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Determination of total cholesterol in serum by liquid chromatography-isotope dilution mass spectrometry.

For an enlarging group of quantitative analytical methods used in the clinical chemistry laboratory, external quality-assessment programs are of crucial importance in guaranteeing the accuracy of the results produced in the routine laboratory. Presently there is a consensus that national quality control assessment programs in clinical chemistry should be based on Reference and Definitive Methods for substrates and drugs [1,2]. Limitations for the development of Reference Methods exist mostly because one accepted technique for the design of such methods is the isotope dilution technique, which requires a final mass spectrometric analysis at a suitable mass of a fragment of the analyte and the isotope-modified internal standard [3]. Until now gas chromatography gas chromatography (GC)

Type of chromatography with a gas mixture as the mobile phase. In a packed column, the packing or solid support (held in a tube) serves as the stationary phase (vapour-phase chromatography, or VPC) or is coated with a liquid stationary phase
 has been the most widely used separation technique, and several such methods for the determination of total cholesterol in serum have been described [4-8]. Attempts have also been made to determine cholesterol by HPLC HPLC high-performance liquid chromatography.

HPLC

high performance liquid chromatography.

HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed
 [9], and mass spectrometry mass spectrometry
 or mass spectroscopy

Analytic technique by which chemical substances are identified by sorting gaseous ions by mass using electric and magnetic fields.
 by direct inlet, after liquid chromatographic chro·mat·o·graph  
n.
An instrument that produces a chromatogram.

tr.v. chro·mat·o·graphed, chro·mat·o·graph·ing, chro·mat·o·graphs
To separate and analyze by chromatography.
 separation and collection of the peak fraction, has been described [10].

Here we describe our new liquid chromatographyisotope dilution mass spectrometry (LC IDMS) (1) method for the determination of cholesterol in serum. The novelty of this method is the separation of the analyte by HPLC previous to mass spectrometry. The evaporation of the eluent eluent

the solution used in elution.
 is done in a particle-beam interface used for coupling the liquid chromatograph chromatograph /chro·mato·graph/ (kro-mat´o-graf)
1. the apparatus used in chromatography.

2. to analyze by chromatography.


chromatograph

1. to analyze by chromatography.

2.
 and the mass spectrometer. Finally selective ion monitoring (SIM) is performed after electron impact (EI) ionization ionization: see ion.
ionization

Process by which electrically neutral atoms or molecules are converted to electrically charged atoms or molecules (ions) by the removal or addition of negatively charged electrons.
. A derivatization prior to analysis on the LC-MS instrument is not required. The results obtained for pooled human sera were compared with those obtained by a slightly modified variant of the gas chromatography (GC) IDMS method used by Siekmann [11]. It was not intended to propose a candidate reference method, but to present an analytical procedure, which may be worked out to a candidate reference method for the determination of total cholesterol in serum.

Principles

The isotope-dilution method for the determination of total cholesterol in serum is based on the addition of identical volume fractions of the internal standard [25,26,27-[sup.13] [C.sub.3]]cholesterol to serum samples and calibrators. The serum is submitted to a basic hydrolysis hydrolysis (hīdrŏl`ĭsĭs), chemical reaction of a compound with water, usually resulting in the formation of one or more new compounds.  to convert the cholesterol esters to free cholesterol. The cholesterol is then transferred to cyclohexane cyclohexane (sī'kləhĕk`sān), C6H12, colorless liquid hydrocarbon. It is a cyclic alkane that melts at 6°C; and boils at 81°C;. It is nearly insoluble in water.  by liquid-liquid extraction. The cyclohexane phase is taken to dryness. For the LC IDMS method, the residue containing the cholesterol is dissolved in ethanol, and this solution is used directly for analysis. The LC separation is performed on a reversed-phase column. Detection is performed by spectrophotometric detection at a wavelength of 210 nm and SIM of the molecular mass ion of cholesterol, m/z = 386, and the respective molecular mass ion of the internal standard, m/z = 389.

In the case of the GC IDMS method, a derivatization of the alcoholic groups to trimethylsilyl ethers has to be done prior to analysis. The GC separation is performed on a nonpolar nonpolar

not having poles; not exhibiting dipole characteristics.
 fused-silica capillary GC column. In this case, the principal isotope ratio measurements are made from the ion abundances of the molecular mass ion of trimethylsilyl cholesterol, m/z = 458, and the respective molecular mass ion of the internal standard, m/z = 461. Standards are made by combining pure unlabeled cholesterol and [25,26,27-[sup.13][C.sub.3]]cholesterol to give one with an unlabeled/ labeled ratio of ~1.0, one standard somewhat lower, and one somewhat higher. These mixtures are evaporated without previous liquid-liquid extraction and processed further like the serum samples. To get the best possible precision, a bracketing technique was applied for both methods [6].

