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Gas Chromatographic
Chromatographic Analysis
Analysis of Sterols
of Plant Plant Sterols

Selected Topics in the Analysis of Lipids

GAS CHROMATOGRAPHIC
ANALYSIS OF PLANT STEROLS

1. Introduction
Jill Winkler-Moser 
Plant sterols (phytosterols) are ubiquitous in plants, and can be classified
either as 4-desmethylsterols, 4-methylsterols, or 4,4’-dimethylsterols. The
phytosterols found in the highest abundance in most plants are sitosterol, campesterol, and stigmasterol
which are all classified as 4-desmethylsterols (Figure 1). However, there are reportedly over 200 different
sterol structures that have been discovered in various plant species [1]. Plant sterols can exist in plants in
their free form, as esters with fatty acids, ferulic acid, or p-coumaric acid, or as glycosides and acylated s tery
glycosides. The most common phytosterols have a double bond at position 5 of the B-ring, (commonly
referred to as Δ5), however, Δ7-phytosterols are also found in many seed oils, while cereals such as corn
wheat, rice, rye, and triticale als o contain appreciable amou nts of saturated sterols , or phytostanols, such as
sitostanol and campestanol [2-4]. See also web pages on plant sterols and oxidized plant sterols
elsewhere on this site.

Figure 1. Structures of som e comm on phytosterols and phytostanols

Phytosterols are well-known for their ability to lower blood cholesterol by competing with absorption o
cholesterol from the diet and reabsorption of bile cholesterol [5]. Phytosterols as food ingredients are
“Generally Recognized As Safe” (GRAS) by the FDA, and they are increasingly incorporated into various
products as functional food ingredients. In the year 2000, the FDA approved a health claim relating
phytosterol es ter or phytostanol es ter consumption to reduced ris k for coronary heart diseas e, in foods such
as margarines and spreads, salad dressings, snack bars, and dietary supplements [6]. However, the FDA
has recently proposed to extend the health claim to include free phytosterols and an increased number of
conventional foods [7]. The European Union (EU) has also approved similar health claims, and has
established labeling guidelines for these foods [8]. Labeling requirement by both the FDA and the EU
include a declaration of the content of plant sterols or s terol esters, and include specifications for phytostero
or phytostanol composition of the esters. Thus, there is increased interest in qualitative and quantitative
analysis of phytosterols in food products.

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Gas chromatography (GC) is the most common method for analyzing phytosterol content and composition
[9]. There are HPLC m ethods available for sep arating and quantifying the various forms of phytosterols, s uch
as free sterols , steryl fatty acid es ters, s teryl glycosides , acylated s teryl glycosides , and hydroxycinnamic acid
esters of sterols [1,10]. However, with over 200 different structures, GC, often combined with mass
spectrometry, is the best and most widely used tool for the chromatographic separation, identification, and
quantification of phytosterols. There are several standardized methods for phytosterol analysis that are
approved by organizations such as the German government [11], the Association of Official Analytica
Chemists (AOAC) [12], American Oil Chemists’ Society (AOCS) [13], and the International Organization for
Standardization (ISO) [14]. Most standardized methods are somewhat tedious in terms of the amount of

glassware, sample, and solvent required and in the number of steps in the procedure, which makes i
difficult to analyze multiple samples at the same time. These procedures have been modified by users to
require smaller sample sizes, less solvent, and to eliminate some of the time-consuming clean-up steps
[15]. However, for the most part, the latest techniques employ the same basic steps that the traditional
methods use: (1) Sample preparation (sample weighing, lipid extraction (optional), addition of interna
standard, acid and/or alkaline hydrolysis, extraction of unsaponifiables, optional sample clean-up/furthe
purification, (2) derivatization, and (3) GC analysis. The intent of this chapter is to describe these steps in
both an informative and practical manner.

