Novel Method For Quantification Research Articles

Detection and quantification of antiviral drug tenofovir using silver nanoparticles and surface enhanced Raman spectroscopy (SERS) with spatially resolved hotspot selection

This study introduces a convenient and ultra-sensitive method of detection and quantification of the antiviral drug, tenofovir (TFV), by surface-enhanced Raman spectroscopy (SERS). Novel spatially resolved instrumentation for spectral acquisition and subsequent statistical analysis for hot spot selection was developed for convenient quantification of TFV in an aqueous matrix. Methods of statistical analysis include the use of partial least squares (PLS) regression vector analysis and spectral ranking by quality indices computed using CHAOS theory. Hydroxylamine-reduced Ag colloidal nanoparticles evaporated to dryness on an aluminum well-plate were used as the SERS substrate. To our knowledge, quantification of TFV down to 25 ng/mL by SERS, comprising clinically relevant concentrations, has not been previously reported. Furthermore, in this work we propose a novel method of quantification of aqueous TFV standards by SERS using statistical treatment of data by PLS and CHAOS theory. Based on these data, we propose future studies to develop a method of TFV detection and quantification in biological samples, beneficial to clinicians for rapid assessment of drug adherence during the treatment and prevention of viral diseases.

A novel method for quantification of cefazolin local tissue concentration in blood, fat, synovium, and bone marrow using liquid chromatography − mass spectrometry

To be effective, the concentration of antibiotic used must exceed the minimum inhibitory concentration (MIC) against infecting organisms at and in the surgical site. Few studies follow antibiotic levels for tissues that are manipulated during surgery. The aim of this work was to develop and validate a novel LC-MS method as well as an efficient extraction technique for the quantification of cefazolin in local tissues and whole blood. This method uses the same efficient extraction method across multiple tissue types affected by orthopedic surgery: blood, fat, synovium, and bone marrow. The ability to quantify cefazolin in these tissues will help identify surgical techniques and antibiotic dosing protocols that better protect patients from infection. The internal standard, 13C2,15N-cefazolin, co-elutes with cefazolin, and was used in calibration curves and tissue extracts as well as for cefazolin recovery and matrix effects. The protocol was rigorously tested, including measurements of reproducibility and calibration curve quality. The recovery of the extraction method ranges from 94% to 113% across all sample types. There is little to no matrix effect on cefazolin signal (98–120%). The developed method was used to determine cefazolin concentrations in tissues of 10 patients undergoing a total knee replacement. Cefazolin blood concentrations were approximately 500 times higher than in adipose, synovium, and bone marrow tissues. This clinical data shows that although the minimum inhibitory concentration is largely surpassed in blood, the concentration of cefazolin in fat, synovium, and bone marrow could be insufficient during a knee replacement. This method of cefazolin quantification will help surgeons optimize antibiotic concentrations in the local tissues during knee replacement surgery and potentially reduce serious post-surgical infections.

Circulating Brain Injury Biomarkers: A Novel Method for Quantification of the Impact on the Brain After Tumor Surgery.

Clinical methods to quantify brain injury related to neurosurgery are scarce. Circulating brain injury biomarkers have recently gained increased interest as new ultrasensitive measurement techniques have enabled quantification of brain injury through blood sampling. To establish the time profile of the increase in the circulating brain injury biomarkers glial fibrillary acidic protein (GFAP), tau, and neurofilament light (NfL) after glioma surgery and to explore possible relationships between these biomarkers and outcome regarding volume of ischemic injury identified with postoperative MRI and new neurological deficits. In this prospective study, 34 adult patients scheduled for glioma surgery were included. Plasma concentrations of brain injury biomarkers were measured the day before surgery, immediately after surgery, and on postoperative days 1, 3, 5, and 10. Circulating brain injury biomarkers displayed a postoperative increase in the levels of GFAP ( P < .001), tau ( P < .001), and NfL ( P < .001) on Day 1 and a later, even higher, peak of NFL at Day 10 ( P = .028). We found a correlation between the increased levels of GFAP, tau, and NfL on Day 1 after surgery and the volume of ischemic brain tissue on postoperative MRI. Patients with new neurological deficits after surgery had higher levels of GFAP and NfL on Day 1 compared with those without new neurological deficits. Measuring circulating brain injury biomarkers could be a useful method for quantification of the impact on the brain after tumor surgery or neurosurgery in general.

