Polycystic mass spectrometer, gas chromatography-mass spectrometer, equilibrium dialysis, chemiluminescence

ovary syndrome (PCOS) is the most common endocrine disease that affects women, (Pannill, 2002). It affects
about 12 – 18% of women worldwide, (Sheehan, 2004) and about
22.4% of women in Jordan, (HealthGRove, 2017) PCOS is
also a leading cause of infertility. Women with PCOS may also be affected with
obesity, amenorrhea, oligomenorrhea, infertility, or androgenic abnormalities.
Those with PCOS are also at increased risk for both diabetes and diabetic
complications and cardiovascular disease, with a risk of a myocardial
infarction “7 times the normal,” (Pannill, 2002). The
purpose of this paper is to determine the abnormal androgen level specifically
testosterone using four different methods including liquid chromatography –
mass spectrometer (LC/MS) (Legro et al., 2010),, gas chromatography – mass
spectrometer (GC/MS) (Stener-Victorin et al., 2010)., chemiluminescence immunoassay (DM Styne and MM
Grumbach, 1978), and equilibrium dialysis, (Witchel et al., 2015). Based on the research, the best method for quantitation of
testosterone in blood serum is GC/MS as it is the most reliable and accurate
method as the procedure includes steps for purifying and stabilizing the
analyte, (McDonald, Matthew, &
Auchus, 2011).

Key words: Polycystic ovary syndrome (PCOS), androgen, testosterone,
liquid chromatography- mass spectrometer, gas chromatography-mass spectrometer,
equilibrium dialysis, chemiluminescence immunoassay. 

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One of the most common diseases
that cause hormonal imbalances in women is polycystic ovary syndrome (PCOS). It
affects 12 – 18 % of women worldwide, (Sheehan,
2004). Prevalence of this disease in Jordan alone is about
22.4%, (HealthGRove,
2017). It is a complex condition as there are many
different signs and symptoms and it varies from woman to woman, (Clinic,
1998 – 2017). The major complications that women may suffer from
include but are not limited to irregular menstrual cycles, infertility,
increased acne and hair growth on the face and body, (Watson
& Rose, 2005 – 2017). Over time, complications may
worsen, especially if the woman becomes overweight or obese. At that time,
complications may be harder to control. From the name “polycystic”, we can
conclude that the woman would suffer from multiple cysts on the ovaries. There
are many other symptoms that may cause this disease such as hormonal
abnormalities (Hailes,

The polycystic ovary syndrome
(PCOS) is a hyper androgenic disorder associated with chronic oligo-anovulation
and polycystic ovarian morphology. It is often associated with psychological
impairments, including depression and other mood disorders and metabolic
derangements, chiefly insulin resistance and compensatory hyperinsulinemia,
which is recognized as a major factor responsible for altered androgen
production and metabolism, (Sirmans
& Pate, 2014). In addition, those with PCOS are also at
increased risk for both diabetes and diabetic complications and cardiovascular
disease, with a risk of a myocardial infarction “7 times the normal,” (Pannill, 2002).The definition of PCOS has led to
an impressive increase of scientific interest in this disorder, which should be
further directed to improve individualized clinical approaches and,
consequently therapeutic strategies (Pasquali
et al., 2011).

Ovaries have two main functions
that include releasing an egg and production of hormones. First, the egg is
surrounded by a group of cells that develops into a small cyst called a
follicle. Now, if a woman is having regular menstrual cycles and ovulation,
once the follicle becomes 20 mm in diameter, it is released and passed through
to the fallopian tube. In the case of polycystic ovaries, they will contain
more than one follicle, which contain an egg each and have not reached its
mature size. Instead, they reach a size of about 2-9 mm in diameter. This ovary
will usually contain twelve of these small follicles or cysts. The ovaries are
usually enlarged and their hormone producing tissue is also increased in
size. The diagnosis is best made by an ultrasound scan, which visualizes the
ovaries and the small cysts/follicles within them. Sometimes however that is
not enough or unclear, therefore blood tests are ordered. These tests give an
implication if there are any changes in hormones that may have caused this
complication (Balen,

This brings us to the second main
function of the ovaries, which is the production of hormones including
oestogens, androgens, and progestogens. Having said that,
women produce these hormones in various proportions (Balen, 2015). Women
diagnosed with PCOS generally have abnormalities in more than one hormone
including testosterone (hormone responsible for hair growth), oestrogen
(stimulates the growth of the endometrium), lethenising hormone (ovulation
hormone), and finally insulin. Insulin is a very crucial hormone that is
involved in utilization of energy from the ingested food. Increased levels of
insulin may cause over production of testosterone and therefore prevents the
natural release of eggs after they have been developed into a mature follicle (Balen,
2015). Hence, the ovary becomes polycystic. The high levels
of insulin have other effects in the body; including stimulating the ovaries to
over produce androgens (mainly testosterone), preventing normal ovulation, (Balen, 2015). The major androgen going to be discussed in this paper is

