Molecularly imprinted sol-gel polymers for the analysis of iprodione fungicide in wine: Synthesis in green solvent.

Iprodione is a fungicide widely used in viticulture in most agricultural countries. It was banned recently in the European community because of its carcinogenic and endocrine disrupting characters. In this work, a cheap analytical method able to monitor iprodione in a white wine was developed. Molecularly imprinted sol-gel polymers (MIS) specific to iprodione and using green solvents were synthesized. An experimental design having the following factors (solvent volume and crosslinker quantity) was used to prepare an optimal MIS. In terms of selectivity, the optimal MIS showed the best partition coefficient towards iprodione in a white wine containing four other competing fungicides (procymidone, pyrimethanil, azoxystrobin and iprovalicarb). A solid phase extraction method using the optimal MIS was optimized and applied to analyse iprodione in a white wine. Low detection and quantification limits were reached 11.7 and 39.1 µg/L respectively.

Simultaneous determination of iprodione, procymidone, and chlorflurenol in lake water and wastewater matrices by GC-MS after multivariate optimization of binary dispersive liquid-liquid microextraction.

This study reports the optimization of a binary dispersive liquid-liquid microextraction method for the determination of iprodione, procymidone, and chlorflurenol by gas chromatography mass spectrometry. The study was aimed at using two extraction solvents to increase the extraction efficiency of all analytes. The binary solvents recorded results higher than the mono-solvents. After examining the effects of main experimental parameters and their interactions by analysis of variance, 200 µL of binary mixture (dichloromethane and 1,2-dichloroethane), 2.5 mL of ethanol, and 15 s vortex were obtained as optimum parameters. The detection and quantification limits calculated for the analytes were found to be between 0.30-1.6 and 1.0-5.3 ng/mL, respectively. Enhancement in detection power calculated as a ratio of the binary extraction detection limit to the detection limit of direct GC-MS analysis was 105-, 214-, and 233-fold for chlorflurenol, iprodione, and procymidone, respectively. In order to check the accuracy of the developed method, recovery study was performed. Water sampled from a lake and two wastewater samples from treatment facilities were spiked at two concentrations, and the percent recovery calculated for the samples ranged between 87 and 116%. These results confirmed the suitability of the method to real samples for accurate determination of the analytes at trace levels.

Doping control study of AICAR in post-race urine and plasma samples from horses.

Acadesine, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, commonly known as AICAR, is a naturally occurring adenosine monophosphate-activated protein kinase (AMPK) activator in many mammals, including humans and horses. AICAR has attracted considerable attention recently in the field of doping control because of a study showing the enhancement of endurance performance in unexercised or untrained mice, resulting in the term 'exercise pill'. Its use has been classified as gene doping by the World Anti-Doping Agency (WADA), and since it is endogenous, it may only be possible to control deliberate administration of AICAR to racehorses after establishment of an appropriate threshold. Herein we report our studies of AICAR in post-race equine urine and plasma samples including statistical studies of AICAR concentrations determined from 1,470 urine samples collected from thoroughbreds and standardbreds and analyzed in Australia, France, and Hong Kong. Quantification methods in equine urine and plasma using liquid chromatography-mass spectrometry were developed by the laboratories in each country. An exchange of spiked urine and plasma samples between the three countries was conducted, confirming no significant differences in the methods. However, the concentration of AICAR in plasma was found to increase upon haemolysis of whole blood samples, impeding the establishment of a suitable threshold in equine plasma. A possible urine screening cut-off at 600 ng/mL for the control of AICAR in racehorses could be considered for adoption. Application of the proposed screening cut-off to urine samples collected after intravenous administration of a small dose (2 g) of AICAR to a mare yielded a short detection time of approximately 4.5 h. Copyright © 2017 John Wiley & Sons, Ltd.

Novel proton MR spectroscopy findings in adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL) deficiency is a rare inborn error of metabolism resulting in accumulation of metabolites including succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (S-Ado) in the brain and other tissues. Patients with ADSL have progressive psychomotor retardation, neonatal seizures, global developmental delay, hypotonia, and autistic features, although variable clinical manifestations may make the initial diagnosis challenging. Two cases of the severe form of the disease are reported here: an 18-month-old boy with global developmental delay, intractable neonatal seizures, progressive cerebral atrophy, and marked hypomyelination, and a 3-month-old girl presenting with microcephaly, neonatal seizures, and marked psychomotor retardation. In both patients in vivo proton magnetic resonance spectroscopy (MRS) showed the presence of S-Ado signal at 8.3 ppm, consistent with a prior report. Interestingly, SAICAr signal was also detectable at 7.5 ppm in affected white matter, which has not been reported in vivo before. A novel splice-site mutation, c.IVS12 + 1/G > C, in the ADSL gene was identified in the second patient. Our findings confirm the utility of in vivo proton MRS in suggesting a specific diagnosis of ADSL deficiency, and also demonstrate an additional in vivo resonance (7.5 ppm) of SAICAr in the cases of severe disease.

