Ion-pair High-performance Liquid Chromatography Research Articles

Electrophoretic characterization of LNP/AAV-encapsulated nucleic acids: Strengths and weaknesses.

The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra- and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non-encapsulated or encapsulated large NAs(>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high-performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.

Ultrasensitive and highly selective determination of iodate by reversed-phase ion pair HPLC–amperometric detector after online electrochemical reduction to iodide

We developed an ultrasensitive and highly selective method to quantify iodate (IO3–) in iodized salt and biological samples. In the high-performance liquid chromatography (HPLC) system, IO3– was injected with sampler and reduced to iodide (I–) online using a Coulomb detector (CD) as a pre-column reactor, retained by C18 column with the help of ion pairs, and then determined by an amperometric detector (AD). For analysis of IO3–, several parameters and reactive conditions of Coulomb detector, including applied potential, pH value, and salt concentration, were optimized to obtain the best reduction efficiency. The optimized HPLC–CD-AD method was found to be linear over a wide range of IO3– concentration (0.05–0.25 μg/mL) with appreciable recovery rates (86.90– 94.70%) of quality controls at excellent detection limits (100 pg) and acceptable variability. The analysis of IO3– in actual samples, such as urine of volunteers, iodized salt, and seawater, discovered that the method has high selectivity and little interference. It can be concluded that the proposed method was ultrasensitive and highly selective in the trace detection of IO3.

ЭФФЕКТЫ ВОЗДЕЙСТВИЯ ХРОНИЧЕСКОЙ АЛКОГОЛИЗАЦИИ И ГИПОДИНАМИИ НА ПОКАЗАТЕЛИ ДОФАМИНЕРГИЧЕСКОЙ СИСТЕМЫ НЕКОТОРЫХ ОТДЕЛОВ ГОЛОВНОГО МОЗГА КРЫС

Purpose of work. To determine the nature of changes in the content of dopamine and its metabolites in the cerebral cortex, hypothalamus and cerebellum of rats with chronic alcohol intoxication (CAI) against the background of physical inactivity (PI). Material and methods. The experiments were carried out on outbred white male rats weighing 180-220 g. Modeling of PI was carried out by placing rats in individual cages-pencil cases, limiting their mobility, for periods of 14 and 28 days. To simulate CAI, the animals were provided with an ethanol solution as the only source of fluid for 14 and 28 days. Determination of the content of the components of the dopaminergic system (tyrosine, 3,4-dioxyphenylalanine, dopamine, homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC) and norepinephrine) was carried out using ion-pair high-performance liquid chromatography. Results. PI for 14 days is accompanied by signs of activation of the dopaminergic system in the hypothalamus and cerebral cortex of rats. With CAI for a period of 28 days, there are signs of a decrease in the activity of the dopaminergic system in the cerebral cortex. The combined effect of PI and CAI for 14 and 28 days leads to an increase in dopamine turnover in the cerebral cortex of rats. Conclusions. With the combined effect of CAI and PI, the most pronounced changes are observed in the cerebral cortex and are accompanied on the 14th day by an increase in the levels of HVA and DOPAC, and on the 28th day by an increase in the concentration of HVA.

Characterizing different types of black fountain pen ink using the chemical approach

Fountain pen ink contains dyes, pigments, or nanoparticles as colorants; water and ethylene glycol or an organic solvent as the vehicle. The dyes in fountain pen ink are usually negatively charged acid dyes. In this study, various types of black fountain pen ink (5 dye-based and 5 nanoparticle-based) were investigated by ultraviolet–visible (UV–Vis) absorption spectroscopy, Ion Pair High Performance Liquid Chromatography Diode-Array Detector (IP-HPLC-DAD), Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS), and Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q/TOF-MS) analysis. UV–Vis spectroscopy of fountain pen ink samples showed different profiles despite their similar color. MALDI-TOF-MS analyses successfully differentiated 10 of the inks. Nanoparticle-based ink, which is usually darker than dye-based ink, showed the same UV absorption at 240 nm. The use of LC-Q/TOF-MS, however, allowed the 5 dye-based fountain pen ink samples to be differentiated from one another. On the basis of the combined results of all analytical methods mentioned above, the discriminating power values were 0.71–1.00. Among them, MALDI-TOF-MS could successfully distinguish fountain pen ink samples with similar black colors and provide an easy analytical approach for the differentiation and identification of various types of fountain pens.

