Between-day Coefficients Of Variation Research Articles

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF EZETIMIBE IN RABBIT PLASMA USING REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objectives: The objective of the present work was to develop an analytical method and validation for the estimation of Ezetimibe in rabbit plasma using high-performance liquid chromatography (HPLC). Methods: A simple, rapid, sensitive, and accurate HPLC method was developed and validated for the quantification of Ezetimibe concentration in rabbit plasma using metoclopramide as an internal standard. Separation was performed on the Xerra C18 column (250 × 4.6 mm 5 μm) using a mobile phase consisting of 0.1% perchloric acid: acetonitrile(55:45 v/v) at a flow rate of 1 ml/min. Validation of the method was performed to demonstrate its selectivity, linearity, precision, accuracy, ruggedness, recovery, and matrix effect. Results: The calibration curves of Ezetimibe were linear over a concentration range of 5–1022 μg/mL. The with-in and between-day coefficients of variation were <10%. The extraction recoveries of Ezetimibe at the three levels of quality control samples were 99.961%, 99.767%, and 99.938%. Conclusion: The method was rapid with a retention time of Ezetimibe and the internal standard observed at 10 min, respectively. The developed method was successfully applied to studying the pharmacokinetics of Ezetimibe in rabbits.

B-041 Evaluation of a Nuclear Magnetic Resonance Spectroscopy-Based Method for Estimating Glomerular Filtration Rate Using Creatinine, Cystatin C, Myo-Inositol, and Valine

Abstract Background Timely and accurate monitoring of renal dysfunction is critical for patients with chronic kidney disease (CKD). Most equations currently used for estimating glomerular filtration rate (GFR) incorporate serum levels of creatinine and/or cystatin C. Here, we evaluate a recently introduced method for estimating GFR that uses nuclear magnetic resonance spectroscopy (NMR) to measure creatinine, myo-inositol, and valine in combination with cystatin C measured by immunoassay, and patient’s age and sex (eGFRNMR). Methods Serum creatinine, valine, and myo-inositol measurements were performed simultaneously by an NMR assay on a Bruker Avance III HD 600 MHz NMR instrument (Bruker Corporation, MA). Cystatin C testing was performed using an immunoturbidimetric assay (Diazyme Laboratories, CA) on a cobas 8000 analyzer (Roche Diagnostics, IN). Within-run and between-day precision studies were performed across 5 days. Method comparison was carried out by testing 86 split serum samples from adult individuals against the numares AG GFR (NMR) assay. Of the 86 samples, 20 were spiked with various concentrations of creatinine, myo-inositol, and valine to reach a wide range of GFR results. An additional method comparison study was performed against Mayo Clinic Laboratories (Rochester, MN) on 59 serum samples from adult patients that had already been tested using the same assay. Measured GFR (mGFR) was also performed on these patients by Mayo Clinic Laboratories using urinary iothalamate clearance. Results Within-run and between-day coefficients of variation (%CV) for eGFRNMR did not exceed 6%. The eGFRNMR measurement range was confirmed at 10 to 157 mL/min/1.73 m2 based on verified analytical measurement ranges of creatinine (0.28–9.8 mg/dL), myo-inositol (39–430 µmol/L), and valine (30–1250 µmol/L). Method comparison studies against both numares AG and Mayo Clinic Laboratories demonstrated excellent concordance (r2 = 0.95 and r2 = 0.97, respectively) and minimal bias (4.1% and −0.3%, respectively) with results ranging from 21 to 157 mL/min/1.73 m2. When the 59 patients who had their GFR measured were staged by their mGFR results according to the CKD classification ranges of <15, 15–29, 30–44, 45–59, 60–89, and ≥90 mL/min/1.73 m2, eGFRNMR showed higher classification concordance with mGFR (69%) than the CKD-EPI 2021 creatinine-cystatin C equation (63%). Conclusion Our evaluation confirms the excellent analytical and clinical performance of the recently introduced eGFRNMR assay, supporting its integration into clinical practice for improved estimation of GFR.

