Determination of artemether and its major metabolite, dihydroartemisinin, in plasma using high-performance liquid chromatography with electrochemical detection

A rapid, selective, sensitive and reproducible HPLC with reductive electrochemical detection for quantitative determination of artemether (ART) and its plasma metabolite, dihydroartemisinin (DHA: alpha and beta isomers) in plasma is described. The procedure involved the extraction of ART, DHA and the internal standard, artemisinin (ARN) with dichloromethane-tert.-methylbutyl ether (1:1, v/v) or n-butyl chloride-ethyl acetate (9:1, v/v). Chromatographic separation was performed with a mobile phase of acetonitrile-water (20:80, v/v) containing 0.1 M acetic acid pH 5.0, running through a microBondapak CN column. The method was capable of separating the two isomeric forms of DHA (alpha, beta). The retention times of alpha-DHA, beta-DHA, ARN and ART were 4.6, 5.9, 7.9 and 9.6 min, respectively. Validation of the assay method was performed using both extraction systems. The two extraction systems produced comparable recoveries of the various analytes. The average recoveries of ART, DHA and ARN over the concentration range 80-640 ng/ml were 86-93%. The coefficients of variation were below 10% for all three drugs (ART, alpha-DHA, ARN). The minimum detectable concentrations for ART and alpha-DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for use in clinical pharmacokinetic study.     

Modulation of the in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, RP-3. III. Enhancement by RP-3 treatment of the anti-sheep red blood cell plaque-forming cell response in rats

Because recent work has shown that neutrophils produce various cytokines and are activated by these agents, this study was undertaken to examine neutrophil participation in immune networks. In our previous work, we demonstrated that delayed type hypersensitivity to SRBC and accompanying mononuclear leukocyte recruitment are inhibited in vivo by selective depletion of neutrophils using a mAb designated RP-3. To elucidate the function of these cells in Ab production, we assessed the splenic plaque-forming cell response to SRBC in a rat model through selective depletion of circulating neutrophils by RP-3. This depletion which occurred at the time of immunization resulted in an increase in the number of anti-SRBC Ab producing cells, as determined by the direct and indirect plaque-forming cell response. This phenomenon was observed only when the Ag was administered i.p. and not with i.v. immunization. These results suggest that neutrophils suppress Ab production in certain situations.     

In vitro selection for different mutational patterns in the HIV-1 reverse transcriptase using high and low selective pressure of the nonnucleoside reverse transcriptase inhibitor HBY 097

Measurement of Na-Ca exchange activity was used to examine subsarcolemmal sodium levels ([Na+]s) in single, voltage-clamped guinea-pig cardiac myocytes while Na-K pump activity was modulated pharmacologically. Changes in Nas were evaluated from phase-plane analysis of the changes in intracellular calcium, measured using the fluorescent indicators Fura-red and Fluo-3. Activation of beta-adrenoceptors with 1 microM isoprenaline resulted in activation of the cAMP-dependent chloride current, but had no effect on the calcium transient mediated via the Na-Ca exchanger, regardless of whether the Na-K pump was active or inhibited (with strophanthidin). The ability of Na-Ca exchange activity to report [Na+]s was demonstrated by the effect of changing the extracellular rubidium concentration from 1 to 5.4 mM to modulate Na-K pump activity. We suggest that beta-adrenergic stimulation does not directly affect either the Na-K pump or the Na-Ca exchanger and that the Na-Ca exchanger can be used as a sensitive indicator of changes in [Na+]s and Na-K pump activity.     

Direct injection analysis of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone in human serum using a semipermeable surface (SPS) HPLC column

The efficacy of combination therapy with cis-diammine-dichloroplatinum (II) (CDDP) and o-(chloroacetyl-carbamoyl) fumagillol (AGM-1470) was evaluated experimentally using a transplantable rat osteosarcoma line, previously established in our laboratory, with a high potential for metastasis. Tumor-bearing male Fischer 344 rats were administered CDDP (2.5 mg/kg) together with, or after discontinuation of, AGM-1470 treatment (10 mg/kg/body weight/week). When CDDP was administered three days after discontinuation of AGM-1470 the most pronounced antimetastatic effects were observed, although the antitumor effect was approximately the same.     

