Repeatability Of Retention Times Research Articles

Analysis of vitamin D and its metabolites in biological samples – Part I: Optimization and comparison of UHPSFC-MS/MS and UHPLC-MS/MS methods

Fat-soluble vitamin D is an essential bioactive compound important for human health. Insufficient vitamin D levels can result not only in bone disease but also in other disorders, such as cancer, metabolic disorders, and diseases related to poor immune function. The current methods commonly used for vitamin D analysis are often applied to determine the levels of the most abundant metabolite in plasma, i.e., 25-OH-D2/D3. These methods do not consider the presence of other hydroxylated and esterified metabolites, including isomers and epimers, which are typically found in low concentrations.In this study, we developed a fast and selective ultra-high performance supercritical fluid chromatography (UHPSFC) method using a 150 mm long 1-amino anthracene (1-AA) column and a mobile phase consisting of carbon dioxide and methanol/isopropanol (1/1, v/v) mixed with 8 % water. After thorough optimization of column temperature and back pressure, the separation of four vitamin D3 esters, vitamin D3 and D2, and eight mono- and di-hydroxylated metabolites, including three groups of isomers, was achieved in 10 min. Two ion sources, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization optimized within this study, were compared in tandem mass spectrometry (MS/MS) detection. No significant sensitivity differences were observed. Subsequently, the same 1-AA column chemistry was examined in ultra-high performance liquid chromatography (UHPLC) as the stationary phase that could hypothetically bring different selectivity in the separation of vitamin D and its metabolites. However, this hypothesis was rejected, and C18 was used as a stationary phase in the final optimized UHPLC-MS/MS method. Despite detailed optimization, the final 15 min UHPLC method was not able to separate di-hydroxylated isomers of vitamin D3, while it enabled better resolution of esterified forms compared to UHPSFC.Optimized methods provided similar repeatability of retention times and peak areas, with RSD < 2 % and 10 %, respectively. The lowest limits of quantification were in the range of 1.2 – 4.9 ng/mL for UHPSFC-APCI-MS/MS, while for UHPLC-APCI-MS/MS, they were typically in the range of 2.6 – 9.6 ng/mL. Based on the obtained results, the UHPSFC-APCI-MS/MS method was the most promising approach for fast, selective, and sensitive analysis that could be applied in the analysis of biological samples with emphasis on the separation of both hydroxylated and esterified metabolites, including isomeric forms.

Development and validation of a rapid GC–MS method for seized drug screening

Controlled substances are one of the most commonly analyzed types of evidence in forensic laboratories. Persistent, high levels of case submissions in recent years have created a need for rapid screening techniques to decrease case backlogs and expedite confirmatory analyses. The most utilized technique for the analysis of seized drugs is gas chromatography mass spectrometry (GC-MS). While this approach typically takes tens of minutes, advances in the technique have enabled approximately 1 min run times with a short column and a rapid (10’s °C/s) temperature program. These advances were used in this work to develop and validate a rapid GC-MS method for screening seized drug samples. Method validation was completed to determine retention time repeatability and reproducibility, limits of detection, identification accuracy, selectivity, and carryover. Validation results showed the utility of this method for accurate and precise qualitative analyses for the screening of seized drugs across seven different drug classes. The optimized method was then applied to 15 real and mock case samples from the Maryland State Police Forensic Sciences Division. Controlled substances, cutting agents, and other diluents were identified from these samples using acceptance criteria of retention time and mass spectral comparisons currently used in casework. Using rapid GC-MS methods in casework could enable faster and more reliable presumptive testing results for the analysis of seized drug exhibits.

Effect of spacer alkyl chain length on retention among three imidazolium stationary phases under various modes in high performance liquid chromatography

