Analysis of Vitamin A Stable Isotope Tracers using LC-MS/MS

Summary

Here, we present a protocol to simultaneously quantify both isotopically labeled and native retinoids in human plasma, using LC-MS/MS, with detection limits of 6 fmol on-column. The method incorporates a simple sample extraction procedure and a short analytical run time, enabling a high throughput of samples.

Abstract

The retinol isotope dilution (RID) method estimates the total body stores (TBS) of vitamin A in humans through stable isotope dilution with the body's vitamin A pool. For this, it is critical to accurately and efficiently determine the plasma isotopic ratio of labeled to non-labeled retinol through mass spectrometry. To avoid extensive and time-consuming extraction and/or purification procedures, such as preparative HPLC and derivatization, LC-MS/MS is employed to conduct fast, sensitive, and simultaneous analysis of labeled and non-labeled retinoids. The method utilizes two levels of detection: (i) an initial mass/charge (m/z) separation of parent (precursor) ions, followed by (ii) detection of fragmented daughter (product) ions. This results in high sensitivity, with retinol detection limits as low as 6 fmol on-column. Despite the advantages of tandem mass spectrometry, a liquid chromatographic separation is required to separate retinol from retinyl esters since the terminal functional groups are lost during ionization, resulting in similar parent (m/z of 269) and daughter ion fragmentation patterns. The article describes the isolation of endogenous and labeled (¹³C or ²H) retinol from plasma by solvent extraction, followed by quantification by LC-MS/MS under atmospheric pressure chemical ionization (APCI) in positive ion mode.

Introduction

Vitamin A is a fat-soluble nutrient essential for human health¹. Vitamin A deficiency increases the susceptibility to severe infectious morbidity and mortality and is estimated to have a global prevalence of 29%, with higher levels in sub-Saharan Africa (48%) and South Asia (44%)². Vitamin A supplementation is an effective strategy to reduce excess child mortality, particularly in high-risk areas such as sub-Saharan Africa³. However, the introduction of semi-annual vitamin A supplementation together with large-scale vitamin A fortification of staple foods and distribution of micronutrient powder, which are often managed through different public and private entities, has the potential to expose individuals to excessive vitamin A intake^{4,5,6,7,8,9,10}. Thus, biomarkers of vitamin A should ideally be able to cover the whole spectrum of vitamin A status, from deficiency to toxicity. The retinol isotope dilution (RID) method is currently the most accurate indirect method for assessing total vitamin A body stores in humans^11,12,13 and has been validated against hepatic vitamin A concentrations, considered the best biochemical marker of vitamin A status^14,15. The method is based on the principle that an oral tracer dose of stable isotope-labeled vitamin A mixes with unlabeled vitamin A (the tracee) in exchangeable body pools. The measurement of plasma retinol specific activity (i.e., the ratio of labeled retinol tracer to total retinol in plasma (tracer/tracee in plasma)) at a specified time post-dosing allows for the estimation of vitamin A status using an RID equation^11,12,13.

To determine accurate and precise concentrations of both retinol tracer and tracee in plasma, various methods have been applied to quantify stable isotope-labeled vitamin A, including gas chromatography-mass spectrometry (GC-MS), isotope ratio mass spectrometry (IRMS), and liquid chromatography tandem mass spectrometry (LC-MS/MS)^{16,17,18,19,20,21,22,23,24,25,26}. Importantly, LC-MS/MS combines high sensitivity, specificity, and operational simplicity with the ability to directly measure both native and labeled retinol in complex matrices. Thus, to avoid time-consuming sample preparation, such as preparative high-performance liquid chromatography (HPLC) for IRMS or derivatization for GC-MS, LC-MS/MS analysis is an ideal choice since this method involves a simple extraction procedure, very low detection limits for stable isotopes, and short runtimes^7,18,19. Here, we present an updated LC-MS/MS method with shorter run time, simple workflows combined with high sensitivity and selectivity as a highly suitable method for high-throughput analysis of samples from large-scale human intervention and observational studies, where the determination of total body stores of vitamin A is required.

Protocol

The study was approved by the Research Ethics Board of the University of the Philippines, Manila (IRB ID: UPMREB 2016-282-01), the Institutional Review Board at UC, Davis (IRB ID: 903681-2), and was registered at ClinicalTrials.gov (ID: NCT03030339). The study was approved by the Ethical Review Committee of the Ghana Health Services (GHS-ERC 012/07/20), and the trial was registered at https://clinicaltrials.gov as NCT04632771. 

