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 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.     

Simple reversed-phase liquid chromatographic assay for simultaneous quantification of free mycophenolic acid and its glucuronide metabolite in human plasma

A reversed-phase HPLC-UV method, involving simple instrumental setup and mobile phase without ion-pairing reagent, was developed and validated for direct simultaneous quantification of free mycophenolic acid (MPA) and its major metabolite MPA-glucuronide (MPAG) in human plasma. Both free MPA and MPAG were isolated from plasma samples using ultrafiltration prior to analysis. Each chromatographic run was completed within 13 min. The optimized method showed good performance in terms of specificity, linearity (r(2)=0.9999), sensitivity (limit of quantitation (LOQ): 0.005 mg/L for MPA; 1 mg/L for MPAG), and intra- and inter-day precision (R.S.D.<7%). This assay was successfully applied to free MPA and MPAG measurements in clinical samples.     

Simple determination of mycophenolic acid in human serum by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic analysis was established to monitor the serum concentration of mycophenolic acid, the active metabolite from mycophenolate mofetil administered for the prophylaxis of acute organ rejection in renal transplantation. The system consisted of two pumps for solvent delivery, a column-switching valve, a precolumn, and a reversed-phase analytical column. The present method enabled us to determine MPA by injecting serum samples directly into HPLC without any pretreatment. The mobile phases with different amounts of organic solvent were delivered to the precolumn and analytical column by separate lines, and samples were applied to the precolumn. The column switching valves were switched automatically following the processes for the elimination of protein and the drug analysis. The peak heights of MPA were linearly related to the concentrations (r=0.999) in the range of 0.1-20 micro g/ml, and the limit of quantification was 0.1 micro g/ml (S/N ratio=3). This method was accurate and reproducible on the basis of the results of recovery (94.0-98.0%) and small coefficient of variations of intra and inter-assay (less than 8.3%).     

Quantification of free mycophenolic acid and its glucuronide metabolite in human plasma by liquid-chromatography using mass spectrometric and ultraviolet absorbance detection

The immunosuppressant drug mycophenolic acid (MPA) and its major metabolite, mycophenolic acid glucuronide (MPAG), are highly bound to albumin. An HPLC-tandem-MS (HPLC/MS/MS) and an HPLC-UV assay were developed to measure free (unbound) concentrations of MPA and MPAG, respectively. Ultrafiltrate was prepared from plasma (500 microl) by ultrafiltration at 3000 x g for 20 min (20 degrees C). Both MPA and MPAG were isolated from ultrafiltrate (100 microl) by acidification and C18 solid-phase extraction. Free MPA was measured by electrospray tandem mass spectrometry using selected reactant monitoring (MPA: m/z 338.2--> 206.9) in positive ionisation mode. Chromatography was performed on a PFPP column (50 mm x 2 mm, 5 microm). Total analysis time was 7 min. The assay was linear over the range 1-200 microg/l with a limit of quantification of 1 microg/l. The inter-day accuracy and imprecision of quality controls (7.5, 40, 150 microg/l) were 94-99% and < 7%, respectively. Free MPAG was chromatographed on a C18 Nova-Pak column (150 mm x 3.9 mm, 5 microm) using a binary gradient over 20 min. The eluent was monitored at 254 nm. The assay was linear over the range 1-50 mg/l with the limit of quantification at 2.5 mg/l. The inter-day accuracy and imprecision of quality controls (5, 20, 45 mg/l) was 101-107% and < 8% (n = 4), respectively. For both methods no interfering substances were found in ultrafiltrate from patients not receiving MPA. The methods described have a suitable dynamic linear range to facilitate the investigation of free MPA and MPAG pharmacokinetics in transplant patients. Further, this is the first reported HPLC-UV method to determine free MPAG concentrations.     

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.     

