Hydrophilic Interaction Liquid Chromatography Method Research Articles

Evaluating the potential of hydrophilic interaction liquid chromatography for collagen peptide mapping analysis

This study presents a systematic approach for developing an innovative hydrophilic interaction liquid chromatography (HILIC) method for collagen peptide mapping analysis. The predominant post-translational modification (PTM) of collagen, proline hydroxylation, introduces polar hydroxyl groups throughout the collagen sequence, making HILIC a promising alternative to classical reversed-phase liquid chromatography (RPLC) approaches. This study employs sixteen model peptides, selected from in silico predicted tryptic peptides with zero missed cleavages and representing diverse physicochemical properties and structural motifs of collagen. The peptides were used as standards to conduct detailed chromatographic evaluation. Various HILIC stationary phases and mobile phases were systematically examined to identify optimal separation conditions for collagen peptides, contributing to a better understanding of peptide behavior in HILIC. The study also explores the effects of sample diluent and injection mode, comparing classical injection with the Performance Optimizing Injection Sequence (POISe), to determine their impact on HILIC performance. Introducing a plug of weak solvent (acetonitrile) prior to sample injection, effectively mitigates the mismatch in eluent strength between the fully aqueous sample diluent (resulting from tryptic digestion) and the mobile phase, addressing issues of peak distortion. Different injection volumes (from 0.5 to 8 µL) and acetonitrile ratios (1:1, 1:2, 1:5 and 1:10) were tested to optimize sample injection and increase sensitivity of collagen tryptic peptides.Following method optimization, HILIC was coupled with mass spectrometry (MS) to evaluate its effectiveness in analyzing collagen-digested samples. This evaluation included the assessment of peptide sequence coverage and the method ability to identify hydroxylation patterns, thereby demonstrating its potential for detailed peptide analysis.

Quantifying skin permeation of a novel metformin lotion using modified hydrophilic interaction liquid chromatography.

Aim: This study aimed to quantify the permeation of metformin (Met) lotion through pig ear skin using high-performance liquid chromatography, specifically hydrophilic interaction liquid chromatography (HILIC), to separate Met from biological contaminants and effectively measure its permeation through skin similar to human skin.Materials & methods: A Franz cell permeation assay was used to assess the permeation kinetics of 6% Met lotion through pig ear skin. Samples were collected at various time points and prepared for high-performance liquid chromatography analysis by removing large biological contaminants. The permeated Met was quantified by monitoring its retention time (RT) at 9min using HILIC, with an acidic, polar mobile phase and a normal-phase column.Results: A distinct Met peak with a RT of approximately 9min was observed in the 6% Met lotion, which was absent in the permeation samples from the 0% Met lotion. This peak (RT 9min) was distinct from the 'biological-contaminants' peaks at RT 2-3min and increased linearly over time, reaching 36.8% of the total applied Met at 24h.Conclusion: These findings demonstrate that the HILIC method effectively separates Met from biological components in pig ear skin, allowing accurate quantification of Met despite the presence of skin lipids and proteins.

Quantitation of Copper Tripeptide in Cosmetics via Fabric Phase Sorptive Extraction Combined with Zwitterionic Hydrophilic Interaction Liquid Chromatography and UV/Vis Detection

The increasing demand for effective cosmetics has driven the development of innovative analytical techniques to ensure product quality. This work presents the development and validation of a zwitterionic hydrophilic interaction liquid chromatography method, coupled with ultraviolet detection, for the quantification of copper tripeptide in cosmetics. A novel protocol for sample preparation was developed using fabric phase sorptive extraction to extract the targeted analyte from the complex cosmetic cream matrix, followed by chromatographic separation on a ZIC®-pHILIC analytical column. A thorough investigation of the chromatographic behavior of the copper tripeptide on the HILIC column was performed during method development. The mobile phase consisted of 133 mM ammonium formate (pH 9, adjusted with ammonium hydroxide) and acetonitrile at a 40:60 (v/v) ratio, with a flow rate of 0.2 mL/min. A design of experiments (DOE) approach allowed precise adjustments to various factors influencing the extraction process, leading to the optimization of the fabric phase sorptive extraction protocol for copper tripeptide analysis. The method demonstrated excellent linearity over a concentration range of 0.002 to 0.005% w/w for copper tripeptide, with a correlation coefficient exceeding 0.998. The limits of detection and quantitation were 5.3 × 10−4% w/w and 2.0 × 10−3% w/w, respectively. The selectivity of the method was verified through successful separation of copper tripeptide from other cream components within 10 min, establishing its suitability for high-throughput quality control of cosmetic formulations.

