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Xcalibur ®
Getting Productive:
Quantitative Analysis
Revision A
XCALI-97063
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Abbreviations
The following abbreviations are used in this and other manuals and in the
online Help.
A ampere
ac alternating current
ADC analog-to-digital converter
AP acquisition processor
APCI atmospheric pressure chemical ionization
API atmospheric pressure ionization
ASCII American Standard Code for Information
Interchange
b bit
B byte (8 b)
baud rate data transmission speed in events per second
°C degrees Celsius
CD compact disc
CD-ROM compact disc read-only memory
cfm cubic feet per minute
CI chemical ionization
CIP carriage and insurance paid to
cm centimeter
cm3 cubic centimeter
CPU central processing unit (of a computer)
CRC cyclic redundancy check
CRM consecutive reaction monitoring
<Ctrl> control key on the terminal keyboard
d depth
Da dalton
DAC digital-to-analog converter
dc direct current
DDS direct digital synthesizer
DEP direct exposure probe
DS data system
DSP digital signal processor
Thermo ______________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ vii
ELECTRON CORPORATION
Read This First
Abbreviations _________________________________________________________ Finnigan Xcalibur
EI electron ionization
EMBL European Molecular Biology Laboratory
<Enter> enter key on the terminal keyboard
ESD electrostatic discharge
ESI electrospray ionization
eV electron volt
f femto (10-15)
°F degrees Fahrenheit
.fasta file extension of a SEQUEST search database file
FOB free on board
ft foot
FTP file transfer protocol
g gram
G giga (109)
GC gas chromatograph; gas chromatography
GC/MS gas chromatograph / mass spectrometer
GND electrical ground
GPIB general-purpose interface bus
GUI graphical user interface
h hour
h height
HPLC high-performance liquid chromatograph
HV high voltage
Hz hertz (cycles per second)
ICIS™ Interactive Chemical Information System
ICL™ Instrument Control Language™
ID inside diameter
IEC International Electrotechnical Commission
IEEE Institute of Electrical and Electronics Engineers
in. inch
I/O input/output
k kilo (103, 1000)
K kilo (210, 1024)
KEGG Kyoto Encyclopedia of Genes and Genomes
kg kilogram
l length
L liter
LAN local area network
lb pound
LC liquid chromatograph; liquid chromatography
LC/MS liquid chromatograph / mass spectrometer
LED light-emitting diode
µ micro (10-6)
m meter
m milli (10-3)
M mega (106)
M+ molecular ion
MB Megabyte (1048576 bytes)
MH+ protonated molecular ion
min minute
mL milliliter
mm millimeter
MS mass spectrometer; mass spectrometry
MS MSn power: where n = 1
MS/MS MSn power: where n = 2
MSn MSn power: where n = 1 through 10
m/z mass-to-charge ratio
n nano (10-9)
NCBI National Center for Biotechnology Information
(USA)
NIST National Institute of Standards and Technology
(USA)
OD outside diameter
Ω ohm
p pico (10-12)
Pa pascal
PCB printed circuit board
PID proportional / integral / differential
P/N part number
P/P peak-to-peak voltage
Typographical Conventions
Typographical conventions have been established for Thermo Electron
San Jose manuals for the following:
• Data input
• Boxed information
• Topic headings
Data Input
Throughout this manual, the following conventions indicate data input and
output via the computer:
• Messages displayed on the screen are represented by capitalizing the
initial letter of each word and by italicizing each word.
• Input that you enter by keyboard is represented in bold face letters.
(Titles of topics, chapters, and manuals also appear in bold face letters.)
• For brevity, expressions such as “choose File | Directories” are used
rather than “pull down the File menu and choose Directories.”
• Any command enclosed in angle brackets < > represents a single
keystroke. For example, “press <F1>” means press the key labeled F1.
• Any command that requires pressing two or more keys simultaneously is
shown with a minus sign connecting the keys. For example, “press
<Shift> - <F1>” means press and hold the <Shift> key and then press the
<F1> key.
• Any button that you click on the screen is represented in bold face letters
and a different font. For example, “click on Close”.
Boxed Information
Information that is important, but not part of the main flow of text, is
displayed in a box such as the one below.
Topic Headings
The following headings are used to show the organization of topics within a
chapter:
Chapter 1
Chapter Name
Reply Cards
Thermo Electron San Jose manuals contain one or two reply cards. All
manuals contain a Customer Registration / Reader Survey card and some
contain a Change of Location card. These cards are located at the front of each
manual.
The Customer Registration / Reader Survey card has two functions. First,
when you return the card, you are placed on the Thermo Electron San Jose
mailing list. As a member of this list, you receive application reports and
technical reports in your area of interest, and you are notified of events of
interest, such as user meetings. Second, it allows you to tell us what you like
and do not like about the manual.
The Change of Location card allows us to track the whereabouts of the
instrument. Fill out and return the card if you move the instrument to another
site within your company or if you sell the instrument. Occasionally, we need
to notify owners of our products about safety or other issues.
Identification
So that Xcalibur can correctly identify each component peak in a
chromatogram, you need to:
• Optimize the chromatogram trace for the component(s) of interest using a
scan filter, mass (or wavelength) range, or trace combination.
• Provide Xcalibur with the expected retention time of each component
together with a time ‘window’ to account for run-to-run variations.
Retention Time
For each component, you supply Xcalibur with an expected retention time and
a time range window describing an allowable deviation from the expected
time. Xcalibur uses this information to determine the retention time window,
within which to look for components within a chromatogram.
In some analyses, there is sufficient variation in retention times to make it
difficult to provide a reliable retention time window. In such cases, you can
compensate for any retention time drift by assigning a retention time
reference. This is a component whose actual retention time is used to
dynamically adjust the expected retention time of other components.
Detection
In many cases, the defined retention time window may contain two or more
peaks. Often, it is not possible to define a sufficiently small retention time
window, or unique quantitation mass that will unambiguously identify a target
component.
Xcalibur uses one of three methods to assess chromatogram peaks and hence
confirm the identify of a component (detection):
Spectrum Using a reference spectrum of the component, Xcalibur
examines the spectrum of each peak within the reten-
tion time window. The peak with the spectrum most
closely matching the reference (within user-specified
tolerances) is assigned to the component.
Highest peak Xcalibur assigns the largest peak (in terms of peak
height) within the retention time window to the compo-
nent.
Nearest RT Xcalibur assigns the peak closest to the expected reten-
tion time to the component.
With Highest Peak and Nearest RT modes, Xcalibur offers an optional
procedure called Ion Ratio Confirmation. This allows you to provide details
for up to five qualifier ions. Xcalibur ensures that these are present within the
assigned chromatogram peak (utilizing user-specified tolerances) before
finally confirming it as the component.
Calibration
To carry out quantitation, you need to evaluate the instrument’s response to
known amounts of the target component.
Response is based on either the height of the chromatogram peak, or more
commonly, the area under the peak’s profile; in both cases taking account of
the detected peak’s baseline. Xcalibur determines the area of chromatogram
peaks by an integration calculation. Figure 1-2 (a) illustrates an integrated
chromatogram peak.
Instrument response is generally measured with several samples commonly
called standards, or calibration standards. These represent a suitably wide
range of concentrations or amounts. Responses to these standards are plotted
in a graph called the calibration curve. See Figure 1-2 (b). This usually
reveals an essentially linear relationship between amount and response,
although more complex relationships are occasionally observed.
Xcalibur fits an equation to the calibration curve by a user chosen method (for
example, a least squares regression). This provides a Response factor - a
comparative measure of the response of the mass spectrometer to a
component. It is based on the amount of sample injected and the resulting
peak area or peak height. Thus, the response factor gives an intuitive (and
quantitative) measure of how responsive or sensitive the mass spectrometer is
to a certain component.
Figure 1-2. (a) Integrated chromatogram peak, and (b) the calibration curve.
Quantitation
Xcalibur achieves quantitation of samples containing unknown amounts of
the target component by first calculating the peak area or height and then
computing and applying the appropriate response to the equation derived
from the calibration curve. This provides an estimate of the amount of the
unknown component. The precision of the measurement depends on the
quality (and to a lesser extent the quantity) of the calibration data.
1.4 Sequences
Each quantitative analysis consists of a number or sequence of samples. The
sequence represents the order of sample analysis, or data acquisition. Xcalibur
processes the sample data, generating calibration curves and performing
quantitation according to the sequence’s bracket type.
Sample Types
A quantitation sequence will contain:
• One or more standards
• One or more unknowns
For more demanding applications, you can also use additional, optional,
sample types called Blanks and QCs.
Standards
A Standard is a sample containing a known amount of all target components.
The purpose of a standard is to measure the response of the instrument to the
target component(s), such that a calibration curve can be computed for each
component.
For the purpose of generating a calibration curve, Xcalibur recognizes and
utilizes several different standard types:
• Standard Clear
• Standard Update
• Standard Bracket
• Start Bracket
• End Bracket
These definitions are a consequence of the various bracket types (see
Brackets) and determine how Xcalibur processes the standards.
Unknowns
An Unknown sample is one containing an unknown amount of the target
components. Xcalibur performs quantitative analysis of any sample defined as
an Unknown.
Blanks
A Blank sample contains no target components, but may well contain ISTD
components when the internal standard quantitation technique is being used.
Xcalibur performs quantitative analysis of any sample defined as a blank
using the same calibration settings as it uses on Unknown samples.
The analysis of a Blank sample:
• Effectively purges all residual components prior to the analysis of the next
sample or sequence.
• Allows you to confirm that there are no residual components (often called
carryover) in the solvent system that can cause erroneous results.
If you use the New Sequence Template dialog box in Sequence Setup, Blank
samples can be associated with Calibration samples (which they both precede
and succeed) and/or QC samples (which they succeed).
QCs
A QC (quality control) sample contains known amounts of one or more
specific target components. QC samples are placed in a sequence so that
quantitation results can be tested for quality assurance purposes. Xcalibur
performs quantitation on QC samples using the same calibration settings as it
uses on Unknown samples. The measured quantity is then compared with the
expected value and an acceptability range. The quantitation of a QC is
classified as PASSED if the difference between the observed and expected
quantities is within the defined tolerance. A QC sample is classified as
FAILED if the difference between the observed and expected quantities is
outside the defined tolerance.
Brackets
Xcalibur organizes quantitation sequences into brackets. Brackets are simply
a way of ordering, grouping and processing standard, unknown, QC and blank
samples. This helps to ensure accurate quantitation by creating and applying
the most up-to-date calibration curve to each unknown sample.
Bracketing basically involves organizing standard samples into two groups,
encompassing Unknown, Blank and QC Samples. The standard samples are
therefore said to bracket (surround) the other sample types. This approach
helps to compensate for any long term systematic variations that may occur
during an analysis.
Calibration and quantitation are performed for each defined bracket in the
sequence. All the standards within a bracket are used to create a calibration
curve, which is then used to quantitate the bracketed samples (Unknowns,
QCs and Blanks). It is important to understand that a sequence represents the
order of sample analysis but not necessarily the processing order. Within a
bracket, Xcalibur processes the raw files for all the standards before
quantifying any other sample types, irrespective of the order of samples
within the sequence.
Xcalibur recognizes four bracketing options:
• None
• Open
• Overlapped
• Non-overlapped
None
With this option, bracketing is not applied. Samples are processed in the order
in which they are submitted in the sequence. For non-bracketed sequences,
Xcalibur uses a named calibration file (.XCAL) to store data representing
each component’s calibration curve. Although in theory it is possible to have a
different calibration file name for every sample, in practice it is usual to have
only one named calibration file per sequence.
