Quantitation With Internal And External Standards
A STANDARDS FILE is simply a set of peak names, retention times, peak areas and amounts (or concentrations) used to quantitate unknown samples. A typical Standards file will have at least two concentrations of standards, generally a high and a low, to bracket the expected concentrations of the samples. A one-level Standards file cheats somewhat by assuming the origin (0 concentration, 0 area) as the lower limit. This works mathematically, but is subject to extrapolation errors. We recommend using at least a two-level Standards file.
ChromGraph allows you to construct a Standards file of up to ten levels, including both external and internal standards, and to update this Standards file automatically. The Standard Addition method for endogenous analytes is supported.
- CREATING A ONE-LEVEL EXTERNAL STANDARDS FILE
- CREATING A MULTI-LEVEL EXTERNAL STANDARDS FILE
- USING AN INTERNAL STANDARD
- INTERACTIVE AND AUTOMATIC STANDARDS UPDATING
- STANDARDS FOR MULTIPLE DETECTORS
- UTILITIES FOR STANDARDS
CREATING A ONE-LEVEL EXTERNAL STANDARDS FILE
A Standards file is constructed from processed data (retention times, peak heights, peak areas), so before creating the Standards file, load the appropriate Data file and process it. (CATS001A.DAT for this example.)
Next choose the DEFINE EXTERNAL STANDARDS section from the Standards Menu:
The DEFINE EXTERNAL STANDARDS dialog box will appear:
Since this will be a new Standards file, click on the << CLEAR OLD button, to erase any old Standards file that may be in RAM. Next enter the following information:
STANDARD LEVEL should be 1, since this is the first level (concentration) of a one-level file.
DEFAULT AMOUNT. If all peaks in the standard injection have the same amount or concentration, enter it here. This is a convenience, as this amount will be brought up automatically in the EDIT STANDARDS section. If all peaks have different concentrations, just ignore this section.
The *-box to the right of Default Amount makes it easier to add new levels to Standard files whose peaks have different concentrations.
UNITS OF AMOUNT. This is a label for the final results. Enter whatever is appropriate (e.g., pg).
DEFAULT % TOLERANCE is the window in time within which a sample peak must fall in order to be identified. For example, if peak 1 in the Standards file has a retention time of 5.0 minutes and a tolerance of 5%, any unknown peak between 4.75 and 5.25 minutes (5 minutes ± 5%) will be identified as peak 1. Default % Tolerance must be greater than zero in order to identify peaks.
UPDATE RETENTION TIMES is used during interactive or automatic updating. It allows the retention times of the new standard injection to be incorporated into the Standards file, and should generally be turned on.
QUANTITATE BY (choose area or height). This option becomes fixed after the first standard is entered; all components of the Standards file must use the same criterion.
ACTION ON STANDARD LEVEL selects the current operation. In this case we will INSERT NEW RESULTS. When updating an existing Standards file with a new injection, use REPLACE OLD RESULTS. DELETE THIS LEVEL is useful when you've accidentally added an extra level, or to clear all levels without losing peak names.
To enter the specified information into the Standards file, click the EDIT EXT. STD. >> button. (OK also enters the information, but does not proceed to the next section.) Information on the level-one standards is automatically entered from the most recently processed Data file, but must be edited. The EDIT EXTERNAL STANDARDS dialog box appears next:
Although the EDIT EXTERNAL STANDARDS dialog box has many options and features, for most purposes the only thing that has to be done here is to name the peaks and perhaps enter an amount. Click on the retention time for the first peak that you wish to incorporate into the Standards file: the retention time will appear in the edit boxes below, and the amount and area will appear on the right. Enter the peak's name in the edit box, and press CHANGE to post it to the listbox. If you used a Default Amount in the Define External Standards dialog, then AMOUNT should be correct. If not, click on it and enter the correct amount in the edit boxes on the right. Click CHANGE LEVEL to post the corrected amount to the listbox.
