Pentafluoropropionic Anhydride Derivatization and GC-MS Analysis of Histamine, Agmatine, Putrescine, and Spermidine: Effects of Solvents and Starting Column Temperature

Gas chromatography–mass spectrometry (GC-MS) is useful for the quantitative determination of the polyamines spermidine (SPD) and putrescine (PUT) and of the biogenic amine agmatine (AGM) in biological samples after derivatization. This GC-MS method involves a two-step extraction with n-butanol and hydrochloric acid, derivatization with pentafluoropropionic anhydride (PFPA) in ethyl acetate, and extraction of the pentafluoropropionic (PFP) derivatives by toluene of SPD, PUT, and AGM. We wanted to extend this GC-MS method for the biogenic amine histamine (HA), but we faced serious problems that did not allow reliable quantitative analysis of HA. In the present work, we addressed this issue and investigated the derivatization of HA and the effects of toluene and ethyl acetate, two commonly used water-insoluble organic solvents in GC-MS, and oven temperature program. Derivatization of unlabelled HA (d0-HA) and deuterium-labelled HA (d4-HA) with PFPA in ethyl acetate (PFPA-EA, 1:4, v/v; 30 min, 65 °C) resulted in the formation of d0-HA-(PFP)2 and d4-HA-(PFP)2 derivatives. d4-HA and 13C4-SPD were used as internal standards for the amines after standardization. Considerable quantitative effects of toluene and ethyl acetate were observed. The starting GC column temperature was also found to influence considerably the GC-MS analysis of HA. Our study shows the simultaneous quantitative analysis of HA as HA-(PFP)2, AGM as AGM-(PFP)3, PUT as PUT-(PFP)2, and SPD as SPD-(PFP)3 derivatives requires the use of ethyl acetate for their extraction and injection into the GC-MS apparatus and a starting GC column temperature of 40 °C instead of 70 °C. The PFP derivatives of HA, AGM, PUT, and SPD were found to be stable in ethyl acetate for several hours at room temperature. Analytically satisfactory linearity, precision, and accuracy were observed for HA, AGM, PUT, and SPD in biologically relevant ranges (0 to 700 pmol). The limits of detection of AGM, PUT, and SPD were about two times lower in ethyl acetate compared to toluene (range, 1–22 fmol). The limits of detection were 1670 fmol for d0-HA and 557 fmol for d4-HA. Despite the improvements achieved in the study for HA, its analysis by GC-MS as a PFP derivative is challenging and less efficient than that of PUT, AGM, and SPD

Pentafluoropropionic Anhydride Derivatization and GC-MS Analysis of Histamine, Agmatine, Putrescine, and Spermidine: Effects of Solvents and Starting Column Temperature

Gas chromatography–mass spectrometry (GC-MS) is useful for the quantitative determination of the polyamines spermidine (SPD) and putrescine (PUT) and of the biogenic amine agmatine (AGM) in biological samples after derivatization. This GC-MS method involves a two-step extraction with n-butanol and hydrochloric acid, derivatization with pentafluoropropionic anhydride (PFPA) in ethyl acetate, and extraction of the pentafluoropropionic (PFP) derivatives by toluene of SPD, PUT, and AGM. We wanted to extend this GC-MS method for the biogenic amine histamine (HA), but we faced serious problems that did not allow reliable quantitative analysis of HA. In the present work, we addressed this issue and investigated the derivatization of HA and the effects of toluene and ethyl acetate, two commonly used water-insoluble organic solvents in GC-MS, and oven temperature program. Derivatization of unlabelled HA (d0-HA) and deuterium-labelled HA (d4-HA) with PFPA in ethyl acetate (PFPA-EA, 1:4, v/v; 30 min, 65 °C) resulted in the formation of d0-HA-(PFP)2 and d4-HA-(PFP)2 derivatives. d4-HA and 13C4-SPD were used as internal standards for the amines after standardization. Considerable quantitative effects of toluene and ethyl acetate were observed. The starting GC column temperature was also found to influence considerably the GC-MS analysis of HA. Our study shows the simultaneous quantitative analysis of HA as HA-(PFP)2, AGM as AGM-(PFP)3, PUT as PUT-(PFP)2, and SPD as SPD-(PFP)3 derivatives requires the use of ethyl acetate for their extraction and injection into the GC-MS apparatus and a starting GC column temperature of 40 °C instead of 70 °C. The PFP derivatives of HA, AGM, PUT, and SPD were found to be stable in ethyl acetate for several hours at room temperature. Analytically satisfactory linearity, precision, and accuracy were observed for HA, AGM, PUT, and SPD in biologically relevant ranges (0 to 700 pmol). The limits of detection of AGM, PUT, and SPD were about two times lower in ethyl acetate compared to toluene (range, 1–22 fmol). The limits of detection were 1670 fmol for d0-HA and 557 fmol for d4-HA. Despite the improvements achieved in the study for HA, its analysis by GC-MS as a PFP derivative is challenging and less efficient than that of PUT, AGM, and SPD

