Within-day Coefficient Of Variation Research Articles

764-P: Time in Range and Time in Tight Range in Tirzepatide-Treated Patients Achieving Normoglycemia in the SURPASS-3 CGM Substudy

Tirzepatide (TZP), a novel once-weekly GIP/GLP-1 receptor agonist, demonstrated superior glycemic control vs insulin degludec (IDeg) in SURPASS-3 CGM. This post-hoc analysis evaluated changes from baseline to Week (Wk) 52 in Time in Range (TIR) (71-180 mg/dL), Time in Tight Range (TITR) (71-140 mg/dL), and glycemic variation (within-day coefficient of variation (CV) and Mean Amplitude of Glycemic Excursions (MAGE)) in TZP-treated patients achieving or not achieving HbA1c <5.7% at Wk 52. Interstitial glucose values were collected by CGM at 5-minute intervals for 7 days at baseline, Wk 24, and Wk 52 in a subset of patients in the SURPASS-3 trial. Analyses used pooled data from all TZP arms (5, 10, 15 mg) only due to limited sample size for IDeg patients achieving HbA1c <5.7% at Wk 52. Patients achieving HbA1c <5.7% were slightly younger, had lower HbA1c and FSG, and higher TITR and TIR vs those not achieving HbA1c <5.7% at baseline (Table). Increases in daily TITR and TIR from baseline (61.02% and 41.12% respectively) were seen in patients achieving HbA1c <5.7% while those not achieving HbA1c <5.7% had increased daily TITR and TIR of 36.62% and 32.40% (Table). Within-day CV and MAGE were reduced in both patient groups but more apparent in those achieving HbA1c <5.7%. In TZP-treated patients, improvements in TIR, TITR, and glycemic variability were more pronounced in those achieving HbA1c <5.7%. Disclosure R.M.Bergenstal: Advisory Panel; Abbott Diabetes, Eli Lilly and Company, Medtronic, Novo Nordisk, Roche Diabetes Care, Zealand Pharma A/S, Consultant; Ascensia Diabetes Care, Bigfoot Biomedical, Inc., CeQur SA, Dexcom, Inc., Hygieia, Onduo LLC, Sanofi, Vertex Pharmaceuticals Incorporated, Research Support; Abbott Diabetes, Dexcom, Inc., Eli Lilly and Company, Insulet Corporation, Medtronic, Novo Nordisk, Sanofi, UnitedHealth Group. Á.Rodríguez: Employee; Eli Lilly and Company, Stock/Shareholder; Eli Lilly and Company. B.D.Benneyworth: Employee; Eli Lilly and Company, Stock/Shareholder; Eli Lilly and Company. C.Nicolay: Employee; Eli Lilly and Company. K.Brown: Employee; Eli Lilly and Company. Funding Eli Lilly and Company

Establishing Pragmatic Analytical Performance Specifications for Blood Beta-Hydroxybutyrate Testing

Currently, no authoritative guidelines exist recommending the analytical performance specification (APS) of blood beta-hydroxybutyrate (BOHB) testing in order to meet the clinical needs of patients. This study has applied existing diabetic ketoacidosis (DKA) BOHB diagnostic thresholds and the recommended rates of fall in BOHB concentrations during DKA treatment to establish pragmatic APSs for BOHB testing. Required analytical performance was based on 2 clinical requirements: (a) to reliably distinguish between non-adjacent DKA BOHB diagnostic categories of <0.6, 0.6 to 1.5, 1.6 to 2.9, and ≥3 mmol/L, and (b) to be assured that a measured 0.5 mmol/L reduction in BOHB indicates the true concentration is at least falling (meaning >0 mmol/L decline). An analytical coefficient of variation (CV) of <21.5% could reliably distinguish all non-adjacent diagnostic categories with >99% certainty, assuming zero bias. In contrast, within-day CVs of 4.9%, 7.0%, and 9.1% at 3 mmol/L BOHB were required to assure truly falling ketone concentrations with 99% (optimal), 95% (desirable), and 90% (minimal) probability, respectively. These CVs are larger at lower BOHB concentrations and smaller at higher concentrations. Reliable tracking of changes in BOHB during DKA treatment largely drives the requirement for analytical performance. These data can be used to guide minimal, desirable, and optimal performance targets for BOHB meters and laboratory assays.

