HPLC conditions

Different transfer techniques and type of interfaces have been developed. Most of the applications involve normal-phase HPLC conditions, although reversed-phase coupled with capillary GC has also been reported. 

HPLC analysis indicated 97.1 area % purity. HPLC conditions Zorbax XDB-C8 column (3.0 x 100mm) eluting with 5-100% MeCN + 0.1% TFA (0.5 mL/min) 220 nm wavelength. 

The interface can be used with a wide range of HPLC conditions, flow rates and mobile phases, both normal and reverse phase, particularly if spray deposition is employed. 

Electrospray is an unusual mass spectrometry technique in that it allows the study of the three-dimensional structure of compounds, particularly proteins, in solution as it is believed that this is relatively unchanged when ions are transferred to the vapour phase. This type of application will be discussed in more detail in Chapter 5 but attention is drawn at this point to the previous comments regarding the effect that the HPLC conditions, such as pH, may have on the appearance of an electrospray spectrum and the conformational deductions that may be made from them. 

The advent of the electrospray interface has allowed the full potential of LC-MS to be achieved. It is now probably the most widely used LC-MS interface as it is applicable to a wide range of polar and thermally labile analytes of both low and high molecular weight and is compatible with a wide range of HPLC conditions. 

In this chapter, seven types of LC-MS interfaces have been described and their performance characteristics compared. Any modifications to the HPLC conditions that are required to allow the interface to operate effectively have been highlighted. 

Figure 13, indicates that the first mole of phenol is released in <30 s, the same elapsed time for the chemiluminescence to reach a maximum intensity. In fact, the measured rate constant r, for the rise in the chemiluminescence emission, is identical to the rate of the first phenol s release from the oxalate ester. Furthermore, the slower rate of release of the second phenol ligand has a rate constant that is identical to the chemiluminescence decay rate f. Thus, the model allows a quantitative analysis of the reaction mechanism, heretofore not available to us. We intend to continue this avenue of investigation in order to optimize the chemiluminescence efficiencies under HPLC conditions and to delineate further the mechanism for peroxy-oxalate chemiluminescence. 

Figure 4C. Chromatogram illustrating the studies conducted on the enzymatic conversion of the PSP toxins to decarbamoyl metabolites (appended with an "M" in these figures). HPLC was used to confirm that the toxin metabolites (labeled "M") were the decarbamoyl form through conversion back to the carbamate form with chlorosulfonyl isocyanate. (See Ref. 15 for HPLC conditions.)...

Figure 6. HPLC separation of the toxins present in various organisms during a dinoflagellate bloom in Quartermaster Harbor, Washington (A) dinoflagellates, (B) mussels, (C) littleneck clams, (D) butter clams. The toxin metabolites ("M") were later found to be the decarbamoyl form. See Ref. 4 for HPLC conditions.)...

The enforcement methods provided by the applicants give basic information about appropriate cleanup steps and specific determination procedures. Typically, direct use of this developmental work occurred when a GC multi-residue method was found appropriate. Owing to the recent developments in the field of MS/MS with atmospheric pressure ionization, an alternative approach for those compounds that can be analyzed by liquid chromatography (LC) will soon be possible. It is important that some fundamental considerations for such method(s) should be agreed at the outset. Considerations include the most suitable extraction solvents and cleanup steps and some standard HPLC conditions. 

Table 1 Example HPLC conditions for the determination of sulfonylurea herbicides by LC/MS/MS...

Bifenox, nitrofen and oxyfluorfen HPLC conditions withpost-columnfluorescence reactor system column, C-18 reversed-phase (25cmx4.6-mm i.d.) temperature, 40 °C flow rate, 1 mL min flow composition, acetonitrile-water (1 4, v/v) (2 min), with increase in acetonitrile at 5%min to 90% acetonitrile to acetonitrile-water... 

InsAument parameters (sheath and auxiliary gas flows, spray voltage, capillary temperature, collision cell gas flow and offset, etc.) should be optimized while infusing a standard of tebuconazole prior to the Arst attempt at analysis. Optimization should be performed at an HPLC Aow rate and composition simulating those present during elution of tebuconazole using each HPLC condition set employed... 

Oligomeric additives with broad MWD tend to be a problem in conventional HPLC conditions. In cases where no interest exists in the oligomer distribution it is common practice to solve the problem by creating a uniform structural unit useful for analysis. For example, isocratic (or gradient) LC-UV was used for the determination of the polymeric light stabiliser Tinuvin 622 in polyolefins using dissolution (toluene)/derivatisation (TBAH)-precipitation (alcohol) the diol formed was quantitatively determined by NPLC [653]. 

HPLC allows a quantitative determination with relatively simple extractions. In many cases, extraction only involves a heating of the commodity with water, followed by filtration and injection onto an HPLC column. In the determination of caffeine, theobromine, and theophylline in cocoa, coffee, or tea, as well as in other foods, there is scarcely a month that passes without a new paper on this assay. Kreiser and Martin provide typical conditions for analysis.28 In their studies, samples were extracted in boiling water and filtered prior to injection onto the HPLC column. The HPLC conditions used a Bondapak reversed phase column and a mobile phase of water methanol acetic acid (74 25 1) with detection at 280 nm. This method is accurate, precise, and conserves time. It has also been adopted by the AOAC as an official method for the determination of theobromine and caffeine in cocoa beans and chocolate products.29... 

FIGURE 7.12 Two-dimensional separation of tryptic digest of BSA in simple 2D HPLC. Conditions as in Fig. 7.11 (reproduced from the reference, Kimuraetal. (2004), with permission from Wiley). 

When using microbial products for mammalian metabolite identification, it is suggested to compare all the analytical data available. For example, slight differences in MS2 or MS3 spectra may indicate that the microbial products are not the same as the mammalian metabolite. Owing to matrix effects, HPLC retention time often varies from run to run, so it is good practice to spike a comparable amount of purified microbial product into the in vitro, in vivo or purified samples that contain the mammalian metabolite of interest. If the microbial metabolite and the mammalian metabolite are the same compound, then they should co-elute under different HPLC conditions, including different solvent pH, and the MS and/or UV peak area would increase accordingly. 

Table 3. Summary of HPLC conditions for the analysis of oxytetracycline... 

BA trans-3.4-dihvdrodiol cannot be separated from BA trans-8.9-dihydrodiol in several HPLC conditions (27-29). Quantification of BA trana-3,4-dihydrodiol by HPLC can only be accomplished after converting the 3,4-dihydrodiol to its diacetate (25.26). The BA trans-3.4-dihydrodiol formed in BA metabolism by liver microsomes from pheno-barbital-treated rats was determined to have a 3R,4R/3S,4S enantiomer ratio of 69 31 (30). Recently we have determined the optical purity of the BA trans-3.4-dihvdrodiol formed in the metabolism of BA by three liver microsomes prepared from untreated rats and rats that had been pretreated with an enzyme inducer. As shown in Table II, cytochrome P-450 isozymes contained in liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats had similar stereoselectivity toward the 3,4-double bond of BA. BA trans-3.4-dihydrodiol is formed via the 3,4-epoxide intermediate (31). 

High performance liquid chromatography has been used to analyze pseudoephedrine hydrochloride and dosage forms containing pseudoephedrine hydrochloride. Table V gives the HPLC conditions used for separations. 

HPLC condition — A Waters reversed-phase HPLC column (Symmetry Shield RP Cl8, 5 pm, 2.1 x 50 mm) was used in conjunction with a Regis SPS guard column (ODS, 5 pm, 100 A,... 

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