Study of ion suppression for phenolic compounds in medicinal plant extracts using liquid chromatography-electrospray tandem mass spectrometry.

A systematic study on the various sources of ion suppression in UHPLC-MS-MS analysis was carried out for 24 phenolic antioxidants in 6 different extracts of medicinal plants from Amazonia. The contributions of matrix effects, mobile-phase additives, analyte co-elution and electric charge competition during ionization to the global ion suppression were evaluated. Herein, the influence of mobile-phase additives on the ionization efficiency was found to be very pronounced, where ion suppression of approximately 90% and ion enhancement effects greater than 400% could be observed. The negative effect caused by the wrong choice of internal standard (IS) on quantitative studies was also evaluated and discussed from the perspective of ion suppression. This work also shows the importance of performing studies with this approach even for very similar matrices, such as varieties of medicinal plants from the same species, because different effects were observed for the same analyte.

Flame and graphite furnace atomic absorption spectrometry for trace element determination in vegetable oils, margarine and butter after sample emulsification.

Trace element analysis plays an important role in oil characterisation and in the detection of oil adulteration because the quality of edible oils and fats is affected by their trace metal content. In this study, the quantification of selected metals in various oils and fats (rice oil, canola oil, sunflower oil, corn oil, soy oil, olive oil, light margarine, regular margarine and butter) was carried out using flame atomic absorption spectrometry (FAAS) and graphite furnace atomic absorption spectrometry (GFAAS) after sample emulsification. FAAS was used to determine the Na, K, Ca, Mg, Zn and Fe levels in the samples, while GFAAS was used for quantifying Cr, Ni, As, Pb, Cd, Cu and Mn, as these elements appeared in the samples at much lower concentrations. Tween-80 and Triton X-100 were employed as surfactants, and emulsions were prepared by a conventional method that involved heating and mixing of the constituents. Complete stabilisation was achieved through magnetic stirring for 15 min at room temperature. The evaluated figures of merit were linearity, accuracy and sensitivity, which were determined by the characteristic concentration and mass. Analysis of spiked samples demonstrated accuracy, which ranged from 90% (Na) to 112% (Fe) for FAAS and from 83% (Cd) to 121% (Pb) for GFAAS measurements. Atomic absorption spectrometry proved to be a promising approach for the analysis of metals in emulsified edible oils and fats. Additionally, under appropriate emulsification conditions (formulation, stirring time and temperature), the emulsions were homogeneous, had excellent stability, and had appropriate viscosity. The proposed method has proved to be simple, sensitive, reproducible, and economical.

Determination of sodium, potassium, calcium, magnesium, zinc, and iron in emulsified egg samples by flame atomic absorption spectrometry.

In this study, oil-in-water formulations were optimized to determine sodium, potassium, calcium, magnesium, zinc, and iron in emulsified egg samples by flame atomic absorption spectrometry (FAAS). This method is simpler and requires fewer reagents when compared with other sample pre-treatment procedures and allows the calibration to be carried out using aqueous standards. Different oily phases such as corn oil, decyl oleate and octyl stearate were tested, as well as Tween 80, Triton X-100 and Triton 114 were analyzed as surfactants. The optimum type and proportion of formulations were determined and their use depended on the element studied. The emulsion preparation was performed by a conventional method that involves mixing both phases at 60 degrees C by magnetic stirring and phase inversion to change the water-to-oil ratio by increasing the volume of the surfactant-water external phase and correspondingly decreasing the volume of internal phase. The accuracy of the method was further confirmed by determining the metals in a whole egg powder CRM and recoveries ranged from 97.5% for Mg to 102.2% for Na, with relative standard deviations lower than 2.3%. The precision of the procedures was determined through repeatability (intra-day precision) and intermediate precision (inter-day). The repeatability presented RSD values lower than 4.2%. The intermediate precision was evaluated using the RSD and F-test. The RSD values to intermediate precision was lower than 5.3% and the computed F-values were lower than tabulated F-values, indicating no significant difference between the results obtained on different days. The proposed method including, sample emulsification for subsequent metal determination for FAAS, has proved to be sensitive, reproducible, simple and economical.