758 A novel method for quantification of major skin metabolites from tapes strips

758 A novel method for quantification of major skin metabolites from tapes strips

PTT-quant: a new method for direct identification and absolute quantification of premature transcription termination events, following the example of bacterial riboswitches

Regulation of gene expression by premature termination of transcription has been well described in all domains of life, including metazoans, yeast, plants, and bacteria. Although methods for identification of such regulatory events by sequencing are available, the focused biochemical studies of the mechanism are hampered by lack of highly sensitive and accurate experimental methods. Here, we propose a new method for absolute quantification of premature transcription termination events, PTT-quant. It is based on highly sensitive two-step digital droplet PCR protocol, coupled with normalized cDNA synthesis attained by site-specific pre-cleavage of investigated transcripts with RNase H. As a consequence, our method enables the reliable and sensitive quantification of both, prematurely terminated and full-length transcripts. By application of our method to investigation of transcriptional riboswitches in Bacillus subtilis, we were able to precisely measure the dynamics of S-adenosylmethionine (SAM) riboswitch induction, which turned to be ~ 23% higher in comparison the results obtained without cDNA synthesis normalization.Key points• A novel method for quantification of premature transcription termination events was established.• PTT-quant measures absolute concentration of full-length and terminated transcripts.• RNase H and the digital droplet PCR technique is used in PTT-quant.

Novel method for quantification of lipoprotein(a)-cholesterol: Implications for improving accuracy of LDL-C measurements

Background and Aims: Current methods for determining “LDL-C” in clinical practice measure the cholesterol content of both LDL and lipoprotein(a) [Lp(a)-C]. Prior studies suggested Lp(a) mass contains ∼30% cholesterol. A high-throughput, sensitive and rapid method to quantitate Lp(a)-C and improve the accuracy of “LDL-C” was developed.

Novel method for quantification of lipoprotein(a)-cholesterol: implications for improving accuracy of LDL-C measurements

Current methods for determining “LDL-C” in clinical practice measure the cholesterol content of both LDL and lipoprotein(a) [Lp(a)-C]. We developed a high-throughput, sensitive, and rapid method to quantitate Lp(a)-C and improve the accuracy of LDL-C by subtracting for Lp(a)-C (LDL-Ccorr). Lp(a)-C is determined following isolation of the Lp(a) on magnetic beads linked to monoclonal antibody LPA4 recognizing apolipoprotein(a). This Lp(a)-C assay does not detect cholesterol in plasma samples lacking Lp(a) and is linear up to 747 nM Lp(a). To validate this method clinically over a wide range of Lp(a) (9.0–822.8 nM), Lp(a)-C and LDL-Ccorr were determined in 21 participants receiving an Lp(a)-specific lowering antisense oligonucleotide and in eight participants receiving placebo at baseline, at 13 weeks during peak drug effect, and off drug. In the groups combined, Lp(a)-C ranged from 0.6 to 35.0 mg/dl and correlated with Lp(a) molar concentration (r = 0.76; P < 0.001). However, the percent Lp(a)-C relative to Lp(a) mass varied from 5.8% to 57.3%. Baseline LDL-Ccorr was lower than LDL-C [mean (SD), 102.2 (31.8) vs. 119.2 (32.4) mg/dl; P < 0.001] and did not correlate with Lp(a)-C. It was demonstrated that three commercially available “direct LDL-C” assays also include measures of Lp(a)-C. In conclusion, we have developed a novel and sensitive method to quantitate Lp(a)-C that provides insights into the Lp(a) mass/cholesterol relationship and may be used to more accurately report LDL-C and reassess its role in clinical medicine.

