Intra-batch Precision Research Articles

An LC‐MS/MS Method Development and Validation for the Quantification of Abametapir in Plasma Samples

ABSTRACTThe major goal of the current research study was to establish a sensitive tandem mass spectrometric method using electrospray ionization and liquid chromatography for quantifying abametapir in biological matrices. An Inertsil ODS chromatographic column with dimensions of 50 × 4.60 mm and 5.0 µm particles size was used to achieve chromatographic elution. Injection volume was set to 10.0 µL. Flow rate and stationary phase oven temperature were retained at 0.70 mL/min and 25.0°C. Isocratic elution was executed with acetonitrile, 0.10% v/v HCOOH, and methanol in a fraction of 20:10:70 (v/v/v) as the mobile phase. On multiple reaction monitoring, precursor‐product ion transitions were monitored at m/z 185.1→106.06 for abametapir and 287.16→269.15 for the abacavir internal standard. The drug's linearity graph had an r2 value of 0.9998 and was rectilinear at concentrations between 2.6 and 104 ng/mL. The inter‐ and intrabatch precision % relative standard deviation values ranged from 2.57 to 4.53. In short, the method that was made has been proven to work and validated as per the regulatory guidelines.

Determination of 22 elements in whole blood by inductively coupled plasma mass spectrometry

Objective: To establish a method for the simultaneous determination of 22 elements, including beryllium, vanadium, chromium, manganese, iron, calcium, magnesium, barium, cobalt, cadmium, copper, zinc, arsenic, selenium, titanium, strontium, nickel, molybdenum, tin, antimony, thallium and lead, in whole blood by inductively coupled plasma mass spectrometry (ICP-MS) . Methods: In September 2023, the analysis conditions were determined by optimizing the detection mode of the instrument, the pretreatment mode and the dilution factor of the samples, etc. Whole blood samples were diluted with a mixture of 0.1% nitric acid and 0.05% triton X-100, and centrifuged at 2000 r/min by high-speed centrifuge for 2 min. The supernatant was taken into inductively coupled plasma mass spectrometer to determine the content of 22 elements, and the detection limit and precision of the method were analyzed. Results: The 22 elements had a good linear relationship in their respective measurement ranges (r=0.9991-0.9999), the detection limit ranged from 0.003 μg/L to 0.012 mg/L. The intra-batch precision ranged from 0.5% to 7.2%, the inter-batch precision ranged from 0.4% to 9.4%, and the average recoveries ranged from 80.6% to 114.9%. Conclusion: ICP-MS method has a good effect on the determination of 22 elements in whole blood. The method is fast and simple, and can be used for clinical detection of multiple elements in whole blood.

Detection of egg white allergy in children by specific IgE microarray chemiluminescence immunoassay

BackgroundAllergen testing has emerged as a pivotal component in prevention and treatment strategies for allergic diseases among children and the utilization of specific IgE (sIgE) through a fully automated chemiluminescent microarray immunoassay (CLMIA) has emerged as a promising trend in the simultaneous detection of multiple allergenic components of children. MethodsThe accuracy and reliability of CLMIA were verified using children’s serum samples that concentrated on allergens. the allergens. The clinical diagnostic practicability of CLMIA was assessed through comprehensive evaluations including measurements of the limit of detection (LOD), intra-batch, and inter-batch precision, linearity analysis, the cross-contamination rate, and the concordance rate with the Phadia system. ResultsAfter the optimization process of CLMIA, the LODs for allergens were calculated to be below 0.01 kU/L, demonstrating the high sensitivity of CLMIA. All components exhibited good linearity within the range of 0.1–100.0 kU/L and the coefficient of determinations (R2 > 0.99). The data of intra-batch precision (<10 %) and inter-batch data (<15 %) illustrated the high reproducibility of CLMIA. The cross-contamination rates for allergens (<0.5 %) showed the high accuracy of CLMIA without interfering. The positive concordance rate between CLMIA and the Phadia system exceeds 90 % with a good negative concordance rate (>85 %) and the Kappa coefficients (>0.8), suggesting the close alignment of CLMIA and the Phadia system and showing the satisfactory clinical potential of CLMIA in children’s allergy disease. ConclusionsThe application of CLMIA has been promising in allergen testing, especially for detecting multiple allergenic components in children.

