Between-run Coefficients Of Variation Research Articles

Performance evaluation of four kits for the detection of neutralizing antibody against SARS-CoV-2 in human serum

To evaluate four novel SARS-CoV-2 neutralizing antibody assay kits' application in neutralizing antibodies of population. Questionnaires from the voluntary participating researchers and selected the qualified questionnaires to analyse. For negative and positive coincidence rate, four novel SARS-Cov-2 neutralization antibody assay kits were tested. For within-run and between-run Precision verification study, four serum samples with two high and two low titer neutralizing antibodies were used to analyse. Based on the questionnaires, 175 qualified samples were divided into two groups. (1) negative neutralizing antibodies group: 31 samples had not been infected with the novel SARS-Cov-2 nor received the vaccine within the past one year; (2) positive neutralizing antibodies group: 144 samples were infected by COVID-19. There was 28 negative and 3 positive neutralizing antibodies of the individuals among the 31 negative samples which based on the questionnaires. The negative rates of 28 negative individules tested by GenScript, Vazyme and Hygeianey were 82.14 %, 60.71 % and 17.85 %, while the positive rates of the 147 positive samples were 93.87 %, 95.23 % and 100 %. The within-run coefficient of variations (C·V) of PBNAs, GenScript, Vazyme and Hygeianey were 11.49 %, 9.12 %, 7.97 % and 7.48 %, while the between-run coefficient of variations (C·V) were 21.37 %, 14.21 %, 12.29 % and 11.78 %. Due to the large within-run and between-run coefficient of variations, PBNAs was not suitable for large-scale promotion, while ELISAs could be leveraged for routine monitoring the titer of neutralizing antibodies against SARS-CoV-2.

A validated UPLC-MS/MS method for the quantification of immunosuppressive drugs in peripheral blood mononuclear cells using liquid-liquid extraction with low temperature purification without complex pretreatment steps

Immunosuppressive drugs (ISDs) are given to avoid the allograft rejection after transplantation. The concentrations of ISDs should be closely monitored owing to their wide inter-individual variability in its pharmacokinetics and narrow therapeutic window. Currently, the whole blood concentration measurement is the major approach of therapeutic drug monitoring of clinical ISDs in organ transplantation. Its correlation with the efficacy of ISDs remains elusive. While the acute rejection after transplantation may occur even when whole-blood ISDs concentrations are within the target range. Since the site of action of ISDs are within the lymphocyte, direct measurement of drug exposure in target cells may more accurately reflect the clinical efficacy of ISDs. Although several methods have been developed for the peripheral blood mononuclear cells (PBMCs) extraction and drug concentration measurement, the complex pre-processing has limited the study of the relationship between intracellular ISDs concentrations and the occurrence of rejection. In this study, the extraction of ISDs in PBMCs was carried out by the liquid-liquid extraction with low temperature purification, without centrifugation. The lower limit of quantitation were 0.2 ng/mL for cyclosporine A, tacrolimus and sirolimus, 1.0 ng/mL for mycophenolic acid, and the within-run and between-run coefficient of variations were both less than 12.4 %. The calibration curves of mycophenolic acid had a linear range (ng/mL): 1.0–128.0 (r2 = 0.9992). The calibration curves of other three ISDs had a linear range (ng/mL): 0.2–20.48 (r2 > 0.9956). A total of 157 clinical samples were analyzed by the UPLC-MS/MS for ISDs concentration in blood or plasma ([ISD]blood or plasma) and the concentration within PBMCs ([ISD]PBMC). Although there was strong association between [ISD]PBMC and [ISD]blood or plasma, the large discrepancies between concentration within [ISD]blood or plasma and [ISD]PBMC were observed in a small proportion of clinical samples. The developed method with short analysis time and little amounts of blood sample can be successfully applied to therapeutic drug monitoring of ISDs in PBMCs for analysis of large numbers of clinical samples and is helpful to explore the clinical value of ISDs concentration in PBMCs.

