Simultaneous determination of buprenorphine, norbuprenorphine and the enantiomers of methadone and its metabolite (EDDP) in human plasma by liquid chromatography/mass spectrometry

A previously reported enantioselective LC-MS assay for the determination of (R)- and (S)-methadone [Met] and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine [EDDP] (the primary metabolite of Met) has been adapted for use in the simultaneous determination of the plasma concentrations of Met, EDDP, buprenorphine (Bu) and norbuprenorphine (norBu). All of the target compounds were separated within 15 min using an alpha1-acid glycoprotein chiral stationary phase, a mobile phase composed of acetonitrile: ammonium acetate buffer [10 mM, pH 7.0] in a ratio of 18:82 (v/v), a flow rate of 0.9 ml/min at 25 degrees C. Deuterium labeled compounds were used as internal standards [d4-Bu, d3-norBu, (R,S)-d3-Met and (R,S)-d3-EDDP] and linear relationships between peak height ratios and drug concentrations were obtained for Bu and norBu in the range 0.2-12 ng/ml with correlation coefficients greater than 0.999. The relative standard deviations (%R.S.D.) for the intra- and inter-day precision of the method were <4.5% and for accuracy was <4.0%. The method was validated and used to analyze plasma samples obtained from opioid dependent methadone-maintained adults enrolled in a research study.     

Simultaneous determination of buprenorphine, norbuprenorphine, and buprenorphine–glucuronide in plasma by liquid chromatography–tandem mass spectrometry

For the first time, an LC–MS–MS method has been developed for the simultaneous analysis of buprenorphine (BUP), norbuprenorphine (NBUP), and buprenorphine–glucuronide (BUPG) in plasma. Analytes were isolated from plasma by C₁₈ SPE and separated by gradient RP-LC. Electrospray ionization and MS–MS analyses were carried out using a PE-Sciex API-3000 tandem mass spectrometer. The m/z 644→m/z 468 transition was monitored for BUPG, whereas for BUP, BUP-d₄, NBUP, and NBUP-d₃ it was necessary to monitor the surviving parent ions in order to achieve the required sensitivity. The method exhibited good linearity from 0.1 to 50 ng/ml (r²≥0.998). Extraction recovery was higher than 77% for BUPG and higher than 88% for both BUP and NBUP. The LOQ was established at 0.1 ng/ml for the three analytes. The method was validated on plasma samples collected in a controlled intravenous and sublingual buprenorphine administration study. Norbuprenorphine–glucuronide was also tentatively detected in plasma by monitoring the m/z 590→m/z 414 transition.     

Simultaneous determination of betamethasone,betamethasone acetate and hydrocortisone in biological fluids using high-performance liquid chromatography

A sensitive, specific, reproducible and rapid high-performance liquid chromatographic method for the simultaneous quantitation of betamethasone, betamethasone 21-acetate and hydrocortisone in biological fluids is described. Hydrocortisone acetate is used as an internal standard and the samples are extracted with dichloromethane before chromatographing on a reversed-phase system. Detection at two ultraviolet wavelengths (254 nm and 240 nm) was used to assess the specificity of the system, and the sensitivity was found to be greater than 10 ng for all steroids. The speed with which this assay can be performed makes it particularly useful for pharmacokinetic studies, and plasma concentration—time profiles resulting from the administration of betamethasone phosphate and betamethasone acetate are presented.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.     

Simultaneous determination of moricizine and its sulphoxidation metabolites in biological fluids by high-performance liquid chromatography

A simultaneous assay for moricizine, its two sulphoxidation metabolites, morizine sulphoxide and moricizine sulphone, using high-performance liquid chromatography (HPLC) is described. The drug and metabolites and clozapine (internal standard) in biological fluids were extracted using pentanesulphonic acid into diethyl ether. The ethereal extract was evaporated to dryness and the residue was redissolved in the mobile phase (methanol-water-triethylamine, 65:35:0.5, v/v). The analyses were performed on a μBondapak reversed-phase C₁₈ column housed in a Waters Z-module, linked to a C₁₈ pre-column, with a run-time of 12 min. The retention times were 2.7, 3.5, 6.2 and 9.7 min for moricizine sulphone, moricizine sulphoxide, moricizine and clozapine, respectively. The recovery of the compounds from plasma ranged from 89.9% for the sulphoxide to 98.1% for clozapine. The limits of detection of the assay for moricizine, moricizine sulphoxide and moricizine sulphone were 20, 10 and 5 ng/ml, respectively.     

