická a manuální integrace, nástroje pro úpravu baselinu a anotace integrací.
- Calibration Curve a Curve Fit Assistant: tvorba a vyhodnocení kalibrační křivky, výběr nejlepšího modelu na základě R2, chyb standardního odchylky a % reziduí, log-log transformace a pásmo důvěry.
- Compounds At a Glance: rychlé přehledové zobrazení chromatogramů více analytů napříč vzorky, identifikace odlehlých signálů a export grafiky.
- Globals Settings: globální parametry metody jako bracketing (žádný, overlapped, sample group), dynamická subtrakce pozadí, multiplikační faktory pro ředění, standard addition, referenční knihovny spekter.
Hlavní výsledky a diskuse
Autoři demonstrují, jak efektivně využít prostředí MassHunter pro řízení dávkování vzorků, optimalizaci integrace a kalibrace. Rychlé filtrování a seskupování analytů i vzorků zvyšuje produktivitu při vyhodnocování rozsáhlých datových sad. Funkce Curve Fit Assistant umožňuje integrovat statistická kritéria výběru ideálního kalibračního modelu a vizualizovat pásmo důvěry, čímž se minimalizuje riziko systematické odchylky.
Přínosy a praktické využití metody
Popisovaná metodika nachází uplatnění v rutinní kvantitativní analýze QA/QC laboratoří, ve farmaceutickém vývoji pro studium stability a degradace léčiv, při monitorování environmentálních vzorků i v potravinářském průmyslu. Automatizované workflow a moduly pro manuální zásahy zajišťují vysokou flexibilitu a reprodukovatelnost výsledků.
Budoucí trendy a možnosti využití
Další rozvoj kvantitativního softwaru lze očekávat v oblasti umělé inteligence a strojového učení pro automatickou detekci a korekci šumu, rozšíření integrace cloudových platforem a LIMS systémů, podpora nových vysokorozlišovacích technologií a širší parametrizace metod pro multiatributovou analýzu proteinů.
Závěr
Agilent MassHunter Quantitative Analysis představuje robustní a modulární platformu pro komplexní kvantitativní zpracování hmotnostně spektrometrických dat. Nabízí nástroje pro optimalizaci metod od nastavení extrakčních oken až po pokročilé kalibrační algoritmy, čímž podporuje vysokou efektivitu a kvalitu výsledků v širokém spektru aplikací analytické chemie.
Obsah byl automaticky vytvořen z originálního PDF dokumentu pomocí AI a může obsahovat nepřesnosti.
Agilent MassHunter
Quantitative Data
Analysis
Presenters: Howard Sanford
Stephen Harnos
MassHunter Quantitation:
Batch Table, Compound
Information Setup,
Calibration Curve and
Globals Settings
1/17/2018
MassHunter Webinar Series
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MassHunter Quantitative Software
Review and Quant Method Optimization
Topics
• Brief Review
• Batch Table Navigation
• Compound Information
- Data review and manual integration
• Calibration Curve
• Working in Compounds At a Glance
• Method Editor Globals Setting
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Review
Three major views in MassHunter Quantitative Analysis.
• Batch At a Glance
• Method Editor
• Compounds At a Glance
Handling MS/MS data QQQ and QTOF.
Handling accurate mass data TOF and QTOF.
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MassHunter Quantitative Software
Batch-at-a-Glance View
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Batch Table
Calibration Curve
Compound Information
Method
Tasks
MassHunter Quantitative Software
Method Editor View
Method > Edit menu or F10 key
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Method Table
Sample
Information
Compound Information
MassHunter Quantitative Software
Compounds at a Glance View
View > Compounds-at-a-Glance
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Editing a Quantitation Method for TOF
Mass Extraction Setup
Method > Edit > Advanced Tasks > Mass Extraction Setup
Allows for a mass range for the extraction of the accurate mass (MZ).
Available MZ Extraction Window Units
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Editing a Quantitation Method for QTOF
Compound Setup & Mass Extraction Setup
QTOF is a combination of MS/MS and accurate mass data
Name
– Compound name
TS
– time segments may be multiple
Transition
– Precursor ion Product ion scan
Scan
– Product Ion
Type
– Target, ISTD, Surrogate or Matrix Spike
Precursor Ion
– mass of the ion
Product Ion
– mass of the ion for the target ion to monitor
RT
– retention time of compound of interest
Ion Polarity
– usually positive
Criteria
– Close RT, Close RT with Qualifiers, Greatest Response or
Greatest Q-Value
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Batch Table
Navigation
Click in a sample row to display data on that sample
Click on Next and Previous icons to move through a batch or use
Hotkeys:
Next Sample
= Alt + Down
Next Compound
= Alt + Right
Previous Sample= Alt + Up
Previous Compound
= Alt + Left
Compound list are frequently long, try the Compound drop down list to go
directly to the desired compound.
