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 μg/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 α, β and γ 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.     

Simultaneous determination of malachite green, gentian violet and their leuco metabolites in catfish or trout tissue by high-performance liquid chromatography with visible detection

A sensitive analytical procedure for the determination of residues of leucomalachite green (LMG)-malachite green (MG) and leucogentian violet (LGV)-gentian violet (GV) in catfish or trout tissue is presented. Frozen (−20°C) fish fillets were cut into small pieces and blended in a Waring blender. A 20-g amount of homogenized fish tissue was extracted with acetonitrile-buffer, partitioned against methylene chloride, and cleaned up on tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 μl (0.8 g equiv.) were chromatographed isocratically in 10 min using an acetonitrile-buffer mobile phase on a short-chain deactivated (SCD) reversed-phase column (250×4.6 mm I.D.) in-line with a post-column PbO₂ oxidation reactor. The PbO₂ post-column reactor efficiently oxidized LMG to the chromatic MG, and LGV to the chromatic GV permitting visible detection at 588 nm for all four compounds. Linearity was demonstrated with standards over the range of 0.5–50 ng per injection. Recoveries of LMG, MG, LGV and GV from catfish tissues fortified at 10 ng/g were 75.4±3.0, 61.3±4.1, 72.6±3.7 and 87.9±2.5, respectively, while trout tissues fortified at 10 ng/g yielded recoveries of 82.6±2.3, 48.6±1.8, 72.1±2.1 and 83.8±4.6 (mean±S.D., N=4), respectively.     

Determination of leukotriene E4 in human urine using liquid chromatography–tandem mass spectrometry

A liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method was developed for the quantitation of urinary leukotriene E₄ (LTE₄). LTE₄ and its internal standard were extracted by solid-phase extraction and analysed using LC–MS–MS in the selected reaction monitoring (SRM) mode. A good linear response over the range of 10 pg to 10 ng was demonstrated. The accuracy of added LTE₄ ranged from 97.0% to 108.0% with a mean and SD of 100.6±2.4%. We detected LTE₄ (63.1±18.7 pg/mg creatinine, n=10) in healthy human urine. This method can be used to determine LTE₄ in biological samples.     

Development of a routine analysis method for liposome encapsulated recombinant interleukin-2

This paper describes the development of an isocratic reversed-phase high-performance liquid chromatographic method for the routine analysis of recombinant interleukin-2 (rIL-2) in liposome samples. The chromatographic system employed a C₄ column maintained at 30°C eluted with 52.5% (w/w) acetonitrile in water, containing 100 mM NaClO₄ and 10 mM HClO₄. To remove phospholipid interference the chromatographic method was combined with a lipid-extraction procedure. No significant loss of rIL-2 was noted upon inclusion of this extraction step. The protein eluted from the column with a capacity factor (k′) of 5.8. The method was validated for robustness, linearity, precision and reproducibility. It was shown that the method was linear over a sample concentration range of 1–100 μg/ml. Upon assessment of the intra-day and inter-day precision, the relative standard deviations (RSD) were within the range of the methodical error (approximately 5%), except at the lower concentration of 10 μg/ml, where the intra-day RSD was relatively high (17.8%). The recovery of rIL-2 upon liposome preparation and subsequent analysis of the samples was in the range 94±9%. The results indicate that the method is suitable for routine quantitation of rIL-2 in liposomal samples.     

Determination of a new antifilarial drug, UMF-058, and mebendazole in whole blood by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for simultaneous quantitative determination of a new antifilarial drug (UMF-058, I) and mebendazole (MBZ) is described. After a simple extraction from whole blood, both compounds were analysed using a C₁₈ Nova Pak reversed-phase column and a mobile phase of methanol—0.05 M ammonium dihydrogenphosphate (50:50, v/v) adjusted to pH 4.0, with ultraviolet detection at 291 nm. The average recoveries of I and MBZ over a concentration range of 25–250 ng/ml were 92.0 ± 7.7 and 84.4 ± 4.4%, respectively. The minimum detectable concentrations in whole blood for I and MBZ were 7 and 6 ng/ml, respectively. This method was found to be suitable for pharmacokinetic studies.     