Iprodione residues and dissipation rates in tobacco leaves and soil.

Field experiments were conducted in two different locations to determine the residue levels and dissipation rates of iprodione in tobacco leaves and soil. Iprodione 50% wettable powder formulation was sprayed once at 12.50 g/ha to study the dissipation behavior and three to four times at 8.33 g/ha (recommended dose) and 12.50 g/ha (1.5 times the recommended field dose) to determine the residue levels of iprodione in tobacco leaves and soil after repeated applications. Iprodione residues in both green tobacco leaves and soil dissipated to about 50% of the initial deposits after 7 days and then further dissipated to more than 90% after 35 days.The dissipation of iprodione followed first order kinetics and the calculated half-life values (T (1/2)) were 5.64-8.80 days in green tobacco leaves and 7.50-9.93 days in soil, respectively. Iprodione residue levels in flue-cured tobacco leaves 21 days after the third and fourth applications ranged from 7.61 to 40.98 mg/kg. Meanwhile, the residues detected in soil decreased to 0.010-0.117 mg/kg 21 days after the last treatment.

A low thermal mass fast gas chromatograph and its implementation in fast gas chromatography mass spectrometry with supersonic molecular beams.

A new type of low thermal mass (LTM) fast gas chromatograph (GC) was designed and operated in combination with gas chromatography mass spectrometry (GC-MS) with supersonic molecular beams (SMB), including GC-MS-MS with SMB, thereby providing a novel combination with unique capabilities. The LTM fast GC is based on a short capillary column inserted inside a stainless steel tube that is resistively heated. It is located and mounted outside the standard GC oven on its available top detector port, while the capillary column is connected as usual to the standard GC injector and supersonic molecular beam interface transfer line. This new type of fast GC-MS with SMB enables less than 1 min full range temperature programming and cooling down analysis cycle time. The operation of the fast GC-MS with SMB was explored and 1 min full analysis cycle time of a mixture of 16 hydrocarbons in the C(10)H(22) up to C(44)H(90) range was achieved. The use of 35 mL/min high column flow rate enabled the elution of C(44)H(90) in less than 45 s while the SMB interface enabled splitless acceptance of this high flow rate and the provision of dominant molecular ions. A novel compound 9-benzylazidanthracene was analyzed for its purity and a synthetic chemistry process was monitored for the optimization of the chemical reaction yield. Biodiesel was analyzed in jet fuel (by both GC-MS and GC-MS-MS) in under 1 min as 5 ppm fatty acid methyl esters. Authentic iprodion and cypermethrin pesticides were analyzed in grapes extract in both full scan mode and fast GC-MS-MS mode in under 1 min cycle time and explosive mixture including TATP, TNT and RDX was analyzed in under 1 min combined with exhibiting dominant molecular ion for TATP. Fast GC-MS with SMB is based on trading GC separation for speed of analysis while enhancing the separation power of the MS via the enhancement of the molecular ion in the electron ionization of cold molecules in the SMB. This paper further discusses several features of fast GC and fast GC-MS and the various trade-offs involved in having powerful and practical fast GC-MS.

Multiresidue method for the determination of 13 pesticides in three environmental matrices: water, sediments and fish muscle.

Pesticides residues in aquatic ecosystems are an environmental concern which requires efficient analytical methods. In this study, we proposed a generic method for the quantification of 13 pesticides (azoxystrobin, clomazone, diflufenican, dimethachlor, carbendazim, iprodion, isoproturon, mesosulfuron-methyl, metazachlor, napropamid, quizalofop and thifensulfuron-methyl) in three environmental matrices. Pesticides from water were extracted using a solid phase extraction system and a single solid-liquid extraction method was optimized for sediment and fish muscle, followed by a unique analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Limits of quantification were below 5 ng L(-1) for water (except for fluroxypyr and iprodion) and ranged between 0.1 ng g(-1) and 57.7 ng g(-1) for sediments and regarding fish, were below 1 ng g(-1) for 8 molecules and were determined between 5 and 49 ng g(-1) for the 5 other compounds. This method was finally used as a new routine practice for environmental research.

