A detailed investigation into the chromatographic retention behaviour and separation of the three regioisomers of the Novel Psychoactive Substance (NPS) methoxphenidine (i.e. 2-, 3- and 4-MXP isomers) has revealed the ionization state of the analyte and stationary phase, to be the controlling factor in dictating which retention mechanism is in operation. At low pH, poor separation and retention was observed. In contrast, at intermediate pH, enhanced retention and separation of the three MXP isomers was obtained; it appeared that there was a synergistic effect between the electrostatic and hydrophobic mechanisms. At high pH, the MXP isomers were retained by hydrophobic retention. Accurate retention time predictions (<0.5%) were achievable using non-linear retention models (3 × 3). This allowed the optimization of the gradient separation of the MXP isomers using a two-dimensional gradient and temperature design space. Prediction errors for peak width and resolution were, in most cases, lower than 5%. The use of linear models (2 × 2) still afforded retention time and resolution accuracies of <2.3 and 11% respectively. A rapid and highly sensitive LC-MS friendly method (i.e. R > 5 within 4 min) was predicted and verified. The developed methodology should be highly suitable for the rapid, specific and sensitive detection and control of MXP regioisomers.

Protein quantification during bioprocess monitoring is essential for biopharmaceutical manufacturing and is complicated by the complex chemical composition of the bioreactor broth. Here we present the early‐stage development and optimization of a polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a hydrolysate‐protein model (mimics clarified bioreactor broth samples) using a standard benchtop laboratory fluorometer. We used UV transmitting polarizers to provide wider range pTSFS spectra for screening of the four different TSFS spectra generated by the measurement: parallel (||), perpendicular (⊥), unpolarized (T) intensity spectra and anisotropy maps. TSFS|| (parallel polarized) measurements were the best for protein quantification compared to standard unpolarized measurements and the Bradford assay. This was because TSFS|| spectra had a better analyte signal to noise ratio (SNR), due to the anisotropy of protein emission. This meant that protein signals were better resolved from the background emission of small molecule fluorophores in the cell culture media. SNR of >5000 was achieved for concentrations of bovine serum albumin/yeastolate 1.2/10 g L–1 with TSFS||. Optimization using genetic algorithm and interval partial least squares based variable selection enabled reduction of spectral resolution and number of excitation wavelengths required without degrading performance. This enables fast (<3.5 min) online/at‐line measurements, and the method had an LOD of 0.18 g L–1 and high accuracy with a predictive error of <9%.