Production of safe cyanobacterial biomass for animal feed using wastewater and drinking water treatment residuals.

The growing interest in microalgae and cyanobacteria biomass as an alternative to traditional animal feed is hindered by high production costs. Using wastewater (WW) as a cultivation medium could offer a solution, but this approach risks introducing harmful substances into the biomass, leading to significant safety concerns. In this study, we addressed these challenges by selectively extracting nitrates and phosphates from WW using drinking water treatment residuals (DWTR) and chitosan. This method achieved peak adsorption capacities of 4.4 mg/g for nitrate and 6.1 mg/g for phosphate with a 2.5 wt% chitosan blend combined with DWTR-nitrogen. Subsequently, these extracted nutrients were employed to cultivate Spirulina platensis, yielding a biomass productivity rate of 0.15 g/L/d, which is comparable to rates achieved with commercial nutrients. By substituting commercial nutrients with nitrate and phosphate from WW, we can achieve a 18 % reduction in the culture medium cost. While the cultivated biomass was initially nitrogen-deficient due to low nitrate levels, it proved to be protein-rich, accounting for 50 % of its dry weight, and contained a high concentration of free amino acids (1260 mg/g), encompassing all essential amino acids. Both in vitro and in vivo toxicity tests affirmed the biomass's safety for use as an animal feed component. Future research should aim to enhance the economic feasibility of this alternative feed source by developing efficient adsorbents, utilizing cost-effective reagents, and implementing nutrient reuse strategies in spent mediums.

Ecological Effects of Benzyl Chloride on Different Korean Aquatic Indigenous Species Using an Artificial Stream Mesocosm Simulating a Chemical Spill.

In this study, an artificial stream mesocosm consisting of a head tank, faster-flowing riffle section, gravel section, pool section, lower-run section, and tail tank was installed to simulate a chemical spill in a river. The responses of freshwater periphyton algae, crustacea (Moina macrocopa), freshwater worm (Limnodrilus hoffmeisteri), benthic midge (Glyptotendipes tokunagai), and fish (Zacco platypus and Aphyocypris chinensis) were observed after exposure to benzyl chloride (classified as an accident preparedness substance, APS) at concentrations of 1, 2, and 4 µL/L for 22.5 h. Higher concentrations increased the inhibition (photosynthetic efficiency decrease) of periphyton algae and the mortality of M. macrocopa, whereas the reproduction of the female cladoceran decreased in the 4 µL/L treatment. Mortality of fish did not occur or was lower (≤20%) at all concentrations; however, toxic symptoms were observed for some time after chemical exposure termination and later, symptoms receded. G. tokunagai mortality increased at all concentrations except the control after seven days, and no significant toxic effects were observed in L. hoffmeisteri. The hazardous concentration of benzyl chloride was calculated as 94 µg/L. This study showed the different sensitivities of each species to benzyl chloride. The findings can assist in environmental risk assessment of APSs after chemical spills to protect Korean aquatic species.

Sensitive Detection and Analysis of Neoantigen-Specific T Cell Populations from Tumors and Blood.

Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.

MATE-Seq: microfluidic antigen-TCR engagement sequencing.

Adaptive immunity is based on peptide antigen recognition. Our ability to harness the immune system for therapeutic gain relies on the discovery of the T cell receptor (TCR) genes that selectively target antigens from infections, mutated proteins, and foreign agents. Here we present a method that selectively labels peptide antigen-specific CD8+ T cells using magnetic nanoparticles functionalized with peptide-MHC tetramers, isolates these specific cells within an integrated microfluidic device, and directly amplifies the TCR genes for sequencing. Critically, the identity of the peptide recognized by the TCR is preserved, providing the link between peptide and gene. The platform requires inputs on the order of just 100 000 CD8+ T cells, can be multiplexed for simultaneous analysis of multiple peptides, and performs sorting and isolation on chip. We demonstrate 1000-fold sensitivity enhancement of detecting antigen-specific TCRs relative to bulk analysis and simultaneous capture of two virus antigen-specific TCRs from a population of T cells.

The potential of a natural biopolymeric flocculant, ε-poly-L-lysine, for harvesting Chlorella ellipsoidea and its sustainability perspectives for cost and toxicity.