The effect of lead on intracellular water content of human red blood cells. Part 1. An experiment in vitro (author's transl)

We observed in vitro experiment that lead increased osmotic resistance of normal human erythrocytes and decreased mean corpuscular volume and intracellular potassium. Though the mechanism of the increased osmotic resistance of erythrocytes caused by lead has not yet been completely clarified, the following hypothesis could be accepted. Lead causes leakage of water from erythrocytes, which means that more water can enter the cell before hemolysis occurs. But there has been no report of direct measurement of the intracellular water content of erythrocytes treated with lead. This paper tried to clarify the relationship between intracellular water and osmotic resistance of lead treated erythrocytes in vitro experiment. The results were: 0.05 mumol/ml of lead increases osmotic resistance, trapped water content and plasma water content of normal human blood and decreases intracellular water content after incubation for 2 hours at 37 degrees C.     

Identification of chondromodulin I as a novel endothelial cell growth inhibitor. Purification and its localization in the avascular zone of epiphyseal cartilage

Cartilage is unique among tissues of mesenchymal origin in that it is resistant to vascular invasion due to an intrinsic angiogenic inhibitor. During endochondral bone formation, however, calcified cartilage formed in the center of the cartilaginous bone rudiment allows vascular invasion, which initiates the replacement of cartilage by bone. The transition of cartilage from the angioresistant to the angiogenic status thus plays a key role in bone formation. However, the molecular basis of this phenotypic transition of cartilage has been obscure. We report here purification of an endothelial cell growth inhibitor from a guanidine extract of bovine epiphyseal cartilage. The N-terminal amino acid sequence indicated that the inhibitor was identical to chondromodulin I (ChM-I), a cartilage-specific growth-modulating factor. Purified ChM-I inhibited DNA synthesis and proliferation of vascular endothelial cells as well as tube morphogenesis in vitro. Expression of ChM-I cDNA in COS7 cells indicated that mature ChM-I molecules were secreted from the cells after post-translational modifications and cleavage from the transmembrane precursor at the predicted processing signal. Recombinant ChM-I stimulated DNA synthesis and proteoglycan synthesis of cultured growth plate chondrocytes, but inhibited tube morphogenesis of endothelial cells. In situ hybridization and immunohistochemical studies indicated that ChM-I is specifically expressed in the avascular zone of cartilage in developing bone, but not present in calcifying cartilage. These results suggest a regulatory role of ChM-I in vascular invasion during endochondral bone formation.     

Toxicokinetics of inhaled 2-butoxyethanol and its major metabolite, 2-butoxyacetic acid, in F344 rats and B6C3F1 mice

2-Butoxyethanol (2BE) is used extensively in the production of cleaning agents and solvents. It is primarily metabolized in the liver to 2-butoxyacetic acid (2BAA), which is believed to be responsible for 2BE toxicities associated with hemolysis of red blood cells. The objective of the study was to characterize the systemic disposition of 2BE and 2BAA in rats and mice during 2-year 2BE inhalation toxicity studies. Male and female F344 rats and B6C3F1 mice (6-7 weeks old) were exposed to target 2BE concentrations of 0, 31.2, 62.5, or 125 ppm (rats), or 0, 62.5, 125, or 250 ppm (mice), by whole-body inhalation for 6 h/day, 5 days/week for up to 18 months. Postexposure blood samples were collected after 1 day, 2 weeks, and 3, 6, 12, and 18 months of exposure. Postexposure 16-h urine samples were collected after 2 weeks and 3, 6, 12, and 18 months of exposure. A separate set of mice was kept in the control chamber and exposed to 2BE for 3 weeks when they were approximately 19 months old. Postexposure blood samples were collected after 1 day and 3 weeks of exposure and 16-h urine samples were collected after 2 weeks of exposure from these aged mice. Blood samples were analyzed for both 2BE and 2BAA and urine samples were analyzed for 2BAA using GC/MS, and their kinetic parameters were estimated through the curve-fitting method using SAS. Systemically absorbed 2BE was rapidly cleared from blood (t1/2-RAT < 10 min; t1/2-MOUSE < 5 min after the 1-day exposure) independent of exposure concentration. Proportional increases in AUC2BE relative to increases in exposure concentration indicated linear 2BE kinetics. In contrast, the rate of 2BAA elimination from blood decreased as the exposure concentration increased. Nonproportional increases in AUC2BAA also indicated that 2BAA is eliminated following dose-dependent, nonlinear kinetics. Overall, mice eliminated both 2BE and 2BAA from blood faster than rats. Sex-related differences in 2BAA elimination were most significant with rats, in that females were less efficient in clearing 2BAA from the blood. Differences in renal excretion of 2BAA are possibly responsible for the sex-related difference in the 2BAA blood profiles in rats. As exposure continued, the rates of elimination for both 2BE and 2BAA decreased in both species, resulting in longer residence times in the blood. When 19-month-old naive mice were exposed to 125 ppm, 2BE was rapidly cleared from the systemic circulation, exhibiting clearance profiles similar to young mice. However, old mice eliminated 2BAA from blood > 10 times slower than young mice after 1-day of exposure. This delayed elimination of 2BAA in old mice was less obvious after 3 weeks of exposure, suggesting that there might be other factors in addition to the age of animals that could influence the apparent difference in 2BAA kinetics between old and young mice. It was concluded that the elimination kinetics of 2BE and 2BAA following repeated 2BE exposure appear to be dependent on species, sex, age, time of exposure, as well as the exposure concentration.     

