Abstract:The crucial role of mobile phase pH for optimizing the separation of a mixture of ionized and non-ionized compounds on a Phenomenex extended pH-range reversed-phase column (Kinetex 5 µm EVO C18) was examined. A previously developed Excel-spreadsheet-based software was used for the whole separation optimization procedure of the sample of interest under isocratic conditions as well as under single linear organic modifier-gradients in different eluent pHs. The importance and the advantages of performing a computer-aided separation optimization compared with a trial-and-error optimization method were realized. Additionally, this study showed that the optimized separation conditions for a given stationary phase may be used to achieve successful separations on new columns of the same type and size. In general, the results of this work could give chromatographers a feel of confidence to establish desired separations of a mixture of ionizable and neutral compounds in reversed-phase columns.

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Background Retinopathy of prematurity (ROP) eye examination screening presupposes adequate mydriasis for an informative fundoscopy of preterm infants at risk, on a weekly basis. Systemic absorption of the instilled mydriatic regimens has been associated with various adverse events in this fragile population. This report aims to present the fully developed protocol of a full-scale trial for testing the hypothesis that the reduced mydriatic drop volume achieves adequate mydriasis while minimizing systemic adverse events. Methods A non-inferiority crossover randomized controlled trial will be performed to study the efficacy and safety of combined phenylephrine 1.67% and tropicamide 0.33% microdrops compared with standard drops in a total of 93 preterm infants requiring ROP screening. Primary outcome will be the pupil diameter at 45 (T45) min after instillation. Pupil diameter at T90 and T120 will constitute secondary efficacy endpoints. Mixed-effects linear regression models will be developed, and the 95% confidence interval approach will be used for assessing non-inferiority. Whole blood samples will be analyzed using hydrophilic liquid chromatography–tandem mass spectrometry method (HILIC–MS/MS), for gathering pharmacokinetic (PK) data on the instilled phenylephrine, at nine specific time points within 3 h from mydriasis. Pooled PK data will be used due to ethical restrictions on having a full PK profile per infant. Heart rate, oxygen saturation, blood pressure measurements, and 48-h adverse events will also be recorded. Discussion This protocol is designed for a study powered to assess non-inferiority of microdrops compared with standard dilating drops. If our hypothesis is confirmed, microdrops may become a useful tool in ROP screening. Trial registration ClinicalTrials.govNCT05043077. Registered on 2 September 2021