Liquid chromatography-tandem mass spectrometry for monitoring vitamin D hydroxymetabolites in human aqueous humor

Fabregat-Cabello, Neus; Darimont, Pierre; Huyghebaert, Loreen; Reynier, Pascal; Annweiler, Cédric; Miléa, Dan; Goff, Caroline Le; Cavalier, Étienne

doi:10.1039/c9ay01896d

Cited by 5 publications

(5 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fabregat-Cabello et al re-injected extracted samples, which were derivatized with Amplifex, after leaving them in the autosampler for 24 h at 5 °C. The authors noticed no significant differences in peak areas [ 19 ]. For 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatives, Lyu et al observed no significant analyte loss when the extract was left for 24 h at room temperature [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

Stability of sample extracts of vitamin D3 metabolites after chemical derivatization for LC–MS/MS analysis

Αλεξανδρίδου

Volmer

2022

Anal Bioanal Chem

View full text Add to dashboard Cite

Liquid chromatography/tandem mass spectrometry (LC–MS/MS) is widely used to determine vitamin D3 metabolites in biological samples. The ionization efficiencies of these metabolites, however, are poor under electrospray ionization conditions. Moreover, the chromatographic separation of multiple vitamin D metabolites and their epimers can be challenging. For these reasons, chemical derivatization reagents are often used to improve sensitivity and selectivity of analysis. While the derivatization schemes have been proven to be very effective, one missing aspect is the investigation of the stability of the chemical derivatization products in stored sample extracts. In this study, we investigated the long-term stability of several vitamin D3 metabolites after 1 and 3 months of storage at − 20 °C. Five vitamin D3 metabolites were examined after derivatization with seven different derivatization reagents. Generally, Amplifex products were the most stable in the long term in our study with 11–20% degraded after 1 month of storage and 14–35% after 3 months. The stabilities for some of the metabolites′ 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD), 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMP-TS), isonicotinoyl chloride (INC) and 4-phenyl-1,2,4-triazoline-3,5-dione acetylated (PTAD-Ac) products were also acceptable after 1 month of storage. Other derivatized metabolites, however, degraded extensively already after 1 month of storage, such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) (54–72% degradation) and 2-nitrosopyridine (PyrNO) (32–100% degradation). Importantly, for every metabolite, there was an optimum derivatization reagent that met the criteria of stability proposed by international regulatory bodies after 1 month of storage. Some derivatives were stable for even up to 3 months of storage, with degradation of less than 15%. Graphical abstract

show abstract

Section: Introductionmentioning

confidence: 99%

Stability of sample extracts of vitamin D3 metabolites after chemical derivatization for LC–MS/MS analysis

Αλεξανδρίδου

Volmer

2022

Anal Bioanal Chem

View full text Add to dashboard Cite

show abstract

“…Ocular barrier epithelial cells are able to convert 25(OH)D 3 to 1,25(OH) 2 D 3 , which could be a protective molecule for the eye and play a role in immune regulation (Alsalem et al, 2014). Moreover, an LC-MS/MS method was developed to determine 24,25(OH) 2 D 3 , 25(OH)D 3 , and 25(OH)D 2 in human aqueous humor after Amplifex derivatization (Fabregat-Cabello et al, 2019), which was the first reported method for determination of vitamin D metabolites in this matrix. All samples showed measurable levels of 24,25(OH) 2 D 3 and/or 25(OH)D 3 , but no 25(OH)D 2 was detected.…”

Section: Biological Samples For Vitamin D Analysis Beyond Serum/plasmamentioning

confidence: 99%

“…Moreover, as vitamin D is present in biological samples at very low concentrations (ng/ml, pg/ml), a preconcentration step is often necessary. Liquid‐liquid extraction (LLE) (Chin et al, 2018; Fabregat‐Cabello et al, 2019; Jumaah et al, 2016; Le et al, 2019; Oberson et al, 2020; Tai & Nelson, 2015; Wang et al, 2015; Yu et al, 2019a) and solid phase extraction (SPE) (Gao et al, 2017; Higashi et al, 2011, 2014; Ogawa et al, 2014; Yoshimura et al, 2019) or online SPE (Kassim et al, 2018) are the most commonly used methods among the many published studies for vitamin D and its analogues. During the last 5 years, improved sample preparation has been applied for vitamin D, such as supported liquid extraction (SLE) (Geib et al, 2016; Jenkinson, Taylor, Hassan‐Smith, et al, 2016; Petruzziello et al, 2017; Satoh et al, 2016; Tang et al, 2017) and hollow fiber‐based liquid phase microextraction (Saber‐Tehrani et al, 2014).…”