Abbott D-dimer assay: analytical performance and diagnostic accuracy in management of venous thromboembolism

This study aimed to assess analytical characteristics and diagnostic accuracy in management of venous thromboembolism (VTE) in the Emergency Department (ED) of the Abbott D-dimer assay applied on the Alinity c clinical chemistry analyzer (Abbott Laboratories, Chicago, IL) compared to the INNOVANCE D-dimer assay (Siemens Healthineers, Marburg, Germany). Precision was determined at three concentration levels following the CLSI EP15-A3 protocol. Method comparison and diagnostic accuracy were assessed using samples obtained from 85 patients who were referred for diagnostic imaging and D-dimer testing due to clinically suspected VTE. Within-run coefficients of variation (CVs) were 3.0%, 0.5% and 0.5% at D-dimer concentrations of 0.54, 1.42 and 2.68 mg/L FEU, while respective between-run CVs were 2.0%, 3.4% and 2.7%, hence fulfilling the desirable biological variation criteria for imprecision (<12.6%). Passing–Bablok regression analysis yielded a small proportional difference between the two compared assays (y = 1.09 (95% confidence interval (CI): 1.01–1.18) x + 0.09 (95%CI: –0.09 to 0.16)), while Bland–Altman analysis showed significant negative absolute (–0.6 mg/L FEU, 95%CI: –0.9 to −0.3) and relative mean bias (–14.1%, 95%CI: –20.3 to −7.9). Spearman’s ρ was 0.979 (95%CI: 0.967–0.986). Inter-assay agreement relative to the cut-off was 92% (kappa coefficient = 0.547 (95%CI: 0.255–0.839)). Diagnostic sensitivity, specificity, positive and negative predictive values of the Abbott assay were 100%, 9.2%, 25.3% and 100%, respectively, compared to the following data for the INNOVANCE assay: 95.0%, 15.4%, 25.7% and 90.9%. Abbott D-dimer assay has shown excellent analytical precision, high comparability with the INNOVANCE D-dimer and high NPV at manufacturer’s cut-off.

Evaluation of a new Electrochemiluminescence Immunoassay System for Measuring High-Sensitivity Cardiac Troponin T.

The aim of this study was to verify the analytical performance of the UD90DT electrochemiluminescence immunoassay system (the UD90DT system) for measuring high-sensitivity cardiac troponin T (hs-cTnT). According to the Clinical and Laboratory Standards Institute guidelines, the imprecision, linearity, reference interval, limit of blank (LoB), limit of detection (LoD), and functional sensitivity (FS) of hs-cTnT using the UD90DT system were verified. The trueness was validated using the Proficiency Testing (PT) materials. The within-run and between-run coefficients of variations (CVs) of two hs-cTnT levels were 7.2% and 1.5%, and 7.1% and 2.6%, respectively. The biases of the PT samples (n = 6) all fell within the allowable total error. The linearity satisfied the requirements, with a slope of 0.9963 and an R12 value of 0.9998. The hs-cTnT levels of the healthy volunteers (n = 20) ranged from 3.0 ng/L to 7.7 ng/L. All blank calibrator measurements (n = 20) fell within the LoD claim, and none of the samples (n = 25) had a LoB value ≤ 3.0 ng/L. The FS was 5.3 ng/L. Furthermore, a good correlation between the UD90DT system and the Cobas e 601 module was observed for hs-cTnT. The analytical performance of hs-cTnT using the UD90DT system is acceptable and satisfies clinical needs.

