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LABORATORY INFORMATION BULLETIN
DFS/ORA No 3991
GCMS Confirmation of Bitertanol in Bananas
by
Carl N. Syvertson and Harold C. Thompson Jr.
Food and Drug Administration
National Center for Toxicological Research
Jefferson AR 72079
Introduction
The method for determination of bitertanol (C20H23N3O2)1 was evaluated to acquire an efficient and practical single residue method since bitertanol is not recovered efficiently by a multi-residue method and because bitertanol method evaluation is indicated in the FDA Five Year Pesticide Research Plan2. The bitertanol method chosen for evaluation required modification3 as well as development of a confirmation step. Because GCMS is a rapid technique for confirming analyte identity and FDA pesticide laboratories now have benchtop GCMS equipment for use on a routine basis, a method was developed for the GCMS confirmation of bitertanol. This method uses extracts from the previously modified determinative method1 and involves selected
ion monitoring of the acetyl derivative of bitertanol. Because the mass spectrum of underivatized bitertanol does not include sufficient peaks for confirmation by selected ion monitoring, derivatization and confirmation of the derivative was necessary. The lowest level examined was 0.1 ppm which is one half the existing regulatory tolerance for bitertanol4.
Method
Sample Preparation - Pipet 1 ml of sample extract from determinative assay1 into a 25 ml separatory funnel. Add 10 ml deionized water and 2 ml methylene chloride. Shake for 2 minutes, drain the methylene chloride portion into a 10 ml graduated centrifuge tube. Repeat the extraction two additional times, then concentrate the combined extract just to dryness under a stream of nitrogen. Add one ml acetic anhydride and two ml Pyridine PlusTM re agent (this reagent is distributed by Alltech for derivatization of sterically hindered alcohols and contains 90% pyridine and 10% 4-dimethylaminopyridine). Heat at 65 OC for 45 min utes. Transfer to a 25 ml separatory funnel and add 10 ml deionized water. Extract three times with 2 ml pentane. Concentrate the combined extract to one half ml under a stream of nitrogen.
Evaluate the extract on a GC-FID to verify that it does not contain gas chromatographable compounds that did not show up on HPLC detectors at levels that will overwhelm the MS ion source. This is performed by injecting 2 æl of extract into a GC-FID that is equipped with a 5 to 12 meter X 0.25mm DB-1 column with a 0.25 æm film thickness, a splitless in jector at 250 oC, a FID at 280 oC, an oven that is initially set at 100 oC then increased at 40 oC/min to a final temperature of 310 oC and is held at this temperature for 20 minutes. The extract is not injected on the GC/MS if there is a peak in the chromatogram that represents ò 250 æg / ml dissolved chromatographable organics.
Instrument Parameters - Use a Hewlett Packard 5890 series II gas chromatograph e quipped with a Hewlett Packard 5971A mass selective detector and with a 30 m X 0.25 mm DB-5 column with a 0.25 æm film thickness for this analysis. Instrument parameters are as follows:
Carrier gas: Set the helium head pressure at 3 psi.
Injector: Splitless injector at 250 oC.
Oven Temperature: Initially set the oven at 100 oC,
increase at 25 oC/min to 310 oC,
hold for 4.5 minutes.
GC/MSD Interface: Set the interface temperature to 280 oC.
Ionization: Use electron ionization (EI) at 70 V.
Delay: Turn on the filament and electron multiplier voltages 3.5 minutes after injection.
Tuning: Auto tune the MSD using ions at m/z 69, 19, and 502 for perfluoro-tributylamine (PFTBA). Meet the following criteria to ensure the accuracy of ion mass assignments and ion abundance measurements. Peak mass assignments do not vary by more than plus or minus 0.20 daltons. Peak widths are between 0.4 and 0.6 daltons.
m/z Ion Abundance Criteria
69 100% Base peak.
70 0.5-1.5 % of mass 69.
219 >30% of mass 69.
220 2-8% of mass 219.
502 >1% of mass 69.
503 5-15% of mass 502.
GC/MS Confirmation of Analyte Identity - Set the instrument for selective ion monitoring using m/z of 112, 141, 154, 168, 170, and 210. Inject 2 æl of derivatized sample extract followed by 2 æl derivatized standard. Generate a background subtracted bitertanol spectrum using the following three steps. First average at least three sample spectra including the apex and an equal number of spectra from either side of the apex. Then produce a background spectrum by averaging at least three scans immediately after the bitertanol peak or use one scan from the valley betweenthe analyte peak and the peak before or after it. Finally, subtract the background spectrum from the average sample spectrum. All of the mass units in the sample should have a signal to noise ratio greater than 3. The relative abundances of m/z 112, 141, 154, 168, and 170 to m/z 210 in the sample should be within ±10% with respect to the base peak of the corresponding relative abundances in the standard. Also, the retention times must match.