The successful production of microalgal biomass requires the precise coordination of many different steps. Cell harvesting is a central process in all methods currently used for the production of microalgal biomass. Therefore, improving the harvesting process itself, and using a harvesting method that is compatible with adjacent steps, is necessary to prevent problems that may occur during downstream processing. This study examined the potential of the cationic biopolymer ε-poly-L-lysine (ε-PLL) for use in the harvest of microalgae (Chlorella ellipsoidea). The effects of ε-PLL concentration and mixing intensity on flocculation efficiency and operating costs were determined. We found that ε-PLL was not toxic to microalgal cells at concentrations of up to 25 mg/L, based on the photosystem II quantum yield. A recovery rate of 95% was achieved using 19 mg/L ε-PLL, and the estimated harvest cost was 20 US$/ton of harvested biomass. Moreover, ε-PLL displayed antimicrobial properties, leaving the harvested biomass intact and pure. Therefore, the use of ε-PLL-induced flocculation appears to be an attractive option when harvesting microalgal biomass for use as low- and high-value commodities for humans or animals.

T cell receptors for the HIV KK10 epitope from patients with differential immunologic control are functionally indistinguishable.

HIV controllers (HCs) are individuals who can naturally control HIV infection, partially due to potent HIV-specific CD8+ T cell responses. Here, we examined the hypothesis that superior function of CD8+ T cells from HCs is encoded by their T cell receptors (TCRs). We compared the functional properties of immunodominant HIV-specific TCRs obtained from HLA-B*2705 HCs and chronic progressors (CPs) following expression in primary T cells. T cells transduced with TCRs from HCs and CPs showed equivalent induction of epitope-specific cytotoxicity, cytokine secretion, and antigen-binding properties. Transduced T cells comparably, albeit modestly, also suppressed HIV infection in vitro and in humanized mice. We also performed extensive molecular dynamics simulations that provided a structural basis for similarities in cytotoxicity and epitope cross-reactivity. These results demonstrate that the differential abilities of HIV-specific CD8+ T cells from HCs and CPs are not genetically encoded in the TCRs alone and must depend on additional factors.

Harvesting and contamination control of microalgae Chlorella ellipsoidea using the bio-polymeric flocculant α-poly-l-lysine.

Microalgae have been extensively studied for the production of various products. However, to date, microalgal biomass has not become economically feasible, mainly due to different issues such as contamination from various sources that occurs during downstream processes, and which leads to low quality biomass with limited application. In this study, to overcome contamination by flocculants and other microorganisms, the cationic biopolymer α-Poly-l-lysine (α-PLL) was applied. The cationic amine moiety and polymeric chain of α-PLL rendered microalgal harvesting efficient. With increasing α-PLL chain length, efficient dose- and time-dependent harvesting was achieved. In addition to efficient flocculation performance, biomass harvested using α-PLL showed suppressed biological contamination through the inherent antimicrobial activity of α-PLL. Thus, it is possible to upgrade the quality and storability of produced microalgal biomass using α-PLL-induced flocculation.

Diffusion tensor imaging of white and grey matter within the spinal cord of normal Beagle dogs: Sub-regional differences of the various diffusion parameters.

Diffusion tensor imaging (DTI) is an advanced diffusion weighted imaging technique that can identify early stage lesions and Wallerian degeneration within the spinal cord; these changes are difficult to recognise on conventional magnetic resonance imaging (MRI). The only DTI parameters previously investigated in dogs are fractional anisotropy and mean diffusivity (MD). The aim of this study was to evaluate multiple DTI parameters in sub-regional areas of the spinal cord in normal Beagles. All imaging data were obtained from the lumbar spinal cord (L1-L3) of ten normal dogs using a 3 Tesla MRI scanner. Transverse multi-shot echo planar imaging sequences (b values = 0 and 800 s/mm(2); 12 directions) were used for DTI. Regions of interest were selected from sub-regions of the white and grey matter, and from the whole spinal cord, in the transverse plane in all DTI maps. The DTI parameters in spinal cord sub-regions in the transverse plane were significantly different amongst the white matter, grey matter and whole spinal cord (P < 0.05 for all DTI parameters except MD), as well as between white matter sub-regions (P < 0.05 for most DTI parameters except radial diffusivity, MD and planar index). DTI-based sub-regional analysis of white and grey matter may be useful for regional evaluation of the dog spinal cord.

