Coefficient Of Variation For Precision Research Articles

A-226 Validation of Calculated Glycerol Corrected Triglycerides

Abstract Background Patients with elevated free glycerol can have falsely elevated triglyceride concentrations known as pseudohypertriglyceridemia. Most clinical laboratory triglycerides methods measure the concentration of glycerol released following the enzymatic hydrolysis of triglycerides to quantitate triglycerides. Thus, increased free glycerol can lead to falsely increased triglyceride concentration. Identifying and measuring the interference is necessary to effectively manage patient care interventions. A glycerol-corrected triglyceride assay was discontinued from Roche Diagnostics in 2021. We created a process to determine glycerol-corrected triglycerides by measuring glycerol directly using an assay from Randox Laboratories. Methods Glycerol (Randox Laboratories) and nonglycerol-blanked triglycerides (Roche Diagnostics) were measured in serum using the Roche Cobas c501. Stability (n=10) and expected values (n=206) were determined using serum from healthy volunteers. Linearity (n=5) and precision (5 replicates on 2 runs over 5 days) were determined using residual clinical serum spiked with exogenous glycerol as necessary to span the analytical measuring range. Glycerol corrected triglyceride concentrations were calculated by subtracting glycerol (mmol/L) from triglyceride (mmol/L) and converting to mg/dL (x88.59). Samples previously measured using the Triglyceride/Glycerol Blanked Reagent (n=53, Roche Diagnostics) were used to assess accuracy. (Figure 1) Results Glycerol measures were stable 3 days refrigerated and 30 days frozen. Precision coefficient of variation was 1.0% within run and 7.1% within lab. Expected values were 10 - 109 mcmol/L, and the method was linear between 2 - 3,971 mcmol/L. The average bias between methods was 11.0% with slope of 1.02 and R2>0.97 (Figure 1). Conclusions Glycerol can be accurately measured using the Randox method and the result can be used to generate an accurate glycerol-corrected triglyceride concentration.

Development and Validation of a High-Performance Liquid Chromatography-Ultraviolet Spectrometry Method for Ampicillin and its Application in Routine Therapeutic Drug Monitoring of Intensive Care Patients.

Ampicillin/sulbactam, a combination of a β-lactam and β-lactamase inhibitor, is widely used in clinical settings. However, therapeutic drug monitoring (TDM) of ampicillin is not commonly performed, particularly in intensive care units (ICUs). The purpose of this study was to develop and validate a rapid and cost-effective high-performance liquid chromatography (HPLC)-ultraviolet spectrometry method to quantify ampicillin in human serum and evaluate its clinical application in ICU patients. Sample cleanup included a protein precipitation protocol, followed by chromatographic separation on a C18 reverse-phase HPLC column within 12.5 minutes using gradient elution of the mobile phase. The assay was validated according to the German Society of Toxicology and Forensic Chemistry criteria. Clinical applications involved the retrospective analysis of TDM data from ICU patients receiving continuous infusion of ampicillin/sulbactam, including the attainment of target ranges and individual predicted and observed pharmacokinetics. The method was robust, with linear relations between the peak area responses and drug concentrations in the range of 2-128 mg/L. The coefficient of variation for precision and the bias for accuracy (both interday and intraday) were less than 10%. Clinical application revealed variable pharmacokinetics of ampicillin in ICU patients (clearance of 0.5-31.2 L/h). TDM-guided dose adjustments achieved good therapeutic drug exposure, with 92.9% of the samples being within the optimal (16-32 mg/L) or quasioptimal (8-48 mg/L) range. This method provides a practical solution for the routine TDM of ampicillin, facilitating individualized dosing strategies to ensure adequate therapeutic drug exposure. Given its simplicity, cost-effectiveness, and clinical relevance, HPLC-ultraviolet spectrometry holds promise for broad implementation in hospital pharmacies and clinical laboratories.

