Aluminum in erythropoietin formulations: lyophilized versus liquid forms.

BACKGROUND: Erythropoietin (EPO) formulations may comprise aluminum (Al) as a contaminant. Due to the toxicity of Al in chronic kidney disease patients, possible sources of Al were investigated. Since EPO formulations are stored in container-closure systems made of glass and rubber, and both contain Al, formulation ingredients may enable its leaching into the solution during shelf-life. METHODS: Individual solutions of formulation ingredients were stored in new glass vials and in contact with the rubber stopper and kept at 4 ± 2 °C. For 12 months, aliquots of each solution were collected for analysis. Fifteen commercial samples of EPO were analyzed for their Al content. Aluminum was determined by atomic absorption spectrometry. RESULTS: Glass and rubber are sources of Al for EPO formulations. Storage assay showed that citrate and phosphate (used as buffers) extracted high amounts of Al from the container/closure parts. The most important difference, however, was found when comparing liquid and lyophilized samples. While in liquid forms the Al level reached 943 µg/L, in lyophilized forms the level did not exceed 20 µg/L. The container system was also confirmed as a source of Al in reconstituted lyophilized samples. Al in reconstituted samples stored in their own vials increased 19-fold in 12 months. Lyophilized powders stored for 2 years in glass vials contained less Al than in 1 month after dissolution. CONCLUSION: The difference in the Al measured in liquid forms of EPO and in lyophilized powders suggests that the latter would be the best pharmaceutical form for CKD patients.

Role of medication in the level of aluminium in the blood of chronic haemodialysis patients.

BACKGROUND: Although dialysis facilities provide high-quality water, abnormal aluminium levels among patients on haemodialysis have still been reported. Since patients with chronic kidney disease are often on multiple medications, medicines may be an extra source of aluminium for them. The degree to which ingesting contaminated medication influenced the level of aluminium in the patients' blood was investigated. METHODS: All medications consumed by a group of patients on regular dialysis treatment were analysed and the total aluminium ingested by each patient was calculated. At the same time, the patients' blood was collected and aluminium was measured. The analyses were carried out by atomic absorption spectrometry. RESULTS: For all drugs consumed, the amount of aluminium ingested versus the blood aluminium level presented no correlation. Since a high level of contamination was presented by injectable iron, insulin and erythropoietin (EPO), another group of patients that received a reduced amount of oral medication was selected. Among them, eight did not receive any injectable drug, five received only EPO and seven injectable iron, EPO and insulin. With these restricted groups, it was possible to show that the injectable administration of contaminated medication increased the Al level in the patients' blood, mainly in relation to iron formulations. CONCLUSION: Among the medications investigated, the injectables are the most significant source of aluminium for patients with renal insufficiency. This extra aluminium intake is reflected in higher aluminium levels in the patients' blood.

A liquid chromatography-atmospheric pressure photoionization tandem mass spectrometric method for the determination of organosulfur compounds in petroleum asphalt cements.

We present a sensitive liquid chromatography-atmospheric pressure photoionization tandem mass spectrometric (UHPLC-APPI-MS/MS) method for the determination of selected organosulfur compounds in Brazilian asphalt cements. It was possible to detect 14 organosulfur compounds of different classes where sulfoxides and sulfones presented higher sensibility in ionization than thiophenes and aromatic sulfides. A dopant-assisted APPI method was also tested, however, when chromatographic flow rate was optimized a decrease in signal was observed for all compounds. PAHs were tested and ruled out as possible interfering compounds and the matrix effect of asphalt cements was within an acceptable range for the quantification of organosulfur compounds. The proposed method was found to have satisfactory linearity and accuracy with recoveries between 83.85 and 110.28% for thianaphthene and 3-methylbenzothiophene, respectively. Therefore, the method allowed the characterization of organosulfur compounds in Brazilian asphalt cements and demonstrated changes in the amount quantified in asphaltenic and maltenic fractions after the RTFOT+SUNTEST aging process.

Efficacy of 2,3-dimercapto-1-propanesulfonic acid (DMPS) and diphenyl diselenide on cadmium induced testicular damage in mice.