Results and Discussion
The first attempt to confirm bitertanol was a simple solvent extraction of the HPLC sample and concentration of the extract to a small volume. This approach worked well as a means of introducing bitertanol into the mass spectrometer with minimal matrix contamination. Yet it fell short of producing confirmable spectra. The EI mass spectrum of bitertanol is very simple, but there are no peaks with relative abundances above 20% (see Figure 1). This poses a problem when trying to select three ions above 20% of the base peak for selected ion monitoring as specified by FDA mass spectra acceptance criteria5.
The first technique examined to obtain a confirmable EI mass spectrum was methane chemi cal ionization. Reproducible fragment ions were obtained but the relative abundances were not consistent enough for definitive confirmation. The variance of ion relative abundances were not even consistent enough between different scans within the same chromatogram. It is possible that consistent relative abundances may be obtained using a GC/MS equipped with a higher pressure chemical ionization ion source. Unfortunately this type of instrument is not currently available to all FDA laboratories that perform pesticide analysis. Therefore, this approach was not pursued further.
After abandoning chemical ionization as a means of confirmation the approach of d erivatizing bitertanol and confirming the derivative was evaluated. Heating with acetic anhydride and a few mild silylating reagents were attempted without success in an effort to derivatize bitertanol. However, silylating reagent containing 90% b is(trimethylsilyl)trifluoroacetamide and 10% trimethylchlorosilane derivatized bitertanol adequately and produced a confirmable EI mass spectrum (see Figure 2). Chromatographyworked well for the derivatized standard. Unfortunately, an unidentified component in the banana matrix was also derivatized. This contaminant destroyed the effectiveness of the column and contaminated the ion source, making it necessary to rejuvenate the column and clean the source. For this reason, silylation was not considered a good idea.
Finally, acetic anhydride and Pyridine PlusTM (a reagent distributed by Alltech for derivatization of sterically hindered alcohols) was used. This derivatization went to completion and produced an acceptable EI mass spectrum (see figure 3). Repeated injections of derivatized fortified sample showed no deterioration of either the chromatography or MS performance.
Electron ionization selected ion monitoring (SIM) was used for all samples. The ions selected were chosen because of the information they could contribute to positive identification of the analyte. A proposed fragmentation scheme that explains a likely source for each of the major fragment ions at 112, 141, 154, 168, 170, and 210 is found in figure 4. The acceptable percentages were determined by evaluating variances in data from analysis of standards.
When in the SIM mode, the Hewlett Packard 5971 mass selective detector has a "high resolution" mode (0.05 mass unit resolution) as well as a "low resolution" mode. The latter uses the peak maximum nearest the selected mass. The "low resolution" mode was used for simplicity of setting up the instrument. This eliminated the need for selecting the exact masses and saved the time setting the instrument up to monitor exact masses after each tune.
Banana samples fortified at 0.1 ppm were checked to verify that bitertanol can be determined in these sample matrices by SIM. The results are reported in table 1. Mass chromatograms of the selected ions show that the signal to noise ratio is well above three for all the ions (see figure 5).
Conclusion
The method is capable of confirming bitertanol in banana extracts. An acetyl derivative is used because it produces a more definitive spectra than underivatized bitertanol. Bitertanol can be confirmed in samples containing ò 0.1 ppm using a SIM technique.
Acknowledgement:
The authors thank Thomas Heinze of the Mass Spectrometry Branch of the Division of Chemistry for his helpful suggestions for the proposed EI fragmentation scheme for acetylated bitertanol.
References:
1] Syvertson, C. N.; Thompson, H. C. Jr.; Laboratory Information Bulletin 3928; Food and Drug Administration, Division of Field Science; Rockville MD 20857.
2] FDA Pesticide Research: Five Year Plan End Time Table: FY 94-FY 98 Feb 93, FDA.
3] Brennecke, R.; Pflanzenschutz-Nachrichten 1988, 41 113-36.
4] Code of Federal Regulations 40, part 180.457.
5] Sphon, James A.; J. Assoc. Off. Anal. Chem. 1978, Vol. 61 No. 5, pp1247-1252.
Figure 1
Full scan EI mass spectrum of underivatized bitertanol.
Figure 2
Full scan of silyl derivatized bitertanol.
Figure 3
Full scan EI mass spectrum of acetyl derivatized bitertanol.
Table 1
Comparison of Standard and Sample EI SIM Data
Standard Sample Difference Within Acceptance
m/z % RA % RA Sample-Standard Range
/_\ % RA (± 10 % of RA)
112 33.1% 42.0% 8.9% Yes
141 20.8% 16.8% -4.0% Yes
154 43.7% 50.5% 6.8% Yes
168 45.3% 51.7% 6.4% Yes
170 41.2% 39.1% -2.1% Yes
210 Base Peak Base Peak N/A Yes
F ig ure 4
Proposed EI fragmentation scheme for acetylated bitertanol.
Figure 5
Mass chromatograms for ions at m/z 112, 141, 154, 168, 170, and 210.
Peak at 12.6 minutes is acetylated bitertanol.