If androgen (testosterone) levels are high, the skin may be
affected. Acne may occur on the face, chest or back. Sometimes, there is also
unwanted hair growth on the face, chest, abdomen, arms and legs, (Balen, 2015). There are many different methods to test the levels of
testosterone in the body. However, in this paper, four major techniques will be
discussed which include, liquid chromatography mass spectrometry (LC/MS (Legro et al., 2010), equilibrium dialysis (Witchel et al., 2015), chemiluminescence immunoassay, and gas chromatography – MS (Stener-Victorin et al., 2010).

Liquid chromatography Mass spectrometry (LC/MS)

The first method used for the detection of testosterone is
liquid chromatography mass spectrometry. According to the Endocrine Society
Position Statement, “… the best prospect for a gold standard lies in the
extraction and chromatography followed by MS or MS-MS in which the chemical
structure of the molecule measured is identified,” (Legro et al., 2010). This is the main reason why the LC/MS method is the most
reliable method  for the purposes of
measuring testosterone levels in blood serum.

            In this technique, 596
women with PCOS were obtained as samples, and 10% of the samples were chosen at
random for duplication. The samples were obtained anonymous that even the lab
personnel did not know that the samples tested were a duplicate. The subjects
were asked to be fasting overnight and off of and hormonal contraceptives and
insulin sensitizing agents, (Legro et al., 2010). In the article cited, the method of LC/MS was used in two
different locations and in different ways.

            First, at Mayo Clinic,
they took about 25 ul of d3- testosterone and added it to a 0.1 ml serum sample
as an internal standard. The mobile phase in this case was acetonitrile. The
sample was then vortexed and incubated for 5 minutes at room temperature. This
is called the protein precipitation step. After that, the supernatant obtained
is extracted using the LC through gradient elution. The resulting fragments
were with analyzed by the mass spectrometer, (Legro et al., 2010).

            On the other hand, a
different clinic called “Quest Diagnostics” also used LC/MS however in a
slightly different matter. They used an analytical sensitivity of 1 ng/dl and
no cross-reactivity with 30 testosterone related steroid compounds. First, the
samples were deproteinized with 10% formic acid into 150 ul of serum. Then 50
ul of the internal standard (1000 ng/dl hexa deuterated testosterone in
methanol) was added. After that the mixture was incubated at room temperature
for 30 minutes. Finally, the mixture was injected into the LC/MS system for
quantification and identification, (Legro et al., 2010).

            Comparing both
experiments to each other, we can conclude that the precision was less with
women who had testosterone values of less than 50 ng/dl and it increased when
the results were higher than 50 ng/dl. However, the blinding of the study
design eliminated bias selection in trials and it is one of the validation
techniques. LC/MS has been recommended as the most preferable assay for
measuring total testosterone after GC/MS, (Legro et al., 2010).  TOF instruments
measure m/z with extreme precision and are very useful for
determining the exact mass of a compound, but historically, these instruments
were not designed for precise quantitation due in part to limited dynamic
range, (Azziz et al., 2009)

Gas Chromatography / Mass Spectrometer

Another method for the separation of testosterone
prior to mass spectrometry is to first employ a chromatographic step such as GC,
which is called the “front end,”(McDonald et al.,
2011). Since ion formation and analysis must occur under high
vacuum, GC was used for their separation prior to MS (GC/MS). That is because
it is very easy to combine these two techniques with respect to vacuum
conditions, (McDonald et al.,

Due to the high resolution of GC and precise
resulting from the MS system, GC/MS has been a very common technique for many
years in the analytical chemistry industry. However, there are some minor
disadvantages to this technique that could be fixed. Consequently, several
steps, including pre-analytical derivatization, are required for analysis by
GC/MS. First, the excreted steroid (testosterone) are separated from salts and
concentrated by solid-phase extraction (SPE). Second, since steroids are mostly
excreted as glucuronide and sulfate conjugates, these charged molecules are
removed chemically or enzymatically by acid or enzyme treatment. Third, the
free hydroxyl groups, and in most cases the ketones as well, are modified
chemically to render the compounds less polar and more volatile. Most commonly,
silylating reagents modify the hydroxyl groups as trimethysilyl or
tert-butyldimethylsilyl ethers. The ketone groups are either converted to silyl
enol ethers with base and the same silylating reagents or converted into oximes
with hydroxylamine before silylating. These derivatives are stable to high
temperatures and more hydrophobic and volatile than the parent steroids, (McDonald et al.,