An effective LC-MS method for the simultaneous determination of a potential anti-rheumatoid arthritis drug, carboxyamidotriazole, and its major metabolite in rat plasma.

Carboxyamidotriazole (CAI) was previously recognized as a well-tolerated anticancer drug. It has also demonstrated significant anti-inflammatory effects in various cell and animal model experiments, prompting its investigation as a potential treatment for rheumatoid arthritis. In this study, the potential biotransformation metabolites of CAI were identified both in vitro and in vivo. A sensitive, specific, and accurate LC-MS method was developed for the quantitative analysis of CAI and its major metabolite, CAI-OH, in rat plasma. CAI, CAI-OH, and telmisartan (used as an internal standard) were separated using a Zorbax SB C18 column. The mobile phase consisted of water (phase A, containing 0.1% formic acid) and acetonitrile (phase B, containing 0.1% formic acid) at a flow rate of 0.2 mL/min. The analytes were examined using a high-resolution mass spectrometer, with detected mass-to-charge ratios of m/z 424.01293 for CAI, m/z 440.00785 for CAI-OH, and m/z 515.24415 for telmisartan. Good linearity was observed within the range of 10-5000 ng/mL. Both inter- and intra-batch precision (relative standard deviation, %) were below 6%, and the accuracy ranged from 94.9% to 106.1%. The analytes remained stable throughout the entire experimental period. This method was successfully applied in a pharmacokinetic study of CAI following oral administration in rats.

Determination of mitomycin C in rat plasma by liquid chromatography-tandem mass spectrometry and its application for determining pharmacokinetics in rat

To develop the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring mitomycin C in rat plasma, samples were processed using solid-phase extraction, with the internal standard being carbamazepine. A reversed phased C18 column was utilized for the LC-MS/MS study, and mobile phases consisting of 0.1 % formic acid in acetonitrile and water were injected into it at a rate of 0.3 mL/min. Multiple reaction monitoring in positive-ion mode with precursor-product ion pairs 335.3 → 242.3 (mitomycin C) and 237.1 → 194.1 (carbamazepine) was employed to quantify the compounds. The linear range in plasma was found to be 10–4000 ng/mL (r2 = 0.992). The inter-batch and intra-batch precision were <14.3 % (LLOQ: 14.7 %) and 13.4 % (LLOQ: 16.1 %), respectively. The recovery and the matrix effect of mitomycin C in plasma were 113 % and 111 %, respectively. Mitomycin C was stable under the conditions of this assay method. In the end, this approach proved effective in a pharmacokinetic investigation with the intravenous and oral administration of mitomycin C to rats.

Simultaneous determination of inflammatory factors SAA and LTF based on stable element labeling and inductively coupled plasma mass spectrometry to aid in the diagnosis of infection

ObjectiveThe clinical value of Serum amyloid A (SAA) and Lactoferrin (LTF) has received significant attention, but their detection methods are inadequate, which limits their application. This study aims to develop a dual detection method based on stable element labeling strategies and inductively coupled plasma mass spectrometry (ICP-MS) for SAA/LTF and to assess whether it can be widely used in clinical practice. MethodsA duplex immunoassay system based on sandwich method was constructed. After optimization, methodological evaluation was performed with the guidelines of Clinical Laboratory Standards Institute (CLSI). Finally, 131 plasma samples were collected to analyze whether the new method is suitable for clinical detection. ResultsThe LoB, LLoQ, ULoQ, and linear range of the assay were 1.09 ng/mL, 3 ng/mL, 1500 ng/mL, 3–1500 ng/mL for SAA and 0.85 ng/mL, 2 ng/mL, 1200 ng/mL, 2–1200 ng/mL for LTF respectively. The recovery rates were 95.01% to 106.26%, the intra-batch precision of low, intermediate, and high-level samples was <8%, and the inter-batch of them was <11%, the deviation of interference test results was less than±10%. The Area Under the Curve (AUC) was 0.9809 for SAA, 0.8599 for LTF, and 0.9986 for combination. ConclusionThe quantitative duplex immunoassay for SAA/LTF has high accuracy, good precision, and high specificity, which meets the clinical testing requirements and can be widely used in clinical practice.