Analysis and stability study of temozolomide using capillary electrophoresis.

The applicability of micellar electrokinetic capillary chromatography (MEKC) for the analysis of temozolomide (TMZ) and its degradants, 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and 5-amino-imidazole-4-carboxamide (AIC) has been studied. Using short-end injection, the analysis of TMZ and its degradants could be performed within 1.2 min. The obtained precision of migration times was better than 1.6 RSD%, and the limit of quantitation (LOQ) was 0.31-0.93 microg/mL. The therapeutic concentration of TMZ in blood samples can be determined after direct sample injection and conventional on-capillary UV detection. The proposed MEKC method was applied to study the stability of TMZ in water and serum at different pH values. It was established that the half-life of the TMZ in vitro serum at room temperature was 33 min, close to the half-life (28 min) obtained in water at pH 7.9.

Analysis of pesticide residues by fast gas chromatography in combination with negative chemical ionization mass spectrometry.

A combination of fast GC with narrow-bore column and bench top quadrupole mass spectrometer (MS) detector in negative chemical ionization (NCI) mode (with methane as reagent gas) is set up and utilized for the ultratrace analysis of 25 selected pesticides. The observed pesticides, belonging to the endocrine disrupting chemicals (EDCs), were from different chemical classes. A comparative study with electron impact (EI) ionization was also carried out (both techniques in selected ion monitoring (SIM) mode). The programmed temperature vaporizer (PTV) injector in solvent vent mode and narrow-bore column (15mx0.15mm I.D.x0.15microm film of 5% diphenyl 95% dimethylsiloxane stationary phase) were used for effective and fast separation. Heptachlor (HPT) as internal standard (I.S.) was applied for the comparison of results obtained from absolute and normalized peak areas. Non-fatty food matrices were investigated. Fruit (apple - matrix-matched standards; orange, strawberry, plum - real samples) and vegetable (lettuce - real sample) extracts were prepared by a quick and effective QuEChERS sample preparation technique. Very good results were obtained for the characterization of fast GC-NCI-MS method analysing EDCs pesticides. Analyte response was linear from 0.01 to 150microgkg(-1) with the R(2) values in the range from 0.9936 to 1.0000 (calculated from absolute peak areas) and from 0.9956 to 1.0000 (calculated from peak areas normalized to HPT). Instrument limits of detection (LODs) and quantification (LOQs) were found at pgmL(-1) level and for the majority of analytes were up to three orders of magnitude lower for NCI compared to EI mode. In both ionization modes, repeatability of measurements expressed as relative standard deviation (RSDs) was less than 10% which is in very good agreement with the criterion of European Union.

Rapid analysis of multiple pesticide residues in fruit-based baby food using programmed temperature vaporiser injection-low-pressure gas chromatography-high-resolution time-of-flight mass spectrometry.

A rapid method using programmed temperature vaporiser injection-low-pressure gas chromatography-high-resolution time-of-flight mass spectrometry (PTV-LP-GC-HR-TOF-MS) for the analysis of multiple pesticide residues in fruit-based baby food was developed. The fast and inexpensive buffered QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction method and "conventional" approach that employs ethyl acetate extraction followed by gel permeation chromatography (GPC) cleanup were employed for sample preparation. A PTV injector in solvent venting mode was used to reduce volume of acetonitrile and acetic acid (from the buffered QuEChERS extracts) that caused higher column bleed without their elimination. Otherwise, the time-to-digital converter would become saturated in HR-TOF-MS. For fast GC separation allowing analysis of 100 analytes within a 7 min runtime, both a high temperature programming rate and vacuum conditions in a megabore GC column were employed. The use of HR-TOF-MS allowed the unbiased identification and reliable quantification of target analytes through the application of a narrow mass window (0.02 Da) for extracting analyte ions and the availability of full spectral information even at very low levels. With only a few exceptions, the lowest calibration levels for the pesticides tested were

Feasibility of LC/TOFMS and elemental database searching as a spectral library for pesticides in food.