Simple determination of urine cefazolin concentration in pediatric patients with urinary tract infections using high-performance liquid chromatography.

Recently, global health concerns regarding increasing multidrug resistance have arisen. This study aimed to develop a simple, inexpensive and rapid high-performance liquid chromatography-ultraviolet (HPLC-UV) method for determining urinary concentrations of a first-generation cephem antibiotic in pediatric patients with urinary tract infections (UTIs). HPLC-UV was used to analyze urinary cefazolin concentrations at a detection wavelength of 254 nm. The assay used contained 10-fold diluted urine with an internal standard (cephapirin). The standard calibration curve for cefazolin was linear in the concentration range of 31.25-500 μg/ml (r2 >0.999). The retention times of cefazolin and the internal standard were 4.2 and 4.9min, respectively. The within- and between-day coefficients of variation were in the concentration ranges 1.2-15.2 and 5.5-19.2%, respectively. The urinary cefazolin concentration of a pediatric patient with a UTI was 1,476.6μg/ml, which was over 700-fold higher than the minimum inhibitory concentration of cefazolin (≤2μg/ml). The developed method is applicable to the confirmation of appropriate use for UTI treatment as therapeutic drug monitoring of cefazolin. Therefore, the findings of this study may contribute to the appropriate use of antibiotics to prevent antimicrobial resistance in pediatric patients with UTIs.

105-LB: Glucose Variability with Second-Generation Basal Insulin Analogs Glargine 300 U/mL and Degludec 100 U/mL, Evaluated by CGM in People with T1D—The InRange Randomized Controlled Trial

Background: InRange has previously demonstrated that insulin glargine 300 U/mL (Gla-300) is non-inferior to insulin degludec 100 U/mL (IDeg-100) in terms of time in glucose range 70--180 mg/dL (primary endpoint) and total glucose coefficient of variation (CV) as measured by CGM in people with T1D. Methods: InRange (NCT04075513) was a multicenter, randomized, active-controlled, parallel-group, 12-week open-label trial comparing Gla-300 vs. IDeg-100 in adults with T1D using 20-day CGM profiles (≥10 days evaluable) . Here we present data for within- and between-day CV. Results: Overall, 343 participants were randomized (172 Gla-300, 171 IDeg-100) : mean ± SD age was 42.8 ± 13.3 years and T1D duration was 20.5 ± 12.8 years. Both within-day and between-day glucose CV at Week 12 were similar with Gla-300 and IDeg-100 (Table) . Within-day glucose CV at Week 12 was below the threshold for glycemic stability (36%) in both groups and was also similar in both treatment groups irrespective of glycemic stability at baseline (CV ≤36% or >36%) . The LS mean within-day glucose CV of participants with CV >36% at baseline reached 33.67 (Gla-300) and 35.38 (IDeg-100) at Week 12. Conclusion: Using clinically relevant CGM metrics, InRange shows that Gla-300 and IDeg-100 provide similar within- and between-day glucose variability in people with T1D. Disclosure R. M. Bergenstal: Advisory Panel; Hygieia, Medtronic, Roche Diabetes Care, Zealand Pharma A/S, Consultant; Abbott Diabetes, Ascensia Diabetes Care, Bigfoot Biomedical, Inc., CeQur SA, Dexcom, Inc., Eli Lilly and Company, Novo Nordisk, Onduo LLC, Sanofi, United HealthCare Services, Inc., Research Support; Abbott Diabetes, Dexcom, Inc., Eli Lilly and Company, Insulet Corporation, Medtronic, Novo Nordisk, Sanofi. T. Battelino: Advisory Panel; Abbott Diabetes, Indigo Diabetes, Lilly Diabetes, Medtronic, Novo Nordisk, Sanofi, Research Support; Medtronic, Novartis Pharmaceuticals Corporation, Novo Nordisk, Zealand Pharma A/S, Speaker’s Bureau; Abbott Diabetes, Dexcom, Inc., Lilly Diabetes, Medtronic, Novo Nordisk, Sanofi, Stock/Shareholder; DreaMed Diabetes, Ltd. S. Edelman: Consultant; Abbott Diabetes, Lilly Diabetes, Sanofi-Aventis U. S., Xeris Pharmaceuticals, Inc. P. Choudhary: Advisory Panel; Abbott Diabetes, Lilly Diabetes, Medtronic, Research Support; Novo Nordisk, Speaker’s Bureau; Dexcom, Inc., Glooko, Inc., Insulet Corporation, Sanofi. T. Danne: Consultant; Abbott, AstraZeneca, Boehringer Ingelheim International GmbH, Dexcom, Inc., Lilly, Medtronic, Novo Nordisk, Roche Pharmaceuticals, Sanofi, Ypsomed AG, Research Support; Abbott, AstraZeneca, Boehringer Ingelheim International GmbH, Dexcom, Inc., Lilly, Medtronic, Novo Nordisk, Roche Pharmaceuticals, Sanofi, Ypsomed AG, Stock/Shareholder; DreaMed Diabetes, Ltd. E. Renard: Consultant; Abbott, AstraZeneca, Boehringer Ingelheim International GmbH, Dexcom, Inc., Insulet Corporation, LifeScan, Lilly, Novo Nordisk, Roche Diabetes Care, Sanofi, Tandem Diabetes Care, Inc. J. Westerbacka: Employee; Sanofi, Stock/Shareholder; Sanofi. B. Mukherjee: Employee; Sanofi, Stock/Shareholder; Sanofi. P. Picard: n/a. V. Pilorget: None. Funding Sanofi