Xcalibur uses two classifications for non-bracketed standards: Standard Clear
and Standard Update. Whenever Xcalibur encounters a Standard Clear in a
sequence, it discards the previous calibration data and starts a new calibration
with this standard. Standard Updates are simply appended to the calibration
file. Effectively, a named calibration file accumulates calibration data from
Standard Update samples until a Standard Clear is encountered in the
sequence.
An illustration of the method used by Xcalibur to process an unordered
non-bracketed sequence is shown in Figure 1-3.
Blank
Standard Clear 1
Standard Update 2 1
5
Standard Update 3
2
Standard Update 4 3
Standard Update 5 4
Blank
Unknown 1
Blank
Standard Update 6
Standard Update 7
1
Unknown 2 5 6
Standard Update 8 7 2
3
Standard Update 9 4
Standard Update 10
Blank
QC 1
QC 2
Blank
Unknown 3 1
Unknown 4 5 6
7 2
Blank 3 9
Standard Clear 11 4
8
Standard Update 12
10
Standard Update 13
Standard Update 14
Standard Update 15
Unknown 5
14
12 11
13
15
If you allow Xcalibur to order your sequence (using the New Sequence
Template dialog box), the samples are arranged as follows:
1. Blank (associated with standards)
2. Standard Clear
3. Standard Updates
4. Blank (associated with standards)
5. QCs
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 1-11
ELECTRON CORPORATION
Quantitative Analysis
Sequences __________________________________________________________ Finnigan Xcalibur
Open
In an open bracket, samples do not need to be ordered in the sequence. There
is only one bracket no matter how many sets of standard samples. All
Standard samples are processed before Blank, QC and Unknown sample
types.
Within Xcalibur, the standards in Open (and Overlapped) brackets are
classified as samples of type: Standard Bracket.
An example of the way Xcalibur arranges an Open sequence is shown in
Figure 1-4.
Blank
Standard Bracket 1
Standard Bracket 2 1
Standard Bracket 3 5 6
7 2
Standard Bracket 4
3 9
Standard Bracket 5 4
Blank 8
10
QC 1
QC 2
Blank
Unknown 1
Unknown 2
Blank
Standard Bracket 6
Standard Bracket 7
Standard Bracket 8
Standard Bracket 9
Standard Bracket 10
Blank
Note that a sequence lists samples in their acquisition order. This is not the
same order used by Xcalibur during the processing of a bracket. Irrespective
of the acquisition sequence order, the processing order is:
1. Standard Bracket samples (in acquisition order)
2. Blank, QC and Unknown samples (in acquisition order)
Overlapped
In overlapped brackets, standard samples (classified as Standard Bracket
samples) are arranged before and after Unknown, Blank and QC samples in
the acquisition sequence. However, standard samples from the end of one
bracket are used as the standard samples for the beginning of the next bracket.
Adjacent brackets therefore overlap because they share standard samples. An
example of a two-bracket, overlapped sequence is shown in Figure 1-5.
Blank
Standard Bracket 1
Standard Bracket 2
1
Standard Bracket 3 7 6
Standard Bracket 4 5 2
10
Standard Bracket 5 9
Blank
Bracket 1 8
3
4
QC 1
QC 2
Blank
Unknown 1
Unknown 2
Blank
Standard Bracket 6
Standard Bracket 7 12 11
Standard Bracket 8 7 6
15
Standard Bracket 9
10
Standard Bracket 10 9
Blank 8 14
QC 3 13
QC 4
Blank
Bracket 2 Unknown 3
Unknown 4
Blank
Standard Bracket 11
Standard Bracket 12
Standard Bracket 13
Standard Bracket 14
Standard Bracket 15
Blank
The first sample in a bracket clears the stored calibration curve data and starts
a new set of calibration data.
As in other bracket types, the Standard samples within each bracket are
processed before Blank, QC and Unknown sample types.
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 1-13
ELECTRON CORPORATION
Quantitative Analysis
Sequences __________________________________________________________ Finnigan Xcalibur
Non-Overlapped
In non-overlapped brackets, Standard samples from one bracket are not
shared with another bracket. The first sample in a bracket clears the stored
calibration curve data and starts a new set of calibration data. As in other
bracket types, Standard samples within each bracket are processed before
Blank, QC and Unknown sample types. Standards are classified as Start
Bracket or End Bracket types to denote the bracket boundaries. An example
of a two bracket non-overlapped sequence is shown in Figure 1-6.
Blank
Start Bracket 1
Start Bracket 2 1
Start Bracket 3 7 6
5 2
Start Bracket 4
10
Start Bracket 5 9
Blank 8 4
3
QC 1
QC 2
Bracket 1 Blank
Levels
Unknown 1
Unknown 2
Blank
End Bracket 6
End Bracket 7 This figure also illustrates
End Bracket 8
the use of calibration
End Bracket 9
levels to define sample
End Bracket 10
amounts. See Levels.
Blank
Blank
Start Bracket 11
Start Bracket 12
12 11
Start Bracket 13 17 16
Start Bracket 14 15
19
Start Bracket 15 20
Blank 18 14
QC 3
Bracket 2 QC 4
13
Blank
Unknown 3 Levels
Unknown 4
Blank
End Bracket 16
End Bracket 17
End Bracket 18
End Bracket 19
End Bracket 20
Blank
Levels
A Level is a user-specified name associating component amounts with
particular Standard or QC samples in a sequence. Calibration and QC levels
are defined in a Processing Method. The individual amounts do not need to be
the same. However, a calibration standard or QC sample injected for a
particular level needs to contain the amounts specified for that level.
In the non-overlapped bracket shown in Figure 1-6, for example, there are
five calibration levels. Also, for example, samples 1, 6, 11 and 16 all belong
to the same calibration level.
Replicates
When Xcalibur orders a bracket for acquisition, it analyzes each standard
sample both before and after the QC and Unknown samples. You can also
specify multiple injections of standards within each bracket. A particular
standard mixture may therefore be analyzed several times within a sequence.
These repeated analyses are called replicates.
In the non-overlapped bracket shown in Figure 1-6, for example, samples 1, 6,
11 and 16 are replicates at the same calibration level.
For non-bracketed sequences, Xcalibur stores information about replicates in
a calibration file. This allows you to choose, during review, which replicates
to include or exclude from a calibration.
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 1-15
ELECTRON CORPORATION
Quantitative Analysis
Quantitation with Xcalibur ________________________________________________ Finnigan Xcalibur
This chapter describes Processing Setup and explains how you can use it to
create a method for automated batch analysis. It leads you through the
parameters required for data processing, calibration, quantitation, reporting
and running additional programs.
The topics in this chapter are as follows:
• Processing Setup
• The Processing Setup Window
• Using Quan View Interactively
• Identification
• Detection
• Levels
• System Suitability
• Reports
• Programs
You can display or hide the View Bar, Components list, Toolbar, and Status
Bar by choosing the appropriate View menu command:
• Choose View > View Bar to display or hide the View Bar
• Choose View > Components list to display or hide the Components list
• Choose View > Toolbar to display or hide the Toolbar
• Choose View > Status Bar to display or hide the Status Bar
If you want to maximize the display of a Processing Setup view, hide all four
of these features.
The Toolbar
The Toolbar contains shortcuts for frequently used menu commands. For
specific information about Processing Setup’s menu commands or Toolbar
buttons refer to Xcalibur’s online Help.
Quan View
Quan view consists of six tabbed pages:
Identification Name components and specify retention time and peak
identification criteria.
Detection Control peak detection and integration in the
chromatogram plot.
Calibration Determine the type of calibration applied to your data.
Levels Define calibration and QC levels and amounts.
System Carry out a sequence of automated chromatographic
Suitability checks that assign a pass or fail qualification to target
peaks.
Processing Setup displays the Chromatogram and Spectrum previews with the
first two pages. You can use these to preview the results of peak identification,
detection and integration in the chromatogram preview. A secondary use of
these previews is to set some of the Quan parameters interactively using an
existing raw file.
Components List
Xcalibur displays the Components list (if enabled) at the far right of the
Processing Method window within a Quan view. It lists all of the component
names defined in the active Processing Method. A processing method may
contain different Identification, Detection, Calibration, Levels and System
Suitability page parameters for each listed component.
To view the parameters for a particular component, click on its name in the
Components list.
To add a new component to the list, select <new> in the Name combo box on
the Identification page. Type the name of the new component and then click
on OK to apply the change. The new component name appears in the Name
list and Components list.
To delete a component from the list, first click on its name in the Components
list. Then, choose Options > Delete component (for example,
Options > Delete Anthracene) and confirm the deletion.
Note. These actions do not affect the saved version of the processing
method. This can only be modified by using the File > Save command.
Xcalibur displays the Apply Changes dialog box (Figure 2-2). In the Apply
Changes dialog box, choose:
• Yes to apply changes.
• No to discard any changes and proceed with the selected action.
• Cancel to abort the intended action and return to the current page
without applying or discarding changes.
Select the Don’t Tell Me About This Again check box to suppress the display
of the Apply Changes dialog box. In future cases where it would normally be
displayed, Xcalibur treats changes according to your final selection in the
dialog box:
• If you clicked on the Yes button, Xcalibur applies changes if validation is
successful and continues with your selected action. If validation fails,
your intended action is aborted and you are returned to Processing Setup
to correct or discard changes made to the page.
• If you clicked on the No button, Xcalibur automatically discards all
changes and continues with your selected action. In such cases, you must
apply changes explicitly, by clicking on the OK button, before you initiate
the action.
Choose Options > Enable Warnings to re-enable this and all other
warning dialog boxes.
If you attempt one of the following actions without applying or discarding
changes:
• Switch to another page
• Switch to another component
• Switch to another View, using either the buttons in the View Bar or the
options on the View menu
• Change chromatography type in the Chromatography Options dialog box
(Options > Chromatography By)
• Change calibration type in the Calibration Options dialog box
(Options > Calibration By)
• Click on the Close button on the title bar
• Choose one of the following menu commands:
• File > Open
• File > <most recently used file list>
• File > Save
• File > Save As
• File > Exit
• File > Import Method
Note. If a processing method is saved when a raw file is present, the raw file
name is saved in the processing method. The associated raw file will be
opened automatically whenever the processing method is opened if you have
selected the On option button in the Auto-Open Raw File group box in the
Settings dialog box.
Previewing Processing
Using a suitable raw file, you can use the Chromatogram and Spectrum
previews to assess processing parameters for the following:
• Peak identification
• Peak detection and integration
For MS data, Xcalibur processes the raw file using the parameters of the
Identification and Detection pages. The chromatogram preview is centered on
the component’s Expected Retention Time value and its display width is
based on its View Width value. It shades all detected peaks and indicates the
start and end of each peak with a blue baseline. Initially, the spectrum shown
in the Spectrum preview will be the one corresponding to the apex scan of the
first detected peak in the chromatogram. If no peak has been detected in the
chromatogram, the chromatogram preview will show the whole raw file and
the spectrum preview will show the spectrum for the first scan in the raw file.