The disadvantages of the
derivatization approach includes the potential for isomeric mixtures of
derivatives, the substantial time required prior to analysis, and the increased
masses of the compounds after derivatization. Acetates or other simpler groups
are sometimes used, particularly for carbon isotope ratio instruments, which
measure isotopic composition of the carbon atoms in the chemical of interest.
Pentafluorobenzyl esters are attractive derivatives for some purposes, because
the pentafluorobenzyl group easily forms a very stable negative ion. Thus,
these compounds are analyzed in negative ion mode using electron capture
negative ionization, yielding highly sensitive spectra with a large molecular
ion (M-H?·), often with minimal or no fragments, (McDonald
et al., 2011).

When there are many compounds to target using GS/MS, SIM method
can be used to increase sensitivity of the system. This SIM method evidently increases
sensitivity by decreasing the noise in obtaining complete mass spectra. SIM
also allows the instrument to focus its power in selected mass regions versus
wasting time and resources scanning through masses not relevant to the
analysis. Modern instruments can be programmed to measure dozens of ions
simultaneously in SIM mode and quantitate target ions at their specified
elution time,” (McDonald et al., 2011).

Analysis using GC

In this method, “DHEA, 5-androstene-3?,
17?-diol (5-DIOL), 4-DIONE, T, DHT, E1, and E2 were measured with a validated
GC-MS system, which uses a 50% phenyl-methyl polysiloxane (DB-17HT) capillary
column and helium as carrier gas. The analytes and internal standard are
detected with an HP5973 quadrupole mass spectrometer equipped with chemical
ionization source. The limits of detection were 0.10 ng/ml for DHEA, 30 pg/ml
for 5-DIOL, 0.05 ng/ml for 4-DIONE, 0.02 ng/ml for T, 5.00 pg/ml for DHT, 5.00
pg/ml for E1, and 1.00 pg/ml for E2”, (Stener-Victorin et al., 2010). Because there are multiple hormones in the mixture, GC/MS was the best technique to use since it provides specific and
accurate separation needed, (Stener-Victorin et al., 2010).

Equilibrium Dialysis

Another method for detecting testosterone
is equilibrium dialysis, (Miller et al., 2004). This method is a specific application of dialysis that is very
important to the study of binding small molecules and ions to proteins. It is
able to perform an interaction without the use of fluorescent or radioactive
tags. The main objective of equilibrium dialysis is to be able to measure the
amount of ligand bound to a macromolecule. This is usually done indirectly as
it is hard to differentiate between the bound and free ligand. Data obtained
from this method provides information on binding parameters of the compounds as
the binding constants and the number of binding capacity, (Scientific, 2017).

Having said that, equilibrium dialysis is considered the gold standard for the
measurement of free testosterone levels in the blood serum. In the human body, sex hormone-binding globulin (SHBG) concentrations control
the amount of testosterone that is free and, hence, bioavailable. Serum free
testosterone (or the related bioavailable testosterone and free androgen index)
is either calculated as the product of the total testosterone concentration
times the free fraction computed from the SHBG concentration or directly
determined by dialysis. Free testosterone determined by equilibrium dialysis is
presumed to be the most sensitive single test for the diagnosis of hyperandrogenemia.
However, free testosterone assays are less well standardized than the total
testosterone assay, which has limited their usefulness, (Witchel et al., 2015).

First, bioavailable T is measured
after precipitation with 50% ammonium sulfate of sex
hormone-binding globulin (SHBG) from the
blood serum. Then the percentage of non-SHBG-bound is T by tracer binding
methods is calculated. On the other hand we can use direct measurements of T in
the supernatant that contains free and albumin-bound T. Because they are
technically more demanding, time-consuming, and expensive, these assays are not
used by most clinical laboratories, but are available from reference
laboratories, (Matsumoto & Bremner,

Chemiluminescence immunoassay 

measurement of serum testosterone in men is related to the investigation of
testicular dysfunction and is used to monitor the treatment of patients with
congenital adrenal hyperplasia. In women however, serum testosterone is useful
evaluating hirsutism, alopecia and menstrual disorder such as the one being
discussed PCOS.  The traditional assay of
plasma testosterone involves extraction of steroids, chromatographic purification,
RIA and ELISA. This system however, is used to establish a CLIA method for
detection of total testosterone in serum, (Grumbach, 1978).