Interaction of aluminium ions with some amino acids present in human blood.

The interaction of aluminium with some amino acids present in human blood was studied combining ion-chromatography (IC), atomic absorption spectrometry (AAS) and ultrafiltration (UF) techniques. An IC system for simultaneous determination of ornithine, lysine, glutamic acid, aspartic acid and tyrosine was developed. By adding aluminium to standard solutions of the amino acids and keeping the pH at 6 and 7 it was possible to verify that aluminium caused a reduction on the amino acid chromatographic signals. Similar experiment, carried out for copper showed the same behaviour (with different percentage of signal reductions) and validated the results for aluminium, considering that the interaction Cu-amino acid is well-established. The AAS analysis of sample fractions (500 microl) after the IC separation showed that aluminium (as copper as well) is not present in the fractions in which the amino acid peaks appear in the chromatogram. These approaches carried out with serum samples after UF showed that part of the "free" fraction of serum aluminium is distributed, besides other ligands, among these amino acids. It was found that in serum the affinity for aluminium followed the sequence Lys>Orn>Tyr>Glu approximately Asp.

Interference in the Limulus amebocyte lysate assay for endotoxin determination in peritoneal dialysis fluids and concentrates for hemodialysis.

The interference of the saline concentration of fluids for peritoneal dialysis and concentrates for hemodialysis on the Limulus amebocyte lysate (LAL) assay for endotoxins was investigated. The experiments were carried out individually with each substance that compose fluids for hemodialysis, to determine the possible inhibition or enhancement effects that they could cause on the LAL assay. The compositions were also assayed to investigate the possibility of synergistic effect. They were assayed by the gel-clot method from two different suppliers, and the samples that showed inhibition effect were also assayed by the chromogenic method. The samples were analysed at successive dilutions, with different LAL sensitivities, to satisfy the endotoxin limits of 5 EU/ml for the concentrate and 0.25 EU/ml for the fluid for dialysis peritoneal. The results showed that the major interference on the gel-clot assay occurs in presence of acetic acid and in concentrates containing acid acetic, even the pH being adjusted between 6.5 and 7.5. However, the test, after an adequate dilution, could be validating for all samples. Chromogenic test can be used for peritoneal dialysis fluids considering a limit of 0.25 EU/ml and sample dilution of eight times, but it cannot be used for concentrates for hemodialysis without further dilution. Considering the results and that the chromogenic is a more time-consuming method, endotoxins in fluids for hemodialysis can be satisfactorily assayed by the gel-clot method.

HPLC-based method using sample pre-column clean-up for the determination of methanethiol and ethanethiol in parenteral amino acid solutions.

A method has been developed for the chromatographic determination of methanethiol (MT) and ethanethiol (ET) as contaminants in amino acid parenteral nutrition (PN) solutions. The clean-up of the samples before chromatographic analysis was investigated by solid-phase extraction (SPE) on pre-columns filled with polyethylene powder (PE), aluminium oxide (AlOx), silica (SiOx), or polyurethane foam (PUF) as adsorbents. The thiols were more efficiently separated from the matrices by SPE on PUF pre-columns. Simultaneous derivatization and elution with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) enabled further discrimination between MT and ET by reversed-phase HPLC with spectrophotometric detection. The retention times for the derivatized MT and ET species were 12.5 and 23.0 min, respectively. Recoveries from spiked PN samples were calculated to be approximately 90%, and the MT and ET content of commercial PN solutions was determined using the methodology described. Detection limits of 15 and 10 microg L(-1) were calculated for MT and ET, respectively.

Influence of the glass packing on the contamination of pharmaceutical products by aluminium. Part II: amino acids for parenteral nutrition.