Characterization of synthetic guide ribonucleic acids through hydrophilic interaction chromatography coupled with mass spectrometry

In this study, we aimed to develop a hydrophilic interaction liquid chromatography (HILIC) method for the analysis of single guide ribonucleic acid (sgRNA), a critical reagent used in CRISPR genome editing. Our results showed that effective profiling of sgRNA can be achieved by suppressing the surface charge of the stationary phase in HILIC. We identified hydrogen bonding as the primary retention mechanism with potential weak partitioning in HILIC separation of large oligonucleotides like 100-mer sgRNA. Moreover, we demonstrated that direct coupling of HILIC with mass spectrometry (MS) allows the intact mass analysis of sgRNA and its impurities with minimal adduct present. Finally, we characterized the post peak shown in the low temperature HILIC and identified it as sgRNA aggregates. Our findings provide valuable insight into the characterization of sgRNA and highlight the potential of HILIC-MS as a powerful analytical tool for relatively large oligonucleotide analysis.

Non-targeted multimodal metabolomics data from ovine rumen fluid fractions.

From an animal health perspective, our understanding of the metabolites in rumen fluid across different host species is poorly understood. Here, we present a metabolomic data set generated using hydrophilic interaction liquid chromatography and semi-polar (C18) chromatography methods coupled to high-resolution mass spectrometry of fractionated ovine rumen samples.

Comparison of chromatographic conditions for the targeted tandem mass spectrometric determination of 354 mammalian metabolites

Metabolomics is becoming increasingly popular in livestock research, but no single analytical method can cover the entire metabolome. As such, we compared similar and complementary chromatographic methods with respect to analyte coverage and chromatographic properties of mammalian metabolites. We investigated 354 biologically relevant primary metabolites from 19 compound classes including amino acids, bile acids, biogenic amines, carboxylic acids, lipids, nucleotides and sugars. A total of 2063 selected reaction monitoring transitions were optimized on a triple quadrupole mass spectrometer. We then determined the retention profiles and peak parameters of our compounds using an anion exchange chromatography (AIC), three reversed-phase (RP) and three hydrophilic interaction liquid chromatography (HILIC) methods. On average, HILIC methods covered 54% of all metabolites with retention factors >1, while average RP coverage was 41%. In contrast to RP, HILIC methods could also retain polar metabolites such as amino acids and biogenic amines. Carboxylic acids, nucleotides, and sugar related compounds were best separated by AIC or zwitterionic pHILIC with alkaline eluents. Combining two complementary HILIC and RP methods increased the library coverage to 92%. By further including important short chain fatty acids, a combination of HILIC, RP and AIC methods achieved a coverage of 97%. The resulting dataset of LC and MS/MS parameters will facilitate the development of tailor-made quantitative targeted LC-MS/MS methods to investigate the mammalian metabolome.

