Quantification of Polyethylene Glycol 400 Excreted in the Urine by MALDI-TOF Mass Spectrometry

Abstract

Polyethylene glycol 400 (PEG 400) was used as a permeability probe to examine the gastrointestinal tract which can be involved in the pathogenesis of some inflammatory and autoimmune diseases. A novel methodology was developed and validated for the quantitation of PEG 400 excreted in human urine after oral administration using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The excretion ratios were determined for the most intense ions corresponding to nine PEG 400 oligomers. The relative error of accuracy was between -6.0% and 8.5%, and the relative standard deviation (RSD) of the precision was below 15%. Our method was successfully applied in a large-scale experimental study involving nearly two hundred volunteers. Due to the large number of measurements, detailed and reliable statistical analysis was performed. No significant difference was found between the male and female group of volunteers at 0.05 significance level, except the two largest PEG oligomers. However, the average excretion ratios of the male volunteers are greater than that of the women for all the nine PEG oligomers, suggesting a difference in the intestinal permeability between men and women.

Keywords: MALDI-TOF; intestinal permeability; polyethylene glycol; quantification.

Figure 1

MALDI-TOF-MS spectrum of PEG 400 with DHB matrix and Na⁺ ionization agent.

Figure 2

MALDI-TOF mass spectrum of a pre-dose human urine sample.

Figure 3

MALDI-TOF spectrum of a pre-dose human urine sample spiked with PEG 400 at 0.900 mg/mL and 50 µL of mPEG. The peaks marked by red symbols denote the mPEG oligomer series.

Figure 4

MALDI camera images of the pre-dose human urine samples spiked with PEG 400 deposited on the MALDI target plate using various matrices: (a) DHB; (b) DCTB; (c) Dithranol; (d) THAP (magnification: 50).

Figure 5

MALDI-TOF mass spectrum of a urine sample from a volunteer following administration of PEG 400 spiked with 50 µL of mPEG as internal standard. The peaks marked by red symbols denote the mPEG oligomer series.

Figure 6

Calibration curve for PEG₁₁. The PEG₁₁/IS ion intensity ratio is plotted against the PEG₁₁ concentration. Error bars represent the standard deviation.

Figure 7

(a) MALDI-TOF mass spectrum of a urine sample from a volunteer following administration of PEG 400 spiked with 50 µL of mPEG as internal standard; (b) Excretion of the PEG_6-14 oligomers calculated by our method based on the spectrum shown to the left; (c,d) Extreme excretion examples.

Figure 8

The average excretion ratios of the PEG_6–14 oligomers based on a sample of about 200 volunteers. The error bars represent the standard deviation of the subjects and the standard error of the mean in the main plot and in the inset, respectively. The red and blue regression curves belong to the average excretion ratios of the female and male volunteers, respectively.