Rapid diagnosis of acute myocardial infarction through integrated microfluidic chips for detection of characteristic targets

Myoglobin (Myo), creatine kinase-MB (CKMB), and cardiac troponin I (cTnI) are crucial biomarkers for diagnosing acute myocardial infarction (AMI) The accurate and rapid detection of these three targets can greatly improve the prognosis of AMI patients. Herein, this study developed a microfluidic immunofluorescence method that can detect all three targets in 10–15 min. Ultrasonic atomization and spray technology are used to modify the surface of the injection-molded microfluidic chip (MFC), which effectively solves the problem of biological cross-linking and antibody immobilization on the MFC surface. In addition, it improves the hydrophilicity of the chip surface, thus enhancing fluid self-driving effect. The linear response towards Myo, CKMB and cTnI range from 5 ng/mL to 500 ng/mL, 1 ng/mL to 70 ng/mL, and 0.05 ng/mL to 30 ng/mL, respectively. The intra-batch precision is ≤ 10%, and the inter-batch precision is ≤ 15%. Furthermore, this method shows good consistency compared with the BECKMAN ACCESS2 chemiluminescent immunoanalyzer. The present work provides an AMI diagnostic method with high sensitivity, good repeatability, high accuracy and simple operation, which can satisfy the needs of clinical diagnosis, and shows promising application prospects.

Pharmacokinetics study of atracurium, dexmedetomidine, midazolam and 1-hydroxymidazolam in patients undergoing acute aortic dissection surgery.

An UPLC-MS/MS method was developed and validated for simultaneous determination of atracurium (ATC), dexmedetomidine (DEX), midazolam (MDZ) and 1-hydroxymidazolam (1-OH-MDZ) and the pharmacokinetics of ATC, DEX, MDZ and 1-OH-MDZ in patients undergoing aortic dissection surgery were investigated. The analytes were extracted by acetonitrile precipitation and separated on an Acquity UPLC BEH C18 column (2.1mm × 50mm, 1.7μm) with a mobile phase of acetonitrile-0.1% formic acid and a gradient mode. In the positive ion mode, the following mass transition pairs were monitored by multiple reaction monitoring (MRM) for the four analytes and IS: m/z 385.1→206.2 for ATC, m/z 201.2→95.1 for DEX, m/z 326.1→291.1 for MDZ, m/z 341.9→324.0 for 1-OH-MDZ, and 284.9→153.9 for diazepam (IS). Seven male patients undergoing aortic dissection surgery received general anesthesia and intravenous administration of ATC, DEX, and MDZ during the surgery. Venous blood was collected at different time points at the end of surgery and after surgery. The concentrations of ATC, DEX, MDZ, and 1-OH-MDZ were detected, and the pharmacokinetic parameters were calculated. The method showed good linearity for each analyte. The inter-batch precision ranged from 1.37% to 9.87% and the intra-batch precision ranged from 2.41% to 10.72%; the accuracy ranged from 94.33% to 104.51%. Finally, the matrix effect, extraction recovery and stability data met the FDA recommended acceptance criteria for validation of bioanalytical methods. The t1/2 of ATC, DEX, MDZ and 1-OH-MDZ was (6.74 ± 2.27) h, (9.55 ± 4.93) h, (10.17 ± 5.35) h, and (6.90 ± 2.38) h, the Cmax, of ATC, DEX, MDZ and 1-OH-MDZ was (1054.20 ± 202.37) ng/mL, (1.93 ± 1.07) ng/mL, (1256.57 ± 389.09) ng/mL, and (1034.39 ± 292.92) ng/mL in patients undergoing aortic dissection surgery, respectively. The developed UPLC-MS/MS method for simultaneous determination of ATC, DEX, MDZ and 1-OH-MDZ in patient plasma was accurate, reproducible, specific. After continuous administration of ATC, DEX, and MDZ to patients undergoing surgery for acute aortic dissection, the pharmacokinetics of ATC, DEX, MDZ and 1-OH-MDZ in patients undergoing aortic dissection surgery were studied.

