Configuration, optimization and evaluation of a novel instrumental platform for automated SPE-LC-MS/MS analysis of drugs in whole blood

The thesis describes the configuration, optimization and evaluation of a novel instrumental platform for fully automated SPE-LC-MS/MS analysis of small molecules, such as drugs, in whole blood. The immunosuppressant Cyclosporine A was chosen as a model analyte, as this drug is predominantly bound to erythrocytes. First, anticoagulated blood is converted into so-called Cell-Disintegrated Blood (CDB) by heat-shock or cryogenic treatment. CDB represents a homogenous blood sample and consists of subcellular particles which do not sediment on standing and do not clog capillaries, sieves or HPLC column packings. For in-line treatment of anticoagulated whole blood, i.e. generation of CDB, a sample mixing unit, two special liquid handling units and two home-made sample processing modules were embedded into a XYZ-autosampler. The module for heat-shock treatment consists of a stainless-steel capillary jacketed with a heating sleeve. Under optimal conditions for sampling and in-line processing of 20 µL of whole blood, it takes 13 seconds at 75 °C to generate CDB. The latter is stored in a holding loop before further treatment. For cryogenic treatment of a blood sample, a stainless-steel processing needle with a large inner diameter was installed in one of the liquid handling units. The autosampler was programmed to introduce the processing needle containing the blood sample (40 µL) into a stand-pipe, which is located in a thermo-flask filled with liquid nitrogen. The processing needle therefore contacts liquid nitrogen and the blood sample is snap-frozen. Optimal conditions were found to be 10 seconds for snap-freezing at -196 °C and 60 seconds for thawing at room temperature. A CDB sample obtained either by heat-shock or cryogenic treatment is further processed by being pumped via a switching-valve through an in-line filter to retain cell nuclei and “cell debris”. It was found that a depth filter packed with spherical hydrophilic silica is optimal. This filter allows at least 200 analysis cycles before it has to be replaced. Next, the CDB sample is pumped on-line via another switching-valve through a SPE column (50 x 0.5 mm ID) at a high flow rate. Due to the special packing material and the very small inner diameter, a high linear flow velocity is achieved and turbulent flow is generated. By this, high-molecular matrix components such as proteins are eluted in the void volume to waste. The low-molecular weight target analyte Cyclosporine A and the Internal Standard Cyclosporine D are retained and extracted by reversed phase partitioning chromatography (RPC). After fractionation of CDB on the SPE column, the analyte and the IS are transferred to a series-connected analytical column and separated from residual matrix components by RPC. Finally, the analyte is detected by a tandem mass spectrometer applying electrospray ionization (ESI) and multiple reaction monitoring (MRM). The optimized method has a total analysis time of less than 11 minutes. The analytical procedure and the instrumental platform were validated for heat-shock treated blood samples with respect to linearity, range (10 - 1000 ng/mL), lower limit of quantitation (10 ng/mL), intra-day and inter-day accuracy and precision, as well as matrix-independent and matrix-dependent recovery (around 100 %). It was shown that the electrospray induced ionization is suppressed by approximately 25 %. These matrix effects, however, can be totally compensated for by the addition of an Internal Standard, i.e. Cyclosporine D. A comparison with a semi-automated SPE-LC-MS/MS method, established in the Institute, revealed a very good agreement. This was shown by Passing and Bablok plots. The robustness of the fully automated SPE-LC-MS/MS analysis platform was monitored during 500 consecutive analysis cycles with heat-shock treated blood samples. The relative standard deviation for the signal response was 15.6 % for Cyclosporine A and 15.2 % for Cyclosporine D. The back pressure of the total system rose only by 52 bar. These findings show that, despite its instrumental and chromatographic complexity, the described analysis platform fulfills the prerequisites to be used in routine clinical-chemical analysis.Die Doktorarbeit beschreibt die Konfiguration, Optimierung und Evaluierung einer neuartigen instrumentellen Plattform für die vollständig automatisierte SPE-LC-MS/MS Analyse von kleinen Molekülen, wie beispielsweise Arzneistoffe, im Vollblut. Das