A high content lipidomics method using scheduled MRM with variable retention time window and relative dwell time weightage

Lipids are highly diverse group of biomolecules that play a pivotal role in biological processes. Lipid compositions of bio-fluids are complex, reflecting a wide range of concentration of different lipid classes with structural diversity within lipid species. Varying degrees of chemical complexity makes their identification and quantification challenging. Newer methods are thus, highly desired for comprehensive analysis of lipid species including identification of structural isomers. Herein, we propose a targeted-MRM method for large scale high-throughput lipidomics analysis using a combination of variable retention time window (variable-RTW) and relative dwell time weightage (relative-DTW) for different lipid species. With this method, we were able to detect more than 1000 lipid species (encompassing 18 lipid classes), including different structural isomers of triglyceride, diglyceride, and phospholipids, in a single-run of 24 minutes. The limit of detection varied between 0.245 pmol/L and 1 nmol/L for different lipid classes with 245 fmol/L being lowest for phosphatidylethanolamine while it was highest for diacylglycerol (1 nmol/L). Similarly, the limit of quantitation varied from 291 fmol/L to 2 nmol/L. The recovery of the method is in the acceptable range and the 849 of lipid species were found to have a coefficient of variance (CV) <30%. Using this method we demonstrate that lipids with ω-3 and ω-6 fatty acid chains are altered in individuals with vitamin B12 deficiency.

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Validated Stability Indicating HPTLC Method for the Estimation of Amoxapine in Different Stress Conditions

Current Pharmaceutical Analysis ◽

10.2174/1573412914666171228163122 ◽

2019 ◽

Vol 15 (3) ◽

pp. 273-279

Author(s):

Shweta G. Rangari ◽

Nishikant A. Raut ◽

Pradip W. Dhore

Keyword(s):

High Performance ◽

Limit Of Detection ◽

Degradation Products ◽

Analysis Data ◽

Peak Height ◽

Good Resolution ◽

Stability Indicating ◽

Limit Of Quantitation ◽

Wide Range ◽

Hptlc Method

Background:The unstable and/or toxic degradation products may form due to degradation of drug which results into loss of therapeutic activity and lead to life threatening condition. Hence, it is important to establish the stability characteristics of drug in various conditions such as in temperature, light, oxidising agent and susceptibility across a wide range of pH values.Introduction:The aim of the proposed study was to develop simple, sensitive and economic stability indicating high performance thin layer chromatography (HPTLC) method for the quantification of Amoxapine in the presence of degradation products.Methods:Amoxapine and its degraded products were separated on precoated silica gel 60F254 TLC plates by using mobile phase comprising of methanol: toluene: ammonium acetate (6:3:1, v/v/v). The densitometric evaluation was carried out at 320 nm in reflectance/absorbance mode. The degradation products obtained as per ICH guidelines under acidic, basic and oxidative conditions have different Rf values 0.12, 0.26 and 0.6 indicating good resolution from each other and pure drug with Rf: 0.47. Amoxapine was found to be stable under neutral, thermal and photo conditions.Results:The method was validated as per ICH Q2 (R1) guidelines in terms of accuracy, precision, ruggedness, robustness and linearity. A good linear relationship between concentration and response (peak area and peak height) over the range of 80 ng/spot to 720 ng/spot was observed from regression analysis data showing correlation coefficient 0.991 and 0.994 for area and height, respectively. The limit of detection (LOD) and limit of quantitation (LOQ) for area were found to be 1.176 ng/mL and 3.565 ng/mL, whereas for height, 50.063 ng/mL and 151.707 ng/mL respectively.Conclusion:The statistical analysis confirmed the accuracy, precision and selectivity of the proposed method which can be effectively used for the analysis of amoxapine in the presence of degradation products.