Quantifying plasma dacarbazine levels in advanced melanoma patients: a liquid chromatography-tandem mass spectrometry performance analysis

The aim of this study was to develop a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying dacarbazine levels in the plasma of advanced melanoma patients, followed by an assessment of its analytical capabilities. The research encompassed the design of a high-performance liquid chromatography (HPLC) system, with the quantitative analysis performed using the multiple reaction monitoring (MRM) techniques and specific ion transition: 181.0 > 152.5 for dacarbazine and 187.1 > 158.6 for the internal standard (IS), dacarbazine-D6. The validation of the method involved an evaluation of parameters including linearity, detection limit, precision, and accuracy. Notably, the linear range extended from 10 to 1,000 µg/L for dacarbazine, and the method exhibited a detection limit of 10 µg/L. The method’s precision, indicated by within-run and between-run coefficients of variation (CV), both being ≤4.2% and ≤8.3%, respectively. Furthermore, the accuracy of measurements, ranging from 86.1% to 99.4%, underscored the method’s reliability. In clinical application, the dacarbazine levels of healthy control (n = 20) were 0.6 ± 0.02 μg/L; 770.9 ± 203.2 μg/mL in early-stage-melanoma patients (n = 22), and 588.7 ± 153.2 μg/mL in advanced melanoma patients (n = 25). The results serve as clinical evidence showing that long-term dacarbazine treatment affects the metabolism of dacarbazine.

Dataset of Partial Analytical Validation of the 1,2-O-Dilauryl-Rac-Glycero-3-Glutaric Acid-(6′-Methylresorufin) Ester (DGGR) Lipase Assay in Equine Plasma

Laboratory assays require analytical validation to prove they are providing accurate results. This dataset describes the partial analytical validation of lipase activity, measured with the 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester (DGGR) lipase assay in equine plasma. Samples with low (approx. 12 U/L), moderately increased (approx. 79 U/L), and markedly increased lipase activity (approx. 298 U/L) were chosen. Linearity was assessed in samples of ascending dilution prepared by mixing samples with low and high lipase activity in different proportions. Repeatability or intra-assay replication was evaluated by measuring each level in 25 replicates within the same run. Reproducibility or inter-assay replication was calculated by measuring each level in five replicates on five consecutive days. The assay was linear in the range of 12–298 U/L (R2 = 0.9998) with a &lt;2.3% deviation from the calculated value at any point. Within-run coefficients of variation were 4.43%, 0.69%, and 1.00% for the low, medium, and high samples, respectively. Between-run coefficients of variation were 3.57%, 1.42%, and 1.16%, respectively. To our knowledge, these are the first published data on the analytical validation of the DGGR lipase assay in horses, which may be of interest to veterinary clinical pathologists and equine clinicians measuring DGGR lipase in equine blood for diagnostic and research purposes.

Analytical validation of the modified Westergren method on the automated erythrocyte sedimentation rate analyzer CUBE 30 touch.

Analytical validation of automated erythrocyte sedimentation rate (ESR) analyzers is necessary prior to their implementation into routine practice. Our aim was toperform the analytical validation of the modified Westergren method applied on the CUBE 30 touch analyzer (Diesse, Siena, Italy). Validation included determination of within-run and between-run precision following the Clinical and Laboratory Standards Institute EP15-A3 protocol, comparison with the reference Westergren method, sample stability assessment at both room temperature and 4°C, after 4, 8 and24-h storage, and checking the extent of hemolysis and lipemia interference. Coefficients of variation (CVs) for within-run precision were 5.2% for the normal and 2.6% for the abnormal range, while between-run CVs were 9.4 and 2.2%, respectively. Comparison with the Westergren method (n=191) yielded Spearman's correlation coefficient of 0.93, noconstant nor proportional difference [y=0.4 (95% CI:-1.7-1.0)+1.06 (95% CI: 1.00-1.14)x] and a non-significant mean absolute bias of-2.6mm (95% CI:-5.3-0.2). Lower comparability was evidenced with increasing ESR values, with both constant and proportional differences for ESR values between 40 and 80mm, and above 80mm. Sample stability was not compromised up to 8-h storage both at room temperature (p=0.054) and 4°C (p=0.421). Hemolysis did not affect ESR measurement up to 1.0g/L of free hemoglobin (p=0.089), while lipemia index above 5.0g/L affects the ESR result (p=0.004). This study proved that CUBE 30 touch provides reliable ESR measurement and satisfactory comparability with the reference Westergren methods, with minor variation related to methodological differences.