The properties of ionic liquids (ILs) greatly influenced the retention of solutes on IL-functionalized stationary phases. In this work, three IL-functionalized stationary phases (Sil-C4Im, Sil-C7Im, and Sil-C10Im) were prepared by modifying silica gel with three ionic liquid silane reagents differing in spacer alkyl chain lengths, which refers to the alkyl chain between the imidazolium and the sulfur atom. The preparation was proved through a range of characterization techniques, including elemental analysis (EA), Fourier transform infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA). The effects of chromatographic conditions, such as acetonitrile content, salt concentration, pH, and column temperature, were studied to explore the retention mechanism of three stationary phases, which indicates that three IL-stationary phases provide interactions with solutes in reverse-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), and ion exchange chromatography (IEC). The column efficiency is 83,686 plates/m for aminobenzoic acid, and the column also has excellent repeatability of retention time with relative standard deviations (RSDs) between 0.24% and 0.34% (n = 12). A variety of analytes, including five nucleosides and nucleobases, six alkylbenzenes, two polycyclic aromatic hydrocarbons (PAHs), and four inorganic anions, were separated well on three IL-functionalized stationary phases. It was found that the spacer alkyl chain length influenced selectivity by comparing the retention of the three IL-functionalized stationary phases.

Sense and nonsense of shifting gradients in on-line comprehensive reversed-phase LC × reversed-phase LC

In on-line comprehensive two-dimensional liquid chromatography (LC × LC), the combination of similar chromatographic modes such as reversed-phase liquid chromatography × reversed-phase liquid chromatography (RPLC × RPLC) usually leads to the partial occupation of the available separation space. A possible solution to circumvent this issue may be to dynamically adjust the gradient elution in the second dimension (2D) throughout the LC × LC analysis. This allows the gradient elution to be tailored to the elution conditions of each fraction instead of using a conventional full gradient program in which the same gradient profile is repeated for each 2D-fraction. In this study, an online RPLC × RPLC method is optimized with shifting gradients in 2D. The logic behind implementing such programs in on-line LC × LC is explained. The optimized method consists of a combination of segmented and shifted gradient modes. It is shown that the retention space coverage can be increased by 50 % compared to a conventional full gradient program, leading to a significant increase in peak capacity (about 35 %). However, such an increase comes at the expense of larger peak widths in 2D and thus lower peak intensities. It is shown here that the use of shifting gradients raises another serious issue related to the repeatability of retention times between two successive 2D-separations.

Persistence, decontamination and dietary risk assessment of propyrisulfuron residue in natural paddy field environment using QuEChERS@UPLC-Q-TOF-MS/MS

A modified QuEChERS technology combined with UPLC-Q-TOF-MS/MS residue analytical method for propyrisulfuron in natural paddy field environment was developed. Propyrisulfuron residues in paddy water, soil, rice plants, brown rice and rice husk samples was extracted with 1.0 % acetic acid-acetonitrile (v/v) solution and separated on a reversed-phase C18 column with a mobile phase of 0.1 % formic acid water-acetonitrile eluted by isometric elution (60/40, v/v), and quantified in high-resolution multiple reaction monitoring (MRMHR) scanning mode providing more accurate confirmatory information. The lower limit of quantification (LOQ) of propyrisulfuron in all the five paddy matrices was in a range of 1.24 ∼ 3.85 µg/kg, and all calibration curves showed good linearity (R2 > 0.991) in a certain concentration range (10 ∼ 1000 µg/kg). At the spiked level of 10 ∼ 200 µg/kg, the intra-day and inter-day average recoveries and relative standard deviation were 82.80 ∼ 106.93 % and 1.81 ∼ 5.51 %, 83.05 ∼ 105.84 % and 2.25 ∼ 4.61 %, respectively, for analysis on three consecutive days, and there was a good retention time repeatability of 15 consecutive injections with RSD of 0.15 ∼ 0.17 %, indicating a high sensitivity, specificity, accuracy and precision. All paddy filed environmental samples frozen at −20 °C were analyzed within 10 weeks after collection for a good storage stability. Propyrisulfuron dissipated in paddy water, soil and rice plants at application doses of 30, 45 and 60 g a.i./ha with half-lives of 1.37 ∼ 1.81, 4.10 ∼ 5.35 and 3.70 ∼ 4.42 days in 2020, 1.44 ∼ 2.04, 4.27 ∼ 5.83 and 3.68 ∼ 4.49 days in 2021, respectively. The final propyrisulfuron residual levels of two years in brown rice, rice husks and rice plants samples prior to harvest were lower than 8.18 µg/kg, and its removal rate was in a range of 1.40 to 98.27 % by several simple available household technologies. Moreover, the chronic dietary risk in brown rice at recommended dose for Chinese residents of different age groups was low and acceptable for daily consumption.

Ion chromatograph with three-dimensional printed absorbance detector for indirect ultraviolet absorbance detection of phosphate in effluent and natural waters.