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Batch Table
Alphabetizing the Compound List
From within the Batch Table
right click, select Arrange
Compounds By > Name
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Batch Table
Messages and Outliers
Messages
Outliers
Red Outlier
– High
(above upper limit)
Select
Outliers
for Display
Blue Outlier
– Low
(below lower limit)
Hover cursor over the outlier or message to display details
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Batch Table Layout Modification
Add/Remove/Move Columns
Use the Add/Remove Columns function to
customize the Batch table.
Note that the columns of data in the Batch
Table are organized into sections.
Columns can be moved only within a section.
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Batch Table Layout Modification
Single and Multiple Compound Modes
Single Compound Mode
Multiple Compound Mode
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Flat Table compounds
across the top.
Batch Table Layout Modification
Compound Table Modes
Compound Table lists by compound rather than sample.
Select Flat Table to see list by sample.
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Batch Table
Compounds Groups
It may be useful to group compounds to organize Batch Table.
Look at parent compound and metabolites.
Group hydrocarbons to Group 1, aromatics to Group 2 and so forth.
Compound groups are generated in the Method Editor.
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Batch Table
Compounds Groups
Yields an Additional Toolbar.
Activated with a right click
to the right of the ‘flags’ or View
> Toolbars > Filtering.
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Batch Table
Samples Groups
Sample Groups are specified in the Batch Table through Add/Remove
Columns.
Each Sample is
assigned to a
group, then only
samples specific
to a group are
displayed and
evaluated.
Samples groups are distinctively different from compound groups.
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Batch Table Layout Modification
Format Columns
How to change the
number of decimal
places.
Alter number formats
1) Exponential
(scientific notation)
2) Fixed point
3) General
4) Significant Figures
Alter Date formats
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Batch Table Layout Modification
Context Menu Properties
Change the Font size
Change the Color scheme
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Export Batch Table
Easy to layout columns and format data, then export into Excel.
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Let’s take a moment
for questions on
Batch Table
Navigation.
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MassHunter Webinar Series
Time for a demo on
Batch Table
Navigation
Next up:
Compound Information
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MassHunter Webinar Series
Compound Information
Target
Quantifier
ISTD
Quantifier
Target
Qualifiers
ISTD Qualifiers
Target
Spectrum
ISTD
Spectrum
Display and access one compound in one sample at a time.
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Compound Information - Toolbar
Sample
and
Compound
Navigation
Next /
Pervious
Un-zooms,
Peak scaling,
Auto Scales x
and y axes
Manual Integration
Functions
Display/Hide
Target,
Qualifiers,
Spectra,
ISTD
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Manual Integrations and MI Toolbar
Click on the Manual Integration icon to place Compound Information in
Manual Integration mode.
Each signal is placed in its own window (no overlay of qualifiers).
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Manual Integration Toolbar
Enable Manual Integration
– this
tool activates the manual integration
toolbar
.
Clear Manual Integration
– this tool
becomes active once a manual
integration is present.
Zero Peak
– this tool sets start and
stop to the same value.
Merge Right/Left Peak
– this tool
merges the peak to the right/left of
the selected peak.
Split Peak and Pick Right/Left
–
this tool splits the peak and then
selects the right or left peak.
Snap Baseline
– this tool places
start/stop integration points on the
baseline.
Drop Baseline
– this tool finds the
lowest end of the peak, then drops
the baseline on the other side of the
peak from it, thereby creating a flat
baseline that avoids negative area.
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Compound Information
Peak Annotation
Activated from Tools > Add-
Ins…
NI
– The peak was not integrated at all by the
computer software.
LT
– The peak in question was inappropriately
integrated to an area less than what it should be
(e.g., Peak area was cut).
GT - The peak in question was inappropriately
integrated to an area greater than what it should be
(e.g., Peak Tailing).
BA
– The baseline had to be adjusted correctly by the analyst.
CO
–The analyst had to split to co-eluting peaks apart that were not (or could
not be) separated by the computer system.
RT
– The retention time for the peak in question has shifted from the expected
retention time.
INT
– There was electronic interference (e.g., Noise).