2. Sample Preparation

The first and most important step in the analysis of phytosterols is sample preparation. Since phytosterols
are a minor component that typically comprises less than 1% of the matrix (but up to 8% in food products
with added phytosterols), the goals of s ample preparation are to is olate the sterol fraction and to convert al
conjugated or esterified phytosterols into free phytosterols for GC analysis. The type of sample preparation
needed depends on the sample matrix (whole plant tissue or extracts, food product, supplement, vegetable
oil, blood serum, etc…) and also on the original form of the phytosterol in the sample. In some samples,
such as blood serum, where phytosterols are present in very low concentrations, lipid extraction to
concentrate the phytosterol-containing lipid fraction away from proteins and other compounds may be a
necessary first step [16]. In other cases, the analyst may want to quantify other lipids in the sample, or the
various forms of phytosterols, i.e. phytosterol ferulates, phytosterol esters, free phytosterols, so a total lipid
extract will also be needed. However, for many matrices, direct alkaline or acid hydrolysis has been used
succes sfully [15,17]. If free sterols and s terol esters are the only sterol forms anticipated in the sam ple, such
as in a refined oil or fat sample, then alkaline hydrolysis (saponification), followed by extraction of the
unsaponifiable material, is sufficient sample preparation for analysis of total phytosterols. However, in
samples such as cereal products, the phytosterols may be trapped within the carbohydrate matrix, or may
have bonds that aren’t hydrolyzed by saponification, such as the glycosidic bonds in steryl glycosides. In this
case, acid hydrolysis prior to saponification is n ecess ary [17].

2a. Sa mple Amount

The sample amount is dependent on the concentration of phytosterols in the sample, the ability to
completely hydrolyze the sample and liberate all of the phytosterols, and the quantification limits of the GC
Therefore, sample weight needs to be optimized for each product. An estimate of the phytosterol
concentration in a sample can usually be determined based on a survey of the literature for the same or
similar sample types. Samples containing at least 50-150 µg of a mixture of phytosterols can easily be
analyzed [18]. Laakso [15] suggests a sample size containing 1 mg phytosterols, but their method is
targeted for foods that are supplem ented with high concentrations of phytosterols. Phillips et al . [16] reported
limi ts of quantitation for individual phytosterols in s erum s amples ranging from 0.2-1.2 µg/ml, corresponding
to injected concentrations that ranged from 1.8-10.8 µg/ml (1.8-10.8 ng/ 1 µl i njection).

2b. Internal Standard

For phytosterol analysis, the addition of an internal s tandard (IS) is neces sary to obtain accurate qual itative
and quantitative results by GC. Since retention tim es o ften sh ift from run to run, the main function of the IS is
to aid in the identification of phytosterols in unknown sam ples , bas ed on the relative retention time (RRT) o
the phytosterol compared to the internal s tandard. In addition, when us ing a s plit injection, which is typically
the case in phytosterol analysis, the amount of sam ple entering the colum n will vary slig htly from injection to
injection, so the IS is us ed to compensate for this variation. Finally, large variations in peak res ponse (area

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for the IS is a good indicator that there is a problem with the extraction protocol or with the GC injection.

The IS should be a compound that is commercially available in pure form, and that is similar in structure to
phytosterols so that it will be extracted quantitatively with the sample phytosterols, and elute from the GC
column within the time frame and with a similar detector response to other phytosterols, without interfering
with the retention time of sample peaks. The IS is typically added to the sample prior to alkaline or acidic
hydrolysis , so that it undergoes the sam e extraction conditions as the samp le phytosterols. However, the IS
may also be added after extraction but prior to derivatization.

The most common internal standards for phytosterol quantification are 5α-cholestane [18]
dihydrocholesterol (5α-cholestan-3β-ol)[19], epicoprostanol (5β-cholestan-3α-ol) [15], and betulin (Lup-
20(29)-ene-3β,28-diol)[11] (Figure 2).

Figure 2. Structure of sterols and other compounds commonly used as internal standards.

