Determination of mycophenolic acid and its phenol glucuronide metabolite in human plasma and urine by high-performance liquid chromatography

Simultaneous determination of mycophenolic acid (MPA) and mycophenolate phenol glucuronide (MPAG) in plasma and urine was accomplished by isocratic HPLC with UV detection. Plasma was simply deproteinated with acetonitrile and concentrated, whereas urine was diluted prior to analysis. Linearity was observed from 0.2 to 50 μg/ml for both MPA and MPAG in plasma and from 1 to 50 μg/ml of MPA and 5 to 2000 μg/ml MPAG in urine with extraction recovery from plasma greater than 70%. Detection limits using 0.25 ml plasma were 0.080 and 0.20 μg/ml for MPA and MPAG, respectively. The method is more rapid and simple than previous assays for MPA and MPAG in biological fluids from patients.     

Rapid and simultaneous determination of mycophenolic acid and its glucuronide conjugate in human plasma by ion-pair reversed-phase high-performance liquid chromatography using isocratic elution

A high-performance liquid chromatographic method has been developed for the simultaneous determination of mycophenolic acid (MPA) and its glucuronide conjugate (MPAG) in human plasma. The method involves protein precipitation with acetonitrile, followed by ion-pair reversed-phase chromatography on C₁₈ column, with a 40 mM tetrabutyl ammonium bromide (TBA)–acetonitrile (65:35, v/v) mobile phase. A 20-μl volume of clear supernatant was injected after centrifugation, and the eluent was monitored at 304 nm. No interference was found either with endogenous substances or with many concurrently used drugs, indicating a good selectivity for the procedure. Calibration curves were linear over a concentration range of 0.5–20.0 μg/ml for MPA and 5–200 μg/ml for MPAG. The accuracy of the method is good, that is, the relative error is below 5%. The intra- and inter-day reproducibility of the analytical method is adequate with relative statistical deviations of 6% or below. The limits of quantification for MPA and MPAG were lower than 0.5 and 5.0 μg/ml, respectively, using 50 μl of plasma. The method was used to determine the pharmacokinetic parameters of MPA and MPAG following oral administration in a patient with renal transplantation.     

Determination of flumequine and 7-hydroxyflumequine in plasma of sheep by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of flumequine and its metabolite 7-hydroxyflumequine in sheep plasma was described. The two compounds were extracted from 100 μl of plasma by liquid–liquid extraction. Aliquots (100 μl) were injected onto the HPLC system and separated on a LiChrospher Select B column with an isocratic system. The compounds were detected by fluorimetric detection for concentrations below 500 μg/l and by UV detection for the concentrations exceeding 500 μg/l. The range of the validated concentrations were 50 000 to 5 μg/l and 500 to 10 μg/l with mean recovery rates of 87±3% and 60±1% for flumequine and 7-hydroxyflumequine, respectively.     

Simultaneous determination of six penicillins in cows'' raw milk by a multiresidue high-performance liquid chromatographic method

A high-performance liquid chromatographic method based on C₁₈ solid-phase extraction and ultraviolet detection at 323 nm of analytes derivatized with benzoic anhydride and 1,2,4-triazole mercuric chloride solution was developed for the simultaneous determination of amoxicillin, penicillin G (benzylpenicillin), ampicillin, oxacillin, cloxacillin and dicloxacillin residues in raw milk. The detection limit of the method was 1.3 μg/l for penicillin G; 1.4 μg/l for amoxicillin, oxacillin and cloxacillin, 1.5 μg/l for ampicillin and 2.7 μg/l for dicloxacillin. The mean recovery was 95–102% for amoxicillin, penicillin G and ampicillin, 92–98% for oxacillin and cloxacillin and 87–94% for dicloxacillin, measured by using an internal standard. The relative repeatability standard deviation was 4–9% on level 4–15 μg/l, respectively, 2–7% on level 30–40 μg/l.     