Continue to name each peak of interest. If there are other peaks present, they may be deleted by highlighting their retention times and clicking the DELETE button. (If you accidentally delete a peak, press the INSERT button immediately to recall it.) The completed dialog box should look like this:
As a final check that all is correct, click the LIST STD. >> button for a listing of your completed Standards file:
Verify that each peak of interest is included in the list, with accurate amounts and areas or heights. Then take the SAVE STANDARDS option from the Standards Menu to name and save the Standards file:
Standards file names can have any combination of characters and spaces. The extension .STA will be appended to the name you choose. Names can theoretically be up to 127 characters long, including the path (thus, C:\BAS2\DATA\mystandards.sta = 28 characters). Some advanced procedures may require shorter names, so we recommend using names with total lengths (including the path) of less than 33 characters.
For display purposes, a filename may be truncated somewhat in various places in the program.
The name of the Standards file will automatically be recorded in the Method, under STANDARDS OPTIONS. Once this is done, all data files processed with this Method will be compared to the standards file, and calculated amounts will be printed in the report.
CREATING A MULTI-LEVEL EXTERNAL STANDARDS FILE
Building a multi-level Standards file is a stepwise extension of building a single-level Standards file. Editing or adding subsequent levels is easier, however, since the peak names have already been entered. (If you've just built the one-level standards file above, skip the next paragraph.)
Begin by loading and processing the Data file for the first level. (Most people find it easier to build the Standards file in ascending order of concentration, so we will adopt that convention.) Then build a one-level Standards file, exactly as above. Save the Standards file, as a precaution.
Next load and process the Data file for the second level. To follow along here, load and process CATS002A.DAT. Then select DEFINE EXTERNAL STANDARDS from the Standards Menu.
IMPORTANT: DO NOT CLEAR THE OLD STANDARDS FOR THE SECOND AND SUBSEQUENT LEVELS! WE WANT TO ADD TO THE PREVIOUS LEVEL, NOT REPLACE IT.
There are only two changes from the first level: STANDARD LEVEL changes from 1 to 2, indicating that this will be the second level, and DEFAULT AMOUNT (if used) changes to reflect the concentration of standards in the second level.
The *-box to the right of Default Amount makes adding levels easier when 1) peaks within levels have different amounts, and 2) all peak amounts in a subsequent level are a constant multiple of amounts in the immediately preceding level. To use this feature, click on the *- box and enter the multiple as the Default Amount. For example, assume level one has four peaks with the following amounts: 100, 50, 250, 75 ng. Clicking the *-box and entering 2.0 as Default Amount causes the following amounts to be automatically entered for the four peaks in level two: 200, 100, 500, 150 ng. This feature should not be checked for the tutorial example.
When the Define External Standards section is completed, click on the EDIT EXT. STD. >> button to add the second level and switch to the editing dialog. The EDIT EXTERNAL STANDARDS dialog box will appear much as it did in level one. But when you click on one of the peak names, both levels of amounts and areas will appear on the right side of the screen:
As in level one, if the peaks comprising level two do not have the same concentrations you may have to do some editing of the amounts. When this is completed, click the LIST STD. >> button. The listing should look somewhat like this:
Again, save these intermediate standards as a precaution.
Subsequent levels can be added exactly as level two was added. For a three-level Standards file, load and process CATS003A.DAT. Then choose DEFINE EXTERNAL STANDARDS from the STANDARDS MENU. Change STANDARD LEVEL to 3 and DEFAULT AMOUNT to 750. Click the EDIT EXT. STD. >> button. The EDIT EXTERNAL STANDARDS dialog box will look like this:
A listing of the Standards file (LIST STD. >>) now looks like this:
Be sure to save the final version of the Standards file.
USING AN INTERNAL STANDARD
An internal standard is used to correct for variable recovery during an extraction step in sample preparation. (If you're not using an extraction step, an internal standard may not be necessary.) A fixed amount of an internal standard is added to each sample before extraction, and to the standards. During processing, the area (or height) of each peak in every run is divided by the area (or height) of the internal standard for that run, to produce a ratio. This process automatically corrects for run-to-run variation in extraction efficiency and chromatographic response (Snyder, L.R. and J.J. Kirkland. 1979. Introduction to modern liquid chromatography. Wiley- Interscience, NY).