Immunsuppressivum Cyclosporin A wurde als Modellanalyt gewählt, da dieser Arzneistoff vorwiegend an Erythrozyten gebunden ist. Zunächst wird antikoaguliertes Blut durch eine Hitze- oder Kälteschock Behandlung in sogenanntes Zell-desintegriertes Blut (Cell-Disintegrated Blood, CDB) überführt. CDB stellt eine homogene Blutprobe dar und besteht aus subzellulären Partikel, die beim Stehen nicht sedimentieren und keine Kapillaren, Siebe und HPLC- Packungsmaterialien verstopfen. Für die in-line Behandlung von antikoagulierten Vollblut, d.h. für die Herstellung von CDB, wurde ein Gerät zum Mischen der Probe, zwei spezielle Bauteile für die Handhabung von Flüssigkeiten und zwei selbst-gebaute Module für die Probenprozessierung in einen XYZ-Probengeber eingebaut. Das Modul für die Hitze-Schock Behandlung besteht aus einer Edelstahlkapillare, die mit einer Heizmanschette ummantelt ist. Unter optimalen Bedingungen für die Probenahme und in-line Prozessierung von 20 µL Vollblut werden 13 Sekunden und 75 °C benötigt um CDB herzustellen. Letzteres wird vor einer weiteren Behandlung in einer Rückhalteschleife gelagert. Für die Tieftemperatur Behandlung einer Blutprobe wurde eine weitlumige Edelstahlnadel zur Prozessierung in eines der Bauteile für die Handhabung von Flüssigkeiten eingebaut. Der Probengeber wurde so programmiert, dass die Nadel, welche die Blutprobe (40 µL) enthält, in ein Steigrohr, welches sich in einem mit flüssigem Stickstoff gefüllten Isolierbehälter befindet, eingeführt wird. Hierdurch wird die Nadel mit flüssigem Stickstoff kontaktiert und die Blutprobe schockgefroren. Als optimale Bedingungen wurden 10 Sekunden für das Schockgefrieren bei -196 °C und 60 Sekunden für das Auftauen bei Raumtemperatur gefunden. Eine CDB Probe, die entweder durch Hitze- oder Kälteschock-Behandlung gewonnen wurde, wird weiter prozessiert, indem sie über ein Schaltventil durch einen in-line Filter gepumpt wird, um Zellkerne und „Zellbruchstücke“ zurückzuhalten. Es stellte sich heraus, dass ein Tiefenfilter, der mit sphärischem hydrophilem Kieselgel gepackt ist, optimal ist. Dieser Filter ermöglicht mindestens 200 Analysen-Zyklen bevor er ausgetauscht werden muss. In einem weiteren Schritt wird die CDB Probe on-line über ein weiteres Schaltventil mit einer hohen Flussrate durch eine SPE Säule (50 x 0.5 mm ID) gepumpt. Aufgrund des speziellen Packungsmaterials und dem sehr kleinen Innendurchmesser wird eine hohe lineare Flussgeschwindigkeit erreicht und eine turbulente Strömung erzeugt. Hierdurch werden hochmolekulare Matrixkomponenten wie beispielsweise Proteine im Totvolumen in den Abfall eluiert. Niedermolekulare Zielanalyte wie Cyclosporin A und der interne Standard Cyclosporin D werden über Umkehrphasen- Verteilungschromatographie (RPC) reteniert und extrahiert. Nach der Fraktionierung von CDB auf der SPE Säule, wird der Analyt und der interne Standard auf eine in Serie geschaltete analytische Säule überführt und von restlichen Matrixbestandteilen über RPC abgetrennt. Zum Schluss wird der Analyt in einem Tandem-Massenspektrometer über eine Elektrospray Ionisation (ESI) und Multiple Reaction Monitoring (MRM) detektiert. Die optimierte Methode weist eine Gesamtanalysezeit von weniger als 11 Minuten auf. Das Analysenverfahren und die instrumentelle Plattform wurden für Hitzeschock behandelte Blutproben hinsichtlich Linearität, Messbereich (10 – 1000 ng/mL), unterer Bestimmungsgrenze (10 ng/mL), Richtigkeit und Präzision innerhalb eines Tages und von Tag zu Tag, sowie Matrix-unabhängiger und Matrix-abhängiger Wiederfindung (um 100 %) validiert. Es konnte gezeigt werden, dass die über Elektrospray induzierte Ionisation um ca. 25 % unterdrückt wird. Diese Matrixeffekte können jedoch durch Zugabe des internen Standards Cyclosporin D vollständig kompensiert werden. Ein Vergleich mit einer teilautomatisierten SPE-LC-MS/MS Routinemethode, die im Institut etabliert ist, ergab eine sehr gute Übereinstimmung. Dies konnte anhand von Passing und Bablok Plots aufgezeigt werden. Die Robustheit der vollständig automatisierten SPE-LC-MS/MS Analysenplattform wurde während 500 aufeinander folgenden Analysezyklen mit Hitzeschock behandelten Blutproben überprüft. Die relative Standardabweichung für das MS-signal betrug 15.6 % für Cyclosporin A und 15.2 % für Cyclosporin D. Der Rückdruck des gesamten Systems stieg nur um 52 bar an. Diese Ergebnisse zeigen, dass – trotz der instrumentellen und chromatographischen Komplexität – die beschriebene Analysenplattform die Anforderungen, die in der klinisch-chemischen Routineanalytik gestellt werden, erfüllt