Epicholesterol (5α-cholesten-3β-ol) has also been used as an IS [16]. 5α-cholestane does not have a
hydroxyl group, so it cannot be si lylated. The advantage of us ing 5α-cholestane a s a s tandard is that it elutes

early so there is little chance of interference with other sterols, and the detector response is very similar to
most phytosterols. However, if there is a problem with the silylation reaction where samples are
incompletely silylated, cholestane will not be affected and therefore will not serve as an indicator of the
problem. Betulin (Lup-20(29)-ene-3β,28-diol) is used by the standardized German method [11], but it has
two hydroxyl groups, s o it may require a longe r time for com plete silylation and it elutes much later than the
other sterols, prolonging analysis times. Dihydrocholesterol is a good choice, but it is a hydrogenation
product of cholesterol, so it could interfere with this peak in sam ples that contain cholesterol and ha ve been
subjected to hydrogenation. Epicoprostanol does not occur naturally in plants, elutes early, and thus does
not usu ally interfere with phytosterol peaks [15].

2c. Acid Hydrolysis

If the sample has a complex protein or carbohydrate matrix and/or contains steryl glycosides, acidic

hydrolysis is necess ary to free the s terols from the glycosidic bond or from the surrounding m atrix [15,17]. In
this case, the IS is added to the weighed sample, the sample is hydrolyzed with hydrochloric acid (~3.5 M)
with reflux at 100ºC, followed by lipid extraction using a non-polar solvent. After solvent removal by rotary
evaporation, saponification of the lipid sample proceeds as described below.

2d. Saponification

Saponification without acid hydrolysis is sufficient sample preparation in refined oil/fat samples or lipid
extracts, where the s terols primarily exist as free s terols or s teryl esters. Sterol esters are hydrolyzed to free
sterols, and the sterols are concentrated in the unsaponifiables fraction, while the triacylglycerols are
converted to fatty acid salts. Under typical hydrolysis conditions, 1-2.5 N ethanolic or methanolic KOH is
added directly to the oil s ample plus IS (or to a lipi d extract obtained after acid hydrolysis), and the reaction is
heated at 60ºC to 80ºC, either in a capped test tube or in a round b ottom flask with a reflux condens er, for 1

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Analysis of Sterols
of Plant Plant Sterols

hr or until completely saponified [11-19]. After saponification, water and/or saturated sodium chloride are
added and the unsa ponifiable material is extracted by mixing vigorously or vortexing with a non-polar sol ven
such as hexane, heptanes, diethyl ether, chloroform, or mixtures thereof. When the phases separate
(centrifugation facilitates phase separation), the fatty acid salts will partition to the polar phase while the
unsaponifiables will remain in the non-polar phase. The extraction of the polar phase with a non-polar
solvent is usually repeated at least once to maximize yield. Rigorous extractions include washing the non-
polar phase with water or saturated salt solutions, but most researchers have been able to omit these time-
and sol vent-consumin g steps and s till obtain quantitative yields . Non-polar extracts are combined, and dried
under a stream of nitrogen or by rotevaporation, and redissolved in an appropriate amount of solvent for

either cleanup by thin-layer chromatography (TLC) or solid-phase extraction (SPE), or directly fo

derivatization. As an alternative to liqui d-liquid extraction of uns aponifiables, the German method [11] us es
aluminium oxide columns to fractionate the unsaponifiables. The soap solution in ethanol is applied to 10 g
alumi na columns and si mply eluted with diethyl ether. In another procedure, saponified oil abs orbed to silica
gel was layered onto a column containing a layer of sodium sulfate and a layer of silica, and the
unsap onifiable material was eluted with a mixture of tert -butylmethyl ether a nd e thyl acetate (1:1, v/v) [20].

