Intrinsic Dual-Emitting Carbon Quantum-Dot-Based Selective Ratiometric Fluorescent Mercaptopurine Detection.

Mercaptopurine (6-MP), an immunosuppressive drug, has been widely prescribed for treating leukemia and autoimmune diseases. The level of the 6-MP drug in body fluids is of great interest due to the severe health problems related to its overdose. This study used a facile microwave preparation route to synthesize carbon quantum dots (CQDs) using glutathione and formamide as carbon sources. The obtained monodispersed quantum dots showed dual fluorescence emission with a sensitive affinity toward the 6-MP drug. The sensor's response was optimized by tuning the temperature, pH, and volume ratio of the probe. The prepared ratiometric fluorescence method showed accurate measurements for determining mercaptopurine in aqueous solutions in the concentration range of 1.4-7.6 mg L-1 with the limit of detection of 1.3 mg L-1. The sensor's performance was assessed in complex solutions, human urine, and human plasma sample and recovery values in the range of 88-127% were obtained. The reliable dual fluorometric sensor showed promising results for 6-MP determination and potential application for the determination of other chemical and biochemical species.

Ratiometric fluorescent nanoprobe based on CdTe/SiO2/folic acid/silver nanoparticles core-shell-satellite assembly for determination of 6-mercaptopurine.

A sensitive and robust fluorescent assay of 6-MP is described which relies on the facile assembly of a fluorescence nanoprobe by design of silica nanosphere encapsulated CdTe quantum dots (CdTe QDs) as scaffold, coupling with chemically tethered folic acid (FA)-protected silver nanoparticles (AgNPs) that function as responsive element. In this way a stable ternary core-shell-satellite nanostructure with dual-emission signals can be established. On binding to the target molecules, 6-MP, FA molecules initially occupied by AgNPs are liberated to give dose-dependent fluorescence emission, which can further form a self-calibration ratiometric fluorescence assay using CdTe QDs as an internal reference. The nanoprobe color vividly changes from red to blue, enabling the direct visual detection. The linear concentration range is 0.15~50 µM with the detection limit of 67 nM. By virtue of the favorable selectivity and robust assays, the nanoprobe was applied to 6-MP detection in urine samples, with recoveries from 97.3 to 106% and relative standard deviations (RSD) less than 5%. Graphical abstract.

An enzymatic ratiometric fluorescence assay for 6-mercaptopurine by using MoS2 quantum dots.

A ratiometric fluorescence method is described for the determination of the anticancer drug 6-mercaptopurine (6-MP). The method is based on the use of fluorescent MoS2 quantum dots (MQDs) and of the enzyme horseradish peroxidase (HRP). In the absence of 6-MP, HRP catalyzes the oxidation of o-phenylenediamine (OPD) by H2O2 to form 2,3-diaminophenazine (DAP). This leads to quenching of the violet fluorescence of MQDs (measured at excitation/emission wavelengths of 360/415 nm), while the strong yellow fluorescence of DAP (peaking at 560 nm) becomes increasingly strong. In the presence of 6-MP, however, it will be preferentially oxidized by the HRP/H2O2 system to form a disulfide dimer. Hence, less H2O2 is available for the oxidation of OPD and less DAP will be formed. This results in the recovery of the violet fluorescence and a decrease of the yellow fluorescence. The ratio of the two signals can be used to quantify either H2O2 or 6-MP. Linear responses are observed for H2O2 in 0.5-140 µM concentration range, and for 6-MP in the 0.5-70 µM concentration range, with detection limits of 0.1 µM and 0.29 µM, respectively. The method was applied to the determination of 6-MP in spiked human urine and gave satisfactory results. Graphical Abstract Schematic of an enzymatic fluorometric method for determination of 6-mercaptopurine (6-MP). It is based on the presence of 6-MP that can inhibit the HRP-catalyzed oxidation of o-phenylenediamine (OPD) to form 2,3-diaminophenazine (DAP). Hence, the fluorescence resonance energy transfer (FRET) between DAP and MoS2 quantum dots (MQDs) is suppressed.