The deleterious effect of acute cadmium-intoxication in mice testes was evaluated. Animals received a single dose of CdCl2 (2.5 or 5 mg/kg, intraperitoneally) and a number of toxicological parameters in mice testes were examined, such as delta-aminolevulinic acid dehydratase (delta-ALA-D) activity, lipid peroxidation, hemoglobin and ascorbic acid contents. Furthermore, the parameters that indicate tissue damage such as plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were also determined. Thus, a possible protective effect of 2,3-dimercapto-1-propane-sulfonic acid (DMPS) and diphenyl diselenide (PhSe)2 were studied. The results demonstrated an inhibition of delta-ALA-D activity, a reduction of ascorbic acid and an increase of lipid peroxidation induced by cadmium, indicating testes damage. Furthermore, we observed an increase of plasma LDH, AST and ALT activities. DMPS (400 mol/kg) and (PhSe)2 (100 micromol/kg) partially protected from the inhibitory effect of 2.5 mg/kg CdCl2 on delta-ALA-D and from the increase of TBARS (thiobarbituric acid reactive species) levels. (PhSe)2 therapy was effective in ameliorate ascorbic acid content when the cadmium dose was 2.5 mg/kg. Treatment with DMPS and (PhSe)2, individually or combined, was inefficient in reducing cadmium-induced plasma LDH and ALT activity increase. The use of combined therapy (DMPS plus (PhSe)2) proved to be efficient in decreasing cadmium levels in testes and in ameliorating plasma AST activity from animals that received the highest dose of cadmium.

Cadmium induced testicular damage and its response to administration of succimer and diphenyl diselenide in mice.

Acute effects of cadmium in mice testes were evaluated. Animals received a single dose of CdCl2 (2.5 mg/kg or 5 mg/kg, intraperitoneally) and a number of toxicological parameters in mice testes were examined such as delta-aminolevulinic acid dehydratase (delta-ALA-D) activity, lipid peroxidation, hemoglobin content and components of the antioxidant defenses (superoxide dismutase (SOD) activity and ascorbic acid concentration). Furthermore, a possible protective effect of meso-2,3-dimercaptosuccinic acid (DMSA) and diphenyl diselenide (PhSe)2 are studied. The results demonstrated inhibition of delta-ALA-D and SOD activities, reduction in ascorbic acid, increase of lipid peroxidation induced by cadmium, indicating testes damage. DMSA (400 micromol/Kg) and (PhSe)2 (100 micromol/Kg) protected inhibitory effect of 2.5 mg/kg CdCl2 on delta-ALA-D and restored the increase of TBARS levels. Otherwise, (PhSe)2 treatment was effective in reducing the increase of TBARS levels induced by 5 mg/kg CdCl2, whereas DMSA and (PhSe)2, in combination, were ineffective in reducing TBARS level. However, these compounds alone or in combination, were unable to protect SOD activity and to improve ascorbic acid levels near to the normal value. The use of combined therapy (DMSA plus (PhSe)2) not proved be better than the monotherapy, in improving toxicological parameters evaluated in this model of testicular damage induced by cadmium.

Accumulation, elimination, and effects of parenteral exposure to aluminum in newborn and adult rats.

Aluminum (Al) delivered to preterm infants via parenteral nutrition may exceed the limit of 5 µg/kg/day set by the US Food and Drug Administration. This study evaluated the effect of the administration of an equivalent amount of Al (0.12 mg/kg/day) to newborn rats. The study included the administration of a higher amount of Al (24.8 mg/kg/day) not only to newborn rats but also to adult (2- and 4-month-old) rats. Aluminum was intraperitoneally administered for a period of 10 days. Newborn animals were evaluated for developmental changes every day starting from the second day after birth. Twenty days after the last administration, 10 animals were killed and their organs were removed; the remainders were killed on day 40. A dosage of 24.8 mg/kg/day was administered to the two groups of adult rats, which were killed following the same protocol after 20 and 40 days. The results of physical parameters and developmental and behavioral tests were not conclusive and no significant differences were observed between the lower and higher Al dose and control groups. The group that received 0.12 mg/kg/day showed significant differences in Al accumulation only in the liver and muscle. The groups that received a higher dose of Al showed an accumulation in all tissues among all age groups studied, but the newborn group showed the greatest accumulation (results for day 20). After 40 days, Al content in all tissues decreased more than 50% in this group, whereas among the adults, the Al content increased or remained constant. An increase in age correlated with a lower elimination rate. Considering the ongoing human Al exposure, along with its age-related elimination rate, Al accumulation in the body may be long-lasting.

