Internal Standard Solution Research Articles

Use of reference materials in coastal monitoring quality assurance

The NOAA National Status and Trends (NS&T) Program determines the current status of, and changes over time in the environmental health of U.S estuarine and coastal waters. Concentrations of organic and inorganic contaminants are determined in bivalves, bottom-dwelling fish and sediments. The quality of the analytical data generated by the NS&T Program is over-seen by the performance-based Quality Assurance Project, which is designed to document sampling protocols, analytical procedures, and laboratory performance, and to reduce intralaboratory and interlaboratory variation. All NS&T cooperating laboratories are required to participate in yearly intercomparison exercises. The analysis of reference materials, such as the National Research Council of Canada's Certified Reference Materials (CRMs) and National Institute of Standards and Technology's (NIST) Standard Reference Materials (SRMs), and of control materials, is required. Because of the need for marine matrix reference materials and standards, NOAA contributed to the production of eight NIST SRMs and seven internal standard solutions. Analytical data from all control materials and all matrix reference materials are reported to the Program office.

TRACE ELEMENT ANALYSIS IN FLOUR

Trace elements in five kinds of flour which were made in Japan. America and Canada, have been measured by means of in-air PIXE. Twelve kinds of trace elements were detected, such as Cl, K, Ca, Sc, Cr, Mn, Fe, Co, Ni, Cu, Zn and Br. The concentration of these elements varied depending on the place where they were made. In order to check the target preparation procedure, two types of sample preparation have been used, such as wet ashing by nitric acid in a microwave oven and making a paste by an internal standard solution. Comparisons of the results of both samples ensures that bromine was not lost during micro-wave oven heating. Analytic fitting of PIXE spectra by a personal computer and a commercial apllication software was successfully used.

The Automated Extraction, Derivatization and GC/MS of Methamphetamine and Amphetamine in Human Urine.

The method for the automatic analysis of methamphetamine (MA) and amphetamine (AP) in the urine was developed using a prepstation in combination with a GC/MS instrument. A prepstation can automate many pretreatment steps, such as solid phase extraction (SPE) and derivatization. Human interaction is only required to load the urine samples into vials, cap them and place them on the auto sampler tray. All of the following steps were automated : 1) An urine sample of 1.5 ml was injected into the SPE cartridge (DAU), where MA and AP were retained. 2) The cartridge was then washed with 1M acetic acid (1 ml) and then with methanol (3 ml). 3) The MA and AP were then eluted using 0.1% sulfric acid-methanol (0.5 ml) from the washed cartridge. 4) The elute was evaporated and dried. Then a 4N sodium hydroxide (0.02 ml) and a 10 ppm diphenylmethane in dichlormethane (0.2 ml) internal standard (I.S.) solution were added and stirred. 5) A derivatizing reagent, N-methyl-bis-trifluoroacetamide (MBTFA) was added into the sample. 6) And finally, the reaction mixture was injected into the GC/MS instrument for analysis. The total analysis time for 10 samples was approximately 10 h and 20 min. The calibration curves, using the I.S. method, demonstrated good linearity throughout the concentration range from 0.1 to 10 μg/ml for both MA and AP. Using a scan mode the detection limits were 20 ng/ml for both MA and AP and using selected ion monitoring (SIM) the limits were 1 ng/ml for MA and 3 ng/ml for AP. The relative standard deviation using the scan mode for a 1 μg/ml sample was 5.9% for MA and 6.0% for AP.

Capillary Gas Chromatographic Determination of Thiabendazole in Citrus and Apple Juices

A rapid and simple procedure for the determination of thiabendazole (TBZ) residue in citrus and apple juices is described. A juice sample is made basic with 2M NaOH and applied to a disposable Extrelut prepacked column. TBZ is eluted with hexane-ethyl acetate (3 + 1) from the column. The eluate is evaporated to dryness under reduced pressure and then dissolved in an internal standard solution. TBZ is monitored without derivatization by capillary gas chromatography with nitrogen-phosphorus detection. The recoveries of TBZ added to fruit juices at 0.05-1.0 micrograms/g were 90-96%. The limit of detection of the method for TBZ was 0.01 micrograms/g. The proposed method is rapid, simple, and sensitive and is applicable to the determination of TBZ in commercial fruit juices.