Analytical Validation of the IMMULITE® 2000 XPi Progesterone Assay for Quantitative Analysis in Ovine Serum.

Monitoring circulating progesterone (P4) concentration is an important component of basic and applied reproduction research and clinical settings. IMMULITE® 2000 XPi (Siemens) is a newly upgraded fully automated immunoassay system marketed for human use to measure concentrations of different analytes including P4. Our objective was therefore to characterize the analytical performance of the IMMULITE® 2000 XPi P4 immunoassay across the reportable range in ovine serum. This validation of analytical performance included determining (1) linearity, (2) precision through within-run, and between-run coefficient of variation (CV) calculations, (3) accuracy through bias calculations for spiking-recovery bias and interlaboratory (range and average based) bias for two laboratories across the reportable range (0.2-40 ng/mL). The average within-run and between-run precision (CV%) across the reportable range of the IMMULITE® 2000 XPi P4 immunoassay for serum P4 concentration were both &lt;5%, ranging between 2-8%. The average Observed Total Analytic Error (TEo) reported here for serum P4 concentration across the reportable range was ~30%, ranging from 14.8-59.4%, regardless of the considered bias. Based on these data we conclude that the automated IMMULITE® 2000 XPi P4 immunoassay provides a precise, accurate, reliable, and safe method for measuring P4 concentration ovine serum.

Analytical validation of 39 clinical chemistry tests and 17 immunoassays on the Alinity analytical system

The aim of this study was to perform the analytical validation of Alinity c and i analyzers (Abbott Laboratories, Chicago, IL, USA) for 39 clinical chemistry tests and 17 immunoassays. Precision was evaluated at least at two concentration levels for 5 days in quintuplicate, following CLSI EP15-A3. Method comparison included parallel analysis of leftover routine samples on Alinity analyzers and the previously used Cobas c501 and e601 (Roche Diagnostics, Mannheim, Germany). Linearity was tested by preparing sequential sample dilutions with high analyte concentration, following the CLSI EP6 document. For clinical chemistry tests, within-run coefficients of variation (CV) were up to 6.0% (beta-2-microglobulin), while between-run CVs up to 5.4% (immunoglobulin M). Among immunoassays, the highest within-run CV was obtained for vitamin B12 (6.9%), while between-run for CA 19-9 (4.3%). Complete agreement with Roche analyzers was observed for 16 (41%) clinical chemistry assays and 6 (35%) immunoassays. Half of all evaluated assays did not meet the desirable biological variation criteria for bias, being especially exceeded for alpha1-antitrypsin, apolipoprotein A1, ceruloplasmin, complement C3 and C4, hemoglobin A1c, lipoprotein (a) and myoglobin, as well as some tumor markers (CA 125, CEA, fPSA, AFP, and ferritin), hormones (cortisol, DHEA-S, insulin) and vitamins (25-OHD). Linearity in the tested ranges was confirmed. Overall, this study revealed that precision criteria derived from manufacturer's claims were not satisfied for all assays while comparison study for some assays yielded differences that imply the need for additional assay evaluation prior to introduction into routine practice.

Labile glycated hemoglobin: an underestimated laboratory marker of short term glycemia.