Real world hypoglycaemia related to glucose variability and Flash glucose scan frequency assessed from global FreeStyle Libre data.

Flash glucose monitoring provides a range of glucose metrics. In the current study, we aim to identify those that indicate that glycaemic targets can be consistently met and contrast the total (t-CV) and within-day coefficient of variation (wd-CV) to guide the assessment of glucose variability and hypoglycaemia exposure. De-identified data from Flash readers were collected. The readers were sorted into 10 equally sized groups of scan frequency followed by quartiles of estimated A1c (eA1c). A similar grouping was performed for the total coefficient of variation (t-CV) and within-day coefficient of variation (wd-CV). In addition, analysis of the association of time below 54 mg/dl and glucose variability measured by t-CV and wd-CV was performed. The dataset included 1002 946 readers. Readers sorted by 10 equal groups of scan rate and quartiles by eA1c, t-CV and wd-CV represented 25 074 readers per group. The association of lower eA1c with higher time in range and reduced time above range was clear. The correlation of eA1c quartiles and time below range was not consistent. An association between glucose variability and hypoglycaemia was found. Both wd-CV and t-CV were associated with time below range. For achieving the consensus target of <1% time below 54 mg/dl, the associated wd-CV and t-CV values were 33.5% and 39.5%, respectively. The type of CV reported by the different continuous glucose monitoring systems should be acknowledged. CV <36% might not be adequate to ensure low hypoglycaemia exposure. To our knowledge, the majority of continuous glucose monitoring reports the t-CV. Appropriate thresholds should be used to identify patients that would probably meet time below range targets (t-CV <40% or wd-CV <34%).

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 &amp;gt;36%) . The LS mean within-day glucose CV of participants with CV &amp;gt;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

728-P: Glycemic Variability of Tirzepatide vs. Insulin Degludec in People with Type 2 Diabetes Using Continuous Glucose Monitoring (SURPASS-3 CGM)

Tirzepatide (TZP) is a novel dual glucose-dependent insulinotropic polypeptide/glucagon-like peptide-1 receptor agonist in development for T2D. In a continuous glucose monitoring (CGM) sub-study of SURPASS-3 (NCT03882970) , participants with T2D on metformin with or without SGLT-2 inhibitors spent up to 91% of time in range (71-180 mg/dL) for TZP (5, 10, 15 mg) vs. 75% for insulin degludec (IDeg) (p&amp;lt;0.001) and within-day coefficient of variation was 16% vs. 24% for TZP vs. IDeg (p&amp;lt;0.001) . Reported here are additional pre-specified glycemic variability (GV) measures at 52 weeks for the sub-study (N=243; mean age of participants, 57 years; T2D duration, 8.8 years; baseline HbA1c, 8.14%) . The between-day GV measured as the mean of daily differences was significantly less for TZP vs. IDeg (TZP 5-15 mg; p&amp;lt;0.001) . The frequency and severity of hypoglycemic events measured by the low blood glucose (BG) index were significantly lower at all doses of TZP vs. IDeg (mg: p&amp;lt;0.05; 5, 15 mg: p&amp;lt;0.001) , as were hyperglycemic events measured by the high BG index compared to IDeg (5 mg: p&amp;lt;0.05; 10, 15 mg: p&amp;lt;0.001) . The BG risk index was significantly less at all doses of TZP (5 mg: p&amp;lt;0.05; 10, 15 mg: p&amp;lt;0.001) (Table) . TZP showed clinically meaningful and significant reductions in the risk of hyperglycemia and hypoglycemia in comparison to IDeg in people with T2D. 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. A.Bartee: None. M.Zeytinoglu: Employee; Eli Lilly and Company. R.Bray: Employee; Eli Lilly and Company. S.Allen: None. K.Brown: Employee; Eli Lilly and Company.

Is the Consensual Threshold for Defining High Glucose Variability Implementable in Clinical Practice?

