Simultaneous measurement of allantoin, uric acid, xanthine and hypoxanthine in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for determining catabolism products of nucleic acids and purines, such as oxypurines (i.e. uric acid, xanthine and hypoxanthine) and allantoin in the blood plasma of ruminants was developed. The plasma was deproteinized with 10% trichloroacetic acid. The method enabled determination of oxypurines without derivatization. Allantoin was determined after conversion with 2,4-dinitrophenylhydrazine to a hydrazone (GLX-DNPH). Separation of converted allantoin, uric acid, xanthine and hypoxanthine derivatives was carried out using two reversed-phase C₁₈ columns. The combination of pre-column derivatization and gradient elution with monitoring of the effluent at 205, 254 and 360 nm provides a simple and selective analytical tool for studying oxypurines and allantoin in plasma. The total run time of the HPLC analysis was 60 min. The recovery of the purine derivatives (i.e. oxypurines and allantoin) added to the plasma was between 95 and 106%. Purine derivatives were stable when the processed samples were stored for 7 days at −10°C. The low values of the intra-assay coefficient of variations (2.5–4.6%) and the low values of the detection limits (0.187–0.004 nmol) point to the satisfactory precision and sensitivity of the method.     

Rapid and sensitive gas-chromatographic determination of caffeine in blood plasma,saliva, and xanthine beverages

A gas chromatographic procedure is reported for the determination of caffeine in plasma, saliva, and xanthine beverages. Using a 75 cm column packed with OV-17, nitrogen-sensitive detection, and 1 ml samples, a suitable limit of analysis (coefficient of variation (CV)=10.2%) of 50 ng/ml was obtained in plasma. Within-day CVs at caffeine concentrations of 0.1–0.5–2.0–7.5–15.0 g/ml in plasma were 7.7–5.6–4.8–3.8–3.4%, respectively. The limit of detection, defined as the injected quantity of caffeine giving rise to a signal to noise ratio of 2, is 40 pg, corresponding to a plasma concentration of 1 ng/ml.The procedure involves addition of the internal standard 7-pentyl theophylline and alkaline extraction of the sample with dichloromethane. The method described rivals any gaschromatographic assay published so far in rapidness and accuracy.Plasma and saliva caffeine concentrations were determined in a healthy male volunteer after swallowing 400 ml of coffee. The calculated pharmacokinetic parameters, assuming complete absorption of caffeine from the G.I. tract, agree well with previously published values.     

High-performance liquid chromatographic determination of selenocysteine with the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid

The method described is based on derivatization of selenocysteine with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid and responds linearly to selenocysteine spiked into plasma. Recovery is insensitive to inter-individual variation or use of serum versus plasma, but is decreased by hemolysis. The derivative is stable for at least three days. The total imprecision of determinations in plasma was 0.8–2.1% (coefficient of variation) over the range of 6–30 μM selenocysteine, with a detection limit of 0.4 μM (3 × S.D.). There was no significant interference from plasma thiols. This appears to be the first report of the selective reaction of free selenocysteine with a fluorescent reagent. This simple method works well in plasma and serum and may be adaptable to other types of samples.     

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.     

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma and whole blood by high-performance liquid chromatography with ultraviolet and fluorescence detection

A method for the simultaneous determination of the three selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, paroxetine and their metabolites in whole blood and plasma was developed. Sample clean-up and separation were achieved using a solid-phase extraction method with C₈ non-endcapped columns followed by reversed-phase high-performance liquid chromatography with fluorescence and ultraviolet detection. The robustness of the solid-phase extraction method was tested for citalopram, fluoxetine, paroxetine, Cl-citalopram and the internal standard, protriptyline, using a fractional factorial design with nine factors at two levels. The fractional factorial design showed two significant effects for paroxetine in whole blood. The robustness testing for citalopram, fluoxetine, Cl-citalopram and the internal standard revealed no significant main effects in whole blood and plasma. The optimization and the robustness of the high-performance liquid chromatographic separation were investigated with regard to pH and relative amount of acetonitrile in the mobile phase by a central composite design circumscribed. No alteration in the elution order and no significant change in resolution for a deviation of ±1% acetonitrile and ±0.3 pH units from the specified conditions were observed. The method was validated for the concentration range 0.050–5.0 μmol/l with fluorescence detection and 0.12–5.0 μmol/l with ultraviolet detection. The limits of quantitation were 0.025 μmol/l for citalopram and paroxetine, 0.050 μmol/l for desmethyl citalopram, di-desmethyl citalopram and citalopram-N-oxide, 0.12 μmol/l for the paroxetine metabolites by fluorescence detection, and 0.10 μmol/l for fluoxetine and norfluoxetine by ultraviolet detection. Relative standard deviations for the within-day and between-day precision were in the ranges 1.4–10.6% and 3.1–20.3%, respectively. Recoveries were in the 63–114% range for citalopram, fluoxetine and paroxetine, and in the 38–95% range for the metabolites. The method has been used for the analysis of whole blood and plasma samples from SSRI-exposed patients and forensic cases.     

