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.     

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.     

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.     

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.     

Determination of betulinic acid in mouse blood, tumor and tissue homogenates by liquid chromatography–electrospray mass spectrometry

A rapid and sensitive high-performance liquid chromatography–electrospray MS method has been developed to determine tissue distribution of betulinic acid in mice. The method involved deproteinization of these samples with 2.5 volumes (v/w) of acetonitrile–ethanol (1:1) and then 5 μl aliquots of the supernatant were injected onto a C₁₈ reversed-phase column coupled with an electrospray MS system. The mobile phase employed isocratic elution with 80% acetonitrile for 10 min; the flow-rate was 0.7 ml/min. The column effluent was analyzed by selected ion monitoring for the negative pseudo-molecular ion of betulinic acid [M−H]⁻ at m/z 455. The limit of detection for betulinic acid in biological samples by this method was approximately 1.4 pg and the coefficients of variation of the assay (intra- and inter-day) were generally low (below 9.1%). When athymic mice bearing human melanoma were treated with betulinic acid (500 mg/kg, i.p.), distribution was as follows: tumor, 452.2±261.2 μg/g; liver, 233.9±80.3 μg/g; lung, 74.8±63.7 μg/g; kidney, 95.8±122.8 μg/g; blood, 1.8±0.5 μg/ml. No interference was noted due to endogenous substances. These methods of analysis should be of value in future studies related to the development and characterization of betulinic acid.     

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.     

Determination of acetylsalicylic acid and salicylic acid in skin and plasma by high-performance liquid chromatography

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C₈ columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water–phosphate buffer (pH 2.5)–acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 μg/cm² and of SA from 0.1 to 5 μg/cm² in tape and to quantities of ASA 0.1 to 2 μg/ml and 1 to 50 μg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 μg/cm²) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 μg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100–200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.     

Determination of aspirin and salicylic acid in transdermal perfusates

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of aspirin and salicylic acid in transdermal perfusates. The compounds were separated on a C₈ Nucleosil column (5 μm, 250×4.6 mm) using a mobile phase containing a mixture of water–acetonitrile–orthophosphoric acid (650:350:2, v/v/v) and a flow-rate of 1 ml/min. The transdermal samples were in phosphate-buffered saline (PBS) and could be injected directly onto the HPLC system. The method was reproducible with inter-day R.S.D. values of no greater than 3.46 and 2.60% for aspirin and salicylic acid, respectively. The method was linear over the concentration range 0.2–5.0 μg/ml and had a limit of detection of 0.05 μg/ml for both compounds. For certain samples, it was necessary to ensure that no transmembrane leakage of the aspirin prodrugs had occurred. In these cases, a gradient was introduced by increasing the acetonitrile content of the mobile phase after the salicylic acid had eluted. The method has been applied to the determination of aspirin and salicylic acid in PBS following in vitro application of the compounds to mouse skin samples.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in rat hepatic microsome after chiral derivatization with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate

A reversed-phase high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat hepatic microsome has been developed. Racemic atenolol was extracted from alkalinized rat hepatic microsome by ethyl acetate. The organic layer was dried with anhydrous sodium sulfate and evaporated using a gentle stream of air. Atenolol racemic compound was derivatized with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate at 35°C for 30 min to form diastereomers. After removal of excess solvent, the diastereomers were dissolved in phosphate buffer (pH 4.6)–acetonitrile (50:30). The diastereomers were separated on a Shimadzu CLC-C18 column (10 μm particle size, 10 cm×0.46 cm I.D.) with a mobile phase of phosphate buffer–methanol–acetonitrile (50:20:30, v/v) at a flow-rate of 0.5 ml/min. A UV–VIS detector was operated at 254 nm. For each enantiomer, the limit of detection was 0.055 μg/ml (signal-to-noise ratio 3) and the limit of quantification (signal-to-noise ratio 10) was 0.145 μg/ml (RSD <10%). In the range 0.145–20 μg/ml, intra-day coefficients of variation were 1.0–7.0% and inter-day coefficients of variation were 0.4–16.5% for each enantiomer. The assay was applied to determine the concentrations of atenolol enantiomers in rat hepatic microsome as a function of time after incubation of racemic atenolol.     