Study of phenanthrenes from their unique mass spectrometric behavior through quantum chemical calculations to liquid chromatographic quantitation

Phenanthrenes and their derivatives have biological relevance owing to their antimicrobial, antioxidant, and cytotoxic effects on cancer cells. They can be efficiently analyzed through ultrahigh-performance liquid chromatography coupled to tandem high-resolution mass spectrometry (UHPLC–MS/HRMS). Herein, we first studied the unique fragmentation behavior of phenanthrenes based on direct infusion MS/HRMS analysis. As a newly described phenomenon, “organ pipe distribution”, we found a structural connection linking their unique fragmentation pattern to serial H radical losses. The bonds responsible for this behavior were identified through quantum chemical calculations using a stepwise approach. Furthermore, the chromatographic aspect of this study was enhanced by developing, validating, and applying a new unscheduled targeted UHPLC–MS/HRMS method for quantifying phenanthrenes in Juncus compressus herb. Targeted compounds were efficiently separated within 4 min upon utilizing the Accucore C30 column, and the unscheduled targeted analytical approach afforded five new isomers. Compounds 1 (effususol), 3 (dehydroeffusol), and 6 (7-hydroxy-1-methyl-2-methoxy-5-vinyl-9,10-dihydrophenanthrene) had their linearity limits determined within 10–5000 nM, and Compounds 2 (effusol), 4 (juncusol), 5 (effususin A), and 7 (compressin A) within 25–5000 nM. The coefficients of variation for precision ranged from 1.4 % to 15.2 %. The obtained matrix effects and accuracy values were also within acceptable ranges. Compounds 2 (effusol) and 3 (dehydroeffusol) were present in both methanolic and dichloromethanolic extracts of Plants 1 and 3 at the highest concentrations. Furthermore, the relationship between phenanthrene fingerprints, obtained through ANOVA statistical analysis of quantitative data, and the geographical location of herbs was also established.

Standardization of Myristicin in Nutmeg (Myristica fragrans Houtt.) Fruit using TLC-Densitometric Method

Background: Myristica fragrans Houtt. (Myristicaceae family), with the main content of myristicin, has been immensely used in herbal medicine. Standardization is essential to ensure the safety of natural extracts and the quality of herbal medicines using various chemical analysis techniques. Method validation is necessary to ascertain the reliability and reproducibility of the method. Myristicin is a member of the phenylpropene group, a natural organic compound found in small amounts in nutmeg fruit, which has pharmacological effects. Objective: This study aims to determine the myristicin content in nutmeg fruit using TLC-Densitometry. Methods: Determination of myristicin in nutmeg fruit extract was performed using TLC-Densitometry with silica GF254 as stationary phase, mobile phase n-hexane: ethyl acetate (8:2 v/v), and spot visualized at 285 nm. In this study, the content of myristicin in nutmeg fruit was determined using compendial methods (AOAC), thus requiring method verification with parameters including selectivity, linearity, precision, LOD, and LOQ. Results: The validation of this method showed good linearity and selectivity with y = 0.0001x + 0.0226 (r = 0.9996) and 1.53 (>1.5), respectively. The LOD and LOQ results were low with values of 0.11 μg/spot and 0.33 μg/spot, respectively. The percentage coefficient of variation for precision was below the requirement value of not more than 4%. The average myristicin content in nutmeg fruit extract was approximately 0.0017 ± 0.0003% (w/w). Conclusion: The developed method was valid and sensitive for the quantification of myristicin content in nutmeg fruit.

A Precise qNMR Method for the Rapid Quantification of Lot-to-Lot Variations in Multiple Quality Attributes of Pentosan Polysulfate Sodium.

Pentosan polysulfate sodium (PPS) is an orphan drug with anticoagulant activity. PPS is prepared from the chemical processing of xylan extracted from beechwood tree to yield a mixture of 4-6kDa polysaccharides. The chain is mainly composed of sulfated xylose (Xyl) with branched 4-O-methyl-glucuronate (MGA). During generic drug development, the quality attributes (QAs) including monosaccharide composition, modification, and length need to be comparable to those found in the reference list drug (RLD). However, the range of QA variation of the RLD PPS has not been well characterized. Here, multiple PPS RLD lots were studied using quantitative NMR (qNMR) and diffusion ordered spectroscopy (DOSY) to quantitate the components in the mixture and to probe both inter- and intra-lot precision variability. The DOSY precision assessed using coefficient of variation (CV) was 6%, comparable to PPS inter-lot CV of 5%. The QAs obtained from 1D qNMR were highly precise with a precision CV < 1%. The inter-lot MGA content was 4.8 ± 0.1%, indicating a very consistent botanical raw material source. Other process-related chemical modification including aldehyde at 0.51 ± 0.04%, acetylation at 3.3 ± 0.2% and pyridine at 2.08 ± 0.06%, varied more than MGA content. The study demonstrated that 1D qNMR is a quick and precise method to reveal ranges of variation in multiple attributes of RLD PPS which can be used to assess equivalency with generic formulations. Interestingly, the synthetic process appeared to introduce more variations to the PPS product than the botanical source of the material.