Bioanalysis of aplidine, a new marine antitumoral depsipeptide, in plasma by high-performance liquid chromatography after derivatization with trans-4′-hydrazino-2-stilbazole

A sensitive bio-analytical assay in plasma of the depsipeptide aplidine is reported, based on reversed-phase liquid chromatography and fluorescence detection of the trans-4′-hydrazino-2-stilbazole (4′H2S) derivative of the analyte. At ambient temperature, two conformations of the depsipeptide are observed in solution due to cis–trans isomerism at the proline–pyruvoyl peptide bond. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, a derivatization with 4′H2S is performed in a water–acetonitrile mixture at pH 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2–100 ng/ml-range, 2 ng/ml being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The identity of the 4′H2S reaction products of aplidine have been confirmed by mass spectrometric analysis. Finally, the method has been employed for a pilot pharmacokinetic study of aplidine in mice which demonstrated its usefulness for pharmacological research.     

Validation of an analytical method for a potent antitumor agent, TZT-1027, in plasma using liquid chromatography–mass spectrometry

A sensitive and specific analytical method for a potent antitumor agent, TZT-1027, in plasma has been developed using liquid chromatography–mass spectrometry (LC–MS) with [²H₄]TZT-1027 as an internal standard (I.S.). A plasma sample was purified by solid-phase extraction on a C₁₈ cartridge, followed by solvent extraction with diethyl ether. The extract was then injected into the LC–MS system. Chromatography was carried out on a C₁₈ reversed-phase column using acetonitrile–0.05% trifluoroacetic acid (TFA) (55:45) as a mobile phase. Mass spectrometric analysis was performed in atmospheric pressure chemical ionization (APCI) mode with positive ion detection, and the protonated molecular ions ([M+H]⁺) of TZT-1027 and I.S. were monitored to allow quantitation. The method was applied to the determination of TZT-1027 in human, monkey, dog, rat and mouse plasma. As far as the sample preparation was concerned, good recoveries (73.5–99.1%) were obtained. The calibration curves were linear over the range of 0.25–100 ng per 1 ml of human, dog and rat plasma, per 0.5 ml of monkey plasma, and per 0.1 ml of mouse plasma. From the intra- and inter-day accuracy and precision, the present method satisfies the accepted criteria for bioanalytical method validation. TZT-1027 was stable when stored below −15°C for 6 months in human plasma and for 3 weeks in plasma from other species. TZT-1027 was also stable in plasma through at least three freeze–thaw cycles.     

Improved high-performance liquid chromatographic analysis of terazosin in human plasma

A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method was developed for the determination of terazosin in human plasma. The method involves a one-step single solvent extraction procedure using dichloromethane with a 0.25 ml plasma sample. Recovery values were all greater than 90% over the concentration range 0.25–100 ng/ml. Terazosin was found to adsorb to glass or plastic tubes, but this could be circumvented by using disposable plastic tubes. Also, rinsing the injector port with methanol after each injection helped to prevent any carry-over effect. The internal standard, prazosin, did not exhibit this problem. The method has a quantification limit of 0.25 ng/ml. The within- and between-day coefficient of variation and accuracy values were all less than 7% over the concentration range 0.25–100 ng/ml and hence the method is suitable for use in pharmacokinetic studies of terazosin.     

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane–isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-μm particle size, C₁₈-bonded silica column and water–sodium acetate–heptanesulfonate–acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4±3.1% for TMB and 89.4±4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10–5000 ng/ml for TMB and 25–25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.     

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.     

Column-switching high-performance liquid chromatographic analysis of BO-2727, a new carbapenem antibiotic, in human plasma and urine by direct injection

A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05–50 μg/ml in plasma and 0.5–100 μg/ml in urine. The limits of quantification for plasma and urine were found to be 0.05 μg/ml and 0.5 μg/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.     

