Determination Of Absolute Concentrations Research Articles

A method to directly analyze free-drug–related species in antibody-drug conjugates without sample preparation

Residual free-drug–related species that are present in antibody-drug conjugates (ADC) are a potential safety risk to patients and are therefore categorized as a critical quality attribute that must be strictly monitored and controlled. Among the many analytical methods developed for free-drug analysis, reversed-phase liquid chromatography (RP-LC) is the most common approach. Conventional RP-LC methods for free-drug analysis, however, involve labor-intensive sample preparation. Here we present a new RP-LC method to directly analyze free-drug–related species in an ADC sample without the need for sample preparation. In our work, free-drug–related species were very well separated from ADC peaks in the chromatography gradient. Typical performance issues observed in conventional RP-LC, such as column fouling, detection interference, and carryover, were not observed or were negligible with this new method. Three options were evaluated for free-drug quantitation: Strohl (2017) [1] use of an external free drug calibration curve for determination of absolute concentration; Perez et al. (2014) [2] calculation of relative percentage based on peak area ratio between free drug and ADC at a characteristic wavelength unique for drug payload; and (Beck et al., 2017) [3] calculation of relative percentage based on peak area ratio between free drug and corrected ADC peak area (at any wavelength). The method with calibration curve provides the highest sensitivity, the best accuracy and precision for determination of free drug present in the ADC. However, the second and third options were simpler because they eliminated the need for an external calibration curve, making them worth exploring. Due to its simplicity and compatibility with mass spectrometry, the new method is also a good choice for direct analysis of stability samples.

Correction to Development of a High-Resolution Laser Absorption Spectroscopy Method with Application to the Determination of Absolute Concentration of Gaseous Elemental Mercury in Air.

Correction to Development of a High-Resolution Laser Absorption Spectroscopy Method with Application to the Determination of Absolute Concentration of Gaseous Elemental Mercury in Air.

Development of a High-Resolution Laser Absorption Spectroscopy Method with Application to the Determination of Absolute Concentration of Gaseous Elemental Mercury in Air.

Isotope dilution-cold-vapor-inductively coupled plasma mass spectrometry (ID-CV-ICPMS) has become the primary standard for measurement of gaseous elemental mercury (GEM) mass concentration. However, quantitative mass spectrometry is challenging for several reasons including (1) the need for isotopic spiking with a standard reference material, (2) the requirement for bias-free passive sampling protocols, (3) the need for stable mass spectrometry interface design, and (4) the time and cost involved for gas sampling, sample processing, and instrument calibration. Here, we introduce a high-resolution laser absorption spectroscopy method that eliminates the need for sample-specific calibration standards or detailed analysis of sample treatment losses. This technique involves a tunable, single-frequency laser absorption spectrometer that measures isotopically resolved spectra of elemental mercury (Hg) spectra of 6 1S0 ← 6 3P1 intercombination transition near λ = 253.7 nm. Measured spectra are accurately modeled from first-principles using the Beer-Lambert law and Voigt line profiles combined with literature values for line positions, line shape parameters, and the spontaneous emission Einstein coefficient to obtain GEM mass concentration values. We present application of this method for the measurement of the equilibrium concentration of mercury vapor near room temperature. Three closed systems are considered: two-phase mixtures of liquid Hg and its vapor and binary two-phase mixtures of Hg-air and Hg-N2 near atmospheric pressure. Within the experimental relative standard uncertainty, 0.9-1.5% congruent values of the equilibrium Hg vapor concentration are obtained for the Hg-only, Hg-air, Hg-N2 systems, in confirmation with thermodynamic predictions. We also discuss detection limits and the potential of the present technique to serve as an absolute primary standard for measurements of gas-phase mercury concentration and isotopic composition.

