Systematic development of an UPLC-MS/MS method for the determination of tricyclic antidepressants in human urine.

Tricyclic antidepressants have been prescribed for the treatment of depression and other disorders since their discovery in the 1950s but have been replaced in recent decades by newer drugs with more favorable side effect profiles. However, for some patients and conditions, tricyclic antidepressants remain the drug of choice. A fast, sensitive, and robust UPLC-MS/MS method for the monitoring of amitriptyline, nortriptyline, imipramine, doxepin, and desipramine in human urine has been developed using a pre-defined and systematic method development approach. The method was developed using sub-2-µm particle technology, providing a state-of-the-art alternative to older methods. Total cycle time was 2.5min. Human urine samples (200µL) were prepared using an Oasis(®) WCX µElution solid-phase extraction plate, which provided good recovery for all analytes (>92%) and low matrix effects (absolute matrix effects <10%). Standard curves were linear over the range 0.02-250ng/mL with r(2) values>0.994. The method was evaluated against current FDA guidelines and was applied to the analysis of patient samples, including an assessment of incurred sample reanalysis (ISR).

Influence of stationary phase chemistry and mobile-phase composition on retention, selectivity, and MS response in hydrophilic interaction chromatography.

A comprehensive retention and selectivity characterization of several hydrophilic interaction chromatography (HILIC) stationary phases was performed with 28 test probes in order to study the influence of particle type, surface chemistry, and mobile-phase pH on chromatographic retention, selectivity, and MS response. Selectivity differences were compared for columns operated at both low and high pH, while ESI-MS was used to study the effects of mobile-phase pH on signal response. Additionally, acetone was explored as a potential alternative to ACN as the weak HILIC solvent. Moderate differences in selectivity were observed on the same column operated at different pH, mostly due to acidic compounds. In addition, the MS response increased when a high pH mobile phase was used, particularly for analytes that were ionized with negative ESI-MS. Even larger selectivity differences were observed for different stationary phases evaluated with the same mobile phase. Acetone was not a suitable replacement for ACN in routine HILIC separations due to differences in selectivity and MS response. Finally, the data from this study were used to establish guidelines for rapid HILIC method development of polar compounds, which is demonstrated with a mixture of histidine dipeptides and organophosphonate nerve agent metabolites.

Simultaneous analysis of morphine-related compounds in plasma using mixed-mode solid phase extraction and UltraPerformance liquid chromatography-mass spectrometry.

A bioanalytical method using mixed-mode solid phase extraction and UltraPerformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the analysis of morphine, morphine-3beta-glucuronide, morphine-6beta-glucuronide, 6-acetylmorphine, morphine N-oxide, and 10-hydroxymorphine in porcine plasma. All six compounds, along with four deuterated internal standards, were simultaneously extracted using mixed-mode strong cation exchange SPE in a 96-well microElution plate format. Due to analyte instability, a neutralizing solvent was used during the elution step to minimize degradation of 6-acetylmorphine. Separation was subsequently performed in 8 minutes on a 2.1 x 100 mm, 1.8 microm C(18 )column designed for retention of extremely polar compounds using a formic acid and methanol gradient. Analytes were detected by positive electrospray ionization in multiple reaction monitoring mode using a fast-scanning triple quadrupole mass spectrometer. Recovery was 73-123% depending on the analyte, and inter-day variability was less than 6%. Linearity was determined in porcine plasma by spiking the analytes prior to SPE. Correlation coefficients were >or= 0.998, and% deviation from the actual concentrations was less than 15%. The lower limit of quantitation (LLOQ) for all compounds was between 0.1 and 0.25 ng/mL.

Effects of extra-column band spreading, liquid chromatography system operating pressure, and column temperature on the performance of sub-2-microm porous particles.