Development of a new method for the determination of thyroxine in serum based on isotope dilution gas chromatography mass spectrometry.

A new gas chromatographic/mass spectrometric method in combination with isotope dilution for the determination of thyroxine in serum is described. Special attention was paid to the methylation step of thyroxine, which was investigated using methanolic HCl, dimethylformamide/dimethylacetal and diazomethane, the latter giving the best results in terms of reproducible isotope ratios. For internal standardization, (13C6)-thyroxine was dissolved in fraction V human albumin solution (70 g l-1). The internal standard-in-albumin solution was mixed with known amounts of thyroxine standard, dissolved in 0.05 M Na2HPO4 buffer at pH 11.6, to give isotope ratios of 0.75, 1.00 and 1.25. The same internal standard solution was also used for isotope dilution of the unknown serum samples. The volume of serum was adapted to give a 1:1 isotope ratio. Sample pretreatment consisted of protein precipitation and a two-step liquid/liquid extraction procedure. After methylation of unlabelled and labelled thyroxine with diazomethane and perfluoroacylation with pentafluoropropionic anhydride and heptafluorobutyric anhydride, respectively, mass spectrometric monitoring was done at m/z 951/957 and 1001/1007. Quantitative determination of thyroxine in five serum samples in duplicate, during three consecutive days, showed a mean overall imprecision of 1.0% and a deviation of +0.4% from the target value as determined by a definitive method.

Determination of lead by flow-injection inductively coupled plasma mass spectrometry comparing several calibration techniques

In this paper we have evaluated the coupling of a modular flow-injection system to an inductively coupled plasma mass spectrometer for lead determinations by direct analysis of wines and urine samples. The flow-injection system used allows an on-line sample dilution and an on-line addition of internal standard and calibrant solution. It was found that particular attention should be paid to the adjustment of the nebulizer gas flow rate in order to obtain maximum sensitivity and precision. In this way and for the experimental set-up described, R.S.D. values ⩽ 1% on the 208Pb + signal for 5 consecutive injections could readily be achieved. The relative merits of external calibration, standard additions and isotope dilution for the calibration of the transient lead signals were compared. It appeared that from the viewpoint of accuracy, precision and flexibility, the standard additions method is the most preferable procedure. Further, it was found that the precision of isotope ratio determinations is degraded in comparison to continuous nebulization. However, matrix-induced mass fractionation on the lead isotope ratios as observed for the continuous nebulization of an aqueous ethanolic (wine) matrix did not occur in flow-injection analysis. The quantitation of lead by direct analysis of undiluted wine and urine samples using this flow-injection method in combination with standard additions calibration resulted in very accurate results (compared to certified and reference values). Excellent relative standard deviations (for 5 consecutive determinations) were obtained ranging from 1% (50 μg 1 −1 Pb in white wine) to 5% (3.6 μg 1 −1 Pb in urine.

Automated determination of midazolam in human plasma by high-performance liquid chromatography using column switching

An automated gradient high-performance liquid chromatographic method using a column-switching technique was developed in order to determine and quantify midazolam (separated from the metabolite α-hydroximidazolam) in human plasma. After dilution with an internal standard (flurazepam) solution, containing 20% acetonitrile, 400 μl of the plasma samples were injected onto a precolumn (17 × 4.6 mm I.D., C 18 Corasil 37–53 μm) and retained. Proteins and polar plasma components were washed out using a 0.1 M sodium hydroxide solution, followed by an equilibration with a phosphate buffer of pH 8.0. After column-switching midazolam and flurazepam were eluted and transferred to the analytical column (RP-select B) in the backflush mode, separated by gradient elution and detected at 230 nm by ultraviolet detection. Precision of replicate analyses on the same day was 1.5% for midazolam and 0.7% for flurazepam. Recovery of midazolam was in the range 80–89% and the detection limit was 2 ng/ml plasma.