For the standard runs, these ratios are fitted to a regression with amount as independent variable. For the samples, the ratios are used to predict amount from the regression. The report from an analysis using an internal standard will contain a column for HeightCF or AreaCF which contains these ratios (times 10,000, for precision).
Begin defining an internal standard by loading the appropriate Method (KITS.MET for this example). Then load the data sets for the standards. For this example, load KITS001A.DAT through KITS004A.DAT.
Next take the CLEAR OLD STANDARDS option from the Standards Menu:
Then process the KITS001A.DAT Data file:
When the report appears, take the DEFINE INTERNAL STANDARD option from the Standards Menu:
In the INTERNAL STANDARD dialog box, click on the retention time of the internal standard (7.03 minutes in this example). Configure the remaining options as follows:
Select AREA or HEIGHT as the parameter for quantitation.
% TOLERANCE is the window of time within which the software will accept a peak as being the internal standard (retention time ± %).
INT. STD. AMOUNT should be set at zero. This tells the software that amounts are to come from external standards, and that the internal standard is to be used for calculating peak ratios within runs.
EXPECTED AREA (or height) is a correction factor that can be used to format the AreaCF (or HeightCF) column of the report. Set this at 10000, and the AreaCF column will report RATIO x 10000. As a verification that the standards have been set up properly, the internal standard identified in each run will have an AreaCF or HeightCF of exactly 10000 (read this as 1.0000).
MINIMUM and MAXIMUM AREAS (or heights) serve both to reject extraneous peaks and to warn you if there was an error in the extraction procedure. The software will not identify a peak in a sample as being the internal standard unless it 1) falls within the window of tolerance, 2) is no larger than the maximum area and 3) is no smaller than the minimum area.
Set the MAXIMUM AREA (or height) slightly greater than the area of the internal standard peak. If the internal standard peak in a sample is larger than this, it indicates an error in the extraction procedure, or a coeluting contaminant. The message 'Internal standard was not found' will appear above the report.
Set the MINIMUM AREA (or height) to a value corresponding to a minimally acceptable extraction efficiency. For example, if you consider anything less than 50% extraction efficiency to indicate an error in the extraction procedure, set the MINIMUM AREA at 50% of the area of the internal standard peak in the first standard. If the internal standard peak in a sample is smaller than this, the message 'Internal standard was not found' will appear above the report.
After defining the internal standard, press the SAVE button and name the standard. This saves the standard and, more importantly, enters its name in the Method, to enable the ratio calculations to proceed.
Since the level-one standard was not processed against the internal standard, this Data file must be processed again. After KITS001A.DAT is reprocessed, the report will contain an AreaCF column:
Remember that these values are ratios x 10000, so the value for the internal standard (peak 3) is read as 1.0000, and the value for peak 1 is read as 0.2042.
Next you must build the external standard file as in the Section on multi-level standards. Choose DEFINE EXTERNAL STANDARDS from the Standards Menu.
IMPORTANT: DO NOT CLEAR OLD STANDARDS NOW!
For the level-one standard, change STANDARD LEVEL to 1, DEFAULT AMOUNT to 100, UNITS OF AMOUNT to pg, DEFAULT % TOLERANCE to 5, and click on UPDATE RETENTION TIMES and INSERT NEW RESULTS. Then press the EDIT EXTERNAL STD. >> button.
In the EDIT EXTERNAL STANDARDS screen above, click on the retention time of each peak of interest, enter its name in the edit boxes, and change the % tolerance or amount if necessary. Delete any peaks of no interest, including the internal standard. Then press the OK button.
Process each subsequent level of the standards, and insert each into the Standards file via the DEFINE EXTERNAL STANDARDS dialog box. The run numbers and amounts (in pg) are as follows for these runs:
| RUN | EPINEPHRINE | NOREPINEPHRINE | INTERNAL STD (DHBA) |
| KITS001A | 100 | 100 | 400 |
| KITS002A | 200 | 200 | 400 |
| KITS003A | 300 | 300 | 400 |
| KITS004A | 400 | 400 | 400 |
When the last level is entered, save the standards file and list it. The listing should look like this:
You can now load and process KITS005A.DAT and KITS006A.DAT, which are simulated unknowns. One has about 150 pg norepinephrine and 350 pg epinephrine, and the other has the opposite amounts.