Determination of phytic acid by gas chromatography–mass spectroscopy: application to biological samples

A GC–MS method is reported for the determination of phytic acid based on purification by anion-exchange chromatography, enzymatic hydrolysis of phytic acid to myo-inositol and derivation to trimethylsilyl derivative, with scyllo-inositol as an internal standard. Analytical features of the method are: limit of detection 9 μg l⁻¹ phytic acid, linear working range 18–500 μg l⁻¹ phytic acid, and coefficient of variation 1.9%. The method has been successfully applied to a variety of biological samples: various rat organs (kidney, liver, brain and bone), human plasma and urine and kidney stones. A comparative study of sample treatments, including deproteization, lipid extraction and the presence of a chelator, is also reported. Phytic acid amounts found in rat organs ranged from 1.07 g kg⁻¹ for bone to 32.0 g kg⁻¹ for brain. Phytic acid in human plasma was of the order of 0.14 mg l⁻¹. In kidney stones, phytic acid was found in calcium containing stones.     

Paired-ion high-performance liquid chromatographic assay for plasma choline

Choline was isolated from deproteinized plasma by cation-exchange chromatography. Isolated choline was directly converted to the 3,5-dinitrobenzoate derivative and was analyzed by paired-ion high-performance liquid chromatography with UV detection at 254 nm. An internal standard, 3-hydroxy-N,N,N-trimethylpropanaminium iodide was used for quantitation of plasma choline.Linearity was achieved from 1–500 nmole/ml with a reproducibility of ± 6%. Plasma choline concentrations below 1 nmole/ml could not be accurately measured while plasma choline concentrations in the μmole/ml range deviated from linearity.     

High-performance liquid chromatographic–tandem mass spectrometric method for the determination of ethionamide in human plasma, bronchoalveolar lavage fluid and alveolar cells

We have developed and validated an accurate, sensitive, and rapid high-performance liquid chromatographic–tandem mass spectrometric method (HPLC–MS–MS) for the determination of ethionamide in plasma, bronchoalveolar fluid (BAL) and alveolar cells (AC). The retention times for ethionamide, clemastine fumarate (internal standard for plasma), promethazine (internal standard for plasma) and propranolol (internal standard for BAL and AC) were approximately 2.62, 1.21, 2.14, and 2.22 min, respectively, with a total run time of 3.2 min. Ethionamide detection for plasma was carried out on a PE Sciex API III (Perkin-Elmer, Foster City, CA, USA). BAL and cell pellets and some plasma specimens were analyzed on a Micromass Quattro LC (Micromass Co., Manchester, UK). The detection limits for ethionamide were 0.05 μg/ml for plasma, and 0.005 μg/ml for BAL supernatants and alveolar cell suspensions.     

Study on measurement of δ-aminolevulinic acid in plasma by high-performance liquid chromatography

A method for the determination of δ-aminolevulinic acid in plasma of lead-exposed workers by high-performance liquid chromatography with fluorescence detection of a fluorescent δ-aminolevulinic acid derivative (2-methylidineamino-3,5-diacetyl-4,6-dimethylpropionic acid) was established. The detection limit of δ-aminolevulinic acid in plasma was 0.01 μg/ml at a signal-to-noise ratio of 5:1. A linear correlation was obtained between the amounts of δ-aminolevulinic acid injected from 0.01 to 0.5 μg/ml (r = 0.999). The recovery of 0.05 and 0.1 μg/ml of δ-aminolevulinic acid added to plasma with various concentrations of δ-aminolevulinic acid in plasma ranged from 80.0 to 100.8%. This method, combined with the use of an automatic sampler, should facilitate the routine measurement of δ-aminolevulinic acid in plasma.     

High-performance liquid chromatographic assay of a new HIV-1 protease inhibitor, LB71350, in the plasma of dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of LB71350 in the plasma of dogs. The analyte was deproteinized with 1.5 volumes of methanol and 0.5 volumes of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a stepwise gradient mixture of acetonitrile and 0.2% triethylamine–HCl with a flow-rate of 1 ml/min and detection at UV 245 nm. The proportion of acetonitrile was kept at 52% for the first 6 min, increased to 100% for the next 0.5 min, kept at 100% for the next 2 min, decreased to 52% for the next 0.5 min, and finally kept at 52% for the next 7 min. The retention time of LB71350 was 6.9 min. The calibration was linear over the concentration range of 0.1–100 mg/l for dog plasma (r>0.997) and the limit of quantitation was 0.1 mg/l using 0.1 ml plasma. The quality control samples were reproducible with acceptable accuracy and precision at 0.1, 1, 10 and 100 mg/l concentrations. The within-day recovery (n=5) was 90.2–93.9%, the between-day recovery (n=5) was 89.5–93.5%, and the absolute between-day recovery (n=5) was 77–81%. The within-day precision (n=5) and between-day precision (n=5) were 2.59–5.82% and 3.17–4.55%, respectively. No interferences from endogenous substances were observed. Taken together, the above HPLC assay method by deproteinization and UV detection was suitable for the determination of LB71350 in the preclinical pharmacokinetics.     