Diabetes mellitus is a major public health problem. Hemoglobin A1c (HbA1c) is a key laboratory parameter in the management of diabetes patients. However, in diabetes monitoring, interpretation of HbA1c results is hampered by the important interindividual variation in red blood cell (RBC) life span. Furthermore, HbA1c only slowly responds to changes in glucose metabolism. Besides HbA1c, there exists a labile HbA1c fraction (l-HbA1c), exhibiting much faster kinetics. As both HbA1c and l-HbA1c are measured by modern standard chromatography, we explored the possibilities of using the l-HbA1c fraction for monitoring glycemia. l-HbA1c and HbA1c fractions were simultaneously assayed on a Tosoh G8 analyzer and expressed as %. l-HbA1c results were compared with serum glucose and HbA1c. Concomitantly, RBC distribution width (RDW) was determined on a Sysmex SN analyzer as a marker for erythrocyte life span. l-HbA1c could be measured with between-run coefficient of variations (CVs) between 2.2 and 2.3%. l-HbA1c correlated with both glycemia (r=0.80) and HbA1c results (r=0.73). In a multiple regression model (r2=0.752), glycemia and HbA1c were the most determining factors. To a lesser extent, RDW correlated with l-HbA1c (r=0.158). Furthermore, the l-HbA1c/HbA1c ratio weakly positively correlated with RDW (r=0.247). L-HBA1c represents an additional marker for monitoring the rapid occurrence of glycemic disorders that escape detection when using only HbA1c and blood glucose. RDW can be used as an indicator of atypical RBCs life span, in which the l-HbA1c fraction may be helpful.

Comparison between an Emerging Point-of-Care Tool for TSH Evaluation and a Centralized Laboratory-Based Method in a Cohort of Patients from Southern Italy.

Endocrine and metabolic disorders are a common condition in Europe and worldwide, and, among these, thyroid dysfunction still remains a problem. The measurement of thyroid stimulating hormone (TSH) levels represents the first-line assay for the assessment of thyroid function. In the present study, we compared serum concentrations of TSH, measured using a commercially available point-of-care test (POCT) method (FastPack® IP) and an established “conventional” laboratory-based method (Beckmann Access 2) in a cohort of patients from Foggia in Southern Italy. A strong correlation (r = 0.994) was found between both methods and was also confirmed by receiver operating characteristic (ROC) curve analysis (0.82). The within-run coefficient of variation (CV) using FastPack® ranged from 4.03% and 8.57% at the TSH concentrations of 39.49 and 0.70 mIU/L, respectively. The between-run CV was 10.34% and 6.33% at the TSH concentrations of 0.87 and 26.55 mIU/L, respectively. The ratios of within- to between-assay CV were 0.83 and 1.06 at the TSH levels of 0.70 and 52.59 mIU/mL, respectively. In this study, we showed that serum TSH levels can be measured in a few minutes and at low-cost in terms of materials and equipment required. We observed that this approach is user-friendly, accurate, reproducible, and suitable for use in the clinic, while also meeting the criteria for effectiveness, impact, efficiency, and sustainability.

Application of enzymatic fluorometric assays to quantify phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in human plasma lipoproteins

Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) are important surface components of plasma lipoproteins, including very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). However, the pathophysiological roles of PC, PE and SM in lipoproteins have not been well characterized owing to the difficulties in quantifying phospholipid classes in lipoproteins. In this study, we assessed the precision and accuracy of the enzymatic fluorometric assays for measuring PC, PE and SM in VLDL, LDL and HDL, which were isolated from human plasma by ultracentrifugation. The within-run coefficients of variation (CV) for the measurements of PC, PE and SM in lipoproteins were 1.5–2.8 %, 1.1–2.4 % and 0.9–2.3 %, respectively, whereas the between-run CVs for the PC, PE and SM assays were 2.7–4.7 %, 2.1–4.5 % and 1.6–3.3 %, respectively. Excellent linearity and almost complete recovery were achieved for all assays measuring PC, PE and SM in VLDL, LDL and HDL. Our preliminary results using these enzymatic fluorometric assays suggested that the phospholipid compositions were different among VLDL, LDL and HDL. In conclusion, we established high-throughput enzymatic fluorometric assays to quantify PC, PE and SM in human plasma VLDL, LDL and HDL, which will be useful for further investigation of pathophysiological roles of phospholipids in lipoproteins.