Knowledge of The HILIC Retention Behaviors of Two Quinolone Antibiotics on Sulfobetaine-Type Zwitterionic Stationary Phases

Fluoroquinolones treat infections of the urinary, respiratory, gastrointestinal, skin, bone, and joint systems. This article describes the development of the hydrophilic interaction liquid chromatography (ZIC-HILIC) method to determine norfl oxacin and ofl oxacin retention characteristics. Separation of all analytes was accomplished using ZIC-HILIC1 and ZIC-HILIC4 columns. The mobile phase was 40 mM sodium acetate water solution in acetonitrile pumped at a 0.7 mL/min fl ow rate. The infl uence of acetonitrile concentration, the presence of salt in the mobile phase, and pH on the retention of norfl oxacin and ofl oxacin in the HILIC mode were investigated in this study. When using ZIC-HILIC columns, it was discovered that the retention factors of the analytes were inversely proportional to the amount of water present in the mobile phase, which is representative of a conventional hydrophilic partitioning mechanism. The ZIC-HILIC based strategies described in this article represent a signifi cant step toward more sensitive drug analysis. We observed that zwitterionic materials could govern the separation of materials, as shown by the infl uence of the chain length between the zwitterion groups, which contributed to the explanation of the variation in the retention period of the materials to be separated.

Miniaturized Two-Dimensional Heart Cutting for LC–MS-Based Metabolomics

Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics usually combines hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) chromatography to cover a wide range of metabolomes, requiring both significant sample consumption and analysis time for separate workflows. We developed an integrated workflow enabling the coverage of both polar and nonpolar metabolites with only one injection of the sample for each ionization mode using heart-cutting trapping to combine HILIC and RP separations. This approach enables the trapping of some compounds eluted from the first chromatographic dimension for separation later in the second dimension. In our case, we applied heart-cutting to non-retained metabolites in the first dimension. For that purpose, two independent miniaturized one-dimensional HILIC and RP methods were developed by optimizing the chromatographic and ionization conditions using columns with an inner diameter of 1 mm. They were then merged into one two-dimensional micro LC-MS method by optimization of the trapping conditions. Equilibration of the HILIC column during elution on the RP column and vice versa reduced the overall analysis time, and the multidimensionality allows us to avoid signal measurements during the solvent front. To demonstrate the benefits of this approach to metabolomics, it was applied to the analysis of the human plasma standard reference material SRM 1950, enabling the detection of hundreds of metabolites without the significant loss of some of them while requiring an injection volume of only 0.5 μL.

Strategies for the Separation and Quantification of Non-Steroidal Anti- Inflammatory Drugs Using ZIC-HILIC-HPLC with UV Detection

Background: Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used worldwide for their analgesic, anti-inflammatory, and antipyretic properties. Objective: The article highlights the development of zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) methods to quantify three NSAIDs as active ingredients in pharmaceutical formulations simultaneously. Method: For NSAIDs analysis, two homemade ZIC-HILIC stationary phases were carried out by adding sulfobetaine monomers to polystyrene-divinylbenzene particles. The methods were developed by examining the effect of HILIC mode parameters like the kind of organic modifier (methanol or acetonitrile), acetonitrile content, pH and concentration of the acetate buffer in the eluent. Results: The experimental data exhibit the predominant mechanism of NSAIDs with two ZICHILIC stationary phases. The different chain lengths in the two ZIC-HILIC stationary phases are employed to investigate NSAIDs retention behaviour. The strategies were evaluated for their specificity, linearity, accuracy, and precision. The linear ranges were 0.01-5.0, 0.05-17.5 and 0.12-13.5 μg ml-1, LOD 0.005. 0.015 and 0.046 μg ml-1 and LOQ 0.015, 0.045 and 0.139 μg ml-1 for nimesulide, tenoxicam and diclofenac, respectively. Conclusion: They represent interactions with three NSAIDs, ZIC-HILIC columns, and mobile phase in a mixed-mode of hydrophilic and hydrophobic interactions. In contrast to the ZICHILIC- 1 stationary phase with a shorter chain length, the ZIC-HILIC-4 stationary phase with a longer chain length exhibited a longer retention time, lower detection and limit of quantitation for three NSAIDs.The verification findings demonstrate the suitability of the proposed ZIC-HILIC methods for quantifying NSAIDs in pharmaceutical formulations.