NADH-fluorescence scattering correction for absolute concentration determination in a liquid tissue phantom using a novel multispectral magnetic-resonance-imaging-compatible needle probe

In this report, a quantitative nicotinamide adenine dinucleotide hydrate (NADH) fluorescence measurement algorithm in a liquid tissue phantom using a fiber-optic needle probe is presented. To determine the absolute concentrations of NADH in this phantom, the fluorescence emission spectra at 465 nm were corrected using diffuse reflectance spectroscopy between 600 nm and 940 nm. The patented autoclavable Nitinol needle probe enables the acquisition of multispectral backscattering measurements of ultraviolet, visible, near-infrared and fluorescence spectra. As a phantom, a suspension of calcium carbonate (Calcilit) and water with physiological NADH concentrations between 0 mmol l−1 and 2.0 mmol l−1 were used to mimic human tissue. The light scattering characteristics were adjusted to match the backscattering attributes of human skin by modifying the concentration of Calcilit. To correct the scattering effects caused by the matrices of the samples, an algorithm based on the backscattered remission spectrum was employed to compensate the influence of multiscattering on the optical pathway through the dispersed phase. The monitored backscattered visible light was used to correct the fluorescence spectra and thereby to determine the true NADH concentrations at unknown Calcilit concentrations. Despite the simplicity of the presented algorithm, the root-mean-square error of prediction (RMSEP) was 0.093 mmol l−1.

Characterization of 3-Aminopropyl Oligosilsesquioxane.

The synthesis routes in the production of polysilsesquioxanes have largely relied upon in situ formations. This perspective often leads to polymers in which their basic structures including molecular weight and functionality are unknown [ Lichtenhan , J. D. ; et al. Silsesquioxane-siloxane copolymers from polyhedral silsesquioxanes Macromolecules , 1993 , 26 , 2141 - 2142 , http://dx.doi.org/10.1021/ma0060a053 ]. For a better understanding of the polysilsesquioxane properties and applications, there is a need to develop more techniques to enable their chemical characterization. An innovative method was developed to determine the molecular weight distribution (MWD) of an oligosilsesquioxane synthesized in-house from (3-aminopropyl)triethoxysilane. This method, which can be applied to other silsesquioxanes, siloxanes, and similar oligomers and polymers, involved separation using high performance liquid chromatography (HPLC) and detection using mass spectrometry (MS) with electrospray ionization (ESI). The novelty of the method lies on the unique determination of the absolute concentrations of the individual homologues present in the sample formulation. The use of absolute concentrations is necessary in estimating the MWD of the formulation when relative percentage, which is based solely on mass spectral ion intensities, becomes irrelevant due to the disproportionate response factors of the homologues. Determination of absolute concentration requires the use of single-homologue calibration standards. Because of commercial unavailability, these standards were prepared by efficient fractionation of the original formulation.

Non-linear effects in quantitative 2D NMR of polysaccharides: Pitfalls and how to avoid them

Quantitative 2D NMR is a powerful analytical tool which is widely used to determine the concentration of small molecules in complex samples. Due to the site-specific response of the 2D NMR signal, the determination of absolute concentrations requires the use of a calibration or standard addition approach, where the analyte acts as its own reference. Standard addition methods, where the targeted sample is gradually spiked with known amounts of the targeted analyte, are particularly well-suited for quantitative 2D NMR of small molecules. This paper explores the potential of such quantitative 2D NMR approaches for the quantitative analysis of a high molecular weight polysaccharide. The results highlight that the standard addition method leads to a strong under-estimation of the target concentration, whatever the 2D NMR pulse sequence. Diffusion measurements show that a change in the macromolecular organization of the studied polysaccharide is the most probable hypothesis to explain the non-linear evolution of the 2D NMR signal with concentration. In spite of this non-linearity – the detailed explanation of which is out of the scope of this paper – we demonstrate that accurate quantitative results can still be obtained provided that an external calibration is performed with a wide range of concentrations surrounding the target value. This study opens the way to a number of studies where 2D NMR is needed for the quantitative analysis of macromolecules.