2e. Clean-Up and Further Purification of Unsaponifiable Lipids

 After sa poni fication, the lipi d extract, calle d “uns aponi fiables ”, ma y contain other lipi ds bes ides stero ls
including hydrocarbons, carotenoids, tocopherols , free fatty acids, and o ther triterpenes. Many researchers
proceed with derivatization and GC analysis without further sample cleanup and do not have problems with
interference by these compounds. This is usually acceptable for the unsaponifiables fraction of a refined fa

or oil sam ple. However, in some s ampl es, s uch as crude oils, it may be necess ary to further purify the stero
fraction from polar uns aponifiable lipids . Silica, C18, and am inopropyl SPE cartridges have all bee n used fo
this purpose. Phillips and co-workers [16] used aminopropyl SPE columns to separate sterols and stanols
from serum unsaponifiable lipids, using chloroform: isopropanol for elution. Toivo and co-workers [17]
demonstrated that equal yields were obtained using either C18 or silica SPE columns and eluting the stero
fraction from ei ther column wi th 5% methanol in chloroform or 1% isop ropanol in hexane, respectively.

Separation of 4-desmethylsterols, 4-methylsterols, and 4,4’-dimethylsterols. The 4,4’-

dimethylsterols and 4-methylsterols are biosynthetic precursors to 4-desmethylsterols, and
thus are usually present at lower levels in mature plant tissues. However, some oils such as
linseed and olive, have relatively high content of 4,4-dimethylsterols [21]. Some of these
compounds co-elute on GC columns with 4-desmethylsterols, necessitating separation of the
three fractions. Column chromatography or TLC were used to isolate the three phytosterol
classes prior to the invention of disposable SPE columns. For example, neutral alumina
columns (30g) were used to chromatograph 1 g unsaponifiable fractions into 4,4’-
dimethylsterols, 4-methylsterols, and 4-desmethylsterols by sequential elution with 80:20
(v/v), 70:30, and 60:40 mixtures of hexane/diethyl ether [22]. The three phytosterol classes can
also be separated into three different zones on a silica TLC plate by developing twice in
hexane/diethyl ether/acetic acid (70:30:1, v/v) [21] or by one development on with a 1:1 (v/v)
mixture of hexane:diethyl ether [11].

Column chromatography can take time to set up so is not very amenable to running multiple
sam ples. TLC is quicker and eas ier to run, but yields can often be low. Therefore, Azadmard-
Damirchi and Dutta [23,24] developed a method for separation of the three phytosterol
classes using disposable silica SPE columns and sequential elution with hexane: diethyl
ether mixtures of increas ing polarity. This method resulted in m uch higher recovery compared
to traditional TLC.

Separation of Δ5-phytosterols from Δ7-phytosterols. Separation of Δ5-sterols from Δ7-

sterols can be difficult when they are both present in significant quantities [20]. Zhang and
coworkers [25] developed a silica gel column chromatography method to sequentially elute
 Δ7-4-m ethylsterols , Δ5-4-des me thyls terols , and Δ7-4-desm ethylsterol s. This me thod could
likely be adapted for use w ith SPE column s.

2f. Derivatization

Sterols are us ually analyzed as either trimethylsilyl (TMS) ethers or as s terol acetates, which improves thei
volatility, peak shape, and response factors. Injection of free sterols results in broader peaks and a lower

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Analysis of Sterols
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FID respons e. Acetylation with acetic anhydride was comm on in the pas t, but s ilylation is the m ore common
derivatization method these days. One silylation reagent used is N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA), often with added 1% trimethylchlorosilane, which acts as a catalyst to improve the reactivity o
hindered hydroxyl groups. N -methyltrimethylsilyltrifluoroacetamid e (MSTFA) is also used, as well as mixtures
of hexamethyldisilazane (HMDS) with TMCS (Tri-Sil HTP™, Thermo-Fisher Scientific, or Hydrox-Sil™,Regis
Morton Grove, IL). Note that while there are several different Tri-Sil mixtures available with differing
compositions (Tri-Sil HTP is HMDS:TMCS:pyridine, Tri-Sil BP is N,O-bis(trimethylsilyl)acetamide
(BSA):pyridine, Tri-Sil TBT is trimethylsilylimidazole (TMSI):BSA:TMCS, and Tri-Sil TP is TMSI:pyridine), in the
literature the reagent is often referred to as 'Tri-Sil', without specifying which variation was used.

