Hollow porous molecularly imprinted polymer for highly selective clean-up followed by influential preconcentration of ultra-trace glibenclamide from bio-fluid.

In the present work, hollow porous molecularly imprinted polymer (HPMIP) was prepared via adopting a sacrificial support approach using glibenclamide (GB) as template, methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate as cross-linker (EGDMA) and mesoporous MCM-48 nanospheres as support. Owing to a short thickness of the foresaid HPMIP, a suitable steric structure was readily available that lead to fast and effective mass transfer of target analyte to sorbent and consequently supply high binding capacity. After ultrasonic-assisted dispersive solid phase extraction of urine sample, the analyte of interest was quantitatively pre-concentrated and determined by high performance liquid chromatography-ultraviolet detection (HPLC-UV). Influence of factors affecting the extraction efficiency such as sonication time, sample pH, sorbent dosage, and volumes of eluent and washing agents as well as their significant interactions were simultaneously optimized by experimental design methodology. Under optimized conditions, present method has linear response over concentration range of 10.0-3000.0µgL-1 in human urine samples with a satisfactory detection limit close to 3.5µgL-1. The inter-day precisions of the current method (coefficient of variation) are lower than 5%, while recoveries are more than 89.5%.

Quantitative determination of metformin, glyburide and its metabolites in plasma and urine of pregnant patients by LC-MS/MS.

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87-99%, and that from urine samples was 85-95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100-0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984-1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy was 86-114% in plasma, and 94-105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine.

Validation of a sensitive LC-MS assay for quantification of glyburide and its metabolite 4-transhydroxy glyburide in plasma and urine: an OPRU Network study.

Glyburide (glibenclamide, INN), a second generation sulfonylurea is widely used in the treatment of gestational diabetes mellitus (GDM). None of the previously reported analytical methods provide adequate sensitivity for the expected sub-nanogram/mL maternal and umbilical cord plasma concentrations of glyburide during pregnancy. We developed and validated a sensitive and low sample volume liquid chromatographic-mass spectrometric (LC-MS) method for simultaneous determination of glyburide (GLY) and its metabolite, 4-transhydroxy glyburide (M1) in human plasma (0.5 mL) or urine (0.1 mL). The limits of quantitation (LOQ) for GLY and M1 in plasma were 0.25 and 0.40 ng/mL, respectively whereas it was 1.06 ng/mL for M1 in urine. As measured by quality control samples, precision (% coefficient of variation) of the assay was <15% whereas the accuracy (% deviation from expected) ranged from -10.1 to 14.3%. We found that the GLY metabolite, M1 is excreted in the urine as the glucuronide-conjugate.

Pharmacokinetics of glibenclamide and its metabolites in diabetic patients with impaired renal function.

BACKGROUND: Glibenclamide (Gb) may provoke long-lasting hypoglycaemic reactions, and one of the known risk factors is impaired renal function. We have demonstrated Gb to have a terminal elimination half-life of 15 h, and the main metabolites have a hypoglycaemic effect. With few exceptions, detailed studies on second generation sulphonylureas in diabetics with impaired renal function are lacking. Therefore, we analysed the pharmacokinetics of Gb and its active metabolites, 4-trans-hydroxyglibenclamide (M1) and 3-cis-hydroxy-glibenclamide (M2) in this patient group. METHODS: Two groups of 11 diabetic patients with impaired renal function (IRF, iohexol clearance range 7-42 ml.min(-1) . 1.73 m(-2)) or normal renal function (NRF, iohexol clearance range 75-140 ml.min(-1) . 1.73 m(-2)) were compared. A single oral 7-mg dose of Gb was administered after overnight fasting. Serum samples and urine collections were obtained over 48 h and 24 h, respectively. Concentrations of Gb, M1 and M2 were determined by a sensitive and selective high-performance liquid chromatography assay. RESULTS: Peak serum values of M1 (24-85 ng.ml(-1) vs 16-57 ng.ml(-1)), M2 (7-22 ng.ml(-1) vs <5-18 ng.ml(-1)) and M1 + M2 (32-100 ng.ml(-1) vs 23-76 ng.ml(-1)) were higher in the IRF group. AUC and Cmax of Gb were lower and the clearance to bioavailability ratio (CL/f) was higher in the IRF group. AUC and Cmax of M1 were higher and CL/f lower in the IRF group. Much lower amounts of M and M2 were excreted in the urine in the IRF group (7.2% vs 26.4% in 24 h). The fraction of the Gb dose excreted as metabolites (fe(met) 0-24 h), ranged between 0.005 and 0.36 and correlated significantly with renal function measured by iohexol clearance. No other pharmacokinetic differences were found. CONCLUSION: The differences in AUC, Cmax and CL/f of Gb may be explained by a higher free fraction in the IRF group which would increase Gb metabolic clearance. The inverse findings regarding M1 may be explained by the fact that the metabolites are primarily eliminated by the kidneys. After a single dose of Gb, neither Gb, M1 nor M2 seemed to accumulate in diabetic subjects with IRF. As only small amounts of M1 and M2 were excreted in the urine, this indicates one or several complementary non-renal elimination routes, e.g. shunting of metabolised Gb to the biliary excretion route and/or enterohepatic recycling of both metabolites and unmetabolised Gb.