Efficient recovery of nitrate and phosphate from wastewater by an amine-grafted adsorbent for cyanobacterial biomass production.

Various types of wastewater have been widely utilized in microalgae and cyanobacteria cultivation for environmental and economic reasons. However, the problems of low cell growth and biomass contamination due to direct use of wastewater remain unresolved. In the present study, nitrate and phosphate were separated from wastewater by adsorption and subsequently used for cyanobacterial biomass production. To this end, an amine-grafted magnetic absorbent was synthesized. The synthesized absorbent recovered ca. 78% nitrate and 93% phosphate from wastewater. Regenerated medium was prepared using recovered nutrients as nitrogen and phosphate sources, which were efficiently assimilated by cyanobacterial culture. Compared to synthetic medium, there was no difference in growth and nutrient removal using regenerated medium. The proposed indirect method of wastewater utilization would prevent contamination of the produced biomass by unfavorable substances, which will broaden its potential applications.

Evaluation of safety and efficacy of adipose-derived stem cells in rat myocardial infarction model using hexadecyl-4-[¹²4I]iodobenzoate for cell tracking.

This study was aimed to evaluate the effect of (124)I-labeling with hexadecyl-4-iodobenzoate (HIB) on gene expression related to cell cycle, DNA repair, transcription, proliferation and differentiation of adipose-derived stem cells (ADSCs). [(124)I]HIB showed high labeling efficiency with ADSCs (51.3±1.3%, 0.3-2.0 Bq/cell) and there is no morphological change of ADSCs. In the microarray analysis of gene expression pattern, differences were not observed between non-labeled and [(124)I]HIB-labeled ADSCs. We demonstrated that (124)I-labeling with HIB did not affect the biological properties of ADSCs.

Adenovirus-mediated expression of human sodium-iodide symporter gene permits in vivo tracking of adipose tissue-derived stem cells in a canine myocardial infarction model.

INTRODUCTION: In vivo tracking of the transplanted stem cells is important in pre-clinical research of stem cell therapy for myocardial infarction. We examined the feasibility of adenovirus-mediated sodium iodide symporter (NIS) gene to cell tracking imaging of transplanted stem cells in a canine infarcted myocardium by clinical single photon emission computed tomography (SPECT). METHODS: Beagle dogs were injected intramyocardially with NIS-expressing adenovirus-transfected canine stem cells (Ad-hNIS-canine ADSCs) a week after myocardial infarction (MI) development. (99m)Tc-methoxyisobutylisonitrile ((99m)Tc-MIBI) and (99m)Tc-pertechnetate ((99m)TcO4(-)) SPECT imaging were performed for assessment of infarcted myocardium and viable stem cell tracking. Transthoracic echocardiography was performed to monitor any functional cardiac changes. RESULTS: Left ventricular ejection fraction (LVEF) was decreased after LAD ligation. There was no significant difference in EF between the groups with the stem cell or saline injection. (125)I uptake was higher in Ad-hNIS-canine ADSCs than in non-transfected ADSCs. Cell proliferation and differentiation were not affected by hNIS-carrying adenovirus transfection. (99m)Tc-MIBI myocardial SPECT imaging showed decreased radiotracer uptake in the infarcted apex and mid-anterolateral regions. Ad-hNIS-canine ADSCs were identified as a region of focally increased (99m)TcO4(-) uptake at the lateral wall and around the apex of the left ventricle, peaked at 2 days and was observed until day 9. CONCLUSIONS: Combination of adenovirus-mediated NIS gene transfection and clinical nuclear imaging modalities enables to trace the fate of transplanted stem cells in infarcted myocardium for translational in vivo cell tracking study for prolonged duration.

A Prospective Randomized Clinical Trial Comparing Bone Union Rate Following Anterior Cervical Discectomy and Fusion Using a Polyetheretherketone Cage: Hydroxyapatite/B-Tricalcium Phosphate Mixture versus Hydroxyapatite/Demineralized Bone Matrix Mixture.

