A sensitive and specific method for measurement of multiple retinoids in human serum with UHPLC-MS/MS

Abstract

Retinol (vitamin A) circulates at 1-4 μM concentration and is easily measured in serum. However, retinol is biologically inactive. Its metabolite, retinoic acid (RA), is believed to be responsible for biological effects of vitamin A, and hence the measurement of retinol concentrations is of limited value. A UHPLC-MS/MS method using isotope-labeled internal standards was developed and validated for quantitative analysis of endogenous RA isomers and metabolites. The method was used to measure retinoids in serum samples from 20 healthy men. In the fed state, the measured concentrations were 3.1 ± 0.2 nM for atRA, 0.1 ± 0.02 nM for 9-cisRA, 5.3 ± 1.3 nM for 13-cisRA, 0.4 ± 0.4 nM for 9,13-dicisRA, and 17.2 ± 6.8 nM for 4oxo-13-cisRA. The concentrations of the retinoids were not significantly different when measured after an overnight fast (3.0 ± 0.1 nM for atRA, 0.09 ± 0.01 nM for 9-cisRA, 3.9 ± 0.2 nM for 13-cisRA, 0.3 ± 0.1 nM for 9,13-dicisRA, and 11.9 ± 1.6 nM for 4oxo-13-cisRA). 11-cisRA and 4OH-RA were not detected in human serum. The high sensitivity of the MS/MS method combined with the UHPLC separation power allowed detection of endogenous 9-cisRA and 4oxo-atRA for the first time in human serum.

Fig. 1.

Representative MS/MS spectra of RA, 4oxo-RA, and 4OH-RA standards and of RA and 4oxo-RA detected in serum samples. The MS/MS spectra of RA isomers and metabolites as clean standards and from serum samples were collected as described in Materials and Methods. The MS/MS spectra representative of all detected isomers are shown for atRA (A), 4oxo-13-cisRA (C), and 4OH-atRA (E) standards. Similar MS/MS spectra were generated for each quantified retinoid in a sample of pooled human serum, and representative MS/MS spectra for atRA (B) and for 4oxo-13-cisRA (D) are shown.

Fig. 2.

Separation of retinoic acid and 4oxo-RA isomers and detection of the retinoids and labeled internal standards in serum. Separation of atRA, 9,13-dicisRA, 9-cisRA, and 13-cisRA standards (A) and detection of the same isomers in serum (C). (B) Separation of 4oxo-atRA and 4oxo-13-cisRA and (D) detection of these compounds in serum. The dotted line in panel (D) also shows the lack of detection of 4OH-RA in extracted serum. All serum samples were prepared by liquid-liquid extraction and analyzed as described in Materials and Methods. (E) Detection of 13-cisRA-d₅ internal standard (at 13.2 min) extracted from serum together with the structure of the labeled compound. The peak at 13.9 min is not a retinoid but a matrix peak. (F) Detection of 4oxo-13-cisRA-d₃ internal standard in serum together with the structure of the internal standard. Panels E and F also show the lack of isomerization of the internal standards in serum. The insets on each panel specify the MS/MS transition used for the specific analyte.

Fig. 3.

Chromatograms of the detected retinoids as standards at determined LLOD. The on-column LLOD and LLOQ values for each compound are shown in the inset table. All retinoids were measured using the MS/MS transitions summarized in Table 1, and the corresponding SRM channels (m/z 301 > 205 for RA isomers, m/z 315 > 159 for 4oxo-RA isomers, and m/z 299 > 157 for 4OH-RA isomers) are shown.

Fig. 4.

Evaluation of the presence of 11-cisRA in serum. (A) Light-protected serum sample without the addition of 11-cisRA and palladium(II)nitrate extracted as described in Materials and Methods. (C) Chromatogram of an aliquot of the same extracted sample spiked with 11-cisRA. Fractions of both samples were treated with palladium(II)nitrate. (B and D) Chromatograms of the serum samples after the reaction. No decrease in 9,13-dicisRA peak between panel A and B is detected, and no increase in atRA peak suggesting that 11-cisRA was not present in serum. In the control reaction, the spiked 11-cisRA was quantitatively converted to atRA (panel C vs. D).

Fig. 5.

Detection of 4OH-RA isomers in human serum using an ACN protein precipitation and centrifugation for sample preparation. Blank serum (A), 5 nM 4OH-atRA and 4OH-9-cisRA spiked into blank serum (B), and a representative human serum sample (C) are shown. The peak at 5.75 min in panel C is likely 4OH-atRA, but the peak area is below LLOQ for this compound.

Fig. 6.

Box and whiskers plots for endogenous retinoid concentrations in serum. The box represents the 25th and 75th percentiles of each group. The whiskers are determined based on 10th and 90th percentiles, and outliers are calculated as 1.5 times the mean of the sample set. All of the retinoid concentrations, with the exception of 4oxo-13-cisRA, are plotted against the left y axis. The 4oxo-13-cisRA concentrations are plotted against the right hand y axis.