You can re-scale the chromatogram or spectrum previews by using:
• Cursor actions (refer to Cursor Actions)
• Toolbar buttons
• Zoom menu commands, either from the top-level menu, or from the
shortcut menu (refer to Using the Toolbar)
Cursor Actions
Within the chromatogram and spectrum previews, you can use the cursor in
three ways:
• A click picks a point on the preview
• A line dragged parallel to any axis picks a range
• A line dragged in any diagonal direction selects an area
The effect of these actions depends on the state of the preview:
• Inactive
• Active and unpinned (each preview has a pin icon in its top right corner)
• Active and pinned
Only one of the previews can be active at any one time. The active preview is
highlighted with a gray border. In the method shown in Figure 2-1, for
example, the spectrum preview is active, but not pinned.
Note. Right click on the active preview to display a shortcut menu with
Display Options (see Customizing the Previews) and Zoom commands.
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 2-11
ELECTRON CORPORATION
Quantitative Processing
Using Quan View Interactively _____________________________________________ Finnigan Xcalibur
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 2-13
ELECTRON CORPORATION
Quantitative Processing
Identification _________________________________________________________ Finnigan Xcalibur
2.4 Identification
Figure 2-4 shows the Identification page of the Quan view. Xcalibur uses the
parameters on this page to:
• Generate a chromatogram from a raw file
• Identify each component peak within the chromatogram
Detector
Detector Type Select the type of detector: MS, Analog, A/D card,
PDA, or UV.
Peak Detection Select the type of peak detection algorithm: Genesis,
Method ICIS, or Avalon.
Filter
The Filter field is enabled if you select an MS detector type. Use this combo
box to specify a scan filter. A scan filter causes processing to be applied to a
subset of the scans in a raw file.
When you load a raw file, Xcalibur lists the scan filters associated with it in
the Filter combo box (Xcalibur creates scan filters from the Instrument
Method during data acquisition). Select a scan filter from the list. Xcalibur
applies the scan filter to the data in the raw file and displays the resulting
filtered chromatogram data in the Chromatogram preview if you click on OK.
For advanced uses, you can type your own filter although you must write it in
the scan filter format. For information about scan filter formats consult the
online Help.
Trace
Use these fields to specify the type of chromatogram you want to use for
qualitative processing. The Trace options depend on your selection of
Detector Type:
• For MS scans, select Mass Range, TIC or Base Peak.
• For Analog data, select from four channels (labeled Analog 1-4).
• For data from an A/D Card, select from four channels (labeled A/D Card
Ch. 1-4).
• For PDA data, select Wavelength Range, Total Scan, or Spectrum
Maximum.
The three Trace list boxes allow you to choose:
• A basic chromatogram type, for example, TIC.
• A logical operator: - and, according to the trace, +. Your selection of an
operator then enables:
• A second chromatogram type to add to, or subtract from, the first trace.
For example, Mass Range. The list contains valid traces which may be
subtracted from, or added to, the trace specified in the first list box.
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In most cases, you will probably use a single trace such as TIC. A second
trace is useful for subtracting contributions to a chromatogram from a solvent
or other noise.
Table 2-3 lists the various MS traces and gives examples of their use.
Table 2-4 lists traces for non-MS detectors.
Table 2-3. MS Traces and combinations
Trace Use
TIC Compiles a chromatogram from all the ions in each MS scan.
Mass Range Compiles a chromatogram from a single mass, or a range of masses in each scan.
This can be a list of masses or ranges separated by commas and summed.
Base Peak Compiles a chromatogram from the most abundant ion within the specified mass
range.
TIC - Mass Allows you to ‘clean up’ a TIC by subtracting a range of background contamination
Range and thereby allowing less abundant masses to have a more significant effect on the
chromatogram. For example, consider data acquired from 50 to 1000 with dominant
solvent or contaminant peaks in the range 50 to 150. Use this trace combination with
Mass Range = 50 to 150.
TIC - Base Useful in situations where the most intense spectral peak throughout the run is due to
Peak a contaminant. Subtracting the base peak from the TIC would then remove this. You
could also use the TIC-mass range combination.
Mass Range - Can be used to remove a variety of background, solvent or contaminant peaks from a
Mass Range chromatogram. Consider an example in which data has been acquired from
m/z 50 to 900: solvent contamination is evident below m/z 150 and there are intense
contaminant peaks in the intermediate range m/z 500 to 600. Use Mass Range
1 = 150 to 900; Mass Range 2 = 500 to 600.
Mass Range + Similar uses to Mass Range – Mass Range above. Considering the same example
Mass Range as above, identical results could be obtained using this trace combination with:
Mass Range 1 = 150 to 499; Mass Range 2 = 601 to 900.
Base Peak - Rarely used. Consider an example in which the most intense peaks in the spectrum
Mass Range are, say, m/z 130 at one point in the chromatogram and m/z 140 at another. If there
are no sample masses in this range BPI– (125 to 145) could remove the effect of
these peaks.
Base Peak + Useful if the Base Peak trace does not show up every chromatogram peak of
Mass Range interest. The mass range of interest can then be added to enhance the spectrum.
Trace Use
Analog x For monitoring any external detector, such as an FID detector, that
provides an analog signal.
Analog x - Analog y For some external detectors that give out an analog signal, such as UV
detectors, it is possible to monitor more than one channel (typically two)
and to set channels to a range, for example, 220 to 500 nm. These
outputs are simple analog voltages (typically 0 to 1V). You could acquire
two channels from the same detector, one a range and one a single
wavelength or smaller range (for example, at a contaminants' specific
wavelength). Then subtract one from the other (for example,
(220 to 500) – (260 to 280) nm).
Analog x + Analog y As Analog x - Analog y above. You could add two channels
corresponding to the wavelengths of two compounds of interest (ranges
cannot be set on some detectors, only single channels).
A/D Card Channel For monitoring any external detector that provides a digital signal. You
can also specify channel combinations.
Wavelength Range PDA detector wavelength range
Wavelength Range + You could add two channels corresponding to the wavelengths of two
Wavelength Range compounds of interest (ranges cannot be set on some detectors, only
single channels).
Wavelength Range – You could acquire two channels from the same detector, one a range
Wavelength Range and one a single wavelength or smaller range (for example, at a
contaminants' specific wavelength) then subtract one from the other, for
example, (220 to 500) – (260 to 280) nm.
Total Scan PDA detector total scan
Total Scan – Wavelength Use this option to subtract a single wavelength or small range (for
Range example, at a contaminants' specific wavelength) from the total scan.
Spectrum Maximum PDA spectrum maximum
Mass or Wavelength
This parameter is available only if you select an MS or PDA detector type in
the Detector Type text box (Figure 2-4).
For an MS detector type, use the Mass text box to specify the mass or mass
range for trace combinations featuring Mass Range or Base Peak traces (for
example, Mass Range, TIC - Base Peak, TIC - Mass Range). If you use Base
Peak ± Mass Range or Mass Range ± Mass Range trace combinations, an
additional Mass (m/z) text box is displayed for you to specify the second mass
range.
For the PDA detector type, use the Wavelength text box in the cases where the
specified Trace combination features Spectrum Maximum or Wavelength
Range to specify the wavelength or wavelength range for the chromatogram.
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Note. You must provide a mass or wavelength range for each enabled Mass
Range or Wavelength Range text box. If a Mass Range or Wavelength Range
text box is blank, Xcalibur will not allow you to save the parameters or
change to another page until you have provided a range (or switched to a
different trace combination which does not involve Mass/Wavelength
Ranges).
Retention Time
These settings define the expected retention time in minutes and the error
window in seconds for component peak elution and detection.
Expected (min) Enter the anticipated retention time for the detection of
the selected component by Xcalibur. The valid range is
dependent upon the configured hardware.
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2.5 Detection
The Detection page (Figure 2-5) consists of two groups of parameters:
• Peak integration
• Peak detection
Peak Detection modes are determined by your selection in the
Chromatography Options dialog box. Xcalibur detects the type of instrument
(LC or GC) connected (when it is run for the first time) and makes this the
default type. Peak Integration parameters are common to both GC and LC
Chromatography modes.
Peak Integration
Xcalibur provides three peak detection algorithms. The ICIS peak detection
algorithm has been designed for MS data and has superior peak detection
efficiency at low MS signal levels. This is the Xcalibur default peak detection
algorithm. The Genesis peak detection algorithm is the original Xcalibur peak
detection algorithm. This algorithm has been provided for backward
compatibility with Xcalibur 1.0 studies. The Avalon peak detection algorithm
supports detectors other than MS, detects negative chromatographic peaks,
and shoulders more accurately than Genesis or ICIS.
For new methods only, you can change the default between the ICIS, the
Genesis, and the Avalon Xcalibur peak detection algorithms at any time for
each type of detector. From the Roadmap view of the Home Page, choose
Tools > Configuration and select the appropriate option on the Detection
page.
For example, the ICIS Peak Integration group box contains the following
options for peak integration:
Smoothing Use this text box to enter the amount of smoothing that
Points Xcalibur applies before integration. The value must be
odd and in the range 1 (minimum smoothing) to 15
(maximum smoothing).
Baseline Use this text box to enter the number of scans over
Window which to look for a local minima. The valid range is 1.0
to 500. The default value is 40 scans.
Area Noise Use this text box to specify the noise level multiplier
Factor used to determine the peak edge after the location of a
peak candidate. The valid range is 1 through 500. The
default multiplier is 5.
Peak Noise Use this text box to specify the noise level multiplier
Factor used to determine the potential peak signal threshold.
The valid multiplier range is 1 through 1000. The
default multiplier is 1.
Enable or disable the Constrain Peak Width check box to adjust integration
parameters for tailing peaks:
Peak height Enter the percentage of the total peak height that a
signal needs to be above the baseline before integration
is turned on or off. The valid range is 0 to 100.0%
Tailing factor Enter the maximum ratio of the trailing edge to the
leading side of a constrained peak. The valid range is
0.5 to 9.0.
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The two graphical display boxes (entitled Min and Max) at the right of the
ICIS Peak Integration group box depict the effect of small and large values for
the selected option as a visual reminder of how the option operates on data.
For example, the boxes in the margin above show the large and small values
for the peak integration parameters, and illustrate their effects on a simple
data representation, not the actual data.
Peak Detection
The ICIS, Genesis and Avalon Peak Detection group boxes contain options
for determining how Xcalibur detects peaks within the retention time window.
There are three options:
Spectrum This option is only available in GC chromatography
mode. It allows you to use a reference spectrum for
component identification. Xcalibur attempts to match
the reference spectrum with a series of unknown
spectra and calculates a score for each comparison.
Highest Peak This option allows you to identify the active component
with the highest peak in the retention time window.
This is the default LC chromatography mode option.
Nearest RT This option allows you to identify the selected
component with the peak having a retention time
nearest to the Expected value.
The following parameter is applicable to all peaks, irrespective of the
detection mode:
Minimum peak This is a signal-to-noise threshold for peak integration.
height Peaks with signal-to-noise less than this value are not
integrated. Peaks with signal-to-noise greater than this
value are integrated. The valid range is 0.0 to 999.0.
Spectrum Detection
The spectrum detection mode is designed specifically for use in gas
chromatography (GC), where peak widths are typically about 6 seconds and
often significantly less. It can therefore be difficult to define a precise
retention time window for a specific peak. If you use a large retention time
window, it is possible that several peaks will be identified within it. In LC,
peak widths are significantly greater and the definition of a retention time
window is generally a simple matter.
Figure 2-7. ICIS Peak Detection group box with spectrum detection
enabled
How it Works
The full spectrum detection procedure is as follows:
1. Xcalibur calculates the component’s predicted retention time range. This
consists of using the parameters you specified on the Identification page
to calculate the expected retention time and window.