Materials and Methods

CHEMICALS

The cholesterol used for preparation of the calibrator calibrator

an instrument for dilating a tubular structure or for determining the caliber of such a structure.
 solutions was Standard Reference Materials (SRM (1) (Storage Resource Management) The management of the storage resources in an organization in order to avoid duplication of files and to determine space utilization across all servers. 911b) purity from NIST (National Institute of Standards & Technology, Washington, DC, www.nist.gov) The standards-defining agency of the U.S. government, formerly the National Bureau of Standards. It is one of three agencies that fall under the Technology Administration (www.technology.  (obtained from Promochem, Wesel, Germany). This material has a purity of 99.8%, (uncertainty 0.1%). The [25,26,27-[sup.13][C.sub.3]]cholesterol used as internal standard in GC IDMS measurements as well as LC IDMS measurements was obtained under MS-3501 from IC Chemikalien, Munich, Germany. The material had a certified 99% isotope enrichment (neither uncertainty of the isotope enrichment nor the purity of the material was given by the supplier). N-Methyl-N-(trimethylsilyl)trifluoroacetamide and silylation-grade pyridine pyridine (pĭr`ĭdēn) or azine (ăz`ēn), C5H5N, colorless, flammable, toxic liquid with a putrid odor. It melts at −42°C; and boils at 115.5°C;.  were obtained from Macherey & Nagel. All other chemicals were of analytical grade and purchased from Merck, Sigma, and Baker. Deionized water Deionized water (DI water or de-ionized water; also spelled deionised water, see spelling differences) is water that lacks ions, such as cations from sodium, calcium, iron, copper and anions such as chloride and bromide.  was prepared with a MilliQ apparatus (Millipore).

SAMPLES

The control materials used for checking accuracy of the methods were human serum SRM 909 with a certified cholesterol concentration of 1415 [+ or -] 46 mg/L; SRM 909b, grade 1 with a certified cholesterol concentration of 1464 [+ or -] 18 mg/L; and SRM 909b, grade 2 with a certified cholesterol concentration of 2353 [+ or -] 30 mg/L from NIST. The other control materials were Precinorm U from Boehringer Mannheim with a target value for the concentration of total cholesterol of 1200 mg/L and Kontrollogen-LP from Behringwerke with a target value of 1330 mg/L. Both control materials were based on processed human sera, and the target value for total cholesterol was determined by use of a GC IDMS method that was not further specified.

For the method comparison pooled sera were used (n = 28). The pools were prepared with serum samples taken after analysis in the Central Laboratory of the Institute of Clinical Chemistry and Pathobiochemistry at the University Hospital of the Technical University Aachen. Samples were obtained from the outpatients' departments as well as the patient care units of the University Hospital. All specimens were collected in Sarstedt monovettes with separation gel. Serum was obtained after centrifugation Centrifugation

A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal
.

INSTRUMENTS AND SETTINGS

LC IDMS. The analysis was performed on a Waters Integrity system (Waters), consisting of an Alliance 2690 chromatography module, a column bypass module, a photodiode A light sensor (photodetector) that allows current to flow in one direction from one side to the other when it absorbs photons (light). The more light, the more the current. Used to detect light pulses in optical fibers and other light-sensitive applications, it works the opposite of a  array detector 996, and a Waters Thermabeam mass detector, equipped with ion-source working unchangeable un·change·a·ble  
adj.
Not to be altered; immutable: the unchangeable seasons.



un·change
 in [El.sup.-] mode. System controlling, data acquisition, and integration were performed with the Waters Millennium Software, Rel. 2.21.

For chromatography a 150 mm X 2 mm Novapak C18 analytical minibore column (Waters Chromatography) was used. The eluent consisted of acetonitrile acetonitrile /ac·e·to·ni·trile/ (as?e-to-ni´tril) a colorless liquid with an etherlike odor used as an extractant, solvent, and intermediate; ingestion or inhalation yields cyanide as a metabolic product.  and isopropyl alcohol isopropyl alcohol: see isopropanol.  (65:35 by vol). The flow rate was 0.3 mL/min. The injection volume was 5 [micro]L for all samples. The duration of one chromatographic run was 7 min.

The helium flow in the particle-beam liquid chromatography-mass spectrometry Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (aka HPLC) with the mass analysis capabilities of mass spectrometry.  interface was 30 mL/min. The nebulizer was heated to 70[degrees]C, and the temperature of expansion region was set to 90[degrees]C. The pressure in the interface was constantly at 67 Pa. The temperature of the ion source An ion source is an electro-magnetic device that is used to create charged particles. These are used primarily within mass spectrometers or particle accelerators. Mass spectrometry  was set to 220[degrees]C, the El energy was set to 70 eV, and the pressure in the ion chamber was [less than or equal to] 0.026 Pa. The voltage settings for the ion optic were 5 V for the ion volume, -42 V for the extraction lens, -20 V for the prequad, and -101 V for the exit lens. The multiplier voltage was set to 1980 V.