Bioactive amines in ingredients and feeds of broilers and storage effects on their levels

To quantify amines in ingredients and in different types of feed and to verify spermine inclusion influence in polyamine levels at storage of experimental diets of broiler, ingredients to formulate diets and commercial starting and growth feeds and were purchased. Spermidine, spermine, putrescine, cadaverine, agmatine, tyramine and histamine were detected by ion pair high performance liquid chromatography in ingredients, commercial diets, control and 0.06 and 012% spermine included formulations. Bioactive amines levels were analysed weekly to initial and growth diets, respectively until 21st and 49th days of storage at 26 ± 3°C and 72% relative humidity. All amines were detected in commercial feed. Spermidine and spermine predominate in soybean, wheat and corn, and tyramine in meat and bone meal. To initial diets averages of cadaverine reduced significantly in day 21, except for 0.06% spermine inclusion; agmatine decreased (p<0.05) in times, except for 0.12% spermine inclusion; spermidine were smaller (p<0.05) at 14 and 21 days and increased significantly with the level of inclusion. In growth diets cadaverine oscillated (p<0.05) between 0.35 at 28 days in control and 1.15 mg.100g-1 in 0.06% spermine inclusion at 49 days. Agmatine were 0.36 to 0.91 mg.100g-1 in control; spermidine vary significantly at 7 days in control and in 0.12% spermine inclusion at 28 days; spermine vary from 1.33 at 42 days to control to 72.55 mg.100g-1 at 7 days in 0.12% spermine inclusion. Polyamine levels were low in ingredients, commercial feed and initial and growth experimental formulations even when stored for 21 or 49 days.

Effective and repeatable chromatographic separation of 5 nucleotides in infant formula milk powder by ion-pair high-performance liquid chromatography–ultraviolet

A robust method using HPLC-UV was developed to improve the accuracy and repeatability of a quantitative method to detect 5 nucleotides (cytidine-monophosphate, uridine monophosphate, adenosine monophosphate, guanine monophosphate, and inosine monophosphate) in infant formulas. The results showed that efficient separation could not be achieved without strict conditions. The proposed method displayed a strong linear relationship (R2 > 0.9999) of single nucleotide in infant formula milk powder in the range of 10 to 1,000 mg/kg, a steady recovery (80.0% ∼110.0%) with relative standard deviation from 0.5% to 3.5%, under strict conditions of hydrophilic C18 column with di-isopropyl at 62.5 ± 2.5°C (± standard deviation), 0.65 ± 0.1 mg/mL tetrabutylammonium bisulfate, and mobile phase of pH of 2.75 ± 0.02. By applying this method on a series of milk products in the Chinese market, we found a few of them exceeded the legal limits of nucleotides.

Innovated formulation of oseltamivir powder for suspension with stability study after reconstitution using a developed ion-pair reversed phase high-performance liquid chromatography method.

ABSTRACTOseltamivir is an antiviral neuraminidase inhibitor used for the treatment and prophylaxis of influenza infection with viruses A and B. The mechanism of oseltamivir antiviral activity is by inhibiting the activity of the viral neuraminidase enzyme present on the surface of the virus, which stops viral replication and infectivity. Oral suspensions of oseltamivir phosphate are dispensed orally capsules and suspension. However, the use of oral suspension for pediatric administration is preferable and is prepared as a powder for suspension. The reconstituted suspension degrades rapidly within a few days. The objective of this work is to establish a stable formulation of oseltamivir phosphate as a suspension and to assure the stability conditions for prolonged use after reconstitution in aqueous form. In addition, this required formulation should maintain a high rate of dissolution, which subsequently leads to higher bioavailability. In this study, oseltamivir forms an inclusion complex with the natural and safe polymer hydroxypropyl beta-cyclodextrin, which resembles a host because its structural cavity carries the oseltamivir molecule in the aqueous preparation and provides a protective property against environmental challengers. In addition, a high-performance liquid chromatography (HPLC) stability-indicating method of analysis has been developed using an ion-pair reversed-phase HPLC technique that is validated for precision, accuracy, reproducibility, and specificity for the determination of oseltamivir in suspension. The results of this work show the relatively long shelf life of the reconstituted oseltamivir oral powder for suspension in the new pediatric formulation, and the developed HPLC method was precisely suitable for stability study.

A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems

Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.

