Simultaneous determination of the camptothecin analogue CPT-11 and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in cancer patients.

CPT-11 (I; 7-ethyl-10-[4-(1-piperidino)-1- piperidino]carbonyloxycamptothecin) is a new anticancer agent currently under clinical development. A sensitive high-performance liquid chromatographic assay suitable for the simultaneous determination of I and its active metabolite SN-38 (II) in human plasma, and their preliminary clinical pharmacokinetics, are described. Plasma samples were processed using a solid-phase (C18) extraction step allowing mean recoveries of I, II and the internal standard camptothecin (III) of 84, 99 and 72%, respectively. The extracts were chromatographed on a C18 reversed-phase column with a mobile phase composed of acetonitrile, phosphate buffer and heptanesulphonic acid, with fluorescence detection. The calibration graphs were linear over a wide range of concentrations (1 ng/ml-10 micrograms/ml), and the lower limit of determination was 1 ng/ml for both I and II. The method showed good precision: the within-day relative standard deviation (R.S.D.) (5-1000 ng/ml) was 13.0% (range 4.9-19.4%) for I and 12.8% (6.7-19.1%) for II; the between-day R.S.D. (5-10,000 ng/ml was 7.9% (5.4-17.5%) for I and 9.7% (3.5-15.1%) for II. Using this assay, plasma pharmacokinetics of both I and II were simultaneously determined in three patients receiving 100 mg/m2 I as a 30-min intravenous infusion. The mean peak plasma concentration of I at the end of the intravenous infusion was 2400 +/- 285 ng/ml (mean +/- standard error of the mean). Plasma decay was triphasic with half-lives alpha, beta and gamma of 5.4 +/- 1.8 min, 2.5 +/- 0.5 h and 20.2 +/- 4.6 h, respectively. The volume of distribution at steady state was 105 +/- 15 l/m2, and the total body clearance was 12.5 +/- 1.9 l/h.m2. The maximum concentrations of the active metabolite II reached 36 +/- 11 ng/ml.     

High-performance liquid chromatographic analysis of 5-ethylpyrimidines and 5-methylpyrimidines in plasma

A high-performance liquid chromatographic (HPLC) method employing a C18 reversed-phase column, a mobile phase of sodium acetate and methanol, and an ultraviolet detector was developed for the analysis of 5-ethylpyrimidines and 5-methylpyrimidines in plasma. Samples were prepared for HPLC by sequential cation-exchange and anion-exchange column chromatography. Linear standard curves were obtained for samples containing 0.05-50 micrograms/ml 5-ethyl-2'-deoxyuridine and 5-ethyluracil, 0.05-10 micrograms ml 5-(1-hydroxyethyl)uracil, and 0.1-50 micrograms/ml thymidine, thymine and 5-hydroxymethyluracil. Applicability of the method to determination of the kinetics of 5-ethyl-2'-deoxyuridine elimination by the isolated perfused rat liver was demonstrated; clearance of the drug was 1.29 ml/min.     

Determination of psychotropic phenylalkylamine derivatives in biological matrices by high-performance liquid chromatography with photodiode-array detection.

Several procedures using high-performance liquid chromatography with photodiode-array detection have been developed to create phytochemical and toxicological profiles of phenylalkylamine derivatives in biological samples (e.g. plant materials and urine). Mescaline-containing cactus samples were extracted with basic methanol, using methoxamine as internal standard; the extraction and clean-up of urine samples were performed on cation-exchange solid-phase extraction columns. The extracts were separated on a 3-micron ODS column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was performed at 198 or 205 nm; peak identity and homogeneity were ascertained by on-line scanning of the UV spectra from 190 to 300 nm. The detection limit of phenylalkylamine derivatives in urine and cactus material was 0.026-0.056 micrograms/ml and 0.04 micrograms/mg, respectively. Following a single oral dose of 1.7 mg/kg methylenedioxymethylamphetamine (MDMA) the concentrations found in urine ranged from 1.48 to 5.05 micrograms/ml MDMA and 0.07-0.90 micrograms/ml methylenedioxyamphetamine (a metabolite of MDMA). The mescaline content of the cactus Trichocereus pachanoi varied between 1.09 and 23.75 micrograms/mg.     

Determination of the anticonvulsant felbamate in beagle dog plasma by high-performance liquid chromatography.

An automated internal standard method for the determination of felbamate in 0.1 ml of plasma from pediatric and adult beagle dogs was developed. Plasma proteins are precipitated with acetonitrile and after centrifugation the supernatant is directly injected on a Spherisorb ODS2, 3 microns, 150 mm x 4.6 mm I.D. column with 25% acetonitrile in aqueous phosphate buffer, pH 6.50, as mobile phase with ultraviolet detection at 210 nm. The run time is 10 min, the linear range is 0.150-150 micrograms/ml felbamate, and the lower limit of quantitation is 0.150 microgram/ml.     