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.

Method development for the quantitative determination of captopril from Caco-2 cell monolayers by using LC-MS/MS

Aim. Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. Although compounds were used in the original Caco-2 cells monolayer assays, compounds have been replaced in most laboratories by the use of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS/MS). Mass spectrometry not only eliminates the need for compounds, but permits the simultaneous measurement of multiple compounds. The measurement of multiple compounds per assay reduces the number of incubations that need to be carried out, thereby increasing the throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add another dimension to Caco-2 assays by facilitating the investigation of the metabolism of compounds by Caco-2 cells. A simple, rapid LC-MS/MS method has been developed for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Materials and methods. Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl. Results. Under these conditions, captopril was eluted at 1.42 min. A linear response function was established at 2 – 200 ng/mL. The regression equation for the analysis was y =0.0187x+0.000248 with coefficient of correction (r2) = 0.9993. According to the Caco-2 test results, captopril showed low permeability. It should be noted that the recovery value is 103.20%. The within-run coefficients of variation ranged between 0.321% and 0.541%. The within-run percentages of nominal concentrations ranged between 99.13% and 101.12%. The between-run coefficients of variation ranged between 0.314% and 0.663%. The between-run percentages of nominal concentrations ranged between 99.17% and 101.03%.The assay values on both the occasions (intra- and inter-day) were found to be within the accepted limits. Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of captopril from Caco-2 cell monolayers.

A Simple and High-Throughput LC-MS-MS Method for Simultaneous Measurement of Nicotine, Cotinine, 3-OH Cotinine, Nornicotine and Anabasine in Urine and Its Application in the General Korean Population.

Measuring nicotine metabolites is the most objective method for identifying smoke exposure. Liquid chromatography--tandem mass spectrometry (LC-MS-MS) can measure multiple metabolites and is sensitive enough to detect low concentrations of metabolites. Therefore, we developed a simple and high-throughput method for measuring nicotine, cotinine, trans-3'-hydroxycotinine (3-OH cotinine), nornicotine and anabasine for population-based studies using LC-MS-MS. Each 30 µL of urine sample was diluted with 90 µL of acetonitrile containing five deuterated internal standards. Chromatographic separation used a C18 column, and LC-MS-MS analysis was performed with a multiple reaction monitoring mode. The chromatographic run time for each sample was 6.5 min. The method was validated by evaluating selectivity, interference, limit of detection, lower limit of quantification, precision, accuracy, linearity, extraction recovery, matrix effect and carryover according to guidelines. Our methods required a short preparation time (∼20 min) while simultaneously measuring five markers for smoking status. No endogenous or exogenous interference was found. Our method showed excellent precision and accuracy: within-run coefficient of variation (CV) 2.9-9.4%, between-run CV 4.8-8.7% and bias -10.1 to 5.3%. Linear dynamic ranges were 1-10,000 ng/mL for nicotine, nornicotine and anabasine; 2-5,000 ng/mL for cotinine and 5-15,000 ng/mL for 3-OH cotinine. Extraction recovery was consistent (87-109%) across concentrations. No significant matrix effect or carryover was observed. The validated method was applied to 849 urine samples. In samples from the 125 current smokers, nicotine, cotinine, 3-OH cotinine, nornicotine and anabasine were detected in 97.6, 99.2, 98.4, 96.8 and 87.2%, respectively. No markers were detected in 93.9% of 609 nonsmokers. The overlapping detection of multiple markers made it possible to identify the smoking status even in current smokers with a low concentration of cotinine. Our LC-MS-MS method using a simple sample preparation technique is sensitive and effective for screening of smoking status in the general population.

