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N-Carbamoylimidazoles dissociate in solution to yield imidazole and an isocyanate that may be reacted with another aryl amine to form an unsymmetrical biaryl urea. This paper investigates the reaction kinetics and the influence of electron withdrawing/donating substituents on the reaction of N-carbamoylimidazoles with aniline. The overall reaction mechanism involves two zwitterionic intermediates, formed during dissociation and upon reaction of the liberated isocyanate with aniline. The rate limiting step for the reaction is a base catalysed proton transfer from the second zwitterionic intermediate. Although electron withdrawing substituents on the aryl group hinder dissociation, they significantly increase reaction rates compared to compounds bearing electron donating substituents. The imidazole liberated upon dissociation catalyses the rate determining step so that reactions of dissociated N-carbamoylimidazoles proceed more rapidly than those involving only isocyanates. In addition, the imidazole eliminates the need for anhydrous reaction conditions. The N-carbamoylimidazole methodology was demonstrated by preparing sorafenib, a biaryl urea kinase inhibitor, in good yield and excellent purity.

BACKGROUND AND PURPOSE Imatinib is a clinically important inhibitor of tyrosine kinases that are dysregulated in chronic myelogenous leukaemia and gastrointestinal stromal tumours. Inter-individual variation in imatinib pharmacokinetics is extensive, and influences drug safety and efficacy. Hepatic cytochrome P450 (CYP) 3A4 has been implicated in imatinib N-demethylation, but the clearance of imatinib decreases during prolonged therapy. CYP3A phenotype correlates with imatinib clearance at the commencement of therapy, but not at steady state. The present study evaluated the possibility that multiple CYPs may contribute to imatinib oxidation in liver. EXPERIMENTAL APPROACH Imatinib biotransformation in human liver microsomes (n= 20) and by cDNA-expressed CYPs was determined by LC–MS. Relationships between imatinib N-demethylation and other drug metabolizing CYPs were assessed. KEY RESULTS N-desmethylimatinib formation was correlated with microsomal oxidation of the CYP3A4 substrates testosterone (ρ= 0.60; P < 0.01) and midazolam (ρ= 0.46; P < 0.05), and the CYP2C8 substrate paclitaxel (ρ= 0.58; P < 0.01). cDNA-derived CYPs 2C8, 3A4, 3A5 and 3A7 supported imatinib N-demethylation, but 10 other CYPs were inactive; in kinetic studies, CYP2C8 was a high-affinity enzyme with a catalytic efficiency ∼15-fold greater than those of CYPs 3A4 and 3A5. The CYP3A inhibitors ketoconazole and troleandomycin, and the CYP2C8 inhibitors quercetin and paclitaxel decreased imatinib oxidation. From molecular modelling, the imatinib structure could be superimposed on a pharmacophore for CYP2C8 substrates. CONCLUSIONS AND IMPLICATIONS CYP2C8 and CYPs 3A contribute to imatinib N-demethylation in human liver. The involvement of CYP2C8 may account in part for the wide inter-patient variation in imatinib pharmacokinetics observed in clinical practice.

The aim of this study was to examine the accuracy of patients’ accounts of their psychoactive substance use in the week before admission to an acute mental health unit. Fifty consecutively admitted patients undertook a semistructured clinician-administered questionnaire for recent substance use. The results of the interview were compared to the results of gas chromatography mass spectrometry/ mass spectrometry. No patient refused to participate, 46 patients were able to complete the interview, and 48 patients provided a blood sample. Six patients had unreported cocaine or amphetamine in their blood. Cannabis and opiates were not detected in the blood of most of the patients who reported using these drugs. The self-reports of the use of prescription sedatives were inaccurate, but self-reports of recent use of caffeine, nicotine, antidepressants, antipsychotics, and carbamazepine had good overall reliability. Patient reports of recent illicit substance use at the point of admission were found to be unreliab...