Determination of tamsulosin in plasma of healthy Chinese male subjects by a novel and simple LC-MS/MS method and its application to pharmacokinetic studies

Tamsulosin, the first highly selective α1-adrenoceptor antagonist, is widely used for urination disorders caused by benign prostatic hyperplasia (BPH). The pharmacokinetics and safety of 0.2 mg tamsulosin hydrochloride sustained-release capsules were evaluated in 60 healthy Chinese male subjects under fasting and fed conditions in this study. A simple, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of tamsulosin in human plasma, which has been applied to pharmacokinetic study. The analyte and internal standard (tamsulosin-d5) were extracted by protein precipitation, and separated on a ZORBAX Eclipse XDB-C18 column (4.6 × 50 mm,1.8 µm; Agilent Tech) using a gradient elution with mobile phases methanol and 5 mM ammonium acetate. The linear range was 0.05-15.0 ng/mL. It showed good selectivity in normal, hyperlipidemic, and hemolyzed blank matrices. The CV (%) of intra-batch precision was <4.4% and the RE (%) of accuracy was in the range of −5.0%–6.7%; the CV (%) of inter-batch precision was <−5.8% and the RE (%) of accuracy was in the range of 1.2%-4.0%. The mean extraction recovery for the analyte was 102.1 ± 3.75% and for the IS was 102.2 ± 2.00%. Two formulations were considered bioequivalent under fasting and fed conditions, and the 90% confidence intervals for the geometric mean test/reference ratios were within the predetermined range of 80%-125%. A single oral dose of 0.2 mg tamsulosin hydrochloride sustained-release capsule was well-tolerated throughout the clinical trial, and no > Grade 1 adverse events (AEs) and serious AEs occurred during the trial.

An HPLC-MS/MS method for determination of sulbactam in human plasma and its pharmacokinetic application in critically ill patients with augmented renal clearance.

A simple, rapid, and specific method has been developed and validated to measure sulbactam in human plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The pharmacokinetic characteristics of sulbactam in critically ill patients with augmented renal clearance were investigated after the repeated administration of cefoperazone-sulbactam (3 g, q8, IV drip, combination ratio of 2:1). Sulbactam plasma concentration was determined using LC-MS/MS with tazobactam used as an internal standard (IS). The method was fully validated with a sensitivity of 0.20 μg/mL, the linear concentration was ranged from 0.20 to 30.0 μg/mL. The intra-batch precision (RSD%) was less than 4.9%, and the accuracy deviation (RE%) ranged from -9.9 to 1.0%; the inter-batch precision (RSD%) was less than 6.2%, and the accuracy deviation (RE%) ranged from -9.2% to 3.7%. The value of the mean matrix factor at the low and high quality control (QC) concentration was 96.8 and 101.0%, respectively. The extraction recovery for QCL and QCH of sulbactam were 92.5 and 87.5%, respectively. Plasma samples and clinical data were collected at 0 (pre dose), 0.25, 0.5, 1, 2, 3, 6, and 8 hours (post dose) from 11 critically ill patients. Pharmacokinetic parameters were determined by non-compartmental analysis (NCA) using Phoenix WinNonlin software. This method was successfully applied to study the pharmacokinetics of sulbactam for critically ill patients. The main pharmacokinetic parameters of sulbactam in augmented renal function and normal renal function groups were summarized as follows: half-life, 1.45 ± 0.66 and 1.72 ± 0.58 hours, area under the concentration-time curve from 0 to 8 hours, 59.1 ± 20.1 and 111.4 ± 23.2 μg × h/mL, drug plasma clearance at steady state, 18.9 ± 7.5 and 9.32 ± 2 .03 L/h, respectively. These results suggested that a higher dose of sulbactam should be used in critically ill patients with augmented renal clearance.

LC-MS/MS method for the quantitation of a dual PI3K/BRD4 inhibitor SF2523 in mouse plasma: Application to plasma protein binding and metabolism studies.

