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PubMed1992

Thermospray liquid chromatographic-mass spectrometric analysis of anti-AIDS nucleosides: quantification of 2',3'-dideoxycytidine in plasma samples.

Thermospray liquid chromatography-mass spectrometry was investigated as a method for quantification of 2',3'-dideoxycytidine (DDC) from human plasma. A stable isotope analog of DDC ([15N2,2H2]DDC) was used as an internal standard. Selected ion monitoring of the protonated molecular ions for DDC and the internal standard was used to record mass chromatograms. The areas of the peaks in the mass chromatograms were used for quantification. The detection limit of DDC in this assay was 50 pg on-column. The calibration curve was linear over the desired range, 0.25-20 ng/ml. The major advantages of this assay over others are: no derivatization, high sensitivity, high specificity and short assay time.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Localization and quantification of the nonspecific esterase in injured skin for timing of wounds.

Histochemical quantification of the nonspecific esterase (NSE) in injured skin was performed using histochemical demonstration of the enzyme and a microspectrophotometric scanning technique on specimens taken from 32 Hartley guinea-pigs and 8 cases of human skin wounds. In all antemortem incisions and lacerations, including those made at the agonal stage, NSE activity could be observed in the dermal tissue of the wound edge. The enzyme activity increases with the antemortem duration of the injuries. Both total content and mean concentration of NSE in the wound edge between antemortem and postmortem wound groups differ significantly (less than 0.01). Multiple range test shows that significant differences (P less than 0.05 or P less than 0.01) of total content of NSE in the wound edge also exist in 0-5 min, 15-30 min, 1-h, 2-h and 4-h antemortem incised wound groups and in 0-min, 5-min, 15-30 min, 1-h and 4-h antemortem laceration groups. The positive NSE reactions in 8 cases of human skin wounds were similar. The study indicates that histochemical quantification of NSE in injured skin is very useful in timing wounds and is exactly applicable in medicolegal practice. According to the different influences of inhibitors on enzymes, it was inferred that the enzyme activity in wound edges was due to B-esterase.

Animals↗

PubMedDate not supplied

In-plane flow velocity quantification along the phase encoding axis in MRI.

In-plane flow quantification in MRI offers the potential for assessing vessel patency, and both volume flow rate and flow velocity. These techniques will have definite future impact on MR angiography. The method used in this paper employs motion artifact suppression technique (MAST) gradients to refocus spins travelling along any of the three imaging axes while encoding the velocity component along the phase encoding axis. This method has several advantages over in-plane flow quantification along the read axis. Primarily, flow voids due to complete spin dephasing can be eliminated (or reduced), wider velocity limits can be measured, and gradients can be designed which are sensitive to only velocity along the phase axis with no additional effect from higher order derivatives, or motion along the read axis. Flow phantom studies, carried out on 19 mm inside diameter glass tubes, have produced accurate results for flow rates ranging from 0.6 gallons per minute (GPM) to 2.5 GPM, corresponding to a mean velocity range from 13.2 cm/sec to 55.3 cm/sec. Reynolds numbers varied from 2,700 to 11,500. Errors were less than or equal to 8% over the range of flow rates studied.

Blood Flow Velocity↗

PubMed1990

A solution hybridization method for quantification of mRNAs: determining the amount and stability of oncogene mRNA.

A solution hybridization method for the quantification of specific mRNAs is described. This assay utilizes complementary RNA probes prepared by in vitro transcription, sandwich hybridization in solution, and affinity-based hybrid collection. The possibility of using this method for crude biological samples without purifying mRNAs makes it ideal when accurate quantification of multiple samples is needed. Human N-myc oncogene transcript was used as a model and as little as 0.24 pg (2 X 10(5) molecules) of N-myc mRNA could be detected. Using this assay it was shown that human neuroblastoma IMR-32 cells contain approximately 500 N-myc mRNA molecules per cell having a half-life of approximately 35 min.