Background Commercial omeprazole paediatric mixtures are not available. The stability of omeprazole mixtures prepared from Probitor capsules or omeprazole powder has not been established. Aim To determine the physicochemical stability of two omeprazole mixtures, prepared from Probitor capsules or omeprazole powder BP, over a 45–day period under refrigeration or at room temperature, shaken or unshaken. Method Omeprazole 2 mg/mL mixtures were prepared using standard procedures by either emptying the contents of Probitor capsules or by adding omeprazole powder to preserved sodium bicarbonate 8.4% solution. Each formulation was stored in 100 mL amber glass bottles under refrigeration (2–8°C) or at room temperature (21 ± 2°C). One bottle of each formulation was shaken and the other not shaken, pH was measured and colour change determined using a visual scale. Omeprazole concentrations were measured using liquid chromatograph tandem mass spectrometry on Days 1, 7, 14, 28 and 46. Results Omeprazole 2 mg/mL mixture prepared from Probitor capsules was stable over 45 days when refrigerated and regularly shaken. When unshaken, the omeprazole concentration decreased rapidly over 7 days. Mixtures prepared from omeprazole powder had lower concentrations compared to the mixtures prepared from Probitor capsules, but tended to be more stable when stored at room temperature. Observed colour changes did not correlate with measured omeprazole concentrations and pH remained constant. Conclusion Omeprazole mixture prepared from Probitor capsules was stable over 45 days, when refrigerated and regularly shaken. Omeprazole concentration markedly decreased in the mixture prepared from omeprazole powder when refrigerated.

Methyl esters of C18 polyunsaturated fatty acids, including gamma-linolenic acid, alpha-linolenic acid and stearidonic acid, were epoxidised using m-chloroperbenzoic acid. Nine monoepoxides were obtained by normal-phase HPLC, identified using LC-MS and NMR, and characterized by NMR spectroscopy and mass spectrometry. This study is focused on structural characterization using LC-MS and LC/APCI/MS/MS. The elution profiles of these monoepoxides in RP-HPLC are determined as 12,13-9,10-6,7-epoxy, 9,10-15,16-12,13-epoxy and 15,16-12,13-9,10-epoxy derivatives of gamma-linolenic, alpha-linolenic and stearidonic acid methyl esters, respectively. The major diagnostic fragmentations in MS/MS identified are postulated to be induced by cleavages of the epoxide ring and alpha-bond cleavage to the epoxy group from [M+H]+ and/or [M+H-MeOH]+.

A highly sensitive and selective liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry assay was developed and validated for simultaneous determination of epimeric budesonide (BUD) and fluticasone propionate (FP) in plasma. The drugs were isolated from human plasma using C18 solid-phase extraction cartridges, and epimeric BUD was acetylated with a mixture of 12.5% acetic anhydride and 12.5% triethylamine in acetonitrile to form the 21-acetyl derivatives following the solid-phase extraction. Deuterium-labelled BUD acetate with an isotopic purity >99% was synthesized and used as the internal standard. The assay was linear over the ranges 0.05–10.0 ng/ml for epimeric BUD, and 0.02–4.0 ng/ml for FP. The inter- and intra-day relative standard deviations were