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Validasi Metode Penentuan Benzena, Toluena dan Xilena pada Sampel Udara dan Tanah Menggunakan Kromatografi Gas

ALCHEMY Jurnal Penelitian Kimia ◽

10.20961/alchemy.15.1.25522.177-189 ◽

2019 ◽

Vol 15 (1) ◽

pp. 177

Author(s):

Aman Sentosa Panggabean ◽

Tika Widyastuti ◽

Noor Hindryawati

Keyword(s):

Gas Chromatography ◽

Retention Time ◽

Limit Of Detection ◽

Soil Samples ◽

Analytical Performance ◽

Air Samples ◽

Limit Of Quantitation ◽

Benzene Toluene ◽

Precision And Accuracy

Penelitian tentang validasi metode penentuan kadar benzena, toluena dan xilena pada sampel udara dan tanah dengan menggunakan kromatografi gas telah dilakukan. Untuk mendapatkan hasil pengukuran yang valid, beberapa parameter penting yang berpengaruh dalam validasi metode telah ditentukan. Beberapa parameter penting yang dilakukan adalah penentuan besaran dasar kromatografi yang meliputi: waktu retensi, kapasitas (k’), faktor selektivitas (α) dan kinerja analitik yang meliputi: penentuan linearitas (r), limit deteksi (LOD), limit kuantitasi (LOQ), presisi dan akurasi. Hasil penelitian penentuan kinerja analitik sangat baik ditunjukkan oleh nilai presisi sebagai % KV < 2/3 nilai KV Horwitz, LOD untuk masing-masing senyawa benzena, toluena dan xilena adalah 0,02 mg/L; 0,59 mg/L dan 0,08 mg/L serta LOQ untuk masing-masing senyawa benzena, toluena dan xilena adalah 0,07 mg/L; 1,99 mg/L dan 0,27 mg/L. Akurasi metode ini sangat baik ditunjukkan dengan nilai presentase perolehan kembali masing-masing senyawa benzena, toluena dan xilena untuk sampel tanah sebesar 102,61 ± 4,61%; 101,65 ± 7,41%; 102,15 ± 4,15%, dan untuk sampel udara masing-masing senyawa sebesar 101,69 ± 5,77%; 102,08 ± 5,43% dan 98,55 ± 5,11%. Berdasarkan hasil penelitian ini, metode kromatografi gas dapat digunakan dalam penentuan benzena, toluena dan xilena pada sampel udara dan tanah dengan memberikan hasil yang valid.Validation Method on The Determination of Benzene, Toluene and Xylene in Air and Soil Samples Using Gas Chromatography. The present study investigated the method for the determination of the content of benzene, toluene, and xylene in air and soil samples using gas chromatography. To obtain a valid measurement result, several important parameters that influence the method validation have been determined. The several important parameters carried out are the determination of the basic chromatographic such as retention time, capacity factor (k'), selectivity (α) and analytical performance measurement includes: the determination of linearity (r), limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy. The result of analytical performance of the research are well verified, in which the value of precision was % CV < 2/3 CV Horwitz value, LOD for benzene, toluene, and xylene compound were 0.02 mg/L, 0.59 mg/L, 0.08 mg/L, respectively, and LOQ for benzene, toluene and xylene compounds was 0.07 mg/L, 1.99 mg/L and 0.27 mg/L respectively. This method achieved high accuration, indicated by a percentage of recovery value of benzene, toluene, and xylene for soil samples of 102.61 ± 4.61%; 101.65 ± 7.41%; 102.15 ± 4.15%, and for air samples was 101.69 ± 5.77%, 102.08 ± 5.43%, and 98.55 ± 5.11% respectively. Based on the results of this research, the method presented in this study can be applied for the determination of benzene, toluene, and xylene using gas chromatography in air and soil samples with valid results.

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Determination of Diazepam in Cream Biscuits by Liquid Chromatography

Journal of AOAC International ◽

10.1093/jaoac/87.3.569 ◽

2004 ◽

Vol 87 (3) ◽

pp. 569-572 ◽

Cited By ~ 2

Author(s):

Priyankar Ghosh ◽

Mudiam Mohanakrishna Reddy ◽

Beedu Sashidhar Rao ◽

Rajendra Kumar Sarin

Keyword(s):