Chromatography and atomic absorption spectrometry for the assessment of heavy metal distribution among amino acids used in parenteral nutrition formulations-studies with cadmium and lead.

The distribution of Cd (II) and Pb (II) among amino acids in parenteral nutrition formulations was investigated by coupling ion-chromatography (HPLC/IC) and electrothermal atomic absorption spectrometry. The methodology was based on ion-exchange separation and fluorimetric amino acid detection after post-column derivatization. Cd (II) and Pb (II) were assayed in 500-µL fractions of the column effluent. The distribution of Cd (II) and Pb (II) in alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), histidine (His), methionine (Met), phenylalanine (Phe), serine (Ser), and threonine (Thr) were analyzed by monitoring changes in the concentration of free amino acids by HPLC/IC. The results indicated that Cd (II) and Pb (II) were distributed according to the following trend: Gly-Cd > Gly-Pb > Ala-Cd > Ala-Pb > His-Cd ∼ His-Pb > Thr-Cd > Thr-Pb > Phe-Cd ∼ Phe-Pb ∼ Asp-Cd ∼ Asp-Pb ∼ Met-Cd ∼ Met-Pb ∼ Glu-Cd ∼ Glu-Pb > Ser-Cd ∼ Ser-Pb. The effects of amino acid concentration and stability constants of amino acid-metal complexes are discussed.

Evidence for aluminum-binding erythropoietin by size-exclusion chromatography coupled to electrothermal absorption atomic spectrometry.

Erythropoietin (EPO) is a glycoprotein that stimulates erythropoiesis and is clinically used for treating anemia during chronic renal failure and for anemia in preterm infants. EPO formulations usually have elevated rates of contamination due to aluminum (Al), which is toxic to both types of patients. Size-exclusion chromatography (SEC) coupled with graphite furnace atomic absorption spectrometry (GF AAS) was employed to separate proteins and to quantify the amount of aluminum present in the elution volume corresponding to EPO and, therefore, to evaluate possible binding. Because EPO formulations contain human serum albumin (HSA), a chromatographic method was optimized for the separation of these proteins. Subsequent to the chromatographic separation, 1-mL fractions of the column effluent were collected, and the Al content in these aliquots was measured by GF AAS. EPO and HSA samples were incubated with Al for 4h at 4°C and 37°C as well as for 16 h at 4°C and 37°C. Afterwards, they were injected into the chromatographic system. These samples were also submitted to ultrafiltration (10 and 50 kDa membranes), and Al was measured in the ultrafiltrates. The results showed that Al was present in the eluent volume corresponding to the EPO peak but not in the HSA peak in the chromatograms. Temperature strengthened the interaction because the Al present in the EPO fraction was 3 times higher at 37°C compared to 4°C. Thirty-eight percent of the Al present in a 2.4 µg/mL EPO standard solution, and approximately 50% of the Al in formulation samples containing approximately 11 µg/mL EPO and either citrate or phosphate, were non-ultrafiltrable, which suggests that EPO is an effective Al acceptor in vitro.

Presence of synthetic pharmaceuticals as adulterants in slimming phytotherapeutic formulations and their analytical determination.

Obesity that is associated with a high consumption of slimming substances is considered a public health problem around the world. In this context, the increasing consumption of phytotherapeutic formulations as alternative obesity treatments has revealed the presence of synthetic pharmaceuticals as adulterants. The illegally added adulterants are frequently anorexic, anxiolytic, and antidepressant pharmaceuticals. This review aims to describe the analytical methodologies utilized for the determination of adulterants in slimming phytotherapeutic formulations. Furthermore, this review describes some important adulteration cases, which occurred mainly in Europe, Asia, Brazil, and the USA.

A new method for the simultaneous determination of 1,4-benzodiazepines and amfepramone as adulterants in phytotherapeutic formulations by voltammetry.