2. Xcalibur compares the reference spectrum with the chromatogram. This
consists of comparing each raw file spectrum across the component’s
retention time range with the reference spectrum and calculating Forward
and Reverse match factors.
3. Xcalibur computes the peak detection function. This consists of utilizing
the Forward and Reverse match values, together with the intensity of the
component’s mass, for each spectrum within the retention time range.
4. Xcalibur carries out peak detection and integration on the chromatogram
plot and peak detection function. Using the parameters on the Detection
page, Xcalibur detects peaks in both the component’s mass chromatogram
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Figure 2-9. Qualifier ion table in the Ion Ratio Confirmation group box
How it Works
The Ion Ratio Confirmation procedure is as follows:
1. Xcalibur generates a mass chromatogram for the quantitation mass(es).
Using the parameters you specified in the Trace, Filter and Mass (m/z)
fields, Xcalibur generates a mass chromatogram for the quantitation
mass(es).
2. Xcalibur carries out peak detection.
If you are using area response, Xcalibur integrates each qualifier ion peak
and ratios it with the quantitation peak area. Xcalibur then compares this
ratio with your specified target ratio. If the calculated ratio is outside of
the target ratio by more than your specified tolerance (Window ±%), the
quantitation peak is rejected and the IRC Flag for the quantitation peak is
set to false.
If you are using Height response, Xcalibur ratios the qualifier ion peak
height with that of quantitation peak. Xcalibur then compares this ratio
with your specified target ratio. If the calculated ratio is outside of the
target ratio by more than your specified tolerance (Window ±%), the
quantitation peak is rejected and the IRC Flag for the quantitation peak is
set to false.
These four steps are repeated for each qualifier ion. All qualifier ratios must
be within the target ratio tolerances for the IRC Flag to be set to true.
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For more information about ICIS, Genesis, and Avalon advanced detection
options, refer to Xcalibur online Help.
Void Time
The Void Time parameter allows you to obtain correct retention times for each
peak. Void time is the time taken by a non-retained compound to elute from
the column. To obtain the correct retention time for each peak either:
• Select the Value (min) option button and enter a value for the void time
(this is subtracted from the elution time for all recorded peaks), or
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• Select the First Peak option button and set the void time to that of the first
detected peak. Xcalibur subtracts this time from the elution time for all
remaining peaks.
Baseline
Xcalibur calculates baseline noise (Genesis Detection only) in an iterative
process using the filtered and smoothed mass chromatogram. The objective of
the noise calculation process is to draw a line through the baseline composed
of a number of points with a noise ratio that is less than a specified tolerance.
Xcalibur uses the calculated baseline noise value throughout the peak
characterization process to determine whether or not baseline adjusted
intensities or heights of measurements are significant. The value is judged
significant if it is greater than the product of noise and S/N threshold
(Genesis Detection page only). Likewise, when values are less than this
product, they are considered baseline values.
The parameters defining this process are:
Baseline and Xcalibur applies this parameter to each peak and
noise window calculates the baseline and baseline noise within this
(min) window (valid range 0.1 to 1000). To ensure an
accurate noise calculation, enter a value that includes
the base width of the peak and an appreciable amount
of baseline. If the window is too small, the baseline will
be positioned up the sides of the peak.
Baseline noise This parameter controls how the baseline is drawn in
tolerance (%) the noise data. The higher the baseline noise tolerance
value, the higher the baseline is drawn through the
noise data. The valid range is 0.0 to 100.0.
Minimum This parameter defines the minimum number of scans
number of that Xcalibur uses in the baseline calculation. A larger
scans in number includes more data in determining an averaged
baseline baseline. The valid range is 2 to 100.0.
Note. The default values are suitable for most analysis requirements. Change
these settings only if standard chromatogram detection and integration
options do not provide the desired result.
Noise Method
Xcalibur uses this advanced parameter to determine how the noise level of the
data is determined by the ICIS peak detection algorithm.
INCOS Noise This option allows you to use a single pass algorithm to
determine the noise level. This is the default noise
method.
Repetitive Noise This option allows you to use a multiple pass algorithm
to determine the noise level. In general, this algorithm
is more accurate in analyzing the noise than the INCOS
Noise method.
RMS This option allows you to use a root mean squared
(RMS) algorithm to determine the noise level. By
default, Xcalibur uses Peak To Peak for the noise
calculation. RMS is automatically selected if you
determine the noise region manually.
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Peak Parameters
The Xcalibur ICIS peak detection algorithm uses the following advanced
peak detection parameters:
Min Peak Width This text box allows you to enter the minimum number
of scans in a peak. The valid range is 0 to 100 scans.
The default value is 3 scans.
Multiplet This text box allows you to enter the minimum
Resolution separation in scans between the apexes of two potential
peaks. This is a criterion to determine if two peaks are
resolved. The valid range is 1 to 500 scans. The default
value is 10 scans.
Area Tail This text box allows you to enter the number of scans
Extension past the peak endpoint to use in averaging the intensity.
The valid range is 0 to 100 scans. The default value is 5
scans.
Area Scan This text box allows you to enter the number of scans
Window on each side of the peak apex to be allowed. The valid
range is 0 to 100 scans. The default value of 0 scans
specifies that all scans from peak start to peak end are
to be included in the area integration.
Data Flags
The Data Flags dialog box (Figure 2-12) allows you to set flags for peak area
and height thresholds. Flags are recorded as true or false in the result file. If
you set a value to zero, the flag will always be false. Flags are reported in
Quan Browser and in printed or exported Reports.
To open the Data Flags dialog box, click on Flags on the Detection page.
Flags are reported as true if they exceed these thresholds:
Area threshold Enter a value for the Area Threshold Data flag. This is
an absolute value of peak area (counts).
Height Enter a value for the Height Threshold Data flag. This
threshold is an absolute value of peak height (counts).
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2.6 Calibration
The Calibration page allows you to assign one of the following types to each
of the components defined on the Identification page:
• Target compound
• ISTD
If you select Target compound, the Target Compounds group box is enabled.
This allows you to:
• Assign an ISTD to the compound
• Carry out an Isotope Contribution Correction
• Select a calibration curve type
• Select the response type
• Specify units
If you select ISTD:
• The ISTD group box becomes active
• The Target Compounds group box is grayed
• The Levels page becomes unavailable
• In the ISTD group box, you provide the amount and units for the ISTD
component.
ISTD options are available only if you have selected the Internal Standard
option button in the Calibration Options dialog box (Figure 2-13). This is the
default option for Xcalibur.
Note. If you select External Standard, any ISTD components in the active
method will be converted to Target Compounds. The ISTD option button
and group box will be grayed. The Amount and Unit information will be
lost. If you select Internal Standard, Xcalibur will prompt you to assign at
least one of the components as an ISTD.
Assigning an ISTD
The following procedure describes how to define a component as an ISTD:
1. Click on a component in the Components list located at the far right of the
Processing Setup window. If this is not visible, choose View >
Components list.
2. Select the ISTD option button in the Component type group box
(Figure 2-14). The ISTD group box is enabled.
3. Use the Amount text box to specify the amount of the internal standard
injected into each sample.
4. Use the Units text box to specify the units of the internal standard injected
into each sample.
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Assigning a Target
The following procedure describes how to define a Target Compound:
Note. When creating an internal standard method, you need to define at least
one component to be an ISTD before you can define any other components
as target compounds.
1. Click on a component in the Components list located at the far right of the
Processing Setup window. If this is not visible, choose View >
Components list.
2. Select the Target Compound option button in the Component type group
box (Figure 2-15). The Target Compounds group box is enabled.
3. Select an Internal Standard (ISTD) for the Target from those listed in the
ISTD combo box.
4. If you want to make calibration corrections for isotope contributions of
the internal standard to the target compound or the target compound to the
internal standard click on the Isotope percent button. This opens the
Correction For Isotope Contribution dialog box (Figure 2-16).
Note. Check that the values in the Correction For Isotope Contribution
dialog box are set to zero if you do not require isotope contribution
correction.
5. Select a Calibration Curve type from those listed in the Calibration Curve
combo box. The options are:
Linear A linear polynomial curve of the following mathematical
form: Y = mX + B, where m is the slope of the curve and B
is the intercept point on the Y-axis.
8. Use the Units text box to enter the units to be displayed on graphs and
reports.
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Repeat this procedure for all the components in the active method that you
wish to define as target compounds.
Isotope Correction
The Correction For Isotope Contribution dialog box (Figure 2-16) allows you
to correct for an impurity in the internal standard compound that elutes at the
same time as the target compound and/or correct for an impurity in the target
compound that elutes at the same time as the internal standard.
Access the dialog box by clicking on Isotope % in the Target Compounds
group box on the Detection page. This is only available for components
defined as Target Compounds.
Note. Check that the values in the Correction For Isotope Contribution
dialog box are set to zero if you do not require isotope contribution
correction.
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Calibration Flag
R-squared Enter a threshold to test the goodness of fit of the
calibration curve. Xcalibur calculates a coefficient of
determination (R-squared) whenever it computes a
calibration curve. If the value is less than the R-squared
threshold, the R-squared flag in the result file is set to
true; otherwise it is set to false.
Quantitation Flags
Detection limit Enter a threshold for the limit of detection. If the
quantified component concentration is less than the
Detection Limit threshold, the Detection Limit flag in
the result file is set to true; otherwise it is set to false.
Linearity limit Enter a threshold for the linearity limit. If the quantified
component concentration is greater than the Linearity
Limit threshold, the Linearity Limit flag in the result
file is set to true; otherwise it is set to false.
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2.7 Levels
The Levels page (Figure 2-18) allows you to define the concentrations of
target compounds in calibration standard samples. A Level is a text ‘label’ for
each of the defined amounts. Cal Levels are associated with calibration
standard samples when you set up a sequence. QC Levels are associated with
QC samples when you set up a sequence.
The Levels page is not available for components defined as ISTDs in the
active method (since they are spiked into samples at a fixed amount as
specified by the Amount text box of the ISTD group box on the Calibration
page).
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Standard Dilution
The Standard Dilution dialog box (Figure 2-19) allows you to enter
calibration level information for all target components simultaneously. To
open the dialog box, choose Options > Standard Dilution from the Levels
page.
At the top of the dialog, the Target Compound Components readback line
displays the total number of target components defined in the processing
method out of the total number of all components in the method.
The Selected Components readback line displays the selected number of
non-ISTD components for Standard Dilution out of the total number of all
components in the method.
• Use the Dilution text boxes to enter the stock dilution factors for each
calibration level. To enter a dilution factor, type the value in the
appropriate Dilution text box. The value must be greater than
0.00000001 and less than or equal to 1.
3. Click on OK to save the new settings and close the dialog box.
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Xcalibur repeats this procedure for all Calibration Levels, and all
components.
Note. The Standard Dilution dialog box is not updated with any changes
made directly on the Levels page. If you use the dialog box to set up a
method, you should ensure that you use it to modify the method
subsequently. Any manual changes made to the Levels page will be lost if
you subsequently use the Standard Dilution dialog box.
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Resolution
The resolution test uses a single parameter - the resolution threshold.
This enables Xcalibur to check the resolution of quantitation peaks against a
threshold value. Resolution testing is based on a comparison of the peak
height with the adjacent valley ‘height’ within the quantitation window.