DNA Probes↗

PubMed1991

Quantification of noncyclooxygenase derived prostanoids as a marker of oxidative stress.

Recently, we discovered there is a unique class of prostaglandin F2-like compounds that are formed in vitro from arachidonoyl-containing lipids in plasma by a free radical-catalyzed mechanism. More recent studies have elucidated that these prostanoids are also produced in vivo in humans by a similar noncyclooxygenase mechanism. Levels of these PGF2 compounds detected by a mass spectrometric assay in normal human plasma and urine range from approximately 5-50 pg/mL and 500-3000 pg/mg creatinine, respectively. Circulating levels of the compounds were shown to increase by as much as 200-fold in animal models of free radical-induced lipid peroxidation. These results suggest that quantification of these prostanoids may provide a new approach to assess oxidative stress in vivo in humans. Potential advantages of this approach are that the mass spectrometric assay has a high degree of sensitivity, accuracy, and specificity and the assay can be used to quantitate these compounds in a variety of biological fluids. In addition, quantification of these compounds is of interest because these compounds possess biological activity. Disadvantages of the assay are the potential of ex vivo formation of these compounds in biological fluids containing lipids and, further, these compounds must be differentiated from PGF2 compounds that are formed via the cyclooxygenase enzyme. In addition, because the levels of these compounds in normal human plasma and urine are relatively high, assaying these compounds in circulating plasma and urine may be somewhat insensitive for the detection of increased production at isolated sites of oxidant injury within the body, in which case sampling near localized sites of their formation may be required.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers↗

PubMed2026

Development and validation of an LC-MS/MS method for the quantification of the KRASG12C inhibitor divarasib.

Divarasib is a newly developed covalent KRASG12C inhibitor, currently under clinical investigation in a phase 3 trial in patients with non-small cell lung cancer (NSCLC). At the moment, very limited pharmacokinetic data are publicly known. However, obtaining more insight into the pharmacokinetic properties of divarasib is important, since this may provide a better understanding of its efficacy and safety risks. Pre-clinical studies have been performed in mouse models to evaluate the effect of drug transporters and drug-metabolizing enzymes on the plasma exposure and tissue distribution of divarasib. Therefore, a reliable quantification method is required. To our knowledge, no bioanalytical assay of divarasib has been published yet. Therefore, in this study we developed and validated an assay to quantify divarasib in human plasma and in eight different mouse-related matrices, and partially in mouse plasma, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was initially evaluated over a concentration range of 1-10,000 nM. However, due to carry-over observed at 10,000 nM, the validated calibration range was established at 1-2000 nM, with matrix-dependent LLOQs of 1-10 nM. Erlotinib was used as an internal standard and acetonitrile was utilized to perform protein precipitation as sample pretreatment. Divarasib demonstrated stability in human plasma and in mouse plasma and tissue homogenates under various experimental conditions. A pilot in vivo study showed the applicability of our validated LC-MS/MS method. Ongoing clinical trials may collect plasma samples, and this developed method enables quantification of divarasib in both mouse and human plasma samples.

Animals↗

PubMed2026

A species-discriminatory aerA TaqMan qPCR assay for rapid quantification of Aeromonas veronii in fish tissues and aquaculture water.