CardiOvascular examination in awake Orangutans (Pongo pygmaeus pygmaeus): Low-stress Echocardiography including Speckle Tracking imaging (the COOLEST method).

IntroductionCardiovascular diseases have been identified as a major cause of mortality and morbidity in Borneo orangutans (Pongo pygmaeus pygmaeus). Transthoracic echocardiography is usually performed under anesthesia in great apes, which may be stressful and increase risks of peri-anesthetic complications in case of cardiac alteration. The aim of the present pilot study was hence to develop a quick and non-stressful echocardiographic method (i.e., the COOLEST method) in awake Borneo orangutans (CardiOvascular examination in awake Orangutans: Low-stress Echocardiography including Speckle Tracking imaging) and assess the variability of corresponding variables.Materials and methodsFour adult Borneo orangutans trained to present their chest to the trainers were involved. A total of 96 TTE examinations were performed on 4 different days by a trained observer examining each orangutan 6 times per day. Each examination included four two-dimensional views, with offline assessment of 28 variables (i.e., two-dimensional (n = 12), M-mode and anatomic M-mode (n = 6), Doppler (n = 7), and speckle tracking imaging (n = 3)), representing a total of 2,688 measurements. A general linear model was used to determine the within-day and between-day coefficients of variation.ResultsMean±SD (minimum-maximum) images acquisition duration was 3.8±1.6 minutes (1.3–6.3). All within-day and between-day coefficients of variation but one (n = 55/56, 98%) were <15%, and most (51/56, 91%) were <10% including those of speckle tracking systolic strain variables (2.7% to 5.4%).DiscussionHeart morphology as well as global and regional myocardial function can be assessed in awake orangutans with good to excellent repeatability and reproducibility.ConclusionsThis non-stressful method may be used for longitudinal cardiac follow-up in awake orangutans.

A Novel UHPLC-MS/MS Method for Measuring 8-iso-Prostaglandin F2α in Bronchoalveolar Lavage Fluid.

In August 2019, the Centers for Disease Control and Prevention (CDC) received the first reports of lung injuries that were eventually termed e-cigarette, or vaping, product use–associated lung injury (EVALI). As part of the investigation, CDC laboratories rapidly developed assays for analyzing substances in bronchoalveolar lavage (BAL) fluid collected from EVALI case patients. This report describes the development and validation of a high-throughput isotope dilution UHPLC-MS/MS method for measuring a major oxidative stress biomarker, 8-iso-prostaglandin F2α (8-isoprostane), in BAL fluid samples. The method showed good sensitivity, 17.6 pg/ml LOD, and requires only 50 μl of sample volume. The method had high throughput with an analytical run time of 11 min. The within-day and between-day coefficient of variation (CV) were below 2%. Accuracy, calculated from spiked recovery, at three spiking levels, ranged from 95.5–101.8%. This novel UHPLC-MS/MS method characterizes oxidative stress in lung epithelial tissue and thus helps to elucidate potential pathologic processes.