A simple and rapid LC-MS/MS method for the simultaneous determination of 15 bile acids in human serum and its application to patients with decompensated cirrhosis.

Liver cirrhosis is currently the twelfth leading cause of death globally and the sixth leading cause of death in China. Its treatment is expensive. Changes in the composition of the serum bile acid pool are sensitive indicators of the severity of liver cirrhosis. In this study, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and used to simultaneously determine 15 bile acids in human serum in patients with decompensated cirrhosis. Sample preparation involved spiking with isotope internal standards (ISs) followed by protein precipitation and the analytical run time was 5 min. The LC-MS/MS method was fully validated according to CLSI C62A and the Consensus of method development and validation of liquid chromatography-tandem mass spectrometry in clinical laboratories. The method achieved an acceptable coefficient of variation for precision (0.83-14.80%) and accuracy (89.39-107.62%). Finally, as proof of applicability, the method was applied to patients with decompensated cirrhosis in routine clinical sample analysis. The degree of variation of different bile acids was clearly shown. These results indicated that abnormal metabolic pathways might play important roles in decompensated cirrhosis.

Implementation of Blood‐Based Biomarker Assays to Deliver Consistent and Comparable Data

AbstractBackgroundImplementing biomarkers for Alzheimer’s Disease (AD) is an important area of research that will enhance the ability to accurately characterize the disease with the goal of earlier detection and treatment. Analytical validation is the first step to determine the measuring range, specificity, precision, and matrix effects of biomarker assays. Studies on analyte stability and performance bridging when key reagents are changed are critical to deliver reliable and consistent data over time. The goal of these studies is to demonstrate the performance of the assays implemented in the NCRAD Biomarker Assay Laboratory (BAL).MethodSamples were collected from patients with AD (N=14) and from healthy control subjects (N=19) by the Indiana Biobank. Samples were assayed on the Quanterix Simoa HD‐X platform using the Simoa Nf‐light Advantage Kit and P‐tau181v2 Advantage Kit to determine neurofilament light chain and P‐tau 181 concentrations. Five samples were chosen to span high, medium, and low concentrations for the validation method. Sets of 3 plates were run on two separate days using kits of the same lot number and same 5 samples. Validation assessed precision, parallelism, protein recovery, dilutional linearity, and sensitivity. In addition, our validation method allowed assessment of intra and inter batch variability. Euroimmun Beta‐Amyloid 1‐40 and 1‐42 ELISA kits were also assessed.ResultThe Nf‐Light Advantage Kit precision data coefficient of variation (CV) was less than 7% within plates and less than 8% percent within batches. The CV for precision between the runs on both days was less than 10%. The P‐tau181v2 Advantage assay precision data CV was less than 7% within plates and less than 10% within batches. The CV for total precision between runs on both days was less than 10%. Dilutional linearity, protein recovery, sensitivity, and parallelism were within acceptable levels.ConclusionThe validation studies of the commercially available Simoa Nf‐light and P‐tau181v2 Advantage Kits demonstrate the performance of these assays as implemented within the NCRAD BAL. The stability and bridging studies illustrate how the assays will be kept performing over time to deliver consistent and reliable data for researchers submitting samples for analysis. Supported by U24 AG021886 and UL1TR002529.

Second‐generation fully automated Elecsys cerebrospinal fluid immunoassays demonstrate high precision, reproducibility, and sample stability suitable for clinical use to aid Alzheimer’s disease diagnosis

