Elemental speciation by liquid chromatography-inductively coupled plasma mass spectrometry with direct injection nebulization.

A new version of the direct injection nebulizer (DIN) is used to interface liquid chromatographic (LC) separations with element-selective detection using inductively coupled plasma mass spectrometry (ICP-MS). The DIN injects all of the sample into the ICP and has a dead volume of less than 1 microliter. Charged species of arsenic and tin are separated as ion pairs on a micro-scale (1 mm i.d.), packed, reversed-phase column. Detection limits are 0.2-0.6 pg for arsenic and 8-10 pg for tin. For methanol + water eluents, the signal is highest at 25% methanol and stays within 25% of this maximum as the methanol fraction is varied from 20 to 80%. Compared with LC-ICP-MS with conventional nebulizers, the absolute detection limits and chromatographic resolution are substantially superior, and the dependence of analyte signal on solvent composition is somewhat less severe with the DIN.     

Validation of direct injection electrospray LC-MS/MS for confirmation of opiates in urine drug testing

A method based on the direct injection of diluted urine for the identification and quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide, ethylmorphine, ethylmorphine-6-glucuronide and 6-acetylmorphine (6AM) in human urine by electrospray ionisation liquid chromatography-tandem mass spectrometry was validated for use as a confirmation procedure in urine drug testing. Four deuterium labelled analogues were used as internal standards: morphine-3-glucuronide-D3, morphine-D3, codeine-D3 and 6AM-D3. Twenty microlitre aliquots of urine were mixed with 80 mul of the internal standard solution in autosampler vials and 10 mul was injected. The chromatographic system consisted of a 2.0 x 100 mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/l formic acid. Two product ions produced from the protonated molecular ions were monitored in the selected reaction monitoring mode. The intra- and inter-assay variability (coefficient of variation) was below 10% at higher levels for all analytes, but at the reporting limits the variation was above 20% for 6AM, morphine-3-glucuronide and codeine-6-glucuronide. Ion suppression occurred early after injection but did not affect the identification and quantification of the analytes in authentic samples. The method was further validated by comparison with a reference gas chromatographic-mass spectrometric method using authentic urine samples. The two methods agreed almost completely (99%) regarding the identified analytes, but for the quantitative results there were slightly lower levels when measuring glucuronides directly as compared to total determination after hydrolysis by gas chromatography-mass spectrometry.We conclude that the presented liquid chromatographic-tandem mass spectrometric method is robust and reliable, and suitable for use as a confirmation method in urine drug testing for opiates     

Flow injection determination of boron, copper, molybdenum, tungsten and zinc in organic matrices with direct current plasma optical emission spectrometry

Summary Flow injection has been applied for organic matrices in direct current plasma atomic emission spectroscopy and a method is described that allows the introduction of 600 l test solution through a loop into a rapidly flowing carrier liquid leading to the nebuliser. The effects of tube length, flow rate, loop size, viscosity and slit width on the analytical signal were first studied, using ethanol as the carrier liquid. This was followed by comparing other carrier liquids and solvents and the procedures were optimised. Limits of detection (in ng/cm³) are as follows: B: 21;Cu: 14; Mo: 28; W: 120 and Zn: 20; these are 5–7 times higher than those obtainable by static methods. A comparison between evaluation through peak height and peak area is also presented.

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Fließinjektions-Bestimmung von B, Cu, Mo, W und Zn in organischen Matrices mit Gleichstom-Plasma-optischer Emissionsspektrometrie

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Dedicated to Prof. G. Tölg on the occasion of his 60th birthday     

Determination of organoarsenic species in blood plasma by HPLC–ICP MS

A study of arsenic speciation in blood plasma of patients undergoing renal dialysis has been performed using HPLC coupled with ICP MS. It was found that the only detectable arsenic species present in the plasma was arsenobetaine. The limit of detection using an injection volume of 175 µl was found to be 0.25 ng of arsenic as arsenobetaine. Spiking experiments demonstrated recoveries of approximately 100%. In the absence of certified reference materials or an alternative technique, we believe this was the best way to demonstrate that the method was reliable and accurate. Arsenobetaine concentrations in pre‐dialysis plasma were similar to those for the healthy volunteers, although after dialysis the concentrations were significantly reduced. It is thus concluded that, except for a few patients, dialysis removed the arsenobetaine efficiently (hence preventing an accumulation of arsenic) and that no biotransformations were occurring. The exceptions to this conclusion were in a few patients whose arsenobetaine levels increased marginally after dialysis, but this was attributed to the levels both pre‐ and post‐dialysis being very close to the detection limit. Copyright © 1999 John Wiley & Sons, Ltd.     

