Ultraviolet Absorbance Detection Research Articles

Selective solid-phase extraction using a molecularly imprinted polymer for the analysis of patulin in apple-based foods.

The aim of this work was to evaluate the use of a molecularly imprinted polymer as a selective solid-phase extraction sorbent for the clean-up and pre-concentration of patulin from apple-based food products. Ultra high pressure liquid chromatography coupled to ultraviolet absorbance detection was used for the analysis of patulin. The molecularly imprinted polymer was applied, for the first time, to the determination of patulin in apple juice, puree and jam samples spiked within the maximum levels specified by the European Commission No. 1881/2006. High recoveries (>77%) were obtained. The method was validated and found to be linear in the range 2-100 μg/kg with correlation coefficients greater than 0.965 and repeatability relative standard deviation below 11% in all cases. Compared with dispersive solid-phase extraction (QuEChERS method) and octadecyl sorbent, the molecularly imprinted polymer showed higher recoveries and selectivity for patulin. The application of Affinisep molecularly imprinted polymer as a selective sorbent material for detection of patulin fulfilled the method performance criteria required by the Commission Regulation No. 401/2006, demonstrating the suitability of the technique for the control of patulin at low ppb levels in different apple-based foods such as juice, puree and jam samples.

Single-Laboratory Validation for the Determination of Flavonoids in Hawthorn Leaves and Finished Products by LC-UV.

Suitably validated analytical methods that can be used to quantify medicinally active phytochemicals in natural health products are required by regulators, manufacturers, and consumers. Hawthorn (Crataegus) is a botanical ingredient in natural health products used for the treatment of cardiovascular disorders. A method for the quantitation of vitexin-2″-O- rhamnoside, vitexin, isovitexin, rutin, and hyperoside in hawthorn leaf and flower raw materials and finished products was optimized and validated according to AOAC International guidelines. A two-level partial factorial study was used to guide the optimization of the sample preparation. The optimal conditions were found to be a 60-minute extraction using 50 : 48 : 2 methanol : water : acetic acid followed by a 25-minute separation using a reversed-phased liquid chromatography column with ultraviolet absorbance detection. The single-laboratory validation study evaluated method selectivity, accuracy, repeatability, linearity, limit of quantitation, and limit of detection. Individual flavonoid content ranged from 0.05 mg/g to 17.5 mg/g in solid dosage forms and raw materials. Repeatability ranged from 0.7 to 11.7 % relative standard deviation corresponding to HorRat ranges from 0.2 to 1.6. Calibration curves for each flavonoid were linear within the analytical ranges with correlation coefficients greater than 99.9 %. Herein is the first report of a validated method that is fit for the purpose of quantifying five major phytochemical marker compounds in both raw materials and finished products made from North American (Crataegus douglasii) and European (Crataegus monogyna and Crataegus laevigata) hawthorn species. The method includes optimized extraction of samples without a prolonged drying process and reduced liquid chromatography separation time.

Quantification of ascorbic acid and acetylsalicylic acid in effervescent tablets by CZE-UV and identification of related degradation products by heart-cut CZE-CZE-MS.