1. Preparation of internal standards

1. Internal standard stock solution: Prepare the internal stock solution at a concentration of 1 mg/mL retinyl acetate in ethanol containing 0.1% butylated hydroxytoluene (BHT; w/v). Divide the stock solutions into aliquots and store them at -80 °C for up to 3 months.
   NOTE: For the internal standard, it is feasible to use either non-labeled retinyl-acetate or labeled retinyl acetate. If a non-labeled retinyl-acetate standard is used, it is vital that baseline resolution of the retinyl acetate and retinol peaks is obtained (see Figure 1).
2. Internal standard working solution: Prepare an internal standard working solution by diluting the stock standard (1 mg/mL) to 1 µg/mL. First, dilute 60 µL of the stock standard with 300 µL of ethanol, followed by diluting 60 µL of this solution in 10,000 µL of ethanol. For each sample, 20 µL of the working internal standard solution is added, containing 20 ng of retinyl-acetate.
   NOTE: Internal standard working solutions can be stored at -20 °C for up to 1 week.

2. Preparation of calibration standards

1. Calibration stock standard: Prepare the calibration stock standard solutions at a concentration of 1 mg/mL retinol in ethanol containing 0.1% BHT (w/v) for both the tracee and the tracer. Aliquot stock solutions and store at -80 °C for up to 3 months. Only use each aliquot once to avoid freeze-thaw cycles.
2. Calibration standard working solution: Take out 100 µL of the calibration stock standard solution and dilute with 10,000 µL of ethanol in a volumetric flask. Determine the concentration of the diluted calibration stock solution at 325 nm in a spectrophotometer using a quartz cuvette (see Table 1 for details).
3. Take an aliquot of the diluted calibration stock standard and adjust the concentration to 8 µM/L for the tracee using 50% ethanol and 50% acetonitrile/dH₂O (70:30 (v/v) and to 2 µM/L for the tracer using 100% ethanol.
4. Prepare duplicate calibration standards at seven different concentrations for the tracee using 50% ethanol and 50% acetonitrile/dH₂O (70:30 (v/v). If 200 µL of plasma is extracted, the tracee calibration standards should range from 6-0.03 µM/L.
   NOTE: For accurate quantification, isotopically labeled retinol and retinyl esters in plasma are quantified using duplicated 7-point calibration curves²⁷. The highest point on the calibration curve (double the highest expected concentration of the tracee and tracer in plasma) considers the concentration factor of the reconstituted extract from the original plasma volume. Refer to Supplementary file 1 for the preparation of the duplicate calibration standards.

3. Extraction of retinoids from plasma

1. Remove plasma samples from -80 °C freezer and defrost at room temperature (approximately 30 min).
2. Once defrosted, vortex each sample and spin for a few seconds to make sure no plasma is left in the lid. Thereafter, place samples on ice.
3. Turn on a refrigerated centrifuge and allow it to cool to 4 °C. Label 15 mL glass tubes with screw caps (subject ID and time point) and pipette 200 µL of deionized water (dH₂O) into each sample tube.
4. From the internal standard working solution, pipette 20 µL of the internal standard mixture into the glass tube.
5. Using a pipette, dispense 200 µL of plasma into the respective tube containing the internal standard and dH₂O.
6. Pipette 400 µL of ethanol into each sample tube and swirl the tube to mix the ethanol with the plasma. Do not vortex to avoid protein residues sticking to the glass wall.
7. For the first hexane extraction, add 2 mL of hexane to each tube and tightly fasten the tube lids.
8. Place the tubes in an orbital shaker and shake at 15,000 rpm for 30 min. Transfer tubes to a refrigerated centrifuge and spin at 1,800 x g for 10 min at 4 °C.
9. Using glass Pasteur pipettes, transfer the supernatant to a clean set of labeled glass test tubes. Take care not to transfer protein precipitate or water.
10. Produce a second hexane extraction by adding a further 2 mL of hexane to the remaining residue, tightly fasten the tube lids, and place the tubes in the orbital shaker and shake at 15,000 rpm for 15 min.
11. Transfer tubes to a refrigerated centrifuge and spin at 1,800 x g for 10 min at 4 °C. Using glass Pasteur pipettes, transfer the supernatant to the glass test tube containing the supernatant of the first hexane extraction (step 3.9) for each sample, respectively.
12. Evaporate the combined hexane extraction solvent (first and second extraction supernatant for each sample) under a gentle stream of N₂ in a darkened fume hood for approximately 30 min. Set the heating block at 35 °C.
13. Resuspend the dried residue in 50 µL of ethanol, vortex briefly, and add 50 µL of mobile phase (70:30 acetonitrile/dH₂O (v/v)) and vortex again.
    NOTE: Resuspension of the dried residue should be performed using 100 µL of ethanol only if the aim is to quantify retinyl-esters.
14. Transfer into labeled 0.5 mL microcentrifuge tubes. Centrifuge at 16,900 x g for 5 min.
15. Transfer 80 µL of the reconstituted residue to amber HPLC vials with 300 µL inserts. Immediately cap the vial to prevent solvent loss due to evaporation.
16. Transfer glass vials into a cryobox, label them with the study name, extraction date, and sample IDs, and store them in a -80 °C freezer until analysis.
    NOTE: Analysis of stored samples must be completed within 3-5 days. A plasma volume of 200 µL is normally sufficient to detect the labeled vitamin A tracer up to 90 days post-dose. However, the plasma volume can be adjusted to 400 µL if the ratios of plasma/water/ethanol are maintained at 1:1:2 by volume.