The effects of extra-column band spreading, LC system operating pressure, and separation temperature were investigated for sub-2-microm particle columns using both a conventional HPLC system as well as a UPLC system. The contributions of both volume- and time-based extra-column effects were analyzed in detail. In addition, the performance difference between columns containing 2.5 and 1.7-microm particles (same stationary phase) was studied. The performance of these columns was compared using a conventional HPLC system and a low dead volume UPLC system capable of routine operation up to 1000 bar. The system contribution to band spreading and the pressure limitations of the conventional HPLC system were found to be the main difficulties that prevented acceptable performance of the sub-2-microm particle columns. Finally, an increase in operating temperature needs to be accompanied by an increase in flow rate to prevent a loss of separation performance. Thus, at a fixed column length, an increase in temperature is not a substitute for the need for very high operating pressures.

The application of novel 1.7 microm ethylene bridged hybrid particles for hydrophilic interaction chromatography.

An un-derivatized 1.7 microm ethylene bridged hybrid (BEH) particle was evaluated for its utility in retaining polar species in hydrophilic interaction chromatography (HILIC), and was compared to a 3 microm un-derivatized silica material. Retentivity as a function of mobile phase pH, polar modifier and ACN content was examined. Also, the efficiency of the two particle substrates was compared by plotting HETP vs. linear velocity. Improved chemical resistance of the un-derivatized BEH particle was compared to un-derivatized silica at pH 5, demonstrating no performance deterioration over the course of 2000 injections for the BEH particle, while the silica particle deteriorated rapidly after 800 injections. Lastly, ESI-MS sensitivity as a function of particle size and separation mode was demonstrated. A 2.2 to 4.7-times higher ESI-MS response was observed on the 1.7 microm particle compared to the 3 microm particle, whereas a 5.6 to 8.8-times higher ESI-MS response was observed using HILIC as when compared to traditional RP chromatography.

Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses.

A systematic, comprehensive strategy that optimizes sample preparation and chromatography to minimize matrix effects in bioanalytical LC/MS/MS assays was developed. Comparisons were made among several sample preparation methods, including protein precipitation (PPT), liquid-liquid extraction (LLE), pure cation exchange solid-phase extraction (SPE), reversed-phase SPE and mixed-mode SPE. The influence of mobile phase pH and gradient duration on the selectivity and sensitivity for both matrix components and basic analytes was investigated. Matrix effects and overall sensitivity and resolution between UPLC technology and HPLC were compared. The amount of specific matrix components, or class of matrix components, was measured in the sample preparation extracts by LC/MS/MS with electrospray ionization (ESI) using both precursor ion scanning mode and multiple reaction monitoring (MRM). PPT is the least effective sample preparation technique, often resulting in significant matrix effects due to the presence of many residual matrix components. Reversed-phase and pure cation exchange SPE methods resulted in cleaner extracts and reduced matrix effects compared to PPT. The cleanest extracts, however, were produced with polymeric mixed-mode SPE (both reversed-phase and ion exchange retention mechanisms). These mixed-mode sorbents dramatically reduced the levels of residual matrix components from biological samples, leading to significant reduction in matrix effects. LLE also provided clean final extracts. However, analyte recovery, particularly for polar analytes, was very low. Mobile phase pH was manipulated to alter the retention of basic compounds relative to phospholipids, whose retention tends to be relatively independent of pH. In addition to the expected resolution, speed and sensitivity benefits of UPLC technology, a paired t-test demonstrated a statistically significant improvement with respect to matrix effects when this technology was chosen over traditional HPLC. The combination of polymeric mixed-mode SPE, the appropriate mobile phase pH and UPLC technology provides significant advantages for reducing matrix effects resulting from plasma matrix components and in improving the ruggedness and sensitivity of bioanalytical methods.

Differences in preparative loadability between the charged and uncharged forms of ionizable compounds.

Ionizable compounds experience a drastic difference in preparative loadability as a function of pH. It can be shown that the preparative loadability of a compound in the ionic form is by a factor of 20 or more inferior to the loadability of the same compound in the unionized form. In this paper, we demonstrate the reason for this behavior, and show practical applications of the principle.