Racecadotril, an anti-secretory medication, has been used as an adjuvant in an oral rehydration therapy for children experiencing severe diarrhea. Racecadotril is quickly converted to thiorphan, an active metabolite, after oral treatment, which mediates all subsequent activities. An efficient and rapid liquid chromatography-tandem mass spectrometry method was developed and fully validated to measure thiorphan in human plasma, using thiorphan-d7 as an internal standard. The extraction method used was protein precipitation while chromatographic separation was achieved using InertSil CN-3 (50 × 2.1 mm, 5 µm column). The assay was linear over the concentration range of 1-200 ng/ml with correlation coefficients of ≥0.9991. The intra- and inter-day precisions were less than 10.0 % for all concentrations investigated. 0.02 % aqueous formic acid and methanol (30:70 v: v) were used as mobile phases, with an analysis time of less than 1 min. This method proved stable under several conditions. The developed method worked well in a three-period pharmacokinetic bioequivalence study after a single oral administration of 100 mg racecadotril to 15 healthy Jordanian volunteers under fasting conditions.

Aims: Cyanoenone triterpenoids penetrate the CNS, exhibiting biological activity via the nuclear factor E2-related factor (Nrf2) pathway. This is the first report on methods for the quantification of cyanonenone triterpenoids\' distribution in various CNS tissues by LC-MS/MS. Materials & methods: The analyte was extracted from brain tissue homogenate using protein precipitation and supported liquid extraction. Results & conclusion: The assay validated a quantification range of 3.00-3000 ng/g in brain tissue samples as low as 5 mg. All parameters, including interference (≤20% at LLOQ) and accuracy/precision (15%, with 20% at LLOQ), met acceptance criteria. This assay supported a CNS distribution study, analyzing more than 10 mouse brain regions successfully.

Sample clean-up with the protein precipitation solvent trichloroacetic acid (TCA), combined with a stable isotope labeled internal standard, is widely used for the analysis of endogenous and exogenous compounds in serum and plasma with liquid chromatography-tandem mass spectrometry (LC-MS/MS). During the application of an assay for methylmalonic acid (MMA), used for routine analysis in patient care, negative long-term side effects of TCA on assay performance were observed. Step-by-step extensive troubleshooting disclosed the limitations of using TCA in MS. After running over 2000 samples with the MMA assay over a course of one year, a black coating formed between the probe and the heater that was traced to the use of TCA. The MMA assay used a C18 column with an isocratic eluent of 95% water (0.1% formic acid) as starting condition, on which TCA was more retained than MMA. Next, concentrations of 2.2% TCA in the prepared serum or plasma sample caused a drop in spray voltage during ionization into the MS. This was caused by the strong acid properties of TCA, resulting in current loss of the spray voltage between the heated electrospray ionization (HESI) needle and the union holder, which had also a grounding function. Replacing the original metal HESI needle with a custom made fussed silica HESI needle or detaching the union from the union holder, eliminated the effect of the drop in spray voltage. In conclusion, TCA can seriously affect the long-term robustness by affecting the source of the MS. We recommend the use of a very low sample injection volume, and/or shifting the mobile phase to waste when TCA is eluting, when using TCA in LC-MS/MS analysis.

This study aimed to establish a simple and sensitive analytical method to simultaneously quantify donepezil (DPZ) and tadalafil (TAD) in rat plasma using lansoprazole (LPZ) as an internal standard (IS) by using liquid chromatography tandem mass spectrometry. The fragmentation pattern of DPZ, TAD, and IS was elucidated using multiple reaction monitoring in electrospray ionization positive ion mode for the quantification of precursor to production at m/z 380.1 → 91.2 for DPZ, m/z 390.2 → 268.1 for TAD, and m/z 370.3 → 252.0 for LPZ. The extracted DPZ and TAD from plasma using acetonitrile-induced protein precipitation was separated using Kinetex C18 (100 × 2.1 mm, 2.6 µm) column with a gradient mobile phase system consisting of 2 mM ammonium acetate and 0.1% formic acid in acetonitrile at a flow rate of 0.25 mL/min for 4 min. The selectivity, lower limit of quantification, linearity, precision, accuracy, stability, recovery, and matrix effect of this developed method was validated according to the guidelines of the U.S. Food and Drug Administration and the Ministry of Food and Drug Safety of Korea. The established method achieved acceptance criteria in all validation parameters, ensuring reliability, reproducibility, and accuracy, and was successfully implemented in a pharmacokinetic study on the co-administration of DPZ and TAD orally in rats.

