Eliminating Preparation of Multisample External Calibration Curves and Dilution of Study Samples Using the Multiple Isotopologue Reaction Monitoring (MIRM) Technique in Quantitative LC-MS/MS Bioanalysis.

Preparation of multisample external calibration curves and dilution of study samples are critical steps in bioanalytical sample processing for quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) based bioanalysis of small-molecule compounds, biotherapeutics, and biomarkers, but they can be time-consuming and prone to error. It is highly desired to simplify or eliminate these two steps in order to improve the assay throughput and robustness. While multisample external calibration curve preparation using authentic matrices can be eliminated with a previously reported in-sample calibration curve (ISCC) approach using multiple isotopologue reaction monitoring (MIRM) of a stable isotopically labeled (SIL) analyte, dilution of study samples is still inevitable due to limited LC-MS/MS assay ranges. In this work, a one-sample multipoint external calibration curve and isotope sample dilution, both using MIRM of an analyte, for quantitative LC-MS/MS based bioanalysis are proposed and demonstrated. By spiking a known amount of an analyte into one blank authentic matrix sample, a one-sample multipoint external calibration curve in an authentic matrix can be established on the basis of the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) and the MS/MS responses in the corresponding MIRM channels. This one-sample multipoint external calibration curve can be used in the same way as the traditional multisample external calibration curve for quantitative LC-MS/MS-based bioanalysis. As isotopic abundance in each MIRM channel can be calculated and measured accurately, isotope sample dilution can be achieved by simply monitoring one or a few of the MIRM channels of the analyte in addition to the most abundant MIRM channel for study samples. While the most abundant MIRM channel (isotopic abundance of 100%) is used for the quantitation of samples having concentrations within the assay calibration curve range, less abundant MIRM channels (isotopic abundance of IA%) can be used for the quantitation of samples having concentrations beyond the assay upper limit of quantitation (ULOQ), resulting in isotope dilution factors (IDF) of 100%/IA%. The approaches of one-sample multipoint external calibration curve and isotope sample dilution were evaluated and demonstrated in this work with an example of the quantitation of daclatasvir in human plasma extracted with liquid-liquid extraction. Using these approaches together with the MIRM-ISCC methodology, accurate and reliable LC-MS/MS bioanalysis can be achieved without the need of preparation of multisample external calibration curve and dilution of study samples.

Systematic comparison of post-column isotope dilution using LC-CO-IRMS with qNMR for amino acid purity determination.

Determination of the purity of a substance traceable to the International System of Units (SI) is important for the production of reference materials affording traceability in quantitative measurements. Post-column isotope dilution using liquid chromatography-chemical oxidation-isotope ratio mass spectrometry (ID-LC-CO-IRMS) has previously been suggested as a means to determine the purity of organic compounds; however, the lack of an uncertainty budget has prevented assessment of the utility this approach until now. In this work, the previously published ID-LC-CO-IRMS methods have not only been improved by direct gravimetric determination of the mass flow of 13C-labelled spike but also a comprehensive uncertainty budget has been established. This enabled direct comparison of the well-characterised ID-LC-CO-IRMS method to quantitative nuclear magnetic resonance spectroscopy (qNMR) for purity determination using valine as the model compound. The ID-LC-CO-IRMS and qNMR methods provided results that were in agreement within the associated measurement uncertainty for the purity of a sample of valine of (97.1 ± 4.7)% and (99.64 ± 0.20)%, respectively (expanded uncertainties, k = 2). The magnitude of the measurement uncertainty for ID-LC-CO-IRMS determination of valine purity precludes the use of this method for determination of purity by direct analysis of the main component in the majority of situations; however, a mass balance approach is expected to result in significantly improved measurement uncertainty.

Journal of Clinical Monitoring and Computing 2017 end of year summary: cardiovascular and hemodynamic monitoring.