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Compound Information
Peak Annotation
The editable file is located in C:\Program
Files\Agilent\MassHunter\Workstation\Quant\bin\AddIns\PeakAnnotations.xml.
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Compound Information
Peak Annotation
The Peak Annotation can also be displayed in the Batch Table.
When the batch is saved, the values are retained.
The MI flag is checked in the Compound
Results.
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Compound Information
Context Menu Integration Parameters
Right click to expose the
context menu.
Most of the features are toolbar
icons.
Integration Parameters can be
used to change the integration
parameters.
Applies ONLY to this compound
in this sample.
Restore Integration Parameters
reverts to the method
integration values.
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Compound Information
Context Menu Properties
Properties allows for
customization of the display.
Can change Fill colors.
Can change Peak labels.
Can change Titles.
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Compound Information
Context Menu Properties
Qualifier Properties can be
changed.
Spectrum Properties can be
altered.
Manual integration
parameters can be varied.
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Signal to Noise
Five algorithms are available
Consult online help for more
information on the algorithms.
Noise regions can be
automatically determined or
individually specified.
The Noise regions can be
displayed in the Compound
Information window under
Properties > Compound
Information (1) > Baseline
Calculation Points.
In this example, 2 noise regions
were determined
—one before
the peak and the other after.
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Let’s take a moment
for questions on
Compound
Information
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MassHunter Webinar Series
Let’s take a moment
for a demo on the
features of Compound
Information
Next up:
Calibration Curve and
Curve Fit Assistant
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MassHunter Webinar Series
Calibration Curve
View ISTD Responses
Change Curve Fit.
R2
Fit to
Levels
(Scaling)
Concentration can be set as relative (to ISTD) or actual.
QC Samples
CC Levels
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Calibration Curve
Disable calibration points
Click on calibration point once to disable and remove from curve.
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Curve Fit Assistant
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Curve Fit Assistant
Sorting Columns
Curve Fit Assistant calculates the mathematical curves.
Best curve fit may be ranked using by
R2, Standard Error, or Max % Residual.
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Curve Fit Assistant
Disabled Points
# of Disabled Points defaults to 3 but column can be filtered.
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Curve Fit Assistant
Confidence Band
Original Fit
(black)
Best Fit
(blue)
Confidence
Band
(blue)
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Calibration Curve
Log Log Plot
Logarithmic
Scale
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Curve Fit Assistant
Accept Assistant Curve
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Calibration Curve
Properties
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Let’s take a moment
for questions on
Calibration Curve and
Curve Fit Assistant
1/17/2018
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MassHunter Webinar Series
Time for a demo on
Calibration Curve and
Curve Fit Assistant
Next up:
Compounds at a Glance
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MassHunter Webinar Series
Compounds-at-a-Glance
High throughput data review environment.
View compounds across multiple samples.
View all compounds within a sample.
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Compounds-at-a-Glance
To start, select View > Compounds-at-a-
Glance…
Choose Layout > Predefined Layout
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Compounds-at-a-Glance
Setup Graphics Wizard
Specify Samples to view in
Compounds-at-a-Glance then
Compounds. By default, all samples
and all compounds are selected.
Specific samples and order can be
modified.
To customize select Layout >
Setup…
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Compounds-at-a-Glance
Setup Graphics Wizard
Define how to Organize the selected
compounds and samples.
Define the Overlay mode.
Review Mode
Sample by Sample
Compound by Compound
Compound Group by Compound
Group
Display Options
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Compounds-at-a-Glance
Setup Graphics Wizard
Finally, define which Outliers
should be highlighted…
Outliers can also be filtered by
Panes without outliers
Panes with outliers
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Compounds-at-a-Glance
Outliers
Outliers are
highlighted in red.
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Compounds-at-a-Glance
Manual Integration Pop Up
Double click for
single pane
access.
Can be accessed
without manual
integration being
activated.
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Compounds-at-a-Glance
Print Preview
Select File > Print Preview to create a chromatogram report.
Export Graphics
give a graphic
image in various
formats.
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Compounds-at-a-Glance
Properties
From the
context menu
select
Properties
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Compounds-at-a-Glance
Save Layout
Some settings in the
Layout screens are not
saved such as
compounds, samples, etc.
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Time for questions on
Compounds at a
Glance
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MassHunter Webinar Series
Let’s watch a demo
on Compounds at a
Glance
Next up:
Global Settings
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MassHunter Webinar Series
Globals Settings
Component of Method Editor.
Global parameters are critical to proper quantitation.