Determination of total mycophenolic acid and its glucuronide metabolite using liquid chromatography with ultraviolet detection and unbound mycophenolic acid using tandem mass spectrometry

Two simple, sensitive and reproducible methods for determination of total mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) as well as unbound MPA (fMPA) was developed by the use of HPLC-UV and LC-MS/MS methods, respectively. For the total MPA/MPAG method, the analytes were extracted using Isolute C(2) solid-phase extraction (SPE) cartridges and analyzed at 254 nm over a Zorbax Rx C(8) column (150 mm x 4.6 mm, 5 microm). The mobile phase was a gradient mixture of methanol and water (containing 0.1% (v/v) phosphoric acid). The total run time was 18 min and the extraction recovery was 77% for MPA and 84% for MPAG. The method was precise and accurate with a lower limit of quantification (LLOQ) of 0.5 mg/l for MPA and 5.0 mg/l for MPAG. For the fMPA method, plasma was subjected to ultrafiltration followed by SPE using C(18) cartridges. Analytical column was the same as the HPLC-UV method and the mobile phase was a gradient composition of methanol:0.05% formic acid with a flow rate of 0.6 ml/min for the first 3 min and 0.7 ml for the last 4 min. The chromatographic method separated MPA from its metabolites MPAG and Acyl-MPAG. Mass transitions in negative ionization mode for MPA and the internal standard, indomethacin were m/z: 319-->190.9 and m/z: 356-->312.2, respectively. The assay was linear in the concentration range of 1-1000 microg/l for fMPA with a LLOQ of 1 microg/l and an accuracy of >95%. The two methods reported have an adequate degree of robustness and dynamic concentration range for the measurement of MPA, MPAG and fMPA for therapeutic drug monitoring purposes or pharmacokinetics investigations.     

Simple and rapid assay for acetaminophen and conjugated metabolites in low-volume serum samples

The use of marker compounds for estimating drug metabolic capacity or pharmacokinetic parameters is common in the biological sciences. Often small laboratory animals are used and thus sample size is a limiting concern. In this report, we describe an assay we developed for measuring the concentration of acetaminophen and its conjugated metabolites in low-volume serum samples. Acetaminophen and metabolites were removed from 10 μl serum samples by a single-step 6% (v/v) perchloric acid deproteination using theophylline as internal standard. Samples were separated in a pH 2.2 sodium sulfate–acetonitrile mobile phase at a flow-rate of 1.5 ml/min on a 15 cm octadecylsilyl column at room temperature. Analytes were detected at a wavelength of 254 nm. The resulting chromatograms showed no interfering peaks from endogenous serum components. The concentration ranges measured were 1.56–200 μg/ml for acetaminophen and acetaminophen sulfate and 3.91–500 μg/ml for acetaminophen glucuronide. The assay was linear in the range of concentrations analyzed. The intra-day and inter-day coefficient of variation ranged from 0.4 to 8.2% and 0.2 to 12.3% for acetaminophen, 0.5 to 12.9% and 0.3 to 16.1% for acetaminophen glucuronide, and 0.4 to 8.1% and 0.2 to 14.3% for acetaminophen sulfate, respectively. Results from the experiments show that acetaminophen and its conjugated metabolites can easily and reproducibly be measured in low-volume serum samples and thus may offer an additional method to measure these compounds when the volume of biological samples may be limited.     