AbstractBackgroundBeta‐amyloid(1–42) (Abeta42) and phosphorylated tau 181 (pTau) cerebrospinal fluid (CSF) biomarker analysis is recommended alongside cognitive evaluation to aid Alzheimer’s disease (AD) diagnosis. For clinical use, CSF biomarker assays should demonstrate high precision/reproducibility and sample stability. This study evaluated precision, reproducibility, and sample stability of second‐generation fully automated Elecsys® CSF immunoassays.MethodFrozen/pooled CSF and PreciControl samples were analyzed using Elecsys β‐Amyloid(1–42) CSF II/Phospho‐Tau (181P) CSF immunoassays on cobas e 601 (Roche Diagnostics International Ltd). pTau/Abeta42 ratios were calculated. Precision was evaluated at one site per Clinical and Laboratory Standards Institute (CLSI) EP02‐A3 (two runs/day in duplicate over 21 days; n=84). Reproducibility was determined per CLSI EP05‐A3 over five days using a single lot across three sites (site‐to‐site)/three lots at one site (lot‐to‐lot). Coefficients of variation (CVs)/standard deviations (SDs) for repeatability/intermediate precision/reproducibility were calculated using variance component analysis. Sample stability was determined from fresh CSF, collected per manufacturer's instructions. Baseline measurements were taken &lt;6 hours after lumbar puncture and follow‐up measurements (from stored aliquots) at defined intervals for ≤8 days at room temperature (RT; 25°C; n=13), ≤15 days at 6°C (n=14), and ≤13 weeks at ‐20°C (n=25). Storage stability was determined from percent recoveries/ordinary linear regression.ResultRepeatability CVs were &lt;2.4% and &lt;1.9% for Abeta42 and pTau immunoassays, respectively, and &lt;2.4% for pTau/Abeta42 ratio. Intermediate precision CVs were &lt;3.3% and &lt;3.7% for Abeta42 and pTau immunoassays, respectively, and &lt;3.9% for pTau/Abeta42 ratio. Site‐to‐site reproducibility CVs were &lt;5.1% and &lt;3.6% for Abeta42 and pTau immunoassays, respectively, and &lt;6.5% for pTau/Abeta42 ratio (Table 1). For the Abeta42 immunoassay, lot‐to‐lot reproducibility CVs were &lt;5.4%, except for one sample pool (mean concentration: 322 pg/mL; SD: 34.1 pg/mL; Table 2). Lot‐to‐lot reproducibility CVs were &lt;3.6% for the pTau immunoassay and &lt;6.0% for pTau/Abeta42 ratio. At maximum sample storage duration (8 days at RT/15 days at 2−8°C), median recoveries of 95%/98% were observed for Abeta42 and 97%/99% for pTau immunoassays. Stability assessment at ‐20°C is ongoing.ConclusionThe Elecsys immunoassays demonstrated high precision/reproducibility, supporting clinical use in AD diagnosis. Conservative recommendations for sample storage are 5 days at RT/14 days at 2−8°C.

Thalidomide measurement in plasma and dried plasma spot by SPE combined with UHPLC-MS/MS for therapeutic drug monitoring.

Aims: To validate an SPE-ultra-HPLC-MS/MS method for thalidomide (THD) measurement in dried plasma spot (DPS). Methods: Extraction included acetonitrile/water clean-up and online SPE. The LOD, LLOQ, linearity, precision, accuracy, recovery, matrix effect, process efficiency, carryover, stability, druginterferenceand dilution integrity were assessed. Results: The method was linear from 50 to 2000ng/mlwith aLOD of 20ng/ml and LLOQ of 50ng/ml. The coefficient of variation for precision was 0.4-7.9% for intra-assay and 1.3-8.9% for interassay, and accuracy was 81.4-97.1%. Adequate matrix effect(100.6-107.0%), recovery(88.7-105.0%)and process efficiency(91.3-109.3%) were registered. DPS was stable for14 days at room temperature and 45°C,and for 4 months at -80°C. The method was applied to quantify THD in both wet plasma and DPS from patients with cutaneous lupus receiving THD treatment. The difference between THD wet plasma and DPS concentration was <15%. Conclusion: The method is suitable to quantify THD in DPS.

Simultaneous determination of serum homocysteine, cysteine, and methionine in patients with schizophrenia by liquid chromatography-tandem mass spectrometry.

Schizophrenia is a debilitating psychiatric disorder affecting approximately 1% of the population worldwide. Disturbances in the homocysteine metabolism are an important factor in the pathophysiology of schizophrenia. In this research, a novel validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification procedure was developed to investigate three significant compounds of homocysteine metabolism: homocysteine, cysteine, and methionine in patients with schizophrenia and healthy controls. Sample preparation involved a reduction with dithiothreitol followed by protein precipitation, and the chromatographic runtime was 2min. The LC-MS/MS method was validated according to CLSI C62-A and the Chinese Guidance for Liquid Chromatography and Mass Spectrometry Clinical Application. The performance of the method was excellent with a coefficient of variation for precision in the range of 0.5-6.9%, an accuracy of 90.4-101.6%. In addition, the practical applicability of the method was demonstrated by applying it in the routine sample analysis for a schizophrenic patient. Increased homocysteine levels and decreased cysteine levels were observed in the patient with schizophrenia. These results indicate that the activity of the transsulfuration pathway may play a key role in the pathogenesis of schizophrenia.