Determination of HP 749, a potential therapeutic agent for Alzheimer's disease, in plasma by high-performance liquid chromatography

N-(n-Propyl)-N-(4-pyridinyl)-1H-indol-1-amine hydrochloride (HP 749, I), a non-receptor-dependent cholinomimetic agent with noradrenergic activity, is a potential agent for the treatment of Alzheimer's disease. Pharmacokinetic studies in animals and humans showed that I was well absorbed and metabolized primarily to the N-despropyl metabolite (P7480, II) after oral administration. To facilitate the kinetic studies, a sensitive and selective high-performance chromatographic assay was developed. I and II are extracted from plasma by a mixture of cyclohexane—ethyl acetate and chromatographed on an isocratic reversed-phase high-performance liquid chromatographic system employing an analytical phenyl column with acetonitrile—ammonium formate as mobile phase. The concentrations of these two compounds, quantitated by internal standardization, are monitored by ultraviolet detection. The method is linear in the plasma assay over a concentration range of 0.5–500 ng/ml for both compounds with a quantitation limit of 0.5 ng/ml. The precision and accuracy of the calibration curves and/or method are less than 10%. The recovery of I and II from plasma is 63–74 and 63–68%, respectively, over a concentration range of 0.5–500 ng/ml.     

Determination of the GABAB receptor agonist CGP 44532 (3-amino-2-hydroxypropylmethylphosphinic acid) in rat plasma after pre-column derivatization by micro-high-performance liquid chromatography combined with negative electrospray tandem mass spectrometry

An assay, based on pre-column derivatization and micro-high-performance liquid chromatography–tandem mass spectrometry, was developed for the determination of the GABA_B agonist CGP 44532 in rat plasma. CGP 44532, a highly polar 3-amino-2(S)-hydroxypropylmethylphosphinic acid, presented difficulties in developing a chromatographic method for the analysis of the compound in rat plasma. Instead of analyzing the target compound directly, it was derivatized prior to separation to a 4-nitrobenzylcarbamate isopropyliden derivative. In order to reach the required quantitation limit, on-line solid-phase extraction was utilized for sample clean-up and reversed-phase micro-column high-performance liquid chromatography, for separation of the plasma samples. The separated compounds were detected by negative electrospray tandem mass spectrometry in selected reaction monitoring mode. The derivatives show good chromatographic and mass spectrometric properties and both the target compound and the internal standard, could be eluted as symmetrical peaks with good signal/noise ratio. The MS–MS detection was selective and sensitive due to the straight fragmentation pattern. After injection of 200-μl sample aliquots, the limit of quantification was 10 ng ml⁻¹. The analytical assay is useable in the range of 10–500 ng ml⁻¹.     

Determination of erythromycin, clarithromycin, roxithromycin, and azithromycin in plasma by high-performance liquid chromatography with amperometric detection

In this study, a high-performance liquid chromatographic method was developed for the quantitative determination of erythromycin (EM), roxithromycin (RXM), and azithromycin (AZM) in rat plasma with amperometric detection under a standardized common condition using clarithromycin (CAM) as an internal standard. This method was also proved to be applicable for the determination of CAM by employing RXM as an internal standard. Each drug was extracted from 150 μl of plasma sample spiked with internal standard under an alkaline condition with tert.-butyl methyl ether. The detector cell potential for the oxidation of the drugs was set at +950 mV. The linearity of the calibration curves were preserved over the concentration ranges of 0.1–10 μg/ml for EM and RXM, and 0.03–3.0 μg/ml for CAM and AZM. Coefficients of variation and relative error were less than 9% and ±7%, respectively. The analytical method presented here was proved to be useful for the investigation of the pharmacokinetic characteristics of EM, CAM, RXM, and AZM in rats.