Anabolic androgenic steroids are testosterone and its derivates. Testosterone is the most important naturally existing sex hormone for men and is used for its anabolic effects providing increased muscle mass. Testosterone is taken orally or by intramuscular injection in its ester form and are available illegally in different forms of esters. Anabolic androgenic steroids are today analyzed only in urine. To differentiate between the human natural testosterone and exogenous supply the quote natural testosterone and epitestosterone is used. Detection of testosterone esters in serum is an unmistakable proof of exogenous supply of testosterone. The aim of this thesis was to find a method for determining testosterone esters in serum and to study an extraction method possible for quantification of testosterone esters in serum. The technique used to separate and identify the Testosterone esters was Liquid Chromatography Tandem Mass Spectrometry Electro Spray Ionisation. Parameters for chromatography and mass detection were optimized for nine testosterone esters and evaluated according to selectivity, resolution and intensity. A method that could be used for determination of testosterone esters in serum was found. The MS-method was set and at least three possible transitions for each testosterone ester were found. The best choice of column proved to be the C18 column where all the esters were separated and seven of them were base-line separated. The C18 column along with methanol and ammonium acetate buffer, 5 mM, pH 5 showed the highest sensitivity for Multiple Reaction Monitoring-detection. A gradient profile for a total runtime of 5.6 minutes was established. Two alternative extraction procedures were tested, with tert-butylmethylether or diethyl ether/ethyl acetate and both seemed to work satisfactory. Analysis of serum proved to work well and no severe interference occurred. Results from the linearity tests indicate that future quantification method in serum will be possible.

Magnetic resonance mammography (MRM) is a promising technique, since it provides high resolution breast imaging with no use of ionising radiation and with inherently good soft tissue discrimination. The addition of dynamic contrast enhancement kinetics of the breast upgraded the method to a great extend, due to highly differentiated malignant vs. benign lesion hemodynamics resulting from the angiogenetic properties of cancerous cells. Straightforward pharmacokinetic analysis, such as the 3TP algorithm, has been implemented in commercially available CAD systems. Quantitative parameters can be extracted that directly correspond to different aspects of the underlying pathology and can be compared to biopsy results. However, there is a general understanding that straightforward pharmacokinetic analysis (3TP model) requires a very demanding imaging protocol in order to be able to measure such parameters accurately. Fitting the experimental data of the dynamic series to simple mathematical models extracting quantitative features provides a means to evaluate and to shrink the big amount of data of the study to one set of images, in order make the diagnostic process faster and more robust. That could facilitate the clinical routine. The dynamic series of the MRM examinations of the 55 patients were analyzed in this study. Radiologists specialized in MRM have identified and characterized all suspicious lesions according to BIRADS lexicon. Dynamic data were fitted pixel-wise to a simple bilinear model to extract washout, time to peak and washin parameters. Subsequently, those parameters were mapped to Hue, Saturation and Value, respectively, of an HSV color model, which was utilized for characterizing the lesions. Also, Hue heterogeneity was qualitatively assessed for the characterization of lesions. In addition, observers evaluated the haemodynamic properties of the lesions with the conventional hand-drawn ROI based technique (Kuhl system). The results of the two methods were then compared to the histological ground truth to derive their classification performance. Classification performance for the proposed and the conventional one was Az=0.880.05 and Az=0.860.05, respectively, by means of ROC analysis. Results indicate no statistically different performance between the two methods, with the proposed one offering time savings and reproducibility. / Η μαγνητική τομογραφία μαστών (MRM) είναι μια πολλά υποσχόμενη τεχνική, αφού προσφέρει απεικόνιση των μαστών με υψηλή διακριτική ικανότητα αλλά και εγγενή ικανότητα διάκρισης διαφόρων τύπων μαλακών ιστών, χωρίς τη χρήση ιοντίζουσας ακτινοβολίας. Η προσθήκη δυναμικής απεικόνισης των ιστών του μαστού με τη χρήση παραμαγνητικής σκιαγραφικής ουσίας στο εξεταστικό πρωτόκολλο ισχυροποίησε τη μέθοδο αφού υπάρχει σημαντική διαφοροποίηση στην αιμοδυναμική συμπεριφορά ανάμεσα σε καλοήθεις και κακοήθεις παθήσεις λόγω του αγγειακού δικτύου που δημιουργούν οι μεταβολικές ανάγκες των καρκινικών κυττάρων. Υπολογιστικά συστήματα αυτόματης διάγνωσης που διατίθενται εμπορικά, πραγματοποιούν φαρμοκοκινητική ανάλυση της δυναμικής συμπεριφοράς των ιστών του μαστού με χρήση μαθηματικών αλγορίθμων όπως ο αλγόριθμος τριών χρονικών σημείων (3TP). Από τέτοιες αναλύσεις εξάγονται ποσοτικές παράμετροι που έχουν ευθεία συσχέτιση με διάφορα χαρακτηριστικά της υποκείμενης παθολογίας και μπορούν να συγκριθούν με τα αποτελέσματα ιστολογικών μελετών. Παρόλα αυτά είναι γενικά αποδεκτό ότι για να επιτευχθεί ακριβής υπολογισμός των παραμέτρων αυτών απαιτείται η επιλογή ειδικού εξεταστικού πρωτοκόλλου με μεγάλες απαιτήσεις σε χρονική διακριτική ικανότητα. Η επιλογή απλών μαθηματικών μοντέλων για τον υπολογισμό ποσοτικών παραμέτρων με έμμεση συνάφεια με την παθολογία μας δίνει τη δυνατότητα να ελαχιστοποιήσουμε τον όγκο δεδομένων που παρέχει η εξέταση σε ένα σετ εικόνων και να κάνουμε τη διαδικασία της διάγνωσης πιο γρήγορη και ασφαλή από ότι παρουσιάζεται σήμερα στην κλινική ρουτίνα. Η σειρά των εικόνων της δυναμικής μελέτης των εξετάσεων μαγνητικής τομογραφίας μαστών 55 ασθενών αναλύθηκαν για αυτή τη μελέτη. Ακτινολόγοι με εξειδίκευση στην εξέταση κατέταξαν όλες τις ανιχνευθείσες παθολογικές περιοχές κατά BIRADS. Έγινε προσέγγιση των πειραματικών τιμών των pixels των δυναμικών σειρών με ένα απλό διγραμμικό μοντέλο και εξάχθηκαν χάρτες ποσοτικών παραμέτρων έκπλυσης σήματος (washout), χρόνου μέγιστης ενίσχυσης (time to peak) και ενίσχυσης (washin). Στη συνέχεια αυτές οι παράμετροι χρησιμοποιήθηκαν σαν απόχρωση (Hue), κορεσμό (Saturation) και ένταση (Value) της χρωματικής κλίμακας HSV. Με αυτή την αντιστοίχηση δημιουργήθηκαν χάρτες λειτουργικής απεικόνισης οι οποίοι και χρησιμοποιήθηκαν για το χαρακτηρισμό της παθολογίας. Για τον τελικό χαρακτηρισμό εκτιμήθηκε ποιοτικά και η ανομοιογένεια των λειτουργικών χαρτών ως προς την απόχρωση. Επιπλέον η αιμοδυναμική συμπεριφορά των ευρημάτων εκτιμήθηκε με τη συμβατική μέθοδο περιγραφής περιοχής ενδιαφέροντος με το χέρι και εξαγωγής καμπύλης σήματος – χρόνου από αυτήν την περιοχή (μέθοδος Kuhl). Τα αποτελέσματα των δυο μεθόδων συγκρίθηκαν με τα αποτελέσματα της ιστολογικής εξέτασης των ελεγχθεισών παθολογιών και υπολογίστηκε η απόδοση της κάθε μεθόδου. Αυτή βρέθηκε Az=0.880.05 για την προτεινόμενη μέθοδο και Az=0.860.05 για την κλασική, με χρήση ανάλυσης ROC. Τα αποτελέσματα αυτά υποδεικνύουν ότι δεν υπάρχει στατιστικώς σημαντική διαφορά μεταξύ των δυο μεθόδων, με την προτεινόμενη να παρουσιάζει κέρδος χρόνου και αυξημένη επαναληψιμότητα.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Apolipoprotein M (apoM) is necessary for the formation of lipid-poor preβ-HDL particles, the initial precursor of HDL and acceptors of cholesterol efflux from peripheral cells. An assay to quantify apoM in serum is not widely-available, hampering the efforts to further understand apoM and to develop therapeutic methods to increase circulating levels of apoM. An antibody-free, high throughput mass spectrometry (MS)-based assay was developed to quantitatively measure apoM from a variety of species including human, mouse, and rat. Apolipoproteins were enriched by selectively binding to Liposorb, an affinity resin, followed by enzymatic digestion. This peptide mixture was separated by HPLC coupled in-line with tandem MS/ MS. Signal intensities from the MS/ MS fragmentation of apoM-specific peptides were measured simultaneously in a targeted method spanning many commonly used species. The same amount of purified human apolipoprotein A-IV uniformly labeled with 15N was spiked into all samples and was used as an internal standard to correct for any variation in sample handling and recovery. Assay variability and accuracy was statistically validated in a three-day spike recovery experiment to determine the working range of the assay. The concentration range for quantification of apoM