Resolution threshold is defined as the ratio:
100 × V/P
where:
V is the horizontal asymptote extended from the target peak’s apex to the
lowest point in the valley between the target peak and a neighboring peak
P is the height of the target peak
The default value for the resolution threshold is 50% and the valid range is 0
to 100%.
Compounds are flagged (R) if any of the target peaks fail to meet the
resolution threshold.
Symmetry
These settings allow you to specify system suitability checks for the
symmetry of quantitation peaks. Symmetry is determined at a specified peak
height and is a measure of how even-sided (symmetrical) a peak is about a
perpendicular dropped from its apex.
The test uses two parameters:
Peak height This text box specifies the Peak Height % at which
Xcalibur measures the symmetry of target peaks. You
can enter any value within the range 1% to 100%.
Xcalibur determines symmetry at the peak height specified in the Peak Height
% text box. For the purposes of the test, a peak is considered symmetrical if:
(Lesser of L and R) × 100 / (Greater of L and R) > Symmetry Threshold %
where:
L is the distance from the left side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height
R is the distance from the right side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height
Measurements of L and R are taken from the raw file without smoothing.
Compounds are flagged (S) if any of the target peaks fail to meet the
symmetry threshold.
Peak Classification
This group consists of 5 classification checks:
• Detect peak width
• Detect tailing
• Detect column overload
• Detect baseline clipping
• Detect minimum signal-to-noise ratio
Peak Width
These settings allow you to specify system suitability checks for the width of
quantitation peaks. The test uses three parameters:
Peak height This text box specifies the Peak Height % at which
Xcalibur tests the width of target peaks. You can enter
any value within the range 0% to 100%. The default
value is 50%.
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Min peak width This text box specifies the minimum peak width, at the
(sec) specified peak height, for the peak width suitability
test. The default value is 1.8. You can set any value in
the range 0 to 30 seconds.
Compounds are flagged (W) if the peak width is less
than the minimum peak width.
Max peak width This text box specifies the maximum peak width, at the
(sec) specified peak height, for the peak width suitability
test. The default value is 3.6. You can set any value in
the range 0 to 30 seconds.
Tailing
These settings allow you to specify system suitability checks for the tailing of
peaks. The test uses two parameters:
Peak height This text box specifies the Peak Height % at which
Xcalibur measures the tailing of target peaks. You can
enter any value within the range 1% to 100%.
Failure This text box specifies the failure threshold for the
threshold tailing test. The valid range is 1 to 100.
Tailing is calculated at the value defined in the Peak Height % text box. For
the purposes of the test, a peak is considered to be excessively tailed if:
R / L > Failure Threshold %
where:
L is the distance from the left side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height
R is the distance from the right side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height
Measurements of L and R are taken from the raw file without smoothing.
Compounds are flagged (T) if any of the target peaks exceed the tailing failure
threshold.
Column Overload
These settings allow you to specify system suitability checks for column
overloading. The test uses two parameters:
Peak height This text box specifies the Peak Height % at which
Xcalibur measures column overloading. You can enter
any value within the range 1% to 100%.
Failure This text box specifies the failure threshold value for
threshold the column overload test. The valid range is 1 to 100.
Baseline Clipping
This test uses a single parameter to check quantitation peaks for baseline
clipping - Number of peak widths for noise detection.
A peak is considered to be baseline clipped if there is no signal (zero
intensity) on either side of the peak within the number of peak widths
specified in the Number Of Peak Widths For Noise Detection text box. The
default value is 1.0 and the permitted range is 0 to 100. The range is truncated
to the quantitation window if the specified number of peak widths extends
beyond the window’s edge.
Compounds are flagged (B) if any of the target peaks fail the
baseline-clipping test. Baseline clipping is often indicative of problems with
the MS detector and associated electronics.
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Quantitative Processing
System Suitability ______________________________________________________ Finnigan Xcalibur
This is the threshold for system suitability testing of the signal-to-noise ratio.
The default value is 3 and the permitted range is 0 to 100. Xcalibur calculates
the signal-to-noise ratio within the quantitation window using only baseline
signal. Any extraneous, minor, detected peaks are excluded from the
calculation.
Compounds are flagged (N) if any of the target peaks fail the signal-to-noise
test.
This feature is active in Processing Setup or Qual Browser only when both of
the following conditions are true:
• A raw data file for a PDA analysis is open
• PDA is selected from the Detector Type list box in the Quan view of
Processing Setup or PDA is selected from the Detector list box in the
Chromatogram Ranges dialog box of Qual Browser
You can determine suitable Peak Purity parameters for raw data by processing
the raw file in Qual Browser; Xcalibur displays the correlation factor in the
active chromatogram view of Qual Browser. Then, you can include this
correlation factor in a Processing Method by using the Quan view and/or the
Qual view of Processing Setup. Finally, you can produce a Peak Purity report
by using the Reports view of Processing Setup when you include the
correlation factor in a Processing Method.
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Quantitative Processing
Peak Purity __________________________________________________________ Finnigan Xcalibur
Scan Threshold
The Scan Threshold text box allows you to specify a minimum value of
intensity for wavelength scans in milliabsorbance units (mAU). A Peak Purity
computation using scan threshold starts with a scan at the apex of the peak
and collects wavelength data from scans on both sides of the apex until the
scan threshold is reached.
Use scan threshold for either symmetrical or asymmetrical peaks. The default
value for scan threshold is 3 mAU. The range of possible values is 0 to
1000 mAU (or 1 AU). In a sample with high background or noise, you might
start with a value for scan threshold of 40 mAU.
Peak Coverage
The Peak Coverage text box allows you to specify a maximum percent value
of the width of the integrated peak. A Peak Purity computation using peak
coverage starts with the scan at the apex of the peak and collects wavelength
data from scans on both sides of the apex until the percent peak coverage is
reached. Use peak coverage for symmetrical peaks. The default value for peak
coverage is 95% of the integrated peak width.
2.10 Reports
Xcalibur’s automated reporting creates comprehensive, high quality, printed
documentation. Xcalibur’s Merlin reporting package uses Microsoft Word
to create report templates, utilizing a palette of Report Objects for insertion at
any point in a page. You can customize reports to suit your own requirements
using Merlin. Reports are specified in the Reports view. (See Figure 2-22.)
The Reports view of Processing Setup lists:
Sample reports The reports issued for each sample.
Summary The summary reports issued after processing of a
reports sequence quantitation bracket (or non-bracketed
sequence).
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Quantitative Processing
Reports _____________________________________________________________ Finnigan Xcalibur
Sample Reports
The Sample Reports list consists of seven columns:
Enable Enable or disable report.
Std Enable to produce report for a Standard sample type.
QC Enable to produce report for a QC sample type.
Unk Enable to produce report for Unknown sample type.
Other Enable to produce report for all other sample types.
Save As Select a report export option. Xcalibur saves the
exported file with the sample file name and the
appropriate extension in the Data folder where result
files are stored. Valid export file types are:
Noneprint only, no exported file
TextASCII text file (*.txt), no printed copy
DocWord XP file (*.doc), no printed copy
HTMLHTML file, (*.html), no printed copy
Report Template Enter the full pathname of the template to be used by
Name Xcalibur in the generation of the report.
Summary Reports
The summary reports list contains three fields:
Enable Enable or disable report.
Save As Select a report export option. Xcalibur saves the
exported file with the file name and the appropriate
extension in the Data folder where result files are
stored. Valid export file types are:
None print only, no exported file
Text ASCII text file (*.txt), no printed copy
Doc Word XP file (*.doc), no printed copy
HTML HTML file, (*.html), no printed copy
Report Template Enter the full pathname of the template to be used by
Name Xcalibur in the generation of the report.
When you have specified a Report Template Name you
can invoke the GRD to edit the report template. To do
this:
1. Click on the cell
2. Right-click to display the shortcut menu
3. Choose Open from the shortcut menu.
You can edit cells and rows in the same manner as described above, in the
topic Sample Reports.
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Quantitative Processing
Programs ____________________________________________________________ Finnigan Xcalibur
2.11 Programs
The Programs view of Processing Setup (Figure 2-23) allows you to list
programs or macros to be run by Xcalibur after the analysis of a sample and
the processing of the resulting data. Xcalibur runs the programs in the listed
order.
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Chapter 3
Automating Analysis
This chapter describes Sequence Setup and explains how to set up and use a
sequence for automated quantitative analysis.
The topics in this chapter are as follows:
• Sequence Setup
• The Sequence Setup Window
• About Sequences
• Creating a New Sequence
• Working with a Sequence
• Running Samples
• Reprocessing Samples
• The Acquisition Queue
Figure 3-1. A simple sequence for the quantitative analysis of five samples
Within Sequence Setup, you are recommended to display the View and
Sequence Editor toolbars. Display or hide a toolbar by choosing the
appropriate View menu command.
For details about Road Map and Plot toolbars and more information about the
use of Home Page, refer to your Getting Started manual.
For specific information about Home Page’s toolbar buttons or menu
commands refer to Xcalibur’s online Help.
Importing a Sequence
Xcalibur allows you to import data into some or all of the columns in a
sequence.
Xcalibur reads comma separated text files with file extension .csv. This file
format can be created by a text editor, such as Microsoft Notepad, or a
spreadsheet program, such as Microsoft Excel.
To import a sequence:
1. Choose File > Import Sequence to open the Import Sequence dialog
box (Figure 3-4).
2. Use the Browse button to select the file for importing. Alternatively, type
the path and file name directly into the Import from File field.
3. In the Select Columns to Import group box, select the sequence columns
to be included in the imported file.
4. Click on All to select all the column options
5. Click on Clear to deselect all the column options
6. Click on OK to import the selected columns of the sequence you have
specified. Xcalibur displays the imported file in Sequence Setup.
Xcalibur generates an ‘invalid file’ message if you attempt to import a file:
• With an incorrect extension or file type, or
• In which the separator character is different from the character currently
set in the International dialog box (see Changing the List Separator
Character).
Calibration File • For first time processing, enter a new name for
the calibration file. Xcalibur will create this during
processing.
• For batch reprocessing, enter a new name for the
calibration file or select an existing calibration file.
Use the Browse button to locate the file.
Specifying Samples
In the Samples group box, you enter details about the number of samples in
the sequence and configure the autosampler.
Enter details about your samples:
Number of Enter the number of samples to be analyzed, for
Samples example, 96 for a single analysis of each sample in a
Surveyor AS tray.
Injections per Enter the number of times each sample is to be
Sample analyzed.
Base Sample ID Enter the identifier code for the sequence. The Base
Sample ID is an alphanumeric prefix to the Sample ID
that Xcalibur applies to each sample in a new sequence.
Xcalibur adds a suffix to the base sample ID starting
with 001. For example, if you enter a base sample ID of
AB12, Xcalibur numbers the first five samples as
follows:
AB12001
AB12002
AB12003
AB12004
AB12005
Select the autosampler configuration:
Tray Type Select the autosampler tray type from the drop-down
list.
Initial Vial Enter the first vial number in the tray.
Number
Re-Use Vial Select this option if you want Xcalibur to use the same
Numbers vial number for replicate samples.
The Select Vials button displays the Vial Selection dialog box (Figure 3-6).
This allows you to create a sequence of samples from individually selected
vials on any of the configured trays. Select or deselect a vial simply by
clicking on it. You can deselect all selected vials (highlighted in blue) by
clicking on the Cancel Selection button in the New Sequence Template dialog
box.
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Automating Analysis
Creating a New Sequence _______________________________________________ Finnigan Xcalibur
Note. The Select Vials feature is only available with suitable autosamplers
such as Surveyor Autosampler.
