HPLC isolation

In a study by Stresser and co-workers, the effect on tumor modulation by 227 has been investigated. HPLC on liver extracts from Fisher 344 rats revealed two major compounds, 3,3 -bisindolylmefliane (133) and a linear trimer, together with a < l(KX)-fold lower content of 4 in comparison with the two major substances. The HPLC isolate was derivatized with /V-methyl-/V-bis(trifluoroacetamide) that, upon MS detection, gave a compound identical to /V,W -ditrifluoroacetylindolo-[3,2-()]carbazole. The content of 4 in this system was estimated to be 0.(XKX)13% of the total dose of 227 given. Thus, it was concluded that the beneficial effect of oral distribution of 227 is due to the total content of derivatives formed (95MI5). 

Merghem, R., Qualitative analysis and HPLC isolation and identification of procya-nidins from Vicia faba, Phytochem. Anal., 15, 95, 2004. 

In an initial series of experiments, [Me-14C] methionine and [8-14C]adenine were injected into D. verrucosa hepatopancreas. After HPLC isolation, xylosyl-MTA was found consistently labelled in particular, in the experiment with labelled methionine 28.0% of the recovered radioactivity was found associated with xylosyl-MTA, while 12.1% was recovered in the adenosylmethionine... 

General Experimental Procedures MPLC fractionation was performed using an Isco CombiFlash, and HPLC isolation was performed using Shimadzu pumps and detector and YMC-Pack ODS-AQ C18 column. 

Pereda-Miranda R, Hemandez-Carlos B (2002) HPLC Isolation and Structural Elucidation of Diastereomeric Niloyl Ester Tetrasaccharides from Mexican Scammony Root. Tetrahedron 58 3145... 

Convergent ligation of cyclic peptide 86b (1.6 mmol) in twofold excess (8 equiv) of 93 (0.1 mmol) with a final concentration of 1-2 mM was performed in H20 at pH 8 with MeCN (50%) as a cosolvent to increase the solubility of 93. The ligation yielded >80% of the four-branch peptide dendrimer 94 (MALDI-MS mJz - calcd 8685.0 found 8685.7) together with <15% of the two- and three-branch dendrimers (MALDI-MS for three-branch dendrimer m/z calcd 6602 found 6604.0). After 45 min, the reaction was terminated and purified by RP-HPLC isolated yield 58%. 

Fig. 12. The 2D [1H,15N] HSQC-NMR spectrum of the HPLC-isolated dien ring-opened complex at pH 4.0. Only the NH2 group of L-MetH was 15N-labelled, and the four sets of crosspeaks (peaks a, a to d, d ) can be assigned to the non-equivalent Pt-NH2 groups in the four diastereomers of [Pt(dienH-A,AO(15N-L-Met-YA012+- All peaks have 2J(NHa, NHb) of ca. 12 Hz, while only peaks a and b have an additional V( a-CH.NHj of ca. 13 Hz. (Adapted...

The pharmacologically active hydroxy metabolites, present either as free or as fatty acid conjugates, will be of prime interest. Sufficient milk volume must be collected to allow HPLC isolation of suspected cannabinoids and off-line mass spectral characterization. 

Refers to the first step only ee values determined by chiral HPLC Isolated yields for two steps... 

Run Product Reagent Solvent Time (h) HPLC Isolated... 

Perform mass analysis of HPLC isolated peptides prior to sequence analysis 39 39%... 

Bouchet, N. Levesque, J. Pousset, J.L. HPLC isolation, identification and quantification of tannins from Guiera senelalensis. Ph3dochem. Anal. 2000, 11, 52-56. 

The purity was lower than desirable for NMR analysis, so further purification was undertaken to facilitate structure elucidation by NMR. The existing analytical methodology contained perchloric acid that was not suitable for preparative HPLC isolation and LC/MS analysis because of safety concerns with concentration of perchloric acid and high probability of damage to the mass spectrometer over time. A mass spectrometry compatible method using a 0.1% acetic acid buffer was selected as a starting point. Minor method development produced a suitable method to separate the reaction components the drug substance, the sulfoxide, and the N-oxide. 

In terms of impact on the project, the N-oxide structural elucidation allowed for an appropriate specification of the degradant, and clinical time lines were not impacted. It is advised in all preparative HPLC isolations that an analytical or preparative scale reinjection is performed to clean up the analyte of interest from the salt. This can include washing the analyte by reversed-phase HPLC (preparative or analytical scale depending on isolate amount) with aqueous phase and ramping up the organic phase to elute the desalted analyte of interest. 