An efficient ratiometric fluorescence sensor based on metal-organic frameworks and quantum dots for highly selective detection of 6-mercaptopurine.

The development of a simple and accurate quantitative method for the determination of 6-mercaptopurine (6-MP) is of great importance because of its serious side effects. Ratiometric fluorescence (RF) sensors are not subject to interference from environmental factors, and exhibit enhanced precision and accuracy. Therefore, a novel RF sensor for the selective detection of 6-MP was developed based on a dual-emission nanosensor. The nanosensor was fabricated by combining a blue-emission metal-organic framework (MOF) NH2-MIL-53(Al) (λem=425nm) with green-emission 3-mercaptopropionic acid-capped CdTe quantum dots (MPA-CdTe QDs) (λem=528nm) under a single excitation wavelength (335nm). Upon addition of 6-MP, the fluorescence of NH2-MIL-53(Al) in the nanohybrid was selectively quenched due to strong inner filter effects, while the fluorescence of the MPA-CdTe QDs was enhanced. The novel RF sensor exhibited higher selectivity towards 6-MP than CdTe QDs alone, and higher sensitivity than MOFs alone. 6-MP could be detected in the range of 0-50µM with a detection limit of 0.15µM (S/N=3). The developed sensor was applied for the determination of 6-MP in human urine samples and satisfactory results were obtained. Overall, a novel and efficient fluorescence-based method was developed for the detection of 6-MP in biosamples.

Simultaneous determination of 6-mercaptopruine, 6-thioguanine and dasatinib as three important anticancer drugs using nanostructure voltammetric sensor employing Pt/MWCNTs and 1-butyl-3-methylimidazolium hexafluoro phosphate.

6-Mercaptopurine, 6-thioguanine and dasatinib are three important anticancer drugs with high adverse effects in human body. In this study, a Pt/MWCNTs-1-butyl-3-methylimidazolium hexafluoro phosphate-modified carbon paste electrode was developed for the simultaneously determination of 6-mercaptopurine, 6-thioguanine and dasatinib for the first time. The Pt/MWCNTs synthesized by polyol method and have been characterized by transmission electron microscopy and X-ray diffraction methods. The obtained data revealed that the electro-oxidation of 6-mercaptopurine, 6-thioguanine and dasatinib is facilitated as a novel voltammetric sensor. After optimization of electrochemical parameters employing this sensor at pH 8.0, the oxidation peak currents for 6-mercaptopurine, 6-thioguanine and dasatinib were found to vary linearly with their concentrations in the range of 0.05-550µM; 0.1-500µM and 5.0-500µM with detection limits of 0.009µM, 0.05µM and 1.0µM respectively using square wave voltammetric method. The modified electrode has been applied for the selective and precise analysis of 6-mercaptopurine, 6-thioguanine and dasatinib in pharmaceutical formulations and urine samples.

Resonance light scattering determination of 6-mercaptopurine coupled with HPLC technique.

A simple, fast, costless, sensitive and selective method of resonance light scattering coupled with HPLC was established for the determination of 6-mercaptopurine in human urine sample. In a Britton-Robinson buffer solution of pH5.5, the formation of coordination complex between 6-mercaptopurine and metal palladium (II) led to enhance the RLS intensity of the system. The RLS signal was detected by fluorescence detector at λ(ex)=λ(em)=315 nm. The analytical parameters were provided by the coupled system, the linear of 6-mercaptopurine response from 0.0615 to 2.40 µg L(-1) and the limit of detection (S/N=3) was 0.05 µg L(-1). The presented method has been applied to determine 6-mercaptopurine in human urine samples which obtained satisfactory results. Moreover, the reaction mechanism and possible reasons for enhancement of RLS were fully discussed.

Determination of urinary 6-mercaptopurine and three of its metabolites by HPLC-UV coupled with the iodine-azide reaction.

