masshunter herramienta clave para la obtención de un mayor ... · revisión de resultados –...

MassHunter Herramienta clave para la obtención de un mayor rendimiento de los datos

Manuel Otero

Ingeniero de Soporte

Especialista en GCMS

MassHunter Quantitative Analysis Software

7000 TQ 7010 TQ

7200 Q-TOF

5975 Series SQ 5977 Series SQ

Flujo de Trabajo para Análisis Cuantitativo

• Crear un batch para las muestras adquiridas

• Crear, abrir y/o editar un método usando los estándares del batch para la

cuantificación

• Aplicar el método y analizar el batch

• Revisión de los resultados – Compound at a Glance

• Generar un informe con una plantilla de los resultados obtenidos

Creación del Batch Usually, a batch file is created in

the same directory as the data

files that will be used.

Name the Batch

Data files to process

Añadir muestras al Batch

1. Select Add Samples to

add samples for analysis to

the batch.

2. Highlight the samples and

press OK.

Use Browse to Copy Samples

to add samples from other

directories to the batch directory.




cuantificación




Creación de Método desde un dato MRM adquirido

New Method from Acquired MRM

Select a calibrator for the Method Editor to use

to start building the method.

• High calibrator for trace level work

• Mid-range for typical applications

Important: It may be necessary to navigate

to the correct directory.

Creación de Método desde un dato SIM adquirido Quant B.07.00 will use compound names and create targets

and qualifiers as it has with MRM data

Requires data file be collected under GCMS Acquisition B.07.01

Prior to B.07.00 software creates one compound for each SIM

ion and names it “Compound_##”

New Method from File – CEF

Start by analyzing a sample in Qualitative Analysis

• Create Compounds using either

- Find Compounds by Formula (All Ions Workflow)

- Find Compounds by Chromatogram Deconvolution

- Find by Integration

• Optionally identify the compounds with a library search and review the search results to ensure proper identity.

• Delete unwanted compounds.

• Export in CEF file format.

In Quant, run New Method from File…

Compound Setup

• MH Quant tomará la información de los compuestos del método de adquisición y

agrupará las transiciones (o iones) con el mismo nombre.

• Usará la transición (o ion) más intenso como Target (Cuantificador) y los demás como

Cualificadores.

Edición del Método

Funcionalidades básicas del Método Cuantitativo • Compound Setup

• Retention Time Setup

• ISTD Setup

• Qualifier Setup

• Calibration Curve Setup

• Global Setup

MRM Compound Setup: Compound Type

Four Compound Types are available:

Target compound

ISTD – Analog of target compound (e.g. isotopically labeled), added to samples in fixed concentration, as

reference for quantitation.

Surrogate – Non-target analyte of known concentration used to measure the efficiency of sample preparation

and analysis.

Matrix Spike – Prepared by adding a known mass of a target compound to a field sample, to measure matrix

interference.

Retention Time Setup

Retention time window:

•Percent of retention time – good for long runs where retention

time shifts are larger late in the run.

•Absolute minutes

Set data extraction windows to qualify

chromatographic peak.

ISTD Setup Mark all ISTDs with the

ISTD Flag

Flagged compounds

can be selected as

the ISTD

ISTD amount

in sample

Time Reference Peaks

must always be present

in the chromatogram.

Expected retention time

of target peak based on

any shifts in time

reference peak.

Concentration Setup from Calibration Samples

Dummy calibration levels

created for compounds. Level

IDs match those defined in the

batch table.

ISTD levels created

correctly.

Manually enter correct

concentrations.

Copy Calibration Levels

If you have multiple compounds for calibration and they have the same

amounts, you can copy calibration levels.

Qualifiers

Target or Quant Ion

Qualifier

If checked, qualifier

area added to target

Calibration Curve

Curve Fit

•Linear

•Quadratic

•Power

•1st order in

•2nd order in

•Average response factors

Curve Fit Origin

•Ignore

•Include

•Force

Curve Fit Weight

•None

•1/x

•1/x^2

•1/y

•1/y^2

•Log

Note: Calibration Curve not available

for viewing until batch is analyzed.

Global Setup

Recognition window

for ISTD’s

flagged as

reference peaks

Recognition

window for ISTD’s

and targets

Qualifier deviation

window


March 2, 2016

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cuantificación




March 2, 2016

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Aplicar el Método al Batch

Click Yes to apply the method to the batch and return to Batch View.

An option is given to perform additional processing after applying, if None is selected the batch will

need to be either Analyzed or Quantitated once in Batch View. Usually either Analyze or Quantitate

will be used to avoid the step.

