Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

Zheng N ，Fung EN ，Buzescu A ，Arnold ME ，Zeng J ... -  《-》

Simultaneous determination of a selective adenosine 2A agonist, BMS-068645, and its acid metabolite in human plasma by liquid chromatography-tandem mass spectrometry--evaluation of the esterase inhibitor, diisopropyl fluorophosphate, in the stabilization

Zeng J ，Onthank D ，Crane P ，Unger S ，Zheng N ，Pasas-Farmer S ，Arnold M ... -  《JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES》

Phospholipids in liquid chromatography/mass spectrometry bioanalysis: comparison of three tandem mass spectrometric techniques for monitoring plasma phospholipids, the effect of mobile phase composition on phospholipids elution and the association of phos

Because plasma phospholipids may cause matrix effects in bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods, it is important to establish optimal mass spectrometric techniques to monitor the fate of phospholipids during method development and application. We evaluated three MS/MS techniques to monitor phospholipids using positive and negative electrospray ionization (ESI). The first technique is based on using positive precursor ion scan of m/z 184, positive neutral loss scan of 141 Da and negative precursor ion scan of m/z 153. The second technique is based on using class-specific positive and negative selected reaction monitoring (SRM) transitions to monitor class-representative phospholipids. The third technique, previously reported, utilizes in-source collision-induced dissociation (CID)-based positive SRM of m/z 184 --> 184. We recommend the all-inclusive technique 1 for use in qualitative assessment of all classes of phospholipids and technique 2 for use in quantitative assessment of class-representative phospholipids. Secondly, we evaluated the elution behaviors of the plasma phospholipids under different reversed-phase mobile phase conditions. The phospholipid-eluting strength of a mobile phase was mainly dependent on the type and amount (%) of the organic eluent and the strength increased in the order of methanol, acetonitrile and isopropyl alcohol. Under the commonly used gradient and isocratic elution schemes in LC/MS/MS bioanalysis, not all the phospholipids are eluted off the column. Thirdly, we investigated the association between phospholipids and matrix effects in positive and negative ESI using basic, acidic and neutral analytes. While the phospholipids caused matrix effects in both positive and negative ESI, the extent of ionization suppression was analyte-dependent and was inversely related to the retention factor and broadness of the phospholipids peaks. The lysophospholipids which normally elute earlier in reversed-phase chromatography are more likely to cause matrix effects compared to the later-eluting phospholipids in spite of the larger concentrations of the latter in plasma.

Xia YQ ，Jemal M 《-》

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry.

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid-liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.

Ismaiel OA ，Zhang T ，Jenkins RG ，Karnes HT ... -  《-》

Systematic evaluation of supported liquid extraction in reducing matrix effect and improving extraction efficiency in LC-MS/MS based bioanalysis for 10 model pharmaceutical compounds.

In past a few years, there has been a large increase in the application of supported liquid extraction (SLE) for LC-MS/MS based bioanalysis due to its distinct practical advantage in reduced time cost, ease of operation and the feasibility for automation. The main purpose of this study was to systematically evaluate supported liquid extraction in reducing matrix effect and improving extraction efficiency/recovery under various extraction conditions with 10 model pharmaceutical compounds in liquid chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS) analysis. Selected compounds have diverse physicochemical properties where logP ranges from 0.1 to 6.24 and pK(a) ranges from 4.0 to 11.1. The factors that may have the impact on the recovery of analytes and phospholipids (PL) were assessed. Over 75% recovery was achieved for every analyte under its respectively optimized extraction conditions where the selection of the polarity of extraction solvent and buffered pH can be critical for efficient recovery. Furthermore, the matrix effect was assessed by postextraction spike and postcolumn infusion method. The matrix effect was considerably reduced for all analytes under most extraction conditions evaluated for SLE, compared with protein precipitation (PPT) method. The correlation between matrix effect and residual phospholipids in sample extract was clearly shown. Although analyte-dependent matrix effect was observed prominently in sample extract prepared by PPT, it was minimized by SLE sample preparation process that effectively removes the majority of phospholipids. Sample extracted by ethyl acetate contained more phospholipids and demonstrated stronger matrix effect than by other organic solvents. Water-miscible organic content, such as methanol and acetonitrile in samples prior to loading has significant impact on PL recovery when eluting with methyl tert-butyl ether. However, isopropanol does not enhance the recovery of PL when adding to dichloromethane for elution. In addition, the compromise between improved extraction efficiency by SLE and reduced matrix effect is sometimes necessary to yield clean extract with acceptable recovery. The effective removal of phospholipids and reduction of matrix effect, while achieving good recovery for all pharmaceutical compounds with diverse physicochemical properties, demonstrated that SLE is a valuable alternative technique to liquid-liquid extraction (LLE) in high throughput LC-MS/MS based bioanalysis.

Jiang H ，Cao H ，Zhang Y ，Fast DM ... -  《-》