Estimating glycemic variability (GV) through within-day coefficient of variation (%CVw) is recommended for patients with type 1 Diabetes (T1D). High GV (hGV) is defined as %CVw > 36%. However, continuous glucose monitoring (CGM) devices provide exclusively total CV (%CVT). We aimed to assess consequences of this disparity. We retrospectively calculated both %CVT and %CVw of consecutive T1D patients from their CGM raw data during 14 days. Patients with hGV with %CVT >36% and %CVw ≤36% were called the "inconsistent GV group". A total of 104 patients were included. Mean ± SD %CVT and %CVw were 42.4 ± 8% and 37.0 ± 7.4% respectively (P < 0.0001). Using %CVT, 81 patients (73.6%) were classified as having hGV, whereas 59 (53.6%) using %CVw (P < 0.0001) corresponding to 22 patients (21%) in the inconsistent GV population. Evaluation of GV through %CV in patients with T1D is highly dependent on the calculation method and then must be standardized.

Optical Coherence Tomography Angiography of Peripapillary Retinal Blood Flow Response to Hyperoxia.

To measure the change in peripapillary retinal blood flow in response to hyperoxia by using optical coherence tomography (OCT) angiography. One eye of each healthy human participants (six) was scanned with a commercial high-speed (70 kHz) spectral OCT. Scans were captured twice after 10-minute exposures to normal breathing (baseline) and hyperoxia. Blood flow was detected by the split-spectrum amplitude-decorrelation angiography (SSADA) algorithm. Peripapillary retinal blood flow index and vessel density were calculated from en face maximum projections of the retinal layers. The experiment was performed on 2 separate days for each participant. Coefficient of variation (CV) was used to measure within-day repeatability and between-day reproducibility. Paired t-tests were used to compare means of baseline and hyperoxic peripapillary retinal blood flow. A decrease of 8.87% ± 3.09% (mean ± standard deviation) in flow index and 2.61% ± 1.50% in vessel density was observed under hyperoxia. The within-day repeatability CV of baseline measurements was 5.75% for flow index and 1.67% for vessel density. The between-day reproducibility CV for baseline flow index and vessel density was 11.1% and 1.14%, respectively. The between-day reproducibility of the hyperoxic response was 3.71% and 1.67% for flow index and vessel density, respectively. Optical coherence tomography angiography with SSADA was able to detect a decrease in peripapillary retinal blood flow in response to hyperoxia. The response was larger than the variability of baseline measurements. The magnitude of an individual's hyperoxic response was highly variable between days. Thus, reliable assessment may require averaging multiple measurements.

Gas chromatography–mass spectrometry microanalysis of alpha- and gamma-tocopherol in plasma and whole blood

BackgroundAssessing vitamin E status in humans is critical for nutritional evaluation and verification of clinical and biological compliance of supplemented subjects. An accurate analytical method for measuring the two main vitamin E isoforms, i.e. α- and γ-tocopherol (α- and γ-TOH) in small volumes of plasma can facilitate the application of this analysis to clinical trials and in situations where a limited amount of sample is available. MethodsWe have developed a micro method, which uses only 5μL plasma, based on isotope dilution, trimethylsilation and GC–MS. The method was validated according to the guidelines of the International Conference on Harmonization of analytical procedures. The method was also applied to 5μL of whole blood for the potential use in conditions were the availability of specimens is limited. ResultsAccurate quantitation of α-TOH and γ-TOH was achieved at levels ≥0.417μM and ≥0.007μM, respectively. Within-day coefficient of variation was 1.31% and 4.70% for α-TOH and γ-TOH, respectively. Between-day coefficient of variation was 1.32% and 2.88% for α-TOH and γ-TOH, respectively. Recovery, assessed at three concentration levels, ranged 98–103% and 100–102% for α-TOH and γ-TOH, respectively. The method allowed the detection of α-TOH and γ-TOH in 5μL whole blood and in membranes of red blood cells washed from 5μL of blood as well.The analytical performance was assessed in plasma from a cohort of Italian healthy subjects (n=205). The mean plasma concentrations were 28.01±6.31 and 0.68±0.48μM (mean±SD) for α-TOH and γ-TOH, respectively. Alpha-TOH correlated with total cholesterol (r=0.617, p<0.0001) and triglycerides (r=0.420, p<0.0001) while γ-TOH correlated modestly with total cholesterol (r=0.213, p<0.0001) but not with triglycerides. γ-TOH, but not α-TOH, was significantly lower in smokers than in non-smokers (0.72±0.50 vs. 0.56±0.37, μM, mean±SD, p=0.017). Given the high sensitivity, the method allowed to be applied to 5μM whole blood without specific modification. ConclusionsThis micro-method represents an analytical advancement in α- and γ-TOH assay that is available to accurately verify the nutritional status and compliance after supplementation in large-scale settings, and to measure the two vitamers in conditions where sample availability is limited.

