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.     

Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.     

Improved and simplified liquid chromatographic assay for adefovir, a novel antiviral drug, in human plasma using derivatization with chloroacetaldehyde

A rapid and simplified chromatographic assay is reported for the quantification of adefovir (PMEA) utilizing derivatization with chloroacetaldehyde. Adefovir is isolated from plasma using protein precipitation with trichloroacetic acid; next, the fluorescent 1,N⁶-etheno derivative is directly formed at 98°C in the buffered extract with chloroacetaldehyde. This derivative is analyzed using isocratic ion-pair liquid chromatography and fluorescence detection at 254 nm for excitation and 425 nm for emission. In the evaluated concentration range (10–1000 ng/ml) precisions ≤5% and accuracies between 95 and 117% were found, using a 0.2-ml volume of plasma. The lower limit of quantification is 10 ng/ml with a intra-assay precision of 16%. The currently reported bioanalytical method is 20–25-fold more sensitive than previously published assays.     

Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography–mass spectrometry

We report here the development and validation of an LC–MS method for quantitation of loperamide (LOP) and its N-demethyl metabolite (DMLOP) in human plasma. O-Acetyl-loperamide (A-LOP) was synthesized by us for use as an internal standard in the assay. After addition of the internal standard, the compounds of interest were extracted with methyl tert.-butylether and separated by HPLC on a C₁₈ reversed-phase column using an acetonitrile–water gradient containing 20 mM ammonium acetate. The three compounds were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in plasma. Analytes were quantitated using positive electrospray ionization in a triple quadrupole mass spectrometer operating in the MS–MS mode. Selected reaction monitoring was used to quantify LOP (m/z 477→266), DMLOP (m/z 463→252) and A-LOP (m/z 519→266) on ions formed by loss of the 4-(p-chlorophenyl)-4-hydroxy-piperidyl group upon low energy collision-induced dissociation. Calibration curves, which were linear over the range 1.04 to 41.7 pmol/ml (LOP) and 1.55 to 41.9 pmol/ml (DMLOP), were run contemporaneously with each batch of samples, along with low (4.2 pmol/ml), medium (16.7 pmol/ml) and high (33.4 pmol/ml) quality control samples. The lower limit of quantitation (LLQ) of LOP and DMLOP was about 0.25 pmol/ml in plasma. The extraction efficiency of LOP and DMLOP from human plasma was 72.3±1.50% (range: 70.7–73.7%) and 79.4±12.8% (64.9–88.8%), respectively. The intra- and inter-assay variability of LOP and DMLOP ranged from 2.1 to 14.5% for the low, medium and high quality control samples. The method has been used successfully to study loperamide pharmacokinetics in adult humans.     

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.     

Analysis of aplidine (dehydrodidemnin B), a new marine-derived depsipeptide, in rat biological fluids by liquid chromatography–tandem mass spectrometry

Aplidine (dehydrodidemnin B) is a new marine-derived depsipeptide with a powerful cytotoxic activity, which is under early clinical investigation in Europe and in the US. In order to investigate the pharmacokinetic properties of this novel drug, an HPLC–tandem mass spectrometry method was developed for the determination of aplidine in biological samples. Didemnin B, a hydroxy analogue, was used as internal standard. After protein precipitation with acetonitrile and extraction with chloroform, aplidine was chromatographed with a RP octadecylsilica column using a water–acetonitrile linear gradient in the presence of formic acid at the flow-rate of 500 μl/min. The method was linear over a 5–100 ng/ml range (LOD=0.5 ng/ml) in plasma and over a 1.25–125 ng/ml range (LOD=0.2 ng/ml) in urine with precision and accuracy below 14.0%. The intra- and inter-day precision and accuracy were below 12.5%. The extraction procedure recoveries for aplidine and didemnin B were 69% and 68%, respectively in plasma and 91% and 87%, respectively in urine. Differences in linearity, LOQ, LOD and recoveries between plasma and urine samples seem to be matrix-dependent. The applicability of the method was tested by measuring aplidine in rat plasma and urine after intravenous treatment.     