Capillary electrophoresis is commonly applied for the analysis of pharmaceutical products due to its high separation efficiency and selectivity. For this purpose, electrospray-ionization-(ESI)-interfering additives or electrolytes are often required, which complicates the identification of impurities and degradation products by mass spectrometry (MS). Here, a capillary zone electrophoresis (CZE) method with ultraviolet (UV) absorption detection for the simultaneous determination and quantification of ascorbic acid and acetylsalicylic acid in effervescent tablets was developed. Related degradation products were identified via CZE-CZE-MS. Systematic optimization yielded 100 mM tricine (pH = 8.8) as appropriate background electrolyte, resulting in baseline separation of ascorbic acid, acetylsalicylic acid, and related anionic UV-active degradation products. The CZE-UV method was successfully validated regarding the guidelines of the Food and Drug Administration. The validated method was applied to trace the degradation rate of the active pharmaceutical ingredients at defined ambient conditions. A heart-cut CZE-CZE-MS approach, including a 4-port-nL-valve, was performed for the identification of the observed degradation products. This 2D setup enables a precise cutting of accurate sample volumes (20nL) and the independent operation of two physically separated CZE dimensions, which is especially beneficial regarding MS detection. Hence, the ESI-interfering tricine electrolyte components were separated from the analytes in a second electrophoretic dimension prior to ESI-MS detection. The degradation products were identified as salicylic acid and mono- and diacetylated ascorbic acid. This setup is expected to be generally applicable for the mass spectrometric characterization of CZE separated analytes in highly ESI-interfering electrolyte systems. Graphical Abstract A CZE-UV method for the quantification of effervescent tablet ingredients and degradation products was developed and validated. In order to identify unknown degradation products separated in the CZE-UV, a 2D heart-cut approach was performed applying a mechanical 4-port-valve. The unknown substances were transferred from the 1st to the 2nd dimension followed by the separation of ESI-interfering tricine from the analytes prior to mass spectrometric detection.

An investigation on the chemical structure of nitrogen and sulfurcodoped carbon nanoparticles by ultra-performance liquid chromatography-tandem mass spectrometry.

A highly fluorescent nitrogen and sulfur codoped carbon nanoparticles (N,S-CNP) sample was obtained by microwave-assisted pyrolysis of citric acid and L-cysteine. After being purified by dialysis, the complexity and chemical composition of N,S-CNP were evaluated by ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) as well as by UPLC coupled with ultraviolet (UV) absorption and fluorescence detection (UPLC-UV/FLD) methods. By using the high-resolution UPLC separation, the N,S-CNP were well fractionated into six fractions within 3.5min. Based on high-accuracy MS and tandem (MS/MS) analyses, the N,S-CNP species were revealed to display various chemical formulas, including (C12H16N2O7S2) n , (C9H13NO8S) n , (C18H20N2O14S2) n , (C18H20N2O12S2) n , (C9H11NO5S) n , and (C9H11NO7S) n . More importantly, our study disclosed unambiguously for the first time that the N,S-CNP species exist as supramolecular clusters with their individual monomer units linked together through noncovalent bonding forces. By using UPLC-UV/FLD analysis, the spectral characteristics of each N,S-CNP species were revealed. Each individual CNP species possesses its unique absorption and PL properties with absorption bands that are redshifted, whereas its emission bands are blueshifted with its elution order. This work highlights the merit of UPLC-MS together with UPLC-UV/FLD to investigate the chemical composition and the spectral properties of each individual N,S-CNP species. It is anticipated that our proposed methodology will openup a new venue in optimizing experimental conditions for producing specific N,S-CNP species of desired composition. Graphical Abstract Carbon nanoparticles synthesized by microwave-assisted pyrolysis of citric acid and L-cysteine exist as supramolecular clusters with their individual monomer units linked together by noncovalent interactions.

25-Hydroxyvitamin D isomerizes to pre-25-hydroxyvitamin D in solution: considerations for calibration in clinical measurements.

Reference standards for the vitamin D metabolites 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, and 3-epi-25-hydroxyvitamin D3 were evaluated using liquid chromatography (LC) with ultraviolet (UV) absorbance and mass spectrometric (MS) detection to assess purity. The chromatograms for solutions of all three 25(OH)D compounds, obtained using a pentafluorophenyl (PFP) stationary phase, revealed peaks that increased in area over time and had MS spectra that were nearly identical to the parent compound, indicating isomers had formed in solution that were unrelated to the reference standard purity. However, when the purity evaluations were completed with a cyanopropyl stationary phase, the isomeric products coeluted with the parent compounds and were not observable. The rates of formation of the isomeric products were found to increase when heated and were confirmed to be pre-25-hydroxyvitamin D compounds using spectral information from both MS detection and nuclear magnetic resonance (NMR) spectroscopy. The rates of conversion of 25(OH)D3 to pre-25(OH)D3 was studied in solutions of ethanol and bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The solutions prepared with BSA/PBS were found to form twice as much pre-25(OH)D3 as the solutions in ethanol. The isomerization of 25(OH)D in solution has implications for calibration of 25(OH)D in clinical measurements, which are discussed.

