A quality improvement study of the implementation and initial results of a pragmatic clinical decision support system in the community pharmacy setting.

BACKGROUND: A six year collaboration between academics, community pharmacists and informaticians, led to the development of nine guidelines for a clinical decision support system, enhancing community pharmacists' ability to address drug-related problems and improve care. AIM: The objective of this study was to assess the effectiveness of clinical decision support system rules in enhancing medication management within the community pharmacy setting. This was achieved through retrospective monitoring of real-world usage and measuring the pharmacotherapeutic impact of the rules. METHOD: In 2019, a retrospective observational evaluation appraised the acceptance rate of the clinical decision support system components in 490 Belgian pharmacies. Among these, 51 pharmacies underwent a longitudinal analysis involving (i) co-prescription of methotrexate and folic acid, (ii) gastroprotection with non-steroidal anti-inflammatory drugs, and (iii) drug combinations causing QT prolongation. The study period spanned one year pre-launch, one year post-launch, and two years post-launch. RESULTS: Of the targeted pharmacies, 80% used 7 of the 9 rules. After four years, methotrexate-folic acid co-prescription increased 4%, reaching 79.8%. Gastroprotection improved by 3% among older patients and 7.47% in younger individuals (< 70 year) with multiple risk factors. The QT prolongation rules faced implementation difficulties. CONCLUSION: Pharmacists' acceptance of the developed rules was high and coincided with a decline in drug-related problems, holding potential public health impact. This real-world data can inform the future implementation of such systems, as it demonstrated the need for more detailed data-gathering and more intensive training of pharmacists in the handling of more complex problems such as QT prolongation.

Improved analyte detectability of proteins and peptide lysates by means of multiple large-volume injection in LC-MS.

A 'multiple (trapping) large-volume injection' approach was developed for the analysis of peptides and proteins. In this way, a maximally 10-fold gain in sensitivity could be achieved. The system involves the use of an automated 10-port switching valve in combination with a 1 mm i.d. trapping/guard column and a 1 mm i.d. x 150 mm analytical column. The optimized multiple injection/loading procedure allows quantitative measurements of peptides and protein lysates. Linear calibration curves (R(2) > or = 0.988) over a minimum of two orders of magnitude were generated for a range of peptide and protein standards with sensitivities equal to or even exceeding, those generally achieved only through increasing miniaturization (quantification limit > or = 0.5 pmol/mL).

LC-MS metabolic profiling of Arabidopsis thaliana plant leaves and cell cultures: optimization of pre-LC-MS procedure parameters.

This study treats the optimization of methods for homogenizing Arabidopsis thaliana plant leaves as well as cell cultures, and extracting their metabolites for metabolomics analysis by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). Absolute recovery, process efficiency and procedure repeatability have been compared between different pre-LC-MS homogenization/extraction procedures through the use of samples fortified before extraction with a range of representative metabolites. Hereby, the magnitude of the matrix effect observed in the ensuing LC-MS based metabolomics analysis was evaluated. Based on relative recovery and repeatability of key metabolites, comprehensiveness of extraction (number of m/z-retention time pairs) and clean-up potential of the approach (minimum matrix effects), the most appropriate sample pre-treatment was adopted. It combines liquid nitrogen homogenization for plant leaves with thermomixer based extraction using MeOH/H(2)O 80/20. As such, an efficient and highly reproducible LC-MS plant metabolomics set-up is achieved, as illustrated by the obtained results for both LC-MS (8.88%+/-5.16 versus 7.05%+/-4.45) and technical variability (12.53%+/-11.21 versus 9.31%+/-6.65) data in a comparative investigation of A. thaliana plant leaves and cell cultures, respectively.

Evaluation of hydrophilic interaction chromatography versus reversed-phase chromatography in a plant metabolomics perspective.

The metabolomics goal, the unbiased relative quantification of all metabolites in a biological system, still lacks a universal analytical approach. In the LC-MS line of approach, one of the major problems encountered is the polar nature of a large group of (plant) metabolites. Here, we investigate the potential of hydrophilic interaction chromatography (HILIC) and compare its qualities with extended polarity RP chromatography. Two opposite LC phase compositions (Atlantis dC18 vs. TSKgel Amide-80) are compared in a plant metabolomics setting. Both performed equally well with regard to retentive capacities, but variation in peak area was about 5% higher for the HILIC approach. Focussing on matrix effects (ME) on the other hand, it was observed that this well-known problem in RP LC-MS metabolomics was not reduced on using hydrophilic interaction chromatography.