Determination of anticancer drug vitamin K3 in plasma by high-performance liquid chromatography

Synthetic vitamin K₃ (VK₃, 2-methyl-1,4-naphthoquinone, or menadione) has been found to exhibit antitumor activity against various human cancer cells at relative high dose. Parallel to our study on the mechanism of VK₃ action and for future clinical trials in Taiwan, we developed a simple, sensitive and accurate high-performance liquid chromatographic method for the determination of VK₃ in biological fluids. VK₃ was extracted from the plasma samples with n-hexane. The chromatographic separation employed an ODS analytical column (5 μm, 250 × 4.6 mm I.D.) with a mobile phase of methanol-water (70:30 v/v) and UV detection at 265 nm. On completely drying of the extraction solution, n-hexane, by a stream of nitrogen, menadione was lost to a great extent. Methanol (70%, 200 μl) was added to the extraction solvent after extraction and centrifugation to prevent the loss of menadione. The absolute recovery was 82.4±7.69% (n = 7). The within-day and between-day calibration curves of VK₃ in plasma in the ranges of interest (0.01–10.00 μg/ml; 0.01–5.00 μg/ml) showed good linearity (r>0.999) and acceptable precision. The limit of quantitation of VK₃ was 10 ng/ml) showed good method has been succesfully applied to a pilot pharmacokinetic study of VK₃ in rabbits receiving an intravenous high-dose bolus injection of 75 mg menadiol sodium diphosphate (Synkayvite). The pharmacokinetic properties of menadione could be described adequately by an open two-compartment model. The mean half-life of menadiol (transformation to menadione) was 2.60±0.12 min. The elimination half-life, volume of distribution and plasma clearance of menadione were 26.3±2.97 min, 25.7±0.78 1, and 0.68±0.10 1/min, respectively.     

A structural transition in egg lecithin-cholesterol bilayers at 12 °C

The thermal coefficient of expansion of egg lecithin bilayer thickness, αd₁, was measured as a function of its cholesterol content up to mole ratio lecithin/cholesterol of 1:1, and over the temperature range 0–40 °C. At all cholesterol contents αd₁ changes abruptly at approximately 12 °C indicating a structural transition at this temperature. Above 12 °C, αd₁ decreases monotonically from −2·10⁻³ for pure egg lecithin to −1·10^–3 at mole ratio 1:1. Below 12 °C αd₁ is walways higher than above 12 °C and shows a sharp, anomalously high value of −6·10⁻³ at the mole ratio 2:1. The results have been interpreted as the movement of cholesterol into the bilayer or the formation of lecithin-cholesterol “complexes” at temperatures below 12 °C. Similar studies with phosphatidylinositol containing cholesterol showed no structural transition and lysolecithin containing cholesterol behaved differently giving two lamellar phases in equilibrium.     

Determination of a new antibronchospastic agent, MX2/120, in guinea pig plasma by high-performance liquid chromatography in a pharmacokinetic study

7-[(2,2-Dimethyl)propyl)]-1-methylxanthine (I, Lab code MX2/120) is a new potent antibronchospastic agent. A rapid and simple HPLC assay for I in guinea pig plasma has been developed. Compound I was extracted from plasma with dichloromethane by a solid-phase extraction procedure, after adding 1,3-dimethyl-7-pentylxanthine at a concentration of 5 μg/ml as the internal standard (I.S.). The extraction residue was redissolved in water—acetonitrile and chromatographed on a RP-18 reversed-phase column. The eluate was monitored by spectrophotometric detection at 280 nm. The method showed good linearity over the range 0.1–20 μg/ml (r = 0.9998) and is precise (C.V. × Student's T-TEST = 1.84%) and accurate (mean recovery ± limit of CONFIDENCE = 100.25 ± 0.34). The HPLC assay was successfully applied to the determination of the pharmacokinetic profile of I after intravenous and oral administration in guinea pigs. The main pharmacokinetic parameters are presented.     

Use of a carrier for quantitation of a new dihydropyridine calcium antagonist (OPC-13340) in human plasma by highly sensitive gas chromatography with negative-ion chemical ionization mass spectrometry

A sensitive gas chromatographic—mass spectrometric method for the quantitation of (±)-methyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (OPC-13340, I), a new dihydropyridine calcium antagonist with a potent and long-acting antihypertensive and antianginal effect, was developed in order to elucidate its pharmacokinetics. Dihydropyridine calcium antagonists have been usually quantified by this technique in the negative-ion chemical ionization mode. However, direct application of this method to quantify trace amounts of I in biological fluids completely failed, owing to its adsorption on the column and oxidation of its dihydropyridine ring. Human plasma containing I and (±)-[²H₅]methyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (II), the internal standard, was extracted with n-hexane—diethyl ether under weakly basic conditions (pH 8). In order to prevent adsorption of the compounds on the column, (±)-[²H₃]ethyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (III), an analogue of I, was added to the extracts as a carrier. In addition, this carrier was also effective in preventing the oxidation of I. The quantitation limit of I in human plasma by this method was found to be less than 30 pg/ml. Thus, the method is sufficiently sensitive to study the pharmacokinetics of I in humans.     

Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction

As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at −20°C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 μl) on a 100-mg C₁₈-endcapped cartridge. Matrix components were eliminated with 750 μl of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 μl of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 μl of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4–20 μg/ml and 2–150 μg/ml for plasma and urine, respectively. THe extraction recovery of temozolomide was 86–90% from plasma and 103–105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37°C. An acidic pH (<5–6) shoul be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.     