A method for low volume and low Se concentration samples and application to paired cerebrospinal fluid and serum samples

The well-known beneficial health effects of Se have demanded the development of rapid and accurate methods for its analysis. A flow injection (FI) method with inductively coupled plasma mass spectrometry (ICP-MS) as a selenium-selective detector was optimized. Flow injection was carried out using a Knauer 1100 smartline inert series liquid chromatograph coupled with a Perkin Elmer DRC II ICP-mass spectrometer. For sample injection a Perkin Elmer electronic valve equipped with a 25 μL sample loop was employed. Before measurement, standards or samples were administered with 1 μg/L rhodium as internal standard for correction of changes in detector response according to changes in sample electrolyte concentration. The method characterization parameters are: LOD (3σ criterion): 26 ng/L, LOQ (10σ criterion): 86 ng/L, linearity: 0.05–>10 μg/L, r²=0.9999, serial or day-to-day precision at 2 μg/L: 4.48% or 5.6%. Accuracy was determined by (a) recovery experiments (CSF spiked with 2 μg/L Se); (b) comparison of FI-ICP-MS measurement with graphite furnace atomic absorption (GFAAS) measurements of 1:10 diluted serum samples; (c) Se determination in urine and serum control materials. Recovery (a) was 101.4%, measurement comparison with GFAAS (b) showed 98.8% (5 serum samples, 1:10 diluted in the range of 0.5–1.3 μg/L, compared to GFAAS determination, which was set to 100%), and accuracy was 96.8% or 105.6% for the serum or urine control material. Analysis time per sample was short and typically below 2 min for the complete measurement, including sample introduction, sample-line purge and quadruplicate Se determination.This method was used to determine Se in cerebrospinal fluid (CSF) and plasma (here parallel to GFAAS) in 35 paired serum and CSF samples. Se determination gave values in the range of 42–130 μg/L for serum and 1.63–6.66 μg/L for CSF. The median for Se in 35 individual CSF samples was 3.28 μg/L, the mean (±SD) was 3.67 (1.35) μg/L, whilst for individual serum samples the median was 81 μg/L and the mean (±SD) was 85 (26) μg/L. When relating the paired Se concentrations of CSF samples to respective serum samples it turned out that Se-CSF (behind blood brain barrier (BBB)) is independent on Se-serum concentration (before BBB).     

Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats

Studies showed that the metabolic unlike the neuroendocrine effects of ghrelin could be abrogated by co-administered unacylated ghrelin. The aim was to investigate the interaction between ghrelin and desacyl ghrelin administered intraperitoneally on food intake and neuronal activity (c-Fos) in the arcuate nucleus in non-fasted rats. Ghrelin (13 μg/kg) significantly increased food intake within the first 30 min post-injection. Desacyl ghrelin at 64 and 127 μg/kg injected simultaneously with ghrelin abolished the stimulatory effect of ghrelin on food intake. Desacyl ghrelin alone at both doses did not alter food intake. Both doses of desacyl ghrelin injected separately in the light phase had no effects on food intake when rats were fasted for 12 h. Ghrelin and desacyl ghrelin (64 μg/kg) injected alone increased the number of Fos positive neurons in the arcuate nucleus compared to vehicle. The effect on neuronal activity induced by ghrelin was significantly reduced when injected simultaneously with desacyl ghrelin. Double labeling revealed that nesfatin-1 immunoreactive neurons in the arcuate nucleus are activated by simultaneous injection of ghrelin and desacyl ghrelin. These results suggest that desacyl ghrelin suppresses ghrelin-induced food intake by curbing ghrelin-induced increased neuronal activity in the arcuate nucleus and recruiting nesfatin-1 immunopositive neurons.     

Simultaneous determination of cholesterol and cholestanol in human serum by high-performance liquid chromatography using 3-(5,6-methylenedioxy-2-phthalimidyl)benzoyl azide as precolumn fluorescent labelling reagent

A fluorescent labelling reagent, 3-(5,6-methylenedioxy-2-phthalimidyl)benzoyl azide, designed for the determination of alcohols by precolumn high-performance liquid chromatography, has been applied to the simultaneous determination of cholesterol and cholestanol in human serum. The reagent reacts with cholesterol and cholestanol at 140°C for 10 min to produce the fluorescent derivatives, which can be separated on a reversed-phase column with acetonitrile—ethanol—water (60:35:7.5, v/v) as eluent. The detection limits for cholesterol and cholestanol were 45 and 50 fmol per injection (20 μl), respectively. The values of cholesterol and cholestanol in normal human sera were 135–212 mg/dl and 137–928 μg/dl, respectively.     

