Chromatographic resolving

Figure 3.26 shows an example of the use of RICs to locate a compound which is not chromatographically resolved from the major components of the mixture and which is not obviously present from an examination of the TIC trace. In this case, integration of these RICs, as shown in Figure 3.28, would allow quantitative measurements to be made on all three components, although with different degrees of precision. 

A high-throughput approach to HPLC-MS/MS for metabolite identify-cation was also described by Dear and co-workers [29], where up to six hydroxylated isomers were chromatographically resolved in 1 min with the overall cycle time reduced to 5 min on a monolithic column (4 mL/min). 

Should be chromatographically resolved from the analyte and any excipients present in the chromatogram of the formulation extract... 

It should incorporate 13C, 15N, and 180 instead of deuterium (2H) as the first choice to minimize the isotope effect and possibility of exchange. Multiple deuterium atoms can result in a significant isotope effect, to the point that the fully labeled form can be chromatographically resolved completely from the native. This means that the fully labeled form is not in the LC-MS or LC-MS/MS ion source at the same time as the analyte, and therefore this standard cannot control ion source events, limiting its ability to mimic the analyte behavior. 

Determinative and confirmatory methods of analysis for PIR residue in bovine milk and liver have been developed, based on HPLC-TS-MS (209). Milk sample preparation consisted of precipitating the milk proteins with acidified MeCN followed by partitioning with a mixture of -butylchloride and hexane, LLE of PIR from aqueous phase into methylene chloride, and SPE cleanup. The dry residue after methylene chloride extraction was dissolved in ammonium hydroxide, and this basic solution was transferred to the top of Cl8 SPE column. The PIR elution was accomplished with TEA in MeOH. For liver, the samples were extracted with trifluoroacetic acid (TFA) in MeCN. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, and then extracted with methylene chloride. The organic solvent was evaporated to dryness, and the residue was dissolved in ammonium acetate. The overall recovery of PIR in milk was 94.5%, RSD of 8.7%, for liver 97.6%, RSD of 5.1 %. A chromatographically resolved stereoisomer of PIR with TS-MS response characteristics identical to PIR was used as an internal standard for the quantitative analysis of the ratio of peak areas of PIR and internal standard in the pro-tonated molecular-ion chromatogram at m/z 411.2. The mass spectrometer was set for an 8 min SIM-MS acquisition. Six samples can be processed and analyzed in approximately 3 hours. 

M. Martin, D. P. Herman and G. Guiochon, Probability distributions of the number of chromatographically resolved peaks and resolvable components in mixtures , Anal. Chem. 58 2200 (1986). 

Monobasic acids are determined by gas chromatographic analysis of the free acids dibasic acids usually are derivatized by one of several methods prior to chromatographing (176,177). Methyl esters are prepared by treatment of the sample with BF,—methanol, H2S04—methanol, or tetramethylammonium hydroxide. Gas chromatographic analysis of silylation products also has been used extensively. Liquid chromatograpliic analysis of free acids or of derivatives also has been used (178). More sophisticated liplc methods have been developed recently to meet the needs for trace analyses in the environment, in biological fluids, and other sources (179,180). Mass spectral identification of both dibasic and monobasic acids usually is done on gas chromatographically resolved derivatives. 

Despite the enormous selectivity provided by multiple-reaction monitoring through targeting individual precursor and product ion mass combinations, interference from cross talk can still confound mixture analysis in LC/MS. Cross talk is defined as contributions from ions of similar or identical mass to other precursor-product ion channels. Cross talk can occur via two distinct pathways. Chemical interference due to in-source fragmentation of metabolites or related substances will yield ions identical to the analyte precursor which pass through the first quadrupole. This type of interference can only be detected if the metabolite is chromatographically resolved from the analyte peak. 

Goal All related substances must be chromatographically resolved from the active substance nothing should coelute with a specified related substance. Unspecified related substances could coelute with each other but must not coelute with the active, specified related substance or an excipient of the formulation. 

Recently, Blount et al. (2000) summarized a methodology to detect di- -butyl phthalate metabolites in urine. In humans or animals, di- -butyl phthalate is metabolized to mono- -butyl phthalate and oxidative products, which are excreted through the urine and feces. Human urine samples are processed by P-glucuronidase hydrolysis (to release the mono phthalate ester) followed by solid-phase extraction. The eluate is concentrated mono- -butyl phthalate is chromatographically resolved by reverse-phase HPLC, detected by negative ion atmospheric pressure chemical ionization (APCI) tandem mass spectrometry, and quantified by isotope dilution. 

Equation (3.27) is the basis for TEQA, because for a given VOC and fixed sample volume in a headspace vial (i.e., and P), Chs is directly proportional to the original concentration of VOC in the aqueous sample, Co - The volume of headspace sampled and injected into a GC is usually held fixed so that the area under the curve of a chromatographically resolved GC peak, is directly proportional to Chs - As discussed in Chapter 2, Eqs. (2.1) and (2.2), with respect to the external mode of instrument calibration, the sensitivity of the HS jC technique is related to the magnitude of the response factor, according to... 

The above three factors broaden chromatographically resolved peaks by contributing a variance for each factor starting with Eq. (4.23) as follows ... 

The fluorescence HPLC detector (HPLC FL) is similar to the UV-absorp-tion HPLC detector (HPLC UV) in that a source of UV radiation is made incident to a micro-flow cell in whieh the chromatographically resolved analyte passes through. However, a photomultiplier tube (PMT) and associated optics are positioned at right angles relative to the incident UV radiation. Lakowicz has articulated just what molecular luminescence is (93) ... 

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