Development of an achiral supercritical fluid chromatography method with ultraviolet absorbance and mass spectrometric detection for impurity profiling of drug candidates. Part II. Selection of an orthogonal set of stationary phases

Impurity profiling of organic products that are synthesized as possible drug candidates requires complementary analytical methods to ensure that all impurities are identified. Supercritical fluid chromatography (SFC) is a very useful tool to achieve this objective, as an adequate selection of stationary phases can provide orthogonal separations so as to maximize the chances to see all impurities.In this series of papers, we have developed a method for achiral SFC-MS profiling of drug candidates, based on a selection of 160 analytes issued from Servier Research Laboratories.In the first part of this study, focusing on mobile phase selection, a gradient elution with carbon dioxide and methanol comprising 2% water and 20mM ammonium acetate proved to be the best in terms of chromatographic performance, while also providing good MS response [1].The objective of this second part was the selection of an orthogonal set of ultra-high performance stationary phases, that was carried out in two steps. Firstly, a reduced set of analytes (20) was used to screen 23 columns. The columns selected were all 1.7–2.5μm fully porous or 2.6–2.7μm superficially porous particles, with a variety of stationary phase chemistries. Derringer desirability functions were used to rank the columns according to retention window, column efficiency evaluated with peak width of selected analytes, and the proportion of analytes successfully eluted with good peak shapes. The columns providing the worst performances were thus eliminated and a shorter selection of columns (11) was obtained. Secondly, based on 160 tested analytes, the 11 columns were ranked again. The retention data obtained on these columns were then compared to define a reduced set of the best columns providing the greatest orthogonality, to maximize the chances to see all impurities within a limited number of runs. Two high-performance columns were thus selected: ACQUITY UPC2 HSS C18 SB and Nucleoshell HILIC.

Development of an achiral supercritical fluid chromatography method with ultraviolet absorbance and mass spectrometric detection for impurity profiling of drug candidates. Part I: Optimization of mobile phase composition

Supercritical fluid chromatography (SFC) is a very useful tool in the purpose of impurity profiling of drug candidates, as an adequate selection of stationary phases can provide orthogonal separations so as to maximize the chances to see all impurities. The purpose of the present work is to develop a method for chemical purity assessment. The first part, presented here, focuses on mobile phase selection to ensure adequate elution and detection of drug-like molecules, while the second part focuses on stationary phase selection for optimal separation and orthogonality.The use of additives in the carbon dioxide – solvent mobile phase in SFC is now commonplace, and enables in particular to increase the number of eluted compounds and to improve peak shapes. The objective of this first part was to test different additives (acids, bases, salts and water) for their chromatographic performance assessed in gradient elution with a diode-array detector, but also for the mass responses obtained with a single-quadrupole mass detector, equipped with an electrospray ionization source (Waters ACQUITY QDa).In this project, we used a selection of one hundred and sixty compounds issued from Servier Research Laboratories to screen a set of columns and additives in SFC with a Waters ACQUITY UPC2 system. The selected columns were all high-performance columns (1.7–1.8μm with totally porous particles or 2.6–2.7μm with superficially porous particles) with a variety of stationary phase chemistries.Initially, eight additives dissolved in the methanol co-solvent were tested on a UPC2 ACQUITY UPC2 HSS C18 SB column. A Derringer desirability function was used to classify the additives according to selected criteria: elution capability, peak shapes, UV baseline drift, and UV and mass responses (signal-to-noise ratios). Following these tests, the two best additives (ammonium acetate and ammonium hydroxide) were tested on a larger number of columns (10) where the two additives appeared to provide very comparable overall scores. However, ammonium acetate was selected for slightly better chromatographic quality.In a second step, we investigated the effects of ammonium acetate concentration (between 0 and 25mM in the methanol co-solvent) on retention and peak efficiency. Two types of silica supports were tested by working with ACQUITY UPC2 HSS C18 SB and BEH columns. 20mM ammonium acetate in methanol with 2% water was finally selected as the best co-solvent composition.