BACKGROUND: The presented method is able to determine 6-mercaptopurine (6-MP), 6-thioguanine, 6-mercaptopurine riboside and 6-thioguanine riboside in urine, and is thereby dedicated to control of thiopurine therapy of children with acute lymphoblastic leukemia. Good separation of the mentioned compounds was achieved on a C18 stationary phase with a sodium azide and sodium heptane sulfonate solution, acetonitrile and water at ratio of 50:1:49 (v/v/v). RESULTS: Coefficient of regression is >0.99 for all linearity ranges. LOD and LOQ are 0.3, 0.4, 0.3, 0.8 and 0.4, 0.6, 0.5 and 0.9 nmol/ml of urine for 6-MP, 6-thioguanine, 6-mercaptopurine riboside and 6-thioguanine riboside, respectively. Intra- and inter-day recovery and RSD are close to 100% and less than 10%, respectively, for all investigated thiopurines. CONCLUSION: The elaborated method was successfully applied for detection and quantitation of 6-MP and its selected metabolites in patients' urine samples.

Determination of 6-mercaptopurine in the presence of uric acid using modified multiwall carbon nanotubes-TiO2 as a voltammetric sensor.

In this work, a multiwall carbon nanotubes modified electrode (prepared by incorporating TiO(2) nanoparticles with p-aminophenol as a mediator) was used as voltammetric sensor for the determination of 6-mercaptopurine (6-MP) in the presence of uric acid (UA). The voltammograms of 6-MP and UA in a mixture can be separated from each other by differential pulse voltammetry with a potential difference of 380 mV at a scan rate of 10 mV s(-1). These conditions are sufficient to allow for the determination of 6-MP and UA both individually and simultaneously. The electrocatalytic currents increase linearly with 6-MP concentration in the ranges of 0.09-350 µmol L(-1) (two linear segments with different slopes). The detection limit for 6-MP was 0.065 µmol L(-1) . The RSD% for 1.0 and 15.0 µmol L(-1) 6-MP were 0.7%, and 1.2%, respectively. The kinetic parameters of the system were determined using electrochemical approaches. The method was successfully applied for the determination of 6-MP in drug sample, and 6-MP plus UA in urine samples.

HPLC analysis of azathioprine metabolites in red blood cells, plasma and urine in renal transplant recipients.

Anemia has been frequently reported in renal transplant recipients receiving azathioprine for immunosuppression and enalapril for treatment of hypertension. During the course of a prospective trial in such patients we determined azathioprine metabolites in erythrocytes, plasma, and urine as well as erythropoietin and hemoglobin levels in order to evaluate a potential interaction between these 2 drugs, possibly leading to anemia. Two specific high performance liquid chromatography (HPLC) methods for determination of azathioprine metabolites, both employing a mercurial cellulose resin for extraction, are presented. One method using a strong anion exchange column allows detection of 6-thioguanosine di- and triphosphate (thioguanine nucleotides) in red blood cells (RBC) with a sensitivity of 30 pmol/100 microliters RBC. 6-mercaptopurine (MP) and 6-thiouric acid (TUA) in plasma and urine were analyzed simultaneously by reversed-phase HPLC with a sensitivity of 5 ng/ml. The average (median values are given) steady state concentrations of thioguanine nucleotides in erythrocytes came to 267 pmol/100 microliters RBC (range 53-613) with and to 246 pmol/100 microliters RBC (range 39-629) without concomitant enalapril medication. Mean plasma concentrations of MP and TUA 3 hours after drug intake came to 14.8 +/- 9.9 ng/ml and 398 +/- 262 ng/ml, respectively, during enalapril comedication. Withdrawal of enalapril did not influence these metabolite levels coming to 15.3 +/- 9.1 and 451 +/- 253 after stopping enalapril treatment. Thioguanine nucleotides in RBCs were neither related to the dose of azathioprine given (r = -0.113, p > 0.05) nor to hemoglobin levels (r = 0.278, p > 0.05). However, azathioprine dose/kg body weight seemed to be related to hemoglobin concentration, with and without enalapril comedication. We conclude that enalapril therapy does not influence the measured azathioprine metabolites, the reported cases of anemia may rather be due to a pharmacodynamic interaction as shown by the significant increase in erythropoietin after withdrawal of enalapril. The assays described here are suitable to study the metabolism of azathioprine in patients with various diseases.