The glutamyl tail length of the cofactor F420 in the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus

The cofactor F420 is synthesized by many different organisms and as a redox cofactor, it plays a crucial role in the redox reactions of catabolic and biosynthetic metabolic pathways. It consists of a deazaflavin structure, which is linked via lactate to an oligoglutamate chain, that can vary in length. In the present study, the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus were cultivated on different carbon sources and their coenzyme F420 composition has been assayed by reversed-phase ion-pair high-performance liquid chromatography with fluorometric detection regarding both, overall cofactor F420 production and distribution of F420 glutamyl tail length. In Methanosarcina thermophila cultivated on methanol, acetate, and a mixture of acetate and methanol, the most abundant cofactors were F420-5 and F420-4, whereby the last digit refers to the number of expressed glutamyl rests. By contrast, in the obligate CO2 reducing Methanoculleus thermophilus the most abundant cofactors were F420-3 and F420-4. In Methanosarcina thermophila, the relative proportions of the expressed F420 tail length changed during batch growth on all three carbon sources. Over time F420-3 and F420-4 decreased while F420-5 and F420-6 increased in their relative proportion in comparison to total F420 content. In contrast, in Methanoculleus thermophilus the relative abundance of the different F420 cofactors remained stable. It was also possible to differentiate the two methanogenic Archaea based on the glutamyl tail length of the cofactor F420. The cofactor F420-5 in concentrations >2% could only be assigned to Methanosarcina thermophila. In all four variants a trend for a positive correlation between the DNA concentration and the total concentration of the cofactor could be shown. Except for the variant Methanosarcinathermophila with acetate as sole carbon source the same could be shown between the concentration of the mcrA gene copy number and the total concentration of the cofactor.

Quantification of Histidine-Containing Dipeptides in Dolphin Serum Using a Reversed-Phase Ion-Pair High-Performance Liquid Chromatography Method

The quantification of histidine-containing dipeptides (anserine, carnosine, and balenine) in serum might be a diagnostic tool to assess the health condition of animals. In this study, an existing reversed-phase ion-pair high-performance liquid chromatography (HPLC)–ultraviolet detection method was improved and validated to quantify serum anserine, carnosine, and balenine levels in the dolphin. The serum was deproteinized with trichloroacetic acid and directly injected into the HPLC system. Chromatographic separation of the three histidine-containing dipeptides was achieved on a TSK–gel ODS-80Ts (4.6 mm × 150 mm, 5 µm) analytical column using a mobile phase of 50 mmol/L potassium dihydrogen phosphate (pH 3.4) containing 6 mmol/L 1-heptanesulfonic acid and acetonitrile (96:4). The standard curve ranged from 0.1 µmol/L to 250 µmol/L. The average accuracy of the intra- and inter-analysis of anserine, carnosine, and balenine was 97–106%. The relative standard deviations of total precision (RSDr) of anserine, carnosine, and balenine in dolphin serum were 5.9%, 4.1%, and 2.6%, respectively. The lower limit of quantification of these compounds was 0.11–0.21 µmol/L. These results indicate that the improved method is reliable and concise for the simultaneous determination of anserine, carnosine, and balenine in dolphin serum, and may be useful for evaluation of health conditions in dolphins. Furthermore, this method can also be applied to other biological samples.

Production of Structured RNA Fragments by In Vitro Transcription and HPLC Purification.

The understanding of the functional importance of RNA has increased enormously in the last decades. This has required research on the RNA molecules themselves, with the concomitant need for obtaining purified RNA samples, such as for structural studies by NMR or other methods. The main method to create labeled and unlabeled RNA, T7 in vitro transcription, suffers from sequence-dependent yield and often low homogeneity for short constructs (<100 nt) and requires laborious purification. Additionally, the design of structured RNA fragments mimicking the structure of a larger biological RNA is often not straightforward. Secondary structure simulations can be used to make reliable predictions about the folding of a particular RNA fragment. In this article, we describe how to design an RNA construct of interest from a larger sequence, and we combine several previously published improvements of the in vitro transcription method, such as the use of 2'-methoxy modifications and dimethyl sulfoxide or the use of tandem repeats, to increase yield and purity of in vitro-transcribed RNA. Together with a high-performance liquid chromatography (HPLC) purification procedure using both reversed-phase ion-pairing and ion-exchange HPLC, we provide a robust protocol to obtain highly pure RNA of short to intermediate length in large quantities. The protocol optimizes yield, especially for RNA starting with nucleotides other than G. At the same time, it is simplified, and the required time is reduced. The protocols described here constitute a versatile pipeline for the production of purified RNA samples and are suitable for users with little experience in liquid chromatography. © 2021 The Authors. Basic Protocol 1: RNA construct design Basic Protocol 2: DNA template production and in vitro transcription Alternate Protocol: Tandem transcription and RNase H cleavage Basic Protocol 3: Reversed-phase ion-pairing HPLC purification Basic Protocol 4: Ion-exchange HPLC purification.