Determination of 5‐hydroxymethyl‐2‐furaldehyde in honey by micellar electrokinetic capillary chromatography

AbstractMethodology using micellar electrokinetic capillary chromatography (MECC) to determine 5‐hydroxymethyl‐2‐furaldehyde (HMF) as a degradation product of carbohydrates in aged and thermal treated honey is described. Sodium dodecyl sulfate was employed as the anion surfactant in a phosphate buffer, pH 7.5. The sample preparation was limited to dissolution of honey in water and addition of an internal standard solution (2‐furylmethylketone). The method was highly selective and exhibited high reporducibility, accuracy, and short analysis time.

Simultaneous determination of Ibuproxam and Ibuprofen in human plasma by HPLC with column switching

A new high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of Ibuproxam and its metabolite Ibuprofen in plasma. The plasma samples were injected onto a LiChrosorb RP-8 precolumn after dilution with an internal standard solution in phosphoric acid. Polar plasma components were eluted with 0.05 M phosphoric acid. After valve switching, the concentrated drugs were eluted in the back-flush mode and separated on an Inertsil ODS-2-column with acetonitrile-methanolphosphate buffer as mobile phase. The method showed excellent precision, accuracy and speed with detection limit of 0.1 μg ml−1. Total analysis time per sample was less than 30 min and the coefficients of variation for intra and inter-assay were less than 3.5%. This method has been successfully applied to plasma samples from human volunteers after oral administration of Ibuproxam.

Chemical analysis of rare earth-iron magnetostriction alloys by ICP-AES using an internal standard.

Rare earth-iron magnetostriction alloy (0.1g) was decomposed with hydrochloric acid and hydrogen peroxide within a few minutes. The solution obtained was diluted to 100ml with water. To 10 ml of this diluted solution, an internal standard solution and hydrochloric acid were added and then diluted to 100 ml again with water. The amounts of Mn, Fe, Tb and Dy were measured by ICP-AES without any concomitant effect. Among the internal standard elements investigated (Sc, Y and La), La was most suitable. Recoveries of each element measured with a synthetic sample solution were over 99.7%. The relative standard deviations (n=5) were 0.04%0.35%.

Isotope dilution gas chromatography/mass spectrometry for cadmium determination in urine.

A stable isotope dilution gas chromatographic/mass spectrometric method using 106Cd as an internal standard is described for determining Cd in urine. In this method, the urine matrix is digested with HNO3 + H2O2 and the Cd is chelated with lithium bis(trifluoroethyl)dithiocarbamate. All isotope ratio measurements are made with an organic mass spectrometer. Overall precision values for the five major isotopes relative to 114Cd are 1 to 3% when 10-ng samples of chelated Cd are analyzed. Accuracy of the method is established by measuring Cd in the NIST freeze-dried urine reference material. A small memory effect is observed when measuring isotope ratios differing by a factor of 10. This can, however, be minimized by optimum addition of the internal standard solution.

Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection

We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C 18 column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 μl of the mobile phase. A 10-μl aliquot of the resulting solution is injected onto a 150 mm × 4.6 mm I.D. column packed with 5-μm octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E 1 = +0.25 V and E 2 = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90–95% which allows the determination of 0.5 μg/l analyte.

Determination of diclofenac in plasma using a fully automated analytical system combining liquid—solid extraction with liquid chromatography

A fully automated analytical system based on liquid—solid extraction combined with column liquid chromatography is described for the determination of diclofenac in plasma. After addition of pH 5 buffer and the internal standard solution to the plasma sample, both sample preparation via a C 18 disposable extraction column and injection were performed by a Gilson ASPEC system. Diclofenac and the internal standard were separated on a reversed-phase column, using methanol—pH 7.2 phosphate buffer (56:44, v/v) as mobile phase at a flow-rate of 0.4 ml/min. The reproducibility and accuracy of the method were acceptable over the concentration range 31–3140 nmol/l in plasma.

