Comparison of the assay to external reference materials.

<p>The oxysterol reference materials were from Referenzinstitut für Bioanalytik and the vitamin D3 reference material was from The National Institute of Standards and Technology. 50pct; 50th percentile of the corresponding LC-MS sub-collective</p><p>Comparison of the assay to external reference materials.</p

Simultaneous Determination of Oxysterols, Cholesterol and 25-Hydroxy-Vitamin D3 in Human Plasma by LC-UV-MS

<div><p>Background</p><p>Oxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers.</p><p>Methods</p><p>A method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated.</p><p>Results</p><p>Our method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 μL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 μg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S)-, 25-, 27-, 7α-hydroxycholesterol (HC) and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis.</p><p>Conclusion</p><p>The method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential biomarkers of disease.</p></div

LC-MS-PDA chromatogram of standards.

<p>The top panel shows the chromatogram of each selected ion-monitoring (SIM) channel with each analyte identified at the peak apex. MS Off/MS On indicated the activation points for the flow control valve. The bottom panel shows the PDA Chromatogram at 204 nm.</p

Side-chain oxygenated oxysterol levels in patient groups.

<p>Distribution of 24-hydroxy cholesterol (24HC; Fig 2A), 25-hydroxy cholesterol (25HC; Fig 2C) and 27-hydroxy cholesterol (27HC; Fig 2E) for healthy controls (HC), multiple sclerosis (MSC) and other neurological diseases (OND). The results in Fig 2B, 2D and 2F correspond to the oxysterols in Fig 2A, 2C and 2E, respectively, but are normalized to total cholesterol (TC) levels. The dot-plots of individual patients are superimposed on the box plots. The solid line in the box plots are the median, the box delineates upper and lower quartiles and the error bar represents the range. Panel E for 27HC and Panel F for 27HC-TC ratio contains the p-value from the Kruskal-Wallis test in the main body. The inset contains a bar graph and p-values from follow-up Mann-Whitney tests.</p

B-Ring oxygenated oxysterol and 25-hydroxy vitamin D3 levels in patient groups.

<p>Distribution of 7α hydroxy cholesterol (7αHC; Fig 3A) and 7 ketocholesterol (7KC; Fig 3C) and 25-hydroxy-vitamin D3 (25-OHVD₃; Fig 3E) for healthy controls (HC), multiple sclerosis (MSC) and other neurological diseases (OND). The results in Fig 3B and 3C correspond to the oxysterols in Fig 3A and 3B, respectively, but are normalized to total cholesterol (TC) levels. The dot-plots for individual patients are superimposed on the box plots. The solid line in the box-plots are the median, the box delineates upper and lower quartiles and the error bar represents the range. Panel A for 7αHC and Panel B for 7αHC-TC ratio contains the p-value from the Kruskal-Wallis test in the main body. The inset contains a bar graph and p-values from follow-up Mann-Whitney tests. Panel E illustrates the agreement between 25-OHVD₃ measured by our method on the y-axis compared to a validated LC-MS/MS method on the x-axis.</p

Analyte systematic name, acronym and the selected ion monitoring (SIM) segment windows used for APCI⁺-MS detection and the wavelengths for photodiode array detection.

<p>Peak retention time and mass to charge ratio (<i>m/z</i>) of the major fragment generated by each analyte are listed.</p><p>Analyte systematic name, acronym and the selected ion monitoring (SIM) segment windows used for APCI⁺-MS detection and the wavelengths for photodiode array detection.</p

Intra- and inter-assay imprecision for method validation samples.

<p>* <i>n</i> = 6 on each of three consecutive days, the maximum daily value is reported</p><p>** <i>n</i> = 18; 6 replicates on each of 3 consecutive days</p><p>Intra- and inter-assay imprecision for method validation samples.</p