PowerPoint Presentation

Thermo Fisher Scientificw/ Dr. Joseph DiBussoloIan White

Goals of InternshipGain experience with basic sample preparation (e.g. protein precipitation, liquid-liquid extraction)Mastery of hardware and software associated with LC/MS system Thermo Scientific Transcend TLX-1, TSQ Quantum Ultra and TSQ QuantivaXcalibur, LCquan and Aria softwareDevelopment and optimization of bioanalytical methods using LC/MS technology to achieve accurate high-throughput results

Worked with blood serum, blood plasma, and urine2

Applications in PharmaPreclinical R&DCandidate identificationQuantity of major and minor componentsClinical TrialsMetabolite screeningBiomarker analysis

Preclinical What have you made, and how much is present?

Pharmacokinetic/Pharmacodynamic studies in preclinical animal models

Look at metabolite screening for patient compliance; making sure patient is using medication as directed is critically important in clinical trials.

Biomarker analysis to assess effect of drug on the body (upregulation/downregulation as a result of treatment)3

See labroots.com

4

Conditions for chromatography; allow analytes to stick to column, elute analytes into mass spectrum, wash column and prepare for next injection5

Significance of ApplicationPatient complianceIndicates whether patient took drug, and how much was takenEthanol use in conjunction to prescription use

Analysis for both precursor (buprenorphrine) and metabolite (norbuprenorphrine) to detect sample doping7

TroubleshootingInterference from succinic acidGeometric isomer of methylmalonic acidButyl-MMA ester derivative

Formation esters and separation by chromatography allow for MMA detection9

Significance of ApplicationBiomarkerIndicates that treatment is working as expectedHigh levels of MMA indicates B12 deficiency

In preclinical/clinical trials, upregulation or downregulation of a biomarker can indicate how the drug affects the body.10

Pregnenolone MethodHigh-Throughput LC-MS/MS Measurement of Pregnenolone in Human Blood Serum for Research PurposesJoe Di Bussolo1, Ian White2, Emily Herman2, Raidiri Castillo3, Amit Shah3 and Hashim Othman3; 1Thermo Fisher Scientific, 2West Chester University of PA, 3Bio-Reference Laboratories.Pregnenolone is a biosynthetic precursor to other steroids such as corticosteroids, androgens and estrogens. Researchers need to measure pregnenolone within an analytical range of 10 to 500 ng/dL of blood serum. This was achieved using a multi-channel ultra high-performance liquid chromatography (UHPLC) utilizing solid-core packed columns with an alkyl-aromatic bonded phase coupled to a triple-quadrupole mass spectrometer (MS) utilizing heated electro-spray ionization (HESI). Sample preparation involved extracting specimens with methyl-t-butyl ether after spiking them with an internal standard (Pregnenolone-D4). After transferring and evaporating each extract to respectively labeled tubes, the residue of each was reacted with hydroxylamine to form oxime derivatives of pregnenolone. Each tube was then dried and the residue was reconstituted with water and methanol and transferred into autosampler vials. Injections were then made into a 4-channel UHPLC system. A 4.5-minute water-to-methanol gradient eluted the analyte and internal standard into the HESI source of the MS. Selective-reaction monitoring within a 1-minute data window produced quantitation and conformation peaks with intra-assay and inter-assay precisions < 6% RSD and carryover < 0.5%. Correlation of specimen results with reference-lab results are in progress. Sample throughput was 13, 26, 39 or 52 injections per hour when multi-channeled across 1, 2, 3 or 4 channels, respectively. Pregnenolone batches were also multi-channeled with estrogen batches which utilized the same MS source conditions.

Method is nearly validated11

MS Analysis of Synthetic Reactions

Important from a synthetic standpoint (quantity and quality of candidate products)12

Project SummaryEtG/EtSMethylmalonic acidSteroids17-OH progesteroneEstrogensPregnenoloneCortisone/ol MS evaluationBPA (w/ Dr. Slusher)Testosterone (w/ Dr. Pruitt, Brennan Harding)Spectral analyses (w/ Dr. Ross)

Thank you!