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RATIONALE: Rivaroxaban is an anticoagulant prescribed to patients who are at risk of medical conditions such as deep-vein thrombosis, pulmonary embolisms, and strokes caused by blood clots. The administration of this drug is monitored to adjust the dosage and evaluate patients' blood concentration. Rapid quantification of this drug in plasma could make it possible to ensure that the dose present in the blood of patients does not represent a danger for the medical intervention to be carried out. METHODS: Liquid chromatography-tandem mass spectrometry is usually employed to quantify rivaroxaban in blood, plasma, and serum. Here, an alternative method of analysis based on laser diode thermal desorption-triple quadrupole mass spectrometry (LDTD-QqQMS) was developed and comprehensively validated. This new method allows the quantification of rivaroxaban in less than 13 s from sample to sample. The extraction of rivaroxaban in human serum was done by a salting-out liquid-liquid extraction with acetonitrile and a saturated sodium chloride solution. RESULTS: The proposed method allows the quantification of rivaroxaban in less than 13 s from sample to sample. During validation, all criteria were respected. The accuracy was <15% of the nominal value, the precision was <15%CV, and the recovery was ≥89.9%. There were no observed carryover or matrix effects. Analysis of the extracted samples established the stability of dry (24 h) and wet samples (1 week) when samples cannot be analyzed immediately, a considerable advantage in a clinical setting. CONCLUSIONS: This method improves sample throughput by more than 1200% compared to liquid chromatography-tandem mass spectrometry methods of analysis of rivaroxaban and decreases analysis costs by reducing solvent consumption and instrument time.
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Rivaroxabán , Espectrometría de Masas en Tándem , Rivaroxabán/sangre , Humanos , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los Resultados , Límite de Detección , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Modelos LinealesRESUMEN
We analyze how pandemic business interruption coverage can be put in place by building on capitalization mechanisms and a portfolio management strategy. As evidenced with COVID-19, pandemics affect economic sectors in differentiated ways: some are very severely affected because their activity is heavily impacted by travel bans and constraints on work organization, while others are more resistant. This opens the door to risk-coverage mechanisms based on a portfolio of financial securities, including long-short positions and options in stock markets. We show that such a strategy allows insurers to offer business interruption coverage in pandemic states, while simultaneously hedging the risks associated with alternating bullish and bearish non-pandemic states. These conclusions contrast sharply with the idea of governments being the only solution to the pandemic insurability problem. They are derived from a theoretical model of corporate risk management, and their practical relevance is illustrated by numerical simulations, using data from the French stock exchange.
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High-field asymmetric waveform ion mobility spectrometry (FAIMS) has gained popularity in the proteomics field for its capability to improve mass spectrometry sensitivity and to decrease peptide co-fragmentation. The recent implementation of FAIMS on Tribrid Orbitrap instruments enhanced proteome coverage and increased the precision of quantitative measurements. However, the FAIMS interface has not been available on older generation Orbitrap mass spectrometers such as the Q-Exactive. Here, we report the integration of the FAIMS Pro device with embedded electrical and gas connections to a Q-Exactive HF mass spectrometer. Proteomic experiments performed on HeLa tryptic digests with the modified mass spectrometer improved signal to noise and reduced interfering ions, resulting in an increase of 42% in peptide identification. FAIMS was also combined with segmented ion fractionation where 100 m/z windows were obtained in turn to further increase the depth of proteome analysis by reducing the proportion of chimeric MS/MS spectra from 50 to 27%. We also demonstrate the application of FAIMS to improve quantitative measurements when using isobaric peptide labeling. FAIMS experiments performed on a two-proteome model revealed that FAIMS Pro provided a 65% improvement in quantification accuracy compared to conventional LC-MS/MS experiments.