An ion chromatography system employing a low-cost three-dimensional printed absorbance detector for indirect ultraviolet detection towards portable phosphate analysis of environmental and industrial waters has been developed. The optical detection cell was fabricated using stereolithography three-dimensional printing of nanocomposite material. Chromatographic analysis and detection of phosphate were carried out using a CS5A 4 × 250mm analytical column with indirect ultraviolet detection using a 255nm light-emitting diode. Isocratic elution using a 0.6mM potassium phthalate eluent combined with 1.44mM sodium bicarbonate was employed at a flow rate of 0.75mL/min. A linear calibration range of 0.5 to 30mg/L PO4 3- applicable to environmental and wastewater analysis was achieved. For retention time and peak area repeatability, relative standard deviation values were 0.68 and 4.09%, respectively. Environmental and wastewater samples were analyzed with the optimized ion chromatography platform and the results were compared to values obtained by an accredited ion chromatograph. For the analysis of environmental samples, relative errors of<14 % were achieved. Recovery analysis was also carried out on both freshwater and wastewater samples and recovery results were within the acceptable range for water analysis using standard ion chromatography methods.

Multi-Component Collaborative Step-by-Step Coloring Strategy to Achieve High-Performance Light-Responsive Color-Switching.

Light‐responsive color‐switching materials (LCMs) are long‐lasting hot fields. However, non‐ideal comprehensive performance (such as color contrast and retention time cannot be combined, unsatisfactory repeatability, and non‐automated coloring mode) significantly hinder their development toward high‐end products. Herein, the development of LCMs that exhibit long retention time, good color contrast, repeatability, and the property of automatic coloring is reported. The realization of this goal stems from the adoption of a bio‐inspired multi‐component collaborative step‐by‐step coloring strategy. Under this strategy, a conventional one‐step photochromic process is divided into a “light+heat” controlled multi‐step process for the fabrication of the desired LCMs. The obtained LCMs can effectively resist the long‐troubled ambient‐light interference and avoid its inherent yellow background, thereby achieving the longest retention time and good repeatability. Multiple colors are generated and ultra‐fast imaging compatible with the laser‐printing technology is also realized. The application potential of the materials in short‐term reusable identity cards, absorptive readers, billboards, and shelf labels is demonstrated. The results reported herein can potentially help in developing and designing various high‐performance, switchable materials that can be used for the production of high‐end products.

Extraction and quantitative determination of bile acids in feces

With rapid advances in gut microbiome research, fecal bile acids are increasingly being monitored as potential biomarkers of diet related disease susceptibility. As such, rapid, robust and reliable methods for their analysis are of increasing importance. Herein is described a simple extraction method for the analysis of bile acids in feces suitable for subsequent quantification by liquid chromatography and tandem mass spectrometry. A C18 column separated the analytes with excellent peak shape and retention time repeatability maintained across 800 injections. The intra-day and inter-day precision and accuracy was greater than 80%. Recoveries ranged from 83.58 to 122.41%. The limit of detection and limit of quantification were in the range 2.5–15 nM, respectively. The optimized method involved extracting bile acids from wet feces with minimal clean up. A second aliquot of fecal material was dried and weighed to correct for water content. Extracting from dried feces showed reduced recovery that could be corrected for by spiking the feces with deuterated standards prior to drying. Storage of the extracts and standards in a refrigerated autosampler prior to analysis on the LC-MS is necessary. Multiple freeze-thaws of both extracts and standards lead to poor recoveries for some bile acids. The method was successfully applied to 100 human fecal samples.

A gas chromatography-flame ionization detection method for direct and rapid determination of small molecule volatile organic compounds in aqueous phase.

It is still difficult to directly detect low content of volatile organic compounds (VOCs) in water samples by gas chromatography (GC) because when water is the only solvent, it would result in the instability and poor repeatability of peak retention time and peak shape. The adverse effects of water on direct GC analysis of VOCs cannot be significantly reduced or eliminated by simply changing the detection condition of GC. However, it was found that the addition of methanol in samples to a certain final proportion, such as 50 or 75% (v/v), could greatly reduce or eliminate the adverse effects of water. By using 75% (v/v) methanol as a solvent, the standard curves of ethanol, acetic acid, acetone, and isopropanol with correlation coefficient (R 2) over 0.99 were successfully plotted by gas chromatography-flame ionization detection (GC-FID) in a certain concentration range, respectively. The results showed that the retention time and peak shape stability of ethanol, acetic acid, acetone, and isopropanol in aqueous solution were greatly improved by the addition of methanol to final concertation of 75% (v/v). To verify the practical application potential of this method, the method was applied to the detection of components in isopropanol fermentation wastewater. The results showed that the method has well applicability and reliability. The key points in the application of this method were also summarized. This GC analysis method would have a wider and better application prospect in the detection of water-soluble organic matters.