A sensitive and selective liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of dual PI3K/BRD4 inhibitor SF2523 in mouse plasma. The analysis was performed on a UPLC system connected to a Shimadzu 8060 mass spectrometer by electrospray ionization in positive multiple reaction monitoring mode. Chromatographic separation was carried out on an ACE Excel C18 column with a gradient elution containing 0.1% formic acid and methanol as the mobile phase. The linearity was conducted in the concentration range 0.1-500 ng/ml for SF2523 in 100 μl of plasma. The inter- and intra-batch precision (RSD) were both lower than 13.5%, with the accuracy (percentage bias) ranging from -10.03 to 11.56%. The validated method was successfully applied to plasma protein binding and in vitro metabolism studies. SF2523 was highly bound to mouse plasma proteins (>95% bound). Utilizing mouse S9 fractions, a total of seven phase I and II metabolites were identified with hydroxylation found to be the major metabolic pathway. Metabolite identification included analysis of retention behaviors, molecular weight changes and MS/MS fragment patterns of SF2523 and the metabolites. This newly developed and validated method allows the rapid and easy determination of the SF2523 concentration with high sensitivity in a low sample volume and can be applied to future pre-clinical studies.

Rapid and quantitative detection of neutrophil gelatinase-associated lipocalin in synovial fluid using fluorescence immunochromatographic test strips for diagnosing prosthetic joint infections

ObjectivesWe developed a rapid and highly sensitive method for quantitatively analyzing neutrophil gelatinase-associated lipocalin (NGAL) levels in synovial fluid and assessed its diagnostic performance for prosthetic joint infection (PJI). Designs or MethodsWe conducted a preliminary analysis of the performance of the developed test strips utilizing clinical specimens to verify their sensitivity, precision, specificity and accuracy. ResultsThe standard curve of the test strip NGAL values was linear. The detection limit and the limit of quantification (LOQ) were 12.37 and 29.49 ng/mL, respectively, and the approximate detection range was 12.37 to 1250 ng/mL. The interbatch and intrabatch precision of the test strips were each less than 10%, and the cross-reaction rate with competitors’ systems was less than 1%. ConclusionsThe test strips can be used for the determination of synovial fluid NGAL levels; the test strips are highly sensitive, precise, specific, and stable. Furthermore, they demonstrated good performance in clinical verification.

Establishment of matrix metalloproteinase 3 time-resolved immunoassay and some potential clinical applications

AimTo develop a highly sensitive time-resolved fluorescence immunoassay (TRFIA) for the detection of serum matrix metalloproteinase-3 (MMP-3) and to assess MMP-3's clinical value in patients with colorectal cancer (CRC).st. MethodsMMP-3 levels were established using the double antibody sandwich technique. The MMP-3 TRFIA technique was developed and optimized, and its linearity, sensitivity, accuracy, specificity, and recovery were assessed. Then, serum concentrations in healthy individuals and patients with CRC were determined by MMP-3 TRFIA. ResultsThe linear range of MMP-3 TRFIA was 0.73–500 ng/mL. MMP-3 TRFIA had an intra-batch precision range of 2.16%–7.10% percent and an inter-batch precision range of 3.99%–11.21%. MMP-3, tumor-associated trypsinogen 2, and AFP had no cross reaction.The recovery is between 90% and 110%, and had no serum interference. Patients with CRC had serum MMP-3 levels (73.95 ± 78.43 ng/mL) that were considerably higher than those of healthy individuals (21.45 ± 11.12 ng/mL), and those with metastasis had serum MMP-3 levels (95.89 ± 76.21 ng/mL) that were considerably higher than those of patients without metastasis (52.74 ± 47.25 ng/mL). ConclusionsA highly sensitive MMP-3 TRFIA assay was successfully developed, and serum MMP-3 may be associated with CRC invasion and metastasis. Therefore, MMP-3 can be used in the auxiliary diagnosis of CRC.