Specifying QC Samples
In the QC group box, select Add QCs if you want to add quality control
samples. Choose between the following QC options:
• After First Calibration Only
• After Every Calibration
Add Blanks Enable this option to add blanks immediately after the
QC samples.
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Automating Analysis
Creating a New Sequence _______________________________________________ Finnigan Xcalibur
Cal File Enter the path and filename of the calibration file.
Alternatively, double-click in the text box and browse
to the appropriate file using the Select Directory dialog
box.
Position Specify the position of the vial on the autosampler tray.
Inj. Vol. Enter the injection volume (in microliters). Xcalibur
sends this volume to the syringe pump. If you do not
enter an injection volume, Xcalibur uses the default
injection volume set in the experiment method.
Level Specify a level if the sample type is QC, Std clear, or
Std Update (you need to create and select a processing
method with Calibration or QC levels before you can
select a level). Double-click in the Level text box to
open the Select Level dialog box. Select a level and
click on OK.
ISTD Corr Amt Enter the correct amount of internal standard used, if
the internal standard amount injected into the sample is
different from the amount specified in the active
Processing Setup. The internal standard response will
then be corrected by the ratio of specified/actual.
Dil Factor Enter the sample dilution factor.
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Automating Analysis
Working with a Sequence ________________________________________________ Finnigan Xcalibur
4. Xcalibur identifies the first selected row as the one to copied, and all
subsequent selected rows as targets for the Fill Down operation. Check
the extent of the range to be filled:
Fill Rows Y to If required, type in a new value for Row Z, the last row
Z using to be filled with Row X duplicates. If X is incorrect,
Row X click on the Cancel button to close the dialog box and
repeat the procedure from step 1.
Inserting a Row
To insert a row:
1. Select the row immediately below where you want to insert a row.
2. Choose Edit > Insert Row. A dialog box now asks for confirmation of
the action Insert above line x?.
3. Click on the Yes button.
4. The inserted row is a copy of the row immediately prior to the row
selected in step 1.
Deleting a Row
To delete a row:
1. Select the row you want to delete
2. Choose Edit > Delete Row. A dialog box now asks for confirmation of
the action Delete line x?.
3. Click on the Yes button.
3. Click on OK.
4. Xcalibur closes the dialog box and highlights the selected row.
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Automating Analysis
Working with a Sequence ________________________________________________ Finnigan Xcalibur
Printing a Sequence
You can print a full sequence or a vial list compiled from the active sequence.
To preview the appearance of the Sequence before printing:
1. Choose File > Print Preview. The Print Selection dialog box is
displayed (Figure 3-10).
2. Select either:
Vial Position List To review the vial list from the active sequence
Full Sequence To preview the active sequence.
Refer to the Xcalibur online Help for a complete description of all controls
contained in the Print dialog box.
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-19
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Automating Analysis
Working with a Sequence ________________________________________________ Finnigan Xcalibur
Exporting a Sequence
You can export a Sequence as a separator delimited text file with a file
extension .csv. This file format can be read by a text editor, such as Microsoft
Notepad, or a spreadsheet program, such as Microsoft Excel.
The exported sequence file contains the current list separator character
(normally a comma) that is set in the Microsoft Windows dialog box (refer to
Changing the List Separator Character).
To export a sequence:
1. Choose File > Export Sequence. The Export Sequence dialog box is
displayed (Figure 3-13).
2. Enter the path and file name of the exported sequence file in the Export
To File text box. The file extension should be .csv Alternatively, you may
use the Browse button to select a path for the exported sequence file.
Xcalibur assigns extension .csv to the exported file.
3. Use the options in the Export Sequence group box to select the sequence
columns to be included in the exported file.
• Click on All to select all the column options
• Click on Clear to deselect all the column options
4. Click on OK to export the selected columns of the active sequence to the
specified file and location.
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Automating Analysis
Working with a Sequence ________________________________________________ Finnigan Xcalibur
Running a Sequence
To run a sequence:
1. Highlight the samples you want to run. Click on the left-most column of
the first sample and drag to the last sample you wish to run.
2. Choose Actions > Run Sequence or click on the Run Sequence
toolbar button.
3. The Run Sequence dialog box is then displayed. Refer to Setting up the
Run below.
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Automating Analysis
Running Samples ______________________________________________________ Finnigan Xcalibur
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-25
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Automating Analysis
Running Samples ______________________________________________________ Finnigan Xcalibur
Changing Instruments
The Change Instruments button on the Run Sequence dialog box opens the
Change Instruments in Use dialog box (Figure 3-15).
To change the status of any instrument in the current configuration, toggle the
In Use field by clicking on it.
To change the Start Instrument assignment, toggle the Start Instrument fields
as appropriate. Only one instrument can be designated as the Start Instrument.
No data are acquired during the execution of a start up or shut down method.
Click on OK to save the settings and close the dialog box. Xcalibur then
places the selected sample(s) in the run queue or starts processing
immediately.
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Automating Analysis
Reprocessing Samples __________________________________________________ Finnigan Xcalibur
Alternatively, type in the correct range. The format is either [Row] for one
sample or [First Row - Last Row] for multiple samples.
2. Select the Quan check box to reprocess quantitative data. Select the
following quantitative processing options:
Peak Select this check box if you want to generate new peak
Detection & detection and integration data.
Integration
Calibration Select this check box if you want to carry out new
calibration calculations using the sequence’s standards.
Quantitation Select this check box if you want to re-calculate the
quantitation data for unknown samples in the sequence.
3. Select the Reports check box if you want to print new reports.
Print Sample Select this check box if you want to generate new
Reports sample reports, based on those listed in the processing
method(s).
Print Select this check box if you want to generate new
Summary summary reports, based on those listed in the
Reports processing method(s).
4. Select the Programs check box if you want to run the post-processing
programs or macros, based on those listed in the processing method(s).
5. Select the Print Methods check box if you want to print the Experiment
and Processing Methods used during batch reprocessing.
6. Select the Create Quan Summary Spreadsheet option if you want
Xcalibur to generate a summary spreadsheet for the reprocessed
sequence.
7. Select the Advanced Options – Replace Sample Info check box if you
want to replace the sample information generated during data acquisition
in the sample headers with new information generated during
reprocessing.
8. Click on OK. Xcalibur initiates batch reprocessing of the selected
samples.
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Automating Analysis
The Acquisition Queue __________________________________________________ Finnigan Xcalibur
Figure 3-17. The Acquisition Queue with the Sample Information window displayed
Managing Tasks
Queue Manager (Figure 3-18) provides additional functions for managing
queued tasks. It is active whenever samples or sequences are queued for
reprocessing. If it is not visible, it may be minimized to the Windows toolbar.
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-31
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Automating Analysis
The Acquisition Queue __________________________________________________ Finnigan Xcalibur
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-33
ELECTRON CORPORATION
Chapter 4
Reviewing Quantitation
This chapter describes Quan Browser and its use for analyzing processed
quantitation sequences. The chapter explains the properties and uses of each
component within the Quan Browser window. It also describes how you can
use Quan Browser to achieve calibration and quantitation reviewing tasks.
Calibration Replicates
Calibration replicates are multiple injections of the calibration mixture at the
same calibration level or amount. These standard samples all contain the same
amount of target compound and therefore correspond to the same calibration
level. Xcalibur allows you to choose which replicates to include or exclude
from the calibration curve by using the Calibration Companion View.
Non-Bracketed Sequence
Xcalibur processes a non-bracketed sequence using a procedure known as the
continuing calibration method. Each time it processes a non-bracketed
sequence it creates or updates the calibration file(s) named in the sequence.
In this way and by avoiding the use of Std Clear, a calibration file can have
replicate data incrementally added to it over any desired period of time,
without the existing, ‘historical’ replicate data being discarded.
Quan Browser breaks down non-bracketed sequences into logical groups,
which are somewhat analogous to brackets. It does this by first ordering the
samples chronologically (with respect to acquisition date and time) and then it
examines the sequence and starts a new group whenever it encounters a
standard. After a non-standard sample has then subsequently been found, the
group ends at the non-standard immediately preceding the next standard
found.
Note that, the first group will always start with the first sample, even if it is
not a standard, and the last group will always end with the last sample.
Further, a Std Clear always starts a new group, even if no intervening
non-standard has been found following one or more Std Updates.
Note. Additional logical groups will be formed, for the situations where
different named calibration files have been specified in the Cal File entries
of the sequence; where each change of name causes a new group to be
formed As the use of multiple named calibration files is not the normal
operation, its usage will not be considered any further in this document, but
should be deducible from the discussions on groups.
As each group is processed, its samples are quantitated against the current
calibration curve. As each standard is encountered, it is processed and either
replaces (sample type set to Std Clear) or adds to (sample type set to Std
Update) the calibration replicate list and a new, calibration curve is generated.
Quan Browser processing closely emulates that of batch processing (either
that directly after acquisition, or subsequently as a batch re-process
operation). However, it has the additional capability that, if a cal file is
specified but cannot be found nor opened by Quan Browser, the message Cal
File Unavailable - Using Embedded Calibration will appear in the
Calibration File edit box and replicate data will be taken from that stored in
the result file. In most cases this will be identical to that contained within the
original calibration file anyway.
Once Quan Browser has set up the groups, they are independent and are
effectively treated as brackets. In other words, changes in one group do not
affect any other group, unlike in batch processing where subsequent groups
may well be affected.
The following list illustrates the procedure (for a single named calibration
file):
Note. If you open Quan Browser with a single Result file it is treated as a
sequence with only one entry and is brought in as an unknown. In order to
show the calibration curve used to quantitate the sample, the replicate list is
created from the embedded information.
Note. You can re-enable this, and all other Don’t Ask Again type dialog
boxes by choosing Options > Enable Warnings.
Click on OK to start the session. Quan Browser now loads the specified
sequence or file and configures the Results Grid using your selected viewing
option.
5. Results Grid
7. Companion View
6. Chromatogram
View
Manual Noise Click on the Manual Noise Region button and then drag
Region the cursor horizontally across the region of the
chromatogram that you want to select as the noise
region. Xcalibur marks the region with a red baseline.
Xcalibur calculates noise based on the data points you
select. Xcalibur uses all selected data points as noise
points and calculates noise based on those points. You
can select the noise region from an individual trace or
different noise regions from multiple traces.
Note. A raw file must be open and a chromatogram
selected for this button to be active.
Delete Manual Click on the Delete Manual Noise Region button and
Noise Region then drag the cursor over the region that was previously
selected as the noise region. Release the mouse button
to delete the noise region.
The next group of buttons are Zoom controls used to adjust the display of the
component chromatogram and companion views. These include the zoom in
and out, for both the vertical and horizontal axes as well as the ‘display all’
button to expand the plot to its limits.
The last button is the application Help button.
Many of Quan Browser’s functions are accessed from shortcut menus from
within the Results Grid, Chromatogram View, or Companion View.
For specific information about Quan Browser’s menu commands or toolbar
buttons refer to Xcalibur’s online Help.
Component List
The Component list displays all the components within the current bracket
sorted by retention time. Click on the name of a component to update the
Chromatogram View and Companion View with data for the selected
component.
Results Grid
The results grid is made up of sequence entries. Each row defines a result file
and associated parameters.
Chromatogram View
The Chromatogram View displays the chromatogram for the currently
selected component from the currently selected result file.
If a filter is stored within the embedded processing method for the current
compound this will be applied to the chromatogram. You can adjust the
chromatogram plot using the Zoom menu commands or toolbar buttons.