Choline-chloride and betaine-based deep eutectic solvents for green extraction of nutraceutical compounds from spent coffee ground

Coffee beans contain several compounds which have shown nutraceutial properties; among them chlorogenic acids (CGAs) are the most studied, have shown nutraceutical properties. These molecules are also present in the waste by-product known as spent coffee grounds (SCG). SCG are generated during the production of coffee drink, whereby roasted, ground coffee beans are heat or steam treated. A HPLC-PDA/ESI-MS method has been developed for the analysis of the most representative CGAs in SCG samples using a green extraction approach based on deep eutectic solvents (DESs). We examined fifteen different DESs based on choline chloride and betaine as hydrogen bond acceptor in combination with different hydrogen bond donors being seven alcohols, two organic acids, one sugar and urea. Fifteen CGAs were detected, identified and quantified for the evaluation of extraction efficiency. A betaine-based DES with triethylene glycol (molar ratio 1:2) was the most effective one at extracting CGAs from SCG also compared to a conventional organic solvent. Thirty percent of water added to DES and sample to solvent ratio 1:15 (g/mL) were selected as the best extraction conditions. The chromatographic method was validated studying LOD, LOQ, retention time and peak area precision, and linearity range. Retention time and peak area repeatability were determined obtaining RSDs values below 0.63 % and 8.51 %, respectively. Inter-day precision was determined obtaining RSDs value below 0.58 % and 8.80 % for retention time and peak area, respectively.

Simultaneous analysis of filgrastim and pegfilgrastim aggregates by size-exclusion chromatography

A new size-exclusion high-performance liquid chromatographic (SE-HPLC) method for the simultaneous analysis of filgrastim and pegfilgrastim aggregates was developed and validated. A cross-linked agarose and dextran column was used at ambient temperature and an alkaline sodium phosphate buffer as mobile phase eliminated non-ideal interactions with the stationary phase. The robustness of the method was assessed by varying injection volumes, flow rates and sample vehicles. Other reliability assessments include calibration curve, intra and inter-day precision and accuracy, repeatability of retention times, application to real in-process production samples and column lifetime. The method exhibited linearity over the concentration of 0.02–4 mg/ml range for filgrastim and pegfilgrastim monomer with a correlation coefficient of greater than 0.999. The lower limit of quantification was 0.02 mg/ml and the limit of detection was 0.005 mg/ml. This SE-HPLC technique has been successfully used for several years and more than 10,000 samples.

The Italian panorama of cannabis light preparation: Determination of cannabinoids by LC-UV

Cannabis light preparations are products derived or containing dried female inflorescences of Cannabis sativa belonging to Chemotype III (THC/CBD ratio <<1); the total THC (THC+THCA) content in the crop must not exceed 0.2 % in accordance with the EU regulation.In Italy the most recent law for industrial hemp (242/2016) states that only for farmers this limit is extended to 0.6 %. On the other hand, the Ministry of the Interior published a note stating that the sale or the presence in the markets of products (inflorescences, concentrates, essences and resins) or plants with concentrations higher than 0.5 % constitutes a crime. In this confusing legislation framework, it is very important to assess the legality of hemp, determining the total amount of THC. To this end a reliable LC-UV analytical method was developed and validated taking into account parameters such as precision, accuracy, linearity, repeatability of peak area and retention time, limit of detection (LOD= 0.002 % for all cannabinoids) and limit of quantification (LOQ= 0.005 % for all cannabinoids). Accuracy was expressed as the relative error (Er%), while precision was measured as the coefficient of variation (CV%). A CV% below 3 % and Er% between ± 6 % were obtained. The linearity was proven in the concentration range 0.005–1 % for THC, THCA and CBN and 0.005 %–50 % for CBD and CBDA.The analytical method was applied to more than nine hundred cannabis light samples.Based on the law 242/2016, only 18 % of the crops are to be considered legal for the market (total THC<0.2 %). If the circular of the Ministry of the Interior should be converted as a proper law, a substantial amount of cannabis light preparations (24 %) would be considered illegal (total THC>0.5 %). On the other hand, the most of the inflorescences (58 %) have a total THC content comprised between 0.2 % and 0.5 %, and it is not clear whether these products could be sold or not. Moreover, Cannabis light products are not authorized for human consumption, even if everybody knows that this is their primary use. In conclusion, the cannabis light panorama in Italy is quite confused and more specific and clear legislation should be proposed.