Establishment and application of quantitative immunoassay method based on stable element labeling and inductively coupled plasma mass spectrometry

Objective: To establish a quantitative immunoassay method based on stable element labeling and inductively coupled plasma mass spectrometry (ICP-MS) for the detection of serum amyloid A (SAA) and evaluate its performance. Methods: An immunoassay system based on sandwich method was established with magnetic bead as carrier and holmium (Ho) as element tags. The binding ratio of hydrophilic streptavidin magnetic beads and biotinylated antibody, the amount of elemental antibody, and the reaction time were optimized to choose the optimal reaction conditions. According to the documents of Clinical and Laboratory Standards Institute (CLSI), the analytical performance was evaluated, including the limit of blank (LOB), linearity, accuracy, specificity, imprecision and interference test. Finally, 82 SAA plasma samples were collected after the turbidimetric inhibition immunoassay, and the newly established method was used for detection. Moreover, the detection results of the two methods were analyzed by Pearson correlation analysis. Results: The optimal binding ratio of hydrophilic streptavidin and biotinylated antibody was 1∶0.15, the amount of Ho-labeled antibody was 3 μl and the incubation time of the two reaction steps was 40 min and 30 min, respectively. The LOB was 0.6 ng/ml. The linearity was good within the range of 0-1 200 μg/L (R2=0.998 9, P<0.001). The inter-batch precision of high-value samples and low-value samples was 9.42% and 7.95%, respectively, and the intra-batch precision was 14.56% and 13.56%, respectively. The recovery was 96.01%-104.76%. The cross-reaction rates with procalcitonin (PCT) and C-reactive protein (CRP) were 0.45% and 0.015%, respectively. When the concentration of triglyceride≤35.5 mg/L, bilirubin≤0.52 mg/L and hemoglobin≤2.4 g/L, the interference bias was less than 10%. The results of 82 SAA plasma samples were 12.65 (4.45, 59.03) mg/L by ICP-MS immunoassay and 18.23 (9.33, 68.72) mg/L by turbidimetric inhibition immunoassay, respectively. The newly established system was well correlated with turbidimetric inhibition immunoassay (R2=0.983, P<0.001). Conclusion: The quantitative immunoassay for SAA with Ho as marker established in this study has high precision, good accuracy, high specificity, and wide linear range, which can meet the clinical testing requirements.

A Simple and Sensitive LC-MS/MS for Quantitation of ICG in Rat Plasma: Application to a Pre-Clinical Pharmacokinetic Study

A selective, sensitive, and rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of ICG in rat plasma. The chromatographic separation was achieved using an ACE excel C18 (3 µm, 50 × 3.0 mm) column, with a mobile phase composition of 0.1% formic acid and 0.1% formic acid in acetonitrile, using a gradient flow at a rate of 0.3 mL/min. The MS was operated at a unit resolution in the multiple reaction monitoring mode, using the precursor ion → product ion combinations of 753.3 → 330.2 m/z (ICG) and 747.45 → 717.50 (Cy7.5 amine) with a run time of 5 min. The assay was linear over a concentration range of 1–1000 ng/mL with a regression coefficient (r2) of 0.998 or better. The inter and intra-batch precision (% relative standard deviation, %RSD) was lower than 13.5%, with accuracy (%Bias) between −10.03% and 11.56%. The ICG was stable under laboratory storage and handling conditions. The validated method was successfully applied to preclinical pharmacokinetic (PK) studies of ICG at a dose of 0.39 mg/kg in rats. PK parameters suggested the highest plasma concentration within 2 min of intravenous dosing with restricted systemic distribution and rapid clearance.

Simultaneous determination of NTB-3119, a novel anti-tuberculosis agent, and its major metabolites in mouse plasma by LC-MS/MS and its application in preclinical pharmacokinetics study