Determination of glyceryl trinitrate in human plasma by gas chromatography—negative ion chemical ionization—selected ion monitoring

A specific, sensitive and accurate quantitation method for glyceryl trinitrate was developed using gas chromatography—negative ion chemical ionization—selected ion monitoring with dichloromethane as a reagent gas. [¹⁵N₃] and [²H₅, ¹⁵N₃] variants were synthesized from non-labelled or [²H₈]glycerol and [¹⁵N]nitric acid. The former variant was used for preventing adsorption of glyceryl trinitrate onto active sites on column materials and the latter was used as an internal standard for quantitation of glyceryl trinitrate in biological fluids by selected ion monitoring. The quantitation limit of this method is 0.1 ng/ml of human plasma. When glyceryl trinitrate was administered intravenously in the dose of 4 μg/kg to patients receiving hypotensive anesthesia for surgical operation, the plasma levels exhibited a biexponential decay. The mean and standard deviation of half-lives of the α and β phases were found to be about 0.41 ± 0.13 and 5.34 ± 1.60 min, respectively.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

Pulmonary formation of prostacyclin in man

The pulmonary formation of prostacyclin (PGI₂), as reflected by the difference in concentration of pulmonary and systematic arterial radioimmunoassayed 6-keto-PGF_1α, was determined in six healthy waking subjects. The systematic arterial 6-keto-PGF_1α levels were low (50 pg/ml), and no evidence of pulmonary formation and release of the compound was noted. In other experiments systemic arterial 6-keto-PGF_1α levels were determined in patients prior to and during artificial ventilation, as well as during and after occlusion of the pulmonary circulation (extra-corporeal circulation, ECC). The arterial 6-keto-PGF_1α concentration prior to artificial ventillation was 17±4 pg/ml, i.e. within the range observed in the healthy subjects. During artificial ventilation the arterial levels of 6-keto-PGF_1α increased to 191±21 pg/ml, suggesting that pulmonary formation of PGI₂ was stimulated. In the patients subjected to ECC with occluded pulmonary circulation the arterial content of 6-keto-PGF_1α was stabilised at an elevated level (120−170 pg/ml). Following re-establishment of the pulmonary circulation the arterial concentrations of 6-keto-PGF_1α increased markedly, to 284±50 pg/ml. It is suggested that the basal pulmonary formation of PGI₂ in man is low or non-existent, and that enhanced formation of the compound in the lungs is a consequence of intervention with normal pulmonary ventilation or perfusion.     

Prostaglandins E and F, and 19-hydroxylated E and F (series I and II) in semen of fertile men : Gas and liquid chromatographic separation with selected ion detection

Low concentrations of prostaglandins (PG) could be related to male clinical infertility although relevant experimental data are scarce. The aim of this work is to establish reliable seminal PG levels in fertile men by rigorous sample control, to prevent degradation, and by rapid and simple extraction and assay procedures.Single semen samples from healthy fertile men were immediately centrifuged (within 30 min of ejaculation) adding PGF_2α D₄ to the seminal plasma as internal standard. The samples were next ultrafiltered and the PGs in the ultrafiltrate were derivatized with a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide and piperidine (1:1) at 60° for 30 min. Optimum gas—liquid chromatographic separation of all of the peaks of interest was achieved on 4 m × 1/4 in. I.D. Dexsil 300 packed columns at 280°. The detection and quantitation of all the peaks of interest depends on the selected ion monitoring of specific masses. The values obtained (in μg/ml, range in parentheses) were: PGEs, 63.5 ± 49.3 (9–164); PGFs, 2.6 ± 1.92 (0.95–6.63); 19-OH PGEs, 592.6 ± 312.5 (142.1–1047); and 19-OH PGFs, 12.66 ± 5.21 (4–19). Individual values for members of both series I and II are also presented.The sample collection and extraction procedures were further checked by high-performance liquid chromatography on a μPorasil column, with individual isolation and collection of all of the PGs, including the 19-OH PGs not previously separated by liquid chromatography.     

Determination of ciprofloxacin levels in chinchilla middle ear effusion and plasma by high-performance liquid chromatography with fluorescence detection

An isocratic high-performance liquid chromatographic method has been developed to determine ciprofloxacin levels in chinchilla plasma and middle ear fluid. Ciprofloxacin and the internal standard, difloxacin, were separated on a Keystone ODS column (100 × 2.1 mm I.D., 5 μm Hypersil) using a mobile phase of 30 mM phosphate buffer (pH 3), 20 mM triethylamine, 20 mM sodium dodecyl sulphate—acetonitrile (60:40, v/v). The retention times were 3.0 min for ciprofloxacin and 5.2 min for difloxacin. This fast, efficient protein precipitation procedure together with fluorescence detection allows a quantification limit of 25 ng/ml with a 50 μl sample size. The detection limit is 5 ng/ml with a signal-to-noise ratio of 5:1. Recoveries (mean ± S.D., n = 5) at 100 ng/ml in plasma and middle ear fluid were 89.4 ± 1.2% and 91.4 ± 1.6%, respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering ciprofloxacin.     