Changes in plasma endothelin-1 and lipid peroxidate levels and amount of superoxidate dismutase in red blood cell in patients with pregnancy-induced hypertension

OBJECTIVE: To evaluate the role of endothelin (ET-1) and lipid peroxide (LPO) in the pathogenesis of pregnancy-induced hypertension (PIH). METHODS: The plasma concentrations of ET-1, LPO and the amount of superoxide dismutase (SOD) in red blood cell were measured by radioimmunoassay, thiobarbituric acid fluorimetric determination and catechol self-oxidation determination respectively in total 95 women (normal non-pregnant women 15, Late pregnant women 20 and patients with PIH 60). RESULTS: (1) The levels of ET-1, LPO and SOD in normal late pregnant women were significantly higher than those in non-pregnant women (P < 0.01), but the LPO/SOD ratio was not significantly different between the two groups. (2) The levels of plasma ET-1 and LPO/SOD ratio in cases with PIH were markedly higher than those in normal late pregnant women, and it increased with the severity of the disease and returned to the levels of normal late pregnant women within 3-7 days after delivery. (3) There was a positive correlation between ET-1 and LPO/SOD ratio in PIH. CONCLUSIONS: The imbalance of oxidation and antioxidation and endothelial cell injury may play an important role in the pathogenesis of PIH.     

Simultaneous determination of thiabendazole and its major metabolite, 5-hydroxythiabendazole, in bovine tissues using gradient liquid chromatography with thermospray and atmospheric pressure chemical ionisation mass spectrometry

Modification of an existing neural structure to support a second function will produce a trade-off between the two functions if they are in some way incompatible. The trade-off between two such sensory functions is modeled here in pyramidal neurons of the gymnotiform electric fish's medullar electrosensory lateral line lobe (ELL). These neurons detect two electric stimulus features produced when a nearby object interferes with the fish's autogenous electric field: (1) amplitude modulation across a cell's entire receptive field and (2) amplitude variation within a cell's receptive field produced by an object's edge. A model of sensory integration shows that detection of amplitude modulation and enhancement of spatial contrast involve an inherent mechanistic trade-off and that the severity of the trade-off depends on the particular algorithm of sensory integration. Electrophysiology data indicate that of the two algorithms for sensory integration modeled here for the gymnotiform fish Brachyhypopomus pinnicaudatus, the algorithm with the better trade-off function is used. Further, the intrinsic trade-off within single cells has been surmounted by the replication of ELL into multiple electrosensory map segments, each specialized to emphasize different sensory features.     