Traditionally, the screening of unknown pesticides in food has been accomplished by GC/MS methods using conventional library-searching routines. However, many of the new polar and thermally labile pesticides are more readily and easily analysed by LC/MS methods and no searchable libraries currently exist (with the exception of some user libraries, which are limited). Therefore, there is a need for LC/MS libraries that can detect pesticides and their degradation products. This paper reports an identification scheme using a combination of LC/MS time-of-flight (accurate mass) and an Access database of 350 pesticides that are amenable to positive ion electrospray. The approach differs from conventional library searching of fragment ions. The concept consists of three parts: (1) initial screening of possible pesticides in actual market-place fruit extracts (apple and orange) using accurate mass and generating an accurate mass via an automatic ion-extraction routine, (2) searching the Access database manually for screening identification of a pesticide, and (3) identification of the suspected compound by accurate mass of at least one fragment ion and comparison of retention time with an actual standard. Imazalil and iprodione were identified in apples and thiabendazole in oranges using this database approach.

Application of comprehensive two-dimensional gas chromatography with nitrogen-selective detection for the analysis of fungicide residues in vegetable samples.

Comprehensive two-dimensional gas chromatography (GCxGC) with nitrogen-phosphorus detection (NPD) has been investigated for the separation and quantitation of fungicides in vegetable samples. The detector gas flows (H(2), N(2) and air) were adjusted to achieve maximum response of signal whilst minimizing peak width. The comparison of different column sets and selection of the temperature program were carried out with a mixture of nine N-containing standard fungicides, eight of which were chlorinated. The results from GCxGC-NPD and GCxGC with micro electron-capture detection (muECD) were compared. External calibrations of fungicides were performed over a concentration range from 1 to 1,000 microgL(-1). The peak area calibration curves generally had regression coefficients of R(2)>0.9980, however for iprodione which was observed to undergo on-column degradation, an R(2) of 0.990 was found. The limit of detection (LOD) and limit of quantitation (LOQ) were less than about 74 and 246 ng L(-1), respectively. The intra-day and inter-day RSD values were measured for solutions of concentration 0.100, 0.500 and 1.50 mg L(-1). For the 0.500 mg L(-1) solution, intra- and inter-day precision of peak area and peak height for most of the pesticides were about 2% and 8%, respectively. Excellent linearity was observed for these standards, from 0.001 to 25.00 mg L(-1). The standard mixture peak positions were identified by using GCxGC with quadrupole mass spectrometry (qMS). To illustrate the potential and the versatility of both GCxGC-NPD and GCxGC-muECD, the method was applied to determination of fungicides in a vegetable extract. Decomposition of one fungicide standard (iprodione) during chromatography elution was readily observed in the two-dimensional (2D) GCxGC plot as a diagonal ridge response in the 2D chromatogram between the degrading compound and the decomposition product.

Development of an SPME-GC-MS/MS method for the determination of pesticides in rainwater: laboratory and field experiments.

A solid-phase microextraction -- coupled to a gas chromatography -- ion trap tandem mass spectrometry (SPME-GC-MS/MS) method was developed for the quantitative determination in rainwater of 8 pesticides amongst the most used in France and 3 triazines metabolites. The main factors affecting the SPME process were studied. Using a 3 mL sample, the method developed showed good linearity for concentrations ranging from 0.05 to 50 microgL(-1) with correlation coefficients between 0.997 and 0.9999 and relative standard deviations (% RSD) below 14%. The study of matrix effects showed that rainwater was too diluted to have any significant influence on the extraction efficiency. To validate the method, a field campaign was carried out on the rain events, which occurred in Strasbourg during a one-year period. The rain concentrations showed patterns of high pesticide concentrations during spring months, which were correlated to the spraying periods of most of these substances.

Evaluation of multiwavelength chromatograms for the quantification of mixtures of pesticides by high-performance liquid chromatography-diode array detection with multivariate calibration.

Three multivariate calibration methods, partial least squares (PLS-1 and PLS-2) and principal component regression, were applied to the simultaneous determination of the five pesticides iprodione, procymidone, chlorothalonil, folpet and triazophos by high-performance liquid chromatography with diode array detection. Such detection gives multiwavelength chromatograms from a single analysis of one sample. In this paper, calibration models at two different wavelengths were developed to resolve mixtures of five pesticides with overlapping chromatographic peaks. The first model, carried out at 220 nm as detector compromise wavelength, yielded satisfactory sensitivity for accurate estimation of the concentration of iprodione, procymidone, chlorothalonil and folpet and the second model, at 200 nm, was used for accurate estimation of triazophos. Both calibration models were evaluated using the chromatograms and first-derivative (1D) chromatograms by predicting the concentrations of independent test set samples. Finally, the proposed 1D calibration models were successfully applied to the determination of these pesticides in groundwater and soil samples. In all cases, the PLS-1 calibration method showed superior quantitative prediction ability than the PLS-2 or principal component regression methods.