Tunable plant-based materials via in vitro cell culture using a Zinnia elegans model

Abstract Current systems for plant-based biomaterial production are inefficient and place unsustainable demands on environmental resources. This work proposes a novel solution to these shortcomings based on selective cultivation of tunable plant tissues using scalable, land-free techniques unconstrained by seasonality, climate, or local resource availability. By limiting biomass cultivation to only desirable plant tissues, ex planta farming promises to improve yields while reducing plant waste and competition for arable land. Employing a Zinnia elegans model system, this work provides the first proof-of-concept demonstration of isolated, tissue-like plant material production in vitro by way of gel-mediated cell culture. Parameters governing cell development and morphology including hormone concentrations, medium pH, and initial cell density are optimized and implemented to demonstrate the tunability of cultured biomaterials at cellular and macroscopic scales. Targeted deposition of cell-doped, nutrient-rich gel scaffolds via casting and 3D bioprinting enable biomaterial growth in near-final form, reducing downstream processing requirements. These investigations demonstrate the implementation of plant cell culture in a new application space, propose novel methods for quantification and evaluation of cell development, and characterize morphological developments in response to critical culture parameters—illustrating the feasibility and potential of the proposed techniques.

The effect of silver nanoparticles on the penetration properties of the skin and quantification of their permeation through skin barrier

Transdermal delivery of drugs represents a non-invasive alternative treatment used not only for skin diseases. As one of the possible penetration enhancing agents, various types of nanoparticles (NPs) could be used. Silver NPs (AgNPs) could be used for some medical purposes considering their antibacterial and antiinflammatory properties. We demonstrate a novel method of quantification of permeated AgNPs, detection of AgNPs dissolving while passing the skin, and examination of interactions between skin and systems with AgNPs. Several AgNPs (exhibiting defined mean diameters of 20, 40, 60, and 100 nm) were added individually to the pure solvents commonly used in pharmaceuticals, namely ethanol, methanol, dimethyl sulfoxide, and demineralized water. AgNP dispersions in different solvents were applied to untreated samples of the skin. Attenuated total reflection technique was used for monitoring the kinetic series of infrared spectra to elucidate the time-dependent changes in the uppermost layer of the skin. The depth profiling spectra series were measured using confocal Raman microspectrometer. All recorded vibrational spectra were evaluated by multivariate statistical methods. A strong influence of AgNP size on the structural changes of the skin surface was evident. The largest changes of the skin structure were caused by the 20-nm and 40-nm AgNPs. Permeation of used AgNPs was studied on vertical Franz diffusion cells with detection of permeated AgNPs by new method based on single-particle inductively coupled plasma mass spectrometry.

A study of curling in rib-knit constructions

Rib-knit fabrics are among the fundamental structures which can be produced on the double-bed knitting machine. Ribs are very popular textiles because of certain crucial properties, i.e. extensive shrinkage in width and dimensional stability of the structure, which are not observed in the single jersey fabrics. In this study, an analysis of the physical and the mechanical properties of balanced rib-knitted fabrics has been presented. The loop structure and dimensions were also quantified. To explain the properties of rib-knitted fabrics, we have proposed and employed the phenomenon of curling. In rib-knitted fabrics, curling happens in the local regions of the fabric. Higher rib structures exhibited greater curling tendencies. Consequently, a novel method of quantification of the curling in ribs was proposed, i.e. the concept of ‘de-curling energy’. This proposed notion of de-curling energy was found to be applicable to all fabric designs. The experimental results were found to be coherent with the proposed theory.

Multiplexed Proteomic Analysis for Diagnosis and Screening of Five Primary Immunodeficiency Disorders From Dried Blood Spots.

Early detection of Primary Immunodeficiencies Disorders (PIDDs) is of paramount importance for effective treatment and disease management. Many PIDDs would be strong candidates for newborn screening (NBS) if robust screening methods could identify patients from dried blood spots (DBS) during the neonatal period. As majority of congenital PIDDs result in the reduction or absence of specific proteins, direct quantification of these target proteins represents an attractive potential screening tool. Unfortunately, detection is often limited by the extremely low protein concentrations in blood cells and limited blood volume present in DBS. We have recently developed a robust novel method for quantification of low abundance proteins in DBS for PIDDs using peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-SRM). Here, we further generated a multiplexed Immuno-SRM panel for simultaneous screening of eight signature peptides representing five PIDD-specific and two cell-type specific proteins from DBS. In samples from 28 PIDD patients including two carriers, representing X-Linked Agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), X-Linked Chronic Granulomatous Disease (XL-CGD), DOCK8 Deficiency and ADA deficiency, peptides representing each disease are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. Also included in the multiplex panel are cell specific markers for platelets (CD42) and Natural Killer Cells (CD56). In patients with WAS, CD42 levels were found to be significantly reduced consistent with characteristic thrombocytopenia. A patient with WAS analyzed before and after bone marrow transplant showed normalized WAS protein and platelet CD42 after treatment highlighting the ability of immuno-SRM to monitor the effects of PIDD treatment. The assay was readily reproduced in two separate laboratories with similar analytical performance and complete agreement in patient diagnosis demonstrating the effective standardized methods. A high-throughput Immuno-SRM method screens PIDD-specific peptides in a 2.5-min runtime meeting high volume NBS workflow requirements was also demonstrated in this report. This high-throughput method returned identical results to the standard Immuno-SRM PIDD panel. Immuno-SRM peptide analysis represents a robust potential clinical diagnostic for identifying and studying PIDD patients from easily collected and shipped DBS and supports a significant potential for early PIDD diagnosis through newborn screening.