Recent developments of proton‐transfer reaction mass spectrometry (PTR‐MS) and its applications in medical research

Proton-transfer reaction mass spectrometry (PTR-MS) allows for real-time, on-line determination of absolute concentrations of volatile organic compounds (VOCs) with a high sensitivity and low detection limits (in the pptv range). The technique utilizes H₃O⁺ ions for proton-transfer reactions with many common VOCs while having little to no reaction with any constituents commonly present in air. Over the past decades, research has greatly improved the applications and instrumental design of PTR-MS. In this article, we give an overview of the development of PTR-MS in recent years and its application in medical research. The theory of PTR-MS and various methods for discriminating isobaric VOCs are also described. We also show several specialized designs of sample inlet system, some of those may make PTR-MS suitable for the detection of aqueous solution and/or non-volatile samples.

In vivo magnetic resonance spectroscopy of cancer

Magnetic resonance (MR) methods provide anatomical, physiological and metabolic assessment of the cancer non- invasively. In vivo MR spectroscopy (MRS) provides information on endogenous metabolites present in tissues which is useful not only for diagnosis but also for monitoring the tumor response to therapy. Technical developments in field of in vivo MRS have resulted in mapping spatial distribution of metabolites (spectroscopic imaging), improvement in spectral sensitivity and resolution, and determination of absolute concentration of metabolites. The localized proton MRS first reported two decades ago has now emerged as a non-invasive methodology, to be used in clinical settings for the evaluation of brain and other tumors. The method has shown tremendous potential in the assessment of cancer of different organs, notably, in brain, breast and prostate cancers. Recent developments in MRS technology has made possible to expand its application to other malignancies such as musculoskeletal system, head and neck, and liver. In this review we briefly summarize the potential applications of MRS in diagnosis, staging, monitoring treatment, guiding biopsies and prediction of malignancy in brain, breast and prostate cancers. We also present briefly the current status and limitations of the applications of MRS in musculoskeletal system and liver, as two examples.

Measurement of absolute concentrations of individual compounds in metabolite mixtures by gradient-selective time-zero 1H-13C HSQC with two concentration references and fast maximum likelihood reconstruction analysis.

Time-zero 2D (13)C HSQC (HSQC(0)) spectroscopy offers advantages over traditional 2D NMR for quantitative analysis of solutions containing a mixture of compounds because the signal intensities are directly proportional to the concentrations of the constituents. The HSQC(0) spectrum is derived from a series of spectra collected with increasing repetition times within the basic HSQC block by extrapolating the repetition time to zero. Here we present an alternative approach to data collection, gradient-selective time-zero (1)H-(13)C HSQC(0) in combination with fast maximum likelihood reconstruction (FMLR) data analysis and the use of two concentration references for absolute concentration determination. Gradient-selective data acquisition results in cleaner spectra, and NMR data can be acquired in both constant-time and non-constant-time mode. Semiautomatic data analysis is supported by the FMLR approach, which is used to deconvolute the spectra and extract peak volumes. The peak volumes obtained from this analysis are converted to absolute concentrations by reference to the peak volumes of two internal reference compounds of known concentration: DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) at the low concentration limit (which also serves as chemical shift reference) and MES (2-(N-morpholino)ethanesulfonic acid) at the high concentration limit. The linear relationship between peak volumes and concentration is better defined with two references than with one, and the measured absolute concentrations of individual compounds in the mixture are more accurate. We compare results from semiautomated gsHSQC(0) with those obtained by the original manual phase-cycled HSQC(0) approach. The new approach is suitable for automatic metabolite profiling by simultaneous quantification of multiple metabolites in a complex mixture.