NTB-3119, a novel benzothiopyranone derivative, has been developed as a potential anti-tuberculosis(TB) drug with strong activity. In this study, three major metabolites of NTB-3119 were firstly identified in vitro and in vivo. A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the quantitative analysis of NTB-3119 and its major metabolites NTB-3190, NTB-3202 and NTB-3204 in mouse plasma. The plasma samples were processed by protein precipitation with organic solvent. NTB-3119, NTB-3190, NTB-3202, NTB-3204 and NTB-4A (Internal Standard, IS) were separated by a Zorbax-SB C18 column (100 mm × 2.1 mm, 3.5 µm) with a gradient mobile phase of acetonitrile/water at a flow rate of 0.25 mL/min. The analytes were detected by electrospray positive ion mode in Parallel Reaction Monitoring (PRM) mode on a high resolution mass spectrum (HRMS, Thermo Q Executive). The monitored transitions were m/z 456.15632 → 360.06137 for NTB-3119, m/z 426.18214 → 246.01891 for NTB-3190, m/z 472.15124 → 360.06143 for NTB-3202, m/z 442.17706 → 246.01903 for NTB-3204 and m/z 337.13691 → 163.02081 for NTB-4A, respectively. Good linearity was conducted in the range of 5–2000 ng/mL for NTB-3119, NTB-3202 and NTB-3204 as well as 2.5–1000 ng/mL for NTB-3190. The inter- and intra-batch precision (RSD%) were both lower than 13.3 %, with the accuracy ranged from 88.0 % to 108.1 %. The analytes were proved to be stable during all samples storage, preparation and analytic procedures. The validated method was successfully applied to study the pharmacokinetics and bioavailability of NTB-3119 after oral treatment in Balb/c mouse.

Pharmacokinetics and tissue distribution of trans-ferulic acid-4-β-glucoside in rats using UPLC-MS/MS.

Trans-ferulic acid-4-β-glucoside (FAG) is a monomer extracted from Radix Aconiti Lateralis Preparata, which is a potential candidate for the prevention and treatment of cold injury. To determine the concentration of FAG in rats, it is essential to develop an ultra-performance liquid chromatography coupled with MS/MS method. Chromatographic separation was achieved by an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7μm). A Xevo triple quadrupole tandem mass spectrometer was used to quantitatively determine FAG in the negative-ion mode. The standard calibration curve was linear over the concentration range of 0.1-100 μg/mL and 0.0626-31.28 μg/g for rat plasma and liver tissue homogenate samples, respectively. The inter- and intra-batch precision (% relative standard deviation) of the assay was ≤8.29%, and accuracy (% relative error) ranged from -7.41 to 10.99%. The matrix effect was between 92.99 and 102.39%. The oral absolute bioavailability of FAG was obtained as 1.80%. The results of tissue distribution suggested that FAG spread rarely in the liver and brown adipose, which was not propitious to exert its ability to treat cold injury. In general, these studies were significant to provide necessary information for further study.

Development and Evaluation of Serum CST1 Detection for Early Diagnosis of Esophageal Squamous Cell Carcinoma.

PurposeOur pilot study has shown that cystatin SN (CST1) protein is highly expressed in esophageal squamous cell carcinoma (ESCC) tissues. We intend to develop a chemiluminescent enzyme immunoassay (CLEIA) available for serum CST1 detection and define the diagnostic value of CST1 detection for early ESCC patients, and establish a panel of CST1 with traditional tumor markers to improve the diagnostic sensitivity for early ESCC.MethodsDetection performance of CLEIA for CST1 was evaluated by linearity, detection limit, accuracy, precision, anti-interference and stability. Diagnostic performance of CST1 for early ESCC was evaluated by detecting CST1 of 112 early ESCC, 107 esophageal benign lesions (EBL), and 151 healthy controls (HC). CEA, CYFRA21-1 and SCC-Ag were detected by chemiluminescence immunoassay (CLIA).ResultsThe linear range and detection limit of CLEIA for CST1 were 6.25–400 pg/mL and 1.35 pg/mL, respectively; the average recovery rate was 102.65%; CVs of intra-batch precision and inter-batch precision were <1/4 TEa and <1/3 TEa, respectively; 8 interferents including 7 common interferents and CST4 had no interference on CST1 detection; stability evaluation showed good sample and reagent stability. The level and positive rate of CST1 in early ESCC group were significantly higher than those in EBL/HC groups (P<0.05). The diagnostic sensitivity of CST1 for early ESCC was 31.25% (specificity 92.64%, AUC 0.654). The diagnostic sensitivity of traditional tumor markers ranged from 16.07% to 28.57%, at >93.0% specificity, and SCC-Ag showed the highest AUC (0.709). Combination of CST1 and CEA, SCC-Ag exhibited the highest AUC up to 0.736 (sensitivity 49.11%, specificity 89.53%).ConclusionCLEIA has excellent detection performance for CST1. CST1 might be a prospective serological biomarker for early diagnosis of ESCC, while combination of CST1 and CEA, SCC-Ag might improve the early diagnostic performance.

