Quantification of All-Trans Retinoic Acid in Type 2 Diabetes Plasma

Title: Quantification of All-Trans Retinoic Acid by Liquid Chromatography–Tandem Mass Spectrometry and Association with Lipid Profile in Patients with Type 2 Diabetes

Journal: Metabolites

Published: 2021

Background

Retinoic acids, particularly all-trans retinoic acid (atRA), are vital vitamin A metabolites with significant roles in human physiology, including immune regulation, cell growth, and differentiation. While atRA is commonly used to treat acne and acute promyelocytic leukemia, its metabolism may also be linked to diseases like Type 2 diabetes. Prior research indicates that atRA can influence diabetic complications by inhibiting NF-κB translocation in kidney cells. However, previous methods to measure atRA in human plasma had limited sensitivity and specificity. This study aimed to develop a highly sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to quantify atRA in plasma samples, assess its stability, and investigate its association with lipid profiles in patients with poorly controlled Type 2 diabetes.

Materials & Methods

Chemicals and Reagents

High-purity solvents (acetonitrile, hexane, methanol, hydrochloric acid, and formic acid) were used. All-trans retinoic acid (atRA) and its deuterated standard (atRA-d5) were obtained from Biomol. Artificial plasma (Biseko®) served as the matrix for method validation. All solutions were stored in amber vials to prevent light degradation.

Calibration Standards

Stock solutions of atRA (500 µg/mL) and atRA-d5 (100 µg/mL) were prepared in DMSO and stored at −80°C. Working solutions were diluted in methanol. Calibration standards (50–3200 pg/mL) were prepared with a constant 500 pg/mL of atRA-d5.

Sample Collection and Extraction

Plasma samples from 20 healthy controls and 39 Type 2 diabetes patients were collected under fasting conditions. After centrifugation, the plasma was stored at −80°C. For extraction, 200 µL plasma was mixed with 100 pg atRA-d5, acidified with HCl, and precipitated with methanol. A liquid–liquid extraction using hexane and ethyl acetate followed. The organic phase was evaporated and reconstituted for analysis.

Chromatography and Mass Spectrometry

Analysis was performed using a Waters® Acquity UPLC with a BEH C18 column (1.7 µm, 2.1 × 50 mm). Gradient elution with formic acid in water and methanol was applied at a flow rate of 0.2 mL/min. Detection was carried out on a XEVO TQ-S mass spectrometer using positive ESI in MRM mode.

Method Validation

The method was validated for linearity, accuracy, precision, recovery, LLOD, and LLOQ. Stability was tested under various temperatures and UV light exposure. Matrix effects were assessed using atRA-d5. Calibration curves showed linearity with R² > 0.99.

Clinical Chemistry and Statistical Analysis

Plasma cholesterol, triglycerides, glucose, and HbA1c levels were analyzed using automated systems. Statistical analysis was performed using GraphPad Prism 7 with unpaired t-tests and Pearson's correlation for associations with lipid parameters.

Results

Fragmentation and Mass Transitions:

atRA was detected using positive ionization mode in mass spectrometry, showing higher sensitivity compared to negative ionization mode.
The primary fragments identified were 283.1 m/z (qualifier) and 122.9 m/z (quantifier) with stable and reproducible fragmentation patterns.

Chromatography:

Efficient separation of atRA was achieved using a reverse-phase C18 column with a water-methanol gradient.
Formic acid enhanced ionization, providing sharp peaks and low noise.
A carry-over effect was managed by running blank samples every 50 injections.

Analytical Specificity:

No interfering compounds were detected using artificial human plasma, confirming the assay's specificity.
Fragmentation patterns of atRA and its deuterated internal standard (atRA-d5) remained stable with high reproducibility.

Linearity and Limits of Detection/Quantification:

Calibration showed excellent linearity (R² > 0.99) within the range of 50–3200 pg/mL.
Lower limits of detection (LLOD) and quantification (LLOQ) were 20 pg/mL and 50 pg/mL, respectively.

Recovery, Precision, and Accuracy:

Extraction recovery was 89.7 ± 9.2%.
Intra- and interday precision showed coefficients of variation (CV) of 9.3% and 14.0%, respectively.
Accuracy was 96.5% (intraday) and 101.2% (interday).

Stability:

atRA levels decreased significantly (~51%) when stored at 4°C for one week.
Multiple freeze-thaw cycles caused minor degradation (~14%).
Exposure to UV light for 3 hours led to ~40% reduction in atRA concentration.

Graph showing calibration range, LLOQ, LLOD, and R². Below, two chromatograms of all-trans retinoic acid (atRA) at LLOQ (top) and LLOD (bottom). Linearity and limitations. (A) Calibration range (50-3200 pg·mL−1), lower limit of quantification (LLOQ) (6.4/1), lower limit of detection (LLOD) (3.2/1), and R². (B) Total ion chromatogram of all-trans retinoic acid (atRA) for LLOQ (top) and LLOD (bottom) (Morgenstern, Jakob, et al., 2021).

(A) MRM chromatogram of atRA. (B) MRM chromatogram of atRA-d5. (C) Total ion chromatogram of atRA in a control subject. (D) Total ion chromatogram of atRA-d5 in the same control subject. Multiple reaction monitoring (MRM) chromatograms and total ion chromatograms of atRA/atRA-d5. (A) Extracted MRM spectrum (quantifier/qualifier) of atRA. (B) Extracted MRM spectrum (quantifier/qualifier) of atRA-d5. (C) Total ion chromatogram of atRA in a control subject (1.02 ng·mL−1). (D) Total ion chromatogram of atRA-d5 in the same control subject as in (C).

Reference

Morgenstern, Jakob, et al. "Quantification of all-trans retinoic acid by liquid chromatography–tandem mass spectrometry and association with lipid profile in patients with type 2 diabetes." Metabolites 11.1 (2021): 60. https://doi.org/10.3390/metabo11010060

* For Research Use Only. Not for use in diagnostic procedures.

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