Oral platelet aggregation inhibitor Ro 48-3657: determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 microl of plasma) and 25 ng/ml (50 microl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

Pharmacokinetics of dilevalol and its conjugates in man. Assay method for plasma, blood, urine and bile samples and preliminary pharmacokinetic studies

The renal and biliary excretion of the beta-adrenoceptor blocking agent dilevalol (CAS 75659-07-3) and its conjugates was examined in a preliminary pharmacokinetic study. Plasma, urine and bile dilevalol concentrations were determined with a simplified procedure that is based on alkaline liquid-liquid extraction using diethyl ether and subsequent reversed-phase HPLC separation of the reconstituted samples (on a PRP-1 stationary phase using a mixture of methanol and pH 9.8 carbonate buffer as mobile phase). Triamterene was used as internal standard. The quantification of the conjugates was accomplished indirectly via enzymatic hydrolysis (glusulase) with and without addition of the beta-glucuronidase inhibitor 1,4-saccharolactone (at a final concentration of 5.5 mmol/l). In the pharmacokinetic study healthy volunteers and cholecystectomised patients with a T-drain received a single oral dose of 200 mg dilevalol. Furthermore, to healthy volunteers an i.v. dose of 60 mg dilevalol was given in order to estimate the absolute bioavailability. From the obtained data the systemic plasma clearance was calculated to be 1708 ml/min. The oral bioavailability was calculated to be 16%. The log concentration-time curves of the metabolites paralleled those of dilevalol in the terminal section with average terminal half-lives of approx. 5 h. In volunteers the fractions of the dose excreted renally were 0.5% for parent drug, 23% for the glucuronide(s) and 8% for the sulfate. The corresponding values found for the patients were not significantly different. In the patients' bile only 1.2% of the total dose were found (0.03% dilevalol, 1.1% dilevalol glucuronide(s), 0.1% dilevalol sulfate).(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational change due to esterification of hydroxy groups in erythromycin A and its major metabolite: analysis of these derivatives with different biological properties using NMR and molecular dynamics (MD) data

A conformational study is performed on the acylated erythromycin and erythralosamine derivatives from comparison between experimental results (NMR) and theoretical calculations by Molecular Dynamics (MD) in attempts to correlate their conformations with their abilities to generate cytochrome P450-nitroso metabolite complexes in vitro. As the 3'-dimethyl-amino function of the desosamine is metabolized and responsible for the interaction with cytochrome P450, its position, mobility and steric hindrance in the proximity of this functional group are related to its biological properties. The major conformations of the lactone ring were termed A (A1, A2, A3) and B (B1, B2), and this macrocycle flexibility induced five different orientations a, b, c, d and e for the desosamine sugar. Conformations A and B differ in many ways but the major change is the inward folding of the C(3) fragment in B. Conformer a exhibits an orientation of the desosamine nearly perpendicular to the macrocycle whereas the two units are in the same plane in conformations c and e. For conformation b, the cladinose unit lifts up above the macrocycle. Conformation d exhibits a turned-back cladinose. In the erythromycin derivatives esterification at the beta position to the N(CH3)2 group of the desosamine reduces the degree of freedom of the macrocyclic lactone ring which corresponds to conformation A only. The desosamine sugar was found to be perpendicular to the macrocycle (a conformer) and both sugar groups are parallel to reduce the steric energy. In the erythralosamine derivatives, the macrocycle is always present as conformation B with the two conformations b and c of the sugar rings. The steric parameters favour the b conformers in which the amino group is tilted up, while in 3,2'-dibenzoylated stacking aromatic attraction stabilizes the planar c conformer. Both isomers are thus shown to adopt well-defined conformations and to be well-adapted for a comparative structure-activity correlation studies. There is a significant relationship between the conformation b and the formation of cytochrome P450-nitroso metabolite complexes.     