Retention Time ◽

Mobile Phase ◽

Limit Of Detection ◽

Linear Regression Analysis ◽

Internal Standard ◽

Reversed Phase ◽

Calibration Plot ◽

Relative Standard ◽

Limit Of Quantitation

Abstract An analytical procedure was developed for the detection and quantitation of diazepam in cream biscuits, which were used to commit crime. The method involves the extraction of diazepam with ethanol at room temperature, and the extract is filtered, evaporated to dryness, and redissolved in the mobile phase, methanol–acetonitrile–tetrahydrofuran–water (15 + 55 + 4 + 26, v/v). The separation is achieved on a C18 reversed-phase column with the mobile phase and diode array detection (λmax) at 230 nm. Medazepam is used as the internal standard is for quantification. The calibration plot for the determination of diazepam is based on linear regression analysis (y = 0.6687x + 0.0372; r2 = 0.995). The limit of detection for diazepam in the biscuit samples was estimated as 600 ng/mL. The limit of quantitation for diazepam was estimated as 1.75 μg/mL. The diazepam detected per piece of biscuit was found to be in the range of 0.27–0.45 mg. Pure diazepam was added to biscuit samples at 3 levels (100 and 500 μg/g, and 1 mg/g), and the recoveries were found to be 95%. The mean retention time of diazepam was 2.7 min and that of medazepam (IS) was 4 min. The relative standard deviations of the diazepam level in the biscuit samples were estimated to be 0.4% for retention time and 1.02% for peak area in intraday analysis, whereas the corresponding values were and 0.61 and 2.34% in interday analysis. The method is rapid and reliable for qualitative and quantitative analysis of cream biscuits laced with diazepam, and it can be used by law enforcement laboratories for routine analysis.

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Comparison of Four C18 Columns for the Liquid Chromatographic Determination of Deoxynivalenol in Naturally Contaminated Wheat

Journal of AOAC International ◽

10.1093/jaoac/90.4.995 ◽

2007 ◽

Vol 90 (4) ◽

pp. 995-999 ◽

Cited By ~ 5

Author(s):

El-Sayed M Abdel-Aal ◽

Kaddus Miah ◽

J Christopher Young ◽

Iwona Rabalski

Keyword(s):

Liquid Chromatography ◽

Retention Time ◽

Limit Of Detection ◽

Charge Ratio ◽

Enzyme Linked Immunosorbent Assay ◽

Atomic Mass Unit ◽

Column Temperature ◽

Mass Unit ◽

Relative Standard ◽

Limit Of Quantitation

Abstract Three long and 1 short reversed-phase C18 columns were compared for separation of deoxynivalenol (DON) in extracts of naturally contaminated wheat samples using liquid chromatography with ultraviolet detection and liquid chromatography/mass spectrometry (LC/MS). Among the 3 long columns used, a Symmetry C18 column with an isocratic solvent mixture of wateracetonitrilemethanol (90 + 5 + 5, v/v/v) gave the best separation for DON without interferences from other compounds in the wheat extracts. The Symmetry short (75 mm) column was comparable with the long column (250 mm) in resolving DON but significantly reduced retention time (i.e., 5.8 versus 16.3 min). Increasing the column temperature from 25 to 45C resulted in a further reduction in retention time. Identity of DON in the wheat extracts and standard solutions was confirmed by LC/MS in the positive ion mode, whereby DON appeared with an (M+1)+ ion at a mass-to-charge ratio of 297 plus fragment ions associated with loss of water and/or a 30 atomic mass unit (amu) CH2O fragment. The Symmetry short column was also capable of separating a mixture of the mycotoxins DON, 15-acetyl-DON, nivalenol, and zearalenone by use of a combination of an isocratic and gradient solvent system. The overall method showed high precision, exhibiting a relative standard deviation of 4.8%, limit of detection of 50 ng/g, and limit of quantitation of 165 ng/g. It was significantly correlated with enzyme-linked immunosorbent assay analysis, indicating its appropriateness for safety and quality assurance of wheat and related grains.

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Stability Indicating HPTLC Method for Sofosbuvir and Daclatasvir in Combination

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/ijpsn.2020.13.6.8 ◽

2020 ◽

Vol 13 (6) ◽

pp. 5234-5242

Author(s):

Mrinalini Damle ◽

Nivedita Pawar

Keyword(s):