Determination of metformin in rat plasma by HILIC-MS/MS combined with Tecan automation and direct injection

Metformin is a well‐known oral antihyperglycemic drug used in treatment of type II diabetes. Analysis of metformin in biological fluids is a challenge owing to its high polarity and small molecular size, which lead to poor retention of metformin on reversed‐phase liquid chromatographic columns. A high‐throughput method was developed and validated for the determination of metformin in rat plasma in support of preclinical toxicology studies, using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC‐MS/MS) and Tecan automated sample preparation. Extracted samples were directly injected onto the unbounded silica column with an aqueous–organic mobile phase. This HILIC‐MS/MS method was validated for accuracy, precision, sensitivity, stability, matrix effect, recovery and calibration range. Acceptable intra‐run and inter‐run assay precision (coefficient of variation ≤ 3.9%) and accuracy (99.0–101.8%) were achieved over a linear range of 50–50,000 ng/mL. Metformin is stable in rat plasma for at least 6 h at room temperature, 147 days at ?70°C and through three freeze (?70°C) and thaw cycles. Metformin is also stable in rat whole blood for at least 2 h at room temperature and in an ice–water bath. The validated method was successfully used in support of several preclinical studies where metformin is dosed together with an investigational drug substance. The ruggedness of the validated method was demonstrated by the incurred sample reproducibility test. Copyright © 2011 John Wiley & Sons, Ltd.     

The determination of zinc in blood plasma by atomic absorption spectrometry

Atomic absorption spectrometry has been used in the analysis of plasma zinc because of its sensitivity and simplicity. Dilution techniques reduce the viscosity of plasma and facilitate direct analysis, but viscosity differences can produce deviations in aspiration rates between sample and standard, and so cause errors. A direct (1 + 4) dilution of plasma with deionized water is suggested. Working zinc standards are prepared in 5% glycerol to approximate the viscosity characteristics and aspiration rates of the diluted plasma samples. The analytical curves for diluted plasma samples and 5% glycerol working standards proved identical. Plasma zinc concentrations are accurately calculated from a daily working curve. The accuracy of the method exceeds 99% and recovery of added inorganic zinc to a pooled plasma averages 99.8%. The precision is primarily limited by baseline drift. A confidence interval of ± 2 μg/100 ml was achieved by means of six contiguous 10s-integration readings. The method is free of nebulizer clogging and matrix interferences and is not subject to significant day-to-day variations. Because the method is accurate, sensitive, reliable and specific, it should be useful in the clinical laboratory.     

On the direct determination of metals in lubricating oils by ICP

New equipment and procedures are evaluated for the direct analysis of metals in lubricating oils without the need for sample pretreatment or dilution. A modified Babington principle nebulizer equipped with a sample heater is shown to be capable of producing aerosols from undiluted oils, which are suitable for introduction into an inductively coupled plasma atomic emission spectrophotometer. Heating the samples immediately prior to nebulization greatly increases the output of aerosol and reduces output variations in emission intensity due to differences in oil manufacture and viscosity. The type of organometallic complex used in the preparation of standards is shown to be unimportant if the plasma observation region is properly chosen. Performance of a conventional plasma geometry and an inverted torch geometry on analysis of field collected oil samples is presented.     

Separation of zinc and copper with cyanoacetamide in the determination of zinc

The new use of cyanoacetamide as a selective masking agent for copper in the determination of microgram amounts of zinc with zincon is described. It is very possible that the unique masking action of this compound may have importance in eliminating the interference of copper in other spectrophotometric and titrimetric procedures for zinc thereby reducing the time required to complete the analysis by decreasing or eliminating laborious separation or extraction techniques. The possibility of using cyanoacetamide as a masking agent for other ions should not be overlooked.     

Flow injection and microwave-oven sample decomposition for determination of copper,zinc and iron in whole blood by atomic absorption spectrometry

A simple and rapid method is proposed for the determination of copper, zinc and iron in whole blood. The injected sample is mineralized in the flow system on passage through a microwve oven and the metals are determined by atomic absorption spectrometry. Prior sample destruction or removal of organic material prior to injection is not necessary. The required volumes for each analysis are 90, 60 and 100 μl for copper, zinc and iron, respectively. The relative standard deviations were less than 3% in all cases. There was good agreement between the results obtained with the flow-injection method and those attained by conventional spectrophotometric measurements.     