Hemodynamic monitoring provides the basis for the optimization of cardiovascular dynamics in intensive care medicine and anesthesiology. The Journal of Clinical Monitoring and Computing (JCMC) is an ideal platform to publish research related to hemodynamic monitoring technologies, cardiovascular (patho)physiology, and hemodynamic treatment strategies. In this review, we discuss selected papers published on cardiovascular and hemodynamic monitoring in the JCMC in 2017.

Journal of Clinical Monitoring and Computing 2015 end of year summary: cardiovascular and hemodynamic monitoring.

Hemodynamic monitoring is essential in critically ill patients. In this regard, the Journal of Clinical Monitoring and Computing (JCMC) has become an ideal platform for publishing cardiovascular and hemodynamic monitoring-related research, as reflected by an increasing number of articles related to this topic and published in the recent years. To highlight this new progress, every New Year the journal prints a descriptive review on some important papers published last year in the JCMC and related to blood, cardiovascular function and hemodynamic monitoring.

Influence of lung injury on cardiac output measurement using transpulmonary ultrasound dilution: a validation study in neonatal lambs.

BACKGROUND: Transpulmonary ultrasound dilution (TPUD) is a promising method for cardiac output (CO) measurement in severely ill neonates. The incidence of lung injury in this population is high, which might influence CO measurement using TPUD because of altered lung perfusion. We evaluated the influence of lung injury on the accuracy and precision of CO measurement using TPUD in an animal model. METHODS: In nine neonatal lambs, central venous and arterial catheters were inserted and connected to the TPUD monitor. Repeated lavages with warmed isotonic saline were performed to gradually induce lung injury. CO measurements with TPUD (COtpud) were compared with those obtained by an ultrasonic transit-time flow probe around the main pulmonary artery (COufp). An increase in oxygenation index was used as an indicator of induced lung injury during the experiment. Post-mortem lung injury was confirmed by histopathological examination. RESULTS: Fifty-five sessions of three paired CO measurements were analysed. The mean COufp was 1.53 litre min(-1) (range 0.66-2.35 litre min(-1)), and the mean COtpud was 1.65 litre min(-1) (range 0.78-2.91 litre min(-1)). The mean bias (standard deviation) between the two methods was 0.13 (0.15) litre min(-1) with limits of agreement of ±0.29 litre min(-1). The overall percentage error was 19.1%. The accuracy and precision did not change significantly during progressive lung injury. Histopathological severity scores were consistent with heterogeneous lung injury. The capability to track changes in CO using TPUD was moderate to good. CONCLUSIONS: The accuracy and precision of CO measurement using TPUD is not influenced in the presence of heterogeneous lung injury in an animal model.

Determination of creatinine in human urine with flow injection tandem mass spectrometry.

BACKGROUND/AIMS: Excretion of urinary compounds in spot urine is often estimated relative to creatinine. For the growing number of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays of urine-excreted molecules, a fast and accurate method for determination of creatinine is needed. METHODS: A high-throughput flow injection tandem mass spectrometry method for exact quantitation of creatinine in urine has been developed and validated. Sample preparation used only two-step dilution for protein precipitation and matrix dilution. Flow injection analysis without chromatographic separation allowed for total run times of 1 min per sample. Creatinine concentrations were quantitated using stable isotope dilution tandem mass spectrometry. Selectivity and coelution-free quantitation were assured by qualifier ion monitoring. RESULTS: Method validation revealed excellent injection repeatability of 1.0% coefficient of variation (CV), intraday precision of 1.2% CV and interday precision of 2.4% CV. Accuracy determined from standard addition experiments was 106.1 ± 3.8%. The linear calibration range was adapted to physiological creatinine concentrations. Comparison of quantitation results with a routinely used method (Jaffé colorimetric assay) proved high agreement (R(2) = 0.9102). CONCLUSIONS: The new method is a valuable addition to the toolbox of LC-MS/MS laboratories where excretion of urinary compounds is studied. The 'dilute and shoot' approach to isotope dilution tandem mass spectrometry makes the new method highly accurate as well as cost- and time-efficient.