Development and validation of a reverse phase HPLC method for the determination of caprylic acid in formulations of therapeutic immunoglobulins and its application to antivenom production

A novel method of high performance liquid chromatography with UV detection for the quantification of caprylic acid in formulations of therapeutic immunoglobulins was developed and validated. Samples have interfering proteins that were removed by ultrafiltration in a centrifugal filter unit of 10 kDa nominal molecular weight limit. Then, compounds present in ultrafiltrates were separated on an Eclipse XDB-C8 5 μm column (150 mm × 4.6 mm i.d.), using a mixture of acetonitrile–water (60:40, v/v) as the mobile phase at a flow rate of 1 mL/min. The UV detection was performed at 210 nm. The method was found to be precise and accurate, with a linearity range from 400 μg/mL to 600 μg/mL (r = 0.9948). The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 13.46 μg/mL and 44.85 μg/mL, respectively. To illustrate the usefulness of the method in the in-process and final quality control for production of therapeutic immunoglobulin formulations, permeates obtained from the industrial diafiltration step in the manufacture of equine-derived snake antivenoms and ten batches of finished product were analyzed.     

Simultaneous high-performance liquid chromatographic determination of a glucuronyl prodrug of doxorubicin, doxorubicin and its metabolites in human lung tissue

A rapid and sensitive method was developed for the simultaneous determination of the new doxorubicin glucuronide prodrug HMR 1826, the parent drug doxorubicin and its metabolites in human lung tissue samples. Homogenization of frozen tissue samples with the micro-dismembrator was followed by a silver nitrate precipitation step. By removing the exceeding silver ions with sodium chloride further purification steps could be omitted. Compounds were separated by isocratic high-performance liquid chromatography on a LiChrospher 100 RP18 column and a mobile phase consisting of citric acid buffer–acetonitrile–methanol–tetrahydrofuran within 30 min and quantified with fluorescence detection. The method showed good recoveries for all compounds (86–99%) and a linear calibration range of 20 ng/g–80 μg/g for doxorubicin and 1–600 μg/g for HMR 1826.     

Determination of benzimidazole residues using liquid chromatography and tandem mass spectrometry

The determination of residues of benzimidazole using liquid chromatography and tandem mass spectrometry (LC–MS–MS) with ion spray ionization is described. Swine muscle tissue was spiked with a mixture of fifteen benzimidazoles, including metabolites of fenbendazole and albendazole. As clean-up procedure, an ethyl acetate extraction followed by solid-phase extraction on styrol-divinyl-benzene cartridge was used. The evaluation was performed by selecting the characteristic product ions for the benzimidazoles and using multiple reaction mode. 2-n-Butylmercaptobenzimidazole was used as internal standard. Blank muscle samples were fortified in the concentration range of 1–22 μg/kg. The limits of detection were below 6 μg/kg and the limits of quantification for most benzimidazoles were below 10 μg/kg. The matrix effect was checked using spiked muscle tissues of cattle and sheep as well as liver of cattle. Practical application will be shown by incurred egg material from laying hens treated with flubendazole. The recovery of the clean-up was mostly above 50% in muscle tissue and 70% in egg yolk.     

Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography

A simple, rapid and reproducible reversed-phase high-performance liquid chromatographic method for the simultaneous determination of benzoic acid (BA), phenylacetic acid (PAA) and their respective glycine conjugates hippuric acid (HA) and phenaceturic acid (PA) in sheep urine is described. The procedure involves only direct injection of a diluted urine sample, thus obviating the need for an extraction step or an internal standard. The compounds were separated on a Nova-Pak C₁₈ column with isocratic elution with acetate buffer (25 mM, pH 4.5)—methanol (95:5). A flow-rate of 1.0 ml/min, a column temperature of 35°C and detection at 230 nm were employed. These conditions were optimized by investigating the effects of pH, molarity, methanol concentration in the mobile phase and column temperature on the resolution of the metabolites. The total analysis time was less than 15 min per sample. At a signal-to-noise ratio of 3 the detection limits for ten-fold diluted urine were 1.0 μg/ml for BA and HA and 5.0 μg/ml for PAA and PA with a 20-μl injection.     