Automated direct determination of chromium in blood and urine by electrothermal atomic absorption spectrometry

A simple direct procedure for the determination of chromium in whole blood and urine by graphite-furnace atomic absorption spectrometry is described. Whole blood samples are diluted with 0.1% Triton-X solution before injection, whereas urine samples are injected directly. Calibration is done by direct comparison against matrix-matched standards. Between-run precision is 5.4% at 154 nmol l^?1 for urine and 3.6% at 142 nmol l^?1 for blood. The detection limits are 3.8 nmol l^?1 for urine and 11.5 nmol l^?1 for blood, each for a 20μl sample. The calibration range extends up to 770 nmol l^?1 for both blood and urine. This allows the determination of chromium in both occupationally exposed and unexposed groups. The graphite-furnace conditions for each matrix are similar. Elimination fo sample pretreatment minimizes the risk of contamination and allows a rapid sample throughput of 50–60 samples per day. The methods described are particularly suited for the screening and surveying of populations occupationally exposed to chromium.     

Isolation of new quinidine metabolites and simultaneous determination of quinidine and its metabolites in blood and urine by direct injection HPLC analysis

Summary New quinidine metabolites, including 10,11-dihydrodiol quinidine N-oxide, 10,11-dihydrodiol quinidine and their glucuronides, were found in human urine. A quinidine monitoring HPLC method including these metabolites, is proposed by the direct injection of body fluid samples onto the precolumn for deproteinization followed by reverse phase separation in the analytical column with a column switching technique. The recovery of spiked quinidine and its metabolites in plasma was quantitative (98–102%) with good reproducibility (C.V.: 1.6–4.0%). Several clinical samples such as whole blood and urine were analyzed by the present method.     

Analysis of undigested honey samples by isotope dilution inductively coupled plasma mass spectrometry with direct injection nebulization (ID-ICP-MS)

Undigested honey samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) using direct injection nebulization (DIN). Samples were diluted 25-fold using dilute HNO₃ to which Triton X-100 was added. Isotope dilution (ID) was used to quantify Ba, Cu, Pb and Zn in these natural matrix samples. DIN parameters were optimized to obtain a stable transient signal enough to determine multiple isotope ratios. The Ba, Cu and Zn isotope ratios were measured in a single sample injection of 50 μl diluted honey and the Pb isotopes in a separate injection. The analysis was characterized by precisions given by R.S.D. values from 0.20 to 1.24% for Ba, 0.49–1.23% for Cu, 0.61–2.28% for Pb and 0.50–1.51% for Zn. The results were in agreement, at the 95% confidence level, with those obtained by analyzing digested samples using external calibration. Using this direct method, 30 real honey samples per hour could be analyzed.     

Determination of thiamine in blood serum and urine by high-performance liquid chromatography with direct injection and post-column derivatization

Thiamine (vitamin B₁) was determined in human serum and urine by HPLC with fluorimetric detection of its oxidation product, thiochrome. The samples were injected directly into the chromatographic system without previous treatment or dilution. A column filled with an ultra-high molecular weight surface-modified polyethylene (PE) was able to separate matrix components from analyte and also to allow a good chromatographic resolution of thiamine. The interaction of thiamine, thiocrome and both matrices (serum and urine) with PE was studied off- and on-line to determine the optimal procedure for vitamin B₁ determination. When carried off-line, matrix adsorption yield was 49 mg serum proteins/g polymer and components of 1000 μl urine/g polymer. In an on-line arrangement, the yield dropped to 10 mg/g and 150 μl/g, respectively. The matrix/analyte separation was carried out in an on-line procedure on a 50×4.6-mm, 25-μm PE column, using a water-sodium phosphate-methanol gradient elution. Part of the matrix was eluted within the first 2 min and thiamine after 3.8 min. The rest of the matrix retained on the column was eluted after thiamine at the last step of the gradient elution. Analysis time was 12 min. The within-day and day-to-day precision gave C.V. varying from 3.6% to 14.5% and recoveries from spiked samples were in the range of 84.8-98.8%.     

A highly sensitive LC-tandem MS assay for the measurement in plasma and in urine of salbutamol administered by nebulization during mechanical ventilation in healthy volunteers

The new-generation nebulizers are commonly used for the administration of salbutamol in mechanically ventilated patients. The different modes of administration and new devices have not been compared. We developed a liquid chromatography-tandem mass spectrometry method for the determination of concentrations as low as 0.05 ng/mL of salbutamol, corresponding to the desired plasma concentration after inhalation. Salbutamol quantification was performed by reverse-phase HPLC. Analyte quantification was performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection ESI in the positive mode. The method was validated over concentrations ranging from 0.05 to 100 ng/mL in plasma and from 0.18 to 135 ng/mL in urine. The method is precise, with mean inter-day coefficient of variation (CV%) within 3.1-8.3% in plasma and 1.3-3.9% in urine, as well as accurate. The proposed method was found to reach the required sensitivity for the evaluation of different nebulizers as well as nebulization modes. The present assay was applied to examine whether salbutamol urine levels, normalized with the creatinine levels, correlated with the plasma concentrations. A suitable, convenient and noninvasive method of monitoring patients receiving salbutamol by mechanical ventilation could be implemented.     