Identification of interleukin-1 and platelet-derived growth factor-B as major mitogens for the spindle cells of Kaposi's sarcoma: a combined in vitro and in vivo analysis

By means of a combined in vitro and in vivo analysis we provide evidence that IL-1 beta and PDGF-B, but not OSM (oncostatin M) or IL-6, are major mitogens for the spindle cells of Kaposi's sarcoma (KS) in vivo. PDGF-B and IL-1 beta stimulated proliferation of cultivated KS spindle cells in vitro. Analysis of gene expression in vivo revealed that both factors as well as the PDGF beta-receptor are present in KS lesions. By contrast, IL-6 had no effect and OSM inhibited proliferation of cultivated KS spindle cells. Again, the effect of these factors on cultivated KS spindle cells in vitro was reflected by the gene expression observed in KS lesions in vivo. Neither the expression of IL-6 receptor nor of OSM could be detected in KS lesions by in situ hybridization. Moreover, in situ hybridization revealed an identical pattern of gene expression in cultivated KS spindle cells and KS spindle cells in vivo with respect to the above-mentioned cytokines [PDGF-B, IL-1 beta, IL-1 alpha, IL-6, OSM] and their receptors [PDGF beta-receptor, gp130, IL-6 receptor, leukemia inhibitory factor (LIF) receptor]. This further supported the suitability of cultivated KS spindle cells as an in vitro model in order to determine which cytokines may activate proliferation of KS spindle cells in vivo.     

Uptake of the food-derived heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its N-hydroxy metabolite into rat pancreatic acini and hepatocytes in vitro

Sinc DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed at relatively high levels in the rat pancreas but not liver, we examined the uptake of PhIP and its N-hydroxy metabolite (N-OH-PhIP) into pancreatic acini and hepatocytes to determine if differential tissue uptake was a factor modulating the formation of PhIP-DNA adducts. In addition, since the precursors of PhIP formation are two amino acids and since various amino acid transporters have been identified in the pancreas, the possible involvement of these transporters in the uptake of PhIP and N-OH-PhIP was investigated. The uptake both heterocyclic compounds into both tissue preparations was rapid, with maximal uptake occurring with 1-2 min. However, PhIP uptake into pancreatic acini was significantly (2-way ANOVA, P < 0.05) greater than uptake of N-OH-PhIP into pancreatic acini and the uptake of both PhIP and N-OH-PhIP into hepatocytes. Although uptake was rapid, efflux of both compounds from both tissue preparations was also rapid. However, the efflux rate constant (1.86 +/- 0.6/min, mean +/- SEM) for PhIP) was significantly lower (Student's t-test, P < 0.05) than that for N-OH-PhIP (4.14 +/- 0.04/min) from pancreatic acini. This, combined with the increased uptake of PhIP into pancreatic acini , suggests that there is substantial but reversible binding of PhIP in the pancreas. The uptake of both PhIP and N-OH-PhIP into pancreatic acini and hepatocytes was not affected by the presence of various amino acids in the incubation buffer, indicating that amino acid transporters are not involved in uptake of these compounds. Furthermore, uptake of both compounds did not appear to be dependent on metabolic energy supply. The above data, together with the high octanol:buffer partition coefficients (logP = 1.322 and 1.301 for PhiP and N-OH-PhIP respectively) suggest that both uptake and efflux of PhIP and N-OH-PhIP are consistent with a process of passive diffusion. The tissue binding characteristics for PhIP in the pancreas may create conditions whereby pancreatic cytochrome P450 1A1 can catalyse the formation of N-OH-PhIP. While N-OH-PhIP is not the ultimate reactive DNA binding species, it has been shown to directly bind to and form DNA adducts. Therefore, it is possible that the apparent selective accumulation of PhIP may contribute to the high level of PhIP-DNA adducts formed in the rat pancreas.     

Preliminary studies on the validity of in vitro measurement of drug toxicity using HeLa cells. III. Lethal action to man of 43 drugs related to the HeLa cell toxicity of the lethal drug concentrations

The human lethal plasma concentrations of 46 drugs were divided by their IC50 for HeLa cells in vitro to make up a series of cytotoxic quotients (CQLv). CQLv was then compared with the recorded lethal action to man of 43 of the drugs. While the 7 drugs with the lowest CQLv values produce a non-cytotoxic interference with neuro-transmission, most of the remaining 36 drugs have a known local or systemic cytotoxicity to man. A majority of the 36 drugs induces a non-specific central nervous system (CNS)-depression at lethal dosage, intermingled with function loss from organs outside CNS in proportion to decreasing drug accumulation in CNS cells and increasing CQLv. The remaining drugs which do not penetrate CNS cells and at lethal dosage induce a widespread injury and function loss of tissues outside the CNS, have a CQLv near unity. Non-specific CNS-depression may thus be the primary human reaction to lethal systemic drug cytotoxicity, while widespread drug injury to various tissues outside CNS--conventionally considered to be cytotoxic in origin--may be the obligatory human reaction to drugs that do not penetrate cells well. The present findings indicate a relevance to human toxicity of the HeLa toxicity for most drugs.     