Determination of LSD in blood by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis (CE) with HeCd laser-induced fluorescence (LIF) detection and its application in forensic toxicology is demonstrated by the determination of

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-lysergic acid diethylamide (LSD) in blood. Following precipitation of proteins, washing of the evaporated supernatant and extraction, the residue was reconstituted in methanol and injected electrokinetically (10 s, 10 kV). The total analysis time for quantification of LSD was 8 min using a citrate–methanol buffer, pH 4.0. With this buffer system it is possible to separate LSD, nor-LSD, iso-LSD and iso-nor-LSD. Using a specific sample preparation, electrokinetic injection, extended light path (bubble cell) capillaries and especially LIF detection (λ_ex 325 nm, λ_em 435 nm), a limit of detection of 0.1–0.2 ng LSD per ml blood could be obtained. The limit of quantitation was about 0.4–0.5 ng/ml. The quantitative evaluation for LSD was carried out using methylergometrine as internal standard. The precision expressed as coefficient of variation (C.V.) and accuracy of the method were <20% and 86–110%, respectively. The application of the method to human blood samples from two forensic cases and a comparison with radioimmunoassay demonstrated that the results were consistent.     

Determination of gestrinone in human serum by liquid chromatography–electrospray tandem mass spectrometry

A rapid, sensitive and specific high-performance liquid chromatography–electrospray tandem mass spectrometric method has been developed for the determination of gestrinone (R 2323) in human serum using mifepristone (RU 486) as an internal standard. R 2323 was extracted from human serum by an ether extraction procedure. Multiple reaction monitoring was used to detect R 2323 and RU 486. The calibration curve was linear over the range of 3.5–177 ng/ml (r²≥0.99) with the limitation of detection of 0.8 ng/ml. The intra-day precision and accuracy, expressed as C.V. and RE, ranged from 2.3–13.7 to −4.8–3.0%. The inter-day precision and accuracy ranged from 5.5–14.8 to −6.7–3.1%. The mean recovery was 91.0% for R 2323, and 90.6% for the internal standard. The method was successfully applied to the pharmacokinetic study of R 2323.     

Quantification of fluoxetine and norfluoxetine serum levels by reversed-phase high-performance liquid chromatography with ultraviolet detection

A rapid and sensitive high-performance liquid chromatography assay method was developed to determine serum fluoxetine and norfluoxetine levels by single extraction of 0.1 ml of serum with sodium hydroxide. The mobile phase (55% acetonitrile–45% distilled water containing 10 mM aqueous triethylamine) was used to separate fluoxetine and norfluoxetine (25–1000 ng/ml, using clomipramine as the internal standard) by ultraviolet detection at 226 nm. The inter- and intra-day variabilities of fluoxetine and norfluoxetine were 13–18%, and the recoveries of both drugs exceeded 89%. This assay method was applied to a pharmacokinetic disposition study of fluoxetine in mice.     

High-performance liquid chromatographic analysis of melatonin in human plasma and rabbit serum with on-line column enrichment

This paper describes the development of a simple and sensitive analytical method for the quantification of melatonin in human plasma and rabbit serum, using standard analytical equipment and on-line column enrichment without prior extraction, clean-up or derivatization. The analytical procedure was found to be accurate, precise and linear. For human plasma, the accuracy was 101% (range 89–106%), and the mean precision was 5% (range 2–9%) for all concentrations (0, 2, 10, 50 and 200 ng/ml) tested (n=6). The accuracy in rabbit serum was 101% (range 90–112%), and the mean precision was 13% (range 8–19%) for all concentrations (0, 2, 10, 50, 200 and 500 ng/ml) tested (n=6). The retention time of melatonin was about 8 min and the total recoveries were found to be approximately 65 and 85%, respectively, for human plasma and rabbit serum. The limit of detection was found to be lower than 1 ng/ml for human plasma and around 2 ng/ml for rabbit serum. The method is, therefore, found to be suitable for melatonin bioavailability studies in rabbits and presumably also in humans.     

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether–isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 μl potassium phosphate (0.1 M, pH 2.2) and 60 μl of the acid solution was injected into a C₁₈ BDS Hypersil analytical column (3 μm, 100×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H₃PO₄)–acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5–100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.     

