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John D. Ragsdale, GC/MS Product Specialist Dioxin/Furan Screening Method John D. Ragsdale, GC/MS Product Specialist Of the 210 possible chlorinated isomers this method addresses the 17 2,3,7,8 substituted dioxins and furans. These are considered to be the most toxic.

GC/MS/MS Dioxin Screening In The Beginning… Current Methodology The Plan Sample Preparation The GC/MS/MS Configuration The data Summary of the presentation

In The Beginning… Interest in the analysis of dioxins and furans have increased due to the contamination of feed for livestock and poultry in Europe. The local sales force has responded by requesting application notes and other literature for this analysis on PolarisQ. Similar requests have come from our Pan-Pacific and Asian sales force. This group also includes a current Finnigan customer directly involved in the analysis of dioxin and furan contaminated samples. The reason the project was started.

Dioxin/Furan Analysis EPA Methods Low Resolution MS 8270 ( full scan ) 8280 ( SIM ) Part per million and part per billion detection limits High Resolution MS 8290 ( SIM ) 1613 ( SIM ) Part per trillion detection limits The current EPA methodology for the analysis of dioxins and furans in various matrices. All of the methods are either single ion monitoring or full scan.

The Plan Sample Preparation Chromatographic Resolution Minimal cleanup required Chromatographic Resolution BPX-5; 25M; 0.15mm ID column Robust Chromatography Split injections (2 ul injection) Specificity MS/MS Deliverables Write SOPs The concept behind a screening method is minimal sample preparation, sensitivity and robustness. To achieve these goals the hardware must be able to withstand repeated injections of “dirty” samples. The term dirty is not used to describe the levels of the target analytes but it is used to describe the amount of co-extractable material from the samples.

Sample Preparation 25 grams of material mixed with 50 grams of sodium sulfate. Soxhlet extraction for 24 hours with 50:50 hexane : methylene chloride. Partition against 4 X 40ml concentrated sulfuric acid. Concentrate to 100 ul of 5X dilution of CS3 (2pg/ul of tetra-isomers) The bare minimum of sample preparation can deliver low part per trillion detection limits in a variety of sample matrices.

Additional Sample Preparation Any additional cleanup will lower ultimate detection limits but is not required for this MS/MS screening technique Gel Permeation Chromatography Removes high molecular weight interferences Acid, neutral, and basic silica gel and carbon Removes nonpolar and polar interferences Alumina, Florosil Removes chlorodiphenyl ethers By increasing the amount of sample preparation one can achieve even lower detection limits and extend the time between maintenance even longer. These steps are the most time consuming and are the cause of the most analyte loss. All of the aforementioned steps can be automated with the purchase of automated sample extraction apparatus and automated sample cleanup equipment. Most dioxin/furan laboratories purchase this equipment when using the high-resolution instrumentation. The cost of these two sample preparation devices surpasses the cost of a fully loaded PolarisQ system.

Dioxin Screening GC/MS/MS System Trace GC with split/splitless inlet PPKD DPFC 700kPa with Manual Split Flow 250 Liter Turbo pump PolarisQ EI only / Vacuum Interlock AS2000 Liquid Autosampler SGE BPX-5, 25 meter, 0.15 mm ID, 0.25 micron film column Restek Silco-Steel split liners w/glass wool plug This is the basic system configuration that was used to generate the data in this presentation. Other options may be added at additional cost but are not required for screening.

Configuration Rationale Split Injections allow for “dirtier” sample introduction. Extracts can be screened without the time and cost of extensive cleanup. MS/MS affords the selectivity to remove matrix interference from “dirty” extracts. By using split injection much less matrix is placed on the column and therefore there is less total material to cause any loss of chromatographic performance or any loss in signal from the detector. By using MS/Ms most of the interfering matrix is removed from the chromatogram and all of the components are easily identifiable. For the preparation of this presentation over 200 injections were made on the system and the only item that was replaced on the system was the injection port septum. The injection port septum requires replace after about 75-100 injections due to the high pressures involved using a 0.15mm ID column.