Clinical ecology and its role in diagnosis of chronic diseases caused by environmental pollution. Indoor air pollution as a major factor

The author discusses the four basic principles of clinical ecology, which include total load, such as exposure to chemicals, effects of physical factors (cold, heat, positive ions, radiation), exposure to bacteria, viruses and fungi, effects of consumed foods and psychological stress. Adaptation is discussed in detail as it pertains to chronic degenerative disease and chemical sensitivity. Lack of understanding of adaptive phenomena leads contemporary physicians to prescribe drugs instead of finding and eliminating the cause of disease to alleviate suffering. Body response to environmental stimuli during adaptive processes is biphasic in nature and is characterized by stimulatory and withdrawal reactions. Symptoms developed during these phases are described in detail. Effects of foods and chemicals on patients' health should be examined and considered before excluding in favour of idiopathic, mental or psychosomatic disturbances. In order to understand the variety of symptoms and signs developed during the process of degenerative disease one has to accept and apply the concept of biological individuality. Each patient should be assessed and dealt with carefully since each represents a unique clinical picture. The author further on discusses indoor air pollution, its causes and effects on health. Particular emphasis is placed on the indoor use of pesticides in the Canadian setting. Four groups of population have been defined as the result of exposure to chemicals and are discussed in brief. The author concludes with a comparison of traditional and environmental medicine in respect to history taking, the stage at which disease is diagnosed, the patient's viewpoint, specialization and therapies.     

Demonstration of neuraminidase activity in human blood serum and human milk using a modified, radioactively labelled alpha1-glycoprotein as substrate

N-Acetylneuramini acid of alpha1-glycoprotein was oxidized with a small molar excess of periodate and reduced with tritium-labelled borohydride. By this method about 50% of the N-acetylneuraminic acid was converted to its radioactively labelled C8-analog and 25% to its C7-analog. Using this modified alpha1-glycoprotein as substrate, minimum neuraminidase concentrations of 10(-18) units/ml, related to the activity of neuraminidase from Vibrio cholerae, could be determined. Neuraminidase activity was demonstrated in 95% of the sera or blood plasma samples from a series of 417 healthy or ill human individuals and in milk samples from 5 different donors. The neuraminidase in both serum and milk had optimal activity at pH 5.5. On an average, 10(-10) neuraminidase units were found in 1 ml serum and 10(-8) units in 1 ml milk. Although the neuraminidase activities in the sera varied, a correlation with definite pathological states is not yet possible. N-Acetylneuraminate pyruvate-lyase activity could not be detected in human serum.     

Novel high-performance liquid chromatographic assay using fluorescence detection for the quantitation of plasma gamma-methylene-10-deazaaminopterin and its major metabolite, 7-hydroxy-gamma-methylene-10-deazaaminopterin, in patients with solid cancers in a phase I trial

Gamma-methylene-10-deazaaminopterin (MDAM), a unique dihydrofolate reductase inhibitor, has demonstrated antitumor activity against a broad spectrum of human solid tumors in preclinical studies. A novel reversed-phase, ion-pair high-performance liquid chromatography (HPLC) assay that uses fluorescence detection has been developed to quantitate levels of MDAM and its major metabolite, 7-hydroxy-gamma-methylene-10-deazaaminopterin (7-OH-MDAM), in human plasma. The recovery of MDAM and 7-OH-MDAM from plasma was >97% by a simple one-step deproteinization process using tetrabutylammonium bromide (TBABr) and methanol. MDAM and 7-OH-MDAM remained stable in plasma over a 28-day test period at ambient temperatures, and neither compound was light-sensitive. The limit of quantitation was 0.005 microM for both MDAM and 7-OH-MDAM. This assay has been found to be simple, sensitive and reproducible in determining plasma concentrations of MDAM and 7-OH-MDAM in patients with solid cancers in a phase I trial.