Multidimensional separation methods for in-depth proteome profiling of low-microgram samples.

Tweetable abstract Multidimensional separation methods with improved sensitivity and peak capacity and throughput allow in-depth proteome profiling of low-microgram samples.

High-resolution mass spectrometry for glycoproteomics.

Tweetable abstract Bottom-up glycoproteomics combined with top-down strategy allows direct analysis of glycoform-mapped glycosylation and its glycans by high-resolution mass spectrometry.

Recent development in hydrophilic interaction liquid chromatography stationary materials for glycopeptide analysis.

Protein glycosylation is one of the most important post-translational modifications, and aberrant glycosylation is associated with the occurrence and development of diseases. Deciphering abnormal glycosylation changes can identify disease-specific signatures to facilitate the discovery of potential disease biomarkers. However, glycosylation analysis is challenging due to the diversity of glycans, heterogeneity of glycosites, and poor electrospray ionization of mass spectrometry. To overcome these obstacles, glycosylation is often elucidated using enriched glycopeptides by removing highly abundant non-glycopeptides. Hydrophilic interaction liquid chromatography (HILIC) is widely used for glycopeptide enrichment due to its excellent selectivity and specificity to hydrophilic glycans and compatibility with mass spectrometry. However, the development of HILIC has lagged far behind hydrophobic interaction chromatography, so efforts to further improve the performance of HILIC are beneficial for glycosylation analysis. This review discusses recent developments in HILIC materials and their advanced applications. Based on the physiochemical properties of glycopeptides, the use of amino acids or peptides as stationary phases showed improved enrichment and separation of glycopeptides. We can envision that the use of glycopeptides as stationary phases would definitely further improve the selectivity and specificity of HILIC for glycosylation analysis.

Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade.

Advances in bioanalytical technology have accelerated the analysis of complex protein glycosylation, which is beneficial to understand glycosylation in drug discovery and disease diagnosis. Due to its biological uniqueness in the course of disease occurrence and development, disease-specific glycosylation requires quantitative characterization of protein glycosylation. We provide a comprehensive review of recent advances in glycosylation analysis, including workflows for glycoprotein digestion, glycopeptide separation and enrichment, and mass spectrometry sequencing. We specifically focus on different strategies for glycopeptide enrichment through physical interaction, chemical oxidation, or metabolic labeling of intact glycopeptides. Recent advances and challenges of O-glycosylation analysis are presented, and the development of improved enrichment methods combining different proteases to analyze O-glycosylation is also proposed.

Technique development of high-throughput and high-sensitivity sample preparation and separation for proteomics.

Sample preparation and separation methods determine the sensitivity and the quantification accuracy of the proteomics analysis. This article covers a comprehensive review of the recent technique development of high-throughput and high-sensitivity sample preparation and separation methods in proteomics research.

Simultaneous determination of multi-class active pharmaceutical ingredients by UHPLC-HRMS.

In this work, a UHPLC-HRMS method using a quadrupole-orbitrap mass spectrometer has been developed for the detection and quantification of 47 compounds. These compounds include a range of chemical structures and properties and are popularly referred to as active pharmaceutical ingredients (API). The APIs selected have historically been incorporated into a variety of products commonly marketed towards acne, hair loss, male erectile dysfunction, and skin whitening. A fast ultrasound-assisted extraction (UAE) procedure without sample cleanup was developed and a high-resolution product ion spectral library was generated for compound verification in complex matrices. Collision energies were optimized for all analytes to overcome the limitations by applying stepped collision energies, such as insufficient fragmentation and excessive fragmentation without molecular ion information. Higher HRMS2 spectra matching scores (0.6 or above) were obtained for the analytes in the tested complex matrices. Eleven representative stable isotopically labeled API analogs were used as internal standards to compensate for the influence of complex matrices, such as shampoo and creams, and as an instrument quality control. One-hundred products with complex matrices were analyzed using the validated UHPLC-HRMS method. Eight APIs (ketoconazole, hydroquinone, salicylic acid, benzocaine, progesterone, azelaic acid, lidocaine, and minoxidil) were identified in 26 out of 100 products ranging from 103 µg/g to 156,000 µg/g.

High-throughput untargeted screening of veterinary drug residues and metabolites in tilapia using high resolution orbitrap mass spectrometry.