Evaluation and Application of ZIC-HILIC Columns Selectivity for Four Angiotensin II Receptor Blockers in Pharmaceutical Formulations

Background: Angiotensin II receptor blockers (ARBs) are helpful medications for treating hypertension and heart failure and have been proposed as a possible alternative to standard angiotensin-converting enzyme inhibitors. Objective: The present article describes and validates sensitive hydrophilic interaction liquid chromatography methods to simultaneously analyse four angiotensin II receptor blockers: valsartan, telmisartan, losartan, and irbesartan. Method: Numerous chromatographic parameters were studied and optimized in detail. As a mobile phase, the best separation was obtained on two handmade columns (ZIC-S1 and ZIC-S4) using acetonitrile/sodium acetate buffer (40 mM, pH 4.75). The detection of target pharmaceuticals was obtained at 220 nm. Results: The linear ranges of target drugs (valsartan, telmisartan, losartan, and irbesartan) were 0.02-4, 0.06-7, 0.005-6 and 0.08-5 μg/mL on the ZIC-S1 and ZIC-S4 columns, respectively. The suggested methods demonstrated high precision (RSD ≤ 1.15), linearity (r2 ≥ 0.9964), and accuracy (between 98.88 and 101%) with detection and quantitation limits of (0.0021-0.0550 and 0.0063-0.1666 μg/mL, respectively) for all target pharmaceuticals. Conclusion: The suggested methods have been validated and applied to pharmaceutical formulations, indicating that it is suitable for therapeutic drug monitoring of these medications.

Evaluation and correction of injection order effects in LC-MS/MS based targeted metabolomics

For large-scale and long-term metabolomics studies that involve a large batch or multiple batches of analyses, batch effects cause nonbiological systematic biases that may lead to false positive or false negative findings. Quantitative monitoring and correction of batch effects is critical to the development of reproducible and robust metabolomics platforms either for untargeted or targeted analyses. To achieve sufficient retention and separation of a broad range of metabolites with diverse chemical structures and physicochemical properties, LC-MS/MS based targeted metabolomics often involves 3 complemented chromatographic separation methods, including reversed-phase liquid chromatography (RP-LC), hydrophilic interaction liquid chromatography (HILIC), and ion-pair liquid chromatography (IP-LC). The purpose of this study is to quantitatively evaluate intra-batch variations or injection order effects of the RP-LC, HILIC, and IP-LC methods for targeted metabolomics analyses, and develop strategies to minimize intra-batch variations and correct injection order effects for problematic metabolites. Both RP-LC and HILIC methods exhibit robust intra-batch reproducibility in 0.2 µM standard mix QC, with ∼96 % of the measured metabolites showing acceptable intra-batch variations (<20 %); whereas, the intra-batch reproducibility for some metabolites in cell matrix QC may be compromised due to stability issue, suboptimal chromatographic retention, and/or matrix effects causing ionization suppression and/or retention instability. The IP-LC method exhibits significant injection order effects, which could be effectively corrected by the developed exponential models of signal drift trends as a function of injection order for individual targeted metabolites.

Mass Spectrometry-Based Untargeted Metabolomics and Lipidomics Platforms to Analyze Cell Culture Extracts.

Metabolites represent the most downstream level of the cellular organization. Hence, an in vitro untargeted metabolomics approach is extremely valuable to deepen the understanding of how endogenous metabolites in cells are altered under a given biological condition. This chapter describes a robust liquid chromatography-high-resolution mass spectrometry-based metabolomics and lipidomics platform applied to cell culture extracts. The analytical workflow includes an optimized sample preparation procedure to cover a wide range of metabolites using liquid-liquid extraction and validated instrumental operation procedures with the implementation of comprehensive quality assurance and quality control measures to ensure high reproducibility. The lipidomics platform is based on reversed-phase liquid chromatography for the separation of slightly polar to apolar metabolites and covers a broad range of lipid classes, while the metabolomics platform makes use of two hydrophilic interaction liquid chromatography methods for the separation of polar metabolites, such as organic acids, amino acids, and sugars. The chapter focuses on the analysis of cultured HepaRG cells that are derived from a human hepatocellular carcinoma; however, the sample preparation and analytical platforms can easily be adapted for other types of cells.