A modified clot-based assay to measure negatively charged procoagulant phospholipids

Growing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL). Current commercial PPL-dependent clotting assays use chemically phospholipid depleted plasma to measure PPL activity. The purpose of our study was to modify the PPL assay by substituting the chemically phospholipid depleted plasma with PPL depleted plasma obtained by ultracentrifugation This in order to get readily access to a sensitive and reliable assay to measure PPL activity in human plasma and cell supernatants. The performance of the assay was tested, including the influence of individual coagulation factors and postprandial lipoproteins and compared to a commercial PPL assay (STA-Procoag-PPL). The two PPL assays displayed similar sensitivity to exogenously added standardized phospholipids. The PPL activity measured by the modified assay strongly correlates with the results from the commercial assay. The intraday- and between-days coefficients of variation ranged from 2–4% depending on the PPL activity in the sample. The modified PPL assay was insensitive to postprandial lipoprotein levels in plasma, as well as to tissue factor (TF) positive EVs from stimulated whole blood. Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF+ EVs.

Establishment of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Immunosuppressant Levels in the Peripheral Blood Mononuclear Cells of Chinese Renal Transplant Recipients.

Monitoring immunosuppressant levels, such as mycophenolic acid (MPA), cyclosporin A (CsA), and tacrolimus (TAC), in peripheral blood mononuclear cells (PBMCs) could be useful in organ transplant patients administered individualized therapy. The authors developed a liquid chromatography-tandem mass spectrometry assay technique to simultaneously determine immunosuppressant levels in PBMCs and assess their pharmacokinetics in Chinese renal allograft recipients. PBMCs were isolated from the whole blood of 27 Chinese renal transplant patients using Ficoll-Paque Plus solution, and cell number was determined; acetonitrile treatment for protein precipitation, and gradient elution was performed on an Agilent Eclipse XDB-C18 column (3.5 μm, 2.1 × 100 mm) with mobile phase: water and methanol (containing 2 mM ammonium formate); flow rate: 0.3 mL·min. The calibration curves of MPA, CsA, and TAC had a linear range (ng·mL): 0.098-39.2 (r = 0.9987), 0.255-102 (r = 0.9969), and 0.028-11.2 (r = 0.9993), respectively. The extraction effects, matrix effects, and mean relative recovery of these immunosuppressants were 70.4%-93.2%, 72.7%-96.5%, and 90.1%-112.4%, respectively. The within-day and between-day coefficients of variation were <15%. The AUC0-12 of MPA in PBMCs correlated well with those in plasma. The level of MPA, CsA, and TAC in PBMCs might be more stable during dosing interval. The derived liquid chromatography-tandem mass spectrometry assay is suitable for simultaneously monitoring different immunosuppressants in PBMCs. Pharmacokinetic of MPA, CsA, and TAC displayed considerable interindividual variability. Intracellular monitoring of immunosuppressants may facilitate individualized therapy for renal allograft recipients.

Detection of urinary prostate specific antigen by a lateral flow biosensor predicting repeat prostate biopsy outcome

This work describes the development and the application of a lateral flow biosensor for the detection of the prostate specific antigen in urine (uPSA). The biosensor allowed uPSA detection in 10 minutes with a limit of detection and a range of quantification respectively of 20 ng mL-1 and 37 – 420 ng mL-1, showing within and between-day coefficients of variation ≤ 13%. It showed 92% of accuracy and an almost perfect concordance with the reference electrochemiluminescence immunoassay method. The biosensor design provides the disappearance of the Test line signal at the cut-off concentration. This was achieved with a double layer sensing strategy, in which gold nanoparticles were functionalized with Staphylococcal protein A – a mediator – instead of anti-PSA antibody. This strategy allow making a fine-tune on the concentration of the specific antibody, obtaining an on/off switch of the Test line at the cut-off value. The cut-off value was also established in this work, based on the distribution of uPSA levels from 140 patients, who were suspected of prostate cancer and who underwent to first biopsy. The clinical application of the biosensor to predict repeat biopsy outcome in 28 patients showed sensitivity, specificity, positive and negative predictive values of 100%, 64%, 74% and 100%, respectively.