Comparison of sample preparation methods, validation of an UPLC–MS/MS procedure for the quantification of cyclosporine A in whole blood sample

Current main methods for therapeutic drug monitoring (TDM) of cyclosporine A (CsA) are immunoassays and liquid chromatography tandem mass spectrometry. The sample pretreatment of these methods is mainly based on extraction of drug which is bound to erythrocytes by divalent heavy metal ions (such as zinc and copper). Although these methods are effective for whole blood drug extraction and measurement, the pollution of heavy metals in sample pretreatment process will have potential negative impact on environment and human health. To overcome the pollution problem, in this study we have developed and validated an UPLC-MS/MS method for CsA determination in whole blood samples using physical pretreatment method. According to the characteristics of erythrocytes, a series of physical pretreatment methods, including sonication, freeze-thaw and osmotic burst, have been developed and evaluated. The results showed that the osmotic burst method was an effective way for drug extraction from erythrocytes. The lower limit of quantitation for CsA was 25 ng/mL, the within-run and between-run coefficient of variations were both less than 11.6 %. The agreement of the UPLC-MS/MS methods using these two sample pretreatment was evaluated by Bland-Altman plot and the two-tailed Student’s T-test. Comparison studies show that the effect of erythrocyte fragmentation by osmotic burst is similar to that of zinc sulfate method. The CsA measurement of 103 whole blood samples obtained by these two UPLC-MS/MS assays were no significant difference. These results demonstrate that the sample pretreatment by osmotic burst method is an eco-friendly and precise method for detecting the whole blood CsA concentration and therapeutic drug monitoring of CsA.

Quantitation of plasma and biliary cefpiramide concentrations in human samples using high-performance liquid chromatography.

Cefpiramide is frequently used to treat biliary infections. However, no bioanalytical method has been validated to quantitate cefpiramide in human samples, particularly in bile. Therefore, this study was conducted to develop a simple, selective and validated high-performance liquid chromatographic method to determine cefpiramide in human plasma and bile. A protein precipitation procedure was used to extract cefpiramide and cefoperazone (internal standard, IS) from 200 μl of plasma and bile. Utilizing a Capcell Pak C18 column (4.6 × 250 mm), cefpiramide and IS were separated using the timed-gradient mobile phase consisting of 0.1 m sodium acetate (pH 5.2) and acetonitrile at a flow rate of 1 ml/min with photodiode array detector (wavelength set at 273 nm). The calibration curves showed linearity at concentrations ranging from 1 to 150 μg/ml in both plasma and bile (r2 > 0.999). The within- and between-run coefficients of variation (CVs) for plasma samples were 0.570-4.43 and 1.10-2.76%, respectively; for bile samples, the within- and between-day precision (CV) was 0.814-6.34 and 2.05-4.00%, respectively. Our newly developed bioanalytical method was successfully employed to quantify cefpiramide concentrations in both plasma and bile at multiple time points in patients with acute cholangitis.

Urinary Concentrations of Phthalate Metabolite Mixtures in Relation to Serum Biomarkers of Thyroid Function and Autoimmunity among Women from a Fertility Center.

Background:Although previous epidemiological studies have explored associations of phthalate metabolites with thyroid function, no studies to date have assessed associations of mixtures with thyroid function and autoimmunity among potentially susceptible subgroups such as subfertile women.Objective:We aimed to explore associations of mixtures of urinary phthalate metabolites with serum markers of thyroid function and autoimmunity.Methods:This cross-sectional study included 558 women attending a fertility center who provided one spot urine and one blood sample at enrollment (2005–2015). We quantified urinary concentrations of eight phthalate metabolites using mass spectrometry, and biomarkers of thyroid function [thyroid-stimulating hormone (TSH), free and total thyroxine (, ) and triiodothyronine (, ), and autoimmunity [thyroid peroxidase and thyroglobulin antibodies (TPOAb and TgAb, respectively)] in serum using electrochemiluminescence assays. We applied principal component analysis (PCA) and Bayesian kernel machine regression (BKMR) to identify the main patterns of urinary phthalate metabolites. We used linear mixed models to assess the association between PCA-derived factor scores in quintiles and serum thyroid function and autoimmunity, adjusting for age, body mass index (BMI), specific gravity (SG), and, for the PCA, other factor scores.Results:We observed two factors using PCA, one representing the di(2-ethylhexyl) (DEHP) and another non-DEHP metabolites. Compared to women in the lowest quintile of the DEHP factor scores, women in the highest quintile had significantly lower serum concentrations of , , , and [absolute difference: ; 95% confidence interval (CI): , ; ; absolute difference: ; 95% CI: , ; ; absolute difference: ; 95% CI: 0.54, ; ; and absolute difference: ; 95% CI: , ; , respectively]. Using BKMR, we observed that mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) was the primary contributor to these negative associations. DEHP and non-DEHP factor scores were not associated with serum TSH, TgAb, or TPOAb.Conclusions:Mixtures of urinary DEHP metabolites were inversely associated with serum biomarkers of thyroid function but not with autoimmunity, which were within normal ranges for healthy adult women. https://doi.org/10.1289/EHP6740