 A basic silylation procedu re using BSTFA:TMCS is to mix <1 to 5 mg purified phytosterols or u nsapon ifiable
material, with 0.2 ml of a 1:1 mixture of pyridine and BSTFA+1%TMCS (or other silylation reagent) in a
capped tube or vial. The reaction can be heated to 60ºC to 70ºC for anywhere up to an hour. We have found
optimal condi tions to be 60ºC for 1 hr (18). However, with s ilylation m ixtures containing HMDS with TMCS
such as Tri-Sil HTP™ and Hydrox-Sil™, derivatization reactions may proceed completely in five to fifteen
minutes at room temperature, according to the product brochures. However, if the hydroxyl group on the
sterols are hindered in a ny way, the mixture m ay need to be he ated to complete the reaction. We have found
anecdotally, that some s terols such as ergos terol and 4,4-dimethylsterols see m to take longer to completely
derivatize than others. If silylation is not complete, two peaks will elute in the chromatogram , rather than one
Since silylation reagents are available in several forms and from various manufacturers, it is important to
optimize the silylation conditions according to the manufacturer ins tructions, the s terols being analyzed, and
the conditions of each individual laboratory.

3. GC Analysis

Once derivatized, phytosterols can immediately be analyzed by GC, or diluted in an organic solvent to an
appropriate concentration and then injected. The TMS-ethers are known to hydrolyze over time, so they
shoul d be analyzed within a few da ys. Although the derivatization reagents elute early and do not interfere
with peaks of interest, we have found that they quickly foul and eventually plug GC injector split ins erts. This
can be prevented by carefully evaporating the derivatization reagents with nitrogen at low temperatures to
prevent any phytosterol ethers from evaporating as well, then redissolving the phytosterols in chloroform
before injecting. This is a tedious extra s tep, but it can prevent instrument downtim e and data los s.

3a. Column Phase

In the past, packed glass GC columns were used for phytosterol analysis, but this column technology has
been replaced with fused s ilica capillary columns due to im provements i n res olution and s ensi tivity. Various
column phases have been used to analyze phytosterols as reviewed by Abidi [10]. In general, most labs
utilize polysiloxane phases of low to mid-polarity. The most common column phase used for general
phytosterol analysis is composed of 95% dimethyl-, 5% diphenyl-polysiloxane, which is available from a
number of manufacturers (RTX-5/Restek, DB-5/J&W, SPB-5 or PTE-5/Supelco, HP-5/Agilent). A slightly more
non-polar phase of 100% dimethyl polysiloxane (RTX-1, DB-1, SPB-1, HP-1, etc.) can be used, but
separation of a sterol peak from its respective stanol peak is not easily achieved with this column phase. A
mid-polarity column may be useful to achieve better resolution of sterols and stanols, or of Δ5-sterols from
 Δ7-s terols . For example , a mi d-pola rity DB-1701 (14% cyanopropyl-phenyl-me thylpolysiloxane) was

suggested by Dutta and Normén [26] for better resolution of Δ5-saturated and unsaturated sterols. We and
others h ave found that baseline separation of s terol/stanol peaks can be achieved with a 95% dimethyl, 5%
diphenyl-polysil oxane column, but sitos terol and Δ5-avenasterol overlap to s ome extent [15,30] (Figure 3). A
higher polarity column may be necessary for the samples that are high in Δ7-sterols. For example, a fused
silica capillary column with a 65% dimethyl-35% diphenyl polysiloxane phase (DB-35) was used to analyze
phytosterols in pumpkin s eed oil, which are mainly compos ed of Δ7-sterols [20,27].

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Figure 3. GC Chromatogram s of TMS-ether Derivatives of Phytosterols on a DB-5 capillary

column (30m x 0.25 m m i.d. x 0.25 µm ). A. A mixture of standards , B. Phytosterols in oil
extracted from corn dis tillers dried grains . Cholestane was the IS. GC conditions are
described in [30].