The Reliability of Neuromuscular and Perceptual Measures Used to Profile Recovery, and the Time-Course of such Responses following Academy Rugby League Match-Play.

In professional academy rugby league (RL) players, this two-part study examined; (A) the within- and between-day reliability of isometric mid-thigh pulls (IMTP), countermovement jumps (CMJ), and a wellness questionnaire (n = 11), and (B) profiled the responses with acceptable reliability (no between-trial differences and between-day coefficient of variation (CV) ≤10% and intraclass correlation coefficient (ICC) ≥0.8) for 120 h (baseline: −3, +24, +48, +72, +96, +120 h) following RL match-play (n = 10). In part A, force at 200, and 250 ms, and peak force (PF) demonstrated acceptable within- (CV%: 3.67–8.41%, ICC: 0.89–0.93) and between-day (CV%: 4.34–8.62%, ICC: 0.87–0.92) reliability for IMTP. Most CMJ variables demonstrated acceptable within-day reliability (CV%: 3.03–7.34%, ICC: 0.82–0.98), but only six (i.e., flight-time, PF, peak power (PP), relative PP, velocity at take-off (VTO), jump-height (JH)) showed acceptable between-day reliability (CV%: 2.56–6.79%, ICC: 0.83–0.91). Only total wellness demonstrated acceptable between-day reliability (CV%: 7.05%, ICC: 0.90) from the questionnaire. In part B, reductions of 4.75% and 9.23% (vs. baseline; 2.54 m∙s−1; 0.33 m) occurred at +24 h for CMJ VTO, and JH, respectively. Acceptable reliability was observed in some, but not all, variables and the magnitude and time-course of post-match responses were test and variable specific. Practitioners should therefore be mindful of the influence that the choice of recovery monitoring tool may have upon the practical interpretation of the data.

Performance evaluation of Siemens Atellica enhanced estradiol assay.

Serum estradiol (E2) levels may be used in the diagnostic and/or monitoring of a broad variety of clinical conditions. The aims of this study were to evaluate the Siemens enhanced estradiol assay (eE2) on Atellica IM 1600 (Siemens Healthineers) and to perform a sample comparison with the Siemens ADVIA Centaur XP (Siemens Healthineers). Within-day and between-day coefficient of variation (CV) were determined using serum sample pools and quality control materials. Six serum samples with decreasing concentrations of E2 were used to assess the limit of quantification. Linearity was evaluated using two different serum samples. Accuracy was evaluated by measuring three certified reference materials. Passing-Bablok regression and Bland-Altman plot were used for comparing Atellica and Centaur XP in 119 serum samples ranging from 45 to 10,059pmol/L. Within-day and between-day imprecision was <6.6%. Accuracy was <6.0% for all values except for 114pmol/L (22%). The study of limit of quantification resulted in an interday imprecision <20%. High correlation between measured and expected estradiol dilution results was observed (R=0.99), with recoveries ranging from 77 to 93%. Comparison study showed good agreement and no significant bias. The study shows that Atellica eE2 assay presents acceptable imprecision and accuracy and correlates well with Centaur XP.