Evaluation of humoral pattern detection performance of Mindray BC-6000PLUS blood analyzer based on WS/T 662-2020.

Manual microscopic examination is the gold standard of humoral cell count test. However, it has some limitations and cannot fully meet clinical needs. Compared with the manual method, the automatic blood cell analyzer has the advantages of a high degree of automation, minimal error, high speed, high precision, and easy standardization. This study intends to verify the detection performance of the body fluid model of the Mindray BC-6000PLUS automatic hematology analyzer. This study was performed in accordance with the International Committee for Standardization in Haematology (ICSH) Hematology Analyzer Evaluation Guide (version 2014) and the requirements of WS/T662-2020 "Clinical humoral examination technique". The humoral white blood cell-body fluid (WBC-BF), humoral red blood cell-body fluid (RBC-BF), monocyte (MN), polymorphonuclear (PMN) were measured to verify the performance indicators of the instrument, including background counting, intra-batch precision, accuracy, carrying contamination rate, and linear range. Referring to the WS/T514-2017 (Establishment and verification of detection capability for clinical laboratory measurement procedures), the limit of blank (LoB) and limit of detection (LoD) values of WBC-BF and RBC-BF in the humoral mode of the instrument were established. The blank count of WBC-BF and RBC-BF, and contamination of Mindray BC-6000PLUS analyzer were zero; the coefficient of variation (CV) of intra-batch precision at different levels of each item was less than 10%. There was a high correlation between instrument test results and manual microscopic examination results (r>0.95). The linear range of the instrument was wide, and the linear verification parameters was good (R2>0.999). The LoB value and LoD value of WBC-BF established by the instrument were 0×109/L and 0.004×109/L, respectively. The LoB value and LoD value of the RBC-BF established by the instrument were 0×1012/L and 0.004×1012/L, respectively. The lower detection limits of WBC-BF and RBC-BF were set as 0.004×109/L and 0.004×1012/L, respectively. All performance indicators of the Mindray BC-6000PLUS automatic blood analyzer met the requirements of the manufacturer's criteria. This instrument can fulfill the requirement of body fluid sample routine test in clinical practice.

MO281THE EVALUATION OF TEST EFFICIENCY ON LOW TITER SERUM PHOSPHOLIPASE A2 RECEPTOR ANTIBODY

Abstract Background and Aims Antibody to phospholipase A2 receptor (PLA2R) was widely detected for a decade as a diagnostic marker of idiopathic membranous nephropathy. Recently, several studies reported that a lower cut-off of serum PLA2R antibody (PLA2RsAb) could improve diagnostic efficacy. The recommend cut-off ranged from 2.0 to 2.7 RU/ml which was considered largely improving the sensitivity of the assay. Other reported that although sensitivity of the measurement might loss to some extent cut-off at 14 RU/ml might of the best clinical performance. However, the lower cut-off was slightly higher than the minimum detection limit, that was 2.0 RU/ml. There was no study evaluated the test efficiency of low titer antibody of the measurement. Method The reference range of PLA2RsAb was validated by testing 40 serum from health volunteer, male/female ratio was 20/20. The samples for intra and inter-batch precision test were prepared by mixing low titer PLA2RsAb serum. The intra-batch precision was performed by repeating six times of the validation sample in one plate. The inter-batch precision was performed by repeating four times of validation samples in one plate on five days. Results The medium age of male was 34 (31, 53) and 46 (31, 54) for female, respectively. PLA2RsAb was ranged from 2.0 to 4.3 RU/ml for male and 2.0 to 4.0 RU/ml for female. The average of intra-batch validation sample was 8.1 ± 1.2 RU/ml. The intra-batch precision was presented by coefficient of variation, that was 15%. The average of inter-batch validation samples were 4.0 ± 0.4 RU/ml and 5.2 ± 0.5 RU/ml, respectively. The inter-batch precision was 10% for both validation serum. Conclusion Healthy population showed low titer PLA2RsAb positive according to a lower cut-off. The validation showed relative high coefficient of variation on intra and inter-batch precision of low titer PLA2RsAb.