Standing Out from the Crowd: The Real Effects of Outliers

We study the impact of outlier opinions – extreme views voiced by individuals – in financial markets. Using analyst forecasts as a laboratory, we show that market participants respond to the arrival of extremely optimistic forecasts, instead of ignoring them as noise. An outlier forecast subsequently moves group consensus and begets more extreme forecasts by peers. Outlier forecasts also generate stronger market reactions from investors, more media coverage, and more conservative management guidance. Further analyses reveal that issuing outlier forecasts increases an analyst’s chance to cover more important clients of his employer. Outlier forecasts are also more likely to take place when an analyst’s reputation cost is lower and information uncertainty is high. These findings suggest that the propensity for expressing extreme views is situational and that personal incentives are the likely cause at play

Three Essays in Entrepreneurial and Corporate Finance

Thesis advisor: Thomas J. ChemmanurMy dissertation is comprised of three chapters. In the first chapter, I analyze the effect of top management changes on subsequent corporate innovation in venture capital-backed private firms using a hand-collected dataset. I find that top management changes are associated with significantly more and higher quality corporate innovation (as measured by their patenting activity). I show that top management changes are likely to be venture-driven and that the effect of top management changes on corporate innovation is stronger for firms where venture capitalists have greater power. An instrumental variable analysis using an exogenous shock to the supply of outside managers available for hire implies a causal effect of top management changes on corporate innovation. I establish that one mechanism through which top management changes enhance corporate innovation is through new management teams hiring more inventors for a given investment size. I also show that both top management changes and corporate innovation have a positive impact on firms' successful exits. In the second chapter, co-authored with Thomas Chemmanur and Karthik Krishnan, we hypothesize that VC-backing garners greater “investor attention” (Merton (1987)) for IPOs, allowing IPO underwriters to perform two information-related roles more efficiently during the book-building and road-show process: information dissemination, where the lead underwriter disseminates noisy information about various aspects of the IPO firm to institutional investors; and information extraction, where the lead underwriter extracts information useful in pricing the IPO firm equity from institutional investors. Using pre-IPO media coverage as a proxy, we show empirically that VC-backed firm IPOs indeed obtain greater investor attention, causally yielding them more favorable IPO characteristics such as higher IPO and secondary market valuations. In the third chapter, co-authored with Thomas Chemmanur, Lei Kong, and Karthik Krishnan, using panel data on top management characteristics and a management quality factor constructed using common factor analysis on individual management quality proxies, we analyze the relation between the human capital or “quality” of firm management and its innovation inputs and outputs. We control for the endogenous matching between firm and management quality using a plausibly exogenous shock to the supply of new managers as an instrument, thereby finding a causal relationship between management quality and innovation activities. We show that higher management quality firms achieve greater innovation output by hiring more and higher quality inventors.Thesis (PhD) — Boston College, 2017.Submitted to: Boston College. Carroll School of Management.Discipline: Finance

Association between statin use and acute pulmonary embolism in intensive care unit patients with sepsis: a retrospective cohort study

IntroductionAcute pulmonary embolism (APE) is a life-threatening medical condition that is frequently encountered and associated with significant incidence and mortality rates, posing a substantial threat to patients’ well-being and quality of life. Sepsis is prominent independent risk factor for the development of APE. Despite recent investigations indicating a reduced APE risk through statin therapy, its impact on patients with sepsis and APE remains unresolved.MethodsThe Medical Information Mart for Intensive Care (MIMIC)-IV database was utilized to identify patients diagnosed with sepsis and APE, irrespective of statin treatment status, as part of this study. The primary study aim was to assess the risk of APE, which was analyzed using multivariate logistic regression models.ResultsThe study encompassed a total of 16,633 participants, with an average age of 64.8 ± 16.2 years. Multivariate logistic regression revealed that septic patients receiving statin therapy in the intensive care unit (ICU) exhibited a 33% reduction in the risk of developing APE (OR = 0.67, 95% CI: 0.52–0.86, p < 0.001). The findings of further analyses, including stratification based on statin usage, dosage, and propensity score matching, consistently reinforced the hypothesis that administering statins to patients with sepsis effectively mitigates their potential APE risk.DiscussionThe results of the study provide compelling evidence in favor of administering statins to septic patients as a prophylactic measure against APE, given that statins may reduce the risk of developing APE, and their anti-APE effect appears to be dose-dependent. Nonetheless, future randomized controlled trials are needed to validate these results