Limit Of Detection ◽

Fluorescent Light ◽

Simultaneous Estimation ◽

Major Advance ◽

Good Resolution ◽

Combination Tablet ◽

Stability Indicating ◽

Limit Of Quantitation ◽

Wide Range ◽

Hptlc Method

Direct acting antiviral agents represent the major advance in treatment of hepatitis C virus (HCV) infection. Daclatasvir with sofosbuvir that are co-administrated once per day oral dose has been reported to achieve a high rate of virological response in patients with HCV genotype 1, 2 or 3. So, the basic objective of a research involved development and validation of stability indicating HPTLC Method for simultaneous estimation of Sofosbuvir and Daclatasvir available in market in the form of combination tablet. Samples were applied on HPTLC aluminium plates precoated with silica gel 60 F254 (250μm thickness). Mobile phase consists of ethyl acetate: isopropanol in the ratio 9:1v/v. A good resolution was observed between peaks of both the drugs. The retention factor for Sofosbuvir is about 0.51 ±0.02. And for Daclatasvir is about 0.30 ± 0.02. Deuterium lamp as a source of radiation at the wavelength of 260 and 318 nm for analysis of Sofosbuvir and Daclatasvir, respectively. The proposed method was validated according to ICH guidelines and the results were acceptable for linearity and range, accuracy, precision, robustness, detection limit and quantitation limit. The calibration curves were linear over a wide range of 200-1000 ng/band (r2= 0.991) for Sofosbuvir and 45-225 ng/band (r2=0.990) for Daclatasvir. The limit of detection was found to be 21.17 ng/band and 4.38 ng/band for SOF and DAC and limit of quantitation was 64.18 ng/band and 13.28 ng/band for SOF and DAC respectively. During stress degradation study, it was observed that the Daclatasvir is degraded less in thermal and photolytic condition but more in basic hydrolysis condition. Sofosbuvir was found to be sensitive to all stress conditions except the fluorescent light. The suggested method was successfully applied for analysis of both drugs and excellent recovery results were obtained. Being simple, fast, robust, and economic, the method could be applied to the quality control and routine stability monitoring of Sofosbuvir and Daclatasvir.

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Small Molecule Permeation Across Membrane Channels: Chemical Modification to Quantify Transport Across OmpF

10.26434/chemrxiv.7040354.v1 ◽

2018 ◽

Author(s):

Jiajun Wang ◽

Jayesh Arun Bafna ◽

Satya Prathyusha Bhamidimarri ◽

Mathias Winterhalter

Keyword(s):

Dwell Time ◽

Covalent Binding ◽

Channel Structure ◽

Ion Current ◽

E Coli ◽

Current Fluctuation ◽

Wide Range ◽

Outer Membrane Channel ◽

Biological Channels ◽

Constriction Region

Biological channels facilitate the exchange of small molecules across membranes, but surprisingly there is a lack of general tools for the identification and quantification of transport (i.e., translocation and binding). Analyzing the ion current fluctuation of a typical channel with its constriction region in the middle does not allow a direct conclusion on successful transport. For this, we created an additional barrier acting as a molecular counter at the exit of the channel. To identify permeation, we mainly read the molecule residence time in the channel lumen as the indicator whether the molecule reached the exit of the channel. As an example, here we use the well-studied porin, OmpF, an outer membrane channel from E. coli. Inspection of the channel structure suggests that aspartic acid at position 181 is located below the constriction region (CR) and we subsequently mutated this residue to cysteine, where else cysteine free and functionalized it by covalent binding with 2-sulfonatoethyl methanethiosulfonate (MTSES) or the larger glutathione (GLT) blockers. Using the dwell time as the signal for transport, we found that both mono-arginine and tri-arginine permeation process is prolonged by 20% and 50% respectively through OmpFE181CMTSES, while the larger sized blocker modification OmpFE181CGLT drastically decreased the permeation of mono-arginine by 9-fold and even blocked the pathway of the tri-arginine. In case of the hepta-arginine as substrate, both chemical modifications led to an identical ‘blocked’ pattern observed by the dwell time of ion current fluctuation of the OmpFwt. As an instance for antibiotic permeation, we analyzed norfloxacin, a fluoroquinolone antimicrobial agent. The modulation of the interaction dwell time suggests possible successful permeation of norfloxacin across OmpFwt. This approach may discriminate blockages from translocation events for a wide range of substrates. A potential application could be screening for scaffolds to improve the permeability of antibiotics.