Simultaneous determination of α-tocopheryl acetate and tocopherols in aquatic organisms and fish feed

In aquaculture, α-tocopheryl acetate (α-TA) is the main source of vitamin E used to fortify fish feed. α-TA in fish is often determined indirectly, i.e., by alkaline hydrolysis, followed by quantitation of “total α-tocopherol” (α-T) and subtraction of the natively present α-T. The aim of this study was to develop an HPLC method for the simultaneous quantitative determination of α-TA and free tocopherols in aquatic organisms and fish feed. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with consecutive UV and fluorescence detection of α-TA and tocopherols, respectively. The peak of the internal standard tocol in the fluorescence trace was used for quantitation. Linearity was achieved over the range of 0.2 to 4.2 μg α-TA per ml extract of Artemia nauplii, which would correspond to 30.7 to 614.4 μg/g dry mass. The within-run coefficient of variation was 1.9% at a level of 310 μg/g dry mass. The recovery of α-TA ranged from 97.7 to 100.8% (concentration=2.1 and 20.5 μg/ml, n=6). The detection limit was about 7 ng and the quantification limit on spiked samples was 0.2 μg/ml. This method was routinely applied to determine α-TA and α-, γ- and δ-tocopherol (α-T, γ-T, δ-T) simultaneously in Artemia, fish feed, shrimp eggs and various other aquatic organisms.     

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)–acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 μg/ml and the urine method was linear from 2 to 40 μg/ml. The method had a detection limit of 1 μg/ml for vanillin and vanillic acid using 5 μl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.     

Quantification of 3,4-methylenedioxymetamphetamine and its metabolites in plasma and urine by gas chromatography with nitrogen–phosphorus detection

A gas chromatographic method with nitrogen–phosphorus detection involving a solid–liquid extraction phase was developed and validated for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in plasma. A modification of this method was validated for the analysis of MDMA, MDA, 4-hydroxy-3-methoxymethamphetamine (HMMA) and, 4-hydroxy-3-methoxyamphetamine (HMA) in urine. Under the analytical conditions described, the limits of detection in plasma and urine were less than 1.6 μg/l and 47 μg/l, respectively, for all the compounds studied. Good linearity was observed in the concentration range evaluated in plasma (5–400 μg/l) and urine (100–2000 μg/l) for all compounds tested. The recoveries obtained from plasma were 85.1% and 91.6% for MDMA and MDA, respectively. Urine recoveries were higher than 90% for MDMA and MDA, 74% for HMMA, and 64% for HMA. Methods have been successfully used in the assessment of plasma and urine concentrations of MDMA and its main metabolites in samples from clinical studies in healthy volunteers.     

Method for the biological monitoring of hexahydrophthalic anhydride by the determination of hexahydrophthalic acid in urine using gas chromatography and selected-ion monitoring

A method for the determination of hexahydrophthalic acid, a metabolite of hexahydrophthalic anhydride, in human urine has been developed. The urine was worked-up by liquid—solid extraction, esterified with boron trifluoride—methanol, and analysed by capillary gas chromatography and selected-ion monitoring. Hexadeuterium-labelled hexahydrophthalic acid was used as the internal standard. The precision was 4% at 0.7 μg/ml and 5% at 0.07 μg/ml. The recovery of the acid for the overall method was 101% at 0.07 μg/ml of urine (with a coefficient of variation of 4%) and 95% at 0.7 μg/ml (coefficient of variation 2%). The limit of detection was 20 ng/ml urine.     

Plasma determination of the novel anticonvulsant d,l-3-hydroxy-3-ethyl-3-phenylpropionamide and preliminary pharmacokinetic studies in the rat

A sensitive gas chromatographic method with flame ionization detection was developed for the analysis in plasma of the novel anticonvulsant d,l-3-hydroxy-3-ethyl-3-phenylpropionamide (HEPP), using d,l-2-hydroxy-2-ethyl-2-phenylacetamide as the internal standard. HEPP was extracted from alkalinized plasma into dichloromethane and quantified after derivatization with bis(trimethylsilyl)-trifluoroacetamide. Standard curves were linear from 0.5 to 50 and from 2 to 100 μg/ml of plasma, using 1.5 and 5 μg of the internal standard, respectively. The lower limit of detection at a signal-to-noise ratio of 3 standard deviations was 0.33 μg/ml of sample. The sensitivity, accuracy and reproducibility of the method were shown to be satisfactory for pharmacokinetic studies of HEPP. After intraperitoneal administration of 50 mg/kg to Wistar rats, the principal kinetic parameters were: absorption half-life = 0.04 h; volume of distribution = 1.32 l/kg; clearance = 4.40 ml/min; peak concentration = 50 μg/ml; peak time = 0.25 h; mean residence time = 4.55 h.     

