Background: Phosphatidylethanol (PEth) is an alcohol use biomarker with higher clinical sensitivity and specificity than commonly used alcohol markers. Since its introduction as a clinical alcohol-marker in 2006, the number of samples sent to our laboratory for the determination of PEth has shown a strong annual increase. This has prompted the need to develop a cost-effective and reliable analytical procedure with high capacity. Methods: An LC-MS/MS method for the determination of PEth 16:0/18:1 with a short turnaround time (3 min) has been evaluated with respect to accuracy, sensitivity, and precision. We compared this method with a previously used HPLC method, as well as a manual and a simplified automated method for sample workup, and investigated potential causes of analytic and preanalytic errors. Results: The method shows limits of detection and quantification of 0.0075 μmol/L (5.2 ng/mL) and <0.05 μmol/L (<35 ng/mL), respectively. During a 2.1-year period, the method has shown a total CV < 8% for control samples (n = 2808) in the range of 0.10 (70) to 3.5 μmol/L (2461 ng/mL). The simplified automated method for sample preparation works equally well as the manual one. No specific and clinically significant causes of preanalytic errors were found. Conclusions: This LC-MS/MS method with automated sample workup is well suited for a clinical laboratory with LC-MS/MS experience and has the capability, proven from several years of use, to produce reliable PEth results in a high-volume laboratory (>50 000 clinical samples/year). IMPACT STATEMENT Alcohol use disorders are a major health problem worldwide, and alcohol markers are an important tool for diagnosis, follow-up, and treatment evaluation. Phosphatidylethanol in whole blood (B-PEth) is a direct alcohol biomarker with higher clinical sensitivity and specificity than commonly used alcohol markers. Evidence presented here showed that B-PEth can be determined by LC-MS/MS preceded by a robotized extraction procedure. The method has a high capacity and is cost-effective and clinically reliable. This information may increase interest in the marker and make it more widely used.

Background: Alcohol use disorders are a major but often unrecognized health problem. Alcohol markers can therefore be of great value for diagnosis, follow-up, and treatment evaluation. Phosphatidylethanol in blood (B-PEth) is an alcohol biomarker with higher clinical sensitivity and specificity than commonly used alcohol markers but has shown a considerable interindividual variation in relation to reported consumption.Methods: An in vitro system was used to investigate factors, which may affect the formation rate of PEth or which may give rise to interindividual variation in the rate of formation. In this system, isolated erythrocytes from 31 individuals were incubated in the presence of various concentrations of ethanol (EtOH). The concentration of PEth and phosphatidylcholine (PC), the parent molecule of PEth, was determined by chromatographic methods.Results: Time, EtOH, and PC concentration were major factors determining the amount of PEth formed. The interindividual variation in PEth formation rate, calculated at an EtOH concentration of 50 mmol/l, showed a coefficient of variation (CV) from 23 to 31% for the different PEth forms studied (PEth 16:0/18:2, total PEth and PEth 16:0/18:1). The concentration of PC was found to be an important determinant of this variation. The formation rate for PEth 16:0/18:2 was somewhat higher than for PEth 16:0/18:1. The formation of PEth 16:0/18:1 but not PEth 16:0/18:2 showed a positive correlation to the concentration of PEth at baseline (endogenous PEth). Calculation of enzyme kinetics for the reaction resulting in the formation of PEth 16:0/18:1 or PEth 16:0/18:2 showed an apparent K m (Michaelis constant) of approximately 160 to 170 mmol/l.Conclusions: Interindividual variation in the formation rate of PEth appears to be a significant but relatively modest source of variation in the relation between B-PEth and reported consumption. Correction for interindividual variation in PC concentrations might substantially reduce the interindividual variability in PEth formation and consequently in B-PEth.

Background: The textile dye mix (TDM) 6.6% pet. contains Disperse Blue (DB) 35, Disperse Yellow 3, Disperse Orange (DO) 1 and 3, Disperse Red 1 and 17, and DB 106 and 124. The most frequent allergen in TDM-positive patients is DO 3. Around 85% of p-phenylenediamine (PPD)-allergic dermatitis patients have shown positive patch test reactions to DO 3. There has been a discussion to exclude DO 3 from TDM 6.6% because of frequent, strong reactions to TDM 6.6% and PPD. Objectives: To study if DO 3 can be omitted from a TDM. Methods: Patch tests were performed on 2250 dermatitis patients with TDM 6.6%, TDM 5.6% pet., TDM 7.0% pet., and PPD 1.0% pet.; 122 patients were also patch tested with DO 3 1.0% pet. Results: Among the 2250 patients patch tested, contact allergy prevalence to TDM 6.6% was 2.4%, to TDM 5.6% 1.8%, and to TDM 7.0% 2.0%. Of the 54 TDM 6.6%positive patients, 55.6% reacted to PPD; as much as 42.2% of PPD-allergic women and 50% of PPD-allergic men reacted to TDM 6.6%. Of the 17 DO 3-positive patients, 94.1% showed a positive reaction to PPD. Conclusion: Results indicate that DO 3 can probably be omitted from TDM, but patch testing with TDM 6.6%, TDM 7.0%, DO 3 1.0%, and PPD 1.0% simultaneously is needed to finally decide whether it is possible or not. K E Y W O R D S baseline series, contact allergy, disperse dyes, Disperse Orange 3, patch testing, pphenylenediamine, simultaneous reactivity, textile dye mix Highlight 1: Of the 2250 patients tested, 54 (2.4%) reacted with a positive reaction to textile dye mix (TDM) 6.6%, 41 (1.8%) to TDM 5.6%, and 46 (2.0%) to TDM 7.0%. Two patients were exclusively positive to TDM 5.6% and seven patients were exclusively positive to TDM 7.0%. Highlight 2: Of the 54 TDM 6.6%-positive patients, 30 (55.6%) also reacted to p-phenylenediamine (PPD). As much as 19 of 45 (42.2%) PPD-allergic women and 7 of 14 (50%) PPD-allergic men also reacted to TDM 6.6%. A total of 17 individuals were positive to Disperse Orange (DO) 3. In 16/17 DO 3-positive individuals (94.1%) a positive reaction to PPD was seen, and in 15/17 (88.2%) a simultaneous positive reaction to TDM 6.6% was noted. Highlight 3: Results indicate that DO 3 can be omitted from TDM, but patch testing with TDM 6.6%, TDM 7.0%, DO 3 1.0%, and PPD 1.0% simultaneously is needed. If DO 3 is excluded from the textile dye mix, and a patient reacts to PPD, which may indicate textile dye allergy, the patient should be questioned about skin problems from textiles and if they do indicate problems, DO 3 should be patch tested on its own, preferably included in a textile series. The patient information leaflet on PPD allergy must in that case be updated with accurate information.

Background: The textile dye mix (TDM) 6.6% in petrolatum contains Disperse Blue (DB) 35, Disperse Yellow 3, Disperse Orange (DO) 1 and 3, Disperse Red 1 and 17, and DB 106 and 124. The most frequent allergen in TDM-positive patients is DO 3. Around 85% of para-phenylenediamine (PPD)-allergic dermatitis patients have been positive to DO 3. There has been a discussion to exclude DO 3 from TDM 6.6% because of strong simultaneous reactions to TDM and PPD.Objectives: To study if DO 3 can be excluded from TDM 6.6%.Methods: Patch tests were performed on 1481 dermatitis patients with TDM 6.6%, TDM 7.0% (without DO 3 but the other disperse dyes at 1.0% each), DO 3 1.0%, and PPD 1.0% pet.