Introduction: Tacrolimus is a macrolide immunosuppressant that has a narrow therapeutic index, displays considerable variability in response, and has the potential for serious drug interactions. Therapeutic drug monitoring and dose individualisation for tacrolimus is complicated but essential. Few studies have investigated the blood distribution and protein binding of tacrolimus and the results of these studies are conflicting. The aim of the present study is to establish and validate methods to investigate the distribution of tacrolimus in human blood. To conduct these studies at clinically relevant concentrations the use of 3 H-dihydro-tacrolimus instead of tacrolimus was investigated. Methods: The use of radiolabelled tacrolimus was validated by conducting studies with a mixture of both labelled and unlabelled drug where tacrolimus was analysed by LC-MS/MS. The in vitro distribution of tacrolimus and 3 H-dihydro-tacrolimus was investigated in blood collected from healthy subjects using Ficoll–Paque reagent and density gradient ultracentrifugation, respectively. The unbound fraction of tacrolimus in plasma was studied using equilibrium dialysis conducted at 37 °C. Results: In blood, tacrolimus was found to be mainly associated with erythrocytes (85.3±1.5%), followed by diluted plasma proteins (14.3±1.5%) and lymphocytes (0.46±0.10%). In plasma, tacrolimus was found to mainly be associated with the soluble protein fraction (61.2±2.5%), high-density lipoproteins (HDL, 28.1±5.4%), low-density lipoproteins (LDL, 7.8±1.6%), and very low-density lipoproteins (VLDL, 1.4±0.3%). The unbound fraction of tacrolimus was found to be only 1.2±0.12%. Statistical comparison indicated that there was no significant difference in the blood distribution and plasma protein binding of 3 H-dihydro-tacrolimus when compared with tacrolimus. Discussion: These results have important implications for therapeutic drug monitoring of tacrolimus and subsequent studies of tacrolimus distribution in transplant recipients.

Low level exposure to organophosphate (OP) pesticides can be determined by the measurement of dialkylphosphate (DAP) metabolites in urine. An analytical method is presented here which can measure the metabolites dimethyl phosphate (DMP), diethyl phosphate (DEP), dimethyl thiophosphate (DMTP), dimethyl dithiophosphate (DMDTP), diethyl thiophosphate (DETP), and diethyl dithiophosphate (DEDTP) at low levels. This was achieved by lyophilization of the urine, derivatization with pentafluorobenzyl bromide (PFBBr) and quantification by negative ion chemical ionization GC/MS-MS. The detection limits for the metabolites were 0.5 microg L(-1) DMP, 0.1 microg L(-1) DEP, 0.1 microg L(-1) DMTP, 0.04 microg L(-1) DMDTP, 0.04 microg L(-1) DETP and 0.02 microg L(-1) DEDTP. The RSD for the analytical method was 4-14% for the six metabolites. The method was used to monitor a group of non-occupationally exposed individuals in Sydney, Australia. The metabolites DMP, DEP, DMTP, DMDTP, DETP and DEDTP occurred in 73, 77, 96, 48, 100 and 2% of the samples with median values of 13, 3, 12,1, 1 and 1 microg L(-1) respectively. The method is simple to use, sensitive and suitable for routine analysis of non-occupational exposure levels. These detection limits are between one and two orders of magnitude lower than those previously reported in the literature.

Aims Pharmacokinetic variability is likely to be a significant factor contributing to the interindividual differences in dose requirements, anti-inflammatory response and side-effects with inhaled corticosteroids (ICS), but there is limited information about the disposition of ICS during regular dosing with a pressurized metered dose inhaler (pMDI). This study uses a mixed effects modelling approach to quantify and compare the interindividual variability in pharmacokinetics of epimeric budesonide (BUD) and fluticasone propionate (FP) after repeat-dose inhalation. Methods This pharmacokinetic substudy was part of a previously published open-label, randomised, placebo-controlled, 7-period crossover study to evaluate the short-term effects on plasma cortisol levels of inhaled BUD (400, 800, 1600 µg twice daily) and FP (375, 750, 1000 µg twice daily) via pMDI in a group of healthy male volunteers. On the fifth day of each high-dose treatment period (BUD 1600 µg twice daily and FP 1000 µg twice daily), venous blood samples were collected in nine subjects prior to the last dose and at 15 min, 30 min, 1, 2, 4, 6 and 8 h postdose for measurement of plasma drug concentrations to determine the pharmacokinetics of epimeric BUD and FP following inhalation. Non-compartmental analysis and a mixed effects model were used to characterize the disposition profiles. Results Both drugs had a rapid absorption half-life (BUD 10 min vs FP 11.3 min), but quite different elimination half-lives (BUD 2.4 h vs FP 7.8 h). Although there were intraindividual differences in the handling of the 22R-and 22S-epimers of BUD, there were no consistent pharmacokinetic differences between the two enantiomers in the group as a whole. Consistent with previous reports of FP’s higher volume of distribution (V) and lower systemic bioavailability (F), the V/F ratio was lower for BUD than FP (498 l vs 8100 l). The parameter with the greatest interindividual variability for both BUD and FP was the rate of systemic absorption from the lung. Conclusions This is the first report describing the pharmacokinetics of epimeric BUD and FP after repeat dose inhalation via pMDI. Three observations may be of clinical relevance: (1) there is considerable intersubject variability in the rate of absorption of both drugs from the lung; (2) in some individuals there was a long t½,z for BUD, resulting in higher and more sustained plasma drug levels in the 4–12 h postdose period than would be predicted from single-dose pharmacokinetic data; and (3) there is evidence of diurnal variation in FP pharmacokinetics, with higher-than-expected plasma drug concentrations in the morning compared with the evening.