Flavin-induced photodecomposition of sulfur-containing amino acids is decisive in the formation of beer lightstruck flavor.

Photooxidation of sulfur-containing amino acids and derivatives readily occurs upon visible-light irradiation in the presence of flavins. The sulfur moiety seems pivotal for interaction, as was determined from kinetic analyses using laser flash photolysis spectroscopy. After photooxidation, the resulting radical intermediates were characterized by addition to a spin trap, followed by electron paramagnetic resonance spectroscopy and evaluation of the coupling constants. The presence of the proposed radical intermediates was strongly supported by the identification of the reaction products using mass spectrometry. Accordingly, feasible degradation pathways for various sulfur-containing amino acids and derivatives were proposed. It was finally proven that flavin-induced photoproduction of sulfhydryl radicals and recombination with a 3-methylbut-2-enyl radical, derived from the photodegradation of hop-derived isohumulones, are decisive in the formation of beer lightstruck flavor.

Simultaneous quantitative analysis of the antimalarials pyrimethamine and sulfamethoxypyrazine in plasma samples using liquid chromatography/tandem mass spectrometry.

The work presented here deals with the development of a quantitative tool for the simultaneous determination of sulfamethoxypyrazine (sulfalene)/pyrimethamine in plasma. The chromatography used only takes 12.5 min, allowing a fast sample turnover time. Relative standard deviation of retention times was never above 3.48% (n = 66). Adequate sample clean-up was achieved by a simple and relatively fast liquid/liquid extraction. In this way, ionisation suppression effects, typical for more simple sample clean-up procedures, could be avoided resulting in absolute plasma effects of maximum -17.1% for sulfalene, -16.1 for the internal standard (IS), and 12% for pyrimethamine. For both pyrimethamine and sulfalene, quadratic calibration curves from 0.00101 to 0.807 microg/mL for pyrimethamine and from 0.271 to 216 microg/mL for sulfalene gave the best fit. Mean coefficients of determination (R2) were 0.9951 (n = 6, CV% 0.39) for pyrimethamine and 0.9942 (n = 6, CV% 0.13) for sulfalene. Precision was below 9.35% for pyrimethamine and 13.9% for sulfalene. Inaccuracy remained below 15% at all cases. The optimised method was used for a time-course study of the sulfalene/pyrimethamine combination concentration in plasma of patients treated with Co-Arinate, a new curative antimalaria-medicine.

The use of tryptic marker-peptides for the quantitative analysis of cystatin C.

The use of marker-peptides, measured by LC-MS/MS, is investigated for the quantitative analysis of proteins. To that end, cystatin C is chosen as a model protein. It not only functions as a proof of concept protein but the growing interest in cystatin C as a new marker of kidney failure provides a practical application at the same time. The use of trypsin-based proteolysis, to obtain so-called marker-peptides, simplifies the quantification of a protein to the quantification of a single or a number of peptides. Reproducibility of the trypsin proteolysis procedure is vital and has been optimised. A number of the marker-peptides obtained are selected for LC-MS(/MS) analysis. They are completely separated by high-pressure LC allowing maximum selectivity and mass spectrometric multiple reaction monitoring sensitivity. By doing so, linear calibration curves can be obtained for cystatin C over two orders of magnitude. Experiments have been performed on a triple quadrupole mass spectrometer by single ion monitoring (maximum sensitivity) as well as by multiple reaction monitoring (maximum specificity).

Capillary liquid chromatography and tandem mass spectrometry for the quantification of enkephalins in cerebrospinal fluid.

A capillary LC-MS/MS system was evaluated for the absolute quantification of enkephalins in cerebrospinal fluid (CSF). On column focusing on a C18 trapping column, in-line with the analytical column, was used for preconcentration. Quantification was performed with a triple quadrupole instrument in the multiple reaction monitoring mode. Weighted linear regression analysis proves to be a good linearity in a dynamic range of two orders of magnitude. The method was validated, yielding calibration curves with correlation coefficients greater than 0.9914. Assay precision and accuracy were evaluated by direct injection of enkephalin fortified artificial CSF (aCSF) samples at three concentration levels. Mean accuracy of analysed concentrations was between 97.63 and 107.6%. LOD and LOQ were assessed at, respectively, 0.5 and 1 pmol/mL. Validation results show that it is feasible, with a capillary LC-MS/MS system, to quantify neuropeptides in the low femtomole range in aCSF. The obtained coefficients of variation, however, indicate that the use of appropriate isotopically labelled internal standards in neuropeptide quantification using narrow bore LC, combined with ESI-MS, may be highly beneficial.