Chromatographic Conditions Injector: 250C Column Flow: 0.9 ml/min Split flow: 10 ml/min Ramp: 150C (1min) to 200C @ 20C/min (1.5min) to 300C @ 3C/min (1.7min) : 40min total A description of the GC conditions used for all of the data in this presentation

Chromatographic Resolution Tetra-Dioxins Tetra-Furans 2,3,7,8 TCDD 2,3,7,8 TCDF Demonstrates the ability to chromatographically separate the 2,3,7,8 substituted dioxins and furans from the other non-toxic dioxins and furans to eliminate false positives. 2,3,7,8 TCDD-IS 2,3,7,8 TCDF-IS

Full Scan, SIM, or MS/MS This slide describes the possible interference that the carbon 13 labeled furan internal standards impart on the native dioxins. Under the chromatographic conditions described in this presentation all of the labeled furan internal standards are chromatographically separated from the 2,3,7,8-substituted dioxins. This phenomenon is shown in the slide as a peak next to the 500 fg/ul 2,3,7,8-TCDD in a later slide. By observing the spectrum one can clearly see that it is not a dioxin but a carbon 13 labeled furan instead because of the ions at m/z 252 and 254.

Full Scan – 10pg/ul in 25g Cow’s milk This is a 25g cow’s milk extract prepared as described earlier in the presentation. This is the type of sample that can be injected over and over again without any loss in performance using the techniques described in the presentation

Dioxins: EPA Method 1613 Linearity to 500 fg/uL to 200 pg/uL for 2,3,7,8-TCDD A calibration curve of 2,3,7,8-TCDD where each calibration level was injected 5 times.

Dioxins: Typical 500 fg/ul injection A typical 500 fg/ul injection of 2,3,7,8-TCDD in pure standard using MS/MS. The large peak to the left of the native TCDD is the interference from the furan mentioned earlier.

Dioxins:Typical Precision for 1613 COMPOUND % RSD 2,3,7,8-TCDD 10.8 1,2,3,7,8-PeCDD 6.4 1,2,3,4,7,8-HxCDD 6.9 1,2,3,6,7,8-HxCDD 9.1 1,2,3,7,8,9-HxCDD 8.7 1,2,3,4,6,7,8-HpCDD 4.9 OCDD 5.2 The calibration curve data from 500 fg/ul to 200 pg/ul for TCDD and 2.5pg/ul to 1000 pg/ul for PeCDD through HpCDD and 5 pg/ul to 2000 pg/ul for OCDD. The internal standards are at 100 pg/ul for each except C13-OCDD which is at 200 pg/ul.

Dioxins:Typical Precision for 1613 ISTD COMPOUND % RSD C13-2,3,7,8-TCDD 4.2 C13-1,2,3,7,8-PeCDD 5.6 C13-1,2,3,4,7,8-HxCDD 8.6 C13-1,2,3,6,7,8-HxCDD 6.9 C131,2,3,7,8,9-HxCDD 7.0 C13-1,2,3,4,6,7,8-HpCDD 8.9 C13-OCDD 9.2 The RSD data from the internal standard areas of the calibration curve mentioned earlier.

Dioxins: Isotope Ratio Stability COMPOUND IONS RATIO % RSD 2,3,7,8-TCDD 259/257 0.97 22.7 1,2,3,7,8-PeCDD 275/277 0.83 10.0 1,2,3,4,7,8-HxCDD 325/327 0.69 20.1 1,2,3,6,7,8-HxCDD 0.66 13.15 1,2,3,7,8,9-HxCDD 14.4 1,2,3,4,6,7,8-HpCDD 361/363 1.35 9.5 OCDD 395/397 1.14 5.7 The ion ratio stability of the MS/MS fragment of each level of chlorination. This could be used for confirmation purposes if necessary.