Contribution to the determination of the chemotherapeutic drug G1 in biological fluids

A sensitive gas chromatographic method for the quantitative determination of the new antibacterial and antifungal drug G1, 1-(5-bromofuran-2-yl)-2-bromo-2-nitroethene, has been optimized. The method involves a fast and single extraction step from spiked serum and urine samples. The G1 drug was quantified using an internal standard method and by means of a nitrogen-selective detector. The results are statistically significant and show that mean levels of G1 as low as 1 μg ml⁻¹ can be measured accurately.     

Simultaneous determination of oxidative hair dye p-phenylenediamine and its metabolites in human and rabbit biological fluids

A liquid chromatography with an electrochemical detector method has been developed for the quantitative measurement for three diamine derivatives (p-phenylenediamine, N,N(')-p-phenylenebisacetamide, and 4-aminoacetanilide) in human urine and rabbit blood, urine, and feces. The detection cell consisted of a glassy carbon electrochemical signal obtained with a supporting electrolyte containing 20% methanol-5mM octylammonium orthophosphate (pH 6.30) as the mobile phase. A comparison of the results obtained from HPLC-UV shows agreement.     

Simultaneous determination of selenomethionine enantiomers in biological fluids by stable isotope dilution gas chromatography-mass spectrometry

A method for the stereoselective determination of D- and L-enantiomers of selenomethionine in mouse plasma was developed using gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS-SIM). DL-[(2)H(3,)(82)Se]selenomethionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. Selenomethionine enantiomers in mouse plasma were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. The intra- and inter-day precision for D- and L-selenomethionine spiked to mouse plasma gave good reproducibility with relative standard deviation of 3% and 3% for D-selenomethionine and 6% and 3% for L-selenomethionine, respectively. The estimated amounts were in good agreement with the actual amounts spiked, the intra- and inter-day relative error being 5% and 2% for D-selenomethionine and 2% and 1% for L-selenomethionine, respectively. The present method is sensitive enough to determine pharmacokinetics of selenomethionine enantiomers.     

The determination of aminoketones in biological fluids

• 1.1. A new chromatographic procedure is described for the determination of aminoacetone and δ-aminolevulinic acid (after conversion to pyrroles) in both tissues and urine following their separation from porphobilinogen.
• 2.2. The method for measurement of both porphobilinogen and aminoketones involves only two chromatographic procedures, both utilizing Dowex-1 acetate resin. It permits concentration of pyrroles derived from both aminoketones, providing for the determination of as little as 20-mμmole quantities of these substances. Reproducibility and recoveries are excellent over a wide range of concentrations.
• 3.3. Aminoacetone pyrrole is retained by the resin primarily by adsorption; δ-aminolevulinic acid pyrrole is bound by an ionic mechanism.
• 4.4. Aminoacetone constitutes a significant portion of the aminoketones in nonporphyric urines.
• 5.5. The administration of relatively large doses of threonine results in an increased urinary excretion of aminoacetone.

     

Simultaneous determination of barbiturates in human biological fluids by direct immersion solid-phase microextraction and gas chromatography-mass spectrometry

Simultaneous determination of seven barbiturates in human whole blood and urine by combining direct immersion solid-phase microextraction (DI-SPME) with gas chromatography-mass spectrometry (GC-MS) is presented. The main parameters affecting the DI-SPME process, such as SPME fibers, salt additives, pHs, extraction temperatures and immersion times were optimized for simultaneous determination of the drugs. The extraction efficiencies were 0.0180-0.988 and 0.0156-2.76% for whole blood and urine, respectively. The regression equations of the drugs showed excellent linearity for both samples; the correlation coefficients (r(2)) were 0.994-0.999. The detection limits for whole blood were 0.05-1 microg x ml(-1), and those for urine 0.01-0.6 microg x ml(-1). Actual quantitation could be made for pentobarbital in whole blood and urine obtained from volunteers, who had been orally administered a therapeutic dose of the drug. The DI-SPME/GC-MS procedure for barbiturates established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology.     