Capillary electrophoretic detection of metabolites in the urine of patients receiving hypoglycemic drug therapy.

Micellar electrokinetic chromatography (MEKC) in tandem with diode array detection (DAD) has been exploited as an analytical method for the separation and detection of sulfonylurea drugs. The ultimate goal is the development of an assay to detect these drugs or their metabolites in urine as a means of diagnosing sulfonylurea drug abuse. Using a separation buffer consisting of 5 mM borate/5 mM phosphate/75 mM sodium cholate, separation of both the second and third generation sulfonylurea drugs can be achieved. The characteristic absorbance spectra associated with each of the third generation drugs, glipizide and glyburide, allow for their identification in mixtures. Coinjection of glyburide, its primary metabolite, hydroxy glyburide, and glipizide demonstrated that the metabolite was resolved from the parent drug but shared its absorbance spectral properties. MEKC analysis of a series of solid phase-extracted urine samples from patients prescribed glipizide or glyburide, as well as from control patients not ingesting the drug, showed that the parent compounds were difficult to detect in the urine. However, the use of DAD allowed for detection of metabolites in the urine of these patients. With glyburide patients, only primary metabolites were detected, while urine from patients on glipizide showed a series of peaks whose absorbance spectra was consistent with the presence of both primary and secondary metabolites. In addition, the intensity of the metabolite peaks corresponded reasonably well with the respective dose and in vivo time interval associated with the urine collection. This study shows that MEKC with DAD has potential for further exploration as a clinical assay for detecting surreptitious abuse of sulfonylurea drugs.

Comparison of the kinetics of glyburide and its active metabolites in humans.

The pharmacokinetics of glyburide (Gb) and its active metabolites, 4-trans-hydroxyglibenclamide (M1) and 3-cis-hydroxyglibenclamide (M2), were compared in eight healthy subjects. After an overnight fast, each subject received a 3.5-mg single dose of Gb, M1 or M2 intravenously in random cross-over order. For comparison, a 3.5-mg oral dose of micronized formulation of Gb was also given in a test. The subjects continued to fast until standard meals were given at 0.5 and 5.5 h after each dose. Serum samples and urine fractions were collected for 10 h. Serum concentrations of Gb, M1 and M2, and urine concentrations of M1 and M2 were determined by a selective and sensitive liquid chromatographic method. The two metabolites had very similar pharmacokinetic profiles, except for volume of distribution and renal clearance. Estimated mean volume of distribution, total and renal clearance of M1 and M2 were 20.8 +/- 8.4 litres, 11.9 +/- 1.7 litres/h, 13.5 +/- 3.7 litres/h and 15.5 +/- 5.5 litres, 10.4 +/- 1.3 litres/h, 8.6 +/- 1.6 litres/h, respectively. Estimates of the volume of distribution and total clearance were significantly higher than those of Gb, which were 7.44 +/- 1.53 litres, 4.42 +/- 0.56 litres/h intravenously and 9.32 +/- 2.79 litres, 4.09 +/- 0.45 litres/h orally. There was no significant difference in total metabolite urine recovery between intravenous or oral administration of Gb, suggesting almost complete oral bioavailability of the micronized glibenclamide formulation.

Detection of hypoglycemic drugs in human urine using micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) is evaluated as a potential analytical method for the separation and detection of a series of sulfonylurea drugs used in the treatment of hyperglycemia. These drugs are often surreptitiously abused, producing extremely low blood glucose levels and symptoms indistinguishable from those associated with an insulin-secreting tumor. Separation buffer containing 50 mM sodium dodecyl sulfate (SDS) was found to be adequate for the MEKC separation of the third generation drugs (glipizide and glyburide) but not the second generation drugs (acetohexamide chlorpropamide, tolazamide, and tolbutamide). At a pH of 8.5 in the presence of 20 mM borate/20 mM phosphate and 150 mM SDS, all seven components were adequately resolved with an analysis time of 17 min. Altering the concentration of the buffering components to either 5 mM borate/5 mM phosphate or 40 mM borate alone reduced the analysis time to less than 10 min with no observable loss in resolution. A series of other micelle-forming surfactants were evaluated, and only sodium cholate provided an improvement over the SDS-based system. Optimal separation was obtained with 75 mM sodium cholate and led to complete analysis with baseline resolution of all seven components in less than 8 min. These conditions were shown to be adequate for the detection of the hypoglycemic drugs spiked into normal urine and in patients taking these drugs. The precision associated with nine consecutive injections of six samples (n = 54) was found to be acceptable with percent coefficient of variance for absolute migration times (MTabs) for all peaks averaging 0.89 with peak area and peak height being 8.49 and 8.26, respectively. The between-sample precision was found to average 0.92% for MTabs and 8.56% and 8.45%, respectively, for the relative peak area and peak height. With a detection limit for the drugs in urine (following extraction) in the 50 ng/mL range, the potential exists for an MEKC-based assay for the detection of sulfonylurea drugs in urine.