Dioxins: Full Scan – 10pg/ul std vs. cow’s milk 2,3,7,8 TCDD 2,3,7,8-TCDD ?? Running the samples in full scan of low resolution SIM does yield a peak in the pure standard but the native TCDD is difficult to detect in the cow’s milk matrix. Internal standard @ 100 pg/ul. Peak to the left of native 2,3,7,8 is contribution of mass 322 from the furan internal standard. 2,3,7,8 TCDD-IS 2,3,7,8 TCDD-IS 1,2,3,4 TCDD-RS 1,2,3,4 TCDD-RS Nonane Cow’s milk

Dioxins: MS/MS – 10pg in nonane 2,3,7,8 TCDD Using MS/MS the peaks are clearly identifiable and the spectrum shows the isotope ratios consistent with the standards run earlier. 2,3,7,8 TCDD-IS 1,2,3,4 TCDD-RS

Dioxins: MS/MS – 10pg/ul in cow’s milk 2,3,7,8 TCDD Even in the cow’s milk the TCDD clearly stands out form the base line and the spectrum is also consistent with the standards. 2,3,7,8 TCDD-IS 1,2,3,4 TCDD-RS

Furans: EPA Method 1613 Linearity from 500 fg/uL to 200 pg/uL for 2,3,7,8-TCDF Same as for the dioxins

Furans: Typical 500 fg/ul Injection Same as for the dioxins except that there is no interference from any other internal standards.

Furans:Typical Precision for 1613 COMPOUND %RSD 2,3,7,8-TCDF 17.4 1,2,3,7,8-PeCDF 5.5 2,3,4,7,8-PeCDF 6.2 1,2,3,4,7,8-HxCDF 3.5 1,2,3,6,7,8-HxCDF 4.0 2,3,4,6,7,8-HxCDF 5.6 1,2,3,7,8,9-HxCDF 6.4 1,2,3,4,6,7,8-HpCDF 4.5 1,2,3,4,7,8,9-HpCDF 5.8 OCDF 5.4 The linearity data from the same concentrations as the dioxins.

Furans:Typical Precision for 1613 ISTD COMPOUND %RSD C13-2,3,7,8-TCDF 4.0 C13-1,2,3,7,8-PeCDF 4.5 C13-2,3,4,7,8-PeCDF 4.7 C13-1,2,3,4,7,8-HxCDF 7.2 C13-1,2,3,6,7,8-HxCDF 5.5 C13-2,3,4,6,7,8-HxCDF 6.2 C13-1,2,3,7,8,9-HxCDF 7.7 C13-1,2,3,4,6,7,8-HpCDF 8.1 C13-1,2,3,4,7,8,9-HpCDF 9.4 C13-OCDD 9.2 The internal standard reproducibility from the label furans

Furans: Isotope Ratio Stability COMPOUND IONS RATIO % RSD 2,3,7,8-TCDF 243/241 0.81 22.7 1,2,3,7,8-PeCDF 275/277 7.1 2,3,4,7,8-PeCDF 0.82 7.0 1,2,3,4,7,8-HxCDF 309/311 0.67 10.4 8.1 1,2,3,6,7,8-HxCDF 0.65 5.5 2,3,4,6,7,8-HxCDF 7.9 1,2,3,4,6,7,8-HpCDF 345/347 1.26 7.3 1,2,3,4,7,8,9-HpCDF 1.29 OCDF 379/381 1.14 5.6 The isotope ratio stability across the entire concentration range of the polychlorinated furans.