Voltammetric determination of cefixime in pharmaceuticals and biological fluids

Electroreduction and adsorption of cefixime was studied in phosphate buffer by cyclic voltammetry (CV), differential pulse cathodic adsorptive stripping voltammetry (DPCAdSV), and square-wave cathodic adsorptive stripping voltammetry (SWCAdSV) at hanging mercury drop electrode (HMDE). These fully validated sensitive and reproducible cathodic adsorptive stripping voltammetric procedures were applied for the trace determination of the bulk drug in pharmaceutical formulations and in human urine. The optimal experimental parameters were as follows: accumulation potential = −0.1 V (vs. Ag/AgCl, 3 M KCl), accumulation time = 50 s, frequency = 140 Hz, pulse amplitude = 0.07 V, and scan increment = 10 mV in phosphate buffer (pH 2.6). The first peak current showed a linear dependence with the drug concentration over the range of 50 ng ml⁻¹ to 25.6 μg ml⁻¹. The achieved limit of detection and limit of quantitation were 3.99 and 13.3 ng ml⁻¹ by SWCAdSV and 7.98 and 26.6 ng ml⁻¹ by DPCAdSV, respectively. The procedure was applied to assay the drug in tablets. Applicability was also tested in urine samples. Peak current was linear with the drug concentration in the range of 1 to 60 μg ml⁻¹ of the urine, and minimum detectability was found to be 12.6 ng ml⁻¹ by SWCAdSV and 58.4 ng ml⁻¹ by DPCAdSV.     

Simple high-performance liquid chromatographic method for the determination of PGT/1A, a new immunostimulating drug, in biological fluids

This paper describes a simple high-performance liquid chromatographic method for the determination of PGT/1A (3- -pyroglutamyl- -thiazolidine-4-carboxylic acid), a new immunostimulating drug, in plasma and urine. The column was packed with LiChrospher-NH₂ (5 μm), the mobile phase was 0.02 M monobasic potassium phosphate (pH 3.2 with concentrated phosphoric acid)—acetonitrile (25:75, v/v), the flow-rate was 1.2 ml/min, the detection wavelength was 210 nm and the apparatus was a Varian Model 5000. Plasma (1 ml) was added to 1.2 ml of acetonitrile and the supernatant injected; the urine was diluted 1:5. The retention time of PGT/1A was 9.4 min in plasma and 9.9 min in urine. The method was validated for recovery, accuracy and reproducibility. The results after intravenous injection in twelve volunteers are also given.     

Enzymatic determination of dehydroepiandrosterone sulfate in biological fluids

A simple method is described for the determination of androgens, particularly dehydroepiandrosterone sulfate (DHEAS), in human plasma and urine. The method does not involve extraction or chromatography. An internal standard is used as reference. The androgens are enzymatically converted to estrogens and these latter compounds can be measured by the enzymatic method previously described [1]. The range of accuracy was between 99 and 103%. The precision of the method was 2.5%. The sensitivity was 0.1 pM per sample. The method is suitable for routine use, and one worker can easily perform twenty assays in one day.     

A liquid chromatographic-electrospray ionization-tandem mass spectrometric method for determination of buprenorphine,its metabolite,norbuprenorphine, and a coformulant,naloxone, that is suitable for in vivo and in vitro metabolism studies

A liquid chromatographic-electrospray ionization-tandem mass spectrometric method has been developed and validated for determination of the antiabuse medication, buprenorphine, its primary metabolite, norbuprenorphine, and a proposed coformulant, naloxone. The method uses deuterated internal standards and a simple liquid-liquid extraction. Mass spectrometry employed selected reaction monitoring of the transitions of m/z 468 to 396 for buprenorphine, 472 to 400 for [2H4]buprenorphine, 414 to 101 for norbuprenorphine, 423 to 110 for [2H9]norbuprenorphine, 328 to 310 for naloxone, and 345 to 327 for its internal standard, [2H3]naltrexone. The method was accurate and precise across the dynamic range of 0.1 to 10 ng/ml. All analytes were stable in human plasma stored at room temperature for up to 24 h and after three freeze-thaw cycles. Reconstituted extracts were stable at -20 degrees C for up to 3 days. In human subjects receiving a sublingual tablet of 8 mg buprenorphine and 2 mg naloxone, buprenorphine and norbuprenorphine were detected for up to 24 h with respective maximum concentrations at 1 and 1.5 h. Maximal concentrations ranged from 2.2 to 2.8 and 1.5 to 2.4 ng/ml for buprenorphine and norbuprenorphine, respectively (i.e., approximately 6 nM). The method detected norbuprenorphine formation in human liver microsomes incubated with 5-82 nM buprenorphine, which encompasses the therapeutic plasma concentration range. When cDNA-expressed P450s were incubated with 21 nM buprenorphine, norbuprenorphine formation was detected for P450s 3A4, as previously described, but also for 3A5, 3A7, and 2C8. Buprenorphine utilization generally exceeded norbuprenorphine formation, suggesting that P450s 2C18, 2C19, 2D6, and 2E1 may also be involved in buprenorphine metabolism to other products. These results suggest this method is suitable for both in vivo and in vitro studies of buprenorphine metabolism to norbuprenorphine.     

