Total cholesterol in serum determined by isotope dilution/mass spectrometry, with liquid-chromatographic separation.

Abstract

We describe an accurate, precise method for determination of total serum cholesterol by isotope dilution/mass spectrometry (IDMS) with liquid-chromatographic separation. After adding [3,4-13C]cholesterol to serum and hydrolyzing the cholesterol esters, we extract the total cholesterol. "High-performance" liquid chromatography (HPLC) is used to separate the extracted cholesterol for measurement by electron-impact mass spectrometry with use of a direct-insertion device. To evaluate the specificity and the accuracy of this method, we also studied the conventional IDMS method, which involves converting cholesterol to the trimethylsilyl ether and assay by gas chromatography-mass spectrometry with use of a capillary column. The coefficient of variation for the HPLC method was a little larger than that for the conventional method, but mean values by each method agreed within 1% for all sera tested.

Cholesterol Content and Methods for Cholesterol Determination in Meat and Poultry

“…It has become the definitive method of analysis, which has been used to certify concentrations of food‐related analytes in SR materials (Ellerbe and others 1989; Welch and others 2001; NIST 2010). The ID/MS was initially evaluated for determining cholesterol in serum only (Gambert and others 1979; Cohen and others 1980; Takatsu and Nishi 1987). The isotopically labeled cholesterol such as cholesterol‐(3,4– 13 C 2 ), cholesterol‐d 7 , or cholesterol‐(25,26,27 13 C 3 ) is added to the sample as an internal standard.…”

“…Cohen and others (1980) described GC‐ID/MS (using cholesterol‐d 7 ) as a highly accurate and precise method for measuring cholesterol in serum with a CV of 0.36%. Takatsu and Nishi (1987), using cholesterol‐(3,4– 13 C 2 ), showed that ID/MS could be coupled with an HPLC system for the same accuracy as GC‐ID/MS (1% difference between HPLC‐ID/MS and GC‐ID/MS); however, the HPLC‐ID/MS had a slightly greater CV. Pelletier and others (1987) similarly proposed an optimized GC‐ID/MS [using cholesterol‐(3,4– 13 C 2 )] for serum cholesterol.…”

“…Most chromatographic methods for cholesterol analysis in meat and poultry were initially developed for blood serum (Abell and others 1952;Gambert and others 1979;Cohen and others 1980;Iwata and others 1987;Pelletier and others 1987;Takatsu and Nishi 1987;Eckfeldt and others 1991;Nakajima and others 1995). The AOAC Intl.…”

“…Before the 1990s, much of the cholesterol data for meat and poultry products were generated using colorimetric measurement (Sweeney and Weihrauch 1976; Bohac and Rhee 1988; Hoelscher and others 1988; Beyer and Jensen 1989; Browning and others (1990); Sweeten and others 1990; Swize and others 1992; Akinwunmi and others 1993). Most chromatographic methods for cholesterol analysis in meat and poultry were initially developed for blood serum (Abell and others 1952; Gambert and others 1979; Cohen and others 1980; Iwata and others 1987; Pelletier and others 1987; Takatsu and Nishi 1987; Eckfeldt and others 1991; Nakajima and others 1995). The AOAC Intl.…”

Analytical methods for cholesterol quantification

“…Chromatographic techniques such as gas (GC, where gas is a mobile phase) and liquid (LC, where liquid is a mobile phase) chromatography are commonly used for cholesterol quantification [ 32 , 51 ]. Those methods are widely accepted to be more reliable, sensitive and accurate than the other methods.…”