A highly sensitive and selective method has been developed for the quantification of fluticasone propionate (FP) in human plasma. The drug was isolated from human plasma using C 18 solid-phase extraction cartridges. The analysis was based on high-performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry (HPLC/APCI/MS), using the 22R epimer of budesonide (BUD) acetate, synthesised using acetic anhydride, as internal standard. The mass spectrometer was operated in APCI mode with selected ions at tune masses of 473.2 and 501.2 m / z , corresponding to the MH + of acetylated (22R)BUD and FP, respectively. The mobile phase used was a mixture of 50% ethanol in water with a flow rate of 0.45 ml min −1 . The system was optimised by tuning the capillary and tube lens with a concentrated solution of FP. The recovery of FP from human plasma was 86.3%. Linearity of response was obtained over the concentration range 0.2–4.0 ng ml −1 . The intra-assay and inter-assay variability were 6.3 and 2.9%, respectively. The lower limit of quantification was 0.2 ng ml −1 when a solid-phase extraction preceded the HPLC/APCI/MS.

The tyrosine kinase inhibitor drug sorafenib is used in the treatment of liver and renal cancers but adverse effects may necessitate dose interruption and under-dosage may lead to therapeutic failure. Sorafenib also undergoes cytochrome P450 (CYP)-dependent biotransformation to the N-oxide and other metabolites. However, although CYPs are major determinants of efficacy and toxicity the roles of these enzymes in the formation of multiple sorafenib metabolites are unclear. In the present study CYP-mediated pathways of sorafenib oxidation in human liver were evaluated. cDNA-expressed CYP3A4 was the major catalyst in the formation of the principal N-oxide and N-hydroxymethyl metabolites of sorafenib, as well as the minor N-desmethyl metabolite. In contrast, CYP3A5 exhibited only ∼5% of the activity of CYP3A4 and eleven other CYPs and three flavin-containing monooxygenases were inactive. In human hepatic microsomes metabolite formation was correlated with CYP3A4-mediated midazolam 1′-hydroxylation, but not with other CYP-specific substrate oxidations. In accord with these findings the CYP3A4 inhibitor ketoconazole selectively inhibited microsomal sorafenib oxidation pathways. From computational modeling studies atoms in the structure of sorafenib that undergo biotransformation were within ∼5.4 of the CYP3A4 heme. Important hydrogen bonding interactions between sorafenib and amino acids Ser-119 and Glu-374 in the active center of CYP3A4 were identified. These findings indicate that sorafenib is oxidized selectively by human CYP3A4. This information could be adapted in individualized approaches to optimize sorafenib safety and efficacy in cancer patients. © 2012 Elsevier Inc.