Separation of minor steviol glycosides using hydrophilic interaction liquid chromatography (HILIC) and off-line two-dimensional reversed-phase liquid chromatography/HILIC methods

Steviol glycosides (SGs) are increasingly used as noncaloric sweeteners. In this study, hydrophilic interaction liquid chromatography (HILIC) methods and an off-line environmentally friendly two-dimensional reversed-phase liquid chromatography/HILIC (2D-RPLC/HILIC) for the separation of SGs were developed. The retention behavior on the polyacrylamide-based silica (PA) column was investigated by separating twelve SGs and comparing with three HILIC columns (Diol, XAmide and Unitary NH2 columns). Ethanol instead of acetonitrile as the weak eluent in HILIC mode was investigated. The PA column exhibited superior hydrophilicity, higher selectivity and better resolution. Two HILIC-UV methods for separation of SGs on a PA column with water/acetonitrile (LOD, 0.0049–0.013 mg/mL; RSD <3.73%; recovery, 95.02–102.5%) or water/ethanol (LOD, 0.010–0.016 mg/mL; RSD <4.52%; recovery, 91.90–97.76%) as elution system were developed. The preparative separation of minor SGs from stevia extracts was investigated by 2D-RPLC/HILIC using a preparative C18 column with the methanol/water mobile phase in the first dimension and a preparative PA column with the ethanol/water mobile phase in the second dimension. Two SGs were isolated and identified. In summary, safer and cheaper ethanol/water binary eluent system increased the diversity of HILIC retention and improved the solubility of SGs, which is greatly beneficial at a preparative scale in the food industry.

Matrix Effect of Diverse Biological Samples Extracted with Different Extraction Ratios on the Detection of β-N-Methylamino-L-Alanine by Two Common LC-MS/MS Analysis Methods.

Neurotoxin β-N-methylamino-L-alanine (BMAA) is hypothesized as an important pathogenic factor for neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC). Comparative study on the accuracy of BMAA analyzed by the regular LC-MS/MS methods is still limited for different biological matrices. In this study, a free-BMAA sample of cyanobacterium and BMAA-containing positive samples of diatom, mussel, scallop, and oyster were extracted with varied extraction ratios (ER) ranging from 1:20 to 1:2000. These extracts were then purified by MCX cartridges. After SPE purification, these different biological samples were analyzed by two common LC-MS/MS analysis methods, a direct analysis without derivatization by a hydrophilic interaction liquid chromatography (HILIC)-MS/MS and pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization combined with a C18 column. The results suggested that the recoveries of BMAA spiked in the cyanobacterial sample were close to 100% in the total soluble form extracts with the ER of 1:100 (g/mL) and the precipitated bound form extracts with the ER of 1:500. The recommended ER for the precipitated bound form of BMAA in diatoms and the total soluble form of BMAA in mollusks are 1:500 and 1:50, respectively. The quantitative results determined by the AQC derivatization method were lower than those determined by the direct analysis of the HILIC method in diatom and mollusk samples. The results of the HILIC method without the derivatization process were closer to the true value of BMAA in cyanobacteria. This work contributes to the performance of the solid-phase extraction (SPE) purification protocol and the accuracy of BMAA analysis by LC-MS/MS in diverse biological samples.