Impurity profiling of Compound Amino Acid Injection (6AA) using ion-pair high performance liquid chromatography coupled with corona-charged aerosol detection and high resolution mass spectrometry

The complex industrial production process of amino acids (AAs) leads to the existence of a certain amount of impurities in Compound Amino Acid Injection (6AA). It is difficult to obtain its comprehensive and systematic impurity profile using conventional ultraviolet (UV) detectors due to lack of a suitable chromophore in the structures of AAs and their impurities. In our study, a universal ion-pair high performance liquid chromatography (HPLC) method combined with high resolution mass spectrometer (HRMS) and charged aerosol detection (CAD) was developed to identify and determine the content of impurities in Compound Amino Acid Injection (6AA), respectively. After optimizing the content of trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA) in the mobile phase on a C18 AQ column, HPLC-CAD method was developed and nine unknown impurities were detected. These impurities were successfully identified using HPLC coupled with orbitrap mass spectrometry and confirmed with their reference substances. The CAD parameters setting was optimized to improve the sensitivity and linearity of the methods before the developed method was validated. The results of validation reflected that the limit of detection (LOD) was approximately 2 ng (corresponding to approximately 0.02 % of L-isoleucine in injection). Under the optimized power function value (PFV) of CAD, the linear range of each impurity was 1 ∼ 200 μg mL−1 (the linear range of one of the impurities with higher content was 2 ∼ 400 μg mL−1) with coefficients of determination (R2) greater than 0.998. The recovery rates for nine impurities were 93.37 % ∼ 110.23 %. This study made full use of the qualitative functions of HRMS and the versatility of CAD, revealing possible impurities in the 6AA injection, which could provide reference for the safety research of it.

Isolation and Characterization of Nitrate Reducing Bacteria for Conversion of Vegetable-Derived Nitrate to ‘Natural Nitrite’

In the US, sodium nitrate is used as a preservative and curing agent in processed meats and is therefore a regulated ingredient. Nitrate reducing bacteria (NRB) can convert vegetable nitrate into nitrite allowing green/clean label status in the US as per the USDA-FSIS definition of ‘natural nitrite’. The current ‘in-liquid’ test tube assay for detecting nitrite is not suitable for screening mixtures of bacteria nor is commercial nitrate broth suitable for growth of many Gram (+) bacteria. M17 broth was therefore used to develop M17-nitrate broth to be inclusive of Gram (+) bacteria. An ‘on-agar’ colony-screening assay was developed to detect the conversion of nitrate to nitrite on agar plates and could detect one NRB+ colony among ~300–500 colonies on a single plate. Samples that might have NRB were spread-plated on M17 agar plates, sandwiched with nitrate agar, and after incubation followed with sequential agar overlays containing the reagents used in the nitrate reduction assay; the appearance of red color zones above colonies indicated the presence of nitrite. NRB derived from various samples were confirmed for nitrate conversion and both nitrate and nitrite were quantified by C8 reversed-phase (RP) ion-pairing high performance liquid chromatography (HPLC) analysis (1 ppm limit of detection). Staphylococcus carnosus, a strain commonly used for nitrate reduction, was able to convert 1100 ppm M17-nitrate broth to 917 ppm nitrite. Staphylococcus caprae and Panteoa agglomerans, NRB isolated using the M17-nitrate agar assay, were also able to ferment the same broth to 916 ppm and 867 ppm nitrite, respectively. This is the first report of an on-agar colony screening assay for the detection and isolation of nitrite reducing bacteria allowing NRB to be readily isolated. This may allow for the identification of new bacteria that may have a more efficient process to generate nitrite, and possibly concomitant with production of additional natural antimicrobials, as vegetable nitrite becomes more widely used to prevent spore germination.