A combination of solid-phase extraction and dispersive solid-phase extraction effectively reduces the matrix interference in liquid chromatography-ultraviolet detection during pyraclostrobin analysis in perilla leaves.

Perilla leaves contain many interfering substances; thus, it is difficult to protect the analytes during identification and integration. Furthermore, increasing the amount of sample to lower the detection limit worsens the situation. To overcome this problem, we established a new method using a combination of solid-phase extraction and dispersive solid-phase extraction to analyze pyraclostrobin in perilla leaves by liquid chromatography with ultraviolet absorbance detection. The target compound was quantitated by external calibration with a good determination coefficient (R(2) = 0.997). The method was validated (in triplicate) with three fortification levels, and 79.06- 89.10% of the target compound was recovered with a relative standard deviation <4. The limits of detection and quantification were 0.0033 and 0.01 mg/kg, respectively. The method was successfully applied to field samples collected from two different areas at Gwangju and Muan. The decline in the resiudue concentrations was best ascribed to a first-order kinetic model with half-lives of 5.7 and 4.6 days. The variation between the patterns was attributed to humidity.

Automated Flow Injection Chromatography for the Rapid Assay of Parabens in Hygiene Wipes

ABSTRACTA fully automated flow injection chromatography (FIC) instrument and method are reported for the determination of methylparaben and propylparaben. The system consisted of a computer-controlled MilliGAT precision pump, a six–way injection valve, a peristaltic pump, a monolithic column, and an ultraviolet absorbance detector. The mobile phase composition, flow rate, and sample volume were optimized. The method was fully validated; the calibration plots were linear from 1.0 × 10−6 to 5.0 × 10−5 mol L−1 for both parabens. The limits of detection were 1.0 × 10−7 mol L−1 for methylparaben and 3.0 × 10−7 mol L−1 for propylparaben. The recoveries for both parabens were 97% and the interlaboratory reproducibility was 2.4% for methylpareben and 4.1% for propylparaben at 1.0 × 10−5 mol L−1. The method was employed for the determination of methylparaben and propylparaben in hygiene wipes.

Investigation of photochemical reactions of saccharides during direct ultraviolet absorbance detection in capillary electrophoresis

Direct UV detection of saccharides in capillary electrophoresis is possible due to a base-catalyzed conversion into UV absorbing substances initiated by the light of the UV detector lamp. In the present study the compounds formed during this reaction were investigated with capillary electrophoresis using an additional UV lamp for online irradiation at a certain distance before the detector resulting in a separation of the conversion products. It was found that for all investigated saccharides (glucose, ribose and sucrose) the major portion of the UV absorption in direct UV detection resulted from one and the same substance. By CE–UV, CE–MS as well as HPLC–MS measurements this substance was demonstrated to be malondialdehyde, present as its conjugate base malonenolate under alkaline conditions. Additional experiments revealed that the conversion reaction is highly sensitive to the residence time under the UV light as malondialdehyde can further degrade into non-UV absorbing products. NMR measurements showed that under alkaline conditions this degradation proceeds slowly even without UV irradiation.

Simultaneous Determination of Cortisol, Cortisone, 6 -Hydroxycortisol and 6 -Hydroxycortisone by HPLC