An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites.

BACKGROUND: Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. METHODS: Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. RESULTS: The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11 ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. CONCLUSIONS: This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue.

Increase in efficiency of media utilization for recombinant protein production in Chinese hamster ovary culture through dilution.

Animal cells are extensively used for the large-scale production of recombinant proteins. Processes and genetically engineered cell lines have been developed to enhance longevity of the culture and increase protein productivity. In this study, we tested the effect of diluting a culture of Chinese hamster ovary (CHO) cells with phosphate-buffered saline (PBS) on cell growth and efficiency of media utilization. An immunoglobulin G-expressing CHO cell line was cultured in CD CHO media followed by dilution of the culture with PBS after the end of the exponential phase. A 28% and 61% increase in protein yield per milliliter of media was observed in the diluted culture in the batch and fed-batch mode with glucose and protein hydrolysate feeding, respectively. To aid in analyzing the potential causes of this observed increase, an unstructured mathematical model was constructed using previously reported kinetics to simulate cell growth, nutrient utilization, and protein production. The model predicts an increase in recombinant protein yield per milliliter of media in PBS diluted cultures under both batch and fed-batch conditions, and suggests that this observed increase could at least partly be due to a decrease in inhibitor concentration in the diluted culture.

Validation of the measurement of haemodialysis access flow using a haemoglobin dilution test.

Monitoring of blood flows in arteriovenous fistulae and arteriovenous grafts is recommended to predict access thrombosis. The ultrasound dilution technique (UDT) is the gold standard. We compare a recently described haemoglobin dilution technique (HDT) with the UDT in measurement of vascular access flow. Access blood flow was measured in 67 stable dialysis patients using HDT by bedside Hemocue (Hemocue AB, Ängelholm, Sweden) and laboratory measurement. Access blood flow was then measured by UDT in the same dialysis session. Median flow rate by UDT was 950 ml/min (IQR 490-1,440 ml/min), by Hemocue HDT 935 ml/min (IQR 475-1,395 ml/min, p = 0.534), and by laboratory haemoglobin HDT 920 ml/min (IQR 463-1,378 ml/min). Bland-Altman plots demonstrated poor agreement between UDT and HDT (limits of agreement for Hemocue HDT -22.7 to 20.1%, for laboratory HDT -21.2 to 20.4%). HDT can be used to measure vascular access flow but requires validation against clinical outcomes before being recommended as an alternative to UDT.

Validation of cardiac output measurement by ultrasound dilution technique with pulmonary artery thermodilution in a pediatric animal model.

Novel COstatus system (Transonic Systems, Inc., NY), based on ultrasound dilution (UD), works off in situ arterial and central venous catheters in pediatric patients to measure cardiac output (CO). The purpose of the present study was to validate CO measurement by UD (COUD) with pulmonary artery (PA) thermodilution (COTD) in a prospective animal study. Ten anesthetized pigs (16-45 kg) were instrumented with pediatric PA, central venous, and peripheral artery catheters. For COUD measurements, normothermic saline (0.5-1.0 ml/kg body weight, up to a maximum of 30 ml) was injected into the venous limb of an arteriovenous loop that was connected between in situ catheters. For COTD measurements, 5-10 ml cold saline was injected into the PA catheter. Sixty-four averaged sets were obtained for comparison. COTD mean was 2.98 ± 1.21 l/min (range 1.33-6.29), and COUD mean was 2.68 ± 1.16 l/min (range 1.33-5.85). This study yielded a correlation r = 0.96, COUD = 0.91*(COTD) - 0.04 l/min; bias was 0.3 l/min with limits of agreement as -0.39 to 0.99 l/min; and the percentage error was 23.73% between the methods. CO measurements by UD agreed well with thermodilution measurements in the pediatric swine model.

[Assessment of cardiac output and intrathoracic blood volume by means of transpulmonary thermodilution and ultrasound dilution: similarities and differences].