Experimental and statistical protocol for the effective validation of chromatographic analytical methods

The validation of analytical methods is of crucial importance in several fields of application. A new protocol for the validation of chromatographic methods has been proposed. The overall protocol is described in a parallel paper, where the case of a multi-targeted gas chromatography – mass spectrometry (GC–MS) method for the determination of androgens in human urine is in-depth discussed. The purpose of this paper is to report the details about the GC–MS separation and detection of the target analytes, and to provide the mathematical formulas needed to perform the validation of the principal parameters. Briefly, the validation protocol foresees the repetition of three calibration curves in three different days, providing a total amount of nine replicates. Such a structured design allows to use the same experiments to•perform a rigorous calibration study, by the evaluation of heteroscedasticity, comparison of several weights and linear/quadratic calibration curves.•determine several parameters which are traditionally computed from dedicated experiments, namely intra- and inter-day accuracy and precision, limit of detection, specificity, selectivity, ion abundance repeatability, and carry over.•Finally, few further experiments are necessary to evaluate the retention time repeatability, matrix effect and extraction recovery.

Establishing repeatability and ruggedness of chiral separations in micellar electrokinetic chromatography mass spectrometry: Combined use of covalently bonded capillary column and molecular micelles

Although micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) using bare silica capillary filled with molecular micelles is an advantageous hyphenated technique for chiral analysis, it is still in the developmental stage. This is mainly because of the lower repeatability of retention time and peak area associated with the difficulty in controlling electroosmotic flow on bare silica capillaries. Besides the lower robustness and electrospray erosion of the fused-silica capillary tip, the lifetime is limited for 10–15 runs per capillary column. We have tested a new MEKC-MS method to eradicate this problem using a covalently bonded 2-acrylamido-2-methyl-1-propane-sulfonic acid (AMPS) column filled with free floating molecular micelles, polysodium N-undecenoxy carbonyl-L-leucinate (poly-L-SUCL). Simultaneous enantiomeric separations and MS/MS detection of three β-blockers [atenolol (ATEN), metoprolol (METO) and, pindolol (PINDO)] was achieved within 25 min with improved column lifetime of at least 45–50 runs. Excellent repeatability of retention time was observed for β-blockers, as evidenced by the relative standard deviation of less than 2% and 3% for intra-capillary and inter-capillary column, respectively. The linear calibration range of both β-blockers was simultaneously established using enantiomers of PINDO as an internal standard. The limit of detection and the lower limit of quantitation were 0.2 μg/mL and 0.5 μg/mL, respectively, for both ATEN and METO. Acceptable recovery of the enantiomeric content of the commercial METO tablet (98-99.5%) and ATEN tablet (89-92.5%) were obtained with adequate system suitability for the precision of peak area (≤10% RSD) under optimum conditions. The developed MEKC-MS approach was extended, which provided broader repeatibility of chiral separation to a panel of primary, secondary and tertiary amines as well as one anionic chiral compound.

Supercritical fluid chromatography–mass spectrometry in routine anti-doping analyses: Estimation of retention time variability under reproducible conditions

