This investigation studied the efficiency of high performance liquid chromatography in the determination of free fatty acids present on the fingertips, and assessed the quantitative relationship between skin fatty acids and the degree of microbial contamination. Automated surgical scrub was utilized to eliminate the microbial contamination. The high performance liquid chromatography provided excellent separation of skin fatty acids for evaluation with the bacterial counts. The fatty acid peaks identified ranged in chain length from C12 through C32. All the fatty acids evaluated showed positive correlation with the bacterial counts with the exception of one acid which had an inverse relationship but none were statistically significant. Finally, the surgical scrub chromatograms showed that the straight chain acids C19 and C21 were lower in concentration than C23 and C25; also, C26 was lower in concentration than C28 and C30. It was evident from the data that fatty acids which have been shown to be bacteriostatic in vitro do not demonstrate the same property on the fingertips. The finding of lower concentration of C19, C21, and C26 than longer chain acids is inconsistent with simple two carbon addition, and indicates there is possible branching at these points in metabolic pathway of fatty acid synthesis. (Author).

A study of parameters (organic content, additives and pH of the mobile phase) to yield good separation and detection of a series of commercially available free fatty acids ranging from C12:0 (lauric acid) to C18:3 (linolenic acid) using HPLC-CAD is undertaken. Working methods using a C18 silica column were assessed by measuring the experimental limit of detection (LOD) and sensitive ranges with consideration to effects of the Charged Aerosol Detector (CAD) temperature and detector voltage. An isocratic method with high content in acetonitrile and low pH was developed that allowed the CAD detection and quantification of the less volatile fatty acids in the range of from around 1 to 5 ng/uL to over 200 ng/uL. A power fit calibration curve was necessitated since the response of the standards did not display a true linear relationship using linear regression analysis. Conditions or mobile phase additives were not found to increase detection of semi-volatile fatty acids such as lauric acid or myristic acid (C14:0). Column bleed was potentially identified as an unexpected additive that resulted in enhanced peak detection, attributed to the formation or stabilization of bigger aerosol particles. The isocratic method was tested for an olive oil standard using an acetonitrile: 0.01M TFA (96.5:3.5) mobile phase, ion voltage at -300 V, and CAD heater setting of 35°C. Using those conditions, separation and detection of major C16 to C18 fatty acids were achieved although palmitic and oleic acids were not completely resolved. The olive oil analysis showed that relative recovery of the major fatty acid components is consistent and supports the use of HPLC-CAD system for a rapid detection of fatty acids at trace levels.

Omega-3 long chain polyunsaturated fatty acid (n-3 LCPUFA) blood levels are a potential risk factor for coronary heart disease, particularly sudden cardiac death. Venipuncture sampling for fatty acid profiling is invasive, requires highly qualified personnel and requires a multi-step protocol to isolate blood fractions. Alternately, the use of whole blood for fatty acid profiling improves analytical throughput and allows sample collection in field research locations by enabling dried blood spotting (DBS). Dried blood spots are advantageous in comparison to venous blood sampling as they require small blood volumes and is relatively inexpensive to collect. However, FA profiles in DBS are commonly expressed qualitatively (% of total fatty acids) and not quantitatively ([mu]g/mL) as finger-tip prick (FTP) sampling usually results in the collection of an unknown volume of blood. Quantitation can be effected by preexisting fatty acid contaminants on DBS collection materials and oxidative losses of sensitive fatty acids such as n-3 LCPUFA due to the increased surface area of DBS samples. Fatty acid quantitation could detect hypo- and hyper-lipidemia in samples that a qualitative only assessments would miss. To address these issues, the relationship between blood volume and blood spot area on 903 Protein Saver Cards (903 PSC) was examined to determine the blood volume associated with a 6mm hole punch and the Mitra Microsampling Device, a product designed to collect 10 [mu]L of blood regardless of hematocrit, was assessed for FA and lipidomic analyses. To determine if fatty acid contaminants were present, the 903 PSC, the Mitra tips and Whatman chromatography paper (also commonly used to collect blood spots) were examined using gas chromatography and ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Finally, the stability of the fatty acids in a DBS sample on Mitra and 903 PSC stored at ambient, 4C, -20C and -80C temperatures with and without antioxidant for 3 months was examined. It was determined that the 6mm punch of 903 PSC contained 9.6 [mu]L of blood and that FA profiles determined from the Mitra samples were comparable to FA profiles from wet blood controls. The Mitra tips could also be used to provide similar lipidomic profiles. The 903 PSC, the Mitra tips and the Whatman paper all contained palmitic and stearic acid as free fatty acids (FFA) while the Mitra tips also had palmitoyl and stearoyl lysophosphatidylcholines (LysoPC) present. With DBS storage, the n-3 LCPUFA biomarkers were the most stable with -80C storage followed by 4C or ambient room temperatures while samples stored at -20C storage had the lowest stability in both antioxidant and no antioxidant conditions, which mirrored previous research examining whole blood storage. In conclusion, quantitative fatty acid determinations of DBS samples are possible. Blood volumes can be estimated using a defined hole punch on the commonly used 903 PSC, or defined by using a Mitra sampling device. Analysis of blank sampling devices is recommended to assess the potential impact of fatty acid and fatty acyl contaminants in any DBS collection materials to be used. Finally, storage conditions need to be a consideration with DBS sample collection as preventative steps such as storage temperature and the use of antioxidants can improve sample stability and ensure data integrity.