A specific and sensitive method based on high-performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) was developed for the simultaneous determination of urinary cortisol (F), cortisone (E), 6β-hydroxycortisol (6β-OHF) and 6β-hydroxycortisone (6β-OHE) using dexamethasone as the internal standard. The method involved solid-phase extraction of the five compounds from urine using Oasis HLB Waters cartridges with an elution solvent of ethyl acetate-diethyl ether (5 mL; 4:1, v/v), followed by 1 mol/L of NaOH (1 mL) and 1.0% acetic acid (1 mL). Separation of the five analytes was achieved within 31 min by using a reversed-phase C18 analytical column (200 × 4.6 mm, 5 µm, Agilent). A UV detector operated at 245 nm was used. According to the method validation, inter-run and intra-run precision was below 9.45% and accuracy ranged from 98.16 to 115.50%. The lower limits of quantitation were 5 ng/mL for four analytes. This is the first HPLC method that can simultaneously determine F, E, 6β-OHF and 6β-OHE in human urine. The assay was applied to research the ratio of (6β-OHF + 6β-OHE)/(F + E) as a non-invasive biomarker for the metabolism of tacrolimus.

Ion-pair chromatography-indirect ultraviolet detection for determination of tetraethyl ammonium using a monolithic column and a packed column

Two methods were developed for the determination of tetraethyl ammonium by ion-pair chromatography-indirect ultraviolet detection using a monolithic column and a packed column with ionic liquid as additive in mobile phase. Chromatographic separations were performed on a monolithic column and a packed column both on reversed phase using imidazolium ionic liquid aqueous solution-ion-pair reagent-organic solvent as mobile phase. The effects of the background ultraviolet absorption reagent, detection wavelength, ion-pair reagent, organic solvent, column temperature and flow rate on the determination of tetraethyl ammonium were investigated. The difference between the two chromatographic columns was compared and the retention rules were discussed. Under the optimized chromatographic conditions, for tetraethyl ammonium on monolithic column and packed column, the retention times were 2.40 and 3.02 min; the detection limits (S/N=3), 0.04 and 0.07 mg/L; the RSDs (n = 5) for peak areas, 0.16% and 0.11%; and the RSDs (n=5) for retention times, 0.02% and 0.01%, respectively. The two methods have been successfully applied to the determination of tetraethyl ammonium ionic liquids synthesized by laboratory. The recoveries of the tetraethyl ammonium after spiking were 98.2% and 99.1%, respectively. The two methods can meet the requirements for the quantitative analysis of tetraethyl ammonium.

Gradient high performance liquid chromatography method for simultaneous determination of ilaprazole and its related impurities in commercial tablets

A methodology (HPLC) proposed in this paper for simultaneously quantitative determination of ilaprazole and its related impurities in commercial tablets was developed and validated. The chromatographic separation was carried out by gradient elution using an Agilent C8 column (4.6 mm × 250 mm, 5 μm) which was maintained at 25 °C. The mobile phase composed of solvent A (methanol) and solvent B (solution consisting 0.02 mmol/l monopotassium phosphate and 0.025 mmol/l sodium hydroxide) was at a flow rate of 1.0 ml/min. The samples were detected and quantified at 237 nm using an ultraviolet absorbance detector. Calibration curves of all analytes from 0.5 to 3.5 μg/ml were good linearity (r ≥ 0.9990) and recovery was greater than 99.5% for each analyte. The lower limit of detection (LLOD) and quantification (LOQ) of this analytical method were 10 ng/ml and 25 ng/ml for all impurities, respectively. The stress studies indicated that the degradation products could not interfere with the detection of ilaprazole and its related impurities and the assay can thus be considered stability-indicating. The method precisions were in the range of 0.41–1.21 while the instrument precisions were in the range of 0.38–0.95 in terms of peak area RSD% for all impurities, respectively. This method is considered stability-indicating and is applicable for accurate and simultaneous measuring of the ilaprazole and its related impurities in commercial enteric-coated tablets.

Effects of diet and strain on mouse serum and tissue retinoid concentrations.