Kinetics of mercaptopurine and thioguanine nucleotides in renal transplant recipients during azathioprine treatment.

The purpose of this study was to examine the pharmacokinetics of mercaptopurine (6-MP) and thioguanine nucleotides (6-TGN) during azathioprine treatment. Plasma profiles and urinary excretion of 6-MP and 6-TGN concentrations in red blood cells (RBCs) were measured repeatedly during the first 3 weeks following transplantation in 10 adults, who had received kidney grafts from living related donors. Mean maximal 6-MP plasma concentration (Cmax) was 340 nmol/L (SD = 290), mean time to Cmax (Tmax) was 2 h (SD = 1.8), and mean area under the plasma concentration-time curve (AUC) was 930 nmol/L/h (SD = 770). The mean fraction of azathioprine dose excreted as 6-MP in urine was 1.32% (SD = 1.11). Up to eightfold variability of Cmax and AUC was observed from day to day within each patient. The correlation between 6-MP AUC and amount excreted in the urine was weak (r = 0.37, 95% CI from 0.02 to 0.64). In this group of patients the observed 6-TGN levels in RBCs were low; maxima during the observation period ranged from undetectable to 250 pmol/8 x 10(8) RBCs. In individual patients, 6-TGN levels were relatively stable throughout the dosing interval ("within-dose-interval-CV" < 19%), even when sharp and high 6-MP peaks in plasma were observed.

Assessment of exposure to chloramphenicol and azathioprine among workers in a South African pharmaceutical plant.

There have been very few published studies that have evaluated exposure to myelotoxic drugs among production workers in pharmaceutical plants. Previous studies have focussed mainly on nurses and evaluated exposure to cytotoxic drugs using urine mutagenicity as a marker of exposure. The aim of this study was to evaluate the exposure of workers involved in the production of chloramphenicol and azathioprine. Exposure was evaluated utilising biological monitoring, biological effect monitoring and environmental monitoring. Biological monitoring included plasma chloramphenicol levels, plasma 6-mercaptopurine and urine 6-thiouric acid levels. These were analysed using high performance liquid chromatography. Myelotoxic effect was assessed by measuring the haematological indices of bone marrow function. The exposed 17 workers were compared to matched controls of equal numbers. Neither substance could be detected in serum nor urine by the analytical methods employed. However, haematological indices demonstrated a significantly decreased mean reticulocyte and neutrophil count in the azathioprine exposed group. Industrial hygiene measurements demonstrated contamination of the air inside the airhood of exposed workers. In conclusion, it is evident that workers involved in the production of both these drugs are at risk of developing adverse health effects. Furthermore, more sensitive analytical methods need to be developed to evaluate absorption of myelotoxic chemicals among occupationally exposed workers.

Pattern of 6-mercaptopurine urinary excretion in children with acute lymphoblastic leukemia: urinary assays as a measure of drug compliance.

A method for the measurement of 6-mercaptopurine (6MP) in urine using high-performance liquid chromatography is described. Urinary excretion of 6MP was measured in 46 children with acute lymphoblastic leukemia. The proportion of unchanged drug excreted after oral dosage in the morning was greater than after an evening dose (5.6 +/- 3.3% vs. 3.3 +/- 2.4%). Possible reasons for the discrepancy are discussed. In all children receiving 6MP in the morning, the drug was detected in urine at 2 and 4 h after ingestion. At 6 h, 6MP was still detectable in 77% of patients, at 8 h in 54%, at 10 h in 12%, and at 12 h in 8%. The reliability of urinary 6MP determination as a measure of drug compliance was assessed in 39 children accustomed to receiving their medication in the evening. 6MP was detected in 81% of first morning urine samples, indicating compliance with medication the preceding evening. The absence of 6MP in first morning urine samples did not necessarily indicate poor compliance because of the variability in 6MP excretion and unpredictable pattern of night voiding in children. The method was therefore a reliable measure of good short-term compliance. It also directed attention toward possible noncompliance in children with negative samples.