4. Preparation of internal standard control samples

1. Prepare the ethanol/mobile phase solution by pipetting 120 µL of ethanol into a microcentrifuge tube and adding 200 µL of (70:30) acetonitrile/dH₂O (v/v) and vortexing.
2. Into three separate 0.6 mL tubes, pipette 20 µL of the internal standard working solution.
3. Add 80 µL of the ethanol/mobile phase solution prepared and vortex. Transfer the three internal standard control samples to amber HPLC vials with 300 µL inserts and immediately cap the vials to prevent solvent loss due to evaporation.

5. Plasma house standards

1. To prepare the house standard samples, obtain sufficient plasma volume to allow analysis of a minimum of three samples per analytical run. The total required plasma volume is determined by the number of samples that need to be analyzed.
2. Aliquot house standard plasma into small cryovials at volumes needed for each analytical run and store at -80 °C.
   NOTE: It is critical that the house standard contains the isotope-labeled retinol tracer used in the unknown samples. Sufficient material can be obtained from pooling volunteer samples into one aliquot.
3. Extract at least three plasma house standard samples for every analytical run at the same volume as unknown plasma samples, as done in step 3.
4. Place the plasma house standards throughout the sample rack with at least 10 unknown plasma samples between them. Use plasma house standards to calculate intra- and inter-day variations.

6. External standard samples

1. Obtain certified external standard samples and store at -80 °C. A widely used certified external standard is the NIST Standard Reference Material SRM 968.
2. Extract at least four plasma external standard samples per analysis at the same volume as unknown plasma samples using step 3. Use external standard samples to verify the accuracy of the obtained results.

7. Manual compound optimization

1. Ensure the APCI Heated Nebulizer probe is installed in the MS source housing. Prepare the infusion solution for each stable isotope tracer at approximately 1 µg/mL.
   NOTE: The solution should be similar to the mobile phase and should contain some acid for protonation. A good solvent to use would be 1:1 acetonitrile/dH₂O with 0.1% formic acid (v/v).
2. Fill a 1 mL Luer-lock Hamilton syringe with the infusion solution, then connect the syringe to the source with PEEK tubing via the grounding union. Set the infusion flow rate to 10 µL/s.
3. Select Manual Tuning and make sure the instrument is in Tune and Calibrate Mode. Start the syringe pump and select Q1 Scan Type in positive mode.
4. Set a lower mass range of 50 Da, and an upper mass range approximately 100 Da above the molecular mass of the target compound.
5. Set cycles to 500, scan rate 200 Da/s. Click Start and observe the signal in the mass spectra window.
6. Wait for the signal to stabilize, then check that the expected m/z peaks are appearing. Consider the [M+H]⁺ and [M+2H]²⁺ peaks since the MS is in positive mode.
7. The target peak height should be around 1e6. The total ion current (TIC) should be 1e7-1e9. Continue infusing until the signal is stable, then click Stop. There should be a distinct peak with a high intensity that is formed from the compound in the infusion solution. The total ion current (TIC) is displayed, which shows the total signal for all ions combined.
   NOTE: the expected mass of ¹²C Retinol is 269 Da, so a range of 50-300 Da is appropriate to show [M+H]⁺ and [M+2H]²⁺ ions.