i. Principle of the test

the Test CLIA test procedure, the testosterone standard or patient serum is
incubated with the testosterone antibody and the testosterone-horseradish
peroxidase conjugate in the anti-mouse IgG coated well. In this solid-phase
system, the antibody bound testosterone will remain on the well while unbound
testosterone will be removed by washing. A chemiluminescence reaction is
developed when the CLIA substrate is mixed with the antibody bound testosterone-horseradish
peroxidase enzyme conjugate. The Related Light Unit (RLU) is proportional to
the amount of enzyme present and is inversely related to the amount of
unlabeled Test in the sample. By reference to a series of Test standards
assayed in the same way, the concentration of Test in the unknown sample is
quantified, (Grumbach, 1978).

Assay Procedure

Secure the desired number of coated wells in the holder. Dispense 25?l of
Reference Standards, specimens, and controls into appropriate wells.

Dispense 50?l of Anti-Test Reagent into each well.

Dispense 50?l of Enzyme Conjugate Reagent into each well. Gently mix for 30

 4. Incubate at room temperature for 60

At the end of the incubation, remove the incubation mixture by emptying the
plate content into a waste container. Rinse and empty the microtiter plate 5
times with wash buffer. Strike the microtiter plate sharply onto absorbent
paper or paper towels to remove all residual water droplets. The volume of each
well is about 300?l.

Dispense 25?l of substrate A, then 25?l of substrate B into each well. Gently
mix for 10 seconds.

Incubate at room temperature in the dark for 10 minutes without shaking, then
read the RLU values with a Luminolmeter,” (Grumbach, 1978).

Calculation Of Results

Calculate the mean value from any duplicate reagents. Where appropriate, the
mean values should be used for plotting. 2009-02 3/5

On linear graph paper plot the RLU (ordinate) for each Reference Standard
against the corresponding concentration of TEST in ng/ml (abscissa) and draw a
calibration curve through the Reference Standard points by connecting the
plotted points with straight lines.

Read the concentration for each control and sample by interpolating on the
calibration curve.

Computer assisted data reduction will simplify these calculations. If automatic
result processing is used, a 4-parameter logistic function curve fitting is
recommended,” (Grumbach, 1978).

iiii. Performance

A. Sensitivity

“The lower detection limit is calculated from
the standard curve by identifying the concentration corresponding to the mean
RLU of standard diluent (based on 10 replicate analyses) subtract 2 SD.
Therefore, the sensitivity of the Autobio Test CLIA kit is not higher than
0.2ng/ml,” (Grumbach, 1978).

B. Specificity

“No interference was detected with the
performance of Autobio Test CLIA upon addition of massive amounts of the following
substances to a human serum pool,” (Grumbach, 1978).

C. Precision

a. Intra-assay Precision:  determined by 20 replicates of each control, (Grumbach, 1978).

b. Inter-assay Precision: determined by duplicates of the each control
in 10 separate runs, (Grumbach, 1978).

E. Accuracy

“The resulting correlation coefficient was 0.9687,” (Grumbach, 1978).

Conclusion, recommendations and future studies

In conclusion, polycystic ovary syndrome (PCOS)
is known to be a complex condition characterized mainly by elevated androgen
levels, menstrual irregularities, and small cysts on one or both ovaries, (Ndefo, Eaton,
& Green, 2013).  The disorder can be physical such as
formation of polycystic ovaries, or predominantly biochemical
hyperandrogenemia. Hyperandrogenism is the main concern as it can cause
inhibition of follicular development, microcysts in the ovaries, anovulation,
and menstrual changes. As mentioned before, PCOS is hormonal disorder with a
potential that can also lead to various diseases such as cardiovascular and
diabetes. It also continues to be a common cause of infertility among women, (Ndefo et al.,
2013). Although signs and symptoms vary the three most common
factors associated with PCOS include ovulation irregularities, increased
androgen levels, and cystic ovaries, (Balen, 2015).

said that, while comparing levels of testosterone in blood serum using the four
techniques in the text (LC/MS, GC/MS, equilibrium dialysis, and
Chemiluminescence immunoassay), we can safely say that the GC/MS is by far the
best technique there is. Although it is a little bit costly and time consuming,
it provides us with accurate and precise measurements of the substance needed
using the additional SIM method described previously, (McDonald et al., 2011).

The wise and foresighted analytical chemist
should adopt this method for clinical investigations related to human biology
in adrenal diseases. Comprehensive evidence-based
guidelines are needed to aid early diagnosis, appropriate investigation,
regular screening and treatment of this common condition to help in prevention
and treatment of women, (Teede, Deeks, & Moran, 2010).