Simple and Rapid Quantification of the Multi-Enzyme Targeting Antifolate Pemetrexed in Human Plasma.

Pemetrexed is an antifolate cytostatic drug that targets multiple enzymes involved in folate biosynthesis and is indicated for treatment of non-small-cell lung cancer and malignant pleural mesothelioma. As evidence for an exposure-response/toxicity relationship is accumulating, dose individualization using therapeutic drug monitoring may be a feasible strategy to optimize treatment. The purpose of this study was to develop a simple, sensitive, high-performance liquid chromatography method with UV detection for quantification of pemetrexed levels in human plasma. The method involves a simple protein precipitation, followed by ultra-performance liquid chromatography with ultraviolet detection at a wavelength of 254 nm. Pemetrexed was separated using a mobile phase with a linear gradient and a run time of only 7 minutes. The assay has been validated over the concentration range 0.25-500 mg/L of pemetrexed. Accuracy for this assay ranged from -4.50% to 1.78%, and the within- and between-run coefficients of variation were <3.57%. Pemetrexed in plasma was proven to be stable for 8 months at -40°C. The bioanalytical method we developed proved to be simple, accurate, precise, and fast. This analytical method is successfully in use for therapeutic drug monitoring and will be used for pharmacokinetic studies.

Analytical Performance of Two Enzymatic Methods for Glycated Albumin.

The measurement of glycemic control among patients with diabetes mellitus is important for predicting the risk of diabetic complications. Glycated hemoglobin (HbA1c) measurements have been used for long-term glycemic control in clinical practice. However, glycated albumin (GA) or glycated serum protein (GSP) is a more reliable indicator of glycemic control in the short term (2 - 4 weeks) and an alternative marker of HbA1c in clinical situations with changing red blood cell (RBC) lifespan. Here, we evaluated an analytical performance of the two enzymatic assays commercially available, Lucica GA-L and Autolab GA, for the determination of GA (%). For each assay, the imprecision was evaluated based on CLSI EP05-A2. In total, serum samples of 283 subjects were simultaneously tested using the two enzymatic assays for method comparison according to CLSI EP09-A3. Some subjects collected the laboratory data for HbA1c. The GA (%) value of the Lucica GA-L assay showed highly reproducible results with within-run, between-run, and total coefficient of variations (CVs) below 2.4%. The Autolab GA assay also showed reliable results with within-run, between-run, and total CVs below 3.9%. The Lucica GA-L assay showed a very high correlation with the Autolab GA assay (r = 0.9993). However, at the median decision point (MDP, 14.3%), the estimated bias of the Autolab GA assay was 4.5%, exceeding the allowable bias (2.9%) accounting for the biological variation. For the correlation analysis between HbA1c and GA (%), the two assays demonstrated the same pattern, with no statistical differences between the two independent correlation coefficients. Both GA assays evaluated in this study showed good precision and excellent correlation, but the comparability at MDP did not meet the acceptance criteria.