UNASSIGNED: The study aimed to develop a rapid, simple and sensitive LC/ESI-MS/MS method to measure prazosin concentration in human plasma and apply bedside sampling in bioequivalence study of two prazosin tablets to resolve the adverse effect of orthostatic hypotension.
UNASSIGNED: The LC/ESI-MS/MS prazosin method was highly sensitive and selective. Bedside sampling reduced the orthostatic hypotension incidence and subject dropout rate.
UNASSIGNED: After sample preparation, prazosin and terazosin (IS) were detected on mass spectrometer operating in multiple reaction monitoring mode using positive ionization. Mobile phase flow rate was set at 0.40 mL/min with sample run time of 1.75 min. The bioanalytical method was validated as per EMEA and FDA guidelines. Bedside sampling was performed in bioequivalence study for the first 4 h after dosing. The three primary pharmacokinetic parameters, Cmax, AUC0-t and AUC0-∞ and 90% confidence interval were determined.
UNASSIGNED: The small injection volume of 1 μL minimized instrumentation contamination and prolonged the analytical column lifespan. Linearity was obtained between 0.5 and 30.0 ng/mL, with coefficient of determination, r2 ≥ 0.99. The mean extraction recovery of prazosin and IS was >92%, with precision value (CV, %) ≤ 10.3%. Only two orthostatic hypotension adverse events were reported. The two prazosin formulations were found to be bioequivalent.
UNASSIGNED: The LC/ESI-MS/MS method has shown robustness and reliability exemplified by the incurred sample re-analysis result. Bedside sampling should be proposed for bioequivalence or pharmacokinetic studies of drugs demonstrating adverse event of orthostatic hypotension.

Etoricoxib is a non-steroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation. The objective of the current study was to develop a sensitive, fast and high-throughput HPLC-ESI-MS/MS method to measure etoricoxib levels in human plasma using a one-step methanol protein precipitation technique. A tandem mass spectrometer equipped with an electrospray ionization (ESI) source operated in a positive mode and multiple reaction monitoring (MRM) were used for data collection. The quantitative MRM transition ions were m/z 359.15 > 279.10 and m/z 363.10 > 282.10 for etoricoxib and IS. The linear range was from 10.00 to 4000.39 ng/mL and the validation parameters were within the acceptance limits of the European Medicine Agency (EMA) and Food and Drug Analysis (FDA) guidelines. The present method was sensitive (10.00 ng/mL with S/N > 40), simple, selective (K prime > 2), and fast (short run time of 2 min), with negligible matrix effect and consistent recovery, suitable for high throughput analysis. The method was used to quantitate etoricoxib plasma concentrations in a bioequivalence study of two 120 mg etoricoxib formulations. Incurred sample reanalysis results further supported that the method was robust and reproducible.

Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathways are tightly regulated multistep chain reactions that involve a wide range of molecular interactions and enzymatic activities. The first signal induced by VEGF binding to VEGFR2, is the activation of the receptor tyrosine kinase and autophosphorylation of intracellular tyrosine residues of the receptor. In endothelial cells, five tyrosine residues in the VEGFR2 intracellular domain are essential in signal transmission and in the respective regulation of cellular processes. Because of their number and their localization on the receptor, it is challenging to locate the proteins with which these tyrosine residues interact that result in further downstream signaling cascades. In this chapter, we describe a method to precipitate phosphotyrosine binding proteins using phosphotyrosine-containing synthetic peptides immobilized to magnetic beads. The identity of the precipitated proteins is determined by mass spectrometry and the findings validated by Western blot. Using this method, we identified and verified two proteins, growth factor receptor binding-2 (GRB2) and phosphoinositide 3\'-kinase (PI3Kp85), binding to the tyrosine 1214 of VEGFR2. Thereby, we can predict the signaling pathways downstream of pY1214.