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Development and Validation of Stability-Indicating RP-HPLC Method for the Estimation of Lenalidomide and its Impurities in Oral Solid Dosage Form

Oriental Journal Of Chemistry ◽

10.13005/ojc/350115 ◽

2019 ◽

Vol 35 (1) ◽

pp. 140-149 ◽

Cited By ~ 1

Author(s):

Somana Siva Prasad ◽

G. V. Krishna Mohan ◽

A. Naga Babu

Keyword(s):

Limit Of Detection ◽

Degradation Products ◽

Pharmaceutical Formulations ◽

Hplc Method ◽

Stability Robustness ◽

Rp Hplc ◽

Mobile Phases ◽

Limit Of Quantitation ◽

Successful Separation ◽

Quality By Design Approach

In this study, a novel, simple and precise RP-HPLC method has been developed for the quantitative analysis of Lenalidomide (LLM) in pharmaceutical formulations using analytical quality by design approach. An X-bridge-C18 column (150 mm × 4.6 mm × 3.5 µ) with mobile phases containing a Potassium dihydrogen orthophosphate anhydrous buffer and methanol in the ratio of (90:10 v/v) and (35:65 v/v) are used for the estimation of LLM and its degradation products. The flow rate of 0.8 mL/min is maintained and all degradation studies are performed at 210 nm using photodiode array (PDA) detector. Method Validation is carried out according to International Council for Harmonisation (ICH) guidelines and the parameters namely; precision, accuracy, specificity, stability, robustness, linearity, limit of quantitation (LOQ) and limit of detection (LOD) are evaluated. The present developed RP-HPLC method shows the purity angle of peaks is less than their threshold angle, signifying that it to be suitable for stability studies. Hence, the developed method can be used for the successful separation of LLM and its impurities in the pharmaceutical dosage formulations.

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Computational Design of a Molecularly Imprinted Polymer for the Biomonitoring of the Organophosphorous Metabolite Chlorferron

Biosensors ◽

10.3390/bios11060192 ◽

2021 ◽

Vol 11 (6) ◽

pp. 192

Author(s):

Bakhtiyar Qader ◽

Issam Hussain ◽

Mark Baron ◽

Rebeca Jiménez-Pérez ◽

Guzmán Gil-Ramírez ◽

...

Keyword(s):

Biological Samples ◽

Limit Of Detection ◽

Computational Design ◽

Signal Change ◽

Highly Sensitive ◽

Limit Of Quantitation ◽

Molecularly Imprinting Polymers ◽

Mip Sensor ◽

Parasitic Mites

Coumaphos is an organophosphorus compound used as insecticide and frequently used by beekeepers for the management of parasitic mites. The most important metabolite, chlorferron (CFN), has been identified in biological samples and foodstuff. The need to quickly identify the presence of typical metabolites, as an indication of interaction with coumaphos has driven the need to produce a highly sensitive electrochemical method for chlorferron analysis, based on molecularly imprinting polymers (MIP) technology. It showed irreversible behaviour with mixed diffusion/adsorption-controlled reactions at the electrode surface. A monoelectronic mechanism of reaction for oxidation has also been suggested. The linear range observed was from 0.158 to 75 µM. Median precision in terms of %RSD around 3% was also observed. For DPV, the limit of detection (LOD) and the limit of quantitation (LOQ) for the CFN-MIP were 0.158 µM and 0.48 µM, respectively. The obtained median % recovery was around 98%. The results were also validated to reference values obtained using GC-MS. Urine and human synthetic plasma spiked with CFN were used to demonstrate the usability of the method in biological samples, showing the potential for biomonitoring. The developed imprinted sensor showed maximum signal change less than 16.8% when related metabolites or pesticide were added to the mix, suggesting high selectivity of the MIP sensor toward CFN molecules. The results from in vitro metabolism of CMP analysed also demonstrates the potential for detection and quantification of CFN in environmental samples. The newly developed CFN-MIP sensor offers similar LoDs than chromatographic methods with shorter analysis time.