Identification of protein targets for mycophenolic acid acyl glucuronide in rat liver and colon tissue

Covalent binding of acyl glucuronides to proteins is considered an initiating event for the organ toxicity of drugs containing a carboxylic acid group. An acyl glucuronide (AcMPAG) of the immunosuppressant mycophenolic acid was described and shown to form covalent adducts with plasma albumin in vivo. The aim of the present investigation was to identify AcMPAG target proteins in the liver and colon of rats treated with mycophenolate mofetil, which may contribute to a better understanding of the mechanisms responsible for the development of side effects during therapy with this drug. Mycophenolate mofetil was administered per os in to Wistar rats (40 mg/kg/day) over 21 days. Proteins in liver and colon homogenates were separated by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. AcMPAG labeled protein spots were detected by Western blotting. After in-gel tryptic digestion of the protein spots from parallel gels (n = 2), peptides were characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. Data base searching identified AcMPAG target proteins. Tryptic peptides with sufficient signal intensities were subjected to post-source decay analysis. Three proteins in the liver (ATPase/ATP synthase (alpha and beta subunits), protein disulfide isomerase A3 and selenium binding protein) and one protein in the colon (selenium binding protein) were identified as targets for AcMPAG. ATPase/ATP synthase and protein disulfide isomerase are essential proteins involved in the control of the energy and redox state of the cells, whereas the physiological role of selenium binding protein is not fully understood. This study shows for the first time the formation of adducts between tissue proteins and AcMPAG. Whether this chemical modification is associated with compromised protein function and drug toxicity remains to be investigated.     

Rapid determination of mycophenolic acid in plasma by reversed-phase high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure plasma concentrations of mycophenolic acid. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a Zorbax Eclipse C(8) reversed-phase column with a flow-rate of 1.5 ml/min, and monitoring the absorbance at 250 nm. Throughput was up to 100 samples in 24 h. Within the investigated concentration ranges of mycophenolic acid (0-100 mg/l), good linearity (r>0.99) was obtained. The method is sensitive (the limit of detection was about 20 microg/l) and precise (for 0.49 mg/l added to plasma, within-run C.V. was 2% and between-run was 4.2%; for 2.88 mg/l, within-run C.V. was 0.35% and between-run C.V. was 0.69%; for 24.38 mg/l, within-run C.V. was 0.77% and between-run C.V. was 3.1%). Analytical recoveries were 96% for 0.5 mg/l mycophenolic acid added to plasma, 100% for 12 mg/l and 102.5% for 24 mg/l.     

Simultaneous determination of mycophenolic acid and its phenolic glucuronide in human plasma using an isocratic high-performance liquid chromatography procedure

Simultaneous determination of mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) in plasma was accomplished by isocratic HPLC with UV detection. After protein precipitation and phase separation with saturated sodium dihydrogenphosphate, chromatographic separation was achieved on a monolithic column "Chromolith Performance RP-18e", with acetonitrile/0.01 M phosphate buffer, pH 3, (25:75, v/v), as the mobile phase; flow rate 3.3 ml/min and measurement at 214 nm. Linearity was verified up to 40 mg/l for MPA and up to 400 mg/l for MPAG. Detection limits based on the analysis of 50 microl plasma were 0.05 and 0.5 mg/l for MPA and MPAG, respectively. Accuracy was 99.6-104% for MPA and 95.6-105% for MPAG and total imprecision (CV) was <7% for both compounds. Analytical recovery was >95% for MPA and MPAG. The method is simple, rapid, accurate and suitable for routine determination of MPA and MPAG in plasma.     

High-performance liquid chromatographic assay for acetaminophen glucuronide in human liver microsomes

A rapid and specific high-performance liquid chromatographic assay was developed for the determination of acetaminophen glucuronide formed by human liver microsomes. In addition, incubation conditions were systematically evaluated. Conditions that yielded the optimal rate of acetaminophen glucuronide formation over various concentrations of acetaminophen (0.15–30 mM) consisted of the following: 0.1 M potassium phosphate buffer, 1 mM magnesium chloride, 30 μg/mg alamethicin, 4 mM uridine 5′-diphosphoglucuronic acid at a pH of 7.1. Alamethicin produced higher and more consistent APAPG formation rates compared to Brij-58. Adding saccharolactone to the incubation medium reduced the velocity of the reaction. Acetaminophen glucuronide, acetaminophen, and the internal standard (paraxanthine), were analyzed on a C₁₈ column with UV detection at 250 nm. The mean correlation coefficient (r²) of the standard curves for acetaminophen glucuronide was >0.99 over the range of 0.1–25 nmol. The intra- and inter-day coefficients of variation were <4%. This method is suitable for in vitro studies using acetaminophen glucuronide formation as an index reaction for UGT activity.     