Method Performance Verification of Anti-GAD65 and Anti-Insulin antibody Assays.

The aim of this study was to evaluate the performance of chemiluminescence immunoassays for anti-GAD65 and anti-insulin antibodies following user verification guidelines. The analytical performance of anti-GAD65 and anti-insulin antibodies using a MAGLUMI 2000 analyzer was verified following user verification guidelines by the Clinical and Laboratory Standards Institute. Performance specifications including precision, linearity, carry-over, cutoffs for positive results, reference intervals, and comparability with pre-existing commercially available radioimmunoassays using patient specimens and certified reference material were verified (coefficients of variation for precision of anti-GAD65 and anti-insulin antibodies were 2.6% and 3.4%, respectively). Comparability assessed using clinical serum specimens showed overall agreement with radioimmunoassay of 87.2% (95% confidence interval 74.8% - 94.0%) for the anti-GAD65 antibody assay and 85.4% (95% confidence interval 71.6% - 93.1%) for the anti-insulin antibody assay. The results of this study verified the analytical performance of MAGLUMI anti-GAD65 and anti-insulin antibody assays for clinical use.

Multicenter Evaluation of a New, Fully Automated Androstenedione Electrochemiluminescence Immunoassay: Precision Analysis, Method Comparison, and Determination of Reference Ranges.

Androstenedione (ASD) levels can aid diagnosis of hyperandrogenism together with other clinical/laboratory findings. We evaluated performance of the new, automated Elecsys® ASD assay vs an ASD isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference measurement procedure and determined reference ranges. Repeatability/intermediate precision were assessed using 3 control levels and 5 human serum pools (n = 75 each; Clinical and Laboratory Standards Institute EP05-A3). Method comparisons vs commercially available immunoassays [IMMULITE ASD (Siemens) and LIAISON ASD (DiaSorin)] and an ID-LC-MS/MS measurement procedure method were conducted using 421 serum samples; Passing-Bablok regression and Pearson's correlation coefficients were calculated. Reference ranges and distribution of values associated with polycystic ovary syndrome (PCOS) were determined in five clinical cohorts using samples from several sites/vendors. Repeatability/intermediate precision coefficients of variation across all sites were 2.01% to 3.91% and 2.43% to 4.30%, respectively (mean ASD: 7.80-34.7 nmol/L). The Elecsys ASD assay showed poor agreement with IMMULITE ASD (slope = 0.459; r = 0.856; n = 320), fair agreement with LIAISON ASD (slope = 0.625; r = 0.984; n = 327), and very good agreement with ID-LC-MS/MS (slope = 1.040; r = 0.996; n = 332). Reference ranges (2.5th-97.5th percentiles) were: children (≤8 years; n = 140), <0.525 to 1.81 nmol/L; males (≥18 years; n = 138), 0.979 to 5.32 nmol/L; and postmenopausal females (n = 140), 0.654 to 3.74 nmol/L. Reference range (5th-95th percentiles) for females with fertile cycle (≥18 years; n = 84) was 1.71 to 4.58 nmol/L. The distribution of values (2.5th-97.5th percentiles) in females with PCOS (n = 125) was 2.26 to 12.1 nmol/L. Elecsys ASD assay demonstrated excellent precision and very good agreement with ID-LC-MS/MS. Reference ranges were established to support results interpretation in routine practice.

A highly sensitive, simple, and fast gas chromatography-mass spectrometry method for the quantification of serum short-chain fatty acids and their potential features in central obesity.

