Analysis of the Nonionic Contrast Agent Iohexol by UHPLC-CMS Method

Iohexol is a widely used nonionic contrast agent employed to enhance the contrast in X-ray imaging. Its low osmolarity allows for rapid renal clearance, preventing reabsorption and further metabolism—thus making iohexol safer to use than other contrast agents. In many clinical imaging applications, contrast agents improve both the contrast and spatial resolution of scans; however, due to their toxicity and potential side effects, analysis of the known concentrations and purity of these agents is crucial for their safe use and accurate diagnosis. This article presents a simple and accurate HPLC-CMS method for the analysis of iohexol.

Experimental method

Experimental reagents

All solvents used in the application are HPLC-grade. Two iodoheptanol standards are employed: one is a certified reference material with a purity of 99.99%, and the other has a purity of ≥95%.

Experimental instruments

All experiments were conducted on the AIS expression® CMS, coupled with the AVANT™ UHPLC system, with parameters as shown in Table 1.

HPLC/UV/MS Analysis of Iodohexol (100 µg/mL)

At room temperature, iohexol undergoes isomerization, and two peaks can be detected in HPLC/UV/MS analysis.

These two peaks (Figure 1A) arise because a large number of iodine atoms are attached to the central benzene ring of iohexol, thereby hindering the rotation of the N-acetyl group on the amide moiety of the benzene ring. These two compounds are essentially “rotamers” that slowly interconvert in aqueous solution at room temperature.

Figure 1: (A) HPLC chromatogram of iohexol (254 nm); (B) Extracted ion chromatogram of protonated iohexol (m/z 821.8); (C) Average mass spectrum at the retention time of 4.06 minutes.

The two peaks were confirmed by mass spectrometry (MS) analysis and exhibited the same mass-to-charge ratio (m/z) of 821.9 (see Figures 1B and 1C). Moreover, no visible differences were observed in their source-induced collision-induced dissociation (CID) mass spectra (see Figures 2A and 2B). In X-ray analysis, both rotamers were found to be present.

Figure 2: (A) Average in-source CID mass spectrum of the peak at a retention time of 3.61 minutes, (B) Average in-source CID mass spectrum of the peak at a retention time of 4.06 minutes.

Purity analysis by HPLC/UV method

By comparing the HPLC responses of iodixanol samples with those of certified iodixanol reference standards at the same concentration, the ratio of peak areas between the sample and the certified standard can be used for rapid analysis of concentration and purity.

The formula for calculating the iodine-hexanol concentration ratio is as follows:

The HPLC chromatograms of the iohexol sample and the certified reference standard are shown in Figures 3A and 3B, respectively.

The average of the sum of the peak areas for the iohexol sample was 714 (Figure 3A), while the average for the certified iohexol reference standard was 740 (Figure 3B). Based on the concentration ratio calculation, the concentration of iohexol in the sample was determined to be 0.0964 mg/mL, with a purity of 96.4%, meeting the specified product purity requirement of ≥95%.

Figure 3: (A) HPLC chromatogram of iohexol sample (0.1 mg/mL) at 254 nm; (B) HPLC chromatogram of iohexol reference standard (0.1 mg/mL) at 254 nm.

HPLC/UV Quantitative Analysis

To more accurately determine the purity of the iohexol sample, calibration curves for the certified reference material of iohexol were established by performing three replicate measurements at five different dilution levels ranging from 25 to 500 μg/mL. The resulting linear calibration curve had a correlation coefficient of 0.9999 (Figure 4), demonstrating excellent linearity.

Figure 4: Calibration Curve for Iohexol by HPLC Chromatography (254 nm)

Using the iohexol calibration curve method, the purity of the iohexol sample was determined to be 97.5%, which exceeds the minimum specified purity of 95%. The measured value obtained via the ratio calculation differed from this result by only 1.1%. Therefore, for certified reference materials, both the calibration curve method and the direct UV response ratio analysis method can be used for determining the purity of organic chemicals that exhibit UV absorption.

Experimental conclusion

The Advion Interchim Scientific® AVANT™ (U)HPLC system provides an accurate analytical method for determining the purity of the contrast agent iohexol. The UHPLC-CMS hyphenated technique not only allows for the identification of the target compound but also enables rapid detection of impurities.

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