The measurement was performed in SIM mode at m/z = 386 (cholesterol) and m/z = 389 ([25,26,27[sup.13][C.sub.3]]cholesterol) with a frequency of 1 scan/s; within one chromatography 420 scans were performed, and the duration of the elution elution /elu·tion/ (e-loo´shun) in chemistry, separation of material by washing; the process of pulverizing substances and mixing them with water in order to separate the heavier constituents, which settle out in solution, from the  of the cholesterol peak was about 50 scans.

GC IDMS. The instrument used was a Fisons MD-800 combined gas chromatograph-quadrupole mass spectrometer (Fisons Instruments), equipped with an El source and a GC8000 series gas chromatograph gas chromatograph
n.
An instrument used in gas chromatography to separate a sample of a volatile substance into its components.
 and autosampler AS800. For instrument controlling and data acquisition the Fisons MassLab Software Rel. 1.30 was used.

The gas chromatography was performed on a Hewlett-Packard Ultra 1 [0.33 [micro]m, 12 m X 0.32 mm (i.d.)] capillary column (IAS See iPlanet Application Server.

1. (computer) IAS - The first modern computer. It had main registers, processing circuits, information paths within the central processing unit, and used Von Neumann's fetch-execute cycle.
, Leipzig, Germany). The carrier gas was helium at 690 kPa (100 psi) at a flow rate of 1 mL/min, the split exit was set at 50 mL/min (1:50), the injector temperature was 320[degrees]C, the oven temperature was isothermal i·so·ther·mal
adj.
Of, relating to, or indicating equal or constant temperatures.



isothermal, isothermic

having the same temperature.
 280[degrees]C, and the interface temperature was set to 290[degrees]C. The sample size injected by the AS800 was constantly set to 1.0 [micro]L.

The temperature in the ion source was set to 200[degrees]C, the El energy was set to 70 eV, and the emission current was set to 235 mA. The voltage settings for the ion optic were 1.4 V for the ion energy, 0.7 V for the repeller, 8 V for lens 1, 79 V for lens 2, 7.4 V for low mass resolution, and 12.2 V for high mass resolution. The multiplier voltage was set to 500 V.

For SIM mode measurements mass detection was set at m/z = 458 [+ or -] 0.25 (cholesterol) and m/z = 461 [+ or -] 0.25 ([25,26,27-[sup.13] [C.sub.3]]cholesterol), the dwell time The time cargo remains in a terminal's in-transit storage area while awaiting shipment by clearance transportation. See also storage.  was set to 0.15 s, and the channel delay to 20 ms, leading to a measurement frequency of 3 scans/s. The data acquisition delay time was set to 2.45 min. Within one chromatography 450 scan were performed, and the duration of the elution of the peak of the cholesterol derivate der·i·vate  
adj.
Derivative.
 was about 30 scans.

PROCEDURES AND MEASUREMENTS

Weighing and pipetting procedures. Cholesterol (SRM 911b) and the isotopically labeled [25,26,27-[sup.13] [C.sub.3]]cholesterol were weighed on a microbalance (Mikrowaage 708501, Fa. Sartorius). This balance has a weight range of 15 mg, and the certified accuracy is 0.5% at 1 mg and 0.15% at 10 mg, respectively, which was checked with calibrated weight prior to each use. All other weighing procedures, including all required calibrations of volumetric volumetric /vol·u·met·ric/ (vol?u-met´rik) pertaining to or accompanied by measurement in volumes.

vol·u·met·ric
adj.
Of or relating to measurement by volume.
 devices, were done on a semimicrobalance (Halbmikrowaage AC 211 S-OCE, Fa. Sartorius). This balance has a single measuring range up to 210 g with a certified reproducibility of [less than or equal to] [+ or -]0.1 mg and certified linearity deviation of [less than or equal to] [+ or -]0.2 mg. All pipetting procedures were performed with Digital Syringe Series 1700 syringes (Fa. Hamilton). Every volume setting used was calibrated gravimetrically before use.

Sample preparation for calibrators, control materials, and pooled sera. The reconstitution of lyophilized control sera was performed as described previously [1]. The calibrators of cholesterol and [25,26,27-[sup.13] [C.sub.3]]cholesterol with a concentration of 1 mg/mL were prepared fresh every day by dissolving 10 mg of solute solute /so·lute/ (sol´ut) the substance dissolved in solvent to form a solution.

sol·ute
n.
 in 10 mL of ethanol. To minimize the effects of the varying accuracy of the syringe with varying pipetting volume, in all cases 100 [micro]L (corresponding to 100 [micro]g of [25,26,27-[sup.13] [C.sub.3]]cholesterol) of internal standard were pipetted, so only the volume of the unknown sample or calibrator had to be varied.