Excretion of primary prostanoids and their metabolites during acute volume expansion

Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF_1α, PGE-M, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE₂ rose (p < 0.05) from 14.2 ± 4.0 to 86.2 ± 20.7, PGF_2α from 60.0 ± 4.9 to 119.8 ± 24.0, 6-keto-PGF_2α from 7.2 ± 1.3 to 51.5 ± 17.0, and txB₂ from 11.2 ± 3.3 to 13.6 ± 3.6 ng/h/1.73 m² ( ) at the maximal urine flow. Except for 6-keto-PGF_1α and TxB₂, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged peroid of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB₂ one can assume that this prostanoid represents renal as well as systemic TxA₂ activity.     

Conformational change of core particles studied by quasielastic light scattering

The diffusion translational coefficient D_T of core particles in monodisperse solutions has been measured by the quasielastic light scattering method in a large scale of salinities over the range 6.10⁻⁴ to 2M Na⁺ or K⁺. The observed values of D_T are independent of particle concentration in the range 0.1–2 mg/ml and do not vary with the scattering vector q corresponding to scattering angles between 40°–120°. When the salinity is progressively raised an increase of D_T from 1.9.10⁻⁷ cm²s⁻¹ to 3.2.10⁻⁷ cm²s⁻¹ was observed at about 2.10⁻³ M NaCl followed by a decrease of D_T beyond 0.6 M NaCl.The various possible causes of the changes of D_T such as interactions between particles or between particles and salt ions are discussed. We show that the single low ionic strength change is due to a conformational transition of the core particles, while the second variation of D_T accompanies the disorganization of the core particles.     

Characterization of a vasoactive intestinal peptide-sensitive adenylate cyclase in rat intestinal epithelial cell membranes

A vasoactive intestinal peptide-sensitive adenylate cyclase in intestinal epithelial cell membranes was characterized. Stimulation of adenylate cyclase activity was a function of vasoactive intestinal peptide concentration over a range of 1 · 10⁻¹⁰−1 · 10⁻⁷ M and was increased six-times by a maximally stimulating concentration of vasoactive intestinal peptide. Half-maximal stimulation was observed with 4.1 ± 0.7 nM vasoactive intestinal peptide. Fluoride ion stimulated adenylate cyclase activity to a higher extent than did vasoactive intestinal peptide. Under standard assay conditions, basal, vasoactive inteetinal peptide- and fluoride-stimulated adenylate cyclase activities were proportional to time of incubation up to 15 min and to membrane concentration up to 60 μg protein per assay. The vasoactive intestinal peptide-sensitive enzyme required 5–10 mM Mg²⁺ and was inhibited by 1 · 10⁻⁵ M Ca²⁺. At sufficiently high concentrations, both ATP (3 mM) and Mg²⁺ (40 mM) inhibited the enzyme.Secretin also stimulated the adenylate cyclase activity from intestinal epithelial cell membranes but its effectiveness was 1/1000 that of vasoactive intestinal peptide. Prostaglandins E₁ and E₂ at 1 · 10⁻⁵ M induced a two-fold increase of cyclic AMP production. Vasoactive intestinal peptide was the most potent stimulator of adenylate cyclase activity, suggesting an important physiological role of this peptide in the cyclic AMP-dependent regulation of the intestinal epithelial cell function.     

Gas chromatographic–mass spectrometric analysis of dichlorobenzene isomers in human blood with headspace solid-phase microextraction

Headspace solid-phase microextraction (HS-SPME) was utilized for the determination of three dichlorobenzene isomers (DCBs) in human blood. In the headspace at 30°C, DCBs were absorbed for 15 min by a 100-μm polydimethylsiloxane (PDMS) fiber. They were then analyzed by capillary column gas chromatography–mass spectrometry (GC–MS). By setting the initial column oven temperature at 20°C, the three isomers were resolved at the baseline level. p-Xylene-d₁₀ was used as the internal standard (I.S.). For quantitation, the molecular ion at m/z 146 for each isomer and the molecular ion at m/z 116 for I.S. were selected. For day-to-day precision, relative standard deviations in the range 3.2–10.7% were found at blood concentrations of 1.0 and 10 μg/ml. Each compound was detectable at a level of at least 0.02 μg per 1 g of whole blood (by full mass scanning). HS-SPME–GC–MS, when performed at relatively low temperatures, was found to be feasible in toxicological laboratories. Using this method, the plasma levels of one patient who had drunk a pesticide-like material were measured.