Trapezius muscle activity variation during computer work performed by individuals with and without neck-shoulder pain

This study aimed at determining the extent to which individuals with neck-shoulder pain and non-symptomatic individuals differ in muscle activation patterns, when performing computer work, as quantified by exposure variation analysis (EVA). As a secondary aim, we also aimed to quantify the day-to-day reliability of EVA variables describing trapezius muscle activation in a non-symptomatic control group. Thirteen touch-typing computer users (pain: n = 5, non-symptomatic: n = 8) completed three pre-selected computer tasks in the laboratory. Upper trapezius muscle activity was recorded using electromyography and analyzed using EVA with five amplitude and five duration categories. Individuals with neck-shoulder pain spent less time at low amplitudes and exhibited longer uninterrupted periods of muscle activation compared to their non-symptomatic counterparts. Thus, non-symptomatic workers tended to switch between exposure levels more often than individuals with pain. For a majority of EVA variables, ICCs ranged from 0.6 to 0.9, and between-days coefficients of variation were between 0.4 and 2.2.

A quantitative LC/MSMS method for determination of edoxaban, a Xa inhibitor and its pharmacokinetic application in patients after total knee arthroplasty.

Edoxaban was extracted from human plasma by simple protein precipitation with acetonitrile, followed by quantitative determination using a liquid chromatography-mass spectrometry method. The recoveries of edoxaban and the internal standard (ticlopidine) from human plasma were >85%, and the within- and between-day coefficients of variation were within 15%. The limit of quantification in human plasma was 1 ng/mL. The concentration of edoxaban in blood decreased at room temperature, but remained unchanged for 1 week at 4°C. On the other hand, the concentration in plasma at both -20 and -80°C remained unchanged for 5 months. These results indicated that blood samples should be centrifuged immediately or stored at 4°C, and that plasma samples should be stored below -20°C until analysis. This method was applied to human plasma obtained from four patients after total knee arthroplasty. Analysis of edoxaban pharmacokinetics demonstrated an absorption time lag of 4h, a maximum concentration of 110 ± 26 ng/mL and an oral clearance of 37 ± 16 L/h. The analytical methods established in this study will be suitable for determining the concentrations of edoxaban in human plasma.

Comparison Between a Manual Squamous Cell Carcinoma Antigen Assay and an Automated Assay in a Clinical Setting.

To compare a manual squamous cell carcinoma antigen (SCCA) assay and an automated SCCA assay in a clinical setting. We included, in this study, a total of 158 specimens that had been tested using the manual SCCA assay. The CanAg SCC EIA assay and the Elecsys SCC were compared for their clinical settings. Within-run and between-day coefficients of variation (CVs, %) were lower than 3%. Comparison of the manual and automated SCCA assays yielded good correlation. The correlation coefficient (R2) between the CanAg SCC EIA and Elecsys SCC assay results was 0.989 (P <.001) and the overall concordance rate was 94.3%. We report that the Elecsys SCC automated SCCA assay yielded performance comparable to that of the manual SCCA assay: the automated assay reduced the number of manual steps and test turnaround time.

Quantitative assessment of systolic and diastolic right ventricular function by echocardiography and speckle-tracking imaging: a prospective study in 104 dogs.

Our aim was (1) to determine the within-day and between-day variability of several indices of systolic and diastolic right ventricular (RV) function by using conventional echocardiography and speckle-tracking echocardiography (STE) (Study 1), (2) to quantify these variables in a large healthy canine population (n = 104) with Doppler-derived estimated systolic pulmonary arterial pressure (SPAP) and left ventricular (LV) function, and (3) to establish the corresponding reference intervals (Study 2). For both studies, RV variables included tricuspid annular plane systolic excursion (TAPSE), right fractional area change (RFAC), STE longitudinal systolic strain (StS) of the RV free wall (RVFW) and of the entire RV (i.e., global RV StS), STE longitudinal systolic RVFW strain rate (SRS), and the diastolic early:late strain rate ratio. All but one within- and between-day coefficients of variation (13/14) were < 15%, the lowest being observed for TAPSE (3.6–9.8%), global RV StS (3.8–9.9%), and RVFW StS (3.7–7.3%). SPAP was weakly and negatively correlated with the TAPSE:body weight ratio (rs = −0.26, p = 0.01) and RVFW SRS (rs = −0.23, p < 0.05). Reference intervals (lower and upper limits with 90% confidence intervals) were provided for all variables. STE provides a non-invasive evaluation of RV function that may be used for clinical investigations in canine cardiology.