Highly Sensitive GC/MS/MS Method for Quantitation of Amino and Nonamino Organic Acids

Metabolite profiling methods are important tools for measurement of metabolite pools in biological systems. While most metabolite profiling methods report relative intensities or depend on a few internal standards representing all metabolites, the ultimate requirement for a quantitative description of the metabolite pool in biological cells and fluids is absolute concentration determination. We report here a high-throughput and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) targeted metabolite profiling method enabling absolute quantification of all detected metabolites. The method is based on methyl chloroformate derivatization and quantification by spiking samples with metabolite standards separately derivatized with deuterated derivatization reagents. The traditional electron impact ionization is replaced with positive chemical ionization since the latter to a much larger extent preserve the molecular ion and other high molecular weight fragments. This made it easier to select unique MS/MS transitions among the many coeluting metabolites. Currently, the novel GC/MS/MS method comprises 67 common primary metabolites of which most belong to the groups of amino and nonamino organic acids. We show the applicability of the method on urine and serum samples. The method is a significant improvement of present methodology for quantitative GC/MS metabolite profiling of amino acids and nonamino organic acids.

Thermal desorption extraction proton transfer reaction mass spectrometer (TDE-PTR-MS) for rapid determination of residual solvent and sterilant in disposable medical devices

Thermal desorption extraction proton transfer reaction mass spectrometer (TDE-PTR-MS) has been exploited to provide rapid determination of residual solvent and sterilant like cyclohexanone (CHX) and ethylene oxide (EO) in disposable medical devices. Two novel methods are proposed for the quantification of residual chemicals in the polyvinyl chloride infusion sets with our homemade PTR-MS. In the first method, EO residue in the solid infusion sets ( y, mg set −1) is derived through the determination of EO gas concentration within its packaging bag ( x, ppm) according to the correlative equation of y = 0.00262 x. In the second one, residual EO and CHX in the solid infusion sets are determined through a time integral of their respective mass emission rates. The validity of the proposed methods is demonstrated by comparison with the experimental results from the exhaustive extraction method. Due to fast response, absolute concentration determination and high sensitivity, the TDE-PTR-MS is suggested to be a powerful tool for the quality inspection of disposable medical devices including the quantitative determination of residual solvent and sterilant like CHX and EO.

An online ultra-high sensitivity Proton-transfer-reaction mass-spectrometer combined with switchable reagent ion capability (PTR + SRI − MS)

Proton-transfer-reaction mass-spectrometry (PTR-MS) developed in the 1990s is used today in a wide range of scientific and technical fields. PTR-MS allows for real-time, online determination of absolute concentrations of volatile (organic) compounds (VOCs) in air with high sensitivity (into the low pptv range) and a fast response time (in the 40–100 ms time regime). Most PTR-MS instruments employed so far use an ion source consisting of a hollow cathode (HC) discharge in water vapour which provides an intense source of proton donor H 3O + ions. As the use of other ions, e.g. NO + and O 2 +, can be useful for the identification of VOCs and for the detection of VOCs with proton affinities (PA) below that of H 2O, selected ion flow tube mass spectrometry (SIFT-MS) with mass selected ions has been applied in these instances. SIFT-MS suffers, however, from at least two orders lower reagent ion counts rates and therefore SIFT-MS suffers from lower sensitivity than PTR-MS. Here we report the development of a PTR-MS instrument using a modified HC ion source and drift tube design, which allows for the easy and fast switching between H 3O +, NO + and O 2 + ions produced in high purity and in large quantities in this source. This instrument is capable of measuring low concentrations (with detection limits approaching the ppqv regime) of VOCs using any of the three reagent ions investigated in this study. Therefore this instrument combines the advantages of the PTR-MS technology (the superior sensitivity) with those of SIFT-MS (detection of VOCs with PAs smaller than that of the water molecule and the capability to distinguish between isomeric compounds). We will first discuss the setup of this new PTR+SRI-MS mass spectrometer instrument, its performance for aromates, aldehydes and ketones (with a sensitivity of up to nearly 1000 cps/ppbv and a detection limit of about several 100 ppqv) and finally give some examples concerning the ability to distinguish structural isomeric compounds.