Measurement and analysis of photonuclear reactions on thick target samples of biological importance

A novel method for quantification of trace elements in herbal samples using photon activation analysis is reported. Seven trace elements have been detected and their concentrations have been estimated from residue yields after the photo-nuclear reaction. This method can complement the conventional neutron activation analysis for trace elemental detection. The data is useful for setting referral standards for quality assurance of herbs and herbal formulations commercially available for therapeutic purposes. This is a relatively simple, novel and sensitive method for trace elemental analysis which can be scaled to suit industrial and statutory requirements of standardization and quality control.

Counting R‐loops: A Novel Quantification Method for Nuclear DNA:RNA Hybrids

R‐loops are three stranded nucleic acid structures that form during transcription; when the nascent strand of RNA invades the replication bubble, hybridizes with the DNA template strand and displaces the non‐template strand. For many years, these DNA:RNA hybrids were thought to only be byproducts of transcription. In recent years, however, it has been shown that R‐loops play many roles in diverse cellular processes, such as immunoglobulin class‐switching recombination, epigenetic regulation, and in DNA replication. R‐loop mis regulation can lead to DNA damage and genomic instability. Additionally, R‐loops have been shown to play a role in several neurodegenerative diseases. Although, our knowledge of R‐loops has grown in the last few years, detection and quantification of R‐loops can still be difficult and inaccurate. Here, we provide a novel method for quantification of nuclear R‐loops based on cellular fractionation and compare it with commonly used R‐loop quantification methods.Support or Funding InformationResearch was funded by HHMI Research Scholars program at NMSU, Cowboys for Cancer Research and the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Establishment of a rapid and simple liquid chromatography tandem mass spectrometry method for measuring aldosterone in urine

Aldosterone (ALD) measurement plays a critical role in screening and diagnosis of primary aldosteronism. A variety of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for serum ALD have been developed. However, the few LC-MS/MS methods for 24 h urinary ALD that have been reported require tedious sample pretreatment procedure. This study aimed to establish a novel method for quantification of ALD in urine by LC-MS/MS. Urine samples from study participants were hydrolyzed and subjected to anion exchange solid phase extraction (SPE) followed by the detection of eluates by the negative electro-spray ionization and multiple reaction monitor modes. The established method was compared with two CLIA methods and 114 participants (M:39, F:75) were recruited to study the distribution of urinary ALD in an apparently healthy population. The pretreatment time was reduced to 4 h and the total run time for each sample was 4.5 min. The linearity of the method was in the range of 2–2000 pg/mL (r > 0.999); within laboratory coefficient variation (CV) and repeatability were both <5% and recovery was within 100% ± 10%. The established method showed good consistency with two CLIA methods with the correlation coefficients of 0.993(DiaSorin) and 0.980(Auto), respectively. In the 114 apparent healthy individuals, urinary ALD was 0.74–17.09 μg/24 h (P2.5–P97.5). The method showed an excellent performance of the method, prompting us to conclude that this method may be adopted in the clinic for routine testing and analysis of ALD in urine samples.

A Novel Method for the Quantification of White Wine Mannoproteins by a Competitive Indirect Enzyme-Linked Lectin Sorbent Assay (CI-ELLSA).