Globals are method parameters.
Globals apply to the whole batch
– all samples and all compounds.
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Globals Settings
Apply Multiplier’s
To avoid flagging target compounds that absent
None or Overlapped or Sample Group
Peak identification within the extraction window
Associates target and qualifiers in min
Semi quant relative to ISTD
Quantitate with Standard Addition
Peak identification within the extraction window
(ISTD with Time Reference Flag checked)
Spectral Reference Library (.reflibrary.xml)
Spectral Pattern Reference Library (.reflibrary.xml)
Part of unified method
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Dynamic Background Subtraction
Globals Settings
Calculated and Final Concentration
Calculated Concentration (Calc. Conc.)
—is the concentration of the
compound as calculated from the calibration curve.
Final Concentration (Final Conc.)
—is the concentration after the
Multiplier has been applied to the Calculated Concentration.
The equations are:
Final Concentration = Calculated Concentration x Multiplier
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Globals Settings
Multiplier
Multiplier = Dilution x Sample Amount Multiplier where
Sample Amount Multiplier = TotalAmt/Amt
All three of the factors are columns in the Sample section of the Batch Table.
By default, they are set to 1, either
explicitly (Dil.) or implicitly (Amt. and Tot. Amt.)
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Globals Settings
Multiplier
The columns (Dil., Amt.,
TotalAmt, and Multiplier) must
be added to the Batch Table.
The Multiplier in the Compound
Results represents the product
applied to the compound.
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No (None) Bracketing allows the user to have sample(s) quantitated
using the average of all the calibration standards in the batch without
regard to order.
[Cal Set A]
[Sample 1 Set]
Avg. Cal (A+B+C)
[Cal Set B]
[Sample 2 Set]
[Cal Set C]
[Sample 3 Set]
Globals Settings
Bracketing type = None
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Overlapped Bracketing allows the user to have sample(s) quantitated
using the average of the calibration standards injected both before and
after the samples injection.
[
Cal Set A]
[Sample 1 Set] Average Calibration (A+B)
[Cal Set B]
[Sample 2 Set]
Average Calibration (B+C)
[Cal Set C]
Globals Settings
Bracketing type = Overlapped
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Used with Stream Select (LC)
When the Bracketing Type Sample Group is implemented, only samples
from a specific Sample Group (specified in the Batch Table) are considered
in the formation of the calibration curve and quantitation of the samples. This
feature would accommodate slight shifts in retention time and/or variations
in instrument responses.
Added from Worklist or Batch Table.
Globals Settings
Bracketing Type = Sample Group
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Globals Settings
Correlation Window
Indicates the retention time relationship of target and ions to one or more
qualifiers.
Tolerance of extracted ions to be
considered a single peak.
Default time of 0.5 min is rather wide.
Typically 0.01 to 0.05 min (0.6 sec to
3 sec)
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Globals Settings
Dynamic Background Subtraction
Removes noise or background ions in TIC, not generally recommended.
Before DBS
After DBS
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Globals Settings
Non Reference & Reference Window
MassHunter Webinar Series
Extract full signal for baseline and noise.
Restrict peak selection to smaller RT window.
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Option to Display Reference RT and Window
Compound Information > Properties > Retention time
Chose which reference points to show and in what color and style.
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Globals Settings
Reference Library
MassHunter Webinar Series
The Reference Library is activated from Library
> Setup Reference Library…
A reference library can be created from a
calibrator or existing library and added to the
quant method to aid in the identification of
compounds.
High level calibrator
should be selected before
entering Method Editor to
obtain better quality
spectra for the reference
library from the sample.
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Globals Settings
Reference Library
MassHunter Webinar Series
Target Scan
Reference Spectrum
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Library Match
Score is column
that can be added
in the Batch Table.
Globals Settings
Relative ISTD
MassHunter Webinar Series
A method of semi quantitation.
It is a global parameter and is applied to every sample in the batch.
Relative ISTD is a concept where the response factor of the ISTD is used
for quantitation.
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Globals Settings
Standard Addition
MassHunter Webinar Series
Standard Addition is activated in Globals Setup in the Method Editor.
The calibration curve is Linear, Ignore Origin and No Weighting.
The non-spiked sample MUST precede the calibrators in the Batch Table.
Quant assumes that the order samples are acquired are the order in which
they are analyzed.
Order in Batch Table is imperative.
Sample, Cal1, Cal 2….
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Training Resources
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Training resources that are available.
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76
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MassHunter Quantitative Analysis
1/17/2018
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