Analysis and Quantification of Diagnostic Serum Markers and Protein Signatures for Gaucher Disease

Novel approaches for the qualitative and quantitative proteomics analysis by nanoscale LC-MS applied to the study of protein expression response in depleted and undepleted serum of Gaucher patients undergoing enzyme replacement therapy are presented. Particular emphasis is given to the method reproducibility of these LC-MS experiments without the use of isotopic labels. The level of chitotriosidase, an established Gaucher biomarker, was assessed by means of an absolute concentration determination technique for alternate scanning LC-MS generated data. Disease associated proteins, including fibrinogens, complement cascade proteins, and members of the high density lipoprotein serum content, were recognized by various clustering methods and sorting and intensity profile grouping of identified peptides. Condition-unique LC-MS protein signatures could be generated utilizing the measured serum protein concentrations and are presented for all investigated conditions. The clustering results of the study were also used as input for gene ontology searches to determine the correlation between the molecular functions of the identified peptides and proteins.

32] Ultrastructural localization and relative quantification of 4-hydroxynonenal-modified proteins in tissues and cell compartments

Publisher Summary The chapter presents methods to localize 4-Hydroxynonenal (HNE)-modified proteins in vivo . Methods for relative quantification of 4HNE-modified proteins in tissues and subcellular compartments are also discussed. These latter analytical methods allow one to compare levels of 4HNE-modified proteins between an experimental and a control group, but do not allow determination of absolute concentrations of 4HNE-modified proteins. Degradation rates of oxidatively damaged proteins may vary among cell types. The cell turnover rate is unique for each cell type. Positive controls should be used in any morphologic analysis. 4HNE is found in large quantities in organs with large amounts of lipofuscin, including seminal vesicle and prostate. Quantitative image analysis indicated an increase in oxidative damage following TPA treatment in nuclei and mitochondria but not in the cytoplasm. The results illustrate the importance of immunomorphologic techniques in quantifying oxidative damage to allow study of important biological problems.

Determination of absolute concentration of nitroxide radical by radio-frequency EPR imaging

The absolute concentrations of a nitroxide radical in samples in a loop-gap resonator (LGR) were determined by using a radio-frequency (about 720 MHz) electron paramagnetic resonance (EPR) imaging system. EPR imaging of phantoms containing a nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (carbamoyl-PROXYL), dissolved in various concentrations of an aqueous sodium chloride solution was made to investigate the influence of dielectric losses and sample position within the LGR. As it was found that these influences on the signal intensity were sufficiently small (less than 6%), it is possible to use identical radical solutions in which the radical is dissolved in a known concentration as an internal marker. Two phantoms containing aqueous solutions of 3 mM (as a marker) and 1, 2, 3, 4, or 5 mM (as a sample) carbamoyl-PROXYL were placed together in the LGR. From EPR images of these phantoms, the absolute concentration of the sample could be calculated by using the gray-scale value (i.e., the signal intensity) of the marker and sample within a small margin of error (about 4%).

Concentration Measurement by Proton NMR Using the ERETIC Method

The ERETIC method (Electronic REference To access In vivo Concentrations) provides a reference signal, synthesized by an electronic device, which can be used for the determination of absolute concentrations. The results presented here demonstrate the accuracy and precision of the method in the case of (1)H high resolution NMR. Five tubes were filled with D(2)O solutions of trimethylamine hydrochloride (TMA) 3.84 mM and sodium lactate at concentrations ranging from 5.25 to 54.11 mM. Results obtained with the ERETIC method were compared to those obtained by using TMA as an internal reference. The standard deviations were the same for the two methods and always lower than 1% of the mean. The accuracy (difference between true value and measured value) was slightly better for the ERETIC method than for the internal reference. No significant variation was observed when the experiments were performed over 56 h. Measurements were repeated once a month during three months. As the values obtained showed a standard deviation of only 3%, we can conclude that the ERETIC method has a good stability and only requires monthly calibration. Furthermore, it must be noted that nothing is added to the sample and that the reference signal frequency can be freely chosen to fall within a transparent region of the spectrum.