Aeromonas veronii is a major bacterial pathogen in freshwater aquaculture, yet rapid species-level quantification remains challenging within the genetically complex genus Aeromonas. We developed a singleplex hydrolysis-probe (TaqMan) quantitative PCR (qPCR) assay targeting an A. veronii-discriminatory region of the aerolysin gene (aerA) and validated it according to MIQE recommendations. Plasmid standards gave a linear range of 2 to 2 × 106 copies/reaction (R2 = 0.9962) with 100.5% amplification efficiency. The endpoint limit of detection was 2 copies per reaction, and 20 copies per reaction was set as the practical reporting limit based on reproducible detection and low intra- and inter-assay variation. Analytical specificity was evaluated with genomic DNA from an 18-strain panel, with reproducible amplification observed only for A. veronii. The assay was further tested in 55 fish-tissue and 11 aquaculture-water DNA extracts. NH8B-1D2 sample-process monitoring was used for matrix-level recovery correction, and tissue and water extraction blanks were undetermined. The aerA target was detected in all tested gill, stomach/intestine, spleen, kidney/head kidney, pond-water filter and Xiamen seawater filter extracts, and in 10/11 liver extracts. Median NH8B-corrected loads were highest in gill among tissues and higher in pond-water filters than in Xiamen seawater filters. A separate Vibrio harveyi inhibition-check assay indicated no obvious amplification-stage inhibition. This assay supports rapid quantification of aerA-positive A. veronii in fish and aquaculture-water matrices.

Animals↗

PubMed2025

Analytical and clinical performance validation of HPV-SEQ, a novel NGS-based liquid biopsy platform for detection and quantification of human papilloma virus circulating tumor DNA.

BACKGROUND: Human papillomavirus (HPV) is the primary causative driver of oropharyngeal squamous cell carcinoma (OPSCC). Accurate detection of HPV-DNA is critical for risk stratification and management of OPSCC. However, assays designed to detect HPV in primary tumors do not allow monitoring of HPV-DNA over time, whereas commercially available droplet digital PCR-based methods for assessment of circulating cell free (cf)HPV-DNA in plasma remain suboptimal, hindering adaptation into clinical practice. We have developed HPV-SEQ, a novel next-generation-sequencing (NGS) based method for detection and quantification of HPV16/18 DNA in plasma of patients with OPSCC. METHODS: The assay uses primers targeting the L1 gene of HPV16 and HPV18 viral genomes and strain specific calibrators at a defined concentration to determine the ratio of native HPV to a known standard, enabling accurate reporting of patient-derived HPV16/18 viral load in a sample. This study was conducted using two different patient populations in addition to healthy donors and contrived material. All experiments were performed to fulfill several applicable analytical, performance and validation guidelines. RESULTS: A thorough analytical characterization and clinical validation of this platform demonstrates that HPV-SEQ detects cfHPV-DNA with exceptional limit of quantification and high precision, providing a foundation for integrating this platform into clinical settings. CONCLUSIONS: This ultra-sensitive HPV profiling method with optimal analytical performance may represent a significant advancement in risk stratification, treatment management, and post-treatment surveillance for patients with OPSCC.

Humans↗

PubMed2026

Thermal analysis techniques for microplastic mass quantification: Methodological challenges and standardization needs.

Microplastics (MPs, 1 μm-5 mm) and nanoplastics (NPs, <1 μm) are ubiquitous contaminants requiring standardized quantification methods. This systematic review evaluates thermal analysis techniques for mass-based MP detection, including pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), thermogravimetry-MS (TGA-MS), thermal extraction desorption-GC-MS (TED-GC-MS), and differential scanning calorimetry (DSC). Database searches (Web of Science, from inception to December 1, 2025) following PRISMA guidelines identified studies across seven environmental matrices (water, soil/sediment, atmosphere, biota, human tissues). We identify critical standardization gaps: inconsistent marker ion selection, unvalidated conversion factors for tire and road wear particles (TRWPs), and the absence of certified reference materials for complex matrices. Py-GC-MS demonstrates versatility but suffers from lipid interference in biological samples; TED-GC-MS offers superior sensitivity (sample capacity ∼200× Py-GC-MS) but lacks real-time chromatographic monitoring. To advance data comparability, we propose: (i) harmonized ion selection hierarchies based on specificity-sensitivity balance, (ii) matrix-specific TRWP quantification protocols, and (iii) inter-laboratory validation using environmental reference materials. This review provides a methodological roadmap for standardizing thermal analysis in MP research.