Multiplex serology of paraneoplastic antineuronal antibodies

Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diagnosing a neurological syndrome as paraneoplastic (70% is not yet known to have cancer) and in directing the search for the underlying neoplasm. At present six onconeural antibodies are considered ‘well characterized’ and recognize the antigens HuD, CDR62 (Yo), amphiphysin, CRMP-5 (CV2), NOVA-1 (Ri), and Ma2. The gold standard of detection is the characteristic immunohistochemical staining pattern on brain tissue sections combined with confirmation by immunoblotting using recombinant purified proteins. Since all six onconeural antibodies are usually analyzed simultaneously and objective cut-off values for these analyses are warranted, we developed a multiplex assay based on Luminex technology. Reaction of serial dilutions of six onconeural standard sera with microsphere-bound antigens showed lower limits of detection than with Western blotting. Using the six standard sera at a dilution of 1:200, the average within-run coefficient of variation (CV) was 4% (range 1.9–7.3%). The average between-run within-day CV was 5.1% (range 2.9–6.7%) while the average between-day CV was 8.1% (range 2.8–11.6%). The shelf-life of the antigen coupled microspheres was at least two months. The sensitivity of the multiplex assay ranged from 83% (Ri) to 100% (Yo, amphiphysin, CV2) and the specificity from 96% (CV2) to 100% (Ri). In conclusion, Luminex-based multiplex serology is highly reproducible with high sensitivity and specificity for the detection of onconeural antibodies. Conventional immunoblotting for diagnosis of onconeural antibodies in the setting of a routine laboratory may be replaced by this novel, robust technology.

Establishment and preliminary clinical application of serum bile acid spectrum analysis by ultra high performance liquid chromatography-tandem mass spectrometry

Objective To establish an ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for detecting the bile acid expression in serum and to explore the relationship between the bile acid spectrum and the obesity. Methods After pre-treatment through solid phase extraction, serum samples were chromatographed by gradient elution on an UPLC HSS T3 column, and then mass spectrometric analysis of bile acid was performed by multiple reaction monitoring (MRM) mode with negative electrospray ionization (ESI).And its methodology performance, including specificity, linearity, sensitivity, imprecision and recovery, were verified according to the guideline of State Food and Drug Administration (SFDA).Furthermore，serum samples from 10 simple obesity subjects and 10 healthy controls were analyzed with this established method, and rank sum test was used for statistical analysis. Results This established UPLC-MS/MS method could simultaneously quantify 14 bile acid subgroups in serum sample, its analytical linear range was 10-20 000 nmol/L.For different bile acid subgroups, the limit of detection (LOD) was 0.02-7.90 nmol/L, limit of quantitation (LOQ) was 0.07-44.20 nmol/L; and its within-day coefficient of variation (CV) was 0.35%-12.41%, between-day CV was 1.11%-13.04%; the relative recovery ratio of this method was 89.8%-114.6%.Some differences were observed in chromatogram between obesity and control subjects, and both free bile acid and conjugated bile acid concentrations in obesity subjects ［0.49 （0.45-1.90）, 1.44（0.84-3.72） μmol/L］ were lower than them in control subjects ［0.98（0.53-3.06）, 1.99（0.67-2.88） μmol/L］, but the difference was not significant (Z=-0.958, -0.801, P>0.05). Conclusions This established UPLC-MS/MS method can efficiently differentiate and quantify 14 bile acid subgroups, and is characterized with broad analytical measurement range, good analytical sensitivity and precision.This method can be applied for the free and conjugated bile acid analysis in clinical specimens.(Chin J Lab Med, 2012，35：253-256) Key words: Obesity; Bile acids and salts; Chromatography; high pressure liquid; Tandem mass spectrometry