An analytical method was developed and validated for simultaneous analysis of one hundred and thirty-seven veterinary drug residues and metabolites from sixteen different classes in tilapia utilizing an improved fully non-targeted way of data acquisition with fragmentation. The automated on-line extraction procedure was achieved in a simple disposable pipet extraction. Ultrahigh-performance liquid chromatography and electrospray ionization quadrupole Orbitrap high-resolution mass spectrometry (UHPLC Q-Orbitrap) was used for the separation and detection of all the analytes. The methodology was validated by taking into consideration the guidelines specified in European SANCO/12571/2013 Guideline 2013 and Commission Decision 2002/657/EC. The extraction recoveries ranged from 81% to 111%. The limits of decision ranged from 0.01 to 2.73 µg kg-1 and the detection capabilities ranged from 0.01 to 4.73 µg kg-1. The one hundred and thirty-seven compounds behave dynamic 0.1-500 µg kg-1, with correlation coefficient >0.99. The fully non-targeted data acquisition way improves both sensitivity and selectivity for the fragments, which is beneficial for screening performance and identification capability. This validated method has been successfully applied on screening of veterinary drug residues and metabolites in muscle of tilapia, an important and intensively produced fish in aquaculture.

Rapid determination of cocamidopropyl betaine impurities in cosmetic products by core-shell hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Cocamidopropyl betaine (CAPB) is a common surfactant widely used in personal care products. Dimethylaminopropylamine (DMAPA) and lauramidopropyldimethylamine (LAPDMA) are two chemicals present as impurities in CAPB and have been reported as skin sensitizers. A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method, using a core shell hydrophilic interaction liquid chromatography (HILIC) column, has been developed to quantify DMAPA and LAPDMA in cosmetic products. Corresponding stable isotopically labeled analogues of the above native compounds were used as internal standards to compensate for matrix effect and for loss of recovery. Each sample was first screened to determine whether the sample needed to be diluted to minimize matrix effects as well as to fit the calibration range. The concept of matrix effect factor (MEF) was introduced to quantitatively evaluate each sample with a unique matrix using the internal standards. Recoveries at three spiking levels of low, medium, and high concentrations ranged from 98.4 to 112% with RSDs less than 5%. This method has been validated and is the first UHPLC-MS/MS method, which uses core shell HILIC column and stable isotopically labeled internal standards to simultaneously determine these two CAPB impurities in cosmetic products.

Simultaneous determination of cosmetics ingredients in nail products by fast gas chromatography with tandem mass spectrometry.

A rapid and sensitive gas chromatography with tandem mass spectrometry (GC-MS/MS) method has been developed and validated to quantitatively determine cosmetic ingredients, such as toluene, N-methylpyrrolidone, 2,4-dihydroxybenzophenone (benzophenone-1, BP-1), and diethylene glycol dimethacrylate, in nail products. In this procedure, test portions were extracted with acetone, followed by vortexing, sonication, centrifugation, and filtration. During the extraction procedure, BP-1 was derivatized making it amenable to GC-MS analysis, using N,O-​bis(trimethylsilyl)​trifluoroacetamide. The four ingredients were quantified by GC-MS/MS in an electron ionization mode. Four corresponding stable isotopically labeled analogues were selected as internal standards, which were added at the beginning of the sample preparation to correct for recoveries and matrix effects. The validated method was used to screen 34 commercial nail products for these four cosmetic ingredients. The most common ingredients detected in the nail products were toluene and BP-1. Toluene was detected in 26 products and ranged from 1.36 to 173,000µg/g. BP-1 ranged from 18.3 to 2,370µg/g in 10 products.

Determination of methylisothiazolinone and methylchloroisothiazolinone in cosmetic products by ultra high performance liquid chromatography with tandem mass spectrometry.

Isothiazolinone biocides are broad-spectrum preservatives that are widely used in cosmetics, household, and industrial products. An increase in the number of cases of allergic contact dermatitis to isothiazolinone preservatives, namely, methylisothiazolinone and methylchloroisothiazolinone, have been recently noticed. The Food and Drug Administration relies on analytical methods to quantify levels of use of cosmetic ingredients and support enforcement action against products that are not in compliance with the law. In this study, an efficient ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the determination of methylisothiazolinone and methylchloroisothiazolinone in selected cosmetic products. The lower limit of quantitation was determined to be 0.1 µg/g for both preservatives. A survey of 24 cosmetic products was conducted and found concentrations of methylisothiazolinone and methylchloroisothiazolinone ranging from not quantified, or below the lower limit of quantitation, to 89.64 µg/g and not quantified to 10.31 µg/g, respectively.

In vitro skin penetration of acetyl hexapeptide-8 from a cosmetic formulation.