Quantitation of N,N,N',N'-tetrakis (2-hydroxypropyl)-ethylenediamine in plasma by gas chromatography

A wide-bore capillary gas chromatographic method with nitrogen-selective thermionic detection is described for the quantitative analysis of N,N,N',N'-tetrakis (2-hydroxypropyl)ethylenediamine (Quadrol) in plasma. N,N,N',N'-tetrakis (2-hydroxybutyl)ethylenediamine is used as an internal standard. Rat or human plasma samples (0.5 ml) are mixed with internal standard, adjusted to alkaline pH and subjected to a single extraction with dichloromethane. Quadrol recovery from plasma typically exceeds 90%. The method is linear over the range 1.0-50 micrograms/ml. The working detection limit is 0.5 microgram/ml and the analysis time is under 7 min. The procedure has been used to obtain plasma concentration versus time data for the evaluation of Quadrol pharmacokinetics in rats.     

Determination of L-753,037 in human plasma by liquid chromatography/turbo ionspray tandem mass spectrometry.

A liquid chromatographic/turbo ionspray tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of L-753,037, a potent endothelin receptor antagonist currently under development for the treatment of cardiovascular diseases, in human plasma. L-753,037 is extracted from 0.5 ml of human plasma using liquid-liquid extraction and analyzed by LC/MS/MS with a turbo ionspray interface. Method validation results showed that this method is very sensitive, reliable, selective and reproducible. L-753,048, an ethoxy analogue of L-753,037, was used as the internal standard. The method has a lower limit of quantitation (LOQ) of 50 pg ml(-1) with a linear calibration range of 0.05-50 ng ml(-1). The intra-day precision and accuracy (n = 5) were measured to be below 10% relative standard deviation (RSD) and between 97.4 and 102.8% of the nominal values, respectively, for all calibration standard concentrations within the calibration curve range. The inter-day precision and accuracy (n = 3 days, 5 replicates per day) were measured to be below 6.5% RSD and between 99.3 and 102.0% of the nominal values, respectively, for all quality control concentrations. The extraction recovery was determined to be approximately 99% on average. The analyte was found to be stable in plasma through three freeze-thaw cycles, for at least 4 h at ambient temperature and for up to 40 days under -20 degrees C freezer storage conditions. The analyte was also shown to be stable for at least 24 h in the reconstitution solution at room temperature and for up to 3 days as a dried extract at 4 degrees C. Additional variations in plasma concentration of the analyte due to the use of different sources of plasma were also evaluated.     

Reversed-phase high-performance liquid chromatography and amperometric detection of 3-O-methyl isoprenaline sulphate: application to studies on the presystemic metabolism of d-isoprenaline in man

A selective method for the determination of 3-O-methyl isoprenaline sulphate in human urine and blood plasma has been developed using reversed-phase high-performance liquid chromatography with amperometric detection. The sulphoconjugate was subjected to acidic hydrolysis and the liberated 3-O-methyl isoprenaline was isolated by organic extraction and conventional cation exchange. An internal standard of 3-O-methyl isoetharine was synthesized from commercially available isoetharine and used to correct for recovery losses. The assay was shown to be linear over the range 5 ng/ml to 20 micrograms/ml with a limit of detection of 2 ng/ml. The reliability of the analytical method was examined together with its applicability to in-vivo studies in man.     

High-performance liquid chromatographic method for the determination of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL-39123) in human plasma and urine

A rapid, sensitive and reliable reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection has been developed for the assay of a novel anti-herpes agent, 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL-39123), in human plasma and urine. The drug and the internal standard, the structural analogue BRL-42377, were extracted from the biological matrix by adsorption on a cation-exchange column and were subsequently eluted under alkaline conditions prior to HPLC. The method is reproducible, with coefficients of variation of ca. 5%, and linear from 0.1 to at least 30 micrograms ml-1 in plasma and from 50 to at least 2000 micrograms ml-1 in urine. The method has been used extensively to measure BRL-39123 in plasma and urine samples generated during clinical studies and is adequate for defining pharmacokinetics at projected therapeutic doses.     

Determination of the anxiolytic agent 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide in whole blood, plasma or urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether-methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 +/- 5.4% (S.D.) and 90.3 +/- 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration-time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.     