Fullerite (a mix of crude C[sub 60] and C[sub 70]) has been hydrogenated using hydrogen radical species (H[sup [sm bullet]]) at elevated temperatures (400[degrees]C) and hydrogen pressures (6.9 MPa (cold)). Iodoethane was employed as the hydrogen radical promoter. Analysis of the reduced fullerite by fast atom bombardment mass spectrometry revealed a mixture of hydrofullerenes comprising mainly C[sub 60]H[sub 36] and C[sub 70]H[sub 36]. No spectral evidence for the ethylation or iodination of the fullerite was found. Prepared under these conditions, the product is only sparingly soluble in common solvents. Solid-state [sup 13]C nuclear magnetic resonance spectroscopy and infrared spectroscopy were used to characterize the reduced fullerite. The structure of the C[sub 60]H[sub 36] produced under these conditions is of lower symmetry than T[sub h] and most likely D[sub 3d] or C[sub 3i] symmetry. 18 refs., 3 figs.

The phenotyping cocktail is a practical approach for phenotyping of cytochrome P450 (CYP) enzymes in vivo. In this study, a liquid chromatography-tandem mass spectrometry method using a dual-extraction approach was developed and validated to quantify 5 selective substrates and their metabolites for the simultaneous phenotyping CYPs 1A2, 2C19, 2C9, 2D6, and 3A4 in patient blood samples. The assay was applied in a pilot study of 11 patients with schizophrenia. Five blood samples were collected before and at 1, 2, 4, and 6 hours after administration of a phenotyping cocktail consisting of 100 mg caffeine, 20 mg omeprazole, 25 mg losartan, 30 mg dextromethorphan, and 2 mg midazolam. The method successfully quantitated the CYP enzyme activities without serious side effects in patients. The ratios of metabolite to parent area under the concentration-time curve values were calculated over the 6-hour postdosage to reflect CYP2D6, CYP3A4, and CYP2C9 activities. The ratios of metabolite to parent plasma concentrations were calculated at 4-hour postdosage for CYP1A2 and at 4- or 6-hour postdose for CYP2C19, respectively. The plasma concentration of midazolam at 4 hours was also estimated as another phenotyping index for CYP3A4 activity. The simultaneous assay of all these analytes in a single matrix (plasma) will increase the feasibility of CYP phenotyping in patients.

The activities of cytochrome P450-derived epoxide metabolites of omega-6 polyunsaturated fatty acids (PUFAs) in cellular homeostasis have generated considerable topical interest, but there is less information on the effects of omega-3 PUFA epoxides. Mass spectroscopic data on the epoxides of the omega-3 PUFA eicosapentaenoic acid (EPA) have been reported but the absence of corresponding NMR data currently hinders their biological assessment. In the present study five monoepoxy derivatives of EPA methyl ester were synthesized by treating EPA methyl ester with m-chloroperbenzoic acid. The individual regioisomers were purified by normal-phase chromatography and characterized by LC-MS/MS and a combination of NMR approaches including (1)H-, (13)C-, (1)H-(1)H-COSY, (1)H-(13)C-HSQC, and (1)H-(13)C-HMBC. The chromatographic properties for these monoepoxides were studied in normal-phase and reversephase-HPLC systems and the MS/MS fragmentation patterns using electrospray ionization were established. This paper also focuses on the NMR characterization of epoxide, olefinic and methylenic moieties and the complete assignments of the isomers.

Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones--desmethoxyyangonin, methysticin and kavain--in rat liver microsomes using diazepam as an internal standard; liquid-liquid extraction was used for sample preparation and analysis was performed on a Shimadzu 10A high-performance liquid chromatography system. The analysis was carried out in reversed-phase mode with a Luna C(18) column (150 x 2.00 mm, 3 microm) at 40 degrees C. The limit of quantitation was 0.1 microg/mL using 0.25 mL of microsomal solution. The assays were linear over the range 0.1-10 microg/mL for desmethoxyyangonin, methysticin and kavain. Quality control samples exhibited good accuracy and precision with relative standard deviations lower than 15% and recoveries between 85 and 105%. The assays exhibited satisfactory performance with high sensitivity for quantifying desmethoxyyangonin, methysticin and kavain in rat liver microsomes and were successfully used to determine the three kavalactones and their microsomal metabolites.

