Optimum settings for gas chromatograph parameters and correct retention time windows for time-sequenced selected ion monitoring mode (SIM) analysis of PCDDs/PCDFs should be established by analyzing a Window Defining Mixture containing the earliest and latest eluting congeners within each homologous group of congeners.
This mixture should be analyzed at regular intervals for routine verification of window settings, and must be analyzed after any intentional change in GC parameter settings, and after any condition or upset that requires that the GC column be disconnected.
As shown in Table 3, the order of elution on a 60-meter DB-5 column (polymethyl [5% phenyl]silicone) is such that five retention time windows can be defined, corresponding to the five levels of chlorine substitution (4 Cl to 8 Cl) without any overlap.
For 2,3,7,8-TCDD and 2,3,7,8-TCDF analysis, a Column Performance Test Mixture containing the target analyte and its neighbouring isomers at equal concentration should be analyzed daily to confirm acceptable chromatographic resolution. It is recommended that these isomers be included in the Window Defining Mixture.
The valley between peaks representing 2,3,7,8-TCDD and its closest neighbouring isomer should be equal to or less than 25% of the 2,3,7,8-TCDD peak height. The corresponding peak valley criterion for 2,3,7,8-TCDF is 30% maximum. These criteria must be satisfied whenever analyses are performed for regulatory compliance purposes. In other cases, results for either of these two congeners must be flagged if the corresponding criterion has not been met.
The acceptable chromatographic resolution for 2,3,7,8-TCDD and 2,3,7,8-TCDF is shown in Figure 1.
Figure 1: Acceptable Chromatographic Resolution for 2,3,7,8-TCDD and 2,3,7,8-TCDF
The linear calibration range for each analyte should be established by running a series of calibration standards before initial sample analysis.
Calibration standards should contain:
- all seventeen 2,3,7,8-substituted PCDD/PCDF congeners;
- the set of labelled surrogate congeners added to samples before processing; and
- the labelled congeners that are added to sample extracts just before GC/MS analysis.
These latter congeners, 13C12-1,2,3,4-TCDD and 13C12-1,2,3,7,8,9-H6CDD, are used for calculating sample surrogate recoveries. In addition to their function as recovery standards, these compounds also serve as reference congeners for assigning sample peak identities on the basis of relative retention time.
Recommended compositions and concentration levels for LRMS and HRMS calibration standards are presented in Table 4 and Table 5.
An internal standard method is recommended for quantitation of sample data. Use of this method relies upon consistent linearity of MS response over the intervals between multi-point calibrations. The method is easily integrated into automated routines for data quantitation.
Internal standard quantitation is based on the use of Relative Response Factors (RRFs). An RRF is the ratio of analyte response factor to the response factor of the corresponding labelled surrogate. These RRFs remain unchanged over the range of concentration for which MS response is linear. Using these RRFs, along with surrogate responses from the sample run, concentrations of PCDD/PCDFs can be calculated directly, without the need to calculate surrogate recoveries. Recoveries should nevertheless be calculated separately and reported, as these values serve to indicate the overall quality of the concentration data being reported.
Isomer-specific RRFs are used for quantitation of 2,3,7,8-substituted congeners when isomer-specific analysis is required. For homologues represented by more than one isomer in the calibration standard solutions, the "homologue-average" RRF is used to quantitate all target analytes that are not 2,3,7,8-substituted congeners. If isomer specific analysis is not required, the "homologue-average" RRFs are used exclusively.
From the initial calibration data, RRF values are calculated for each native analyte at each standard concentration level. The Relative Standard Deviation (RSD) of these RRF values for each native analyte must be less than 20%. If this criterion is met, then the calibration data is considered to be linear, and mean RRF values are used for quantitation of subsequent PCDD/PCDF data.
The established calibration must be verified by analyzing a calibration verification standard (LCS3 in Table 4 and HCS3 in Table 5) at least once during every 12-hour period in which samples are being analyzed.
Verification standard results are calculated in the same manner as an actual sample, using the mean RRF values obtained from the initial calibration. The calculated concentration for each native PCDD/PCDF congener must be within 20% of its actual known value. The calculated recovery of each surrogate compound must be within the range of 75 to 125%. Remedial action is required if any native or surrogate compound fails this verification test.
Whenever new calibration standard solutions are prepared, and at least twice a year, the accuracy of the calibration standards should be verified against certified reference materials, e.g., National Institute of Standard and Technology, Reference Material for 2,3,7,8-TCDD concentration (NIST,1985).
Sample components are identified as PCDD/PCDF only if GC/MS data satisfy the following criteria.
- Peak responses for each of the two selected molecular cluster ions must be at least three times the background noise level (S/N ≥ 3).
- For LRMS, the chlorine isotope ratio for the two molecular cluster ions must be within ±20% of the correct ratio. For HRMS, the criterion is ±15%.
- Peak maxima for the molecular cluster ions must coincide within two seconds.
- COCl-loss must be indicated when analyte concentration is sufficient for detection (for LRMS only).
- For combustion emission and industrial waste samples, monitoring for chlorinated diphenyl ether interference is recommended. Reported sample concentrations of PCDFs must be flagged with an "E" if any peak recorded in the chlorinated diphenyl ether channel maximizes within two seconds of the retention time of an apparent PCDF congener, and response for the ether ion exceeds 5% of the sum of the peak areas for the two monitored furan ions.
- Sample components are identified as 2,3,7,8-substituted congeners if the following applicable criterion is also satisfied.
- In the case of a congener for which a labelled analogue is present in the surrogate spiking mixture, all native and surrogate ion peak maxima must be coincident within three seconds.
- In the case of a congener for which a labelled analogue is not present in the surrogate spiking mixture, peak maxima for both molecular cluster ions must be coincident with each other within two seconds, and both peaks must be within three seconds of the expected retention time for that congener. Expected retention time is determined on the basis of known relative retention times for these congeners.
For HRMS analysis, the mass spectrometer should be operated in a mass drift correction mode and must be tuned, using PKF (perflurokerosene), to achieve a resolution of at least 10 000 (10% valley definition). Hard copied verification of these measurements (peak shape and peak width) must be available (see Environment Canada, 1992 for calibration procedures).
If the estimation of toxicity equivalents (TEQ) is required, the International Toxicity Equivalency Factors given in Table 6 should be used.
