Preparative HPLC, isolation

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... 

Phosphate Esters. The phosphorylation of sucrose using sodium metaphosphate has been reported (78). Lyoptulization of a sodium metaphosphate solution of sucrose at pH 5 for 20 hours followed by storage at 80°C for five days produced a mixture of sucrose monophosphates. These products were isolated by preparative hplc, with a calculated yield of 27% based on all organic phosphate as sucrose monoesters. Small proportions of glucose and fmctose were also formed. 

Samples of PB-DPE were subjected to RP-HPLC producing the chromatographic patterns shown in Figure 1. The two major peaks of Penta (Fig. lA, peaks 1 and 2), were isolated by preparative HPLC and analyzed by NMR. The spectra (Table 1) were in excellent agreement with previously published data (refs. 3-6). The two HPLC peaks were identified as 2,2, 4,4 tetrabromo DPF and 2,2, 4,4, 5 pentabromo DPF, respectively. 

Of the five remaining peaks (Figure IB, peaks 5 to 9), three (peaks 5,7 and 9), were easily separated from the octa samples by preparative HPLC, and the two remaining (peaks 6 and 8) were isolated from high-melting (Figure 1C). 

Sequence information for the remaining fragments was obtained by Edman degradation (see Section 5.3.1 above) after isolation of the individual peptides using preparative HPLC - the chromatographic resolution being sufficient to allow this, and thus enabled the complete sequence to be determined. 

Prior to semi-preparative HPLC, matrix compounds may be present that require a purification step. In some cases, high sugar or salt contents and also pectic-like substances may preclude concentration and isolation precipitation using 2-propanol or ethanol is then required. Subsequent filtration will yield a pigment solution and a colorless filtrate that may be rinsed with a mixture of one part water and two parts alcohol for complete discoloration. The filtrate volume will be reduced under vacuum before further purification. Usually, precipitation precedes desalting. 

Forthright isolation of standard substances from known sources may be achieved by analytical and/or semi-preparative HPLC. Although it appears promising with respect to obtainable pigment yields, countercurrent chromatography has been applied only once for red beets but lacked sufficient separation efficiency. "... 

Hotchkiss, Jr., A. T., Haines, R. M., and Hicks, K. B., Improved gram-quantity isolation of malto-oligosaccharides by preparative HPLC, Curb. Res., 242, 1, 1993. 

The major components are series of homologous trimers, tetramers, and pentamers of the three acids 44-46, along with smaller quantities of dimers, hexamers, and heptamers. Furthermore, the secretion contains several isomers of each oligomer, furnishing a combinatorial library of several hundred macro-cyclic polyamines [51, 52]. Using repeated preparative HPLC fractionation, the most abundant trimeric, tetrameric and pentameric earliest-eluting compounds were isolated. One and two-dimensional H NMR spectroscopic analyses showed that these molecules were the symmetric macrocyclic lactones 48, 49, and 50 (m, n, o, p, q=7) derived from three, four or five units, respectively, of acid 46. Moreover, using preparative HPLC and NMR methods, various amide isomers, such as 53,54, and 55 (Fig. 9) were also isolated and characterized [51,52]. 

Once an assessment on a particular impurity has been made all process-related compounds will be examined to confirm that the impurity of interest is indeed an unknown. An easy way of doing this is to compare the retention times of known process-related compounds to that in question. If this analysis confirms that the compound is an unknown, the next step would be to obtain an LC-MS on the compound. Mass spectrometry provides structural information which aids in determining structure. In some cases, mass spectrometry will be enough to identify the compound. In other cases, more complicated methods like LC-NMR are needed or the impurity will need to be isolated in order to obtain additional information. Compounds that are not purified often contain high levels of by-products and can be used for this purpose. Alternatively, mother liquors from crystallizations also contain levels of by-products. Other ways of obtaining larger quantities of impurities include flash chromatography which is typically used for normal phase separations or preparative HPLC which is more common for reversed phase methods. Once a suitable quantity of the compound in question has been obtained a full characterization can be carried out to identify it. 

LC-NMR is a powerful technique especially for the analysis of compounds which cannot be isolated by preparative HPLC. For the identification of impurities in API from unpublished work from this laboratory, impurities at a 1% level from drug substances can be identified on a 600 MHz NMR spectrometer using the stop-flow mode. 

Recently, an improved pretreatment of the plant material combined with preparative HPLC separation has been published for the isolation of anhydrovincaleukoblastine (8) from the leaves of C. roseus 27). It has also be shown that the yield of 8 could be enhanced by treatment of acidic aqueous extracts of C. roseus with sodium borohydride, suggesting that... 

Refluxing benzene solutions of Cjq in the presence of a 20-fold excess of BujSnH leads to hydrostannylation (Scheme 6.15) [73]. Multiple additions can also take place. To maximize the yield of the monoadduct CgoHSnBuj (24), the time dependence of the reaction was followed quantitatively by HPLC. After about 4 h, the concentration of the monoadduct 24 reaches its maximum. Compound 24 can be isolated by preparative HPLC on a Cjg-reversed-phase stationary phase with CHCI3-CH3CN (60 40, v/v) as eluent. The structure of C5oHSnBu3 (24) was determined by NMR spectroscopy and other methods, showing that a 1,2-addition takes place regio-selectively (Scheme 6.15) [73]. 

Dried 7 was dissolved in H20 (4mL) and piperidine (0.44 mL) was added (final concentration 1M). After 4h at 20 °C the mixture was adjusted to pH 3.0 at 0°C as before and then diluted with 0.1% TFA (10 mL). The deprotected material was then isolated by preparative HPLC using a 0-30% gradient (same eluants as above) over 30 min with a flow rate of 20mL-min 1 to give the trifluoroacetyl salt yield 370 mg (94%). 

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