A sensitive, rapid LC-MS/MS assay has been developed and validated for the simultaneous quantification of CPT-11 and its two principal metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38), and 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxy-camptothecin (APC) in human liver microsomal fractions and plasma. The method was linear over the ranges of 1.56-100 ng/mL, 3.13-150 ng/mL, and 0.78-100 ng/mL for CPT-11, SN-38, and APC, respectively. The total run time was 7.0 min. This assay offers advantages in terms of expediency, recovery of analytes, and suitability for the analysis of CPT-11 and its metabolites in various biological fluids.

This study investigates the association of liposomal amphotericin B (L-AmB) with plasma proteins and its impact on the pharmacokinetics of L-AmB in paediatric patients with malignant diseases.Paediatric oncology patients (n = 39) who received multiple-doses of L-AmB were recruited into this study. The association of the drug with plasma lipoprotein was investigated using single vertical spin density gradient ultracentrifugation and quantitated with a validated HPLC assay. The unbound amphotericin B (AmB) in the plasma was separated by ultrafiltration and determined with a validated LC/MS/MS assay.The ex vivo lipoprotein distribution of L-AmB found that 68.3 +/- 11.8% of the drug was associated with the high density lipoprotein (HDL) fraction, which demonstrated a significant inverse correlation with posterior Bayesian estimates of L-AmB clearance (r = -0.690, p0.01). The average of unbound fraction of AmB in plasma of patients administered with L-AmB was 0.005, but its relationship with L-AmB clearance did not reach a statistical significance.L-AmB displays different lipoprotein distribution profile from that of the conventional AmB formulation, with L-AmB preferentially associated with HDL in plasma. The inverse correlation of L-AmB clearance to its HDL distribution contributes to the difference in the pharmacokinetic profile of L-AmB.

Fullerite (a mix of crude C[sub 60] and C[sub 70]) has been hydrogenated using hydrogen radical species (H[sup [sm bullet]]) at elevated temperatures (400[degrees]C) and hydrogen pressures (6.9 MPa (cold)). Iodoethane was employed as the hydrogen radical promoter. Analysis of the reduced fullerite by fast atom bombardment mass spectrometry revealed a mixture of hydrofullerenes comprising mainly C[sub 60]H[sub 36] and C[sub 70]H[sub 36]. No spectral evidence for the ethylation or iodination of the fullerite was found. Prepared under these conditions, the product is only sparingly soluble in common solvents. Solid-state [sup 13]C nuclear magnetic resonance spectroscopy and infrared spectroscopy were used to characterize the reduced fullerite. The structure of the C[sub 60]H[sub 36] produced under these conditions is of lower symmetry than T[sub h] and most likely D[sub 3d] or C[sub 3i] symmetry. 18 refs., 3 figs.

A routine gas chromatographic (GC) method is described for the analysis of dialkylphosphate metabolites in the urine of workers occupationally exposed to organophosphorus insecticides. The procedure involves derivatizing a freeze-dried urine sample with pentafluorobenzyl bromide and then determining the metabolites using dual capillary column GC with flame photometric detection.