Humans↗

PubMed1985

Experimental catecholamine-induced myocardial necrosis. I. Morphology, quantification and regional distribution of acute contraction band lesions.

Acute histologic and ultrastructural changes, quantification and regional distribution (transmural, circumferential, transaxial) of damaged myocells from anesthetized, open-chested dogs following one hour of intravenous infusions of saline or increasing doses of isoproterenol (0.1, 1.0, 2.5 micrograms/kg/min) or norepinephrine (4.0 micrograms/kg/min) were investigated. Two predominant subsets of acute contraction band lesions were produced: 'paradiscal' involving aggregation of less than 15 sarcomeres adjacent to the intercalated disc and 'holocytic' involving coagulation of groups of adjoining sarcomeres into transverse bands interspersed with areas of myofibrillar rhexis throughout the cell. Both lesions were distributed as isolated cells or as small foci of myocells surrounded by normal myocardium. Quantification of 'paradiscal' and 'holocytic' contraction band lesions/mm2 of area was used as an index of the severity of catecholamine-induced necrosis. Numbers of 'paradiscal' myocells increased with increasing doses of isoproterenol, while 'holocytic' myocells were not present in any significant numbers until 1.0 microgram/kg/min) and increased further at 2.5 micrograms/kg/min. 'Paradiscal' myocells with both isoproterenol (2.5 micrograms/kg/min) and norepinephrine were distributed with the greatest number in the inner third of the free wall. This gradient was not significant for 'holocytic' lesions. There was, generally, no significant difference in distribution of either type of lesion around the circumference. However, transaxially there was a higher frequency of 'paradiscal' lesions at the apex with norepinephrine. The lesions were identical and present in comparable numbers at both the highest dose of isoproterenol and with norepinephrine. Thus, these two catecholamines result in a similar cardiotoxicity, each with two predominant subsets of lesions, despite their hemodynamic diversities.

Animals↗

PubMed1985

Quantification of deposition of neutrophilic granulocytes on vascular grafts in dogs with 111In-labeled granulocytes.

A new radioisotopic technique has been developed for quantification of deposition of neutrophilic granulocytes on vascular grafts. Nine healthy mongrel dogs underwent bilateral femoral artery resection and reconstruction with grafts of femoral vein and Gore-Tex. Pure granulocytes that had been separated from whole blood by centrifugal elutriation were labeled with 111In-tropolone in plasma. The granulocyte harvesting efficiency was 25 +/- 12%, and the labeling efficiency was 87 +/- 7%. Three hours after injection of labeled granulocytes and 2 hours after reperfusion, the grafts were harvested and cut into several segments for study of areas of anastomoses and midsections. On the basis of the radioactivity in the blood and in anastomotic and graft sections, the area of graft sections, and the neutrophilic granulocyte and differential leukocyte counts, the number of neutrophilic granulocytes adherent to a unit area and the total number of neutrophilic granulocytes on graft sections were calculated. These quantifications of the deposition of neutrophilic granulocytes indicated that the midsections of Gore-Tex grafts retained more neutrophilic granulocytes than did the midsections of vein grafts. Although the anastomotic areas retained more neutrophilic granulocytes than did the midsections of vein grafts, the opposite finding prevailed for the Gore-Tex grafts. A major fraction of neutrophilic granulocytes on Gore-Tex grafts was incorporated into thrombus. Semiquantitative information obtained by scintigraphy of the deposition of neutrophilic granulocytes on vascular grafts also confirmed this observation.

Animals↗

PubMed1985

Non-Hodgkin's lymphoma classification: ultrastructural morphometric studies for the quantification of nuclear compartments in situ.