A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma

Introduction Assessment of the procoagulant effect of snake venoms is important for understanding their effects. The aim of this study was to develop a simple automated method to measure clotting times to assess procoagulant venoms. Methods A turbidimetric assay was developed which monitors changes in optical density when plasma and venom are mixed. Plasma was added simultaneously to venom solutions in a 96 well microtitre plate. After mixing, the optical density at 340 nm was monitored in a microplate reader every 30 s over 30 min. The clotting time was defined as the lag time until the absorbance sharply increased. The turbidimetric method was compared to manual measurement of the clotting time defined as the time when a strand of fibrin can be drawn out of the mixture. The two methods were done simultaneously, with the same venom and plasma, and compared by plotting the manual versus turbidimetric clotting times. Within-day and between-day runs were done and the coefficient of variation (CV) was calculated. Results Plots comparing manual clotting times to the lag time in the turbidimetric assay showed good correlation between the two methods for brown snake ( Pseudonaja textilis) venom, including 24 determinations in triplicate over six days for seven different venom concentrations. Good correlation was also found for four other venoms: tiger snake ( Notechis scutatus), Carpet viper ( Echis carinatus), Russell's viper ( Daboia russelii) and Malaysian pit piper ( Calloselasma rhodostoma). Between-day CV was in the range 10–20% for both methods, while within-day CV < 10%. Discussion The turbidimetric assay appears to be a simple and convenient automated method for the measurement of clotting times to assess the effects of procoagulant venoms.

Diurnal variation of biopyrrin excretion in random urine specimens is not corrected by creatinine

Circulating bilirubin is thought to function as a physiological antioxidant. One of the decomposition products of this process is the biopyrrins, which include two regioisomers: biotripyrrin-a (1,14,15,17-tetrahydro-2,7,13-trimethyl-1,14-deoxy-3-vinyl-16H-tripyrrin-8,12-dipropionic acid) and biotripyrrin-b (1,14,15,17-tetrahydro-3,7,13-trimethyl-1,14-deoxy-3-vinyl-16H-tripyrrin-8,12-dipropionic acid). We measured biopyrrins in random urine specimens and investigated whether the biopyrrin values obtained were valid when expressed as a ratio of the creatinine (Cr) concentrations. All of the random urine specimens collected over 48 hr were from presumably healthy adults. We measured the biopyrrins by means of an enzyme-linked immunosorbent assay (ELISA) using an anti-bilirubin monoclonal antibody. When the values were expressed in terms of the ratio to Cr, the within-day coefficient of variation (%CV) of the excretion of biopyrrins was reduced to 27%+/-10% (P<0.05) from 59%+/-27%. However, assay values on random or spot urine specimens were unreliable because of the large %CV. The biopyrrin concentrations only in the first-morning-urine specimens in terms of both absolute amounts and ratios to Cr significantly reflected those in a 24-hr urine specimen (P<0.001). Concentrations in a random urine specimen voided at the second collection or later did not correlate with the concentration in a 24-hr urine specimen (P>0.05), even if their values were corrected by Cr. The amounts of biopyrrins excreted in 24-hr urine specimens were significantly correlated with the 24-hr cortisol excretion (P<0.001) but not to uropepsin (P>0.05).

Determination of urea kinetics by isotope dilution with [13C]urea and gas chromatography-isotope ratio mass spectrometry (GC-IRMS) analysis.

1. Stable urea isotopes can be used to study urea kinetics in humans. The use of stable urea isotopes for studying urea kinetic parameters in humans on a large scale is hampered by the high costs of the labelled material. We devised a urea dilution for measurement of the distribution volume, production rate and clearance of urea in healthy subjects and renal failure patients using the inexpensive single labelled [13C]urea isotope with subsequent analysis by headspace chromatography-isotope ratio MS (GC-IRMS) of the [13C]urea enrichment. 2. The method involves measurement of the molar percentage excess of [13C]urea in plasma samples taken over a 4 h period after an intravenous bolus injection of [13C]urea. During the sample processing procedure, the plasma samples together with calibration samples containing a known molar percentage excess of [13C]urea are acidified with phosphoric acid to remove endogenous CO2, and are subsequently incubated with urease to convert the urea present in the plasma samples into CO2. The 13C enrichment of the generated CO2 is analysed by means of GC-IRMS. This method allows measurement of the molar percentage excess of [13C]urea to an accuracy of 0.02%. 3. Reproducibility studies showed that the sample processing procedure [within-run coefficient of variation (CV) < 2.8% and between-run CV < 8.8%] and the GC-IRMS analysis (within-day CV < 1.3% and between-day CV < 1.3%) could be repeated with good reproducibility. 4. In clinical urea kinetic studies in a healthy subject and in a renal failure patient without residual renal function, reproducible values of the distribution volume, production rate and clearance of urea were determined using minimal amounts of [13C]urea (25-50 mg). 5. Because only low [13C]urea enrichments are needed in this urea dilution method using GC-IRMS analysis, the costs of urea kinetic studies are reduced considerably, especially in patients with renal failure.