A fast and simple ion-pair high performance liquid chromatography method for analysis of primary bile salts in in vitro digested bean samples

Bile salts (BS) play a key role in cholesterol and lipid metabolism as well as in many other key metabolic pathways. High performance liquid chromatography (HPLC) is the most common technique used to analyze BS in diverse type of samples. However, current HPLC analysis methods used to analyze and quantify single BS in in vitro digested samples showed poor separation of a complex mixture of BS. In this article, we improved a standard method originally used for quantifying individual BS in food samples subjected to in vitro digestion. We also adapted a method previously developed for BS examination in human blood samples to the analysis of these molecules in chyme samples obtained during simulated gastrointestinal digestion. Our method was simple and achieved a fast and successful separation and quantification of four primary BS (sodium salts of taurocholic, glycocholic, taurochenodeoxycholic and glycochenodeoxycholic acids).•A method used to analyze bile salts in human blood samples has been adapted to separate and quantify four primary bile salts in in vitro digested bean samples.•Addition of an ion-pair reagent led to complete separation of glycine and taurine conjugates of chenodeoxycholic and cholic acids within 10 min, and achieved good peak symmetry.•The minimum BS concentration that could be measured was as low as 0.03125mM.

Development and validation of an ion-pair HPLC chromatography for simultaneous determination of lactone and carboxylate forms of SN-38 in nanoparticles

A simple and reproducible reversed-phase ion-pair high performance liquid chromatography (HPLC) method using isocratic elution with UV absorbance detection was developed and validated for the simultaneous determination of carboxylate (C) and lactone (L) forms of SN-38 (active metabolite of irinotecan) in nanoparticles (NPs). Reversed phase (RP) chromatography was performed on a C18 column with an ion-pair solution, KH2PO4 buffer solution and acetonitrile, under a mobile phase 30:30:40 (v/v) and at a flow rate of 1 mL/min. Detection was performed at 265 nm and sharp peaks were obtained for C and L forms of SN-38 at retention times of 2.9 ± 0.01 and 5.1 ± 0.01 min respectively. Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 4 to 100 μg/mL; the regression coefficient was 0.9999 for L form and 0.9998 for C form. The optimized method showed good performance in terms of specificity, linearity, detection and quantitation limits, precision and accuracy in accordance with the International Conference on Harmonization (ICH) Q2 (R1) guidelines. This assay was demonstrated to be applicable for routine quantitation of SN-38 in PLGA NPs.

Simultaneous determination of bromhexine HCl and baicalin in Chinese compound medicine by a reversed-phase ion-pair HPLC

In this article, a reversed-phase ion-pair high-performance liquid chromatography (HPLC) method was established for simultaneous separation and quantitative analysis of bromhexine HCl (BHH) and baicalin in Chinese Ke-Chuan-Ling compound capsule. The influence of the mobile phase composition on the retention of the analytes was investigated. The optimal determination conditions consisted of acetonitrile/water/phosphoric acid/triethylamine (22/78/0.1/0.1, v/v/v/v) mobile phase with the addition of tetra-n-butylammonium bromide (TBA) (0.7 mmol L-1, terminal concentration) as pairing ion, and a detection wavelength at 225 nm. In this case, baicalin and bromhexine HCl were eluted at 21 min and 27 min respectively and were separated successfully from other ingredients. Excellent linear relationship between the peak area and concentration was obtained in the range of 0.2-15.0 μg mL-1 for bromhexine HCl and 0.1-12.0 μg mL-1 for baicalin. The limit of detection of BHH was 0.1 μg mL-1 and it was 0.05 μg mL-1 for baicalin. The average recoveries of bromhexine HCl and baicalin were 97.7% and 99.1%, respectively. From the results it can be concluded that this method is simple, versatile, and reliable for the quality control of Chinese traditional medicines containing baicalin and/or bromhexine HCl.

Determination of Synthetic Dyes in Various Food Samples of Iran’s Market and their Risk Assessment of Daily Intake

THE AIM of this study was to measure the level of synthetic food dyes used in different foodstuffs of Iran’s markets via ion-pair high-performance liquid chromatography diode array detector (Ion-Pair HPLC-DAD). The chronic daily intake (CDI), target hazard quotient (THQ) and hazard index (HI) of synthetic dyes for Iranian population were calculated. Based on obtained results, from 74 foodstuff samples tested, only five synthetic dyes (Quinoline, Sunset yellow, Carmoisine, Allura Red and Brilliant Blue) were detected. Sunset yellow was the most commonly used synthetic colorant (45%). The highest concentration of synthetic colors were identified in fruit snacks (361 mgkg-1) followed by jelly product (161.9 mgkg-1), jelly powder (125.5 mgkg-1), ice products (104 mgL-1) and drinks (99.8 mgL-1). To the determine risk of exposure of Iranian consumers to five identified colors in all food product samples the CDI and THQ values were respectively lower than acceptable daily intake (ADI) recommended by WHO/FAO and one. Also, the HI values of all identified colors were lower than one, that did not represent a sever public health risk for synthetic color intake through assessed food samples in Iran’s market. Nevertheless, the results highlight the importance of a more attention monitoring of these dyes by the public and food health authorities Iran.