3b. Chromatography Conditions

Every analyst will likely need to optimize GC conditions depending on their analytical needs , equipment, and
sam ple type. In general, a typical phytosterol chrom atography method will include a spli t injection (with spl i
ratios varying from 1:15 to 1:100), injector temperature from 250ºC-300ºC, initial column temperature, 250
300ºC, and either isocratic or temperature programmed oven heating, and flame ionization detection (FID)
with the detector temperature set to 28 0ºC-325ºC (Table 1).

Table 1. Column and GC Conditions Used for Phytosterol Analysis

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a
Reference number 
b
Column dimensions are length x inner diameter x column phase thickness

3c. Elution Order 

The elution order is influenced by many structural features s uch as the number of carbons on the side chain
the degree of saturation and location of double bonds in the rings and/or in the side chain, their orientation
(cis vs trans), and the number of methyl groups at the 4-position [10,28,29]. For example, in general
phytosterols with an ethyl group at the 24-position in the side chain will have longer retention times than
those with a methyl group, and phytosterols with a double bond at the 5-position elute before the saturated
counterpart, and before the counterpart with a double bond at the 7-position. In addition, sterols with a
double bond at the 22-position elute earlier than sterols with no double bonds in the side chain. The elution
order for some of the most common 4-desmethylsterols is: cholesterol < brassicasterol < campesterol <
campes tanol < stigma sterol < si tosterol < sitos tanol < Δ5-avenas terol (Figure 3).

3d. Identification

Phytosterol peaks are identified using flam e-ionization detection (FID) by comparing the retention times (RT

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of sterol peaks to that of pure standards. A few sterol standards including cholesterol and cholestero
derivatives and analogues, campesterol, campestanol, stigmasterol, sitosterol, sitostanol, brassicasterol
fucosterol, cycloartenol, and ergosterol are available from commercial vendors such as Sigma, Steraloids
Matreya, Research Plus, and Avanti Polar Lipids, to name a few. Most of the less common phytosterols are
not comme rcially available. In the absen ce of pure standards , identification of unknown phytosterol peaks in
a sample can sometimes be a puzzle. However, using a variety of techniques, analysts can usually piece
together enough information to identify unknown peaks with some confidence. First, the elution
characteristics and relative retention times (RRT) can be us ed to give clues about the s tructural features and
identity of unknown peaks [28]. RRT for many phytosterols are found in the literature, but differences in the

internal s tandard used, GC conditions, and form of phytosterol (free, TMS-ether, or acetate) may com plicate
usin g this data alone to identify an unknown peak.

Therefore, GC-mass spectrometry (GC-MS) is a valuable aid for identifying unknown phytosterol peaks
[31,32] as well as for confirming the identification and purity of identified phytosterols. Electron impact (EI)
ionization in the positive mode, usually with an ionizing voltage of 70 eV, is the most common ionization
method us ed for GC-MS analysis of phytosterols. With free s terols and TMS-ethers of s terols, this technique
usually gives the molecular ion as well as characteristic ion fragments that are useful for identification
[31,32]. The mas s s pectra obtained are usual ly fairly consis tent, so that comparis ons can be m ade with data
obtained in different laboratories. A few representative mass spectra are available on this webs ite here...

3e. Methods of Quantification

Most m odern GC control and analysis software packages have built-in capabilities to automatically integrate
peaks, generate standard curves or response factors, and provide users with output that has already been
integrated and calculated based on the standard curves or response factor. As mentioned above
quantification of phytosterols by GC is mos t accurate and reproducible when an internal standard is used in
combination with generating a standard curve or response factors with pure standards. This requires
obtaining standards and determining their purity by running them by themselves on the GC and determin ing
the purity as the %peak area for all non-s olvent peaks, s ince som etimes the purity is different from the claim
on the label. If a pure standard is not available, then response factors cannot be determined. To solve this
problem, m ost res earchers us e the respons e factor (or standard curve) of the nearest peak with an available
standard. However, the use of theoretical correction factors is also di scus sed below .