Superoxide electrode based on covalently immobilized cytochrome c: modelling studies

We have recently described an optimised electrode for the detection of enzymatic and cellular superoxide (O 2 •−) production based on cytochrome c immobilized covalently at a surface-modified gold electrode and applied this system to the study of free radical production by activated human glioblastoma cells. In this paper we report the development of a mathematical model for the O 2 •− electrode responding to enzymically produced O 2 •− which should enable the determination of absolute concentrations of O 2 •− in biological systems when this electrode is employed for direct, real-time monitoring of free radical release and interactions.

Use of Commercially Available Hemoglobin Standards to Quantitatively Calibrate a High Performance Liquid Chromatography Method

High performance liquid chromatography (HPLC) has proven to be an extremely useful analytical technique to separate and identify different types of hemoglobins, particularly A, C, F and S in blood samples, and compute their relative percentages. Such data provide useful information in the diagnosis of hemoglobinopathies including sickle cell anemia, beta-thalassemia, hemoglobin C disease,etc. In the present investigation, we have explored the determination of absolute concentrations of individual hemoglobins in g/dL and recommend it as an additional parameter which could be included as part of clinical data. Possible correlations between g/dL hemoglobin and the severity of a specific hemoglobinopathy could be established and aid in the diagnosis and/or treatment of such diseases. Several commercially available hemoglobin standards were analyzed and evaluated for use in creating g/dL calibration graphs for the HPLC. Appropriate calibration procedures have been developed and are presented. Also, linear regression graphs and sample chromatograms are included. Preliminary results obtained demonstrate the feasibility of using commercially available hemoglobin standards to calibrate an HPLC method for the estimation of absolute hemoglobin concentrations.

Kinetic Retrieval of Eosinophil Cationic Protein, Hyaluronan, Secretory IgA, Albumin, and Urea During BAL in Healthy Subjects

Determination of absolute concentrations of various soluble components of the epithelial lining fluid (ELF) may be valuable to estimate inflammatory activities within the underlying lung tissue. Internal standards may then be used as markers of dilution of bronchoalveolar lavage (BAL). The aim of this study was to determine whether different dwell times would affect the relationship between the concentrations of any of the three potential internal standards (secretory IgA [SIgA], albumin, and urea), and the concentrations of two potential markers of inflammation (eosinophil cationic protein [ECP] and hyaluronan [HA]) in BAL. A series of aliquots of BAL fluid were aspirated every 60 s up to 8 min after a bolus instillation of saline solution in 20 healthy subjects (10 smokers). The BAL concentrations of albumin and urea increased with time, consistent with continuous diffusion from the body water pool, absorption of the BAL fluid, or both. The rate constant of diffusion was 1,000 times higher for urea than for albumin (3.38 x 10(-1) and 3.64 x 10(-4), respectively), reflecting the difference in molecular weights, and in agreement with the notion that albumin and urea appeared in BAL fluid by a rate-limited procedure related to osmotic transfer. Biexponential increases of SIgA were recorded, suggesting a two-compartmental origin of this compound, normally located to mucosal membranes and presumed to be dissolved in ELF. Time-dependent increases in BAL fluid of HA also were recorded, but on the other hand, the ECP concentrations tended to level off after an initial increase, suggesting that the bulk of ECP appeared in BAL by a nonosmotic mechanism. We conclude that the kinetics of these three internal standards in BAL fluid differs greatly from each other and from the kinetics of the two selected markers of inflammation. Consequently, internal standards for determination of absolute concentrations of markers of inflammation in ELF should be carefully selected because of the requirement of matched kinetics of the markers.

The pixe analytical technique: Principle and applications

We will first recall the principles of the particle induced X-ray emission (PIXE) technique and compare it to the electron microprobe. Then we will describe the experimental set-up needed for PIXE measurements, emphasizing the construction problems for a PIXE microprobe. The data treatment procedure will be discussed: unfolding of X-ray spectra, determination of absolute concentration, … Finally, we will present examples of application, in particular in mineralogy and environmental science, as performed in our laboratory.