Quality evaluation of Lycium barbarum L. fruits from different regions in China based on 2-O-β-D-glucopyranosyl-L-ascorbic acid

The selection of a quality marker is crucial for quality control of Lycium barbarum L. fruits, which are widely used in the food and drug industries, especially as herbal medicine, and functional food. However, using betaine, the quality marker in the current standard, for discrimination of L. barbarum fruits lacks specificity. In this study, a vitamin C derivative, 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG), was first used as an antioxidant biomarker instead of betaine for the quality control of L. barbarum fruits from different regions. Three-factor-three-level response surface methodology was used to optimize extraction conditions, which indicated that the optimal conditions of AA-2βG were 75% ethanol as extraction solvent, 1:25 of solvent-to-solid ratio, and 30 min for extraction time. Hydrophilic interaction liquid chromatography method was applied for the quantification of AA-2βG. The results showed that the content of AA-2βG in L. barbarum fruits from different regions was above 9 mg/g except for samples of NLB 8, NLB 30 and NLB 74. All samples were classified into three clusters with Hierarchical Cluster Analysis, and most of them belong to cluster Ⅲ with the content of AA-2βG in the range of 9.04–11.8 mg/g.

Hydrophilic Interaction Liquid Chromatography Coupled with Fluorescence Detection (HILIC-FL) for the Quantitation of Octreotide in Injection Forms

Octreotide is a synthetic cyclic octapeptide analogue of somatostatin-14. It is mainly administered for the treatment of acromegaly, severe diarrhea, and neuroendocrine neoplasias. In this work, a hydrophilic interaction liquid chromatography (HILIC) method with fluorescence (FL) detection was developed and validated for the quantitation of octreotide in solutions for injection. Chromatographic separation was performed on an XBridge®-HILIC analytical column under isocratic elution with a short chromatographic run time of less than 10 min. The mobile phase consisted of ammonium bicarbonate 8.6 mM (pH 8.1)/acetonitrile 35/65 (v/v). The high sensitivity and selectivity of the fluorescence detection, with the excitation wavelength (λexcitation) set at 280 nm and the emission wavelength set at (λemission) 330 nm, enabled a simple sample preparation procedure that included only dilution steps. The calibration curve showed good linearity with a correlation coefficient greater than 0.998. The method was successfully applied to the analysis of commercially available octreotide injection forms.

Development and validation of a novel HILIC method for the quantification of low-levels of cuprizone in cuprizone-containing chow

Cuprizone is an amide compound that has been wildly used in various animal studies, such as in the investigation of remyelination in mouse model. It is important to control the amount of cuprizone dosed in animals to be consistent as different amounts may lead to different clinical observations. Cuprizone is usually administrated as a minor component (i.e., 0.3%) of a mixture with powdered or pelleted rodent chow. Its low content, combined with the complex nature of chow, represents a significant challenge for the quantification of cuprizone in the mixture. To the best of our knowledge, no method has been reported in the literature so far. In this study, a simple, selective, and sensitive hydrophilic interaction liquid chromatographic method was developed for the quantification of cuprizone in cuprizone pre-clinical formulations. The analytical method comprises a fast ultrasound assisted extraction with acetonitrile/water as a solvent followed by gradient separation using a Waters Xbridge HILIC column with 0.1% TFA in water and acetonitrile as mobile phases and UV detection at 220 nm. The specificity, linearity, accuracy, repeatability, and limit of quantitation (LOQ) of the method were established. The method was determined to be linear in the range of 10–200 μg/mL. Accuracy was assessed by spiking a chow placebo with various amounts of a cuprizone reference standard to achieve target concentration levels and the recoveries were within the acceptance criterion of 90–110% of the target concentrations. Repeatability was demonstrated at the nominal concentration of 100 µg/mL and LOQ level of 2.5 μg/mL. This method has been demonstrated to be suitable for its intended use and has been successfully applied to the quantification of low levels of cuprizone in chow formulations. It was found that the cuprizone content in chow could varied significantly between batches and the potential causes of the variability were investigated.