UNLABELLED: The aim of the study is to compare results of the assessment of cardiac output and intrathoracic blood volume by two methods--transpulmonary (TTD) and ultrasound (UTD) thermodilution. MATERIALS AND METHODS: The prospective study included 58 patients (sepsis, septic shock, acute respiratory distress syndrome, intracranial haemorrhages), which underwent femoral artery catheterization with "Pulsiocath" 5Fr catheter (PICCO technology). For the means of ultrasound the catheter was connected to the central venous catheter by an arteriovenous loop. Sensors on arterial and venous ends of the loop registered the time and the volume of the indicator, blood properties and the ultrasound curve. Cooled (0 to 8 C) 5% glucose solution was used as an indicator for TTD, while heated (up to 37C) 0.9% NaCl solution was used as an indicator for the ultrasound. The cardiac output (CO) was measured by TTD and UTD, the global end diastolic volume (GEDV) by TTD, its analogue total end diastolic volume (TEDV) by UTD, intrathoracic blood volume (ITBV) by TTD and central blood volume (CBV) by UTD. 218 pairs of measurements were conducted. Oscillations of CO (TTD) were 2.76-16.3 l/min (8.6 +/- 2.48 l/min) and of CO (UTD)--2.92-18.1 l/min (8.72 +/- 2.65 l/min). There was a strong correlation between CO (TTD) and CO (UTD). The systematic mistake was 0.12 l/min, percentage based mistake--20.9%. ITBV correlated with CBV. There was a big systematic mistake found, which measured as much as 323 ml, the percentage based mistake was 36.5%. The correlation between GEDV and TEDV was (r = 0.70, p < 0.01). The TTD ejection fraction (23.2 +/- 5.6%) was lower (p < 0.01), than by UTD (57.8 +/- 15.2%). RESULTS: Both methods demonstrate close values of CO. GEDV was higher than TEDV and physiological heart volume. The absolute values of GEDV and ITBV measured by TTD are higher than the actual ones, although they reflect the changes of blood volume and can be used as dynamic preload parameters.

Ventriculoperitoneal shunt malfunction diagnosis based on substance dilution.

OBJECTIVE: Current methods for the diagnosis of ventriculoperitoneal (VP) shunt malfunction lack specific standards; therefore, it may be missed or misdiagnosed. Hence, providing a reliable diagnostic method will help improve the accuracy of preoperative decision-making. Therefore, the aim of the study was to provide a new method for the diagnosis of VP shunt malfunction. METHODS: After in vitro testing, we enrolled a total of 12 patients with VP shunt malfunction. Before revision surgery, 0.1 mL of a 5% sodium valproate (SV) solution was injected into the reservoir; 0.1 mL of the cerebrospinal fluid (CSF) was withdrawn 20 minutes later from the reservoir to measure the SV concentration. The process was repeated on the seventh day after surgery and compared with the preoperative results. RESULTS: The mean ±â€Šstandard deviation preoperative SV concentration in the cerebrospinal fluid was greater than the postoperative concentration (5967.8 ±â€Š1281.3 vs 391.1 ±â€Š184.6 µg/mL, P = .001). CONCLUSION: The proposed method is a reliable, safe, and relatively simple alternative for the diagnosis of VP shunt malfunction and further provides a reference for treatment.

2-layer based microfluidic concentration generator by hybrid serial and volumetric dilutions.