Thalidomide enantiomers: determination in biological samples by HPLC and vancomycin-CSP

Thalidomide is a racemate with potentially different pharmacokinetics and pharmacodynamics of the component (+)-(R)- and (-)-(S)-thalidomide enantiomers. As part of a project on the adjunctive effects of thalidomide and cytotoxic agents, a method for the chiral separation and quantitation of thalidomide was developed and validated. Thalidomide in relevant serum and tissue homogenate samples was stabilized by buffering with an equal volume of citrate-phosphate buffer (pH 2, 0.2M), and stored at -80 degrees C pending assay. The thalidomide enantiomers, extracted from the samples with diethyl ether, were well separated on a chiral HPLC column of vancomycin stationary phase and a mobile phase of 14% acetonitrile in 20 mM ammonium formate adjusted to pH 5.4; their concentrations were determined with phenacetin as internal standard at 220 nm detection. Over a thalidomide concentration range of 0.1-20 microg/ml, assay precision was 1-5% (CV) for both enantiomers, and calibration curves were linear with all correlation coefficients being >0.99. The estimated limit of quantification for both enantiomers was 0.05 microg/ml with 0.2-0.6 ml serum samples. Thalidomide in rat and human serum, acidified and stored as described above, was found to be chemically and chirally stable over 1 year. The method has been successfully applied to serum samples from human patients undergoing thalidomide treatment for mesothelioma, and to serum, blood and tissue samples from a laboratory rodent model using transplanted 9l gliosarcoma. Enantioselectivity in thalidomide pharmacokinetics has been found, thereby reinforcing the need for considering the relevance of chirality in thalidomide pharmacology.     

Liquid chromatographic–electrospray tandem mass spectrometric determination of novel antifungal agents in plasma

A rapid, selective, sensitive and reproducible HPLC–electrospray tandem mass spectrometric method has been developed for the analysis of novel triazole antifungal agents, SYN-2869 and its derivatives (SYN-2836, SYN-2903 and SYN-2921), in rat plasma using SYN-2506 as an internal standard. Isolation of these compounds from plasma and sample desalting were performed by a simple extraction procedure involving protein precipitation, vacuum-drying and reconstitution with acetonitrile. For all the agents, linearity was observed over the range of 10–10 000 ng/ml (r≥0.996) and the limit of quantitation was 10 ng/ml using a 100-μl plasma volume. A measurement rate of 400–500 samples/day/instrument could be achieved using this method.     

Simultaneous determination of verapamil and norverapamil in biological samples by high-performance liquid chromatography using ultraviolet detection

In this paper we develop an high-performance liquid chromatographic method with ultraviolet detection for the determination of verapamil and its primary metabolite norverapamil in biological samples. Both compounds, as well as the internal standard, imipramine, were extracted from alkalinised blood, with n-hexane–isobutyl alcohol, back-extracted into 0.01 M phosphoric acid and determined using a reversed-phase column and ultraviolet monitoring at 210 nm. The average coefficient of variation obtained over the concentration range of 1–1000 ng/ml is about 3%. The detection limit is below 5 ng/ml for both compounds, and extraction recoveries close to 80%. The method was applied to a pharmacokinetic study of the drug and its active metabolite and used to analyse blood samples from verapamil treated rabbits.     

Rapid determination of citalopram in human plasma by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of citalopram in human plasma is presented. The sample preparation involved liquid–liquid extraction of citalopram with hexane–isoamyl alcohol (98:2 v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on a cyano column (45×4.6 mm, 5 μm particles), the mobile phase consisted of an acetonitrile–phosphate buffer, pH 6.0 (50:50, v/v). The run time was 2.6 min. The fluorimetric detector was set at an excitation wavelength of 236 nm and an emission wavelength of 306 nm. Verapamil was used as the internal standard. The limit of quantitation was 0.96 ng/ml using 1 ml of plasma. Within- and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 6%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

A validated high-performance liquid chromatographic assay for the simultaneous determination of denaverine and its N-monodemethyl metabolite in human plasma