STUDY DESIGN: Prospective randomized noninferiority trial. PURPOSE: To evaluate whether the union rate of anterior cervical discectomy and fusion (ACDF) using a polyetheretherketone (PEEK) cage filled with a mixture of hydroxyapatite (HA) and demineralized bone matrix (DBM) is inferior to that of a mixture of ß-tricalcium phosphate (ß-TCP) and HA. OVERVIEW OF LITERATURE: There have been no clinical trials investigating the outcomes of a mixture of HA and DBM in a PEEK cage in ACDF. METHODS: Eighty-five eligible patients were randomly assigned to group B (n=43), in which a PEEK cage with a mixture of HA and DBM was used, or group C (n=42), in which a PEEK cage with a mixture of HA and ß-TCP was used. The primary study endpoint was the fusion rate, which was assessed with dynamic radiographs and computed tomography (CT) scans. Secondary endpoints included pain intensity using a visual analogue scale, functional outcome using a neck disability index score, laboratory tests of inflammatory profiles, and the infection rate. RESULTS: Seventy-seven patients (38 in group B and 39 in group C) were included in the final analysis. One year postoperatively, bone fusion was achieved in 87% of group B patients and 87% of group C patients on dynamic radiographs, and 87% of group B patients and 72% of group C patients on CT scans (p=1.00 and 0.16, respectively). There were also no between-groups differences with respect to the secondary endpoints. CONCLUSIONS: A HA/DBM mixture inside a PEEK cage can provide noninferior outcomes compared to a HA/TCP mixture in ACDF.

Longitudinal monitoring adipose-derived stem cell survival by PET imaging hexadecyl-4-¹²4I-iodobenzoate in rat myocardial infarction model.

This study aims to monitor how the change of cell survival of transplanted adipose-derived stem cells (ADSCs) responds to myocardial infarction (MI) via the hexadecyl-4-(124)I-iodobenzoate ((124)I-HIB) mediated direct labeling method in vivo. Stem cells have shown the potential to improve cardiac function after MI. However, monitoring of the fate of transplanted stem cells at target sites is still unclear. Rat ADSCs were labeled with (124)I-HIB, and radiolabeled ADSCs were transplanted into the myocardium of normal and MI model. In the group of (124)I-HIB-labeled ADSC transplantation, in vivo imaging was performed using small-animal positron emission tomography (PET)/computed tomography (CT) for 9 days. Twenty-one days post-transplantation, histopathological analysis and apoptosis assay were performed. ADSC viability and differentiation were not affected by (124)I-HIB labeling. In vivo tracking of the (124)I-HIB-labeled ADSCs was possible for 9 and 3 days in normal and MI model, respectively. Apoptosis of transplanted cells increased in the MI model compared than that in normal model. We developed a direct labeling agent, (124)I-HIB, and first tried to longitudinally monitor transplanted stem cell to MI. This approach may provide new insights on the roles of stem cell monitoring in living bodies for stem cell therapy from pre-clinical studies to clinical trials.

Metabolic engineering of Ralstonia eutropha for the production of polyhydroxyalkanoates from sucrose.

A sucrose utilization pathway was established in Ralstonia eutropha NCIMB11599 and R. eutropha 437-540 by introducing the Mannheimia succiniciproducens MBEL55E sacC gene that encodes ß-fructofuranosidase. These engineered strains were examined for the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], respectively, from sucrose as a carbon source. It was found that ß-fructofuranosidase excreted into the culture medium could hydrolyze sucrose to glucose and fructose, which were efficiently used as carbon sources by recombinant R. eutropha strains. When R. eutropha NCIMB11599 expressing the sacC gene was cultured in nitrogen-free chemically defined medium containing 20 g/L of sucrose, a high P(3HB) content of 73.2 wt% could be obtained. In addition, R. eutropha 437-540 expressing the Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene accumulated P(3HB-co-21.5 mol% LA) to a polymer content of 19.5 wt% from sucrose by the expression of the sacC gene and the Escherichia coli ldhA gene. The molecular weights of P(3HB) and P(3HB-co-21.5 mol%LA) synthesized in R. eutropha using sucrose as a carbon source were 3.52 × 10(5) (Mn ) and 2.19 × 10(4) (Mn ), respectively. The engineered R. eutropha strains reported here will be useful for the production of polyhydroxyalkanoates (PHAs) from sucrose, one of the most abundant and relatively inexpensive carbon sources.