Sensitive Determination of Piritramide in Human Plasma by High-Performance Liquid Chromatography

Abstract

A selective and sensitive method for the determination of piritramide in human plasma is described. After addition of 50 μl of 2 M ammonia and 20 μl of aqueous promethazine solution (100 ng/10 μ1) as an internal standard, 1 ml of plasma was extracted with 5 ml of toluene (extraction efficiency: 93.9 × 2.6%; mean × S. D.; n = 5). HPLC was performed with a phenyl hypersil NC-04 column, particle size 5 μm, 250 × 4 mm I. D.; mobile phase: 8 parts of acetonitrile and 2 parts of 10 mM potassium phosphate buffer (pH 3. 3). The flow rate was set to 2 ml/min and the column temperature was 22°C. The assay was linear in a concentration range of 3.75 ? 3000 ng/ml (r = 0.999), with a lower limit of detection of 3 ng/ml. The precision was determined using spiked plasma samples (15 ng/ml; 300 ng/ml), with coefficients of variation of 6.1 and 5.9% (intraday; n = 5) and 6.5 and 0.2% (interday; n = 3). In the range of 5.6 ? 1500 ng/ml, the accuracy of the assay was 2.82%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or postoperative analgesia.     

Pre-column derivatization of sisomicin with o-phthalaldehyde-beta-mercaptopropionic acid and its application to sensitive high-performance liquid chromatographic determination with fluorimetric detection

The stability of the o-phthalaldehyde (OPA) derivatives of sisomicin obtained using beta-mercaptopropionic acid was investigated by reversed-phase high-performance liquid chromatography. One of the fluorescent derivatives of sisomicin was stable at least for 6 h in 50% methanol under the optimal conditions used (OPA concentration, pH and temperature). When plasma samples spiked with sisomicin were analysed, the response was linear in the calibration range 136-900 pg of sisomicin per injected volume (40 microliters). As little as 0.06 micrograms of sisomicin per 1 ml of plasma could be detected with signal-to-noise ratio greater than or equal to 2. For plasma samples spiked with 0.2 micrograms/ml sisomicin, the recovery was 97.1 +/- 6.6% (mean +/- S.D., n = 5) with a within-run coefficient of variation of 6.8% and a day-to-day coefficient of variation of 7.2%. The method was also applied to plasma samples from rabbit after a subcutaneous injection of 1 mg/kg sisomicin.     

Determination of Arteether in Plasma Using a Simple and Rapid High-Performance Liquid Chromatographic Assay

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) assay for the determination of the antimalarial drug arteether in plasma was developed and validated in this report. Perchloric acid was used in this method as a plasma protein precipitant and to attain an acidic medium suitable for the decomposition of arteether to a derivative possessing UV absorption. This derivative and the internal standard (progesterone) were separated from the plasma on a 10 μm μ-Bondapack C₁₈ reversed-phase column at ambient temperature with a mobile phase composed of acetonitrile:water (60:40 v/v) and at a flow rate of 1.5 ml/min. The effluent was monitored at 254 nm with a UV detector. Linear relation between drug concentrations and peak height ratios of arteether derivative to the internal standard was achieved in the range of 0.25-10 μg/ml arteether with a detection limit of 50 ng/ml arteether in plasma. The within-day and between-days precisions were evaluated using 3 different concentrations of arteether. The values of the coefficients of variation were 1.35-1.68% and 1.65-2.82% for within-day and between-day, respectively. This method was applied to determine some pharmacokinetic parameters of arteether after intramuscular injection of 50 mg/kg arteether oily solution to rabbits.     

High-performance liquid chromatographic analysis of hippuric acid in human blood plasma

A method has been developed for the isocratic high-performance liquid chromatographic analysis of hippuric acid in human blood plasma. After the addition of an internal standard (3-methoxysalicylic acid), plasma samples (1 ml) were made alkaline and extracted stepwise with methylene chloride and ethyl acetate. The detection limit was 50 pmol of hippuric acid per ml of plasma. The concentrations of hippuric acid in plasma from house painters (n = 8), with long-term exposure to solvent vapours from alkyd paints, were in the range 1-21 nmol/mol (median 11 nmol/ml). These values were statistically significantly higher than those for controls (n = 9): 2-8 nmol/ml (median 3 nmol/ml).     

Determination of glycyrrhetinic acid in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method has been developed for measuring 18 beta-glycyrrhetinic acid (GRA) in human plasma in the range of 0.1-3 micrograms/ml. The acetate ester of GRA is added to the plasma as an internal standard, plasma proteins are denatured with urea to release GRA, and the GRA and the internal standard are extracted in an ion-pairing solid-phase extraction process. An isocratic, reversed-phase HPLC separation is used, followed by ultraviolet absorbance detection at 248 nm. The results from the analysis of five GRA-fortified plasma pools show a mean relative standard deviation of 7% and are accurate to within 10%. With evaporative concentration of the extract, the limit of detection for GRA in plasma is approximately 10 ng/ml.     

