µPESI-MS/MS System for Screening and Quantitating Drugs in Plasma Samples.

Recently, we developed a novel microprobe electrospray ionization (µPESI) source and its coupled MS (µPESI-MS/MS) system. Here, we aimed to widely validate the µPESI-MS/MS method for quantitative analysis of drugs in plasma samples. Furthermore, the relationship between the quantitative performance of the µPESI-MS/MS method and the physicochemical properties of target drugs was analyzed. The µPESI-MS/MS methods for quantitative analysis of 5 representative drugs with a relatively wide range of molecular weight, pKa, and log P values were developed and validated. The results showed that the linearity, accuracy, and precision of these methods met the requirements of the European Medicines Agency (EMA) guidance. Then a total of 75 drugs from plasma samples were primarily detected using the µPESI-MS/MS methods, among which 48 drugs could be quantitatively measured. Logistics regression suggested that drugs with significantly greater log P and physiological charge had better quantitative performance using the µPESI-MS/MS method. Collectively, these results clearly demonstrate the practical application of the µPESI-MS/MS system as a rapid approach to the quantitative analysis of drugs in plasma samples.

An effective pretreatment technique based on multi-walled carbon nanotubes to reduce the matrix effect in plasma samples analyzed by a new type probe electrospray ionization method.

The quantitative analysis of drug plasma samples plays an important role in the drug development and drug clinical use. Our research team developed a new electrospray ion source-Micro probe electrospray ionization (µPESI) in the early stage, which was combined with mass spectrometry (µPESI-MS/MS) showing good qualitative and quantitative analysis performance. However, matrix effect severely interfered the sensitivity in µPESI-MS/MS analysis. To solve this problem, we recently developed a Solid-phase purification method based on multi-walled carbon nanotubes (MWCNTs), which was used for removing matrix interfering substances (especially phospholipid compounds) in the preparation of plasma samples, so as to reduce the matrix effect. In this study, aripiprazole (APZ), carbamazepine (CBZ) and omeprazole (OME) were used as representative analytes, the quantitative analysis related to the plasma samples spiked with the analytes above and the mechanism of the MWCNTs to reduce matrix effect were both investigated. Compared with the ordinary protein precipitation, MWCNTs could reduced the matrix effect for several to dozens of times, which resulting from the removement of phospholipid compounds from the plasma samples by MWCNTs in the selective adsorption manner. We further validated the linearity, precision and accuracy of this pretreatment technique by the µPESI-MS/MS method. These parameters all met the requirements of FDA guidelines. It was showed that MWCNTs have a good application prospect in the drug quantitative analysis of plasma samples using the µPESI-ESI-MS/MS method.

Covalent Bonding and Coulomb Repulsion-Guided AuNP Array: A Tunable and Reusable Substrate for Metabolomic Characterization of Lung Cancer Patient Sera.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) has gained increased attention in the metabolic characterization of human biofluids. However, the stability and reproducibility of nanoparticle-based substrates remain two of the biggest challenges in high-salt environments. Here, by controlling the extent of Coulomb repulsion of 26 nm positively charged AuNPs, a homogeneous layer of covalently bonded AuNPs on a coverslip with tunable interparticle distances down to 16 nm has been successfully fabricated to analyze small biomolecules in human serum. Compared with the self-assembled AuNP array, the covalently bonded AuNP array showed superior performances on stability, reproducibility, and sensitivity in high-salt environments. The stable attachment of AuNPs maintained a detection reproducibility with a RSD less than 12% and enabled the reusability of the array for 10 experiments without significant signal deterioration (<15%) and carryover effects. Moreover, the closely positioned AuNPs allowed the coupling of photoinduced plasmons to generate an enhanced electric field, which promotes the generation of excited electrons to facilitate the desorption/ionization processes instead of the heat dissipation, thus enhancing the detection sensitivity with detection limits down to the femtomole level. Combined with machine learning methods, the AuNP array has been successfully applied to discover seven biomarkers for differentiating early-stage lung cancer patients from healthy controls. It is anticipated that this simple approach of developing robust AuNP arrays can also be extended to other types of NP arrays for wider applications of SALDI-MS technology.

