Fit-for-Purpose Quality Control System in Continuous Bioanalysis During Long-Term Pediatric Studies.

Pharmacokinetic studies are key to evidence-based pharmacotherapy. The reliability of pharmacokinetic parameters is closely related to the quality of bioanalytical data. Bioanalytical method validation is fully described by regulatory guidelines; however, it is conducted just once. To ensure reliability and comparability of clinical data, appropriate quality control systems must be enforced to monitor post-validation bioanalytical runs. While single bioanalytical run evaluation is described in international guidelines, somehow, the long-term reproducibility of the bioanalytical method is unattended; it becomes pivotal with the involvement of pediatric population. Therefore, a customized quality control system was developed that addresses regulatory requirements and encompasses the specific demands of pediatric research. It consisted of continuous multi-parameter assessment, including calibration curves, quality control samples, incurred sample reanalysis, and internal standard data. The recommendations provided by the guidelines were combined with the additional Westgard rules, statistical evaluation, and graphical observations. The applicability of the developed quality control system was investigated by using data from three pediatric clinical trials, where the system was able to identify 16% of all analytical runs as invalid. Using a pooled standard deviation provided a better estimate of long-term reproducibility by calculating the %CV, which ranged from 3.6 to 10.3% at all quality control levels. Irrespective of the difficulties encountered owing to vulnerable pediatric populations, the incurred sample reanalysis fulfilled the regulatory requirement of at least 67%. This quality control approach ensured reliable and comparable results over a whole 31-month duration in relation to pediatric studies.

Structural characterization of photodegradation products of enalapril and its metabolite enalaprilat obtained under simulated environmental conditions by hybrid quadrupole-linear ion trap-MS and quadrupole-time-of-flight-MS.

In the environment, organic micropollutants such as pharmaceuticals can be degraded via various biotic and abiotic transformation routes. In surface waters, for example, photodegradation may constitute a relevant natural attenuation process for drug residues that have been discharged from sewage treatment facilities. In the present work, the photochemical fate of the prodrug enalapril (376 Da, C20H28N2O5) and its active metabolite enalaprilat (348 Da, C18H24N2O5), a hypotensive cardioprotector previously reported to occur in contaminated rivers, was investigated in aqueous media under the influence of irradiation generated by a sunlight simulator. The experiments yielded three detectable photodegradates for enalapril (346 Da, 2 x 207 Da) whereas the photolysis of enalaprilat went hand in hand with the intermittent buildup of one photodegradate (304 Da). Fragmentation patterns of the parent compounds were established on a hybrid quadrupole-linear ion trap-mass spectrometer exploiting its MS3 capabilities. Accurate mass measurements recorded on a hybrid quadrupole-time-of-flight instrument in MS/MS mode allowed us to propose elemental compositions for the molecular ions of the degradates (346 Da, C19H26N2O4; 207 Da, C12H17NO2; 304 Da, C17H24N2O3) as well as of their fragment ions. Based on these complementary data sets from the two distinct mass spectrometric instruments, plausible structures were postulated for the four photodegradates. The compounds formed by enalapril corresponded to the loss of formaldehyde out of the proline residue (346 Da), cleavage of the central amide bond (207 Da) followed by migration of the ethylester side chain (207 Da) while decarboxylation of the free carboxylic acid was described for enalaprilat (304 Da). The study emphasized the potential of sunlight for breaking down an environmentally relevant drug and its metabolite.

Experimental design optimization of a capillary zone electrophoresis method for the screening of several diuretics and ACE inhibitors.

Experimental design methodologies are applied to the development of a capillary zone electrophoretic method for the separation of the angiotensin-converting enzyme inhibitor enalapril and its derivative enalaprilat and the diuretics xipamide and hydrochlorothiazide. The effects of pH, buffer concentration, proportion of boric acid in the mixed boric acid-potassium dihydrogen phosphate background electrolyte, temperature, applied voltage, and percentage of organic modifier are studied. Critical factors are identified in a screening design (a 2(6-2) fractional factorial design), and afterwards, optimal conditions for the separation are reached by means of an optimization design (a 2(2) + 2 x 2 + k central composite design). The studied response is the resolution between peaks. The four studied compounds can be separated in less than 3.5 min using an electrolyte of 20mM boric acid-potassium dihydrogen phosphate (75:25, v/v) with 5% MeOH adjusted to pH 8.0 with KOH, at a potential of 30 kV. The detection wavelength and temperature are 206 nm and 35 degrees C, respectively.

RP-HPLC and NMR study of cis-trans isomerization of enalaprilat.

The angiotensin converting enzyme inhibitor, enalaprilat can exist in solution as cis and trans conformers which interconvert around the amide bond at room temperature. A HPLC with UV detection was performed to study the influence of various chromatographic operational conditions on both rotamers separation and elution of enalaprilat as a single peak. In addition nuclear overhauser enhancement difference was used for the identification of the conformers. The isomer ratio integrated from the obtained 1H NMR result were 71.5:28.5 and 76:24 at 298 and 279 K, respectively where the trans was the major form.