The presence of aluminium in amino acids parenteral nutrition solutions can be related to the affinity of the amino acids for aluminium present in glass containers used for storage. For this study solutions of 19 amino acids used in parenteral nutrition were stored individually in glass flasks and the aluminium measured at determined time intervals. Solutions of complexing agents for aluminium, as ethylene-diaminetetraacetic acid, nitrilotriacetic acid, citrate, oxalate and fluoride ions were also stored in the same flasks and the aluminium measured during the same time interval. The measurements were made by electrothermal atomic absorption spectrometry. The aluminium content of the glass containers was also measured. The results showed that the glasses have from 0.6% to 0.8% Al. Only solutions of cysteine, cystine, aspartic acid and glutamic acid became contaminated by aluminium. As the same occurred with the complexing agents, aluminum can be released from glass due to an affinity of the substances for aluminium. Comparing the action of complexing agents and amino acids for which the stability constants of aluminium complex are known, it is possible to relate the magnitude of the stability constant with the aluminium leached from glass, the higher the stability constant, the higher the aluminium released. The analysis of commercial formulations with and without cysteine, cystine, glutamic acid or aspartic acid stored in glass containers confirms that the presence of these amino acids combined with the age of the soLution are, at least partially, responsible for the aluminium contamination. The resuLts demonstrated that the contamination is an ongoing process due to the presence of aluminium in glass combined with the affinity of some amino acids for this element.

Influence of the glass packing on the contamination of pharmaceutical products by aluminium. Part I: salts, glucose, heparin and albumin.

The presence of aluminium (Al) in pharmaceutical products used parenterally as sodium and potassium chlorides, glucose, heparin and albumin were investigated with respect to their storage in glass containers. As glasses can have aluminium in their composition, the aluminium may be released from the glass into the solution. The action of the substances above mentioned were investigated storing their solutions in glass and plastic containers, and measuring the aluminium in solution at determined time intervals. The aluminium present in the commercial pharmaceutical products, stored in both plastic and glass containers were also measured. All glass containers were analysed to determine their aluminium content. The aluminium determinations were done by atomic absorption spectrometry. The resuLts showed that aluminium is present in all analysed glasses in a percentage of 0.6 to 3%. Although all substances already have a residual aluminium contamination, the major contribution comes from the glass containers in which their solutions were stored. The contamination arising from glass depends too much on the nature of the substance. While the salts extracted about 400 microg Al/l in 60 days, glucose extracted 150 microg Al/l, and albumin and heparin about 500 microg Al/l in the same time interval. Commercial solutions of glucose contain about 10 microg Al/l when stored in polyethylene and from 350 to 1,000 microg Al/l when in glass ampules. Considering all commercial products, solutions stored in plastic containers contained no more than 20 microg Al/l whereas in glass the aluminium contamination reached 1,000 microg/l, and in all of them the aluminium increases with the age of the product.

Effect of aluminum on delta-aminolevulinic acid dehydratase from mouse blood.

The objective of this study was to investigate aluminum deposition in whole blood and plasma of mice and the activity of blood delta-aminolevulinic acid dehydratase (ALA-D) after in vitro and in vivo exposure to this element. In vitro experiments showed activation and inhibition of the enzyme activity when 0.01-5.0 mM of aluminum sulphate were used (IC(50): 1.31 mM). Treatment with citrate and aluminum plus citrate increased ALA-D activity in vivo and the increase in enzyme activity was parallel to the increase in aluminum content in blood and plasma. These results show that aluminum has a distinct effect on ALA-D activity: first, at relatively lower concentrations it activated, and at high concentration it inhibited, blood ALA-D in vitro; second, it activated the enzyme when administered to drinking water. One important toxicological finding of the present report is that the apparent irrelevant addition of citrate to the drinking water significantly increased the level of aluminum in blood and plasma. Thus, in order to predict more accurately the extent of human exposure to aluminum it would be advantageous to consider the level of citrate ingestion and not exclusively the aluminum level in water or food.

Voltammetric determination of volatile free sulfide and alkylthiols as contaminants in parenteral amino acid solutions.

