Method optimization of hydrophilic interaction chromatography separation of nucleotides using design of experiment approaches I: Comparison of several zwitterionic columns.

A systematic method in hydrophilic interaction chromatography (HILIC) was developed for the separation of four monophosphate nucleotides using design of experiment (DOE) approaches. Three HPLC parameters, the buffer concentration (ammonium acetate concentration), gradient time, and temperature, were evaluated within the quality design framework, and the effects on chromatographic parameters were investigated. Four zwitterionic columns (ZIC-HILIC, ZIC-cHILIC, NUCLEODUR HILIC, and PC HILIC) were used to separate four nucleotides, and the HPLC conditions for each column were successfully optimized, although PC HILIC did not give peaks that were suitable for optimization. In addition, it was proved that optimized HPLC conditions differed from column to column even when the same types of zwitterionic sulfobetaine-functionalized columns were applied. This tendency was explained by differences in the separation characteristics of each column, the thickness of the water-enriched layer on the surface of the silica supports, and the pH. DOE for development of the HPLC method provides an effective explanation of the interactions among the variable parameters, especially in HILIC mode. Finally, a robust analytical method could be established by setting the optimum parameters. Among the employed columns, ZIC-cHILIC provided the widest range of suitable analytical conditions. NUCLEODUR HILIC was difficult to build a robust analytical method since the elution order of cytidine monophosphate and guanosine monophosphate was reversed.

Intelligent peak deconvolution through in-depth study of the data matrix from liquid chromatography coupled with a photo-diode array detector applied to pharmaceutical analysis.

Multivariate curve resolution-alternating least squares (MCR-ALS) method was investigated for its potential to accelerate pharmaceutical research and development. The fast and efficient separation of complex mixtures consisting of multiple components, including impurities as well as major drug substances, remains a challenging application for liquid chromatography in the field of pharmaceutical analysis. In this paper we suggest an integrated analysis algorithm functioning on a matrix of data generated from HPLC coupled with photo-diode array detector (HPLC-PDA) and consisting of the mathematical program for the developed multivariate curve resolution method using an expectation maximization (EM) algorithm with a bidirectional exponentially modified Gaussian (BEMG) model function as a constraint for chromatograms and numerous PDA spectra aligned with time axis. The algorithm provided less than ±1.0% error between true and separated peak area values at resolution (Rs) of 0.6 using simulation data for a three-component mixture with an elution order of a/b/c with similarity (a/b)=0.8410, (b/c)=0.9123 and (a/c)=0.9809 of spectra at peak apex. This software concept provides fast and robust separation analysis even when method development efforts fail to achieve complete separation of the target peaks. Additionally, this approach is potentially applicable to peak deconvolution, allowing quantitative analysis of co-eluted compounds having exactly the same molecular weight. This is complementary to the use of LC-MS to perform quantitative analysis on co-eluted compounds using selected ions to differentiate the proportion of response attributable to each compound.