The condensed chromatin distribution in the nuclei of lymphocytes in non-Hodgkin's lymphoma (NHL) is a key element, along with nuclear size and shape, in the classification of this disease for therapeutic and prognostic purposes. This report describes the ultrastructural comparative quantification of the condensed chromatin and the interchromatinic (nuclear matrix or euchromatin) region in the nuclei of mitogen-stimulated human peripheral T lymphocytes and mouse spleen B lymphocytes, human germinal center lymphocytes, and lymphocytes in ten cases of NHL of a variety of subtypes. The sequential morphologic nuclear changes induced in lymphocytes by mitogens are reflected in human germinal center lymphocyte populations. The common features include the changes in the distribution and volume of condensed chromatin aggregates, as well as the fact that the major increments in nuclear volume during lymphocyte transformation result from increases in the volume of the interchromatinic region. In all subtypes of NHL analyzed morphometrically, subpopulations of lymphocytes were identified in which mean nuclear, condensed chromatin, and interchromatinic volumes were more or less equivalent to those of normal lymphocyte subsets in germinal centers in reactive hyperplasia. However, in NHL the abnormal cytologic characteristics of the nucleus result, at least in part, from a complex interplay of condensed chromatin distribution and amount, and the size of the interchromatinic region. Further complexity is introduced by the fact that in NHL these two nuclear compartments can independently be normal, increased, or reduced in size. Morphometric quantification of lymphocytes in NHL indicates that the interchromatinic (matrix) region of the nucleus is the key element in establishing the nuclear volume of neoplastic lymphocytes. The structural and functional, ribonucleoprotein interchromatinic region of the nucleus was visualized in normal and neoplastic lymphocytes by regressive uranyl-EDTA staining. Quantitative morphometric analysis indicates that the cytologic appearance of neoplastic lymphocytes, even within subtypes of NHL, is heterogeneous and that condensed chromatin quantity and distribution may be more critical than nuclear size in distinguishing between certain subtypes of NHL. Improvements in the classification of NHL will occur only with understanding of the alterations in the biologic mechanisms controlling gross nuclear organization and the morphologic events of the various differentiation pathways available to antigen-stimulated lymphocytes.

Animals↗

PubMed1988

Screening, confirmation and quantification of boldenone sulfate in equine urine after administration of boldenone undecylenate (Equipoise).

Methods for screening by thin-layer chromatography, quantification by high-performance liquid chromatography with ultraviolet detection and confirmation by gas chromatography-mass spectrometry of boldenone sulfate in equine urine after administration of boldenone undecylenate (Equipoise) are presented. Sample work-up was done with C18 liquid-solid extraction followed by solvolytic cleavage of the sulfate ester. Confirmatory evidence of boldenone sulfate in equine urine was obtained from 2 h to 42 days following a therapeutic intramuscular dose of Equipoise. The use of 19-nortestosterone sulfate as the internal standard for quantification of boldenone sulfate is discussed.

Animals↗

PubMed1988

Improved high-performance liquid chromatographic assay for the quantification of 5-bromo-2'-deoxyuridine and 5-bromouracil in plasma.

A sensitive and specific procedure using high-performance liquid chromatography (HPLC) was developed for the quantification of 5-bromo-2'-deoxyuridine (BUdR) and 5-bromouracil (BU) in plasma. BUdR and BU were first extracted with a mixture of ethyl acetate and 2-propanol from plasma presaturated with solid ammonium sulfate. Following evaporation of the organic extract, the remaining residue was reconstituted in saturated ammonium sulfate solution, washed with a mixture of n-pentane-methylene chloride and re-extracted with the original solvent mixture. The organic extract was evaporated, reconstituted in mobile phase and chromatographed on a regular-bore ODS HPLC column using ultraviolet absorbance detection. The BUdR and BU quantification limits were both 0.1 microM, the mean intra-assay coefficients of variation were 5.0 and 5.6%, respectively, and the mean inter-assay coefficients of variation were 5.4 and 10.7%, respectively. This method was used to determine steady-state femoral arterial and hepatic venous plasma concentrations of BUdR and BU in a patient receiving a continuous intravenous infusion of BUdR (20 mg/kg per day).