Equal Response Factors. Epicoprostanol, dihydrocholesterol, and 5α-cholestane have all

been reported to have similar FID response to phytosterols, and therefore have been used
without determining response factors (RF) or developing a standard curve for the individual
phytosterols [15]. Their equal response may depend on the instrumental conditions of each
lab, and should therefore be verified by plotting the ratio of peak areas for phytosterol
standards to the IS on the Y-axis vs the ratio of the phytosterol amount to the IS amount over 
the on the X-axis. The response of sample concentration vs peak area should be linear, and
the slope of the curve s hould be very near to 1.0. If the respo nse factors are equal, then the
amount or concentration for each phytosterol is calculated using the following form ula:

Eq. 1  CP = [AP x CIS]/AIS

where CP is the phytosterol amo unt or concentration, CIS is the internal standard am ount or 

concentration, AP is the area of the phytosterol peak, and AIS is the area of the internal
standard peak.

Standard Curve. If the respons e factors are not equal for IS and a s tandard, as we have found
for a few phytosterols, especially campesterol, then a standard curve can be developed as
described ab ove, generating a line wi th the equation:

Eq. 2   AP/AIS = m x CP/[CIS + b]

where m = the slope of the line (effectively the empirical response factor for the phytosterol)
and b is the y-intercept. Thus the am ount of phytosterol would be calculated as:

Eq. 3  CP = [CIS/m] x [(AP/AIS) – b]

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Note that when developing this type of standard curve, the IS amount or concentration is
usually left the same, while the phytosterol standard concentration is added in varying
amounts within the range expected in subsequent samples.

Single Point Response Factors. In lieu of developing a standard curve for each s tandard, one
could determine a s ingle point respon se factor (RF) at one concentration for each standard.

Eq. 4  RF = [AP/AIS]/[CP/CIS]

The calculated RF could then be used i n the above equation to determine the am ount of each
phytosterol in a s ample [16].

Theoretical Correction Factors. Theoretical correction factors (TCF) are bas ed on the finding
that the relative molar res ponse of a compound detected by FID is proportional to the number 
of carbons in a hydrocarbon [33]. All non-carbonyl carbons in a compound’s structure are
considered active carbons that contribute to the FID response. Costin and coworkers [34]
tested the use of TCF for the quantitation of TMS-ether derivatives of stigmasterol,
campesterol, and sitosterol using epicoprostanol as an internal standard. They determined
that the TMS carbons contribute to the FID response, and thus should be included in the
calculation of TCF. They also demonstrated that for all of the sterols that they tested, other 
than campesterol, the TCF were similar to the empirically determined response factors. This
may have been affected by the fact that the purity of campesterol standards is often less than

90%. The TCF is calculated as [34]:

Eq. 5  TCF = Active carbon molecular weight/Total sterol molecular weight

Then, the sterol TCF relative to the IS TCF is calculated as:

Eq. 6  Relative TCFP = TCFIS/TCFsterol

Thereafter, the am ount of each phytosterol can be determined by:

Eq. 7  CP = [AP x CIS x Relative TCF]/AIS

The advantages of using TCF are that they are simpler and faster than developing standard
curves for each phytosterol standard, and can be used for accurate quantification of 
phytosterols where there is no pure standard available, as long as the phytosterol is
accurately identified. However, Costin and coworkers [34] also demonstrated that the GC
injection type (split vs. cold-on-column) affected the precision of results, and thus cautioned
that GC conditions should be optimized to improve results using TCF for quantification. TCF
The AOCS are used in the official AOCS method for the ana lysis of the fatty acid com position of vegetable
Lipid Library oils [35], but they are still not utilized m uch for the analysis of phytosterols.

Home References

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Oils & Fats
Chemistry, technology, history,
and as commodi ties
News
+ li nks to useful data sources, a
blog and a calendar of events
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Jill K. Winkler-Moser 

USDA, ARS, NCAUR, Functional Foods

Research Unit, 1815 N Uni versity Street,
Peoria, IL 61611, U.S.A.

email: Jill.moser@ars.usda.gov

Updated: April 27th , 2011 Credits/disclaimer © AOCS

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