Validation of an analytical method for determination of eight food dyes in beverage and fruit roll-ups by ion-pair HPLC-DAD

Background and aims: Synthetic dyes are widely used as food additives to avoid the loss of original dye in processed foods and to make the foods more attractive to consumers. For simultaneous determination of 8 most commonly used synthetic colors in beverage and foodstuff, an efficient, selective, and sensitive method is suggested. Methods: To analyze food colors in different beverages and fruit roll-ups, a method using Ion-pair high-performance liquid chromatography with diode array detector was suggested and validated. The separation of dyes from beverage and foodstuff was done by solid-phase extraction (SPE). The ultrasound-assisted solvent extraction method was used to extract dye from fruit roll-ups. Results: The limit of detection and the limit of quantification ranged from 0.036 to 0.07 and 0.098 to 0.2 µg/mL, respectively. IP-HPLC-DAD method was validated using precision (RSD %) and accuracy (Recovery %) of two concentrations of 0.5 and 1 µg/mL in terms of intra- and inter-day. SPE method was also validated using intra- and inter-day precision and accuracy. The most commonly detected dye in the tested samples was carmoisine with a concentration of 386 µg/g. Additionally, the concentration of dye was higher than the permitted level in 28.6% of beverages, 40% of edible ice products, and 100% of fruit roll-ups (Lavashak). Conclusion: This method is an effective, appropriate, accessible, reliable and safe analytic method to analyze eight food dyes

New Analytical Approach for The Determination of Calcium Phosphate Dibasic and Tribasic in Processed Food by Comparison of Ion Chromatography With High-Performance Liquid Chromatography.

An analytical method to measure solubilized orthophosphate ions (HPO42− and PO43− ) from the water-insoluble food additives calcium phosphate dibasic (DCP) and calcium phosphate tribasic (TCP) in processed foods was optimized by comparing ion chromatography (IC) coupled with DS6 conductivity detector (Cond.) and high-performance liquid chromatography (HPLC) with Evaporative light scattering detector (ELSD) methods. The ion-pairing HPLC method could analyze calcium and phosphate ions successively. However, this method exhibited low reproducibility after approximately 48 hours of measurements. The IC method was established as an effective method of measuring orthophosphate ions with high reproducibility using distilled water and KOH solution as the mobile phase with a Dionex column. Matrix-based limit of detections (LOD) and limit of quantifications (LOQ) for snacks and cereals were estimated in the range of 0.01–0.91 µg/mL and 0.21–2.74 µg/mL, respectively. In inter-day and intra-day tests, the calculated precision (%RSD) and accuracy (recovery %) ranged from 0.5% to 6.6% and 82% to 117%, respectively, in both food samples. The levels of DCP or TCP could be analyzed in various positive food samples, and the developed IC method demonstrated good applicability in the analysis of DCP and TCP in collected processed foods.

Impurity profiling of l-aspartic acid and glycine using high-performance liquid chromatography coupled with charged aerosol and ultraviolet detection

For the compendial related substances test of l-aspartic acid (Asp) and glycine (Gly), two separate reversed-phase ion-pair high-performance liquid chromatography methods coupled with charged aerosol and ultraviolet detection were developed. Separation of all putative impurities, in particular of the related carboxylic and amino acids, was achieved using volatile perfluorocarboxylic acids as ion-pairing reagents on a polar embedded C18 stationary phase. It was shown that an adjustment of the evaporation temperature of the charged aerosol detector (CAD) was an efficient strategy for meeting the required quantitation limits, when dealing with non-volatile analytes. It was also demonstrated that the usage of a two detector setup can be beneficial for extending the detection range and providing accurate quantitation of low level impurities (LOQs from 5 to 50 ng on column). Both methods were validated with accordance to ICH guideline Q2(R1) assessing specificity, linearity, accuracy, precision, and robustness. Several batches of Asp and Gly were tested for related substances using the developed methods. The purity of each sample was higher than 99.7 %. Coupled charged aerosol and UV detection proved to be a more simple, robust and selective alternative to established derivatization procedures such as the Amino-Acid-Analyser (AAA) for the impurity profiling of amino acids, and should thus be considered for implementation into pharmacopoeial monographs in the future.