Furans: Full Scan – 10pg/ul std vs. cow’s milk 2,3,7,8 TCDF 2,3,7,8 TCDF ??? 2,3,7,8 TCDF-IS Just like with the dioxins, the peaks are identifiable in the nonane data but not really detectable in the cow’s milk 2,3,7,8 TCDF-IS Cow’s Milk Nonane

Furans: MS/MS – 10pg/ul in nonane 2,3,7,8 TCDF The furan peak is easily seen with MS/MS and the spectrum matches the standards run earlier. 2,3,7,8 TCDF-IS

Furans: MS/MS – 10pg/ul in cow’s milk 2,3,7,8 TCDF The furan is easily identifiable using MS/MS even in the cow’s milk matrix. 2,3,7,8 TCDF-IS

Spiked Matrix @ 2pg/ul or 4 pg/g (ppt) Cow’s milk Soil Coal fly ash The three matricies were extracted and then spiked with a 1:5 dilution of method 1613 CS 3 (10pg/ul of TCDD/TCDF). This translates into 2 pg/ul of material in the matrix and based on 25 grams of extract in 100 ul of standard the TCDD/TCDF analytes are at a concentration of 4 pg/g or 4 parts per trillion. 12 injections were made for each matrix with standards and blanks in between for a total of 60 injections. Approximately 45 hours of running samples.

Dioxin: 2 pg/ul in 25g Cow’s Milk 2,3,7,8 TCDF 2,3,7,8 TCDD 2,3,7,8 TCDF-IS 1,2,3,4 TCDD-RS 2 pg/ul spike in 25 grams cow’s milk. Internal standard @ 20 pg/ul Since 12 injections of each of the matricies was performed this represents the last of the series to show the excellent recovery even after 45 hours. 2,3,7,8 TCDD-IS TCDF TCDD

Dioxin: 2 pg/ul in 25g Soil TCDF TCDD 2,3,7,8 TCDF 2,3,7,8 TCDD TCDF-IS 1,2,3,4 TCDD-RS 2 pg/ul spike in 25 grams soil. Internal standard @ 20 pg/ul. Large peak to the left of 2,3,7,8-TCDD is another isomer of TCDD. Since 12 injections of each of the matricies was performed this represents the last of the series to show the excellent recovery even after 45 hours. 2,3,7,8 TCDD-IS TCDF TCDD

2 pg/ul in 25g Coal Fly Ash TCDF TCDD 2,3,7,8 TCDF 2,3,7,8 TCDD TCDF-IS 1,2,3,4 TCDD-RS A 2 pg/ul spike in 25 grams of coal fly ash obtained from NIST. Internal standard @ 20 pg/ul. Since 12 injections of each of the matricies was performed this represents the last of the series to show the excellent recovery even after 45 hours. 2,3,7,8 TCDD-IS TCDF TCDD

Summary of analyte recovery Matrix Average (pg/ul) % RSD (n = 12) Nonane 2.18 10.2 Soil 1.91 9.7 Milk 2.28 7.6 Coal Fly Ash 2.05 2,3,7,8-TCDD (2 pg/ul spike) Summarizing the analyte recovery of 60 injections of matrix for a 2 pg/ul spike of 2,3,7,8-TCDD and 2,3,7,8-TCDF. 2,3,7,8-TCDF (2 pg/ul spike) Matrix Average (pg/ul) % RSD (n = 12) Nonane 1.84 7.8 Soil 1.90 11.5 Milk 1.92 Coal Fly Ash 1.95 5.6

Summary of isotope ratios Matrix Average (cali = 0.97) % RSD (n = 12) Nonane 0.93 11.6 Soil 0.83 17.1 Milk 1.06 20.4 Coal Fly Ash 0.99 24.3 2,3,7,8-TCDD 259/257 Summarizing the isotope ratio of 60 injections of matrix for a 2 pg/ul spike of 2,3,7,8-TCDD and 2,3,7,8-TCDF. This shows a good correlation between the isotope ratios obtained from the calibration curve and the ratio obtained from a matrix. 2,3,7,8-TCDF 243/241 Matrix Average (cali = 0.81) % RSD (n = 12) Nonane 0.80 20.8 Soil 0.82 20.6 Milk 0.87 17.6 Coal Fly Ash 0.89 19.7