Mannoproteins (MPs) are cell wall proteoglycans released in wine by yeast during fermentation and ageing on lees, a procedure used for the production of several wines to enrich them in these components with consequences from both a technological and sensory point of view. Given the significance that wine MPs have for wine quality, winemakers would welcome a simple and accurate method for their quantification, as this would allow them to have a better control of this aspect at different winemaking stages. This study develops and validates a novel, simple and accurate method for MPs quantification in white wines based on a competitive indirect enzyme-linked lectin sorbent assay (CI-ELLSA), using the highly mannosylated yeast invertase as the standard. The method utilizes the lectin concanavalin A (ConA) as the immobilized ligand for MPs, and peroxidase, an enzyme rich in mannose, as the competitor for ConA. After addition of the peroxidase substrate, the intensity of the signal produced by the activity of this enzyme (absorbance at 450 nm) is inversely proportional to the amount of mannosylated proteins in the sample. Results have been validated on several wine styles including still, sparkling and sweet wines.

A novel method for quantification of beam's-eye-view tumor tracking performance.

In-treatment imaging using an electronic portal imaging device (EPID) can be used to confirm patient and tumor positioning. Real-time tumor tracking performance using current digital megavolt (MV) imagers is hindered by poor image quality. Novel EPID designs may help to improve quantum noise response, while also preserving the high spatial resolution of the current clinical detector. Recently investigated EPID design improvements include but are not limited to multi-layer imager (MLI) architecture, thick crystalline and amorphous scintillators, and phosphor pixilation and focusing. The goal of the present study was to provide a method of quantitating improvement in tracking performance as well as to reveal the physical underpinnings of detector design that impact tracking quality. The study employs a generalizable ideal observer methodology for the quantification of tumor tracking performance. The analysis is applied to study both the effect of increasing scintillator thickness on a standard, single-layer imager (SLI) design as well as the effect of MLI architecture on tracking performance. The present study uses the ideal observer signal-to-noise ratio (d') as a surrogate for tracking performance. We employ functions which model clinically relevant tasks and generalized frequency-domain imaging metrics to connect image quality with tumor tracking. A detection task for relevant Cartesian shapes (i.e., spheres and cylinders) was used to quantitate trackability of cases employing fiducial markers. Automated lung tumor tracking algorithms often leverage the differences in benign and malignant lung tissue textures. These types of algorithms (e.g., soft-tissue localization - STiL) were simulated by designing a discrimination task, which quantifies the differentiation of tissue textures, measured experimentally and fit as a power-law in trend (with exponent β) using a cohort of MV images of patient lungs. The modeled MTF and NPS were used to investigate the effect of scintillator thickness and MLI architecture on tumor tracking performance. Quantification of MV images of lung tissue as an inverse power-law with respect to frequency yields exponent values of β=3.11 and 3.29 for benign and malignant tissues, respectively. Tracking performance with and without fiducials was found to be generally limited by quantum noise, a factor dominated by quantum detective efficiency (QDE). For generic SLI construction, increasing the scintillator thickness (gadolinium oxysulfide - GOS) from a standard 290μm to 1720μm reduces noise to about 10%. However, 81% of this reduction is appreciated between 290 and 1000μm. In comparing MLI and SLI detectors of equivalent individual GOS layer thickness, the improvement in noise is equal to the number of layers in the detector (i.e., 4) with almost no difference in MTF. Further, improvement in tracking performance was slightly less than the square-root of the reduction in noise, approximately 84-90%. In comparing an MLI detector with an SLI with a GOS scintillator of equivalent total thickness, improvement in object detectability is approximately 34-39%. We have presented a novel method for quantification of tumor tracking quality and have applied this model to evaluate the performance of SLI and MLI EPID designs. We showed that improved tracking quality is primarily limited by improvements in NPS. When compared to very thick scintillator SLI, employing MLI architecture exhibits the same gains in QDE, but by mitigating the effect of optical Swank noise, results in more dramatic improvements in tracking performance.