There is a concern that peptides in cosmetic creams marketed as anti-aging/anti-wrinkle may penetrate into the deep layers of the skin and potentially stimulate biological activity. Claims for one cosmetic peptide, acetyl hexapeptide-8 (Ac-EEMQRR-amide), suggest interference with neuromuscular signaling as its anti-wrinkle mechanism of action. Therefore, the skin penetration of commercially available Ac-EEMQRR-amide from a cosmetic formulation (oil-in-water (O/W) emulsion) was determined in hairless guinea pig (HGP) and human cadaver skin assembled into in vitro diffusion cells. An O/W emulsion containing 10% Ac-EEMQRR-amide was applied to skin at a dose of 2 mg/cm(2). After a 24-h exposure, the skin surface was washed to remove unabsorbed peptide. Skin disks were tape stripped to determine the amount of peptide in the stratum corneum. Removal of the stratum corneum layers was verified by confocal microscopy. The epidermis was heat separated from the dermis and each skin fraction was homogenized. Skin penetration of Ac-EEMQRR-amide was measured in skin layers by hydrophilic interaction liquid chromatography with tandem mass spectrometry using electrospray ionization (ESI) in the positive mode. Stable isotopically labeled hexapeptides were used as internal standards for the quantitation of native hexapeptides to correct for matrix effects associated with ESI. The results (percent of applied dose) showed that the majority of the Ac-EEMQRR-amide was washed from the surface of both HGP and human skin. Ac-EEMQRR-amide that penetrated skin remained mostly in the stratum corneum of HGP (0.54%) and human (0.22%) with the peptide levels decreasing as each layer was removed by tape stripping. Total Ac-EEMQRR-amide found in the epidermis of HGP and human skin was similar at 0.01%. No peptide was detected in the dermis or buffer collected underneath the skin for both human and HGP. There was no hexapeptide metabolite (H2N-EEMQRR-amide) detected in any layers of HGP skin, human skin or buffer collected underneath the skin. This skin penetration data will be useful for evaluating the safety of cosmetic products containing small peptide cosmetic ingredients.

Determination of prostaglandin analogs in cosmetic products by high performance liquid chromatography with tandem mass spectrometry.

A method was developed and validated for the determination of 16 prostaglandin analogs in cosmetic products. The QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, Safe) liquid-liquid extraction method, typically used for pesticide residue analysis, was utilized as the sample preparation technique. The prostaglandin analogs were chromatographically separated and quantified using high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Thirty-one cosmetic products were surveyed, and 13 products were determined to contain a prostaglandin analog with amounts ranging from 27.4 to 297µg/g. The calculated concentrations for the cosmetic products were in a similar range when compared to the concentrations of three different prostaglandin analog-containing prescription products.

Rapid determination of hexapeptides by hydrophilic interaction LC-MS/MS for in vitro skin-penetration studies.

BACKGROUND: A sensitive analytical method is needed for assessing penetration of topically applied peptides for in vitro skin-penetration studies. RESULTS: A rapid hydrophilic interaction LC (HILIC)-MS/MS method for analyzing the polar peptides Ac-EEMQRR-amide and H2N-EEMQRR-amide in various skin layers and matrices has been developed and evaluated. The matrices included emulsion, receptor fluids, cotton-tipped applicators, stratum corneum tape strips, epidermis and dermis of the skin. Stable isotopically labeled analogues were used as internal standards to correct for recovery and matrix effects. A HILIC-SPE procedure was optimized to minimize significant ion suppression in the more complex matrices. CONCLUSION: This HILIC-MS/MS method is applicable to the determination of Ac-EEMQRR-amide and H2N-EEMQRR-amide in complex skin samples and other matrices generated during in vitro skin-penetration studies.

Rapid determination of parabens in personal care products by stable isotope GC-MS/MS with dynamic selected reaction monitoring.

In this study, a rapid and sensitive analytical method for the determination of methyl-, ethyl-, propyl-, and butyl esters of para-hydroxy benzoic acid (parabens) in personal care products was developed and fully validated. Test portions were extracted with methanol followed by vortexing, sonication, centrifugation, and filtration without derivatization. The four parabens were quantified by GC-MS/MS in the electron ionization mode. Four corresponding isotopically labeled parabens were selected as internal standards, which were added at the beginning of the sample preparation and used to correct for recovery and matrix effects. Sensitivity, extraction efficiency, and recovery of the respective analytes were evaluated. The coefficients of determination (r(2)) were all greater than 0.995 for the four parabens investigated. The recoveries ranged from 97 to 107% at three spiked levels and a one-time (single) extraction efficiency greater than 97% was obtained. This method has been applied to screen 26 personal care products. This is the first time that a unique GC-MS/MS method with dynamic selected reaction monitoring and confirmation of analytes has been used to determine these parabens in cosmetic personal care products.

A perspective on high throughput analysis of pesticide residues in foods.

The screening of pesticide residues plays a vital role in food safety. Applications of high throughput analytical procedures are desirable for screening a large number of pesticides and food samples in a time-efficient and cost-effective manner. This review discusses how sample throughput of pesticide analysis could be improved with an emphasis on sample preparation, instrumentation and data analysis.