using this assay was 11.2-500 nM, whereas average concentration of human apoM measured from a large sampling (n>100) was 370 nM. This assay was used to measure changes in apoM in mouse serum from a pre-clinical study that was designed to evaluate the effects of a microsomal triglyceride transfer protein (MTTP) inhibitor. All measured lipoproteins and apolipoproteins showed a dose-dependent decrease in concentration and the response of apoM closely followed the response of HDL. In a clinical application of the assay, apoM was measured in human serum to evaluate the effects of two cholesterol-lowering compounds, a statin drug and an experimental PPAR-α agonist. ApoM levels did not change with PPAR-α agonist or combination treatments, but significantly decreased with atorvastatin. The measurement of apoM provided additional information on the effects of these drug treatments that previously could not be measured. The availability of a quantitative assay for apoM provides a valuable tool in the development of cardio-protective therapeutics and understanding the mechanisms of these drugs. / Monarch LifeSciences, Eli Lilly and Company

As ectothermic marine megafauna, sea turtle physiology and ecology are tightly intertwined with temperature, seasonality, and oceanography. Identifying how turtles respond when exposed to cold water, how they adapt to cold environments when they need to explore cold environments in order to forage, and what foraging resources are exploited by sea turtles are all components central to their conservation. Cold-stunning is a well-documented phenomenon that occurs when water induced decreases in sea turtle body temperature cause turtles to become immobilized and wash ashore. While most cold-stunned turtles are rescued and rehabilitated, we do not know whether cold-stunning is an acute transient occurrence, or a symptom of a bigger environmental problem. Further, while in some environments avoiding cold water is preferential, in other habitats, sea turtles need to inhabit cold environments in order to forage. Along the Eastern Pacific Rim, discrete upwelling locations are characterized by high primary productivity and unusually cold water. In these environments, avoidance is not possible and sea turtles require physiological adaptions to mitigate body temperature decreases in cold water. Little is known about how turtles handle upwelling environments, despite the fact that sea turtles remain in these habitats regardless of water temperature fluctuations. Because upwelling habitats provide increased nutrient presence, and sea turtles are opportunistic foragers, quantification of diet composition will further our understanding of why sea turtles remain in cold water environments year-round. Diet composition in multiple populations of cohabitating sea turtles revealed partitioning that results in reduced inter-specific competition. Further, flexibility in diets provides a wide range of ecosystem services central to habitat resiliency. Therefore, conservation of endangered sea turtles requires complete ecosystem conservation, and complete understanding of the interconnectivity of sea turtles and their environments is crucial.<br>

O aproveitamento de proteína de pescado como coprodutos do processo de industrialização, constitui-se em uma alternativa para a elaboração de produtos com elevada qualidade nutricional. A carne mecanicamente separada (CMS), produzida a partir de carcaças do descarte do processamento de tilápia híbrida vermelha (CMSV), Oreochromis niloticus var. Red Stirling e de tilápia preta (CMSP), Oreochromis niloticus, foram caracterizadas física, química, microbiológica e sensorialmente. Em um primeiro estudo avaliou-se a eficiência de dois tipos de equipamentos de extração mecânica da CMS, rosca- sem-fim e cinta-tambor. Posteriormente, avaliou-se a qualidade da CMS congelada durante 90 dias de armazenamento. Em seguida avaliou-se os atributos sensoriais de cor, odor e aparência da CMSV e CMSP congelada. Para a avaliação do sabor da CMS, foi adicionado ao molho de tomate com CMSV armazenada por 15 e 90 dias e a aceitação avaliada por consumidores. Foi realizado um estudo para avaliação das características de funcionalidade da CMS reestruturada da tilápia híbrida vermelha. O separador tipo rosca-sem-fim apresentou melhor rendimento de extração e controle da temperatura do processo, quando comparado ao separador