The type of integration used is displayed in the results grid but can be
overridden. The three types are Method Settings, User Settings and Manual
Integration. You can change the Integration method by using commands on
the shortcut menu within the Chromatogram View.
Companion View
The Companion View has two display modes:
Calibration Displays the calibration curve for the current bracket
Curve
Spectrum Plot Displays the spectrum at the selected retention time.
Initially the display shows the spectrum at the apex of
the current peak in the chromatogram view.
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 4-11
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Reviewing Quantitation
The Quan Browser Window ______________________________________________ Finnigan Xcalibur
There are two viewing configurations. These are determined by your choice in
the Viewing Sample Types dialog box at startup, or by your choice in the
Options menu.
If the Options menu viewing preference is set to View Stds and QCs, the
results grid has 3 pages:
All All Standard and QC samples
Stds Standard samples only
QCs QC samples only
If the viewing preference is set to View All, the results grid will have 5 pages:
All All Standard, QC, Blank and Unknown samples
Stds Standard samples only
QCs QC samples only
Blanks Blank samples only
Unknowns Unknown samples only
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Reviewing Quantitation
The Results Grid ______________________________________________________ Finnigan Xcalibur
Bracket/Group in Use
For bracketed sequences, this combo box lists the available brackets in
sequential order. Xcalibur selects the first bracket in the list when the file is
first loaded into Quan Browser, and displays the samples within this bracket
in the results grid.
If you load a non-bracketed sequence, the samples are broken into logical
groups (refer to How Quan Browser Works). The combo box lists the
available groups.
Selecting a new bracket or group from the combo list refills the results grid
with the samples from the selected bracket or group. Xcalibur updates all the
other Views and dialog boxes automatically.
Calibration File
This read only text box shows the calibration method applied to the current
bracket or group.
If the calibration information for the current bracket was obtained from the
embedded processing method and not from a separate cal file, the text box
displays Embedded Calibration.
For non-bracketed sequences, the text box displays the name of the calibration
file associated with the current Group in the sequence. To change the
calibration file, choose File > Replace Calibration. This option is not
available for bracketed sequences.
ISTD Area Integrated area (or height) under the Internal Standard
(or ISTD Height) peak (count secs or counts)
Area Ratio The area (or height) ratio between the selected peak and
(or Height Ratio) the Internal standard
Specified The amount of the component at the Cal or QC level
Amount
Calculated Amount of component as determined by the response
Amount ratio and calibration curve
% Diff Percentage difference between the calculated amount
and the specified amount
% RSD Relative Standard Deviation of the difference between
the calculated amount and the specified amount as
expressed as a percentage of the specified value
Peak Status Low is displayed if the %Difference is < 0, High is
displayed if the %Difference is > 0 and Fail is
displayed if the %Difference is greater than the QC fail
percentage test value.
Level The name of the calibration or QC level of the sample.
Units The units defined in the processing method for quantity
or concentration.
RT Retention time in minutes at the peak apex.
Sample ID Unique sample identification string given to this
sample when the sequence was prepared in Sequence
Setup.
Exclude Indicates if the sample point is to be included or
excluded from the calibration curve for the current
bracket or group.
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Reviewing Quantitation
The Results Grid ______________________________________________________ Finnigan Xcalibur
If a sample is shared between two brackets you cannot change its Sample
Type. Xcalibur will also notify you when a sample is part of two overlapping
brackets if you attempt to change its Integration Type, Level or Exclude state.
You can then either confirm or cancel the change.
Select the check box for a column heading to display it; clear the check box to
hide the column.
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Reviewing Quantitation
The Results Grid ______________________________________________________ Finnigan Xcalibur
The first sorting criteria is fixed to be Sample type. The second and third sort
criteria can be any of the remaining columns, even if the column is currently
hidden.
Click on the Save As Default button if you want to replace the default sorting
criteria with your new selections.
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Reviewing Quantitation
Chromatogram View ____________________________________________________ Finnigan Xcalibur
Set Peak to Not Allows you to override the identification method and
Found Status classify the current component as Not Found. When
used, the chromatogram view re-displays over the
whole acquisition range.
Reset Scaling Allows you to reset the chromatogram scaling to show
the full peak in a normalized window.
If the component peak is not found then the dialog box consists of a single tab
labeled No Peak. This displays the text No Peak Found: Cannot show Peak
Info.
The Peak Information dialog box is read only. If you select other components
or samples, the dialog box is updated with peak information for the displayed
component chromatogram peak.
Info Page
The peak Info page (Figure 4-7) displays the following properties:
Left (min) Left point in minutes of integration baseline
Apex (min) Location of apex in minutes
Right (min) Right point in minutes of integration baseline
Height Height at peak apex
Area (cts-secs) Area measured in count seconds
Baseline Baseline height directly beneath the apex
Base Peak (m/z) Mass to charge ratio of ion with largest response
Signal to Noise Measured signal-to-noise
Expected RT Expected retention time in minutes of peak
(min)
ISTD Response Area (or Height) of internal standard peak
Response Ratio Ratio of this peak’s area (or height) to the ISTD peak’s
area (or height)
Calculated Amount in sample as calculated with response ratio and
Amount calibration curve
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Reviewing Quantitation
Chromatogram View ____________________________________________________ Finnigan Xcalibur
Flags Page
The Flags page (Figure 4-8) displays information about peak detection.
The detection threshold flags (defined in Processing Setup in the Data Flags
dialog box accessed from the method’s Detection page) are:
Area True if the peak area exceeds the defined absolute peak
area.
Height True if the peak height exceeds the defined absolute
peak height.
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Reviewing Quantitation
Chromatogram View ____________________________________________________ Finnigan Xcalibur
Suitability Page
The Suitability page (Figure 4-10) displays the results of specific tests that
may have been performed (as determined by the System Suitability
parameters in the processing method) on the component peak to determine its
suitability to be considered a valid peak.
Spectrum Page
The Spectrum page (Figure 4-11) displays the current spectrum at the Apex
retention time. No adjustments can be made to the plot.
Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 4-25
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Reviewing Quantitation
Chromatogram View ____________________________________________________ Finnigan Xcalibur
Figure 4-12. More Info page of Peak Information dialog box for a
qualifier ion
Ion Coelution Indicates whether the selected qualifier ion passes the
Test Passed Coelution test. To do this, its mass chromatogram must
have a peak apex within the Qualifier Coelution
Window specified on the processing method’s
Detection page (see Chapter 2).
If the qualifier ion fails the Coelution test, the Ion Ratio
test is not performed and the Ion Ratio Test group box
is not displayed.
Ion Ratio Test Indicates whether the selected qualifier ion passes the
Passed Ion Ratio test. The Target ratio % and Absolute
Window % parameters display the results of the test.
Target Ratio The calculated Target Ratio Percentage. See Chapter 2
for more details.
Absolute The calculated Absolute window Percentage. See
Window Chapter 2 for more details.
Chro Page
The Chro page (Figure 4-13) displays the qualifier ion mass chromatogram
within the component peak window. No adjustments can be made to the plot.
Figure 4-13. Chro page of Peak Information dialog box for a qualifier ion
Info Page
The parameters in the Peak Info group box (Figure 4-14) are the same as those
described for the standard peak and qualifier ion Info page.
See Chapter 2 or the online Help for a more detailed description of these
parameters and Spectrum detection.
Note. If the main component peak was detected using Spectrum detection
Xcalibur displays the standard Info page with the Spectrum Results group
box on an additional tabbed page called More Info.
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Chromatogram View ____________________________________________________ Finnigan Xcalibur
Figure 4-14. Info page of Peak Information dialog box for a Spectrum
candidate
Chro Page
The Chro page displays a TIC plot for the Spectrum candidate. The plot has
the width used by the component peak display.
Spectrum Page
The Spectrum page is effectively the same as that described for a standard
peak. It displays the spectrum corresponding to the apex of the spectrum
candidate chromatogram.
Within Quan Browser, Xcalibur allows you to apply unique peak detection
parameters to the chromatogram using the User Identification Settings dialog
box. This duplicates the parameters available in the Identification and
Detection pages in Quan View of Processing Setup, allowing you to adjust
and test the effect of different values. You can:
• Save the settings in a Quan Browser file (*.XQN). Choose File > Save or
File > Save As.
• Export the User Settings as a full Processing Method using the File >
Export Method menu command.
To open the User Identification Settings dialog box, right click on the
Chromatogram View and select User Peak Detection Settings from the
shortcut menu.
The User Identification Settings dialog box for the ICIS peak detection
algorithm consists of the following tabbed pages:
Identification Parameters used by Xcalibur to identify the selected
component in the chromatogram.
Detection Settings used by Xcalibur to confirm peak detection.
ICIS Integration ICIS peak detection algorithm parameters applied to
the component peak.
ICIS Advanced ICIS advanced parameters used by Xcalibur during
peak identification and integration.
Flags Detection flagging thresholds applied to the selected
component to validate detection.
The User Identification Settings dialog box for the Genesis peak detection
algorithm consists of the following tabbed pages:
Identification Parameters used by Xcalibur to identify the selected
component in the chromatogram.
Detection Settings used by Xcalibur to confirm peak detection.
Genesis Genesis peak detection algorithm parameters applied to
Integration the component peak.
Genesis Genesis advanced parameters used by Xcalibur during
Advanced peak identification and integration.
Flags Detection flagging thresholds applied to the selected
component to validate detection.
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Chromatogram View ____________________________________________________ Finnigan Xcalibur
The User Identification Settings dialog box for the Avalon peak detection
algorithm consists of the following tabbed pages:
Identification Parameters used by Xcalibur to identify the selected
component in the chromatogram.
Detection Settings used by Xcalibur to confirm peak detection.
Avalon Avalon peak detection algorithm parameters
Integration applied to the component peak.
Flags Detection flagging thresholds applied to the selected
component to validate detection.
Identification Page
Xcalibur uses the parameters on the Identification page (Figure 4-15) to:
• Generate a chromatogram from raw data
• Identify the component within the chromatogram
The parameters are identical to those on the Identification page of Quan View
in Processing Setup. These are described in Chapter 2, in the topic
Identification.
Detection Page
Xcalibur uses the parameters on the Detection page (Figure 4-16) to confirm
the identity of the component within the retention time window defined by the
Identification settings. The options available on this page depend on the
Chromatography mode selected in the original processing method used to
generate the raw data (refer to Chapter 2).
The parameters are identical to those in the Peak Detection group box on the
Detection page of Quan View in Processing Setup. These are described in
Chapter 2, in the topic Peak Detection.
The controls on the Detection page vary depending upon whether you are
using a GC or LC and also whether the detection method is Spectrum, Highest
Peak, or Nearest RT. For more information, refer to Xcalibur online Help.
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Chromatogram View ____________________________________________________ Finnigan Xcalibur
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Chromatogram View ____________________________________________________ Finnigan Xcalibur
Flags Page
Xcalibur applies the parameters on the Flags page (Figure 4-19) to test the
validity of detected peaks.
The parameters are identical to those in the Data Flags dialog box accessed
from the Detection page of Quan View in Processing Setup. These are
described in Chapter 2, in the topic Data Flags.
For more information about these settings, refer to Xcalibur online Help.
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Calibration Companion View ______________________________________________ Finnigan Xcalibur
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Calibration Companion View ______________________________________________ Finnigan Xcalibur
Type Page
The Type page (Figure 4-21) displays the component type, target compound
or ISTD. For a target compound, you can change the ISTD to be used with it.