We present a 2-layer based microfluidic concentration generator by a hybrid of a serial and a volumetric dilution for dose-response experiments in drug screening. The hybrid dilution method using 2-layer based microfluidic network significantly reduces the total number of cascaded serial dilution stages. The proposed strategy is capable of generating a large number of universal stepwise monotonic concentrations with a wide range of logarithmic and linear scales. We have studied an equivalent electrical circuit to that of the 2-layer based microfluidic network, where the only variable parameter is channel length. We have designed a microfluidic dilution generator simultaneously covering 14 doses with a combination of 4-order logarithmic and 4-point linear concentrations. The design has been verified by a commercial circuit analysis software (e.g., P-Spice) for the electrical circuit analysis and a computational fluid dynamics software (e.g., CFD-ACE+) for the microfluidic circuit analysis. As a real-life application of the proposed dilution generator, we have successfully performed a dose-response experiment using MCF-7 human breast cancer cells. We expect that the proposed dilution method will be useful to study not only high throughput drug screening but also optimization in biology, chemistry, medicine, and material sciences.

Experimental demonstration of masking phenomena between competing odorants via an air dilution sensory test.

To simulate the occurrence of masking phenomena with the aid of an air dilution sensory (ads) test, two types of odorant mixtures were prepared: (1) M(2) with two individual odorants [H(2)S and acetaldehyde (AA)] and (2) M(6) with six individual odorants (H(2)S and five aldehydes). The test results derived for samples containing single individual odorants at a wide range of concentrations are initially used to define the empirical relationship between the dilution-to-threshold (D/T) ratio and odor intensity (OI) scaling. Based on these relationships, the D/T ratios were estimated for each odorant with the same intensity as the synthetic mixture. The relative contribution of each odorant to such mixture is then assessed by comparing the estimated and measured D/T values. This stepwise test confirmed the dominance of certain compounds at a given OI rating. In the case of M(2), H(2)S showed sensitive detection at high OI range, while AA did so at low end. The pattern of a competing relationship is also seen consistently from M(6) between AA (low) and iso-valeraldehyde (IA: high OI range). The overall results thus suggest that the masking phenomena between strong odorants should proceed under competing relationships, if released at the same time.

Evaluation of the accuracy and precision of a new generation indirect calorimeter in canopy dilution mode.

BACKGROUND & AIMS: Indirect calorimetry (IC) is the only way to measure in real time energy expenditure (EE) and to optimize nutrition support in acutely and chronically ill patients. Unfortunately, most of the commercially available indirect calorimeters are rather complex to use, expensive and poorly accurate and precise. Therefore, an innovative device (Q-NRG®, COSMED, Rome, Italy) that matches clinicians' needs has been developed as part of the multicenter ICALIC study supported by the two academic societies ESPEN and ESICM. The aim of this study was to evaluate the accuracy and intra- and inter-unit precision of this new device in canopy dilution mode in vitro and in spontaneously breathing adults. METHODS: Accuracy and precision of oxygen consumption (VO2) and carbon dioxide production (VCO2) measurements were evaluated in vitro and in 15 spontaneously breathing healthy adults by interchanging three Q-NRG® units in a random order. In vitro validation was performed by gas exchange simulation using high-precision gas mixture and mass flow controller. Accuracy was calculated as error of measured values against expected ones based on volume of gas infused. Respiratory coefficient (RQ) accuracy was furthermore assessed using the ethanol-burning test. To evaluate the intra- and inter-unit precisions, the coefficient of variation (CV% = SD/Mean*100) was calculated, respectively, from the mean ± SD or the mean ± SD of the three mean values of VO2, VCO2, RQ and EE measured by each Q-NRG® units. In vivo accuracy measurement of the Q-NRG® was assessed by simultaneous comparison with mass spectrometry (MS) gas analysis, using Bland-Altman plot, Pearson correlation and paired t-test (significance level of p = 0.05). RESULTS: In vitro evaluation of the Q-NRG® accuracy showed measurement errors <1% for VO2, VCO2 and EE and <1.5% for RQ. Evaluation of the intra- and inter-unit precision showed CV% ≤1% for VO2 and EE and CV% ≤1.5% for VCO2 and RQ measurements, except for one Q-NRG® unit where CV% was 2.3% for VO2 and 3% for RQ. Very good inter-unit precision was confirmed in vivo with CV% equal to 2.4%, 3%, 2.8% and 2.3% for VO2, VCCO2, RQ and EE, respectively. Comparison with MS showed correlation of 0.997, 0.987, 0.913 and 0.997 for VO2, VCO2, RQ and EE respectively (p ≤ 0.05). Mean deviation of paired differences was 1.6 ± 1.4% for VO2, -1.5 ± 2.5% for VCO2, -3.1 ± 2.6% for RQ and 0.9 ± 1.4% for EE. CONCLUSION: Both in vitro and in vivo measurements of VO2, VCO2, RQ and EE on three Q-NRG® units showed minimal differences compared to expected values and MS and very low intra- and inter-unit variability. These results confirm the very good accuracy and precision of the Q-NRG® indirect calorimeter in canopy dilution mode in spontaneously breathing adults.