Internal standards. Aliquots of 100 [micro]L of the internal standard, the labeled [25,26,27-[sup.13][C.sub.3]]cholesterol solution, were placed in Reacti-vial test tubes. Then aliquots of 75 [micro]L (standard 1) or 125 [micro]L (standard 2) of unlabeled cholesterol were added, and the tubes were gently swirled. Two samples were required for the determination of the isotope ratio in the pure unlabeled and the labeled cholesterol. For this we placed 200 [micro]L of unlabeled cholesterol in one vial and 200 [micro]L of the labeled [25,26,27-13 C3]cholesterol in another. The ethanol was removed under a stream of nitrogen at 60[degrees]C. For LC IDMS measurements the residue was dissolved in 100 [micro]L of ethanol. For GC IDMS measurements we dissolved the residue in 50 [micro]L of N-methyl-N-(trimethylsilyl)trifluoroacetamide/pyridine, and the derivatization was performed for 30 min at 60[degrees]C.

Control materials, pooled sera. Aliquots of 100 [micro]L of the internal standard, the labeled [25,26,27-[sup.13][C.sub.3]]cholesterol solution, were placed in test tubes. Then appropriate aliquots of the control material or pooled serum pooled serum
n.
Serum obtained from a number of individuals and mixed together. Also called pooled blood serum.
 were added volumetrically vol·u·met·ric  
adj.
Of or relating to measurement by volume.



[volu(me) + -metric.]


vol
 to give an isotope ratio of ~1.0, and the tubes' contents were gently swirled.

To prepare a set for the total cholesterol determination, we then added to each of the test tubes 150 [micro]L of an aqueous potassium hydroxide potassium hydroxide, chemical compound with formula KOH. Pure potassium hydroxide forms white, deliquescent crystals. For commercial and laboratory use it is usually in the form of white pellets.  solution (8.9 mol/L) and 1 mL of ethanol. This mixture was gently swirled and then heated at 50[degrees]C for 3 h. To check for complete hydrolysis, the hydrolysis was performed in a separate experiment by adding 300 [micro]L of the aqueous 8.9 mol/L potassium hydroxide solution and 1 mL of ethanol, swirling, and finally heating at 50[degrees]C for 6 h. After hydrolysis, 1 mL of deionized water and 2 mL of cyclohexane were added. After continuous shaking for 5 min the cyclohexane phase was transferred to Reacti-vials. The samples were dried and derivatized as described for the internal standard.

Calibration and calculation for the determination of cholesterol in serum with the GC IDMS and LC IDMS methods. For the measurement of the unknown samples, each sample of a control material or serum was measured in triplicate bracketed by triplicate measurements of standard 1 (isotope ratio ~0.75) and standard 2 (isotope ratio ~1.25) in either the order: lower weight ratio standard, sample, higher weight ratio standard. This measurement was then repeated in the reversed order. The three observed intensity ratios were acceptable only if the CV was <0.5%, then they were averaged. If this could not be achieved, the measurement of the standard, control, or unknown sample was discarded. The quantity of analyte in the sample was calculated by linear interpolation Linear interpolation is a method of curve fitting using linear polynomials. It is heavily employed in mathematics (particularly numerical analysis), and numerous applications including computer graphics. It is a simple form of interpolation.  of the measured ratio of the sample between the measured ratios of the standards with the known weight ratios as described elsewhere [6]. In every series two values for each control material or serum sample were obtained by this procedure, and these two values were averaged.

Results

SPECIFICITY AND DETECTION LIMIT OF THE LC IDMS AND GC IDMS METHODS

The mass spectra of cholesterol and [25,26,27-[sup.13] [C.sub.3]]cholesterol obtained for LC-MS in scan mode with 1 scan/s, with the same settings as in the SIM experiments, were identical to those previously published [10]. The spectra show characteristics similar to those published earlier [8]. Fig. 1 shows the UV-chromatogram at a wavelength of 210 nm as well as the SIM chromatograms at the molecular mass ions m/z = 386 and m/z = 389 for an extract of a serum sample. The interferences observable in the UV-chromatogram are not visible in the SIM measurements.

The El ionization was sensitive enough for detection of the underivatized cholesterol, and the peak of the molecular ion was the base peak in the mass spectrum; in the case of the trimethylsilyl-derivatized cholesterol the base peak was at m/z = 329 for the unlabeled cholesterol as expected from previous publications [12], but the detection limit at m/z = 458 was nearly identical to that at m/z = 329. The signal-to-noise ratios were comparable, the retention time of the cholesterol in the LC IDMS method was 5.44 min, and the retention time of the cholesterol derivative in the GC IDMS method was 4.15 min.

We determined the isotope ratios of the pure unlabeled cholesterol and the pure [25,26,27-[sup.13][C.sub.3]]cholesterol from the two samples containing only one type of cholesterol, which were required in the further calculations. The pure unlabeled cholesterol had a peak intensity ratio ([A.sub.389]/ [A.sub.386]) of 0.00242, the trimethylsilyl derivative a peak intensity ratio ([A.sub.461]/[A.sub.458]) of 0.0344; the pure labeled cholesterol had a peak intensity ratio ([A.sub.386]/[A.sub.389]) of 0.00646, the trimethylsilyl derivative a peak intensity ratio ([A.sub.458]/[A.sub.461]) of 0.0175.

