Vid Klinisk kemi och farmakologi, Specialkemi, vid Skånes universitetssjukhus, Lund, utförs kvantifiering med LC-MS/MS av antipsykotiska och antidepressiva läkemedelskoncentrationer i serum med acetonitril (ACN) som mobilfas. ACN är på grund av sin höga elueringsstyrka ett av de vanligaste organiska lösningsmedlen vid reversed phase (RP) kromatografi, men uppvisar samtidigt en hög toxicitet med risk för stora leveransproblem. I syfte att reducera mängden ACN undersöktes därför möjligheten till ett mobilfasbyte till metanol (MeOH). Ordinarie metod jämfördes med tre nyutvecklade metoder med MeOH-baserad mobilfas. I en av metoderna ändrades endast mobilfas och elueringsgradient, medan två av metoderna även använde andra sorters RP-kolonner med anpassade elueringsgradienter. Samtliga analyter uppvisade godkänd separation och retention vid eluering med MeOH, men stor fluktuation från referensmetod sågs vid kvantifieringen av flera analyter, däribland olanzapin, desmetylolaznapin och mirtazapin. Liknande avvikelser med avseende på regression och kvantifieringsdifferens observerades vid eluering med andra sorters RP-kolonner. Detta indikerar att vidare optimering av andra vätskekromatografiska och masspektrometriska parametrar bör utföras innan metoderna kan valideras. / The quantification of antipsychotics and antidepressants in human serum with LC-MS/MS is usually performed with acetonitrile (ACN) as mobile phase. ACN is one of the most common organic solvents in reversed phase (RP) chromatography thanks to its high elution strength but is also highly toxic and can at times suffer from major delivery problems. The present study investigated the possibility of replacing ACN with methanol (MeOH) as primary organic solvent with the purpose of reducing the total ACN-usage. Three newly developed methods for chromatographic analysis of antipsychotic and antidepressant drugs using MeOH as organic solvent as part of the mobile phase were compared to the routine method in regard to analyte separation and retention, as well as the relative quantification of substance. In one of the methods only the mobile phase and elution gradient was changed whereas different types of RP columns were applied in addition to the changed mobile phase in the other two. All substances showed acceptable separation and retention when eluted with MeOH but displayed large fluctuations in the quantification of the analyzed substances, more specifically olanzapine, desmethylolanzapine and mirtazapine, in comparison to the reference method. Similar deviations in terms of regression and quantification bias were observed when analytes were eluted with MeOH through other types of RP columns. This indicates that further optimization of other parameters relating to the chromatography and mass spectrometer should be performed before validation.

Rigid poly[hydroxyethyl acrylate-co-poly(ethylene glycol) diacrylate] (Poly(HEA-co-PEGDA) monoliths were synthesized inside 75-µm i.d. capillaries by one-step UV-initiated copolymerization using methanol and ethyl ether as porogens. The optimized monolithic column was evaluated for hydrophobic interaction chromatography (HIC) of standard proteins. Six proteins were separated within 20 min with high resolution using a 20 min elution gradient, resulting in a peak capacity of 54. The performance of this monolithic column for HIC was comparable or superior to the performance of columns packed with small particles. Monoliths synthesized solely from PEGDA were also found to show excellent performance in HIC of proteins. Continuing efforts showed that rigid monoliths could be synthesized from PEGDA or poly(ethylene glycol) dimethacrylates (PEGDMA) containing different ethylene glycol chain lengths for HIC of proteins. Effects of PEG chain length, bi-porogen ratio and reaction temperature on monolith morphology and back pressure were investigated. Monoliths prepared from PEGDA 258 were found to provide the best chromatographic performance with respect to peak capacity and resolution. An optimized PEGDA 258 monolithic column was able to separate proteins using a 20-min elution gradient with a peak capacity of 62. The preparation of these in situ polymerized single-monomer monolithic columns was highly reproducible. The single-monomer synthesis approach clearly improves column-to-column reproducibility.The highly crosslinked monolith networks resulting from single crosslinking monomers were found to enhance the surface area of the monolith and concentrations of mesopores. Thus, monolithic columns were developed from four additional crosslinking monomers, i.e., bisphenol A dimethacrylate (BADMA), bisphenol A ethoxylate diacrylate (BAEDA, EO/phenol = 2 or 4) and pentaerythritol diacrylate monostearate (PDAM) for RPLC of small molecules. Gradient elution of alkyl benzenes and alkyl parabens was achieved with high resolution using all monolithic columns. Porogen selection for the BADMA and PDAM was investigated in detail with the intention of obtaining data that could possiblly lead to a rational method for porogen selection.

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