INTERACTIVE AND AUTOMATIC STANDARDS UPDATING
Once set up, even multi-level Standards files containing internal and external standards may be easily updated with information from fresh standard injections.
To INTERACTIVELY UPDATE a Standards file, first do the relevant chromatographic runs, then load the Data files into RAM. Process the first-level Data file, then take the DEFINE EXTERNAL STANDARDS option in the Standards Menu:
IMPORTANT: DO NOT CLEAR THE OLD STANDARDS!
Change the STANDARD LEVEL to 1, and click on REPLACE OLD RESULTS. Click the OK button and level one is done. All the peak names, amounts, and tolerances from the old Standards file are carried into the new file. Only areas (or heights) and retention times are updated. For updating to work properly, the retention times of the new standards must fall within the % Tolerance of the old standards. Information about the internal standard does not require updating.
Repeat the updating procedure for the remaining levels, remembering to first process the Data files. Be sure to indicate the appropriate level of each update. After the last level is entered, LIST the new Standards file to check for errors, then SAVE it.
AUTOMATIC UPDATING of standards can be accomplished with a Queue. The Queue can be set up so the first few Data files update the Standards file, and the remaining Data files are quantitated against the updated Standards file:
This Queue is set up to update a three-level Standards file, then process 97 samples. For clarity, use a separate line in the Queue for each standard level to be updated. For each level, indicate the Data file name and run number, and which level is to be replaced, then click on the RPL (replace) button. Click the UNKnown button when entering lines for unknowns.
NOTE: The run numbers and standard levels in the queue will increment as the queue is run. Therefore, you should save the queue just after you create it, so its original form will be available for subsequent use.
STANDARDS FOR MULTIPLE DETECTORS
There are often several detectors in use, and therefore several channels of data, for each sample. These may be different types of detectors (e.g., electrochemical, ultraviolet and fluorescence) or the same type of detector at different gains. In either case, response will differ among detectors, so you need a separate Standards file for each detector.
In order to make a separate Standards file for each detector, simply build each one as before. Start with detector A, since it's easier to build the Standards files in a logical sequence. Load the Data files for detector A, process the Data files and build the Standards file.
Both the Standards file for detector A, and the Method for detector A, must be differentiated from those for the remaining detectors. The Standards file must be separated because response differs among detectors. The Methods must be separated because 1) processing must be optimized for each detector individually, and 2) since the Method specifies which Standards file to use, the only way to ensure that the data from each detector are compared to the appropriate standards is to use a different Method for each detector.
Use an easily remembered code to differentiate Methods and Standards files by detector. For example, after building the Standards file for detector A, save it with a name associated with detector A, let's say GOATA. This automatically enters its name in the Standards Options section of the Method. Now save the Method with the name GOATA.
Next load the Data files for detector B, optimize processing for this detector, and build a Standards file. Save this file as GOATB, and save the Method file as GOATB. Do the same for detector C, calling the relevant Standards and Method files GOATC.
There will now be three Method files on disk: each one is optimized for processing the output of its associated detector, and each one will call the appropriate Standards file for quantitation. To start processing, simply define a Queue listing each Method and the appropriate Data files for each Method to process.
The following Queue adds a level of complexity to this; it will automatically update a three-level Standards file for each of two detectors, and then process sample Data files:
UTILITIES FOR STANDARDS
The Standards Menu contains several options that facilitate working with Standards files:
LOAD, LIST and SAVE have the usual meanings. GRAPH STANDARDS and STATISTICS & UNKNOWNS allow you to interactively work with the Standards file.
GRAPH STANDARDS opens a dialog box to graph the completed Standards file. This is used as a visual check for linearity or errors. Click on up to 5 peaks, select the line options, and click on OK:
STATISTICS & UNKNOWNS opens a dialog box that reports statistical information about the standard curve for each peak, and allows you to enter areas or heights of unknowns and obtain calculated amounts:
Simply click on the peak of interest to obtain statistics on the standard curve. To estimate the amount of an unknown, enter its height or area in the edit box, and click EVALUATE >>.