Determination of cholesterol and cortisone absorption in polyurethane : I. Methodology using size-exclusion chromatography and dual detection

A size-exclusion chromatographic method is described for measuring the absorption of the steroid-based lipids cholesterol and cortisone into Pellethane 2363, a polyurethane used in biomedical implants. The method uses refractometry and ultraviolet diode-array detection, with tetrahydrofuran as the mobile phase. Using an injection volume of 150 μl, the lower limit of accurate measurement for cholesterol (refractive index detection) was 6 μg/ml with a lower limit of detection, based on a 2:1 signal-to-noise ratio, of 0.15 μg (1 μg/ml). For cortisone (ultraviolet detection), the lower accurate limit was 0.6 μg/ml with a lower limit of 0.015 μg (0.1 μg/ml). The results show that after 44 h, 2037 μg/g cholesterol and 3131 μg/g cortisone were absorbed by the polyurethane. The method eliminates extensive sample manipulation and is sensitive to low levels of lipid in the presence of a high-molecular-mass synthetic polymer.     

High-performance liquid chromatographic assay with a simple extraction procedure for sensitive quantification of mycophenolic acid in rat and human plasma

We describe a novel sensitive and simplified gradient HPLC assay for quantification of the immunosuppressant mycophenolic acid (MPA) in rat and human plasma. In contrast to previously reported MPA assays, our method used a single step extraction comprising addition of acetonitrile, which contained phenolphthalein glucoronic acid as internal standard, for protein precipitation. Linearity: 0.1–100 μg/ml (r²>0.999), mean recoveries: MPA 98.0%, internal standard 105.2%, mean intra-day precision: 4.3%, mean day-to-day precision: 4.3%, mean day-to-day accuracy: −1.5%. Sensitivity was sufficient to allow for quantification of mycophenolic acid in as little as 50 μl plasma.     

Determination of acrylamide and glycidamide in rat plasma by reversed-phase high performance liquid chromatography

Acrylamide is a widely used monomer that produces peripheral neuropathy. It is metabolized to the epoxide, glycidamide, which is also considered to be neurotoxic. A new reversed-phase high-performance liquid chromatography (HPLC) method is described that permits simultaneous determination of acrylamide and glycidamide in rat plasma. Samples were deproteinized with acetonitrile and chromatography was performed using isocratic elution and UV absorption detection. The limits of detection for acrylamide and glycidamide were 0.05 and 0.25 μg/ml in plasma, respectively, and recovery of both analytes was greater than 90%. The assay was linear from 0.1 to 100 μg/ml for acrylamide and from 0.5 to 100 μg/ml for glycidamide. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration–time profiles of acrylamide and glycidamide in the plasma of acrylamide-treated rats.     

Glucocorticoid inhibition of estrus in ovariectomized pigs: Relationship to progesterone action

Synthetic glucocorticoids and progesterone were evaluated for their inhibitory action on estrus in ovariectomized pigs treated with estrogen. Triamcinolone acetonide (˜70 μg/kg BW), and dexamethasone (˜140 μg/kg BW) inhibited the estrous response to estradiol benzoate when these glucocorticoids were given during a period of 5 days before to 4 days after estradiol benzoate. The minimum effective dosage of progesterone that would inhibit estrus when given concurrently with estradiol benzoate was 600 (μg/kg BW. When triamcinolone acetonide (30 μg/kg BW) or dexamethasone (125 or 150 μg/kg BW) was given as a single injection in combination with progesterone (100 μg/kg BW), estrous response to estradiol benzoate was again inhibited. These steroids at these dosages had no significant effect when either was administered alone. Based on these results, the inhibitory action of these glucocorticoids on estrus in pigs is additive to the action of progesterone, and we suggest that triamcinolone acetonide and dexamethasone inhibit estrus through mechanisms related to those of progesterone.