Section: Progress In Sample Preparation Techniques For Ms Analysis Of...mentioning

confidence: 99%

Analysis of vitamin D metabolic markers by mass spectrometry: Recent progress regarding the “gold standard” method and integration into clinical practice

Αλεξανδρίδου

Schorr

Stokes

et al. 2021

Mass Spectrometry Reviews

View full text Add to dashboard Cite

Liquid chromatography/tandem mass spectrometry is firmly established today as the gold standard technique for analysis of vitamin D, both for vitamin D status assessments as well as for measuring complex and intricate vitamin D metabolic fingerprints. While the actual mass spectrometry technology has seen only incremental performance increases in recent years, there have been major, very impactful changes in the front‐ and back‐end of MS‐based vitamin D assays; for example, the extension to new types of biological sample matrices analyzed for an increasing number of different vitamin D metabolites, novel sample preparation techniques, new powerful chemical derivatization reagents, as well the continued integration of high resolution mass spectrometers into clinical laboratories, replacing established triple‐quadrupole instruments. At the same time, the sustainability of mass spectrometry operation in the vitamin D field is now firmly established through proven analytical harmonization and standardization programs. The present review summarizes the most important of these recent developments.

show abstract

“…In this study, we investigated a wide range of commercially available derivatization reagents and compared analytical figures of merit such as sensitivity, chromatographic selectivity and resolution, derivatization time, ease of implementation, and analysis cost: 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), Amplifex, and 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4triazoline-3,5-dione (DMEQ-TAD), which have been widely used [8][9][10][11][12][13][14][15][16][17][18][19][20][21] for vitamin D metabolites. Isonicotinoyl chloride (INC) and 2-nitrosopyridine (PyrNO) have also been reported for vitamin D 3 measurements [22][23][24].…”

Section: Introductionmentioning

confidence: 99%

Comparing derivatization reagents for quantitative LC–MS/MS analysis of a variety of vitamin D metabolites

2023

View full text Add to dashboard Cite

The present study systematically compares the sensitivity and selectivity of the analysis of multiple vitamin D metabolites after chemical derivatization using different reagents for liquid chromatography-tandem mass spectrometry (LC–MS/MS). Generally, chemical derivatization is applied to vitamin D metabolites to increase the ionization efficiency, which is particularly important for very low abundant metabolites. Derivatization can also improve the selectivity of the LC separation. A wide variety of derivatization reagents has been reported in recent years, but information on their relative performance and applicability to different vitamin D metabolites is, unfortunately, not available in the literature. To fill this gap, we investigated vitamin D3, 3β-25-hydroxyvitamin D3 (3β-25(OH)D3), 3α-25-hydroxyvitamin D3 (3α-25(OH)D3), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and compared response factors and selectivity after derivatizing with several important reagents, including four dienophile reagents (4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD), Amplifex, 2-nitrosopyridine (PyrNO)) as well as two reagents targeting hydroxyl groups: isonicotinoyl chloride (INC) and 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). In addition, a combination of dienophiles and hydroxyl group reagents was examined. For LC separations, reversed-phase C-18 and mixed-mode pentafluorophenyl HPLC columns using different compositions of the mobile phase were compared. With respect to detection sensitivity, the optimum derivatization reagent for the profiling of multiple metabolites was Amplifex. Nevertheless, FMP-TS, INC, PTAD, or PTAD combined with an acetylation reaction showed very good performance for selected metabolites. These reagent combinations provided signal enhancements on the order of 3- to 295-fold depending on the compound. Chromatographic separation of the dihydroxylated vitamin D3 species was readily achieved using any of the derivatization reactions, while for 25(OH)D3 epimers, only PyrNO, FMP, INC, and PTAD combined with acetylation enabled complete separation. In conclusion, we believe this study can serve as a useful reference for vitamin D laboratories, to help analytical and clinical scientists decide which derivatization reagent to choose for their application.

show abstract

Liquid chromatography-tandem mass spectrometry for monitoring vitamin D hydroxymetabolites in human aqueous humor

Abstract: A stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the determination of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 25-hydroxyvitamin D3/D2 (25(OH)D3/D2) in human aqueous humor samples.

Cited by 5 publications

References 18 publications

Stability of sample extracts of vitamin D3 metabolites after chemical derivatization for LC–MS/MS analysis

Stability of sample extracts of vitamin D3 metabolites after chemical derivatization for LC–MS/MS analysis

Analysis of vitamin D metabolic markers by mass spectrometry: Recent progress regarding the “gold standard” method and integration into clinical practice

Comparing derivatization reagents for quantitative LC–MS/MS analysis of a variety of vitamin D metabolites

Contact Info

Product

Resources

About