do tipo cinta-tambor, embora ambos os processos tenham permitido a obtenção de CMS com teores de umidade, proteína e lipídeo adequados para o processamento de coprodutos. A avaliação da vida útil indicou que coprodutos da tilápia híbrida vermelha (CMSV) e tilápia preta (CMSP) apresentaram padrões microbiológicos, valores de pH e oxidação lipídica aceitáveis ao longo do estudo, mesmo apresentado uma descoloração ao longo do armazenamento. A avaliação sensorial para o estabelecimento da vida útil (teste de diferença do controle com provador treinado) foi realizada em períodos até 90 dias. Aos 15 dias de armazenamento, os provadores detectaram diferença significativa, porém o limite sensorial estabelecido pelos provadores (moderadamente diferente do controle) foi atingido aos 60 dias para a CMS da tilápia preta e aos 90 dias para a CMS de tilápia vermelha. A aceitação teve boa resposta, porém a intenção de compra apresentou queda de 19% para o molho adicionado de CMS estocada durante 90 dias. A CMSV é uma matéria prima, com elevado teor proteico (12%) e elevado teor lipídico (15%) e pode ser utilizada para elaboração de diferentes coprodutos. Foi possível obter produto reestruturado com boa funcionalidade tecnológica com adição de 100% de CMS de tilápia híbrida vermelha, o que demonstra a viabilidade de sua utilização, onde apresentaram respostas satisfatórias de rendimento, capacidade de retenção de água e força de penetração no reestruturado. Conclui-se que a CMS das variedades de tilápia estudadas representam um coproduto de qualidade a ser aproveitado pela indústria. Além de contribuir para para fornecer dados, junto a outros trabalhos, para se criar um padrão de identidade e qualidade da CMS de pescado, o que é de suma importância para o controle de qualidade da indústria processadora do coproduto. / The use of fish protein resulting from the industrial process constitutes an alternative to the development of co-products with nutritional quality. Mechanically recovered meat (MRM), produced from carcasses of Red hybrid tilapia, Oreochromis niloticus var. Red Stirling and black tilapia Oreochromis niloticus were characterized in terms of physical, chemical, microbiological and sensory aspects. The first study evaluated the efficiency of two mechanical extraction devices of MRM, the screw type and the belt and drum type. Later assessed the quality of frozen MRM for 90 days of storage. Then it was evaluated the sensory attributes of color, odor and appearance of defrozen MRM. The resulting MRM was used in the development of a tomato sauce and its acceptance with consumers was analyzed. A performance study of MRM restructured of red hybrid tilapia was conducted. The worm screw-type separator presented better extraction yield when compared to belt-drum type separator, but both processes allowed obtaining MRM with levels of moisture, protein and lipids suitable for processing co-products. Both MRM presented microbiological and oxidative stability after 90 days of storage. The sensory analysis of control difference for up to 90 days showed significant difference to that at 15 days of storage. The team of tasters set the value 4 (appearance, color and odor moderately different from the control) as limiting MRM shelf life to 60 days for MRM of the Nile tilapia and 90 days for MRM of the red tilapia. The tomato sauce with the addition of tilapia formulated with MRM of the red tilapia stored for 15 and 90 days was well accepted by consumers, but purchase intent dropped 19% for the product obtained from MRM with 90 days of storage. The MRM from the red hybrid tilapia is a raw material with high protein content, average 12%, and high lipid content, average 15%, and can be used to prepare different co-products of tilapia. It was possible to obtain restructured product with addition of 100% of MRM from the red hybrid tilapia with good processing characteristics, which shows viability of production with satisfactory responses of performance, water retention capacity and penetration force of restructured fish meat. We conclude that the CMS of the studied tilapia varieties represent a quality co-product to be used by the industry. Besides contributing to to provide data , along with other works , to create a standard of identity and quality of fish CMS , which is of paramount importance for the quality control of the co-product processing industry.