New high-performance liquid chromatographic method for amphotericin B analysis using an internal standard.

A simple and reproducible HPLC method for the analysis of amphotericin B (AmB) in serum, lung and liver using natamycin as the internal standard was developed. AmB and natamycin were extracted from serum, lung and liver and were separated using an isocratic elution from C18 reversed-phase column. The mobile phase consisted of acetonitrile-10 mM acetate buffer pH 4.0 (37:63, v/v). The HPLC system had two detectors in series. One was set at 303 nm and the other at 383 nm for the detection of natamycin and AmB, respectively. The retention times of AmB and natamycin were 15 and 6 min, respectively. The recovery efficiency was 96%-70%. The limit of quantification was 0.1 microgram/ml. The assay was reproducible, the within-day coefficient of variation (n = 6) was < 8% for serum, lungs and liver. The between-day variability (n = 6) was < 7.7% for serum, liver and lungs at 1 microgram/ml or 1 microgram/g tissue concentration. The assay was linear within the range 1-40 micrograms/ml (r2 = 0.99).

A Simple High-performance Liquid Chromatographic Assay for Loracarbef in Human Plasma

Abstract A simple, rapid and reproducible high-performance liquid chromatographic (HPLC) method for the determination of loracarbef in human plasma has been developed and evaluated. Plasma protein was precipitated with ammonium sulfate. The drug and the internal standard (Cefetamet) were eluted from a μ-bondapak C-18 column with a mobile phase consisting of acetonitrile:methanol:water:glacial acetic acid (2.5:17.5:79.2:0.8%, v/v). The column eluent was monitored at 265 nm. Quantification was achieved by the measurement of the peak-height ratio of the analyte to the internal standard and the limit of quantification for loracarbef in plasma is 0.5 ug/ml. The within-day coefficient of variation (CV) ranged from 2.28% to 3.67%, and between-day CV from 2.38% to 5.59% at three different concentrations. The absolute recoveries ranged from 91.1% to 93.88%, and the relative recoveries from 93.4% to 108% at three different concentrations. Preliminary stability tests showed that loracarbef is stable for at least 5-w...

Quantification of Muscle Tissue Magnesium and Potassium Using Atomic Absorption Spectrometry

Abstract Flame atomic absorption spectrometry (AAS) has been used to measure magnesium (Mg2+) and potassium (K+) contents of wistar rat myocardium and skeletal muscle. Ten to twenty mg of tissue was homogenized in 10 mM hydrochloric acid, centrifuged, and the clear supernatant was diluted three-fold in deionized distilled water before determination of Mg2+ and K+ concentrations. The calibration curves of both ions were linear from 0.25 to 2.0 mg/L for Mg2+ and from 1.5 to 15.0 mg/L for K+. The mean values (±SD) of Mg2+ and K+ contents for the analyzed cardiac muscle were 371.97±12.16 mg/Kg of wet tissue with a coefficient of variation (CV) of 3.3%, and 4571.02±359.74 mg/Kg with CV of 7.9%, respectively. Those for various skeletal muscles varied between 323.35-420.60 mg/Kg Mg2+, with CV ranging from 3.2% to 8.7%, and 4883.84-8156.68 mg/Kg K+, with CV ranging from 4.4% to 10.1%. The within-day CV were 2.8% to 3.8% for Mg2+ and 3.2% to 5.9% for K+, whereas day-to-day CV were 6.2% to 10.2% for Mg2+ and 8.7% to 10.5% for K+. Recoveries were 92.7% to 104.9% for Mg2+, and 93.2% to 106.1% for K+. Values for Mg2+ obtained by the proposed method were significantly different from those obtained by a published tissue digestion method, those for K+ were not significantly different.