Determination of glibenclamide and its two major metabolites in human serum and urine by column liquid chromatography.

A simple reversed-phase liquid chromatographic method for the measurement of low concentrations of glibenclamide (glyburide) and its two major metabolites, 4-trans- and 3-cis-hydroxyglibenclamide, in human serum and urine has been developed. The compounds were extracted with n-hexane-dichloromethane (1:1). The UV detection wavelength was 203 nm. The minimum detectable serum level of glibenclamide was 1 ng ml (2 nM), and the relative standard deviation was 8.9% (n = 9). When maximum sensitivity was desired the metabolites were chromatographed separately. Metabolites in urine were measured by the same method after five-fold sample dilution. The utility of the method was tested on a healthy volunteer who ingested 3.5 mg of glibenclamide. The parent drug was present in the serum for at least 18 h, and the metabolites in the urine for at least 24 h.

Uniform elimination pattern for glibenclamide in healthy Caucasian males.

It has been shown, that the elimination rate of intravenously administered tolbutamide shows considerable interindividual variation, due to strong genetic influence. These pharmacogenetic differences in the metabolic clearance rate of tolbutamide may result in drug failure in fast-eliminators, and it may cause an increased incidence of side effects in slow-eliminators. It is not known whether different "pharmacogenetics" exist for other sulfonylurea drugs. Therefore, we have injected glibenclamide (0.02 mg/kg body weight) intravenously in 52 male, normal weight, healthy volunteers. Serum glibenclamide levels were followed for up to 24 h. The terminal phase half-life of glibenclamide was 2.46 +/- 0.67 h (mean +/- SD), total drug clearance was 48.7 +/- 11.0 ml.min-1 and the slow disposition phase rate constant was 0.30 +/- 0.08 h-1. From the individual data of each subject frequency histograms were developed for these and other kinetic parameters and tested with a Kolmogoroff-Smirnoff-test for unimodal normal distribution. There was no significant (p greater than 0.05) deviation from the unimodal normal distribution for these parameters. In contrast to the pharmacogenetics of tolbutamide metabolism the present data indicate that glibenclamide follows an uniform elimination pattern in healthy caucasian males.

High-performance liquid chromatographic determination of glibenclamide in human plasma and urine.

A selective and sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma or urine has been developed. With glibornuride as internal standard, acid-buffered plasma or urine was extracted with benzene. The organic layer was evaporated and the residue was dissolved in equilibrated mobile phase (acetonitrile-phosphate buffer 0.01 M pH 3.5, 50:50). An aliquot of 20 microliters was chromatographed on a Spherisorb ODS reversed-phase column, and quantitation was achieved by monitoring the ultraviolet absorbance at 225 nm. The response was linear (0-1000 ng/ml) and the detection limit was 5-10 ng/ml in plasma or urine. The within-assay variation was less than or equal to 10%. No interferences from metabolites or endogenous constituents could be noted. The utility of the method was demonstrated by analysing glibenclamide in samples from diabetic subjects on therapeutic doses of the drug.

Pharmacokinetic disposition of 14C-glyburide in patients with varying renal function.

The pharmacokinetics of 14C-labeled glyburide were studied in 13 men with varying degrees of renal impairment. Patients received a single, 5 mg oral dose of glyburide as a solution (10 microCi/ml/mg) after a high-carbohydrate breakfast. Serial plasma and breath samples were collected for 48 hours and urine and feces were collected for 5 to 7 days. Patients with normal to moderately impaired renal function (creatinine clearance [CLCR] of 29 to 131 ml/min/1.7 m2) had glyburide plasma t1/2 values of 2.0 to 5.0 hours, with no relationship between CLCR and glyburide clearance. One subject with severe renal impairment (CLCR = 5 ml/min/1.7 m2) had decreased glyburide clearance that resulted in a t1/2 of 11 hours. The elimination of metabolites was more dependent on renal status but was only significantly affected in the patient with severe renal impairment.

Pharmacokinetics of glyburide.

The pharmacokinetics of glyburide can be described by a two-compartment open model. Terminal-phase descriptions are formulation-dependent and are complicated by apparent food-associated mobilization of drug from the stomach. Some researchers indicate a possible third (or deep) compartment, but this appears to be an artifact of a nonspecific drug assay. Although no accumulation has been observed in short-term studies, further investigation of the possibility of drug accumulation with long-term therapy is needed. Distribution of glyburide is affected by high affinity for serum albumin (99 percent bound), and elimination of the drug appears to be evenly divided between biliary and renal routes. The biologic half-life of glyburide is not significantly correlated with renal function in subjects with creatinine clearances of 30 ml/minute/1.7 m2 or more. Gradations in disease state, multiple sites of sulfonylurea action, and inter-patient variability have made it difficult to relate biologic response to glyburide serum levels.