Automatic determination of diltiazem and desacetyldiltiazem in human plasma using liquid-solid extraction on disposable cartridges coupled to HPLC — Part I: Optimization of the HPLC system and method validation

A sensitive and automated method for the analysis of diltiazem and desacetyldiltiazem in plasma has been developed using liquid-solid extraction (LSE) on disposable extraction cartridges (DECs) in combination with HPLC. After isolation from plasma, the analytes are separated on a highly deactivated octyl silica column with a mobile phase of methanol-0.05 M phosphate buffer (pH 7.4) (62:38, v/v). The analytes are monitored photometrically at 238 nm. The complete preparation of the plasma sample as well as the injection of the final extract on to the analytical column are performed automatically by means of a sample processor equipped with a robotic arm to which is attached a needle dispensing the different liquids. The internal standard solution is first added to the plasma sample. The DEC is then conditioned successively with methanol and phosphate buffer (pH 7.4). A 1.0-ml volume of sample containing the internal standard solution is applied on an extraction cartridge filled with cyanopropyl silica (50 mg). After the DEC has been washed with the same buffer, the analytes are eluted with 0.16 ml of methanol. A 0.14-ml volume of buffer is then passed through the DEC and 0.25 ml of the final extract is injected onto the HPLC column. The absolute recoveries of the drugs are about 90% and the limit of detection for diltiazem is 0.8 ng ml-1. Relative standard deviations of 2.6% (within-day) and 3.7% (between-day) have been obtained for this compound at a plasma concentration of 50 ng ml-1.

External-source contamination during extraction—distillation in isotope-ratio analysis of soil inorganic nitrogen

The sources of contamination introduced during the extraction, distillation and drying phases of isotope-ratio analysis of soil inorganic nitrogen (ammonium and nitrite + nitrate) were identified, and the individual amounts of contaminants were quantified by isotope dilution. The procedure involves addition of internal standard solutions of 15N-labelled ammonium and nitrite to reagent blanks which are carried through each stage of the analysis at the same time as the test samples. Potassium chloride extractants, filter-papers, distillation reagents and atmospheric ammonia all contributed to dilution of the sample 15N. Some materials tested contained sufficient contaminants to cause large errors in the determination of sample 15N abundance. Both the amount and isotopic composition of contaminants can be determined by the isotope-dilution procedure, which permits the measured sample 15N abundance to be corrected for contamination.

ALKALOID CONTENT OF REED CANARYGRASS (Phalaris arundinaceae L.) AS DETERMINED BY GAS-LIQUID CHROMATOGRAPHY

A technique was developed for the quantification of gramine, hordenine and 5-methoxy-N,N-dimethyl tryptamine (5MeO-DMT) in reed canarygrass (RCG) using gas–liquid chromatography. One gram of freeze-dried, ground RCG sample was extracted with methanol containing 1% ammonium hydroxide. Following solvent evaporation, the residue was extracted with dilute hydrochloric acid (0.01 N). The pH of the extract was then raised and stabilized by adding 0.5 M sodium tetraborate-boric acid buffer pH 9. The internal standard solution (0.25 mg mL−1) n-nonadecane was added and the extract was purified by partitioning with chloroform. The chloroform was then evaporated and the alkaloids were derivatized with bis(trimethylsilyl)-trifluoroacetamide and effectively separated on a gas chromatographic column packed with 2% OV-7 on Chromosorb W. Derivatization was effective at 100 °C (20 min). Extraction of the sample with methanol was maximal at 30-minute extraction time and the recovery of all the alkaloids studied was shown to average 90%. Development of this procedure provides a means to measure RCG gramine, hordenine and 5MeO-DMT concentrations in the presence or absence of tryptamines or β-carbolines. Initial growth and regrowth of five cultivars of RCG from two locations were evaluated for gramine, hordenine and 5MeO-DMT content using this technique. No differences were detected in hordenine content. Gramine content was higher (P < 0.05) in Rival than Venture, with Castor, Palaton and Vantage being intermediate. None of the cultivars contained detectable levels of 5MeO-DMT. There were no differences (P > 0.05) in hordenine content between initial growth and regrowth; however, gramine content of regrowth was higher (P < 0.05) than initial growth.Key words: Gramine, hordenine, 5-methoxy-N,N-dimethyl tryptamine, reed canarygrass, Phalaris arundinaceae L.