[FIGURE 1 OMITTED]

INTERFERENCE OF ENDOGENOUS AND EXOGENOUS STEROIDS

We tested the GC IDMS method and the LC IDMS method for the possible interference of 7-dehydrocholesterol, 5[alpha]-cholest-7-en-3[beta]-ol (lathosterol), lanosterol, [beta]-sitoserol, ergosterol ergosterol /er·gos·te·rol/ (er-gos´te-rol) a sterol occurring mainly in yeast and forming ergocalciferol (vitamin D2) on ultraviolet irradiation or electronic bombardment.

er·gos·ter·ol
n.
, cholest-4,6-dien-3-one, coprostan3-ol, 25-hydroxycholesterol, cholesterol-5[alpha],6[alpha]-epoxide, 4-cholesten-3-one, 5-cholesten-3-one, and dihydrocholesterol. In the GC IDMS method none of the steroids studied interfered, as was expected from earlier studies [12]. In the LC IDMS only lathosterol with a retention time of 5.49 min and a molecular mass of 386.7 could interfere with the determination of the unlabeled cholesterol, the actual signal of lathosterol at m/z = 386 being 30.1% of that of an equal amount of cholesterol. Coprostan-3-ol with a retention time of 5.28 min and a molecular mass of 388.7 could interfere with the determination of the labeled cholesterol, the signal of coprostan-3-ol at m/z = 386 being 1.9% and at m/z = 389 8.9% compared with that of an equal amount of cholesterol. All other steroids tested could not interfere, because they eluted well separated from the cholesterol in HPLC.

MEMORY EFFECTS

We tested the analytical systems used for LC IDMS and GC IDMS for the existence of memory effects. If an unexpected memory effect were present, then the measured isotope ratio of a sample would be influenced by the history of samples measured previously. So we routinely measured on both systems sequences of five determinations of a sample of unlabeled cholesterol, followed by five determinations of the isotope-labeled cholesterol and repeated this all five times. We never observed a drift in the isotope ratios for the unlabeled or the labeled cholesterol with both IDMS methods. If a memory effect were present, then it should be detectable at least in this situation, measuring a sequence of samples with the most extreme isotope ratios possible.

LINEARITY OF THE LC IDMS METHOD

We tested the linearity of the relationship between the mass ratios (c/[c.sup.*]) of unlabeled and labeled cholesterol and the isotope ratio, calculated from the area [A.sub.386] under the peak obtained in the SIM chromatogram chromatogram /chro·mato·gram/ (kro-mat´o-gram) the record produced by chromatography.

chro·mat·o·gram
n.
The pattern of separated substances obtained by chromatography.
 at m/z = 386 and from the area [A.sub.389] under the peak obtained in the SIM chromatogram at m/z = 389. The isotope ratio was corrected for the isotope ratio [f.sub.1] = 0.00646 of the pure labeled cholesterol and the isotope ratio [f.sub.2] = 0.00242 of the pure unlabeled cholesterol, both determined in the previous section, by the formula

([A.sub.386] - [f.sub.1] x [A.sub.389])/([A.sub.389] - [f.sub.2] x [A.sub.386]) (1)

The linearity was tested in the range of mass ratios (c/[c.sub.*]) of unlabeled and labeled cholesterol between 0.25 and 2.0. The data, shown in Fig. 2, demonstrate that linearity is given for isotope ratios between 0.25 and 2.0. The relationship is Y = (-0.0005 [+ or -] 0.0116) + (1.1404 [+ or -] 0.0094) x (c/[c.sup.*]) with a correlation coefficient Correlation Coefficient

A measure that determines the degree to which two variable's movements are associated.

The correlation coefficient is calculated as:
 of r2 = 0.9998, the intercept of the regression line Noun 1. regression line - a smooth curve fitted to the set of paired data in regression analysis; for linear regression the curve is a straight line
regression curve
 is not significantly deviating from the origin, and there was no intrinsic nonlinearity observable in the range of mass ratios tested.

STANDARD STABILITY

The stability of the mass ratio between days was calculated from the measured, corrected isotope ratios Y found for the lower and for the higher standard. The statistic was done for the first measurement of the standards on each day. For the lower standard we found for 20 days a mean of 0.7514 and a SD of 0.0021 (CV = 0.28%); for the higher standard we found under the same conditions a mean of 1.2529 and a SD of 0.0033 (CV = 0.26%).

[FIGURE 2 OMITTED]

STANDARD CONSISTENCY

For the assessment of the standard consistency we used every day two sets of two standards, one set for the calibration of the method and an independent second set with the same isotope ratios for measuring standard recovery. The second set of standards was treated like unknown samples, and the results were calculated as for unknown samples. The data were evaluated as described elsewhere [5]. In the LC IDMS we never found differences between the calculated mass ratio (c/[c.sup.*]) of unlabeled and labeled cholesterol and the weighed-in mass ratio (c/[c.sup.*]) of unlabeled and labeled cholesterol >0.20%.