Aged↗

PubMed1986

Plasma quantification of quazepam and its 2-oxo and N-desmethyl metabolites by capillary gas chromatography.

The authors have developed a gas chromatographic method for the simultaneous quantification of quazepam in plasma and its two main metabolites, 2-oxoquazepam and N-desmethylquazepam. This method involves an extraction from plasma using butyl acetate, and an analysis by electron-capture detection on a CP-Sil 5 WSCOT capillary column. Intra- and inter-day precision and accuracy were better than 10% for each of these three compounds, even near their detection limit estimated at 0.2 ng/ml. Linearity proved satisfactory between 0.2 and 60-70 ng/ml. For endogenous plasma components, adequate specificity was achieved. Despite some inconveniences, a long analysis time, a progressive saturation of the column owing to a low oven temperature, and a relatively short life-span of the CP-Sil 5 columns, this method was the only one available in the literature for the quantification of quazepam and its metabolites from the same plasma sample. It was successfully applied to phase I studies in healthy volunteers.

Anti-Anxiety Agents↗

PubMed1988

Sensitive quantification of pseudoephedrine in human plasma and urine by high-performance liquid chromatography.

An assay for the selective quantification of pseudoephedrine in human plasma and urine was developed using high-performance liquid chromatography with UV detection at 205 nm. Analyte and internal standard were extracted from alkaline plasma or urine into a mixture of n-hexane and diethyl ether, and the organic phase was back-extracted into dilute acid. The chromatographic system comprises microparticulate cyanopropyl-silica as stationary phase and a ternary solvent mixture with ion-pair reagents as mobile phase. Using 0.25 ml plasma, the lower limit of quantification was 25 ng/ml with excellent linearity up to 1000 ng/ml. In urine, the calibration ranged from 2.5 to 100 micrograms/ml. The selectivity of the method was demonstrated for several pharmaceuticals with similar structures. The validated method was applied to a pharmacokinetic study with a single oral dose of 100 mg of pseudoephedrine in two galenic formulations. Precision and accuracy data of the assay and calculated pharmacokinetic parameters are presented.

Adolescent↗

PubMed1989

Capillary gas chromatographic-mass spectrometric method for the identification and quantification of some benzodiazepines and their unconjugated metabolites in plasma.

A gas chromatographic-mass spectrometric method for the identification and/or quantification of diazepam, clobazam, flunitrazepam, triazolam, midazolam, oxazepam and lorazepam and some of their desmethylated and hydroxylated metabolites in plasma is described. Benzodiazepines were extracted from plasma with butyl acetate at pH 9; the hydroxylated compounds were then silylated with N,O-bis (trimethylsilyltrifluoroacetamide). Analysis was performed using a compact mass-selective detector operating in the electron-impact mode. Depending on the concentration, identification was performed either by direct comparison of the observed mass spectra with reference spectra or by the relative intensities of the most intense and characteristic ions in the selected-ion monitoring (SIM) mode. Quantification was performed in the SIM mode using the most intense ion. The intra-assay precision and accuracy were better than 5-6%; linearity was satisfactory up to 1-2 micrograms/ml. The detection limit was 1-5 ng/ml for most of the benzodiazepines. This method can be easily used in clinical situations when a safe and rapid response is essential for patient treatment.

Benzodiazepines↗

PubMed1987

Quantification of 5- and 15-HPETE's by direct chemical ionization mass spectrometry.

This report describes the application of direct chemical ionization mass spectrometry (DCIMS) to the identification and quantification of 5- and 15-HPETEs. A unique feature of the method is use of a polyimide-coated fused silica fiber that allows vaporization of the hydroperoxides, with very low excess energy, into the plume of the chemical ionization reagent gas plasma. Mass spectra are obtained that allow identification of the nonreduced and nonderivatized free acid forms of 5- and 15-HPETE as well as their quantification from 1 microgram to 100 picograms.

Animals↗