Application of porous carbon for solid-phase extraction of dicarboxyimide fungicide residues from wines in combination with high-resolution capillary gas chromatography and gas chromatography-mass spectrometry.

Solid-phase extraction with a novel porous carbon sorbent CARB GR was used for the clean-up step of dicarboxyimide fungicides residues from variety of Slovak grape wines with subsequent capillary gas chromatography-flame ionization detection, -electron-capture detection (ECD) and -mass spectrometry-ion-trap detection (MS-ITD) analysis. Recovery was tested at various concentration levels of vinclozolin and iprodione in standard solutions (R = 80-97%, R.S.D. < or = 5). The value of recovery in spiked wines is dependent on concentration level (studied in the range of 5.9 micrograms/1-1.96 mg/l) and on the variety of wine (R = 80-96%; R.S.D. = 3-5%). Limits of quantitation (for sample volume 50 ml) were determined to be with GC-ECD for both fungicides in ppt range and with GC-MS-ITD in the multiple ion detection mode monitoring in ptt range for vinclozolin and ppb range for iprodione. Concentration levels of vinclozolin residues were determined in treated wines (with 0.1% Ronilan 50 WP) as well as iprodione residues (with 0.15% Rovral 50 WP) and a strong dependence on the protective term before the harvest is shown.

A sensitive assay for the aminoimidazole-containing drug GP531 in plasma using liquid chromatography with amperometric electrochemical detection: a new class of electroactive compounds.

Aminoimidazole-containing compounds have been found to be electroactive and can be detected by amperometric electrochemical detection (ECD) with a high degree of sensitivity. A liquid chromatography (LC) method using ECD was developed for measuring plasma concentrations of the aminoimidazole-containing drug GP531, a potent adenosine-regulating agent. Plasma samples were extracted with 2-propanol and analyzed by LC under isocratic conditions using a mobile phase of methanol-sodium phosphate (pH 6.3; 3.3 mM) (32:68, v/v). The potential of the glassy carbon working electrode was set at +800 mV. The limit of quantitation was 12.5 ng ml-1 of GP531 using 100 microliters of plasma. The method was used to define the pharmacokinetics of GP531 in monkey following i.v. administration.

Capillary electrophoresis for screening of adenylosuccinate lyase deficiency.

We report a new screening method for adenylosuccinate lyase (ASase) deficiency using capillary electrophoresis (CE). This enzyme defect causes secondary autism and psychomotor retardation in early childhood. In all body fluids of these patients, two succinylpurine metabolites can be found that are normally not detectable: succinyladenosine and succinylaminoimidazole carboxamide (SAICA) riboside. A Beckman P/ACE 2050 capillary electrophoresis system was used with a 47.1 cm capillary, 75 microns ID, and the P/ACE Beckman UV absorbance detector. Untreated urine, injected for 1 s, was separated in a pH 8.63 borate buffer at 20 kV. The two succinylpurines (migration times 13.36 and 13.60 min) were detected at 254 nm only in urine of patients with ASase deficiency but not in control samples.

Clinical pharmacokinetics of high-dose DTIC.

The pharmacokinetics of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC, dacarbazine) given at a dose of 850-1,980 mg/m2 as a 10- to 30-min infusion was studied in cancer patients, and the plasma concentration-time curves were adjusted to a two-compartment model, with a mean t1/2 alpha value of 0.17 h (range, 0.1-0.26 h) and a mean t1/2 beta value of 2 h (range, 1.5-2.7 h) being found. The mean volume of the central compartment of (Vc) and the apparent volume of distribution (VB) were 0.42 1 kg-1 (range, 0.24-0.54 1 kg-1) and 1.49 1 kg-1 (range, 0.88-1.74 1 kg-1), respectively. The mean total body clearance of DTIC was 0.58 1 kg-1 h-1 (range, 0.26-0.82 1 kg-1 h-1), and the mean renal clearance was 0.28 1 kg-1 h-1 (range, 0.17-0.49 1 kg-1 h-1). Unchanged DTIC recovered from urine within 24 h varied from 11% to 63% of the delivered dose, with an inverse correlation being found between the DTIC dose and the amount excreted. The metabolite aminoimidazole carboxamide (AICA) was detectable in plasma from the start of DTIC infusion, and its concentration-time curve showed a monophasic decay, exhibiting a mean t1/2 value of 3.25 h (range, 1.77-5.82 h). Mean AICA renal clearance was 0.15 1 kg-1 h-1 (range, 0.05-0.32 1 kg-1 h-1). The amount of AICA excreted in urine increased with increasing DTIC dose and varied from 1.2% to 13.6% of the delivered DTIC dose. Both DTIC distribution and disposition and AICA production and renal excretion seemed to be limited after high DTIC doses as compared with the pharmacokinetics of low-dose DTIC. Nonlinear pharmacokinetics for high-dose DTIC could not be clearly excluded.