Pharmacokinetics of oral 6-mercaptopurine: relationship between plasma levels and urine excretion of parent drug.

Plasma levels and cumulative urine excretion of 6-mercaptopurine (6-MP) were measured using a specific and sensitive high-performance liquid chromatographic assay in seven children with acute lymphoblastic leukemia (ALL) as well as in one healthy volunteer. The dose of 6-MP varied in the range of 25-75 mg/m2 of body surface area and was administered with a standard breakfast. A 4- to 11-fold variation between individuals was found in the pharmacokinetic parameters: peak concentration, time to reach peak, area under the plasma concentration-time curve (AUC), and fraction of dose excreted in the urine. Three repeated determinations in one individual revealed that AUC also varied more than sixfold following an overnight fast. In three individuals, the reducing agents glutathione (10 mg/kg) and ascorbic acid (15 mg/kg) were coadministered with 6-MP to evaluate their possible role in the protection of 6-MP from oxidation and degradation in the intestinal lumen. No consistent effect was observed, however, on the AUCs of either of these agents. A clear relationship was found between AUCs and the 24-h urinary excretion of unchanged drug (r = 0.9381), indicating that determinations of 6-MP in the urine may replace the painful procedure of repeated blood sampling. Further studies are necessary to determine the factors contributing to the unpredictable plasma levels following oral doses of 6-MP and to determine the value of pharmacokinetic monitoring in ALL patients.

HPLC analysis of 6-mercaptopurine and metabolites in extracellular body fluids.

A convenient HPLC assay, which allows for the simultaneous measurement in extracellular fluids of 6-mercaptopurine and four of its metabolites, 6-thioguanine, 6-mercaptopurine riboside, 6-thioxanthine and 6-thiouric acid is described. Solid phase extraction allows for the clean isolation of analytes from plasma, urine or cerebrospinal fluid. The simultaneous determination of 6-mercaptopurine and some of its major metabolites in extracellular fluids may contribute to the monitoring of patient compliance, bioavailability, and individual variation in metabolism and absorption.

6-mercaptopurine: high-dose 24-h infusions in goats.

In vitro investigations have indicated the need for both prolonged exposure to 6-mercaptopurine (6MP) and the use of high concentrations to achieve maximal cell kill. After the customary oral administration the bioavailability of 6MP appeared to be low, and i.v. bolus injections resulted in short-lived high concentrations of 6MP, so prolonged infusions seemed rational. To test the feasibility of this approach 24-h infusions were given to goats. We used our improved HPLC method to quantitate 6MP and 6MP riboside (6MPR) in plasma, CSF, and urine. The concentrations of 6MPR were in excess of those of 6MP. Since 6MPR can easily be converted to 6MP, 6MPR acts as a depot for 6MP. Penetration of both 6MP and 6MPR into CSF was excellent. Of the total dose administered, 38% to 68% could be accounted for in the urine, with about equal amounts of 6MP and 6MPR. At doses of 20 and 10 mg kg-1 h-1 total concentrations of 6MP and 6MPR in excess of 100 microM were reached during 24-h infusions. However, all three experimental animals died due to toxicity. A dose of 2 mg kg-1 h-1 was tolerated; the total steady state concentration of 6MP and 6MPR in two experiments was about 10 microM. We conclude that the prolonged infusion of 6MP is feasible, and in view of the excellent penetration of 6MP and 6MPR into CSF, studies using prolonged infusions of thiopurines are warranted in man.

Formation of 6-thioguanine and 6-mercaptopurine from their 9-alkyl derivatives in mice.