High-performance liquid chromatographic method for the determination of salicylic acid and its metabolites in urine by direct injection

A direct injection method has been developed for the determination of salicylic acid and its metabolites in urine. Urine samples are treated with hydroxylamine to convert salicyl acyl glucuronide to salicylhydroxamic acid, which can be accurately quantitated by direct injection into a high-performance liquid chromatographic system along with salicylic acid, gentisic acid and salicyluric acid. Salicyl phenolic glucuronide is quantitated by difference after hydrochloric acid hydrolysis at 65 degrees C with no loss of salicylic acid by sublimation or hydrolytic loss of salicyluric acid. This method has been applied to urine samples from human subjects and the results are discussed.     

Sample preparation in the determination of metals in oil and petroleum products by ICP MS

Comparative elemental analysis of the Tengiz oil and diesel fuel is performed by inductively coupled plasma mass-spectrometry with autoclave digestion, digestion, dilution with organic solvents, and also rotating coiled columns (RCCs). The advantages and drawbacks of each of the listed sample preparation techniques for the separation of microelements are discussed. In contrast to the other versions, the use of RCCs is shown to provide a unique opportunity to preconcentrating microelements from oil and petroleum products into tiny volumes of aqueous solutions (10 mL of 0.5 M HNO₃). The eluate prepared can be used in the subsequent analysis by ICP MS with no extra sample preparation steps. The RCC preconcentration of elements from oil and petroleum products makes it possible to determine metals in concentrations from μg/kg to ng/kg.     

High-performance liquid chromatographic analysis of tiaprofenic acid and its metabolites in plasma and urine by direct injection

A rapid, convenient, sensitive and selective reversed-phase high-performance liquid chromatographic method was developed to measure tiaprofenic acid, its reduced and oxidized metabolites and their conjugates in biological fluids. The method involved direct injections of plasma and urine samples into the chromatograph before and after alkaline hydrolysis of the conjugates. Concentrations as low as 0.5 micrograms/ml of the drug in plasma and urine were quantifiable. The method was suitable for analysis of tiaprofenic acid and its metabolites in biological fluids after administration of therapeutic doses. Several other non-steroidal anti-inflammatory drugs which were applied to the system did not interfere with the assay.     

Rapid determination of zinc and iron in foods by flow-injection analysis with flame atomic-absorption spectrophotometry and slurry nebulization

A rapid method of determining zinc and iron in food by flame atomic-absorption spectrophotometry with slurry nebulization into an air-acetylene flame has been developed. A V-groove, clog-free Babington-type nebulizer, coupled to a single-line flow-injection analysis (FIA) system, was employed to introduce the slurry into the spray chamber. Under the FIA conditions described, an injection frequency of 120/hr is possible, with negligible carry-over and memory effects. The calibration graphs were obtained by using various concentrations (up to 0.1 g/ml) of white bean homogenate as standards, rather than solutions. The method has been applied to various kinds of foods, including grains, vegetables, fruits and sausage. Homogenization of semi-prepared samples to form slurries took only 4 min. Relative deviations between results by the slurry and solution methods for both elements averaged 2-3%. Detection limits by the slurry method were 0.3 mug/ml Zn and 0.6 mug/ml Fe.     

Rapid determination of piracetam in human plasma and cerebrospinal fluid by micellar electrokinetic chromatography with sample direct injection

A simple micellar electrokinetic chromatography (MEKC) method with UV detection at 200 nm for analysis of piracetam in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of piracetam from biological matrix was performed at 25 degrees C using a background electrolyte consisting of Tris buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Several parameters affecting the separation of the drug from biological matrix were studied, including the pH and concentrations of the Tris buffer and SDS. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Using imidazole as an internal standard (IS), the linear ranges of the method for the determination of piracetam in plasma and in CSF were all between 5 and 500 microg/mL; the detection limit of the drug in plasma and in CSF (signal-to-noise ratio=3; injection 0.5 psi, 5s) was 1.0 microg/mL. The applicability of the proposed method for determination of piracetam in plasma and CSF collected after intravenous administration of 3g piracetam every 6h and oral administration 1.2g every 6h in encephalopathy patients with aphasia was demonstrated.