Generalized serial dilution module for monotonic and arbitrary microfluidic gradient generators.

In this paper, we propose a generalized serial dilution module for universal microfluidic concentration gradient generators including N cascaded-mixing stages in a stepwise manner. Desired concentrations were generated by means of controlled volumetric mixing ratios of two merging solutions in each stage. The flow rates were adjusted by controlling channel length, which is proportional to fluidic resistance in each channel. A generalized mathematical model for generating any complex concentration and output flow rate gradients is presented based on the fact that there is an analogy between microfluidic circuits and electrical circuits. The pressure drop corresponds to a voltage drop, the flow rate to an electrical current, and the flow resistance to an electrical resistance. A simple equivalent electrical circuit model was generalized, and in the model each channel segment was represented by an electrical resistance. As a result of the mathematical modelling, the only variable parameter in the generalized serial dilution module was the channel length. By the use of the generalized serial dilution module with N = 4, three types of microfluidic gradient generators for linear, logarithmic and Gaussian gradients were successfully designed and tested. The proposed strategy is capable of generating universal monotonic gradients with a single module or arbitrary gradients with multiple modules ranging from linear to complex non-linear shapes of concentration gradients as well as arbitrary output flow rate gradients in a stepwise manner. The simple universal gradient generation technology using the generalized serial dilution module will find widespread use in the greater chemical and biological community, and address many challenges of gradient-dependent phenomena.

A microfluidic diluter based on pulse width flow modulation.

We demonstrate that pulse width flow modulation (PWFM) can be used to design fast, accurate, and precise multistage dilution modules for microfluidic devices. The PWFM stage unit presented here yields 10-fold dilution, but several PWFM stages can be connected in series to yield higher-order dilutions. We have combined two stages in a device thus capable of diluting up to 100-fold, and we have experimentally determined a set of rules that can be conveniently utilized to design multistage diluters. Microfabrication with resist-based molds yielded geometrical channel height variances of 7% (22.9(16) microm) with corresponding hydraulic resistance variances of approximately 20%. Pulsing frequencies, channel lengths, and flow pressures can be chosen within a wide range to establish the desired diluter properties. Finally, we illustrate the benefits of on-chip dilution in an example application where we investigate the effect of the Ca(2+) concentration on a phospholipid bilayer spreading from a membrane reservoir onto a SiO(2) surface. This is one of many possible applications where flexible concentration control is desirable.

Isotope dilution LC-ESI-MS/MS and low resolution selected reaction monitoring as a tool for the accurate quantification of urinary testosterone.