Several 9-alkyl, 6-thiopurines have been reported to have more favorable therapeutic indexes than do the parent drugs, 6-mercaptopurine (MP) and 6-thioguanine (TG). Some of these compounds were reported to be active against cells in culture resistant to 6-thiopurines, and it has been assumed that their mechanisms of action may differ from those of TG and MP. 9-(n-Butyl)-6-thioguanine was essentially inactive toward Chinese hamster ovary cells in vitro when compared with TG (50% effective dose, 250 and 1 microM, respectively). However, lethal doses of 9-(n-butyl)-6-thioguanine and TG in mice were similar when these agents were given i.p. daily for 9 consecutive days (50% lethal dose, 13 and 9 mg/kg/day). Similar organ toxicities were observed upon histopathological examination of dying animals. The cumulative, daily urinary excretion of TG was virtually identical in mice given 20- and 10-mg/kg/day of doses of 9-(n-butyl)-6-thioguanine or TG, respectively, for 9 days. The TG formed was identified by ultraviolet light (340 nm) detection following separation on a reverse phase high performance liquid chromatography system and by fluorescent detection of the permanganate oxidation product separated on a strong anion-exchange system. Dealkylation of 9-(n-butyl)-6-mercaptopurine and 9-ethyl-6-mercaptopurine also occurred in AKR mice. At near equitoxic doses, the daily cumulative urinary excretion of MP from 9-(n-butyl)-6-mercaptopurine and 9-ethyl-6-mercaptopurine was about 20-30% of that observed in mice receiving MP. The MP was confirmed in each case by enzymatic peak-shift of MP to 6-thiouric acid and ultraviolet light detection using the high performance liquid chromatography systems referred to above. The results suggest that these 9-alkyl derivatives serve as prodrugs for TG and MP, a finding that explains a number of their pharmacological and toxicological properties.

Pharmacokinetics of elimination of 6-mercaptopurine in the urine of children with acute lymphoblastic leukemia.

6-Mercaptopurine (6-MP)-induced sodium azide--iodine reaction was adapted for determination of 6-MP in urine of four children treated with single oral doses of the drug in tablets. Open one-compartment body model was assumed, and first-order elimination rate constants (K) and biological half-life times (t0,5) were calculated, and their precision was determined by statistical treatment.

Quantitation of 6-mercaptopurine in biologic fluids using high-performance liquid chromatography: a selective and novel procedure.

A simple, selective, sensitive and rapid procedure is described for the quantitation of 6-mercaptopurine (6-MP) in biological fluids. A sensitivity of at least 5 ng/ml is easily achieved in plasma on a reversed-phase octadecylsilane (C18) column using a high-performance liquid chromatography system following an initial protein precipitation and a clean-up step. Mean extractability of the drug from plasma following this procedure is greater than 98% and the overall coefficient of variation for the assay is below 6%. Plasma levels of 6-MP were measured in a rhesus monkey for 12 h following an intravenous administration of a single bolus dose (4 mg/kg) of 6-MP.

Comparative bioavailability and pharmacokinetic studies of azathioprine and 6-mercaptopurine in the rhesus monkey.

Azathioprine (AZA) is cleaved in vivo by glutathione to 6-mercpatopurine (6-MP). 6-MP plasma levels were measured by HPLC in four male rhesus monkeys following oral and iv doses of the two drugs. Following iv 6-MP administration, 6-MP levels were described by a two-compartment body model; mean terminal half-life; plasma clearance (CLp), and volume of distribution (Vdss) were 41.6 +/- 12.1 min, 48.4 +/- 15.4 ml/min/kg, and 1.76 +/- 0.64 liters/kg, respectively. 8-Hydroxymercpatopurine (8-OHMP) was identified as a metabolites of AZA. 8-OHMP had a CLp twice that for 6-MP, while its Vdss was similar to that for 6-MP. After an iv dose, AZA is converted to 6-MP to the extent of 15%. The conversion of AZA to 6-MP and 8-OHMP was independent of the route of administration. Differences in AZA and 6-MP kinetics among the monkeys were attributed to differences in individual aldehyde oxidase and xanthine oxidase levels.