O aproveitamento de proteína de pescado como coprodutos do processo de industrialização, constitui-se em uma alternativa para a elaboração de produtos com elevada qualidade nutricional. A carne mecanicamente separada (CMS), produzida a partir de carcaças do descarte do processamento de tilápia híbrida vermelha (CMSV), Oreochromis niloticus var. Red Stirling e de tilápia preta (CMSP), Oreochromis niloticus, foram caracterizadas física, química, microbiológica e sensorialmente. Em um primeiro estudo avaliou-se a eficiência de dois tipos de equipamentos de extração mecânica da CMS, rosca- sem-fim e cinta-tambor. Posteriormente, avaliou-se a qualidade da CMS congelada durante 90 dias de armazenamento. Em seguida avaliou-se os atributos sensoriais de cor, odor e aparência da CMSV e CMSP congelada. Para a avaliação do sabor da CMS, foi adicionado ao molho de tomate com CMSV armazenada por 15 e 90 dias e a aceitação avaliada por consumidores. Foi realizado um estudo para avaliação das características de funcionalidade da CMS reestruturada da tilápia híbrida vermelha. O separador tipo rosca-sem-fim apresentou melhor rendimento de extração e controle da temperatura do processo, quando comparado ao separador do tipo cinta-tambor, embora ambos os processos tenham permitido a obtenção de CMS com teores de umidade, proteína e lipídeo adequados para o processamento de coprodutos. A avaliação da vida útil indicou que coprodutos da tilápia híbrida vermelha (CMSV) e tilápia preta (CMSP) apresentaram padrões microbiológicos, valores de pH e oxidação lipídica aceitáveis ao longo do estudo, mesmo apresentado uma descoloração ao longo do armazenamento. A avaliação sensorial para o estabelecimento da vida útil (teste de diferença do controle com provador treinado) foi realizada em períodos até 90 dias. Aos 15 dias de armazenamento, os provadores detectaram diferença significativa, porém o limite sensorial estabelecido pelos provadores (moderadamente diferente do controle) foi atingido aos 60 dias para a CMS da tilápia preta e aos 90 dias para a CMS de tilápia vermelha. A aceitação teve boa resposta, porém a intenção de compra apresentou queda de 19% para o molho adicionado de CMS estocada durante 90 dias. A CMSV é uma matéria prima, com elevado teor proteico (12%) e elevado teor lipídico (15%) e pode ser utilizada para elaboração de diferentes coprodutos. Foi possível obter produto reestruturado com boa funcionalidade tecnológica com adição de 100% de CMS de tilápia híbrida vermelha, o que demonstra a viabilidade de sua utilização, onde apresentaram respostas satisfatórias de rendimento, capacidade de retenção de água e força de penetração no reestruturado. Conclui-se que a CMS das variedades de tilápia estudadas representam um coproduto de qualidade a ser aproveitado pela indústria. Além de contribuir para para fornecer dados, junto a outros trabalhos, para se criar um padrão de identidade e qualidade da CMS de pescado, o que é de suma importância para o controle de qualidade da indústria processadora do coproduto. / The use of fish protein resulting from the industrial process constitutes an alternative to the development of co-products with nutritional quality. Mechanically recovered meat (MRM), produced from carcasses of Red hybrid tilapia, Oreochromis niloticus var. Red Stirling and black tilapia Oreochromis niloticus were characterized in terms of physical, chemical, microbiological and sensory aspects. The first study evaluated the efficiency of two mechanical extraction devices of MRM, the screw type and the belt and drum type. Later assessed the quality of frozen MRM for 90 days of storage. Then it was evaluated the sensory attributes of color, odor and appearance of defrozen MRM. The resulting MRM was used in the development of a tomato sauce and its acceptance with consumers was analyzed. A performance study of MRM restructured of red hybrid tilapia was conducted. The worm screw-type separator presented better extraction yield when compared to belt-drum type separator, but both processes allowed obtaining MRM with levels of moisture, protein and lipids suitable for processing co-products. Both MRM presented microbiological and oxidative stability after 90 days of storage. The sensory analysis of control difference for up to 90 days showed significant difference to that at 15 days of storage. The team of tasters set the value 4 (appearance, color and odor moderately different from the control) as limiting MRM shelf life to 60 days for MRM of the Nile tilapia and 90 days for MRM of the red tilapia. The tomato sauce with the addition of tilapia formulated with MRM of the red tilapia stored for 15 and 90 days was well accepted by consumers, but purchase intent dropped 19% for the product obtained from MRM with 90 days of storage. The MRM from the red hybrid tilapia is a raw material with high protein content, average 12%, and high lipid content, average 15%, and can be used to prepare different co-products of tilapia. It was possible to obtain restructured product with addition of 100% of MRM from the red hybrid tilapia with good processing characteristics, which shows viability of production with satisfactory responses of performance, water retention capacity and penetration force of restructured fish meat. We conclude that the CMS of the studied tilapia varieties represent a quality co-product to be used by the industry. Besides contributing to to provide data , along with other works , to create a standard of identity and quality of fish CMS , which is of paramount importance for the quality control of the co-product processing industry.

<div> <div> <div> <p>The exploratory profiling and quantitative bioassays of lipids, small metabolites, and peptides have always been challenging tasks. The most popular instrument platform deployed to solve these problems is chromatography coupled with mass spectrometry. However, it requires large amounts of instrument time, intensive labor, and frequent maintenance, and usually produces results with bias. Thus, the pace of exploratory research is one of poor efficacy and low throughput. The work in this dissertation provides two practical tactics to address these problems. The first solution is multiple reaction monitoring profiling (MRM-profiling), a new concept intended to shift the exploratory research from current identification-centered metabolomics and lipidomics to functional group screening by taking advantage of precursor ion scan and product ion scan. It is also demonstrated that MRM-profiling is capable of quantifying the relative amount of lipids within the same subclass. Besides, an application of the whole workflow to investigate the strain-level differences of bacteria is described. The results have zeroed in on several potential lipid biomarkers and corresponding MRM transitions. The second strategy is aimed to increase the throughput of targeted bioassays by conducting induced nanoelectrospray ionization (nESI) in batch mode. A novel prototype instrument named "Dip-and-Go" system is presented. Characterization of its ability to carry out reaction screening and bioassays exhibits the versatility of the system. The distinct electrophoretic cleaning mechanism contributes to the removal of salt during ionization, which assures the accuracy of measurement.</p></div></div></div>

Current screening tests for type 2 diabetes mellitus (T2DM) identify less than 50% of undiagnosed T2DM patients and provide no information about how the disease will develop in prediabetic patients. Here, twenty-nine protein glycation sites were quantified after tryptic digestion of plasma samples at the peptide level using tandem mass spectrometry and isotope-labelled peptides as internal standard. The glycation degrees were determined in three groups, i.e., 48 patients with a duration of T2DM exceeding ten years, 48 non-diabetic individuals matched for gender, BMI, and age, and 20 prediabetic men. In long-term controlled diabetic patients, 27 glycated peptides were detected at significantly higher levels, providing moderate diagnostic accuracies (ACCs) from 61 to 79%, allowing a subgrouping of patients in three distinct clusters. Moreover, a feature set of one glycated peptides and six established clinical parameters provided an ACC of 95%. The same number of clusters was identified in prediabetic males (ACC of 95%) using a set of eight glycation sites (mostly from serum albumin). All patients present in one cluster showed progression of prediabetic state or advanced towards diabetes in the following five years. Overall, the studied glycation sites appear to be promising biomarkers for subgrouping prediabetic patients to estimate their risk for the development of T2DM.

No / Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.