Isocratic reverse-phase liquid chromatography assay for amino acid metabolic disorders using eluates of dried blood spots

A high-performance liquid chromatographic method for the simultaneous determination of the amino acids methionine, valine, tryptophan, phenylalanine, isoleucine, and leucine extracted from dried blood spots used in neonatal screening is described. The amino acids are eluted from a 3-mm filter paper disc of dried blood with an absolute ethanol:norleucine internal standard solution (1.5:1, v/v), derivatized with o-phthalaldehyde prior to injection, and separated on a C-18 reversephase column with subsequent fluorescent detection. The analysis time is under 9 min at the described sample dilution and the assay is linear from 15 to 300 μmol/liter for five of the amino acids and from 15 to 500 μmol/liter for valine. The interrun coefficients of variation are <10% (except for tryptophan) and the analytical recoveries exceed 85%. Results from patient samples correlate well with those from a Waters Pico-Tag amino acid analysis system and no apparent interferences were encountered. The rapid analysis time and the specificity of the assay will facilitate the presumptive diagnosis of the inherited amino acidopathies phenylketonuria, maple syrup urine disease, and homocystinuria/methioninemia as well as monitoring blood levels of diagnosed patients.

Simultaneous determination of cefoxitin, cefuroxime, cephalexin and cephaloridine in plasma using HPLC and a column-switching technique

A new high performance liquid chromatographic method was developed using a column-switching technique for the simultaneous determination of cephalexin, cefuroxime, cefoxitin and cephaloridine in plasma. The plasma samples were injected onto a precolumn packed with Corasil RP C18 (37–50 μm) after simple dilution with an internal standard solution in 0.01 M acetate buffer (pH 3.5). Polar plasma components were washed out using 0.01 M acetate buffer (pH 3.5). After valve switching, the concentrated drugs were desorbed in back-flush mode and separated on a Partisil ODS-3 column using acetonitrile in 0.02 M acetate buffer (pH 4.3) (15∶85, v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed with a detection limit of 0.5 μg/ml. The total analysis time per sample was less than 25 min, and the mean coefficients of variation for intra- and inter-assay were both less than 4.9 %.

Liquid-chromatographic determination of amiodarone and N-desethylamiodarone in serum

We have developed a stable and simple normal-phase liquid-chromatographic method for simultaneously measuring amiodarone and its metabolite, N-desethylamiodarone, within 8 min. The chromatographic system consists of a 15 cm x 3.9 mm Waters "Resolve" silica column and a mobile phase of ammonium sulfate (17 mmol/L, pH 6.8) and methanol (8/92 by vol), pumped at 1.8 mL/min and monitored at 254 nm. After 250 microL of serum is mixed with 100 microL of 0.36 mol/L NaH2PO4, 100 microL of the internal standard solution (L8040, 6 mg/L), and 200 microL of isopropyl ether, the mixture is vortex-mixed and centrifuged. Fifty microliters of the organic layer is injected onto the column. Relative recovery was 100% over the assay range of 0.1 to 20.0 mg/L for both compounds. Within-run and total (day-to-day) CVs were 3% and 7% for amiodarone and 5% and 8% for N-desethylamiodarone, respectively.

Simultaneous determination of pantothenic acid and hopantenic acid in biological samples and natural products by gas chromatography-mass fragmentography

A method for the simultaneous determination of pantothenic acid and hopantenic acid in plasma samples was developed using gas chromatography-mass spectrometry with multiple ion detection. Plasma samples were directly purified without deproteinization on an ion-exchange resin, and the eluate was extracted with ethyl acetate under acidic conditions. The organic layer was evaporated to dryness under a stream of nitrogen, and the residue was dissolved in an internal standard solution. Pantothenic and hopantenic acids were converted into their trimethylsilyl derivatives by treating with bis(trimethylsilyl)trifluoroacetamide. Aliquots of this solution were injected into the gas chromatograph-mass spectrometer, which was equipped with a wide-bore fused-silica column (DB-17) and analysed by the multiple ion detection method. The detection limits for pantothenic acid and hopantenic acid in plasma were 1 ng/ml each at a signal-to-noise ratio of 5. This method was applied to a study of the assay of pantothenic acid and hopantenic acid in biological samples and natural products.