High-level conversion of L-lysine into 5-aminovalerate that can be used for nylon 6,5 synthesis.

L-Lysine is a potential feedstock for the production of bio-based precursors for engineering plastics. In this study, we developed a microbial process for high-level conversion of L-lysine into 5-aminovalerate (5AVA) that can be used as a monomer in nylon 6,5 synthesis. Recombinant Escherichia coli WL3110 strain expressing Pseudomonas putida delta-aminovaleramidase (DavA) and lysine 2-monooxygenase (DavB) was grown to high density in fed-batch culture and used as a whole cell catalyst. High-density E. coli WL3110 expressing DavAB, grown to an optical density at 600 nm (OD600 ) of 30, yielded 36.51 g/L 5AVA from 60 g/L L-lysine in 24 h. Doubling the cell density of E. coli WL3110 improved the conversion yield to 47.96 g/L 5AVA from 60 g/L of L-lysine in 24 h. 5AVA production was further improved by doubling the L-lysine concentration from 60 to 120 g/L. The highest 5AVA titer (90.59 g/L; molar yield 0.942) was obtained from 120 g/L L-lysine by E. coli WL3110 cells grown to OD600 of 60. Finally, nylon 6,5 was synthesized by bulk polymerization of ϵ-caprolactam and δ-valerolactam prepared from microbially synthesized 5AVA. The hybrid system demonstrated here has promising possibilities for application in the development of industrial bio-nylon production processes.

Positron emission tomography/computed tomography imaging features of renal cell carcinoma and pulmonary metastases in a dog.

A 9-year-old spayed female cocker spaniel dog was referred for hematuria. A large abdominal mass and multiple pulmonary nodules were identified radiographically. A whole-body 2-deoxy-2-[(18)F]fluoro-d-glucose positron emission tomography/computed tomography (PET/CT) scan revealed intensely increased uptake in a renal mass and the pulmonary nodules. Renal cell carcinoma was diagnosed on histological examination.

Caractéristiques de la tomographie à émission de positrons et de l'imagerie à tomographie par ordinateur des métastases du carcinome cellulaire rénal et pulmonaire chez un chien. Une chienne Cocker spaniel stérilisée âgée de 9 ans a été référée pour l'hématurie. Une grande masse abdominale et des nodules pulmonaires multiples ont été identifiés par radiographie. Un balayage par tomographie à émission de positrons et par tomographie par ordinateur de l'ensemble du corps en utilisant le fluoro-d-2-désoxie-2-glucose [18F] a révélé le captage accru intensif dans une masse rénale et des nodules pulmonaires. Le carcinome des cellules rénales a été diagnostiqué à l'examen histologique.(Traduit par Isabelle Vallières).

Symptomatic steno-occlusion of cerebral arteries and subsequent ischemic events in patients with acute ischemic stroke.

BACKGROUND: We aimed to assess the impact of symptomatic steno-occlusion (SYSO) of cerebral arteries and its characteristics on subsequent ischemic event (SIE) in patients with acute ischemic stroke. METHODS: Using a prospective stroke registry database, we identified consecutive patients with ischemic stroke who were hospitalized within 48 hours of symptom onset. SYSO denoted significant stenosis or occlusion of major cerebral arteries with ischemic lesions at the corresponding arterial territories and was characterized by its location and severity. Primary outcome was SIE that was defined as ischemic progression or recurrence within 1 year. RESULTS: In total, 1546 patients (age, 67.4 ± 13.0 years; median National Institutes of Health Stroke Scale score, 4) were enrolled in this study. The cumulative risk of SIE was 14.5% at 7 days, 14.9% at 14 days, 15.5% at 90 days, and 16.9% at 1 year. Patients with SYSO had significantly higher SIE rates compared with those without SYSO (23.0% versus 11.6%). Of the characteristics of SYSO, the location, not the severity, was significantly associated with SIE (P < .001 and P = .186, respectively). Multiple (adjusted hazard ratio, 5.85; 95% confidence interval, 1.81-18.85), intracranial internal carotid artery (ICA) (3.54; 1.21-8.21), and extracranial ICA SYSO (2.88; 1.01-8.21) raised the risk of SIE. CONCLUSIONS: Subsequent cerebral ischemic events (progression or recurrence) after an acute ischemic stroke occur mostly within several days of stroke onset and is associated with the location, but not the severity, of symptomatic steno-occlusion of cerebral arteries.