8. Product ion (MS2) scan / CE spectra check

1. Set the Start mass at a lower mass, e.g., 50 Da, and the Stop mass just above the Q1 mass (e.g., 269.1 for ¹²C Retinol). Set Cycles to 5 and change collision energy (CE) to 10. Check for the Q1 mass as the precursor ion. If there are no smaller fragments appearing, increase CE to 20.
2. As the CE increases, more product ions will be formed. Continue to increase CE until the precursor ion on the right of the spectra is completely gone. Increase the CE even further to see potential secondary fragmentation.

9. MRM scan

1. From the MS2 scan, select the three most abundant or most appropriate product ions to take forward for further optimization.
2. For each product ion, enter the Q1 (precursor) mass and the Q3 (product) masses found. Set Dwell Time to 200 ms. Name each fragment accordingly (e.g., 12C_Frag_93).
3. Select collision energy and identify the optimal CE value by using the ramp feature. Populate the CE column with the optimized CE value for each fragment.
4. Optimize Declustering Potential (DP) optimized after optimizing CE to minimize isobaric interferences. Optimize Collision Cell Exit Potential (CXP) for one transition at a time.

10. Source/gas optimization

1. These parameters cannot be automatically optimized. If you need to improve the signal intensity, perform a manual optimization process. Change one parameter at a time and compare if the signal increased, stayed the same, or decreased.

11. Liquid chromatography conditions

1. For chromatography, use a binary mobile phase gradient of 0.1% (v/v) formic acid in dH₂O (A) and 0.1% (v/v) formic acid in acetonitrile (B).
2. Depending on the LC system applied, use either a 100 mm x 2.1 mm (3 µm) ABZ PLUS column fitted with a 4 mm x 2 mm C18 cartridge (for HPLC), or a 100 mm x 2.1 mm (1.7 µm) Premier BEH Shield fitted with a RP18 FIT column (for ultra-high-pressure chromatography (UPLC)) for the analysis of retinol. Maintain columns at a temperature of either 40 °C or 50 °C, respectively.
3. Inject 10 µL of the plasma extract on-column. Elute analytes at a flow rate of 0.4 mL/min. For HPLC systems, the linear gradient is: 60% B to 95% B in 6.0 min; 100% B from 6.1 min to 13.0 min; 100% B to 60% B from 13.1 to 14.0 min; and 60% B from 14.0 to 17.0 min. For UPLC systems, the linear gradient depends on the research question. If only retinol is to be determined, the gradient is: 40% B to 60% B in 1.0 min; 60% B to 95% B from 1.0 min to 6.0 min; 100% B from 6.5 min to 7.5 min; 100% B to 40% B from 7.5 to 7.6 min; and 40% B from 7.6 to 8.5 min. If the aim is to also analyze retinyl-esters alongside retinol, the linear gradient is: 60% B to 95% B in 3.0 min; 100% B from 3.5 min to 6.5 min; 100% B to 60% B from 6.5 to 7.0 min; and 60% B from 7.0 to 8.5 min.

12. MS/MS conditions

1. Perform MS/MS analysis under atmospheric pressure chemical ionisation (APCI) in positive ion mode. APCI is chosen over electrospray ionization as it provides a higher signal intensity, a broader linear range, and offers lower background noise²⁶.
2. For respective mass transitions for [¹²C]retinol, [¹³C₁₀]retinol, and [²H₆]retinol and their respective esters/acetate with selected reaction monitoring (SRM) parameters for the API4000 and the QTRAP 5500 mass spectrometer, check Table 2, Table 3, and Figure 2.
   NOTE: Selected ion monitoring for retinol, retinyl esters, and retinyl acetate was the same fragment ion of m/z 269 for APCI quantitation, since this ion corresponds to the loss of water, fatty acid, or acetic acid from the protonated molecules. Since the LC-MS/MS is able to separate the retinol peaks from its esters, a single injection allows the determination of isotope enrichment in both the retinol and retinyl-ester fractions in one sample.