SAMPLE PREPARATION

The hydrolysis procedure used in this work is similar to that described elsewhere [6,10]. The main difference is the much larger excess of hydrolyzing reagent used here. So we only checked whether the simultaneous doubling of the hydrolyzing reagent and the hydrolysis time had an impact on the results obtained. Each sample was hydrolyzed in a separate experiment with a doubled amount of hydrolyzing reagent and was incubated for 6 instead of 3 h. In no case could a difference be observed between the results obtained for both hydrolysis procedures.

We also checked for the possible influence of the sample preparation method on the standards. We performed experiments in which we prepared standards like unknown samples. No differences could be observed between the measured isotope ratios of the standards used for calibration and prepared normally and the standards prepared like serum samples or control materials.

PRECISION AND ACCURACY OF THE LC IDMS METHOD

Table 1 shows the day-to-day precision for five different serum-based control materials, obtained from 10 independent series on separate days. For the three control materials obtained from NIST, the CVs are <1.0%. For the other control materials the imprecision is slightly >1.0%. This could be caused by the vial-to-vial variabilities of the control material rather than by LC IDMS method itself. The control materials obtained from NIST were also used as controls for the GC IDMS method. In that case CVs <1.0% were obtained also.

The accuracy of the LC IDMS method for the determination of total cholesterol in serum was checked with the five different serum-based control materials, for which the target values were based on Definitive Methods in the case of the NIST materials and on GC IDMS Reference Method-based target values supplied by the manufacturer. For all tested control materials the bias (from 10 independent series on separate days) was <1.0% and in all cases lower than the CV. In the GC IDMS method, the bias of the three control materials SRM 909, SRM B1, and SRM B2 was <1.0% as well.

For the method comparison 28 pooled human sera were used and measured independently with the LC IDMS method presented and GC IDMS as a Reference Method. Fig. 3 shows the CVs, obtained for each serum pool from five determinations with both methods. The mean imprecision was 0.66% (range 0.26-1.21%) for the GC IDMS method and 0.72% (range 0.31-1.17%) for the LC IDMS method. The mean results for the 28 pooled human sera, obtained by our LC IDMS method, were compared with the chosen GC IDMS Reference Method. Fig. 4a shows the correlation of the results including the graph of the following linear relationship obtained by the method of Passing and Bablok [13]: [C.sub.Lc IDMs] = 0.993 X [C.sub.GC IDMs] - 0.15 mg/L; 95% confidence interval confidence interval,
n a statistical device used to determine the range within which an acceptable datum would fall. Confidence intervals are usually expressed in percentages, typically 95% or 99%.
 for the slope: 0.978-1.008; 95% confidence interval for the intercept: -25.8 mg/L to +25.9 mg/L; correlation coefficient r: 1.000; standard error of the estimates [S.sub.y|x]: 1.375.

[FIGURE 3 OMITTED]

In Fig. 4b the relative deviations of the results obtained with the LC IDMS method from those obtained with the GC IDMS Reference Method [11] are presented graphically. For 32% of the samples the relative deviation between the LC IDMS and the GC IDMS method was <0.5%; for 93% of the samples the relative deviation did not exceed 2.0%.

Discussion

The aim of this study was the evaluation of an isotope dilution method for the determination of total cholesterol in serum by HPLC-particle beam-El-mass spectrometry as an analytical method. This type of coupling liquid chromatography to mass spectrometry has not been used for the development of isotope dilution methods before. HPLC separations of cholesterol have been described before [10], so the only problem was the compatibility of the eluent system with a particle-beam interface used for coupling the liquid chromatograph with the mass spectrometer [14]. After optimization of the pneumatic and thermal variables of the particle-beam interface, the El ionization was sensitive enough for the detection of the underivatized cholesterol. As has been already expected from the results presented [10], the peak of the cholesterol in SIM chromatograms obtained by the LC IDMS is much broader compared with that obtained by GC IDMS. On the other hand, in both methods neither the peak shape nor the baseline noise deteriorated the quality of the quantification process. A great disadvantage of the LC IDMS is that much more analyte has to be introduced in the instrument. This is most likely because of the particle-beam interface and will limit the range of applicability of the LC IDMS approach presented in this work. The study of possible interference of exogenous or endogenous steroids showed that the cholesterol precursor lathosterol is the most interfering steroid, but its concentration in serum is <0.3% of the concentration of the cholesterol itself [15]. Because the relative intensity of lathosterol at m/z = 386 is only 30% of that obtained for an equal amount of cholesterol, this interference is not of practical significance. Coprostanol had even a lower relative intensity compared with cholesterol and, like lathosterol, has not been reported to be present in concentrations in serum that would alter the results obtained by the CDC See Control Data, century date change and Back Orifice.