NMR data were required to differentiate between these proposed structures. Given the deadline, the most efficient technique for sample isolation was reversed-phase preparative HPLC isolation, scaling up the existing analytical reversed-phase method. With two or more possible structures, synthesis is typically too time- and resource-intensive. This would be twice the effort in that the CF2H and dehydration products would both need to be synthesized. 

Case Study B.5 Preparative HPLC Isolation from a Retained Process Sample and Characterization Using LC/MS and NMR Characterization... 

Case Study B.8 Preparative HPLC Isolation from Retained Process Samples and Characterization by LC/MS and NMR... 

Instrumentation. HPLC isolations were performed on a Beckman 421A system using a Vydac column (C-18, 4.6 x 250 mm). Liquid secondary ion mass spectra (LSIMS) were recorded in the positive ion mode on a Kratos (Manchester, UK) MS-50S mass spectrometer equipped with a 23 kG magnet and post-acceleration detector. The LSIMS ion source has been described elsewhere (28). A Cs+ ion beam of energy 10 keV was used as the primary beam (21). Spectra were recorded (300 sec per decade) with a Gould ES-1000 electrostatic recorder. Tandem MS experiments were performed on a Kratos Concept IIHH (Manchester, UK) four sector instrument of EBEB... 

To gain further insights into the chemical structure of the compound causing the sweet taste, fraction III-5 was separately collected and analyzed by RP-HPLC. Isolated fractions were analyzed using a diode array detector and a mass spectrometer. The compound exhibiting sweet taste activity upon degustation showed a molecular mass of 197 Da and exhibited two UV-Vis absoiption maxima at 251 and 328 nm when measured at pH 8.2, or a sole maximum at 298 nm when measured at pH 3.5. 

Porter, P. M., W. L. Banwart, and J. J. Hassett, HPLC isolation and GC-MS identification of genistein, daidzein, and coumes-trol from unhydrolyzed soybean root extracts, Env. Exp. Bot.,... 

Pereda-Miranda R, Bah M (2003) Biodynamic constituents in the Mexicem morning glories Purgative remedies transcending boundaries. Curr Top Med Chem 3 111-131 Pereda-Miranda R, Hemandez-Carlos B (2002) HPLC isolation and structural elucidation of diastereomeric ndoyl ester tetrasaccharides from Mexican scammony root. Tetrahedron 58 31453154... 

NMR spectra were measured with a Varian Unity 300 FT-NMR spectrometer. Liquid chromatography-atmospheric chemical ionization-mass spectrometry (LC-APCl-MS) in positive and negative ion modes was performed using a Hitachi M-1000 spectrometer. Unknown metabolites were isolated and purified from the grape fruits extracts with solid phase extraction method (Porapak Q) and HPLC. Isolated metabolites were analyzed by free form or derivatized (methylated, acetylated) form for identification. All of the non-radiolabelled reference standards 1-6 are synthesized in our laboratory and their chemical structures are shown in Figure 2. 

Over the years a number of techniques and approaches have proved to be useful tools to successfully isolate low-level impiu ities and degradants. TLC is most useful when an impurity or degradant is identifiable by LC-MS and above the 1% level. In cases, where NMR analysis is essential for identification, semipreparative SFC, semipreparative HPLC, and flash chromatography are more suitable techniques. Please refer to the Handbook of Pharmaceutical Analysis, 1st ed. for a more in-depth explanation of TLC and flash chromatography s use for impurity isolation. As mentioned earlier, SPE and liquid-liquid extractions are at times incorporated into the process. These tools can quickly convert bulk supply materials into a more suitable form for SFC or HPLC injection. SPE is also a useful tool in dewatering and desalting final RP-HPLC isolated materials obtained from solvents containing mobile-phase additives. 

Fraction volumes are on the order of 200 mL to 1 L or more. A low 0.1% level impurity, with poor sample loadability and a complicated sample matrix can aggravate the situation with regards to project turnaround times and total solvent costs. Impurity fraction solution stability issues exacerbate the situation even more. For example, substituted benzylic alcohols can dehydrate under acidic HPLC conditions, or carboxylic esters can hydrolyze in aqueous mobile phase. A RP-HPLC isolation can yield solvent costs on the order of 50-200 with a turnaround time of approximately 1 week to yield the quality sample necessary for NMR analysis. 

At about this time, the online Angewandte manuscript of Drew, Lawrence, and Sherbum appeared. This reported an intermolecular RCDA approach to a mixture of kingianins and the HPLC isolation of kingianins A, D, and F. This paper demonstrated the feasibility of preparing pre-kingianins by... 

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