The aim of this study was to estimate the retention time variability under reproducible conditions of an SFC-MS analytical method for routine anti-doping analyses. For this purpose, a set of 51 doping agents, as neat standards and spiked in diluted urine, was used to assess their retention times variability over a period of four months, as well as the column inter-batch reproducibility. Three UHPSFC stationary phases have been employed, the Acquity UPC2 Torus 2-Picolylamine (2-PIC), UPC2 Viridis BEH and Acquity UPLC HSS C18 SB. Four columns, per column chemistry, have been purchased to represent three different production lots, with a total of twelve columns employed in this study. The two columns from the same lot were applied to the first part of the study (repeatability), whereas the representative of three different lots were employed in the second part (robustness). In terms of organic modifier, a mixture of 98% MeOH and 2% water containing 20 mM ammonium formate was selected in order to limit the formation of methyl-silyl ethers on the surface of the silica particles, thus potentially improving the repeatability of retention times. A comparison with an UHPLC reference analytical method was made with the same set of analytes.The average relative standard deviations (RSD%), represented in split violin plots, illustrate how two of the UHPSFC columns assessed in this study were able to generate an excellent repeatability of retention times, with results that are in a similar range of those generated by UHPLC. Moreover, the Torus 2-PIC has proven to be the best of the three stationary phases, with an impressive RSD% of 0.5% in diluted urine relative to the inter-month variability. Finally, the inter-batch reproducibility assessment has highlighted a good reproducibility of the same stationary phase belonging to different production lots for all three column chemistries assessed, with the Viridis BEH silica generating an RSD% of 0.7% in diluted urine. Higher values of RSD (%) were found for Torus 2-PIC and HSS C18 SB, respectively of 1.0% and 1.6%.

Detection of recombinant insulins in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry after immunoaffinity purification based on monolithic microcolumns.

This work describes an analytical procedure based on automated affinity purification followed by liquid chromatography-electrospray tandem mass spectrometry with a conventional triple quadrupole analyzer, in order to detect synthetic insulins (Apidra®, Humalog®, Levemir®, NovoRapid®, and Tresiba®) in human urine. Sample preparation included ultrafiltration followed by immunoaffinity purification on monolithic microcolumns. Chromatographic separation was performed by a C18 microbore column, while mass spectrometric identification of the analytes was achieved by a triple quadrupole mass spectrometer under positive ion electrospray ionization and acquisition mode in selected reaction monitoring. Identification of the synthetic insulins was performed by selecting at least two characteristic ion transitions for each analyte. The newly developed method was validated in terms of specificity, recovery, matrix effect, sensitivity, robustness, and repeatability of retention times and relative ion transition abundance. The specificity and the reproducibility of the relative retention times and the relative abundance of the characteristic ion transitions selected was confirmed to be fit for purposes of ensuring the unambiguous identification of all target analytes, also in the forensic field. The extraction yield was estimated at greater than 60% and the matrix effect smaller than 35%. The lower limits of detection were in the range of 0.02-0.05ng/mL, proving the method to be sufficiently sensitive to detect the abuse of insulins in cases where they are used as performance-enhancing agents in sport. The applicability of the developed method was assessed by the analysis of urine samples obtained from diabetic subjects treated with Tresiba® and/or Humalog®, whose presence was confirmed in urine samples collected after the administration of therapeutic doses. Graphical abstract A hybrid assay comprising MSIA-based immunoextraction combined with liquid chromatography-electrospray tandem mass spectrometry was developed and validated for the detection of recombinant insulins in human urine.

Determination of methylxanthines in tea samples by HPLC method

Abstract Methylxanthines such as caffeine, theophylline, theobromine are significant and widespread psychoactive substances. We developed the isocratic method with optimum composition of the mobile phase 90 % water: 10 % acetonitrile and confirmed repeatability of retention times and peak areas. The developed HPLC method was applied to determine the content of methylxanthines in selected types of black and green teas available on the market. Of the black teas (tea bags), the highest concentration of theobromine was found in Ceylon tea (18.98 mg.L−1). The highest concentration of caffeine was in a cup of Earl Gray tea (254.09 mg.L−1). Among loose black teas, the highest content of both theobromine and caffeine was found in Pu Erh Superior tea, where the theobromine content was 24.62 mg.L−1 and the caffeine content was 520.67 mg.L−1. Of green powder teas, highest caffeine content (306.46 mg.L−1) was in Shizuoka Matcha Premium and the highest content of theobromine (8.45 mg.L−1) was found in GABA Midori. From the loose green tea, the highest concentration of theobromine (12.85 mg.L−1) was in Lung Ching West Lake. The highest caffeine content (484.85 mg.L−1) was in Gyokuro Shizuoka Premium Tea. In both types of teas the amount of theobromine and caffeine was quantified, but the presence of theophylline was not proven. Data on contents of these metabolites in tea products are highly informative for consumers.