Simple reversed-phase ion-pair liquid chromatography assay for the simultaneous determination of mycophenolic acid and its glucuronide metabolite in human plasma and urine

A simple and reproducible reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method using isocratic elution with UV absorbance detection is presented for the simultaneous quantitation of mycophenolic acid (MPA) and MPA-glucuronide (MPAG) in human plasma and urine. The sample preparation procedures involved simple protein precipitation for plasma and 10-fold dilution for urine. Each analytical run was completed within 15min, with MPAG and MPA being eluted at 3.8 and 11.4min, respectively. The optimized method showed good performance in terms of specificity, linearity, detection and quantitation limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.     

Sensitive and convenient high-performance liquid chromatographic method for the determination of mitomycin C in human plasma

An improved high-performance liquid chromatographic assay for the cytostatic drug mitomycin C in plasma is presented. The principal steps are precipitation of plasma proteins with acetonitrile, lyophilization of the supernatant and reversed-phase chromatography on a Hypersil ODS 5 μm column with 0.01 M NaH₂PO₄ buffer (pH 6.5)-methanol (70:30, v/v) in isocratic mode. At a flow-rate of 1.3 ml/min a column pressure of 180–220 bar resulted. Porfiromycin served as internal standard. UV detection was performed at 365 nm. Quantitation limit based on a coefficient of variation <10% in intra- and inter-day assay was 5 μg/l mitomycin C, detection limit based on a signal-to-noise ratio of 3 was 1 μg/l. Recovery was 100% and linearity was shown for the whole range of concentration (1–500 μg/l). None of the five drugs used during chemoembolisation interfered with the assay in vitro. The assay meets the requirements for pharmacokinetic studies of mitomycin C in patients as regards sensitivity and ease of use.     

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C₄, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm². The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.     

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.     

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller–Hinton broth for pharmacokinetic/pharmacodynamic studies

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C₈ 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.     

Sensitive and specific determination of clindamycin in human serum and bone tissue applying liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry

A method for the quantification of clindamycin in human serum and in human bone tissue samples applying high-performance liquid chromatography with atmospheric pressure chemical ionization–mass spectrometry (APCI–MS) is presented. Lincomycin is used as the internal standard. Serum samples are prepared only by protein precipitation with acetonitrile. Bone tissue samples have to be crushed and homogenized in extraction buffer prior to analysis. The chromatographic separation is achieved on an RP-18 stationary phase with 0.02% trifluoroacetic acid in water 60%/acetonitrile 40% v/v as mobile phase. The limits of quantification are 0.1 μg/ml for serum samples and 0.1 μg/g for bone tissue samples. The coefficients of variation for the assays are 4.48 and 8.41% at the limit of quantification for serum and bone tissue samples, respectively. Bone tissue samples as small as 50 mg can be used.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C₁₈ reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

Study of the solid-phase extraction of diclofenac sodium, indomethacin and phenylbutazone for their analysis in human urine by liquid chromatography

A selective semi-automated solid-phase extraction (SPE) of the non-steroidal anti-inflammatory drugs diclofenac sodium, indomethacin and phenylbutazone from urine prior to high-performance liquid chromatography was investigated. The drugs were recovered from urine buffered at pH 5.0 using C₁₈ Bond-Elut cartridges as solid sorbent material and mixtures of methanol–aqueous buffer or acetonitrile–aqueous buffer as washing and elution solvents. The extracts were chromatographed on a reversed-phase ODS column using 10 mM acetate buffer (pH 4.0)–acetonitrile (58:42, v/v) as the mobile phase, and the effluent from the column was monitored at 210 nm with ultraviolet detection. Absolute recoveries of the anti-inflammatory drugs within the range 0.02–1.0 μg/ml were about 85% for diclofenac and indomethacin, and 50% for phenylbutazone without any interference from endogenous compounds of the urine. The within-day and between-day repeatabilities were in all cases less than 5% and 10%, respectively. Limits of detection were 0.007 μg/ml for diclofenac sodium and indomethacin and 0.035 μg/ml for phenylbutazone, whereas limits of quantitation were 0.02 μg/ml for diclofenac and indomethacin and 0.1 μg/ml for phenylbutazone.