LC-MS/MS analysis of phosphatidylethanol in dried blood spots versus conventional blood specimens

Abstract

Phosphatidylethanol (PEth), which is formed extrahepatically by the action of phospholipase D on phosphatidylcholine in the presence of ethanol, has been suggested as a promising marker of alcohol misuse. Analysis of dried blood spots (DBS) is particularly advantageous for the determination of delicate analytes such as PEth. Therefore, measurement of PEth species (18:1/18:1, 16:0/18:1) in DBS versus whole blood was performed to ascertain whether respective results are directly comparable. Samples were obtained from subjects (n = 40) undergoing alcohol detoxification treatment. Analysis involved liquid-liquid extraction from both, DBS and whole blood (100 μL, respectively), with phosphatidylpropanol as the internal standard. Extracts were subjected to LC gradient separation using multiple reaction monitoring of deprotonated molecules. Results from measurements of corresponding DBS and whole blood specimens were compared by estimating the respective mean values and by a Bland and Altman analysis. Concentrations of PEth 18:1/18:1 ranged from 46.1 to 3,360 ng/mL in whole blood (mean, 461.7 ng/mL) and from 35.8 to 3,360 ng/mL in DBS (mean, 457.6 ng/mL); for PEth 16:0/18:1, concentrations were from 900 to 213,000 ng/mL (mean, 23,375 ng/mL) and 922-213,000 ng/mL (mean, 23,470 ng/mL) in blood and DBS, respectively. Estimated mean differences were -4.3 ng/mL for PEth 18:1/18:1 and 95.8 ng/mL for PEth 16:0/18:1. The Bland-Altman plot of both PEth species showed that the variation around the mean difference was similar all through the range of measured values and that all differences except one were within the limits of agreement. It could be shown that the determination of PEth species in DBS is as reliable as in whole blood samples. This assay may facilitate monitoring of alcohol misuse.

Quantification of phosphatidylethanol 16:0/18:1, 18:1/18:1, and 16:0/16:0 in venous blood and venous and capillary dried blood spots from patients in alcohol withdrawal and control volunteers

“…Somewhat lower percentages were observed (between 55 and 78%) for the V-DBS and the C-DBS methods. Similar percentages, ranging from 68 to 91% [15] and 56 to 76% for PEth 16:0/18:1 [18] and from 27 and 43% for PEth 18:1/18:1) [15], were reported earlier for other DBS-based methods. The basis for this somewhat lower extraction efficiency is not known.…”

“…Because the nonpolar part of PEths tends to interact very strongly with the nonpolar hydrocarbon phase of a reversed phase column, the use of a more polar phase (i.e. C8 [14,18,20], C4 [7,15] or phenyl [16]) instead of a C18 phase allows to decrease the retention of PEths [14]. The use of less polar solvents, such as tetrahydrofuran (index polarity = 4.0), isopropanol (index polarity = 3.9) or methanol (index polarity = 5.1) instead of acetonitrile (index polarity = 5.8) also improved the elution of PEths using a reversed phase column.…”

“…Passing-Bablok regressions indicated a good overall correlation (R>0.962), no systematic differences (95% CI of the intercept values include the zero value) and no proportional differences, although 1 was just not included in the 95% CI of the slope in 3 out of 9 comparisons. In literature, agreement between venous blood and V-DBS concentrations has been assessed using Bland-Altman analysis for PEth 18:1/18:1 and PEth 16:0/18:1 [15,18]. One study showed good agreement, with a mean difference of 95.8 ng/mL (RSD=3.0%) and -4.…”

“…The most used extraction technique is a liquid-liquid extraction (LLE) with hexane [5][6][7][11][12][13][14][15][16][17] (or heptane [8]) after stepwise addition of blood to isopropanol and the internal standard (IS) solution. Some methods added water [6], borate buffer pH 9 [5] or sodium acetate buffer pH 5 [15] to dilute the blood. Some publications reported other types of sample preparation, such as protein precipitation with methanol [18] or protein precipitation followed by an online-solid phase extraction [19].…”

“…Also alcohol markers such as ethyl glucuronide, ethyl sulphate and PEths have been determined, starting from DBS ( [15,18,21,[23][24][25]; reviewed by Sadones et al, 2014 [26]). Since 2011, two publications have reported on the quantification of PEth 16:0/18:1 [15,18] and PEth 18:1/18:1 [15] in V-DBS samples, while only one [21] reported on the analysis of C-DBS samples (detection of PEth 16:0/18:1 in newborns to detect prenatal alcohol exposure). V-DBS are prepared by spotting a fixed volume of venous blood onto a filter paper, whereas C-DBS are generated by direct collection of blood drops appearing after a finger or heel prick onto a filter paper.…”