Short-chain fatty acids (SCFAs) are the main gut microbe metabolites, which have no more than six carbons. SCFAs are an emerging biomarker in metabolic diseases, including central obesity. Commonly, SCFAs are measured in fecal samples, where they are highly abundant, but here they do not reflect direct interactions with related organs. Serum SCFAs are assumed to be more associated with metabolic disease than fecal SCFAs, albeit at very low concentrations. The aim of the present study is to develop a highly sensitive, simple, and fast method for measuring six SCFAs in the serum by gas chromatography-mass spectrometry (GCMS). The serum is mixed with meta-phosphoric acid and 2,2-dimethylbutyric acid, followed by homogenization and centrifugation. Supernatant is then injected into the fused silica capillary column. The method is linear from 0.12-500μmol/L for all SCFAs with an accuracy of 90-117%. The total coefficient of variation for precision ranges from 3.8 to 14.1%. A preliminary study is performed with 32 centrally obese subjects and 17 lean subjects. The mean values of all SCFAs, including acetic, propionic, isobutyric, butyric, isovaleric, and valeric acid, in the centrally obese subjects are significantly higher compared with lean subjects. A significant correlation also exists between all SCFAs, with the waist circumference indicating that serum SCFAs have potential features with respect to metabolic diseases, especially central obesity. The validated GCMS method provides highly sensitive, fast, simple, and reliable SCFA quantitation in the serum and demonstrates the potential features of circulating SCFAs in central obesity.

A Novel Bioanalytical Method for Determination of Inotodiol Isolated from Inonotus Obliquus and Its Application to Pharmacokinetic Study.

In this study, we developed a bioanalytical method using liquid chromatography coupled to triple quadrupole tandem mass spectrometry (LC-MS/MS) to apply to a pharmacokinetic study of inotodiol, which is known for its anti-cancer activity. Plasma samples were prepared with alkaline hydrolysis, liquid–liquid extraction, and solid-phase extraction. Inotodiol was detected in positive mode with atmospheric pressure chemical ionization by multiple-reaction monitoring mode using LC-MS/MS. The developed method was validated with linearity, accuracy, and precision. Accuracy ranged from 97.8% to 111.9%, and the coefficient of variation for precision was 1.8% to 4.4%. The developed method was applied for pharmacokinetic study, and the mean pharmacokinetic parameters administration were calculated as follows: λz 0.016 min−1; T1/2 49.35 min; Cmax 2582 ng/mL; Cl 0.004 ng/min; AUC0–t 109,500 ng×min/mL; MRT0–t 32.30 min; Vd 0.281 mL after intravenous administration at dose of 2 mg/kg and λz 0.005 min−1; T1/2 138.6 min; Tmax 40 min; Cmax 49.56 ng/mL; AUC0–t 6176 ng×in/mL; MRT0–t 103.7 min after oral administration. The absolute oral bioavailability of inotodiol was 0.45%, similar to nonpolar phytosterols. Collectively, this is the first bioanalytical method and pharmacokinetic study for inotodiol.

Preliminary evaluation of eight less frequent endocrine assays designed for MAGLUMI 800 chemiluminescence immunoanalyzer

Transition to new analytical systems and methods requires end-user verification to ensure acceptability for routine use. Our aim was to verify precision of MAGLUMI 800 immunoassay analyzer for 17-hydroxyprogesterone (17-OHP), 25-hydroxy vitamin D (25(OH)D), aldosterone, androstenedione, growth hormone (GH), insulin-like growth factor 1 (IGF-1), insulin-like growth factor-binding protein 3 (IGFBP-3) and renin, as well as to assess their comparability with the routinely used assays. Precision was evaluated at two levels following the CLSI EP15-A2 protocol. Method comparison included parallel analysis of 40 routine samples for each assay on MAGLUMI 800 and the routinely used automated or manual immunoassays. Within-run coefficients of variation (CV) ranged from 0.8% (androstenedione) to 14.5% (aldosterone), between-run CVs from 1.0% (IGFBP-3) to 12.8% (renin), while within-laboratory (total) precision CVs were from 2.1% (IGFBP-3) to 14.9% (renin). All assays with the exception of IGF-1 and 25(OH)D at the low concentration control level, satisfied biological variation criteria for imprecision. Passing-Bablok regression showed proportional difference for 17-OHP and aldosterone, constant for androstenedione, while both constant and proportional difference was revealed for 25(OH)D, GH and IGF-1. Statistically significant relative biases higher than the desirable biological variation acceptance criteria were observed for 17-OHP, 25(OH)D, aldosterone, androstenedione and IGF-1. The evaluated assays need further assessment as well as verification of reference intervals in order to be suitable for introduction into routine practice in our laboratory. Our study clearly demonstrates that we are still far from achieving immunoassay standardization and comparability of results.