CDC - Control Data Corporation
 Reference Method for cholesterol [16].

[FIGURE 4 OMITTED]

As has been stated X10], the use of an isotope-labeled internal standard differing only by 2 amu from the unlabeled cholesterol leads to a nonlinearity between the corrected peak intensity ratio Y and (c/[c.sup.*]). The use of an internal standard differing by 3 amu from the unlabeled compound prevents this effect nearly completely.

We chose two standards and bracketing as the calibration method, as described previously [6]. The use of two standards proved to be sufficient, the standards were consistent within-run, and the isotope ratios obtained for the standards were stable for at least 1 month with a CV <0.3% in the measured isotope ratios. The sample preparation technique did not influence the measurement of isotope ratios of standards prepared like serum samples. As stated earlier [6], even shorter periods of time for hydrolysis and lower concentrations of the hydrolyzing reagent lead to a total hydrolysis of cholesterol esters. We did not observe any differences between aliquots hydrolyzed under different conditions. The error introduced by the uncertainty of the used standard materials as well as the error introduced by the weighing procedure are in the order of magnitude A change in quantity or volume as measured by the decimal point. For example, from tens to hundreds is one order of magnitude. Tens to thousands is two orders of magnitude; tens to millions is three orders of magnitude, etc.  of 0.1-0.2%. Overall the imprecision of the LC IDMS method was [less than or equal to] 1.2% for all control materials studied, <1% for NIST materials. For all control materials the bias was lower than the CV of the results. Furthermore in the case of the NIST control sera with certified uncertainty of the target value, the bias found was lower than this uncertainty, which represents the highest bias allowed (Table 1).

The new method was tested against an GC IDMS Reference Method for the determination of total cholesterol in serum. For 28 pooled human sera we found a very good correlation between the results obtained with the new method and the Reference Method. For 93% of the samples the difference of the results for both analytical methods did not exceed 2.0%. Neither the imprecision nor the bias between the methods depended on the concentration of the pooled serum samples used. Even for the highest concentration sample (4351 mg/L) measured by LC IDMS, the bias was 2.04% and was therefore not excluded from the statistical analysis; the relevance of a precise measurement in this range of concentrations is nevertheless limited. The between-run imprecisions for these serum pools did not exceed those obtained for the control materials.

In this study a new analytical technique was used for the development of an isotope dilution method. The liquid chromatographic separation method is applicable to a wide variety of dissolved organic molecules [14], and no derivatization procedure is required previous to analysis, making the sample preparation more simple than in the GC-MS method. The comparison of accuracy and precision of the new LC IDMS method with an GC IDMS method chosen as reference did not show any substantial advantage of the GC IDMS method. The method presented in this study may be considered as a prototype of isotope dilution methods applicable to analytes for which the sample preparation required for gas chromatography is complicated or a volatile derivative does not exist.

References

[1.] Boutwell JH, ed. Proceedings of a conference on national understanding for the development of reference materials and methods in clinical chemistry. Washington DC: AACC, 1978.

[2.] Stamm D. Reference materials and reference methods in clinical chemistry. J Clin Chem Clin Biochem 1979;17:283-97.

[3.] NRSCC NRSCC National Remote Sensing Center of China 1-T. Tentative guidelines for the development of Definitive Methods in clinical chemistry for the National Reference System in Clinical Chemistry. Villanova, PA: National Committee for Clinical Laboratory Standards, 1982.

[4.] Cohen cohen
 or kohen

(Hebrew: “priest”) Jewish priest descended from Zadok (a descendant of Aaron), priest at the First Temple of Jerusalem. The biblical priesthood was hereditary and male.
 A, Hertz HS, Mandel J, Paule RC, Schaffer R, Sniegoski LT, et al. Total serum cholesterol by isotope dilution mass spectrometry: a candidate definitive method. Clin Chem 1980;26:854-60.

[5.] Ellerbe P, Meiselman S, Sniegoski LT, Welch MJ, White E V. Determination of serum cholesterol by a modification of the isotope dilution mass spectrometric definitive method. Anal Chem 1989;61:1710-23.

[6.] Pelletier 0, Wright LA, Breckenridge WC. Isotope dilution/mass spectrometry of serum cholesterol with [3,4-[sup.13]C]cholesterol: proposed definitive method. Clin Chem 1987;33:1403-11.

[7.] Bjorkhem I, Blomstrand R, Svensson L. Serum cholesterol determination by mass fragmentography. Clin Chim Acta 1974;54: 185-93.

[8.] Siekmann L, Huskes KP, Breuer H. Determination of cholesterol in serum using mass fragmentography--a Reference Method in clinical chemistry. Z Anal Chem 1976;279:145-6.

[9.] Duncan IW, Culbreth PH, Burtis CA. Determination of free, total, and esterified cholesterol by high-performance liquid chromatography. J Chromatogr 1979;162:281-92.