High-performance liquid chromatographic separation of noreximide and norendimide in bulk material, tablets and animal feed

Noreximide, a sedative, is generally contaminated to some extent with its endo-isomer, norendimide, which produces excitation. A high-performance liquid chromatographic assay was developed to separate and quantitate these compounds on a 5-microns Ultrasphere ODS column with methanol-water (20:30) as mobile phase and detection at 254 nm. Assay of mixtures of these compounds in bulk material and tablets utilized isoniazide as internal standard. Peak area ratios were linear (r = 0.9999) over 1.4-66.2 micrograms of injected noreximide and 0.2-8.4 micrograms of injected norendimide. Overall percent recovery from simulated tablets containing noreximide alone was 99.6 +/- 0.8% (S.D., n = 3). Overall percent recoveries (+/- S.D.) from tablets containing a mixture of these compounds were 98.9 +/- 0.5% and 102.3 +/- 1.1% for noreximide and norendimide, respectively (n = 3). Noreximide in animal feed for long-term pharmacological studies was isolated by ether extraction and after work up, subjected to the same procedure, except that theophylline was the internal standard. Peak area ratios were linear over 0.2-19.3 micrograms of injected noreximide (r = 0.9999). Overall percent recoveries (+/- S.D., n = 3) of noreximide from spiked animal feed were 97.4 +/- 1.4% and 99.0 +/- 0.5% at the 500- and 5000-ppm levels, respectively. Limits of detection at the 95% confidence level (0.01 a.u.f.s., 20-microliters sample volume injected) were 1.67 microgram/ml and 2.56 micrograms/ml of noreximide and norendimide, respectively, in the final test solution.     

High-performance liquid chromatographic determination of 1,2-diethyl-3-hydroxypyridin-4-one and its 2-(1-hydroxyethyl) metabolite in rat blood.

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the analysis of 1,2-diethyl-3-hydroxypyridin-4-one (CP94, I) and its 2-(1-hydroxyethyl) metabolite (II) in rat blood is described. I, II and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95, III) were extracted into dichloromethane (3 x 5 ml, with the addition of 1 g of sodium chloride) from blood (0.25 ml plus 0.75 ml of pH 7.0 morpholinopropanesulphonic acid buffer). Extractability approached 100% for I and III, and approximately 65% for II under these conditions. Chromatographic analysis was carried out using a Hypercarb porous graphitised carbon HPLC column (10 cm x 0.46 cm). The mobile phase was 14:86 (v/v) acetonitrile-NaH2PO4 buffer (10 mM, containing 2 mM EDTA, pH adjusted to 3 with phosphoric acid) and detection was by ultraviolet at 280 nm. Calibration curves were linear (correlation coefficient greater than 0.99) and reproducible over the concentration range 0-80 micrograms/ml and the coefficient of variation was less than 16% even at low (1 microgram/ml) concentrations. The minimum quantifiable level was 0.5 microgram/ml for both I and II.     

Determination of the monocyclic beta-lactam antibiotic carumonam in plasma and urine by ion-pair and ion-suppression reversed-phase high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatography method has been developed for the determination of the new monocyclic beta-lactam antibiotic carumonam in plasma and urine. The method for plasma involves protein precipitation with acetonitrile and removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantification are achieved using a mobile phase based on either ion-suppression or ion-pair chromatography on a reversed-phase column with UV detection. The limit of determination is 0.5 micrograms/ml plasma, using a 0.5-ml specimen, and 25 micrograms/ml urine, using a 50-microliter specimen. The inter-assay reproducibility is generally better than 4% when an internal standard is used. Since beta-lactam antibiotics may degrade on storage, close attention must be paid to the stability of these drugs in biological fluids; novel measures to prevent degradation on storage are described. The assay has been successfully applied to the analysis of several thousand samples from pharmacokinetic studies, including a study involving patients with impaired renal function.     

Sensitive determination of piritramide in human plasma by gas chromatography.

A selective and sensitive method for the determination of piritramide in human plasma is described. A 1-ml aliquot of plasma was extracted with 10 ml of hexane-isoamyl alcohol (99.5:0.5, v/v) (extraction efficiency 86%) after addition of 50 microliters of 2 M ammonia and 20 microliters of aqueous strychnine solution (100 ng per 10 microliters) as internal standard. Gas chromatography was performed with J&W DB-1, 30 m x 0.53 mm I.D. separation column, film thickness 1.5 microns, using an nitrogen-phosphorus-sensitive detector. The assay was linear in the concentration range 3.75-2250 ng/ml (r = 0.999), with a lower limit of detection of 1-2 ng/ml. The precision was determined using spiked plasma samples (10 and 50 ng/ml), with coefficients of variation of 3.5 and 3.1% (intra-day; n = 5) and 4.6 and 4.1% (inter-day; n = 4). In the range 3.75-150 ng/ml, the accuracy of the assay was 3.36%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or post-operative analgesia.     

Determination of aniracetam and its main metabolite, N-anisoyl-GABA, in human plasma by high-performance liquid chromatography

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.