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PERFORMANCE CHARACTERISTICS OF A FRET-BASED IMMUNOASSAY FOR QUANTITATION OF INFLIXIMAB ON A POINT-OF-CARE INSTRUMENT SYSTEM

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa347.135 ◽

2021 ◽

Vol 27 (Supplement_1) ◽

pp. S57-S57

Author(s):

Edgar Ong ◽

Ruo Huang ◽

Richard Kirkland ◽

Michael Hale ◽

Larry Mimms

Keyword(s):

Whole Blood ◽

Limit Of Detection ◽

Point Of Care ◽

Quantitative Detection ◽

Therapeutic Drug ◽

Analytical Sensitivity ◽

Sample Type ◽

Hook Effect ◽

Limit Of Quantitation ◽

Assay Precision

Abstract Introduction A fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassay was developed for the quantitative detection of infliximab (IFX) and biosimilars for use in therapeutic drug monitoring using only 20 µL of fingerstick whole blood or serum at the point-of-care. The Procise IFX assay and ProciseDx analyzer are CE-marked. Studies were performed to characterize analytical performance of the Procise IFX assay on the ProciseDx analyzer. Methods Analytical testing was performed by spiking known amounts of IFX into negative serum and whole blood specimens. Analytical sensitivity was determined using limiting concentrations of IFX. Linearity was determined by testing IFX across the assay range. Hook effect was assessed at IFX concentrations beyond levels expected to be found within a patient. Testing of assay precision, cross-reactivity and potential interfering substances, and biosimilars was performed. The Procise IFX assay was also compared head-to-head with another CE-marked assay: LISA-TRACKER infliximab ELISA test (Theradiag, France). The accuracy of the Procise IFX assay is established through calibrators and controls traceable to the WHO 1st International Standard for Infliximab (NIBSC code: 16/170). Results The Procise IFX assay shows a Limit of Blank, Limit of Detection, and Lower Limit of Quantitation (LLoQ) of 0.1, 0.2, and 1.1 µg/mL in serum and 0.6, 1.1, and 1.7 µg/mL in whole blood, respectively. The linear assay range was determined to be 1.7 to 77.2 µg/mL in serum and whole blood. No hook effect was observed at an IFX concentration of 200 µg/mL as the value reported as “>ULoQ”. Assay precision testing across 20 days with multiple runs and reagent lots showed an intra-assay coefficient of variation (CV) of 2.7%, an inter-assay CV of <2%, and a total CV of 3.4%. The presence of potentially interfering/cross-reacting substances showed minimal impact on assay specificity with %bias within ±8% of control. Testing of biosimilars (infliximab-dyyb and infliximab-abda) showed good recovery. A good correlation to the Theradiag infliximab ELISA was obtained for both serum (slope=1.01; r=0.99) and whole blood (slope=1.01; r=0.98) samples (Figure 1). Conclusion Results indicate that the Procise IFX assay is sensitive, specific, and precise yielding results within 5 minutes from both whole blood and serum without the operator needing to specify sample type. Additionally, it shows very good correlation to a comparator assay that takes several hours and sample manipulation to yield results. This makes the Procise IFX assay ideal for obtaining fast and accurate IFX quantitation, thus allowing for immediate drug level dosing decisions to be made by the physician during patient treatment.

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Siphon-Controlled Automation on a Lab-on-a-Disc Using Event-Triggered Dissolvable Film Valves

Biosensors ◽

10.3390/bios11030073 ◽

2021 ◽

Vol 11 (3) ◽

pp. 73

Author(s):

Brian D. Henderson ◽

David J. Kinahan ◽

Jeanne Rio ◽

Rohit Mishra ◽

Damien King ◽

...

Keyword(s):

Risk Factor ◽

Limit Of Detection ◽

C Reactive Protein ◽

Cvd Risk ◽

Low Resource Settings ◽

Low Resource ◽

Reactive Protein ◽

Limit Of Quantitation ◽

Chemiluminescent Immunoassay ◽

Event Triggered

Within microfluidic technologies, the centrifugal microfluidic “Lab-on-a-Disc” (LoaD) platform offers great potential for use at the PoC and in low-resource settings due to its robustness and the ability to port and miniaturize ‘wet bench’ laboratory protocols. We present the combination of ‘event-triggered dissolvable film valves’ with a centrifugo-pneumatic siphon structure to enable control and timing, through changes in disc spin-speed, of the release and incubations of eight samples/reagents/wash buffers. Based on these microfluidic techniques, we integrated and automated a chemiluminescent immunoassay for detection of the CVD risk factor marker C-reactive protein displaying a limit of detection (LOD) of 44.87 ng mL−1 and limit of quantitation (LoQ) of 135.87 ng mL−1.

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