13. Quantification of retinol

1. Quantify [¹²C] retinol, [²H₆] retinol, and [¹³C₁₀] retinol sample concentrations against their respective calibration standards.
2. Determine extraction losses using the recovery of the internal standard concentrations for each sample against the internal standard control samples.
3. Perform confirmation of plasma retinol concentrations using the National Institute of Standards and Technology (NIST) standard reference material (SRM) 968.

Results

To illustrate the effectiveness of the analytical method to analyze stable isotope-labeled retinol across different chromatographic systems, we are presenting results from two different studies.

For using the API4000 with upfront HPLC, plasma samples from 111 Filipino children aged 12 to 18 months, who received a 400 µg (1.17 µmol) [¹³C₁₀]retinyl acetate oral dose on day 0, followed by a 6 mL blood sample collection at 4 days post-dose, were analyzed^7,28. Mean plasma total retinol concentrations were 1.1 µmol/L, and mean [¹³C₁₀] retinol was 0.007 µmol/L. Since the background noise level within the m/z 279 to 100 transition had an average peak height of ~50 cps (Figure 1), the level of detection (LOD) and quantitation (LOQ) for [¹³C₁₀] retinol were set at 150 cps and 500 cps, corresponding to 6 fmol and 19 fmol on column, respectively. Since good baseline resolution was obtained between the [¹²C] retinol and the applied internal standard of retinyl-acetate, there was no need to use a stable isotope-labeled retinyl-acetate.

A faster chromatographic separation can be achieved using UPLC in combination with the QTRAP 5500, with run times below 10 min. Depending on the research question, two different separation methods can be applied; (i) if the aim is to determine native and isotope labeled retinol applied with a non-labeled internal standard of retinyl-acetate, retention times between retinol (4.37 min) and retinyl-acetate (5.33 min) give good baseline separation (Figure 3); (ii) if the aim is to separate native and isotope labeled retinol alongside native and labeled retinyl-esters, good separation can be achieved within a 8.5 min run (Figure 4). However, due to the close retention times between retinol (2.10 min) and retinyl acetate (2.43 min), it is advisable to use isotopically labeled retinyl acetate ([²H₄] retinyl acetate) instead. In this example, serum samples were analyzed from 80 Ghanaian women of reproductive age who received a 2 mg retinol equivalent dose of [²H₆] retinyl acetate and [¹³C₁₀] retinyl acetate, followed by blood sample collection at 21 days post-dose. Mean serum total retinol concentrations were 1.89 µmol/L, with mean [²H₆] retinol at 0.027 µmol/L and mean [¹³C₁₀] retinol at 0.025 µmol/L.

The presented method achieved a low level of detection (LOD = 6 fmol; LOQ = 19 fmol on column), demonstrating high sensitivity and specificity for detecting stable isotope-labeled retinol with clear baseline separation between native retinol and internal standards. Furthermore, the method worked effectively with both HPLC and UPLC setups, despite differences in retention times. The use of isotopically labeled internal standards ([²H₄] retinyl acetate) ensured accurate quantification under faster UPLC conditions, if retinyl-esters need to be determined. Validation against NIST-certified reference standards confirmed accuracy, whilst intra-day and inter-day coefficients of variation (2.3% and 4.4%, respectively) demonstrated excellent precision and reproducibility. Successful analysis of plasma/serum samples from two distinct populations (Filipino children and Ghanaian women) with different dosing protocols shows robustness and practical utility in diverse nutritional studies.

Figure 1: LC-MS/MS chromatogram using the API4000 mass spectrometer. The chromatogram of serum retinol was obtained 4 days after oral ingestion of 0.4 mg of [¹³C₁₀] retinyl acetate. Selected reaction monitoring (SRM) of m/z 269 to 93 shows the separation of [¹²C] retinol (RT = 5.06 min) and [¹²C] retinyl acetate (RT = 5.83 min) as an internal standard (IS). The [¹³C₁₀] retinol signal at m/z 279 to 100 sufficiently exceeds both the limit of detection (LOD) and the limit of quantitation (LOQ). Chromatographic conditions: (A) binary mobile phase gradient of 0.1% (v/v) formic acid in dH2O and (B) 0.1% (v/v) formic acid in acetonitrile; flow rate of 0.4 mL/min with a linear gradient of: 60% B to 95% B in 6.0 min; 100% B from 6.1 min to 13.0 min; 100% B to 60% B from 13.1 to 14.0 min; and 60% B from 14.0 to 17.0 min; 100 mm x 2.1 mm (3 µm) ABZ PLUS column, fitted with a 4 mm x 2 mm C18 cartridge maintained at 40 °C. Please click here to view a larger version of this figure.

