HPLC assay

The British Pharmacopoeia specifies a biological assay for the sodium salt of rifamycia SV [14897-39-3]. It also specifies a spectrophotometric assay for rifampicia (201). The United States Pharmacopeia requires an hplc assay for rifampin (202). 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

Acidimetric, spectrophotometric and HPLC assays were developed for determination of 2,3,5,6,7,8-hexahydro-l//-pyrido[l,2-c]pyrimidine-l,3-diones 135 (98M133). Its solubility properties were also characterized. Resolution of the enantiomers of 4-phenyl-2- 4-[4-(2-pyrimidinyl)piperazi-nyl]butyl perhydropyrido[l,2-c]pyrimidine-l,3-dione was achieved on hep-takis(2-N, V-dimethylcarbamoyl)- 6-cyclodextrines (01 JC(A)249). 

The structure of the last eluting peak (Fig. ID, peak 10), structure is easily deduced to be 2,2, 3,3, 4,4, 5,5, 6,6 decabromo DPF from the bromine content of the pure sample, lack of proton spectrum and long retention time. Indeed, a recrystallized standard serves as reference material for a quantitative HPLC assay to be described elsewhere. 

A) solvent effects on the fluorescence emission, and (5) the effects of additional reagents and catalysts normally encountered in HPLC assays. 

In contrast, in HPLC assays the chromatographic separation and the integration of the resulting analyte peak normally are just as or even more error-prone than is the preparation of the solutions here it would be acceptable to simply reinject the same sample solution in order to obtain a quasi-independent measurement. Two independent weighings and duplicate injection for each solution is a commonly applied rule. 

Figure 4.16 (left). Trendlines for the various compwnents. The three scales are different %, ppm, resp. %). (right). Total impurities (columns 1-6, including water of crystallization, versus the HPLC assay of the major compound (column 7). The circle marks the hypothetically pure compound 3.2% water of crystallization, but no other impurities. The arrow indicates the percentage of impurities expected (for this simple linear model) to remain in the product after all solvents and excess water have been driven off. 

The HPLC assay is fully coupled to the impurities A-D on the assumption that there is a direct competition between the major component and some impurity-producing reaction pathways. The basis-value 99 was introduced to simulate other concentration losses that were not accounted for by impurities A-D. 

JUNGLEl.dat Section 2.1 Three parameters (impurity content, HPLC assay, and titration assay) were measured on five batches of a raw material. 

NPYR Rat Liver microsomes HPLC assay for a-hydroxylation 15... 

Figure 5.1. Results for scavenger screen. Percent Pd remaining in the supernatant, as determined by ICP/MS, is shown in the bars for room temperature (dark) and 65°C (light). Percent product remaining in supernatant, as determined by HPLC assay, is shown, for 65°C data set only, as A.

Treatment of a mixture of alcohol 10 and chiral imidate 67 with catalytic TfOH only afforded a 1.2 to 1.3 1 mixture of 18 19 in a combined HPLC assay yield of 91%. Clearly, under these conditions, the reaction was proceeding under an SN1 reaction pathway. The use of other acid catalysts (TMSOTf, HC1, H2S04, TFA, MsOH) in a variety of solvent systems and under a number of reaction conditions did not improve the diastereomeric ratio of 18 19 (typically 1.2 1), or simply resulted in no reaction. 

Comparative study of fluorescence CYPs assays of niclosamide with that obtained by conventional HPLC assay using human liver microsomes and recombinant CYPs was developed [72]. 

FIGURE 7.9 (A) Upper traces show HPLC/MS/MS mass chromatograms for a set of six test compounds (total HPLC assay time ca. 3 min). (B) Lower traces show UPLC/MS/MS mass chromatograms for the same set of six test compounds (total UPLC assay time ca. 1 min). (Source Adapted from Yu, K. et al., Rapid Com-mun. Mass Spectrom., 2006, 20, 544. With permission of John Wiley Sons.)... 

Designing High-Throughput HPLC Assays for Small and Biological Molecules... 

The UHPLC approach permits a significant enhancement over the conventional HPLC assay through a factor 50 in the chemical-physics determination throughput, especially if hyphenation of UHPLC with MS detection is considered. 

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