Validation of plasma amino acid profile using UHPLC-mass spectrometer (QDa) as a screening method in a metabolic disorders reference centre: Performance and accreditation concerns

IntroductionAmino acid (AA) analysis in plasma is essential for diagnosis and monitoring of inborn errors of metabolism (IEM). The efficacy of patient management is governed by the rapidity of AA profile availability, along with the robustness of the method. French quality guidelines and progress made in analytical techniques have led biologists to develop AA profile exploration via mass spectrometry (MS). ObjectivesThe aim of this study was to validate an analytical method with a single quadrupole mass spectrometer (MS) and to suggest reference values in regard to French quality and IEM society recommendations. Design and methodsPlasma samples from patients were deproteinised and derivatised with AccqTag™ reagent. Analysis was performed by reverse-phase chromatography coupled to QDA detector. We evaluated accuracy, intra-days and inter-days precision and limit of quantification by the β-expectation tolerance interval method for 27 AA. Method comparison was performed with the standard method (ion exchange chromatography, IEC) on Jeol Aminotac® and to tandem MS. Reference values were established on AA concentrations of the cohort of patients who had no IEM. ResultsOur method allowed the separations of almost all amino acids with a total run time of 12 min. Separation of isoleucine and alloisoleucine was incomplete (R = 0.55) but without impact on biological interpretation. Precision, accuracy and quantification were satisfactory (intra-days coefficient of variation (CV) was <5%, inter-days precision CV <10% and accuracy <15%). The limits of quantification were validated between 1 and 900 µmol/L. Results were comparable between the new method and IEC. ConclusionUltimately, we validated a rapid method on plasma for quantifying 27 amino acids that can be used in routine practice, according to French quality laboratories and SFEIM (French Society of Inborn Error of Metabolism) recommendations. Furthermore, estimated reference values were similar to those found in published studies focusing on other methods. Despite a lower specificity compared to tandem MS, the simplicity and rapidity of our method are the main advantage of this technique and place it as a major tool in IEM diagnosis and management.

Performance verification of a new fibrinogen assay.

Plasma fibrinogen (FIB), also known as factor I, plays a key role in the coagulation process. FIB testing in a clinical laboratory is crucial for coagulation screening and thrombolytic therapy. Here, we assessed the performance of a new, Chinese-made coagulation analyzer in the detection of FIB by comparing its precision and clinical feasibility with that of an imported system. Blood samples were collected and plasmas were separated. The precision, linearity, reference interval, carryover rate, clinically reportable range, and clinical applicability of the domestic coagulation analyzer for FIB assay were assessed and validated based on the documents or industry standards issued by the United States Clinical and Laboratory Standards Institute (CLSI). The within-batch precision CVs (coefficient of variation) for the low- and high-level specimens were 2.92% and 0.24%, respectively, while the total precision CVs were 3.05% and 1.81%, respectively; all of them met the experimental requirements. The linear range was validated to cover 1.0-6.5 g/L, and a good linear relationship was obtained within the measurement range (R2=0.9998). The reference interval was verified for adults and the carryover rate was also evaluated to be 0.68%. The clinically reportable range was 0.33-13.0 g/L. With a sample size of 180 cases, the methodological comparison showed a correlation coefficient (r) of 0.9886 between Mindray ExC810 and Sysmex CS5100. Furthermore, when the level of FIB was higher than 4.0 g/L or lower than 1.0 g/L, the 2 systems had an agreement rate of 100%. Mindray ExC810 has a good performance for FIB assay in terms of precision, linearity, reference interval, carryover rate, and clinically reportable range. Methodological study showed that Mindray ExC810 has good agreement with Sysmex CS5100 and meets the requirements of laboratory testing. Therefore, Mindray ExC810 is suitable for FIB assay in clinical laboratory.

Separation and Quantification of Superwarfarin Rodenticide Diastereomers-Bromadiolone, Difenacoum, Flocoumafen, Brodifacoum, and Difethialone-in Human Plasma.