Figure 2: APCI-MS/MS product ion mass spectra. Flow-injection APCI-MS/MS product ion mass spectra of (A) m/z 269 [¹²C] retinol, (B) m/z 273 [²H₄] retinol, (C) m/z 275 [²H₆] retinol, and (D) m/z 279 [¹³C₁₀] retinol in positive ion mode. Please click here to view a larger version of this figure.

Figure 3: LC-MS/MS chromatogram using the QTRAP 5500 mass spectrometer. LC-MS/MS chromatogram of a typical extract from a plasma house standard sampled 21 d after oral ingestion of 1.0 mg of [²H₆] retinyl acetate and 90 d after oral ingestion of 1.0 mg of [¹³C₁₀] retinyl acetate. Selected reaction monitoring (SRM) of m/z 269→93 shows the [¹²C] retinol peak (RT = 4.37 min) and retinyl acetate (RT = 5.33 min) as internal standard (IS); the [²H₆] retinol (RT = 4.36 min) at m/z 275→96; and the [¹³C₁₀] retinol peak (RT = 4.38) at m/z 279→100. Chromatographic conditions: (A) binary mobile phase gradient of 0.1% (v/v) formic acid in dH₂O and (B) 0.1% (v/v) formic acid in acetonitrile; flow rate of 0.4 ml/min with a linear gradient of: 40% B to 60% B in 1.0 min; 60% B to 95% B from 1.0 min to 6.0 min; 100% B from 6.5 min to 7.5 min; 100% B to 40% B from 7.5 to 7.6 min; and 40% B from 7.6 to 8.5 min; 100 mm x 2.1 mm (1.7 µm) AcquityTM Premier BEH Shield RP18 VanGuard FIT column, maintained at 50°C. Please click here to view a larger version of this figure.

Figure 4: LC-MS/MS chromatogram of retinol and retinyl-esters using the QTRAP 5500 mass spectrometer. LC-MS/MS chromatogram of a typical extract from a plasma house standard sampled 6 h after oral ingestion of 1.0 mg of [²H₆] retinyl acetate and 90 d after oral ingestion of 1.0 mg of [¹³C₁₀] retinyl acetate. Selected reaction monitoring (SRM) of m/z 269→93 shows the [¹²C]retinol peak (RT = 2.10 min); the [²H₄]retinyl acetate at m/z 273→94 (RT = 2.43 min) as internal standard (IS); the separation of [²H₆]retinol (RT = 2.09 min) and [²H₆]retinyl palmitate (RT = 5.11 min) peaks at m/z 275→96; and the [¹³C₁₀]retinol peak (RT = 2.10) at m/z 279→100. Chromatographic conditions: (A) binary mobile phase gradient of 0.1% (v/v) formic acid in dH₂O and (B) 0.1% (v/v) formic acid in acetonitrile; flow rate of 0.4 ml/min with a linear gradient of: 60% B to 95% B in 3.0 min; 100% B from 3.5 min to 6.5 min; 100% B to 60% B from 6.5 to 7.0 min; and 60% B from 7.0 to 8.5 min; 100 mm x 2.1 mm (1.7 µm) AcquityTM Premier BEH Shield RP18 VanGuard FIT column, maintained at 50 °C. Please click here to view a larger version of this figure.

Parameters
Molar absorptivity (ɛ) of retinol in ethanol:	52,770 M-1 cm-1
Dilution factor:	100 (100µL of stock calibration standard in 10mL of ethanol)
Measured average absorbance:	0.575
Calculation
Concentration (mol) of [12C]retinol in ethanol:	Concentration (mol) = Absorption / ɛ x Length of cuvette
	= 0.575 / 52770 M-1 cm-1 x 1 cm = 1.09 x 10-5 M
Correcting for dilution:	1.09 x 10-5 M x 100 = 1.09 x 10-3 M or 1.09 mM

Table 1: Determination of the calibration standard retinol stock concentration.