The use of synthetic pharmaceuticals in phytotherapeutics can be defined as an illegal practice, since these compounds are normally present as non-declared compounds in the phytotherapeutical formulations. This work aims to show the development of an analytical method based on adsorptive cathodic stripping voltammetry (AdCSV) for the simultaneous determination of 1,4-benzodiazepines and amfepramone. The developed method was used to measure seven benzodiazepines (clonazepam, flurazepam, alprazolam, midazolam, medazepam, chlordiazepoxide, and diazepam) and amfepramone in slimming formulations that have been commercialized in Brazil. This method permits the screening of adulterant classes in a single voltammetric run by using a hanging mercury drop electrode as a working electrode and Ringer buffer (pH 10.0) as a supporting electrolyte. Recovery values ranging from 92.0% to 117.0% demonstrate the reliability of the method in the determination of adulterants in real samples. Among the 12 samples studied by the proposed method, 4 were demonstrated to be adulterated by 1,4-benzodiazepines.

Voltammetric determination of Se(IV) and Se(VI) in saline samples--studies with seawater, hydrothermal and hemodialysis fluids.

Determination of Se(IV) and Se(VI) in high saline media was investigated by cathodic stripping voltammetry (CSV). The voltammetric method was applied to assay selenium in seawater, hydrothermal and hemodialysis fluids. The influence of ionic strength on selenium determination is discussed. The CSV method was based on the co-electrodeposition of Se(IV) with Cu(II) ions and Se(VI) determined by difference after sample UV-irradiation for photolytic selenium reduction. UV-irradiation was also used as sample pre-treatment for organic matter decomposition. Detection limit of 0.030 microg L(-1) (240 s deposition time) and relative standard deviation (RSD) of 6.19% (n=5) for 5.0 microg L(-1) of Se(IV) were calculated. Linear calibration range for selenium was observed from 1.0 to 100.0 microg L(-1). Concerning the pre-treatment step, best results were obtained by using 60 min UV-irradiation interval in H(2)O(2)/HCl medium. Se(VI) was reduced to the Se(IV) electroactive species with recoveries between 91.7% and 112.9%. Interferents were also investigated.

Determination of aluminum as contaminant in dialysis concentrates by adsorptive cathodic stripping voltammetry.

This paper describes a stripping voltammetric method for the determination of aluminum as contaminant in dialysis concentrates. It is based on the adsorptive deposition of the complex Al-1,2-dihydroxyanthraquinone-3-sulfonic acid (DASA) at the hanging mercury drop electrode (HMDE) at -0.9V (versus Ag/AgCl) and its cathodic stripping during the potential scan. The method was optimized concerning the main electrochemical parameters that influence the voltammetric determination in highly saline solutions (sample dilution factor, deposition time and DASA concentration). The linear range for the measurements was from

HPLC based method using sample precolumn cleanup for the determination of triazines and thiolcarbamates in hemodialysis saline solutions.

Solid-phase extraction (SPE) procedures for cleanup and preconcentration followed by HPLC-UV method were investigated for the simultaneous determination of seven low-dosed pesticides in saline concentrates for hemodialysis. The target compounds were ametryn, desmetryn, prometryn, terbutryn, molinate, triallate and butylate. Polyethylene (three different types), teflon, polyurethane and polystyrene, in powder form, were investigated as adsorbents for solid-phase extraction of the analytes from the saline samples. Quantification was performed at 222nm and the analytes were separated on a LiChrosorb RP-18 (5mum, 125mm x 4mm i.d.) column using gradient elution with water/acetonitrile as mobile phase. The duration each chromatographic run was 18min including column reconditioning. The efficiency of the different SPE substrates for retaining the analytes from the highly concentrated saline (HCS) samples was discussed. The best performance was achieved with polystyrene as SPE material considering preconcentration factor, precolumn clogging, reusing capability and similarity between the mobile phases for SPE and HPLC procedures. Analyte concentrations as low as 1mugL(-1) could be determined in spiked HCS samples after preconcentration on polystyrene SPE precolumns. Recoveries between 98.7 and 102.2% were obtained from commercial spiked samples. Detection limits ranging from 4.8 (for prometryn) to 46mugL(-1) (for butylate) were calculated (without preconcentration). The within-day relative standard deviations (n = 9) ranged from 2.3 to 4.8%.