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of denaverine and its N-monodemethyl metabolite (MD 6) in human plasma is described. The assay involves the extraction with an n-heptane–2-propanol mixture (9:1, v/v) followed by back extraction into 12.5% (w/w) phosphoric acid. The analytes of interest and the internal standard were separated on a Superspher RP8 column using a mobile phase of acetonitrile–0.12 M NH₄H₂PO₄–tetrahydrofuran (24:17.2:1, v/v), adjusted to pH 3 with 85% (w/w) phosphoric acid. Ultraviolet detection was used at an operational wavelength of 220 nm. The retention times of MD 6, denaverine and the internal standard were 5.1, 6.3 and 10.2 min, respectively. The assay was validated according to international requirements and was found to be specific, accurate and precise with a linear range of 2.5–150 ng/ml for denaverine and MD 6. Extraction recoveries for denaverine and MD 6 ranged from 44 to 49% and from 42 to 47%, respectively. The stability of denaverine and MD 6 in plasma was demonstrated after 24 h storage at room temperature, after three freeze–thaw cycles and after 7 months frozen storage below −20°C. The stability of processed samples in the autosampler at room temperature was confirmed after 24 h storage. The analytical method has been applied to analyses of plasma samples from a pharmacokinetic study in man.     

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 μl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C₁₈ reversed-phase column with a mobile phase consisting of water–acetonitrile (71:29, v/v) containing 0.05 M Na₂HPO₄ and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1–500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.     

Rapid and simple high-performance liquid chromatographic determination of nimesulide in human plasma

A high-performance liquid chromatographic method for the quantitation of nimesulide in human plasma is presented. The method is based on protein precipitation with methanol and reversed-phase chromatography with spectrophotometric detection at 404 nm. The separation was performed on a Nucleosil 120-5 C₁₈, 50×4-mm I.D. column and the mobile phase consisted of acetonitrile–methanol–15 mM potassium dihydrogenphosphate buffer, pH 7.3 (30:5:65, v/v). Only 250 μl of plasma are used for sample preparation and no internal standard is necessary. The limit of quantitation is 80 ng/ml and the calibration curve is linear up to 10 000 ng/ml. More than 20 samples can be analysed within 1 h. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 8%. The assay was used for pharmacokinetic studies.     

Simultaneous determination of artemether and its major metabolite dihydroartemisinin in plasma by gas chromatography–mass spectrometry-selected ion monitoring

A sensitive, selective, and reproducible GC–MS–SIM method was developed for determination of artemether (ARM) and dihydroartemisinin (DHA) in plasma using artemisinin (ART) as internal standard. Solid phase extraction was performed using C₁₈ Bond Elut cartridges. The analysis was carried out using a HP-5MS 5% phenylmethylsiloxane capillary column. The recoveries of ARM, DHA and ART were 94.9±1.6%, 92.2±4.1% and 81.3±1.2%, respectively. The limit of quantification in plasma was 5 ng/ml (C.V.≤17.4% for ARM and 15.2% for DHA). Calibration curves were linear with R²≥0.988. Within day coefficients of variation were 3–10.4% for ARM and 7.7–14.5% for DHA. Between day coefficients of variations were 6.5–15.4% and 7.6–14.1% for ARM and DHA. The method is currently being used for pharmacokinetic studies. Preliminary data on pharmacokinetics showed C_max of 245.2 and 35.6 ng/ml reached at 2 and 3 h and AUC_0–8h of 2463.6 and 111.8 ngh/ml for ARM and DHA, respectively.     

Determination of gabapentin in plasma by high-performance liquid chromatography

A rapid and simple method for determination of the novel antiepileptic compound gabapentin [1-(aminomethyl)cyclohexaneacetic acid] in plasma is described. Blank human plasma was spiked with gabapentin (1.0–10.0 μg/ml) and internal standard [1-(aminomethyl)-cycloheptaneacetic acid; 5.0 μg/ml]. Individual samples were treated with 2 M perchloric acid, centrifuged and then derivatised with o-phthalaldehyde-3-mercaptopropionic acid. Separation was achieved on a Beckman Ultrasphere 5 μm reversed-phase column with mobile phase consisting of 0.33 M acetate buffer (pH 3.7; containing 100 mg/l EDTA)-methanol-acetonitrile (40:30:30, v/v). Eluents were monitored by fluorescence spectroscopy with excitation and emission wavelengths of 330 and 440 nm, respectively. The calibration curve for gabapentin in plasma was linear (r=0.9997) over the concentration range 1.0–10.0 μg/ml. Recovery was seen to be 90%. The inter- and intra-assay variations for three different gabapentin concentrations were 10% throughout. The lower limit of quantitation was found to be 0.5 μg/ml. Chromatography was unaffacted by a range of commonly employed antiepileptic drugs or selected amino acids.