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Proteómica , Espectrometría de Masas en Tándem , Cromatografía Liquida , Humanos , Espectrometría de Movilidad Iónica , IonesRESUMEN
Quantification of nutritional biomarkers is crucial to accurately assess the dietary intake of different classes of (poly)phenols in large epidemiological studies. High-throughput analysis is mandatory to apply this methodology in large cohorts. However, the current validated methods to quantify (poly)phenols metabolites in biological fluids use ultra performance liquid chromatography (UPLC), leading to analysis time of several minutes per sample. To significantly reduce the run time, we developed and validated a method to quantify in urine the flavan-3-ols biomarkers, phenyl-γ-valerolactones (PVLs), using laser diode thermal desorption (LDTD). This mass spectrometry source allows direct introduction of sample extracts, resulting in analysis time of less than 10 s per sample. Also, to encompass the problem associated with the cost and availability of sulfated and glucuronide analytical standards, urine samples were subjected to enzymatic hydrolysis. Creatinine was also quantified to normalize the results obtained from the urinary spot. Results obtained with LDTD-MS/MS were cross-validated by UPLC-MS/MS using 155 urine samples. Coefficient of correlation was above 0.975 for PVLs and creatinine. For all analytes, the accuracy was between 90% and 113% by LDTD-MS/MS. Altogether, sample preparation was fully automated to demonstrate the application potential of this method to large cohorts.
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Rayos Láser , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida , Creatinina , Fenoles , Biomarcadores , Cromatografía Líquida de Alta PresiónRESUMEN
We develop an epidemionomic model that jointly analyzes the health and economic responses to the COVID-19 crisis and to the related containment and public health policy measures implemented in Luxembourg. The model has been used to produce nowcasts and forecasts at various stages of the crisis. We focus here on two key moments in time, namely the deconfinement period following the first lockdown, and the onset of the second wave. In May 2020, we predicted a high risk of a second wave that was mainly explained by the resumption of social life, low participation in large-scale testing, and reduction in teleworking practices. Simulations conducted 5 months later reveal that managing the second wave with moderately coercive measures has been epidemiologically and economically effective. Assuming a massive third (or fourth) wave will not materialize in 2021, the real GDP loss due to the second wave will be smaller than 0.4 percentage points in 2020 and 2021.
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COVID-19 , Control de Enfermedades Transmisibles , Humanos , Luxemburgo/epidemiología , Política Pública , SARS-CoV-2RESUMEN
The laser diode thermal desorption (LDTD) ionization source allows ultrafast and sensitive analysis of small molecules by mass spectrometry. Signal enhancement in LDTD has been observed when coating the surface of sample microwells with a solution of ethylenediaminetetraacetic acid (EDTA) or nitrilotriacetic acid. Here we present a quantitative analysis of signal enhancement using solutions of diverse commercial proteins (lysozyme, immunoglobulin G, albumin, and fibrinogen) as coatings. Results showed that compounds with polar chemical functions such as carboxylic acid, sulfonyl, and nitro had signal enhancement factors, in most cases higher than 10, when using any of the tested proteins as coating agent. Analysis of variance revealed that immunoglobulin G and fibrinogen gave the best results. However, the signal enhancement factors obtained with these proteins were not superior to those observed with EDTA. To explain the signal enhancement effect of proteins, analysis by scanning electron microscopy of dried samples on the microwell sample plates was carried out. Images showed that salicylic acid, one of the compounds with the highest observed signal enhancement, formed a thick layer when applied directly on the uncoated surface, but it formed small crystals (<1 µm) in the presence of protein or EDTA coatings. Further crystallographic studies using powder X-ray diffraction showed that the crystalline form of salicylic acid is modified in the presence of EDTA. Salicylic acid when mixed with EDTA had a higher percentage of amorphous phase (38.1%) than without EDTA (23.1%). These results appear to confirm that the diminution of crystal size of analytes and the increase of amorphous phase are implicated in signal enhancement effect observed in LDTD using microwell surface coatings. To design better coatings and completely elucidate the signal enhancement effect in LDTD, more studies are necessary to understand the effects of coatings on the ionization of analytes.
