# Development and validation of HPLC-Fluorescence method for pentosidine analysis in fingernails as a biomarker in diabetic patients

Dinh Hieu Nguyen, Karima Ille, Kamel Mohammedi, Vincent Rigalleau,
Michael Howsam, Frederic J. Tessier

► To cite this version:

Dinh Hieu Nguyen, Karima Ille, Kamel Mohammedi, Vincent Rigalleau, Michael Howsam, et al. Development and validation of HPLC-Fluorescence method for pentosidine analysis in fingernails as a biomarker in diabetic patients. IMARS15, Sep 2024, Maastricht, Netherlands. hal-05267120

HAL Id: hal-05267120

https://hal.science/hal-05267120v1

Submitted on 18 Sep 2025

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# Development and validation of HPLC-Fluorescence method for pentosidine analysis in fingernails as a biomarker in diabetic patients

## CONTACT INFORMATION:

Dinh Hieu Nguyen, PhD student, UMR 1167, faculty of medicine, University of Lille

Email: dinh-hieu.nguyen.etu@univ-lille.fr

Dinh Hieu Nguyen,¹,² Karina Ille,¹ Kamel Mohammedi,³ Vincent Rigalleau,³ Michael Howsam,¹ Frédéric J. Tessier.¹

1. University of Lille, INSERM, CHU Lille, Institut Pasteur de Lille, UMR 1167 - RID-AGE - F - 59000 Lille, France.

2. University of Science and Technology of Hanoi (USTH), Vietnam Academic Science and Technology (VAST), 18 Hoang Quoc Viet, Nghia Do, Cau Giay, Hanoi, Vietnam.

3. Department of Endocrinology, Diabetology, nutrition, CHU de Bordeaux, Hôpital Haut-Lévêque, 33600 Pessac, France.

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## DIABETES AND HbA1c TEST

Diabetes, characterized by high blood glucose levels, can lead to destructive complications and increase morbidity and mortality [1].

Traditionally, the glycated hemoglobin (HbA1c) test is used to examine glycemic control over the preceding 90-120 days, and it has some power to access the risk of diabetic complications.

However, there are limitations to this test, not only in its use for glycemic control but also as a predictive tool for diabetic complications, highlighting the need of an alternative biomarker [2].

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## DIABETES BIOMARKERS IN FINGERNAILS ?

Fingernails, composed mainly of keratin proteins, are gaining attention as a surrogate matrix for reflecting medium-term glycation status, offering several advantages over blood samples [3].

Formation of pentosidine, a well-characterized AGE with fluorescence properties, can be accelerated rapidly under hyperglycemia conditions.

Therefore, we have developed a new method for the quantification of pentosidine in fingernails using HPLC-Fluorescence to investigate its potential as a biomarker for prognosis of diabetic complications.

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## MATERIALS & METHODS

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1 mL 6 M HCl

Reduced
or non-reduced

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110°C; 21 hours
ACID
HYDROLYSIS

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3-4 hours Speed-Vac
CONCENTRATION

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200 µL 12,5 mM NFPA
RESOLUBILIZATION

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25 mM citric acid
FILTRATION
& DILUTION

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ex. - em.: 325 - 385 nm
HPLC-FLUO
ANALYSIS

AU

Pentosidine

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DIABETIC: n = 91

HEALTHY: n = 7

LC - MS/MS
ANALYSIS

## METHOD DEVELOPMENT

The method showed good linearity (0-200 ng/mL), accuracy, precision, repeatability, recovery (> 94%); LOD = 0.02 ng/mL; LOQ = 0.05 ng/mL

<figure><img src="image_14.png" /><figcaption>EFFECT OF REDUCTION ON
QUANTIFICATION OF PENTOSIDINE</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>RECOVERY IN DIFFERENT STOKAGE
CONDITIONS AFTER 6 MONTHS</figcaption></figure>

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## PENTOSIDINE RELATIONSHIPS

The pentosidine level, expressed in mmol/mol lysine, was used to assess the correlation with other parameters such as age, current HbA1c, furosine, and CML.

<figure><img src="image_17.png" /></figure>

## POTENTIAL CLINICAL USES

This HPLC-Fluo method to measure pentosidine complements our existing LC-MS/MS to determine amino acids and other fingernail AGEs, providing a more complete view of glycation status in keratin nails.

Fingernail pentosidine and other AGEs could be used as complementary biomarkers with conventional HbA1c, for the assessment of long-term glycemic control and may offer insights into diabetic complications.

## ON-GOING EXPERIMENTS

Effect of different storage conditions (temperature, duration) on nail samples.

More nail samples from healthy and diabetic subjects will be analyzed to reveal the relationship of pentosidine with other parameters.

Compare nail concentrations of pentosidine and other AGEs with the incidence of Type 2 diabetes-related complications.

## REFERENCES:

[1] IDF diabetes atlas. Seventh edition. Brussels: International Diabetes Federation; 2015.

[2] Dorcely B, Katz K, Jagannathan R, Chiang SS, Oluwadare B, Goldberg IJ, et al. Novel biomarkers for prediabetes, diabetes, and associated complications. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2017;Volume 10:345-61. https://doi.org/10.2147/DMSO.S100074.

[3] Jaramillo Ortiz S, Howsam M, van Aken EH, Delanghe JR, Boulanger E, Tessier FJ. Biomarkers of disease in human nails: a comprehensive review. Critical Reviews in Clinical Laboratory Sciences 2022;59:125-41. https://doi.org/10.1080/10408363.2021.1991882.

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