Equilibration for Electrospray Ionization Mass Spectrometry in Quantitative Analysis.

Electrospray ionization mass spectrometry (ESI-MS) is widely used in drug development, therapeutic drug monitoring, and other fields. However, unstable mass spectral signals, especially during the initial stages of instrument operation, plague analysts. Generally, in quantitative experiments, the stability of response can be achieved by running the analytical system for some time. However, the equilibration time required for the responses of different compounds to stabilize has been elusive. To investigate the response stability of the ESI-MS system, 72 compounds with different physicochemical properties were employed on three systems, and flow injection analysis was performed in positive ion mode. With the use of 5.00% (response stable factor, RSF) as the stability limit, about 80% of the compounds were stable within 60 min. Under a 2.00% criterion, the stabilization time was significantly longer. The stabilization time varies with different instruments and physicochemical properties of the compounds. When positive ion detection is performed in an acidic mobile phase, the octanol-water partition coefficient (Log P), molecular weight, and molar volume can all affect the time required to stabilize the response. In general, it is necessary to balance the ESI-MS system for an appropriate time before sample detection, especially for the analysis of compounds with strong hydrophilicity, small molecular weight, or small molar volume under the conditions above.

Signal Drift in Liquid Chromatography Tandem Mass Spectrometry and Its Internal Standard Calibration Strategy for Quantitative Analysis.

The present project studied the signal drift in liquid chromatography tandem mass spectrometry (LC-MS/MS) and proposed a strategy for compensating such drift. In the study, four 4-component groups were repeatedly run on different LC-MS/MS systems for over 12 h to investigate the dependence of signal drift on time and hardware systems. The 4-component groups each consisted of (1) an analyte, (2) a stable isotope labeled analyte, (3) a compound with similar structure to the analyte, and (4) a compound with dissimilar structure. All of the species showed significant signal drift, generally more than 25% over 12 h. The analyte and its stable isotope labeled analog always have the same drifting pattern including the trends and direction from one LC-MS/MS system to another. Signal drift was also found to be concentration dependent. Our experiments further proved that a conventional stable isotope labeled internal standard in LC-MS/MS quantification would not compensate the variations caused by concentration-dependent signal drift. An ideal internal standard for LC-MS/MS has both identical structure and similar concentration to the analyte. For that, we proposed a new internal standard strategy, pseudo internal standard (Pseudo IS), for LC-MS/MS quantification. Pseudo IS could effectively compensate signal drift in spite of its significant time, system, and concentration dependencies.

Development, Validation, and Application of a New Method To Correct the Nonlinearity Problem in LC-MS/MS Quantification Using Stable Isotope-Labeled Internal Standards.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become an indispensable tool for bioanalysis. To quantify a small molecule with LC-MS/MS, a stable isotope-labeled analyte is routinely used as the internal standard. However, cross signal contributions between the analyte and its stable isotope labeled internal standard (SIL-IS) could cause problems when the signal response of the LC-MS/MS system is nonlinear. In the present work, we try to illustrate how the "cross talk" between the analyte and its SIL-IS may cause problems for a nonlinear system. We assume that the instrumental responses toward the analyte and its SIL-IS are the same. When the calibration curve is nonlinear, the addition of a SIL-IS would practically move the response of the analyte up along the parabolic line causing a change in the signal strength of the analyte (usually decrease). The more the SIL-IS is added, the larger change the analyte signal would become. Such a problem would only be corrected by making the calibration curve linear. To this end, we proposed a component equation (CE) as the calibration for nonlinearity correction. In this study, we contrasted the accuracy of CE with the common quantitative method using two drugs whose mass spectrometric responses are linear and nonlinear, respectively. The acceptable accuracy results demonstrated that the CE calibration was comparable with the regular quantitative SIL-IS method with a proper weighting factor and much better than that without weighting. Therefore, CE calibration may provide another reliable way for LC-MS/MS quantification.