High-Performance Liquid Chromatographic Method for the Quantitative Determination of Pheniramine in Plasma

Abstract A simple, sensitive and reproducible high performance liquid chromatographic (HPLC) method for the determination of pheniramine in plasma has been developed and validated. The assay is performed after single extraction of pheniramine and amitriptyline - (internal standard) from alkalinized plasma into ether. The drug and the internal standard were eluted from a μ-Bondapak C18 column at 40°C with a mobile phase consisting of methanol: water (62:38%, v/v) adjusted with phosphoric acid to an apparent pH 3.5 at a flow rate of 1.2 ml/min. The effluent was monitored with an ultraviolet detector set at 262 nm. Standard curves for the analyte in plasma were linear (r>0.999) in the range of 20–400 ng/ml and the minimum detectable concentration in plasma is 10 ng/ml. The within-day coefficient of variation (CV) ranged from 3.57% to 6.51% at three different concentrations. The between-day CVs varied from 5.03% to 7.84%. The absolute recoveries of pheniramine ranged from 94% to 96.9% and the relative recover...

Application of an organic solvent extraction to the determination of catechol-O-methyltransferase activity by high-performance liquid chromatography in human mononuclear cells

We report here a method for measuring mononuclear cell catechol-O-methyltransferase (COMT) activity which is ideally adapted to clinical studies. The method measures the O-methylation of dopamine to 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine. Whole mononuclear cell sonicate is incubated with saturating concentrations of dopamine, S-adenosyl- l-methionine and magnesium chloride in sodium—potassium phosphate buffer at pH 7.3. An organic solvent extraction using ethyl acetate is then used for product separation, followed by high-performance liquid chromatography with electrochemical detection for product separation and quantification. This method allows both O-methylated products, 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine, to be isolated and quantified separately. The apparent Michaelis constants for dopamine and S-adenosyl- l-methionine using this method are similar to values reported previously (0.51 and 14 μ M, respectively). The optimal concentration of magnesium chloride is eight to ten times higher than previously reported. No endogenous inhibitors were apparent using this assay. The within-day coefficient of variation using this method is 7% when measuring 3-methoxytyramine and 5% when measuring 4-methoxy-3-hydroxyphenethylamine. The between-day coefficient of variation is 11%. Mononuclear cell COMT activity can be detected using protein concentrations as low as 0.75 mg/ml, corresponding to 2–3 ml of whole blood. The small amount of blood required per sample allows multiple sample analysis from a single patient, including infants.

Determination of Plasma Levels of Bupivacaine by High-Performance Liquid Chromatography

Abstract We evaluate a method for determination of bupivacaine using HPLC, and the possibility of pharmacological interferences produced by seven commonly used drugs administered before, during and after surgery: diacepam, midazolam, epinephrine, naloxone, flumacenil, atropine and ephedrine. The method has an average recovery of 102.8 +/− 5.4%. The detection limit is 0.125 μg/mL. The within-day coefficient of variation is 5.88% and the between-day coefficient of variation is 15%. We haven't found drug interferences at generally encountered serum concentrations.

Rapid and sensitive gas-chromatographic determination of caffeine in blood plasma, saliva, and xanthine beverages

A gas chromatographic procedure is reported for the determination of caffeine in plasma, saliva, and xanthine beverages. Using a 75 cm column packed with OV-17, nitrogen-sensitive detection, and 1 ml samples, a suitable limit of analysis (coefficient of variation (CV) = 10.2%) of 50 ng/ml was obtained in plasma. Within-day CVs at caffeine concentrations of 0.1-0.5-2.0-7.5-15.0 micrograms/ml in plasma were 7.7-5.6-4.8-3.8-3.4%, respectively. The limit of detection, defined as the injected quantity of caffeine giving rise to a signal to noise ratio of 2, is 40 pg, corresponding to a plasma concentration of 1 ng/ml. The procedure involves addition of the internal standard 7-pentyl theophylline and alkaline extraction of the sample with dichloromethane. The method described rivals any gaschromatographic assay published so far in rapidness and accuracy. Plasma and saliva caffeine concentrations were determined in a healthy male volunteer after swallowing 400 ml of coffee. The calculated pharmacokinetic parameters, assuming complete absorption of caffeine from the G.I. tract, agree well with previously published values.