For ISTDs, you can change the Amount and Units.
The parameters are identical to those in the Component Type and ISTD group
boxes on the Calibration page of Quan View in Processing Setup. These are
described in Chapter 2, Calibration.
Curve Page
The Curve page (Figure 4-22) allows you to change the way Xcalibur
calculates and plots the calibration curve from the data points.
Calibration Use this list box to change the type of algorithm applied
Curve to fit the data points. The available types are Linear,
Quadratic, Linear Log-Log, Quadratic Log-Log,
Average RF, Point-to-Point, Cubic Spline and Locally
Weighted
Weighting Use these option buttons to change the weighting
applied to the individual data points. The available
types include Equal, 1/x, 1/x^2, 1/y, 1/y^2 and 1/s^2
Response Use these option buttons to change the component
response used in the calibration curve: area or height.
Units Use this text box to change the units label used in the
Calibration Companion View, on the Levels page, and
in reports.
These parameters are identical to those in the Target Compounds group box
on the Calibration page of Quan View in Processing Setup. These are
described in more detail in Chapter 2, Calibration.
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Calibration Companion View ______________________________________________ Finnigan Xcalibur
Levels Page
The Levels page (Figure 4-23) allows you to change Calibration and QC level
names and their associated amounts. It is not available for ISTD components
(the page displays the message This component does not use levels).
These parameters are identical to those on the Levels page of Quan View in
Processing Setup. These are described in more detail in Chapter 2, Levels.
Isotope % Page
The Isotope% page (Figure 4-24) allows you to correct data for:
• An impurity in the internal standard compound that elutes at the same
time as the target compound.
• An impurity in the target compound that elutes at the same time as the
internal standard.
These parameters are identical to those in the Correction For Isotope
Contribution dialog box, accessed from the Calibration page of Quan View in
Processing Setup. These are described in more detail in Chapter 2,
Calibration.
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Calibration Companion View ______________________________________________ Finnigan Xcalibur
Flags Page
The Flags page (Figure 4-25) allows you to change the threshold values for
calibration and quantitation flags for the selected compound. An entered value
of 0 will force the flag to be false.
These parameters are identical to those in the Data Flags dialog box, accessed
from the Calibration page of Quan View in Processing Setup. These are
described in more detail in Chapter 2, Calibration.
If you edit any of the values in the Quantitation Flags group, Xcalibur checks
that the relationships between the four fields are maintained. If an entry in one
parameter forces a change to occur in another, Xcalibur displays the
Automatic Adjustment warning dialog box (Figure 4-26).
To open the dialog box, right click on the Calibration Companion View and
choose Exclusion List from the shortcut menu.
The dialog box lists all the replicates used in the current bracket or group and
their exclusion status. Levels are listed under the following headings:
Level Shows the name for the Level
Expected Displays the expected amount for Level
% Diff Shows the percentage difference between measured
and expected amounts
Exclude Denotes excluded levels by the word Yes
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Calibration Companion View ______________________________________________ Finnigan Xcalibur
Pin the spectrum companion view if you want to display spectra from other
regions of the chromatogram. Any click within the Chromatogram View will
then result in the Spectrum Companion View being updated with a spectrum
corresponding to the scan at the ‘clicked’ retention time.
In a pinned Spectrum View you can also use the cursor to rescale the plot. The
Zoom menu commands and toolbar buttons are also effective.
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Reports _____________________________________________________________ Finnigan Xcalibur
4.8 Reports
To generate reports for the current sequence either:
• Click on the Reports toolbar button.
• Choose View > Reports dialog.
The Reports dialog box (Figure 4-28) duplicates the Reports view in
Processing Setup. When opened, it displays the reports specified in the
processing method associated with the active sequence. The displayed
parameters may change as you select different brackets.
For a description of the Sample Reports and Summary Reports tables see
Chapter 2, Reports view. The Reports dialog box features the following
additional parameters:
Include Sample Select this check box if you want to include
Reports sample reports in any print run.
Include Summary Select this check box if you want to include
Reports summary reports in any print run.
Select Samples Click on this button to open the Select Report
Samples dialog box. This allows you to choose
samples in the sequence for report generation and
printing.
Print Reports Click on this button to initiate report generation
and printing as defined in the dialog box.
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Quan Browser Procedures _______________________________________________ Finnigan Xcalibur
Editing a Sequence
To review and edit an existing Sequence, do the following:
Inspect the Sequence. Ensure that the correct raw files are listed in the Results
Grid. Make sure that each raw file in the Sequence is properly associated with
a calibration level, QC level, blank, or unknown.
1. To remove raw files from the sequence:
a. Select the row(s) in the Sequence that you want to delete.
b. Right-click on the mouse in the Sequence to display a menu.
c. Finally, left-click on the Delete Selected Samples command to delete
the selected row(s) in the Sequence.
2. To add raw files to the Sequence:
a. Select the row in the Sequence above where you want the new row
(sample) to be located.
b. Right-click on the mouse in the Results Grid to display a menu.
c. Select the Add Sample command to open the Open Result File dialog
box.
d. Locate the raw file you want to add to the Sequence, and then click on
the Open button to open the Add Sample dialog box.
e. Specify sample information in the Add Sample dialog box, and then
click on the OK button.
3. To change the sample type:
a. Click on the Sample Type list box arrow to display a list of sample
type options.
b. Select the new sample type. Quan Browser displays the new sample
type in the Sample Type list box.
4. To save the sequence with all current detection and calibration settings,
choose File > Save or File > Save As. The resulting Xcalibur Quan
file (extension .XQN) contains all the necessary information required to
recreate the current Quan Browser session.
Reviewing Samples
Use the following procedure to review and rework samples:
1. Select a component. Xcalibur automatically updates the Result List,
Chromatogram and Companion Views.
2. Click on the Standards tab to display calibration standards results.
3. Inspect the calibration curve in the companion view. If it is not currently
displayed either:
• Choose View > Set Companion View >
Show Calibration Curve, or
• Right click in the Chromatogram View and choose
Set Companion View > Show Calibration Curve from the
shortcut menu.
4. Inspect the calibration curve according to the criteria used in your
laboratory.
5. Select the first row in the Results Grid.
6. Check the peak detection and integration fields in the Result Grid for peak
detection and integration problems. Make sure that the selected data file
corresponds to the correct level and sample type.
7. Inspect the plot in the Chromatogram View.
• Make sure that Xcalibur found the peak. Xcalibur shades found peaks
gray and marks the starting and ending points with square integration
markers.
• Make sure that Xcalibur integrated the peak properly. Check that the
shaded area accurately represents the contribution of the component
to the chromatogram.
8. If necessary, modify the peak detection and integration settings:
• Right-click on the mouse in the Chromatogram View and select User
Peak Detection Settings. This opens the User Identification Settings
dialog box.
• If you want to change the detection method, modify the settings in the
Detection tab.
• If you have problems with noise in the peak, unresolved peaks, or
peak tailing, modify the settings in the Integration tab.
• If baseline noise is interfering with peak identification or integration,
modify the settings in the Advanced tab. Advanced options should
only be used if the standard options do not provide sufficiently
selective detection criteria.
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Quan Browser Procedures _______________________________________________ Finnigan Xcalibur
Reviewing a Chromatogram
Use the following procedure to review a chromatogram:
1. Right click in the Chromatogram View and choose Show Peak Info.
This opens the Peak Information dialog box. Review the chromatogram
peak data:
• The properties of the detected peak on the Peak Info page.
• Integration information and flags on the Peak Flags page.
• System Suitability test results on the Peak Suitability page.
• The spectrum for the peak apex scan on the Peak Spectrum page.
2. You can adjust the chromatogram in the following ways:
• To change detection or integration parameters refer to Modifying
Detection and Identification.
• To manually integrate peaks refer to Integrating Chromatogram
Peaks Manually.
• To change chromatogram peak labeling, right click in the
Chromatogram View and choose Peak Labeling. Select the labels
and label styles you require in the Display Options dialog box.
3. If you want to view spectra for the peak, display the Spectrum
Companion View. Right click in the Chromatogram View and choose
Set Companion View > Show Spectrum Plot from the shortcut
menu.
4. View spectra across the chromatogram. Pin the Spectrum Companion
View. Click on points of interest in the chromatogram to view the
corresponding spectrum.
5. To carry out a detailed qualitative analysis of the chromatogram, export
the results file to Qual Browser. Right click on the Results Grid and
choose Send to Qual Browser.
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Quan Browser Procedures _______________________________________________ Finnigan Xcalibur
Figure 4-30. Chromatogram (a) shows a partially integrated peak and chromatogram (b) shows a
manual integration of the peak achieved by dragging the baseline endpoint to a new
location.
You use the second way when Xcalibur has not detected the peak of choice, as
follows:
1. Right click on the Chromatogram View again and select Manually Add
Peak. Xcalibur changes the cursor shape to denote the mode.
2. In the chromatogram, click on one side of the peak you want to manually
integrate, and holding down the mouse button, drag across the peak to
define the point on the other side.
Repeat the procedure for other samples and components as required. If you
want to restore automatic peak detection and integration, right click in the
Chromatogram View and choose Method Settings or User Settings.
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Index
Finnigan Xcalibur ________________________________________________________________________
Index
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Index
___________________________________________________________________ Finnigan Xcalibur
R S
removing jobs from the acquisition queue, 3-32 S/N threshold, 2-30
Repetitive Noise option, 2-31 sample
Replace Calibration command, 4-14 ID, 4-15
replicate samples, 3-11 name, 4-14
replicates, 1-15, 4-3 position, 3-17
reports type, 4-14
exporting, 2-56, 2-57 Sample ID text box, 3-4, 3-13
from processing method, 3-27 Sample Information window, 3-31
sample, 2-56 Sample Type option box, 3-4, 3-13
template, 2-56, 2-57 samples
Reports command, 4-9, 4-10 editing in Quan Browser, 4-49
Reports dialog box, 4-46 ID, 3-13, 3-17
Select Samples button, 4-47 name, 3-5
Reports view, 2-55 QC, 3-13
reprocessing, 3-28 removing from the acquisition queue, 3-32
rescaling a preview display, 2-13 replicate, 3-11
Reset Scaling command, 4-20, 4-36, 4-45 reports, 2-56
resolution suitability test, 4-25 running, 3-23
resolution threshold, 2-48 type, 3-4, 3-13
response, 2-38 unknowns, 3-11
response factor, 1-5 volume, 3-5
Response High flag, 4-23 weight, 3-5
Response Low flag, 4-23 Saturated flag, 4-22
Response OK flag, 4-23 saturation suitability test, 4-25
Result List Column Hiding dialog box, 4-17 Save command, 4-9
results grid, 4-10 saving
changing the sort order, 4-17 page parameters in Processing Setup, 2-5
column headings, 4-14 sequence in Sequence Setup, 3-5
context menu, 4-16 scan filters, 1-4, 2-15
displaying columns, 4-17 Scan Threshold text box
editing a sequence, 4-48 peak purity, 2-54
editing samples, 4-49 Select Directory dialog box, 3-13
hiding columns, 4-17 Select Report Samples dialog box, 4-47
working in, 4-13 Select Samples button, 4-46
results review, 4-2 semi-quantitative analysis, 1-2
resuming the acquisition queue, 3-32 Send To Qual Browser command, 4-16
retention time, 1-3, 1-4 separator character, 3-9
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