A method for the simultaneous voltammetric determination of free sulfide and volatile alkythiols (methanethiol/ethanethiol) existing as contaminant in parenteral nutrition (PN) solutions was developed. The volatile sulfides (dihydrogensulfide and alkylthiols) formed in the formulations were distilled over 45 min at 47 degrees C in a purpose-made Conway cell. The analytes were quantified by differential pulse cathodic stripping voltametry (DPCSV) at the hanging mercury-drop electrode and determined, simultaneously as dihydrogensulfide and alkylthiols using a 60 s preconcentration time at -300 mV (versus Ag/AgCl, Cl(-) 3.0 mol l(-1)). The voltammetric signals were directly linear proportional to the sulfides concentrations in the PN solutions in the range 3-20 mumol l(-1) and detection limits of about 2 mumol l(-1) were calculated. Recoveries of sulfides from PN solutions in the range 90-110% were calculated using the method and nine commercial PN formulations were analyzed. Methanethiol and ethanethiol were quantified from their composite signals, and the mutual influence of the analytes on the DPCSV measurements is discussed.

Polarographic determination of cyanide as contaminant in pralidoxime mesylate formulations.

A method for the polarographic determination of cyanide as contaminant in pralidoxime mesylate (PM) formulations was developed. The volatile cyanide formed in the formulations was stabilized as tetracyanonickelate (TCN) anion complex after reaction with ammoniacal Ni(II) solution. The stable TCN anion complex (K(stb)=10(31)) was determined by anodic stripping voltammetry at the hanging mercury drop electrode (HMDE). The polarographic signal was proportional to the cyanide concentration and the high concentration of PM did not interfere. The linear range of calibration was from 1.2 to 16 mug cyanide with r=0.998. The RSD was 1.3% (n=5) for 2.4 mug cyanide and a detection limit of 0.8 mug cyanide was calculated. The proposed method is adequate as a quality control of PM formulations.

Cyanide determination in biological fluids using a microdiffusion method with a flow system and polarographic detection.

The report describes a method for the automated polarographic determination of cyanide as tetracyanonickelate (II) anion complex in a gas-diffusion flow system. The volatile cyanide, existing in whole blood, plasma and urine samples, was measured after gas-diffusion using 8 x 10-5 mol l-1 hexaaminenickel solution as acceptor. The linear range of calibration, for measurements at the hanging mercury-drop electrode (HMDE), was from 0.1 to 2.0 micrograms cyanide with r = 0.998. The RSD was, respectively, 3.4 and 1.2% (n = 5) for 0.4 microgram cyanide measured with and without the flow-system configuration. Detection limits of 7.4 microgram l-1 were calculated using the flow system and the method was compared with the classical method using Cavet flasks. Parameters that affect the cyanide determination in the proposed method, such as acceptor solution, pH, flow rate and temperature, were investigated.

Liquid chromatography with ultraviolet absorbance detection of ethylenethiourea in blood serum after microwave irradiation as an auxiliary cleanup step.

The report describes a method of deproteinizing serum that combines the action of acids with microwave irradiation. The acid concentration is ten times smaller than in the usual acid deproteinization method and the results are similar. The main advantages of the proposed method are the maintenance of the pH of the supernatant at around 5, and the reduction of the concentration of the deproteinizing agent. The procedure was applied to the determination of ethylenethiourea in serum samples by HPLC with spectrophotometric detection. Using 0.5 ml of serum, 0.04 microgram of ethylenethiourea were determined with recoveries between 89 and 109%.

Automated polarographic determination of sulfide as contaminant in parenteral amino acid solutions.

A method for the automated polarographic determination of sulfide in a gas-diffusion flow system was developed. The volatile sulfide, existing as a contaminant in parenteral amino acid solutions, was measured after gas diffusion using 0.1 mol l-1 NaOH solution as acceptor. The linear range of calibration, for measurements at a dropping-mercury electrode (DME), was from 5 to 100 micrograms of sulfide with r = 0.999 and RSD = 7.5% (n = 5) for 10 micrograms of sulfide. For measurements at the hanging mercury-drop electrode (HMDE), with a preconcentration time of 30 s, the linear range of calibration was from 0.9 to 20 micrograms of sulfide with r = 0.998 and RSD = 5.8% (n = 5) for 2 micrograms of sulfide. Detection limits of 59 and 11 micrograms l-1 at the DME and HMDE, respectively, were calculated and the recoveries of sulfide from spiked samples were 91.5-104.5%. Parameters that affect the sulfide determination using this method, such as the organic content of the matrix, pH, flow rate and sample size, were investigated.