Analyte	MRM transitions (m/z)	Declustering potential (V)	Entrance potential (V)	Collision energy (eV)	Collision exit potential (V)
[12C]-retinol/esters	269→93	51	10	27	6
[2H4]retinyl acetate	273→94	51	10	27	6
[2H6]retinol/esters	275→96	51	10	27	6
[13C10]-retinol/esters	279→100	41	10	27	6

Table 2: Selected reaction monitoring (SRM) parameters. Parameters for [¹²C] retinol, [¹³C₁₀] retinol, and [²H₆] retinol and their respective esters using the API4000

Analyte	MRM transitions (m/z)	Declustering potential (V)	Entrance potential (V)	Collision energy (eV)	Collision exit potential (V)
[12C]-retinol/esters	269→93	94	10	33	8
[2H4]retinyl acetate	273→94	54	10	20	11
[2H6]retinol/esters	275→96	69	10	25	3
[13C10]-retinol/esters	279→100	102	10	35	9

Table 3: Selected reaction monitoring (SRM) parameters. Parameters for [¹²C] retinol, [¹³C₁₀] retinol, and [²H₆] retinol and their respective esters using the QTRAP 5500

Supplementary File 1: Preparation of calibration standards at seven different concentrations for the tracer Please click here to download this File.

Discussion

The presented LC-MS/MS method offers extremely high sensitivity, with detection limits as low as 6 fmol/L, thereby enabling investigators to use as little as 100 - 200 µL of plasma¹⁹. This targeted tandem MS method is particularly powerful for confirmation of retinoid isotopologue identity when both quantifier and qualifier product ions are utilized¹⁸. However, since isotope-labeled retinoids are present at substantially lower concentrations than physiological non-labeled retinoids, plasma samples containing labeled retinol and retinyl esters may require an additional concentration step. This necessity depends on factors such as the timing of the post-dose application blood sample, the concentration of oral tracer, and the sensitivity of the LC-MS/MS system. Historically, higher doses of applications were used to overcome the lower sensitivity of earlier mass spectrometers, but recent advancements have facilitated the use of lower doses¹³. In the presented examples, a higher oral dose increases the level of stable isotope-labeled retinol even at later time points, i.e., at 21 days versus 4 days.

Since the terminal functional groups of retinyl esters are lost during ionization, the same parent m/z of 269 and similar daughter ion fragmentation patterns are observed for both retinol and retinyl esters. Therefore, chromatographic separation is crucial to determine their separate concentrations¹⁸. Furthermore, due to the high natural abundance of ¹³C stable isotopes in endogenous retinol isotopologues, it is recommended to incorporate more than three ¹³C atoms into the retinol tracer to avoid the need to correct for baseline natural ¹³C enrichment of retinol in human plasma²⁹.

Plasma samples require processing prior to isotope analysis in two steps. First, plasma samples need to be deproteinized, followed by the extraction of retinol and retinyl esters using a non-polar solvent, which creates a biphasic system^18,19,20. Although a clear phase separation is obtained using the above protocol, it is critical that the interphase in the biphasic system is not disturbed or collected, as this will increase the drying time and will reduce the accuracy of the analysis. Alternatively, our group has also explored the application of a single-phase extraction procedure for retinoids²⁰. For this, 200 µL plasma samples are pipetted into a 2 mL plastic micro-centrifuge tube, and retinoids are extracted using a combination of ethanol and ethyl acetate. Applying this extraction protocol allows the analysis of retinol and more polar retinoids, such as retinoic acid isomers, with equivalent recovery compared to the biphasic extraction, and is also comparable to the one-step acetonitrile extraction method of Kane and Napoli³⁰.