A new analytical method for the quantification of testosterone in human urine samples by isotope dilution mass spectrometry is proposed. A standard solution of 13C2-testosterone is added to the samples at the beginning of the sample preparation procedure and then the measurements are carried out by UHPLC-ESI-MS/MS. In the proposed method, the resolution of the first quadrupole of the tandem MS instrument is reduced to transmit the whole precursor ion cluster to the collision cell and measure the isotopic distribution of the in-cell product ions with a small number of SRM transitions. The construction of a methodological calibration graph is avoided using a labelled analogue previously characterised in terms of concentration and isotopic enrichment in combination with multiple linear regression. In this way, the molar fractions of natural and labelled testosterone are calculated in each sample injection and the amount of endogenous testosterone computed from the known amount of labelled analogue. Recovery values between 97 and 107% and precisions between 0.4 and 3.7% (as %RSD) were obtained for testosterone concentrations in urine in the range of 1 to 8 ng g-1. The proposed low resolution SRM methodology was compared for the analysis of human urine samples with the traditional IDMS method based on a calibration graph and the IDMS method based on multiple linear regression combined with standard resolution SRM. A similar accuracy and precision was obtained by the three tested approaches. However, using the low resolution SRM method there was no need to resort to calibration graphs or to specific dedicated software to calculate isotopic distributions by tandem MS and a higher sensitivity was obtained. The proposed low resolution SRM method was successfully applied to the analysis of the certified freeze-dried human urine NMIA MX005.

Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides.

DNA contains not only canonical nucleotides but also a variety of modifications of the bases. In particular, cytosine and adenine are frequently modified. Determination of the exact quantity of these noncanonical bases can contribute to the characterization of the state of a biological system, e.g., determination of disease or developmental processes, and is therefore extremely important. Here, we present a workflow that includes detailed description of critical sample preparation steps and important aspects of mass spectrometry analysis and validation. In this protocol, extraction and digestion of DNA by an optimized spin-column and enzyme-based method are described. Isotopically labeled standards are added in the course of DNA digestion, which allows exact quantification by isotope dilution mass spectrometry. To overcome the major bottleneck of such analyses, we developed a short (~14-min-per-sample) ultra-HPLC (UHPLC) and triple quadrupole mass spectrometric (QQQ-MS) method. Easy calculation of the modification abundance in the genome is possible with the provided evaluation sheets. Compared to alternative methods, the quantification procedure presented here allows rapid, ultrasensitive (low femtomole range) and highly reproducible quantification of different nucleosides in parallel. Including sample preparation and evaluation, quantification of DNA modifications can be achieved in less than a week.

Continuous and intermittent cardiac output measurement in hyperdynamic conditions: pulmonary artery catheter vs. lithium dilution technique.

OBJECTIVE: This study aimed to assess the level of agreement of both intermittent cardiac output monitoring by the lithium dilution technique (CO(Li)) and continuous cardiac output monitoring (PulseCO(Li)) using the arterial pressure waveform with intermittent thermodilution using a pulmonary artery catheter (CO(PAC)). DESIGN: Prospective, single-center evaluation. SETTING: University Hospital Intensive Care Unit. PATIENTS: Patients (n=23) receiving liver transplantation. INTERVENTION: Pulmonary artery catheters were placed in all patients and CO(PAC) was determined using thermodilution. CO(Li) and PulseCO(Li) measurements were made using the LiDCO system. MEASUREMENTS AND MAIN RESULTS: Data were collected after intensive care unit admission and every 8h until the 48th hour. A total of 151 CO(PAC), CO(Li) and PulseCO(Li) measurements were analysed. Bias and 95% limit of agreement were 0.11lmin(-1) and -1.84 to + 2.05 lmin(-1) for CO(PAC) vs. CO(Li) (r=0.88) resulting in an overall percentage error of 15.6%. Bias and 95% limit of agreement for CO(PAC) vs. PulseCO(Li) were 0.29 lmin(-1) and -1.87 to + 2.46 lmin(-1) (r=0.85) with a percentage error of 16.8%. Subgroup analysis revealed a percentage error of 15.7% for CO(PAC) vs. CO(Li) and 15.1% for CO(PAC) vs. PulseCO(Li) for data pairs less than 8 lmin(-1), and percentage errors of 15.5% and 18.5% respectively for data pairs higher than 8 lmin(-1). CONCLUSION: In patients with hyperdynamic circulation, intermittent and continuous CO values determined using the LiDCO system showed good agreement with those obtained by intermittent pulmonary artery thermodilution.