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Laser-diode thermal desorption (LDTD) is an ionization source usually coupled to triple quadrupole mass spectrometry (QqQMS) and specifically designed for laboratories requiring high-throughput analysis. It has been observed that surface coatings on LDTD microwell plates can improve the sensitivity of the analysis of small polar molecules. The objective of the present study is to understand and quantify the effect of microwell surface coatings on signal intensity of small organic molecules of clinical, environmental, and forensic interest. Experiments showed that the peak areas of diclofenac, chloramphenicol, salicylic acid, and 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol obtained by LDTD-QqQMS increased by up to 3 orders of magnitude when using microwells coated with ethylenediaminetetraacetic acid (EDTA). Tests with different chelating agents and polytetrafluoroethylene as microwell surface coatings showed that nitrilotriacetic acid gave significantly higher peak areas for five out of the nine compounds that showed signal enhancement using chelating agents as coatings. Scanning electron microscopy studies of EDTA-coated and uncoated microwells showed that analytes deposited in the former formed more uniform and thinner films than in the latter. The enhancement effect of surface coatings in LDTD-QqQMS was explained mainly by the formation of homogenous and thinner layers of nanocrystals of analytes that are easier to desorb thermally than the layers formed when the analytes dry in direct contact with the bare stainless-steel surface. Chemisorption of some analytes to the stainless-steel surface of the microwell plate appeared to be a minor factor. Surface coatings widen the number of compounds analyzable by LDTD-QqQMS and can also improve sensitivity and limits of detection.
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The move toward label-free screening in drug discovery has increased the demand for mass spectrometry (MS)-based analysis. Here we investigated the approach of coupling acoustic sample deposition (ASD) with laser diode thermal desorption (LDTD)-tandem mass spectrometry (MS/MS). We assessed its use in a cytochrome P450 (CYP) inhibition assay, where a decrease in metabolite formation signifies CYP inhibition. Metabolite levels for 3 CYP isoforms were measured as CYP3A4-1'-OH-midazolam, CYP2D6-dextrorphan, and CYP2C9-4'-OH-diclofenac. After incubation, samples (100 nL) were acoustically deposited onto a stainless steel 384-LazWell plate, then desorbed by an infrared laser directly from the plate surface into the gas phase, ionized by atmospheric pressure chemical ionization (APCI), and analyzed by MS/MS. Using this method, we achieved a sample analysis speed of 2.14 s/well, with bioanalytical performance comparable to the current online solid-phase extraction (SPE)-based MS method. An even faster readout speed was achieved when postreaction sample multiplexing was applied, where three reaction samples, one for each CYP, were transferred into the same well of the LazWell plate. In summary, LDTD coupled with acoustic sample deposition and multiplexing significantly decreased analysis time to 0.7 s/sample, making this MS-based approach feasible to support high-throughput screening (HTS) assays.