Analyze always rebuilds the calibration

curve and then calculates analyte

concentrations.

Quantitate does not rebuild the

calibration curve but calculates

concentrations based on the existing

curve.

Integrate simply calculates the response

for compounds in the batch. It does not

use the calibration curve nor obtain final

concentrations.

No processing will be

performed.

Abrir Método existente y Aplicarlo - Rutina

Quant methods may be … • a standalone file (.quantmethod.xml). • embedded in a universal method

folder (.M). • embedded in a batch file (.batch.bin).

Abrir Método existente y Aplicarlo - Rutina Analyze Batch

Analyze Batch will • Extract all signals (SIM or EIC) from the data file. • Integrate the signals to detect peaks for quantifier and qualifier. • Create calibration curves based on calibrators. • Calculate a concentration based on the calibration curves. • Perform numerous QC tests (determine Outliers).

Analyze Batch

Analyze the batch – Table values calculated based on method and calibration curve.

Batch Table

with results

Calibration Curve Compound Information




cuantificación




Revisión de Resultados – Batch-at-a-Glance

Batch Table with results

Calibration Curve

Compound Information

Revisión de Resultados – Tabla de Resultados

These icons filter

outliers in the

display.

In Results Information, the Quantitation Message is a human-readable string that

contains a list of error messages that result from a compound's quantitation.

Blue = low / Red = high

“Outliers” define and specify results of known problem samples/substances that fall outside predefined conditions.

Outliers – Gran Variedad de Funciones de Chequeo

Revisión de Resultados – Compound Information

Compound

Integration

Qualifier

Information

Spectrum

Information

ISTD

Integration (Quntitation:

Part of Agilent’s

Parameterless

Integration)

ISTD Qualifier

Information

ISTD Spectrum

Information

Compound information displays all relevant information in one view.

• Uncertainty Bands

• Actual Ratio

• Manual Integration

Revisión de Resultados – Curva de Calibración

View ISTD responses or display QC samples - click either the ISD or QC button.

Change Curve Fit

R2.

Zoom in/out button

(right click/drag)

Concentration can be set as relative (to ISTD) or actual.

An example of Batch Information is this view of the Calibration Curve. The Curve fit can

be changed and data can be upd instantaneously.

After editing the

calibration

curve, you must

reanalyze the

batch.

Revisión de Resultados - Compounds-at-a-Glance • High throughput data review environment

• View compounds across multiple samples

• View all compounds within a sample

Revisión de Resultados - Compounds-at-a-Glance Specify Samples to view in Compounds-at-a-Glance then

Compounds. By default, all samples and all compounds

are selected.

Define how to Organize the

selected compounds and

samples.

Revisión de Resultados - Compounds-at-a-Glance

Define which

Outliers should be

highlighted…

Filter to show ONLY outliers, NO

outliers, or ALL.

Revisión de Resultados - Compounds-at-a-Glance

Manual Integration toolbar available

Apply integrations to

multiple

chromatograms

simultaneously.

Outliers are highlighted in red.

Revisión de Resultados - Compounds-at-a-Glance Pop up manual integration tool

Double-click

single pane to

access.

Revisión de Resultados - Compounds-at-a-Glance To zero multiple peaks:

1. Use Ctrl to select multiple peaks.

2. Select Zero Peak.




cuantificación




Generación de Informes

Results must be saved to the batch file before reports can be generated. The Report Method file defines the templates and graphics options to use while generating reports. Report Methods may be standalone files (.quantreportmethod.xml) or embedded in a method folder (.M) Samples/Compounds and Generate setting are not stored in the report method file.

Generación de Informes – Pantillas Predefinidas

INDEX_QuantReports_B_07_00.xlsx can be found on the MassHunter Quantitative Analysis Supplemental software CD in the Report Templates\Quant\Index\ReportIndex-En folder or by installing Supplemental CD onto the hard drive.

Filter by Type

Ejemplo de Informe

Template is QuantReport_RefLibraryMatchScore_B_06_00

Conclusiones

Flujo de trabajo de análisis cuantitativo

Creación de método de diferentes maneras dependiendo del tipo

y número de compuestos

Revisión de resultados muy intuitiva y visual - Outliers

Compund at a Glance: herramienta para la revisión de resultados

muy potente y eficaz, especialmente para gran número de

compuestos

Generación de informes con gran variedad de plantillas

masshunter herramienta clave para la obtención de un mayor ... · revisión de resultados –...

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