Given that retinoids are susceptible to oxidation and isomerization when exposed to UV light and heat, it is crucial to perform the analysis as quickly as possible, and general precautions should be taken during the collection, handling, and storage of plasma³⁰. Traditional methods for analyzing stable isotope-labeled retinoids employ time-consuming isolation and/or derivatizations. GC-MS analysis requires isolating the retinol fraction from human serum using high-performance liquid chromatography (HPLC), followed by the derivatization of retinol²². Although derivatization is not needed for GC-C-IRMS, prior separation of retinol using two separate HPLC purification steps is recommended^17,25,31, and the determination of baseline natural ¹³C enrichment of retinol in human plasma in the sample population is also required^17,32. In contrast, the presented LC-MS/MS method has several advantages. It avoids extensive and time-consuming extraction and/or purification procedures, enabling fast, sensitive, and simultaneous analysis of retinyl esters and free retinol^16,18,26,32. Furthermore, the method is effective because it provides high accuracy with low variability and is adaptable to different LC-MS/MS systems.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Solvents
Absolute ethanol HPLC grade	Fisher	10428671
Acetonitrile HPLC grade	Fisher	10660131
Hexane 95% n-Hexane for HPLC CertiFied HPLC	Fisher	H/0406/17
Isopropanol HPLC grade	VWR	20880.32
Isopropanol HPLC grade	VWR	20880.32
Methanol 99% 2.5 L	Thermo Scientific	11367996
Standards/isotopes
all-trans-retinol	BioXtra	95144
Retinyl acetate	Sigma	PHR1236
Stable isotope labelled retinol:	ReseaChem/Buchem BV
Stable isotope labelled retinyl acetate:	ReseaChem/Buchem BV	N/A	A range of stable isotopes can be used for the RID method. A list of different commercially available isotopes are given in Table 1.
Vitamin A  (19,19,19,20,20,20-D6)
Vitamin A  (8,9,10,11,12,13,14,15,19,20-13C10)
Vitamin A acetate (10,19,19,19-D4)
Vitamin A acetate (19,19,19,20,20,20-D6)
Vitamin A acetate (8,9,10,11,12,13,14,15,19,20-13C10)
Equipment
5ml Adjustable solvent dispenser	Sigma-Aldrich	Z627607-1EA
ABI 4000 LC-MS/MS.	AB Sciex
Analytical weighing scales	Fisher	13592970
Capillary pistons (pipette tips) CP100 10x96 TIPACK	Gilson	FD148314
Capillary pistons CP250ST 6x96	Gilson	F148714
Fume cabinet
Microman E M100E Pipette (positive displacement)	Gilson	FD10004
Microman E M250E Pipette (positive displacement)	Gilson	FD10005
Pipetman P100, 10-100ul, metal ejector	Gilson	F114057M
Pipetman P1000, 100-1000ul, metal ejector	Gilson	F114059M
Pipetman P20, 2-20ul, metal ejector	Gilson	F144056M
QTRAP 5500 LC-MS/MS	AB Sciex
Refrigerated Centrifuge 5810 R	Eppendorf
Sonicating water bath	VWR	USC100T
Spectrophotometer	Thermo Fisher Scientific	A51119600C
Techne sample concentrator and heat block.	VWR
Vibrax VXR Basic Shaker	IKA	0002819002
Vortex mixer; MS3 basic IKA	Fisher	MPR-555-050L
VX2 test tube attachment - werke typ VX2	IKA	0000568900
Consumables
1.5ml microcentrifuge tubes	Fisher	FB74323
100 mm x 2.1 mm (1.7 μm) AcquityTM Premier BEH Shield RP18 VanGuard FIT column	Waters	186009498
100 mm x 2.1 mm (3 µm) Supelcosil ABZ PLUS column	Supelco	57917
1000ul pipette tips	Starlab	S1122-1830
100ml volumetric flasks	Fisher	11323644	Pyrex Borosilicate Glass Class A Certified Volumetric Flask
200ul pipette tips	Starlab	S1120-8810
4 mm x 2 mm SecurityGuard C18 cartridge	Phenomenex	AJ0-4286
50ml volumetric flasks	Fisher	11313644	Pyrex Borosilicate Glass Class A Certified Volumetric Flask
9mm amber glass vials with inserts	Fisher	11583680
9mm vial screw thread caps	Fisher	13206419
Cardboard cryobox	Starlab	A9623-8181
Hellma cuvette UV quartz	SLS	CEL1600
Nitrogen gas (highest purity and oxygen-free)	BOC
Pasteur Pipette	Fisher	11546963
Pyrex culture tubes 100x16mm	Corning	CLS9944916
Screw caps PTFE liner 16mm	Corning	CLS999815
Vial glass amber 2ml PTFE caps	Fisher	11573542
Vial screw cap 9mm silicone white/PTFE red	Thermo	13206419