The relationship between dietary vitamin A and all-trans-retinoic acid levels in serum and tissues had not been quantified. We determined the impact of dietary vitamin A on retinoid levels in serum, liver, kidney, testis, and epididymal white adipose of five mouse strains: AKR/J; BALB/cByJ; C3H/HeJ; C57BL/6J; 129S1/SvImJ. Retinoids were quantified in mice fed copious vitamin A (lab chow, ≥20 IU/g) followed by one month feeding a vitamin A-sufficient diet (4 IU/g), or after three generations of feeding a vitamin A-sufficient diet. Retinol and retinyl esters were measured by high-performance liquid chromatography with ultraviolet absorbance detection. All-trans-retinoic acid was quantified by liquid chromatography tandem mass spectrometry. The amounts of dietary vitamin A had long-term strain-specific effects on tissue retinyl ester, retinol and all-trans-retinoic acid concentrations. Three generations of feeding a vitamin A-sufficient diet decreased all-trans-retinoic acid in most tissues of most strains, in some cases more than 60%, compared to a diet with copious vitamin A. With both diets, all-trans-retinoic acid concentrations maintained an order of liver ≈ testis > kidney > white adipose tissue ≈ serum. Neither retinol nor all-trans-retinoic acid in serum reflected all-trans-retinoic acid concentrations in tissues. Strain and tissue-specific differences in retinol and all-trans-retinoic acid altered by different amounts of dietary vitamin A could have profound effects on retinoid action. This would be the case especially with the increased all-trans-retinoic acid values associated with the amounts of vitamin A and its precursors (carotenoids) in chow diets.

The Analysis for Fluroxypyr Ester by Liquid Chromatography

The analysis method for fluroxypyr ester by high performance liquid chromatography (HPLC) is established in this paper. This method is based on the chemical and physical properties of fluroxypyr ester to optimize determination conditions such as the sample solvent, mobile phase and chromatographic column and detection wavelength. The different mass fraction of samples were analyzed by the following conditionds: the mobile phase is methanol-water, and the column is stainless steel filled with packing of Hypersil BDS C18 5 μm, which size is 250 x 4.6 mm (i, d), and the solvent is methanol, and the detector is ultraviolet absorption detector, and the flow velocity is 1.0 mL/min, and the wavelength is 235 nm. The experimental results show that the standard addition recovery is 99.3% ~ 100.3%, the standard deviation is 0.1%, the regression equation is y = 491.78 + 501.31 x, and the linear correlation coefficient is 0.9997.

Automation Highlights from the Literature

Automation Highlights from the Literature

Determination of piperidinium ionic liquid cations by ion-pair chromatography-indirect ultraviolet detection

A method was developed for the determination of piperidinium cations by ion-pair chromatography with indirect ultraviolet detection. Chromatographic separation was performed on a reversed-phase C18 column using background ultraviolet absorption reagent-ion-pair reagent/organic solvent as mobile phase. The effects of the background ultraviolet absorption reagent, detection wavelength, ion-pair reagent, organic solvent, column temperature and flow rate on the determination of piperidinium cations were investigated and the retention rules were studied. The optimized chromatographic conditions for the determination of piperidinium cations were as follows: mobile phase, 0.5 mmol/L 4-aminophenol hydrochloride-0.1 mmol/L 1-heptanesulfonic acid sodium aqueous solution/methanol (80:20, v/v); detection wavelength, 210 nm; column temperature, 30 degrees C; flow rate, 1.0 mL/min. Under these conditions, the three piperidinium cations were baseline separated within 4 min. The detection limits (S/N = 3) of the piperidinium cations were 0.137-0.545 mg/L. The relative standard deviations (n = 5) for peak area and retention time were 0.72% and 0.37% respectively. The method has been successfully applied to the determination of piperidinium cations in ionic liquids synthesized by chemistry laboratory. The recoveries of piperidinium cations after spiking were 97.0%-98.4%. The method is simple, rapid, reproducible, linear, and can meet the quantitative analysis requirement for the determination of piperidinium cations.