Assessment of shared decision making in pediatrics: Developing German scales for patients aged 7-18 years, parents and parent-proxy reports (CollaboRATE(pediatric))

Objective: To develop parsimonious German scales (CollaboRATE(pediatric)) to assess shared decision making (SDM) with patients aged 7-18 years, parents and parents on behalf of their children (parentproxy reports), and to establish comprehensibility and preliminary face validity. Methods: Based on an existing SDM scale for adults (CollaboRATE) we developed CollaboRATE(pediatric) in a two-step approach: (1) team-based translation of the English CollaboRATE scale into German and adaptation for pediatric patients, parents and parent-proxy reports, followed by (2) iterative revisions of the CollaboRATE(pediatric) scales based on cognitive interviews with patients and parents until comprehensibility and preliminary face validity were established. Results: Taking into account seven problem areas identified in four rounds of cognitive interviews (e.g., item complexity) we developed CollaboRATE(pediatric) scales for patients, parents and parent-proxy reports. By iteratively revising items we were able to resolve all problem areas and achieved full comprehensibility and intended interpretation of all items. Conclusion: The scales enable the parsimonious assessment of SDM with pediatric patients and parents as well as comparisons between the two groups. Future empirical work will establish the psychometric performance of CollaboRATE(pediatric). Practice Implications: CollaboRATE(pediatric) can be used in quality improvement initiatives to foster the comprehensive implementation of SDM in pediatrics. (C) 2020 Published by Elsevier B.V

Study protocol: evaluation of the addictive potential of e-cigarettes (EVAPE): neurobiological, sociological, and epidemiological perspectives

Background: Tobacco use is the largest preventable cause of diseases and deaths; reducing tobacco intake is, therefore, an urgent public health goal. In recent years, e-cigarettes have been marketed as a 'healthier' alternative to tobacco smoking, whilst product features have evolved tremendously in the meantime. A lively scientific debate has developed regarding the potential benefits and risks of e-cigarettes although, surprisingly, there are few studies investigating the addictive potential of nicotine-containing e-cigarettes. The present work comprises three work packages investigating the addictive potential of e-cigarettes from different perspectives: (1) the neurobiological addictive potential of e-cigarettes; (2) the experience and perception of dependence symptoms among users of e-cigarettes in a social context; and (3) the epidemiological perspective regarding factors influencing the potential for dependence. Methods: Work package I: the neurobiological study will investigate the key elements of addiction in e-cigarettes compared to tobacco cigarettes using neurobiological and neuropsychological correlates associated with craving, incentive motivation, cue reactivity and attentional bias. Work package II: the sociological study part examines selfreports on the experience and perception of dependence symptoms in a social context, using focus group interviews and the analysis of posts in online discussion forums on e-cigarettes. Work package III: the epidemiological study part focuses on tolerance development and the role of psychosocial and product factors by analyzing longitudinal data from the International Tobacco Control Policy Evaluation Project (ITC). Discussion: The present study offers a chosen mix of three methodological approaches, thereby comprehensively examining core symptoms of positive and negative reinforcement in addiction. Whether e-cigarettes are as reinforcing and addictive as combustible tobacco cigarettes is an important public health issue with implications for prevention and treatment programs

Evaluation of a rebound tonometer (Tonovet®) in clinically normal cat eyes

Objective: To determine the accuracy of and to establish reference values for a rebound tonometer (Tonovet ) in normal feline eyes, to compare it with an applanation tonometer (Tonopen Vet ) and to evaluate the effect of topical anesthesia on rebound tonometry. Procedures: Six enucleated eyes were used to compare both tonometers with direct manometry. Intraocular pressure (IOP) was measured in 100 cats to establish reference values for rebound tonometry. Of these, 22 cats were used to compare rebound tonometry with and without topical anesthesia and 33 cats to compare the rebound and applanation tonometers. All evaluated eyes were free of ocular disease. Results: Both tonometers correlated well with direct manometry. The best agreement with the rebound tonometer was achieved between 25–50 mmHg. The applanation tonometer was accurate at pressures between 0 and 30 mmHg. The mean IOP in clinically normal cats was 20.74 mmHg with the rebound tonometer and 18.4 mmHg with the applanation tonometer. Topical anesthesia did not significantly affect rebound tonometry. Conclusions: As the rebound tonometer correlated well with direct manometry in the clinically important pressure range and was well tolerated by cats, it appears suitable for glaucoma diagnosis. The mean IOP obtained with the rebound tonometer was 2–3 mmHg higher than that measured with the applanation tonometer. This difference is within clinically acceptable limits, but indicates that the same type of tonometer should be used in follow-up examinations in a given cat