Simultaneous determination of free mycophenolic acid and its glucuronide in serum of patients under mycophenolate mophetil therapy by ion-pair reversed-phase liquid chromatography with diode array UV detection

An high performance liquid chromatography (HPLC)-UV method for the simultaneous determination of the free forms of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) in human serum samples was developed for the first time. Chromatographic separation was performed on octadecylsilane based stationary phase in combination with a mobile phase of methanol/buffered tetrabutylammonium (TBA) salt mixture. Sample pretreatment consisted of an ultrafiltration step followed by clean-up/enrichment on a C(18) solid-phase extraction (SPE) cartridge. Average recoveries of (99.7 +/- 0.2)% and (64.1 +/- 6.9)% for free MPA and MPAG, respectively, were estimated in the concentration range from 0.5 to 10 microg/ml. The within-day and between-days coefficients of variation were 0.4 and 0.8% for free MPA (0.1 microg/ml spiking level) and 0.8 and 1.6% for free MPAG (5 microg/ml spiking level), respectively. The linear ranges for free MPA and MPAG were 0.06-1 and 0.2-10microg/ml, respectively. Detection limits of 4 and 17 ng/ml for free MPA and MPAG were estimated in spiked serum. The same HPLC method was also capable of a simultaneous determination of the total concentration of MPA and MPAG when coupled to a proper sample pretreatment step. The potential of the method is demonstrated by excretion kinetics measurement in serum of patients receiving MMF therapy.     

Manual and automated (robotic) high-performance liquid chromatography methods for the determination of mycophenolic acid and its glucuronide conjugate in human plasma

A manual and an automated (Zymark PyTechnology robot) HPLC method for simultaneous determination of plasma mycophenolic acid (MPA) and its glucuronide conjugate (MPAG) are described here. Both methods are reproducible and accurate, and both are equivalent in all respects, including quantification limits (MPA, 0.100 μg/ml; MPAG, 4.00 μg/ml), range (using 0.05–0.5 ml of plasma: MPA, 0.0500–20.0 μg/aliquot; MPAG, 2.00–200 μg/aliquot), precision, and accuracy. MPA and MPAG were stable under the conditions used with both methods. Results from aliquots of paired control samples, analyzed by the manual method over three years at six analytical laboratories, showed excellent agreement in precision and accuracy.     

Determination of ranitidine and its metabolites in human urine by reversed-phase ion-pair high-performance liquid chromatography

A method using ion-pair high-performance liquid chromatography is presented for determining ranitidine, ranitidine N-oxide, ranitidine S-oxide and desmethyl ranitidine in the urine from four volunteers, given on separte occasions an intravenous and oral dose of 100 mg ranitidine. This method has been used to study the metabolism and pharmacokinetics of ranitidine by man. It was found that the elimination half-life of ranitidine ranged from 110–246 min. The mean renal clearance of ranitidine in these four volunteers was 512 ml/min.     

Determination of ebrotidine and its metabolites in human urine by reversed-phase ion-pair high-performance liquid chromatography

Ebrotidine is a new H₂-receptor antagonist with powerful antisecretory activity, demonstrated gastroprotection and the ability to inhibit protease and lipase activities of Helicobacter pylori. As a tool in the clinical pharmacokinetic study of ebrotidine, an analytical method for the simultaneous determination of ebrotidine an its metabolites in human urine was developed. An ion-pair reversed-phase HPLC separation using 1-hexanesulfonic acid and acetonitrile as mobile phase with gradient elution was optimized. In addition, several procedures of preconcentration and clean-up were tested, including solid-phase and liquid—liquid extraction, the mixture dichloromethane—2-propanol (9:1, v/v) at pH 11 being the most efficient. The quality parameters of the whole analytical method were established, the calibration curves were linear over the range studied (1–200 μg/ml) and the reproducibility of the method was high (inter-day R.S.D. values lower than 4.4%).The limits of detection were between 26 and 110 ng/ml of urine for ebrotidine and its metabolites. The method was applied to the analysis of urine collected from two volunteers during 96 h following oral administration of ebrotidine at a dose of 400 mg.     

Simultaneous determination of theophylline and its major metabolites in urine by reversed-phase ion-pair high-performance liquid chromatography

A new, highly selective high-performance liquid-chromatographic (HPLC) assay for theophylline and its major metabolites in urine is described. The method utilizes an ion-pair extraction followed by separation and quantitation by reversed-phase ion-pair gradient-elution HPLC. Comparison with several other methods showed that interferences were present in too many blank urine samples to allow for the accurate quantitation of the metabolites of theophylline by direct injection-isocratic HPLC assays. Sample processing involving ion-pair complexing and extraction together with gradient-elution systems is recommended for accurate pharmacokinetic studies.     

