TRANSTORNO BIPOLAR DO HUMOR E DEPENDÊNCIA DE SUBSTÂNCIAS PSICOATIVAS (1) Unidade de Pesquisa em Álcool e Drogas (UNIAD) – Universidade Federal de São Paulo (UNIFESP) (2) Programa de Doenças Afetivas (PRODAF) – Universidade Federal de São Paulo (UNIFESP) O transtorno bipolar e o uso indevido de substâncias psicoativas são doenças com alto potencial de limitação de autonom
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High Throughput LDTD-MS/MS IC50
Determination of CYP Inhibition in HLM
Keywords: High-throughput, CYP inhibition assay, Human Liver Microsomes, Metabolites
The LDTD is a shotgun approach where the sample is introduced into the mass spectrometer without High throughput analysis of CYP 1A2 / 2C9 / chromatographic step, the separation being achieved by operating the mass spectrometer in MS/MS mode. Probe : Phenacetin, Diclofenac, Tolbutamide, The specificity of the method is therefore achieved by S-mephenytoin, Bufurolol, Dextromethorphan, selecting specific MS/MS transitions for each target Midazolam, Testosterone and Nifedipine ; Inhibitors : Furafylline (1A2), Sulfaphenazole increase dramatically the analytical speed. (2C9), Ticlopidine (2C19), Quinidine (2D6) and Samples Preparation
Acetaminophene (1A2) analyzed in negative
Table 1 shows the incubation conditions of the CYP
probes and inhibitors used in this study. The protein
IC50 comparable with LC-MS/MS and LDTD-MS/MS methods ; concentration was 0.25 mg/mL of HLM prepared in a pH 7.4 phosphate buffer. Reactions were initiated by Instrumentation
adding NADPH and all samples were incubated in 300-µL round bottom 96-well plates at 37 0C for 10 min. LDTD ion source, T-960 (Phytronix Technologies) The incubations were quenched by adding equal interface on a TSQ VantageTM triple quadrupole LC-MS/MS : After vortexing and centrifuging, the
supernatant were transferred to a 96-well injection plate. The resulting samples were analyzed by the Roche Palo Alto in house LC-MS/MS (Sciex API 4000, instrumentation conditions not listed). Bucetin was Introduction
In early drug discovery, identifying potential drug candidates is a common practice. One important step LDTD-MS/MS : After vortexing and centrifuging, the
in this discovery process is to identify drug-drug supernatant were dissolved with four (4) volume of interactions. The most widespread procedure for this acetonitrile and 2µL were transferred to a 96-well is to perform cytochrome P450 (CYP) inhibition assays LazWell plate. The solvent was evaporated to dryness using human liver microsomes (HLM). The most at room temperature. This dilution step was necessary commonly used method for analyzing CYP inhibition to reduce the unvolatile content into the final dry assay samples is LC-MS/MS. However, this method is samples. The resulting dry samples were analyzed in time-consuming and represents the bottleneck in this LDTD-MS/MS. Labelled internal standard were used type of assay. To increase the throughput, we propose for OH-midazolam and OH-diclofenac only. Table 1 Incubation conditions of the CYP assay.
Inhibitor / Highest
Isozyme / Probe substrate
[email protected] All rights reserved ( Tolbutamide, S-Mephenytoin and Testosterone). This Table 2 SRM transition and APCI mode.
could from a mass-effect into the well which seems to affect the thermal desorption. Diluting the final extract Compound
should lower this effect leading to more accurate Table 3 Summary of IC50 values obtained.
Isoform LC-MS/MS LDTD-MS/MS
Results and Discussion
Operating without chromatographic separation in APCI without liquid mobile phase, the LDTD in-source fragmentation needs to be evaluated. Each probe Ketoconazole (CYP3A4 : Midazolam , 2uM)
Ketoconazole (CYP3A4 : Midazolam , 2uM)
were analyzed in matrix at the working concentration (2 to 100 µM, see Table 2) and the corresponding
In (+)APCI a signal of Acetaminophen (MW 151.2 g mol-1) SRM was observed when a blank containing 50 µM of Phenacetin (MW 179.2 g mol-1) was analyzed. The signal intensity correspond to a Phenacetin conversion into Acetaminophen of 1.5 % Figure 1 Representative inhibition curves for the CYP
affecting the IC50 determination. The same analysis has performed in (-)APCI and under these conditions the observed conversion was 10-times lower. The IC was calculated and the value obtained was comparable High throughput advantage of LDTD-MS/MS over
to the LC-MS/MS method. Therefore, in LDTD-
MS/MS, the Acetaminophen should be monitored in
The reproducibility has been evaluated in LDTD- (-)APCI to get accurate IC
MS/MS by running 4 to 6 replicates of the same 50 determination.
sample. The reproducibility has been calculated from the % RSD. and being operated without using an internal standard, The reproducibility is comparable to IC50 determination and accuracy
the one obtained in LC-MS/MS, however it should be Data were fitted using Xlfitting and representative inhibition curves obtained in LDTD-MS/MS are
presented in Figure 1. Calculated IC50 values are
The analytical speed of the LDTD which allows 4 reported in Table 3. The calculated IC50 values as well
seconds per sample is 63-times faster then traditional as inhibition curves were comparable to the results LC-MS/MS method (252 seconds / sample). The analytical speed provided by the LDTD allow to performance from the LDTD-MS/MS system. Using an increase the CYP inhibition assays throughput without internal standard (CYP 2C9 for 4’-OH-diclofenac and compromising the IC50 determination according to the CYP 3A4 for 1’-OH-midazolam) allows to get better accuracy with the LDTD. On the other hand, We Acknoledgments
observe more discrepancy results between LDTD and LC-MS/MS IC The authors would like to acknowledge the work of Limin He, Jae Chang and Jane Huang from the DMPK group at Roche Palo Alto, Palo Alto, CA, USA for [email protected] All rights reserved
UNIVERSIDAD DE SAN CARLOS DE GUATEMALA CENTRO UNIVERSITARIO DE OCCIDENTE DIVISIÓN DE CIENCIAS DE LA SALUD Curso de Ciencias Clínicas I CARRERA DE MEDICO Y CIRUJANO PRIMER AÑO. 2014 . Semana 1, documento 2. ÉTICA MÉDICA ÉTICA: La palabra ética deviene del latín ETHOS que se refiere al MODO DE SER O CARACTER ADQUIRIDO POR EL HOMBRE. El diccionario de la lengua defin