Low cost 235 nm ultra-violet light-emitting diode-based absorbance detector for application in a portable ion chromatography system for nitrite and nitrate monitoring

A low cost, UV absorbance detector incorporating a 235 nm light emitting diode (LED) for portable ion chromatography has been designed and fabricated to achieve rapid, selective detection of nitrite and nitrate in natural waters. The optical cell was fabricated through micromilling and solvent vapour bonding of two layers of poly (methyl methacrylate) (PMMA). The cell was fitted within a 3D printed housing and the LED and photodiode were aligned using 3D printed holders. Isocratic separation and selective detection of nitrite and nitrate was achieved in under 2.5 min using the 235 nm LED based detector and custom electronics. The design of the new detector assembly allowed for effective and sustained operation of the deep UV LED source at a low current (<10 mA), maintaining consistent and low LED temperatures during operation, eliminating the need for a heat sink. The detector cell was produced at a fraction of the cost of commercial optical cells and demonstrated very low stray light (0.01%). For retention time and peak area repeatability, RSD values ranged from 0.75 to 1.10 % and 3.06–4.19 %, respectively. Broad dynamic linear ranges were obtained for nitrite and nitrate, with limits of detection at ppb levels. The analytical performance of the IC set up with optical cell was compared to that of an ISO-accredited IC through the analysis of five various water samples. Relative errors not exceeding 6.86% were obtained for all samples. The detector was also coupled to a low pressure, low cost syringe pump to assess the potential for use within a portable analytical system. RSD values for retention time and peak area using this simple configuration were <1.15% and <3.57% respectively, highlighting repeatability values comparable to those in which a commercial HPLC pump was used.

Separation of Inorganic Anions and Phenolic Compounds Using Tetraethylene Oxide-Bonded Stationary Phases in Capillary Liquid Chromatography

Tetraethylene glycol monomethyl ether (TEGMM) and tetraethylene glycol (TEG) modified silica were synthesized and used in capillary liquid chromatography as stationary phases. The stationary phases were prepared by chemically bonding TEGMM or TEG on silica via reaction with 3-glycidyloxypropyltrimethoxysilane. The present stationary phases were successfully used for the separation of several inorganic anions and phenolic compounds. The TEG-modified silica stationary phase had a better selectivity and higher retention for five anions compared to TEGMM-modified silica stationary phase. The repeatability of retention time for the five anions was satisfactory on both stationary phases. By using 2 mM sodium chloride as eluent, the relative standard deviation values were in ranging from 0.68–3.21 and 2.00–2.16% for analytes in the TEG-and TEGMM-bonded stationary phase, respectively. It was found that the TEG-bonded stationary phase had hydrophilic properties due to the presence of the hydroxyl group at the end of the TEG chains.

Development and validation of a simple LC-MS/MS method for the simultaneous quantitative determination of trimethylamine-N-oxide and branched chain amino acids in human serum.

Serum branched chain amino acids and trimethylamine-N-oxide are monitored as potential indicators of diabetes and cardiovascular health respectively. A rapid method for their simultaneous determination using liquid chromatography and tandem mass spectrometry is described here. Branched chain amino acids and trimethylamine-N-oxide were quantified based on their specific MS/MS fragments using a selected reaction monitoring approach. A number of columns were tested for their ability to separate the analytes. A C18-PFP column separated the analytes in just 4minutes, and resulted in excellent peak shape and retention time repeatability, and was therefore chosen as the optimal column. A second column, the Intrada Amino Acid column, was chosen for comparison and validation experiments as it provided an orthogonal separation mechanism. The intra-day and inter-day precision and accuracy were less than 12% for trimethylamine-N-oxide and less than 6% for the branched chain amino acids. Recoveries, where serum was spiked with three different concentrations of the analytes, ranged from 97 to 113%. The LODs and LOQs for trimethylamine-N-oxide were 1 and 6ng/mL, for leucine and isoleucine were 4 and 8ng/mL, and for valine were 5 and 15ng/mL, respectively. The C18-PFP column method was validated using the Intrada Amino Acid column method and percentage agreement for all four analytes was within 10%. Sample preparation was minimal, and use of labelled internal standards accounted for matrix effects. The method was successfully applied to human plasma samples. Graphical abstract ᅟ.