Chromatographic analysis of purine precursors in mouse L1210 leukemia.

A number of antagonists of nucleotide metabolism with anti-cancer activity affect the de novo purine pathway. To determine the biochemical mechanisms of cytotoxicity of these drugs, assay procedures have been developed for measurement of the levels of intermediates proximal to IMP in the pathway for de novo purine biosynthesis in mouse L1210 leukemia cells. Purine precursors have been synthesized in vitro from [14C]glycine using enzymes from chicken liver. These 14C-labeled intermediates have been used as marker compounds to define retention times for metabolites of leukemia cells separated by HPLC and the chromatographic mobilities of these intermediates after two-dimensional thin-layer chromatography. These new chromatographic procedures have been used in combination to determine the steady-state concentrations for purine precursors in mouse L1210 leukemia cells in the exponential phase of growth: N-formylglycineamide ribotide (16 microM); N-formylglycineamidine ribotide (4.7 microM); 5-aminoimidazole ribotide (4.0 microM); 4-carboxy-5-aminoimidazole ribotide (0.46 microM); N-succino-5-aminoimidazole-4-carboxamide ribotide (11 microM); 5-aminoimidazole-4-carboxamide ribotide (16 microM); 5-formamidoimidazole-4-carboxamide ribotide (2.7 microM); and IMP (57 microM). The metabolic effects of tiazofurin (25 microM) upon mouse L1210 leukemia cells growing in culture define a "metabolic crossover point" at the reaction catalyzed by IMP dehydrogenase (EC 1.1.1.205) which confirms previous reports of inhibition of this enzyme.

Purine de novo synthesis as the basis of synergism of methotrexate and 6-mercaptopurine in human malignant lymphoblasts of different lineages.

Methotrexate (MTX) causes an inhibition of purine de novo synthesis (PDNS), resulting in increased intracellular availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in human malignant lymphoblasts with an active PDNS. Normal bone marrow cells and peripheral blood lymphocytes lack this capacity. The increased levels of PRPP can be used for enhanced incorporation of 6-mercaptopurine (6MP), indicating a potential time-, sequence- and dose-dependent synergism of both drugs. The effects of 0.02 microM and 0.2 microM MTX on the PDNS of MOLT-4 (T-), RAJI (B-) and KM-3 (non-B-non-T-) human malignant lymphoblasts were studied with respect to PRPP levels, aminoimidazolecarboxamide ribonucleosidemonophosphate (AICAR) levels and the incorporation of labeled glycine into purine metabolites. These results were correlated with the activity of the PDNS (labeled glycine incorporation) and the purine salvage pathway (labeled hypoxanthine incorporation) in untreated cells. Inhibition of PDNS by 0.02 microM MTX was complete in KM-3 cells with a moderately active PDNS and salvage pathway. RAJI cells, with a relatively low PDNS and high salvage pathway, demonstrated an incomplete, but increasing inhibition of PDNS, whereas inhibition of PDNS in MOLT-4 cells with both pathways active was minimal and recovered in time. Treatment with 0.2 microM MTX resulted in a complete inhibition of PDNS in all cell lines. After treatment with MTX an enhanced incorporation of labeled hypoxanthine and 6MP was noticed, confirming the potential rescue from MTX cytotoxicity by hypoxanthine and a potential synergism of MTX and 6MP on cytotoxicity. The enhanced incorporation of 6MP was more obvious in RAJI and KM-3 cells in comparison with MOLT-4 cells. These data demonstrate the important role of both the activities of the PDNS and the purine salvage pathway in malignant lymphoblasts of different subclasses with respect to the synergism of MTX and 6MP.