A validated method for analyzing polyunsaturated free fatty acids from dried blood spots using LC–MS/MS

Omega–3 and omega–6 polyunsaturated free fatty acids (PUFA–FFA) are precursors to potent downstream lipid mediators that are regulators of inflammation. We describe the development and validation of a novel and sensitive method for quantification of individual PUFA–FFA in a dried blood spot using liquid chromatography tandem mass spectrometry (LC–MS/MS). Lipids were extracted from dried blood spot and six individual PUFA–FFA were quantified by LC–MS/MS using stable isotope dilution analysis with deuterated internal standards. PUFA–FFA concentrations in blood samples from 30 subjects were measured using the new method and compared to the traditional approach of thin layer chromatography followed by gas chromatography with flame ionization detection (TLC–GC). Responses for each PUFA–FFA were linear throughout a range of concentrations expected in clinical samples. Intra–day and inter–day variations for all PUFA–FFA were ≤ 16%. The concentrations of all PUFA–FFA measured by LC–MS/MS were positively correlated with measures of the same PUFA–FFA obtained by a traditional TLC–GC method. This novel method for the quantification of PUFA–FFA extracted from dried blood is sensitive and precise, and accurately measures levels of biologically important PUFA–FFA in blood.

Monitoring of Wild Pseudomonas Biofilm Strain Conditions Using Statistical Characterization of Scanning Electron Microscopy Images

The present paper proposes a novel method of quantification of the variation in biofilm architecture, in correlation with the alteration of growth conditions that include, variations of substrate and conditioning layer. The polymeric biomaterial serving as substrates are widely used in implants and indwelling medical devices, while the plasma proteins serve as the conditioning layer. The present method uses descriptive statistics of FESEM images of biofilms obtained during a variety of growth conditions. We aim to explore here the texture and fractal analysis techniques, to identify the most discriminatory features which are capable of predicting the difference in biofilm growth conditions. We initially extract some statistical features of biofilm images on bare polymer surfaces, followed by those on the same substrates adsorbed with two different types of plasma proteins, viz. Bovine serum albumin (BSA) and Fibronectin (FN), for two different adsorption times. The present analysis has the potential to act as a futuristic technology for developing a computerized monitoring system in hospitals with automated image analysis and feature extraction, which may be used to predict the growth profile of an emerging biofilm on surgical implants or similar medical applications.

Quantification of microRNAs directly from body fluids using a base-stacking isothermal amplification method in a point-of-care device.

MicroRNAs have been proposed to be a class of biomarkers of disease as expression levels are significantly altered in various tissues and body fluids when compared to healthy controls. As such, the detection and quantification of microRNAs is imperative. While many methods have been established for quantification of microRNAs, they typically rely on time consuming handling such as RNA extraction, purification, or ligation. Here we describe a novel method for quantification of microRNAs using direct amplification in body fluids without upstream sample preparation. Tested with a point-of-care device (termed Gene-Z), the presence of microRNA promotes base-stacking hybridization, and subsequent amplification between two universal strands. The base-stacking approach, which was achieved in <60min, provided a sensitivity of 1.4 fmol per reaction. Tested in various percentages of whole blood, plasma, and faeces, precision (coefficient of variation=2.6%) was maintained and comparable to amplification in pristine samples. Overall, the developed method represents a significant step towards rapid, one-step detection of microRNAs.

Quantification of Warfarin in Dried Rat Plasma Spots by High-Performance Liquid Chromatography with Tandem Mass Spectrometry.

This paper presents the development and validation of a novel method for quantification of the oral anticoagulant drug warfarin in dried plasma spots (DPS) by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Blood plasma was chosen as a biological fluid to preclude the influence of the hematocrit on the results of the analysis. A 30 μL sample of rat plasma was placed onto Whatman 903 Protein Saver Card and was allowed to dry. A single DPS is sufficient for preparing eight 3.2 mm discs, each containing approximately 1.5–1.6 μL of plasma. Warfarin extraction from one 3.2 mm disc was carried out by adding 200 μL of the acetonitrile : water mixture (1 : 1, v/v) containing 10 mM NH4COOH (pH 4.0), with incubation on a shaker at 1000 rpm for 1 h at 25°C. After chromatographic separation, warfarin and coumachlor (an internal standard) were measured using negative-ion multiple-reaction monitoring with ion transitions m/z 307 → 161 for warfarin and m/z 341 → 161 for the internal standard. The working range of this method is 10–10,000 ng/mL. Within this range, intra- and interday variability of precision and accuracy was <13% and recovery was 82–99%. The results indicate that the new method requires only small plasma samples and may be useful for pharmacokinetic research on warfarin.