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Inhibidores Enzimáticos del Citocromo P-450/química , Descubrimiento de Drogas/métodos , Bioensayo/métodos , Calibración , Ensayos Analíticos de Alto Rendimiento/métodos , Extracción en Fase Sólida/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
VasoCare therapy, which involves the administration of autologous blood following the ex vivo exposure to physico-chemical stressors, has been shown to modulate immune responses. Since immune mechanisms have been recognized to be pivotal in the pathogenesis of atherosclerosis, we hypothesized that VasoCare treatment would inhibit atherosclerosis in LDL-R (-/-) mice. Three groups of LDL-R (-/-) mice were studied: a control group that was fed normal chow (Group I) and no other treatment; a control group that received a high cholesterol (HC) diet for 8 weeks (group II) with sham saline injections; and a third group (III) that received HC diet for 8 weeks and VasoCare treatment initiated after four weeks of HC feeding. Atherosclerotic area (AA), relative to total aortic area (TA), was assessed after 8 weeks of HC feeding by oil red O staining, and cross sectional plaque area at the level of the aortic valve leaflets was determined by quantitative morphometry. HC mice exhibited substantial aortic lipid deposition which was profoundly reduced in the VasoCare treated animals (AA/TA ratios in group II: 0.32+/-0.15 vs. group III: 0.17+/-0.06; P<0.05). This was associated with a significant decrease in cross sectional area of plaque in the aortic sinuses. VasoCare therapy also reduced the xanthoma formation and limb swelling characteristic of this animal model. However, cholesterol levels, measured by an enzymatic assay, showed similar marked increases in total serum cholesterol (CHO) in the animals receiving HC diet alone and those receiving the HC diet and VasoCare treatment [group I: 5.4+/-0.8 mM, group II: 46.7+/-3.6 mM, and group III: 44.7+/-2.8 mM (P<0.01 vs. group I)]. We conclude that VasoCare treatment inhibits progression of atherosclerotic lesions in a murine model of human familial hypercholesterolemia by a mechanism independent of cholesterol lowering.
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Enfermedad de la Arteria Coronaria/terapia , Inmunoterapia , Actinas/sangre , Actinas/inmunología , Animales , Especificidad de Anticuerpos/inmunología , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Colesterol en la Dieta/efectos adversos , LDL-Colesterol/sangre , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/etiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Endotelina-1/inmunología , Endotelina-1/metabolismo , Hiperlipidemias/sangre , Hiperlipidemias/etiología , Hiperlipidemias/terapia , Inmunohistoquímica , Metabolismo de los Lípidos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Cardiovasculares , Músculo Liso Vascular/metabolismo , Proyectos Piloto , Receptores de LDL/deficiencia , Linfocitos T/inmunología , Linfocitos T/metabolismo , Resultado del Tratamiento , Xantomatosis/sangre , Xantomatosis/etiología , Xantomatosis/terapiaRESUMEN
INTRODUCTION: Convertases (PCs), especially PC5, have been detected in various layers of atherosclerotic and injured arteries. We postulate that PCs could be important enzymes in vascular disease thus studied PC5 expression in a porcine balloon and stent coronary arterial vascular injury model. METHODS: Immunohistochemistry and in situ hybridization of slides of porcine arteries from paraffin blocks were studied 1, 7, 14 and 28 days post injury. RESULTS: Immunohistochemistry studies show expression of PC5 in control artery endothelial cells, weak medial smooth muscle cell (SMC) staining and strong staining in the small nerves of the adventitia. At 7, 14 and 28 days postinjury, there is strong positive PC5 staining of the neointimal cells and the adventitial vasa vasora and myofibroblasts. Colocalization immunohistochemistry confirms the smooth muscle staining properties of the myofibroblast-like cells in both these locations. Single-label immunohistochemistry studies show the same cells to stain strongly positive with TGF-B, PDGF, matrix metalloproteinase-2 (MMP-2) and MMP-9. CONCLUSION: PC5 may be involved in the process of arterial injury via its effect on growth factors (GFs) and mediators. These preliminary observations suggest that the convertases, especially PC5, represent a target for future study in the process of arterial injury.