Validated Spectrophtometric Method for Simultaneous Determination of Buprenorphine and Naloxone in Pharmaceutical Dosage Forms.

Buprenorphine is a partial mu agonist and kappa antagonist which is used for the treatment of pain and opioid addiction. A mixture of buprenorphine hydrochloride and naloxone hydrochloride has been approved for the treatment of opioid dependence. In this study a third order derivative spectrophotometric method based on zero-crossing technique has been used for the simultaneous determination of buprenorphine hydrochloride and naloxone hydrochloride in tablets. The measurements were carried out at wavelengths of 257.8 (zero-crossing point of naloxone hydrochloride) and 252.2 nm (zero-crossing point of buprenorphice hydrochloride) for buprenorphine hydrochloride and naloxone hydrochloride, respectively in the third order derivative spectra obtained in methanol and 0.1 M NaOH (50:50) as solvent. The method was found to be linear in the range of 20-80 µg/mL for buprenorphine hydrochloride and 5-20 µg/mL for naloxone hydrochloride. The within-day and between-day coefficient of variation and error values were less than 2.5% and 1.8%, respectively. The proposed method was successfully used for simultaneous determination of these drugs in pharmaceutical dosage form without any interference from excipients or need to prior separation before analysis.

Quantification of the steady-state plasma concentrations of clozapine and N-desmethylclozapine in Japanese patients with schizophrenia using a novel HPLC method and the effects of CYPs and ABC transporters polymorphisms.

Background This study developed a novel high-performance liquid chromatography (HPLC) method for the simultaneous quantification of clozapine and its active metabolite, N-desmethylclozapine, in human plasma and investigated the effects of various factors, including genetic polymorphisms in cytochrome P450 (CYP) 2D6, CYP3A5, ABCB1 and ABCG2, on the steady-state plasma trough concentrations (C0) of clozapine and N-desmethylclozapine in Japanese patients with schizophrenia. Methods Forty-five patients had been receiving fixed doses of clozapine for at least four weeks. The CYP2D6 ( CYP2D6*2, CYP2D6*5, CYP2D6*10), CYP3A5 ( CYP3A5*3), ABCB1 (1236C > T, 2677G > T/A, 3435C > T) and ABCG2 (421 C > A) genotypes were identified by polymerase chain reaction. Results The within- and between-day coefficients of variation (CV) were less than 11.0%, and accuracy was within 9.0% over the linear range from 10 to 2500 ng/mL for both analytes, and their LOQs were each 10 ng/mL. The median C0/dose (C0/D) ratios of clozapine were significantly higher in patients with the ABCG2 421 A allele than in those with the 421 C/C genotype ( P = 0.010). However, there were no significant differences in C0/D ratios of clozapine and N-desmethylclozapine among ABCB1, CYP2D6 or CYP3A5 genotypes. In multiple regression analysis, including polymorphisms, age, body weight and biochemical data of patients, the ABCG2 polymorphism alone was correlated with the C0/D ratios of clozapine ( R2 = 0.139, P = 0.016). Conclusions Among the various CYPs and drug transporters, BCRP appeared to most strongly influence clozapine exposure. Knowledge of the patient's ABCG2 421 C > A genotype before initiating therapy may be useful when making dosing decisions aimed at achieving optimal clozapine exposure.

Feasibility, Within-Day and Between-Day Variability of Transthoracic Echocardiography in Sloths (Bradypus Variegatus and Choloepus Hoffmanni)