Superwarfarins, second-generation long-acting anticoagulant rodenticides, are 4-hydroxycoumarin analogues of warfarin that contain a large hydrophobic side chain. These compounds contain two chiral centers and are synthesized for commercial use as two pairs of diastereomer. To support studies of superwarfarin pharmacokinetics and other efforts to improve clinical care for poisoning victims, a quantitative assay was developed for the measurement of diastereomer of bromadiolone, difenacoum, flocoumafen, brodifacoum, and difethialone in human plasma. Based on ultrahigh-pressure liquid chromatography-tandem mass-spectrometry (UHPLC-MS/MS), this method was validated according to U.S. Food and Drug Administration (FDA) guidelines. Sample preparation involved simple protein precipitation followed by reversed phase UHPLC, which resolved all five pairs of cis/trans diastereomer in less than 10 min. Superwarfarins were measured using negative ion electrospray followed by selected-reaction monitoring on a triple quadrupole mass spectrometer. Calibration curves covered 3-4 orders of magnitude with linear regression coefficients of >0.999. The lower limits of quantitation were from 0.013 to 2.41 ng/mL, and intra-day and inter-day accuracy and precision coefficients of variation were <12%. A 10-min UHPLC-MS/MS assay was developed and validated for the separation and quantitative analysis of the pairs of diastereomer of five superwarfarins in human plasma. This method was used to identify and measure superwarfarins and their cis/trans diastereomers in plasma obtained from patients treated for coagulopathy following consumption of contaminated synthetic cannabinoid products.

Pharmacokinetics and bioavailability of hapepunine in mice by ultra-performance liquid chromatography–tandem mass spectrometry

An ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was established to determine the hapepunine in mouse blood, and the pharmacokinetics of hapepunine after intravenous (1.0 mg/kg) and intragastric (2.5, 5, and 10 mg/kg) administrations was studied. Delavinone was used as an internal standard. The UPLC ethylene bridged hybrid (BEH) C18 column was used for chromatographic separation. The mobile phase consisted of acetonitrile and 0.1% formic acid with a gradient elution flow rate of 0.4 mL/min. Multiple reaction monitoring (MRM) mode was used for quantitative analysis of hapepunine in electrospray ionization (ESI) positive interface. Proteins from mouse blood were removed by acetonitrile precipitation. The verification method was established in accordance with the US Food and Drug Administration (FDA) bioanalytical method validation guidelines. In the concentration range of 1–1000 ng/mL, the hapepunine in the mouse blood was linear (r2 &gt; 0.995), and the lower limit of quantification was 1.0 ng/mL. In the mouse blood, the intra-day precision coefficient of variation (CV) was less than 12%, the inter-day precision CV was less than 14%. The accuracy ranged from 91.7% to 109.3%. The average recovery was higher than 76.7%, and the matrix effect was between 86.0% and 106.4%. The UPLC–MS/MS method was sensitive, rapid, and selective and was successfully applied to the pharmacokinetic study of hapepunine in mice. The absolute bioavailability of hapepunine was 22.0%.

Validation of UV-Vis Spectrophotometric Method of Quercetin in Ethanol Extract of Tamarind Leaf

Tamarindus indica L is a medicinal plant that has many benefits. One of the chemical compounds contained therein is the flavonoid quercetin type. The number of herbal products on the market makes the quality assurance of herbal products need to be done by performing the assay of the active compounds using validated methods. This study aims to validate the assay method quercetin in the extract of tamarind leaves. Tamarind leaf extract was macerated with hexane; then, it was re-macerated with 70% ethanol. The extract was concentrated using a rotary evaporator. The assay was performed using the UV-Vis Spectrophotometry method, and parameter validation specified in this study, including linearity, LOD, LOQ, precision, and accuracy. Quercetin level obtained in extracts of tamarind leaves was at 21.52 mg/g. Based on the test method validation, the correlation coefficient (r) was 0.9999, the regression function coefficients (Vx0) was 0.59545%, LOD 0.1515 ppm, LOQ 0.4592 ppm, coefficient of variation precision was less than 2%, and recoveries range was in 97-103%. Tamarindus indica L is a medicinal plant that has many benefits. One of the chemical compounds contained therein is the flavonoid quercetin type. The number of herbal products on the market makes the quality assurance of herbal products need to be done by performing the assay of the active compounds using validated methods. This study aims to validate the assay method quercetin in the extract of tamarind leaves. Tamarind leaf extract was macerated with hexane; then, it was re-macerated with 70% ethanol. The extract was concentrated using a rotary evaporator. The assay was performed using the UV-Vis Spectrophotometry method, and parameter validation specified in this study, including linearity, LOD, LOQ, precision, and accuracy. Quercetin level obtained in extracts of tamarind leaves was at 21.52 mg/g. Based on the test method validation, the correlation coefficient (r) was 0.9999, the regression function coefficients (Vx0) was 0.59545%, LOD 0.1515 ppm, LOQ 0.4592 ppm, coefficient of variation precision was less than 2%, and recoveries range was in 97-103%.