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Arterias/enzimología , Reestenosis Coronaria/enzimología , Vasos Coronarios/enzimología , Proproteína Convertasa 5/metabolismo , Angioplastia de Balón/métodos , Animales , Arterias/patología , Biomarcadores/análisis , Enfermedad Coronaria/patología , Reestenosis Coronaria/patología , Vasos Coronarios/lesiones , Vasos Coronarios/patología , Modelos Animales de Enfermedad , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Técnica del Anticuerpo Fluorescente Indirecta , Hibridación in Situ , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Stents , PorcinosRESUMEN
In the last decade the quantitation of immunosuppressive drugs has seen vast improvements in analytical methods, optimizing time, accuracy of analysis and cost. Laser Diode Thermal Desorption (LDTD) coupled to Atmospheric Pressure Chemical ionization-tandem mass spectrometry (APCI-MS/MS) represents a technological breakthrough that removes the chromatographic separation step and thereby significantly increases the analytical throughput for the quantitation of cyclosporin A (CsA) in whole blood for therapeutic drug monitoring (TDM). A simple protein precipitation step was used prior to depositing 5 µL of the extract on a 96-well LazWell™ plate and CsA was quantified by LDTD-APCI-MS/MS. The laser pattern was set to ramp from 0 to 45% laser power within 2 s. The APCI parameters were set to negative needle voltage (-2 µA), carrier gas temperature (30°C) and air flow rate (3 L min(-1)). The negative ion single reaction monitoring transitions for CsA and its internal standard cyclosporin D (CsD) were respectively m/z 1201.1/1088.9 and m/z 1214.8/1102.8; obtained with a collision energy of -40 V. The analysis was achieved within 9 s from sample to sample. The extraction procedure yielded high recovery (92%; RSD=9.4%, n=6). The lower limit of quantitation was fixed at the first level of calibration: 23.5 ng mL(-1) (accuracy=112.3%; RSD=9.6%; n=6) and a blank+6 point linear regression up to 965 ng mL(-1) was used. Using 4 levels of quality control (QC), intra-day assays (n=6) ranged from 93.5 to 95.7% (bias) and from 3.4 to 13.1% (RSD) while inter-day assays (n=6) ranged from 92.9 to 105.3% (bias) and from 4.9 to 7.5% (RSD). An inter-sample contamination of CsA of 2.3% was calculated that was considered negligible with respect to the range of CsA concentrations. Whole blood samples (120) from patients under CsA treatment were analyzed by LDTD-APCI-MS/MS and HPLC-ESI-MS/MS, the gold standard reference method for CsA quantification. Both methods agreed (P≥0.99), with a coefficient of correlation of 0.99 (95% confidence interval 0.982-0.991). The Passing-Bablok regression revealed no significant deviation from linearity (Cusum test, P=0.11). This method seems suitable for use in TDM of CsA.
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Cromatografía Líquida de Alta Presión , Ciclosporina/sangre , Inmunosupresores/sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , Calibración , Cromatografía Líquida de Alta Presión/normas , Ciclosporina/normas , Humanos , Inmunosupresores/normas , Control de Calidad , Estándares de Referencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/normas , Espectrometría de Masas en Tándem/normasRESUMEN
An ultra-fast, reliable and sensitive analytical method enabling high-throughput quantitative analysis of pharmaceutical compounds in human plasma is described. The quantitative work was performed on one of our compound currently under clinical trial by employing a deuterated internal standard (IS). Plasma samples were treated on solid phase micro-extraction (SPME) plates prior their analysis by laser diode thermal desorption and atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD/APCI-MS/MS) in positive mode. The sample analysis run time was 10s as compared to the 7 min obtained for the validated LC-MS/MS method. The limit of quantification (LOQ) of the method was estimated at 1 ng/mL. The calibration graphs were linear with a regression coefficient R(2) > 0.997. The data of the partial validation show that LDTD/APCI-MS/MS assay was highly reproducible and selective. In addition, the deviations for intra and inter assay accuracy and precision data were within 15% at all quality control levels. The LDTD/APCI-MS/MS method was successfully applied to the analysis of clinical samples and the data obtained were consistent with those found with a validated LC-MS/MS assay. This work demonstrates that LDTD/APCI-MS/MS could be used for the ultra-fast and reliable quantitative analysis of pharmaceutical compounds in human plasma without using the separation step commonly associated with the LC-MS/MS assay.