Simple and simultaneous determination for 12 phenothiazines in human serum by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the 12 phenothiazines (chlorpromazine, fluphenazine, levomepromazine, perazine, perphenazine, prochlorperazine, profenamine, promethazine, propericiazine, thioproperazine, thioridazine and trifluoperazine) in human serum using HPLC/UV. The separation was achieved using a C(18) reversed-phase column (250 mm x 4.6 mm I.D., particle size 5 microm, Inersil ODS-SP). The mobile phase, consisting of acetonitrile-methanol-30 mM NaH(2)PO(4) (pH 5.6) (300:200:500, v/v/v), was delivered at a flow rate of 0.9 mL/min and UV detection was carried out at 250 nm. The recoveries of the 12 phenothiazines spiked into serum samples were 87.6-99.8%. Regression equations for the 12 phenothiazines showed excellent linearity, with detection limits of 3.2-5.5 ng/mL for serum. The inter-day and intra-day coefficients of variation for serum samples were commonly below 8.8%. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic purposes. This sensitive and selective method offers the opportunity for simultaneous screening and quantification of almost all phenothiazines available in Japan for the purposes of clinical and forensic applications.     

Simultaneous determination of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in human serum and plasma by high-performance liquid chromatography

A HPLC method was developed for the determination of the metabolites of coumarin and 7-hydroxycoumarin in plasma and serum. Separation was based on gradient elution of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, coumarin and finally 4-hydroxycoumarin (which is used as an internal standard). Standards, prepared in plasma or serum, and samples were treated with trichloroacetic acid, mixed and centrifuged. The supernatant was removed and analyzed by reversed-phase high-performance liquid chromatography on a C₁₈ column. The limit of detection was 50 ng/ml for 7-hydroxycoumarin and 200 ng/ml for coumarin and 7-hydroxycoumarin glucuronide. The linear range was 0.5–100 μg/ml for each of the analytes. The percentage relative standard deviation about the mean measured concentrations were all below 10%. There was no statistical difference between the standard curves prepared in plasma or serum. The method developed was applied to the determination of each of the three compounds in serum, after the administration of 7-hydroxycoumarin, and in plasma after the administration of coumarin. The concentrations of total 7-hydroxycoumarin in the serum samples were also determined by another HPLC method and the results were compared. There was no statistical difference between the results determined.     

Rapid assay for gamma-carboxyglutamic acid in urine and bone by precolumn derivatization and reversed-phase liquid chromatography

A reversed-phase high-performance liquid chromatographic assay for the analysis of gamma-carboxyglutamic acid (Gla) in urine and bone protein hydrolyzates is described. The method employs precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Gla was quantified by reference to an internal standard (beta-carboxyaspartic acid). The "within-run" coefficient of variation of the assay for Gla in urine was between 2.1 and 3.4%, and that for bone protein hydrolyzates was 3.2%. The "between-run" coefficient of variation ranged from 4.1 to 5.5%. There was good agreement between the measurement of urinary Gla by high-performance liquid chromatography and amino acid analyzer. Free Gla could not be detected in serum.     

Improvement of ethyl glucuronide determination in human urine and serum samples by solid-phase extraction

An improved method for the determination of ethyl glucuronide (EtG) in human serum and urine was developed using solid-phase extraction (SPE) and gas chromatography (GC) with mass spectrometric detection (MS). EtG was isolated from serum and urine using aminopropyl SPE columns after deproteination with perchloric acid and hydrochloric acid, respectively. The chromatographic separation was performed on a DB 1701 fused-silica column. At a signal-to-noise ratio of 3:1, a quantification limit of 173 and 560 ng/ml and a detection limit of 37 and 168 ng/ml could be determined for serum and urine, respectively. This indicates high specificity and sensitivity of the described method. The mean absolute recovery was 85%, while intra- and inter-day precision of the assay were all less than 7.5%. The linearity of the calibration curves was satisfying as indicated by correlation coefficients of >0.993. The presented method provides the basis for determination and identification of EtG in human serum and urine samples in a low-concentration range for monitoring alcohol consumption during treatment for alcohol dependence and comorbid alcohol abuse of psychotherapy patients.