Confirmatory analyses

Contrary to the enforcement methods, additional confirmatory analysis is not necessary where it is demonstrated that the primary residue method is specific to the analyte(s) and the source of the analyte(s) is known. 

For the confirmatory procedure, it is recommended that the sponsor develop spectral data based on at least three structurally specific ions that completely define the marker residue molecule. These ions may or may not include the molecular ion. The use of water loss and isotopic ions is usually unacceptable and CVM concurrence should be sought when water loss ions or isotopic ions are selected for the confirmatory analysis. The proposed fragment ion structures should be consistent with the fragmentation pattern, and justification for specificity of selected ions or scan range should be included. All confirmation criteria should be specified in the standard operating procedure. 

To determine the residue levels of dinitroaniline herbicides, GC/NPD or GC/ECD is used in general. An aliquot of GC-ready sample solution is injected into the gas chromatograph under the conditions outlined below. Further confirmatory analysis is carried out using gas chromatography/mass spectrometry (GC/MS) in the selected-ion monitoring (SIM) mode. 

B. HPLC operating conditions for confirmatory analysis on the Quattro II triple-quadrupole mass spectrometer... 

Collector (collects samples of the suspect aerosol for analysis by the JBPDS, and for confirmatory analysis by supporting laboratories in the Communications Zone and the continental United States). 

MTBE can be analyzed for with U.S. EPA SW-846 Method 8015 or 8021 however, 8021 has lower detection limits, is subject to less interference in highly contaminated samples and tends to be more economical by providing BTEX data in the same analysis. Concerns about coelution with some alkanes requires at least one confirmatory analysis with SW-846 Method 8260 per site. 

Figure 5A, B shows the isotopic distribution, of protonated bosentan (C27H30N5O6S, Mr 552.6) with a mass resolution of 0.5 and 0.1 at FWHM, respectively. It is worthwhile to observe the mass shift of the most abundant ion from m/z 552.2006 to m/z 552.1911. This value does not change with a mass resolving power of 15 000 (Fig. 1.5C) or even 500000 (Fig. 1.5D). Accurate mass measurements are essential to obtain the elemental composition of unknown compounds or for confirmatory analysis. An important aspect in the calculation of the exact mass of a charged ion is to count for the loss of the electron for the protonated molecule [M+H]+. The mass of the electron is about 2000 times lower than of the proton and corresponds to 9.10956 x 10 kg. The exact mass of protonated bosentan without counting the electron loss is 552.1917 units, while it is 552.1911 units with counting the loss of the electron. This represents an error of about 1 ppm.

Elemental composition S 23.76%, Cl 52.54%, 0 23.70%. The compound can be dissolved in an organic solvent, such as toluene, and analyzed by GC using an ECD or FPD type detector or by the GC/MS (a confirmatory analysis). Also, a small amount of compound is decomposed in water (slow reaction) and the products, HCl and H2SO4, are measured by ion chromatography or by wet methods... 

The FDA/FSIS survey lasted to 1997. Only 1 of the 499 eyeball samples gave a positive result with the ELISA screen, but the presence of clenbuterol could not be confirmed in this sample. Confirmatory analysis showed that this animal contained residues of the -agonist fenoterol. 

In addition, a total of 146 sheep urine and 87 chicken muscle samples from birds sold in local markets and originating from Brazil, Denmark, France, and Turkey were tested for residues of diethylstilbestrol and ethinylestradiol (41). Although some of the samples were positive to both analytes by an immunochemical screening assay, confirmatory analysis by GC-mass spectroscopy (MS) showed that none of the samples contained residues of the examined steroids. 

Coupling chromatographic procedures with immunochemical techniques can also provide a very sensitive and specific analytical system for either determinative or confirmatory analysis. If the antibody used is very specific for the analyte of interest and the antibody reactivity is known to be sensitive to small variations in the structure of the analyte tested, positive reactions with the method are strongly indicative that an analyte of defined structural characteristics is present in the sample. Full rigorous confirmation, however, would depend on further analysis by mass spectrometry, which is the method of choice in confirmatory analysis. Mass spectrometry gives specific information on the identity and structure of the compound of interest. Coupled with chromatographic techniques it becomes a very powerful confirmatory tool for both quantitative and qualitative assessment of drug residues in foods. 

Coupling of liquid chromatography with mass spectrometry provides further unequivocal online spectrometric identification of individual analytes at the very low residue levels required by regulatory agencies for confirmatory analysis... 

Electrochemical detection is better suited to the analysis of erythromycin and lincomycin. This method of detection has been applied for the determination of erythromycin A (139) and lincomycin (154) residues in salmon tissues. Liquid chromatography coupled with mass spectrometry is particularly suitable for confirmatory analysis of the nonvolatile macrolides and lincosamides. Typical applications of this technique are through thermospray mass spectrometry, which has been used to monitor pirlimycin in bovine milk and liver (141,142), and chemical ionization, which has been applied for identification of tilmicosin (151) in bovine muscle, and for identification of spiramycin, tylosin, tilmicosin, erythromycin, and josamycin residues in the same tissue (150). 