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Evaluación de Medicamentos/métodos , Preparaciones Farmacéuticas/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Calibración , Evaluación de Medicamentos/instrumentación , Evaluación de Medicamentos/normas , Humanos , Límite de Detección , Preparaciones Farmacéuticas/administración & dosificación , Estándares de Referencia , Microextracción en Fase SólidaRESUMEN
Sulfonamides are antibiotic compounds widely used in the dairy industry. Their presence in diary milk poses a risk to public health and may also contribute to the spread of antibiotic resistance in bacteria. Sulfonamide residues in dairy milk were quantified by tandem mass spectrometry (MS/MS) using a novel ionization source based on laser diode thermal desorption-negative mode atmospheric pressure chemical ionization (LDTD-APCI(-)). Seven sulfonamides spiked in milk were extracted with acetonitrile, which yielded high recoveries (77.5-101.5%). Calibration curves in the matrix showed good linearity (0.9977 >or= R(2) >or= 0.9658) over the dynamic range (1.6-500 microg L(-1)), and limits of quantitation were between 2 and 14 microg L(-1), lower than or of the same magnitude as maximum residue criteria set by several regulatory agencies (10-100 ng L(-1)). In addition, the run time using the LDTD-MS/MS system was 30 s per sample, as compared to actual methods running from 7 to 84 min for the same sulfonamide residue compounds, which gave the method the high screening throughput capacity necessary for monitoring milk production.
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Antibacterianos/química , Residuos de Medicamentos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Leche/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Sulfonamidas/química , Espectrometría de Masas en Tándem/métodos , Animales , Bovinos , Ensayos Analíticos de Alto Rendimiento/instrumentación , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
Real-time monitoring of benzene, toluene, ethylbenzene, and xylenes (BTEX) in ambient air is essential for the early warning detection associated with the release of these hazardous chemicals and in estimating the potential exposure risks to humans and the environment. We have developed a tandem mass spectrometry (MS/MS) method for continuous real-time determination of ambient trace levels of BTEX. The technique is based on the sampling of air via an atmospheric pressure inlet directly into the atmospheric pressure chemical ionization (APCI) source. The method is linear over four orders of magnitude, with correlation coefficients greater than 0.996. Low limits of detection in the range 1-2 microg/m(3) are achieved for BTEX. The reliability of the method was confirmed through the evaluation of quality parameters such as repeatability and reproducibility (relative standard deviation below 8% and 10%, respectively) and accuracy (over 95%). The applicability of this method to real-world samples was evaluated through measurements of BTEX levels in real ambient air samples and results were compared with a reference GC-FID method. This direct APCI-MS/MS method is suitable for real-time analysis of BTEX in ambient air during regulation surveys as well as for the monitoring of industrial processes or emergency situations.
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This paper describes the development of a high-throughput method for the analysis of cytochrome P450 (CYP) inhibition assay incubation samples using laser diode thermal desorption interfaced with atmospheric pressure chemical ionization mass spectrometry (LDTD-APCI-MS). Data for the CYP isoforms 3A4, 2D6, 2C9, and 1A2 from competitive inhibition assays are shown. The potential for inhibition of the CYP isoforms was measured by monitoring the level of the metabolites 6beta-hydroxytestosterone (3A4), dextrorphan (2D6), 4'-hydroxydiclofenac (2C9), and acetaminophen (1A2) formed in the presence of drug candidates using an eight-point titration. The analytical method involves plating of the inhibition samples on specially designed 96-well plates with stainless steel bottoms, followed by direct analysis using the LDTD source. Validation of the LDTD-MS method was performed by testing for interferences, reproducibility, dynamic range, ion suppression, and the ability of the source to produce comparable results to previously validated LC-MS methods. IC50 values for each CYP isoform using 33 different test compounds showed excellent agreement between LDTD-APCI-MS and LC-MS methods and literature values where available. Assay analysis time using the LDTD-APCI source is reduced to less than 30 min for a single 96-well plate compared to greater than 10 h using the LC-MS method. The LDTD-APCI-MS and LC-MS methods and results are compared and limitations and future potential for LDTD-APCI-MS are discussed.