High-performance liquid chromatographic method for determination of gentamicin in biological fluids

A selective and sensitive method for the determination of gentamicin in plasma and urine by high-performance liquid chromatography has been developed. Following deproteinization, the gentamicin is reacted with fluorescamine to produce a fluorescent derivative. This reaction mixture is directly chromatographed on a cation-exchange column using as mobile phase acetonitrile—phosphoric acid (7:3). The gentamicin components elute as a single peak. Using 0.1 ml of plasma, quantitation of gentamicin concentrations as low as 1 mg/l are possible. Possible interference from other aminoglycosides and antibiotics is discussed.     

Simultaneous determination of buprenorphine and its prodrug, buprenorphine propionate, by high-performance liquid chromatography with fluorescence detection: application to pharmacokinetic studies in rabbits

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the simultaneous determination of buprenorphine and buprenorphine propionate in human and animal blood. Buprenorphine propionate was also proven to be a prodrug of buprenorphine. It was comprised of only a one-step extraction procedure with ethyl acetate and normal-phase chromatography on a Betasil Silica column. The recoveries of buprenorphine and buprenorphine propionate were above 84%. Calibration graphs were linear for buprenorphine over the concentration range 2-1500 ng/ml and for buprenorphine propionate over the concentration range 20-1500 ng/ml with a coefficient of variation, both within- and between-day, or less than 10% at any level. The limits of quantitation of buprenorphine and buprenorphine propionate in human or animal blood were 2.0 and 20 ng/ml, respectively, based on a single-to-noise ratio of 3. The method has been successfully applied to pharmacokinetic studies of buprenorphine and buprenorphine propionate in rabbits. The results demonstrated that buprenorphine propionate was rapidly and totally converted to its parent drug, buprenorphine, following intravenous administration. Buprenorphine propionate is a prodrug of buprenorphine.     

A viscosity-dependent affinity sensor for continuous monitoring of glucose in biological fluids

We present a viscometric affinity biosensor for continuous monitoring of glucose in biological fluids such as blood and plasma. The sensing principle of this chemico-mechanical sensor is based upon the viscosity variation of a sensitive fluid with glucose concentration. Basically, this device includes both an actuating and a sensing piezoelectric diaphragms as well as a flow-resistive microchannel. In order to confine the sensitive fluid and allow glucose diffusion into the sensor, a free-standing alumina nanoporous membrane is also used as size-selective interface. Measurements carried out at nominal temperatures of 25 and 37 °C reveal that this sensor topology exhibits a high resolution in the current range of physiological blood glucose concentrations, i.e. 2-20 mM. In addition, complete reversibility was also demonstrated for at least 3 days. Finally, measurements performed in human blood serum confirm that this sensor fulfils all basic requirements for a use in continuous glucose monitoring of biological fluids.     

Mass fragmentographic determination of xanthine and hypoxanthine in biological fluids

The method presented for the simultaneous determination of xanthine and hypoxanthine, uses mass-fragmentography in the electron impact (EI) mode, after the gas chromatographic separation of butylated derivatives. Butylation, rather than methylation, is used in order to avoid interference coming from exogenous caffeine, which is frequently encountered. [7,9-¹⁵N]Xanthine is used as the internal standard, and for each sample, a blank is obtained by xanthine oxidase reaction. In the biological fluids studied the sensitivity was about 50 ng/ml.