[10.] Takatsu A, Nishi S. Total cholesterol by isotope dilution/mass spectrometry, with liquid chromatographic separation. Clin Chem 1987;33:1113-7.

[11.] Siekmann L. Reference Methods for total cholesterol and total glycerol glycerol, glycerin, glycerine, or 1,2,3-propanetriol (prō`pāntrī'ŏl), CH2OHCHOHCH2OH, colorless, odorless, sweet-tasting, syrupy liquid. . Eur J Clin Chem Clin Biochem 1991;29:277-9.

[12.] Kinter M, Herold DA, Hundley J, Wills MR, Savory J. Measurement of cholesterol in serum by gas chromatography/mass spectrometry at moderate mass resolution with a nonendogenous cholesterol isomer as internal standard. Clin Chem 1988;34:531-4.

[13.] Passing M, Bablok W. A new biometrical procedure for testing the equality of measurements from two different analytical methods. Application of linear regression Linear regression

A statistical technique for fitting a straight line to a set of data points.
 procedures for method comparison studies in clinical chemistry. J Clin Chem Clin Biochem 1983;21:709-20.

[14.] Creaser CS, Stygall JW. Particle beam particle beam
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A beam of atoms or subatomic particles that have been accelerated by a particle accelerating device, aimed by magnets, and focused by a lens.

Noun 1.
 liquid chromatographymass spectrometry: instrumentation and applications [Review]. Analyst 1993;118:1467-80.

[15.] Duane WC. Serum lathosterol levels in human subjects reflect changes in whole body cholesterol synthesis induced by lovastatin lovastatin /lo·va·stat·in/ (lo´vah-stat?in) an antihyperlipidemic agent that acts by inhibiting cholesterol synthesis, used in the treatment of hypercholesterolemia and other forms of dyslipidemia and to lower the risks associated with  but not dietary cholesterol. J Lipid Res 1995;36:343-8.

[16.] Ellerbe P, Myers GL, Cooper G, Hertz HS, Sniegoski LT, Welch MJ, White E. A comparison of results for cholesterol in human serum obtained by the Reference Method and by the Definitive Method of the National Reference System for Cholesterol. Clin Chem 1990; 36:370-5.

RUEDIGER KOCK,* BERT (Bit Error Rate Test) An analysis of network transmission efficiency that computes the percentage of bits received in error from the total number sent.  DELVOUX, and HELMUT GREILING

Institute for Clinical Chemistry and Pathobiochemistry, Medical Faculty, University of Technology Aachen, Pauwelsstr. 30, D-52057 Aachen, Germany.

* Author for correspondence. Fax 49-241-88-88-512.

(1) Nonstandard non·stan·dard  
adj.
1. Varying from or not adhering to the standard: nonstandard lengths of board.

2.
 abbreviations: LC IDMS, liquid chromatography isotope dilution mass spectrometry; GC IDMS, gas chromatography isotope dilution mass spectrometry; SIM, selective ion monitoring; El, electron impact; GC-MS, gas chromatography mass spectrometry.

Received March 25, 1997; revision accepted June 6, 1997.
Table 1. Day-to-day precision and accuracy of the GC IDMS and the
new LC IDMS method for the determination of total cholesterol in
five different control materials in n = 10 independent series on
separate days.

                        Target value

 Control material    mg/L   Uncertainty, %

SRM 909              1416        3.25
SRM 909b grade 1     1464        1.23
SRM 909b grade 2     2353        1.28
Precinorm U          1200       na (a)
Kontrollogen-LP      1330         na

                                GC IDMS method

 Control material    mg/L, (mean [+ or -] SD)   CV, %   Bias, %

SRM 909                   1410 [+ or -] 6        0.56     0.42
SRM 909b grade 1          1459 [+ or -] 8        0.55     0.34
SRM 909b grade 2          2342 [+ or -] 12       0.51     0.47
Precinorm U                       na              na       na
Kontrollogen-LP                   na              na       na

                                    LC IDMS method

 Control material    mg/L, (mean [+ or -] SD)   CV, %   Bias, %

SRM 909                   1420 [+ or -] 8        0.56     0.28
SRM 909b grade 1          1469 [+ or -] 13       0.89     0.34
SRM 909b grade 2          2337 [+ or -] 22       0.93     0.68
Precinorm U               1201 [+ or -] 14       1.17     0.08
Kontrollogen-LP           1320 [+ or -] 17       1.29     0.75

(a) na, not applicable.

The uncertainty (%) has been calculated from the absolute uncertainty
of the target value certified for the NIST materials. The bias is
calculated as: bias in % = 100 x [absolute value of [C.sub.mean],
GC IDMS or LC IDMS] - [C.sub.targetvaluel]]/[C.sub.target value].
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Title Annotation:Lipids and Lipoproteins
Author:Kock, Ruediger; Delvoux, Bert; Greiling, Helmut
Publication:Clinical Chemistry
Date:Oct 1, 1997
Words:5541
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