Bradypus variegatus (Xenarthra, Bradypodidae) and Choloepus hoffmanni (Xenarthra, Megalonychidae) are sloths from the tropical rain forest of the Caribbean and Pacific regions of Costa Rica. Because of their unique physiology and behavior, sloths have intrigued scientists, and several studies have already been conducted regarding blood pressure and heart rate variations. However, the use of transthoracic echocardiography (TTE) in these species has never been reported. The aim of the present study was therefore to determine the feasibility, and then the within-day (repeatability) and between-day (reproducibility) variability of TTE in sloths. A total of 36 TTE examinations (including a total of 1080 measurements) with continuous ECG monitoring were performed on 4 different days by a trained observer on 6 healthy, adult, sedated Choloepus hoffmanni (sexually intact females, age, mean ± SD [minmax] 5.1 ± 1.3 years [4.0-7.5]) from the Costa Rica Animal Rescue Centre. Standard transthoracic M-mode and two-dimensional mode measurements included left ventricular (LV) end-diastolic and endsystolic diameters and thicknesses, the LV fractional shortening, the E point-to-septal separation, the left atrium-to-aorta ratio, and the right atrial width-to-the left atrial width ratio. Pulsed-wave Doppler parameters included peak systolic aortic and pulmonary flow velocities as well as early and late diastolic mitral flow velocities. A general linear model was used to determine the within-day and between-day coefficients of variation (CV). The main results were the following: all measurements could be performed at each TTE examination. Most within- and between-day CV values (90%) were <15%, the lowest being observed for the right atrial width-to-left atrial width ratio (2.2%). These results suggest that TTE is feasible and reliable in sloths, and therefore can be part of the cardiovascular exploration in these species. Further studies are now required to determine the corresponding reference intervals.

Development and validation of bioanalytical UHPLC-UV method for simultaneous analysis of unchanged fenofibrate and its metabolite fenofibric acid in rat plasma: Application to pharmacokinetics

A simple, precise, selective and fast ultra-high performance liquid chromatography (UHPLC-UV) method has been developed and validated for the simultaneous determination of a lipid regulating agent fenofibrate and its metabolite fenofibric acid in rat plasma. The chromatographic separation was carried out on a reversed-phase Acquity® BEH C18 column using methanol–water (65:35, v/v) as the mobile phase. The isocratic flow was 0.3ml/min with rapid run time of 2.5min and UV detection was at 284nm. The method was validated over a concentration range of 100–10000ng/ml (r2⩾0.9993). The selectivity, specificity, recovery, accuracy and precision were validated for determination of fenofibrate/fenofibric acid in rat plasma. The lower limits of detection and quantitation of the method were 30 and 90ng/ml for fenofibrate and 40 and 100ng/ml for fenofibric acid, respectively. The within and between-day coefficients of variation were less than 5%. The validated method has been successfully applied to measure the plasma concentrations in pharmacokinetics study of fenofibrate in an animal model to illustrate the scope and application of the method.

Noninvasive measurement of renal oxygen extraction fraction under the influence of respiratory challenge.

To demonstrate the feasibility of using a susceptibility-based magnetic resonance imaging (MRI) technique for measuring renal oxygen extraction fraction (OEF) changes under the influence of carbogen (97% O2 , 3% CO2 ) breathing. Eight New Zealand White rabbits were included in this study with local animal care committee approval. For OEF measurement, an asymmetric spin echo (ASE) sequence was used to acquire source images and a susceptibility model was utilized for OEF estimation at 3.0T. Within-session and between-day tests were conducted to evaluate the reproducibility of this OEF measurement. OEF changes were measured under respiratory challenge with alternated air and carbogen (97% O2 , 3% CO2 ) breathing. For comparison, blood samples were collected for the measurement of pO2 . The within-session coefficients of variation (CVs) of renal OEF measurements were 6.62% in cortex and 5.92% in medulla, while between-day CVs were 7.52% in cortex and 8.03% in medulla. Under carbogen breathing, renal OEFs decreased significantly from 0.32 ± 0.03 to 0.28 ± 0.02 (P < 0.01) in cortex, and from 0.34 ± 0.04 to 0.31 ± 0.03 (P < 0.01) in medulla. No statistical difference of relative OEF change was seen between cortex and medulla (P = 0.93). In addition, negative correlation between renal OEF and blood pO2 was found (r = 0.68 (P < 0.05) in cortex, and r = 0.64 (P < 0.05) in medulla). This study demonstrates the feasibility of using a susceptibility-based OEF measurement method for the evaluation of renal oxygenation changes induced by carbogen breathing. J. Magn. Reson. Imaging 2016;44:230-237.