Validation of FLAIR hyperintense lesions as imaging biomarkers to predict the outcome of acute stroke after intra-arterial thrombolysis following intravenous tissue plasminogen activator.

BACKGROUND: Intravenous tissue plasminogen activator (tPA) given within 4.5 h of symptom onset is accepted as the standard treatment of ischemic stroke. Persistent occlusion of cerebral arteries despite intravenous thrombolysis and unremitting neurologic deficits lead us to consider additional intra-arterial approaches. The aim of this study was to elucidate the potential of fluid-attenuated inversion recovery (FLAIR) MRI performed during or immediately after intravenous thrombolysis for predicting clinical outcomes of subsequent intra-arterial thrombolysis. METHODS: With a prospective stroke registry database of patients hospitalized in our institution from January 2004 to February 2010, we identified ischemic stroke patients with the following conditions: (1) presentation within 2.5 h of onset, (2) treated with intravenous tPA based on brain CT, (3) persistent occlusion on subsequent MRI/MR angiography, including a FLAIR sequence, and (4) eventually treated with intra-arterial thrombolysis. Demographic, clinical and laboratory findings including initial National Institutes of Health Stroke Scale (NIHSS), follow-up NIHSS at the 7th day or discharge, modified Rankin scale (mRS) score at 3 months, and symptomatic hemorrhagic transformation were captured. FLAIR images were reviewed by 2 investigators blinded to clinical information independently and dichotomized into the absence and presence of FLAIR change within the diffusion-restriction lesions. RESULTS: Of the 57 patients who met these conditions, FLAIR-hyperintense lesions (FHL) were observed in 32 (56.1%). The FHL-negative group was 69.1 ± 12.1 years old on average and the FHL-positive group 67.3 ± 11.0 years old. In both groups, hypertension was the most common vascular risk factor, cardioembolic stroke was the most common subtype, and distal middle cerebral artery was the most common site of occlusion. The incidence of symptomatic hemorrhagic transformation was 4.0% in the FHL-negative group and 9.4% in the FHL-positive group (p = 0.62). NIHSS scores of 0-1 on the 7th day of hospitalization or at discharge were observed in 36% of the FHL-negative group and in 9.4% of the FHL-positive group; mRS scores of 0-1 at 3 months was 32% in the FHL-negative group and 21% in the FHL-positive group. An ordinal logistic regression analysis showed that the presence of FHL was associated with higher 7-day NIHSS scores (adjusted for relevant covariates) but not with higher 3-month mRS scores. CONCLUSIONS: This study suggests that the FHL might be used as imaging biomarker to predict outcomes for additional intra-arterial thrombolysis in patients treated with intravenous tPA.

Direct measurement of active thiol metabolite levels of clopidogrel in human plasma using tris(2-carboxyethyl)phosphine as a reducing agent by LC-MS/MS.

A simple, robust, and rapid LC-MS/MS method has been developed and validated for the simultaneous quantitation of clopidogrel and its active metabolite (AM) in human plasma. Tris(2-carboxyethyl)phosphine (TCEP) was used as a reducing agent to detect the AM as a disulfide-bonded complex with plasma proteins. Mixtures of TCEP and human plasma were deproteinized with acetonitrile containing 10 ng/mL of clopidogrel-d4 as an internal standard (IS). The mixtures were separated on a C18 RP column with an isocratic mobile phase consisting of 0.1% formic acid in acetonitrile and water (90:10, v/v) at a flow rate of 0.3 mL/min. Detection and quantification were performed using ESI-MS. The detector was operated in selected reaction-monitoring mode at m/z 322.0→211.9 for clopidogrel, m/z 356.1→155.2 for the AM, and m/z 326.0→216.0 for the IS. The linear dynamic range for clopidogrel and its AM were 0.05-20 and 0.5-200 ng/mL, respectively, with correlation coefficients (r) greater than 0.9976. Precision, both intra- and interday, was less than 8.26% with an accuracy of 87.6-106%. The validated method was successfully applied to simultaneously analyze clinical samples for clopidogrel and its AM.