A simple, fast and sensitive LC-MS/MS method was developed to quantify terazosin in human plasma. The mobile phase consisted of acetonitrile-0.1% (v/v) formic acid (70:30, v/v). Prazosin was used as internal standard (IS). As deproteinization agent, acetonitrile produced a clean sample. A higher response intensity with more symmetrical peak was obtained using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7 μm) compared with Kinetex XB-C18 (100 × 2.1 mm, 2.6 µm) column. The response of terazosin and IS were approximately two times in citrate phosphate dextrose (CPD) plasma compared with dipotassium ethylenediaminetetraacetic acid (K2EDTA) plasma. Plasma calibration curve was linear from 1.0 to 100.0 ng/mL, with coefficient of determination r2 ≥ 0.99. The within-run and between-run precision values (CV, %) were <5.2% and <7.8%, while accuracy values were 102.8-112.7% and 103.4-112.2%. The extended run accuracy was 98.6-102.8% and precision (CV, %) 4.3-10.4%. The recovery of analyte was >98% and IS >94%. Terazosin in plasma kept at benchtop was stable for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h, for 7 freeze-thaw cycles and in freezer for 140 days. Terazosin and IS stock standard solutions were stable for 140 days at room temperature and in the chiller. The high throughput method was successfully utilized to measure 935 samples in a bioequivalence study of terazosin.

ASK120067, an oral irreversible tyrosine kinase inhibitor (TKI) targeting the epidermal growth factor receptor (EGFR), is formulated for the management of patients with non-small cell lung cancer (NSCLC) who harbor T790M resistant and EGFR active mutations. Two rapid and high-throughput methods based on liquid chromatography-tandem mass spectrometry to detect ASK120067 and its primary metabolite CCB4580030 in human plasma were developed and applied in the clinical trials. A protein precipitation method using acetonitrile coupled with a gradient elution separation in a BEH C18 column (1.7 µm, 2.1 × 50 mm) was used to process plasma and separation analytes. The chromatographic separation was performed on the mobile phase of 5 mM ammonium acetate in water with 0.1% formic acid (A) and acetonitrile (B), and the flow rate was 0.4 mL/min. The multiple reaction monitoring (MRM) mode was selected to monitor the precursor-to-product ion transitions of m/z 546.2 → m/z 431.2 for ASK120067 and m/z 532.1 → m/z 420.2 for CCB4580030 at the positive ionization mode. The precision and accuracy of the two methods for ASK1200067 and CCB4580030 were within acceptable range for the linear range in 5.00-5000 ng/mL and 0.500-500 ng/mL, respectively. Further stabilities for the two analytes and internal standard were also investigated covered the entire experimental process beginning from harvesting whole blood to plasma extraction and analysis. ASK120067 was then administered without issue onto a dose-escalation, the first-in-human Phase I clinical trial in Chinese NSCLC patients to determine the pharmacokinetics of oral ASK120067 administration.

A simple, rapid, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method was developed to determine sitagliptin in human plasma. Diphenhydramine HCl was used as internal standard (IS). The chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7) fitted with UHPLC Guard Poroshell 120 EC-C18 (5 × 2.1mmID, 2.7 µm). The mobile phase consisted of 0.1% v/v formic acid and methanol (45:55, v/v) run at a flow rate of 0.45 mL/min at 30 °C. Methanol produced relatively cleaner plasma sample as deproteinization agent. Polytetrafluoroethylene membrane was preferred over nylon membrane as the former produced clear plasma samples. The standard calibration curve was linear over the concentration range of 5-500.03 ng/mL. The within-run precision was 0.53-7.12% and accuracy 87.09-105.05%. The between-run precision was 4.74-11.68% and accuracy 95.02-97.36%. The extended run precision was 3.60-6.88% and accuracy 93.18-95.82%. The recovery of analyte and IS was consistent. Sitagliptin in plasma was stable at benchtop (short term) for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h (post-preparative), after 7 freeze-thaw cycles (-20 ± 10 °C), and 62 days in the freezer (-20 ± 10 °C). Both sitagliptin (analyte) and IS stock solutions were stable for 62 days when kept at room temperature (25 ± 4 °C) and in chiller (2-8 °C). The validated method was successfully applied to a bioequivalence study of two sitagliptin formulations involving 26 healthy Malaysian volunteers.