Confirmatory analysis of suspected liquid chromatographic peaks is usually accomplished by a photodiode array detector that continuously collects spectral data during the chromatographic separation and further compares the spectrum (200-550 nm) of the eluted suspected compound with that of a standard (37, 38, 66, 161, 163, 166-168, 178, 180, 181). Online absorbance ratio techniques combined witlr off-line thin-layer chromatography have been also reported (171). Although tliese confirmation techniques are relatively simple, their sensitivity is not generally adequate to identify trace levels of residual nitrofurans in edible animal products. 

Coupling of liquid chromatography with mass spectrometry allows unequivocal online spectrometric identification of all nitrofurans at the very low residue concentrations required by regulatory agencies for confirmatory analysis in animal-derived foods. Typical examples of mass spectrometry applications in confirming nitrofuran residues in edible animal products employ thermospray (174, 176), ionspray (166), or atmospheric pressure chemical ionization (157) interfaces. 

Confirmatory analysis of suspected liquid chromatographic peaks can be accomplished by a photodiode array detector that continuously collects spectral data during the chromatographic separation and further compares the spectrum (200-450 nm) of tire eluted suspected compound with that of a standard (66,... 

The number of detectors that are sensitive and selective enough to be applied online with LC is limited because the solvents used are not compatible, as in the case of immunochemical detection after reversed- or normal-phase LC. The technology of coupling is still under development and not yet available in a large number of laboratories not specialized in techniques such as LC-MS. Therefore, LC separations are frequently followed by offline detection. Confirmatory analysis of suspected liquid chromatographic peaks can be made possible by coupling liquid chromatography with mass spectrometry. Atmospheric-pressure chemical ionization LC-MS has been employed for the identification of six steroid hormones in bovine tissues (448). 

The sample must be representative and of sufficient size to allow adequate analysis and to allow a repeat analysis and any confirmatory analysis. 

D. Duebelbeis, S. Kapila, T. E. Clevenger and A. F. Yanders, Application of a two-dimensional reaction chromatography system for confirmatory analysis of Chlordane constituents in environmental samples. Chemosphere 20 1401-1408 (1990). 

Pearce and Lushnikova [78] used semipreparative HPLC method for the isolation of the three omeprazole metabolites produced by the fungi. Incubation of Cunninghamella elegans ATCC 9245 and omeprazole allowed putative fungal metabolite to be isolated in sufficient quantities for structural elucidation. The metabolites structures were identified by a combination of LC/MS and NMR spectrometric experiments. These isolates are used as reference standards in the confirmatory analysis of mammalian metabolites of omeprazole. In the LC/MS and LC/MS/MS analysis, components were separated by reversed-phase HPLC on a Hypersyl HyPurity (15 cm x 4.6 mm, 5 /im) column. The mobile phase consisted... 

A similar full scan MS approach was presented by Tang et al. for screening of quartemary ammonium compounds and quantification of ipratropium in horse urine [24] (Table 5). However, they used an IT mass spectrometer that additionally allowed confirmatory analysis of the drug by MS/MS experiments for verification of HPLC peak-drug assignment. 

Its largest field of application, which will focus on this chapter, is the multiresidual analysis in biological samples. In so-called confirmatory analyses, LC-MS, or better LC-MS/MS, provides an unequivocal identification, due to the high selectivity of the MS detector. The results of Systematic Toxicological Screening Analysis (STA) or General Unknown Screening (GUS), other applications of LC-MS, needs of subsequent confirmatory analysis to be used in a court [52, 55]. 

Maralikova B, Weinmann W (2004) Confirmatory analysis for drugs of abuse in plasma and urine by high-performance liquid chromatography-tandem mass spectrometry with respect to criteria for compound identification. J Chromatogr B Analyt Technol Biomed Life Sci 811(1) 21—30. doi 10.1016/j.jchromb.2004.04.039 SI570-0232(04)00642-7 [pii]... 

The modem GC data system will produce a report of peaks detected with the retention time, peak area, and peak height. In order to identify the analytes of interest and quantify the data, a series of calibration standards are required to be analyzed followed by samples. The calibration standards will identify retention times for analytes, surrogates, and internal standards. With the exception of MS analysis, compounds are identified in chromatograms based solely on their retention time. Positive confirmation can be done by analyzing the same sample extract on a different type (polarity) of GC column. If the compound is detected at the same concentration from both GC columns, then the data can be reported (e.g., US EPA Method 8081—OC Pesticides—requires analysis on a DB-5 column with confirmatory analysis on a DB-17 column). For MS analysis, multiple ion chromatograms... 

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