Simultaneous determination of total homocysteine and cysteine in human plasma by capillary zone electrophoresis with pH-mediated sample stacking

An accurate, simple and sensitive method for simultaneous determination of total homocysteine (Hcy) and total cysteine (Cys) in human plasma has been developed and validated. Hcy and Cys play a major role in metabolism and cellular homeostasis. An elevated level of plasma Hcy is considered an important risk factor or marker for several diseases, in particular cardiovascular disease, while Cys is involved in a variety of important cellular functions such as protein synthesis, detoxification and metabolism. The proposed analytical procedure consists of reduction of title thiol dimers and unsymmetrical disulfides with tris(2-carboxyethyl)phosphine, and derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) followed by pH-mediated stacking capillary electrophoresis separation and ultraviolet-absorbance detection of Hcy-CMQT and Cys-CMQT derivatives at 355 nm. Effective baseline electrophoretic separation was achieved using a standard fused-silica capillary (effective length 91.5 cm, 75 μm id) and 0.1 mol L−1, lithium acetate buffer adjusted to pH 4.75. The limit of quantification (signal to noise ratio of 9) for derivatized Hcy and Cys in plasma was 2 μmol L−1. The calibration curves obtained for Hcy and Cys in plasma showed linearity in the range 2–20 μmol L−1, with R2 = 0.9986 and 20–300 μmol L−1, with R2 = 0.9998, respectively. The relative standard deviation of the points of the calibration curve was lower than 7%. The method can be used for a routine monitoring of total Hcy and total Cys levels in plasma of human subjects.

An assessment of 25-hydroxyvitamin D measurements in comparability studies conducted by the Vitamin D Metabolites Quality Assurance Program

BackgroundThe National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health Office of Dietary Supplements, established the first accuracy-based program for improving the comparability of vitamin D metabolite measurements, the Vitamin D Metabolites Quality Assurance Program. MethodsThe study samples were human serum or plasma Standard Reference Materials (SRMs) with 25-hydroxyvitamin D values that were determined at NIST. Participants evaluated the materials using immunoassay (IA), liquid chromatography (LC) with mass spectrometric detection, and LC with ultraviolet absorbance detection. NIST evaluated the results for concordance within the participant community as well as trueness relative to the NIST value. ResultsFor the study materials that contain mostly 25-hydroxyvitamin D3 (25(OH)D3),the coefficient of variation (CV) for the participant results was consistently in the range from 7% to 19%, and the median values were biased high relative to the NIST values. However, for materials that contain significant concentrations of both 25-hydroxyvitamin D2 (25(OH)D2) and 25(OH)D3, the median IA results were biased lower than both the LC and the NIST values, and the CV was as high as 28%. The first interlaboratory comparison results for SRM 972a Vitamin D Metabolites in Human Serum are also reported. ConclusionsRelatively large within-lab and between-lab variability hinders conclusive assessments of bias and accuracy.

Ultrasound‐assisted Extraction with LC–TOF/MS Identification and LC–UV Determination of Imazamox and its Metabolites in Leaves of Wheat Plants

imazamox is a herbicide used in many legominous and cereal crops. There are few methods in the literature for determination of imazamox and its metabolites in plants because of the lack of commercial standards or owing to expensive and/or complex synthesis. To develop a method based on liquid chromatography and ultraviolet absorption detection for simultaneous determination of imazamox and its metabolites in plants. Sample preparation was based on ultrasound-assisted extraction (70 W power and duty cycle of 0.7 s/s for 10 min) with subsequent filtration of the extracts and clean-up and concentration prior to chromatographic separation and detection at 240 nm. The chromatographic analysis was completed in 30 min using a Luna® HILIC column. Identification and confirmatory analysis of the presence of imazamox and its metabolites in extracts from treated plants was performed by LC-TOF/MS in high resolution mode for precursor ions. The metabolites were quantified using a surrogate approach based on an imazamox standard. The method was validated by analysing wheat samples treated with 200 g per hectare of active ingredient imazamox. The linear dynamic range of the calibration curve was within 0.27-600 µg/mL, with a correlation coefficient of 0.998 and precision--studied at 0.1 and 2 µg/mL--of 2.9% and 5.0% for repeatability, and 4.7% and 6.9% for reproducibility, respectively. The analytical characteristics of the method make it recommendable for evaluating the metabolism of imazamox in plants.