The photodegradation of amiloride hydrochloride in deaerated aqueous solution at 30 degrees C in the pH range 4.5-11.0 was studied by spectrophotometry and reversed-phase HPLC. The neutral form of the drug present in alkaline solution degraded approximately 3-fold faster than the cationic form. The quantum yields for photodegradation of neutral amiloride and its conjugate acid were determined using ferrioxalate actinometry to be 0.023+/-0.002 and 0. 009+/-0.001, respectively. The initial photoreaction involves dechlorination of amiloride and the major product is N-amidino-3, 5-diamino-6-hydroxylpyrazine-carboxamide, established by UV, 1H and 13C NMR, and chemical ionization-mass spectrometry. The mechanism of photolysis is postulated to involve cation radical formation that facilitates the dechlorination step. The photosensitizing activity of amiloride hydrochloride was tested by measuring (a) the rate of oxygen uptake in the presence of singlet oxygen substrates, 2, 5-dimethylfuran or l-histidine, and (b) the rate of free radical polymerization of acrylamide, at 30 degrees C in aqueous solution. Photosensitization by amiloride was concluded to occur predominantly via singlet oxygen rather than a free radical mechanism. However, amiloride is a much weaker photosensitizer than other diuretics such as frusemide and hydrochlorothiazide, tested under the same experimental conditions.

A highly sensitive and selective method has been developed for the simultaneous quantification of 22R- and 22S-epimers of budesonide in human plasma. The drug was isolated from human plasma using C18 solid-phase extraction cartridges and was acetylated with a mixture of 12.5% acetic anhydride and 12.5% triethylamine in acetonitrile to form the 21-acetyl derivatives. Deuterium-labelled budesonide was synthesized and determined to have an isotopic purity > 99%. This was used as the internal standard. Epimers were quantified by automated liquid chromatography-atmospheric pressure chemical ionization mass spectrometry, operating in selected ion mode at m/z 473.2 and m/z 476.2. Linear responses were observed for both epimers over the range 0.25 to 10.0 ng/ml. The average recoveries of 22R- and 22S-epimers of budesonide from human plasma were 87.4% and 87.0%, respectively. The lower limit of quantification for each epimer was 0.25 ng/ml, corresponding to 50.0 pg of analyte on column. Within- and between-day coefficients of variation were 8.6% and 4.0%, respectively.

The use of chemical ionization mass spectrometry for the characterization of analogues of methotreate, N-[4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid, has been studied. With methane as reactant gas, abundant [MH]+ ions were generally not produced. However, with ammonia, especially in conjunction with thermal desorption from a platinum wire, significant amounts of [MH]+ ions were formed by methotrexate, the α-, γ- and diamide analogues, and series of α- and γ-monoalkylamide derivatives. The t-butyl esters of the various monoamides behaved similarly to the corresponding monoamides, except for the ready loss of isobutylene. Fragment ions from both methane and ammonia chemical ionization were formed by cleavages ‘benzylic’ to the pteridine ring, by bond breakage at the amide bond between the aminobenzoyl and glutamyl moieties, and by fragmentations on both sides of this amide bond. Fragment ions from these processes, in conjunction with further disintegration of the glutamyl moiety, are diagnostic of the pteridine, aminobenzoyl and glutamyl moieties of the analogues. Examples of application to structural determination are given.

Breast cancer incidence is lower and survival is longer in Asian women residing in Japan, China, or the Philippines than Caucasian women residing in the United States. Phytoestrogen intake has been examined as a possible reason for the disparity in breast cancer incidence and survival. This study examined the association between phytoestrogen intake prior to diagnosis of breast cancer and indicators of breast cancer prognosis (tumor size, estrogen and progesterone receptor status, histological grade, lymphovascular invasion, nodal spread, and stage) in 128 women, aged 40-79 yr, newly diagnosed with invasive breast cancer. After controlling for significant confounding factors, higher intakes of phytoestrogens were associated with favorable indicators of breast cancer. In women with higher intakes of phytoestrogens, there was a 32% reduction in the odds of being diagnosed with any stage of cancer other than stage 1 (95% confidence interval, CI = 0.49-0.93; P = 0.02), a 38% reduction in odds of being diagnosed with positive lymphovascular invasion (95% CI = 0.40-0.95; P = 0.03), and a 66% increase in the odds of being diagnosed with a positive progesterone receptor (95% CI = 1.06-2.58; P = 0.03). We conclude that phytoestrogen intake prior to diagnosis may improve prognosis of breast cancer.