Design of experiments in optimization and validation of hydrophilic interaction liquid chromatography method for determination of amlodipine besylate and its impurities

Abstract A new and rapid hydrophilic interaction liquid chromatographic method has been developed for the quantitative analysis of amlodipine besylate and its specific impurities (D, E, and F). For development of this method, a systematic approach which includes Design of Experiments methodology was applied. For the method optimization, Box–Behnken design and specific way Derringer's desirability function were applied. They provided identification of the optimal chromatographic conditions on the basis of obtained mathematical models and graphical procedures (three D graphs). The optimal chromatographic conditions were the analytical column ZORBAX NH2 (250 × 4.6 mm, 5 µm particle size); mobile phase consisted of acetonitrile-water phase (50 mM ammonium acetate, pH adjusted to 4.0 with glacial acetic acid) (90.5:9.5 v/v); column temperature 30 °C, mobile phase flow rate 1 mL min−1, wavelength of detection 230 nm. As other validation parameters were also found to be suitable, the possibility to apply the proposed method for the determination of amlodipine besylate and its impurities in any laboratory under different circumstances has been proven.

Efficient Sub-1 Minute Analysis of Selected Biomarker Catecholamines by Core-Shell Hydrophilic Interaction Liquid Chromatography (HILIC) with Nanomolar Detection at a Boron-Doped Diamond (BDD) Electrode

A rapid, sensitive method for the separation of catecholamine biomarkers (CAs), of importance in traumatic brain injury (TBI) and in Parkinson’s disease (PD), has been successfully developed using hydrophilic interaction liquid chromatography (HILIC). Dopamine (DA), epinephrine (EPI), and norepinephrine (NE) are known to be three to fivefold elevated above normal in traumatic brain injury (TBI) patients. HILIC facilitates the rapid and efficient separation of these polar biomarkers, which can be poorly retained by reversed-phase liquid chromatography (RPLC), while electrochemical detection (ECD) at the boron-doped diamond (BDD) electrode provides enhanced nanomolar detection. Three HILIC columns were compared, namely the superficially porous (core-shell) Z-HILIC column and the Z-cHILIC and Z-HILIC fully porous columns. The core-shell Z-HILIC showed the highest efficiency with a rapid separation within 60 s. The HILIC method utilizing the core-shell Z-HILIC column was initially optimized for the simultaneous analysis of DA, EPI, and NE using UV detection. The advantages of using the BDD electrode over UV detection were explored, and the improved limits of detection (LODs, S/N = 3) measured were 40, 50, and 50 nM for DA, EPI, and NE, respectively. Method validation is reported in terms of the linearity, repeatability, reproducibility, and LODs. Furthermore, the proposed method was successfully applied to the real sample analysis of urinary CAs following phenylboronic acid (PBA) solid phase extraction (SPE) pretreatment.

Quantitation of Acetyl Hexapeptide-8 in Cosmetics by Hydrophilic Interaction Liquid Chromatography Coupled to Photo Diode Array Detection

Bioactive peptides are gaining more and more popularity in the research and development of cosmetic products with anti-aging effect. Acetyl hexapeptide-8 is a hydrophilic peptide incorporated in cosmetics to reduce the under-eye wrinkles and the forehead furrows. Hydrophilic interaction liquid chromatography (HILIC) is the separation technique of choice for analyzing peptides. In this work, a rapid HILIC method coupled to photodiode array detection operated at 214 nm was developed, validated and used to determine acetyl-hexapeptide-8 in cosmetics. Chromatography was performed on a Xbridge® HILIC BEH analytical column using as mobile phase a 40 mM ammonium formate water solution (pH 6.5)-acetonitrile mixture 30:70, v/v at flow rate 0.25 mL min−1. The assay was linear over the concentration range 20 to 30 μg mL−1 for the cosmetic formulations and 0.004 to 0.007% (w/w) for the cosmetic cream. The limits of quantitation for acetyl hexapeptide-8 were 1.5 μg mL−1 and 0.002% (w/w) for the assay of cosmetic formulations and cosmetic creams, respectively. The method was applied to the analysis of cosmetic formulations and anti-wrinkle cosmetic creams.